Wnt signaling is involved in human articular chondrocyte de-differentiation in vitro.

PubMed

Sassi, N; Laadhar, L; Allouche, M; Zandieh-Doulabi, B; Hamdoun, M; Klein-Nulend, J; Makni, S; Sellami, S

2014-01-01

Osteoarthritis is the most prevalent form of arthritis in the world. Certain signaling pathways, such as the wnt pathway, are involved in cartilage pathology. Osteoarthritic chondrocytes undergo morphological and biochemical changes that lead to chondrocyte de-differentiation. We investigated whether the Wnt pathway is involved in de-differentiation of human articular chondrocytes in vitro. Human articular chondrocytes were cultured for four passages in the presence or absence of IL-1 in monolayer or micromass culture. Changes in cell morphology were monitored by light microscopy. Protein and gene expression of chondrocyte markers and Wnt pathway components were determined by Western blotting and qPCR after culture. After culturing for four passages, chondrocytes exhibited a fibroblast-like morphology. Collagen type II and aggrecan protein and gene expression decreased, while collagen type I, matrix metalloproteinase 13, and nitric oxide synthase expressions increased. Wnt molecule expression profiles changed; Wnt5a protein expression, the Wnt target gene, c-jun, and in Wnt pathway regulator, sFRP4 increased. Treatment with IL-1 caused chondrocyte morphology to become more filament-like. This change in morphology was accompanied by extinction of col II expression and increased col I, MMP13 and eNOS expression. Changes in expression of the Wnt pathway components also were observed. Wnt7a decreased significantly, while Wnt5a, LRP5, β-catenin and c-jun expressions increased. Culture of human articular chondrocytes with or without IL-1 not only induced chondrocyte de-differentiation, but also changed the expression profiles of Wnt components, which suggests that the Wnt pathway is involved in chondrocyte de-differentiation in vitro.

Murine Mesenchymal Stem Cell Commitment to Differentiation is Regulated by Mitochondrial Dynamics

PubMed Central

Forni, Maria Fernanda; Peloggia, Julia; Trudeau, Kyle; Shirihai, Orian; Kowaltowski, Alicia J.

2015-01-01

Mouse skin mesenchymal stem cells (msMSCs) are dermis CD105+CD90+CD73+CD29+CD34− mesodermal precursors which, after in vitro induction, undergo chondro, adipo and osteogenesis. Extensive metabolic reconfiguration has been found to occur during differentiation, and the bioenergetic status of a cell is known to be dependent on the quality and abundance of the mitochondrial population, which may be regulated by fusion and fission. However, little is known regarding the impact of mitochondrial dynamics on the differentiation process. We addressed this knowledge gap by isolating MSCs from Swiss female mice, inducing these cells to differentiate into osteo, chondro and adipocytes and measuring changes in mass, morphology, dynamics and bioenergetics. Mitochondrial biogenesis was increased in adipogenesis, as evaluated through confocal microscopy, citrate synthase activity and mtDNA content. The early steps of adipo and osteogenesis involved mitochondrial elongation, as well as increased expression of mitochondrial fusion proteins Mfn1 and 2. Chondrogenesis involved a fragmented mitochondrial phenotype, increased expression of fission proteins Drp1, Fis1 and 2 and enhanced mitophagy. These events were accompanied by profound bioenergetic alterations during the commitment period. Moreover, knockdown of Mfn2 in adipo and osteogenesis and the overexpression of a dominant negative form of Drp1 during chondrogenesis resulted in a loss of differentiation ability. Overall, we find that mitochondrial morphology and its regulating processes of fission/fusion are modulated early on during commitment, leading to alterations in the bioenergetic profile that are important for differentiation. We thus propose a central role for mitochondrial dynamics in the maintenance/commitment of mesenchymal stem cells. PMID:26638184

ALS/FTLD-linked TDP-43 regulates neurite morphology and cell survival in differentiated neurons

DOE Office of Scientific and Technical Information (OSTI.GOV)

Han, Jeong-Ho; Yu, Tae-Hoon; Ryu, Hyun-Hee

2013-08-01

Tar-DNA binding protein of 43 kDa (TDP-43) has been characterized as a major component of protein aggregates in brains with neurodegenerative diseases such as frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). However, physiological roles of TDP-43 and early cellular pathogenic effects caused by disease associated mutations in differentiated neurons are still largely unknown. Here, we investigated the physiological roles of TDP-43 and the effects of missense mutations associated with diseases in differentiated cortical neurons. The reduction of TDP-43 by siRNA increased abnormal neurites and decreased cell viability. ALS/FTLD-associated missense mutant proteins (A315T, Q331K, and M337V) were partially mislocalizedmore » to the cytosol and neurites when compared to wild-type and showed abnormal neurites similar to those observed in cases of loss of TDP-43. Interestingly, cytosolic expression of wild-type TDP-43 with mutated nuclear localization signals also induced abnormal neurtie morphology and reduction of cell viability. However, there was no significant difference in the effects of cytosolic expression in neuronal morphology and cell toxicity between wild-type and missense mutant proteins. Thus, our results suggest that mislocalization of missense mutant TDP-43 may contribute to loss of TDP-43 function and affect neuronal morphology, probably via dominant negative action before severe neurodegeneration in differentiated cortical neurons. Highlights: • The function of nuclear TDP-43 in neurite morphology in mature neurons. • Partial mislocalization of TDP-43 missense mutants into cytosol from nucleus. • Abnormal neurite morphology caused by missense mutants of TDP-43. • The effect of cytosolic expression of TDP-43 in neurite morphology and in cell survival.« less

Optimising parameters for the differentiation of SH-SY5Y cells to study cell adhesion and cell migration.

PubMed

Dwane, Susan; Durack, Edel; Kiely, Patrick A

2013-09-11

Cell migration is a fundamental biological process and has an important role in the developing brain by regulating a highly specific pattern of connections between nerve cells. Cell migration is required for axonal guidance and neurite outgrowth and involves a series of highly co-ordinated and overlapping signalling pathways. The non-receptor tyrosine kinase, Focal Adhesion Kinase (FAK) has an essential role in development and is the most highly expressed kinase in the developing CNS. FAK activity is essential for neuronal cell adhesion and migration. The objective of this study was to optimise a protocol for the differentiation of the neuroblastoma cell line, SH-SY5Y. We determined the optimal extracellular matrix proteins and growth factor combinations required for the optimal differentiation of SH-SY5Y cells into neuronal-like cells and determined those conditions that induce the expression of FAK. It was confirmed that the cells were morphologically and biochemically differentiated when compared to undifferentiated cells. This is in direct contrast to commonly used differentiation methods that induce morphological differentiation but not biochemical differentiation. We conclude that we have optimised a protocol for the differentiation of SH-SY5Y cells that results in a cell population that is both morphologically and biochemically distinct from undifferentiated SH-SY5Y cells and has a distinct adhesion and spreading pattern and display extensive neurite outgrowth. This protocol will provide a neuronal model system for studying FAK activity during cell adhesion and migration events.

Regulation of C. elegans presynaptic differentiation and neurite branching via a novel signaling pathway initiated by SAM-10

PubMed Central

Zheng, Qun; Schaefer, Anneliese M.; Nonet, Michael L.

2011-01-01

Little is known about transcriptional control of neurite branching or presynaptic differentiation, events that occur relatively late in neuronal development. Using the Caenorhabditis elegans mechanosensory circuit as an in vivo model, we show that SAM-10, an ortholog of mammalian single-stranded DNA-binding protein (SSDP), functions cell-autonomously in the nucleus to regulate synaptic differentiation, as well as positioning of, a single neurite branch. PLM mechanosensory neurons in sam-10 mutants exhibit abnormal placement of the neurite branch point, and defective synaptogenesis, characterized by an overextended synaptic varicosity, underdeveloped synaptic morphology and disrupted colocalization of active zone and synaptic vesicles. SAM-10 functions coordinately with Lim domain-binding protein 1 (LDB-1), demonstrated by our observations that: (1) mutations in either gene show similar defects in PLM neurons; and (2) LDB-1 is required for SAM-10 nuclear localization. SAM-10 regulates PLM synaptic differentiation by suppressing transcription of prk-2, which encodes an ortholog of the mammalian Pim kinase family. PRK-2-mediated activities of SAM-10 are specifically involved in PLM synaptic differentiation, but not other sam-10 phenotypes such as neurite branching. Thus, these data reveal a novel transcriptional signaling pathway that regulates neuronal specification of neurite branching and presynaptic differentiation. PMID:21115607

Regulation of C. elegans presynaptic differentiation and neurite branching via a novel signaling pathway initiated by SAM-10.

PubMed

Zheng, Qun; Schaefer, Anneliese M; Nonet, Michael L

2011-01-01

Little is known about transcriptional control of neurite branching or presynaptic differentiation, events that occur relatively late in neuronal development. Using the Caenorhabditis elegans mechanosensory circuit as an in vivo model, we show that SAM-10, an ortholog of mammalian single-stranded DNA-binding protein (SSDP), functions cell-autonomously in the nucleus to regulate synaptic differentiation, as well as positioning of, a single neurite branch. PLM mechanosensory neurons in sam-10 mutants exhibit abnormal placement of the neurite branch point, and defective synaptogenesis, characterized by an overextended synaptic varicosity, underdeveloped synaptic morphology and disrupted colocalization of active zone and synaptic vesicles. SAM-10 functions coordinately with Lim domain-binding protein 1 (LDB-1), demonstrated by our observations that: (1) mutations in either gene show similar defects in PLM neurons; and (2) LDB-1 is required for SAM-10 nuclear localization. SAM-10 regulates PLM synaptic differentiation by suppressing transcription of prk-2, which encodes an ortholog of the mammalian Pim kinase family. PRK-2-mediated activities of SAM-10 are specifically involved in PLM synaptic differentiation, but not other sam-10 phenotypes such as neurite branching. Thus, these data reveal a novel transcriptional signaling pathway that regulates neuronal specification of neurite branching and presynaptic differentiation.

Diagnosis and management of congenital dyserythropoietic anemias.

PubMed

Gambale, Antonella; Iolascon, Achille; Andolfo, Immacolata; Russo, Roberta

2016-03-01

Congenital dyserythropoietic anemias (CDAs) are inherited disorders hallmarked by chronic hyporegenerative anemia, relative reticulocytopenia, hemolytic component and iron overload. They represent a subtype of the inherited bone marrow failure syndromes, characterized by impaired differentiation and proliferation of the erythroid lineage. Three classical types were defined by marrow morphology, even if the most recent classification recognized six different genetic types. The pathomechanisms of CDAs are different, but all seem to involve the regulation of DNA replication and cell division. CDAs are often misdiagnosed, since either morphological abnormalities or clinical features can be commonly identified in other clinically-related anemias. However, differential diagnosis is essential for guiding both follow up and management of the patients.

Regulation of osteogenesis by micro/nano hierarchical titanium surfaces through a Rock-Wnt5a feedback loop.

PubMed

Yu, Yonglin; Shen, Xinkun; Liu, Junjie; Hu, Yan; Ran, Qichun; Mu, Caiyun; Cai, Kaiyong

2018-05-28

Titanium substrates with micro/nano hierarchical features could positively mediate the osteogenesis of a titanium implant; nevertheless, the underlying molecular mechanism needs to be further revealed. In this work, we fabricated a micro/nano hierarchically structured Ti (MNT) sample and attempted to evaluate its topography-mediated biological effects and potential molecular mechanisms in vitro. The results proved that MNT could not only affect cell morphology and osteogenic differentiation, but also regulate ROCK activity cell biological functions of osteoblasts involved in ROCK activation, β-catenin accumulation, and high-Wnt5a expression in respect to topographical features. Moreover, blockade of ROCK activation resulted in significant inhibition of cell differentiation and Wnt5a expression. Furthermore, the anti-Wnt5a significantly down-regulated ROCK activity. In short, these results indicate the important role of ROCK-Wnt5a feedback loop in regulating cell differentiation by topographies. Copyright © 2018. Published by Elsevier B.V.

Atomic Force Microscopy Reveals a Morphological Differentiation of Chromobacterium violaceum Cells Associated with Biofilm Development and Directed by N-Hexanoyl-L-Homoserine Lactone

PubMed Central

Kamaeva, Anara A.; Vasilchenko, Alexey S.; Deryabin, Dmitry G.

2014-01-01

Chromobacterium violaceum abounds in soil and water ecosystems in tropical and subtropical regions and occasionally causes severe and often fatal human and animal infections. The quorum sensing (QS) system and biofilm formation are essential for C. violaceum's adaptability and pathogenicity, however, their interrelation is still unknown. C. violaceum's cell and biofilm morphology were examined by atomic force microscopy (AFM) in comparison with growth rates, QS-dependent violacein biosynthesis and biofilm biomass quantification. To evaluate QS regulation of these processes, the wild-type strain C. violaceum ATCC 31532 and its mini-Tn5 mutant C. violaceum NCTC 13274, cultivated with and without the QS autoinducer N-hexanoyl-L-homoserine lactone (C6-HSL), were used. We report for the first time the unusual morphological differentiation of C. violaceum cells, associated with biofilm development and directed by the QS autoinducer. AFM revealed numerous invaginations of the external cytoplasmic membrane of wild-type cells, which were repressed in the mutant strain and restored by exogenous C6-HSL. With increasing bacterial growth, polymer matrix extrusions formed in place of invaginations, whereas mutant cells were covered with a diffusely distributed extracellular substance. Thus, quorum sensing in C. violaceum involves a morphological differentiation that organises biofilm formation and leads to a highly differentiated matrix structure. PMID:25111599

Atomic force microscopy reveals a morphological differentiation of chromobacterium violaceum cells associated with biofilm development and directed by N-hexanoyl-L-homoserine lactone.

PubMed

Kamaeva, Anara A; Vasilchenko, Alexey S; Deryabin, Dmitry G

2014-01-01

Chromobacterium violaceum abounds in soil and water ecosystems in tropical and subtropical regions and occasionally causes severe and often fatal human and animal infections. The quorum sensing (QS) system and biofilm formation are essential for C. violaceum's adaptability and pathogenicity, however, their interrelation is still unknown. C. violaceum's cell and biofilm morphology were examined by atomic force microscopy (AFM) in comparison with growth rates, QS-dependent violacein biosynthesis and biofilm biomass quantification. To evaluate QS regulation of these processes, the wild-type strain C. violaceum ATCC 31532 and its mini-Tn5 mutant C. violaceum NCTC 13274, cultivated with and without the QS autoinducer N-hexanoyl-L-homoserine lactone (C6-HSL), were used. We report for the first time the unusual morphological differentiation of C. violaceum cells, associated with biofilm development and directed by the QS autoinducer. AFM revealed numerous invaginations of the external cytoplasmic membrane of wild-type cells, which were repressed in the mutant strain and restored by exogenous C6-HSL. With increasing bacterial growth, polymer matrix extrusions formed in place of invaginations, whereas mutant cells were covered with a diffusely distributed extracellular substance. Thus, quorum sensing in C. violaceum involves a morphological differentiation that organises biofilm formation and leads to a highly differentiated matrix structure.

Tryptophan promotes morphological and physiological differentiation in Streptomyces coelicolor.

PubMed

Palazzotto, Emilia; Renzone, Giovanni; Fontana, Pietro; Botta, Luigi; Scaloni, Andrea; Puglia, Anna Maria; Gallo, Giuseppe

2015-12-01

The molecular mechanisms regulating tryptophan biosynthesis in actinomycetes are poorly understood; similarly, the possible roles of tryptophan in the differentiation program of microorganism life-cycle are still underexplored. To unveil the possible regulatory effect of this amino acid on gene expression, an integrated study based on quantitative teverse transcription-PCR (qRT-PCR) and proteomic approaches was performed on the actinomycete model Streptomyces coelicolor. Comparative analyses on the microorganism growth in a minimal medium with or without tryptophan supplementation showed that biosynthetic trp gene expression in S. coelicolor is not subjected to a negative regulation by the presence of the end product. Conversely, tryptophan specifically induces the transcription of trp genes present in the biosynthetic gene cluster of the calcium-dependent antibiotic (CDA), a lipopeptide containing D- and L-tryptophan residues. In addition, tryptophan stimulates the transcription of the CDA gene cluster regulator cdaR and, coherently, CDA production. Surprisingly, tryptophan also promotes the production of actinorhodin, another antibiotic that does not contain this amino acid in its structure. Combined 2D-DIGE and nano liquid chromatography electrospray linear ion trap tandem mass spectrometry (LC-ESI-LIT-MS/MS) analyses revealed that tryptophan exerts a growth-stage-dependent global effect on S. coelicolor proteome, stimulating anabolic pathways and promoting the accumulation of key factors associated with morphological and physiological differentiation at the late growth stages. Phenotypic observations by scanning electron microscopy and spore production assays demonstrated an increased sporulation in the presence of tryptophan. Transcriptional analysis of catabolic genes kynA and kynB suggested that the actinomycete also uses tryptophan as a carbon and nitrogen source. In conclusion, this study originally provides the molecular basis underlying the stimulatory effect of tryptophan on the production of antibiotics and morphological development program of this actinomycete.

ROCK inhibitor Y-27632 increases thaw-survival rates and preserves stemness and differentiation potential of human Wharton's jelly stem cells after cryopreservation.

PubMed

Gauthaman, Kalamegam; Fong, Chui-Yee; Subramanian, Arjunan; Biswas, Arijit; Bongso, Ariff

2010-12-01

The ROCK inhibitor Y-27632 inhibits apoptosis and increases proliferation of frozen-thawed cells. We examined the role of Y-27632 on human umbilical cord Wharton's jelly stem cells (hWJSCs) for (1) thaw-survival (2) proliferation and (3) preservation of stemness and differentiation potential after cryopreservation. hWJSCs were allotted to 4 groups [Gp I: Untreated hWJSC controls; Gp II: Pretreatment with Y-27632 (10 μM) for 24 h before freezing; Gp III: Y-27632 (10 μM) in freezing medium and Gp IV: Pretreatment with Y-27632 (10 μM) for 24 h and inclusion in freezing medium]. All groups were frozen using a rapid freezing method and stored at -196°C in liquid nitrogen for 90 days before evaluation for apoptosis, cell proliferation, stemness and differentiation. After thawing, Groups II, III and IV showed improved cell attachment, increased thaw-survival (live/dead cell counts) and increased cell proliferation (Trypan blue and MTT assay) compared to controls. CD marker stemness profiles, morphology and normal karyotypes were maintained in the treatment groups after thawing and there was no obvious evidence of apoptosis (Annexin V-FITC and TUNEL assays). After thawing, qRT-PCR demonstrated up-regulation of the anti-apoptotic BCL2 gene and down-regulation of the pro-apoptotic BAX gene and cell cycle regulators (P53 and P21) in the treatment groups. Treated frozen-thawed hWJSCs from all groups differentiated into a neuronal phenotype (neuronal morphology and expression of GFAP, β-3 tubulin and SOX2). Increased thaw-survival and retention of stemness and differentiation potential in hWJSCs following cryopreservation is useful for their storage in cord blood banks for future regenerative medicine purposes.

Dbl3 drives Cdc42 signaling at the apical margin to regulate junction position and apical differentiation

PubMed Central

Zihni, Ceniz; Munro, Peter M.G.; Elbediwy, Ahmed; Keep, Nicholas H.; Terry, Stephen J.; Harris, John

2014-01-01

Epithelial cells develop morphologically characteristic apical domains that are bordered by tight junctions, the apical–lateral border. Cdc42 and its effector complex Par6–atypical protein kinase c (aPKC) regulate multiple steps during epithelial differentiation, but the mechanisms that mediate process-specific activation of Cdc42 to drive apical morphogenesis and activate the transition from junction formation to apical differentiation are poorly understood. Using a small interfering RNA screen, we identify Dbl3 as a guanine nucleotide exchange factor that is recruited by ezrin to the apical membrane, that is enriched at a marginal zone apical to tight junctions, and that drives spatially restricted Cdc42 activation, promoting apical differentiation. Dbl3 depletion did not affect junction formation but did affect epithelial morphogenesis and brush border formation. Conversely, expression of active Dbl3 drove process-specific activation of the Par6–aPKC pathway, stimulating the transition from junction formation to apical differentiation and domain expansion, as well as the positioning of tight junctions. Thus, Dbl3 drives Cdc42 signaling at the apical margin to regulate morphogenesis, apical–lateral border positioning, and apical differentiation. PMID:24379416

The genetics of colony form and function in Caribbean Acropora corals.

PubMed

Hemond, Elizabeth M; Kaluziak, Stefan T; Vollmer, Steven V

2014-12-17

Colonial reef-building corals have evolved a broad spectrum of colony morphologies based on coordinated asexual reproduction of polyps on a secreted calcium carbonate skeleton. Though cnidarians have been shown to possess and use similar developmental genes to bilaterians during larval development and polyp formation, little is known about genetic regulation of colony morphology in hard corals. We used RNA-seq to evaluate transcriptomic differences between functionally distinct regions of the coral (apical branch tips and branch bases) in two species of Caribbean Acropora, the staghorn coral, A. cervicornis, and the elkhorn coral, A. palmata. Transcriptome-wide gene profiles differed significantly between different parts of the coral colony as well as between species. Genes showing differential expression between branch tips and bases were involved in developmental signaling pathways, such as Wnt, Notch, and BMP, as well as pH regulation, ion transport, extracellular matrix production and other processes. Differences both within colonies and between species identify a relatively small number of genes that may contribute to the distinct "staghorn" versus "elkhorn" morphologies of these two sister species. The large number of differentially expressed genes supports a strong division of labor between coral branch tips and branch bases. Genes involved in growth of mature Acropora colonies include the classical signaling pathways associated with development of cnidarian larvae and polyps as well as morphological determination in higher metazoans.

SIGNALS AND REGULATORS THAT GOVERN STREPTOMYCES DEVELOPMENT

PubMed Central

McCormick, Joseph R.; Flärdh, Klas

2012-01-01

Streptomyces coelicolor is the genetically best characterized species of a populous genus belonging to the Gram-positive Actinobacteria. Streptomycetes are filamentous soil organisms, well known for the production of a plethora of biologically active secondary metabolic compounds. The Streptomyces developmental life cycle is uniquely complex, and involves coordinated multicellular development with both physiological and morphological differentiation of several cell types, culminating in production of secondary metabolites and dispersal of mature spores. This review presents a current appreciation of the signaling mechanisms used to orchestrate the decision to undergo morphological differentiation, and the regulators and regulatory networks that direct the intriguing development of multigenomic hyphae, first to form specialized aerial hyphae, and then to convert them into chains of dormant spores. This current view of S. coelicolor development is destined for rapid evolution as data from “-omics” studies shed light on gene regulatory networks, new genetic screens identify hitherto unknown players, and the resolution of our insights into the underlying cell biological processes steadily improve. PMID:22092088

A pathway to bone: signaling molecules and transcription factors involved in chondrocyte development and maturation

PubMed Central

Kozhemyakina, Elena; Lassar, Andrew B.; Zelzer, Elazar

2015-01-01

Decades of work have identified the signaling pathways that regulate the differentiation of chondrocytes during bone formation, from their initial induction from mesenchymal progenitor cells to their terminal maturation into hypertrophic chondrocytes. Here, we review how multiple signaling molecules, mechanical signals and morphological cell features are integrated to activate a set of key transcription factors that determine and regulate the genetic program that induces chondrogenesis and chondrocyte differentiation. Moreover, we describe recent findings regarding the roles of several signaling pathways in modulating the proliferation and maturation of chondrocytes in the growth plate, which is the ‘engine’ of bone elongation. PMID:25715393

Optimising parameters for the differentiation of SH-SY5Y cells to study cell adhesion and cell migration

PubMed Central

2013-01-01

Background Cell migration is a fundamental biological process and has an important role in the developing brain by regulating a highly specific pattern of connections between nerve cells. Cell migration is required for axonal guidance and neurite outgrowth and involves a series of highly co-ordinated and overlapping signalling pathways. The non-receptor tyrosine kinase, Focal Adhesion Kinase (FAK) has an essential role in development and is the most highly expressed kinase in the developing CNS. FAK activity is essential for neuronal cell adhesion and migration. Results The objective of this study was to optimise a protocol for the differentiation of the neuroblastoma cell line, SH-SY5Y. We determined the optimal extracellular matrix proteins and growth factor combinations required for the optimal differentiation of SH-SY5Y cells into neuronal-like cells and determined those conditions that induce the expression of FAK. It was confirmed that the cells were morphologically and biochemically differentiated when compared to undifferentiated cells. This is in direct contrast to commonly used differentiation methods that induce morphological differentiation but not biochemical differentiation. Conclusions We conclude that we have optimised a protocol for the differentiation of SH-SY5Y cells that results in a cell population that is both morphologically and biochemically distinct from undifferentiated SH-SY5Y cells and has a distinct adhesion and spreading pattern and display extensive neurite outgrowth. This protocol will provide a neuronal model system for studying FAK activity during cell adhesion and migration events. PMID:24025096

Development of sodium channel protein during chemically induced differentiation of neuroblastoma cells.

PubMed

Baumgold, J; Spector, I

1987-04-01

We have previously shown that the [3H]saxitoxin binding site of the sodium channel is expressed independently of the [125I]scorpion toxin binding site in chick muscle cultures and in rat brain. In the present work, we studied the development of the sodium channel protein during chemically induced differentiation of N1E-115 neuroblastoma cells, using [3H]saxitoxin binding, [125I]scorpion toxin binding, and 22Na uptake techniques. When grown in their normal culture medium, these cells are mostly undifferentiated, bind 90 +/- 10 fmol of [3H]saxitoxin/mg of protein and 112 +/- 14 fmol of [125I]scorpion toxin/mg protein, and, when stimulated with scorpion toxin and batrachotoxin, take up 70 +/- 5 nmol of 22Na/min/mg of protein. Cells treated with dimethyl sulfoxide (DMSO) or hexamethylene-bis-acetamide (HMBA) differentiate morphologically within 3 days. At this time, the [3H]saxitoxin binding, the [125I]scorpion toxin binding, and the 22Na uptake values are not very different from those of undifferentiated cells. With subsequent time in DMSO or HMBA, these values continue to increase, a result indicating that the main period of sodium channel expression occurs well after the cells have assumed the morphologically differentiated state. The data indicate that the expression of sodium channels and morphological differentiation are independently regulated neuronal properties, that the attainment of morphological differentiation is necessary but not in itself sufficient for full expression of the sodium channel proteins, and that, in contrast to the chick muscle cultures and rat brain, the [3H]saxitoxin site and [125I]scorpion toxin site appear to be coregulated in N1E-115 cells.

Brain cytoplasmic RNA 1 suppresses smooth muscle differentiation and vascular development in mice.

PubMed

Wang, Yung-Chun; Chuang, Ya-Hui; Shao, Qiang; Chen, Jian-Fu; Chen, Shi-You

2018-04-13

The cardiovascular system develops during the early stages of embryogenesis, and differentiation of smooth muscle cells (SMCs) is essential for that process. SMC differentiation is critically regulated by transforming growth factor (TGF)-β/SMAD family member 3 (SMAD3) signaling, but other regulators may also play a role. For example, long noncoding RNAs (lncRNAs) regulate various cellular activities and events, such as proliferation, differentiation, and apoptosis. However, whether long noncoding RNAs also regulate SMC differentiation remains largely unknown. Here, using the murine cell line C3H10T1/2, we found that brain cytoplasmic RNA 1 (BC1) is an important regulator of SMC differentiation. BC1 overexpression suppressed, whereas BC1 knockdown promoted, TGF-β-induced SMC differentiation, as indicated by altered cell morphology and expression of multiple SMC markers, including smooth muscle α-actin (αSMA), calponin, and smooth muscle 22α (SM22α). BC1 appeared to block SMAD3 activity and inhibit SMC marker gene transcription. Mechanistically, BC1 bound to SMAD3 via RNA SMAD-binding elements (rSBEs) and thus impeded TGF-β-induced SMAD3 translocation to the nucleus. This prevented SMAD3 from binding to SBEs in SMC marker gene promoters, an essential event in SMC marker transcription. In vivo , BC1 overexpression in mouse embryos impaired vascular SMC differentiation, leading to structural defects in the artery wall, such as random breaks in the elastic lamina, abnormal collagen deposition on SM fibers, and disorganized extracellular matrix proteins in the media of the neonatal aorta. Our results suggest that BC1 is a suppressor of SMC differentiation during vascular development. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Kidins220/ARMS depletion is associated with the neural-to Schwann-like transition in a human neuroblastoma cell line model.

PubMed

Rogers, Danny A; Schor, Nina F

2013-03-10

Peripheral neuroblastic tumors exist as a heterogeneous mixture of neuroblastic (N-type) cells and Schwannian stromal (S-type) cells. These stromal cells not only represent a differentiated and less aggressive fraction of the tumor, but also have properties that can influence the further differentiation of nearby malignant cells. In vitro neuroblastoma cultures exhibit similar heterogeneity with N-type and S-type cells representing the neuroblastic and stromal portions of the tumor, respectively, in behavior, morphology, and molecular expression patterns. In this study, we deplete kinase D-interacting substrate of 220kD (Kidins220) with an shRNA construct and thereby cause morphologic transition of the human SH-SY5Y neuroblastoma cell line from N-type to S-type. The resulting cells have similar morphology and expression profile to SH-EP1 cells, a native S-type cell line from the same parent cell line, and to SH-SY5Y cells treated with BrdU, a treatment that induces S-type morphology. Specifically, both Kidins220-deficient SH-SY5Y cells and native SH-EP1 cells demonstrate down-regulation of the genes DCX and STMN2, markers for the neuronal lineage. We further show that Kidins220, DCX and STMN2 are co-down-regulated in cells of S-type morphology generated by methods other than Kidins220 depletion. Finally, we report that the association of low Kidins220 expression with S-type morphology and low DCX and STMN2 expression is demonstrated in spontaneously occurring human peripheral neuroblastic tumors. We propose that Kidins220 is critical in N- to S-type transition of neural crest tumor cells. Copyright © 2013 Elsevier Inc. All rights reserved.

Morphological and proteomic analysis of early stage of osteoblast differentiation in osteoblastic progenitor cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hong, Dun; Orthopedic Department, Taizhou Hospital, Wenzhou Medical College, Linhai, Zhejiang 317000; Chen, Hai-Xiao, E-mail: Hxchen-1@163.net

Bone remodeling relies on a dynamic balance between bone formation and resorption, mediated by osteoblasts and osteoclasts, respectively. Under certain stimuli, osteoprogenitor cells may differentiate into premature osteoblasts and further into mature osteoblasts. This process is marked by increased alkaline phosphatase (ALP) activity and mineralized nodule formation. In this study, we induced osteoblast differentiation in mouse osteoprogenitor MC3T3-E1 cells and divided the process into three stages. In the first stage (day 3), the MC3T3-E1 cell under osteoblast differentiation did not express ALP or deposit a mineralized nodule. In the second stage, the MC3T3-E1 cell expressed ALP but did not formmore » a mineralized nodule. In the third stage, the MC3T3-E1 cell had ALP activity and formed mineralized nodules. In the present study, we focused on morphological and proteomic changes of MC3T3-E1 cells in the early stage of osteoblast differentiation - a period when premature osteoblasts transform into mature osteoblasts. We found that mean cell area and mean stress fiber density were increased in this stage due to enhanced cell spreading and decreased cell proliferation. We further analyzed the proteins in the signaling pathway of regulation of the cytoskeleton using a proteomic approach and found upregulation of IQGAP1, gelsolin, moesin, radixin, and Cfl1. After analyzing the focal adhesion signaling pathway, we found the upregulation of FLNA, LAMA1, LAMA5, COL1A1, COL3A1, COL4A6, and COL5A2 as well as the downregulation of COL4A1, COL4A2, and COL4A4. In conclusion, the signaling pathway of regulation of the cytoskeleton and focal adhesion play critical roles in regulating cell spreading and actin skeleton formation in the early stage of osteoblast differentiation.« less

The expression and activity of thioredoxin reductase 1 splice variants v1 and v2 regulate the expression of genes associated with differentiation and adhesion

PubMed Central

Nalvarte, Ivan; Damdimopoulos, Anastasios E.; Rüegg, Joëlle; Spyrou, Giannis

2015-01-01

The mammalian redox-active selenoprotein thioredoxin reductase (TrxR1) is a main player in redox homoeostasis. It transfers electrons from NADPH to a large variety of substrates, particularly to those containing redox-active cysteines. Previously, we reported that the classical form of cytosolic TrxR1 (TXNRD1_v1), when overexpressed in human embryonic kidney cells (HEK-293), prompted the cells to undergo differentiation [Nalvarte et al. (2004) J. Biol. Chem. 279, 54510–54517]. In the present study, we show that several genes associated with differentiation and adhesion are differentially expressed in HEK-293 cells stably overexpressing TXNRD1_v1 compared with cells expressing its splice variant TXNRD1_v2. Overexpression of these two splice forms resulted in distinctive effects on various aspects of cellular functions including gene regulation patterns, alteration of growth rate, migration and morphology and susceptibility to selenium-induced toxicity. Furthermore, differentiation of the neuroblastoma cell line SH-SY5Y induced by all-trans retinoic acid (ATRA) increased both TXNRD1_v1 and TXNRD1_v2 expressions along with several of the identified genes associated with differentiation and adhesion. Selenium supplementation in the SH-SY5Y cells also induced a differentiated morphology and changed expression of the adhesion protein fibronectin 1 and the differentiation marker cadherin 11, as well as different temporal expression of the studied TXNRD1 variants. These data suggest that both TXNRD1_v1 and TXNRD1_v2 have distinct roles in differentiation, possibly by altering the expression of the genes associated with differentiation, and further emphasize the importance in distinguishing each unique action of different TrxR1 splice forms, especially when studying the gene silencing or knockout of TrxR1. PMID:26464515

In-vitro analysis of Quantum Molecular Resonance effects on human mesenchymal stromal cells

PubMed Central

Sella, Sabrina; Adami, Valentina; Amati, Eliana; Bernardi, Martina; Chieregato, Katia; Gatto, Pamela; Menarin, Martina; Pozzato, Alessandro; Pozzato, Gianantonio; Astori, Giuseppe

2018-01-01

Electromagnetic fields play an essential role in cellular functions interfering with cellular pathways and tissue physiology. In this context, Quantum Molecular Resonance (QMR) produces waves with a specific form at high-frequencies (4–64 MHz) and low intensity through electric fields. We evaluated the effects of QMR stimulation on bone marrow derived mesenchymal stromal cells (MSC). MSC were treated with QMR for 10 minutes for 4 consecutive days for 2 weeks at different nominal powers. Cell morphology, phenotype, multilineage differentiation, viability and proliferation were investigated. QMR effects were further investigated by cDNA microarray validated by real-time PCR. After 1 and 2 weeks of QMR treatment morphology, phenotype and multilineage differentiation were maintained and no alteration of cellular viability and proliferation were observed between treated MSC samples and controls. cDNA microarray analysis evidenced more transcriptional changes on cells treated at 40 nominal power than 80 ones. The main enrichment lists belonged to development processes, regulation of phosphorylation, regulation of cellular pathways including metabolism, kinase activity and cellular organization. Real-time PCR confirmed significant increased expression of MMP1, PLAT and ARHGAP22 genes while A2M gene showed decreased expression in treated cells compared to controls. Interestingly, differentially regulated MMP1, PLAT and A2M genes are involved in the extracellular matrix (ECM) remodelling through the fibrinolytic system that is also implicated in embryogenesis, wound healing and angiogenesis. In our model QMR-treated MSC maintained unaltered cell phenotype, viability, proliferation and the ability to differentiate into bone, cartilage and adipose tissue. Microarray analysis may suggest an involvement of QMR treatment in angiogenesis and in tissue regeneration probably through ECM remodelling. PMID:29293552

The TUNEL assay suggests mandibular regression by programmed cell death during presoldier differentiation in the nasute termite Nasutitermes takasagoensis

NASA Astrophysics Data System (ADS)

Toga, Kouhei; Yoda, Shinichi; Maekawa, Kiyoto

2011-09-01

Termite soldiers are the most specialized caste of social insects in terms of their morphology and function. Soldier development requires increased juvenile hormone (JH) titer and the two molts via a presoldier stage. These molts are accompanied by dramatic morphological changes, including the exaggeration and regression of certain organs. Soldiers of the most apical termitid subfamily Nasutitermitinae possess not only a horn-like frontal tube, called the nasus, for the projection of defensive chemicals from the frontal gland reservoir but also regressed mandibles. Although candidate genes regulating soldier mandibular growth were reported in a relatively basal termite species, the regulatory mechanisms of mandibular regression remain unknown. To clarify these mechanisms, we performed morphological and histological examinations of the mandibles during soldier differentiation in Nasutitermes takasagoensis. Mandibular size reduced dramatically during soldier differentiation, and mandibular regression occurred just prior to the presoldier molt. Spotted TUNEL signals were observed in regressing mandibles of presoldiers, suggesting that the regression involved programmed cell death. Because soldiers of N. takasagoensis possess exaggerated organs (nasus and frontal gland), the present results suggest that JH-dependent regressive mechanisms exist in the mandibles without interfering with the formation of the exaggerated organs.

Transcriptional profiling of predator-induced phenotypic plasticity in Daphnia pulex.

PubMed

Rozenberg, Andrey; Parida, Mrutyunjaya; Leese, Florian; Weiss, Linda C; Tollrian, Ralph; Manak, J Robert

2015-01-01

Predator-induced defences are a prominent example of phenotypic plasticity found from single-celled organisms to vertebrates. The water flea Daphnia pulex is a very convenient ecological genomic model for studying predator-induced defences as it exhibits substantial morphological changes under predation risk. Most importantly, however, genetically identical clones can be transcriptionally profiled under both control and predation risk conditions and be compared due to the availability of the sequenced reference genome. Earlier gene expression analyses of candidate genes as well as a tiled genomic microarray expression experiment have provided insights into some genes involved in predator-induced phenotypic plasticity. Here we performed the first RNA-Seq analysis to identify genes that were differentially expressed in defended vs. undefended D. pulex specimens in order to explore the genetic mechanisms underlying predator-induced defences at a qualitatively novel level. We report 230 differentially expressed genes (158 up- and 72 down-regulated) identified in at least two of three different assembly approaches. Several of the differentially regulated genes belong to families of paralogous genes. The most prominent classes amongst the up-regulated genes include cuticle genes, zinc-metalloproteinases and vitellogenin genes. Furthermore, several genes from this group code for proteins recruited in chromatin-reorganization or regulation of the cell cycle (cyclins). Down-regulated gene classes include C-type lectins, proteins involved in lipogenesis, and other families, some of which encode proteins with no known molecular function. The RNA-Seq transcriptome data presented in this study provide important insights into gene regulatory patterns underlying predator-induced defences. In particular, we characterized different effector genes and gene families found to be regulated in Daphnia in response to the presence of an invertebrate predator. These effector genes are mostly in agreement with expectations based on observed phenotypic changes including morphological alterations, i.e., expression of proteins involved in formation of protective structures and in cuticle strengthening, as well as proteins required for resource re-allocation. Our findings identify key genetic pathways associated with anti-predator defences.

Signals of Ezh2, Src, and Akt Involve in Myostatin-Pax7 Pathways Regulating the Myogenic Fate Determination during the Sheep Myoblast Proliferation and Differentiation

PubMed Central

Li, Li; Liu, Ruizao; Zhang, Li; Zhao, Fuping; Lu, Jian; Zhang, Xiaoning; Du, Lixin

2015-01-01

Myostatin and Pax7 have been well documented individually, however, the mechanism by which Myostatin regulates Pax7 is seldom reported. Here, based on muscle transcriptome analysis in Texel (Myostatin mutant) and Ujumqin (wild type) sheep across the five fetal stages, we constructed and examined the Myostatin-Pax7 pathways in muscle. Then we validated the signals by RNAi in the proliferating and differentiating sheep myoblasts in vitro at mRNA, protein, and cell morphological levels. We reveal that Myostatin signals to Pax7 at least through Ezh2, Src, and Akt during the sheep myoblast proliferation and differentiation. Other signals such as p38MAPK, mTOR, Erk1/2, Wnt, Bmp2, Smad, Tgfb1, and p21 are most probably involved in the Myostatin-affected myogenic events. Myostatin knockdown significantly reduces the counts of nucleus and myotube, but not the fusion index of myoblasts during cell differentiation. In addition, findings also indicate that Myostatin is required for normal myogenic differentiation of the sheep myoblasts, which is different from the C2C12 myoblasts. We expand the regulatory network of Myostatin-Pax7 pathways and first illustrate that Myostatin as a global regulator participates in the epigenetic events involved in myogenesis, which contributes to understand the molecular mechanism of Myostatin in regulation of myogenesis. PMID:25811841

Regulation of T-type Ca2+ channel expression by herpes simplex virus-1 infection in sensory-like ND7 cells

PubMed Central

Zhang, Qiaojuan; Hsia, Shao-Chung

2017-01-01

Infection of sensory neurons by herpes simplex virus (HSV)-1 disrupts electrical excitability, altering pain sensory transmission. Because of their low threshold for activation, functional expression of T-type Ca2+ channels regulates various cell functions, including neuronal excitability and neuronal communication. In this study, we have tested the effect of HSV-1 infection on the functional expression of T-type Ca2+ channels in differentiated ND7-23 sensory-like neurons. Voltage-gated Ca2+ currents were measured using whole cell patch clamp recordings in differentiated ND7-23 neurons under various culture conditions. Differentiation of ND7-23 cells evokes a significant increase in T-type Ca2+ current densities. Increased T-type Ca2+ channel expression promotes the morphological differentiation of ND7-23 cells and triggers a rebound depolarization. HSV-1 infection of differentiated ND7-23 cells causes a significant loss of T-type Ca2+ channels from the membrane. HSV-1 evoked reduction in the functional expression of T-type Ca2+ channels is mediated by several factors, including decreased expression of Cav3.2 T-type Ca2+ channel subunits and disruption of endocytic transport. Decreased functional expression of T-type Ca2+ channels by HSV-1 infection requires protein synthesis and viral replication, but occurs independently of Egr-1 expression. These findings suggest that infection of neuron-like cells by HSV-1 causes a significant disruption in the expression of T-type Ca2+ channels, which can results in morphological and functional changes in electrical excitability. PMID:28639215

Drug-activated multiple pathways of defensin mRNA regulation in HL-60 cells are defined by reversed roles of participating protein kinases.

PubMed

Herwig, S; Su, Q; Tempst, P

1998-10-01

Defensin transcription in HL-60 promyelocytic leukemia cells is greatly enhanced during retinoic acid (RA)-induced differentiation. We have probed this regulatory pathway by selective modulation of various kinase activities. Induction was potentiated by elevated cAMP and attenuated by protein kinase C inhibition, entirely correlated to enhanced or blocked morphological differentiation, respectively. Yet, defensin mRNA was also induced in undifferentiated HL-60 cells, but not in others, by cAMP alone. By contrast, modulators that cooperated with RA had adverse effects on the normal capacity of dimethyl sulfoxide to up regulate these transcripts as well. Thus, defensin mRNA accumulation can be selectively uncoupled from maturation stage; and transcript levels may be regulated by multiple pathways, each independently acted upon by different chemical inducers.

Expression of transcription factors during sodium phenylacetate induced erythroid differentiation in K562 cells.

PubMed

Rath, A V; Schmahl, G E; Niemeyer, C M

1997-01-01

During 15 days of treatment of K562 cells with sodium phenylacetate, we observed an increase in the cellular hemoglobin concentration with a similar increase in the expression of gamma-globin mRNA. Morphological studies demonstrated characteristic features of erythroid differentiation and maturation. At the same time there was no change in the level of expression of the cell surface antigenes CD33, CD34, CD45, CD71 and glycophorin A. Likewise, the level of expression of the erythroid transcription factors GATA-1, GATA-2, NF-E2, SCL and RBTN2, all expressed in untreated K562 cells, did not increase during sodium phenylacetate induced erythroid differentiation. The expression of the nuclear factors Evi-1 and c-myb, known to inhibit erythroid differentiation, did not decrease. We conclude that sodium phenylacetate treatment of K562 cells increases gamma-globin mRNA and induces cell maturation as judged by morphology without affecting the expression of the erythroid transcription factors, some of which are known to be involved in the regulation of beta-like globin genes.

Time-dependent regulation of morphological changes and cartilage differentiation markers in the mouse pubic symphysis during pregnancy and postpartum recovery.

PubMed

Castelucci, Bianca Gazieri; Consonni, Sílvio Roberto; Rosa, Viviane Souza; Sensiate, Lucimara Aparecida; Delatti, Paula Cristina Rugno; Alvares, Lúcia Elvira; Joazeiro, Paulo Pinto

2018-01-01

Animal models commonly serve as a bridge between in vitro experiments and clinical applications; however, few physiological processes in adult animals are sufficient to serve as proof-of-concept models for cartilage regeneration. Intriguingly, some rodents, such as young adult mice, undergo physiological connective tissue modifications to birth canal elements such as the pubic symphysis during pregnancy; therefore, we investigated whether the differential expression of cartilage differentiation markers is associated with cartilaginous tissue morphological modifications during these changes. Our results showed that osteochondral progenitor cells expressing Runx2, Sox9, Col2a1 and Dcx at the non-pregnant pubic symphysis proliferated and differentiated throughout pregnancy, giving rise to a complex osteoligamentous junction that attached the interpubic ligament to the pubic bones until labour occurred. After delivery, the recovery of pubic symphysis cartilaginous tissues was improved by the time-dependent expression of these chondrocytic lineage markers at the osteoligamentous junction. This process potentially recapitulates embryologic chondrocytic differentiation to successfully recover hyaline cartilaginous pads at 10 days postpartum. Therefore, we propose that this physiological phenomenon represents a proof-of-concept model for investigating the mechanisms involved in cartilage restoration in adult animals.

Parallel RNAi screens across different cell lines identify generic and cell type-specific regulators of actin organization and cell morphology.

PubMed

Liu, Tao; Sims, David; Baum, Buzz

2009-01-01

In recent years RNAi screening has proven a powerful tool for dissecting gene functions in animal cells in culture. However, to date, most RNAi screens have been performed in a single cell line, and results then extrapolated across cell types and systems. Here, to dissect generic and cell type-specific mechanisms underlying cell morphology, we have performed identical kinome RNAi screens in six different Drosophila cell lines, derived from two distinct tissues of origin. This analysis identified a core set of kinases required for normal cell morphology in all lines tested, together with a number of kinases with cell type-specific functions. Most significantly, the screen identified a role for minibrain (mnb/DYRK1A), a kinase associated with Down's syndrome, in the regulation of actin-based protrusions in CNS-derived cell lines. This cell type-specific requirement was not due to the peculiarities in the morphology of CNS-derived cells and could not be attributed to differences in mnb expression. Instead, it likely reflects differences in gene expression that constitute the cell type-specific functional context in which mnb/DYRK1A acts. Using parallel RNAi screens and gene expression analyses across cell types we have identified generic and cell type-specific regulators of cell morphology, which include mnb/DYRK1A in the regulation of protrusion morphology in CNS-derived cell lines. This analysis reveals the importance of using different cell types to gain a thorough understanding of gene function across the genome and, in the case of kinases, the difficulties of using the differential gene expression to predict function.

Dopamine is a key regulator in the signalling pathway underlying predator-induced defences in Daphnia

PubMed Central

Weiss, Linda C.; Leese, Florian; Laforsch, Christian; Tollrian, Ralph

2015-01-01

The waterflea Daphnia is a model to investigate the genetic basis of phenotypic plasticity resulting from one differentially expressed genome. Daphnia develops adaptive phenotypes (e.g. morphological defences) thwarting predators, based on chemical predator cue perception. To understand the genomic basis of phenotypic plasticity, the description of the precedent cellular and neuronal mechanisms is fundamental. However, key regulators remain unknown. All neuronal and endocrine stimulants were able to modulate but not induce defences, indicating a pathway of interlinked steps. A candidate able to link neuronal with endocrine responses is the multi-functional amine dopamine. We here tested its involvement in trait formation in Daphnia pulex and Daphnia longicephala using an induction assay composed of predator cues combined with dopaminergic and cholinergic stimulants. The mere application of both stimulants was sufficient to induce morphological defences. We determined dopamine localization in cells found in close association with the defensive trait. These cells serve as centres controlling divergent morphologies. As a mitogen and sclerotization agent, we anticipate that dopamine is involved in proliferation and structural formation of morphological defences. Furthermore, dopamine pathways appear to be interconnected with endocrine pathways, and control juvenile hormone and ecdysone levels. In conclusion, dopamine is suggested as a key regulator of phenotypic plasticity. PMID:26423840

Novel developmental biology-based protocol of embryonic stem cell differentiation to morphologically sound and functional yet immature hepatocytes.

PubMed

Bukong, Terence N; Lo, Tracie; Szabo, Gyongyi; Dolganiuc, Angela

2012-05-01

Liver diseases are common in the United States and often require liver transplantation; however, donated organs are limited and thus alternative sources for liver cells are in high demand. Embryonic stem cells (ESC) can provide a continuous and readily available source of liver cells. ESC differentiation to liver cells is yet to be fully understood and comprehensive differentiation protocols are yet to be defined. Here, we aimed to achieve human (h)ESC differentiation into mature hepatocytes using defined recombinant differentiation factors and metabolites. Embryonic stem cell H1 line was sub-cultured on feeder layer. We induced hESCs into endodermal differentiation succeeded by early/late hepatic specification and finally into hepatocyte maturation using step combinations of Activin A and fibroblast growth factor (FGF)-2 for 7 days; followed by FGF-4 and bone morphogenic protein 2 (BMP2) for 7 days, succeeded by FGF-10 + hepatocyte growth factor 4 + epidermal growth factor for 14 days. Specific inhibitors/stimulators were added sequentially throughout differentiation. Cells were analysed by PCR, flow cytometry, microscopy or functional assays. Our hESC differentiation protocol resulted in viable cells with hepatocyte shape and morphology. We observed gradual changes in cell transcriptome, including up-regulation of differentiation-promoting GATA4, GATA6, POU5F1 and HNF4 transcription factors, steady levels of stemness-promoting SOX-2 and low levels of Nanog, as defined by PCR. The hESC-derived hepatocytes expressed alpha-antitrypsin, CD81, cytokeratin 8 and low density lipoprotein (LDL) receptor. The levels of alpha-fetoprotein and proliferation marker Ki-67 in hESC-derived hepatocytes remained elevated. Unlike stem cells, the hESC-derived hepatocytes performed LDL uptake, produced albumin and alanine aminotransferase and had functional alcohol dehydrogenase. We report a novel protocol for hESC differentiation into morphological and functional yet immature hepatocytes as an alternative method for hepatocyte generation. © 2012 John Wiley & Sons A/S.

The -omics Era- Toward a Systems-Level Understanding of Streptomyces

PubMed Central

Zhou, Zhan; Gu, Jianying; Du, Yi-Ling; Li, Yong-Quan; Wang, Yufeng

2011-01-01

Streptomyces is a group of soil bacteria of medicinal, economic, ecological, and industrial importance. It is renowned for its complex biology in gene regulation, antibiotic production, morphological differentiation, and stress response. In this review, we provide an overview of the recent advances in Streptomyces biology inspired by -omics based high throughput technologies. In this post-genomic era, vast amounts of data have been integrated to provide significant new insights into the fundamental mechanisms of system control and regulation dynamics of Streptomyces. PMID:22379394

Cellular responses during morphological transformation in Azospirillum brasilense and Its flcA knockout mutant.

PubMed

Hou, Xingsheng; McMillan, Mary; Coumans, Joëlle V F; Poljak, Anne; Raftery, Mark J; Pereg, Lily

2014-01-01

FlcA is a response regulator controlling flocculation and the morphological transformation of Azospirillum cells from vegetative to cyst-like forms. To understand the cellular responses of Azospirillum to conditions that cause morphological transformation, proteins differentially expressed under flocculation conditions in A. brasilense Sp7 and its flcA knockout mutant were investigated. Comparison of 2-DE protein profiles of wild-type (Sp7) and a flcA deletion mutant (Sp7-flcAΔ) revealed a total of 33 differentially expressed 2-DE gel spots, with 22 of these spots confidently separated to allow protein identification. Analysis of these spots by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and MASCOT database searching identified 48 proteins (≥10% emPAI in each spot). The functional characteristics of these proteins included carbon metabolism (beta-ketothiolase and citrate synthase), nitrogen metabolism (Glutamine synthetase and nitric oxide synthase), stress tolerance (superoxide dismutase, Alkyl hydroperoxidase and ATP-dependent Clp protease proteolytic subunit) and morphological transformation (transducer coupling protein). The observed differences between Sp7 wild-type and flcA- strains enhance our understanding of the morphological transformation process and help to explain previous phenotypical observations. This work is a step forward in connecting the Azospirillum phenome and genome.

Cellular Responses during Morphological Transformation in Azospirillum brasilense and Its flcA Knockout Mutant

PubMed Central

Coumans, Joëlle V. F.; Poljak, Anne; Raftery, Mark J.; Pereg, Lily

2014-01-01

FlcA is a response regulator controlling flocculation and the morphological transformation of Azospirillum cells from vegetative to cyst-like forms. To understand the cellular responses of Azospirillum to conditions that cause morphological transformation, proteins differentially expressed under flocculation conditions in A. brasilense Sp7 and its flcA knockout mutant were investigated. Comparison of 2-DE protein profiles of wild-type (Sp7) and a flcA deletion mutant (Sp7-flcAΔ) revealed a total of 33 differentially expressed 2-DE gel spots, with 22 of these spots confidently separated to allow protein identification. Analysis of these spots by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and MASCOT database searching identified 48 proteins (≥10% emPAI in each spot). The functional characteristics of these proteins included carbon metabolism (beta-ketothiolase and citrate synthase), nitrogen metabolism (Glutamine synthetase and nitric oxide synthase), stress tolerance (superoxide dismutase, Alkyl hydroperoxidase and ATP-dependent Clp protease proteolytic subunit) and morphological transformation (transducer coupling protein). The observed differences between Sp7 wild-type and flcA − strains enhance our understanding of the morphological transformation process and help to explain previous phenotypical observations. This work is a step forward in connecting the Azospirillum phenome and genome. PMID:25502569

The ROR2 tyrosine kinase receptor regulates dendritic spine morphogenesis in hippocampal neurons.

PubMed

Alfaro, Iván E; Varela-Nallar, Lorena; Varas-Godoy, Manuel; Inestrosa, Nibaldo C

2015-07-01

Wnt signaling regulates synaptic development and function and contributes to the fine-tuning of the molecular and morphological differentiation of synapses. We have shown previously that Wnt5a activates non-canonical Wnt signaling to stimulate postsynaptic differentiation in excitatory hippocampal neurons promoting the clustering of the postsynaptic scaffold protein PSD-95 and the development of dendritic spines. At least three different kinds of Wnt receptors have been associated with Wnt5a signaling: seven trans-membrane Frizzled receptors and the tyrosine kinase receptors Ryk and ROR2. We report here that ROR2 is distributed in the dendrites of hippocampal neurons in close proximity to synaptic contacts and it is contained in dendritic spine protrusions. We demonstrate that ROR2 is necessary to maintain dendritic spine number and morphological distribution in cultured hippocampal neurons. ROR2 overexpression increased dendritic spine growth without affecting the density of dendritic spine protrusions in a form dependent on its extracellular Wnt binding cysteine rich domain (CRD) and kinase domain. Overexpression of dominant negative ROR2 lacking the extracellular CRD decreased spine density and the proportion of mushroom like spines, while ROR2 lacking the C-terminal and active kinase domains only affected spine morphology. Our results indicate a crucial role of the ROR2 in the formation and maturation of the postsynaptic dendritic spines in hippocampal neurons. Copyright © 2015 Elsevier Inc. All rights reserved.

ER-mitochondria contacts control surface glycan expression and sensitivity to killer lymphocytes in glioma stem-like cells.

PubMed

Bassoy, Esen Yonca; Kasahara, Atsuko; Chiusolo, Valentina; Jacquemin, Guillaume; Boydell, Emma; Zamorano, Sebastian; Riccadonna, Cristina; Pellegatta, Serena; Hulo, Nicolas; Dutoit, Valérie; Derouazi, Madiha; Dietrich, Pierre Yves; Walker, Paul R; Martinvalet, Denis

2017-06-01

Glioblastoma is a highly heterogeneous aggressive primary brain tumor, with the glioma stem-like cells (GSC) being more sensitive to cytotoxic lymphocyte-mediated killing than glioma differentiated cells (GDC). However, the mechanism behind this higher sensitivity is unclear. Here, we found that the mitochondrial morphology of GSCs modulates the ER-mitochondria contacts that regulate the surface expression of sialylated glycans and their recognition by cytotoxic T lymphocytes and natural killer cells. GSCs displayed diminished ER-mitochondria contacts compared to GDCs. Forced ER-mitochondria contacts in GSCs increased their cell surface expression of sialylated glycans and reduced their susceptibility to cytotoxic lymphocytes. Therefore, mitochondrial morphology and dynamism dictate the ER-mitochondria contacts in order to regulate the surface expression of certain glycans and thus play a role in GSC recognition and elimination by immune effector cells. Targeting the mitochondrial morphology, dynamism, and contacts with the ER could be an innovative strategy to deplete the cancer stem cell compartment to successfully treat glioblastoma. © 2017 The Authors.

MADS-Box Genes and Gibberellins Regulate Bolting in Lettuce (Lactuca sativa L.)

PubMed Central

Han, Yingyan; Chen, Zijing; Lv, Shanshan; Ning, Kang; Ji, Xueliang; Liu, Xueying; Wang, Qian; Liu, Renyi; Fan, Shuangxi; Zhang, Xiaolan

2016-01-01

Bolting in lettuce is promoted by high temperature and bolting resistance is of great economic importance for lettuce production. But how bolting is regulated at the molecular level remains elusive. Here, a bolting resistant line S24 and a bolting sensitive line S39 were selected for morphological, physiological, transcriptomic and proteomic comparisons. A total of 12204 genes were differentially expressed in S39 vs. S24. Line S39 was featured with larger leaves, higher levels of chlorophyll, soluble sugar, anthocyanin and auxin, consistent with its up-regulation of genes implicated in photosynthesis, oxidation-reduction and auxin actions. Proteomic analysis identified 30 differentially accumulated proteins in lines S39 and S24 upon heat treatment, and 19 out of the 30 genes showed differential expression in the RNA-Seq data. Exogenous gibberellins (GA) treatment promoted bolting in both S39 and S24, while 12 flowering promoting MADS-box genes were specifically induced in line S39, suggesting that although GA regulates bolting in lettuce, it may be the MADS-box genes, not GA, that plays a major role in differing the bolting resistance between these two lettuce lines. PMID:28018414

Cellular, ultrastructural and molecular analyses of epidermal cell development in the planarian Schmidtea mediterranea.

PubMed

Cheng, Li-Chun; Tu, Kimberly C; Seidel, Chris W; Robb, Sofia M C; Guo, Fengli; Sánchez Alvarado, Alejandro

2018-01-15

The epidermis is essential for animal survival, providing both a protective barrier and cellular sensor to external environments. The generally conserved embryonic origin of the epidermis, but the broad morphological and functional diversity of this organ across animals is puzzling. We define the transcriptional regulators underlying epidermal lineage differentiation in the planarian Schmidtea mediterranea, an invertebrate organism that, unlike fruitflies and nematodes, continuously replaces its epidermal cells. We find that Smed-p53, Sox and Pax transcription factors are essential regulators of epidermal homeostasis, and act cooperatively to regulate genes associated with early epidermal precursor cell differentiation, including a tandemly arrayed novel gene family (prog) of secreted proteins. Additionally, we report on the discovery of distinct and previously undescribed secreted organelles whose production is dependent on the transcriptional activity of soxP-3, and which we term Hyman vesicles. Copyright © 2017 Elsevier Inc. All rights reserved.

Sox9 regulates cell proliferation and is required for Paneth cell differentiation in the intestinal epithelium

PubMed Central

Bastide, Pauline; Darido, Charbel; Pannequin, Julie; Kist, Ralf; Robine, Sylvie; Marty-Double, Christiane; Bibeau, Frédéric; Scherer, Gerd; Joubert, Dominique; Hollande, Frédéric; Blache, Philippe; Jay, Philippe

2007-01-01

The HMG-box transcription factor Sox9 is expressed in the intestinal epithelium, specifically, in stem/progenitor cells and in Paneth cells. Sox9 expression requires an active β-catenin–Tcf complex, the transcriptional effector of the Wnt pathway. This pathway is critical for numerous aspects of the intestinal epithelium physiopathology, but processes that specify the cell response to such multipotential signals still remain to be identified. We inactivated the Sox9 gene in the intestinal epithelium to analyze its physiological function. Sox9 inactivation affected differentiation throughout the intestinal epithelium, with a disappearance of Paneth cells and a decrease of the goblet cell lineage. Additionally, the morphology of the colon epithelium was severely altered. We detected general hyperplasia and local crypt dysplasia in the intestine, and Wnt pathway target genes were up-regulated. These results highlight the central position of Sox9 as both a transcriptional target and a regulator of the Wnt pathway in the regulation of intestinal epithelium homeostasis. PMID:17698607

ROCK1 and 2 differentially regulate actomyosin organization to drive cell and synaptic polarity

PubMed Central

Badoual, Mathilde; Asmussen, Hannelore; Patel, Heather; Whitmore, Leanna; Horwitz, Alan Rick

2015-01-01

RhoGTPases organize the actin cytoskeleton to generate diverse polarities, from front–back polarity in migrating cells to dendritic spine morphology in neurons. For example, RhoA through its effector kinase, RhoA kinase (ROCK), activates myosin II to form actomyosin filament bundles and large adhesions that locally inhibit and thereby polarize Rac1-driven actin polymerization to the protrusions of migratory fibroblasts and the head of dendritic spines. We have found that the two ROCK isoforms, ROCK1 and ROCK2, differentially regulate distinct molecular pathways downstream of RhoA, and their coordinated activities drive polarity in both cell migration and synapse formation. In particular, ROCK1 forms the stable actomyosin filament bundles that initiate front–back and dendritic spine polarity. In contrast, ROCK2 regulates contractile force and Rac1 activity at the leading edge of migratory cells and the spine head of neurons; it also specifically regulates cofilin-mediated actin remodeling that underlies the maturation of adhesions and the postsynaptic density of dendritic spines. PMID:26169356

The Histone Acetyltransferase MOF is a Key Regulator of the Embryonic Stem Cell Core Transcriptional Network

PubMed Central

Li, Xiangzhi; Li, Li; Pandey, Ruchi; Byun, Jung S.; Gardner, Kevin; Qin, Zhaohui; Dou, Yali

2012-01-01

SUMMARY Pluripotent embryonic stem cells (ESCs) maintain self-renewal and the potential for rapid response to differentiation cues. Both ESC features are subject to epigenetic regulation. Here we show that histone acetyltransferase Mof plays an essential role in the maintenance of ESC self-renewal and pluripotency. ESCs with Mof deletion lose characteristic morphology, alkaline phosphatase (AP) staining and differentiation potential. They also have aberrant expression of core transcription factors Nanog, Oct4 and Sox2. Importantly, the phenotypes of Mof null ESCs can be partially suppressed by Nanog overexpression, supporting that Mof functions as an upstream regulator of Nanog in ESCs. Genome-wide ChIP sequencing and transcriptome analyses further demonstrate that Mof is an integral component of ESC core transcription network and Mof primes genes for diverse developmental programs. Mof is also required for Wdr5 recruitment and H3 K4 methylation at key regulatory loci, highlighting complexity and interconnectivity of various chromatin regulators in ESCs. PMID:22862943

Thyroid Hormone Acts Locally to Increase Neurogenesis, Neuronal Differentiation, and Dendritic Arbor Elaboration in the Tadpole Visual System

PubMed Central

Thompson, Christopher K.

2016-01-01

Thyroid hormone (TH) regulates many cellular events underlying perinatal brain development in vertebrates. Whether and how TH regulates brain development when neural circuits are first forming is less clear. Furthermore, although the molecular mechanisms that impose spatiotemporal constraints on TH action in the brain have been described, the effects of local TH signaling are poorly understood. We determined the effects of manipulating TH signaling on development of the optic tectum in stage 46–49 Xenopus laevis tadpoles. Global TH treatment caused large-scale morphological effects in tadpoles, including changes in brain morphology and increased tectal cell proliferation. Either increasing or decreasing endogenous TH signaling in tectum, by combining targeted DIO3 knockdown and methimazole, led to corresponding changes in tectal cell proliferation. Local increases in TH, accomplished by injecting suspensions of tri-iodothyronine (T3) in coconut oil into the midbrain ventricle or into the eye, selectively increased tectal or retinal cell proliferation, respectively. In vivo time-lapse imaging demonstrated that local TH first increased tectal progenitor cell proliferation, expanding the progenitor pool, and subsequently increased neuronal differentiation. Local T3 also dramatically increased dendritic arbor growth in neurons that had already reached a growth plateau. The time-lapse data indicate that the same cells are differentially sensitive to T3 at different time points. Finally, TH increased expression of genes pertaining to proliferation and neuronal differentiation. These experiments indicate that endogenous TH locally regulates neurogenesis at developmental stages relevant to circuit assembly by affecting cell proliferation and differentiation and by acting on neurons to increase dendritic arbor elaboration. SIGNIFICANCE STATEMENT Thyroid hormone (TH) is a critical regulator of perinatal brain development in vertebrates. Abnormal TH signaling in early pregnancy is associated with significant cognitive deficits in humans; however, it is difficult to probe the function of TH in early brain development in mammals because of the inaccessibility of the fetal brain in the uterine environment and the challenge of disambiguating maternal versus fetal contributions of TH. The external development of tadpoles allows manipulation and direct observation of the molecular and cellular mechanisms underlying TH's effects on brain development in ways not possible in mammals. We find that endogenous TH locally regulates neurogenesis at developmental stages relevant to circuit assembly by affecting neural progenitor cell proliferation and differentiation and by acting on neurons to enhance dendritic arbor elaboration. PMID:27707971

Apelin: an endogenous peptide essential for cardiomyogenic differentiation of mesenchymal stem cells via activating extracellular signal-regulated kinase 1/2 and 5.

PubMed

Wang, Li; Zhu, Zhi-Ming; Zhang, Ning-Kun; Fang, Zhi-Rong; Xu, Xiao-Hong; Zheng, Nan; Gao, Lian-Ru

2016-05-01

Growing evidence has shown that apelin/APJ system functions as a critical mediator of cardiac development as well as cardiovascular function. Here, we investigated the role of apelin in the cardiomyogenic differentiation of mesenchymal stem cells derived from Wharton's jelly of human umbilical cord in vitro. In this research, we used RNA interference methodology and gene transfection technique to regulate the expression of apelin in Wharton's jelly-derived mesenchymal stem cells and induced cells with a effective cardiac differentiation protocol including 5-azacytidine and bFGF. Four weeks after induction, induced cells assumed a stick-like morphology and myotube-like structures except apelin-silenced cells and the control group. The silencing expression of apelin in Wharton's jelly-derived mesenchymal stem cells decreased the expression of several critical cardiac progenitor transcription factors (Mesp1, Mef2c, NKX2.5) and cardiac phenotypes (cardiac α-actin, β-MHC, cTnT, and connexin-43). Meanwhile, endogenous compensation of apelin contributed to differentiating into cells with characteristics of cardiomyocytes in vitro. Further experiment showed that exogenous apelin peptide rescued the cardiomyogenic differentiation of apelin-silenced mesenchymal stem cells in the early stage (1-4 days) of induction. Remarkably, our experiment indicated that apelin up-regulated cardiac specific genes in Wharton's jelly-derived mesenchymal stem cells via activating extracellular signal-regulated kinase (ERK) 1/2 and 5. © 2016 International Federation for Cell Biology.

Induction and characterization of morphologic mutants in a natural Saccharomyces cerevisiae strain.

PubMed

Barberio, Claudia; Bianchi, Lucia; Pinzauti, Francesca; Lodi, Tiziana; Ferrero, Iliana; Polsinelli, Mario; Casalone, Enrico

2007-02-01

Saccharomyces cerevisiae is a good model with which to study the effects of morphologic differentiation on the ecological behaviour of fungi. In this work, 33 morphologic mutants of a natural strain of S. cerevisiae, obtained with UV mutagenesis, were selected for their streak shape and cell shape on rich medium. Two of them, showing both high sporulation proficiency and constitutive pseudohyphal growth, were analysed from a genetic and physiologic point of view. Each mutant carries a recessive monogenic mutation, and the two mutations reside in unlinked genes. Flocculation ability and responsiveness to different stimuli distinguished the two mutants. Growth at 37 degrees C affected the cell but not the colony morphology, suggesting that these two phenotypes are regulated differently. The effect of ethidium bromide, which affects mitochondrial DNA replication, suggested a possible "retrograde action" of mitochondria in pseudohyphal growth.

Mitochondrial Dynamics Impacts Stem Cell Identity and Fate Decisions by Regulating a Nuclear Transcriptional Program.

PubMed

Khacho, Mireille; Clark, Alysen; Svoboda, Devon S; Azzi, Joelle; MacLaurin, Jason G; Meghaizel, Cynthia; Sesaki, Hiromi; Lagace, Diane C; Germain, Marc; Harper, Mary-Ellen; Park, David S; Slack, Ruth S

2016-08-04

Regulated mechanisms of stem cell maintenance are key to preventing stem cell depletion and aging. While mitochondrial morphology plays a fundamental role in tissue development and homeostasis, its role in stem cells remains unknown. Here, we uncover that mitochondrial dynamics regulates stem cell identity, self-renewal, and fate decisions by orchestrating a transcriptional program. Manipulation of mitochondrial structure, through OPA1 or MFN1/2 deletion, impaired neural stem cell (NSC) self-renewal, with consequent age-dependent depletion, neurogenesis defects, and cognitive impairments. Gene expression profiling revealed ectopic expression of the Notch self-renewal inhibitor Botch and premature induction of transcription factors that promote differentiation. Changes in mitochondrial dynamics regulate stem cell fate decisions by driving a physiological reactive oxygen species (ROS)-mediated process, which triggers a dual program to suppress self-renewal and promote differentiation via NRF2-mediated retrograde signaling. These findings reveal mitochondrial dynamics as an upstream regulator of essential mechanisms governing stem cell self-renewal and fate decisions through transcriptional programming. Copyright © 2016 Elsevier Inc. All rights reserved.

Shavenbaby Couples Patterning to Epidermal Cell Shape Control

PubMed Central

Fernandes, Isabelle; Roch, Fernando; Payre, François

2006-01-01

It is well established that developmental programs act during embryogenesis to determine animal morphogenesis. How these developmental cues produce specific cell shape during morphogenesis, however, has remained elusive. We addressed this question by studying the morphological differentiation of the Drosophila epidermis, governed by a well-known circuit of regulators leading to a stereotyped pattern of smooth cells and cells forming actin-rich extensions (trichomes). It was shown that the transcription factor Shavenbaby plays a pivotal role in the formation of trichomes and underlies all examined cases of the evolutionary diversification of their pattern. To gain insight into the mechanisms of morphological differentiation, we sought to identify shavenbaby's downstream targets. We show here that Shavenbaby controls epidermal cell shape, through the transcriptional activation of different classes of cellular effectors, directly contributing to the organization of actin filaments, regulation of the extracellular matrix, and modification of the cuticle. Individual inactivation of shavenbaby's targets produces distinct trichome defects and only their simultaneous inactivation prevent trichome formation. Our data show that shavenbaby governs an evolutionarily conserved developmental module consisting of a set of genes collectively responsible for trichome formation, shedding new light on molecular mechanisms acting during morphogenesis and the way they can influence evolution of animal forms. PMID:16933974

Alcohols inhibit translation to regulate morphogenesis in C. albicans

PubMed Central

Egbe, Nkechi E.; Paget, Caroline M.; Wang, Hui; Ashe, Mark P.

2015-01-01

Many molecules are secreted into the growth media by microorganisms to modulate the metabolic and physiological processes of the organism. For instance, alcohols like butanol, ethanol and isoamyl alcohol are produced by the human pathogenic fungus, Candida albicans and induce morphological differentiation. Here we show that these same alcohols cause a rapid inhibition of protein synthesis. More specifically, the alcohols target translation initiation, a complex stage of the gene expression process. Using molecular techniques, we have identified the likely translational target of these alcohols in C. albicans as the eukaryotic translation initiation factor 2B (eIF2B). eIF2B is the guanine nucleotide exchange factor for eIF2, which supports the exchange reaction where eIF2.GDP is converted to eIF2.GTP. Even minimal regulation at this step will lead to alterations in the levels of specific proteins that may allow the exigencies of the fungus to be realised. Indeed, similar to the effects of alcohols, a minimal inhibition of protein synthesis with cycloheximide also causes an induction of filamentous growth. These results suggest a molecular basis for the effect of various alcohols on morphological differentiation in C. albicans. PMID:25843913

Aberrant levels of histone H3 acetylation induce spermatid anomaly in mouse testis.

PubMed

Dai, Lei; Endo, Daisuke; Akiyama, Naotaro; Yamamoto-Fukuda, Tomomi; Koji, Takehiko

2015-02-01

Histone acetylation is involved in the regulation of chromatin structure and gene function. We reported previously that histone H3 acetylation pattern is subject to dynamic changes and limited to certain stages of germ cell differentiation during murine spermatogenesis, suggesting a crucial role for acetylation in the process. In the present study, we investigated the effects of hyper- and hypo-acetylation on spermatogenesis. Changes in acetylation level were induced by either in vivo administration of sodium phenylbutyrate, a histone deacetylase inhibitor, or by knockdown of histone acetyltransferases using short hairpin RNA plasmids transfection. Administration of sodium phenylbutyrate induced accumulation of acetylated histone H3 at lysine 9 and lysine 18 in round spermatids, together with spermatid morphological abnormalities and induction of apoptosis through a Bax-related pathway. Knockdown of steroid receptor coactivator 1, a member of histone acetyltransferases, but not general control of amino acid synthesis 5 nor elongator protein 3 by in vivo electroporation of shRNA plasmids, reduced acetylated histone H3 at lysine 9 in round spermatids, and induced morphological abnormalities. We concluded that the proper regulation of histone H3 acetylation levels is important for spermatid differentiation and complex chromatin remodeling during spermiogenesis.

Developmental palaeobiology of trilobite eyes and its evolutionary significance

NASA Astrophysics Data System (ADS)

Thomas, A. T.

2005-06-01

Understanding of the calcified composite eyes of trilobites, the oldest preserved visual system, has advanced greatly in recent decades. Three types of trilobite eye occur, the more derived abathochroal and schizochroal types having evolved neotenically from holochroal eyes. Comparative morphology and phylogenetic considerations suggest that all three eye-types were underlain by common developmental systems. So far, understanding of these systems has been based entirely on morphological data from fossils, particularly the way the visual surface grew and the patterning of lens emplacement. Lenses characteristically form a hexagonal array comprising horizontal rows and, conspicuously in schizochroal eyes, dorso-ventral files. Because individual trilobites sometimes have eyes with different numbers of files, file number must reflect the operation of a developmental programme rather than being under immediate genetic control. An empirical developmental model has been devised to describe trilobite eye development, with separate rules dealing with the initiation of lens emplacement, growth and differentiation of the visual surface, and the termination of lens emplacement. Rarely, trilobites may have visual surfaces of normal size, but which lack lenses. This confirms that visual surface growth must have been regulated separately from lens emplacement, and is a feature that cannot be accounted for by the existing developmental model. Such a developmental separation is one of a number of similarities shared with Drosophila, the modern arthropod in which eye development is best understood. Many aspects of eye development are conserved in the Euarthropoda, and in bilaterian metazoans in general. A revised model for trilobite eye development is proposed using extant phylogenetic bracketing, interpreting morphological data from the fossils in the context of the hierarchy of developmental controls now becoming known from living animals. This new model suggests that overall eye shape and size did not require differential growth of the generative zone, as previously thought, and that no separate instruction was needed to specify the termination of lens emplacement. Instead, these features were regulated directly, by controlling the proliferation of cells making up the nascent visual surface. A process documented from Drosophila, which involves the selective inhibition of cells in front of a wave-like front of differentiation, and that is regulated by widely conserved genes, can be used to explain how the trilobite visual surface became differentiated. The model implies also that changes in hormonally regulated developmental pathways known from recent arthropods may have been responsible for the development of abathochroal and schizochroal eyes, and for heterochronic secondary eye reduction and blindness in trilobites.

Proteins associated with critical sperm functions and sperm head shape are differentially expressed in morphologically abnormal bovine sperm induced by scrotal insulation.

PubMed

Shojaei Saadi, Habib A; van Riemsdijk, Evine; Dance, Alysha L; Rajamanickam, Gayathri D; Kastelic, John P; Thundathil, Jacob C

2013-04-26

The objective was to investigate expression patterns of proteins in pyriform sperm, a common morphological abnormality in bull sperm. Ejaculates were collected from sexually mature Holstein bulls (n=3) twice weekly for 10 weeks (pre-thermal insult samples). Testicular temperature was elevated in all bulls by scrotal insulation for 72 consecutive hours during week 2. Total sperm proteins were extracted from pre- and post-thermal insult sperm samples and subjected to two-dimensional gel electrophoresis. Among the protein spots detected, 131 spots were significantly expressed (False Detection Rate <0.01) with ≥ 2 fold changes between normal and pyriform sperm. Among them, 25 spots with ≥ 4 fold difference in expression patterns were identified using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Expression of several proteins involved in sperm capacitation, sperm-egg interaction and sperm cytoskeletal structure was decreased in pyriform sperm, whereas proteins regulating antioxidant activity, apoptosis and metabolic activity were increased. Contents of reactive oxygen species and ubiquitinated proteins were higher in pyriform sperm. In addition to understanding the molecular basis of functional deficiencies in sperm with specific morphological abnormalities, comparing normal versus morphologically abnormal sperm appeared to be a suitable experimental model for identifying important sperm functional proteins. To our knowledge, this study is the first report on differential expression of proteins in pyriform bovine sperm versus morphologically normal sperm. We report that expression of several proteins involved in sperm capacitation, sperm-egg interaction and sperm cytoskeletal structure was decreased in pyriform sperm, whereas proteins which regulate antioxidant activity, apoptosis and metabolic activity were increased. Contents of reactive oxygen species and ubiquitinated proteins were higher in pyriform sperm. In addition to understanding the molecular basis of functional deficiencies in sperm with specific morphological abnormalities, our results suggest that comparing normal versus morphologically abnormal sperm appeared to be a suitable experimental model for identifying important sperm functional proteins. Copyright © 2013 Elsevier B.V. All rights reserved.

Markers of Developmentally Regulated Programmed Cell Death and Their Analysis in Cereal Seeds.

PubMed

Domínguez, Fernando; Cejudo, Francisco Javier

2018-01-01

Programmed cell death (PCD) is a key process for the development and differentiation of multicellular organisms, which is characterized by well-defined morphological and biochemical features. These include chromatin condensation, DNA degradation and nuclear fragmentation, with nucleases and proteases playing a relevant function in these processes. In this chapter we describe methods routinely used for the analysis of hallmarks of developmentally regulated PCD in cereal seed tissues, which are based on agarose and polyacrylamide gel electrophoresis, in situ staining of DNA fragmentation, and cell-free assays of relevant enzymatic activities.

Dynamic changes in gene expression during human trophoblast differentiation.

PubMed

Handwerger, Stuart; Aronow, Bruce

2003-01-01

The genetic program that directs human placental differentiation is poorly understood. In a recent study, we used DNA microarray analyses to determine genes that are dynamically regulated during human placental development in an in vitro model system in which highly purified cytotrophoblast cells aggregate spontaneously and fuse to form a multinucleated syncytium that expresses placental lactogen, human chorionic gonadotropin, and other proteins normally expressed by fully differentiated syncytiotrophoblast cells. Of the 6918 genes present on the Incyte Human GEM V microarray that we analyzed over a 9-day period, 141 were induced and 256 were downregulated by more than 2-fold. The dynamically regulated genes fell into nine distinct kinetic patterns of induction or repression, as detected by the K-means algorithm. Classifying the genes according to functional characteristics, the regulated genes could be divided into six overall categories: cell and tissue structural dynamics, cell cycle and apoptosis, intercellular communication, metabolism, regulation of gene expression, and expressed sequence tags and function unknown. Gene expression changes within key functional categories were tightly coupled to the morphological changes that occurred during trophoblast differentiation. Within several key gene categories (e.g., cell and tissue structure), many genes were strongly activated, while others with related function were strongly repressed. These findings suggest that trophoblast differentiation is augmented by "categorical reprogramming" in which the ability of induced genes to function is enhanced by diminished synthesis of other genes within the same category. We also observed categorical reprogramming in human decidual fibroblasts decidualized in vitro in response to progesterone, estradiol, and cyclic AMP. While there was little overlap between genes that are dynamically regulated during trophoblast differentiation versus decidualization, many of the categories in which genes were strongly activated also contained genes whose expression was strongly diminished. Taken together, these findings point to a fundamental role for simultaneous induction and repression of mRNAs that encode functionally related proteins during the differentiation process.

Down-regulation of the non-coding RNA H19 and its derived miR-675 is concomitant with up-regulation of insulin-like growth factor receptor type 1 during neural-like differentiation of human bone marrow mesenchymal stem cells.

PubMed

Farzi-Molan, Asghar; Babashah, Sadegh; Bakhshinejad, Babak; Atashi, Amir; Fakhr Taha, Masoumeh

2018-03-07

The differentiation of human bone marrow mesenchymal stem cells (BMSCs) into specific lineages offers new opportunities to use the therapeutic efficiency of these pluripotent cells in regenerative medicine. Multiple lines of evidence have revealed that non-coding RNAs play major roles in the differentiation of BMSCs into neural cells. Here, we applied a cocktail of neural inducing factors (NIFs) to differentiate BMSCs into neural-like cells. Our data demonstrated that during neurogenic induction, BMSCs obtained a neuron-like morphology. Also, the results of gene expression analysis by qRT-PCR showed progressively increasing expression levels of neuron-specific enolase (NSE) as well as microtubule-associated protein 2 (MAP-2) and immunocytochemical staining detected the expression of these neuron-specific markers along differentiated BMSC bodies and cytoplasmic processes, confirming the differentiation of BMSCs into neuronal lineages. We also compared differences in the expression levels of the long non-coding RNA (lncRNA) H19 and H19-derived miR-675 between undifferentiated and neurally differentiated BMSCs and found that during neural differentiation down-regulation of the lncRNA H19/miR-675 axis is concomitant with up-regulation of insulin-like growth factor type-1 (IGF-1R), a well-established target of miR-675 involved in neurogenesis. The findings of the current study provide support for the hypothesis that miR-675 may confer functionality to H19, suggesting a key role for this miRNA in the neural differentiation of BSMCs. However, further investigation is required to gain deeper insights into the biological roles of this miRNA in the complex process of neurogenesis. © 2018 International Federation for Cell Biology.

Differentiation induction of mouse embryonic stem cells into sinus node-like cells by suramin

PubMed Central

Wiese, Cornelia; Nikolova, Teodora; Zahanich, Ihor; Sulzbacher, Sabine; Fuchs, Joerg; Yamanaka, Satoshi; Graf, Eva; Ravens, Ursula; Boheler, Kenneth R.; Wobus, Anna M.

2015-01-01

Background Embryonic stem (ES) cells differentiate into cardiac phenotypes representing early pacemaker-, atrial-, ventricular-, and sinus node-like cells, however, ES-derived specification into sinus nodal cells is not yet known. By using the naphthylamine derivative of urea, suramin, we were able to follow the process of cardiac specialization into sinus node-like cells. Methods Differentiating mouse ES cells were treated with suramin (500 μM) from day 5 to 7 of embryoid body formation, and cells were analysed for their differentiation potential via morphological analysis, flow cytometry, RT-PCR, immunohistochemistry and patch clamp analysis. Results Application of suramin resulted in an increased number of cardiac cells, but inhibition of neuronal, skeletal muscle and definitive endoderm differentiation. Immediately after suramin treatment, a decreased mesendoderm differentiation was found. Brachyury, FGF10, Wnt8 and Wnt3a transcript levels were significantly down-regulated, followed by a decrease in mesoderm- and cardiac progenitor-specific markers BMP2, GATA4/5, Wnt11, Isl1, Nkx2.5 and Tbx5 immediately after removal of the substance. With continued differentiation, a significant up-regulation of Brachyury, FGF10 and GATA5 transcript levels was observed, whereas Nkx2.5, Isl1, Tbx5, BMP2 and Wnt11 levels were normalized to control levels. At advanced differentiation stages, sinus node-specific HCN4, Tbx2 and Tbx3 transcript levels were significantly up-regulated. Immunofluorescence and patch-clamp analysis confirmed the increased number of sinus node-like cells, and electrophysiological analysis revealed a lower number of atrial- and ventricular-like cardiomyocytes following suramin treatment. Conclusion We conclude that the interference of suramin with the cardiac differentiation process modified mesoderm- and cardiac-specific gene expression resulting in enhanced formation of sinus node-like cells. PMID:19775764

Metabolic reprogramming during neuronal differentiation.

PubMed

Agostini, M; Romeo, F; Inoue, S; Niklison-Chirou, M V; Elia, A J; Dinsdale, D; Morone, N; Knight, R A; Mak, T W; Melino, G

2016-09-01

Newly generated neurons pass through a series of well-defined developmental stages, which allow them to integrate into existing neuronal circuits. After exit from the cell cycle, postmitotic neurons undergo neuronal migration, axonal elongation, axon pruning, dendrite morphogenesis and synaptic maturation and plasticity. Lack of a global metabolic analysis during early cortical neuronal development led us to explore the role of cellular metabolism and mitochondrial biology during ex vivo differentiation of primary cortical neurons. Unexpectedly, we observed a huge increase in mitochondrial biogenesis. Changes in mitochondrial mass, morphology and function were correlated with the upregulation of the master regulators of mitochondrial biogenesis, TFAM and PGC-1α. Concomitant with mitochondrial biogenesis, we observed an increase in glucose metabolism during neuronal differentiation, which was linked to an increase in glucose uptake and enhanced GLUT3 mRNA expression and platelet isoform of phosphofructokinase 1 (PFKp) protein expression. In addition, glutamate-glutamine metabolism was also increased during the differentiation of cortical neurons. We identified PI3K-Akt-mTOR signalling as a critical regulator role of energy metabolism in neurons. Selective pharmacological inhibition of these metabolic pathways indicate existence of metabolic checkpoint that need to be satisfied in order to allow neuronal differentiation.

Metabolic reprogramming during neuronal differentiation

PubMed Central

Agostini, M; Romeo, F; Inoue, S; Niklison-Chirou, M V; Elia, A J; Dinsdale, D; Morone, N; Knight, R A; Mak, T W; Melino, G

2016-01-01

Newly generated neurons pass through a series of well-defined developmental stages, which allow them to integrate into existing neuronal circuits. After exit from the cell cycle, postmitotic neurons undergo neuronal migration, axonal elongation, axon pruning, dendrite morphogenesis and synaptic maturation and plasticity. Lack of a global metabolic analysis during early cortical neuronal development led us to explore the role of cellular metabolism and mitochondrial biology during ex vivo differentiation of primary cortical neurons. Unexpectedly, we observed a huge increase in mitochondrial biogenesis. Changes in mitochondrial mass, morphology and function were correlated with the upregulation of the master regulators of mitochondrial biogenesis, TFAM and PGC-1α. Concomitant with mitochondrial biogenesis, we observed an increase in glucose metabolism during neuronal differentiation, which was linked to an increase in glucose uptake and enhanced GLUT3 mRNA expression and platelet isoform of phosphofructokinase 1 (PFKp) protein expression. In addition, glutamate–glutamine metabolism was also increased during the differentiation of cortical neurons. We identified PI3K–Akt–mTOR signalling as a critical regulator role of energy metabolism in neurons. Selective pharmacological inhibition of these metabolic pathways indicate existence of metabolic checkpoint that need to be satisfied in order to allow neuronal differentiation. PMID:27058317

The effects of dexamethasone, ascorbic acid, and β-glycerophosphate on osteoblastic differentiation by regulating estrogen receptor and osteopontin expression.

PubMed

Park, Jun-Beom

2012-03-01

Ascorbic acid (AA), β-glycerophosphate (GP), and dexamethasone (DEX) are the compounds known to favor the expression of the osteoblastic phenotype in several bone cell systems. In this report, the combination effects of differentiation agents on osteoprecursor cells were evaluated. The effect on cell proliferation was determined by a cell viability test with morphologic analysis. Differentiation and mineralization were evaluated using an alkaline phosphatase activity test and alizarin red-S staining. Protein expressions related to bone formation, such as transforming growth factor-beta (TGF-β), estrogen receptor-alpha (ER-α), and osteopontin (OPN) were evaluated by using a Western blot analysis. AA and GP provided an inductive effect for differentiation of osteoprecusor cells, while short-term application of DEX seemed to lead to a dose-dependent increase of cellular differentiation. Long-term use of DEX seemed to reduce mineralization. These effects may seem to be regulated by the expression of ER-α, OPN, and TGF-β. Further studies related to this mechanism within the in vivo model may be necessary to ascertain greater detail. Copyright © 2012 Elsevier Inc. All rights reserved.

Embryotoxic and pharmacologic potency ranking of six azoles in the rat whole embryo culture by morphological and transcriptomic analysis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dimopoulou, Myrto, E-mail: myrto.dimopoulou@wur.nl

Differential gene expression analysis in the rat whole embryo culture (WEC) assay provides mechanistic insight into the embryotoxicity of test compounds. In our study, we hypothesized that comparative analysis of the transcriptomes of rat embryos exposed to six azoles (flusilazole, triadimefon, ketoconazole, miconazole, difenoconazole and prothioconazole) could lead to a better mechanism-based understanding of their embryotoxicity and pharmacological action. For evaluating embryotoxicity, we applied the total morphological scoring system (TMS) in embryos exposed for 48 h. The compounds tested showed embryotoxicity in a dose-response fashion. Functional analysis of differential gene expression after 4 h exposure at the ID{sub 10} (effectivemore » dose for 10% decreased TMS), revealed the sterol biosynthesis pathway and embryonic development genes, dominated by genes in the retinoic acid (RA) pathway, albeit in a differential way. Flusilazole, ketoconazole and triadimefon were the most potent compounds affecting the RA pathway, while in terms of regulation of sterol function, difenoconazole and ketoconazole showed the most pronounced effects. Dose-dependent analysis of the effects of flusilazole revealed that the RA pathway related genes were already differentially expressed at low dose levels while the sterol pathway showed strong regulation at higher embryotoxic doses, suggesting that this pathway is less predictive for the observed embryotoxicity. A similar analysis at the 24-hour time point indicated an additional time-dependent difference in the aforementioned pathways regulated by flusilazole. In summary, the rat WEC assay in combination with transcriptomics could add a mechanistic insight into the embryotoxic potency ranking and pharmacological mode of action of the tested compounds. - Highlights: • Embryonic exposure to azoles revealed concentration-dependent malformations. • Transcriptomics could enhance the mechanistic knowledge of embryotoxicants. • Retinoic acid gene set identifies early embryotoxic responses to azoles. • Toxic versus pharmacologic potency determines functional efficacy.« less

Endocrine regulation of predator-induced phenotypic plasticity.

PubMed

Dennis, Stuart R; LeBlanc, Gerald A; Beckerman, Andrew P

2014-11-01

Elucidating the developmental and genetic control of phenotypic plasticity remains a central agenda in evolutionary ecology. Here, we investigate the physiological regulation of phenotypic plasticity induced by another organism, specifically predator-induced phenotypic plasticity in the model ecological and evolutionary organism Daphnia pulex. Our research centres on using molecular tools to test among alternative mechanisms of developmental control tied to hormone titres, receptors and their timing in the life cycle. First, we synthesize detail about predator-induced defenses and the physiological regulation of arthropod somatic growth and morphology, leading to a clear prediction that morphological defences are regulated by juvenile hormone and life-history plasticity by ecdysone and juvenile hormone. We then show how a small network of genes can differentiate phenotype expression between the two primary developmental control pathways in arthropods: juvenoid and ecdysteroid hormone signalling. Then, by applying an experimental gradient of predation risk, we show dose-dependent gene expression linking predator-induced plasticity to the juvenoid hormone pathway. Our data support three conclusions: (1) the juvenoid signalling pathway regulates predator-induced phenotypic plasticity; (2) the hormone titre (ligand), rather than receptor, regulates predator-induced developmental plasticity; (3) evolution has favoured the harnessing of a major, highly conserved endocrine pathway in arthropod development to regulate the response to cues about changing environments (risk) from another organism (predator).

The Role of Hedgehog-Interacting Protein in Maintaining Cavernous Nerve Integrity and Adult Penile Morphology

PubMed Central

Angeloni, Nicholas L.; Bond, Christopher W.; Monsivais, Diana; Tang, Yi; Podlasek, Carol A.

2010-01-01

Introduction Sonic hedgehog (SHH) is an essential regulator of smooth muscle apoptosis in the penis that has significant clinical potential as a therapy to suppress post-prostatectomy apoptosis, an underlying cause of erectile dysfunction (ED). Thus an understanding of how SHH signaling is regulated in the adult penis is essential to move the field of ED research forward and to develop new treatment strategies. We propose that hedgehog-interacting protein (HIP), which has been shown to bind SHH protein and to play a role in SHH regulation during embryogenesis of other organs, is a critical regulator of SHH signaling, penile morphology, and apoptosis induction. Aims We have examined HIP signaling in the penis and cavernous nerve (CN) during postnatal differentiation of the penis, in CN-injured, and a diabetic model of ED. Methods HIP localization/abundance and RNA abundance were examined by immunohistochemical (IHC) analysis and real-time reverse transcriptase-polymerase chain reaction (RT-PCR) in Sprague-Dawley rats between the ages of 7 and 92 days old, in CN-injured Sprague-Dawley rats and in BioBreeding/Worcester diabetic rats. HIP signaling was perturbed in the pelvic ganglia and in the penis and TUNEL assay was performed in the penis. CN tie, lidocaine, and anti-kinesin experiments were performed to examine HIP signaling in the CN and penis. Results In this study we are the first to demonstrate that HIP undergoes anterograde transport to the penis via the CN, that HIP perturbation in the pelvic ganglia or the penis induces apoptosis, and that HIP plays a role in maintaining CN integrity, penile morphology, and SHH abundance. Conclusions These studies are significant because they show HIP involvement in cross-talk (signaling) between the pelvic ganglia and penis, which is integral for maintenance of penile morphology and they suggest a mechanism of how nerves may regulate target organ morphology and function. PMID:19515211

miR-200 family promotes podocyte differentiation through repression of RSAD2

PubMed Central

Li, Zhigui; Yin, Hongqiang; Hao, Shuang; Wang, Lifeng; Gao, Jing; Tan, Xiaoyue; Yang, Zhuo

2016-01-01

Mature podocytes are highly differentiated cells with several characteristic phenotypic features that are involved in the glomerular filtration function. During kidney development, a series of changes of the morphological characteristics and cellular functions may happen in podocytes. The miR-200 family functions in various biological and pathological processes. But the underlying molecular mechanisms of miR-200 family that functions in podocyte differentiation remain poorly understood. Herein is shown that miR-200a, miR-200b and miR-429 are significantly upregulated during the differentiation of podocytes, with highest upregulation of miR-200a. In these cells, restraint of miR-200 family by RNA interference assay revealed a prominent inhibition of cell differentiation. More intriguingly, miR-200 family directly inhibited the radical S-adenosyl methionine domain-containing protein 2 (RASD2) expression. Moreover, further upregulation of RSAD2 combining with restraint of miR-200 family revealed a promotion of podocyte dedifferentiation and proliferation. In addition, the expression of RSAD2 is consistent with that of in vitro podocyte differentiation in prenatal and postnatal mouse kidney, and significantly down-regulated during the kidney development. Together, these findings indicate that miR-200 family may potentially promote podocyte differentiation through repression of RSAD2 expression. Our data also demonstrate a novel role of the antiviral protein RSAD2 as a regulator in cell differentiation. PMID:27251424

A novel stilbene-like compound that inhibits melanoma growth by regulating melanocyte differentiation and proliferation.

PubMed

Stueven, Noah A; Schlaeger, Nicholas M; Monte, Aaron P; Hwang, Sheng-Ping L; Huang, Cheng-Chen

2017-12-15

Melanoma is the most aggressive form of skin cancer. Current challenges to melanoma therapy include the adverse effects from immunobiologics, resistance to drugs targeting the MAPK pathway, intricate interaction of many signal pathways, and cancer heterogeneity. Thus combinational therapy with drugs targeting multiple signaling pathways becomes a new promising therapy. Here, we report a family of stilbene-like compounds called A11 that can inhibit melanoma growth in both melanoma-forming zebrafish embryos and mouse melanoma cells. The growth inhibition by A11 is a result of mitosis reduction but not apoptosis enhancement. Meanwhile, A11 activates both MAPK and Akt signaling pathways. Many A11-treated mouse melanoma cells exhibit morphological changes and resemble normal melanocytes. Furthermore, we found that A11 causes down-regulation of melanocyte differentiation genes, including Pax3 and MITF. Together, our results suggest that A11 could be a new melanoma therapeutic agent by inhibiting melanocyte differentiation and proliferation. Copyright © 2017 Elsevier Inc. All rights reserved.

Alternative splicing mechanisms orchestrating post-transcriptional gene expression: intron retention and the intron-rich genome of apicomplexan parasites.

PubMed

Lunghi, Matteo; Spano, Furio; Magini, Alessandro; Emiliani, Carla; Carruthers, Vern B; Di Cristina, Manlio

2016-02-01

Apicomplexan parasites including Toxoplasma gondii and Plasmodium species have complex life cycles that include multiple hosts and differentiation through several morphologically distinct stages requiring marked changes in gene expression. This review highlights emerging evidence implicating regulation of mRNA splicing as a mechanism to prime these parasites for rapid gene expression upon differentiation. We summarize the most important insights in alternative splicing including its role in regulating gene expression by decreasing mRNA abundance via 'Regulated Unproductive Splicing and Translation'. As a related but less well-understood mechanism, we discuss also our recent work suggesting a role for intron retention for precluding translation of stage specific isoforms of T. gondii glycolytic enzymes. We additionally provide new evidence that intron retention might be a widespread mechanism during parasite differentiation. Supporting this notion, recent genome-wide analysis of Toxoplasma and Plasmodium suggests intron retention is more pervasive than heretofore thought. These findings parallel recent emergence of intron retention being more prevalent in mammals than previously believed, thereby adding to the established roles in plants, fungi and unicellular eukaryotes. Deeper mechanistic studies of intron retention will provide important insight into its role in regulating gene expression in apicomplexan parasites and more general in eukaryotic organisms.

NFIX Regulates Neural Progenitor Cell Differentiation During Hippocampal Morphogenesis

PubMed Central

Heng, Yee Hsieh Evelyn; McLeay, Robert C.; Harvey, Tracey J.; Smith, Aaron G.; Barry, Guy; Cato, Kathleen; Plachez, Céline; Little, Erica; Mason, Sharon; Dixon, Chantelle; Gronostajski, Richard M.; Bailey, Timothy L.; Richards, Linda J.; Piper, Michael

2014-01-01

Neural progenitor cells have the ability to give rise to neurons and glia in the embryonic, postnatal and adult brain. During development, the program regulating whether these cells divide and self-renew or exit the cell cycle and differentiate is tightly controlled, and imbalances to the normal trajectory of this process can lead to severe functional consequences. However, our understanding of the molecular regulation of these fundamental events remains limited. Moreover, processes underpinning development of the postnatal neurogenic niches within the cortex remain poorly defined. Here, we demonstrate that Nuclear factor one X (NFIX) is expressed by neural progenitor cells within the embryonic hippocampus, and that progenitor cell differentiation is delayed within Nfix−/− mice. Moreover, we reveal that the morphology of the dentate gyrus in postnatal Nfix−/− mice is abnormal, with fewer subgranular zone neural progenitor cells being generated in the absence of this transcription factor. Mechanistically, we demonstrate that the progenitor cell maintenance factor Sry-related HMG box 9 (SOX9) is upregulated in the hippocampus of Nfix−/− mice and demonstrate that NFIX can repress Sox9 promoter-driven transcription. Collectively, our findings demonstrate that NFIX plays a central role in hippocampal morphogenesis, regulating the formation of neuronal and glial populations within this structure. PMID:23042739

Regulation of Mesenchymal Stem Cell Differentiation by Nanopatterning of Bulk Metallic Glass.

PubMed

Loye, Ayomiposi M; Kinser, Emily R; Bensouda, Sabrine; Shayan, Mahdis; Davis, Rose; Wang, Rui; Chen, Zheng; Schwarz, Udo D; Schroers, Jan; Kyriakides, Themis R

2018-06-08

Mesenchymal stem cell (MSC) differentiation is regulated by surface modification including texturing, which is applied to materials to enhance tissue integration. Here, we used Pt 57.5 Cu 14.7 Ni 5.3 P 22.5 bulk metallic glass (Pt-BMG) with nanopatterned surfaces achieved by thermoplastic forming to influence differentiation of human MSCs. Pt-BMGs are a unique class of amorphous metals with high strength, elasticity, corrosion resistance, and an unusual plastic-like processability. It was found that flat and nanopattened Pt-BMGs induced osteogenic and adipogenic differentiation, respectively. In addition, osteogenic differentiation on flat BMG exceeded that observed on medical grade titanium and was associated with increased formation of focal adhesions and YAP nuclear localization. In contrast, cells on nanopatterned BMGs exhibited rounded morphology, formed less focal adhesions and had mostly cytoplasmic YAP. These changes were preserved on nanopatterns made of nanorods with increased stiffness due to shorter aspect ratios, suggesting that MSC differentiation was primarily influenced by topography. These observations indicate that both elemental composition and nanotopography can modulate biochemical cues and influence MSCs. Moreover, the processability and highly tunable nature of Pt-BMGs enables the creation of a wide range of surface topographies that can be reproducibly and systematically studied, leading to the development of implants capable of engineering MSC functions.

Low-molecular-weight fucoidan regulates myogenic differentiation through the mitogen-activated protein kinase pathway in C2C12 cells.

PubMed

Kim, Kui-Jin; Lee, Ok-Hwan; Lee, Boo-Yong

2011-12-01

Low-molecular-weight fucoidan (LMWF) has been broadly studied in recent years due to its numerous biological properties. Nevertheless, there have been no reports about the effects of LMWF on myogenic differentiation (MyoD). The objective of the present study was to demonstrate the impact of LMWF on myogenesis in C2C12 cells. The ultimate aim was to establish whether LMWF regulates myogenesis similar to heparin as a partial regulator of myogenesis. LMWF was prepared at a minimal size by ultra-filtration compared with crude fucoidan. We treated C2C12 cells with LMWF and/or heparin during MyoD. The data from the present study are the first to suggest that LMWF suppresses the expression of the myogenic regulatory factors and the myocyte enhancer factors as well as the morphological changes that occur during differentiation. Additionally, the expression of the mitogen-activated protein kinase (MAPK) family was significantly inhibited by LMWF when compared with controls. The LMWF-treated group showed significantly decreased expression of reactive oxygen species (ROS) enzymes compared with control cells. Heparin was used as a positive control because it has been reported to activate MyoD. Taken together, these results suggest that LMWF might regulate MyoD through the MAPK pathway and by regulating ROS activity in C2C12 cells.

Rho-associated protein kinase regulates subcellular localisation of Angiomotin and Hippo-signalling during preimplantation mouse embryo development.

PubMed

Mihajlović, Aleksandar I; Bruce, Alexander W

2016-09-01

The differential activity of the Hippo-signalling pathway between the outer- and inner-cell populations of the developing preimplantation mouse embryo directs appropriate formation of trophectoderm and inner cell mass (ICM) lineages. Such distinct signalling activity is under control of intracellular polarization, whereby Hippo-signalling is either supressed in polarized outer cells or activated in apolar inner cells. The central role of apical-basolateral polarization to such differential Hippo-signalling regulation prompted us to reinvestigate the role of potential upstream molecular regulators affecting apical-basolateral polarity. This study reports that the chemical inhibition of Rho-associated kinase (Rock) is associated with failure to form morphologically distinct blastocysts, indicative of compromised trophectoderm differentiation, and defects in the localization of both apical and basolateral polarity factors associated with malformation of tight junctions. Moreover, Rock-inhibition mediates mislocalization of the Hippo-signalling activator Angiomotin (Amot), to the basolateral regions of outer cells and is concomitant with aberrant activation of the pathway. The Rock-inhibition phenotype is mediated by Amot, as RNAi-based Amot knockdown totally rescues the normal suppression of Hippo-signalling in outer cells. In conclusion, Rock, via regulating appropriate apical-basolateral polarization in outer cells, regulates the appropriate activity of the Hippo-signalling pathway, by ensuring correct subcellular localization of Amot protein in outer cells. Copyright © 2016 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

BMP signaling controls buckling forces to modulate looping morphogenesis of the gut.

PubMed

Nerurkar, Nandan L; Mahadevan, L; Tabin, Clifford J

2017-02-28

Looping of the initially straight embryonic gut tube is an essential aspect of intestinal morphogenesis, permitting proper placement of the lengthy small intestine within the confines of the body cavity. The formation of intestinal loops is highly stereotyped within a given species and results from differential-growth-driven mechanical buckling of the gut tube as it elongates against the constraint of a thin, elastic membranous tissue, the dorsal mesentery. Although the physics of this process has been studied, the underlying biology has not. Here, we show that BMP signaling plays a critical role in looping morphogenesis of the avian small intestine. We first exploited differences between chicken and zebra finch gut morphology to identify the BMP pathway as a promising candidate to regulate differential growth in the gut. Next, focusing on the developing chick small intestine, we determined that Bmp2 expressed in the dorsal mesentery establishes differential elongation rates between the gut tube and mesentery, thereby regulating the compressive forces that buckle the gut tube into loops. Consequently, the number and tightness of loops in the chick small intestine can be increased or decreased directly by modulation of BMP activity in the small intestine. In addition to providing insight into the molecular mechanisms underlying intestinal development, our findings provide an example of how biochemical signals act on tissue-level mechanics to drive organogenesis, and suggest a possible mechanism by which they can be modulated to achieve distinct morphologies through evolution.

BMP signaling controls buckling forces to modulate looping morphogenesis of the gut

PubMed Central

Nerurkar, Nandan L.; Mahadevan, L.; Tabin, Clifford J.

2017-01-01

Looping of the initially straight embryonic gut tube is an essential aspect of intestinal morphogenesis, permitting proper placement of the lengthy small intestine within the confines of the body cavity. The formation of intestinal loops is highly stereotyped within a given species and results from differential-growth–driven mechanical buckling of the gut tube as it elongates against the constraint of a thin, elastic membranous tissue, the dorsal mesentery. Although the physics of this process has been studied, the underlying biology has not. Here, we show that BMP signaling plays a critical role in looping morphogenesis of the avian small intestine. We first exploited differences between chicken and zebra finch gut morphology to identify the BMP pathway as a promising candidate to regulate differential growth in the gut. Next, focusing on the developing chick small intestine, we determined that Bmp2 expressed in the dorsal mesentery establishes differential elongation rates between the gut tube and mesentery, thereby regulating the compressive forces that buckle the gut tube into loops. Consequently, the number and tightness of loops in the chick small intestine can be increased or decreased directly by modulation of BMP activity in the small intestine. In addition to providing insight into the molecular mechanisms underlying intestinal development, our findings provide an example of how biochemical signals act on tissue-level mechanics to drive organogenesis, and suggest a possible mechanism by which they can be modulated to achieve distinct morphologies through evolution. PMID:28193855

Morphological, Genome and Gene Expression Changes in Newly Induced Autopolyploid Chrysanthemum lavandulifolium (Fisch. ex Trautv.) Makino.

PubMed

Gao, Ri; Wang, Haibin; Dong, Bin; Yang, Xiaodong; Chen, Sumei; Jiang, Jiafu; Zhang, Zhaohe; Liu, Chen; Zhao, Nan; Chen, Fadi

2016-10-09

Autopolyploidy is widespread in higher plants and plays an important role in the process of evolution. The present study successfully induced autotetraploidys from Chrysanthemum lavandulifolium by colchicine. The plant morphology, genomic, transcriptomic, and epigenetic changes between tetraploid and diploid plants were investigated. Ligulate flower, tubular flower and leaves of tetraploid plants were greater than those of the diploid plants. Compared with diploid plants, the genome changed as a consequence of polyploidization in tetraploid plants, namely, 1.1% lost fragments and 1.6% novel fragments occurred. In addition, DNA methylation increased after genome doubling in tetraploid plants. Among 485 common transcript-derived fragments (TDFs), which existed in tetraploid and diploid progenitors, 62 fragments were detected as differentially expressed TDFs, 6.8% of TDFs exhibited up-regulated gene expression in the tetraploid plants and 6.0% exhibited down-regulation. The present study provides a reference for further studying the autopolyploidization role in the evolution of C. lavandulifolium. In conclusion, the autopolyploid C. lavandulifolium showed a global change in morphology, genome and gene expression compared with corresponding diploid.

Leukocyte-associated immunoglobulin-like receptor-1 is expressed on human megakaryocytes and negatively regulates the maturation of primary megakaryocytic progenitors and cell line

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xue, Jiangnan, E-mail: xuejinagnan@263.net; Zhang, Xiaoshu; Zhao, Haiya

Research highlights: {yields} LAIR-1 is expressed on human megakaryocytes from an early stage. {yields} Up-regulation of LAIR-1 negatively regulates megakaryocytic differentiation of cell line. {yields} LAIR-1 negatively regulates the differentiation of primary megakaryocytic progenitors. -- Abstract: Leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1) is an inhibitory collagen receptor which belongs to the immunoglobulin (Ig) superfamily. Although the inhibitory function of LAIR-1 has been extensively described in multiple leukocytes, its role in megakaryocyte (MK) has not been explored so far. Here, we show that LAIR-1 is expressed on human bone marrow CD34{sup +}CD41a{sup +} and CD41a{sup +}CD42b{sup +} cells. LAIR-1 is also detectable inmore » a fraction of human cord blood CD34{sup +} cell-derived MK that has morphological characteristics of immature MK. In megakaryoblastic cell line Dami, the membrane protein expression of LAIR-1 is up-regulated significantly when cells are treated with phorbol ester phorbol 12-myristate 13-acetate (PMA). Furthermore, cross-linking of LAIR-1 in Dami cells with its natural ligand or anti-LAIR-1 antibody leads to the inhibition of cell proliferation and PMA-promoted differentiation when examined by the MK lineage-specific markers (CD41a and CD42b) and polyploidization. In addition, we also observed that cross-linking of LAIR-1 results in decreased MK generation from primary human CD34{sup +} cells cultured in a cytokines cocktail that contains TPO. These results suggest that LAIR-1 is a likely candidate for an early marker of MK differentiation, and provide initial evidence indicating that LAIR-1 serves as a negative regulator of megakaryocytopoiesis.« less

Increased intracellular pH is necessary for adult epithelial and embryonic stem cell differentiation

PubMed Central

Azimova, Dinara R.

2016-01-01

Despite extensive knowledge about the transcriptional regulation of stem cell differentiation, less is known about the role of dynamic cytosolic cues. We report that an increase in intracellular pH (pHi) is necessary for the efficient differentiation of Drosophila adult follicle stem cells (FSCs) and mouse embryonic stem cells (mESCs). We show that pHi increases with differentiation from FSCs to prefollicle cells (pFCs) and follicle cells. Loss of the Drosophila Na+–H+ exchanger DNhe2 lowers pHi in differentiating cells, impairs pFC differentiation, disrupts germarium morphology, and decreases fecundity. In contrast, increasing pHi promotes excess pFC cell differentiation toward a polar/stalk cell fate through suppressing Hedgehog pathway activity. Increased pHi also occurs with mESC differentiation and, when prevented, attenuates spontaneous differentiation of naive cells, as determined by expression of microRNA clusters and stage-specific markers. Our findings reveal a previously unrecognized role of pHi dynamics for the differentiation of two distinct types of stem cell lineages, which opens new directions for understanding conserved regulatory mechanisms. PMID:27821494

Synergetic effect of topological cue and periodic mechanical tension-stress on osteogenic differentiation of rat bone mesenchymal stem cells.

PubMed

Liu, Yao; Yang, Guang; Ji, Huanzhong; Xiang, Tao; Luo, En; Zhou, Shaobing

2017-06-01

Mesenchymal stem cells (MSCs) are able to self-renew and differentiate into tissues of mesenchymal origin, making them to be significant for cell-based therapies, such as metabolic bone diseases and bone repair. Regulating the differentiation of MSCs is significant for bone regeneration. Electrospun fibers mimicking natural extracellular matrix (ECM), is an effective artificial ECM to regulate the behaviors and fates of MSCs. The aligned electrospun fibers can modulate polar cell pattern of bone mesenchymal stem cells, which leads to more obvious osteogenic differentiation. Apart from the topographic effect of electrospun fibers, mechanical cues can also intervene the cell behaviors. In this study, the osteogenic differentiation of rat bone mesenchymal stem cells was evaluated, which were cultured on aligned/random electrospun fiber mats materials under mechanical tension intervention. Scanning electron microscope and immune-fluorescent staining were used to directly observe the polarity changing of cellular morphology and cytoskeleton. The results proved that aligned electrospun fibers could be more conducive to promote osteogenic differentiation of rat bone mesenchymal stem cells and this promotion of osteogenic differentiation was enhanced by tension intervention. These results were correlated to the quantitative real-time PCR assay. In general, culturing rat bone mesenchymal stem cells on electrospun fibers under the intervention of mechanical tension is an effective way to mimic a more real cellular microenvironment. Copyright © 2017 Elsevier B.V. All rights reserved.

Interconnected network motifs control podocyte morphology and kidney function.

PubMed

Azeloglu, Evren U; Hardy, Simon V; Eungdamrong, Narat John; Chen, Yibang; Jayaraman, Gomathi; Chuang, Peter Y; Fang, Wei; Xiong, Huabao; Neves, Susana R; Jain, Mohit R; Li, Hong; Ma'ayan, Avi; Gordon, Ronald E; He, John Cijiang; Iyengar, Ravi

2014-02-04

Podocytes are kidney cells with specialized morphology that is required for glomerular filtration. Diseases, such as diabetes, or drug exposure that causes disruption of the podocyte foot process morphology results in kidney pathophysiology. Proteomic analysis of glomeruli isolated from rats with puromycin-induced kidney disease and control rats indicated that protein kinase A (PKA), which is activated by adenosine 3',5'-monophosphate (cAMP), is a key regulator of podocyte morphology and function. In podocytes, cAMP signaling activates cAMP response element-binding protein (CREB) to enhance expression of the gene encoding a differentiation marker, synaptopodin, a protein that associates with actin and promotes its bundling. We constructed and experimentally verified a β-adrenergic receptor-driven network with multiple feedback and feedforward motifs that controls CREB activity. To determine how the motifs interacted to regulate gene expression, we mapped multicompartment dynamical models, including information about protein subcellular localization, onto the network topology using Petri net formalisms. These computational analyses indicated that the juxtaposition of multiple feedback and feedforward motifs enabled the prolonged CREB activation necessary for synaptopodin expression and actin bundling. Drug-induced modulation of these motifs in diseased rats led to recovery of normal morphology and physiological function in vivo. Thus, analysis of regulatory motifs using network dynamics can provide insights into pathophysiology that enable predictions for drug intervention strategies to treat kidney disease.

Interconnected Network Motifs Control Podocyte Morphology and Kidney Function

PubMed Central

Azeloglu, Evren U.; Hardy, Simon V.; Eungdamrong, Narat John; Chen, Yibang; Jayaraman, Gomathi; Chuang, Peter Y.; Fang, Wei; Xiong, Huabao; Neves, Susana R.; Jain, Mohit R.; Li, Hong; Ma’ayan, Avi; Gordon, Ronald E.; He, John Cijiang; Iyengar, Ravi

2014-01-01

Podocytes are kidney cells with specialized morphology that is required for glomerular filtration. Diseases, such as diabetes, or drug exposure that causes disruption of the podocyte foot process morphology results in kidney pathophysiology. Proteomic analysis of glomeruli isolated from rats with puromycin-induced kidney disease and control rats indicated that protein kinase A (PKA), which is activated by adenosine 3′,5′-monophosphate (cAMP), is a key regulator of podocyte morphology and function. In podocytes, cAMP signaling activates cAMP response element–binding protein (CREB) to enhance expression of the gene encoding a differentiation marker, synaptopodin, a protein that associates with actin and promotes its bundling. We constructed and experimentally verified a β-adrenergic receptor–driven network with multiple feedback and feedforward motifs that controls CREB activity. To determine how the motifs interacted to regulate gene expression, we mapped multicompartment dynamical models, including information about protein subcellular localization, onto the network topology using Petri net formalisms. These computational analyses indicated that the juxtaposition of multiple feedback and feedforward motifs enabled the prolonged CREB activation necessary for synaptopodin expression and actin bundling. Drug-induced modulation of these motifs in diseased rats led to recovery of normal morphology and physiological function in vivo. Thus, analysis of regulatory motifs using network dynamics can provide insights into pathophysiology that enable predictions for drug intervention strategies to treat kidney disease. PMID:24497609

PKC delta and NADPH oxidase in retinoic acid-induced neuroblastoma cell differentiation.

PubMed

Nitti, Mariapaola; Furfaro, Anna Lisa; Cevasco, Claudia; Traverso, Nicola; Marinari, Umberto Maria; Pronzato, Maria Adelaide; Domenicotti, Cinzia

2010-05-01

The role of reactive oxygen species (ROS) in the regulation of signal transduction processes has been well established in many cell types and recently the fine tuning of redox signalling in neurons received increasing attention. With regard to this, the involvement of NADPH oxidase (NOX) in neuronal pathophysiology has been proposed but deserves more investigation. In the present study, we used SH-SY5Y neuroblastoma cells to analyse the role of NADPH oxidase in retinoic acid (RA)-induced differentiation, pointing out the involvement of protein kinase C (PKC) delta in the activation of NOX. Retinoic acid induces neuronal differentiation as revealed by the increased expression of MAP2, the decreased cell doubling rate, and the gain in neuronal morphological features and these events are accompanied by the increased expression level of PKC delta and p67(phox), one of the components of NADPH oxidase. Using DPI to inhibit NOX activity we show that retinoic acid acts through this enzyme to induce morphological changes linked to the differentiation. Moreover, using rottlerin to inhibit PKC delta or transfection experiments to overexpress it, we show that retinoic acid acts through this enzyme to induce MAP2 expression and to increase p67(phox) membrane translocation leading to NADPH oxidase activation. These findings identify the activation of PKC delta and NADPH oxidase as crucial steps in RA-induced neuroblastoma cell differentiation. 2010 Elsevier Inc. All rights reserved.

Depolarization Alters Phenotype, Maintains Plasticity of Predifferentiated Mesenchymal Stem Cells

PubMed Central

Sundelacruz, Sarah; Levin, Michael

2013-01-01

Although adult stem cell transplantation has been implemented as a therapy for tissue repair, it is limited by the availability of functional adult stem cells. A potential approach to generate stem and progenitor cells may be to modulate the differentiated status of somatic cells. Therefore, there is a need for a better understanding of how the differentiated phenotype of mature cells is regulated. We hypothesize that bioelectric signaling plays an important role in the maintenance of the differentiated state, as it is a functional regulator of the differentiation process in various cells and tissues. In this study, we asked whether the mature phenotype of osteoblasts and adipocytes derived from human mesenchymal stem cells (hMSCs) could be altered by modulation of their membrane potential. hMSC-derived osteoblasts and adipocytes were depolarized by treatment with ouabain, a Na+/K+ ATPase inhibitor, or by treatment with high concentrations of extracellular K+. To characterize the effect of voltage modulation on the differentiated state, the depolarized cells were evaluated for (1) the loss of differentiation markers; (2) the up-regulation of stemness markers and stem properties; and (3) differences in gene expression profiles in response to voltage modulation. hMSC-derived osteoblasts and adipocytes exhibited significant down-regulation of bone and fat tissue markers in response to depolarization, despite the presence of differentiation-inducing soluble factors, suggesting that bioelectric signaling overrides biochemical signaling in the maintenance of cell state. Suppression of the osteoblast or adipocyte phenotype was not accompanied by up-regulation of genes associated with the stem state. Thus, depolarization does not activate the stem cell genetic signature and, therefore, does not induce a full reprogramming event. However, after transdifferentiating the depolarized cells to evaluate for multi-lineage potential, depolarized osteoblasts demonstrated improved ability to achieve correct adipocyte morphology compared with nondepolarized osteoblasts. The present study thus demonstrates that depolarization reduces the differentiated phenotype of hMSC-derived cells and improves their transdifferentiation capacity, but does not restore a stem-like genetic profile. Through global transcript profiling of depolarized osteoblasts, we identified pathways that may mediate the effects of voltage signaling on cell state, which will require a detailed mechanistic inquiry in future studies. PMID:23738690

A high-content morphological screen identifies novel microRNAs that regulate neuroblastoma cell differentiation

PubMed Central

Zhao, Zhenze; Ma, Xiuye; Hsiao, Tzu-Hung; Lin, Gregory; Kosti, Adam; Yu, Xiaojie; Suresh, Uthra; Chen, Yidong; Tomlinson, Gail E.; Pertsemlidis, Alexander; Du, Liqin

2014-01-01

Neuroblastoma, the most common extracranial solid tumor of childhood, arises from neural crest cell precursors that fail to differentiate. Inducing cell differentiation is an important therapeutic strategy for neuroblastoma. We developed a direct functional high-content screen to identify differentiation-inducing microRNAs, in order to develop microRNA-based differentiation therapy for neuroblastoma. We discovered novel microRNAs, and more strikingly, three microRNA seed families that induce neuroblastoma cell differentiation. In addition, we showed that microRNA seed families were overrepresented in the identified group of fourteen differentiation-inducing microRNAs, suggesting that microRNA seed families are functionally more important in neuroblastoma differentiation than microRNAs with unique sequences. We further investigated the differentiation-inducing function of the microRNA-506-3p/microRNA-124-3p seed family, which was the most potent inducer of differentiation. We showed that the differentiation-inducing function of microRNA-506-3p/microRNA-124-3p is mediated, at least partially, by down-regulating expression of their targets CDK4 and STAT3. We further showed that expression of miR-506-3p, but not miR-124-3p, is dramatically upregulated in differentiated neuroblastoma cells, suggesting the important role of endogenous miR-506-3p in differentiation and tumorigenesis. Overall, our functional screen on microRNAs provided the first comprehensive analysis on the involvements of microRNA species in neuroblastoma cell differentiation and identified novel differentiation-inducing microRNAs. Further investigations are certainly warranted to fully characterize the function of the identified microRNAs in order to eventually benefit neuroblastoma therapy. PMID:24811707

Silencing hyperoxia-induced C/EBPα in neonatal mice improves lung architecture via enhanced proliferation of alveolar epithelial cells

PubMed Central

Yang, Guang; Hinson, Maurice D.; Bordner, Jessica E.; Lin, Qing S.; Fernando, Amal P.; La, Ping; Wright, Clyde J.

2011-01-01

Postnatal lung development requires proliferation and differentiation of specific cell types at precise times to promote proper alveolar formation. Hyperoxic exposure can disrupt alveolarization by inhibiting cell growth; however, it is not fully understood how this is mediated. The transcription factor CCAAT/enhancer binding protein-α (C/EBPα) is highly expressed in the lung and plays a role in cell proliferation and differentiation in many tissues. After 72 h of hyperoxia, C/EBPα expression was significantly enhanced in the lungs of newborn mice. The increased C/EBPα protein was predominantly located in alveolar type II cells. Silencing of C/EBPα with a transpulmonary injection of C/EBPα small interfering RNA (siRNA) prior to hyperoxic exposure reduced expression of markers of type I cell and differentiation typically observed after hyperoxia but did not rescue the altered lung morphology at 72 h. Nevertheless, when C/EBPα hyperoxia-exposed siRNA-injected mice were allowed to recover for 2 wk in room air, lung epithelial cell proliferation was increased and lung morphology was restored compared with hyperoxia-exposed control siRNA-injected mice. These data suggest that C/EBPα is an important regulator of postnatal alveolar epithelial cell proliferation and differentiation during injury and repair. PMID:21571903

Oligodendroglial p130Cas Is a Target of Fyn Kinase Involved in Process Formation, Cell Migration and Survival

PubMed Central

Gonsior, Constantin; Binamé, Fabien; Frühbeis, Carsten; Bauer, Nina M.; Hoch-Kraft, Peter; Luhmann, Heiko J.; Trotter, Jacqueline; White, Robin

2014-01-01

Oligodendrocytes are the myelinating glial cells of the central nervous system. In the course of brain development, oligodendrocyte precursor cells migrate, scan the environment and differentiate into mature oligodendrocytes with multiple cellular processes which recognize and ensheath neuronal axons. During differentiation, oligodendrocytes undergo dramatic morphological changes requiring cytoskeletal rearrangements which need to be tightly regulated. The non-receptor tyrosine kinase Fyn plays a central role in oligodendrocyte differentiation and myelination. In order to improve our understanding of the role of oligodendroglial Fyn kinase, we have identified Fyn targets in these cells. Purification and mass-spectrometric analysis of tyrosine-phosphorylated proteins in response to overexpressed active Fyn in the oligodendrocyte precursor cell line Oli-neu, yielded the adaptor molecule p130Cas. We analyzed the function of this Fyn target in oligodendroglial cells and observed that reduction of p130Cas levels by siRNA affects process outgrowth, the thickness of cellular processes and migration behavior of Oli-neu cells. Furthermore, long term p130Cas reduction results in decreased cell numbers as a result of increased apoptosis in cultured primary oligodendrocytes. Our data contribute to understanding the molecular events taking place during oligodendrocyte migration and morphological differentiation and have implications for myelin formation. PMID:24586768

Relationship between nanotopographical alignment and stem cell fate with live imaging and shape analysis

NASA Astrophysics Data System (ADS)

Newman, Peter; Galenano-Niño, Jorge Luis; Graney, Pamela; Razal, Joselito M.; Minett, Andrew I.; Ribas, João; Ovalle-Robles, Raquel; Biro, Maté; Zreiqat, Hala

2016-12-01

The topography of a biomaterial regulates cellular interactions and determine stem cell fate. A complete understanding of how topographical properties affect cell behavior will allow the rational design of material surfaces that elicit specified biological functions once placed in the body. To this end, we fabricate substrates with aligned or randomly organized fibrous nanostructured topographies. Culturing adipose-derived stem cells (ASCs), we explore the dynamic relationship between the alignment of topography, cell shape and cell differentiation to osteogenic and myogenic lineages. We show aligned topographies differentiate cells towards a satellite cell muscle progenitor state - a distinct cell myogenic lineage responsible for postnatal growth and repair of muscle. We analyze cell shape between the different topographies, using fluorescent time-lapse imaging over 21 days. In contrast to previous work, this allows the direct measurement of cell shape at a given time rather than defining the morphology of the underlying topography and neglecting cell shape. We report quantitative metrics of the time-based morphological behaviors of cell shape in response to differing topographies. This analysis offers insights into the relationship between topography, cell shape and cell differentiation. Cells differentiating towards a myogenic fate on aligned topographies adopt a characteristic elongated shape as well as the alignment of cells.

Protein carboxyl methylation increases in parallel with differentiation of neuroblastoma cells.

PubMed

Kloog, Y; Axelrod, J; Spector, I

1983-02-01

Cells of mouse neuroblastoma clone N1E-115 in the confluent phase of growth can catalyze the formation of endogenous protein carboxyl methyl esters, using a protein carboxyl methylase and membrane-bound methyl acceptor proteins. The enzyme is localized predominantly in the cytosol of the cells and has a molecular weight of about 20,000 daltons. Treatment of the cells with dimethylsulfoxide (DMSO) or hexamethylene-bisacetamide (HMBA), agents that induce morphological and electrophysiological differentiation, results in a marked increase in protein carboxyl methylase activity. Maximal levels are reached 6-7 days after exposure to the agents, a time course that closely parallels the development of electrical excitability mechanisms in these cells. Serum deprivation also causes neurite outgrowth but does not enhance electrical excitability or enzyme activity. The capacity of membrane-bound neuroblastoma protein(s) to be carboxyl methylated is increased by the differentiation procedures that have been examined. However, the increase in methyl acceptor proteins induced by DMSO or HMBA is the largest, and its time course parallels electrophysiological differentiation. In contrast, serum deprivation induced a small increase that reached maximal levels within 24 h. The data suggest that increased protein carboxyl methylation is a developmentally regulated property of neuroblastoma cells and that at least two groups of methyl acceptor proteins are induced during differentiation: a minor group related to morphological differentiation, and a major group that may be related to ionic permeability mechanisms of the excitable membrane.

Long non-coding RNAs regulate effects of β-crystallin B2 on mouse ovary development.

PubMed

Gao, Qian; Ren, Hanxiao; Chen, Mingkun; Niu, Ziguang; Tao, Haibo; Jia, Yin; Zhang, Jianrong; Li, Wenjie

2016-11-01

β-crystallin B2 (CRYBB2) knockout mice exhibit morphological and functional abnormalities in the ovary. Long non‑coding RNAs (lncRNAs) regulate gene transcription and translation, and epigenetic modification of genomic DNA. The present study investigated the role of lncRNAs in mediating the effects of CRYBB2 in the regulation of ovary development in mice. In the current study, ovary tissues from wild‑type (WT) and CRYBB2 knockout mice were subjected to lncRNA and mRNA microarray profiling. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed to group the differentially expressed lncRNAs into regulated gene pathways and functions. The correlation matrix method was used to establish a network of lncRNA and mRNA co‑expression. Quantitative reverse transcription-polymerase chain reaction (RT‑qPCR) was used to verify expression of a number of these differentially expressed lncRNAs and mRNAs. There were 157 differentially expressed lncRNAs and 1,085 differentially expressed mRNAs between ovary tissues from WT and CRYBB2 knockout mice. The GO and KEGG analyses indicated that these differentially expressed lncRNAs and mRNAs were important in Ca2+ signaling and ligand and receptor interactions. The correlation matrix method established an lncRNA and mRNA co‑expression network, consisting of 53 lncRNAs and 45 mRNAs with 98 nodes and 75 connections. RT‑qPCR confirmed downregulation of lncRNA A‑30‑P01019163 expression, which further downregulated its downstream gene purinergic receptor P2X, ligand‑gated ion channel, 7 (P2rx7) expression in ovary tissues from CRYBB2 knockout mice. In conclusion, CRYBB2 regulates expression of different lncRNAs to influence ovary development. lncRNA A‑30‑P01019163 may affect ovarian cell cycle and proliferation by regulating P2rx7 expression in the ovary.

Transgelin is a TGFβ-inducible gene that regulates osteoblastic and adipogenic differentiation of human skeletal stem cells through actin cytoskeleston organization

PubMed Central

Elsafadi, M; Manikandan, M; Dawud, R A; Alajez, N M; Hamam, R; Alfayez, M; Kassem, M; Aldahmash, A; Mahmood, A

2016-01-01

Regenerative medicine is a novel approach for treating conditions in which enhanced bone regeneration is required. We identified transgelin (TAGLN), a transforming growth factor beta (TGFβ)-inducible gene, as an upregulated gene during in vitro osteoblastic and adipocytic differentiation of human bone marrow-derived stromal (skeletal) stem cells (hMSC). siRNA-mediated gene silencing of TAGLN impaired lineage differentiation into osteoblasts and adipocytes but enhanced cell proliferation. Additional functional studies revealed that TAGLN deficiency impaired hMSC cell motility and in vitro transwell cell migration. On the other hand, TAGLN overexpression reduced hMSC cell proliferation, but enhanced cell migration, osteoblastic and adipocytic differentiation, and in vivo bone formation. In addition, deficiency or overexpression of TAGLN in hMSC was associated with significant changes in cellular and nuclear morphology and cytoplasmic organelle composition as demonstrated by high content imaging and transmission electron microscopy that revealed pronounced alterations in the distribution of the actin filament and changes in cytoskeletal organization. Molecular signature of TAGLN-deficient hMSC showed that several genes and genetic pathways associated with cell differentiation, including regulation of actin cytoskeleton and focal adhesion pathways, were downregulated. Our data demonstrate that TAGLN has a role in generating committed progenitor cells from undifferentiated hMSC by regulating cytoskeleton organization. Targeting TAGLN is a plausible approach to enrich for committed hMSC cells needed for regenerative medicine application. PMID:27490926

Distinct requirements of wls, wnt9a, wnt5b and gpc4 in regulating chondrocyte maturation and timing of endochondral ossification

PubMed Central

Ling, Irving TC; Rochard, Lucie; Liao, Eric C.

2017-01-01

Formation of the mandible requires progressive morphologic change, proliferation, differentiation and organization of chondrocytes preceding osteogenesis. The Wnt signaling pathway is involved in regulating bone development and maintenance. Chondrocytes that are fated to become bone require Wnt to polarize and orientate appropriately to initiate the endochondral ossification program. Although the canonical Wnt signaling has been well studied in the context of bone development, the effects of non-canonical Wnt signaling in regulating the timing of cartilage maturation and subsequent bone formation in shaping ventral craniofacial structure is not fully understood.. Here we examined the role of the non-canonical Wnt signaling pathway (wls, gpc4, wnt5b and wnt9a) in regulating zebrafish Meckel’s cartilage maturation to the onset of osteogenic differentiation. We found that disruption of wls resulted in a significant loss of craniofacial bone, whereas lack of gpc4, wnt5b and wnt9a resulted in severely delayed endochondral ossification. This study demonstrates the importance of the non-canonical Wnt pathway in regulating coordinated ventral cartilage morphogenesis and ossification. PMID:27908786

[Proliferation and morphological differentiation of neurblastoma cells in cultured under the effect of avermectins].

PubMed

Miakisheva, S N; Kostenko, M A; Driniaev, V A; Mosin, V A

2001-01-01

The effect of natural avermectin complex (Aversectin C) and Abamectin on the processes of proliferation and morphological differentiation of the neural cells was studied using N1E-115 murine neuroblastoma cells (clone C-1300) as a model. Aversectin C in concentrations 10(-7)-10(-8) was shown to induce morphological differentiation of cultured nervous cells. Treatment with Abamectin resulted in the changes of proliferation pattern of the cells. Morphological differentiation of the cultured nervous cells treated with Aversectin C was associated with electrophysiological one.

Dietary Quercetin Attenuates Adipose Tissue Expansion and Inflammation and Alters Adipocyte Morphology in a Tissue-Specific Manner

PubMed Central

Forney, Laura A.; Lenard, Natalie R.; Stewart, Laura K.

2018-01-01

Chronic inflammation in adipose tissue may contribute to depot-specific adipose tissue expansion, leading to obesity and insulin resistance. Dietary supplementation with quercetin or botanical extracts containing quercetin attenuates high fat diet (HFD)-induced obesity and insulin resistance and decreases inflammation. Here, we determined the effects of quercetin and red onion extract (ROE) containing quercetin on subcutaneous (inguinal, IWAT) vs. visceral (epididymal, EWAT) white adipose tissue morphology and inflammation in mice fed low fat, high fat, high fat plus 50 μg/day quercetin or high fat plus ROE containing 50 μg/day quercetin equivalents for 9 weeks. Quercetin and ROE similarly ameliorated HFD-induced increases in adipocyte size and decreases in adipocyte number in IWAT and EWAT. Furthermore, quercetin and ROE induced alterations in adipocyte morphology in IWAT. Quercetin and ROE similarly decreased HFD-induced IWAT inflammation. However, quercetin and red onion differentially affected HFD-induced EWAT inflammation, with quercetin decreasing and REO increasing inflammatory marker gene expression. Quercetin and REO also differentially regulated circulating adipokine levels. These results show that quercetin or botanical extracts containing quercetin induce white adipose tissue remodeling which may occur through inflammatory-related mechanisms. PMID:29562620

Tumor Cell Plasticity in Uveal Melanoma

PubMed Central

Folberg, Robert; Arbieva, Zarema; Moses, Jonas; Hayee, Amin; Sandal, Tone; Kadkol, ShriHari; Lin, Amy Y.; Valyi-Nagy, Klara; Setty, Suman; Leach, Lu; Chévez-Barrios, Patricia; Larsen, Peter; Majumdar, Dibyen; Pe’er, Jacob; Maniotis, Andrew J.

2006-01-01

The histological detection of laminin-rich vasculogenic mimicry patterns in human primary uveal melanomas is associated with death from metastases. We therefore hypothesized that highly invasive uveal melanoma cells forming vasculogenic mimicry patterns after exposure to a laminin-rich three-dimensional microenvironment would differentially express genes associated with invasive and metastatic behavior. However, we discovered that genes associated with differentiation (GDF15 and ATF3) and suppression of proliferation (CDKNa1/p21) were up-regulated in highly invasive uveal melanoma cells forming vasculogenic mimicry patterns, and genes associated with promotion of invasive and metastatic behavior such as CD44, CCNE2 (cyclin E2), THBS1 (thrombospondin 1), and CSPG2 (chondroitin sulfate proteoglycan; versican) were down-regulated. After forming vasculogenic mimicry patterns, uveal melanoma cells invaded only short distances, failed to replicate, and changed morphologically from the invasive epithelioid to the indolent spindle A phenotype. In human tissue samples, uveal melanoma cells within vasculogenic mimicry patterns assumed the spindle A morphology, and the expression of Ki67 was significantly reduced in adjacent melanoma cells. Thus, the generation of vasculogenic mimicry patterns is accompanied by dampening of the invasive and metastatic uveal melanoma genotype and phenotype and underscores the plasticity of these cells in response to cues from the microenvironment. PMID:17003493

Changes in global gene expression during in vitro decidualization of rat endometrial stromal cells

PubMed Central

Vallejo, Griselda; Maschi, Darío; Citrinovitz, Ana Cecilia Mestre; Aiba, Kazuhiro; Maronna, Ricardo; Yohai, Victor; Ko, Minoru S. H.; Beato, Miguel; Saragüeta, Patricia

2009-01-01

During the preimplantation phase of pregnancy the endometrial stroma differentiates into decidua, a process that implies numerous morphological changes and is an example of physiological transdifferentiation. Here we show that UIII rat endometrial stromal cells cultured in the presence of calf serum acquired morphological features of decidual cells and expressed decidual markers. To identify genes involved in decidualization we compared gene expression patterns of control and decidualized UIII cells using cDNA microarray. We found 322 annotated genes exhibiting significant differences in expression (>3 fold, FDR > 0.005), of which 312 have not been previously related to decidualization. Analysis of overrepresented functions revealed that protein synthesis, gene expression and chromatin architecture and remodeling are the most relevant modified functions during decidualization. Relevant genes are also found in the functional terms differentiation, cell proliferation, signal transduction, and matrix/structural proteins. Several of these new genes involved in decidualization (Csdc2, Trim27, Eef1a1, Bmp1, Wt1, Aes, Gna12, and Men1) are shown to be also regulated in uterine decidua during normal pregnancy. Thus, the UIII cell culture model will allow future mechanistic studies to define the transcriptional network regulating reprogramming of stromal cells into decidual cells. PMID:19780023

Differential growth factor control of bone formation through osteoprogenitor differentiation.

PubMed

Chaudhary, L R; Hofmeister, A M; Hruska, K A

2004-03-01

The osteogenic factors bone morphogenetic protein (BMP-7), platelet-derived growth factor (PDGF)-BB, and fibroblast growth factor (FGF-2) regulate the recruitment of osteoprogenitor cells and their proliferation and differentiation into mature osteoblasts. However, their mechanisms of action on osteoprogenitor cell growth, differentiation, and bone mineralization remain unclear. Here, we tested the hypothesis that these osteogenic agents were capable of regulating osteoblast differentiation and bone formation in vitro. Normal human bone marrow stromal (HBMS) cells were treated with BMP-7 (40 ng ml(-1)), PDGF-BB (20 ng ml(-1)), FGF-2 (20 ng ml(-1)), or FGF-2 plus BMP-7 for 28 days in a serum-containing medium with 10 mM beta-glycerophosphate and 50 microg ml(-1) ascorbic acid. BMP-7 stimulated a morphological change to cuboidal-shaped cells, increased alkaline phosphatase (ALKP) activity, bone sialoprotein (BSP) gene expression, and alizarin red S positive nodule formation. Hydroxyapatite (HA) crystal deposition in the nodules was demonstrated by Fourier transform infrared (FTIR) spectroscopy only in BMP-7- and dexamethasone (DEX)-treated cells. DEX-treated cells appeared elongated and fibroblast-like compared to BMP-7-treated cells. FGF-2 did not stimulate ALKP, and cell morphology was dystrophic. PDGF-BB had little or no effect on ALKP activity and biomineralization. Alizarin Red S staining of cells and calcium assay indicated that BMP-7, DEX, and FGF-2 enhanced calcium mineral deposition, but FTIR spectroscopic analysis demonstrated no formation of HA similar to human bone in control, PDGF-BB-, and FGF-2-treated samples. Thus, FGF-2 stimulated amorphous octacalcium phosphate mineral deposition that failed to mature into HA. Interestingly, FGF-2 abrogated BMP-7-induced ALKP activity and HA formation. Results demonstrate that BMP-7 was competent as a sole factor in the differentiation of human bone marrow stromal cells to bone-forming osteoblasts confirmed by FTIR examination of mineralized matrix. Other growth factors, PDGF, and FGF-2 were incompetent as sole factors, and FGF-2 inhibited BMP-7-stimulated osteoblast differentiation.

Transcriptomic Analysis Reveals Mechanisms of Sterile and Fertile Flower Differentiation and Development in Viburnum macrocephalum f. keteleeri

PubMed Central

Lu, Zhaogeng; Xu, Jing; Li, Weixing; Zhang, Li; Cui, Jiawen; He, Qingsong; Wang, Li; Jin, Biao

2017-01-01

Sterile and fertile flowers are an important evolutionary developmental (evo-devo) phenotype in angiosperm flowers, playing important roles in pollinator attraction and sexual reproductive success. However, the gene regulatory mechanisms underlying fertile and sterile flower differentiation and development remain largely unknown. Viburnum macrocephalum f. keteleeri, which possesses fertile and sterile flowers in a single inflorescence, is a useful candidate species for investigating the regulatory networks in differentiation and development. We developed a de novo-assembled flower reference transcriptome. Using RNA sequencing (RNA-seq), we compared the expression patterns of fertile and sterile flowers isolated from the same inflorescence over its rapid developmental stages. The flower reference transcriptome consisted of 105,683 non-redundant transcripts, of which 5,675 transcripts showed significant differential expression between fertile and sterile flowers. Combined with morphological and cytological changes between fertile and sterile flowers, we identified expression changes of many genes potentially involved in reproductive processes, phytohormone signaling, and cell proliferation and expansion using RNA-seq and qRT-PCR. In particular, many transcription factors (TFs), including MADS-box family members and ABCDE-class genes, were identified, and expression changes in TFs involved in multiple functions were analyzed and highlighted to determine their roles in regulating fertile and sterile flower differentiation and development. Our large-scale transcriptional analysis of fertile and sterile flowers revealed the dynamics of transcriptional networks and potentially key components in regulating differentiation and development of fertile and sterile flowers in Viburnum macrocephalum f. keteleeri. Our data provide a useful resource for Viburnum transcriptional research and offer insights into gene regulation of differentiation of diverse evo-devo processes in flowers. PMID:28298915

Dynamic Hydrostatic Pressure Promotes Differentiation of Human Dental Pulp Stem Cells

PubMed Central

Yu, V; Damek-Poprawa, M.; Nicoll, S. B.; Akintoye, S.O.

2009-01-01

The masticatory apparatus absorbs high occlusal forces, but uncontrolled parafunctional or orthodontic forces damage periodontal ligament (PDL), cause pulpal calcification, pulp necrosis and tooth loss. Morphology and functional differentiation of connective tissue cells can be controlled by mechanical stimuli but effects of uncontrolled forces on intra-pulpal homeostasis and ability of dental pulp stem cells (DPSCs) to withstand direct external forces are unclear. Using dynamic hydrostatic pressure (HSP), we tested the hypothesis that direct HSP disrupts DPSC survival and odontogenic differentiation. DPSCs from four teenage patients were subjected to HSP followed by assessment of cell adhesion, survival and recovery capacity based on odontogenic differentiation, mineralization and responsiveness to bone morphogenetic protein-2 (BMP-2). HSP down-regulated DPSC adhesion and survival but promoted differentiation by increasing mineralization, in vivo hard tissue regeneration and BMP-2 responsiveness despite reduced cell numbers. HSP-treated DPSCs displayed enhanced odontogenic differentiation, an indication of favorable recovery from HSP-induced cellular stress. PMID:19555657

NPH4, a Conditional Modulator of Auxin-Dependent Differential Growth Responses in Arabidopsis1

PubMed Central

Stowe-Evans, Emily L.; Harper, Reneé M.; Motchoulski, Andrei V.; Liscum, Emmanuel

1998-01-01

Although sessile in nature, plants are able to use a number of mechanisms to modify their morphology in response to changing environmental conditions. Differential growth is one such mechanism. Despite its importance in plant development, little is known about the molecular events regulating the establishment of differential growth. Here we report analyses of the nph4 (nonphototropic hypocotyl) mutants of Arabidopsis that suggest that the NPH4 protein plays a central role in the modulation of auxin-dependent differential growth. Results from physiological studies demonstrate that NPH4 activity is conditionally required for a number of differential growth responses, including phototropism, gravitropism, phytochrome-dependent hypocotyl curvature, apical hook maintenance, and abaxial/adaxial leaf-blade expansion. The nph4 mutants exhibited auxin resistance and severely impaired auxin-dependent gene expression, indicating that the defects associated with differential growth likely arise because of altered auxin responsiveness. Moreover, the auxin signaling events mediating phototropism are genetically correlated with the abundance of the NPH4 protein. PMID:9847100

Propylthiouracil, independent of its antithyroid effect, promotes vascular smooth muscle cells differentiation via PTEN induction.

PubMed

Chen, Wei-Jan; Pang, Jong-Hwei S; Lin, Kwang-Huei; Lee, Dany-Young; Hsu, Lung-An; Kuo, Chi-Tai

2010-01-01

Propylthiouracil (PTU), independent of its antithyroid effect, is recently found to have an antiatherosclerotic effect. The aim of this study is to determine the impact of PTU on phenotypic modulation of vascular smooth muscle cells (VSMCs), as phenotypic modulation may contribute to the growth of atherosclerotic lesions and neointimal formation after arterial injury. Propylthiouracil reduced neointimal formation in balloon-injured rat carotid arteries. In vitro, PTU may convert VSMCs from a serum-induced dedifferentiation state to a differentiated state, as indicated by a spindle-shaped morphology and an increase in the expression of SMC differentiation marker contractile proteins, including calponin and smooth muscle (SM)-myosin heavy chain (SM-MHC). Transient transfection studies in VSMCs demonstrated that PTU induced the activity of SMC marker genes (calponin and SM-MHC) promoters, indicating that PTU up-regulates these genes expression predominantly at the transcriptional level. Furthermore, PTU enhanced the expression of PTEN and inhibition of PTEN by siRNA knockdown blocked PTU-induced activation of contractile proteins expression and promoter activity. In the rat carotid injury model, PTU reversed the down-regulation of contractile proteins and up-regulated PTEN in the neointima induced by balloon injury. Propylthiouracil promotes VSMC differentiation, at lest in part, via induction of the PTEN-mediated pathway. These findings suggest a possible mechanism by which PTU may contribute to its beneficial effects on atherogenesis and neointimal formation after arterial injury.

Vitamin D signaling regulates oral keratinocyte proliferation in vitro and in vivo

PubMed Central

YUAN, FENG-NING F.; VALIYAPARAMBIL, JAYASANKER; WOODS, MICHAEL C.; TRAN, HUY; PANT, RIMA; ADAMS, JOHN S.; MALLYA, SANJAY M.

2014-01-01

The secosteroidal hormone 1,25-dihyroxyvitamin D [1,25(OH)2D3] and its receptor, the vitamin D receptor (VDR), are crucial regulators of epidermal proliferation and differentiation. However, the effects of 1,25(OH)2D3-directed signaling on oral keratinocyte pathophysiology have not been well studied. We examined the role of 1,25(OH)2D3 in regulating proliferation and differentiation in cultured oral keratinocytes and on the oral epithelium in vivo. Using lentiviral-mediated shRNA to silence VDR, we generated an oral keratinocyte cell line with stable knockdown of VDR expression. VDR knockdown significantly enhanced proliferation and disrupted calcium- and 1,25(OH)2D3-induced oral keratinocyte differentiation, emphasizing the anti-proliferative and pro-differentiation effects of 1,25(OH)2D3 in oral keratinocytes. Using vitamin D3-deficient diets, we induced chronic vitamin D deficiency in mice as evidenced by decreased serum 25-hydroxyvitamin D (25OHD) concentrations. The vitamin D-deficient mice manifested increased proliferation of the tongue epithelium, but did not develop any morphological or histological abnormalities in the oral epithelium, suggesting that vitamin D deficiency alone is insufficient to alter oral epithelial homeostasis and provoke carcinogenesis. Immunohistochemical analyses of human and murine oral squamous cell carcinomas showed increased VDR expression. Overall, our results provide strong support for a crucial role for vitamin D signaling in oral keratinocyte pathophysiology. PMID:24626468

Differential protein expression during colonic adaptation in ultra-short bowel rats

PubMed Central

Jiang, Hai-Ping; Chen, Tao; Yan, Guang-Rong; Chen, Dan

2011-01-01

AIM: To investigate the proteins involved in colonic adaptation and molecular mechanisms of colonic adaptation in rats with ultra-short bowel syndrome (USBS). METHODS: Sprague Dawley rats were randomly assigned to three groups: USBS group (10 rats) undergoing an approximately 90%-95% small bowel resection; sham-operation group (10 rats) undergoing small bowel transaction and anastomosis; and control group (ten normal rats). Colon morphology and differential protein expression was analyzed after rats were given post-surgical enteral nutrition for 21 d. Protein expression in the colonic mucosa was analyzed by two-dimensional electrophoresis (2-DE) in all groups. Differential protein spots were detected by ImageMaster 2D Platinum software and were further analyzed with matrix-assisted laser desorption/ionization-time-of-flight/time-of-flight-mass spectrometric (MALDI-TOF/TOF-MS) analysis. RESULTS: The colonic mucosal thickness significantly increased in the USBS group compared with the control group (302.1 ± 16.9 μm vs 273.7 ± 16.0 μm, P < 0.05). There was no statistically significant difference between the sham-operation group and control group (P > 0.05). The height of colon plica markedly improved in USBS group compared with the control group (998.4 ± 81.2 μm vs 883.4 ± 39.0 μm, P < 0.05). There was no statistically significant difference between the sham-operation and control groups (P > 0.05). A total of 141 differential protein spots were found in the USBS group. Forty-nine of these spots were down-regulated while 92 protein spots were up-regulated by over 2-folds. There were 133 differential protein spots in USBS group. Thirty of these spots were down-regulated and 103 were up-regulated. There were 47 common differential protein spots among the three groups, including 17 down-regulated protein spots and 30 up-regulated spots. Among 47 differential spots, eight up-regulated proteins were identified by MALDI-TOF/TOF-MS. These proteins were previously reported to be involved in sugar and fat metabolism, protein synthesis and oxidation reduction, which are associated with colonic adaption. CONCLUSION: Eight proteins found in this study play important roles in colonic compensation and are associated with sugar and fat metabolism, protein synthesis, and molecular chaperoning PMID:21633663

Differential protein expression during colonic adaptation in ultra-short bowel rats.

PubMed

Jiang, Hai-Ping; Chen, Tao; Yan, Guang-Rong; Chen, Dan

2011-05-28

To investigate the proteins involved in colonic adaptation and molecular mechanisms of colonic adaptation in rats with ultra-short bowel syndrome (USBS). Sprague Dawley rats were randomly assigned to three groups: USBS group (10 rats) undergoing an approximately 90%-95% small bowel resection; sham-operation group (10 rats) undergoing small bowel transaction and anastomosis; and control group (ten normal rats). Colon morphology and differential protein expression was analyzed after rats were given post-surgical enteral nutrition for 21 d. Protein expression in the colonic mucosa was analyzed by two-dimensional electrophoresis (2-DE) in all groups. Differential protein spots were detected by ImageMaster 2D Platinum software and were further analyzed with matrix-assisted laser desorption/ionization-time-of-flight/time-of-flight-mass spectrometric (MALDI-TOF/TOF-MS) analysis. The colonic mucosal thickness significantly increased in the USBS group compared with the control group (302.1 ± 16.9 μm vs 273.7 ± 16.0 μm, P < 0.05). There was no statistically significant difference between the sham-operation group and control group (P > 0.05). The height of colon plica markedly improved in USBS group compared with the control group (998.4 ± 81.2 μm vs 883.4 ± 39.0 μm, P < 0.05). There was no statistically significant difference between the sham-operation and control groups (P > 0.05). A total of 141 differential protein spots were found in the USBS group. Forty-nine of these spots were down-regulated while 92 protein spots were up-regulated by over 2-folds. There were 133 differential protein spots in USBS group. Thirty of these spots were down-regulated and 103 were up-regulated. There were 47 common differential protein spots among the three groups, including 17 down-regulated protein spots and 30 up-regulated spots. Among 47 differential spots, eight up-regulated proteins were identified by MALDI-TOF/TOF-MS. These proteins were previously reported to be involved in sugar and fat metabolism, protein synthesis and oxidation reduction, which are associated with colonic adaption. Eight proteins found in this study play important roles in colonic compensation and are associated with sugar and fat metabolism, protein synthesis, and molecular chaperoning.

Taraxinic acid, a hydrolysate of sesquiterpene lactone glycoside from the Taraxacum coreanum NAKAI, induces the differentiation of human acute promyelocytic leukemia HL-60 cells.

PubMed

Choi, Jung-Hye; Shin, Kyung-Min; Kim, Na-Young; Hong, Jung-Pyo; Lee, Yong Sup; Kim, Hyoung Ja; Park, Hee-Juhn; Lee, Kyung-Tae

2002-11-01

The present work was performed to elucidate the active moiety of a sesquiterpene lactone, taraxinic acid-1'-O-beta-D-glucopyranoside (1). from Taraxacum coreanum NAKAI on the cytotoxicity of various cancer cells. Based on enzymatic hydrolysis and MTT assay, the active moiety should be attributed to the aglycone taraxinic acid (1a). rather than the glycoside (1). Taraxinic acid exhibited potent antiproliferative activity against human leukemia-derived HL-60. In addition, this compound was found to be a potent inducer of HL-60 cell differentiation as assessed by a nitroblue tetrazolium reduction test, esterase activity assay, phagocytic activity assay, morphology change, and expression of CD 14 and CD 66 b surface antigens. These results suggest that taraxinic acid induces the differentiation of human leukemia cells to monocyte/macrophage lineage. Moreover, the expression level of c-myc was down-regulated during taraxinic acid-dependent HL-60 cell differentiation, whereas p21(CIP1) and p27(KIP1) were up-regulated. Taken together, our results suggest that taraxinic acid may have potential as a therapeutic agent in human leukemia.

Signaling hierarchy regulating human endothelial cell development.

PubMed

Kelly, Melissa A; Hirschi, Karen K

2009-05-01

Our present knowledge of the regulation of mammalian endothelial cell differentiation has been largely derived from studies of mouse embryonic development. However, unique mechanisms and hierarchy of signals that govern human endothelial cell development are unknown and, thus, explored in these studies. Using human embryonic stem cells as a model system, we were able to reproducibly and robustly generate differentiated endothelial cells via coculture on OP9 marrow stromal cells. We found that, in contrast to studies in the mouse, bFGF and VEGF had no specific effects on the initiation of human vasculogenesis. However, exogenous Ihh promoted endothelial cell differentiation, as evidenced by increased production of cells with cobblestone morphology that coexpress multiple endothelial-specific genes and proteins, form lumens, and exhibit DiI-AcLDL uptake. Inhibition of BMP signaling using Noggin or BMP4, specifically, using neutralizing antibodies suppressed endothelial cell formation; whereas, addition of rhBMP4 to cells treated with the hedgehog inhibitor cyclopamine rescued endothelial cell development. Our studies revealed that Ihh promoted human endothelial cell differentiation from pluripotent hES cells via BMP signaling, providing novel insights applicable to modulating human endothelial cell formation and vascular regeneration for human clinical therapies.

The bovine endometrial epithelial cells promote the differentiation of trophoblast stem-like cells to binucleate trophoblast cells.

PubMed

Li, Xiawei; Li, Zhiying; Hou, Dongxia; Zhao, Yuhang; Wang, Chen; Li, Xueling

2016-12-01

Endometrial epithelial cells (EECs) cultured in vitro are valuable tools for investigating embryo implantation and trophoblast differentiation. In this study, we have established the bovine EECs and trophoblast stem-like (TS) coculture system, and used it to investigate the binucleate cell formation of ungulates. The EECs was derived from the uterine horn ipsilateral to the corpus luteum by using collagenase I and deoxyribonuclease I, which exhibited typical epithelial morphology and were expressing bovine uterine epithelial marker such as IFNAR1, IFNAR2, Erα, PGR, ESR1 and KRT18. The cells immunostained positively by epithelial and trophectoderm marker cytokeratin 18 (KRT18) and stromal marker vimentin antibodies, and the KRT18 positive cells reached 99 %. The EECs can be cultured for up to 20 passages in vitro with no significant morphology changes and uterine epithelial marker gene expression alteration. The bTS cells were established in a dual inhibitor system and exhibited typical trophoblast stem cell characteristics. When bTS cells were cultured with EECs, the bTS cells adhered to the EECs as adhering to feeder cells. Binucleate cells began appearing on day 4 of coculture and reached approximately 18.47 % of the differentiated cells. Quantitative real-time PCR or immunofluorescence analyses were performed on bTS cells cocultured at day 6 and day 12. The results showed that the expression level of KRT18 was down-regulated while the expression level of trophoblast differentiation marker MASH2, HAND1, GCM1 and CDX2 was up-regulated in bTS cells. In conclusion, bovine EECs can be obtained from the uterine horn ipsilateral to the corpus luteum via treatment with collagenase I and deoxyribonuclease I, and the EECs-bTS cells coculture system presents an ideal tool for studying the differentiation of bTS cells to trophoblast binucleate cells.

CTCF-Mediated and Pax6-Associated Gene Expression in Corneal Epithelial Cell-Specific Differentiation

PubMed Central

Tsui, Shanli; Wang, Jie; Wang, Ling; Dai, Wei; Lu, Luo

2016-01-01

Background The purpose of the study is to elicit the epigenetic mechanism involving CCCTC binding factor (CTCF)-mediated chromatin remodeling that regulates PAX6 gene interaction with differentiation-associated genes to control corneal epithelial differentiation. Methods Cell cycle progression and specific keratin expressions were measured to monitor changes of differentiation-induced primary human limbal stem/progenitor (HLS/P), human corneal epithelial (HCE) and human telomerase-immortalized corneal epithelial (HTCE) cells. PAX6-interactive and differentiation-associated genes in chromatin remodeling mediated by the epigenetic factor CTCF were detected by circular chromosome conformation capture (4C) and ChIP (Chromatin immunoprecipitation)-on-chip approaches, and verified by FISH (Fluorescent in situ hybridization). Furthermore, CTCF activities were altered by CTCF-shRNA to study the effect of CTCF on mediating interaction of Pax6 and differentiation-associated genes in corneal epithelial cell fate. Results Our results demonstrated that differentiation-induced human corneal epithelial cells expressed typical corneal epithelial characteristics including morphological changes, increased keratin12 expression and G0/G1 accumulations. Expressions of CTCF and PAX6 were suppressed and elevated following the process of differentiation, respectively. During corneal epithelial cell differentiation, differentiation-induced RCN1 and ADAM17 were found interacting with PAX6 in the process of CTCF-mediated chromatin remodeling detected by 4C and verified by ChIP-on-chip and FISH. Diminished CTCF mRNA with CTCF-shRNA in HTCE cells weakened the interaction of PAX6 gene in controlling RCN1/ADAM17 and enhanced early onset of the genes in cell differentiation. Conclusion Our results explain how epigenetic factor CTCF-mediated chromatin remodeling regulates interactions between eye-specific PAX6 and those genes that are induced/associated with cell differentiation to modulate corneal epithelial cell-specific differentiation. PMID:27583466

Degradation-mediated cellular traction directs stem cell fate in covalently crosslinked three-dimensional hydrogels

NASA Astrophysics Data System (ADS)

Khetan, Sudhir; Guvendiren, Murat; Legant, Wesley R.; Cohen, Daniel M.; Chen, Christopher S.; Burdick, Jason A.

2013-05-01

Although cell-matrix adhesive interactions are known to regulate stem cell differentiation, the underlying mechanisms, in particular for direct three-dimensional encapsulation within hydrogels, are poorly understood. Here, we demonstrate that in covalently crosslinked hyaluronic acid (HA) hydrogels, the differentiation of human mesenchymal stem cells (hMSCs) is directed by the generation of degradation-mediated cellular traction, independently of cell morphology or matrix mechanics. hMSCs within HA hydrogels of equivalent elastic moduli that permit (restrict) cell-mediated degradation exhibited high (low) degrees of cell spreading and high (low) tractions, and favoured osteogenesis (adipogenesis). Moreover, switching the permissive hydrogel to a restrictive state through delayed secondary crosslinking reduced further hydrogel degradation, suppressed traction, and caused a switch from osteogenesis to adipogenesis in the absence of changes to the extended cellular morphology. Furthermore, inhibiting tension-mediated signalling in the permissive environment mirrored the effects of delayed secondary crosslinking, whereas upregulating tension induced osteogenesis even in the restrictive environment.

Hydrophilic polyurethane matrix promotes chondrogenesis of mesenchymal stem cells☆

PubMed Central

Nalluri, Sandeep M.; Krishnan, G. Rajesh; Cheah, Calvin; Arzumand, Ayesha; Yuan, Yuan; Richardson, Caley A.; Yang, Shuying; Sarkar, Debanjan

2016-01-01

Segmental polyurethanes exhibit biphasic morphology and can control cell fate by providing distinct matrix guided signals to increase the chondrogenic potential of mesenchymal stem cells (MSCs). Polyethylene glycol (PEG) based hydrophilic polyurethanes can deliver differential signals to MSCs through their matrix phases where hard segments are cell-interactive domains and PEG based soft segments are minimally interactive with cells. These coordinated communications can modulate cell–matrix interactions to control cell shape and size for chondrogenesis. Biphasic character and hydrophilicity of polyurethanes with gel like architecture provide a synthetic matrix conducive for chondrogenesis of MSCs, as evidenced by deposition of cartilage-associated extracellular matrix. Compared to monophasic hydrogels, presence of cell interactive domains in hydrophilic polyurethanes gels can balance cell–cell and cell–matrix interactions. These results demonstrate the correlation between lineage commitment and the changes in cell shape, cell–matrix interaction, and cell–cell adhesion during chondrogenic differentiation which is regulated by polyurethane phase morphology, and thus, represent hydrophilic polyurethanes as promising synthetic matrices for cartilage regeneration. PMID:26046282

Jmy regulates oligodendrocyte differentiation via modulation of actin cytoskeleton dynamics.

PubMed

Azevedo, Maria M; Domingues, Helena S; Cordelières, Fabrice P; Sampaio, Paula; Seixas, Ana I; Relvas, João B

2018-05-06

During central nervous system development, oligodendrocytes form structurally and functionally distinct actin-rich protrusions that contact and wrap around axons to assemble myelin sheaths. Establishment of axonal contact is a limiting step in myelination that relies on the oligodendrocyte's ability to locally coordinate cytoskeletal rearrangements with myelin production, under the control of a transcriptional differentiation program. The molecules that provide fine-tuning of actin dynamics during oligodendrocyte differentiation and axon ensheathment remain largely unidentified. We performed transcriptomics analysis of soma and protrusion fractions from rat brain oligodendrocyte progenitors and found a subcellular enrichment of mRNAs in newly-formed protrusions. Approximately 30% of protrusion-enriched transcripts encode proteins related to cytoskeleton dynamics, including the junction mediating and regulatory protein Jmy, a multifunctional regulator of actin polymerization. Here, we show that expression of Jmy is upregulated during myelination and is required for the assembly of actin filaments and protrusion formation during oligodendrocyte differentiation. Quantitative morphodynamics analysis of live oligodendrocytes showed that differentiation is driven by a stereotypical actin network-dependent "cellular shaping" program. Disruption of actin dynamics via knockdown of Jmy leads to a program fail resulting in oligodendrocytes that do not acquire an arborized morphology and are less efficient in contacting neurites and forming myelin wraps in co-cultures with neurons. Our findings provide new mechanistic insight into the relationship between cell shape dynamics and differentiation in development. © 2018 Wiley Periodicals, Inc.

miR-200 Regulates Endometrial Development During Early Pregnancy

PubMed Central

Mainigi, Monica A.; Word, R. Ann; Kraus, W. Lee; Mendelson, Carole R.

2016-01-01

For successful embryo implantation, endometrial stromal cells must undergo functional and morphological changes, referred to as decidualization. However, the molecular mechanisms that regulate implantation and decidualization are not well defined. Here we demonstrate that the estradiol- and progesterone-regulated microRNA (miR)-200 family was markedly down-regulated in mouse endometrial stromal cells prior to implantation, whereas zinc finger E-box binding homeobox-1 and -2 and other known and predicted targets were up-regulated. Conversely, miR-200 was up-regulated during in vitro decidualization of human endometrial stromal cells. Knockdown of miR-200 negatively affected decidualization and prevented the mesenchymal-epithelial transition-like changes that accompanied decidual differentiation. Notably, superovulation of mice and humans altered miR-200 expression. Our findings suggest that hormonal alterations that accompany superovulation may negatively impact endometrial development and decidualization by causing aberrant miR-200 expression. PMID:27533790

Probing Mechanoregulation of Neuronal Differentiation by Plasma Lithography Patterned Elastomeric Substrates

NASA Astrophysics Data System (ADS)

Nam, Ki-Hwan; Jamilpour, Nima; Mfoumou, Etienne; Wang, Fei-Yue; Zhang, Donna D.; Wong, Pak Kin

2014-11-01

Cells sense and interpret mechanical cues, including cell-cell and cell-substrate interactions, in the microenvironment to collectively regulate various physiological functions. Understanding the influences of these mechanical factors on cell behavior is critical for fundamental cell biology and for the development of novel strategies in regenerative medicine. Here, we demonstrate plasma lithography patterning on elastomeric substrates for elucidating the influences of mechanical cues on neuronal differentiation and neuritogenesis. The neuroblastoma cells form neuronal spheres on plasma-treated regions, which geometrically confine the cells over two weeks. The elastic modulus of the elastomer is controlled simultaneously by the crosslinker concentration. The cell-substrate mechanical interactions are also investigated by controlling the size of neuronal spheres with different cell seeding densities. These physical cues are shown to modulate with the formation of focal adhesions, neurite outgrowth, and the morphology of neuroblastoma. By systematic adjustment of these cues, along with computational biomechanical analysis, we demonstrate the interrelated mechanoregulatory effects of substrate elasticity and cell size. Taken together, our results reveal that the neuronal differentiation and neuritogenesis of neuroblastoma cells are collectively regulated via the cell-substrate mechanical interactions.

GLABROUS INFLORESCENCE STEMS (GIS) is required for trichome branching through gibberellic acid signaling in Arabidopsis.

PubMed

An, Lijun; Zhou, Zhongjing; Su, Sha; Yan, An; Gan, Yinbo

2012-02-01

Cell differentiation generally corresponds to the cell cycle, typically forming a non-dividing cell with a unique differentiated morphology, and Arabidopsis trichome is an excellent model system to study all aspects of cell differentiation. Although gibberellic acid is reported to be involved in trichome branching in Arabidopsis, the mechanism for such signaling is unclear. Here, we demonstrated that GLABROUS INFLORESCENCE STEMS (GIS) is required for the control of trichome branching through gibberellic acid signaling. The phenotypes of a loss-of-function gis mutant and an overexpressor showed that GIS acted as a repressor to control trichome branching. Our results also show that GIS is not required for cell endoreduplication, and our molecular and genetic study results have shown that GIS functions downstream of the key regulator of trichome branching, STICHEL (STI), to control trichome branching through the endoreduplication-independent pathway. Furthermore, our results also suggest that GIS controls trichome branching in Arabidopsis through two different pathways and acts either upstream or downstream of the negative regulator of gibbellic acid signaling SPINDLY (SPY).

Bacillus subtilis sporulation: regulation of gene expression and control of morphogenesis.

PubMed Central

Errington, J

1993-01-01

Bacillus subtilis sporulation is an adaptive response to nutritional stress and involves the differential development of two cells. In the last 10 years or so, virtually all of the regulatory genes controlling sporulation, and many genes directing the structural and morphological changes that accompany sporulation, have been cloned and characterized. This review describes our current knowledge of the program of gene expression during sporulation and summarizes what is known about the functions of the genes that determine the specialized biochemical and morphological properties of sporulating cells. Most steps in the genetic program are controlled by transcription factors that have been characterized in vitro. Two sporulation-specific sigma factors, sigma E and sigma F, appear to segregate at septation, effectively determining the differential development of the mother cell and prespore. Later, each sigma is replaced by a second cell-specific sigma factor, sigma K in the mother cell and sigma G in the prespore. The synthesis of each sigma factor is tightly regulated at both the transcriptional and posttranslational levels. Usually this regulation involves an intercellular interaction that coordinates the developmental programmes of the two cells. At least two other transcription factors fine tune the timing and levels of expression of genes in the sigma E and sigma K regulons. The controlled synthesis of the sigma factors and other transcription factors leads to a spatially and temporally ordered program of gene expression. The gene products made during each successive stage of sporulation help to bring about a sequence of gross morphological changes and biochemical adaptations. The formation of the asymmetric spore septum, engulfment of the prespore by the mother cell, and formation of the spore core, cortex, and coat are described. The importance of these structures in the development of the resistance, dormancy, and germination properties of the spore is assessed. Images PMID:8464402

Insulin-like growth factor 1 can promote the osteogenic differentiation and osteogenesis of stem cells from apical papilla.

PubMed

Wang, Sainan; Mu, Jinquan; Fan, Zhipeng; Yu, Yan; Yan, Ming; Lei, Gang; Tang, Chunbo; Wang, Zilu; Zheng, Yangyu; Yu, Jinhua; Zhang, Guangdong

2012-05-01

Insulin-like growth factor 1 (IGF-1) plays an important role in the regulation of tooth root development, and stem cells from apical papilla (SCAPs) are responsible for the formation of root pulp and dentin. To date, it remains unclear whether IGF-1 can regulate the function of SCAPs. In this study, SCAPs were isolated and purified from human immature root apex, and stimulated by 100 ng/mL exogenous IGF-1. The effects of IGF-1 on the proliferation and differentiation of SCAPs were subsequently investigated. IGF-1 treated SCAPs presented the morphological and ultrastructural changes. Cell proliferation, alkaline phosphatase (ALP) activity and mineralization capacity of SCAPs were increased by IGF-1. Western blot and quantitative RT-PCR analyses further demonstrated that the expression of osteogenic-related proteins and genes (e.g., alkaline phosphatase, runt-related transcription factor 2, osterix, and osteocalcin) was significantly up-regulated in IGF-1 treated SCAPs, whereas the expression of odontoblast-specific markers (e.g., dentin sialoprotein and dentin sialophosphoprotein) was down-regulated by IGF-1. In vivo results revealed that IGF-1 treated SCAPs mostly gave birth to bone-like tissues while untreated SCAPs mainly generated dentin-pulp complex-like structures after transplantation. The present study revealed that IGF-1 can promote the osteogenic differentiation and osteogenesis capacity of SCAPs, but weaken their odontogenic differentiation and dentinogenesis capability, indicating that IGF-1 treated SCAPs can be used as a potential candidate for bone tissue engineering. Copyright © 2011 Elsevier B.V. All rights reserved.

Topography of calcium phosphate ceramics regulates primary cilia length and TGF receptor recruitment associated with osteogenesis.

PubMed

Zhang, Jingwei; Dalbay, Melis T; Luo, Xiaoman; Vrij, Erik; Barbieri, Davide; Moroni, Lorenzo; de Bruijn, Joost D; van Blitterswijk, Clemens A; Chapple, J Paul; Knight, Martin M; Yuan, Huipin

2017-07-15

The surface topography of synthetic biomaterials is known to play a role in material-driven osteogenesis. Recent studies show that TGFβ signalling also initiates osteogenic differentiation. TGFβ signalling requires the recruitment of TGFβ receptors (TGFβR) to the primary cilia. In this study, we hypothesize that the surface topography of calcium phosphate ceramics regulates stem cell morphology, primary cilia structure and TGFβR recruitment to the cilium associated with osteogenic differentiation. We developed a 2D system using two types of tricalcium phosphate (TCP) ceramic discs with identical chemistry. One sample had a surface topography at micron-scale (TCP-B, with a bigger surface structure dimension) whilst the other had a surface topography at submicron scale (TCP-S, with a smaller surface structure dimension). In the absence of osteogenic differentiation factors, human bone marrow stromal cells (hBMSCs) were more spread on TCP-S than on TCP-B with alterations in actin organization and increased primary cilia prevalence and length. The cilia elongation on TCP-S was similar to that observed on glass in the presence of osteogenic media and was followed by recruitment of transforming growth factor-β RII (p-TGFβ RII) to the cilia axoneme. This was associated with enhanced osteogenic differentiation of hBMSCs on TCP-S, as shown by alkaline phosphatase activity and gene expression for key osteogenic markers in the absence of additional osteogenic growth factors. Similarly, in vivo after a 12-week intramuscular implantation in dogs, TCP-S induced bone formation while TCP-B did not. It is most likely that the surface topography of calcium phosphate ceramics regulates primary cilia length and ciliary recruitment of p-TGFβ RII associated with osteogenesis and bone formation. This bioengineering control of osteogenesis via primary cilia modulation may represent a new type of biomaterial-based ciliotherapy for orthopedic, dental and maxillofacial surgery applications. The surface topography of synthetic biomaterials plays important roles in material-driven osteogenesis. The data presented herein have shown that the surface topography of calcium phosphate ceramics regulates mesenchymal stromal cells (e.g., human bone marrow mesenchymal stromal cells, hBMSCs) with respect to morphology, primary cilia structure and TGFβR recruitment to the cilium associated with osteogenic differentiation in vitro. Together with bone formation in vivo, our results suggested a new type of biomaterial-based ciliotherapy for orthopedic, dental and maxillofacial surgery by the bioengineering control of osteogenesis via primary cilia modulation. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Transcriptome analysis of the painted lady butterfly, Vanessa cardui during wing color pattern development.

PubMed

Connahs, Heidi; Rhen, Turk; Simmons, Rebecca B

2016-03-31

Butterfly wing color patterns are an important model system for understanding the evolution and development of morphological diversity and animal pigmentation. Wing color patterns develop from a complex network composed of highly conserved patterning genes and pigmentation pathways. Patterning genes are involved in regulating pigment synthesis however the temporal expression dynamics of these interacting networks is poorly understood. Here, we employ next generation sequencing to examine expression patterns of the gene network underlying wing development in the nymphalid butterfly, Vanessa cardui. We identified 9, 376 differentially expressed transcripts during wing color pattern development, including genes involved in patterning, pigmentation and gene regulation. Differential expression of these genes was highest at the pre-ommochrome stage compared to early pupal and late melanin stages. Overall, an increasing number of genes were down-regulated during the progression of wing development. We observed dynamic expression patterns of a large number of pigment genes from the ommochrome, melanin and also pteridine pathways, including contrasting patterns of expression for paralogs of the yellow gene family. Surprisingly, many patterning genes previously associated with butterfly pattern elements were not significantly up-regulated at any time during pupation, although many other transcription factors were differentially expressed. Several genes involved in Notch signaling were significantly up-regulated during the pre-ommochrome stage including slow border cells, bunched and pebbles; the function of these genes in the development of butterfly wings is currently unknown. Many genes involved in ecdysone signaling were also significantly up-regulated during early pupal and late melanin stages and exhibited opposing patterns of expression relative to the ecdysone receptor. Finally, a comparison across four butterfly transcriptomes revealed 28 transcripts common to all four species that have no known homologs in other metazoans. This study provides a comprehensive list of differentially expressed transcripts during wing development, revealing potential candidate genes that may be involved in regulating butterfly wing patterns. Some differentially expressed genes have no known homologs possibly representing genes unique to butterflies. Results from this study also indicate that development of nymphalid wing patterns may arise not only from melanin and ommochrome pigments but also the pteridine pigment pathway.

Gap junctional intercellular communication is required to maintain embryonic stem cells in a non-differentiated and proliferative state.

PubMed

Todorova, Mariana G; Soria, Bernat; Quesada, Ivan

2008-02-01

Pluripotent embryonic stem (ES) cells are capable of maintaining a self-renewal state and have the potential to differentiate into derivatives of all three embryonic germ layers. Despite their importance in cell therapy and developmental biology, the mechanisms whereby ES cells remain in a proliferative and pluripotent state are still not fully understood. Here we establish a critical role of gap junctional intercellular communication (GJIC) and connexin43 (Cx43) in both processes. Pharmacological blockers of GJIC and Cx43 down-regulation by small interfering RNA (siRNA) caused a profound inhibitory effect on GJIC, as evidenced by experiments of fluorescence recovery after photobleaching. This deficient intercellular communication in ES cells induced a loss of their pluripotent state, which was manifested in morphological changes, a decrease in alkaline phosphatase activity, Oct-3/4 and Nanog expression, as well as an up-regulation of several differentiation markers. A decrease in the proliferation rate was also detected. Under these conditions, the formation of embryoid bodies from mouse ES cells was impaired, although this inhibition was reversible upon restoration of GJIC. Our findings define a major function of GJIC in the regulation of self-renewal and maintenance of pluripotency in ES cells. (c) 2007 Wiley-Liss, Inc.

Midgut morphological changes and autophagy during metamorphosis in sand flies.

PubMed

Malta, Juliana; Heerman, Matthew; Weng, Ju Lin; Fernandes, Kenner M; Martins, Gustavo Ferreira; Ramalho-Ortigão, Marcelo

2017-06-01

During metamorphosis, holometabolous insects undergo significant remodeling of their midgut and become able to cope with changes in dietary requirements between larval and adult stages. At this stage, insects must be able to manage and recycle available food resources in order to develop fully into adults, especially when no nutrients are acquired from the environment. Autophagy has been previously suggested to play a crucial role during metamorphosis of the mosquito. Here, we investigate the overall morphological changes of the midgut of the sand fly during metamorphosis and assess the expression profiles of the autophagy-related genes ATG1, ATG6, and ATG8, which are associated with various steps of the autophagic process. Morphological changes in the midgut start during the fourth larval instar, with epithelial degeneration followed by remodeling via the differentiation of regenerative cells in pre-pupal and pupal stages. The changes in the midgut epithelium are paired with the up-regulation of ATG1, ATG6 and ATG8 during the larva-adult transition. Vein, a putative epidermal growth factor involved in regulating epithelial midgut regeneration, is also up-regulated. Autophagy has further been confirmed in sand flies via the presence of autophagosomes residing within the cytoplasmic compartment of the pupal stages. An understanding of the underlying mechanisms of this process should aid the future management of this neglected tropical vector.

alpha2-chimaerin, a Cdc42/Rac1 regulator, is selectively expressed in the rat embryonic nervous system and is involved in neuritogenesis in N1E-115 neuroblastoma cells.

PubMed

Hall, C; Michael, G J; Cann, N; Ferrari, G; Teo, M; Jacobs, T; Monfries, C; Lim, L

2001-07-15

Neuronal differentiation involves Rac and Cdc42 GTPases. alpha-Chimaerin, a Rac/Cdc42 regulator, occurs as alpha1- and alternatively spliced Src homology 2 (SH2) domain-containing alpha2-isoforms. alpha2-chimaerin mRNA was highly expressed in the rat embryonic nervous system, especially in early postmitotic neurons. alpha1-chimaerin mRNA was undetectable before embryonic day 16.5. Adult alpha2-chimaerin mRNA was restricted to neurons within specific brain regions, with highest expression in the entorhinal cortex. alpha2-chimaerin protein localized to neuronal perikarya, dendrites, and axons. The overall pattern of alpha2-chimaerin mRNA expression resembles that of cyclin-dependent kinase regulator p35 (CDK5/p35) which participates in neuronal differentiation and with which chimaerin interacts. To determine whether alpha2-chimaerin may have a role in neuronal differentiation and the relevance of the SH2 domain, the morphological effects of both chimaerin isoforms were investigated in N1E-115 neuroblastoma cells. When plated on poly-lysine, transient alpha2-chimaerin but not alpha1-chimaerin transfectants formed neurites. Permanent alpha2-chimaerin transfectants generated neurites whether or not they were stimulated by serum starvation, and many cells were enlarged. Permanent alpha1-chimaerin transfectants displayed numerous microspikes and contained F-actin clusters, a Cdc42-phenotype, but generated few neurites. In neuroblastoma cells, alpha2-chimaerin was predominantly soluble with some being membrane-associated, whereas alpha1-chimaerin was absent from the cytosol, being membrane- and cytoskeleton-associated, paralleling their subcellular distribution in brain. Transient transfection with alpha2-chimaerin mutated in the SH2 domain (N94H) generated an alpha1-chimaerin-like phenotype, protein partitioned in the particulate fraction, and in NGF-stimulated pheochromocytoma cell line 12 (PC12) cells, neurite formation was inhibited. These results indicate a role for alpha2-chimaerin in morphological differentiation for which its SH2 domain is vital.

A cascade of Ca2+/calmodulin-dependent protein kinases regulates the differentiation and functional activation of murine neutrophils

PubMed Central

Gaines, Peter; Lamoureux, James; Marisetty, Anantha; Chi, Jeffrey; Berliner, Nancy

2008-01-01

Objective The function of neutrophils as primary mediators of innate immunity depends on the activity of granule proteins and critical components of the NADPH oxidase complex. Expression of their cognate genes is regulated during neutrophil differentiation by a complex network of intracellular signaling pathways. In this study we have investigated the role of two members of the calcium/calmodulin-dependent protein kinase (CaMK) signaling cascade, CaMKI-like kinase (CKLiK) and CaMKKα, in regulating neutrophil differentiation and functional activation. Materials and Methods Mouse myeloid cell lines were used to examine the expression of a CaMK cascade in developing neutrophils and to examine the effects of constitutive activation versus inhibition of CaMKs on neutrophil maturation. Results Expression of CaMKKα was shown to increase during neutrophil differentiation in multiple cell lines, whereas expression of CKLiK increased as multipotent progenitors committed to promyelocytes but then decreased as cells differentiated into mature neutrophils. Expression of constitutively active CKLiKs did not affect morphologic maturation, but caused dramatic decreases in both respiratory burst responses and chemotaxis. This loss of neutrophil function was accompanied by reduced secondary granule and gp91phox gene expression. The CaMK inhibitor KN93 attenuated cytokine-stimulated proliferative responses in promyelocytic cell lines, and inhibited the respiratory burst. Similar data were observed with the CaMKKα inhibitor, STO-609. Conclusions Overactivation of a cascade of CaMKs inhibits neutrophil maturation, suggesting that these kinases play an antagonistic role during neutrophil differentiation, but at least one CaMK is required for myeloid cell expansion and functional activation. PMID:18400360

Episome-generated N-myc antisense RNA restricts the differentiation potential of primitive neuroectodermal cell lines.

PubMed Central

Whitesell, L; Rosolen, A; Neckers, L M

1991-01-01

Neuroectodermal tumors of childhood provide a unique opportunity to examine the role of genes potentially regulating neuronal growth and differentiation because many cell lines derived from these tumors are composed of at least two distinct morphologic cell types. These types display variant phenotypic characteristics and spontaneously interconvert, or transdifferentiate, in vitro. The factors that regulate transdifferentiation are unknown. Application of antisense approaches to the transdifferentiation process has allowed us to explore the precise role that N-myc may play in regulating developing systems. We now report construction of an episomally replicating expression vector designed to generate RNA antisense to part of the human N-myc gene. Such a vector is able to specifically inhibit N-myc expression in cell lines carrying both normal and amplified N-myc alleles. Inhibition of N-myc expression blocks transdifferentiation in these lines, with accumulation of cells of an intermediate phenotype. A concomitant decrease in growth rate but not loss of tumorigenicity was observed in the N-myc nonamplified cell line CHP-100. Vector-generated antisense RNA should allow identification of genes specifically regulated by the proto-oncogene N-myc. Images PMID:1996098

Perspectives on the mathematics of biological patterning and morphogenesis

NASA Astrophysics Data System (ADS)

Garikipati, Krishna

2017-02-01

A central question in developmental biology is how size and position are determined. The genetic code carries instructions on how to control these properties in order to regulate the pattern and morphology of structures in the developing organism. Transcription and protein translation mechanisms implement these instructions. However, this cannot happen without some manner of sampling of epigenetic information on the current patterns and morphological forms of structures in the organism. Any rigorous description of space- and time-varying patterns and morphological forms reduces to one among various classes of spatio-temporal partial differential equations. Reaction-transport equations represent one such class. Starting from simple Fickian diffusion, the incorporation of reaction, phase segregation and advection terms can represent many of the patterns seen in the animal and plant kingdoms. Morphological form, requiring the development of three-dimensional structure, also can be represented by these equations of mass transport, albeit to a limited degree. The recognition that physical forces play controlling roles in shaping tissues leads to the conclusion that (nonlinear) elasticity governs the development of morphological form. In this setting, inhomogeneous growth drives the elasticity problem. The combination of reaction-transport equations with those of elasto-growth makes accessible a potentially unlimited spectrum of patterning and morphogenetic phenomena in developmental biology. This perspective communication is a survey of the partial differential equations of mathematical physics that have been proposed to govern patterning and morphogenesis in developmental biology. Several numerical examples are included to illustrate these equations and the corresponding physics, with the intention of providing physical insight wherever possible.

Slit/Robo1 signaling regulates neural tube development by balancing neuroepithelial cell proliferation and differentiation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Guang; Li, Yan; Wang, Xiao-yu

2013-05-01

Formation of the neural tube is the morphological hallmark for development of the embryonic central nervous system (CNS). Therefore, neural tube development is a crucial step in the neurulation process. Slit/Robo signaling was initially identified as a chemo-repellent that regulated axon growth cone elongation, but its role in controlling neural tube development is currently unknown. To address this issue, we investigated Slit/Robo1 signaling in the development of chick neCollege of Life Sciences Biocentre, University of Dundee, Dundee DD1 5EH, UKural tube and transgenic mice over-expressing Slit2. We disrupted Slit/Robo1 signaling by injecting R5 monoclonal antibodies into HH10 neural tubes tomore » block the Robo1 receptor. This inhibited the normal development of the ventral body curvature and caused the spinal cord to curl up into a S-shape. Next, Slit/Robo1 signaling on one half-side of the chick embryo neural tube was disturbed by electroporation in ovo. We found that the morphology of the neural tube was dramatically abnormal after we interfered with Slit/Robo1 signaling. Furthermore, we established that silencing Robo1 inhibited cell proliferation while over-expressing Robo1 enhanced cell proliferation. We also investigated the effects of altering Slit/Robo1 expression on Sonic Hedgehog (Shh) and Pax7 expression in the developing neural tube. We demonstrated that over-expressing Robo1 down-regulated Shh expression in the ventral neural tube and resulted in the production of fewer HNK-1{sup +} migrating neural crest cells (NCCs). In addition, Robo1 over-expression enhanced Pax7 expression in the dorsal neural tube and increased the number of Slug{sup +} pre-migratory NCCs. Conversely, silencing Robo1 expression resulted in an enhanced Shh expression and more HNK-1{sup +} migrating NCCs but reduced Pax7 expression and fewer Slug{sup +} pre-migratory NCCs were observed. In conclusion, we propose that Slit/Robo1 signaling is involved in regulating neural tube development by tightly coordinating cell proliferation and differentiation during neurulation. - Highlights: ► The role of Slit/Robo1 signaling was investigated with chick and mouse models. ► Disturbance of Slit/Robo1 signaling resulted in neural tube defects. ► Slit/Robo1 signaling regulated the proliferation of neural tube cells. ► Slit/Robo1 signaling modulated the differentiation of neural tube cells. ► Slit/Robo1 signaling balanced the proliferation and differentiation of neural tube.« less

Epithelial-mesenchymal transition in colonies of rhesus monkey embryonic stem cells: a model for processes involved in gastrulation.

PubMed

Behr, Rüdiger; Heneweer, Carola; Viebahn, Christoph; Denker, Hans-Werner; Thie, Michael

2005-01-01

Rhesus monkey embryonic stem (rhES) cells were grown on mouse embryonic fibroblast (MEF) feeder layers for up to 10 days to form multilayered colonies. Within this period, stem cell colonies differentiated transiently into complex structures with a disc-like morphology. These complex colonies were characterized by morphology, immunohistochemistry, and marker mRNA expression to identify processes of epithelialization as well as epithelial-mesenchymal transition (EMT) and pattern formation. Typically, differentiated colonies were comprised of an upper and a lower ES cell layer, the former growing on top of the layer of MEF cells whereas the lower ES cell layer spread out underneath the MEF cells. Interestingly, in the central part of the colonies, a roundish pit developed. Here the feeder layer disappeared, and upper layer cells seemed to ingress and migrate through the pit downward to form the lower layer while undergoing a transition from the epithelial to the mesenchymal phenotype, which was indicated by the loss of the marker proteins E-cadherin and ZO-1 in the lower layer. In support of this, we found a concomitant 10-fold upregulation of the gene Snail2, which is a key regulator of the EMT process. Conversion of epiblast to mesoderm was also indicated by the regulated expression of the mesoderm marker Brachyury. An EMT is a characteristic process of vertebrate gastrulation. Thus, these rhES cell colonies may be an interesting model for studies on some basic processes involved in early primate embryogenesis and may open new ways to study the regulation of EMT in vitro.

Neurogenic transdifferentiation of human adipose-derived stem cells? A critical protocol reevaluation with special emphasis on cell proliferation and cell cycle alterations.

PubMed

Kompisch, Kai Michael; Lange, Claudia; Steinemann, Doris; Skawran, Britta; Schlegelberger, Brigitte; Müller, Reinhard; Schumacher, Udo

2010-11-01

Adipose-derived stem cells (ASCs) are reported to display multilineage differentiation potential, including neuroectodermal pathways. The aim of the present study was to critically re-evaluate the potential neurogenic (trans-)differentiation capacity of ASCs using a neurogenic induction protocol based on the combination of isobutylmethylxanthine (IBMX), indomethacin and insulin. ASCs isolated from lipo-aspirate samples of five healthy female donors were characterized and potential neurogenic (trans-)differentiation was assessed by means of immunohistochemistry and gene expression analyses. Cell proliferation and cell cycle alterations were studied, and the expression of CREB/ATF transcription factors was analyzed. ASCs expressed CD59, CD90 and CD105, and were tested negative for CD34 and CD45. Under neurogenic induction, ASCs adopted a characteristic morphology comparable to neur(on)al progenitors and expressed musashi1, β-III-tubulin and nestin. Gene expression analyses revealed an increased expression of β-III-tubulin, GFAP, vimentin and BDNF, as well as SOX4 in induced ASCs. Cell proliferation was significantly reduced under neurogenic induction; cell cycle analyses showed a G2-cell cycle arrest accompanied by differential expression of key regulators of cell cycle progression. Differential expression of CREB/ATF transcription factors could be observed on neurogenic induction, pointing to a decisive role of the cAMP-CREB/ATF system. Our findings may point to a potential neurogenic (trans-)differentiation of ASCs into early neur(on)al progenitors, but do not present definite evidence for it. Especially, the adoption of a neural progenitor cell-like morphology must not automatically be misinterpreted as a specific characteristic of a respective (trans-)differentiation process, as this may as well be caused by alterations of cell cycle progression.

Tlr7 deletion alters expression profiles of genes related to neural function and regulates mouse behaviors and contextual memory.

PubMed

Hung, Yun-Fen; Chen, Chiung-Ya; Li, Wan-Chen; Wang, Ting-Fang; Hsueh, Yi-Ping

2018-06-07

The neuronal innate immune system recognizes endogenous danger signals and regulates neuronal development and function. Toll-like receptor 7 (TLR7), one of the TLRs that trigger innate immune responses in neurons, controls neuronal morphology. To further assess the function of TLR7 in the brain, we applied next generation sequencing to investigate the effect of Tlr7 deletion on gene expression in hippocampal and cortical mixed cultures and on mouse behaviors. Since previous in vivo study suggested that TLR7 is more critical for neuronal morphology at earlier developmental stages, we analyzed two time-points (4 and 18 DIV) to represent young and mature neurons, respectively. At 4 DIV, Tlr7 KO neurons exhibited reduced expression of genes involved in neuronal development, synaptic organization and activity and behaviors. Some of these Tlr7-regulated genes are also associated with multiple neurological and neuropsychiatric diseases. TLR7-regulated transcriptomic profiles differed at 18 DIV. Apart from neuronal genes, genes related to glial cell development and differentiation became sensitive to Tlr7 deletion at 18 DIV. Moreover, Tlr7 KO mice exhibited altered behaviors in terms of anxiety, aggression, olfaction and contextual fear memory. Electrophysiological analysis further showed an impairment of long-term potentiation in Tlr7 KO hippocampus. Taken together, these results indicate that TLR7 regulates neural development and brain function, even in the absence of infectious or pathogenic molecules. Our findings strengthen evidence for the role of the neuronal innate immune system in fine-tuning neuronal morphology and activity and implicate it in neuropsychiatric disorders. Copyright © 2018 Elsevier Inc. All rights reserved.

Estrogen and the Dietary Phytoestrogen Resveratrol as Regulators of the Rho GTPase Rac in Breast Cancer Metastasis

DTIC Science & Technology

2011-09-01

cancer patient, express breast differentiation-specific proteins , and secrete milk lipids [45]. Therefore, the simplest conclusion is that MDA-MB-435...chromosomes; expresses milk proteins and lipids; and when transfected with the nm23 metastasis suppressor gene, MDA-MB-435 cells show the morphologic... proteins and secrete milk lipids [44]. Since the patient had no evidence of melanoma but was diagnosed with only a breast carcinoma; and, since

FoxP2 protein levels regulate cell morphology changes and migration patterns in the vertebrate developing telencephalon.

PubMed

Garcia-Calero, Elena; Botella-Lopez, Arancha; Bahamonde, Olga; Perez-Balaguer, Ariadna; Martinez, Salvador

2016-07-01

In the mammalian telencephalon, part of the progenitor cells transition from multipolar to bipolar morphology as they invade the mantle zone. This associates with changing patterns of radial migration. However, the molecules implicated in these morphology transitions are not well known. In the present work, we analyzed the function of FoxP2 protein in this process during telencephalic development in vertebrates. We analyzed the expression of FoxP2 protein and its relation with cell morphology and migratory patterns in mouse and chicken developing striatum. We observed FoxP2 protein expressed in a gradient from the subventricular zone to the mantle layer in mice embryos. In the FoxP2 low domain cells showed multipolar migration. In the striatal mantle layer where FoxP2 protein expression is higher, cells showed locomoting migration and bipolar morphology. In contrast, FoxP2 showed a high and homogenous expression pattern in chicken striatum, thus bipolar morphology predominated. Elevation of FoxP2 in the striatal subventricular zone by in utero electroporation promoted bipolar morphology and impaired multipolar radial migration. In mouse cerebral cortex we obtained similar results. FoxP2 promotes transition from multipolar to bipolar morphology by means of gradiental expression in mouse striatum and cortex. Together these results indicate a role of FoxP2 differential expression in cell morphology control of the vertebrate telencephalon.

MicroRNAs regulate the main events in rice drought stress response by manipulating the water supply to shoots.

PubMed

Fard, Ehsan Mohseni; Bakhshi, Behnam; Farsi, Mohammad; Kakhki, Amin Mirshamsi; Nikpay, Nava; Ebrahimi, Mohammad Ali; Mardi, Mohsen; Salekdeh, Ghasem Hosseini

2017-10-24

MicroRNAs (miRNAs) are small endogenous regulatory RNAs that are involved in a variety of biological processes related to proliferation, development, and response to biotic and abiotic stresses. miRNA profiles of rice (Oryza sativa L. cv. IR64.) leaves in a partial root zone drying (PRD) system were analysed using a high-throughput sequencing approach to identify miRNAs associated with drought signalling. The treatments performed in this study were as follows: well-watered ("wet" roots, WW), wherein both halves of the pot were watered daily; drought ("dry" roots, DD), wherein water was withheld from both halves of the pot; and well-watered/drought ("wet" and "dry" roots, WD), wherein one half of each pot was watered daily, the same as in WW, and water was withheld from the other part, the same as in DD. High-throughput sequencing enabled us to detect novel miRNAs and study the differential expression of known miRNAs. A total of 209 novel miRNAs were detected in this study. Differential miRNA profiling of the DD, WD and WW conditions showed differential expression of 159 miRNAs, among which 83, 44 and 32 miRNAs showed differential expression under both DD and WD conditions. The detection of putative targets of the differentially expressed miRNAs and investigation of their functions showed that most of these genes encode transcription factors involved in growth and development, leaf morphology, regulation of hormonal homeostasis, and stress response. The most important differences between the DD and WD conditions involved regulation of the levels of hormones such as auxin, cytokinin, abscisic acid, and jasmonic acid and also regulation of phosphor homeostasis. Overall, differentially expressed miRNAs under WD conditions were found to differ from those under DD conditions, with such differences playing a role in adaptation and inducing the normal condition. The mechanisms involved in regulating hormonal homeostasis and involved in energy production and consumption were found to be the most important regulatory pathways distinguishing the DD and WD conditions.

Rhizoid differentiation of Spirogyra is regulated by substratum.

PubMed

Ikegaya, Hisato; Sonobe, Seiji; Murakami, Kohei; Shimmen, Teruo

2008-11-01

Some species of Spirogyra can anchor to substratum with rod- or rosette-shaped rhizoid (hapteron). The rhizoid differentiation can be induced by cutting algal filaments in a laboratory. Requirement of contact stimulation for rhizoid differentiation has been reported (Nagata in Plant Cell Physiol 14:531-541, 1973a). However, the control mechanism of rhizoid morphology has not been elucidated. When cut filaments were incubated on the glass surface, start of tip growth, secretion of lectin-binding material and callose synthesis were observed. In the absence of contact to the glass surface, none of above phenomena was induced. Systematic analysis showed that rosette-shaped rhizoid was formed only on the hydrophobic substratum. On the hydrophobic substratum, both Bandeiraea (Griffonia) simplicifolia lectin and jacalin strongly stained the rhizoids. On the hydrophilic substratum, however, only Bandeiraea (Griffonia) simplicifolia lectin strongly stained the rhizoids.

RotundRacGAP Functions with Ras during Spermatogenesis and Retinal Differentiation in Drosophila melanogaster

PubMed Central

Bergeret, Evelyne; Pignot-Paintrand, Isabelle; Guichard, Annabel; Raymond, Karine; Fauvarque, Marie-Odile; Cazemajor, Michel; Griffin-Shea, Ruth

2001-01-01

Our analysis of rotund (rn) null mutations in Drosophila melanogaster revealed that deletion of the rn locus affects both spermatid and retinal differentiation. In the male reproductive system, the absence of RnRacGAP induced small testes, empty seminal vesicles, short testicular cysts, reduced amounts of interspermatid membrane, the absence of individualization complexes, and incomplete mitochondrial condensation. Flagellar growth continued within the short rn null cysts to produce large bulbous terminations of intertwined mature flagella. Organization of the retina was also severely perturbed as evidenced by grossly misshapen ommatidia containing reduced numbers of photoreceptor and pigment cells. These morphological phenotypes were rescued by genomic rnRacGAP transgenes, demonstrating that RnRacGAP function is critical to spermatid and retinal differentiation. The testicular phenotypes were suppressed by heterozygous hypomorphic mutations in the Dras1 and drk genes, indicating cross talk between RacGAP-regulated signaling and that of the Ras pathway. The observed genetic interactions are consistent with a model in which Rac signaling is activated by Ras and negatively regulated by RnRacGAP during spermatid differentiation. RnRacGAP and Ras cross talk also operated during retinal differentiation; however, while the heterozygous hypomorphic drk mutation continued to act as a suppressor of the rn null mutation, the heterozygous hypomorphic Dras1 mutation induced novel retinal phenotypes. PMID:11509670

Drp1 levels constitutively regulate mitochondrial dynamics and cell survival in cortical neurons.

PubMed

Uo, Takuma; Dworzak, Jenny; Kinoshita, Chizuru; Inman, Denise M; Kinoshita, Yoshito; Horner, Philip J; Morrison, Richard S

2009-08-01

Mitochondria exist as dynamic networks that are constantly remodeled through the opposing actions of fusion and fission proteins. Changes in the expression of these proteins alter mitochondrial shape and size, and may promote or inhibit the propagation of apoptotic signals. Using mitochondrially targeted EGFP or DsRed2 to identify mitochondria, we observed a short, distinctly tubular mitochondrial morphology in postnatal cortical neurons in culture and in retinal ganglion cells in vivo, whereas longer, highly interconnected mitochondrial networks were detected in cortical astrocytes in vitro and non-neuronal cells in the retina in vivo. Differential expression patterns of fusion and fission proteins, in part, appear to determine these morphological differences as neurons expressed markedly high levels of Drp1 and OPA1 proteins compared to non-neuronal cells. This finding was corroborated using optic tissue samples. Moreover, cortical neurons expressed several splice variants of Drp1 including a neuron-specific isoform which incorporates exon 3. Knockdown or dominant-negative interference of endogenous Drp1 significantly increased mitochondrial length in both neurons and non-neuronal cells, but caused cell death only in cortical neurons. Conversely, depletion of the fusion protein, Mfn2, but not Mfn1, caused extensive mitochondrial fission and cell death. Thus, Drp1 and Mfn2 in normal cortical neurons not only regulate mitochondrial morphology, but are also required for cell survival. The present findings point to unique patterns of Drp1 expression and selective vulnerability to reduced levels of Drp1 expression/activity in neurons, and demonstrate that the regulation of mitochondrial dynamics must be tightly regulated in neurons.

Drp1 levels constitutively regulate mitochondrial dynamics and cell survival in cortical neurons

PubMed Central

Uo, Takuma; Dworzak, Jenny; Kinoshita, Chizuru; Inman, Denise M.; Kinoshita, Yoshito; Horner, Philip J.; Morrison, Richard S.

2009-01-01

Mitochondria exist as dynamic networks that are constantly remodeled through the opposing actions of fusion and fission proteins. Changes in the expression of these proteins alter mitochondrial shape and size, and may promote or inhibit the propagation of apoptotic signals. Using mitochondrially targeted EGFP or DsRed2 to identify mitochondria, we observed a short, distinctly tubular mitochondrial morphology in postnatal cortical neurons in culture and in retinal ganglion cells in vivo, whereas longer, highly interconnected mitochondrial networks were detected in cortical astrocytes in vitro and non-neuronal cells in the retina in vivo. Differential expression patterns of fusion and fission proteins, in part, appear to determine these morphological differences as neurons expressed markedly high levels of Drp1 and OPA1 proteins compared to non-neuronal cells. This finding was corroborated using optic tissue samples. Moreover, cortical neurons expressed several splice variants of Drp1 including a neuron-specific isoform which incorporates exon 3. Knockdown or dominant negative interference of endogenous Drp1 significantly increased mitochondrial length in both neurons and non-neuronal cells, but caused cell death only in cortical neurons. Conversely, depletion of the fusion protein, Mfn2, but not Mfn1, caused extensive mitochondrial fission and cell death. Thus, Drp1 and Mfn2 in normal cortical neurons not only regulate mitochondrial morphology, but are also required for cell survival. The present findings point to unique patterns of Drp1 expression and selective vulnerability to reduced levels of Drp1 expression/activity in neurons, and demonstrate that the regulation of mitochondrial dynamics must be tightly regulated in neurons. PMID:19445933

Isolated Fungal Promoters and Gene Transcription Terminators and Methods of Protein and Chemical Production in a Fungus

DOEpatents

Dai, Ziyu; Lasure, Linda L.; Magnuson, Jon K.

2008-11-11

The present invention encompasses isolated gene regulatory elements and gene transcription terminators that are differentially expressed in a native fungus exhibiting a first morphology relative to the native fungus exhibiting a second morphology. The invention also encompasses a method of utilizing a fungus for protein or chemical production. A transformed fungus is produced by transforming a fungus with a recombinant polynucleotide molecule. The recombinant polynucleotide molecule contains an isolated polynucleotide sequence linked operably to another molecule comprising a coding region of a gene of interest. The gene regulatory element and gene transcription terminator may temporally and spatially regulate expression of particular genes for optimum production of compounds of interest in a transgenic fungus.

Isolated fungal promoters and gene transcription terminators and methods of protein and chemical production in a fungus

DOEpatents

Dai, Ziyu; Lasure, Linda L.; Magnuson, Jon K.

2008-11-11

The present invention encompasses isolated gene regulatory elements and gene transcription terminators that are differentially expressed in a native fungus exhibiting a first morphology relative to the native fungus exhibiting a second morphology. The invention also encompasses a method of utilizing a fungus for protein or chemical production. A transformed fungus is produced by transforming a fungus with a recombinant polynucleotide molecule. The recombinant polynucleotide molecule contains an isolated polynucleotide sequence linked operably to another molecule comprising a coding region of a gene of interest. The gene regulatory element and gene transcription terminator may temporally and spatially regulate expression of particular genes for optimum production of compounds of interest in a transgenic fungus.

Isolated fungal promoters and gene transcription terminators and methods of protein and chemical production in a fungus

DOEpatents

Dai, Ziyu; Lasure, Linda L; Magnuson, Jon K

2014-05-27

The present invention encompasses isolated gene regulatory elements and gene transcription terminators that are differentially expressed in a native fungus exhibiting a first morphology relative to the native fungus exhibiting a second morphology. The invention also encompasses a method of utilizing a fungus for protein or chemical production. A transformed fungus is produced by transforming a fungus with a recombinant polynucleotide molecule. The recombinant polynucleotide molecule contains an isolated polynucleotide sequence linked operably to another molecule comprising a coding region of a gene of interest. The gene regulatory element and gene transcription terminator may temporally and spatially regulate expression of particular genes for optimum production of compounds of interest in a transgenic fungus.

Lysobacter enzymogenes uses two distinct cell-cell signaling systems for differential regulation of secondary-metabolite biosynthesis and colony morphology.

PubMed

Qian, Guoliang; Wang, Yulan; Liu, Yiru; Xu, Feifei; He, Ya-Wen; Du, Liangcheng; Venturi, Vittorio; Fan, Jiaqin; Hu, Baishi; Liu, Fengquan

2013-11-01

Lysobacter enzymogenes is a ubiquitous environmental bacterium that is emerging as a potentially novel biological control agent and a new source of bioactive secondary metabolites, such as the heat-stable antifungal factor (HSAF) and photoprotective polyene pigments. Thus far, the regulatory mechanism(s) for biosynthesis of these bioactive secondary metabolites remains largely unknown in L. enzymogenes. In the present study, the diffusible signal factor (DSF) and diffusible factor (DF)-mediated cell-cell signaling systems were identified for the first time from L. enzymogenes. The results show that both Rpf/DSF and DF signaling systems played critical roles in modulating HSAF biosynthesis in L. enzymogenes. Rpf/DSF signaling and DF signaling played negative and positive effects in polyene pigment production, respectively, with DF playing a more important role in regulating this phenotype. Interestingly, only Rpf/DSF, but not the DF signaling system, regulated colony morphology of L. enzymgenes. Both Rpf/DSF and DF signaling systems were involved in the modulation of expression of genes with diverse functions in L. enzymogenes, and their own regulons exhibited only a few loci that were regulated by both systems. These findings unveil for the first time new roles of the Rpf/DSF and DF signaling systems in secondary metabolite biosynthesis of L. enzymogenes.

Lysobacter enzymogenes Uses Two Distinct Cell-Cell Signaling Systems for Differential Regulation of Secondary-Metabolite Biosynthesis and Colony Morphology

PubMed Central

Qian, Guoliang; Wang, Yulan; Liu, Yiru; Xu, Feifei; He, Ya-Wen; Du, Liangcheng; Venturi, Vittorio; Fan, Jiaqin; Hu, Baishi

2013-01-01

Lysobacter enzymogenes is a ubiquitous environmental bacterium that is emerging as a potentially novel biological control agent and a new source of bioactive secondary metabolites, such as the heat-stable antifungal factor (HSAF) and photoprotective polyene pigments. Thus far, the regulatory mechanism(s) for biosynthesis of these bioactive secondary metabolites remains largely unknown in L. enzymogenes. In the present study, the diffusible signal factor (DSF) and diffusible factor (DF)-mediated cell-cell signaling systems were identified for the first time from L. enzymogenes. The results show that both Rpf/DSF and DF signaling systems played critical roles in modulating HSAF biosynthesis in L. enzymogenes. Rpf/DSF signaling and DF signaling played negative and positive effects in polyene pigment production, respectively, with DF playing a more important role in regulating this phenotype. Interestingly, only Rpf/DSF, but not the DF signaling system, regulated colony morphology of L. enzymgenes. Both Rpf/DSF and DF signaling systems were involved in the modulation of expression of genes with diverse functions in L. enzymogenes, and their own regulons exhibited only a few loci that were regulated by both systems. These findings unveil for the first time new roles of the Rpf/DSF and DF signaling systems in secondary metabolite biosynthesis of L. enzymogenes. PMID:23974132

The insulator protein BEAF-32 is required for Hippo pathway activity in the terminal differentiation of neuronal subtypes.

PubMed

Jukam, David; Viets, Kayla; Anderson, Caitlin; Zhou, Cyrus; DeFord, Peter; Yan, Jenny; Cao, Jinshuai; Johnston, Robert J

2016-07-01

The Hippo pathway is crucial for not only normal growth and apoptosis but also cell fate specification during development. What controls Hippo pathway activity during cell fate specification is incompletely understood. In this article, we identify the insulator protein BEAF-32 as a regulator of Hippo pathway activity in Drosophila photoreceptor differentiation. Though morphologically uniform, the fly eye is composed of two subtypes of R8 photoreceptor neurons defined by expression of light-detecting Rhodopsin proteins. In one R8 subtype, active Hippo signaling induces Rhodopsin 6 (Rh6) and represses Rhodopsin 5 (Rh5), whereas in the other subtype, inactive Hippo signaling induces Rh5 and represses Rh6. The activity state of the Hippo pathway in R8 cells is determined by the expression of warts, a core pathway kinase, which interacts with the growth regulator melted in a double-negative feedback loop. We show that BEAF-32 is required for expression of warts and repression of melted Furthermore, BEAF-32 plays a second role downstream of Warts to induce Rh6 and prevent Rh5 fate. BEAF-32 is dispensable for Warts feedback, indicating that BEAF-32 differentially regulates warts and Rhodopsins. Loss of BEAF-32 does not noticeably impair the functions of the Hippo pathway in eye growth regulation. Our study identifies a context-specific regulator of Hippo pathway activity in post-mitotic neuronal fate, and reveals a developmentally specific role for a broadly expressed insulator protein. © 2016. Published by The Company of Biologists Ltd.

Regulation of raft-dependent endocytosis

PubMed Central

Lajoie, P; Nabi, IR

2007-01-01

Abstract Raft-dependent endocytosis is in large part defined as the cholesterol-sensitive, clathrin-independent internalization of ligands and receptors from the plasma membrane. It encompasses the endocytosis of caveo-lae, smooth plasmalemmal vesicles that form a subdomain of cholesterol and sphingolipid-rich lipid rafts and that are enriched for caveolin-1. While sharing common mechanisms, like cholesterol sensitivity, raft endocytic routes show differential regulation by various cellular components including caveolin-1, dynamin-2 and regulators of the actin cytoskeleton. Dynamin-dependent raft pathways, mediated by caveolae and morphologically equivalent non-caveolin vesicular intermediates, are referred to as caveolae/raft-dependent endocytosis. In contrast, dynamin-independent raft pathways are mediated by non-caveolar intermediates. Raft-dependent endocytosis is regulated by tyrosine kinase inhibitors and, through the regulation of the internalization of various ligands, receptors and effectors, is also a determinant of cellular signaling. In this review, we characterize and discuss the regulation of raft-dependent endocytic pathways and the role of key regulators such as caveolin-1. PMID:17760830

REGULATION OF GEOGRAPHIC VARIABILITY IN HAPLOID:DIPLOD RATIOS OF BIPHASIC SEAWEED LIFE CYCLES(1).

PubMed

da Silva Vieira, Vasco Manuel Nobre de Carvalho; Santos, Rui Orlando Pimenta

2012-08-01

The relative abundance of haploid and diploid individuals (H:D) in isomorphic marine algal biphasic cycles varies spatially, but only if vital rates of haploid and diploid phases vary differently with environmental conditions (i.e. conditional differentiation between phases). Vital rates of isomorphic phases in particular environments may be determined by subtle morphological or physiological differences. Herein, we test numerically how geographic variability in H:D is regulated by conditional differentiation between isomorphic life phases and the type of life strategy of populations (i.e. life cycles dominated by reproduction, survival or growth). Simulation conditions were selected using available data on H:D spatial variability in seaweeds. Conditional differentiation between ploidy phases had a small effect on the H:D variability for species with life strategies that invest either in fertility or in growth. Conversely, species with life strategies that invest mainly in survival, exhibited high variability in H:D through a conditional differentiation in stasis (the probability of staying in the same size class), breakage (the probability of changing to a smaller size class) or growth (the probability of changing to a bigger size class). These results were consistent with observed geographic variability in H:D of natural marine algae populations. © 2012 Phycological Society of America.

β-Catenin Signaling Biases Multipotent Lingual Epithelial Progenitors to Differentiate and Acquire Specific Taste Cell Fates

PubMed Central

Gaillard, Dany; Xu, Mingang; Liu, Fei; Millar, Sarah E.; Barlow, Linda A.

2015-01-01

Continuous taste bud cell renewal is essential to maintain taste function in adults; however, the molecular mechanisms that regulate taste cell turnover are unknown. Using inducible Cre-lox technology, we show that activation of β-catenin signaling in multipotent lingual epithelial progenitors outside of taste buds diverts daughter cells from a general epithelial to a taste bud fate. Moreover, while taste buds comprise 3 morphological types, β-catenin activation drives overproduction of primarily glial-like Type I taste cells in both anterior fungiform (FF) and posterior circumvallate (CV) taste buds, with a small increase in Type II receptor cells for sweet, bitter and umami, but does not alter Type III sour detector cells. Beta-catenin activation in post-mitotic taste bud precursors likewise regulates cell differentiation; forced activation of β-catenin in these Shh+ cells promotes Type I cell fate in both FF and CV taste buds, but likely does so non-cell autonomously. Our data are consistent with a model where β-catenin signaling levels within lingual epithelial progenitors dictate cell fate prior to or during entry of new cells into taste buds; high signaling induces Type I cells, intermediate levels drive Type II cell differentiation, while low levels may drive differentiation of Type III cells. PMID:26020789

β-Catenin Signaling Biases Multipotent Lingual Epithelial Progenitors to Differentiate and Acquire Specific Taste Cell Fates.

PubMed

Gaillard, Dany; Xu, Mingang; Liu, Fei; Millar, Sarah E; Barlow, Linda A

2015-05-01

Continuous taste bud cell renewal is essential to maintain taste function in adults; however, the molecular mechanisms that regulate taste cell turnover are unknown. Using inducible Cre-lox technology, we show that activation of β-catenin signaling in multipotent lingual epithelial progenitors outside of taste buds diverts daughter cells from a general epithelial to a taste bud fate. Moreover, while taste buds comprise 3 morphological types, β-catenin activation drives overproduction of primarily glial-like Type I taste cells in both anterior fungiform (FF) and posterior circumvallate (CV) taste buds, with a small increase in Type II receptor cells for sweet, bitter and umami, but does not alter Type III sour detector cells. Beta-catenin activation in post-mitotic taste bud precursors likewise regulates cell differentiation; forced activation of β-catenin in these Shh+ cells promotes Type I cell fate in both FF and CV taste buds, but likely does so non-cell autonomously. Our data are consistent with a model where β-catenin signaling levels within lingual epithelial progenitors dictate cell fate prior to or during entry of new cells into taste buds; high signaling induces Type I cells, intermediate levels drive Type II cell differentiation, while low levels may drive differentiation of Type III cells.

Suppression subtractive hybridization and comparative expression analysis to identify developmentally regulated genes in filamentous fungi.

PubMed

Gesing, Stefan; Schindler, Daniel; Nowrousian, Minou

2013-09-01

Ascomycetes differentiate four major morphological types of fruiting bodies (apothecia, perithecia, pseudothecia and cleistothecia) that are derived from an ancestral fruiting body. Thus, fruiting body differentiation is most likely controlled by a set of common core genes. One way to identify such genes is to search for genes with evolutionary conserved expression patterns. Using suppression subtractive hybridization (SSH), we selected differentially expressed transcripts in Pyronema confluens (Pezizales) by comparing two cDNA libraries specific for sexual and for vegetative development, respectively. The expression patterns of selected genes from both libraries were verified by quantitative real time PCR. Expression of several corresponding homologous genes was found to be conserved in two members of the Sordariales (Sordaria macrospora and Neurospora crassa), a derived group of ascomycetes that is only distantly related to the Pezizales. Knockout studies with N. crassa orthologues of differentially regulated genes revealed a functional role during fruiting body development for the gene NCU05079, encoding a putative MFS peptide transporter. These data indicate conserved gene expression patterns and a functional role of the corresponding genes during fruiting body development; such genes are candidates of choice for further functional analysis. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Cutaneous Malignant Melanoma With Rhabdoid Morphology and Smooth Muscle Differentiation: A Challenging Histopathologic Diagnosis.

PubMed

Prieto-Torres, Lucía; Alegría-Landa, Victoria; Llanos, Concepción; Córdoba, Alicia; Kutzner, Heinz; Requena, Luis

2017-05-01

Divergent differentiation or metaplastic change is a rare feature exhibited occasionally in malignant melanoma (MM), which is characterized by the development of morphologically, immunochemically, and/or ultrastructurally nonmelanocytic cells within the tumor. Smooth muscle differentiation in MM is an exceedingly rare phenomenon reported only in a few cases in the literature. We report the case of a 69-year-old woman who presented with a pure dermal amelanotic MM with smooth muscle cell differentiation and an area of rhabdoid morphology, which made the accurate histopathologic diagnostic of MM challenging.

Troglitazone induces differentiation in Trypanosoma brucei

DOE Office of Scientific and Technical Information (OSTI.GOV)

Denninger, Viola; Figarella, Katherine; Schoenfeld, Caroline

2007-05-15

Trypanosoma brucei, a protozoan parasite causing sleeping sickness, is transmitted by the tsetse fly and undergoes a complex lifecycle including several defined stages within the insect vector and its mammalian host. In the latter, differentiation from the long slender to the short stumpy form is induced by a yet unknown factor of trypanosomal origin. Here we describe that some thiazolidinediones are also able to induce differentiation. In higher eukaryotes, thiazolidinediones are involved in metabolism and differentiation processes mainly by binding to the intracellular receptor peroxisome proliferator activated receptor {gamma}. Our studies focus on the effects of troglitazone on bloodstream formmore » trypanosomes. Differentiation was monitored using mitochondrial markers (membrane potential, succinate dehydrogenase activity, inhibition of oxygen uptake by KCN, amount of cytochrome transcripts), morphological changes (Transmission EM and light microscopy), and transformation experiments (loss of the Variant Surface Glycoprotein coat and increase of dihydroliponamide dehydrogenase activity). To further investigate the mechanisms responsible for these changes, microarray analyses were performed, showing an upregulation of expression site associated gene 8 (ESAG8), a potential differentiation regulator.« less

Regulation of neuromuscular junction organization by Rab2 and its effector ICA69 in Drosophila.

PubMed

Mallik, Bhagaban; Dwivedi, Manish Kumar; Mushtaq, Zeeshan; Kumari, Manisha; Verma, Praveen Kumar; Kumar, Vimlesh

2017-06-01

The mechanisms underlying synaptic differentiation, which involves neuronal membrane and cytoskeletal remodeling, are not completely understood. We performed a targeted RNAi-mediated screen of Drosophila BAR-domain proteins and identified islet cell autoantigen 69 kDa (ICA69) as one of the key regulators of morphological differentiation of the larval neuromuscular junction (NMJ). We show that Drosophila ICA69 colocalizes with α-Spectrin at the NMJ. The conserved N-BAR domain of ICA69 deforms liposomes in vitro Full-length ICA69 and the ICAC but not the N-BAR domain of ICA69 induce filopodia in cultured cells. Consistent with its cytoskeleton regulatory role, ICA69 mutants show reduced α-Spectrin immunoreactivity at the larval NMJ. Manipulating levels of ICA69 or its interactor PICK1 alters the synaptic level of ionotropic glutamate receptors (iGluRs). Moreover, reducing PICK1 or Rab2 levels phenocopies ICA69 mutation. Interestingly, Rab2 regulates not only synaptic iGluR but also ICA69 levels. Thus, our data suggest that: (1) ICA69 regulates NMJ organization through a pathway that involves PICK1 and Rab2, and (2) Rab2 functions genetically upstream of ICA69 and regulates NMJ organization and targeting/retention of iGluRs by regulating ICA69 levels. © 2017. Published by The Company of Biologists Ltd.

Spinal neurons require Islet1 for subtype-specific differentiation of electrical excitability

PubMed Central

2014-01-01

Background In the spinal cord, stereotypic patterns of transcription factor expression uniquely identify neuronal subtypes. These transcription factors function combinatorially to regulate gene expression. Consequently, a single transcription factor may regulate divergent development programs by participation in different combinatorial codes. One such factor, the LIM-homeodomain transcription factor Islet1, is expressed in the vertebrate spinal cord. In mouse, chick and zebrafish, motor and sensory neurons require Islet1 for specification of biochemical and morphological signatures. Little is known, however, about the role that Islet1 might play for development of electrical membrane properties in vertebrates. Here we test for a role of Islet1 in differentiation of excitable membrane properties of zebrafish spinal neurons. Results We focus our studies on the role of Islet1 in two populations of early born zebrafish spinal neurons: ventral caudal primary motor neurons (CaPs) and dorsal sensory Rohon-Beard cells (RBs). We take advantage of transgenic lines that express green fluorescent protein (GFP) to identify CaPs, RBs and several classes of interneurons for electrophysiological study. Upon knock-down of Islet1, cells occupying CaP-like and RB-like positions continue to express GFP. With respect to voltage-dependent currents, CaP-like and RB-like neurons have novel repertoires that distinguish them from control CaPs and RBs, and, in some respects, resemble those of neighboring interneurons. The action potentials fired by CaP-like and RB-like neurons also have significantly different properties compared to those elicited from control CaPs and RBs. Conclusions Overall, our findings suggest that, for both ventral motor and dorsal sensory neurons, Islet1 directs differentiation programs that ultimately specify electrical membrane as well as morphological properties that act together to sculpt neuron identity. PMID:25149090

Diamagnetic levitation promotes osteoclast differentiation from RAW264.7 cells.

PubMed

Sun, Yu-Long; Chen, Zhi-Hao; Chen, Xiao-Hu; Yin, Chong; Li, Di-Jie; Ma, Xiao-Li; Zhao, Fan; Zhang, Ge; Shang, Peng; Qian, Ai-Rong

2015-03-01

The superconducting magnet with a high magnetic force field can levitate diamagnetic materials. In this study, a specially designed superconducting magnet with large gradient high magnetic field (LGHMF), which provides three apparent gravity levels (μg, 1 g, and 2 g), was used to study its influence on receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast differentiation from preosteoclast cell line RAW264.7. The effects of LGHMF on the viability, nitric oxide (NO) production, morphology in RAW264.7 cells were detected by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method, the Griess method, and the immunofluorescence staining, respectively. The changes induced by LGHMF in osteoclast formation, mRNA expression, and bone resorption were determined by tartrate-resistant acid phosphatase staining, semiquantity PCR, and bone resorption test, respectively. The results showed that: 1) LGHMF had no lethal effect on osteoclast precursors but attenuated NO release in RAW264.7 cells. 2) Diamagnetic levitation (μg) enhanced both the formation and bone resorption capacity of osteoclast. Moreover, diamagnetic levitation up-regulated mRNA expression of RANK, Cathepsin K, MMP-9, and NFATc1, while down-regulated RunX2 in comparison with controls. Furthermore, diamagnetic levitation induced obvious morphological alterations in osteoclast, including active cytoplasmic peripheral pseudopodial expansion, formation of pedosome belt, and aggregation of actin ring. 3) Magnetic field produced by LGHMF attenuated osteoclast resorption activity. Collectively, LGHMF with combined effects has multiple effects on osteoclast, which attenuated osteoclast resorption with magnetic field, whereas promoted osteoclast differentiation with diamagnetic levitation. Therefore, these findings indicate that diamagnetic levitation could be used as a novel ground-based microgravity simulator, which facilitates bone cell research of weightlessness condition.

Differential KrasV12 protein levels control a switch regulating lung cancer cell morphology and motility

PubMed Central

Schäfer, C.; Mohan, A.; Burford, W.; Driscoll, M. K.; Ludlow, A. T.; Wright, W. E.; Shay, J. W.; Danuser, G.

2016-01-01

Introduction Oncogenic Kras mutations are important drivers of lung cancer development and metastasis. They are known to activate numerous cellular signaling pathways implicated in enhanced proliferation, survival, tumorigenicity and motility during malignant progression. Objectives Most previous studies of Kras in cancer have focused on the comparison of cell states in the absence or presence of oncogenic Kras mutations. Here we show that differential expression of the constitutively active mutation KrasV12 has profound effects on cell morphology and motility that drive metastatic processes. Methods The study relies on lung cancer cell transformation models, patient-derived lung cancer cell lines, and human lung tumor sections combined with molecular biology techniques, live-cell imaging and staining methods. Results Our analysis shows two cell functional states driven by KrasV12 protein levels: a non-motile state associated with high KrasV12 levels and tumorigenicity, and a motile state associated with low KrasV12 levels and cell dissemination. Conversion between the states is conferred by differential activation of a mechano-sensitive double-negative feedback between KrasV12/ERK/Myosin II and matrix-adhesion signaling. KrasV12 expression levels change upon cues such as hypoxia and integrin-mediated cell-matrix adhesion, rendering KrasV12 levels an integrator of micro-environmental signals that translate into cellular function. By live cell imaging of tumor models we observe shedding of mixed high and low KrasV12 expressers forming multi-functional collectives with potentially optimal metastatic properties composed of a highly mobile and a highly tumorigenic unit. Discussion Together these data highlight previously unappreciated roles for the quantitative effects of expression level variation of oncogenic signaling molecules in conferring fundamental alterations in cell function regulation required for cancer progression. PMID:29057096

Two-Dimensional Magnesium Phosphate Nanosheets Form Highly Thixotropic Gels That Up-Regulate Bone Formation.

PubMed

Laurenti, Marco; Al Subaie, Ahmed; Abdallah, Mohamed-Nur; Cortes, Arthur R G; Ackerman, Jerome L; Vali, Hojatollah; Basu, Kaustuv; Zhang, Yu Ling; Murshed, Monzur; Strandman, Satu; Zhu, Julian; Makhoul, Nicholas; Barralet, Jake E; Tamimi, Faleh

2016-08-10

Hydrogels composed of two-dimensional (2D) nanomaterials have become an important alternative to replace traditional inorganic scaffolds for tissue engineering. Here, we describe a novel nanocrystalline material with 2D morphology that was synthesized by tuning the crystallization of the sodium-magnesium-phosphate system. We discovered that the sodium ion can regulate the precipitation of magnesium phosphate by interacting with the crystal's surface causing a preferential crystal growth that results in 2D morphology. The 2D nanomaterial gave rise to a physical hydrogel that presented extreme thixotropy, injectability, biocompatibility, bioresorption, and long-term stability. The nanocrystalline material was characterized in vitro and in vivo and we discovered that it presented unique biological properties. Magnesium phosphate nanosheets accelerated bone healing and osseointegration by enhancing collagen formation, osteoblasts differentiation, and osteoclasts proliferation through up-regulation of COL1A1, RunX2, ALP, OCN, and OPN. In summary, the 2D magnesium phosphate nanosheets could bring a paradigm shift in the field of minimally invasive orthopedic and craniofacial interventions because it is the only material available that can be injected through high gauge needles into bone defects in order to accelerate bone healing and osseointegration.

Genome-wide expression profiling of in vivo-derived bloodstream parasite stages and dynamic analysis of mRNA alterations during synchronous differentiation in Trypanosoma brucei

PubMed Central

Kabani, Sarah; Fenn, Katelyn; Ross, Alan; Ivens, Al; Smith, Terry K; Ghazal, Peter; Matthews, Keith

2009-01-01

Background Trypanosomes undergo extensive developmental changes during their complex life cycle. Crucial among these is the transition between slender and stumpy bloodstream forms and, thereafter, the differentiation from stumpy to tsetse-midgut procyclic forms. These developmental events are highly regulated, temporally reproducible and accompanied by expression changes mediated almost exclusively at the post-transcriptional level. Results In this study we have examined, by whole-genome microarray analysis, the mRNA abundance of genes in slender and stumpy forms of T.brucei AnTat1.1 cells, and also during their synchronous differentiation to procyclic forms. In total, five biological replicates representing the differentiation of matched parasite populations derived from five individual mouse infections were assayed, with RNAs being derived at key biological time points during the time course of their synchronous differentiation to procyclic forms. Importantly, the biological context of these mRNA profiles was established by assaying the coincident cellular events in each population (surface antigen exchange, morphological restructuring, cell cycle re-entry), thereby linking the observed gene expression changes to the well-established framework of trypanosome differentiation. Conclusion Using stringent statistical analysis and validation of the derived profiles against experimentally-predicted gene expression and phenotypic changes, we have established the profile of regulated gene expression during these important life-cycle transitions. The highly synchronous nature of differentiation between stumpy and procyclic forms also means that these studies of mRNA profiles are directly relevant to the changes in mRNA abundance within individual cells during this well-characterised developmental transition. PMID:19747379

Hydrophilic polyurethane matrix promotes chondrogenesis of mesenchymal stem cells.

PubMed

Nalluri, Sandeep M; Krishnan, G Rajesh; Cheah, Calvin; Arzumand, Ayesha; Yuan, Yuan; Richardson, Caley A; Yang, Shuying; Sarkar, Debanjan

2015-09-01

Segmental polyurethanes exhibit biphasic morphology and can control cell fate by providing distinct matrix guided signals to increase the chondrogenic potential of mesenchymal stem cells (MSCs). Polyethylene glycol (PEG) based hydrophilic polyurethanes can deliver differential signals to MSCs through their matrix phases where hard segments are cell-interactive domains and PEG based soft segments are minimally interactive with cells. These coordinated communications can modulate cell-matrix interactions to control cell shape and size for chondrogenesis. Biphasic character and hydrophilicity of polyurethanes with gel like architecture provide a synthetic matrix conducive for chondrogenesis of MSCs, as evidenced by deposition of cartilage-associated extracellular matrix. Compared to monophasic hydrogels, presence of cell interactive domains in hydrophilic polyurethanes gels can balance cell-cell and cell-matrix interactions. These results demonstrate the correlation between lineage commitment and the changes in cell shape, cell-matrix interaction, and cell-cell adhesion during chondrogenic differentiation which is regulated by polyurethane phase morphology, and thus, represent hydrophilic polyurethanes as promising synthetic matrices for cartilage regeneration. Copyright © 2015 Elsevier B.V. All rights reserved.

Differentiation of endosperm transfer cells of barley: a comprehensive analysis at the micro-scale.

PubMed

Thiel, Johannes; Riewe, David; Rutten, Twan; Melzer, Michael; Friedel, Swetlana; Bollenbeck, Felix; Weschke, Winfriede; Weber, Hans

2012-08-01

Barley endosperm cells differentiate into transfer cells (ETCs) opposite the nucellar projection. To comprehensively analyse ETC differentiation, laser microdissection-based transcript and metabolite profiles were obtained from laser microdissected tissues and cell morphology was analysed. Flange-like secondary-wall ingrowths appeared between 5 and 7 days after pollination within the three outermost cell layers. Gene expression analysis indicated that ethylene-signalling pathways initiate ETC morphology. This is accompanied by gene activity related to cell shape control and vesicle transport, with abundant mitochondria and endomembrane structures. Gene expression analyses indicate predominant formation of hemicelluloses, glucuronoxylans and arabinoxylans, and transient formation of callose, together with proline and 4-hydroxyproline biosynthesis. Activation of the methylation cycle is probably required for biosynthesis of phospholipids, pectins and ethylene. Membrane microdomains involving sterols/sphingolipids and remorins are potentially involved in ETC development. The transcriptional activity of assimilate and micronutrient transporters suggests ETCs as the main uptake organs of solutes into the endosperm. Accordingly, the endosperm grows maximally after ETCs are fully developed. Up-regulated gene expression related to amino acid catabolism, C:N balances, carbohydrate oxidation, mitochondrial activity and starch degradation meets high demands for respiratory energy and carbohydrates, required for cell proliferation and wall synthesis. At 10 days after pollination, ETCs undergo further differentiation, potentially initiated by abscisic acid, and metabolism is reprogrammed as shown by activated storage and stress-related processes. Overall, the data provide a comprehensive view of barley ETC differentiation and development, and identify candidate genes and associated pathways. © 2012 The Authors. The Plant Journal © 2012 Blackwell Publishing Ltd.

SCAPs Regulate Differentiation of DFSCs During Tooth Root Development in Swine

PubMed Central

Wu, Xiaoshan; Hu, Lei; Li, Yan; Li, Yang; Wang, Fu; Ma, Ping; Wang, Jinsong; Zhang, Chunmei; Jiang, Canhua; Wang, Songlin

2018-01-01

The tooth root transmits and balances occlusal forces through the periodontium to the alveolar bone. The periodontium, including the gingiva, the periodontal ligament, the cementum and the partial alveolar bone, derives from the dental follicle (DF), except for the gingiva. In the early developmental stages, the DF surrounds the tooth germ as a sphere and functions to promote tooth eruption. However, the morphological dynamics and factors regulating the differentiation of the DF during root elongation remain largely unknown. Miniature pigs are regarded as a useful experimental animal for modeling in craniofacial research because they are similar to humans with respect to dentition and mandible anatomy. In the present study, we used the third deciduous incisor of miniature pig as the model to investigate the factors influencing DF differentiation during root development. We found that the DF was shaped like a crescent and was located between the root apical and the alveolar bone. The expression levels of WNT5a, β-Catenin, and COL-I gradually increased from the center of the DF (beneath the apical foramen) to the lateral coronal corner, where the DF differentiates into the periodontium. To determine the potential regulatory role of the apical papilla on DF cell differentiation, we co-cultured dental follicle stem cells (DFSCs) with stem cells of the apical papilla (SCAPs). The osteogenesis and fibrogenesis abilities of DFSCs were inhibited when being co-cultured with SCAPs, suggesting that the fate of the DF can be regulated by signals from the apical papilla. The apical papilla may sustain the undifferentiated status of DFSCs before root development finishes. These data yield insight into the interaction between the root apex and surrounding DF tissues in root and periodontium development and shed light on the future study of root regeneration in large mammals. PMID:29511365

Chd8 mediates cortical neurogenesis via transcriptional regulation of cell cycle and Wnt signaling

PubMed Central

Durak, Omer; Gao, Fan; Kaeser-Woo, Yea Jin; Rueda, Richard; Martorell, Anthony J.; Nott, Alexi; Liu, Carol Y.; Watson, L. Ashley; Tsai, Li-Huei

2016-01-01

De novo mutations in CHD8 are strongly associated with autism spectrum disorder (ASD), however the basic biology of CHD8 remains poor understood. Here we report that Chd8 knockdown during cortical development results in defective neural progenitor proliferation and differentiation that ultimately manifests in abnormal neuronal morphology and behaviors in adult mice. Transcriptome analysis revealed that while Chd8 stimulates the transcription of cell cycle genes, it also precludes the induction of neural specific genes by regulating the expression of PRC2 complex components. Furthermore, knockdown of Chd8 disrupts the expression of key transducers of Wnt signaling, and enhancing Wnt signaling rescues the transcriptional and behavioral deficits caused by Chd8 knockdown. We propose that these roles of Chd8 and the dynamics of Chd8 expression during development help negotiate the fine balance between neural progenitor proliferation and differentiation. Together, these observations provide new insights into the neurodevelopmental role of Chd8. PMID:27694995

Essential role of citron kinase in cytokinesis of spermatogenic precursors.

PubMed

Cunto, Ferdinando Di; Imarisio, Sara; Camera, Paola; Boitani, Carla; Altruda, Fiorella; Silengo, Lorenzo

2002-12-15

During spermatogenesis, the first morphological indication of spermatogonia differentiation is incomplete cytokinesis, followed by the assembly of stable intercellular cytoplasmic communications. This distinctive feature of differentiating male germ cells has been highly conserved during evolution, suggesting that regulation of the cytokinesis endgame is a crucial aspect of spermatogenesis. However, the molecular mechanisms underlying testis-specific regulation of cytokinesis are still largely unknown. Citron kinase is a myotonin-related protein acting downstream of the GTPase Rho in cytokinesis control. We previously reported that Citron kinase knockout mice are affected by a complex neurological syndrome caused by cytokinesis block and apoptosis of specific neuronal precursors. In this report we show that, in addition, these mice display a dramatic testicular impairment, with embryonic and postnatal loss of undifferentiated germ cells and complete absence of mature spermatocytes. By contrast, the ovaries of mutant females appear essentially normal. Developmental analysis revealed that the cellular depletion observed in mutant testes is caused by increased apoptosis of undifferentiated and differentiating precursors. The same cells display a severe cytokinesis defect, resulting in the production of multinucleated cells and apoptosis. Our data indicate that Citron kinase is specifically required for cytokinesis of the male germ line.

Neural stem cell proliferation and differentiation in the conductive PEDOT-HA/Cs/Gel scaffold for neural tissue engineering.

PubMed

Wang, Shuping; Guan, Shui; Xu, Jianqiang; Li, Wenfang; Ge, Dan; Sun, Changkai; Liu, Tianqing; Ma, Xuehu

2017-09-26

Engineering scaffolds with excellent electro-activity is increasingly important in tissue engineering and regenerative medicine. Herein, conductive poly(3,4-ethylenedioxythiophene) doped with hyaluronic acid (PEDOT-HA) nanoparticles were firstly synthesized via chemical oxidant polymerization. A three-dimensional (3D) PEDOT-HA/Cs/Gel scaffold was then developed by introducing PEDOT-HA nanoparticles into a chitosan/gelatin (Cs/Gel) matrix. HA, as a bridge, not only was used as a dopant, but also combined PEDOT into the Cs/Gel via chemical crosslinking. The PEDOT-HA/Cs/Gel scaffold was used as a conductive substrate for neural stem cell (NSC) culture in vitro. The results demonstrated that the PEDOT-HA/Cs/Gel scaffold had excellent biocompatibility for NSC proliferation and differentiation. 3D confocal fluorescence images showed cells attached on the channel surface of Cs/Gel and PEDOT-HA/Cs/Gel scaffolds with a normal neuronal morphology. Compared to the Cs/Gel scaffold, the PEDOT-HA/Cs/Gel scaffold not only promoted NSC proliferation with up-regulated expression of Ki67, but also enhanced NSC differentiation into neurons and astrocytes with up-regulated expression of β tubulin-III and GFAP, respectively. It is expected that this electro-active and bio-active PEDOT-HA/Cs/Gel scaffold will be used as a conductive platform to regulate NSC behavior for neural tissue engineering.

Adipogenesis of bovine perimuscular preadipocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Taniguchi, Masaaki; Le Luo Guan; Zhang Bing

2008-02-01

In this study, non-transformed progeny adipofibroblasts, derived from mature adipocyte dedifferentiation, was used as a novel in vitro model to study adipogenic gene expression in cattle. Adipofibroblasts from dedifferentiated mature perimuscular fat (PMF) tissue were cultured with differentiation stimulants until the cells exhibited morphological differentiation. Treated cells were harvested from day 2 to 16 for RNA extraction, whereas control cells were cultured without addition of stimulants. Results from time course gene expression assays by quantitative real-time PCR revealed that peroxisome proliferator-activated receptor gamma (PPAR-{gamma}), sterol regulatory element binding protein 1 (SREBP-1) and their six down-stream genes were co-expressed at daymore » 2 post-differentiation induction. When compared to other adipogenesis culture systems, the adipogenic gene expression of bovine PMF adipofibroblasts culture was different, especially to the rodent model. Collectively, these results demonstrated PPAR-{gamma} and SREBP-1 cooperatively play a key role to regulate the re-differentiation of bovine adipofibroblasts, during early conversion stages in vitro.« less

Regulation of RAW 264.7 macrophages behavior on anodic TiO2 nanotubular arrays

NASA Astrophysics Data System (ADS)

Yao, Shenglian; Feng, Xujia; Li, Wenhao; Wang, Lu-Ning; Wang, Xiumei

2017-12-01

Titanium (Ti) implants with TiO2 nanotubular arrays on the surface could regulate cells adhesion, proliferation and differentiation to determine the bone integration. Additionally, the regulation of immune cells could improve osteogenesis or lead in appropriate immune reaction. Thus, we evaluate the behavior of RAW264.7 macrophages on TiO2 nanotubular arrays with a wide range diameter (from 20 to 120 nm) fabricated by an electrochemical anodization process. In this work, the proliferation, cell viability and cytokine/chemokine secretion were evaluated by CCK-8, live/dead staining and ELISA, respectively. SEM and confocal microscopy were used to observe the adhesion morphology. Results showed that the small size nanotube surface was benefit for the macrophages adhesion and proliferation, while larger size surface could reduce the inflammatory response. These findings contribute to the design of immune-regulating Ti implants surface that supports successful implantation.

Square cell packing in the Drosophila embryo through spatiotemporally regulated EGF receptor signaling

PubMed Central

Tamada, Masako; Zallen, Jennifer A.

2015-01-01

Summary Cells display dynamic and diverse morphologies during development, but the strategies by which differentiated tissues achieve precise shapes and patterns are not well understood. Here we identify a developmental program that generates a highly ordered square cell grid in the Drosophila embryo through sequential and spatially regulated cell alignment, oriented cell division, and apicobasal cell elongation. The basic leucine zipper transcriptional regulator Cnc is necessary and sufficient to produce a square cell grid in the presence of a midline signal provided by the EGF receptor ligand, Spitz. Spitz orients cell divisions through a Pins/LGN-dependent spindle positioning mechanism and controls cell shape and alignment through a transcriptional pathway that requires the Pointed ETS domain protein. These results identify a strategy for producing ordered square cell packing configurations in epithelia and reveal a molecular mechanism by which organized tissue structure is generated through spatiotemporally regulated responses to EGF receptor activation. PMID:26506305

In vitro differentiation of rat bone marrow mesenchymal stem cells into hepatocytes.

PubMed

Feng, Zhihui; Li, Changying; Jiao, Shuxian; Hu, Bin; Zhao, Lin

2011-01-01

To investigate the mechanism and regulation of differentiation from bone marrow mesenchymal stem cells (BMSCs) into hepatocytes and to find a new source for therapies of hepatic diseases. We isolated BMSCs for subsequent differentiation in the presence of hepatocyte growth factor (HGF) or beta-nerve growth factor (beta-NGF). Cell morphology was observed and cell surface phenotypings were detected by flow cytometry. a1-antitrypsin (AAT) expression of the hepatocytes was confirmed by immunocytochemistry and albumin expression was validated by real time PCR and western blotting. The expression of high-affinity nerve growth factor receptor (TrkA) and the activation of Erk pathway were detected by western blotting. Hepatocyte functional activity was confirmed by uptake of indocyanine green (ICG) assay. Small round cells appeared in the presence of HGF on day 10 or beta-NGF on day 12. Differentiated cells expressed albumin and had functional characteristics of hepatocytes, such as uptake of ICG. BMSCs were positive for TrkA. HGF and beta-NGF significantly upregulated the protein levels of phospho-Erk. BMSCs could differentiate into hepatocytes in the differentiation media including HGF or beta-NGF. Combination of HGF and beta-NGF significantly increased the efficiency of hepatic differentiation.

Morphological Characters and Transcriptome Profiles Associated with Black Skin and Red Skin in Crimson Snapper (Lutjanus erythropterus)

PubMed Central

Zhang, Yan-Ping; Wang, Zhong-Duo; Guo, Yu-Song; Liu, Li; Yu, Juan; Zhang, Shun; Liu, Shao-Jun; Liu, Chu-Wu

2015-01-01

In this study, morphology observation and illumina sequencing were performed on two different coloration skins of crimson snapper (Lutjanus erythropterus), the black zone and the red zone. Three types of chromatophores, melanophores, iridophores and xanthophores, were organized in the skins. The main differences between the two colorations were in the amount and distribution of the three chromatophores. After comparing the two transcriptomes, 9200 unigenes with significantly different expressions (ratio change ≥ 2 and q-value ≤ 0.05) were found, of which 5972 were up-regulated in black skin and 3228 were up-regulated in red skin. Through the function annotation, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of the differentially transcribed genes, we excavated a number of uncharacterized candidate pigment genes as well as found the conserved genes affecting pigmentation in crimson snapper. The patterns of expression of 14 pigment genes were confirmed by the Quantitative real-time PCR analysis between the two color skins. Overall, this study shows a global survey of the morphological characters and transcriptome analysis of the different coloration skins in crimson snapper, and provides valuable cellular and genetic information to uncover the mechanism of the formation of pigment patterns in snappers. PMID:26569232

Morphological Characters and Transcriptome Profiles Associated with Black Skin and Red Skin in Crimson Snapper (Lutjanus erythropterus).

PubMed

Zhang, Yan-Ping; Wang, Zhong-Duo; Guo, Yu-Song; Liu, Li; Yu, Juan; Zhang, Shun; Liu, Shao-Jun; Liu, Chu-Wu

2015-11-12

In this study, morphology observation and illumina sequencing were performed on two different coloration skins of crimson snapper (Lutjanus erythropterus), the black zone and the red zone. Three types of chromatophores, melanophores, iridophores and xanthophores, were organized in the skins. The main differences between the two colorations were in the amount and distribution of the three chromatophores. After comparing the two transcriptomes, 9200 unigenes with significantly different expressions (ratio change ≥ 2 and q-value ≤ 0.05) were found, of which 5972 were up-regulated in black skin and 3228 were up-regulated in red skin. Through the function annotation, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of the differentially transcribed genes, we excavated a number of uncharacterized candidate pigment genes as well as found the conserved genes affecting pigmentation in crimson snapper. The patterns of expression of 14 pigment genes were confirmed by the Quantitative real-time PCR analysis between the two color skins. Overall, this study shows a global survey of the morphological characters and transcriptome analysis of the different coloration skins in crimson snapper, and provides valuable cellular and genetic information to uncover the mechanism of the formation of pigment patterns in snappers.

Homeobox gene distal-less is required for neuronal differentiation and neurite outgrowth in the Drosophila olfactory system

PubMed Central

Plavicki, Jessica; Mader, Sara; Pueschel, Eric; Peebles, Patrick; Boekhoff-Falk, Grace

2012-01-01

Vertebrate Dlx genes have been implicated in the differentiation of multiple neuronal subtypes, including cortical GABAergic interneurons, and mutations in Dlx genes have been linked to clinical conditions such as epilepsy and autism. Here we show that the single Drosophila Dlx homolog, distal-less, is required both to specify chemosensory neurons and to regulate the morphologies of their axons and dendrites. We establish that distal-less is necessary for development of the mushroom body, a brain region that processes olfactory information. These are important examples of distal-less function in an invertebrate nervous system and demonstrate that the Drosophila larval olfactory system is a powerful model in which to understand distal-less functions during neurogenesis. PMID:22307614

Cyclic stretch-induced the cytoskeleton rearrangement and gene expression of cytoskeletal regulators in human periodontal ligament cells.

PubMed

Wu, Yaqin; Zhuang, Jiabao; Zhao, Dan; Zhang, Fuqiang; Ma, Jiayin; Xu, Chun

2017-10-01

This study aimed to explore the mechanism of the stretch-induced cell realignment and cytoskeletal rearrangement by identifying several mechanoresponsive genes related to cytoskeletal regulators in human PDL cells. After the cells were stretched by 1, 10 and 20% strains for 0.5, 1, 2, 4, 6, 12 or 24 h, the changes of the morphology and content of microfilaments were recorded and calculated. Meanwhile, the expression of 84 key genes encoding cytoskeletal regulators after 6 and 24 h stretches with 20% strain was detected by using real-time PCR array. Western blot was applied to identify the protein expression level of several cytoskeletal regulators encoded by these differentially expressed genes. The confocal fluorescent staining results confirmed that stretch-induced realignment of cells and rearrangement of microfilaments. Among the 84 genes screened, one gene was up-regulated while two genes were down-regulated after 6 h stretch. Meanwhile, three genes were up-regulated while two genes were down-regulated after 24 h stretch. These genes displaying differential expression included genes regulating polymerization/depolymerization of microfilaments (CDC42EP2, FNBP1L, NCK2, PIKFYVE, WASL), polymerization/depolymerization of microtubules (STMN1), interacting between microfilaments and microtubules (MACF1), as well as a phosphatase (PPP1R12B). Among the proteins encoded by these genes, the protein expression level of Cdc42 effector protein-2 (encoded by CDC42EP2) and Stathmin-1 (encoded by STMN1) was down-regulated, while the protein expression level of N-WASP (encoded by WASL) was up-regulated. The present study confirmed the cyclic stretch-induced cellular realignment and rearrangement of microfilaments in the human PDL cells and indicated several force-sensitive genes with regard to cytoskeletal regulators.

Endometrial carcinomas with significant mucinous differentiation associated with higher frequency of k-ras mutations: a morphologic and molecular correlation study.

PubMed

Xiong, Jinjun; He, Mai; Jackson, Cynthia; Ou, Joyce J; Sung, C James; Breese, Virgina; Steinhoff, Margaret M; Quddus, M Ruhul; Tejada-Berges, Trevor; Lawrence, W Dwayne

2013-09-01

K-ras gene product in the mitogen-activated protein kinase/extracellular signal-regulated kinase pathway is critical in the development of certain types of malignancies. K-ras mutation-associated pancreatic and ovarian carcinomas often display mucinous differentiation. Previous studies have shown that k-ras mutation is found in 10% to 30% of endometrial carcinomas. We investigated k-ras mutations in several morphologic subtypes of endometrial carcinomas with particular emphasis on various degrees of mucinous differentiation. Genomic DNA was extracted from formalin-fixed paraffin-embedded (FFPE) tissue sections. Polymerase chain reaction amplification for k-ras codons 12 and 13 were performed, followed by sequencing using capillary electrophoresis. The Fisher exact test is used to compare the prevalent difference of k-ras mutation among the groups. P < 0.05 was considered significant. K-ras mutations were detected in 8 (80%) of 10 mucinous carcinomas, 12 (67%) of 18 endometrioid carcinomas (ECs) with significant mucinous differentiation (ECMD), 4 (25%) of 16 ECs, and 1 (9%) of 11 serous carcinomas. The differences were statistically significant between mucinous carcinomas versus EC (P < 0.01) and ECMD versus EC (P < 0.05). The findings suggest that mucinous carcinoma and endometrioid carcinoma with significant mucinous component are more likely to be associated with k-ras mutation. Potential clinical implications of k-ras mutation lies in the management of recurrent or higher-stage endometrial mucinous tumors, which would not be responsive to treatment protocols containing epidermal growth factor receptor inhibitors.

Effects of non-ablative fractional erbium glass laser treatment on gene regulation in human three-dimensional skin models.

PubMed

Amann, Philipp M; Marquardt, Yvonne; Steiner, Timm; Hölzle, Frank; Skazik-Voogt, Claudia; Heise, Ruth; Baron, Jens M

2016-04-01

Clinical experiences with non-ablative fractional erbium glass laser therapy have demonstrated promising results for dermal remodelling and for the indications of striae, surgical scars and acne scars. So far, molecular effects on human skin following treatment with these laser systems have not been elucidated. Our aim was to investigate laser-induced effects on skin morphology and to analyse molecular effects on gene regulation. Therefore, human three-dimensional (3D) organotypic skin models were irradiated with non-ablative fractional erbium glass laser systems enabling qRT-PCR, microarray and histological studies at same and different time points. A decreased mRNA expression of matrix metalloproteinases (MMPs) 3 and 9 was observed 3 days after treatment. MMP3 also remained downregulated on protein level, whereas the expression of other MMPs like MMP9 was recovered or even upregulated 5 days after irradiation. Inflammatory gene regulatory responses measured by the expression of chemokine (C-X-C motif) ligands (CXCL1, 2, 5, 6) and interleukin expression (IL8) were predominantly reduced. Epidermal differentiation markers such as loricrin, filaggrin-1 and filaggrin-2 were upregulated by both tested laser optics, indicating a potential epidermal involvement. These effects were also shown on protein level in the immunofluorescence analysis. This novel standardised laser-treated human 3D skin model proves useful for monitoring time-dependent ex vivo effects of various laser systems on gene expression and human skin morphology. Our study reveals erbium glass laser-induced regulations of MMP and interleukin expression. We speculate that these alterations on gene expression level could play a role for dermal remodelling, anti-inflammatory effects and increased epidermal differentiation. Our finding may have implications for further understanding of the molecular mechanism of erbium glass laser-induced effects on human skin.

Increasing Avermectin Production in Streptomyces avermitilis by Manipulating the Expression of a Novel TetR-Family Regulator and Its Target Gene Product.

PubMed

Liu, Wenshuai; Zhang, Qinling; Guo, Jia; Chen, Zhi; Li, Jilun; Wen, Ying

2015-08-01

Avermectins produced by Streptomyces avermitilis are commercially important anthelmintic agents. The detailed regulatory mechanisms of avermectin biosynthesis remain unclear. Here, we identified SAV3619, a TetR-family transcriptional regulator designated AveT, to be an activator for both avermectin production and morphological differentiation in S. avermitilis. AveT was shown to indirectly stimulate avermectin production by affecting transcription of the cluster-situated activator gene aveR. AveT directly repressed transcription of its own gene (aveT), adjacent gene pepD2 (sav_3620), sav_7490 (designated aveM), and sav_7491 by binding to an 18-bp perfect palindromic sequence (CGAAACGKTKYCGTTTCG, where K is T or G and Y is T or C and where the underlining indicates inverted repeats) within their promoter regions. aveM (which encodes a putative transmembrane efflux protein belonging to the major facilitator superfamily [MFS]), the important target gene of AveT, had a striking negative effect on avermectin production and morphological differentiation. Overexpression of aveT and deletion of aveM in wild-type and industrial strains of S. avermitilis led to clear increases in the levels of avermectin production. In vitro gel-shift assays suggested that C-5-O-B1, the late pathway precursor of avermectin B1, acts as an AveT ligand. Taken together, our findings indicate positive-feedback regulation of aveT expression and avermectin production by a late pathway intermediate and provide the basis for an efficient strategy to increase avermectin production in S. avermitilis by manipulation of AveT and its target gene product, AveM. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

[Role of CD2-associated protein in podocyte differentiation.].

PubMed

Jiang, Hua-Jun; Chang, Ying; Zhu, Zhong-Hua; Liu, Jian-She; Deng, An-Guo; Zhang, Chun

2008-02-25

To study the cellular changes and the potential role of CD2-associated protein (CD2AP) in podocyte differentiation, conditionally immortalized murine podocyte cell line was cultured in RPMI 1640 medium under permissive condition at 33 °C. After transfection with CD2AP small interfering RNA (siRNA) the cells were shifted to non-permissive condition at 37 °C. Simultaneously, untransfected cells were taken as differentiation control. The podocyte proliferation rate was determined by MTT method. The expressions of CD2AP, WT1, synaptopodin and nephrin mRNAs were examined by RT-PCR. CD2AP, WT1 and nephrin protein expressions were examined by Western blot. The distribution of CD2AP, nephrin, F-actin and tubulin in differentiated and undifferentiated podocytes was detected by laser scanning confocal microscopy. The results showed: (1) CD2AP, WT1 and nephrin were stably expressed in differentiated and undifferentiated podocytes while synaptopodin was only expressed in differentiated podocytes. (2) CD2AP and nephrin mRNA and protein expressions were up-regulated during podocyte differentiation (P<0.05). (3) CD2AP and tubulin were distributed in the cytoplasm and perinulcear region in undifferentiated podocytes, and F-actin was predominantly localized to a cortical belt and paralleled to the cell axis. Under differentiation condition, CD2AP distribution profile was presented as peripheral accumulation, tubulin took on fascicular style and F-actin extended into foot processes in podocytes. CD2AP colocalized with nephrin and F-actin in undifferentiated podocytes. (4) After transfection with CD2AP siRNA, the expression of CD2AP was partially inhibited and cell growth was arrested; Synaptopodin, the differentiation podocyte marker, was apparently down-regulated; The differentiation of podocytes was delayed. The results demonstrate that podocyte differentiation is accompanied by cytoskeleton rearrangement and cell morphology change. CD2AP might play an essential role in podocyte differentiation.

Mechanical influence of tissue culture plates and extracellular matrix on mesenchymal stem cell behavior: A topical review.

PubMed

Tatullo, Marco; Marrelli, Massimo; Falisi, Giovanni; Rastelli, Claudio; Palmieri, Francesca; Gargari, Marco; Zavan, Barbara; Paduano, Francesco; Benagiano, Vincenzo

2016-03-01

Tissue engineering applications need a continuous development of new biomaterials able to generate an ideal cell-extracellular matrix interaction. The stem cell fate is regulated by several factors, such as growth factors or transcription factors. The most recent literature has reported several publications able to demonstrate that environmental factors also contribute to the regulation of stem cell behavior, leading to the opinion that the environment plays the major role in the cell differentiation.The interaction between mesenchymal stem cells (MSCs) and extracellular environment has been widely described, and it has a crucial role in regulating the cell phenotype. In our laboratory (Tecnologica Research Institute, Crotone, Italy), we have recently studied how several physical factors influence the distribution and the morphology of MSCs isolated from dental pulp, and how they are able to regulate stem cell differentiation. Mechanical and geometrical factors are only a small part of the environmental factors able to influence stem cell behavior, however, this influence should be properly known: in fact, this assumption must be clearly considered during those studies involving MSCs; furthermore, these interactions should be considered as an important bias that involves an high number of studies on the MSCs, since in worldwide laboratories the scientists mostly use tissue culture plates for their experiments. © The Author(s) 2015.

Differential morphology and image processing.

PubMed

Maragos, P

1996-01-01

Image processing via mathematical morphology has traditionally used geometry to intuitively understand morphological signal operators and set or lattice algebra to analyze them in the space domain. We provide a unified view and analytic tools for morphological image processing that is based on ideas from differential calculus and dynamical systems. This includes ideas on using partial differential or difference equations (PDEs) to model distance propagation or nonlinear multiscale processes in images. We briefly review some nonlinear difference equations that implement discrete distance transforms and relate them to numerical solutions of the eikonal equation of optics. We also review some nonlinear PDEs that model the evolution of multiscale morphological operators and use morphological derivatives. Among the new ideas presented, we develop some general 2-D max/min-sum difference equations that model the space dynamics of 2-D morphological systems (including the distance computations) and some nonlinear signal transforms, called slope transforms, that can analyze these systems in a transform domain in ways conceptually similar to the application of Fourier transforms to linear systems. Thus, distance transforms are shown to be bandpass slope filters. We view the analysis of the multiscale morphological PDEs and of the eikonal PDE solved via weighted distance transforms as a unified area in nonlinear image processing, which we call differential morphology, and briefly discuss its potential applications to image processing and computer vision.

Induction of temporally dissociated morphological and physiological differentiation of N1E-115 cells.

PubMed

Cosgrove, C; Cobbett, P

1991-07-01

Clonal cells derived from neural tumors have been widely used to study the processes of neuronal differentiation in vitro. The murine neuroblastoma clone N1E-115 has recently been shown to differentiate morphologically in response to removal of serum from the culture medium. In the present study, the nature and time course of electrophysiological differentiation of N1E-115 cells maintained in serum-free medium was examined. Differentiated cells had a higher resting potential and lower input conductance than nondifferentiated cells. Differentiated but not nondifferentiated cells generated current evoked action potentials, and differentiated cells fired spontaneous, repetitive action potentials after 13 days in serum-free medium. The rate of potential change during the depolarizing and repolarizing phases of the action potential became faster as the duration of maintenance of cells in serum-free medium increased. Remarkably, morphological differentiation appeared to be complete after exposure to serum-free medium for 5 days but electrophysiological differentiation was not complete until 13 days in this medium.

Regulation of c- and N-myc expression during induced differentiation of murine neuroblastoma cells.

PubMed

Larcher, J C; Vayssière, J L; Lossouarn, L; Gros, F; Croizat, B

1991-04-01

Using clones N1E-115 and N1A-103 from mouse neuroblastoma C1300, a comparative analysis of c- and N-myc gene expression was undertaken both in proliferating cells and in cultures exposed to conditions which induce differentiation. Under the latter conditions, while N1E-115 cells extend abundant neurites and express many biochemical features of mature neurons, clone N1A-103 stops dividing and expresses certain neurospecific markers but is unable to differentiate morphologically. In both clones, chemical agents, i.e. 1-methyl cyclohexane carboxylic acid (CCA) or dimethyl sulfoxide (DMSO), induce a decrease in c-myc expression. Similar results were found for N-myc gene in N1E-115 cells, but in contrast, in clone N1A-103, N-myc expression is increased with CCA and not modified with DMSO. Globally, this study favours the hypothesis that changes in c-myc expression would correspond to cell division blockade and differentiation, while modulations in N-myc are more closely related to an early phase of terminal differentiation.

A Unique Fungal Two-Component System Regulates Stress Responses, Drug Sensitivity, Sexual Development, and Virulence of Cryptococcus neoformans

PubMed Central

Bahn, Yong-Sun; Kojima, Kaihei; Cox, Gary M.

2006-01-01

The stress-activated mitogen-activated protein kinase (MAPK) pathway is widely used by eukaryotic organisms as a central conduit via which cellular responses to the environment effect growth and differentiation. The basidiomycetous human fungal pathogen Cryptococcus neoformans uniquely uses the stress-activated Pbs2-Hog1 MAPK system to govern a plethora of cellular events, including stress responses, drug sensitivity, sexual reproduction, and virulence. Here, we characterized a fungal “two-component” system that controls these fundamental cellular functions via the Pbs2-Hog1 MAPK cascade. A typical response regulator, Ssk1, modulated all Hog1-dependent phenotypes by controlling Hog1 phosphorylation, indicating that Ssk1 is the major upstream signaling component of the Pbs2-Hog1 pathway. A second response regulator, Skn7, governs sensitivity to Na+ ions and the antifungal agent fludioxonil, negatively controls melanin production, and functions independently of Hog1 regulation. To control these response regulators, C. neoformans uses multiple sensor kinases, including two-component–like (Tco) 1 and Tco2. Tco1 and Tco2 play shared and distinct roles in stress responses and drug sensitivity through the Hog1 MAPK system. Furthermore, each sensor kinase mediates unique cellular functions for virulence and morphological differentiation. Our findings highlight unique adaptations of this global two-component MAPK signaling cascade in a ubiquitous human fungal pathogen. PMID:16672377

Investigating the underlying mechanism of Saccharomyces cerevisiae in response to ethanol stress employing RNA-seq analysis.

PubMed

Li, Ruoyun; Xiong, Guotong; Yuan, Shukun; Wu, Zufang; Miao, Yingjie; Weng, Peifang

2017-11-03

Saccharomyces cerevisiae has been widely used for wine fermentation and bio-fuels production. A S. cerevisiae strain Sc131 isolated from tropical fruit shows good fermentation properties and ethanol tolerance, exhibiting significant potential in Chinese bayberry wine fermentation. In this study, RNA-sequence and RT-qPCR was used to investigate the transcriptome profile of Sc131 in response to ethanol stress. Scanning Electron Microscopy were carried out to observe surface morphology of yeast cells. Totally, 937 genes were identified differential expressed, including 587 up-regulated and 350 down-regulated genes, after 4-h ethanol stress (10% v/v). Transcriptomic analysis revealed that, most genes involved in regulating filamentous growth or pseudohyphal growth were significantly up-regulated in response to ethanol stress. The complex protein quality control machineries, Hsp90/Hsp70 and Hsp104/Hsp70/Hsp40 based chaperone system combining with ubiquitin-proteasome proteolytic pathway were both activated to recognize and degrade misfolding proteins. Genes related to biosynthesis and metabolism of two well-known stress-responsive substances trehalose and ergosterol were generally up-regulated, while genes associated with amino acids biosynthesis and metabolism processes were differentially expressed. Moreover, thiamine was also important in response to ethanol stress. This research may promote the potential applications of Sc131 in the fermentation of Chinese bayberry wine.

Sodium and calcium currents in neuroblastoma x glioma hybrid cells before and after morphological differentiation by dibutyryl cyclic AMP.

PubMed

Bodewei, R; Hering, S; Schubert, B; Wollenberger, A

1985-04-01

Sodium and calcium inward currents (INa and ICa) were measured in neuroblastoma X glioma hybrid cells of clones 108CC5 and 108CC15 by a single suction pipette method for internal perfusion and voltage clamp. Morphologically undifferentiated, exponentially growing cells were compared with cells differentiated by cultivation with 1 mmol/l dibutyryl cyclic AMP. Outward currents were eliminated by perfusing the cells with a K+-free solution. Voltage dependence and ion selectivity as well as steady state inactivation characteristics of INa and ICa resembled those of differentiated mouse neuroblastoma cells, clone N1E-115 (Moolenaar and Spector 1978, 1979). These parameters were identical in undifferentiated and differentiated cells of both clones. After differentiation the average density of the peak sodium and calcium currents was increased two and four-fold, respectively, in both cell lines. Our data indicate that exponentially growing, morphologically undifferentiated 108CC5 and 108CC15 neuroblastoma X glioma hybrid cells possess functional Na+ and Ca2+ channels undistinguishable from those of non-proliferating cells of these clones differentiated morphologically by treatment with dibutyryl cyclic AMP. That Na+ and Ca2+ spikes were not detected by other authors in these cells prior to morphological differentiation by dibutyryl cyclic AMP may be attributed to the fact that at the low resting membrane potential measured the Na+ and Ca2+ channels are inactivated.

Induction of Fas-Mediated Apoptosis by Interferon-γ is Dependent on Granulosa Cell Differentiation and Follicular Maturation in the Rat Ovary.

PubMed

Lee, Hye-Jeong; Kim, Ji Young; Park, Ji Eun; Yoon, Yong-Dal; Tsang, Benjamin K; Kim, Jong-Min

2016-12-01

Fas ligand (FasL) and its receptor Fas have been implicated in granulosa cell apoptosis during follicular atresia. Although interferon-gamma (IFN-γ) is believed to be involved in the regulation Fas expression in differentiated granulosa or granulosa-luteal cells, the expression of this cytokine and its role in the regulation of the granulosa cell Fas/FasL system and apoptosis during follicular maturation have not been thoroughly investigated. In the present study, we have examined the presence of IFN-γ in ovarian follicles at different stage of development by immunohistochemistry and related their relative intensities with follicular expression of Fas and FasL, and with differences in granulosa cell sensitivity to Fas activation by exogenous agonistic Anti-Fas monoclonal antibody (Fas mAb). Although IFN-γ immunostaining was detectable in oocyte and granulosa cells in antral follicles, most intense immunoreactivity for the cytokine was observed in these cells of preantral follicles. Intense immunoreactivity for IFN-γ was most evident in granulosa cells of atretic early antral follicles where increased Fas and FasL expression and apoptosis were also observed. Whereas low concentrations of IFN-γ (10-100 U/mL) significantly increased Fas expression in undifferentiated granulosa cells (from preantral or very early antral follicles) in vitro , very higher concentrations (≥ 1,000 U/mL) were required to up-regulate of Fas in differentiated cells isolated from eCG-primed (antral) follicles. Addition of agonistic Fas mAb to cultures of granulosa cells at the two stages of differentiation and pretreated with IFN-γ (100 U/mL) elicited morphological and biochemical apoptotic features which were more prominent in cells not previously exposed to the gonadotropin in vivo . These findings suggested that IFN-γ is an important physiologic intra-ovarian regulator of follicular atresia and plays a pivotal role in regulation of expression of Fas receptor and subsequent apoptotic response in undifferentiated (or poorly differentiated) granulosa cells at an early (penultimate) stage of follicular development.

Induction of Fas-Mediated Apoptosis by Interferon-γ is Dependent on Granulosa Cell Differentiation and Follicular Maturation in the Rat Ovary

PubMed Central

Lee, Hye-Jeong; Kim, Ji Young; Park, Ji Eun; Yoon, Yong-Dal; Tsang, Benjamin K.; Kim, Jong-Min

2016-01-01

ABSTRACT Fas ligand (FasL) and its receptor Fas have been implicated in granulosa cell apoptosis during follicular atresia. Although interferon-gamma (IFN-γ) is believed to be involved in the regulation Fas expression in differentiated granulosa or granulosa-luteal cells, the expression of this cytokine and its role in the regulation of the granulosa cell Fas/FasL system and apoptosis during follicular maturation have not been thoroughly investigated. In the present study, we have examined the presence of IFN-γ in ovarian follicles at different stage of development by immunohistochemistry and related their relative intensities with follicular expression of Fas and FasL, and with differences in granulosa cell sensitivity to Fas activation by exogenous agonistic Anti-Fas monoclonal antibody (Fas mAb). Although IFN-γ immunostaining was detectable in oocyte and granulosa cells in antral follicles, most intense immunoreactivity for the cytokine was observed in these cells of preantral follicles. Intense immunoreactivity for IFN-γ was most evident in granulosa cells of atretic early antral follicles where increased Fas and FasL expression and apoptosis were also observed. Whereas low concentrations of IFN-γ (10-100 U/mL) significantly increased Fas expression in undifferentiated granulosa cells (from preantral or very early antral follicles) in vitro, very higher concentrations (≥ 1,000 U/mL) were required to up-regulate of Fas in differentiated cells isolated from eCG-primed (antral) follicles. Addition of agonistic Fas mAb to cultures of granulosa cells at the two stages of differentiation and pretreated with IFN-γ (100 U/mL) elicited morphological and biochemical apoptotic features which were more prominent in cells not previously exposed to the gonadotropin in vivo. These findings suggested that IFN-γ is an important physiologic intra-ovarian regulator of follicular atresia and plays a pivotal role in regulation of expression of Fas receptor and subsequent apoptotic response in undifferentiated (or poorly differentiated) granulosa cells at an early (penultimate) stage of follicular development. PMID:28144637

Cytoskeletal Reorganization Drives Mesenchymal Condensation and Regulates Downstream Molecular Signaling.

PubMed

Ray, Poulomi; Chapman, Susan C

2015-01-01

Skeletal condensation occurs when specified mesenchyme cells self-organize over several days to form a distinctive cartilage template. Here, we determine how and when specified mesenchyme cells integrate mechanical and molecular information from their environment, forming cartilage condensations in the pharyngeal arches of chick embryos. By disrupting cytoskeletal reorganization, we demonstrate that dynamic cell shape changes drive condensation and modulate the response of the condensing cells to Fibroblast Growth Factor (FGF), Bone Morphogenetic Protein (BMP) and Transforming Growth Factor beta (TGF-β) signaling pathways. Rho Kinase (ROCK)-driven actomyosin contractions and Myosin II-generated differential cell cortex tension regulate these cell shape changes. Disruption of the condensation process inhibits the differentiation of the mesenchyme cells into chondrocytes, demonstrating that condensation regulates the fate of the mesenchyme cells. We also find that dorsal and ventral condensations undergo distinct cell shape changes. BMP signaling is instructive for dorsal condensation-specific cell shape changes. Moreover, condensations exhibit ventral characteristics in the absence of BMP signaling, suggesting that in the pharyngeal arches ventral morphology is the ground pattern. Overall, this study characterizes the interplay between cytoskeletal dynamics and molecular signaling in a self-organizing system during tissue morphogenesis.

Cytoskeletal Reorganization Drives Mesenchymal Condensation and Regulates Downstream Molecular Signaling

PubMed Central

Ray, Poulomi; Chapman, Susan C.

2015-01-01

Skeletal condensation occurs when specified mesenchyme cells self-organize over several days to form a distinctive cartilage template. Here, we determine how and when specified mesenchyme cells integrate mechanical and molecular information from their environment, forming cartilage condensations in the pharyngeal arches of chick embryos. By disrupting cytoskeletal reorganization, we demonstrate that dynamic cell shape changes drive condensation and modulate the response of the condensing cells to Fibroblast Growth Factor (FGF), Bone Morphogenetic Protein (BMP) and Transforming Growth Factor beta (TGF-β) signaling pathways. Rho Kinase (ROCK)-driven actomyosin contractions and Myosin II-generated differential cell cortex tension regulate these cell shape changes. Disruption of the condensation process inhibits the differentiation of the mesenchyme cells into chondrocytes, demonstrating that condensation regulates the fate of the mesenchyme cells. We also find that dorsal and ventral condensations undergo distinct cell shape changes. BMP signaling is instructive for dorsal condensation-specific cell shape changes. Moreover, condensations exhibit ventral characteristics in the absence of BMP signaling, suggesting that in the pharyngeal arches ventral morphology is the ground pattern. Overall, this study characterizes the interplay between cytoskeletal dynamics and molecular signaling in a self-organizing system during tissue morphogenesis. PMID:26237312

Diet regulates liver autophagy differentially in murine acute Trypanosoma cruzi infection

PubMed Central

Lizardo, Kezia; Almonte, Vanessa; Law, Calvin; Aiyyappan, Janeesh Plakkal; Cui, Min-Hui; Nagajyothi, Jyothi F

2017-01-01

Chagas disease is a tropical parasitic disease caused by the protozoan Trypanosoma cruzi, which affects about 10 million people in its endemic regions of Latin America. After the initial acute stage of infection, 60–80% of infected individuals remain asymptomatic for several years to a lifetime; however, the rest develop the debilitating symptomatic stage, which affects the nervous system, digestive system and heart. The challenges of Chagas disease have become global due to immigration. Despite well documented dietary changes accompanying immigration, as well as a transition to a western style diet in the Chagas endemic regions, the role of host metabolism in the pathogenesis of Chagas disease remains underexplored. We have previously used a mouse model to show that host diet is a key factor regulating cardiomyopathy in Chagas disease. In this study we investigated the effect of a high fat diet on liver morphology and physiology, lipid metabolism, immune signaling, energy homeostasis, and stress responses in the murine model of acute T. cruzi infection. Our results indicate that in T. cruzi infected mice diet differentially regulates several liver processes, including autophagy, a stress response mechanism, with corresponding implications for human Chagas disease patients. PMID:27987056

The directed differentiation of human iPS cells into kidney podocytes.

PubMed

Song, Bi; Smink, Alexandra M; Jones, Christina V; Callaghan, Judy M; Firth, Stephen D; Bernard, Claude A; Laslett, Andrew L; Kerr, Peter G; Ricardo, Sharon D

2012-01-01

The loss of glomerular podocytes is a key event in the progression of chronic kidney disease resulting in proteinuria and declining function. Podocytes are slow cycling cells that are considered terminally differentiated. Here we provide the first report of the directed differentiation of induced pluripotent stem (iPS) cells to generate kidney cells with podocyte features. The iPS-derived podocytes share a morphological phenotype analogous with cultured human podocytes. Following 10 days of directed differentiation, iPS podocytes had an up-regulated expression of mRNA and protein localization for podocyte markers including synaptopodin, nephrin and Wilm's tumour protein (WT1), combined with a down-regulation of the stem cell marker OCT3/4. In contrast to human podocytes that become quiescent in culture, iPS-derived cells maintain a proliferative capacity suggestive of a more immature phenotype. The transduction of iPS podocytes with fluorescent labeled-talin that were immunostained with podocin showed a cytoplasmic contractile response to angiotensin II (AII). A permeability assay provided functional evidence of albumin uptake in the cytoplasm of iPS podocytes comparable to human podocytes. Moreover, labeled iPS-derived podocytes were found to integrate into reaggregated metanephric kidney explants where they incorporated into developing glomeruli and co-expressed WT1. This study establishes the differentiation of iPS cells to kidney podocytes that will be useful for screening new treatments, understanding podocyte pathogenesis, and offering possibilities for regenerative medicine.

Nicotinamide N-methyltransferase expression in SH-SY5Y neuroblastoma and N27 mesencephalic neurones induces changes in cell morphology via ephrin-B2 and Akt signalling

PubMed Central

Thomas, M G; Saldanha, M; Mistry, R J; Dexter, D T; Ramsden, D B; Parsons, R B

2013-01-01

Nicotinamide N-methyltransferase (NNMT, E.C. 2.1.1.1) N-methylates nicotinamide to produce 1-methylnicotinamide (MeN). We have previously shown that NNMT expression protected against neurotoxin-mediated cell death by increasing Complex I (CxI) activity, resulting in increased ATP synthesis. This was mediated via protection of the NDUFS3 subunit of CxI from degradation by increased MeN production. In the present study, we have investigated the effects of NNMT expression on neurone morphology and differentiation. Expression of NNMT in SH-SY5Y human neuroblastoma and N27 rat mesencephalic dopaminergic neurones increased neurite branching, synaptophysin expression and dopamine accumulation and release. siRNA gene silencing of ephrin B2 (EFNB2), and inhibition of Akt phosphorylation using LY294002, demonstrated that their sequential activation was responsible for the increases observed. Incubation of SH-SY5Y with increasing concentrations of MeN also increased neurite branching, suggesting that the effects of NNMT may be mediated by MeN. NNMT had no significant effect on the expression of phenotypic and post-mitotic markers, suggesting that NNMT is not involved in determining phenotypic fate or differentiation status. These results demonstrate that NNMT expression regulates neurone morphology in vitro via the sequential activation of the EFNB2 and Akt cellular signalling pathways. PMID:23764850

Nicotinamide N-methyltransferase expression in SH-SY5Y neuroblastoma and N27 mesencephalic neurones induces changes in cell morphology via ephrin-B2 and Akt signalling.

PubMed

Thomas, M G; Saldanha, M; Mistry, R J; Dexter, D T; Ramsden, D B; Parsons, R B

2013-06-13

Nicotinamide N-methyltransferase (NNMT, E.C. 2.1.1.1) N-methylates nicotinamide to produce 1-methylnicotinamide (MeN). We have previously shown that NNMT expression protected against neurotoxin-mediated cell death by increasing Complex I (CxI) activity, resulting in increased ATP synthesis. This was mediated via protection of the NDUFS3 subunit of CxI from degradation by increased MeN production. In the present study, we have investigated the effects of NNMT expression on neurone morphology and differentiation. Expression of NNMT in SH-SY5Y human neuroblastoma and N27 rat mesencephalic dopaminergic neurones increased neurite branching, synaptophysin expression and dopamine accumulation and release. siRNA gene silencing of ephrin B2 (EFNB2), and inhibition of Akt phosphorylation using LY294002, demonstrated that their sequential activation was responsible for the increases observed. Incubation of SH-SY5Y with increasing concentrations of MeN also increased neurite branching, suggesting that the effects of NNMT may be mediated by MeN. NNMT had no significant effect on the expression of phenotypic and post-mitotic markers, suggesting that NNMT is not involved in determining phenotypic fate or differentiation status. These results demonstrate that NNMT expression regulates neurone morphology in vitro via the sequential activation of the EFNB2 and Akt cellular signalling pathways.

Digital gene expression analysis of corky split vein caused by boron deficiency in 'Newhall' Navel Orange (Citrus sinensis Osbeck) for selecting differentially expressed genes related to vascular hypertrophy.

PubMed

Yang, Cheng-Quan; Liu, Yong-Zhong; An, Ji-Cui; Li, Shuang; Jin, Long-Fei; Zhou, Gao-Feng; Wei, Qing-Jiang; Yan, Hui-Qing; Wang, Nan-Nan; Fu, Li-Na; Liu, Xiao; Hu, Xiao-Mei; Yan, Ting-Shuai; Peng, Shu-Ang

2013-01-01

Corky split vein caused by boron (B) deficiency in 'Newhall' Navel Orange was studied in the present research. The boron-deficient citrus exhibited a symptom of corky split vein in mature leaves. Morphologic and anatomical surveys at four representative phases of corky split veins showed that the symptom was the result of vascular hypertrophy. Digital gene expression (DGE) analysis was performed based on the Illumina HiSeq™ 2000 platform, which was applied to analyze the gene expression profilings of corky split veins at four morphologic phases. Over 5.3 million clean reads per library were successfully mapped to the reference database and more than 22897 mapped genes per library were simultaneously obtained. Analysis of the differentially expressed genes (DEGs) revealed that the expressions of genes associated with cytokinin signal transduction, cell division, vascular development, lignin biosynthesis and photosynthesis in corky split veins were all affected. The expressions of WOL and ARR12 involved in the cytokinin signal transduction pathway were up-regulated at 1(st) phase of corky split vein development. Furthermore, the expressions of some cell cycle genes, CYCs and CDKB, and vascular development genes, WOX4 and VND7, were up-regulated at the following 2(nd) and 3(rd) phases. These findings indicated that the cytokinin signal transduction pathway may play a role in initiating symptom observed in our study.

Optical projection tomography reveals dynamics of HEV growth after immunization with protein plus CFA and features shared with HEVs in acute autoinflammatory lymphadenopathy.

PubMed

Kumar, Varsha; Chyou, Susan; Stein, Jens V; Lu, Theresa T

2012-01-01

The vascular-stromal compartment of lymph nodes is important for lymph node function, and high endothelial venules (HEVs) play a critical role in controlling the entry of recirculating lymphocytes. In autoimmune and autoinflammatory diseases, lymph node swelling is often accompanied by apparent HEV expansion and, potentially, targeting HEV expansion could be used therapeutically to limit autoimmunity. In previous studies using mostly flow cytometry analysis, we defined three differentially regulated phases of lymph node vascular-stromal growth: initiation, expansion, and the re-establishment of vascular quiescence and stabilization. In this study, we use optical projection tomography to better understand the morphologic aspects of HEV growth upon immunization with ovalbumin/CFA (OVA/CFA). We find HEV elongation as well as modest arborization during the initiation phase, increased arborization during the expansion phase, and, finally, vessel narrowing during the re-establishment of vascular quiescence and stabilization. We also examine acutely enlarged autoinflammatory lymph nodes induced by regulatory T cell depletion and show that HEVs are expanded and morphologically similar to the expanded HEVs in OVA/CFA-stimulated lymph nodes. These results reinforce the idea of differentially regulated, distinct phases of vascular-stromal growth after immunization and suggest that insights gained from studying immunization-induced lymph node vascular growth may help to understand how the lymph node vascular-stromal compartment could be therapeutically targeted in autoimmune and autoinflammatory diseases.

[Expression of ICAT and Wnt signaling-related proteins in the monocytic differentiation of HL-60 cells induced by a new steroidal drug NSC67657].

PubMed

Wang, J S; Wang, W J; Wang, T; Zhang, Y

2016-04-01

To investigate the expression of mRNA and proteins of β-catenin, TCF-4 (ICAT) and Wnt signaling pathway-related genes in the monocytic differentiation of acute myeloid leukemia HL-60 cells induced by a new steroidal drug NSC67657. Wright's staining and α-NBE staining were used to observe the differentiation of HL-60 cells after 5 days of 10 μmol/L NSC67657 treatment. Flow cytometry (FCM) was used to detect the differentiation and cell cycles. The expressions of mRNA and proteins of ICAT and Wnt signaling pathway-related factors, including β-catenin, TCF-4, c-myc, cyclin D1 and TCF-1 before and after differentiation, were detected by RT-PCR and Western blot. Morphological observation showed that NSC67657 induced monocytic differentiation of HL-60 cells. At 5 days after 10 μmol/L NSC67657 treatment, the number of CD14(+) HL-60 cells was (94.37±2.84)%, significantly higher than the (1.31±0.09)% in control group (P<0.01). The flow cytometry assay revealed that NSC67657 induced (76.46±2.83)% of G1/G0 phase arrest, significantly higher than that of (59.40±5.42)% in the control group (P<0.05), while the S phase cells were of (18.76±0.98)%, significantly lower than that of (34.38±2.61) % in the control group (P<0.05). The NSC67657 treatment also up-regulated the expression of ICAT mRNA and protein, and down-regulated the expression of β-catenin mRNA and protin (P<0.01 for all). However, the nuclear expression of β-catenin was down-regulated (P<0.01). The NSC67657 treatment induced nonsignificant alterations of TCF-4 mRNA, total protein and nuclear protein in the HL-60 cells (P>0.05 for all). The target genes of Wnt signaling pathway, including c-myc, cyclinD1 and TCF-1 mRNA and proteins in the HL-60 cells were significantly down-regulated after NSC67657 treatment (P<0.05). The new steroidal drug NSC67657 induces monocytic differentiation of HL-60 cells, and down-regulates the expression of β-catenin and target genes of Wnt signaling pathway. These results indicate that Wnt signaling pathway may be directly or indirectly involved in the monocytic differentiation process of HL-60 cells.

Transcriptional dynamics of a conserved gene expression network associated with craniofacial divergence in Arctic charr.

PubMed

Ahi, Ehsan Pashay; Kapralova, Kalina Hristova; Pálsson, Arnar; Maier, Valerie Helene; Gudbrandsson, Jóhannes; Snorrason, Sigurdur S; Jónsson, Zophonías O; Franzdóttir, Sigrídur Rut

2014-01-01

Understanding the molecular basis of craniofacial variation can provide insights into key developmental mechanisms of adaptive changes and their role in trophic divergence and speciation. Arctic charr (Salvelinus alpinus) is a polymorphic fish species, and, in Lake Thingvallavatn in Iceland, four sympatric morphs have evolved distinct craniofacial structures. We conducted a gene expression study on candidates from a conserved gene coexpression network, focusing on the development of craniofacial elements in embryos of two contrasting Arctic charr morphotypes (benthic and limnetic). Four Arctic charr morphs were studied: one limnetic and two benthic morphs from Lake Thingvallavatn and a limnetic reference aquaculture morph. The presence of morphological differences at developmental stages before the onset of feeding was verified by morphometric analysis. Following up on our previous findings that Mmp2 and Sparc were differentially expressed between morphotypes, we identified a network of genes with conserved coexpression across diverse vertebrate species. A comparative expression study of candidates from this network in developing heads of the four Arctic charr morphs verified the coexpression relationship of these genes and revealed distinct transcriptional dynamics strongly correlated with contrasting craniofacial morphologies (benthic versus limnetic). A literature review and Gene Ontology analysis indicated that a significant proportion of the network genes play a role in extracellular matrix organization and skeletogenesis, and motif enrichment analysis of conserved noncoding regions of network candidates predicted a handful of transcription factors, including Ap1 and Ets2, as potential regulators of the gene network. The expression of Ets2 itself was also found to associate with network gene expression. Genes linked to glucocorticoid signalling were also studied, as both Mmp2 and Sparc are responsive to this pathway. Among those, several transcriptional targets and upstream regulators showed differential expression between the contrasting morphotypes. Interestingly, although selected network genes showed overlapping expression patterns in situ and no morph differences, Timp2 expression patterns differed between morphs. Our comparative study of transcriptional dynamics in divergent craniofacial morphologies of Arctic charr revealed a conserved network of coexpressed genes sharing functional roles in structural morphogenesis. We also implicate transcriptional regulators of the network as targets for future functional studies.

Meisoindigo is a promising agent with in vitro and in vivo activity against human acute myeloid leukemia.

PubMed

Lee, Chin-Cheng; Lin, Che-Pin; Lee, Yueh-Lun; Wang, Giueng-Chueng; Cheng, Yuan-Chih; Liu, H Eugene

2010-05-01

Meisoindigo, a derivative of Indigo naturalis, has been used in China for chronic myeloid leukemia. In vitro cell line studies have shown that this agent might induce apoptosis and myeloid differentiation of acute myeloid leukemia (AML). In this study, we explored its mechanisms and potential in AML. NB4, HL-60, and U937 cells and primary AML cells were used to examine its effects and the NOD/SCID animal model was used to evaluate its in vivo activity. Meisoindigo inhibited the growth of leukemic cells by inducing marked apoptosis and moderate cell-cycle arrest at the G(0)/G(1) phase. It down-regulated anti-apoptotic Bcl-2, and up-regulated pro-apoptotic Bak and Bax and cell-cycle related proteins, p21and p27. Furthermore, it induced myeloid differentiation, as demonstrated by morphologic changes, up-regulation of CD11b, and increased nitroblue tetrazolium reduction activity in all cell lines tested. In addition, meisoindigo down-regulated the expression of human telomerase reverse transcriptase and enhanced the cytotoxicity of conventional chemotherapeutic agents, cytarabine and idarubicin. As with the results from cell lines, meisoindigo also induced apoptosis, up-regulated p21 and p27, and down-regulated Bcl-2 in primary AML cells. The in vivo anti-leukemic activity of meisoindigo was also demonstrated by decreased spleen size in a dose-dependent manner. Taking these results together, meisoindigo is a potential agent for AML.

Surface Curvature Differentially Regulates Stem Cell Migration and Differentiation via Altered Attachment Morphology and Nuclear Deformation

PubMed Central

Werner, Maike; Blanquer, Sébastien B. G.; Haimi, Suvi P.; Korus, Gabriela; Dunlop, John W. C.; Duda, Georg N.; Grijpma, Dirk. W.

2016-01-01

Signals from the microenvironment around a cell are known to influence cell behavior. Material properties, such as biochemical composition and substrate stiffness, are today accepted as significant regulators of stem cell fate. The knowledge of how cell behavior is influenced by 3D geometric cues is, however, strongly limited despite its potential relevance for the understanding of tissue regenerative processes and the design of biomaterials. Here, the role of surface curvature on the migratory and differentiation behavior of human mesenchymal stem cells (hMSCs) has been investigated on 3D surfaces with well‐defined geometric features produced by stereolithography. Time lapse microscopy reveals a significant increase of cell migration speed on concave spherical compared to convex spherical structures and flat surfaces resulting from an upward‐lift of the cell body due to cytoskeletal forces. On convex surfaces, cytoskeletal forces lead to substantial nuclear deformation, increase lamin‐A levels and promote osteogenic differentiation. The findings of this study demonstrate a so far missing link between 3D surface curvature and hMSC behavior. This will not only help to better understand the role of extracellular matrix architecture in health and disease but also give new insights in how 3D geometries can be used as a cell‐instructive material parameter in the field of biomaterial‐guided tissue regeneration. PMID:28251054

Transfection of gene regulation nanoparticles complexed with pDNA and shRNA controls multilineage differentiation of hMSCs.

PubMed

Kim, Hye Jin; Yi, Se Won; Oh, Hyun Jyung; Lee, Jung Sun; Park, Ji Sun; Park, Keun-Hong

2018-05-29

Overexpression and knockdown of specific proteins can control stem cell differentiation for therapeutic purposes. In this study, we fabricated RUNX2, SOX9, and C/EBPα plasmid DNAs (pDNAs) and ATF4-targeting shRNA (shATF4) to induce osteogenesis, chondrogenesis, and adipogenesis of human mesenchymal stem cells (hMSCs). The pDNAs and shATF4 were complexed with TRITC-gene regulation nanoparticles (GRN). Osteogenesis-related gene expression was reduced at early (12 h) and late (36 h) time points after co-delivery of shATF4 and SOX9 or C/EBPα pDNA, respectively, and osteogenesis was inhibited in these hMSCs. By contrast, osteogenesis-related genes were highly expressed upon co-delivery of RUNX2 and ATF4 pDNAs. DEX in GRN enhanced chondrogenic differentiation. Expression of osteogenesis-, chondrogenesis-, and adipogenesis-related genes was higher in hMSCs transfected with NPs complexed with RUNX2 and ATF4 pDNAs, shATF4 and SOX9 pDNA, and shATF4 and C/EBPα pDNA for 72 h than in control hMSCs, respectively. Moreover, delivery of these NPs also increased expression of osteogenesis-, chondrogenesis-, and adipogenesis-related proteins. These alterations in expression led to morphological changes, indicating that hMSCs differentiated into osteoblasts, chondrocytes, and adipose cells. Copyright © 2018 Elsevier Ltd. All rights reserved.

Genome-Wide Reprogramming of Transcript Architecture by Temperature Specifies the Developmental States of the Human Pathogen Histoplasma

PubMed Central

Gilmore, Sarah A.; Voorhies, Mark; Gebhart, Dana; Sil, Anita

2015-01-01

Eukaryotic cells integrate layers of gene regulation to coordinate complex cellular processes; however, mechanisms of post-transcriptional gene regulation remain poorly studied. The human fungal pathogen Histoplasma capsulatum (Hc) responds to environmental or host temperature by initiating unique transcriptional programs to specify multicellular (hyphae) or unicellular (yeast) developmental states that function in infectivity or pathogenesis, respectively. Here we used recent advances in next-generation sequencing to uncover a novel re-programming of transcript length between Hc developmental cell types. We found that ~2% percent of Hc transcripts exhibit 5’ leader sequences that differ markedly in length between morphogenetic states. Ribosome density and mRNA abundance measurements of differential leader transcripts revealed nuanced transcriptional and translational regulation. One such class of regulated longer leader transcripts exhibited tight transcriptional and translational repression. Further examination of these dually repressed genes revealed that some control Hc morphology and that their strict regulation is necessary for the pathogen to make appropriate developmental decisions in response to temperature. PMID:26177267

Genome-Wide Reprogramming of Transcript Architecture by Temperature Specifies the Developmental States of the Human Pathogen Histoplasma.

PubMed

Gilmore, Sarah A; Voorhies, Mark; Gebhart, Dana; Sil, Anita

2015-07-01

Eukaryotic cells integrate layers of gene regulation to coordinate complex cellular processes; however, mechanisms of post-transcriptional gene regulation remain poorly studied. The human fungal pathogen Histoplasma capsulatum (Hc) responds to environmental or host temperature by initiating unique transcriptional programs to specify multicellular (hyphae) or unicellular (yeast) developmental states that function in infectivity or pathogenesis, respectively. Here we used recent advances in next-generation sequencing to uncover a novel re-programming of transcript length between Hc developmental cell types. We found that ~2% percent of Hc transcripts exhibit 5' leader sequences that differ markedly in length between morphogenetic states. Ribosome density and mRNA abundance measurements of differential leader transcripts revealed nuanced transcriptional and translational regulation. One such class of regulated longer leader transcripts exhibited tight transcriptional and translational repression. Further examination of these dually repressed genes revealed that some control Hc morphology and that their strict regulation is necessary for the pathogen to make appropriate developmental decisions in response to temperature.

Arf6 guanine-nucleotide exchange factor, cytohesin-2, interacts with actinin-1 to regulate neurite extension.

PubMed

Torii, Tomohiro; Miyamoto, Yuki; Nakamura, Kazuaki; Maeda, Masahiro; Yamauchi, Junji; Tanoue, Akito

2012-09-01

Proper regulation of morphological changes in neuronal cells is essential for their differentiation. Complex signaling mechanisms mediate a variety of morphological changes such as formation of neurites. It is well established that a number of small GTPases control neurite behavior before the connection with the target tissue. However, their regulatory mechanisms remain to be fully understood. Here, we show that the Arf6 guanine-nucleotide exchange factor (GEF), cytohesin-2 (CYTH2), interacts with the cytoskeletal protein actinin-1 (ACTN1) and regulates neurite extension in N1E-115 cells used as the model. Knockdown of ACTN1, as well as that of CYTH2, in cells inhibits cellular Arf6 activity and neurite extension. The C-terminal polybasic region of CYTH2 participates in interacting directly with the EFh2 domain of ACTN1. Expression of CYTH2 mutant deficient of the EFh2 domain in cells also inhibits Arf6 activation and neurite extension. Furthermore, FRET analysis detects that the respective interactive region peptides, tagged with cell-permeable short peptides, greatly decrease Arf6 activation at growth cones in a time-dependent manner. Collectively, the signaling through CYTH2 and ACTN1 properly regulates neurite extension in N1E-115 cells, demonstrating the unexpected interaction of CYTH2 and ACTN1 in the regulation of cellular Arf6 activity involved in neurite extension. Copyright © 2012 Elsevier Inc. All rights reserved.

A natural diarylheptanoid promotes neuronal differentiation via activating ERK and PI3K-Akt dependent pathways.

PubMed

Tang, G; Dong, X; Huang, X; Huang, X-J; Liu, H; Wang, Y; Ye, W-C; Shi, L

2015-09-10

Neuronal differentiation is a critical developmental process that determines accurate synaptic connection and circuit wiring. A wide variety of naturally occurring compounds have been shown as promising drug leads for the generation and differentiation of neurons. Here we report that a diarylheptanoid from the plant Alpinia officinarum, 7-(4-hydroxyphenyl)-1-phenyl-4E-hepten-3-one (Cpd 1), exhibited potent activities in neuronal differentiation and neurite outgrowth. Cpd 1 induced differentiation of neuroblastoma Neuro-2a cells into a neuron-like morphology, and accelerated the establishment of axon-dendrite polarization of cultured hippocampal neurons. Moreover, Cpd 1 promoted neurite extension in both Neuro-2a cells and neurons. We showed that the effects of Cpd 1 on neuronal differentiation and neurite growth were specifically dependent on the activation of extracellular signal-regulated kinases (ERKs) and phosphoinositide 3-kinase (PI3K)-Akt signaling pathways. Importantly, intraperitoneal administration of Cpd 1 promoted the differentiation of new-born progenitor cells into mature neurons in the adult hippocampal dentate gyrus. Collectively, this study identifies a naturally occurring diarylheptanoid with beneficial effects on neuronal differentiation and neurite outgrowth in vitro and in vivo. Copyright © 2015 IBRO. Published by Elsevier Ltd. All rights reserved.

Notch Signaling Regulates Late-Stage Epidermal Differentiation and Maintains Postnatal Hair Cycle Homeostasis

PubMed Central

Lin, Hsien-Yi; Kao, Cheng-Heng; Lin, Kurt Ming-Chao; Kaartinen, Vesa; Yang, Liang-Tung

2011-01-01

Background Notch signaling involves ligand-receptor interactions through direct cell-cell contact. Multiple Notch receptors and ligands are expressed in the epidermis and hair follicles during embryonic development and the adult stage. Although Notch signaling plays an important role in regulating differentiation of the epidermis and hair follicles, it remains unclear how Notch signaling participates in late-stage epidermal differentiation and postnatal hair cycle homeostasis. Methodology and Principal Findings We applied Cre/loxP system to generate conditional gene targeted mice that allow inactivation of critical components of Notch signaling pathway in the skin. Rbpj, the core component of all four Notch receptors, and Pofut1, an essential factor for ligand-receptor interactions, were inactivated in hair follicle lineages and suprabasal layer of the epidermis using the Tgfb3-Cre mouse line. Rbpj conditional inactivation resulted in granular parakeratosis and reactive epidermal hyperplasia. Pofut1 conditional inactivation led to ultrastructural abnormalities in the granular layer and altered filaggrin processing in the epidermis, suggesting a perturbation of the granular layer differentiation. Disruption of Pofut1 in hair follicle lineages resulted in aberrant telogen morphology, a decrease of bulge stem cell markers, and a concomitant increase of K14-positive keratinocytes in the isthmus of mutant hair follicles. Pofut1-deficent hair follicles displayed a delay in anagen re-entry and dysregulation of proliferation and apoptosis during the hair cycle transition. Moreover, increased DNA double stand breaks were detected in Pofut1-deficent hair follicles, and real time PCR analyses on bulge keratinocytes isolated by FACS revealed an induction of DNA damage response and a paucity of DNA repair machinery in mutant bulge keratinocytes. Significance our data reveal a role for Notch signaling in regulating late-stage epidermal differentiation. Notch signaling is required for postnatal hair cycle homeostasis by maintaining proper proliferation and differentiation of hair follicle stem cells. PMID:21267458

miRNAs involved in the development and differentiation of fertile and sterile flowers in Viburnum macrocephalum f. keteleeri.

PubMed

Li, Weixing; He, Zhichong; Zhang, Li; Lu, Zhaogeng; Xu, Jing; Cui, Jiawen; Wang, Li; Jin, Biao

2017-10-13

Sterile and fertile flowers are important evolutionary developmental phenotypes in angiosperm flowers. The development of floral organs, critical in angiosperm reproduction, is regulated by microRNAs (miRNAs). However, the mechanisms underpinning the miRNA regulation of the differentiation and development of sterile and fertile flowers remain unclear. Here, based on investigations of the morphological differences between fertile and sterile flowers, we used high-throughput sequencing to characterize the miRNAs in the differentiated floral organs of Viburnum macrocephalum f. keteleeri. We identified 49 known miRNAs and 67 novel miRNAs by small RNA (sRNA) sequencing and bioinformatics analysis, and 17 of these known and novel miRNA precursors were validated by polymerase chain reaction (PCR) and Sanger sequencing. Furthermore, by comparing the sequencing results of two sRNA libraries, we found that 30 known and 39 novel miRNA sequences were differentially expressed, and 35 were upregulated and 34 downregulated in sterile compared with fertile flowers. Combined with their predicted targets, the potential roles of miRNAs in V. macrocephalum f. keteleeri flowers include involvement in floral organogenesis, cell proliferation, hormonal pathways, and stress responses. miRNA precursors and targets were further validated by quantitative real-time PCR (qRT-PCR). Specifically, miR156a-5p, miR156g, and miR156j expression levels were significantly higher in fertile flowers than in sterile flowers, while SPL genes displayed the opposite expression pattern. Considering that the targets of miR156 are predicted to be SPL genes, we propose that miR156 may be involved in the regulation of stamen development in V. macrocephalum f. keteleeri. We identified miRNAs differentially expressed between fertile and sterile flowers in V. macrocephalum f. keteleeri and provided new insights into the important regulatory roles of miRNAs in the differentiation and development of fertile and sterile flowers.

Regulation of dorso‐ventral polarity by the nerve cord during annelid regeneration: A review of experimental evidence

PubMed Central

Boilly‐Marer, Yolande; Bely, Alexandra E.

2017-01-01

Abstract An important goal for understanding regeneration is determining how polarity is conferred to the regenerate. Here we review findings in two groups of polychaete annelids that implicate the ventral nerve cord in assigning dorso‐ventral polarity, and specifically ventral identity, to the regenerate. In nereids, surgical manipulations indicate that parapodia develop where dorsal and ventral body wall territories contact. Without a nerve cord at the wound site, the regenerate differentiates no evident polarity (with no parapodia) and only dorsal identity, while with two nerve cords the regenerate develops a twinned dorso‐ventral axis (with four parapodia per segment instead of the normal two). In sabellids, a striking natural dorso‐ventral inversion in parapodial morphology occurs along the body axis and this inversion is morphologically correlated with the position of the nerve cord. Parapodial inversion also occurs in segments in which the nerve cord has been removed, even without any segment amputation. Together, these data strongly support a role for the nerve cord in annelid dorso‐ventral pattern regulation, with the nerve cord conferring ventral identity. PMID:28616245

Mediating human stem cell behaviour via defined fibrous architectures by melt electrospinning writing.

PubMed

Eichholz, Kian F; Hoey, David A

2018-05-29

The architecture within which cells reside is key to mediating their specific functions within the body. In this study, we use melt electrospinning writing (MEW) to fabricate cell micro-environments with various fibrous architectures to study their effect on human stem cell behaviour. We designed, built and optimised a MEW apparatus and used it to fabricate four different platform designs of 10.4±2μm fibre diameter, with angles between fibres on adjacent layers of 90°, 45°, 10° and R (random). Mechanical characterisation was conducted via tensile testing, and human skeletal stem cells (hSSCs) were seeded to scaffolds to study the effect of architecture on cell morphology and mechanosensing (nuclear YAP). Cell morphology was significantly altered between groups, with cells on 90° scaffolds having a lower aspect ratio, greater spreading, greater cytoskeletal tension and nuclear YAP expression. Long term cell culture studies were then conducted to determine the differentiation potential of scaffolds in terms of alkaline phosphatase activity, collagen and mineral production. Across these studies, an increased cell spreading in 3-dimensions is seen with decreasing alignment of architecture correlated with enhanced osteogenesis. This study therefore highlights the critical role of fibrous architecture in regulating stem cell behaviour with implications for tissue engineering and disease progression. This is the first study which has investigated the effect of controlled fibrous architectures fabricated via melt electrospinning writing on cell behaviour and differentiation. After optimising the process and characterising scaffolds via SEM and tensile testing, cells were seeded to fibrous scaffolds with various micro-architectures and studied in terms of cell morphology. Nuclear YAP expression was further investigated as a marker of cell shape, cytoskeletal tension and differentiation potential. In agreement with these early markers, long term cell culture studies revealed for the first time that a 90° fibrous architecture is optimal for the osteogenic differentiation of skeletal stem cells. This is the first study to investigate the effect of controlled fibrous material architectures fabricated via melt electrospinning writing on cell shape, mechanosignalling and differentiation. After optimising the biofabrication process and characterising scaffolds via SEM and tensile testing, cells were seeded to fibrous scaffolds with various micro-architectures and studied in terms of cell shape. Nuclear YAP expression was further investigated as a marker of cytoskeletal tension and differentiation potential. In agreement with these early markers, long term cell culture studies revealed for the first time that a 90° fibrous architecture is optimal for the osteogenic differentiation of skeletal stem cells, by driving a spread morphology and nuclear translocation of YAP in 3 dimensions . Copyright © 2018 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Human platelet lysate is a successful alternative serum supplement for propagation of monocyte-derived dendritic cells.

PubMed

Švajger, Urban

2017-04-01

Clinical protocols for dendritic cell (DC) generation from monocytes require the use of animal serum-free supplements. Serum-free media can also require up to 1% of serum supplementation. In addition, recommendations based on the 3Rs (Refinement, Reduction, Replacement) principle also recommend the use of non-animal sera in in vitro studies. The aim of this study was to explore the potential use of platelet lysate (PL) for generation of optimally differentiated DCs from monocytes. Cells were isolated from buffy coats from healthy volunteers using immunomagnetic selection. DCs were differentiated in RPMI1640 supplemented with either 10% fetal bovine serum (FBS), 10% AB serum or 10% PL with the addition of granulocyte monocyte colony stimulating factor and interleukin-4. Generated DCs were assessed for their morphology, viability, endocytotic capacity, surface phenotype (immature, mature and tolerogenic DCs) and activation of important signaling pathways. DC function was evaluated on the basis of their allostimulatory capacity, cytokine profile and ability to induce different T-helper subsets. DCs generated with PL displayed normal viability, morphology and endocytotic capacity. Their differentiation and maturation phenotype was comparable to FBS-cultured DCs. They showed functional plasticity and up-regulated tolerogenic markers in response to their environment. PL-cultured mature DCs displayed unhindered allostimulatory potential and the capacity to induce Th1 responses. The use of PL allowed for activation of crucial signaling proteins associated with DC differentiation and maturation. This study demonstrates for the first time that human PL represents a successful alternative to FBS in differentiation of DCs from monocytes. DCs display the major phenotypic and functional characteristics compared with existing culture protocols. Copyright © 2017 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Influence of in vitro biomimicked stem cell 'niche' for regulation of proliferation and differentiation of human bone marrow-derived mesenchymal stem cells to myocardial phenotypes: serum starvation without aid of chemical agents and prevention of spontaneous stem cell transformation enhanced by the matrix environment.

PubMed

Kim, Jae Hyung; Shin, Sang-Hyun; Li, Tian Zhu; Suh, Hwal

2016-01-01

Niche appears important for preventing the spontaneous differentiation or senescence that cells undergo during in vitro expansion. In the present study, it was revealed that human bone marrow-derived mesenchymal stem cells (hBM-MSCs) undergo senescence-related differentiation into the myocardial lineage in vitro without any induction treatment. This phenomenon occurred over the whole population of MCSs, much different from conventional differentiation with limited frequency of occurrence, and was accompanied by a change of morphology into large, flat cells with impeded proliferation, which are the representative indications of MSC senescence. By culturing MSCs under several culture conditions, it was determined that induction treatment with 5-azacytidine was not associated with the phenomenon, but the serum-starvation condition, under which proliferation is severely hampered, caused senescence progression and upregulation of cardiac markers. Nevertheless, MSCs gradually developed a myocardial phenotype under normal culture conditions over a prolonged culture period and heterogeneous populations were formed. In perspectives of clinical applications, this must be prevented for fair and consistent outcomes. Hence, the biomimetic 'niche' was constituted for hBM-MSCs by cultivating on a conventionally available extracellular matrix (ECM). Consequently, cells on ECM regained a spindle-shape morphology, increased in proliferation rate by two-fold and showed decreased expression of cardiac markers at both the mRNA and protein levels. In conclusion, the outcome indicates that progression of MSC senescence may occur via myocardial differentiation during in vitro polystyrene culture, and this can be overcome by employing appropriate ECM culture techniques. Copyright © 2013 John Wiley & Sons, Ltd.

Biocompatible, Biodegradable, and Electroactive Polyurethane-Urea Elastomers with Tunable Hydrophilicity for Skeletal Muscle Tissue Engineering.

PubMed

Chen, Jing; Dong, Ruonan; Ge, Juan; Guo, Baolin; Ma, Peter X

2015-12-30

It remains a challenge to develop electroactive and elastic biomaterials to mimic the elasticity of soft tissue and to regulate the cell behavior during tissue regeneration. We designed and synthesized a series of novel electroactive and biodegradable polyurethane-urea (PUU) copolymers with elastomeric property by combining the properties of polyurethanes and conducting polymers. The electroactive PUU copolymers were synthesized from amine capped aniline trimer (ACAT), dimethylol propionic acid (DMPA), polylactide, and hexamethylene diisocyanate. The electroactivity of the PUU copolymers were studied by UV-vis spectroscopy and cyclic voltammetry. Elasticity and Young's modulus were tailored by the polylactide segment length and ACAT content. Hydrophilicity of the copolymer films was tuned by changing DMPA content and doping of the copolymer. Cytotoxicity of the PUU copolymers was evaluated by mouse C2C12 myoblast cells. The myogenic differentiation of C2C12 myoblasts on copolymer films was also studied by analyzing the morphology of myotubes and relative gene expression during myogenic differentiation. The chemical structure, thermal properties, surface morphology, and processability of the PUU copolymers were characterized by NMR, FT-IR, gel permeation chromatography (GPC), thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), X-ray diffraction (XRD), scanning electron microscopy (SEM), atomic force microscopy (AFM), and solubility testing, respectively. Those biodegradable electroactive elastic PUU copolymers are promising materials for repair of soft tissues such as skeletal muscle, cardiac muscle, and nerve.

Dedifferentiated Liposarcoma of the Retroperitoneum With Heterologous Osteosarcomatous Differentiation and a Striking Aneurysmal Bone Cyst-Like Morphology.

PubMed

Van Haverbeke, Carole; Van Dorpe, Jo; Lecoutere, Evelyne; Flucke, Uta; Ferdinande, Liesbeth; Creytens, David

2017-06-01

A 69-year-old woman with a 10-year medical history of recurrent retroperitoneal dedifferentiated liposarcoma presented with a 3-cm large hemorrhagic and multicystic left-sided retroperitoneal mass. Histopathological examination of the resected specimen showed a heterogeneous, high-grade mesenchymal nonlipogenic tumor with areas of osteoblastic/osteosarcomatous differentiation and aneurysmal bone cyst-like features. Based on the clinical presentation, the morphology, and the supportive immunohistochemical and molecular findings (MDM2 overexpression and amplification of the MDM2 gene, respectively), a diagnosis of a dedifferentiated liposarcoma with heterologous osteosarcomatous differentiation and an aneurysmal bone cyst-like morphology was made. To the best of our knowledge, this is the first description of aneurysmal bone cyst-like morphology in dedifferentiated liposarcoma, further expanding the broad morphological spectrum of dedifferentiated liposarcoma.

Spliceosome Protein (SRp) Regulation of Glucocorticoid Receptor Isoforms and Glucocorticoid Response in Human Trabecular Meshwork Cells

PubMed Central

Jain, Ankur; Wordinger, Robert J.; Yorio, Thomas; Clark, Abbot F.

2012-01-01

Purpose. Glaucoma is a leading cause of visual impairment and blindness, with elevated intraocular pressure (IOP) as a major causative risk factor. Glucocorticoid (GC) therapy causes morphologic and biochemical changes in the trabecular meshwork (TM), an ocular tissue involved in regulating IOP, which can lead to the development of glaucoma in susceptible individuals (steroid responders). Steroid responders comprise 40% of the general population and are at higher risk of developing glaucoma. In addition, a majority of glaucoma patients are steroid responders. Differential distribution of various isoforms of GC receptor (GR) may be responsible for this heterogeneity in the steroid response. The alternatively spliced GRβ isoform acts as dominant negative regulator of classical GRα transcriptional activity. mRNA splicing is mediated by spliceosomes, which include serine-arginine rich proteins (SRps). The purpose of this study was to determine whether specific SRps regulate levels of these isoforms and thereby GC response in TM cells. Methods. Quantitative RT-PCR, Western blot analysis, and immunocytochemistry were used to determine the differential expression of different SRps (SRp20, 30c, and 40) in human normal and glaucomatous TM cell strains. Bioinformatics was used to find putative binding sites for SRp20 and SRp40 on exon 9 of the GR gene. A peptide modulator of splicing (bombesin) and SRp expression vectors were used to modulate SRp levels and determine their effects on GRα/GRβ ratios as well as dexamethasone (DEX) responsiveness via GRE- luciferase reporter activity, fibronectin, and myocilin induction in TM cells. Results. SRp20, SRp30c, and SRp40 regulate GR splicing and the GC response in TM cells. Modulation of SRp levels altered the GRβ/α ratio that correlated with DEX responsiveness. Bombesin decreased SRp20; increased SRp30c, SRp40 levels, and GRβ/α ratio, and suppressed DEX response in TM cells. Conclusions. Relative levels of SRp20, SRp30c, and SRp40 in TM cells control differential expression of the two alternatively spliced isoforms of the GR and thereby regulate GC responsiveness. Different levels and/or activities of these SRps may account for differential GC sensitivity among the normal and glaucoma populations. PMID:22205602

A gradient of auxin and auxin-dependent transcription precedes tropic growth responses.

PubMed

Esmon, C Alex; Tinsley, Amanda G; Ljung, Karin; Sandberg, Goran; Hearne, Leonard B; Liscum, Emmanuel

2006-01-03

Plants, although sessile, can reorient growth axes in response to changing environmental conditions. Phototropism and gravitropism represent adaptive growth responses induced by changes in light direction and growth axis orientation relative to gravitational direction, respectively. The nearly 80-year-old Cholodny-Went theory [Went, F. W. & Thimann, K. V. (1937) Phytohormones (Macmillan, New York)] predicts that formation of a gradient of the plant morphogen auxin is central to the establishment of tropic curvature. Loss of tropic responses in seedling stems of Arabidopsis thaliana mutants lacking the auxin-regulated transcriptional activator NPH4/ARF7 has further suggested that a gradient of gene expression represents an essential output from the auxin gradient. Yet the molecular identities of such output components, which are likely to encode proteins directly involved in growth control, have remained elusive. Here we report the discovery of a suite of tropic stimulus-induced genes in Brassica oleracea that are responsive to an auxin gradient and exhibit morphologically graded expression concomitant with, or before, observable curvature responses. These results provide compelling molecular support for the Cholodny-Went theory and suggest that morphologically graded transcription represents an important mechanism for interpreting tropically stimulated gradients of auxin. Intriguingly, two of the tropic stimulus-induced genes, EXPA1 and EXPA8, encode enzymes involved in cell wall extension, a response prerequisite for differential growth leading to curvatures, and are up-regulated before curvature in the flank that will elongate. This observation suggests that morphologically graded transcription likely leads to the graded expression of proteins whose activities can directly regulate the establishment and modulation of tropic curvatures.

Uniform modeling of bacterial colony patterns with varying nutrient and substrate

NASA Astrophysics Data System (ADS)

Schwarcz, Deborah; Levine, Herbert; Ben-Jacob, Eshel; Ariel, Gil

2016-04-01

Bacteria develop complex patterns depending on growth condition. For example, Bacillus subtilis exhibit five different patterns depending on substrate hardness and nutrient concentration. We present a unified integro-differential model that reproduces the entire experimentally observed morphology diagram at varying nutrient concentrations and substrate hardness. The model allows a comprehensive and quantitative comparison between experimental and numerical variables and parameters, such as colony growth rate, nutrient concentration and diffusion constants. As a result, the role of the different physical mechanisms underlying and regulating the growth of the colony can be evaluated.

Proteomic changes associated with deletion of the Magnaporthe oryzae conidial morphology-regulating gene COM1

PubMed Central

2010-01-01

Background The rice blast disease caused by Magnaporthe oryzae is a major constraint on world rice production. The conidia produced by this fungal pathogen are the main source of disease dissemination. The morphology of conidia may be a critical factor in the spore dispersal and virulence of M. oryzae in the field. Deletion of a conidial morphology regulating gene encoding putative transcriptional regulator COM1 in M. oryzae resulted in aberrant conidial shape, reduced conidiation and attenuated virulence. Results In this study, a two-dimensional gel electrophoresis/matrix assisted laser desorption ionization- time of flight mass spectrometry (2-DE/MALDI-TOF MS) based proteomics approach was employed to identify the cellular and molecular components regulated by the COM1 protein (COM1p) that might contribute to the aberrant phenotypes in M. oryzae. By comparing the conidial proteomes of COM1 deletion mutant and its isogenic wild-type strain P131, we identified a potpourri of 31 proteins that exhibited statistically significant alterations in their abundance levels. Of these differentially regulated proteins, the abundance levels of nine proteins were elevated and twelve were reduced in the Δcom1 mutant. Three proteins were detected only in the Δcom1 conidial proteome, whereas seven proteins were apparently undetectable. The data obtained in the study suggest that the COM1p plays a key role in transcriptional reprogramming of genes implicated in melanin biosynthesis, carbon and energy metabolism, structural organization of cell, lipid metabolism, amino acid metabolism, etc. Semi-quantitative RT-PCR analysis revealed the down-regulation of genes encoding enzymes involved in melanin biosynthesis in the COM1 mutant. Conclusions Our results suggest that the COM1p may regulate the transcription of genes involved in various cellular processes indispensable for conidial development and appressorial penetration. These functions are likely to contribute to the effects of COM1p upon the aberrant phenotypes of M. oryzae. Reviewers This article is reviewed by George V. Shpakovski, Karthikeyan Sivaraman (nominated by M. Madan Babu) and Lakshminarayan M. Iyer. PMID:21040590

Habitat partitioning and morphological differentiation: the Southeast Asian Draco lizards and Caribbean Anolis lizards compared.

PubMed

Ord, Terry J; Klomp, Danielle A

2014-06-01

Sympatric species that initially overlap in resource use are expected to partition the environment in ways that will minimize interspecific competition. This shift in resource use can in turn prompt evolutionary changes in morphology. A classic example of habitat partitioning and morphological differentiation are the Caribbean Anolis lizards. Less well studied, but nevertheless striking analogues to the Anolis are the Southeast Asian Draco lizards. Draco and Anolis have evolved independently of each other for at least 80 million years. Their comparison subsequently offers a special opportunity to examine mechanisms of phenotypic differentiation between two ecologically diverse, but phylogenetically distinct groups. We tested whether Draco shared ecological axes of differentiation with Anolis (e.g., habitat use), whether this differentiation reflected interspecific competition, and to what extent adaptive change in morphology has occurred along these ecological axes. Using existing data on Anolis, we compared the habitat use and morphology of Draco in a field study of allopatric and sympatric species on the Malay Peninsula, Borneo and in the Philippines. Sympatric Draco lizards partitioned the environment along common resource axes to the Anolis lizards, especially in perch use. Furthermore, the morphology of Draco was correlated with perch use in the same way as it was in Anolis: species that used wider perches exhibited longer limb lengths. These results provide an important illustration of how interspecific competition can occur along common ecological axes in different animal groups, and how natural selection along these axes can generate the same type of adaptive change in morphology.

Granulocyte-macrophage colony-stimulating factor induces the differentiation of murine erythroleukaemia cells into dendritic cells.

PubMed Central

Cao, X; Zhao, Y; Yu, Y; Wang, Y; Zhang, M; Zhang, W; Wang, J

1998-01-01

Dendritic cells (DC) are professional antigen-presenting cells (APC) within the immune system and antigen-pulsed DC can be used as an effective vaccine for active immunotherapy of cancer. Granulocyte-macrophage colony-stimulating factor (GM-CSF) plays an important role in the generation of DC. We previously showed that GM-CSF can induce murine erythroleukaemia cells (FBL-3) to differentiate into monocyte-like cells. To develop a new vaccinating method to stimulate the host immune response to leukaemia, we further investigate whether FBL-3 cells induced by GM-CSF can differentiate into DC in the present study. After being treated with GM-CSF, FBL-3 cells expressed high levels of 33D1 and NLDC-145, which are the specific markers of DC. The expression of MHC-II, B7-1, B7-2 and vascular cell adhesion molecule-1 (VCAM-1) was up-regulated markedly; the typical morphology of DC were also observed by electron microscopy. Functionally, the GM-CSF-induced FBL-3 cells could apparently stimulate the proliferation of naive allogeneic and autologous T lymphocytes and induce the generation of specific CTL more efficiently than the wild-type FBL-3 cells. Mice immunized with GM-CSF-induced FBL-3 cells could resist the subsequent challenge with the wild-type FBL-3 cells. Collectively, these data indicate that GM-CSF differentiates murine erythroleukaemia cells into DC phenotypically, morphologically and functionally. FBL-3-derived DC can be used as a new type of vaccine. Our results may have important implications for the immunotherapy of leukaemia. Images Figure 3 Figure 4 PMID:9767469

A chromatin link to caste identity in the carpenter ant Camponotus floridanus

PubMed Central

Simola, Daniel F.; Ye, Chaoyang; Mutti, Navdeep S.; Dolezal, Kelly; Bonasio, Roberto; Liebig, Jürgen; Reinberg, Danny; Berger, Shelley L.

2013-01-01

In many ant species, sibling larvae follow alternative ontogenetic trajectories that generate striking variation in morphology and behavior among adults. These organism-level outcomes are often determined by environmental rather than genetic factors. Therefore, epigenetic mechanisms may mediate the expression of adult polyphenisms. We produced the first genome-wide maps of chromatin structure in a eusocial insect and found that gene-proximal changes in histone modifications, notably H3K27 acetylation, discriminate two female worker and male castes in Camponotus floridanus ants and partially explain differential gene expression between castes. Genes showing coordinated changes in H3K27ac and RNA implicate muscle development, neuronal regulation, and sensory responses in modulating caste identity. Binding sites of the acetyltransferase CBP harbor the greatest caste variation in H3K27ac, are enriched with motifs for conserved transcription factors, and show evolutionary expansion near developmental and neuronal genes. These results suggest that environmental effects on caste identity may be mediated by differential recruitment of CBP to chromatin. We propose that epigenetic mechanisms that modify chromatin structure may help orchestrate the generation and maintenance of polyphenic caste morphology and social behavior in ants. PMID:23212948

De novo comparative transcriptome analysis of genes involved in fruit morphology of pumpkin cultivars with extreme size difference and development of EST-SSR markers.

PubMed

Xanthopoulou, Aliki; Ganopoulos, Ioannis; Psomopoulos, Fotis; Manioudaki, Maria; Moysiadis, Theodoros; Kapazoglou, Aliki; Osathanunkul, Maslin; Michailidou, Sofia; Kalivas, Apostolos; Tsaftaris, Athanasios; Nianiou-Obeidat, Irini; Madesis, Panagiotis

2017-07-30

The genetic basis of fruit size and shape was investigated for the first time in Cucurbita species and genetic loci associated with fruit morphology have been identified. Although extensive genomic resources are available at present for tomato (Solanum lycopersicum), cucumber (Cucumis sativus), melon (Cucumis melo) and watermelon (Citrullus lanatus), genomic databases for Cucurbita species are limited. Recently, our group reported the generation of pumpkin (Cucurbita pepo) transcriptome databases from two contrasting cultivars with extreme fruit sizes. In the current study we used these databases to perform comparative transcriptome analysis in order to identify genes with potential roles in fruit morphology and fruit size. Differential Gene Expression (DGE) analysis between cv. 'Munchkin' (small-fruit) and cv. 'Big Moose' (large-fruit) revealed a variety of candidate genes associated with fruit morphology with significant differences in gene expression between the two cultivars. In addition, we have set the framework for generating EST-SSR markers, which discriminate different C. pepo cultivars and show transferability to related Cucurbitaceae species. The results of the present study will contribute to both further understanding the molecular mechanisms regulating fruit morphology and furthermore identifying the factors that determine fruit size. Moreover, they may lead to the development of molecular marker tools for selecting genotypes with desired morphological traits. Copyright © 2017. Published by Elsevier B.V.

Reconstructing the regulatory circuit of cell fate determination in yeast mating response.

PubMed

Shao, Bin; Yuan, Haiyu; Zhang, Rongfei; Wang, Xuan; Zhang, Shuwen; Ouyang, Qi; Hao, Nan; Luo, Chunxiong

2017-07-01

Massive technological advances enabled high-throughput measurements of proteomic changes in biological processes. However, retrieving biological insights from large-scale protein dynamics data remains a challenging task. Here we used the mating differentiation in yeast Saccharomyces cerevisiae as a model and developed integrated experimental and computational approaches to analyze the proteomic dynamics during the process of cell fate determination. When exposed to a high dose of mating pheromone, the yeast cell undergoes growth arrest and forms a shmoo-like morphology; however, at intermediate doses, chemotropic elongated growth is initialized. To understand the gene regulatory networks that control this differentiation switch, we employed a high-throughput microfluidic imaging system that allows real-time and simultaneous measurements of cell growth and protein expression. Using kinetic modeling of protein dynamics, we classified the stimulus-dependent changes in protein abundance into two sources: global changes due to physiological alterations and gene-specific changes. A quantitative framework was proposed to decouple gene-specific regulatory modes from the growth-dependent global modulation of protein abundance. Based on the temporal patterns of gene-specific regulation, we established the network architectures underlying distinct cell fates using a reverse engineering method and uncovered the dose-dependent rewiring of gene regulatory network during mating differentiation. Furthermore, our results suggested a potential crosstalk between the pheromone response pathway and the target of rapamycin (TOR)-regulated ribosomal biogenesis pathway, which might underlie a cell differentiation switch in yeast mating response. In summary, our modeling approach addresses the distinct impacts of the global and gene-specific regulation on the control of protein dynamics and provides new insights into the mechanisms of cell fate determination. We anticipate that our integrated experimental and modeling strategies could be widely applicable to other biological systems.

Gene trapping in differentiating cell lines: regulation of the lysosomal protease cathepsin B in skeletal myoblast growth and fusion.

PubMed

Gogos, J A; Thompson, R; Lowry, W; Sloane, B F; Weintraub, H; Horwitz, M

1996-08-01

To identify genes regulated during skeletal muscle differentiation, we have infected mouse C2C12 myoblasts with retroviral gene trap vectors, containing a promoterless marker gene with a 5' splice acceptor signal. Integration of the vector adjacent to an actively transcribed gene places the marker under the transcriptional control of the endogenous gene, while the adjacent vector sequences facilitate cloning. The vector insertionally mutates the trapped locus and may also form fusion proteins with the endogenous gene product. We have screened several hundred clones, each containing a trapping vector integrated into a different endogenous gene. In agreement with previous estimates based on hybridization kinetics, we find that a large proportion of all genes expressed in myoblasts are regulated during differentiation. Many of these genes undergo unique temporal patterns of activation or repression during cell growth and myotube formation, and some show specific patterns of subcellular localization. The first gene we have identified with this strategy is the lysosomal cysteine protease cathepsin B. Expression from the trapped allele is upregulated during early myoblast fusion and downregulated in myotubes. A direct role for cathepsin B in myoblast growth and fusion is suggested by the observation that the trapped cells deficient in cathepsin B activity have an unusual morphology and reduced survival in low-serum media and undergo differentiation with impaired cellular fusion. The phenotype is reproduced by antisense cathepsin B expression in parental C2C12 myoblasts. The cellular phenotype is similar to that observed in cultured myoblasts from patients with I cell disease, in which there is diminished accumulation of lysosomal enzymes. This suggests that a specific deficiency of cathepsin B could contribute to the myopathic component of this illness.

CLOCK regulates mammary epithelial cell growth and differentiation

PubMed Central

Crodian, Jennifer; Suárez-Trujillo, Aridany; Erickson, Emily; Weldon, Bethany; Crow, Kristi; Cummings, Shelby; Chen, Yulu; Shamay, Avi; Mabjeesh, Sameer J.; Plaut, Karen

2016-01-01

Circadian clocks influence virtually all physiological processes, including lactation. Here, we investigate the role of the CLOCK gene in regulation of mammary epithelial cell growth and differentiation. Comparison of mammary morphology in late-pregnant wild-type and ClockΔ19 mice, showed that gland development was negatively impacted by genetic loss of a functional timing system. To understand whether these effects were due, in part, to loss of CLOCK function in the gland, the mouse mammary epithelial cell line, HC11, was transfected with short hairpin RNA that targeted Clock (shClock). Cells transfected with shClock expressed 70% less Clock mRNA than wild-type (WT) HC11 cultures, which resulted in significantly depressed levels of CLOCK protein (P < 0.05). HC11 lines carrying shClock had four-fold higher growth rates (P < 0.05), and the percentage of cells in G1 phase was significantly higher (90.1 ± 1.1% of shClock vs. 71.3 ± 3.6% of WT-HC11) following serum starvation. Quantitative-PCR (qPCR) analysis showed shClock had significant effects (P < 0.0001) on relative expression levels of Ccnd1, Wee1, and Tp63. qPCR analysis of the effect of shClock on Fasn and Cdh1 expression in undifferentiated cultures and cultures treated 96 h with dexamethasone, insulin, and prolactin (differentiated) found levels were reduced by twofold and threefold, respectively (P < 0.05), in shClock line relative to WT cultures. Abundance of CDH1 and TP63 proteins were significantly reduced in cultures transfected with shClock. These data support how CLOCK plays a role in regulation of epithelial cell growth and differentiation in the mammary gland. PMID:27707717

Reactive oxygen species activate differentiation gene transcription of acute myeloid leukemia cells via the JNK/c-JUN signaling pathway.

PubMed

Lam, Chung Fan; Yeung, Hoi Ting; Lam, Yuk Man; Ng, Ray Kit

2018-05-01

Reactive oxygen species (ROS) and altered cellular redox status are associated with many malignancies. Acute myeloid leukemia (AML) cells are maintained at immature state by differentiation blockade, which involves deregulation of transcription factors in myeloid differentiation. AML cells can be induced to differentiate by phorbol-12-myristate-13-acetate (PMA), which possesses pro-oxidative activity. However, the signaling events mediated by ROS in the activation of transcriptional program during AML differentiation has not been fully elucidated. Here, we investigated AML cell differentiation by treatment with PMA and ROS scavenger N-acetyl-l-cysteine (NAC). We observed elevation of intracellular ROS level in the PMA-treated AML cells, which correlated with differentiated cell morphology and increased CD11b + mature cell population. The effect of PMA can be abolished by NAC co-treatment, supporting the involvement of ROS in the process. Moreover, we demonstrated that short ROS elevation mediated cell cycle arrest, but failed to activate myeloid gene transcription; whereas prolonged ROS elevation activated JNK/c-JUN signaling pathway. Inhibition of JNK suppressed the expression of key myeloid transcriptional regulators c-JUN, SPI-1 and MAFB, and prevented AML cells from undergoing terminal differentiation. These findings provide new insights into the crucial role of JNK/c-Jun signaling pathway in the activation of transcriptional program during ROS-mediated AML differentiation. Copyright © 2018 Elsevier Ltd. All rights reserved.

Spatial structure of morphological and neutral genetic variation in Brook Trout

USGS Publications Warehouse

Kazyak, David C.; Hilderbrand, Robert H.; Keller, Stephen R.; Colaw, Mark C.; Holloway, Amanda E.; Morgan, Raymond P.; King, Timothy L.

2015-01-01

Brook Trout Salvelinus fontinalis exhibit exceptional levels of life history variation, remarkable genetic variability, and fine-scale population structure. In many cases, neighboring populations may be highly differentiated from one another to an extent that is comparable with species-level distinctions in other taxa. Although genetic samples have been collected from hundreds of populations and tens of thousands of individuals, little is known about whether differentiation at neutral markers reflects phenotypic differences among Brook Trout populations. We compared differentiation in morphology and neutral molecular markers among populations from four geographically proximate locations (all within 24 km) to examine how genetic diversity covaries with morphology. We found significant differences among and/or within streams for all three morphological axes examined and identified the source stream of many individuals based on morphology (52.3% classification efficiency). Although molecular and morphological differentiation among streams ranged considerably (mean pairwise FST: 0.023–0.264; pairwise PST: 0.000–0.339), the two measures were not significantly correlated. While in some cases morphological characters appear to have diverged to a greater extent than expected by neutral genetic drift, many traits were conserved to a greater extent than were neutral genetic markers. Thus, while Brook Trout exhibit fine-scale spatial patterns in both morphology and neutral genetic diversity, these types of biological variabilities are being structured by different ecological and evolutionary processes. The relative influences of genetic drift versus selection and phenotypic plasticity in shaping morphology appear to vary among populations occupying nearby streams.

Sprouty / FGF signaling regulates the proximal-distal feather morphology and the size of dermal papillae

PubMed Central

Yue, Zhicao; Jiang, Ting Xin; Wu, Ping; Widelitz, Randall B; Chuong, Cheng Ming

2013-01-01

In a feather, there are distinct morphologies along the proximal-distal axis. The proximal part is a cylindrical stalk (calamus), whereas the distal part has barb and barbule branches. Here we focus on what molecular signaling activity can modulate feather stem cells to generate these distinct morphologies. We demonstrate the drastic tissue remodeling during feather cycling which includes initiation, growth and resting phases. In the growth phase, epithelial components undergo progressive changes from the collar growth zone to the ramogenic zone, to maturing barb branches along the proximal- distal axis. Mesenchymal components also undergo progressive changes from the dermal papilla, to the collar mesenchyme, to the pulp along the proximal- distal axis. Over-expression of Spry4, a negative regulator of receptor tyrosine kinases, promotes barb branch formation at the expense of the epidermal collar. It even induces barb branches from the follicle sheath (equivalent to the outer root sheath in hair follicles). The results are feathers with expanded feather vane regions and small or missing proximal feather shafts (the calamus). Spry4 also expands the pulp region while reducing the size of dermal papillae, leading to a failure to regenerate. In contrast, over-expressing Fgf10 increases the size of the dermal papillae, expands collar epithelia and mesenchyme, but also prevents feather branch formation and feather keratin differentiation. These results suggest that coordinated Sprouty/FGF pathway activity at different stages is important to modulate feather epidermal stem cells to form distinct feather morphologies along the proximal-distal feather axis. PMID:23000358

Comparative studies of differential expression of chitinolytic enzymes encoded by chiA, chiB, chiC and nagA genes in Aspergillus nidulans.

PubMed

Pusztahelyi, T; Molnár, Z; Emri, T; Klement, E; Miskei, M; Kerékgyárto, J; Balla, J; Pócsi, I

2006-01-01

N-Acetyl-D-glucosamine, chito-oligomers and carbon starvation regulated chiA, chiB, and nagA gene expressions in Aspergillus nidulans cultures. The gene expression patterns of the main extracellular endochitinase ChiB and the N-acetyl-beta-D-glucosaminidase NagA were similar, and the ChiB-NagA enzyme system may play a morphological and/or nutritional role during autolysis. Alterations in the levels of reactive oxygen species or in the glutathione-glutathione disulfide redox balance, characteristic physiological changes developing in ageing and autolyzing fungal cultures, did not affect the regulation of either the growth-related chiA or the autolysis-coupled chiB genes although both of them were down-regulated under diamide stress. The transcription of the chiC gene with unknown physiological function was repressed by increased intracellular superoxide concentration.

Antifungal Activity of Eucalyptus Oil against Rice Blast Fungi and the Possible Mechanism of Gene Expression Pattern.

PubMed

Zhou, Li-Jun; Li, Fu-Rong; Huang, Li-Jie; Yang, Zhi-Rong; Yuan, Shu; Bai, Lin-Han

2016-05-12

Eucalyptus oil possesses a wide spectrum of biological activity, including anti-microbial, fungicidal, herbicidal, acaricidal and nematicidal properties. We studied anti-fungal activities of the leaf oil extracted from Eucalyptus. grandis × E. urophylla. Eleven plant pathogenic fungi were tested based on the mycelium growth rates with negative control. The results showed that Eucalyptus oil has broad-spectrum inhibitory effects toward these fungi. Remarkable morphological and structural alterations of hypha have been observed for Magnaporthe grisea after the treatment. The mRNA genome array of M. grisea was used to detect genes that were differentially expressed in the test strains treated by the Eucalyptus oil than the normal strains. The results showed 1919 genes were significantly affected, among which 1109 were down-regulated and 810 were up-regulated (p < 0.05, absolute fold change >2). According to gene ontology annotation analysis, these differentially expressed genes may cause abnormal structures and physiological function disorders, which may reduce the fungus growth. These results show the oil has potential for use in the biological control of plant disease as a green biopesticide.

Splicing factor SRSF3 is crucial for hepatocyte differentiation and metabolic function

PubMed Central

Sen, Supriya; Jumaa, Hassan; Webster, Nicholas J.G.

2015-01-01

SR family RNA binding proteins regulate splicing of nascent RNAs in vitro but their physiological role in vivo is largely unexplored, as genetic deletion of many SR protein genes results in embryonic lethality. Here we show that SRSF3HKO mice carrying a hepatocyte-specific deletion of Srsf3 (homologous to human SRSF3/SRp20) have a disrupted hepatic architecture and show pre- and postnatal growth retardation. SRSF3HKO mice exhibit impaired hepatocyte maturation with alterations in glucose and lipid homeostasis characterized by reduced glycogen storage, fasting hypoglycemia, increased insulin sensitivity and reduced cholesterol synthesis. We identify various splicing alterations in the SRSF3HKO liver that explain the in vivo phenotype. In particular, loss of SRSF3 causes aberrant splicing of Hnf1α, Ern1, Hmgcs1, Dhcr7 and Scap genes, which are critical regulators of glucose and lipid metabolism. Our study provides the first evidence for a SRSF3-driven genetic programme required for morphological and functional differentiation of hepatocytes that may have relevance for human liver disease and metabolic dysregulation. PMID:23299886

Transcriptomic and epigenomic characterization of the developing bat wing

PubMed Central

Eckalbar, Walter L.; Schlebusch, Stephen A.; Mason, Mandy K.; Gill, Zoe; Parker, Ash V.; Booker, Betty M.; Nishizaki, Sierra; Muswamba-Nday, Christiane; Terhune, Elizabeth; Nevonen, Kimberly; Makki, Nadja; Friedrich, Tara; VanderMeer, Julia E.; Pollard, Katherine S.; Carbone, Lucia; Wall, Jeff D.; Illing, Nicola; Ahituv, Nadav

2016-01-01

Bats are the only mammals capable of powered flight, but little is known about the genetic determinants that shape their wings. Here, we generated a genome for Miniopterus natalensis and performed RNA-seq and ChIP-seq (H3K27ac, H3K27me3) on its developing forelimb and hindlimb autopods at sequential embryonic stages to decipher the molecular events that underlie bat wing development. Over 7,000 genes and several lncRNAs, including Tbx5-as1 and Hottip, were differentially expressed between forelimb, hindlimb and different stages. ChIP-seq identified thousands of regions that are differentially modified in forelimb versus hindlimb. Comparative genomics found 2,796 bat-accelerated regions within H3K27ac peaks, several of which cluster near limb-associated genes. Pathway analyses revealed multiple ribosomal proteins and known limb patterning signaling pathways as differentially regulated, and implicated increased forelimb mesenchymal condensations with differential growth. Combined, our work outlines multiple genetic components that contribute to bat wing formation, providing a genomic blueprint for this morphological innovation. PMID:27019111

Enteric neural crest cells regulate vertebrate stomach patterning and differentiation.

PubMed

Faure, Sandrine; McKey, Jennifer; Sagnol, Sébastien; de Santa Barbara, Pascal

2015-01-15

In vertebrates, the digestive tract develops from a uniform structure where reciprocal epithelial-mesenchymal interactions pattern this complex organ into regions with specific morphologies and functions. Concomitant with these early patterning events, the primitive GI tract is colonized by the vagal enteric neural crest cells (vENCCs), a population of cells that will give rise to the enteric nervous system (ENS), the intrinsic innervation of the GI tract. The influence of vENCCs on early patterning and differentiation of the GI tract has never been evaluated. In this study, we report that a crucial number of vENCCs is required for proper chick stomach development, patterning and differentiation. We show that reducing the number of vENCCs by performing vENCC ablations induces sustained activation of the BMP and Notch pathways in the stomach mesenchyme and impairs smooth muscle development. A reduction in vENCCs also leads to the transdifferentiation of the stomach into a stomach-intestinal mixed phenotype. In addition, sustained Notch signaling activity in the stomach mesenchyme phenocopies the defects observed in vENCC-ablated stomachs, indicating that inhibition of the Notch signaling pathway is essential for stomach patterning and differentiation. Finally, we report that a crucial number of vENCCs is also required for maintenance of stomach identity and differentiation through inhibition of the Notch signaling pathway. Altogether, our data reveal that, through the regulation of mesenchyme identity, vENCCs act as a new mediator in the mesenchymal-epithelial interactions that control stomach development. © 2015. Published by The Company of Biologists Ltd.

Neuroblastoma differentiation involves the expression of two isoforms of the alpha-subunit of Go.

PubMed

Brabet, P; Pantaloni, C; Rodriguez, M; Martinez, J; Bockaert, J; Homburger, V

1990-04-01

The regulation of GTP-binding proteins (G proteins) was examined during the course of differentiation of neuroblastoma N1E-115 cells. N1E-115 cell membranes possess three Bordetella pertussis toxin (PTX) substrates assigned to alpha-subunits (G alpha) of Go (a G protein of unknown function) and "Gi (a G protein inhibitory to adenylate cyclase)-like" proteins and one substrate of Vibrio cholerae toxin corresponding to an alpha-subunit of Gs (a G protein stimulatory to adenylate cyclase). In undifferentiated cells, only one form of Go alpha was found, having a pI of 5.8 Go alpha content increased by approximately twofold from the undifferentiated state to 96 h of cell differentiation. This is mainly due to the appearance of another Go alpha form having a pI of 5.55. Both Go alpha isoforms have similar sizes on sodium dodecyl sulfate-polyacrylamide gels, are recognized by polyclonal antibodies to bovine brain Go alpha, are ADP-ribosylated by PTX, and are covalently myristylated in whole N1E-115 cells. In addition, immunofluorescent staining of N1E-115 cells with Go alpha antibodies revealed that association of Go alpha with the plasma membrane appears to coincide with the expression of the most acidic isoform and morphological cell differentiation. In contrast, the levels of both Gi alpha and Gs alpha did not significantly change, whereas that of the common beta-subunit increased by approximately 30% over the same period. These results demonstrate specific regulation of the expression of Go alpha during neuronal differentiation.

Proteomic Analysis of Human Adipose Derived Stem Cells during Small Molecule Chemical Stimulated Pre-neuronal Differentiation

PubMed Central

Santos, Jerran; Milthorpe, Bruce K; Herbert, Benjamin R; Padula, Matthew P

2017-01-01

Background Adipose derived stem cells (ADSCs) are acquired from abdominal liposuction yielding a thousand fold more stem cells per millilitre than those from bone marrow. A large research void exists as to whether ADSCs are capable of transdermal differentiation toward neuronal phenotypes. Previous studies have investigated the use of chemical cocktails with varying inconclusive results. Methods Human ADSCs were treated with a chemical stimulant, beta-mercaptoethanol, to direct them toward a neuronal-like lineage within 24 hours. Quantitative proteomics using iTRAQ was then performed to ascertain protein abundance differences between ADSCs, beta-mercaptoethanol treated ADSCs and a glioblastoma cell line. Results The soluble proteome of ADSCs differentiated for 12 hours and 24 hours was significantly different from basal ADSCs and control cells, expressing a number of remodeling, neuroprotective and neuroproliferative proteins. However toward the later time point presented stress and shock related proteins were observed to be up regulated with a large down regulation of structural proteins. Cytokine profiles support a large cellular remodeling shift as well indicating cellular distress. Conclusion The earlier time point indicates an initiation of differentiation. At the latter time point there is a vast loss of cell population during treatment. At 24 hours drastically decreased cytokine profiles and overexpression of stress proteins reveal that exposure to beta-mercaptoethanol beyond 24 hours may not be suitable for clinical application as our results indicate that the cells are in trauma whilst producing neuronal-like morphologies. The shorter treatment time is promising, indicating a reducing agent has fast acting potential to initiate neuronal differentiation of ADSCs. PMID:28844130

Proteomic Analysis of Human Adipose Derived Stem Cells during Small Molecule Chemical Stimulated Pre-neuronal Differentiation.

PubMed

Santos, Jerran; Milthorpe, Bruce K; Herbert, Benjamin R; Padula, Matthew P

2017-11-30

Adipose derived stem cells (ADSCs) are acquired from abdominal liposuction yielding a thousand fold more stem cells per millilitre than those from bone marrow. A large research void exists as to whether ADSCs are capable of transdermal differentiation toward neuronal phenotypes. Previous studies have investigated the use of chemical cocktails with varying inconclusive results. Human ADSCs were treated with a chemical stimulant, beta-mercaptoethanol, to direct them toward a neuronal-like lineage within 24 hours. Quantitative proteomics using iTRAQ was then performed to ascertain protein abundance differences between ADSCs, beta-mercaptoethanol treated ADSCs and a glioblastoma cell line. The soluble proteome of ADSCs differentiated for 12 hours and 24 hours was significantly different from basal ADSCs and control cells, expressing a number of remodeling, neuroprotective and neuroproliferative proteins. However toward the later time point presented stress and shock related proteins were observed to be up regulated with a large down regulation of structural proteins. Cytokine profiles support a large cellular remodeling shift as well indicating cellular distress. The earlier time point indicates an initiation of differentiation. At the latter time point there is a vast loss of cell population during treatment. At 24 hours drastically decreased cytokine profiles and overexpression of stress proteins reveal that exposure to beta-mercaptoethanol beyond 24 hours may not be suitable for clinical application as our results indicate that the cells are in trauma whilst producing neuronal-like morphologies. The shorter treatment time is promising, indicating a reducing agent has fast acting potential to initiate neuronal differentiation of ADSCs.

Myostatin inhibits myosatellite cell proliferation and consequently activates differentiation: evidence for endocrine-regulated transcript processing.

PubMed

Garikipati, Dilip K; Rodgers, Buel D

2012-10-01

Myostatin is a potent negative regulator of muscle growth in mammals. Despite high structural conservation, functional conservation in nonmammalian species is only assumed. This is particularly true for fish due to the presence of several myostatin paralogs: two in most species and four in salmonids (MSTN-1a, -1b, -2a, and -2b). Rainbow trout are a rich source of primary myosatellite cells as hyperplastic muscle growth occurs even in adult fish. These cells were therefore used to determine myostatin's effects on proliferation whereas our earlier studies reported its effects on quiescent cells. As in mammals, recombinant myostatin suppressed proliferation with no changes in cell morphology. Expression of MSTN-1a was several fold higher than the other paralogs and was autoregulated by myostatin, which also upregulated the expression of key differentiation markers: Myf5, MyoD1, myogenin, and myosin light chain. Thus, myostatin-stimulated cellular growth inhibition activates rather than represses differentiation. IGF-1 stimulated proliferation but had minimal and delayed effects on differentiation and its actions were suppressed by myostatin. However, IGF-1 upregulated MSTN-2a expression and the processing of its transcript, which is normally unprocessed. Myostatin therefore appears to partly mediate IGF-stimulated myosatellite differentiation in rainbow trout. This also occurs in mammals, although the IGF-stimulated processing of MSTN-2a transcripts is highly unique and is indicative of subfunctionalization within the gene family. These studies also suggest that the myokine's actions, including its antagonistic relationship with IGF-1, are conserved and that the salmonid gene family is functionally diverging.

Yap1 is dispensable for self-renewal but required for proper differentiation of mouse embryonic stem (ES) cells.

PubMed

Chung, HaeWon; Lee, Bum-Kyu; Uprety, Nadima; Shen, Wenwen; Lee, Jiwoon; Kim, Jonghwan

2016-04-01

Yap1 is a transcriptional co-activator of the Hippo pathway. The importance of Yap1 in early cell fate decision during embryogenesis has been well established, though its role in embryonic stem (ES) cells remains elusive. Here, we report that Yap1 plays crucial roles in normal differentiation rather than self-renewal of ES cells. Yap1-depleted ES cells maintain undifferentiated state with a typical colony morphology as well as robust alkaline phosphatase activity. These cells also retain comparable levels of the core pluripotent factors, such as Pou5f1 and Sox2, to the levels in wild-type ES cells without significant alteration of lineage-specific marker genes. Conversely, overexpression of Yap1 in ES cells promotes nuclear translocation of Yap1, resulting in disruption of self-renewal and triggering differentiation by up-regulating lineage-specific genes. Moreover, Yap1-deficient ES cells show impaired induction of lineage markers during differentiation. Collectively, our data demonstrate that Yap1 is a required factor for proper differentiation of mouse ES cells, while remaining dispensable for self-renewal. © 2016 The Authors.

In Vitro Hepatic Trans-Differentiation of Human Mesenchymal Stem Cells Using Sera from Congestive/Ischemic Liver during Cardiac Failure

PubMed Central

Bishi, Dillip Kumar; Mathapati, Santosh; Cherian, Kotturathu Mammen; Guhathakurta, Soma; Verma, Rama Shanker

2014-01-01

Cellular therapy for end-stage liver failures using human mesenchymal stem cells (hMSCs)-derived hepatocytes is a potential alternative to liver transplantation. Hepatic trans-differentiation of hMSCs is routinely accomplished by induction with commercially available recombinant growth factors, which is of limited clinical applications. In the present study, we have evaluated the potential of sera from cardiac-failure-associated congestive/ischemic liver patients for hepatic trans-differentiation of hMSCs. Results from such experiments were confirmed through morphological changes and expression of hepatocyte-specific markers at molecular and cellular level. Furthermore, the process of mesenchymal-to-epithelial transition during hepatic trans-differentiation of hMSCs was confirmed by elevated expression of E-Cadherin and down-regulation of Snail. The functionality of hMSCs-derived hepatocytes was validated by various liver function tests such as albumin synthesis, urea release, glycogen accumulation and presence of a drug inducible cytochrome P450 system. Based on these findings, we conclude that sera from congestive/ischemic liver during cardiac failure support a liver specific microenvironment for effective hepatic trans-differentiation of hMSCs in vitro. PMID:24642599

Effects of salinity on the cellular physiological responses of Natrinema sp. J7-2

PubMed Central

Mei, Yunjun; Liu, Huan; Zhang, Shunxi; Yang, Ming; Hu, Chun; Zhang, Jian; Shen, Ping; Chen, Xiangdong

2017-01-01

The halophilic archaea (haloarchaea) live in hyersaline environments such as salt lakes, salt ponds and marine salterns. To cope with the salt stress conditions, haloarchaea have developed two fundamentally different strategies: the "salt-in" strategy and the "compatible-solute" strategy. Although investigation of the molecular mechanisms underlying the tolerance to high salt concentrations has made outstanding achievements, experimental study from the aspect of transcription is rare. In the present study, we monitored cellular physiology of Natrinema sp. J7-2 cells incubated in different salinity media (15%, 25% and 30% NaCl) from several aspects, such as cellular morphology, growth, global transcriptome and the content of intracellular free amino acids. The results showed that the cells were polymorphic and fragile at a low salt concentration (15% NaCl) but had a long, slender rod shape at high salt concentrations (25% and 30% NaCl). The cells grew best in 25% NaCl, mediocre in 30% NaCl and struggled in 15% NaCl. An RNA-seq analysis revealed differentially expressed genes (DEGs) in various salinity media. A total of 1,148 genes were differentially expressed, consisting of 719 DEGs (348 up-regulated and 371 down-regulated genes) between cells in 15% vs 25% NaCl, and 733 DEGs (521 up-regulated and 212 down-regulated genes) between cells in 25% vs 30% NaCl. Moreover, 304 genes were commonly differentially expressed in both 15% vs 25% and 25% vs30% NaCl. The DEGs were enriched in different KEGG metabolic pathways, such as amino acids, glycerolipid, ribosome, nitrogen, protoporphyrin, porphyrin and porhiniods. The intracellular predominant free amino acids consisted of the glutamate family (Glu, Arg and Pro), aspartate family (Asp) and aromatic amino acids (Phe and Trp), especially Glu and Asp. PMID:28926633

Calcium-dependent depletion zones in the cortical microtubule array coincide with sites of, but do not regulate, wall ingrowth papillae deposition in epidermal transfer cells

PubMed Central

Zhang, Hui-ming; Talbot, Mark J.; McCurdy, David W.; Patrick, John W.; Offler, Christina E.

2015-01-01

Trans-differentiation to a transfer-cell morphology is characterized by the localized deposition of wall ingrowth papillae that protrude into the cytosol. Whether the cortical microtubule array directs wall ingrowth papillae formation was investigated using a Vicia faba cotyledon culture system in which their adaxial epidermal cells were spontaneously induced to trans-differentiate to transfer cells. During deposition of wall ingrowth papillae, the aligned cortical microtubule arrays in precursor epidermal cells were reorganized into a randomized array characterized by circular depletion zones. Concurrence of the temporal appearance, spatial pattern, and size of depletion zones and wall ingrowth papillae was consistent with each papilla occupying a depletion zone. Surprisingly, microtubules appeared not to regulate construction of wall ingrowth papillae, as neither depolymerization nor stabilization of cortical microtubules changed their deposition pattern or morphology. Moreover, the size and spatial pattern of depletion zones was unaltered when the formation of wall ingrowth papillae was blocked by inhibiting cellulose biosynthesis. In contrast, the depletion zones were absent when the cytosolic calcium plumes, responsible for directing wall ingrowth papillae formation, were blocked or dissipated. Thus, we conclude that the depletion zones within the cortical microtubule array result from localized depolymerization of microtubules initiated by elevated cytosolic Ca2+ levels at loci where wall ingrowth papillae are deposited. The physiological significance of the depletion zones as a mechanism to accommodate the construction of wall ingrowth papillae without compromising maintenance of the plasma membrane–microtubule inter-relationship is discussed. PMID:26136268

Diversity in global gene expression and morphology across a watercress (Nasturtium officinale R. Br.) germplasm collection: first steps to breeding

PubMed Central

Payne, Adrienne C.; Clarkson, Graham J.J.; Rothwell, Steve; Taylor, Gail

2015-01-01

Watercress (Nasturtium officinale R. Br.) is a nutrient intense, leafy crop that is consumed raw or in soups across the globe, but for which, currently no genomic resources or breeding programme exists. Promising morphological, biochemical and functional genomic variation was identified for the first time in a newly established watercress germplasm collection, consisting of 48 watercress accessions sourced from contrasting global locations. Stem length, stem diameter and anti-oxidant (AO) potential varied across the accessions. This variation was used to identify three extreme contrasting accessions for further analysis. Variation in global gene expression was investigated using an Affymetrix Arabidopsis ATH1 microarray gene chip, using the commercial control (C), an accession selected for dwarf phenotype with a high AO potential (dwarfAO, called ‘Boldrewood’) and one with high AO potential alone. A set of transcripts significantly differentially expressed between these three accessions, were identified, including transcripts involved in the regulation of growth and development and those involved in secondary metabolism. In particular, when differential gene expression was compared between C and dwarfAO, the dwarfAO was characterised by increased expression of genes encoding glucosinolates, which are known precursors of phenethyl isothiocyanate, linked to the anti-carcinogenic effects well-documented in watercress. This study provides the first analysis of natural variation across the watercress genome and has identified important underpinning information for future breeding for enhanced anti-carcinogenic properties and morphology traits in this nutrient-intense crop. PMID:26504575

Novel hydrated graphene ribbon unexpectedly promotes aged seed germination and root differentiation

NASA Astrophysics Data System (ADS)

Hu, Xiangang; Zhou, Qixing

2014-01-01

It is well known that graphene (G) induces nanotoxicity towards living organisms. Here, a novel and biocompatible hydrated graphene ribbon (HGR) unexpectedly promoted aged (two years) seed germination. HGR formed at the normal temperature and pressure (120 days hydration), presented 17.1% oxygen, 0.9% nitrogen groups, disorder-layer structure, with 0.38 nm thickness ribbon morphology. Interestingly, there were bulges around the edges of HGR. Compared to G and graphene oxide (GO), HGR increased seed germination by 15% root differentiation between 52 and 59% and enhanced resistance to oxidative stress. The metabonomics analysis discovered that HGR upregulated carbohydrate, amino acid, and fatty acids metabolism that determined secondary metabolism, nitrogen sequestration, cell membrane integrity, permeability, and oxidation resistance. Hexadecanoic acid as a biomarker promoted root differentiation and increased the germination rate. Our discovery is a novel HGR that promotes aged seed germination, illustrates metabolic specificity among graphene-based materials, and inspires innovative concepts in the regulation of seed development.

Dynamic Cytology and Transcriptional Regulation of Rice Lamina Joint Development1[OPEN

PubMed Central

2017-01-01

Rice (Oryza sativa) leaf angle is determined by lamina joint and is an important agricultural trait determining leaf erectness and, hence, the photosynthesis efficiency and grain yield. Genetic studies reveal a complex regulatory network of lamina joint development; however, the morphological changes, cytological transitions, and underlying transcriptional programming remain to be elucidated. A systemic morphological and cytological study reveals a dynamic developmental process and suggests a common but distinct regulation of the lamina joint. Successive and sequential cell division and expansion, cell wall thickening, and programmed cell death at the adaxial or abaxial sides form the cytological basis of the lamina joint, and the increased leaf angle results from the asymmetric cell proliferation and elongation. Analysis of the gene expression profiles at four distinct developmental stages ranging from initiation to senescence showed that genes related to cell division and growth, hormone synthesis and signaling, transcription (transcription factors), and protein phosphorylation (protein kinases) exhibit distinct spatiotemporal patterns during lamina joint development. Phytohormones play crucial roles by promoting cell differentiation and growth at early stages or regulating the maturation and senescence at later stages, which is consistent with the quantitative analysis of hormones at different stages. Further comparison with the gene expression profile of leaf inclination1, a mutant with decreased auxin and increased leaf angle, indicates the coordinated effects of hormones in regulating lamina joint. These results reveal a dynamic cytology of rice lamina joint that is fine-regulated by multiple factors, providing informative clues for illustrating the regulatory mechanisms of leaf angle and plant architecture. PMID:28500269

Dynamic Cytology and Transcriptional Regulation of Rice Lamina Joint Development.

PubMed

Zhou, Li-Juan; Xiao, Lang-Tao; Xue, Hong-Wei

2017-07-01

Rice ( Oryza sativa ) leaf angle is determined by lamina joint and is an important agricultural trait determining leaf erectness and, hence, the photosynthesis efficiency and grain yield. Genetic studies reveal a complex regulatory network of lamina joint development; however, the morphological changes, cytological transitions, and underlying transcriptional programming remain to be elucidated. A systemic morphological and cytological study reveals a dynamic developmental process and suggests a common but distinct regulation of the lamina joint. Successive and sequential cell division and expansion, cell wall thickening, and programmed cell death at the adaxial or abaxial sides form the cytological basis of the lamina joint, and the increased leaf angle results from the asymmetric cell proliferation and elongation. Analysis of the gene expression profiles at four distinct developmental stages ranging from initiation to senescence showed that genes related to cell division and growth, hormone synthesis and signaling, transcription (transcription factors), and protein phosphorylation (protein kinases) exhibit distinct spatiotemporal patterns during lamina joint development. Phytohormones play crucial roles by promoting cell differentiation and growth at early stages or regulating the maturation and senescence at later stages, which is consistent with the quantitative analysis of hormones at different stages. Further comparison with the gene expression profile of leaf inclination1 , a mutant with decreased auxin and increased leaf angle, indicates the coordinated effects of hormones in regulating lamina joint. These results reveal a dynamic cytology of rice lamina joint that is fine-regulated by multiple factors, providing informative clues for illustrating the regulatory mechanisms of leaf angle and plant architecture. © 2017 American Society of Plant Biologists. All Rights Reserved.

The head-regeneration transcriptome of the planarian Schmidtea mediterranea.

PubMed

Sandmann, Thomas; Vogg, Matthias C; Owlarn, Suthira; Boutros, Michael; Bartscherer, Kerstin

2011-08-16

Planarian flatworms can regenerate their head, including a functional brain, within less than a week. Despite the enormous potential of these animals for medical research and regenerative medicine, the mechanisms of regeneration and the molecules involved remain largely unknown. To identify genes that are differentially expressed during early stages of planarian head regeneration, we generated a de novo transcriptome assembly from more than 300 million paired-end reads from planarian fragments regenerating the head at 16 different time points. The assembly yielded 26,018 putative transcripts, including very long transcripts spanning multiple genomic supercontigs, and thousands of isoforms. Using short-read data from two platforms, we analyzed dynamic gene regulation during the first three days of head regeneration. We identified at least five different temporal synexpression classes, including genes specifically induced within a few hours after injury. Furthermore, we characterized the role of a conserved Runx transcription factor, smed-runt-like1. RNA interference (RNAi) knockdown and immunofluorescence analysis of the regenerating visual system indicated that smed-runt-like1 encodes a transcriptional regulator of eye morphology and photoreceptor patterning. Transcriptome sequencing of short reads allowed for the simultaneous de novo assembly and differential expression analysis of transcripts, demonstrating highly dynamic regulation during head regeneration in planarians.

The head-regeneration transcriptome of the planarian Schmidtea mediterranea

PubMed Central

2011-01-01

Background Planarian flatworms can regenerate their head, including a functional brain, within less than a week. Despite the enormous potential of these animals for medical research and regenerative medicine, the mechanisms of regeneration and the molecules involved remain largely unknown. Results To identify genes that are differentially expressed during early stages of planarian head regeneration, we generated a de novo transcriptome assembly from more than 300 million paired-end reads from planarian fragments regenerating the head at 16 different time points. The assembly yielded 26,018 putative transcripts, including very long transcripts spanning multiple genomic supercontigs, and thousands of isoforms. Using short-read data from two platforms, we analyzed dynamic gene regulation during the first three days of head regeneration. We identified at least five different temporal synexpression classes, including genes specifically induced within a few hours after injury. Furthermore, we characterized the role of a conserved Runx transcription factor, smed-runt-like1. RNA interference (RNAi) knockdown and immunofluorescence analysis of the regenerating visual system indicated that smed-runt-like1 encodes a transcriptional regulator of eye morphology and photoreceptor patterning. Conclusions Transcriptome sequencing of short reads allowed for the simultaneous de novo assembly and differential expression analysis of transcripts, demonstrating highly dynamic regulation during head regeneration in planarians. PMID:21846378

Circadian Rhythms Regulate Amelogenesis

PubMed Central

Zheng, Li; Seon, Yoon Ji; Mourão, Marcio A.; Schnell, Santiago; Kim, Doohak; Harada, Hidemitsu; Papagerakis, Silvana; Papagerakis, Petros

2013-01-01

Ameloblasts, the cells responsible for making enamel, modify their morphological features in response to specialized functions necessary for synchronized ameloblast differentiation and enamel formation. Secretory and maturation ameloblasts are characterized by the expression of stage-specific genes which follows strictly controlled repetitive patterns. Circadian rhythms are recognized as key regulators of development and diseases of many tissues including bone. Our aim was to gain novel insights on the role of clock genes in enamel formation and to explore the potential links between circadian rhythms and amelogenesis. Our data shows definitive evidence that the main clock genes (Bmal1, Clock, Per1 and Per2) oscillate in ameloblasts at regular circadian (24h) intervals both at RNA and protein levels. This study also reveals that two markers of ameloblast differentiation i.e. amelogenin (Amelx; a marker of secretory ameloblasts) and kallikrein-related peptidase 4 (Klk4, a marker of maturation ameloblasts) are downstream targets of clock genes. Both, Amelx and Klk4 show 24h oscillatory expression patterns and their expression levels are up-regulated after Bmal1 over-expression in HAT-7 ameloblast cells. Taken together, these data suggest that both the secretory and the maturation stage of amelogenesis might be under circadian control. Changes in clock genes expression patterns might result in significant alterations of enamel apposition and mineralization. PMID:23486183

Different level of population differentiation among human genes.

PubMed

Wu, Dong-Dong; Zhang, Ya-Ping

2011-01-14

During the colonization of the world, after dispersal out of African, modern humans encountered changeable environments and substantial phenotypic variations that involve diverse behaviors, lifestyles and cultures, were generated among the different modern human populations. Here, we study the level of population differentiation among different populations of human genes. Intriguingly, genes involved in osteoblast development were identified as being enriched with higher FST SNPs, a result consistent with the proposed role of the skeletal system in accounting for variation among human populations. Genes involved in the development of hair follicles, where hair is produced, were also found to have higher levels of population differentiation, consistent with hair morphology being a distinctive trait among human populations. Other genes that showed higher levels of population differentiation include those involved in pigmentation, spermatid, nervous system and organ development, and some metabolic pathways, but few involved with the immune system. Disease-related genes demonstrate excessive SNPs with lower levels of population differentiation, probably due to purifying selection. Surprisingly, we find that Mendelian-disease genes appear to have a significant excessive of SNPs with high levels of population differentiation, possibly because the incidence and susceptibility of these diseases show differences among populations. As expected, microRNA regulated genes show lower levels of population differentiation due to purifying selection. Our analysis demonstrates different level of population differentiation among human populations for different gene groups.

Wrinkling pattern evolution of cylindrical biological tissues with differential growth.

PubMed

Jia, Fei; Li, Bo; Cao, Yan-Ping; Xie, Wei-Hua; Feng, Xi-Qiao

2015-01-01

Three-dimensional surface wrinkling of soft cylindrical tissues induced by differential growth is explored. Differential volumetric growth can cause their morphological stability, leading to the formation of hexagonal and labyrinth wrinkles. During postbuckling, multiple bifurcations and morphological transitions may occur as a consequence of continuous growth in the surface layer. The physical mechanisms underpinning the morphological evolution are examined from the viewpoint of energy. Surface curvature is found to play a regulatory role in the pattern evolution. This study may not only help understand the morphogenesis of soft biological tissues, but also inspire novel routes for creating desired surface patterns of soft materials.

Hls5 regulated erythroid differentiation by modulating GATA-1 activity.

PubMed

Endersby, Raelene; Majewski, Ian J; Winteringham, Louise; Beaumont, Jennifer G; Samuels, Amy; Scaife, Robin; Lim, Esther; Crossley, Merlin; Klinken, S Peter; Lalonde, Jean-Philippe

2008-02-15

Hemopoietic lineage switch (Hls) 5 and 7 were originally isolated as genes up-regulated during an erythroid-to-myeloid lineage switch. We have shown previously that Hls7/Mlf1 imposes a monoblastoid phenotype on erythroleukemic cells. Here we show that Hls5 impedes erythroid maturation by restricting proliferation and inhibiting hemoglobin synthesis; however, Hls5 does not influence the morphology of erythroid cells. Under the influence of GATA-1, Hls5 relocates from cytoplasmic granules to the nucleus where it associates with both FOG-1 and GATA-1. In the nucleus, Hls5 is able to suppress GATA-1-mediated transactivation and reduce GATA-1 binding to DNA. We conclude that Hls5 and Hls7/Mlf1 act cooperatively to induce biochemical and phenotypic changes associated with erythroid/myeloid lineage switching.

Vesicular glutamate transporters play a role in neuronal differentiation of cultured SVZ-derived neural precursor cells

PubMed Central

Sánchez-Mendoza, Eduardo H.; Bellver-Landete, Victor; Arce, Carmen; Doeppner, Thorsten R.; Hermann, Dirk M.

2017-01-01

The role of glutamate in the regulation of neurogenesis is well-established, but the role of vesicular glutamate transporters (VGLUTs) and excitatory amino acid transporters (EAATs) in controlling adult neurogenesis is unknown. Here we investigated the implication of VGLUTs in the differentiation of subventricular zone (SVZ)-derived neural precursor cells (NPCs). Our results show that NPCs express VGLUT1-3 and EAAT1-3 both at the mRNA and protein level. Their expression increases during differentiation closely associated with the expression of marker genes. In expression analyses we show that VGLUT1 and VGLUT2 are preferentially expressed by cultured SVZ-derived doublecortin+ neuroblasts, while VGLUT3 is found on GFAP+ glial cells. In cultured NPCs, inhibition of VGLUT by Evans Blue increased the mRNA level of neuronal markers doublecortin, B3T and MAP2, elevated the number of NPCs expressing doublecortin protein and promoted the number of cells with morphological appearance of branched neurons, suggesting that VGLUT function prevents neuronal differentiation of NPCs. This survival- and differentiation-promoting effect of Evans blue was corroborated by increased AKT phosphorylation and reduced MAPK phosphorylation. Thus, under physiological conditions, VGLUT1-3 inhibition, and thus decreased glutamate exocytosis, may promote neuronal differentiation of NPCs. PMID:28493916

Involvement of CRF2 signaling in enterocyte differentiation

PubMed Central

Ducarouge, Benjamin; Pelissier-Rota, Marjolaine; Powell, Rebecca; Buisson, Alain; Bonaz, Bruno; Jacquier-Sarlin, Muriel

2017-01-01

AIM To determine the role of corticotropin releasing factor receptor (CRF2) in epithelial permeability and enterocyte cell differentiation. METHODS For this purpose, we used rat Sprague Dawley and various colon carcinoma cell lines (SW620, HCT8R, HT-29 and Caco-2 cell lines). Expression of CRF2 protein was analyzed by fluorescent immunolabeling in normal rat colon and then by western blot in dissociated colonic epithelial cells and in the lysates of colon carcinoma cell lines or during the early differentiation of HT-29 cells (ten first days). To assess the impact of CRF2 signaling on colonic cell differentiation, HT-29 and Caco-2 cells were exposed to Urocortin 3 recombinant proteins (Ucn3, 100 nmol/L). In some experiments, cells were pre-exposed to the astressin 2b (A2b) a CRF2 antagonist in order to inhibit the action of Ucn3. Intestinal cell differentiation was first analyzed by functional assays: the trans-cellular permeability and the para-cellular permeability were determined by Dextran-FITC intake and measure of the transepithelial electrical resistance respectively. Morphological modifications associated to epithelial dysfunction were analyzed by confocal microscopy after fluorescent labeling of actin (phaloidin-TRITC) and intercellular adhesion proteins such as E-cadherin, p120ctn, occludin and ZO-1. The establishment of mature adherens junctions (AJ) was monitored by following the distribution of AJ proteins in lipid raft fractions, after separation of cell lysates on sucrose gradients. Finally, the mRNA and the protein expression levels of characteristic markers of intestinal epithelial cell (IEC) differentiation such as the transcriptional factor krüppel-like factor 4 (KLF4) or the dipeptidyl peptidase IV (DPPIV) were performed by RT-PCR and western blot respectively. The specific activities of DPPIV and alkaline phosphatase (AP) enzymes were determined by a colorimetric method. RESULTS CRF2 protein is preferentially expressed in undifferentiated epithelial cells from the crypts of colon and in human colon carcinoma cell lines. Furthermore, CRF2 expression is down regulated according to the kinetic of HT-29 cell differentiation. By performing functional assays, we found that Ucn3-induced CRF2 signaling alters both para- and trans-cellular permeability of differentiated HT-29 and Caco-2 cells. These effects are partly mediated by Ucn3-induced morphological changes associated with the disruption of mature AJ in HT-29 cells and tight junctions (TJ) in Caco-2 cells. Ucn3-mediated activation of CRF2 decreases mRNA and protein expression levels of KLF4 a transcription factor involved in IEC differentiation. This signaling is correlated to a down-regulation of key IEC markers such as DPPIV and AP, at both transcriptional and post-transcriptional levels. CONCLUSION Our findings suggest that CRF2 signaling could modulate IEC differentiation. These mechanisms could be relevant to the stress induced epithelial alterations found in inflammatory bowel diseases. PMID:28811708

Involvement of CRF2 signaling in enterocyte differentiation.

PubMed

Ducarouge, Benjamin; Pelissier-Rota, Marjolaine; Powell, Rebecca; Buisson, Alain; Bonaz, Bruno; Jacquier-Sarlin, Muriel

2017-07-28

To determine the role of corticotropin releasing factor receptor (CRF2) in epithelial permeability and enterocyte cell differentiation. For this purpose, we used rat Sprague Dawley and various colon carcinoma cell lines (SW620, HCT8R, HT-29 and Caco-2 cell lines). Expression of CRF2 protein was analyzed by fluorescent immunolabeling in normal rat colon and then by western blot in dissociated colonic epithelial cells and in the lysates of colon carcinoma cell lines or during the early differentiation of HT-29 cells (ten first days). To assess the impact of CRF2 signaling on colonic cell differentiation, HT-29 and Caco-2 cells were exposed to Urocortin 3 recombinant proteins (Ucn3, 100 nmol/L). In some experiments, cells were pre-exposed to the astressin 2b (A2b) a CRF2 antagonist in order to inhibit the action of Ucn3. Intestinal cell differentiation was first analyzed by functional assays: the trans-cellular permeability and the para-cellular permeability were determined by Dextran-FITC intake and measure of the transepithelial electrical resistance respectively. Morphological modifications associated to epithelial dysfunction were analyzed by confocal microscopy after fluorescent labeling of actin (phaloidin-TRITC) and intercellular adhesion proteins such as E-cadherin, p120ctn, occludin and ZO-1. The establishment of mature adherens junctions (AJ) was monitored by following the distribution of AJ proteins in lipid raft fractions, after separation of cell lysates on sucrose gradients. Finally, the mRNA and the protein expression levels of characteristic markers of intestinal epithelial cell (IEC) differentiation such as the transcriptional factor krüppel-like factor 4 (KLF4) or the dipeptidyl peptidase IV (DPPIV) were performed by RT-PCR and western blot respectively. The specific activities of DPPIV and alkaline phosphatase (AP) enzymes were determined by a colorimetric method. CRF2 protein is preferentially expressed in undifferentiated epithelial cells from the crypts of colon and in human colon carcinoma cell lines. Furthermore, CRF2 expression is down regulated according to the kinetic of HT-29 cell differentiation. By performing functional assays, we found that Ucn3-induced CRF2 signaling alters both para- and trans-cellular permeability of differentiated HT-29 and Caco-2 cells. These effects are partly mediated by Ucn3-induced morphological changes associated with the disruption of mature AJ in HT-29 cells and tight junctions (TJ) in Caco-2 cells. Ucn3-mediated activation of CRF2 decreases mRNA and protein expression levels of KLF4 a transcription factor involved in IEC differentiation. This signaling is correlated to a down-regulation of key IEC markers such as DPPIV and AP, at both transcriptional and post-transcriptional levels. Our findings suggest that CRF2 signaling could modulate IEC differentiation. These mechanisms could be relevant to the stress induced epithelial alterations found in inflammatory bowel diseases.

Gene expression networks underlying ovarian development in wild largemouth bass (Micropterus salmoides).

PubMed

Martyniuk, Christopher J; Prucha, Melinda S; Doperalski, Nicholas J; Antczak, Philipp; Kroll, Kevin J; Falciani, Francesco; Barber, David S; Denslow, Nancy D

2013-01-01

Oocyte maturation in fish involves numerous cell signaling cascades that are activated or inhibited during specific stages of oocyte development. The objectives of this study were to characterize molecular pathways and temporal gene expression patterns throughout a complete breeding cycle in wild female largemouth bass to improve understanding of the molecular sequence of events underlying oocyte maturation. Transcriptomic analysis was performed on eight morphologically diverse stages of the ovary, including primary and secondary stages of oocyte growth, ovulation, and atresia. Ovary histology, plasma vitellogenin, 17β-estradiol, and testosterone were also measured to correlate with gene networks. Global expression patterns revealed dramatic differences across ovarian development, with 552 and 2070 genes being differentially expressed during both ovulation and atresia respectively. Gene set enrichment analysis (GSEA) revealed that early primary stages of oocyte growth involved increases in expression of genes involved in pathways of B-cell and T-cell receptor-mediated signaling cascades and fibronectin regulation. These pathways as well as pathways that included adrenergic receptor signaling, sphingolipid metabolism and natural killer cell activation were down-regulated at ovulation. At atresia, down-regulated pathways included gap junction and actin cytoskeleton regulation, gonadotrope and mast cell activation, and vasopressin receptor signaling and up-regulated pathways included oxidative phosphorylation and reactive oxygen species metabolism. Expression targets for luteinizing hormone signaling were low during vitellogenesis but increased 150% at ovulation. Other networks found to play a significant role in oocyte maturation included those with genes regulated by members of the TGF-beta superfamily (activins, inhibins, bone morphogenic protein 7 and growth differentiation factor 9), neuregulin 1, retinoid X receptor, and nerve growth factor family. This study offers novel insight into the gene networks underlying vitellogenesis, ovulation and atresia and generates new hypotheses about the cellular pathways regulating oocyte maturation.

Separation of integrin-dependent adhesion from morphological changes based on differential PLC specificities.

PubMed

Wooten, D K; Teague, T K; McIntyre, B W

1999-01-01

In normal lymphocytes an inside-out signal up-regulating integrin adhesion is followed by a ligand-mediated outside-in cell spreading signal. Protein kinase C (PKC) inhibition blocks lymphocyte adherence to and spreading on fibronectin. In contrast, putative PLC inhibitors yield distinct differences with respect to adhesion and morphology. The phosphatidylinositol-specific phospholipase C (PLC) inhibitor neomycin blocked spreading of CD3/CD28-activated T cells on fibronectin by disrupting adhesion. Furthermore, when an additional inside-out signal for fibronectin adhesion is unnecessary such as with HPB-ALL T leukemic or phorbol-myristate-acetate-treated normal T cells, neomycin treatment does not alter adhesion or morphology. However, the phosphatidylcholine-specific PLC inhibitor D609 abrogates cell spreading without affecting adhesion to fibronectin in these cells as well as the CD3/CD28-activated T cells. These results strongly suggest that inside-out signaling for the integrin alpha4beta1 in lymphocytes proceeds through phosphatidylinositol-specific PLC and PKC, whereas the outside-in signal utilizes phosphatidylcholine-specific PLC and PKC.

Photomorphogenic responses to ultraviolet-B light.

PubMed

Jenkins, Gareth I

2017-11-01

Exposure to ultraviolet B (UV-B) light regulates numerous aspects of plant metabolism, morphology and physiology through the differential expression of hundreds of genes. Photomorphogenic responses to UV-B are mediated by the photoreceptor UV RESISTANCE LOCUS8 (UVR8). Considerable progress has been made in understanding UVR8 action: the structural basis of photoreceptor function, how interaction with CONSTITUTIVELY PHOTOMORPHOGENIC 1 initiates signaling and how REPRESSOR OF UV-B PHOTOMORPHOGENESIS proteins negatively regulate UVR8 action. In addition, recent research shows that UVR8 mediates several responses through interaction with other signaling pathways, in particular auxin signaling. Nevertheless, many aspects of UVR8 action remain poorly understood. Most research to date has been undertaken with Arabidopsis, and it is important to explore the functions and regulation of UVR8 in diverse plant species. Furthermore, it is essential to understand how UVR8, and UV-B signaling in general, regulates processes under natural growth conditions. Ultraviolet B regulates the expression of many genes through UVR8-independent pathways, but the activity and importance of these pathways in plants growing in sunlight are poorly understood. © 2017 John Wiley & Sons Ltd.

Recrudescence Mechanisms and Gene Expression Profile of the Reproductive Tracts from Chickens during the Molting Period

PubMed Central

Ahn, Suzie E.; Lim, Chul-Hong; Lee, Jin-Young; Bae, Seung-Min; Kim, Jinyoung; Bazer, Fuller W.; Song, Gwonhwa

2013-01-01

The reproductive system of chickens undergoes dynamic morphological and functional tissue remodeling during the molting period. The present study identified global gene expression profiles following oviductal tissue regression and regeneration in laying hens in which molting was induced by feeding high levels of zinc in the diet. During the molting and recrudescence processes, progressive morphological and physiological changes included regression and re-growth of reproductive organs and fluctuations in concentrations of testosterone, progesterone, estradiol and corticosterone in blood. The cDNA microarray analysis of oviductal tissues revealed the biological significance of gene expression-based modulation in oviductal tissue during its remodeling. Based on the gene expression profiles, expression patterns of selected genes such as, TF, ANGPTL3, p20K, PTN, AvBD11 and SERPINB3 exhibited similar patterns in expression with gradual decreases during regression of the oviduct and sequential increases during resurrection of the functional oviduct. Also, miR-1689* inhibited expression of Sp1, while miR-17-3p, miR-22* and miR-1764 inhibited expression of STAT1. Similarly, chicken miR-1562 and miR-138 reduced the expression of ANGPTL3 and p20K, respectively. These results suggest that these differentially regulated genes are closely correlated with the molecular mechanism(s) for development and tissue remodeling of the avian female reproductive tract, and that miRNA-mediated regulation of key genes likely contributes to remodeling of the avian reproductive tract by controlling expression of those genes post-transcriptionally. The discovered global gene profiles provide new molecular candidates responsible for regulating morphological and functional recrudescence of the avian reproductive tract, and provide novel insights into understanding the remodeling process at the genomic and epigenomic levels. PMID:24098561

The cytoskeletal arrangements necessary to neurogenesis

PubMed Central

Compagnucci, Claudia; Piemonte, Fiorella; Sferra, Antonella; Piermarini, Emanuela; Bertini, Enrico

2016-01-01

During the process of neurogenesis, the stem cell committed to the neuronal cell fate starts a series of molecular and morphological changes. The understanding of the physio-pathology of mechanisms controlling the molecular and morphological changes occurring during neuronal differentiation is fundamental to the development of effective therapies for many neurologic diseases. Unfortunately, our knowledge of the biological events occurring in the cell during neuronal differentiation is still poor. In this study, we focus preliminarily on the relevance of the cytoskeletal rearrangements, which earlier drive the morphology of the neuronal precursors, and later the migrating/mature neurons. In fact, neuritogenesis, neurite branching, outgrowth and retraction are seminal to the development of a fully functional nervous system. With this in mind, we highlight the importance of iPSC technology to study the processes of cytoskeletal-driven morphological changes during neuronal differentiation. PMID:26760504

Morphological Analysis of Live Undifferentiated Cells Derived from Induced Pluripotent Stem Cells.

PubMed

Osawa, Yukihiko; Miyamoto, Tomoyuki; Ohno, Setsuyo; Ohno, Eiji

2018-01-01

Induced pluripotent stem (iPS) cells possess pluripotency and self-renewal ability. Therefore, iPS cells are expected to be useful in regenerative medicine. However, iPS cells form malignant immature teratomas after transplantation into animals, even after differentiation induction. It has been suggested that undifferentiated cells expressing Nanog that remain after differentiation induction are responsible for teratoma formation. Various methods of removing these undifferentiated cells have therefore been investigated, but few methods involve morphological approaches, which may induce less cell damage. In addition, for cells derived from iPS cells to be applied in regenerative medicine, they must be alive. However, detailed morphological analysis of live undifferentiated cells has not been performed. For the above reasons, we assessed the morphological features of live undifferentiated cells remaining after differentiation induction as a basic investigation into the clinical application of iPS cells. As a result, live undifferentiated cells remaining after differentiation induction exhibited a round or oval cytoplasm about 12 μm in diameter and a nucleus. They exhibited nucleo-cytoplasmic (N/C) ratio of about 60% and eccentric nuclei, and they possessed partially granule-like structures in the cytoplasm and prominent nucleoli. Although they were similar to iPS cells, they were smaller than live iPS cells. Furthermore, very small cells were present among undifferentiated cells after differentiation induction. These results suggest that the removal of undifferentiated cells may be possible using the morphological features of live iPS cells and undifferentiated cells after differentiation induction. In addition, this study supports safe regenerative medicine using iPS cells.

Three regulators of G protein signaling differentially affect mating, morphology and virulence in the smut fungus Ustilago maydis.

PubMed

Moretti, Marino; Wang, Lei; Grognet, Pierre; Lanver, Daniel; Link, Hannes; Kahmann, Regine

2017-09-01

Regulators of G protein signaling (RGS) proteins modulate heterotrimeric G protein signaling negatively. To broaden an understanding of the roles of RGS proteins in fungal pathogens, we functionally characterized the three RGS protein-encoding genes (rgs1, rgs2 and rgs3) in the phytopathogenic fungus Ustilago maydis. It was found that RGS proteins played distinct roles in the regulation of development and virulence. rgs1 had a minor role in virulence when deleted in a solopathogenic strain. In crosses, rgs1 was dispensable for mating and filamentation, but was required for teliospore production. Haploid rgs2 mutants were affected in cell morphology, growth, mating and were unable to cause disease symptoms in crosses. However, virulence was unaffected when rgs2 was deleted in a solopathogenic strain, suggesting an exclusive involvement in pre-fusion events. These rgs2 phenotypes are likely connected to elevated intracellular cAMP levels. rgs3 mutants were severely attenuated in mating, in their response to pheromone, virulence and formation of mature teliospores. The mating defect could be traced back to reduced expression of the transcription factor rop1. It was speculated that the distinct roles of the three U. maydis RGS proteins were achieved by direct modulation of the Gα subunit-activated signaling pathways as well as through Gα-independent functions. © 2017 John Wiley & Sons Ltd.

Artificial Symmetry-Breaking for Morphogenetic Engineering Bacterial Colonies.

PubMed

Nuñez, Isaac N; Matute, Tamara F; Del Valle, Ilenne D; Kan, Anton; Choksi, Atri; Endy, Drew; Haseloff, Jim; Rudge, Timothy J; Federici, Fernan

2017-02-17

Morphogenetic engineering is an emerging field that explores the design and implementation of self-organized patterns, morphologies, and architectures in systems composed of multiple agents such as cells and swarm robots. Synthetic biology, on the other hand, aims to develop tools and formalisms that increase reproducibility, tractability, and efficiency in the engineering of biological systems. We seek to apply synthetic biology approaches to the engineering of morphologies in multicellular systems. Here, we describe the engineering of two mechanisms, symmetry-breaking and domain-specific cell regulation, as elementary functions for the prototyping of morphogenetic instructions in bacterial colonies. The former represents an artificial patterning mechanism based on plasmid segregation while the latter plays the role of artificial cell differentiation by spatial colocalization of ubiquitous and segregated components. This separation of patterning from actuation facilitates the design-build-test-improve engineering cycle. We created computational modules for CellModeller representing these basic functions and used it to guide the design process and explore the design space in silico. We applied these tools to encode spatially structured functions such as metabolic complementation, RNAPT7 gene expression, and CRISPRi/Cas9 regulation. Finally, as a proof of concept, we used CRISPRi/Cas technology to regulate cell growth by controlling methionine synthesis. These mechanisms start from single cells enabling the study of morphogenetic principles and the engineering of novel population scale structures from the bottom up.

Differentiation of lepidoptera scale cells from epidermal stem cells followed by ecdysone-regulated DNA duplication and scale secreting.

PubMed

Yuan, Shenglei; Huang, Wuren; Geng, Lei; Beerntsen, Brenda T; Song, Hongsheng; Ling, Erjun

2017-01-01

Integuments are the first line to protect insects from physical damage and pathogenic infection. In lepidopteran insects, they undergo distinct morphology changes such as scale formation during metamorphosis. However, we know little about integument development and scale formation during this stage. Here, we use the silkworm, Bombyx mori, as a model and show that stem cells in the integument of each segment, but not intersegmental membrane, divide into two scale precursor cells during the spinning stage. In young pupae, the scale precursor cell divides again. One of the daughter cells becomes a mature scale-secreting cell that undergoes several rounds of DNA duplication and the other daughter cell undergoes apoptosis later on. This scale precursor cell division is crucial to the development and differentiation of scale-secreting cells because scale production can be blocked after treatment with the cell division inhibitor paclitaxel. Subsequently, the growth of scale-secreting cells is under the control of 20-hydroxyecdysone but not juvenile hormone since injection of 20-hydroxyecdysone inhibited scale formation. Further work demonstrated that 20-hydroxyecdysone injection inhibits DNA duplication in scale-secreting cells while the expression of scale-forming gene ASH1 was down-regulated by BR-C Z2. Therefore, this research demonstrates that the scale cells of the silkworm develops through stem cell division prior to pupation and then another wave of cell division differentiates these cells into scale secreting cells soon after entrance into the pupal stage. Additionally, DNA duplication and scale production in the scale-secreting cells were found to be under the regulation of 20-hydroxyecdysone.

Endoderm development in Caenorhabditis elegans: the synergistic action of ELT-2 and -7 mediates the specification→differentiation transition.

PubMed

Sommermann, Erica M; Strohmaier, Keith R; Maduro, Morris F; Rothman, Joel H

2010-11-01

The transition from specification of cell identity to the differentiation of cells into an appropriate and enduring state is critical to the development of embryos. Transcriptional profiling in Caenorhabditis elegans has revealed a large number of genes that are expressed in the fully differentiated intestine; however, no regulatory factor has been found to be essential to initiate their expression once the endoderm has been specified. These gut-expressed genes possess a preponderance of GATA factor binding sites and one GATA factor, ELT-2, fulfills the expected characteristics of a key regulator of these genes based on its persistent expression exclusively in the developing and differentiated intestine and its ability to bind these regulatory sites. However, a striking characteristic of elt-2(0) knockout mutants is that while they die shortly after hatching owing to an obstructed gut passage, they nevertheless contain a gut that has undergone complete morphological differentiation. We have discovered a second gut-specific GATA factor, ELT-7, that profoundly synergizes with ELT-2 to create a transcriptional switch essential for gut cell differentiation. ELT-7 is first expressed in the early endoderm lineage and, when expressed ectopically, is sufficient to activate gut differentiation in nonendodermal progenitors. elt-7 is transcriptionally activated by the redundant endoderm-specifying factors END-1 and -3, and its product in turn activates both its own expression and that of elt-2, constituting an apparent positive feedback system. While elt-7 loss-of-function mutants lack a discernible phenotype, simultaneous loss of both elt-7 and elt-2 results in a striking all-or-none block to morphological differentiation of groups of gut cells with a region-specific bias, as well as reduced or abolished gut-specific expression of a number of terminal differentiation genes. ELT-2 and -7 synergize not only in activation of gene expression but also in repression of a gene that is normally expressed in the valve cells, which immediately flank the termini of the gut tube. Our results point to a developmental strategy whereby positive feedback and cross-regulatory interactions between two synergistically acting regulatory factors promote a decisive and persistent transition of specified endoderm progenitors into the program of intestinal differentiation. Copyright © 2010 Elsevier Inc. All rights reserved.

Differentiation of umbilical cord derived mesenchymal stem cells to hepatocyte cells by transfection of miR-106a, miR-574-3p, and miR-451.

PubMed

Khosravi, Maryam; Azarpira, Negar; Shamdani, Sara; Hojjat-Assari, Suzzan; Naserian, Sina; Karimi, Mohammad Hossein

2018-08-15

Studying the profile of micro RNAs (miRs) elucidated the highest expressed miRs in hepatic differentiation. In this study, we investigated to clarify the role of three embryonic overexpressed miRs (miR-106a, miR-574-3p and miR-451) during hepatic differentiation of human umbilical cord derived mesenchymal stem cells (UC-MSCs). We furthermore, aimed to explore whether overexpression of any of these miRs alone is sufficient to induce the differentiation of the UC-MSCs into hepatocyte-like cells. UC-MSCs were transfected either alone or together with miR-106a, miR-574-3p and miR-451 and their potential hepatic differentiation and alteration in gene expression profile, morphological changes and albumin secretion ability were investigated. We found that up-regulation of any of these three miRs alone cannot induce expression of all hepatic specific genes. Transfection of each miR alone, led to Sox17, FoxA2 expression that are related to initiation step of hepatic differentiation. However, concurrent ectopic overexpression of three miRs together can induce UC-MSCs differentiation into functionally mature hepatocytes. These results show that miRs have the capability to directly convert UC-MSCs to a hepatocyte phenotype in vitro. Copyright © 2018. Published by Elsevier B.V.

Fetal bovine serum enables cardiac differentiation of human embryonic stem cells.

PubMed

Bettiol, Esther; Sartiani, Laura; Chicha, Laurie; Krause, Karl Heinz; Cerbai, Elisabetta; Jaconi, Marisa E

2007-10-01

During development, cardiac commitment within the mesoderm requires endoderm-secreted factors. Differentiation of embryonic stem cells into the three germ layers in vitro recapitulates developmental processes and can be influenced by supplements added to culture medium. Hence, we investigated the effect of fetal bovine serum (FBS) and KnockOut serum replacement (SR) on germ layers specification and cardiac differentiation of H1 human embryonic stem cells (hESC) within embryoid bodies (EB). At the time of EB formation, FBS triggered an increased apoptosis. As assessed by quantitative PCR on 4-, 10-, and 20-day-old EB, FBS promoted a faster down-regulation of pluripotency marker Oct4 and an increased expression of endodermal (Sox17, alpha-fetoprotein, AFP) and mesodermal genes (Brachyury, CSX). While neuronal and hematopoietic differentiation occurred in both supplements, spontaneously beating cardiomyocytes were only observed in FBS. Action potential (AP) morphology of hESC-derived cardiomyocytes indicated that ventricular cells were present only after 2 months of culture. However, quantification of myosin light chain 2 ventricular (mlc2v)-positive areas revealed that mlc2v-expressing cardiomyocytes could be detected already after 2 weeks of differentiation, but not in all beating clusters. In conclusion, FBS enabled cardiac differentiation of hESC, likely in an endodermal-dependent pathway. Among cardiac cells, ventricular cardiomyocytes differentiated over time, but not as the predominant cardiac cell subtype.

Double-bromo and extraterminal (BET) domain proteins regulate dendrite morphology and mechanosensory function

PubMed Central

Bagley, Joshua A.; Yan, Zhiqiang; Zhang, Wei; Wildonger, Jill

2014-01-01

A complex array of genetic factors regulates neuronal dendrite morphology. Epigenetic regulation of gene expression represents a plausible mechanism to control pathways responsible for specific dendritic arbor shapes. By studying the Drosophila dendritic arborization (da) neurons, we discovered a role of the double-bromodomain and extraterminal (BET) family proteins in regulating dendrite arbor complexity. A loss-of-function mutation in the single Drosophila BET protein encoded by female sterile 1 homeotic [fs(1)h] causes loss of fine, terminal dendritic branches. Moreover, fs(1)h is necessary for the induction of branching caused by a previously identified transcription factor, Cut (Ct), which regulates subtype-specific dendrite morphology. Finally, disrupting fs(1)h function impairs the mechanosensory response of class III da sensory neurons without compromising the expression of the ion channel NompC, which mediates the mechanosensitive response. Thus, our results identify a novel role for BET family proteins in regulating dendrite morphology and a possible separation of developmental pathways specifying neural cell morphology and ion channel expression. Since the BET proteins are known to bind acetylated histone tails, these results also suggest a role of epigenetic histone modifications and the “histone code,” in regulating dendrite morphology. PMID:25184680

The Arabidopsis minE mutation causes new plastid and FtsZ1 localization phenotypes in the leaf epidermis

PubMed Central

Fujiwara, Makoto T.; Kojo, Kei H.; Kazama, Yusuke; Sasaki, Shun; Abe, Tomoko; Itoh, Ryuuichi D.

2015-01-01

Plastids in the leaf epidermal cells of plants are regarded as immature chloroplasts that, like mesophyll chloroplasts, undergo binary fission. While mesophyll chloroplasts have generally been used to study plastid division, recent studies have suggested the presence of tissue- or plastid type-dependent regulation of plastid division. Here, we report the detailed morphology of plastids and their stromules, and the intraplastidic localization of the chloroplast division-related protein AtFtsZ1-1, in the leaf epidermis of an Arabidopsis mutant that harbors a mutation in the chloroplast division site determinant gene AtMinE1. In atminE1, the size and shape of epidermal plastids varied widely, which contrasts with the plastid phenotype observed in atminE1 mesophyll cells. In particular, atminE1 epidermal plastids occasionally displayed grape-like morphology, a novel phenotype induced by a plastid division mutation. Observation of an atminE1 transgenic line harboring an AtMinE1 promoter::AtMinE1-yellow fluorescent protein fusion gene confirmed the expression and plastidic localization of AtMinE1 in the leaf epidermis. Further examination revealed that constriction of plastids and stromules mediated by the FtsZ1 ring contributed to the plastid pleomorphism in the atminE1 epidermis. These results illustrate that a single plastid division mutation can have dramatic consequences for epidermal plastid morphology, thereby implying that plastid division and morphogenesis are differentially regulated in epidermal and mesophyll plastids. PMID:26500667

Morphological features of different polyploids for adaptation and molecular characterization of CC-NBS-LRR and LEA gene families in Agave L.

PubMed

Tamayo-Ordóñez, M C; Rodriguez-Zapata, L C; Narváez-Zapata, J A; Tamayo-Ordóñez, Y J; Ayil-Gutiérrez, B A; Barredo-Pool, F; Sánchez-Teyer, L F

2016-05-20

Polyploidy has been widely described in many Agave L. species, but its influence on environmental response to stress is still unknown. With the objective of knowing the morphological adaptations and regulation responses of genes related to biotic (LEA) and abiotic (NBS-LRR) stress in species of Agave with different levels of ploidy, and how these factors contribute to major response of Agave against environmental stresses, we analyzed 16 morphological trials on five accessions of three species (Agave tequilana Weber, Agave angustifolia Haw. and Agave fourcroydes Lem.) with different ploidy levels (2n=2x=60 2n=3x=90, 2n=5x=150, 2n=6x=180) and evaluated the expression of NBS-LRR and LEA genes regulated by biotic and abiotic stress. It was possible to associate some morphological traits (spines, nuclei, and stomata) to ploidy level. The genetic characterization of stress-related genes NBS-LRR induced by pathogenic infection and LEA by heat or saline stresses indicated that amino acid sequence analysis in these genes showed more substitutions in higher ploidy level accessions of A. fourcroydes Lem. 'Sac Ki' (2n=5x=150) and A. angustifolia Haw. 'Chelem Ki' (2n=6x=180), and a higher LEA and NBS-LRR representativeness when compared to their diploid and triploid counterparts. In all studied Agave accessions expression of LEA and NBS-LRR genes was induced by saline or heat stresses or by infection with Erwinia carotovora, respectively. The transcriptional activation was also higher in A. angustifolia Haw. 'Chelem Ki' (2n=6x=180) and A. fourcroydes 'Sac Ki' (2n=5x=150) than in their diploid and triploid counterparts, which suggests higher adaptation to stress. Finally, the diploid accession A. tequilana Weber 'Azul' showed a differentiated genetic profile relative to other Agave accessions. The differences include similar or higher genetic representativeness and transcript accumulation of LEA and NBS-LRR genes than in polyploid (2n=5x=150 and 2n=6x=180) Agave accessions, thus suggesting a differentiated selection pressure for overcoming the lower ploidy level of the diploid A. tequilana Weber 'Azul'. Copyright © 2016 Elsevier GmbH. All rights reserved.

Glycoconjugates reveal diversity of human neural stem cells (hNSCs) derived from human induced pluripotent stem cells (hiPSCs).

PubMed

Kandasamy, Majury; Roll, Lars; Langenstroth, Daniel; Brüstle, Oliver; Faissner, Andreas

2017-06-01

Neural stem cells (NSCs) have the ability to self-renew and to differentiate into various cell types of the central nervous system. This potential can be recapitulated by human induced pluripotent stem cells (hiPSCs) in vitro. The differentiation capacity of hiPSCs is characterized by several stages with distinct morphologies and the expression of various marker molecules. We used the monoclonal antibodies (mAbs) 487 LeX , 5750 LeX and 473HD to analyze the expression pattern of particular carbohydrate motifs as potential markers at six differentiation stages of hiPSCs. Mouse ESCs were used as a comparison. At the pluripotent stage, 487 LeX -, 5750 LeX - and 473HD-related glycans were differently expressed. Later, cells of the three germ layers in embryoid bodies (hEBs) and, even after neuralization of hEBs, subpopulations of cells were labeled with these surface antibodies. At the human rosette-stage of NSCs (hR-NSC), LeX- and 473HD-related epitopes showed antibody-specific expression patterns. We also found evidence that these surface antibodies could be used to distinguish the hR-NSCs from the hSR-NSCs stages. Characterization of hNSCs FGF-2/EGF derived from hSR-NSCs revealed that both LeX antibodies and the 473HD antibody labeled subpopulations of hNSCs FGF-2/EGF . Finally, we identified potential LeX carrier molecules that were spatiotemporally regulated in early and late stages of differentiation. Our study provides new insights into the regulation of glycoconjugates during early human stem cell development. The mAbs 487 LeX , 5750 LeX and 473HD are promising tools for identifying distinct stages during neural differentiation.

A Multistate Toggle Switch Defines Fungal Cell Fates and Is Regulated by Synergistic Genetic Cues

PubMed Central

Anderson, Matthew Z.; Porman, Allison M.; Wang, Na; Mancera, Eugenio; Bennett, Richard J.

2016-01-01

Heritable epigenetic changes underlie the ability of cells to differentiate into distinct cell types. Here, we demonstrate that the fungal pathogen Candida tropicalis exhibits multipotency, undergoing stochastic and reversible switching between three cellular states. The three cell states exhibit unique cellular morphologies, growth rates, and global gene expression profiles. Genetic analysis identified six transcription factors that play key roles in regulating cell differentiation. In particular, we show that forced expression of Wor1 or Efg1 transcription factors can be used to manipulate transitions between all three cell states. A model for tristability is proposed in which Wor1 and Efg1 are self-activating but mutually antagonistic transcription factors, thereby forming a symmetrical self-activating toggle switch. We explicitly test this model and show that ectopic expression of WOR1 can induce white-to-hybrid-to-opaque switching, whereas ectopic expression of EFG1 drives switching in the opposite direction, from opaque-to-hybrid-to-white cell states. We also address the stability of induced cell states and demonstrate that stable differentiation events require ectopic gene expression in combination with chromatin-based cues. These studies therefore experimentally test a model of multistate stability and demonstrate that transcriptional circuits act synergistically with chromatin-based changes to drive cell state transitions. We also establish close mechanistic parallels between phenotypic switching in unicellular fungi and cell fate decisions during stem cell reprogramming. PMID:27711197

Morphological and molecular diagnostics of Phytoseiulus persimilis and Phytoseiulus macropilis (Acari: Phytoseiidae).

PubMed

Okassa, Mireille; Tixier, Marie-Stéphane; Kreiter, Serge

2010-11-01

This study focuses on the diagnostics of two natural enemy species, belonging to the genus Phytoseiulus in the family Phytoseiidae (sub-family Amblyseiinae): P. macropilis and P. persimilis. These two species are of primary importance in biological control all over the world. However, they are morphologically very similar and specific diagnostics is difficult. This study utilizes mitochondrial molecular markers (12S rRNA and Cytb mtDNA) to differentiate these two species. Morphological analyses showed significant differences between P. persimilis and P. macropilis for 17 morphological characters of the 32 considered. However, despite these significant differences, the ranges of all characters overlap. Only the serration of the macroseta on the basitarsus (StIV) allows the differentiation between P. persimilis and P. macropilis. Despite these small morphological differences, molecular results, for both mitochondrial DNA fragments considered (rRNA and Cytb mtDNA), showed a clear delineation between the specimens of P. macropilis and P. persimilis. This study emphasizes (i) that only one morphological character (serration of the seta StIV) clearly separates these two species, and (ii) the usefulness of an automatical molecular and simple diagnostic tool for accurate differentiation of the two species and ensure the morphological diagnostics. Further studies are proposed, including more DNA sequences especially for P. macropilis.

Influence of differentiation on muscarinic receptors in N1E 115 neuroblastoma cells.

PubMed

Buyse, M A; Lefebvre, R A; Fraeyman, N H

1989-01-01

The effect of inducing morphological differentiation in N1E 115 mouse neuroblastoma cells on the number of muscarinic receptors and the ligand binding affinity was investigated using the lipophylic quinuclidinyl benzylate and the hydrophylic N-methylscopolamine as tritiated ligands. Induction of morphological differentiation was accompanied by a two- to three-fold increase of the number of receptors when assayed in a broken cell preparation; the ligand binding affinity was unaffected by differentiation. Using intact cells, this increase was not paralleled by a similar increase in binding sites accessible for N-methylscopolamine, which binds preferentially to extracellular sites.

Intersection of transfer cells with phloem biology—broad evolutionary trends, function, and induction

PubMed Central

Andriunas, Felicity A.; Zhang, Hui-Ming; Xia, Xue; Patrick, John W.; Offler, Christina E.

2013-01-01

Transfer cells (TCs) are ubiquitous throughout the plant kingdom. Their unique ingrowth wall labyrinths, supporting a plasma membrane enriched in transporter proteins, provides these cells with an enhanced membrane transport capacity for resources. In certain plant species, TCs have been shown to function to facilitate phloem loading and/or unloading at cellular sites of intense resource exchange between symplasmic/apoplasmic compartments. Within the phloem, the key cellular locations of TCs are leaf minor veins of collection phloem and stem nodes of transport phloem. In these locations, companion and phloem parenchyma cells trans-differentiate to a TC morphology consistent with facilitating loading and re-distribution of resources, respectively. At a species level, occurrence of TCs is significantly higher in transport than in collection phloem. TCs are absent from release phloem, but occur within post-sieve element unloading pathways and particularly at interfaces between generations of developing Angiosperm seeds. Experimental accessibility of seed TCs has provided opportunities to investigate their inductive signaling, regulation of ingrowth wall formation and membrane transport function. This review uses this information base to explore current knowledge of phloem transport function and inductive signaling for phloem-associated TCs. The functional role of collection phloem and seed TCs is supported by definitive evidence, but no such information is available for stem node TCs that present an almost intractable experimental challenge. There is an emerging understanding of inductive signals and signaling pathways responsible for initiating trans-differentiation to a TC morphology in developing seeds. However, scant information is available to comment on a potential role for inductive signals (auxin, ethylene and reactive oxygen species) that induce seed TCs, in regulating induction of phloem-associated TCs. Biotic phloem invaders have been used as a model to speculate on involvement of these signals. PMID:23847631

Calcium-dependent depletion zones in the cortical microtubule array coincide with sites of, but do not regulate, wall ingrowth papillae deposition in epidermal transfer cells.

PubMed

Zhang, Hui-ming; Talbot, Mark J; McCurdy, David W; Patrick, John W; Offler, Christina E

2015-09-01

Trans-differentiation to a transfer-cell morphology is characterized by the localized deposition of wall ingrowth papillae that protrude into the cytosol. Whether the cortical microtubule array directs wall ingrowth papillae formation was investigated using a Vicia faba cotyledon culture system in which their adaxial epidermal cells were spontaneously induced to trans-differentiate to transfer cells. During deposition of wall ingrowth papillae, the aligned cortical microtubule arrays in precursor epidermal cells were reorganized into a randomized array characterized by circular depletion zones. Concurrence of the temporal appearance, spatial pattern, and size of depletion zones and wall ingrowth papillae was consistent with each papilla occupying a depletion zone. Surprisingly, microtubules appeared not to regulate construction of wall ingrowth papillae, as neither depolymerization nor stabilization of cortical microtubules changed their deposition pattern or morphology. Moreover, the size and spatial pattern of depletion zones was unaltered when the formation of wall ingrowth papillae was blocked by inhibiting cellulose biosynthesis. In contrast, the depletion zones were absent when the cytosolic calcium plumes, responsible for directing wall ingrowth papillae formation, were blocked or dissipated. Thus, we conclude that the depletion zones within the cortical microtubule array result from localized depolymerization of microtubules initiated by elevated cytosolic Ca(2+) levels at loci where wall ingrowth papillae are deposited. The physiological significance of the depletion zones as a mechanism to accommodate the construction of wall ingrowth papillae without compromising maintenance of the plasma membrane-microtubule inter-relationship is discussed. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Genetic link between Cabeza, a Drosophila homologue of Fused in Sarcoma (FUS), and the EGFR signaling pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shimamura, Mai; Kyotani, Akane; Insect Biomedical Research Center, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585

2014-08-01

Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease that causes progressive muscular weakness. Fused in Sarcoma (FUS) that has been identified in familial ALS is an RNA binding protein that is normally localized in the nucleus. However, its function in vivo is not fully understood. Drosophila has Cabeza (Caz) as a FUS homologue and specific knockdown of Caz in the eye imaginal disc and pupal retina using a GMR-GAL4 driver was here found to induce an abnormal morphology of the adult compound eyes, a rough eye phenotype. This was partially suppressed by expression of the apoptosis inhibitor P35. Knockdownmore » of Caz exerted no apparent effect on differentiation of photoreceptor cells. However, immunostaining with an antibody to Cut that marks cone cells revealed fusion of these and ommatidia of pupal retinae. These results indicate that Caz knockdown induces apoptosis and also inhibits differentiation of cone cells, resulting in abnormal eye morphology in adults. Mutation in EGFR pathway-related genes, such as rhomboid-1, rhomboid-3 and mirror suppressed the rough eye phenotype induced by Caz knockdown. Moreover, the rhomboid-1 mutation rescued the fusion of cone cells and ommatidia observed in Caz knockdown flies. The results suggest that Caz negatively regulates the EGFR signaling pathway required for determination of cone cell fate in Drosophila. - Highlights: • Knockdown of Cabeza induced rough eye phenotype. • Knockdown of Cabeza induced fusion of cone cells in pupal retinae. • Knockdown of Cabeza induced apoptosis in pupal retinae. • Mutation in EGFR pathway-related genes suppressed the rough eye phenotype. • Cabeza may negatively regulate the EGFR pathway.« less

Zebrafish no isthmus reveals a role for pax2.1 in tubule differentiation and patterning events in the pronephric primordia.

PubMed

Majumdar, A; Lun, K; Brand, M; Drummond, I A

2000-05-01

Pax genes are important developmental regulators and function at multiple stages of vertebrate kidney organogenesis. In this report, we have used the zebrafish pax2.1 mutant no isthmus to investigate the role for pax2.1 in development of the pronephros. We demonstrate a requirement for pax2.1 in multiple aspects of pronephric development including tubule and duct epithelial differentiation and cloaca morphogenesis. Morphological analysis demonstrates that noi(- )larvae specifically lack pronephric tubules while glomerular cell differentiation is unaffected. In addition, pax2.1 expression in the lateral cells of the pronephric primordium is required to restrict the domains of Wilms' tumor suppressor (wt1) and vascular endothelial growth factor (VEGF) gene expression to medial podocyte progenitors. Ectopic podocyte-specific marker expression in pronephric duct cells correlates with loss of expression of the pronephric tubule and duct-specific markers mAb 3G8 and a Na(+)/K(+) ATPase (&agr;)1 subunit. The results suggest that the failure in pronephric tubule differentiation in noi arises from a patterning defect during differentiation of the pronephric primordium and that mutually inhibitory regulatory interactions play an important role in defining the boundary between glomerular and tubule progenitors in the forming nephron.

Differentiation of Human Mesenchymal Stem Cells into Insulin Producing Cells by Using A Lentiviral Vector Carrying PDX1.

PubMed

Allahverdi, Amir; Abroun, Saied; Jafarian, Arefeh; Soleimani, Masoud; Taghikhani, Mohammad; Eskandari, Fatemeh

2015-01-01

Type I diabetes is an immunologically-mediated devastation of insulin producing cells (IPCs) in the pancreatic islet. Stem cells that produce β-cells are a new promising tool. Adult stem cells such as mesenchymal stem cells (MSCs) are self renewing multi potent cells showing capabilities to differentiate into ectodermal, mesodermal and endodermal tissues. Pancreatic and duodenal homeobox factor 1 (PDX1) is a master regulator gene required for embryonic development of the pancreas and is crucial for normal pancreatic islets activities in adults. We induced the over-expression of the PDX1 gene in human bone marrow MSCs (BM-MSCs) by Lenti-PDX1 in order to generate IPCs. Next, we examine the ability of the cells by measuring insulin/c-peptide production and INSULIN and PDX1 gene expressions. After transduction, MSCs changed their morphology at day 5 and gradually differentiated into IPCs. INSULIN and PDX1 expressions were confirmed by real time polymerase chain reaction (RT-PCR) and immunostaining. IPC secreted insulin and C-peptide in the media that contained different glucose concentrations. MSCs differentiated into IPCs by genetic manipulation. Our result showed that lentiviral vectors could deliver PDX1 gene to MSCs and induce pancreatic differentiation.

Acrylamide inhibits cellular differentiation of human neuroblastoma and glioblastoma cells.

PubMed

Chen, Jong-Hang; Chou, Chin-Cheng

2015-08-01

This study explores human neuroblastoma (SH-SY5Y) and human glioblastoma (U-1240 MG) cellular differentiation changes under exposure to acrylamide (ACR). Differentiation of SH-SY5Y and U-1240 MG cells were induced by retinoic acid (RA) and butyric acid (BA), respectively. Morphological observations and MTT assay showed that the induced cellular differentiation and cell proliferation were inhibited by ACR in a time- and dose-dependent manner. ACR co-treatment with RA attenuated SH-SY5Y expressions of neurofilament protein-L (NF-L), microtubule-associated protein 1b (MAP1b; 1.2 to 0.7, p < 0.001), MAP2c (2.2 to 0.8, p < 0.05), and Janus kinase1 (JAK1; 1.9 to 0.6, p < 0.001), while ACR co-treatment with BA attenuated U-1240 MG expressions of glial fibrillary acidic protein (GFAP), MAP1b (1.2 to 0.6, p < 0.001), MAP2c (1.5 to 0.7, p < 0.01), and JAK1 (2.1 to 0.5, p < 0.001), respectively. ACR also decreased the phosphorylation of extracellular-signal-regulated kinases (ERK) and c-Jun N-terminal kinases (JNK) in U-1240 MG cells, while caffeine reversed this suppression of ERK and JNK phosphorylation caused by ACR treatment. These results showed that RA-induced neurogenesis of SH-SY5Y and BA-induced astrogliogenesis of U-1240 MG cells were attenuated by ACR and were associated with down-regulation of MAPs expression and JAK-STAT signaling. Copyright © 2015 Elsevier Ltd. All rights reserved.

In Vitro Cardiomyogenic Potential of Human Amniotic Fluid Stem Cells

PubMed Central

Guan, Xuan; Delo, Dawn M.; Atala, Anthony; Soker, Shay

2010-01-01

Stem cell therapy for damaged cardiac tissue is currently limited by a number of factors, including the inability to obtain sufficient cell numbers, the potential tumorigenicity of certain types of stem cells, and the possible link between stem cell therapy and the development of malignant arrhythmias. In this study, we investigated whether human amniotic fluid-derived stem (hAFS) cells could be a potential source of cells for cardiac cell therapy by testing the in vitro differentiation capabilities. Undifferentiated hAFS cells express several cardiac genes, including the transcription factor mef2, the gap junction connexin43, and H- and N-cadherin. A 24-hour incubation with 5-aza-2′–deoxycytidine (5-AZA-dC) induced hAFS cell differentiation along the cardiac lineage. Evidence for this differentiation included morphological changes, up-regulation of cardiac-specific genes (cardiac troponin I and cardiac troponin T) and redistribution of connexin43, as well as down-regulation of the stem cell marker SRY-box 2 (sox2). When co-cultured with neonatal rat cardiomyocytes (NRCs), hAFS cells formed both mechanical and electrical connections with the NRCs. Dye transfer experiments showed that calcein dye could be transferred from NRCs to hAFS cells through cellular connections. The gap junction connexin 43 likely involved in the communication between the two cell types, because 12-O-Tetradecanoylphorbol 13-acetate (TPA) could partially block cellular crosstalk. We conclude that hAFS cells can be differentiated into a cardiomyocyte-like phenotype and can establish functional communication with NRCs. Thus, hAFS cells may potentially be used for cardiac cell therapy. PMID:20687122

Morphologic Differentiation of Viruses beyond the Family Level

PubMed Central

Goldsmith, Cynthia S.

2014-01-01

Electron microscopy has been instrumental in the identification of viruses by being able to characterize a virus to the family level. There are a few cases where morphologic or morphogenesis factors can be used to differentiate further, to the genus level. These include viruses in the families Poxviridae, Reoviridae, Retroviridae, Herpesviridae, Filoviridae, and Bunyaviridae. PMID:25502324

Differentiating among Students: The Value Added of a Dynamic Assessment of Morphological Problem-Solving

ERIC Educational Resources Information Center

Stanfa, Kathleen M.

2010-01-01

In this study, a dynamic assessment was used to measure the morphological analysis skills of participating sixth graders. The primary aim of this research was to examine the utility of this dynamic assessment and specifically to identify whether this measure can differentiate among students based on reading proficiency. Twenty-seven…

Differentiation of Aedes aegypti and Aedes notoscriptus (Diptera: Culicidae) eggs using scanning electron microscopy.

PubMed

Faull, Katherine J; Williams, Craig R

2016-05-01

Aedes notoscriptus and Aedes aegypti are both peri-domestic, invasive container-breeding mosquitoes. While the two potential arboviral vectors are bionomically similar, their sympatric distribution in Australia is limited. In this study, analyses of Ae. aegypti and Ae. notoscriptus eggs were enabled using scanning electron microscopy. Significant variations in egg length to width ratio and outer chorionic cell field morphology between Ae. aegypti and Ae. notoscriptus enabled distinction of the two species. Intraspecific variations in cell field morphology also enabled differentiation of the separate populations of both species, highlighting regional and global variation. Our study provides a comprehensive comparative analysis of inter- and intraspecific egg morphological and morphometric variation between two invasive container-breeding mosquitoes. The results indicate a high degree of intraspecific variation in Ae. notoscriptus egg morphology when compared to the eggs of Ae. aegypti. Comparative morphological analyses of Ae. aegypti and Ae. notoscriptus egg attributes using SEM allows differentiation of the species and may be helpful in understanding egg biology in relation to biotope of origin. Copyright © 2016 Elsevier Ltd. All rights reserved.

Double-bromo and extraterminal (BET) domain proteins regulate dendrite morphology and mechanosensory function.

PubMed

Bagley, Joshua A; Yan, Zhiqiang; Zhang, Wei; Wildonger, Jill; Jan, Lily Yeh; Jan, Yuh Nung

2014-09-01

A complex array of genetic factors regulates neuronal dendrite morphology. Epigenetic regulation of gene expression represents a plausible mechanism to control pathways responsible for specific dendritic arbor shapes. By studying the Drosophila dendritic arborization (da) neurons, we discovered a role of the double-bromodomain and extraterminal (BET) family proteins in regulating dendrite arbor complexity. A loss-of-function mutation in the single Drosophila BET protein encoded by female sterile 1 homeotic [fs(1)h] causes loss of fine, terminal dendritic branches. Moreover, fs(1)h is necessary for the induction of branching caused by a previously identified transcription factor, Cut (Ct), which regulates subtype-specific dendrite morphology. Finally, disrupting fs(1)h function impairs the mechanosensory response of class III da sensory neurons without compromising the expression of the ion channel NompC, which mediates the mechanosensitive response. Thus, our results identify a novel role for BET family proteins in regulating dendrite morphology and a possible separation of developmental pathways specifying neural cell morphology and ion channel expression. Since the BET proteins are known to bind acetylated histone tails, these results also suggest a role of epigenetic histone modifications and the "histone code," in regulating dendrite morphology. © 2014 Bagley et al.; Published by Cold Spring Harbor Laboratory Press.

Insights from ecological niche modeling on the taxonomic distinction and niche differentiation between the black-spotted and red-spotted tokay geckoes (Gekko gecko).

PubMed

Zhang, Yueyun; Chen, Chongtao; Li, Li; Zhao, Chengjian; Chen, Weicai; Huang, Yong

2014-09-01

The black-spotted tokay and the red-spotted tokay are morphologically distinct and have largely allopatric distributions. The black-spotted tokay is characterized by a small body size and dark skin with sundry spots, while the red-spotted tokay has a relatively large body size and red spots. Based on morphological, karyotypic, genetic, and distribution differences, recent studies suggested their species status; however, their classifications remain controversial, and additional data such as ecological niches are necessary to establish firm hypotheses regarding their taxonomic status. We reconstructed their ecological niches models using climatic and geographic data. We then performed niche similarity tests (niche identity and background tests) and point-based analyses to explore whether ecological differentiation has occurred, and whether such differences are sufficient to explain the maintenance of their separate segments of environmental ranges. We found that both niche models of the black- and the red-spotted tokay had a good fit and a robust performance, as indicated by the high area under the curve (AUC) values ("black" = 0.982, SD = ± 0.002, "red" = 0.966 ± 0.02). Significant ecological differentiation across the entire geographic range was found, indicating that the involvement of ecological differentiation is important for species differentiation. Divergence along the environmental axes is highly associated with climatic conditions, with isothermality being important for the "black" form, while temperature seasonality, precipitation of warmest quarter, and annual temperature range together being important for the "red" form. These factors are likely important factors in niche differentiation between the two forms, which result in morphological replacement. Overall, beside morphological and genetic differentiation information, our results contribute to additional insights into taxonomic distinction and niche differentiation between the black- and the red-spotted tokay.

Circadian rhythms regulate amelogenesis.

PubMed

Zheng, Li; Seon, Yoon Ji; Mourão, Marcio A; Schnell, Santiago; Kim, Doohak; Harada, Hidemitsu; Papagerakis, Silvana; Papagerakis, Petros

2013-07-01

Ameloblasts, the cells responsible for making enamel, modify their morphological features in response to specialized functions necessary for synchronized ameloblast differentiation and enamel formation. Secretory and maturation ameloblasts are characterized by the expression of stage-specific genes which follows strictly controlled repetitive patterns. Circadian rhythms are recognized as key regulators of the development and diseases of many tissues including bone. Our aim was to gain novel insights on the role of clock genes in enamel formation and to explore the potential links between circadian rhythms and amelogenesis. Our data shows definitive evidence that the main clock genes (Bmal1, Clock, Per1 and Per2) oscillate in ameloblasts at regular circadian (24 h) intervals both at RNA and protein levels. This study also reveals that the two markers of ameloblast differentiation i.e. amelogenin (Amelx; a marker of secretory stage ameloblasts) and kallikrein-related peptidase 4 (Klk4, a marker of maturation stage ameloblasts) are downstream targets of clock genes. Both, Amelx and Klk4 show 24h oscillatory expression patterns and their expression levels are up-regulated after Bmal1 over-expression in HAT-7 ameloblast cells. Taken together, these data suggest that both the secretory and the maturation stages of amelogenesis might be under circadian control. Changes in clock gene expression patterns might result in significant alterations of enamel apposition and mineralization. Copyright © 2013 Elsevier Inc. All rights reserved.

PSD-95 promotes synaptogenesis and multiinnervated spine formation through nitric oxide signaling.

PubMed

Nikonenko, Irina; Boda, Bernadett; Steen, Sylvain; Knott, Graham; Welker, Egbert; Muller, Dominique

2008-12-15

Postsynaptic density 95 (PSD-95) is an important regulator of synaptic structure and plasticity. However, its contribution to synapse formation and organization remains unclear. Using a combined electron microscopic, genetic, and pharmacological approach, we uncover a new mechanism through which PSD-95 regulates synaptogenesis. We find that PSD-95 overexpression affected spine morphology but also promoted the formation of multiinnervated spines (MISs) contacted by up to seven presynaptic terminals. The formation of multiple contacts was specifically prevented by deletion of the PDZ(2) domain of PSD-95, which interacts with nitric oxide (NO) synthase (NOS). Similarly, PSD-95 overexpression combined with small interfering RNA-mediated down-regulation or the pharmacological blockade of NOS prevented axon differentiation into varicosities and multisynapse formation. Conversely, treatment of hippocampal slices with an NO donor or cyclic guanosine monophosphate analogue induced MISs. NOS blockade also reduced spine and synapse density in developing hippocampal cultures. These results indicate that the postsynaptic site, through an NOS-PSD-95 interaction and NO signaling, promotes synapse formation with nearby axons.

VEGF and VEGFB Play Balancing Roles in Adipose Differentiation, Gene Expression, and Function.

PubMed

Jin, Honghong; Li, Dan; Wang, Xutong; Jia, Jia; Chen, Yang; Yao, Yapeng; Zhao, Chunlan; Lu, Xiaodan; Zhang, Shujie; Togo, Jacques; Ji, Yan; Zhang, Luqing; Feng, Xuechao; Zheng, Yaowu

2018-05-01

Obesity is the result of abnormal adipose development and energy metabolism. Using vascular endothelial growth factor (VEGF) B-knockout and inducible VEGF downregulation mouse models, we have shown that VEGFB inactivation caused expansion of white adipose, whitening of brown adipose, an increase in fat accumulation, and a reduction in energy consumption. At the same time, expression of the white adipose-associated genes was increased and brown adipose-associated genes decreased. VEGF repression, in contrast, induced brown adipose expansion and brown adipocyte development in white adipose, increased energy expenditure, upregulated brown adipose-associated genes, and downregulated white adipose-associated genes. When VEGFB-knockout and VEGF-repressed mice are crossed together, VEGF and VEGFB can counteractively regulate large numbers of genes and efficiently reverse each other's roles. These genes, under counteractive VEGF and VEGFB regulations, include transcription factors, adhesion molecules, and metabolic enzymes. This balancing role is confirmed by morphologic and functional changes. This study reports that VEGF and VEGFB counteractively regulate adipose development and function in energy metabolism.

Role of transcriptional regulation in the evolution of plant phenotype: A dynamic systems approach.

PubMed

Rodríguez-Mega, Emiliano; Piñeyro-Nelson, Alma; Gutierrez, Crisanto; García-Ponce, Berenice; Sánchez, María De La Paz; Zluhan-Martínez, Estephania; Álvarez-Buylla, Elena R; Garay-Arroyo, Adriana

2015-03-02

A growing body of evidence suggests that alterations in transcriptional regulation of genes involved in modulating development are an important part of phenotypic evolution, and this can be documented among species and within populations. While the effects of differential transcriptional regulation in organismal development have been preferentially studied in animal systems, this phenomenon has also been addressed in plants. In this review, we summarize evidence for cis-regulatory mutations, trans-regulatory changes and epigenetic modifications as molecular events underlying important phenotypic alterations, and thus shaping the evolution of plant development. We postulate that a mechanistic understanding of why such molecular alterations have a key role in development, morphology and evolution will have to rely on dynamic models of complex regulatory networks that consider the concerted action of genetic and nongenetic components, and that also incorporate the restrictions underlying the genotype to phenotype mapping process. Developmental Dynamics, 2015. © 2015 Wiley Periodicals, Inc. © 2015 Wiley Periodicals, Inc.

Loss of proliferation and differentiation capacity of aged human periodontal ligament stem cells and rejuvenation by exposure to the young extrinsic environment.

PubMed

Zheng, Wei; Wang, Shi; Ma, Dandan; Tang, Liang; Duan, Yinzhong; Jin, Yan

2009-09-01

The application of periodontal ligament stem cells (PDLSCs) may be effective for periodontal regenerative therapy. As tissue regenerative potential may be negatively regulated by aging, whether aging and its microenvironment modify human PDLSCs remains a question. In this study, we compared the proliferation and differentiation capacity of PDLSCs obtained from young and aged donors. Then, we exposed aged PDLSCs to young periodontal ligament cell-conditioned medium (PLC-CM), and young PDLSCs were exposed to aged PLC-CM. Morphological appearance, colony-forming assay, cell cycle analysis, osteogenic and adipogenic induction media, gene expression of cementoblast phenotype, and in vivo differentiation capacities of PDLSCs were evaluated. PDLSCs obtained from aged donors exhibited decreased proliferation and differentiation capacity when compared with those from young donors. Young PLC-CM enhanced the proliferation and differentiation capacity of PDLSCs from aged donors. Aged PDLSCs induced by young PLC-CM showed enhanced tissue-regenerative capacity to produce cementum/periodontal ligament-like structures, whereas young PDLSCs induced by aged PLC-CM transplants mainly formed connective tissues. To our knowledge, this is the first study to mimic the developmental microenvironment of PDLSCs in vitro, and our data suggest that age influences the proliferation and differentiation potential of human PDLSCs, and that the activity of human PDLSCs can be modulated by the extrinsic microenvironment.

Effects of Structural Properties of Electrospun TiO2 Nano-fiber Meshes on their Osteogenic Potential

PubMed Central

Wang, Xiaokun; Gittens, Rolando A.; Song, Rosemary; Tannenbaum, Rina; Olivares-Navarrete, Rene; Schwartz, Zvi; Chen, Haifeng; Boyan, Barbara D.

2011-01-01

Ideal outcomes in the field of tissue engineering and regenerative medicine involve biomaterials that can enhance cell differentiation and production of local factors for natural tissue regeneration without the use of systemic drugs. Biomaterials typically used in tissue engineering applications include polymeric scaffolds that mimic the 3-D structural environment of the native tissue, but these are often functionalized with proteins or small peptides to improve their biological performance. For bone applications, titanium (Ti) implants, or more appropriately the titania (TiO2) passive oxide layer formed on their surface, have been shown to enhance osteoblast differentiation in vitro and to promote osseointegration in vivo. In this study we evaluated the effect on osteoblast differentiation of pure TiO2 nano-fiber meshes with different surface micro-roughness and nano-fiber diameters, prepared by the electrospinning method. MG63 cells were seeded on TiO2 meshes, and cell number, differentiation markers and local factor production were analyzed. The results showed that cells grew throughout the entire surfaces and with similar morphology in all groups. Cell number was sensitive to surface micro-roughness, whereas cell differentiation and local factor production was regulated by both surface roughness and nano-fiber diameter. These results indicate that scaffold structural cues alone can be used to drive cell differentiation and create an osteogenic environment without the use of exogenous factors. PMID:22075122

[Regulation of moxibustion for expression of gastric mucosa cell-related marker protein in rats with acute gastric ulcer].

PubMed

Yang, Zong-Bao; Wang, Chen-Guang; Gong, An; Xie, Yu-feng; Liu, Qiong; Yang, Qing

2013-11-01

To explore relevant material basis of moxibustion for recovering gastric mucosal lesion. METHODL Forty-five SD rats were randomly divided into a normal goup, a model group, an acupoint group and a control group, 15 rats in the model group and 10 rats in the rest three groups. Except the normal group, binding and cold stress method were used to establish gastric mucosa injury model. The suspended moxibustion was applied in the acupoint group and control group at acupoints of the stomach meridian ("Liangmen" (ST 21) and "Zusanli" (ST36) and control acupoints (Laterally 1cm next to the "Liangmen" (ST 21) and Zusanli" (ST36), once a day, consectutively for 12 days. After 12 days, morphology of gastric mucosal was observed under optical microscope; protein fingerprints of gastric mucosa cell in rats were detected by protein fingerprint technology, weak cation chip and weak anion chip. Also mass to charge ratio of differential proteins in groups were compared and analyzed. Compared with the model group, index of gastric mucosal lesion in the acupoint group was reduced and its morphology was obviously improved (P<0.05). Campared with control group, index and morphology of gastric mucosal lesion were significantly improved in the acupoint group (P<0.05). According to test of weak cation chip, there was four marker proteins that had expression differences, indicating moxibustion at acupoints of stomach meridian could inrease expression of three marker protein whose molecular weight was 1354Da, 5692Da and 8432Da (all P<0.05) while reduce expression of marker protein with molecular weight of 3287Da (_<0.05). According to test of weak anion chip, moxibustion at acupoints of stomach meridian could increase expression of three marker proteins whose molecular weight was 2412 Da, 3026Da and 6475 Da (allP<0.05). Moxibustion at acupoints of the stomach meridian could regulate differential expression of gastric mucosa cell-related marker protein in rats with acute gastric ulcer and recover gastric mucosal lesion, it's effect is better than that of the points of laterally 1 cm next to acupoint.

Loss of MyoD and Myf5 in Skeletal Muscle Stem Cells Results in Altered Myogenic Programming and Failed Regeneration.

PubMed

Yamamoto, Masakazu; Legendre, Nicholas P; Biswas, Arpita A; Lawton, Alexander; Yamamoto, Shoko; Tajbakhsh, Shahragim; Kardon, Gabrielle; Goldhamer, David J

2018-03-13

MyoD and Myf5 are fundamental regulators of skeletal muscle lineage determination in the embryo, and their expression is induced in satellite cells following muscle injury. MyoD and Myf5 are also expressed by satellite cell precursors developmentally, although the relative contribution of historical and injury-induced expression to satellite cell function is unknown. We show that satellite cells lacking both MyoD and Myf5 (double knockout [dKO]) are maintained with aging in uninjured muscle. However, injured muscle fails to regenerate and dKO satellite cell progeny accumulate in damaged muscle but do not undergo muscle differentiation. dKO satellite cell progeny continue to express markers of myoblast identity, although their myogenic programming is labile, as demonstrated by dramatic morphological changes and increased propensity for non-myogenic differentiation. These data demonstrate an absolute requirement for either MyoD or Myf5 in muscle regeneration and indicate that their expression after injury stabilizes myogenic identity and confers the capacity for muscle differentiation. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

Gene expression in bovine rumen epithelium during weaning identifies molecular regulators of rumen development and growth.

PubMed

Connor, Erin E; Baldwin, Ransom L; Li, Cong-jun; Li, Robert W; Chung, Hoyoung

2013-03-01

During weaning, epithelial cell function in the rumen transitions in response to conversion from a pre-ruminant to a true ruminant environment to ensure efficient nutrient absorption and metabolism. To identify gene networks affected by weaning in bovine rumen, Holstein bull calves were fed commercial milk replacer only (MRO) until 42 days of age, then were provided diets of either milk + orchardgrass hay (MH) or milk + grain-based calf starter (MG). Rumen epithelial RNA was extracted from calves sacrificed at four time points: day 14 (n = 3) and day 42 (n = 3) of age while fed the MRO diet and day 56 (n = 3/diet) and day 70 (n = 3/diet) while fed the MH and MG diets for transcript profiling by microarray hybridization. Five two-group comparisons were made using Permutation Analysis of Differential Expression® to identify differentially expressed genes over time and developmental stage between days 14 and 42 within the MRO diet, between day 42 on the MRO diet and day 56 on the MG or MH diets, and between the MG and MH diets at days 56 and 70. Ingenuity Pathway Analysis (IPA) of differentially expressed genes during weaning indicated the top 5 gene networks involving molecules participating in lipid metabolism, cell morphology and death, cellular growth and proliferation, molecular transport, and the cell cycle. Putative genes functioning in the establishment of the rumen microbial population and associated rumen epithelial inflammation during weaning were identified. Activation of transcription factor PPAR-α was identified by IPA software as an important regulator of molecular changes in rumen epithelium that function in papillary development and fatty acid oxidation during the transition from pre-rumination to rumination. Thus, molecular markers of rumen development and gene networks regulating differentiation and growth of rumen epithelium were identified for selecting targets and methods for improving and assessing rumen development and function, particularly in the growing calf.

Cell type dependent morphological adaptation in polyelectrolyte hydrogels governs chondrogenic fate.

PubMed

Raghothaman, Deepak; Leong, Meng Fatt; Lim, Tze Chiun; Wan, Andrew C A; Ser, Zheng; Lee, Eng Hin; Yang, Zheng

2016-04-04

Repair of critical-size articular cartilage defects typically involves delivery of cells in biodegradable, 3D matrices. Differences in the developmental status of mesenchymal stem cells (MSCs) and terminally differentiated mature chondrocytes might be a critical factor in engineering appropriate 3D matrices for articular cartilage tissue engineering. This study examined the relationship between material-driven early cell morphological adaptations and chondrogenic outcomes, by studying the influence of aligned collagen type I (Col I) presentation on chondrocytes and MSC in interfacial polyelectrolyte complexation (IPC)-based hydrogels. In the absence of Col I, both chondrocytes and MSCs adopted rounded cell morphology and formed clusters, with chondrocyte clusters favoring the maintenance of hyaline phenotype, while MSC clusters differentiated to fibro-superficial zone-like chondrocytes. Encapsulated chondrocytes in IPC-Col I hydrogel adopted a fibroblastic morphology forming fibro-superficial zone-like phenotype, which could be reversed by inhibiting actin polymerization using cytochalasin D (CytD). In contrast, adoption of fibroblastic morphology by encapsulated MSCs in IPC-Col I facilitated superior chondrogenesis, generating a mature, hyaline neocartilage tissue. CytD treatment abrogated the elongation of MSCs and brought about a single cell-like state, resulting in insignificant chondrogenic differentiation, underscoring the essential requirement of providing matrix environments that are amenable to cell-cell interactions for robust MSC chondrogenic differentiation. Our study demonstrates that MSCs and culture-expanded chondrocytes favour differential microenvironmental niches and emphasizes the importance of designing biomaterials that meet cell type-specific requirements, in adopting chondrocyte or MSC-based approaches for regenerating hyaline, articular cartilage.

Diversity among African Pygmies

PubMed Central

Ramírez Rozzi, Fernando V.; Sardi, Marina L.

2010-01-01

Although dissimilarities in cranial and post-cranial morphology among African pygmies groups have been recognized, comparative studies on skull morphology usually pull all pygmies together assuming that morphological characters are similar among them and different with respect to other populations. The main aim of this study is to compare cranial morphology between African pygmies and non-pygmies populations from Equatorial Africa derived from both the Eastern and the Western regions in order to test if the greatest morphological difference is obtained in the comparison between pygmies and non-pygmies. Thirty three-dimensional (3D) landmarks registered with Microscribe in four cranial samples (Western and Eastern pygmies and non-pygmies) were obtained. Multivariate analysis (generalized Procrustes analysis, Mahalanobis distances, multivariate regression) and complementary dimensions of size were evaluated with ANOVA and post hoc LSD. Results suggest that important cranial shape differentiation does occur between pygmies and non-pygmies but also between Eastern and Western populations and that size changes and allometries do not affect similarly Eastern and Western pygmies. Therefore, our findings raise serious doubt about the fact to consider African pygmies as a homogenous group in studies on skull morphology. Differences in cranial morphology among pygmies would suggest differentiation after divergence. Although not directly related to skull differentiation, the diversity among pygmies would probably suggest that the process responsible for reduced stature occurred after the split of the ancestors of modern Eastern and Western pygmies. PMID:21049030

Diversity among African pygmies.

PubMed

Ramírez Rozzi, Fernando V; Sardi, Marina L

2010-10-26

Although dissimilarities in cranial and post-cranial morphology among African pygmies groups have been recognized, comparative studies on skull morphology usually pull all pygmies together assuming that morphological characters are similar among them and different with respect to other populations. The main aim of this study is to compare cranial morphology between African pygmies and non-pygmies populations from Equatorial Africa derived from both the Eastern and the Western regions in order to test if the greatest morphological difference is obtained in the comparison between pygmies and non-pygmies. Thirty three-dimensional (3D) landmarks registered with Microscribe in four cranial samples (Western and Eastern pygmies and non-pygmies) were obtained. Multivariate analysis (generalized Procrustes analysis, Mahalanobis distances, multivariate regression) and complementary dimensions of size were evaluated with ANOVA and post hoc LSD. Results suggest that important cranial shape differentiation does occur between pygmies and non-pygmies but also between Eastern and Western populations and that size changes and allometries do not affect similarly Eastern and Western pygmies. Therefore, our findings raise serious doubt about the fact to consider African pygmies as a homogenous group in studies on skull morphology. Differences in cranial morphology among pygmies would suggest differentiation after divergence. Although not directly related to skull differentiation, the diversity among pygmies would probably suggest that the process responsible for reduced stature occurred after the split of the ancestors of modern Eastern and Western pygmies.

A Suppressive Antagonism Evidences Progesterone and Estrogen Receptor Pathway Interaction with Concomitant Regulation of Hand2, Bmp2 and ERK during Early Decidualization

PubMed Central

Mestre-Citrinovitz, Ana C.; Kleff, Veronika; Vallejo, Griselda

2015-01-01

Progesterone receptor and estrogen receptor participate in growth and differentiation of the different rat decidual regions. Steroid hormone receptor antagonists were used to study steroid regulation of decidualization. Here we describe a suppressive interaction between progesterone receptor (onapristone) and estrogen receptor (ICI182780) antagonists and their relation to a rescue phenomenon with concomitant regulation of Hand2, Bmp2 and p-ERK1/2 during the early decidualization steps. Phenotypes of decidua development produced by antagonist treatments were characterized by morphology, proliferation, differentiation, angiogenesis and expression of signaling molecules. We found that suppression of progesterone receptor activity by onapristone treatment resulted in resorption of the implantation sites with concomitant decrease in progesterone and estrogen receptors, PCNA, KI67 antigen, DESMIN, CCND3, CX43, Prl8a2, and signaling players such as transcription factor Hand2, Bmp2 mRNAs and p-ERK1/2. Moreover, FGF-2 and Vegfa increased as a consequence of onapristone treatment. Implantation sites from antagonist of estrogen receptor treated rats developed all decidual regions, but showed an anomalous blood vessel formation at the mesometrial part of the decidua. The deleterious effect of onapristone was partially counteracted by the impairment of estrogen receptor activity with rescue of expression levels of hormone steroid receptors, proliferation and differentiation markers, and the induction of a probably compensatory increase in signaling molecules Hand2, Bmp2 and ERK1/2 activation compared to oil treated controls. This novel drug interaction during decidualization could be applied to pathological endometrial cell proliferation processes to improve therapies using steroid hormone receptor targets. PMID:25897495

A paper-based scaffold for enhanced osteogenic differentiation of equine adipose-derived stem cells.

PubMed

Petersen, Gayle F; Hilbert, Bryan J; Trope, Gareth D; Kalle, Wouter H J; Strappe, Padraig M

2015-11-01

We investigated the applicability of single layer paper-based scaffolds for the three-dimensional (3D) growth and osteogenic differentiation of equine adipose-derived stem cells (EADSC), with comparison against conventional two-dimensional (2D) culture on polystyrene tissue culture vessels. Viable culture of EADSC was achieved using paper-based scaffolds, with EADSC grown and differentiated in 3D culture retaining high cell viability (>94 %), similarly to EADSC in 2D culture. Osteogenic differentiation of EADSC was significantly enhanced in 3D culture, with Alizarin Red S staining and quantification demonstrating increased mineralisation (p < 0.0001), and an associated increase in expression of the osteogenic-specific markers alkaline phosphatase (p < 0.0001), osteopontin (p < 0.0001), and runx2 (p < 0.01). Furthermore, scanning electron microscopy revealed a spherical morphology of EADSC in 3D culture, compared to a flat morphology of EADSC in 2D culture. Single layer paper-based scaffolds provide an enhanced environment for the in vitro 3D growth and osteogenic differentiation of EADSC, with high cell viability, and a spherical morphology.

Histone modifications and chromatin dynamics: a focus on filamentous fungi

PubMed Central

Brosch, Gerald; Loidl, Peter; Graessle, Stefan

2008-01-01

The readout of the genetic information of eukaryotic organisms is significantly regulated by modifications of DNA and chromatin proteins. Chromatin alterations induce genome-wide and local changes in gene expression and affect a variety of processes in response to internal and external signals during growth, differentiation, development, in metabolic processes, diseases, and abiotic and biotic stresses. This review aims at summarizing the roles of histone H1 and the acetylation and methylation of histones in filamentous fungi and links this knowledge to the huge body of data from other systems. Filamentous fungi show a wide range of morphologies and have developed a complex network of genes that enables them to use a great variety of substrates. This fact, together with the possibility of simple and quick genetic manipulation, highlights these organisms as model systems for the investigation of gene regulation. However, little is still known about regulation at the chromatin level in filamentous fungi. Understanding the role of chromatin in transcriptional regulation would be of utmost importance with respect to the impact of filamentous fungi in human diseases and agriculture. The synthesis of compounds (antibiotics, immunosuppressants, toxins, and compounds with adverse effects) is also likely to be regulated at the chromatin level. PMID:18221488

Neogenin, a regulator of adult hippocampal neurogenesis, prevents depressive-like behavior.

PubMed

Sun, Dong; Sun, Xiang-Dong; Zhao, Lu; Lee, Dae-Hoon; Hu, Jin-Xia; Tang, Fu-Lei; Pan, Jin-Xiu; Mei, Lin; Zhu, Xiao-Juan; Xiong, Wen-Cheng

2018-01-08

Adult neurogenesis in hippocampal dentate gyrus (DG) is a complex, but precisely controlled process. Dysregulation of this event contributes to multiple neurological disorders, including major depression. Thus, it is of considerable interest to investigate how adult hippocampal neurogenesis is regulated. Here, we present evidence for neogenin, a multifunctional transmembrane receptor, to regulate adult mouse hippocampal neurogenesis. Loss of neogenin in adult neural stem cells (NSCs) or neural progenitor cells (NPCs) impaired NSCs/NPCs proliferation and neurogenesis, whereas increased their astrocytic differentiation. Mechanistic studies revealed a role for neogenin to positively regulate Gli1, a crucial downstream transcriptional factor of sonic hedgehog, and expression of Gli1 into neogenin depleted NSCs/NPCs restores their proliferation. Further morphological and functional studies showed additional abnormities, including reduced dendritic branches and spines, and impaired glutamatergic neuro-transmission, in neogenin-depleted new-born DG neurons; and mice with depletion of neogenin in NSCs/NPCs exhibited depressive-like behavior. These results thus demonstrate unrecognized functions of neogenin in adult hippocampal NSCs/NPCs-promoting NSCs/NPCs proliferation and neurogenesis and preventing astrogliogenesis and depressive-like behavior, and suggest neogenin regulation of Gli1 signaling as a possible underlying mechanism.

Influence of Oxygen Tension on Dopaminergic Differentiation of Human Fetal Stem Cells of Midbrain and Forebrain Origin

PubMed Central

Krabbe, Christina; Bak, Sara Thornby; Jensen, Pia; von Linstow, Christian; Martínez Serrano, Alberto; Hansen, Claus; Meyer, Morten

2014-01-01

Neural stem cells (NSCs) constitute a promising source of cells for transplantation in Parkinson's disease (PD), but protocols for controlled dopaminergic differentiation are not yet available. Here we investigated the influence of oxygen on dopaminergic differentiation of human fetal NSCs derived from the midbrain and forebrain. Cells were differentiated for 10 days in vitro at low, physiological (3%) versus high, atmospheric (20%) oxygen tension. Low oxygen resulted in upregulation of vascular endothelial growth factor and increased the proportion of tyrosine hydroxylase-immunoreactive (TH-ir) cells in both types of cultures (midbrain: 9.1±0.5 and 17.1±0.4 (P<0.001); forebrain: 1.9±0.4 and 3.9±0.6 (P<0.01) percent of total cells). Regardless of oxygen levels, the content of TH-ir cells with mature neuronal morphologies was higher for midbrain as compared to forebrain cultures. Proliferative Ki67-ir cells were found in both types of cultures, but the relative proportion of these cells was significantly higher for forebrain NSCs cultured at low, as compared to high, oxygen tension. No such difference was detected for midbrain-derived cells. Western blot analysis revealed that low oxygen enhanced β-tubulin III and GFAP expression in both cultures. Up-regulation of β-tubulin III was most pronounced for midbrain cells, whereas GFAP expression was higher in forebrain as compared to midbrain cells. NSCs from both brain regions displayed less cell death when cultured at low oxygen tension. Following mictrotransplantation into mouse striatal slice cultures predifferentiated midbrain NSCs were found to proliferate and differentiate into substantial numbers of TH-ir neurons with mature neuronal morphologies, particularly at low oxygen. In contrast, predifferentiated forebrain NSCs microtransplanted using identical conditions displayed little proliferation and contained few TH-ir cells, all of which had an immature appearance. Our data may reflect differences in dopaminergic differentiation capacity and region-specific requirements of NSCs, with the dopamine-depleted striatum cultured at low oxygen offering an attractive micro-environment for midbrain NSCs. PMID:24788190

Persistent Low Level of Osterix Accelerates Interleukin-6 Production and Impairs Regeneration after Tissue Injury

PubMed Central

Baek, Wook-Young; Park, Seung-Yoon; Kim, Yeo Hyang; Lee, Min-A; Kwon, Tae-Hwan; Park, Kwon-Moo; de Crombrugghe, Benoit; Kim, Jung-Eun

2013-01-01

Osterix (Osx) is an essential transcription factor for osteoblast differentiation and bone formation. Osx knockout show a complete absence of bone formation, whereas Osx conditional knockout in osteoblasts produce an osteopenic phenotype after birth. Here, we questioned whether Osx has a potential role in regulating physiological homeostasis. In Osx heterozygotes expressing low levels of Osx in bones, the expression levels of pro-inflammatory cytokines were significantly elevated, indicating that reduced Osx expression may reflect an inflammatory-prone state. In particular, the expression of interleukin-6, a key mediator of chronic inflammation, was increased in Osx heterozygotes and decreased in Osx overexpressing osteoblasts, and transcriptionally down-regulated by Osx. Although no significant differences were revealed in renal morphology and function between Osx heterozygotes and wild-type under normoxic conditions, recovery of kidneys after ischemic damage was remarkably delayed in Osx heterozygotes, as indicated by elevated blood urea nitrogen and creatinine levels, and by morphological alterations consistent with acute tubular necrosis. Eventually, protracted low Osx expression level caused an inflammatory-prone state in the body, resulting in the enhanced susceptibility to renal injury and the delayed renal repair after ischemia/reperfusion. This study suggests that the maintenance of Osx expression in bone is important in terms of preventing the onset of an inflammatory-prone state. PMID:23922826

Hepatogenic and neurogenic differentiation of bone marrow mesenchymal stem cells from abattoir-derived bovine fetuses

PubMed Central

2014-01-01

Background Mesenchymal stem cells (MSC) are multipotent progenitor cells characterized by their ability to both self-renew and differentiate into tissues of mesodermal origin. The plasticity or transdifferentiation potential of MSC is not limited to mesodermal derivatives, since under appropriate cell culture conditions and stimulation by bioactive factors, MSC have also been differentiated into endodermal (hepatocytes) and neuroectodermal (neurons) cells. The potential of MSC for hepatogenic and neurogenic differentiation has been well documented in different animal models; however, few reports are currently available on large animal models. In the present study we sought to characterize the hepatogenic and neurogenic differentiation and multipotent potential of bovine MSC (bMSC) isolated from bone marrow (BM) of abattoir-derived fetuses. Results Plastic-adherent bMSC isolated from fetal BM maintained a fibroblast-like morphology under monolayer culture conditions. Flow cytometric analysis demonstrated that bMSC populations were positive for MSC markers CD29 and CD73 and pluripotency markers OCT4 and NANOG; whereas, were negative for hematopoietic markers CD34 and CD45. Levels of mRNA of hepatic genes α-fetoprotein (AFP), albumin (ALB), alpha1 antitrypsin (α1AT), connexin 32 (CNX32), tyrosine aminotransferase (TAT) and cytochrome P450 (CYP3A4) were up-regulated in bMSC during a 28-Day period of hepatogenic differentiation. Functional analyses in differentiated bMSC cultures evidenced an increase (P < 0.05) in albumin and urea production and glycogen storage. bMSC cultured under neurogenic conditions expressed NESTIN and MAP2 proteins at 24 h of culture; whereas, at 144 h also expressed TRKA and PrPC. Levels of MAP2 and TRKA mRNA were up-regulated at the end of the differentiation period. Conversely, bMSC expressed lower levels of NANOG mRNA during both hepatogenic and neurogenic differentiation processes. Conclusion The expression patterns of linage-specific markers and the production of functional metabolites support the potential for hepatogenic and neurogenic differentiation of bMSC isolated from BM of abattoir-derived fetuses. The simplicity of isolation and the potential to differentiate into a wide variety of cell lineages lays the foundation for bMSC as an interesting alternative for investigation in MSC biology and eventual applications for regenerative therapy in veterinary medicine. PMID:25011474

Decreased serotonin level during pregnancy alters morphological and functional characteristics of tonic nociceptive system in juvenile offspring of the rat.

PubMed

Butkevich, Irina P; Khozhai, Ludmila I; Mikhailenko, Victor A; Otellin, Vladimir A

2003-11-13

Serotonin (5-HT) contributes to the prenatal development of the central nervous system, acting as a morphogen in the young embryo and later as a neurotransmitter. This biologically active agent influences both morphological and biochemical differentiation of raphe neurons, which give rise to the descending serotonergic paths that regulate the processing of acutely evoked nociceptive inputs. The involvement of 5-HT in the prenatal development of tonic nociceptive system has not been studied. In the present study we evaluated the effects of a single injection (400 mg/kg, 2 ml, i.p.) of the 5-HT synthesis inhibitor, para-chlorophenylalanine (pCPA), given to pregnant rats during the critical period fetal serotonin development. The functional integrity of the tonic nociceptive response was investigated in 25 day old rats using the classic formalin test. Morphological analysis of brain structures involved in formalin-induced pain and 5-HT levels in the heads of 12-day embryos were also evaluated. Embryonic levels of 5-HT were significantly lowered by the treatment. The juvenile rats from pCPA-treated females showed altered brain morphology and cell differentiation in the developing cortex, hippocampus, raphe nuclei, and substantia nigra. In the formalin test, there were significant decreases in the intensity and duration of the second phase of the formalin-induced response, characterizing persistent, tonic pain. The extent of impairments in the brain structures correlated positively with the level of decrease in the behavioral responses. The data demonstrate the involvement of 5-HT in the prenatal development of the tonic nociceptive system. The decreased tonic component of the behavioral response can be explained by lower activity of the descending excitatory serotonergic system originating in the raphe nuclei, resulting in decreased tonic pain processing organized at the level of the dorsal horn of the spinal cord.

ERK1/2 mediates glucose-regulated POMC gene expression in hypothalamic neurons.

PubMed

Zhang, Juan; Zhou, Yunting; Chen, Cheng; Yu, Feiyuan; Wang, Yun; Gu, Jiang; Ma, Lian; Ho, Guyu

2015-04-01

Hypothalamic glucose-sensing neurons regulate the expression of genes encoding feeding-related neuropetides POMC, AgRP, and NPY - the key components governing metabolic homeostasis. AMP-activated protein kinase (AMPK) is postulated to be the molecular mediator relaying glucose signals to regulate the expression of these neuropeptides. Whether other signaling mediator(s) plays a role is not clear. In this study, we investigated the role of ERK1/2 using primary hypothalamic neurons as the model system. The primary neurons were differentiated from hypothalamic progenitor cells. The differentiated neurons possessed the characteristic neuronal cell morphology and expressed neuronal post-mitotic markers as well as leptin-regulated orexigenic POMC and anorexigenic AgRP/NPY genes. Treatment of cells with glucose dose-dependently increased POMC and decreased AgRP/NPY expression with a concurrent suppression of AMPK phosphorylation. In addition, glucose treatment dose-dependently increased the ERK1/2 phosphorylation. Blockade of ERK1/2 activity with its specific inhibitor PD98059 partially (approximately 50%) abolished glucose-induced POMC expression, but had little effect on AgRP/NPY expression. Conversely, blockade of AMPK activity with its specific inhibitor produced a partial (approximately 50%) reversion of low-glucose-suppressed POMC expression, but almost completely blunted the low-glucose-induced AgRP/NPY expression. The results indicate that ERK1/2 mediated POMC but not AgRP/NPY expression. Confirming the in vitro findings, i.c.v. administration of PD98059 in rats similarly attenuated glucose-induced POMC expression in the hypothalamus, but again had little effect on AgRP/NPY expression. The results are indicative of a novel role of ERK1/2 in glucose-regulated POMC expression and offer new mechanistic insights into hypothalamic glucose sensing. © 2015 Society for Endocrinology.

System analysis of metabolism and the transcriptome in Arabidopsis thaliana roots reveals differential co-regulation upon iron, sulfur and potassium deficiency.

PubMed

Forieri, Ilaria; Sticht, Carsten; Reichelt, Michael; Gretz, Norbert; Hawkesford, Malcolm J; Malagoli, Mario; Wirtz, Markus; Hell, Ruediger

2017-01-01

Deprivation of mineral nutrients causes significant retardation of plant growth. This retardation is associated with nutrient-specific and general stress-induced transcriptional responses. In this study, we adjusted the external supply of iron, potassium and sulfur to cause the same retardation of shoot growth. Nevertheless, limitation by individual nutrients resulted in specific morphological adaptations and distinct shifts within the root metabolite fingerprint. The metabolic shifts affected key metabolites of primary metabolism and the stress-related phytohormones, jasmonic, salicylic and abscisic acid. These phytohormone signatures contributed to specific nutrient deficiency-induced transcriptional regulation. Limitation by the micronutrient iron caused the strongest regulation and affected 18% of the root transcriptome. Only 130 genes were regulated by all nutrients. Specific co-regulation between the iron and sulfur metabolic routes upon iron or sulfur deficiency was observed. Interestingly, iron deficiency caused regulation of a different set of genes of the sulfur assimilation pathway compared with sulfur deficiency itself, which demonstrates the presence of specific signal-transduction systems for the cross-regulation of the pathways. Combined iron and sulfur starvation experiments demonstrated that a requirement for a specific nutrient can overrule this cross-regulation. The comparative metabolomics and transcriptomics approach used dissected general stress from nutrient-specific regulation in roots of Arabidopsis. © 2016 John Wiley & Sons Ltd.

Morphological and functional differentiation in BE(2)-M17 human neuroblastoma cells by treatment with Trans-retinoic acid

PubMed Central

2013-01-01

Background Immortalized neuronal cell lines can be induced to differentiate into more mature neurons by adding specific compounds or growth factors to the culture medium. This property makes neuronal cell lines attractive as in vitro cell models to study neuronal functions and neurotoxicity. The clonal human neuroblastoma BE(2)-M17 cell line is known to differentiate into a more prominent neuronal cell type by treatment with trans-retinoic acid. However, there is a lack of information on the morphological and functional aspects of these differentiated cells. Results We studied the effects of trans-retinoic acid treatment on (a) some differentiation marker proteins, (b) types of voltage-gated calcium (Ca2+) channels and (c) Ca2+-dependent neurotransmitter ([3H] glycine) release in cultured BE(2)-M17 cells. Cells treated with 10 μM trans-retinoic acid (RA) for 72 hrs exhibited marked changes in morphology to include neurite extensions; presence of P/Q, N and T-type voltage-gated Ca2+ channels; and expression of neuron specific enolase (NSE), synaptosomal-associated protein 25 (SNAP-25), nicotinic acetylcholine receptor α7 (nAChR-α7) and other neuronal markers. Moreover, retinoic acid treated cells had a significant increase in evoked Ca2+-dependent neurotransmitter release capacity. In toxicity studies of the toxic gas, phosgene (CG), that differentiation of M17 cells with RA was required to see the changes in intracellular free Ca2+ concentrations following exposure to CG. Conclusion Taken together, retinoic acid treated cells had improved morphological features as well as neuronal characteristics and functions; thus, these retinoic acid differentiated BE(2)-M17 cells may serve as a better neuronal model to study neurobiology and/or neurotoxicity. PMID:23597229

Morphological and functional differentiation in BE(2)-M17 human neuroblastoma cells by treatment with Trans-retinoic acid.

PubMed

Andres, Devon; Keyser, Brian M; Petrali, John; Benton, Betty; Hubbard, Kyle S; McNutt, Patrick M; Ray, Radharaman

2013-04-18

Immortalized neuronal cell lines can be induced to differentiate into more mature neurons by adding specific compounds or growth factors to the culture medium. This property makes neuronal cell lines attractive as in vitro cell models to study neuronal functions and neurotoxicity. The clonal human neuroblastoma BE(2)-M17 cell line is known to differentiate into a more prominent neuronal cell type by treatment with trans-retinoic acid. However, there is a lack of information on the morphological and functional aspects of these differentiated cells. We studied the effects of trans-retinoic acid treatment on (a) some differentiation marker proteins, (b) types of voltage-gated calcium (Ca2+) channels and (c) Ca2+-dependent neurotransmitter ([3H] glycine) release in cultured BE(2)-M17 cells. Cells treated with 10 μM trans-retinoic acid (RA) for 72 hrs exhibited marked changes in morphology to include neurite extensions; presence of P/Q, N and T-type voltage-gated Ca2+ channels; and expression of neuron specific enolase (NSE), synaptosomal-associated protein 25 (SNAP-25), nicotinic acetylcholine receptor α7 (nAChR-α7) and other neuronal markers. Moreover, retinoic acid treated cells had a significant increase in evoked Ca2+-dependent neurotransmitter release capacity. In toxicity studies of the toxic gas, phosgene (CG), that differentiation of M17 cells with RA was required to see the changes in intracellular free Ca2+ concentrations following exposure to CG. Taken together, retinoic acid treated cells had improved morphological features as well as neuronal characteristics and functions; thus, these retinoic acid differentiated BE(2)-M17 cells may serve as a better neuronal model to study neurobiology and/or neurotoxicity.

Anchoring of protein kinase A by ERM (ezrin-radixin-moesin) proteins is required for proper netrin signaling through DCC (deleted in colorectal cancer).

PubMed

Deming, Paula B; Campbell, Shirley L; Stone, Jamie B; Rivard, Robert L; Mercier, Alison L; Howe, Alan K

2015-02-27

Netrin-1, acting through its principal receptor DCC (deleted in colorectal cancer), serves as an axon guidance cue during neural development and also contributes to vascular morphogenesis, epithelial migration, and the pathogenesis of some tumors. Several lines of evidence suggest that netrin-DCC signaling can regulate and be regulated by the cAMP-dependent protein kinase, PKA, although the molecular details of this relationship are poorly understood. Specificity in PKA signaling is often achieved through differential subcellular localization of the enzyme by interaction with protein kinase A anchoring proteins (AKAPs). Here, we show that AKAP function is required for DCC-mediated activation of PKA and phosphorylation of cytoskeletal regulatory proteins of the Mena/VASP (vasodilator-stimulated phosphoprotein) family. Moreover, we show that DCC and PKA physically interact and that this association is mediated by the ezrin-radixin-moesin (ERM) family of plasma membrane-actin cytoskeleton cross-linking proteins. Silencing of ERM protein expression inhibits DCC-PKA interaction, DCC-mediated PKA activation, and phosphorylation of Mena/VASP proteins as well as growth cone morphology and neurite outgrowth. Finally, although expression of wild-type radixin partially rescued growth cone morphology and tropism toward netrin in ERM-knockdown cells, expression of an AKAP-deficient mutant of radixin did not fully rescue growth cone morphology and switched netrin tropism from attraction to repulsion. These data support a model in which ERM-mediated anchoring of PKA activity to DCC is required for proper netrin/DCC-mediated signaling. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

The Guanine Nucleotide Exchange Factor Tiam1 Affects Neuronal Morphology; Opposing Roles for the Small GTPases Rac and Rho

PubMed Central

van Leeuwen, Frank N.; Kain, Hendrie E.T.; van der Kammen, Rob A.; Michiels, Frits; Kranenburg, Onno W.; Collard, John G.

1997-01-01

The invasion-inducing T-lymphoma invasion and metastasis 1 (Tiam1) protein functions as a guanine nucleotide exchange factor (GEF) for the small GTPase Rac1. Differentiation-dependent expression of Tiam1 in the developing brain suggests a role for this GEF and its effector Rac1 in the control of neuronal morphology. Here we show that overexpression of Tiam1 induces cell spreading and affects neurite outgrowth in N1E-115 neuroblastoma cells. These effects are Rac-dependent and strongly promoted by laminin. Overexpression of Tiam1 recruits the α6β1 integrin, a laminin receptor, to specific adhesive contacts at the cell periphery, which are different from focal contacts. Cells overexpressing Tiam1 no longer respond to lysophosphatidic acid– induced neurite retraction and cell rounding, processes mediated by Rho, suggesting that Tiam1-induced activation of Rac antagonizes Rho signaling. This inhibition can be overcome by coexpression of constitutively active RhoA, which may indicate that regulation occurs at the level of Rho or upstream. Conversely, neurite formation induced by Tiam1 or Rac1 is further promoted by inactivating Rho. These results demonstrate that Rac- and Rho-mediated pathways oppose each other during neurite formation and that a balance between these pathways determines neuronal morphology. Furthermore, our data underscore the potential role of Tiam1 as a specific regulator of Rac during neurite formation and illustrate the importance of reciprocal interactions between the cytoskeleton and the extracellular matrix during this process. PMID:9348295

Genetic Dissection of Leaf Development in Brassica rapa Using a Genetical Genomics Approach1[W

PubMed Central

Xiao, Dong; Wang, Huange; Basnet, Ram Kumar; Zhao, Jianjun; Lin, Ke; Hou, Xilin; Bonnema, Guusje

2014-01-01

The paleohexaploid crop Brassica rapa harbors an enormous reservoir of morphological variation, encompassing leafy vegetables, vegetable and fodder turnips (Brassica rapa, ssp. campestris), and oil crops, with different crops having very different leaf morphologies. In the triplicated B. rapa genome, many genes have multiple paralogs that may be regulated differentially and contribute to phenotypic variation. Using a genetical genomics approach, phenotypic data from a segregating doubled haploid population derived from a cross between cultivar Yellow sarson (oil type) and cultivar Pak choi (vegetable type) were used to identify loci controlling leaf development. Twenty-five colocalized phenotypic quantitative trait loci (QTLs) contributing to natural variation for leaf morphological traits, leaf number, plant architecture, and flowering time were identified. Genetic analysis showed that four colocalized phenotypic QTLs colocalized with flowering time and leaf trait candidate genes, with their cis-expression QTLs and cis- or trans-expression QTLs for homologs of genes playing a role in leaf development in Arabidopsis (Arabidopsis thaliana). The leaf gene BRASSICA RAPA KIP-RELATED PROTEIN2_A03 colocalized with QTLs for leaf shape and plant height; BRASSICA RAPA ERECTA_A09 colocalized with QTLs for leaf color and leaf shape; BRASSICA RAPA LONGIFOLIA1_A10 colocalized with QTLs for leaf size, leaf color, plant branching, and flowering time; while the major flowering time gene, BRASSICA RAPA FLOWERING LOCUS C_A02, colocalized with QTLs explaining variation in flowering time, plant architectural traits, and leaf size. Colocalization of these QTLs points to pleiotropic regulation of leaf development and plant architectural traits in B. rapa. PMID:24394778

Cooperative activity of GABP with PU.1 or C/EBPε regulates lamin B receptor gene expression, implicating their roles in granulocyte nuclear maturation1

PubMed Central

Malu, Krishnakumar; Garhwal, Rahul; Pelletier, Margery G. H.; Gotur, Deepali; Halene, Stephanie; Zwerger, Monika; Yang, Zhong-Fa; Rosmarin, Alan G.; Gaines, Peter

2016-01-01

Nuclear segmentation is a hallmark feature of mammalian neutrophil differentiation, but the mechanisms that control this process are poorly understood. Gene expression in maturing neutrophils requires combinatorial actions of lineage-restricted and more widely expressed transcriptional regulators. Examples include interactions of the widely expressed ETS transcription factor, GA-binding protein (GABP), with the relatively lineage-restricted ETS factor, PU.1, and with CCAAT enhancer binding proteins, C/EBPα and C/EBPε. Whether such cooperative interactions between these transcription factors also regulate the expression of genes encoding proteins that control nuclear segmentation is unclear. We investigated the roles of ETS and C/EBP family transcription factors in regulating the gene encoding the lamin B receptor (LBR), an inner nuclear membrane protein whose expression is required for neutrophil nuclear segmentation. Although C/EBPε was previously shown to bind the Lbr promoter, surprisingly, we found that neutrophils derived from Cebpe null mice exhibited normal Lbr gene and protein expression. Instead, GABP provided transcriptional activation through the Lbr promoter in the absence of C/EBPε, and activities supported by GABP were greatly enhanced by either C/EBPε or PU.1. Both GABP and PU.1 bound Ets sites in the Lbr promoter in vitro, and in vivo within both early myeloid progenitors and differentiating neutrophils. These findings demonstrate that GABP, PU.1, and C/EBPε cooperate to control transcription of the gene encoding LBR, a nuclear envelope protein that is required for the characteristic lobulated morphology of mature neutrophils. PMID:27342846

Psychiatric risk factor ANK3/Ankyrin-G nanodomains regulate the structure and function of glutamatergic synapses

PubMed Central

Smith, Katharine R.; Kopeikina, Katherine J.; Fawcett-Patel, Jessica M.; Leaderbrand, Katherine; Gao, Ruoqi; Schürmann, Britta; Myczek, Kristoffer; Radulovic, Jelena; Swanson, Geoffrey T.; Penzes, Peter

2014-01-01

Summary Recent evidence implicates glutamatergic synapses as key pathogenic sites in psychiatric disorders. Common and rare variants in the ANK3 gene, encoding ankyrin-G, have been associated with bipolar disorder, schizophrenia, and autism. Here we demonstrate that ankyrin-G is integral to AMPAR-mediated synaptic transmission and maintenance of spine morphology. Using super-resolution microscopy we find that ankyrin-G forms distinct nanodomain structures within the spine head and neck. At these sites, it modulates mushroom spine structure and function, likely as a perisynaptic scaffold and barrier within the spine neck. Neuronal activity promotes ankyrin-G accumulation in distinct spine subdomains, where it differentially regulates NMDA receptor-dependent plasticity. These data implicate subsynaptic nanodomains containing a major psychiatric risk molecule, ankyrin-G, as having location-specific functions, and opens directions for basic and translational investigation of psychiatric risk molecules. PMID:25374361

A central role for vesicle trafficking in epithelial neoplasia: Intracellular highways to carcinogenesis

PubMed Central

Goldenring, James R.

2014-01-01

Epithelial cell carcinogenesis involves the loss of polarity, alteration of polarized protein presentation, dynamic cell morphology changes, increased proliferation and increased cell motility and invasion. Elements of membrane vesicle trafficking underlie all of these processes. Specific membrane trafficking regulators, including Rab small GTPases, through the coordinated dynamics of intracellular trafficking along cytoskeletal pathways, determine cell surface presentation of proteins and overall function of both differentiated and neoplastic cells. While mutations in vesicle trafficking proteins may not be direct drivers of transformation, elements of the machinery of vesicle movement play critical roles in the phenotypes of neoplastic cells. Therefore, the regulators of membrane vesicle trafficking decisions are critical mediators of the full spectrum of cell physiologies driving cancer cell biology, including initial loss of polarity, invasion and metastasis. Targeting of these fundamental intracellular processes may provide important points for manipulation of cancer cell behaviour. PMID:24108097

Dynamic Reciprocity in the Wound Microenvironment

PubMed Central

Schultz, Gregory S.; Davidson, Jeffrey M.; Kirsner, Robert S.; Bornstein, Paul; Herman, Ira M.

2011-01-01

Here, we define dynamic reciprocity (DR) as an ongoing, bidirectional interaction amongst cells and their surrounding microenvironment. In the review, we posit that DR is especially meaningful during wound healing as the DR-driven biochemical, biophysical and cellular responses to injury play pivotal roles in regulating tissue regenerative responses. Such cell-extracellular matrix interactions not only guide and regulate cellular morphology, but cellular differentiation, migration, proliferation, and survival during tissue development, including e.g. embryogenesis, angiogenesis, as well as during pathologic processes including cancer diabetes, hypertension and chronic wound healing. Herein, we examine DR within the wound microenvironment while considering specific examples across acute and chronic wound healing. This review also considers how a number of hypotheses that attempt to explain chronic wound pathophysiology, which may be understood within the DR framework. The implications of applying the principles of dynamic reciprocity to optimize wound care practice and future development of innovative wound healing therapeutics are also briefly considered. PMID:21362080

Rac1 and Rac3 have opposing functions in cell adhesion and differentiation of neuronal cells.

PubMed

Hajdo-Milasinović, Amra; Ellenbroek, Saskia I J; van Es, Saskia; van der Vaart, Babet; Collard, John G

2007-02-15

Rac1 and Rac3 are highly homologous members of the Rho small GTPase family. Rac1 is ubiquitously expressed and regulates cell adhesion, migration and differentiation in various cell types. Rac3 is primarily expressed in brain and may therefore have a specific function in neuronal cells. We found that depletion of Rac1 by short interference RNA leads to decreased cell-matrix adhesions and cell rounding in neuronal N1E-115 cells. By contrast, depletion of Rac3 induces stronger cell adhesions and dramatically increases the outgrowth of neurite-like protrusions, suggesting opposite functions for Rac1 and Rac3 in neuronal cells. Consistent with this, overexpression of Rac1 induces cell spreading, whereas overexpression of Rac3 results in a contractile round morphology. Rac1 is mainly found at the plasma membrane, whereas Rac3 is predominantly localized in the perinuclear region. Residues 185-187, present in the variable polybasic rich region at the carboxyl terminus are responsible for the difference in phenotype induced by Rac1 and Rac3 as well as for their different intracellular localization. The Rac1-opposing function of Rac3 is not mediated by or dependent on components of the RhoA signaling pathway. It rather seems that Rac3 exerts its function through negatively affecting integrin-mediated cell-matrix adhesions. Together, our data reveal that Rac3 opposes Rac1 in the regulation of cell adhesion and differentiation of neuronal cells.

Sex, genes, and heat: triggers of diversity.

PubMed

Western, P S; Sinclair, A H

2001-11-01

In vertebrates, sex is determined by a surprising variety of mechanisms. In many reptiles, the primary testis or ovary-determining trigger is regulated by egg incubation temperature. This temperature dependent sex determining (TSD) mechanism occurs in all crocodilians and marine turtles examined to date and is common in terrestrial turtles and viviparous lizards (Ewert et al. 1994. J Exp Zool 270:3-15; Lang and Andrews. 1994. J Exp Biol 270:28-44; Mrosovsky. 1994. J Exp Zool 270:16-27; Pieau. 1996. Bioessays 18:19-26; Viets et al. 1994. J Exp Zool 270:45-56; Wibbels et al. 1998. J Exp Zool 281:409-416). In contrast, sex in mammals and birds is determined chromosomally (CSD). Despite these differences, morphological development of the gonads in all these vertebrate groups appears to have been conserved through evolution. Therefore, the genetic mechanisms triggering sex determination appear not to have been conserved through evolution, although the basic genetic pathway controlling the morphological differentiation of the gonads appears to have been conserved. Copyright 2001 Wiley-Liss, Inc.

Fresh Garlic Extract Induces Growth Arrest and Morphological Differentiation of MCF7 Breast Cancer Cells

PubMed Central

DiCarlo, Stephen E.; Reddy, Thipparthi R.

2012-01-01

Consumption of diets rich in fruits and vegetables is often associated with a reduced risk of developing cancer, particularly breast cancer. Considering that 1 in 8 women in the United States will develop breast cancer in the course of her lifetime, dietary manipulation could have a major impact on the incidence of breast cancer. We report here that fresh extracts of garlic (not boiled) arrested the growth and altered the morphology of MCF7 breast cancer cells. Deregulated levels of E-cadherin, cytokeratin8/18, and β-catenin correlated with the altered phenotype. We propose that early down-regulation of cyclin D1, reduced phosphorylation of ERK1, and increased phosphorylation of eIF2-α triggered the phenotypical changes. Reduced expression of hsp27 and sam68 and elevated levels of Rb and p21 further contributed to the sustained growth reduction. These findings provide a better understanding of the cellular responses to dietary supplements and provide potential options to treat breast cancer. PMID:23050048

Mitochondrial Control by DRP1 in Brain Tumor Initiating Cells

PubMed Central

Xie, Qi; Wu, Qiulian; Horbinski, Craig M.; Flavahan, William A.; Yang, Kailin; Zhou, Wenchao; Dombrowski, Stephen M.; Huang, Zhi; Fang, Xiaoguang; Shi, Yu; Ferguson, Ashley N.; Kashatus, David F.; Bao, Shideng; Rich, Jeremy N.

2015-01-01

Brain tumor initiating cells (BTICs) coopt the neuronal high affinity GLUT3 glucose transporter to withstand metabolic stress. Here, we investigated another mechanism critical to brain metabolism, mitochondrial morphology. BTICs displayed mitochondrial fragmentation relative to non-BTICs, suggesting that BTICs have increased mitochondrial fission. The essential mediator of mitochondrial fission, dynamin-related protein 1 (DRP1), was activated in BTICs and inhibited in non-BTICs. Targeting DRP1 using RNA interference or pharmacologic inhibition induced BTIC apoptosis and inhibited tumor growth. Downstream, DRP1 activity regulated the essential metabolic stress sensor, AMP-activated protein kinase (AMPK), and AMPK targeting rescued the effects of DRP1 disruption. Cyclin-dependent kinase 5 (CDK5) phosphorylated DRP1 to increase its activity in BTICs, whereas Ca2+–calmodulin-dependent protein kinase 2 (CAMK2) inhibited DRP1 in non-BTICs, suggesting tumor cell differentiation induces a regulatory switch in mitochondrial morphology. DRP1 activation correlates with poor prognosis in glioblastoma, suggesting mitochondrial dynamics may represent a therapeutic target for BTICs. PMID:25730670

In vitro-microenvironment directs preconditioning of human chorion derived MSC promoting differentiation of OPC-like cells.

PubMed

Periasamy, Ramesh; Surbek, Daniel V; Schoeberlein, Andreina

2018-06-01

The loss of oligodendrocyte progenitor cells (OPC) is a hallmark of perinatal brain injury. Our aim was to develop an in vitro culture condition for human chorion-derived mesenchymal stem cells (MSC) that enhances their stem cell properties and their capability to differentiate towards OPC-like cells. MSC were grown either in serum replacement medium (SRM) or serum-containing medium (SM) and tested for their morphology, proliferation, secretome, migration, protein expression and differentiation into OPC-like cells. MSC cultured in SRM condition have distinct morphology/protein expression profile, increased cell proliferation/migration and capacity to differentiate into OPC-like cells. Copyright © 2018 Elsevier Ltd. All rights reserved.

miRNA Signature and Dicer Requirement during Human Endometrial Stromal Decidualization In Vitro

PubMed Central

Estella, Carlos; Herrer, Isabel; Moreno-Moya, Juan Manuel; Quiñonero, Alicia; Martínez, Sebastián; Pellicer, Antonio; Simón, Carlos

2012-01-01

Decidualization is a morphological and biochemical transformation of endometrial stromal fibroblast into differentiated decidual cells, which is critical for embryo implantation and pregnancy establishment. The complex regulatory networks have been elucidated at both the transcriptome and the proteome levels, however very little is known about the post-transcriptional regulation of this process. miRNAs regulate multiple physiological pathways and their de-regulation is associated with human disorders including gynaecological conditions such as endometriosis and preeclampsia. In this study we profile the miRNAs expression throughout human endometrial stromal (hESCs) decidualization and analyze the requirement of the miRNA biogenesis enzyme Dicer during this process. A total of 26 miRNAs were upregulated and 17 miRNAs downregulated in decidualized hESCs compared to non-decidualized hESCs. Three miRNAs families, miR-181, miR-183 and miR-200, are down-regulated during the decidualization process. Using miRNAs target prediction algorithms we have identified the potential targets and pathways regulated by these miRNAs. The knockdown of Dicer has a minor effect on hESCs during in vitro decidualization. We have analyzed a battery of decidualization markers such as cell morphology, Prolactin, IGFBP-1, MPIF-1 and TIMP-3 secretion as well as HOXA10, COX2, SP1, C/EBPß and FOXO1 expression in decidualized hESCs with decreased Dicer function. We found decreased levels of HOXA10 and altered intracellular organization of actin filaments in Dicer knockdown decidualized hESCs compared to control. Our results provide the miRNA signature of hESC during the decidualization process in vitro. We also provide the first functional characterization of Dicer during human endometrial decidualization although surprisingly we found that Dicer plays a minor role regulating this process suggesting that alternative biogenesis miRNAs pathways must be involved in human endometrial decidualization. PMID:22911744

Neural Differentiation of Mesenchymal Stem Cells on Scaffolds for Nerve Tissue Engineering Applications.

PubMed

Quintiliano, Kerlin; Crestani, Thayane; Silveira, Davi; Helfer, Virginia Etges; Rosa, Annelise; Balbueno, Eduardo; Steffens, Daniela; Jotz, Geraldo Pereira; Pilger, Diogo André; Pranke, Patricia

2016-11-01

Scaffolds produced by electrospinning act as supports for cell proliferation and differentiation, improved through the release of neurotrophic factors. The objective of this study was to develop aligned and random nanofiber scaffolds with and without nerve growth factor to evaluate the potential of mesenchymal stem cells (MSCs) for neural differentiation. Nanofiber morphology, diameter, degradability, cell morphology, adhesion, proliferation, viability, cytotoxicity, and neural differentiation were performed to characterize the scaffolds. The expression for nestin, β-III tubulin, and neuron-specific enolase was also evaluated. The scaffolds demonstrated a satisfactory environment for MSC growth, being nontoxic. The MSCs cultivated on the scaffolds were able to adhere and proliferate. The evaluation of neural differentiation indicated that in all groups of scaffolds the MSCs were able to upregulate neural gene expression.

Morphological similarity and ecological overlap in two rotifer species.

PubMed

Gabaldón, Carmen; Montero-Pau, Javier; Serra, Manuel; Carmona, María José

2013-01-01

Co-occurrence of cryptic species raises theoretically relevant questions regarding their coexistence and ecological similarity. Given their great morphological similitude and close phylogenetic relationship (i.e., niche retention), these species will have similar ecological requirements and are expected to have strong competitive interactions. This raises the problem of finding the mechanisms that may explain the coexistence of cryptic species and challenges the conventional view of coexistence based on niche differentiation. The cryptic species complex of the rotifer Brachionus plicatilis is an excellent model to study these questions and to test hypotheses regarding ecological differentiation. Rotifer species within this complex are filtering zooplankters commonly found inhabiting the same ponds across the Iberian Peninsula and exhibit an extremely similar morphology-some of them being even virtually identical. Here, we explore whether subtle differences in body size and morphology translate into ecological differentiation by comparing two extremely morphologically similar species belonging to this complex: B. plicatilis and B. manjavacas. We focus on three key ecological features related to body size: (1) functional response, expressed by clearance rates; (2) tolerance to starvation, measured by growth and reproduction; and (3) vulnerability to copepod predation, measured by the number of preyed upon neonates. No major differences between B. plicatilis and B. manjavacas were found in the response to these features. Our results demonstrate the existence of a substantial niche overlap, suggesting that the subtle size differences between these two cryptic species are not sufficient to explain their coexistence. This lack of evidence for ecological differentiation in the studied biotic niche features is in agreement with the phylogenetic limiting similarity hypothesis but requires a mechanistic explanation of the coexistence of these species not based on differentiation related to biotic niche axes.

Effects of nicotine in the presence and absence of vitamin E on morphology, viability and osteogenic gene expression in MG-63 osteoblast-like cells.

PubMed

Torshabi, Maryam; Esfahrood, Zeinab Rezaei; Gholamin, Parisan; Karami, Elahe

2016-11-01

Evidence shows that oxidative stress induced by nicotine plays an important role in bone loss. Vitamin E with its antioxidative properties may be able to reverse the effects of nicotine on bone. This study aimed to assess the effects of nicotine in the presence and absence of vitamin E on morphology, viability and osteogenic gene expression in MG-63 (osteosarcoma) human osteoblast-like cells. We treated the cells with 5 mM nicotine. The viability and morphology of cells were evaluated respectively using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium (MTT) and crystal violet assays. The effect of nicotine on osteogenic gene expression in MG-63 cells was assessed by real-time reverse-transcription polymerase chain reaction of osteoblast markers, namely, alkaline phosphatase, osteocalcin and bone sialoprotein. The results revealed that survival and proliferation of MG-63 cells were suppressed following exposure to nicotine, and cytoplasm vacuolization occurred in the cells. Nicotine significantly down-regulated the expression of osteogenic marker genes. Such adverse effects on morphology, viability and osteogenic gene expression of MG-63 cells were reversed by vitamin E therapy. In conclusion, vitamin E supplementation may play a role in proliferation and differentiation of osteoblasts, and vitamin E can be considered as an anabolic agent to treat nicotine-induced bone loss.

Major Differences in Hypoxia Tolerance and P38 Regulation Among Different Renal Cells.

PubMed

Shi, Qianqian; Shi, Jian; Luo, Fengbao; Song, Guanglai; He, Xiaozhou; Xia, Ying

2018-01-01

Mitogen-activated protein kinases (MAPKs) are involved in the cellular response to hypoxia and their dysregulation may contribute to the progression and pathology of diverse human renal diseases. Recent studies suggest that the regulation of MAPK responses to hypoxic stress may be different in different cells, even within the same organ. However, it is unclear if MAPKs are differentially regulated in different renal cells in hypoxia. This work was carried out to clarify this fundamental issue. We cultured normal rat kidney epithelial (NRK-52E) cells, human kidney epithelial (HK-2) cells and human renal cell adenocarcinoma (769-P) cells simultaneously under normoxia and hypoxia (1% O2) for 24-72 hours. The protein levels of P-ERK1/2, ERK1/2, P-p38, p38 and eEF2K were detected by western blotting. The morphology of all cells was examined using light microscopy. Under the same hypoxic condition, P-ERK1/2 was up-regulated in all renal cells. Meanwhile,P-p38 in NRK-52E cells was markedly increased after hypoxia for 24-72 hours, while it appeared to show no appreciable change in HK-2 and 769-P cells exposed to hypoxia for 24-48 hours and significantly decreased in these cells after 72 hours hypoxia. On the other hand, hypoxia markedly down-regulated the expression of eukaryotic elongation factor-2 kinase (eEF2K) in all three cells. Under microscopy, NRK-52E cells had no visible injury after 72 hours hypoxia, while HK-2 and 769-P cells were mostly damaged under the same condition. Our data suggest that in response to prolonged hypoxic stress, ERK1/2 and p38 are differentially regulated in three renal cells, while eEF2K is largely down-regulated in all of these cells. © 2018 The Author(s). Published by S. Karger AG, Basel.

Gene Expression Networks Underlying Ovarian Development in Wild Largemouth Bass (Micropterus salmoides)

PubMed Central

Martyniuk, Christopher J.; Prucha, Melinda S.; Doperalski, Nicholas J.; Antczak, Philipp; Kroll, Kevin J.; Falciani, Francesco; Barber, David S.; Denslow, Nancy D.

2013-01-01

Background Oocyte maturation in fish involves numerous cell signaling cascades that are activated or inhibited during specific stages of oocyte development. The objectives of this study were to characterize molecular pathways and temporal gene expression patterns throughout a complete breeding cycle in wild female largemouth bass to improve understanding of the molecular sequence of events underlying oocyte maturation. Methods Transcriptomic analysis was performed on eight morphologically diverse stages of the ovary, including primary and secondary stages of oocyte growth, ovulation, and atresia. Ovary histology, plasma vitellogenin, 17β-estradiol, and testosterone were also measured to correlate with gene networks. Results Global expression patterns revealed dramatic differences across ovarian development, with 552 and 2070 genes being differentially expressed during both ovulation and atresia respectively. Gene set enrichment analysis (GSEA) revealed that early primary stages of oocyte growth involved increases in expression of genes involved in pathways of B-cell and T-cell receptor-mediated signaling cascades and fibronectin regulation. These pathways as well as pathways that included adrenergic receptor signaling, sphingolipid metabolism and natural killer cell activation were down-regulated at ovulation. At atresia, down-regulated pathways included gap junction and actin cytoskeleton regulation, gonadotrope and mast cell activation, and vasopressin receptor signaling and up-regulated pathways included oxidative phosphorylation and reactive oxygen species metabolism. Expression targets for luteinizing hormone signaling were low during vitellogenesis but increased 150% at ovulation. Other networks found to play a significant role in oocyte maturation included those with genes regulated by members of the TGF-beta superfamily (activins, inhibins, bone morphogenic protein 7 and growth differentiation factor 9), neuregulin 1, retinoid X receptor, and nerve growth factor family. Conclusions This study offers novel insight into the gene networks underlying vitellogenesis, ovulation and atresia and generates new hypotheses about the cellular pathways regulating oocyte maturation. PMID:23527095

The biology of spermatogenesis: the past, present and future

PubMed Central

Cheng, C. Yan; Mruk, Dolores D.

2010-01-01

The physiological function of spermatogenesis in Caenorhabditis elegans, Drosophila melanogaster and mammals is to produce spermatozoa (1n, haploid) that contain only half of the genetic material of spermatogonia (2n, diploid). This half number of chromosomes from a spermatozoon will then be reconstituted to become a diploid cell upon fertilization with an egg, which is also haploid. Thus, genetic information from two parental individuals can be passed onto their offspring. Spermatogenesis takes place in the seminiferous epithelium of the seminiferous tubule, the functional unit of the mammalian testis. In mammals, particularly in rodents, the fascinating morphological changes that occur during spermatogenesis involving cellular differentiation and transformation, mitosis, meiosis, germ cell movement, spermiogenesis and spermiation have been well documented from the 1950s through the 1980s. During this time, however, the regulation of, as well as the biochemical and molecular mechanisms underlying these diverse cellular events occurring throughout spermatogenesis, have remained largely unexplored. In the past two decades, important advancements have been made using new biochemical, cell and molecular biology techniques to understand how different genes, proteins and signalling pathways regulate various aspects of spermatogenesis. These include studies on the differentiation of spermatogonia from gonocytes; regulation of spermatogonial stem cells; regulation of spermatogonial mitosis; regulation of meiosis, spermiogenesis and spermiation; role of hormones (e.g. oestrogens, androgens) in spermatogenesis; transcriptional regulation of spermatogenesis; regulation of apoptosis; cell–cell interactions; and the biology of junction dynamics during spermatogenesis. The impact of environmental toxicants on spermatogenesis has also become an urgent issue in the field in light of declining fertility levels in males. Many of these studies have helped investigators to understand important similarities, differences and evolutionary relationships between C. elegans, D. melanogaster and mammals relating to spermatogenesis. In this Special Issue of the Philosophical Transactions of the Royal Society B: Biological Sciences, we have covered many of these areas, and in this Introduction, we highlight the topic of spermatogenesis by examining its past, present and future. PMID:20403863

β-Catenin Knockdown Affects Mitochondrial Biogenesis and Lipid Metabolism in Breast Cancer Cells.

PubMed

Vergara, Daniele; Stanca, Eleonora; Guerra, Flora; Priore, Paola; Gaballo, Antonio; Franck, Julien; Simeone, Pasquale; Trerotola, Marco; De Domenico, Stefania; Fournier, Isabelle; Bucci, Cecilia; Salzet, Michel; Giudetti, Anna M; Maffia, Michele

2017-01-01

β-catenin plays an important role as regulatory hub in several cellular processes including cell adhesion, metabolism, and epithelial mesenchymal transition. This is mainly achieved by its dual role as structural component of cadherin-based adherens junctions, and as a key nuclear effector of the Wnt pathway. For this dual role, different classes of proteins are differentially regulated via β-catenin dependent mechanisms. Here, we applied a liquid chromatography-mass spectrometry (LC-MS/MS) approach to identify proteins modulated after β-catenin knockdown in the breast cancer cell line MCF-7. We used a label free analysis to compare trypsin-digested proteins from CTR (shCTR) and β-catenin knockout cells (shβcat). This led to the identification of 98 differentially expressed proteins, 53 of them were up-regulated and 45 down-regulated. Loss of β-catenin induced morphological changes and a significant modulation of the expression levels of proteins associated with primary metabolic processes. In detail, proteins involved in carbohydrate metabolism and tricarboxylic acid cycle were found to be down-regulated, whereas proteins associated to lipid metabolism were found up-regulated in shβcat compared to shCTR. A loss of mitochondrial mass and membrane potential was also assessed by fluorescent probes in shβcat cells with respect to the controls. These data are consistent with the reduced expression of transcriptional factors regulating mitochondrial biogenesis detected in shβcat cells. β-catenin driven metabolic reprogramming resulted also in a significant modulation of lipogenic enzyme expression and activity. Compared to controls, β-catenin knockout cells showed increased incorporation of [1- 14 C]acetate and decreased utilization of [U- 14 C]glucose for fatty acid synthesis. Our data highlight a role of β-catenin in the regulation of metabolism and energy homeostasis in breast cancer cells.

Transcriptomic analysis reveals the roles of gibberellin-regulated genes and transcription factors in regulating bolting in lettuce (Lactuca sativa L.).

PubMed

Liu, Xueying; Lv, Shanshan; Liu, Ran; Fan, Shuangxi; Liu, Chaojie; Liu, Renyi; Han, Yingyan

2018-01-01

A cool temperature is preferred for lettuce cultivation, as high temperatures cause premature bolting. Accordingly, exploring the mechanism of bolting and preventing premature bolting is important for agriculture. To explore this relationship in depth, morphological, physiological, and transcriptomic analyses of the bolting-sensitive line S39 at the five-leaf stage grown at 37°C were performed in the present study. Based on paraffin section results, we observed that S39 began bolting on the seventh day at 37°C. During bolting in the heat-treated plants, GA3 and GA4 levels in leaves and the indoleacetic acid (IAA) level in the stem reached a maximum on the sixth day, and these high contents were maintained. Additionally, bolting begins in the fifth day after GA3 treatment in S39 plants, GA3 and GA4 increased and then decreased, reaching a maximum on the fourth day in leaves. Similarly, IAA contents reached a maximum in the stem on the fifth day. No bolting was observed in the control group grown at 25°C, and significant changes were not observed in GA3 and GA4 levels in the controls during the observation period. RNA-sequencing data implicated transcription factors (TFs) in regulating bolting in lettuce, suggesting that the high GA contents in the leaves and IAA in the stem promote bolting. TFs possibly modulate the expression of related genes, such as those encoding hormones, potentially regulating bolting in lettuce. Compared to the control group, 258 TFs were identified in the stem of the treatment group, among which 98 and 156 were differentially up- and down-regulated, respectively; in leaves, 202 and 115 TFs were differentially up- and down-regulated, respectively. Significant changes in the treated group were observed for C2H2 zinc finger, AP2-EREBP, and WRKY families, indicating that these TFs may play important roles in regulating bolting.

The effect of Platelet Lysate on osteoblast proliferation associated with a transient increase of the inflammatory response in bone regeneration.

PubMed

Ruggiu, Alessandra; Ulivi, Valentina; Sanguineti, Francesca; Cancedda, Ranieri; Descalzi, Fiorella

2013-12-01

Platelet Lysate (PL) contains a cocktail of growth factors and cytokines, which actively participates in tissue repair and its clinical application has been broadly described. The aim of this study was to assess the regenerative potential of PL for bone repair. We demonstrated that PL stimulation induces a transient increase of the inflammatory response in quiescent human osteoblasts, via NF-kB activation, COX-2 induction, PGE2 production and secretion of pro-inflammatory cytokines. Furthermore, we showed that long-term PL stimulation enhances proliferation of actively replicating osteoblasts, without affecting their differentiation potential, along with changes of cell morphology, resulting in increased cell density at confluence. In confluent resting osteoblasts, PL treatment induced resumption of proliferation, change in cell morphology and increase of cell density at confluence. A burst of PL treatment (24-h) was sufficient to trigger such processes in both conditions. These results correlated with up-regulation of the proliferative and survival pathways ERKs and Akt and with cell cycle re-activation via induction of CyclinD1 and phosphorylation of Rb, following PL stimulation. Our findings demonstrate that PL treatment results in activation and expansion of resting osteoblasts, without affecting their differentiation potential. Therefore PL represents a good therapeutic candidate in regenerative medicine for bone repair. Copyright © 2013 Elsevier Ltd. All rights reserved.

MiT Family Translocation-Associated Renal Cell Carcinoma: A Contemporary Update With Emphasis on Morphologic, Immunophenotypic, and Molecular Mimics.

PubMed

Magers, Martin J; Udager, Aaron M; Mehra, Rohit

2015-10-01

Translocation-associated renal cell carcinoma (t-RCC) is a relatively uncommon subtype of renal cell carcinoma characterized by recurrent gene rearrangements involving the TFE3 or TFEB loci. TFE3 and TFEB are members of the microphthalmia transcription factor (MiT) family, which regulates differentiation in melanocytes and osteoclasts, and MiT family gene fusions activate unique molecular programs that can be detected immunohistochemically. Although the overall clinical behavior of t-RCC is variable, emerging molecular data suggest the possibility of targeted approaches to advanced disease. Thus, distinguishing t-RCC from its morphologic, immunophenotypic, and molecular mimics may have important clinical implications. The differential diagnosis for t-RCC includes a variety of common renal neoplasms, particularly those demonstrating clear cell and papillary features; in addition, because of immunophenotypic overlap and/or shared molecular abnormalities (ie, TFE3 gene rearrangement), a distinctive set of nonepithelial renal tumors may also warrant consideration. Directed ancillary testing is an essential aspect to the workup of t-RCC cases and may include a panel of immunohistochemical stains, such as PAX8, pancytokeratins, epithelial membrane antigen, carbonic anhydrase IX, HMB-45, and Melan-A. Dual-color, break-apart fluorescent in situ hybridization for TFE3 or TFEB gene rearrangement may be helpful in diagnostically challenging cases or when molecular confirmation is needed.

Early prenatal androgen exposure reduces testes size and sperm concentration in sheep without altering neuroendocrine differentiation and masculine sexual behavior.

PubMed

Scully, C M; Estill, C T; Amodei, R; McKune, A; Gribbin, K P; Meaker, M; Stormshak, F; Roselli, C E

2018-01-01

Prenatal androgens are largely responsible for growth and differentiation of the genital tract and testis and for organization of the control mechanisms regulating male reproductive physiology and behavior. The aim of the present study was to evaluate the impact of inappropriate exposure to excess testosterone (T) during the first trimester of fetal development on the reproductive function, sexual behavior, and fertility potential of rams. We found that biweekly maternal T propionate (100 mg) treatment administered from Day 30-58 of gestation significantly decreased (P < 0.05) postpubertal scrotal circumference and sperm concentration. Prenatal T exposure did not alter ejaculate volume, sperm motility and morphology or testis morphology. There was, however, a trend for more T-exposed rams than controls to be classified as unsatisfactory potential breeders during breeding soundness examinations. Postnatal serum T concentrations were not affected by prenatal T exposure, nor was the expression of key testicular genes essential for spermatogenesis and steroidogenesis. Basal serum LH did not differ between treatment groups, nor did pituitary responsiveness to GnRH. T-exposed rams, like control males, exhibited vigorous libido and were sexually attracted to estrous females. In summary, these results suggest that exposure to exogenous T during the first trimester of gestation can negatively impact spermatogenesis and compromise the reproductive fitness of rams. Copyright © 2017 Elsevier Inc. All rights reserved.

Xin, an actin binding protein, is expressed within muscle satellite cells and newly regenerated skeletal muscle fibers.

PubMed

Hawke, Thomas J; Atkinson, Daniel J; Kanatous, Shane B; Van der Ven, Peter F M; Goetsch, Sean C; Garry, Daniel J

2007-11-01

Xin is a muscle-specific actin binding protein of which its role and regulation within skeletal muscle is not well understood. Here we demonstrate that Xin mRNA is robustly upregulated (>16-fold) within 12 h of skeletal muscle injury and is localized to the muscle satellite cell population. RT-PCR confirmed the expression pattern of Xin during regeneration, as well as within primary muscle myoblast cultures, but not other known stem cell populations. Immunohistochemical staining of single myofibers demonstrate Xin expression colocalized with the satellite cell marker Syndecan-4 further supporting the mRNA expression of Xin in satellite cells. In situ hybridization of regenerating muscle 5-7 days postinjury illustrates Xin expression within newly regenerated myofibers. Promoter-reporter assays demonstrate that known myogenic transcription factors [myocyte enhancer factor-2 (MEF2), myogenic differentiation-1 (MyoD), and myogenic factor-5 (Myf-5)] transactivate Xin promoter constructs supporting the muscle-specific expression of Xin. To determine the role of Xin within muscle precursor cells, proliferation, migration, and differentiation analysis using Xin, short hairpin RNA (shRNA) were undertaken in C2C12 myoblasts. Reducing endogenous Xin expression resulted in a 26% increase (P < 0.05) in cell proliferation and a 20% increase (P < 0.05) in myoblast migratory capacity. Skeletal muscle myosin heavy chain protein levels were increased (P < 0.05) with Xin shRNA administration; however, this was not accompanied by changes in myoglobin protein (another marker of differentiation) nor overt morphological differences relative to differentiating control cells. Taken together, the present findings support the hypothesis that Xin is expressed within muscle satellite cells during skeletal muscle regeneration and is involved in the regulation of myoblast function.

The proliferation and differentiation of primary pig preadipocytes is suppressed when cultures are incubated at 37°Celsius compared to euthermic conditions in pigs

PubMed Central

Bohan, Amy E; Purvis, Katelyn N; Bartosh, Julia L; Brandebourg, Terry D

2014-01-01

Given similarities in metabolic parameters and cardiovascular physiology, the pig is well positioned as a biomedical model for metabolic disease and obesity in humans. Better understanding molecular mechanisms governing porcine adipocyte hyperplasia may provide insight into the regulation of adipose tissue development that is useful both when considering the pig as a commodity and when extrapolating porcine data to human disease. Primary cultures of pig stromal-vascular cells have served as a useful tool for investigating factors that regulate preadipocyte proliferation and differentiation. However, such cultures have generally been maintained at 37°C in vitro despite euthermia being 39°C in pigs. To address potential concerns about the physiological relevance of culturing primary pig preadipocytes under what would be hypothermic conditions in vivo, the objective of this study was to investigate the effect of culture temperature on the proliferation and differentiation of pig preadipocytes in primary culture. Culturing primary preadipocytes at 37 rather than 39°C decreases their proliferation rates based upon cleavage of the tetrazolium salt, MTT (P < 0.001), reduction of resazurin (P < 0.001), and daily cell counts (P < 0.001). Likewise, culturing primary porcine preadipocytes at 37°C suppressed their adipogenic potential based upon monitoring adipogenesis morphologically, biochemically, and via the expression of mRNA encoding adipogenic marker genes. Collectively, these data indicate the proliferation and differentiation of primary pig preadipocytes is suppressed when cultures are incubated at 37°C compared to normal body temperature of pigs. This may confound investigation of factors that impact adipocyte hyperplasia in the pig. PMID:26317057

Compound-specific effects of diverse neurodevelopmental toxicants on global gene expression in the neural embryonic stem cell test (ESTn)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Theunissen, P.T., E-mail: Peter.Theunissen@rivm.nl; Department of Toxicogenomics, Maastricht University, Maastricht; Robinson, J.F.

Alternative assays for developmental toxicity testing are needed to reduce animal use in regulatory toxicology. The in vitro murine neural embryonic stem cell test (ESTn) was designed as an alternative for neurodevelopmental toxicity testing. The integration of toxicogenomic-based approaches may further increase predictivity as well as provide insight into underlying mechanisms of developmental toxicity. In the present study, we investigated concentration-dependent effects of six mechanistically diverse compounds, acetaldehyde (ACE), carbamazepine (CBZ), flusilazole (FLU), monoethylhexyl phthalate (MEHP), penicillin G (PENG) and phenytoin (PHE), on the transcriptome and neural differentiation in the ESTn. All compounds with the exception of PENG altered ESTnmore » morphology (cytotoxicity and neural differentiation) in a concentration-dependent manner. Compound induced gene expression changes and corresponding enriched gene ontology biological processes (GO–BP) were identified after 24 h exposure at equipotent differentiation-inhibiting concentrations of the compounds. Both compound-specific and common gene expression changes were observed between subsets of tested compounds, in terms of significance, magnitude of regulation and functionality. For example, ACE, CBZ and FLU induced robust changes in number of significantly altered genes (≥ 687 genes) as well as a variety of GO–BP, as compared to MEHP, PHE and PENG (≤ 55 genes with no significant changes in GO–BP observed). Genes associated with developmentally related processes (embryonic morphogenesis, neuron differentiation, and Wnt signaling) showed diverse regulation after exposure to ACE, CBZ and FLU. In addition, gene expression and GO–BP enrichment showed concentration dependence, allowing discrimination of non-toxic versus toxic concentrations on the basis of transcriptomics. This information may be used to define adaptive versus toxic responses at the transcriptome level.« less

DCGL v2.0: an R package for unveiling differential regulation from differential co-expression.

PubMed

Yang, Jing; Yu, Hui; Liu, Bao-Hong; Zhao, Zhongming; Liu, Lei; Ma, Liang-Xiao; Li, Yi-Xue; Li, Yuan-Yuan

2013-01-01

Differential co-expression analysis (DCEA) has emerged in recent years as a novel, systematic investigation into gene expression data. While most DCEA studies or tools focus on the co-expression relationships among genes, some are developing a potentially more promising research domain, differential regulation analysis (DRA). In our previously proposed R package DCGL v1.0, we provided functions to facilitate basic differential co-expression analyses; however, the output from DCGL v1.0 could not be translated into differential regulation mechanisms in a straightforward manner. To advance from DCEA to DRA, we upgraded the DCGL package from v1.0 to v2.0. A new module named "Differential Regulation Analysis" (DRA) was designed, which consists of three major functions: DRsort, DRplot, and DRrank. DRsort selects differentially regulated genes (DRGs) and differentially regulated links (DRLs) according to the transcription factor (TF)-to-target information. DRrank prioritizes the TFs in terms of their potential relevance to the phenotype of interest. DRplot graphically visualizes differentially co-expressed links (DCLs) and/or TF-to-target links in a network context. In addition to these new modules, we streamlined the codes from v1.0. The evaluation results proved that our differential regulation analysis is able to capture the regulators relevant to the biological subject. With ample functions to facilitate differential regulation analysis, DCGL v2.0 was upgraded from a DCEA tool to a DRA tool, which may unveil the underlying differential regulation from the observed differential co-expression. DCGL v2.0 can be applied to a wide range of gene expression data in order to systematically identify novel regulators that have not yet been documented as critical. DCGL v2.0 package is available at http://cran.r-project.org/web/packages/DCGL/index.html or at our project home page http://lifecenter.sgst.cn/main/en/dcgl.jsp.

Increased curvature of hollow fiber membranes could up-regulate differential functions of renal tubular cell layers.

PubMed

Shen, Chong; Meng, Qin; Zhang, Guoliang

2013-08-01

Tissue engineering devices as in vitro cell culture systems in scaffolds has encountered the bottleneck due to their much lower cell functions than real tissues/organs in vivo. Such situation has been improved in some extent by mimicking the cell microenvironments in vivo from either chemical or physical ways. However, microenvironmental curvature, commonly seen in real tissues/organs, has never been manipulated to regulate the cell performance in vitro. In this regard, this paper fabricated polysulfone membranes with or without polyethylene glycol modification to investigate the impact of curvature on two renal tubular cells. Regardless the varying membrane curvatures among hollow fiber membranes of different diameters and flat membrane of zero curvature, both renal cells could well attach at 4 h of seeding and form similar confluent layers at 6 days on each membrane. Nevertheless, the renal cells on hollow fibers, though showing confluent morphology as those on flat membranes, expressed higher renal functions and, moreover, the renal functions significantly increased with the membrane curvature among hollow fibers. Such upregulation on functions was unassociated with mass transport barrier of hollow fibers, because the cultures on lengthwise cut hollow fibers without mass transfer barrier showed same curvature effect on renal functions as whole hollow fibers. It could be proposed that the curvature of hollow fiber membrane approaching to the large curvature in kidney tubules increased the mechanical stress in the renal cells and thus might up-regulate the renal cell functions. In conclusion, the increase of substrate curvature could up-regulate the cell functions without altering the confluent cell morphology and this finding will facilitate the design of functional tissue engineering devices. Copyright © 2013 Wiley Periodicals, Inc.

Regulation of mitochondrial bioenergetics by the non-canonical roles of mitochondrial dynamics proteins in the heart.

PubMed

Wang, Wang; Fernandez-Sanz, Celia; Sheu, Shey-Shing

2018-05-01

Recent advancement in mitochondrial research has significantly extended our knowledge on the role and regulation of mitochondria in health and disease. One important breakthrough is the delineation of how mitochondrial morphological changes, termed mitochondrial dynamics, are coupled to the bioenergetics and signaling functions of mitochondria. In general, it is believed that fusion leads to an increased mitochondrial respiration efficiency and resistance to stress-induced dysfunction while fission does the contrary. This concept seems not applicable to adult cardiomyocytes. The mitochondria in adult cardiomyocytes exhibit fragmented morphology (tilted towards fission) and show less networking and movement as compared to other cell types. However, being the most energy-demanding cells, cardiomyocytes in the adult heart possess vast number of mitochondria, high level of energy flow, and abundant mitochondrial dynamics proteins. This apparent discrepancy could be explained by recently identified new functions of the mitochondrial dynamics proteins. These "non-canonical" roles of mitochondrial dynamics proteins range from controlling inter-organelle communication to regulating cell viability and survival under metabolic stresses. Here, we summarize the newly identified non-canonical roles of mitochondrial dynamics proteins. We focus on how these fission and fusion independent roles of dynamics proteins regulate mitochondrial bioenergetics. We also discuss potential molecular mechanisms, unique intracellular location, and the cardiovascular disease relevance of these non-canonical roles of the dynamics proteins. We propose that future studies are warranted to differentiate the canonical and non-canonical roles of dynamics proteins and to identify new approaches for the treatment of heart diseases. This article is part of a Special issue entitled Cardiac adaptations to obesity, diabetes and insulin resistance, edited by Professors Jan F.C. Glatz, Jason R.B. Dyck and Christine Des Rosiers. Copyright © 2017 Elsevier B.V. All rights reserved.

GSK126 alleviates the obesity phenotype by promoting the differentiation of thermogenic beige adipocytes in diet-induced obese mice.

PubMed

Wu, Xiaohui; Wang, Yuying; Wang, Yingmei; Wang, Xinli; Li, Jianqiang; Chang, Kaixuan; Sun, Cheng; Jia, Zhen; Gao, Song; Wei, Jiachang; Xu, Jiuhang; Xu, Yuqiao; Li, Qing

2018-06-18

A close relationship between epigenetic regulation and obesity has been demonstrated in several recent studies. Histone methyltransferase enhancer of Zeste homolog 2 (Ezh2), which mainly catalyzes trimethylation of histone H3K27 to form H3K27me3 was found to be required for the differentiation of white and brown adipocytes in vitro. Here, we investigated the effects of the Ezh2-specific inhibitor GSK126 in a mouse model of obesity induced by a high-fat diet (HFD). We found that GSK126 treatment reduced body fat, improved glucose tolerance, increased lipolysis and improved cold tolerance in mice by promoting the differentiation of thermogenic beige adipocytes. Moreover, we discovered that GSK126 inhibited the differentiation of white adipocytes, and the decrease of Ezh2 enzymatic activity and H3K27me3 also changed the morphology of brown adipocytes but did not alter the expression of thermogenic genes in these cells. Our results indicated that GSK126 was a novel chemical inducer of beige adipocytes and may be a potential therapeutic agent for the management of obesity. Furthermore, they also prompted that Ezh2 and H3K27me3 play different roles in the differentiation of the white, brown, and beige adipocytes in vivo. Copyright © 2018 Elsevier Inc. All rights reserved.

An Integrated Genomic Approach for Rapid Delineation of Candidate Genes Regulating Agro-Morphological Traits in Chickpea

PubMed Central

Saxena, Maneesha S.; Bajaj, Deepak; Das, Shouvik; Kujur, Alice; Kumar, Vinod; Singh, Mohar; Bansal, Kailash C.; Tyagi, Akhilesh K.; Parida, Swarup K.

2014-01-01

The identification and fine mapping of robust quantitative trait loci (QTLs)/genes governing important agro-morphological traits in chickpea still lacks systematic efforts at a genome-wide scale involving wild Cicer accessions. In this context, an 834 simple sequence repeat and single-nucleotide polymorphism marker-based high-density genetic linkage map between cultivated and wild parental accessions (Cicer arietinum desi cv. ICC 4958 and Cicer reticulatum wild cv. ICC 17160) was constructed. This inter-specific genetic map comprising eight linkage groups spanned a map length of 949.4 cM with an average inter-marker distance of 1.14 cM. Eleven novel major genomic regions harbouring 15 robust QTLs (15.6–39.8% R2 at 4.2–15.7 logarithm of odds) associated with four agro-morphological traits (100-seed weight, pod and branch number/plant and plant hairiness) were identified and mapped on chickpea chromosomes. Most of these QTLs showed positive additive gene effects with effective allelic contribution from ICC 4958, particularly for increasing seed weight (SW) and pod and branch number. One robust SW-influencing major QTL region (qSW4.2) has been narrowed down by combining QTL mapping with high-resolution QTL region-specific association analysis, differential expression profiling and gene haplotype-based association/LD mapping. This enabled to delineate a strong SW-regulating ABI3VP1 transcription factor (TF) gene at trait-specific QTL interval and consequently identified favourable natural allelic variants and superior high seed weight-specific haplotypes in the upstream regulatory region of this gene showing increased transcript expression during seed development. The genes (TFs) harbouring diverse trait-regulating QTLs, once validated and fine-mapped by our developed rapid integrated genomic approach and through gene/QTL map-based cloning, can be utilized as potential candidates for marker-assisted genetic enhancement of chickpea. PMID:25335477

Proliferation and differentiation of osteoblastic cells on titanium modified by ammonia plasma immersion ion implantation

NASA Astrophysics Data System (ADS)

Liu, Fei; Li, Bin; Sun, Junying; Li, Hongwei; Wang, Bing; Zhang, Shailin

2012-03-01

We report here a new method of titanium surface modification through ammonia (NH3) plasma immersion ion implantation (PIII) technique and its effect on the cellular behaviors of MC3T3-E1 osteoblastic cells. The NH3 PIII-treated titanium substrates (NH3-Ti) were characterized by X-ray photoelectron (XPS), which showed that NH3-Ti had a nitrogen-rich surface. However, there was no significant difference between the surface morphology of NH3-Ti and unmodified Ti. When MC3T3-E1 cells were cultured on NH3-Ti substrates, it was found that cell proliferation was accelerated at 4 and 7 days of culture. Meanwhile, cell differentiation was evaluated using type I collagen (COL I), osteocalcin (OC) and bone sialoprotein (BSP) as differentiation markers. It was found that expression of COL I and OC genes was up-regulated on NH3-Ti substrates. However, no significant difference was found in BSP gene expression between NH3-Ti and unmodified Ti substrates. Therefore, findings from this study indicate that surface modification of titanium through NH3 PIII favors osteoblastic proliferation and differentiation and as a result, it may be used to improve the biocompatibility of Ti implants in vivo.

Basal Lamina Mimetic Nanofibrous Peptide Networks for Skeletal Myogenesis

NASA Astrophysics Data System (ADS)

Yasa, I. Ceren; Gunduz, Nuray; Kilinc, Murat; Guler, Mustafa O.; Tekinay, Ayse B.

2015-11-01

Extracellular matrix (ECM) is crucial for the coordination and regulation of cell adhesion, recruitment, differentiation and death. Therefore, equilibrium between cell-cell and cell-matrix interactions and matrix-associated signals are important for the normal functioning of cells, as well as for regeneration. In this work, we describe importance of adhesive signals for myoblast cells’ growth and differentiation by generating a novel ECM mimetic peptide nanofiber scaffold system. We show that not only structure but also composition of bioactive signals are important for cell adhesion, growth and differentiation by mimicking the compositional and structural properties of native skeletal muscle basal lamina. We conjugated laminin-derived integrin binding peptide sequence, “IKVAV”, and fibronectin-derived well known adhesive sequence, “RGD”, into peptide nanostructures to provide adhesive and myogenic cues on a nanofibrous morphology. The myogenic and adhesive signals exhibited a synergistic effect on model myoblasts, C2C12 cells. Our results showed that self-assembled peptide nanofibers presenting laminin derived epitopes support adhesion, growth and proliferation of the cells and significantly promote the expression of skeletal muscle-specific marker genes. The functional peptide nanofibers used in this study present a biocompatible and biodegradable microenvironment, which is capable of supporting the growth and differentiation of C2C12 myoblasts into myotubes.

Basal Lamina Mimetic Nanofibrous Peptide Networks for Skeletal Myogenesis

PubMed Central

Yasa, I. Ceren; Gunduz, Nuray; Kilinc, Murat; Guler, Mustafa O.; Tekinay, Ayse B.

2015-01-01

Extracellular matrix (ECM) is crucial for the coordination and regulation of cell adhesion, recruitment, differentiation and death. Therefore, equilibrium between cell-cell and cell-matrix interactions and matrix-associated signals are important for the normal functioning of cells, as well as for regeneration. In this work, we describe importance of adhesive signals for myoblast cells’ growth and differentiation by generating a novel ECM mimetic peptide nanofiber scaffold system. We show that not only structure but also composition of bioactive signals are important for cell adhesion, growth and differentiation by mimicking the compositional and structural properties of native skeletal muscle basal lamina. We conjugated laminin-derived integrin binding peptide sequence, “IKVAV”, and fibronectin-derived well known adhesive sequence, “RGD”, into peptide nanostructures to provide adhesive and myogenic cues on a nanofibrous morphology. The myogenic and adhesive signals exhibited a synergistic effect on model myoblasts, C2C12 cells. Our results showed that self-assembled peptide nanofibers presenting laminin derived epitopes support adhesion, growth and proliferation of the cells and significantly promote the expression of skeletal muscle-specific marker genes. The functional peptide nanofibers used in this study present a biocompatible and biodegradable microenvironment, which is capable of supporting the growth and differentiation of C2C12 myoblasts into myotubes. PMID:26555958

Nicotinamide: a vitamin able to shift macrophage differentiation toward macrophages with restricted inflammatory features.

PubMed

Weiss, Ronald; Schilling, Erik; Grahnert, Anja; Kölling, Valeen; Dorow, Juliane; Ceglarek, Uta; Sack, Ulrich; Hauschildt, Sunna

2015-11-01

The differentiation of human monocytes into macrophages is influenced by environmental signals. Here we asked in how far nicotinamide (NAM), a vitamin B3 derivative known to play a major role in nicotinamide adenine dinucleotide (NAD)-mediated signaling events, is able to modulate monocyte differentiation into macrophages developed in the presence of granulocyte macrophage colony-stimulating factor (GM-MØ) or macrophage colony-stimulating factor (M-MØ). We found that GM-MØ undergo biochemical, morphological and functional modifications in response to NAM, whereas M-MØ were hardly affected. GM-MØ exposed to NAM acquired an M-MØ-like structure while the LPS-induced production of pro-inflammatory cytokines and COX-derived eicosanoids were down-regulated. In contrast, NAM had no effect on the production of IL-10 or the cytochrome P450-derived eicosanoids. Administration of NAM enhanced intracellular NAD concentrations; however, it did not prevent the LPS-mediated drain on NAD pools. In search of intracellular molecular targets of NAM known to be involved in LPS-induced cytokine and eicosanoid synthesis, we found NF-κB activity to be diminished. In conclusion, our data show that vitamin B3, when present during the differentiation of monocytes into GM-MØ, interferes with biochemical pathways resulting in strongly reduced pro-inflammatory features. © The Author(s) 2015.

Inducible growth mode switches influence Valonia rhizoid differentiation.

PubMed

Elvira, Paul Rommel; Sekida, Satoko; Okuda, Kazuo

2013-02-01

Cell differentiation and cell type commitment are an integral part of plant growth and development. Investigations on how environmental conditions affect the formation of shoots, roots, and rhizoids can help illustrate how plants determine cell fate and overall morphology. In this study, we evaluated the role of substratum and light on rhizoid differentiation in the coenocytic green alga, Valonia aegagropila. Elongating rhizoids displayed varying growth modes and cell shape upon exposure to different substrata and light conditions. It was found that soft substrata and dark incubation promoted rhizoid elongation via tip growth while subsequent exposure to light prevented tip growth and instead induced swelling in the apical region of rhizoids. Swelling was accompanied by the accumulation of protoplasm in the rhizoid tip through expansion of the cell wall and uninhibited cytoplasmic streaming. Subsequent diffuse growth led to the transformation from slender, rod-shaped rhizoids into spherical thallus-like structures that required photosynthesis. Further manipulation of light regimes caused vacillating cell growth redirections. An elongating V. aegagropila rhizoid cell thus appears capable of growth mode switching that is regulated by immediate environmental conditions thereby influencing ultimate cell shape and function. This is the first description of inducible, multiple growth mode shifts in a single intact plant cell that directly impact its differentiation.

Impact of low oxygen tension on stemness, proliferation and differentiation potential of human adipose-derived stem cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Choi, Jane Ru; Pingguan-Murphy, Belinda; Wan Abas, Wan Abu Bakar

2014-05-30

Highlights: • Hypoxia maintains the stemness of adipose-derived stem cells (ASCs). • ASCs show an increased proliferation rate under low oxygen tension. • Oxygen level as low as 2% enhances the chondrogenic differentiation potential of ASCs. • HIF-1α may regulate the proliferation and differentiation activities of ASCs under hypoxia. - Abstract: Adipose-derived stem cells (ASCs) have been found adapted to a specific niche with low oxygen tension (hypoxia) in the body. As an important component of this niche, oxygen tension has been known to play a critical role in the maintenance of stem cell characteristics. However, the effect of O{submore » 2} tension on their functional properties has not been well determined. In this study, we investigated the effects of O{sub 2} tension on ASCs stemness, differentiation and proliferation ability. Human ASCs were cultured under normoxia (21% O{sub 2}) and hypoxia (2% O{sub 2}). We found that hypoxia increased ASC stemness marker expression and proliferation rate without altering their morphology and surface markers. Low oxygen tension further enhances the chondrogenic differentiation ability, but reduces both adipogenic and osteogenic differentiation potential. These results might be correlated with the increased expression of HIF-1α under hypoxia. Taken together, we suggest that growing ASCs under 2% O{sub 2} tension may be important in expanding ASCs effectively while maintaining their functional properties for clinical therapy, particularly for the treatment of cartilage defects.« less

The Effect of Quercetin on the Osteogenesic Differentiation and Angiogenic Factor Expression of Bone Marrow-Derived Mesenchymal Stem Cells

PubMed Central

Zhou, Yuning; Wu, Yuqiong; Jiang, Xinquan; Zhang, Xiuli; Xia, Lunguo; Lin, Kaili; Xu, Yuanjin

2015-01-01

Bone marrow-derived mesenchymal stem cells (BMSCs) are widely used in regenerative medicine in light of their ability to differentiate along the chondrogenic and osteogenic lineages. As a type of traditional Chinese medicine, quercetin has been preliminarily reported to promote osteogenic differentiation in osteoblasts. In the present study, the effects of quercetin on the proliferation, viability, cellular morphology, osteogenic differentiation and angiogenic factor secretion of rat BMSCs (rBMSCs) were examined by MTT assay, fluorescence activated cell sorter (FACS) analysis, real-time quantitative PCR (RT-PCR) analysis, alkaline phosphatase (ALP) activity and calcium deposition assays, and Enzyme-linked immunosorbent assay (ELISA). Moreover, whether mitogen-activated protein kinase (MAPK) signaling pathways were involved in these processes was also explored. The results showed that quercetin significantly enhanced the cell proliferation, osteogenic differentiation and angiogenic factor secretion of rBMSCs in a dose-dependent manner, with a concentration of 2 μM achieving the greatest stimulatory effect. Moreover, the activation of the extracellular signal-regulated protein kinases (ERK) and p38 pathways was observed in quercetin-treated rBMSCs. Furthermore, these induction effects could be repressed by either the ERK inhibitor PD98059 or the p38 inhibitor SB202190, respectively. These data indicated that quercetin could promote the proliferation, osteogenic differentiation and angiogenic factor secretion of rBMSCs in vitro, partially through the ERK and p38 signaling pathways. PMID:26053266

Genetic divergence in the small Indian mongoose (Herpestes auropunctatus), a widely distributed invasive species.

PubMed

Thulin, Carl-Gustaf; Simberloff, Daniel; Barun, Arijana; McCracken, Gary; Pascal, Michel; Islam, M Anwarul

2006-11-01

The combination of founder events, random drift and new selective forces experienced by introduced species typically lowers genetic variation and induces differentiation from the ancestral population. Here, we investigate microsatellite differentiation between introduced and native populations of the small Indian mongoose (Herpestes auropunctatus). Many expectations based on introduction history, such as loss of alleles and relationships among populations, are confirmed. Nevertheless, when applying population assignment methods to our data, we observe a few specimens that are incorrectly assigned and/or appear to have a mixed ancestry, despite estimates of substantial population differentiation. Thus, we suggest that population assignments of individuals should be viewed as tentative and that there should be agreement among different algorithms before assignments are applied in conservation or management. Further, we find no congruence between previously reported morphological differentiation and the sorting of microsatellite variation. Some introduced populations have retained much genetic variation while others have not, irrespective of morphology. Finally, we find alleles from the sympatric grey mongoose (Herpestes edwardsii) in one small Indian mongoose within the native range, suggesting an alternative explanation for morphological differentiation involving a shift in female preferences in allopatry.

Transcriptome analysis of Petunia axillaris flowers reveals genes involved in morphological differentiation and metabolite transport

PubMed Central

Amano, Ikuko; Kitajima, Sakihito; Suzuki, Hideyuki; Koeduka, Takao

2018-01-01

The biosynthesis of plant secondary metabolites is associated with morphological and metabolic differentiation. As a consequence, gene expression profiles can change drastically, and primary and secondary metabolites, including intermediate and end-products, move dynamically within and between cells. However, little is known about the molecular mechanisms underlying differentiation and transport mechanisms. In this study, we performed a transcriptome analysis of Petunia axillaris subsp. parodii, which produces various volatiles in its corolla limbs and emits metabolites to attract pollinators. RNA-sequencing from leaves, buds, and limbs identified 53,243 unigenes. Analysis of differentially expressed genes, combined with gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses, showed that many biological processes were highly enriched in limbs. These included catabolic processes and signaling pathways of hormones, such as gibberellins, and metabolic pathways, including phenylpropanoids and fatty acids. Moreover, we identified five transporter genes that showed high expression in limbs, and we performed spatiotemporal expression analyses and homology searches to infer their putative functions. Our systematic analysis provides comprehensive transcriptomic information regarding morphological differentiation and metabolite transport in the Petunia flower and lays the foundation for establishing the specific mechanisms that control secondary metabolite biosynthesis in plants. PMID:29902274

Demonstration of intermediate cells during human prostate epithelial differentiation in situ and in vitro using triple-staining confocal scanning microscopy.

PubMed

van Leenders, G; Dijkman, H; Hulsbergen-van de Kaa, C; Ruiter, D; Schalken, J

2000-08-01

In human prostate epithelium, morphologically basal and luminal cells can be discriminated. The basal cell layer that putatively contains progenitor cells of the secretory epithelium is characterized by the expression of keratins (K) 5 and 14. Luminal cells represent the secretory compartment of the epithelium and express K8 and 18. We developed a technique for the simultaneous analysis of K5, 14, and 18 to identify intermediate cell stages in the prostate epithelium and to study the dynamic aspects of its differentiation in vitro. Nonmalignant prostate tissue and primary epithelial cultures were immunohistochemically characterized using triple staining with antibodies for K5, K14, and K18. Antibodies for K18 and K5 were conjugated directly with fluorochromes Alexa 488 and 546. K14 was visualized indirectly with streptavidin-Cy5. Keratin expression was analyzed by confocal scanning microscopy. The occurrence of exocrine and neuroendocrine differentiation in culture was determined via antibodies to prostate-specific antigen (PSA), chromogranin A, and serotonin. We found that basal cells expressed either K5(++)/14(++)/18+ or K5(++)/18+. The majority of luminal cells expressed K18(++), but colocalization of K5+/18(++) were recognized. Epithelial monolayer cultures predominantly revealed the basal cell phenotype K5(++)/14(++)/18+, whereas intermediate subpopulations expressing K5+/14+/18(++) and K5+/18(++) were also identified. On confluence, differentiation was induced as multicellular gland-like buds, and extensions became evident on top of the monolayer. These structures were composed of K18(++)- and K5+/18(+)-positive cell clusters surrounded by phenotypically basal cells. Few multicellular structures and cells in the monolayer showed exocrine differentiation (PSA+), but expression of chromogranin A and serotonin was absent. We conclude that simultaneous evaluation of keratin expression is useful for analyzing epithelial differentiation in the prostate. During this process, putative stem cells phenotypically resembling K5(++)/14(++)/18+ differentiate toward luminal cells (K18(++)) via intermediate cell stages, as identified by up-regulation of K18 and down-regulation of K5 and 14.

Gα73B is a downstream effector of JAK/STAT signalling and a regulator of Rho1 in Drosophila haematopoiesis.

PubMed

Bausek, Nina; Zeidler, Martin P

2014-01-01

JAK/STAT signalling regulates many essential developmental processes including cell proliferation and haematopoiesis, whereas its inappropriate activation is associated with the majority of myeloproliferative neoplasias and numerous cancers. Furthermore, high levels of JAK/STAT pathway signalling have also been associated with enhanced metastatic invasion by cancerous cells. Strikingly, gain-of-function mutations in the single Drosophila JAK homologue, Hopscotch, result in haemocyte neoplasia, inappropriate differentiation and the formation of melanised haemocyte-derived 'tumour' masses; phenotypes that are partly orthologous to human gain-of-function JAK2-associated pathologies. Here we show that Gα73B, a novel JAK/STAT pathway target gene, is necessary for JAK/STAT-mediated tumour formation in flies. In addition, although Gα73B does not affect haemocyte differentiation, it does regulate haemocyte morphology and motility under non-pathological conditions. We show that Gα73B is required for constitutive, but not injury-induced, activation of Rho1 and for the localisation of Rho1 into filopodia upon haemocyte activation. Consistent with these results, we also show that Rho1 interacts genetically with JAK/STAT signalling, and that wild-type levels of Rho1 are necessary for tumour formation. Our findings link JAK/STAT transcriptional outputs, Gα73B activity and Rho1-dependent cytoskeletal rearrangements and cell motility, therefore connecting a pathway associated with cancer with a marker indicative of invasiveness. As such, we suggest a mechanism by which JAK/STAT pathway signalling may promote metastasis.

TGFβ1-induced down-regulation of microRNA-138 contributes to epithelial-mesenchymal transition in primary lung cancer cells.

PubMed

Zhang, Fang; Li, Tiepeng; Han, Lu; Qin, Peng; Wu, Zhao; Xu, Benling; Gao, Quanli; Song, Yongping

2018-02-19

The existence of cancer stem cells within the tumor could lead to cancer therapy resistance. TGFβ1 is considered as one of the most powerful players in the generation of CSCs through induction of epithelial-mesenchymal transition in different types of cancer including lung cancer, however, the detailed mechanisms by which TGFβ1 contribute to EMT induction and CSC maintenance remains unclear. Here, we showed primary lung cancer cells treated by TGFβ1 exhibit mesenchymal features, including morphology and expression of mesenchymal marker in a time-dependent manner. We also observed long-term TGFβ1 exposure leads to an enrichment of a sub-population of CD44 + CD90 + cells which represent CSCs in lung cancer cells. Moreover, the differential expression microRNAs between CSCs and non-CSCs were identified using next-generation sequencing to screen key miRNAs which might contribute to TGFβ1-induced EMT and CSCs generation. Among those differentially expressed miRNAs, the expression of microRNA-138 was time-dependently down-regulated by TGFβ1 treatment. We further demonstrated primary lung cancer cells, in which we knockdown the expression of miR-138, exhibit mesenchymal phenotypes and stem cell properties. Taken together, these findings indicate TGFβ1-induced down-regulation of microRNA-138 contributes to EMT in primary lung cancer cells, and suggest that miR-138 might serve as a potential therapeutic target. Copyright © 2018 Elsevier Inc. All rights reserved.

Regulation of Pleiotrophin, Midkine, Receptor Protein Tyrosine Phosphatase β/ζ, and Their Intracellular Signaling Cascades in the Nucleus Accumbens During Opiate Administration

PubMed Central

Laorden, María Luisa; Milanés, María Victoria

2016-01-01

Background: Most classes of addictive substances alter the function and structural plasticity of the brain reward circuitry. Midkine (MK) and pleiotrophin (PTN) are growth/differentiation cytokines which, similarly to neurotrophins, play an important role in repair, neurite outgrowth, and cell differentiation. PTN or MK signaling through receptor protein tyrosine phosphatase β/ζ (RPTPβ/ζ), leads to the activation of extracellular signal-regulated kinases and thymoma viral proto-oncogene. This activation induces morphological changes and modulates addictive behaviors. Besides, there is increasing evidence that during the development of drug addiction, astrocytes contribute to the synaptic plasticity by synthesizing and releasing substances such as cytokines. Methods: In the present work we studied the effect of acute morphine administration, chronic morphine administration, and morphine withdrawal on PTN, MK, and RPTPβ/ζ expression and on their signaling pathways in the nucleus accumbens. Results: Present results indicated that PTN, MK, and RPTPβ/ζ levels increased after acute morphine injection, returned to basal levels during chronic opioid treatment, and were up-regulated again during morphine withdrawal. We also observed an activation of astrocytes after acute morphine injection and during opiate dependence and withdrawal. In addition, immunofluorescence analysis revealed that PTN, but not MK, was overexpressed in astrocytes and that dopaminoceptive neurons expressed RPTPβ/ζ. Conclusions: All these observations suggest that the neurotrophic and behavioral adaptations that occur during opiate addiction could be, at least partly, mediated by cytokines. PMID:26164717

Regulation of Pleiotrophin, Midkine, Receptor Protein Tyrosine Phosphatase β/ζ, and Their Intracellular Signaling Cascades in the Nucleus Accumbens During Opiate Administration.

PubMed

García-Pérez, Daniel; Laorden, María Luisa; Milanés, María Victoria

2015-07-11

Most classes of addictive substances alter the function and structural plasticity of the brain reward circuitry. Midkine (MK) and pleiotrophin (PTN) are growth/differentiation cytokines which, similarly to neurotrophins, play an important role in repair, neurite outgrowth, and cell differentiation. PTN or MK signaling through receptor protein tyrosine phosphatase β/ζ (RPTPβ/ζ), leads to the activation of extracellular signal-regulated kinases and thymoma viral proto-oncogene. This activation induces morphological changes and modulates addictive behaviors. Besides, there is increasing evidence that during the development of drug addiction, astrocytes contribute to the synaptic plasticity by synthesizing and releasing substances such as cytokines. In the present work we studied the effect of acute morphine administration, chronic morphine administration, and morphine withdrawal on PTN, MK, and RPTPβ/ζ expression and on their signaling pathways in the nucleus accumbens. Present results indicated that PTN, MK, and RPTPβ/ζ levels increased after acute morphine injection, returned to basal levels during chronic opioid treatment, and were up-regulated again during morphine withdrawal. We also observed an activation of astrocytes after acute morphine injection and during opiate dependence and withdrawal. In addition, immunofluorescence analysis revealed that PTN, but not MK, was overexpressed in astrocytes and that dopaminoceptive neurons expressed RPTPβ/ζ. All these observations suggest that the neurotrophic and behavioral adaptations that occur during opiate addiction could be, at least partly, mediated by cytokines. © The Author 2015. Published by Oxford University Press on behalf of CINP.

Differentially-dimensioned furrow formation by zygotic gene expression and the MBT

PubMed Central

Xie, Yi

2018-01-01

Despite extensive work on the mechanisms that generate plasma membrane furrows, understanding how cells are able to dynamically regulate furrow dimensions is an unresolved question. Here, we present an in-depth characterization of furrow behaviors and their regulation in vivo during early Drosophila morphogenesis. We show that the deepening in furrow dimensions with successive nuclear cycles is largely due to the introduction of a new, rapid ingression phase (Ingression II). Blocking the midblastula transition (MBT) by suppressing zygotic transcription through pharmacological or genetic means causes the absence of Ingression II, and consequently reduces furrow dimensions. The analysis of compound chromosomes that produce chromosomal aneuploidies suggests that multiple loci on the X, II, and III chromosomes contribute to the production of differentially-dimensioned furrows, and we track the X-chromosomal contribution to furrow lengthening to the nullo gene product. We further show that checkpoint proteins are required for furrow lengthening; however, mitotic phases of the cell cycle are not strictly deterministic for furrow dimensions, as a decoupling of mitotic phases with periods of active ingression occurs as syncytial furrow cycles progress. Finally, we examined the turnover of maternal gene products and find that this is a minor contributor to the developmental regulation of furrow morphologies. Our results suggest that cellularization dynamics during cycle 14 are a continuation of dynamics established during the syncytial cycles and provide a more nuanced view of developmental- and MBT-driven morphogenesis. PMID:29337989

Spontaneous Differentiation of Human Mesenchymal Stem Cells on Poly-Lactic-Co-Glycolic Acid Nano-Fiber Scaffold.

PubMed

Sonomoto, Koshiro; Yamaoka, Kunihiro; Kaneko, Hiroaki; Yamagata, Kaoru; Sakata, Kei; Zhang, Xiangmei; Kondo, Masahiro; Zenke, Yukichi; Sabanai, Ken; Nakayamada, Shingo; Sakai, Akinori; Tanaka, Yoshiya

2016-01-01

Mesenchymal stem cells (MSCs) have immunosuppressive activity and can differentiate into bone and cartilage; and thus seem ideal for treatment of rheumatoid arthritis (RA). Here, we investigated the osteogenesis and chondrogenesis potentials of MSCs seeded onto nano-fiber scaffolds (NFs) in vitro and possible use for the repair of RA-affected joints. MSCs derived from healthy donors and patients with RA or osteoarthritis (OA) were seeded on poly-lactic-glycolic acid (PLGA) electrospun NFs and cultured in vitro. Healthy donor-derived MSCs seeded onto NFs stained positive with von Kossa at Day 14 post-stimulation for osteoblast differentiation. Similarly, MSCs stained positive with Safranin O at Day 14 post-stimulation for chondrocyte differentiation. Surprisingly, even cultured without any stimulation, MSCs expressed RUNX2 and SOX9 (master regulators of bone and cartilage differentiation) at Day 7. Moreover, MSCs stained positive for osteocalcin, a bone marker, and simultaneously also with Safranin O at Day 14. On Day 28, the cell morphology changed from a spindle-like to an osteocyte-like appearance with processes, along with the expression of dentin matrix protein-1 (DMP-1) and matrix extracellular phosphoglycoprotein (MEPE), suggesting possible differentiation of MSCs into osteocytes. Calcification was observed on Day 56. Expression of osteoblast and chondrocyte differentiation markers was also noted in MSCs derived from RA or OA patients seeded on NFs. Lactic acid present in NFs potentially induced MSC differentiation into osteoblasts. Our PLGA scaffold NFs induced MSC differentiation into bone and cartilage. NFs induction process resembled the procedure of endochondral ossification. This finding indicates that the combination of MSCs and NFs is a promising therapeutic technique for the repair of RA or OA joints affected by bone and cartilage destruction.

MicroRNAs in Honey Bee Caste Determination

PubMed Central

Ashby, Regan; Forêt, Sylvain; Searle, Iain; Maleszka, Ryszard

2016-01-01

The cellular mechanisms employed by some organisms to produce contrasting morphological and reproductive phenotypes from the same genome remains one of the key unresolved issues in biology. Honeybees (Apis mellifera) use differential feeding and a haplodiploid sex determination system to generate three distinct organismal outcomes from the same genome. Here we investigate the honeybee female and male caste-specific microRNA and transcriptomic molecular signatures during a critical time of larval development. Both previously undetected and novel miRNAs have been discovered, expanding the inventory of these genomic regulators in invertebrates. We show significant differences in the microRNA and transcriptional profiles of diploid females relative to haploid drone males as well as between reproductively distinct females (queens and workers). Queens and drones show gene enrichment in physio-metabolic pathways, whereas workers show enrichment in processes associated with neuronal development, cell signalling and caste biased structural differences. Interestingly, predicted miRNA targets are primarily associated with non-physio-metabolic genes, especially neuronal targets, suggesting a mechanistic disjunction from DNA methylation that regulates physio-metabolic processes. Accordingly, miRNA targets are under-represented in methylated genes. Our data show how a common set of genetic elements are differentially harnessed by an organism, which may provide the remarkable level of developmental flexibility required. PMID:26739502

Morphology and phylogeny of Triadinium polyedricum (Pouchet) Dodge (Dinophyceae) from Korean coastal waters

NASA Astrophysics Data System (ADS)

Shin, Hyeon Ho; Li, Zhun; Kim, Eun Song; Youn, Joo Yeon; Jeon, Seul Gi; Oh, Seok Jin; Lim, Weol-Ae

2016-12-01

To identify features that can be used to differentiate Triadinium polyedricum from other related species, such as Fukuyoa paulensis and Alexandrium species, the detailed morphology and phylogeny of T. polyedricum collected from Korean coastal waters were investigated. The cells had a plate formula of Po, 3', 7″, 5‴, 1p and 2″″, which is consistent with morphological descriptions in previous reports. Large subunit ribosomal DNA sequences also revealed that T. polyedricum from Korean coastal waters is identical to previously recorded isolates. T. polyedricum is morphologically characterized by a ventral pore in the 1″ plate that is comparable to F. paulensis and Alexandrium species. This result indicates that the location and presence of this ventral pore seems suitable for differentiating T. polyedricum from other related species.

Morphogenetic circuitry regulating growth and development in the dimorphic pathogen Penicillium marneffei.

PubMed

Boyce, Kylie J; Andrianopoulos, Alex

2013-02-01

Penicillium marneffei is an emerging human-pathogenic fungus endemic to Southeast Asia. Like a number of other fungal pathogens, P. marneffei exhibits temperature-dependent dimorphic growth and grows in two distinct cellular morphologies, hyphae at 25°C and yeast cells at 37°C. Hyphae can differentiate to produce the infectious agents, asexual spores (conidia), which are inhaled into the host lung, where they are phagocytosed by pulmonary alveolar macrophages. Within macrophages, conidia germinate into unicellular yeast cells, which divide by fission. This minireview focuses on the current understanding of the genes required for the morphogenetic control of conidial germination, hyphal growth, asexual development, and yeast morphogenesis in P. marneffei.

Evaluation of the effects of a VEGFR-2 inhibitor compound on alanine aminotransferase gene expression and enzymatic activity in the rat liver

PubMed Central

2011-01-01

Background Traditional assessment of drug-induced hepatotoxicity includes morphological examination of the liver and evaluation of liver enzyme activity in serum. The objective of the study was to determine the origin of drug-related elevation in serum alanine aminotransferase (ALT) activity in the absence of morphologic changes in the liver by utilizing molecular and immunohistochemical techniques. Methods Sixteen female Sprague-Dawley rats were divided into 2 groups (control and treated, n = 4 per group) and treated rats were dosed orally twice daily (400 mg/kg/day) for 7 days with a VEGFR-2 compound (AG28262), which in a previous study caused ALT elevation without morphological changes. Serum of both treated and control animals were evaluated on day 3 of treatment and at day 8. Three separate liver lobes (caudate, right medial, and left lateral) were examined for determination of ALT tissue activity, ALT gene expression and morphological changes. Results ALT activity was significantly (p < 0.01) elevated on day 3 and further increased on day 8. Histologic changes or increase in TUNEL and caspase3 positive cells were not observed in the liver lobes examined. ALT gene expression in the caudate lobe was significantly up-regulated by 63%. ALT expression in the left lateral lobe was not significantly affected. Statistically significant increased liver ALT enzymatic activity occurred in the caudate (96%) and right medial (41%) lobes but not in the left lateral lobe. Conclusions AG28262, a VEFG-r2 inhibitor, causes an increase in serum ALT, due in part to both gene up-regulation. Differences between liver lobes may be attributable to differential distribution of blood from portal circulation. Incorporation of molecular data, such as gene and protein expression, and sampling multiple liver lobes may shed mechanistic insight to the evaluation of hepatotoxicity. PMID:21846403

Evaluation of the effects of a VEGFR-2 inhibitor compound on alanine aminotransferase gene expression and enzymatic activity in the rat liver.

PubMed

Fuentealba, Carmen; Bera, Monali; Jessen, Bart; Sace, Fred; Stevens, Greg J; Trajkovic, Dusko; Yang, Amy H; Evering, Winston

2011-08-17

Traditional assessment of drug-induced hepatotoxicity includes morphological examination of the liver and evaluation of liver enzyme activity in serum. The objective of the study was to determine the origin of drug-related elevation in serum alanine aminotransferase (ALT) activity in the absence of morphologic changes in the liver by utilizing molecular and immunohistochemical techniques. Sixteen female Sprague-Dawley rats were divided into 2 groups (control and treated, n = 4 per group) and treated rats were dosed orally twice daily (400 mg/kg/day) for 7 days with a VEGFR-2 compound (AG28262), which in a previous study caused ALT elevation without morphological changes. Serum of both treated and control animals were evaluated on day 3 of treatment and at day 8. Three separate liver lobes (caudate, right medial, and left lateral) were examined for determination of ALT tissue activity, ALT gene expression and morphological changes. ALT activity was significantly (p < 0.01) elevated on day 3 and further increased on day 8. Histologic changes or increase in TUNEL and caspase3 positive cells were not observed in the liver lobes examined. ALT gene expression in the caudate lobe was significantly up-regulated by 63%. ALT expression in the left lateral lobe was not significantly affected. Statistically significant increased liver ALT enzymatic activity occurred in the caudate (96%) and right medial (41%) lobes but not in the left lateral lobe. AG28262, a VEFG-r2 inhibitor, causes an increase in serum ALT, due in part to both gene up-regulation. Differences between liver lobes may be attributable to differential distribution of blood from portal circulation. Incorporation of molecular data, such as gene and protein expression, and sampling multiple liver lobes may shed mechanistic insight to the evaluation of hepatotoxicity.

Development of Morphological Awareness in Young Chinese Readers: Comparing Poor Comprehenders and Good Comprehenders

ERIC Educational Resources Information Center

Zhang, Haomin

2017-01-01

The present study explored the relationship between reading comprehension skill and morphological awareness among young Chinese readers. The objectives were to investigate whether morphological awareness is a critical cognitive and linguistic ability to differentiate poor comprehenders and good comprehenders and to further scrutinize the…

The Gem GTP-binding protein promotes morphological differentiation in neuroblastoma.

PubMed

Leone, A; Mitsiades, N; Ward, Y; Spinelli, B; Poulaki, V; Tsokos, M; Kelly, K

2001-05-31

Gem is a small GTP-binding protein within the Ras superfamily whose function has not been determined. We report here that ectopic Gem expression is sufficient to stimulate cell flattening and neurite extension in N1E-115 and SH-SY5Y neuroblastoma cells, suggesting a role for Gem in cytoskeletal rearrangement and/or morphological differentiation of neurons. Consistent with this potential function, in clinical samples of neuroblastoma, Gem protein was most highly expressed within cells which had differentiated to express ganglionic morphology. Gem was also observed in developing trigeminal nerve ganglia in 12.5 day mouse embryos, demonstrating that Gem expression is a property of normal ganglionic development. Although Gem expression is rare in epithelial and hematopoietic cancer cell lines, constitutive Gem levels were detected in several neuroblastoma cell lines and could be further induced as much as 10-fold following treatment with PMA or the acetylcholine muscarinic agonist, carbachol.

Genetic and morphologic differentiation of Bolbophorus confusus and B. levantinus (Digenea: Diplostomatidae), based on rDNA SSU polymorphism and SEM.

PubMed

Dzikowski, R; Levy, M G; Poore, M F; Flowers, J R; Paperna, I

2003-12-29

Metacercariae of Bolbophorus species are serious pathogens of farmed fish. Molecular diagnostic tools, capable of identifying and differentiating these parasites, may assist in the development of rationale control strategies. The rDNA 18S (small sub-unit: SSU) genes of adult B. confusus and B. levantinus obtained from a pelican, Pelecanus onocrotalus, and a night heron, Nycticorax nycticorax, respectively, were amplified, sequenced, and aligned. Based on this alignment, we developed a genetic differentiation assay between B. confusus and B. levantinus. These 2 species were compared genetically with the North American species B. damnificus and Bolbophorus sp. ('Type 2'). The relationship between species is outlined and discussed. In addition to the molecular study, specimens of B. confusus and B. levantinus were compared morphologically, using scanning electron microscopy. Morphologic analysis revealed interspecific differences in details of the holdfast organ and the position of the acetabulum.

Functions of TAM RTKs in regulating spermatogenesis and male fertility in mice.

PubMed

Chen, Yongmei; Wang, Huizhen; Qi, Nan; Wu, Hui; Xiong, Weipeng; Ma, Jing; Lu, Qingxian; Han, Daishu

2009-10-01

Mice lacking TYRO3, AXL and MER (TAM) receptor tyrosine kinases (RTKs) are male sterile. The mechanism of TAM RTKs in regulating male fertility remains unknown. In this study, we analyzed in more detail the testicular phenotype of TAM triple mutant (TAM(-/-)) mice with an effort to understand the mechanism. We demonstrate that the three TAM RTKs cooperatively regulate male fertility, and MER appears to be more important than AXL and TYRO3. TAM(-/-) testes showed a progressive loss of germ cells from elongated spermatids to spermatogonia. Young adult TAM(-/-) mice exhibited oligo-astheno-teratozoospermia and various morphological malformations of sperm cells. As the mice aged, the germ cells were eventually depleted from the seminiferous tubules. Furthermore, we found that TAM(-/-) Sertoli cells have an impaired phagocytic activity and a large number of differentially expressed genes compared to wild-type controls. By contrast, the function of Leydig cells was not apparently affected by the mutation of TAM RTKs. Therefore, we conclude that the suboptimal function of Sertoli cells leads to the impaired spermatogenesis in TAM(-/-) mice. The results provide novel insight into the mechanism of TAM RTKs in regulating male fertility.

Molecular and morphological differentiation between two Miocene-divergent lineages of Amazonian shrimps, with the description of a new species (Decapoda, Palaemonidae, Palaemon)

PubMed Central

Carvalho, Fabrício Lopes; Magalhães, Célio; Mantelatto, Fernando Luis

2014-01-01

Abstract Palaemon carteri (Gordon, 1935) and Palaemon ivonicus (Holthuis, 1950) are morphologically similar species of South American freshwater shrimps. Past studies have questioned the taxonomic status of both species, which are supposed to have partially sympatric geographic distributions in the Amazon basin. We analyzed a 550 bp fragment of the mitochondrial 16S rRNA gene from these Amazonian Palaemon species as well as from 11 palaemonids as the outgroup. Additionally, we checked diagnostic characters of the genus and family as well as other morphological characters that have been little explored before. Palaemon carteri and Palaemon ivonicus are allocated in two sister lineages, with wide genetic divergence and little morphological differentiation. The divergence time between these lineages was estimated as approximately 10 million years ago. Both molecular and morphological data support the taxonomic validity of both Palaemon carteri and Palaemon ivonicus, refuting the hypothesis of synonymy. In addition, a new species, Palaemon yuna sp. n., closely related to Palaemon ivonicus, is described. Our findings indicate that these species can be differentiated using the projection of the anterolateral margin and anterolateral spine of the first antennular segment, shape of the rostrum, and relative size of the appendix masculina. PMID:25561832

Developmental studies of the lamprey and hierarchical evolutionary steps towards the acquisition of the jaw

PubMed Central

Kuratani, Shigeru

2005-01-01

The evolution of animal morphology can be understood as a series of changes in developmental programs. Among vertebrates, some developmental stages are conserved across species, representing particular developmental constraints. One of the most conserved stages is the vertebrate pharyngula, in which similar embryonic morphology is observed and the Hox code is clearly expressed. The oral developmental program also appears to be constrained to some extent, as both its morphology and the the Hox-code-default state of the oropharyngeal region are well conserved between the lamprey and gnathostome embryos. These features do not by themselves explain the evolution of jaws, but should be regarded as a prerequisite for evolutionary diversification of the mandibular arch. By comparing the pharyngula morphology of the lamprey and gnathostomes, it has become clear that the oral pattern is not entirely identical; in particular, the positional differentiation of the rostral ectomesenchyme is shifted between these animals. Therefore, the jaw seems to have arisen as an evolutionary novelty by overriding ancestral constraints, a process in which morphological homologies are partially lost. This change involves the heterotopic shift of tissue interaction, which appears to have been preceded by the transition from monorhiny to diplorhiny, as well as separation of the hypophysis. When gene expression patterns are compared between the lamprey and gnathostomes, cell-autonomously functioning genes tend to be associated with identical cell types or equivalent anatomical domains, whereas growth-factor-encoding genes have changed their expression domains during evolution. Thus, the heterotopic evolution may be based on changes in the regulation of signalling-molecule-encoding genes. PMID:16313390

[Bone metabolism and cardiovascular function Update. Vascular calcification as a manifestation of bone-vascular axis].

PubMed

Kurabayashi, Masahiko

2014-07-01

Vascular calcification is the major cause of cardiovascular morbidity and mortality in the patients with type 2 diabetes, chronic kidney disease and in aging patients. Regardless of the morphology and location, most evidence indicates that vascular calcification involves an organized process recapitulating many cellular and molecular events that govern skeletal bone formation. While the large body of evidence that osteoblastic and osteochondrocytic cells contribute to vascular calcification, it remains unclear how osteoclasts are differentiated from their precursors and how osteoclasts play a role in calcium reabsorption in calcifying arteries. It is reassuring that calcium paradox is not merely due to the calcium shift from bone to artery wall, but is likely due to the differential response of both osteoblasts and osteoclasts to oxidative stress between bone and artery. To date, many studies have highlighted the important role for RANK/RANKL/OPG axis as unifying theme for the apparently opposite regulation of calcification between two tissues.

Functionally distinct smiles elicit different physiological responses in an evaluative context.

PubMed

Martin, Jared D; Abercrombie, Heather C; Gilboa-Schechtman, Eva; Niedenthal, Paula M

2018-03-01

When people are being evaluated, their whole body responds. Verbal feedback causes robust activation in the hypothalamic-pituitary-adrenal (HPA) axis. What about nonverbal evaluative feedback? Recent discoveries about the social functions of facial expression have documented three morphologically distinct smiles, which serve the functions of reinforcement, social smoothing, and social challenge. In the present study, participants saw instances of one of three smile types from an evaluator during a modified social stress test. We find evidence in support of the claim that functionally different smiles are sufficient to augment or dampen HPA axis activity. We also find that responses to the meanings of smiles as evaluative feedback are more differentiated in individuals with higher baseline high-frequency heart rate variability (HF-HRV), which is associated with facial expression recognition accuracy. The differentiation is especially evident in response to smiles that are more ambiguous in context. Findings suggest that facial expressions have deep physiological implications and that smiles regulate the social world in a highly nuanced fashion.

The mouse F3/contactin glycoprotein

PubMed Central

Bizzoca, Antonella; Corsi, Patrizia

2009-01-01

F3/Contactin is an immunoglobulin superfamily component expressed in the nervous tissue of several species. Here we focus on the structural and functional properties of its mouse relative, on the mechanisms driving its regulated expression and on its developmental role. F3/Contactin is differentially expressed in distinct populations of central and peripheral neurons and in some non-neuronal cells. Accordingly, the regulatory region of the underlying gene includes promoter elements undergoing differential activation, associated with an intricate splicing profile, indicating that transcriptional and posttranscriptional mechanisms contribute to its expression. Transgenic models allowed to follow F3/Contactin promoter activation in vivo and to modify F3/Contactin gene expression under a heterologous promoter, which resulted in morphological and functional phenotypes. Besides axonal growth and pathfinding, these concerned earlier events, including precursor proliferation and commitment. This wide role in neural ontogenesis is consistent with the recognized interaction of F3/Contactin with developmental control genes belonging to the Notch pathway. PMID:19372728

Ngn3+ endocrine progenitor cells control the fate and morphogenesis of pancreatic ductal epithelium

PubMed Central

Magenheim, Judith; Klein, Allon M.; Stanger, Ben Z.; Ashery-Padan, Ruth; Sosa-Pineda, Beatriz; Gu, Guoqiang; Dor, Yuval

2013-01-01

Summary During pancreas development, endocrine and exocrine cells arise from a common multipotent progenitor pool. How these cell fate decisions are coordinated with tissue morphogenesis is poorly understood. Here we have examined ductal morphology, endocrine progenitor cell fate and Notch signaling in Ngn3−/− mice, which do not produce islet cells. Ngn3 deficiency results in reduced branching and enlarged pancreatic duct-like structures, concomitant with Ngn3 promoter activation throughout the ductal epithelium and reduced Notch signaling. Conversely, forced generation of surplus endocrine progenitor cells causes reduced duct caliber and an excessive number of tip cells. Thus, endocrine progenitor cells normally provide a feedback signal to adjacent multipotent ductal progenitor cells that activates Notch signaling, inhibits further endocrine differentiation and promotes proper morphogenesis. These results uncover a novel layer of regulation coordinating pancreas morphogenesis and endocrine/exocrine differentiation, and suggest ways to enhance the yield of beta-cells from stem cells. PMID:21888903

PSD-95 promotes synaptogenesis and multiinnervated spine formation through nitric oxide signaling

PubMed Central

Nikonenko, Irina; Boda, Bernadett; Steen, Sylvain; Knott, Graham; Welker, Egbert; Muller, Dominique

2008-01-01

Postsynaptic density 95 (PSD-95) is an important regulator of synaptic structure and plasticity. However, its contribution to synapse formation and organization remains unclear. Using a combined electron microscopic, genetic, and pharmacological approach, we uncover a new mechanism through which PSD-95 regulates synaptogenesis. We find that PSD-95 overexpression affected spine morphology but also promoted the formation of multiinnervated spines (MISs) contacted by up to seven presynaptic terminals. The formation of multiple contacts was specifically prevented by deletion of the PDZ2 domain of PSD-95, which interacts with nitric oxide (NO) synthase (NOS). Similarly, PSD-95 overexpression combined with small interfering RNA–mediated down-regulation or the pharmacological blockade of NOS prevented axon differentiation into varicosities and multisynapse formation. Conversely, treatment of hippocampal slices with an NO donor or cyclic guanosine monophosphate analogue induced MISs. NOS blockade also reduced spine and synapse density in developing hippocampal cultures. These results indicate that the postsynaptic site, through an NOS–PSD-95 interaction and NO signaling, promotes synapse formation with nearby axons. PMID:19075115

Molecular layer interneurons of the cerebellum: developmental and morphological aspects.

PubMed

Sotelo, Constantino

2015-10-01

During the past 25 years, our knowledge on the development of basket and stellate cells (molecular layer interneurons [MLIs]) has completely changed, not only regarding their origin from the ventricular zone, corresponding to the primitive cerebellar neuroepithelium, instead of the external granular layer, but above all by providing an almost complete account of the genetic regulations (transcription factors and other genes) involved in their differentiation and synaptogenesis. Moreover, it has been shown that MLIs' precursors (dividing neuroblasts) and not young postmitotic neurons, as in other germinal neuroepithelia, leave the germinative zone and migrate all along a complex and lengthy path throughout the presumptive cerebellar white matter, which provides suitable niches exerting epigenetic influences on their ultimate neuronal identities. Recent studies carried out on the anatomical-functional properties of adult MLIs emphasize the importance of these interneurons in regulating PC inhibition, and point out the crucial role played by electrical synaptic transmission between MLIs as well as ephaptic interactions between them and Purkinje cells at the pinceaux level, in the regulation of this inhibition.

Microtubule nucleation and organization in dendrites

PubMed Central

Delandre, Caroline; Amikura, Reiko; Moore, Adrian W.

2016-01-01

ABSTRACT Dendrite branching is an essential process for building complex nervous systems. It determines the number, distribution and integration of inputs into a neuron, and is regulated to create the diverse dendrite arbor branching patterns characteristic of different neuron types. The microtubule cytoskeleton is critical to provide structure and exert force during dendrite branching. It also supports the functional requirements of dendrites, reflected by differential microtubule architectural organization between neuron types, illustrated here for sensory neurons. Both anterograde and retrograde microtubule polymerization occur within growing dendrites, and recent studies indicate that branching is enhanced by anterograde microtubule polymerization events in nascent branches. The polarities of microtubule polymerization events are regulated by the position and orientation of microtubule nucleation events in the dendrite arbor. Golgi outposts are a primary microtubule nucleation center in dendrites and share common nucleation machinery with the centrosome. In addition, pre-existing dendrite microtubules may act as nucleation sites. We discuss how balancing the activities of distinct nucleation machineries within the growing dendrite can alter microtubule polymerization polarity and dendrite branching, and how regulating this balance can generate neuron type-specific morphologies. PMID:27097122

Neurogenic and cardiomyogenic differentiation of mesenchymal stem cells isolated from minipig bone marrow.

PubMed

Kumar, B Mohana; Maeng, Geun-Ho; Lee, Yeon-Mi; Kim, Tae-Ho; Lee, Jeong-Hyeon; Jeon, Byeong-Gyun; Ock, Sun-A; Yoo, Jae-Gyu; Rho, Gyu-Jin

2012-10-01

The present study investigated the potential of minipig bone marrow-mesenchymal stem cells (BM-MSCs) to differentiate in vitro into neuron- and cardiomyocyte-like cells. Isolated BM-MSCs exhibited a fibroblast-like morphology, expressed CD29, CD44 and CD90, and differentiated into osteocytes, adipocytes and chondrocytes. Upon induction in two different neuronal specific media, most of BM-MSCs acquired the distinctive morphological features and positively stained for nestin, neurofilament-M (NF-M), neuronal nuclei (NeuN), β-tubulin, galactocerebroside (Gal-C) and glial fibrillary acidic protein (GFAP). Expression of nestin, GFAP and NF-M was further demonstrated by RT-PCR and RT-qPCR. Following cardiomyogenic induction, MSCs exhibited a stick-like morphology with extended cytoplasmic processes, and formed cluster-like structures. The expression of cardiac specific markers α-smooth muscle actin, cardiac troponin T, desmin and α-cardiac actin was positive for immunofluorescence staining, and further confirmed by RT-PCR and RT-qPCR. In conclusion, our results showed the in vitro differentiation ability of porcine BM-MSCs into neuron-like and cardiomyocyte-like cells. Copyright © 2011 Elsevier Ltd. All rights reserved.

Immunohistochemical study of Ulex europaeus agglutinin 1 (UEA-1) binding of megakaryocytes in bone marrow biopsy specimens: demonstration of heterogeneity in staining pattern reflecting the stages of differentiation.

PubMed

Liu, S M; Li, C Y

1996-01-01

During differentiation, megakaryocytes undergo nuclear endoreplication, an increase in cell size, cytoplasmic granulation, and release of platelets. The changes in highly lobulated nuclei with varying degree of polyploidy and increasing cell size are easily recognized morphologically. However, the actual cytoplasmic changes are more difficult to perceive morphologically. With the peroxidase-antiperoxidase (PAP) method using UEA-1 as the binding protein to the alpha-L-fucose of glycoprotein synthesized by megakaryocytes, we observed significant variation in cytoplasmic staining of megakaryocytes in routinely processed bone marrow biopsy sections. A total of 3344 megakaryocytes in bone marrow sections from 10 patients with nonhematologic diseases and from 10 patients with idiopathic thrombocytopenic purpura (ITP) was studied. According to the intensity and pattern of cytoplasmic staining, we divided megakaryocytes into at least six groups: (1) low granular (LG), (2) diffuse granular (DG), (3) diffuse dense granular (DDG), (4) marginal granular (MG), (5) denuded (DMK), and (6) endomitotic (EndoM). Most of the megakaryocytes were DG (mean, 42.75% +/- 19.21%) and DDG (mean, 50.25% +/- 21.23%). In correlation with nuclear morphology and cell size, it appears that substances binding to UEA-1 are located in the paranuclear region in early megakaryocytes and produce a low granular focal staining pattern (LG cells). Next, the granules spread throughout the cytoplasm (DG cells) and increase in quantity (DDG). This is followed by migration of granules to the periphery of the cytoplasm (MG cells) and is associated with the liberation of platelets and eventual formation of DMK megakaryocytes. Endomitosis, regulated by unknown factors, occurred in the MG stage. In comparing the group with nonhematologic disease (mean DG, 35.4% +/- 18.48%; DDG, 58.4% +/- 21.8%) and the group with ITP (mean DG, 50.1% +/- 17.82%; DDG, 42.1% +/- 18.12%), we found an increasing proportion of DG megakaryocytes in ITP, which suggests a left-shifted maturation of megakaryocytes. By understanding the staining pattern seen in the different stages of megakaryocytic differentiation, UEA-1 staining may be a practical method for studying megakaryocytopoiesis in routinely processed paraffin sections of bone marrow biopsy samples.

Neural Differentiation in HDAC1-Depleted Cells Is Accompanied by Coilin Downregulation and the Accumulation of Cajal Bodies in Nucleoli.

PubMed

Krejčí, Jana; Legartová, Soňa; Bártová, Eva

2017-01-01

Cajal bodies (CBs) are important compartments containing accumulated proteins that preferentially regulate RNA-related nuclear events, including splicing. Here, we studied the nuclear distribution pattern of CBs in neurogenesis. In adult brains, coilin was present at a high density, but CB formation was absent in the nuclei of the choroid plexus of the lateral ventricles. Cells of the adult hippocampus were characterized by a crescent-like morphology of coilin protein. We additionally observed a 70 kDa splice variant of coilin in adult mouse brains, which was different to embryonic brains and mouse pluripotent embryonic stem cells (mESCs), characterized by the 80 kDa standard variant of coilin. Here, we also showed that depletion of coilin is induced during neural differentiation and HDAC1 deficiency in mESCs caused coilin accumulation inside the fibrillarin-positive region of the nucleoli. A similar distribution pattern was observed in adult brain hippocampi, characterized by lower levels of both coilin and HDAC1. In summary, we observed that neural differentiation and HDAC1 deficiency lead to coilin depletion and coilin accumulation in body-like structures inside the nucleoli.

Specific knockdown of Oct4 and beta2-microglobulin expression by RNA interference in human embryonic stem cells and embryonic carcinoma cells.

PubMed

Matin, Maryam M; Walsh, James R; Gokhale, Paul J; Draper, Jonathan S; Bahrami, Ahmad R; Morton, Ian; Moore, Harry D; Andrews, Peter W

2004-01-01

We have used RNA interference (RNAi) to downregulate beta2-microglobulin and Oct4 in human embryonal carcinoma (hEC) cells and embryonic stem (hES) cells, demonstrating that RNAi is an effective tool for regulating specific gene activity in these human stem cells. The knockdown of Oct4 but not beta2-microglobulin expression in both EC and ES cells resulted in their differentiation, as indicated by a marked change in morphology, growth rate, and surface antigen phenotype, with respect to SSEA1, SSEA3, and TRA-1-60 expression. Expression of hCG and Gcm1 was also induced following knockdown of Oct4 expression, in both 2102Ep hEC cells and in H7 and H14 hES cells, consistent with the conclusion that, as in the mouse, Oct4 is required to maintain the undifferentiated stem cell state, and that differentiation to trophectoderm occurs in its absence. NTERA2 hEC cells also differentiated, but not to trophectoderm, suggesting their equivalence to a later stage of embryogenesis than other hEC and hES cells.

Global gene response in Saccharomyces cerevisiae exposed to silver nanoparticles.

PubMed

Niazi, Javed H; Sang, Byoung-In; Kim, Yeon Seok; Gu, Man Bock

2011-08-01

Silver nanoparticles (AgNPs), exhibiting a broad size range and morphologies with highly reactive facets, which are widely applicable in real-life but not fully verified for biosafety and ecotoxicity, were subjected to report transcriptome profile in yeast Saccharomyces cerevisiae. A large number of genes accounted for ∼3% and ∼5% of the genome affected by AgNPs and Ag-ions, respectively. Principal component and cluster analysis suggest that the different physical forms of Ag were the major cause in differential expression profile. Among 90 genes affected by both AgNPs and Ag-ions, metalloprotein mediating high resistance to copper (CUP1-1 and CUP1-2) were strongly induced by AgNPs (∼45-folds) and Ag-ions (∼22-folds), respectively. A total of 17 genes, responsive to chemical stimuli, stress, and transport processes, were differentially induced by AgNPs. The differential expression was also seen with Ag-ions that affected 73 up- and 161 down-regulating genes, and most of these were involved in ion transport and homeostasis. This study provides new information on the knowledge for impact of nanoparticles on living microorganisms that can be extended to other nanoparticles.

Conditional ablation of the Notch2 receptor in the ocular lens

PubMed Central

Saravanamuthu, Senthil S.; Le, Tien T.; Gao, Chun Y.; Cojocaru, Radu I.; Pandiyan, Pushpa; Liu, Chunqiao; Zhang, Jun; Zelenka, Peggy S.; Brown, Nadean L.

2011-01-01

Notch signaling is essential for proper lens development, however the specific requirements of individual Notch receptors have not been investigated. Here we report the lens phenotypes of Notch2 conditionally mutant mice, which exhibited severe microphthalmia, reduced pupillary openings, disrupted fiber cell morphology, eventual loss of the anterior epithelium, fiber cell dysgenesis, denucleation defects, and cataracts. Notch2 mutants also had persistent lens stalks as early as E11.5, and aberrant DNA synthesis in the fiber cell compartment by E14.5. Gene expression analyses showed that upon loss of Notch2, there were elevated levels of the cell cycle regulators Cdkn1a (p21Cip1), Ccnd2 (CyclinD2), and Trp63 (p63) that negatively regulates Wnt signaling, plus down-regulation of Cdh1 (E-Cadherin). Removal of Notch2 also resulted in an increased proportion of fiber cells, as was found in Rbpj and Jag1 conditional mutant lenses. However, Notch2 is not required for AEL proliferation, suggesting that a different receptor regulates this process. We found that Notch2 normally blocks lens progenitor cell death. Overall, we conclude that Notch2-mediated signaling regulates lens morphogenesis, apoptosis, cell cycle withdrawal, and secondary fiber cell differentiation. PMID:22173065

Isolation, culture and chondrogenic differentiation of canine adipose tissue- and bone marrow-derived mesenchymal stem cells--a comparative study.

PubMed

Reich, Christine M; Raabe, Oksana; Wenisch, Sabine; Bridger, Philip S; Kramer, Martin; Arnhold, Stefan

2012-06-01

In the dog, mesenchymal stem cells (MSCs) have been shown to reside in the bone marrow (bone marrow-derived mesenchymal stem cells: BM-MSCs) as well as in the adipose tissue (adipose tissue-derived stem cells: ADSCs). Potential application fields for these multipotent MSCs in small animal practice are joint diseases as MSCs of both sources have shown to possess chondrogenic differentiation ability. However, it is not clear whether the chondrogenic differentiation potential of cells of these two distinct tissues is truly equal. Therefore, we compared MSCs of both origins in this study in terms of their chondrogenic differentiation ability and suitability for clinical application. BM-MSCs harvested from the femoral neck and ADSCs from intra-abdominal fat tissue were examined for their morphology, population doubling time (PDT) and CD90 surface antigen expression. RT-PCR served to assess expression of pluripotency marker Oct4 and early differentiation marker genes. Chondrogenic differentiation ability was compared and validated using histochemistry, transmission electron microscopy (TEM) and quantitative RT-PCR. Both cell populations presented a highly similar morphology and marker expression in an undifferentiated stage except that freshly isolated ADSCs demonstrated a significantly faster PDT than BM-MSCs. In contrast, BM-MSCs revealed a morphological superior cartilage formation by the production of a more abundant and structured hyaline matrix and higher expression of lineage specific genes under the applied standard differentiation protocol. However, further investigations are necessary in order to find out if chondrogenic differentiation can be improved in canine ADSCs using different protocols and/or supplements.

Human neuroblastoma SH-SY5Y cells show increased resistance to hyperthermic stress after differentiation, associated with elevated levels of Hsp72.

PubMed

Cheng, Lesley; Smith, Danielle J; Anderson, Robin L; Nagley, Phillip

2011-01-01

Terminally differentiated neurones in the central nervous system need to be protected from stress. We ask here whether differentiation of progenitor cells to neurones is accompanied by up-regulation of Hsp72, with acquisition of enhanced thermotolerance. Human neuroblastoma SH-SY5Y cells were propagated in an undifferentiated form and subsequently differentiated into neurone-like cells. Thermotolerance tests were carried out by exposure of cells to various temperatures, monitoring nuclear morphology as index of cell death. Abundance of Hsp72 was measured in cell lysates by western immunoblotting. The differentiation of SH-SY5Y cells was accompanied by increased expression of Hsp72. Further, in both cell states, exposure to mild hyperthermic stress (43°C for 30 min) increased Hsp72 expression. After differentiation, SH-SY5Y cells were more resistant to hyperthermic stress compared to their undifferentiated state, correlating with levels of Hsp72. Stable exogenous expression of Hsp72 in SH-SY5Y cells (transfected line 5YHSP72.1, containing mildly elevated levels of Hsp72), led to enhanced resistance to hyperthermic stress. Hsp72 was found to be inducible in undifferentiated 5YHSP72.1 cells; such heat-treated cells displayed enhanced thermotolerance. Treatment of cells with KNK437, a suppressor of Hsp72 induction, resulted in acute thermosensitisation of all cell types tested here. Hsp72 has a major role in the enhanced hyperthermic resistance acquired during neuronal differentiation of SH-SY5Y cells. These findings model the requirement in intact organisms for highly differentiated neurones to be specially protected against thermal stress.

Manganese- and 1-methyl-4-phenylpyridinium-induced neurotoxicity display differences in morphological, electrophysiological and genome-wide alterations: implications for idiopathic Parkinson's disease.

PubMed

Mythri, Rajeswara Babu; Raghunath, Narayana Reddy; Narwade, Santosh Chandrakant; Pandareesh, Mirazkar Dasharatha Rao; Sabitha, Kollarkandi Rajesh; Aiyaz, Mohamad; Chand, Bipin; Sule, Manas; Ghosh, Krittika; Kumar, Senthil; Shankarappa, Bhagyalakshmi; Soundararajan, Soundarya; Alladi, Phalguni Anand; Purushottam, Meera; Gayathri, Narayanappa; Deobagkar, Deepti Dileep; Laxmi, Thenkanidiyoor Rao; Srinivas Bharath, Muchukunte Mukunda

2017-11-01

Idiopathic Parkinson's disease and manganese-induced atypical parkinsonism are characterized by movement disorder and nigrostriatal pathology. Although clinical features, brain region involved and responsiveness to levodopa distinguish both, differences at the neuronal level are largely unknown. We studied the morphological, neurophysiological and molecular differences in dopaminergic neurons exposed to the Parkinson's disease toxin 1-methyl-4-phenylpyridinium ion (MPP + ) and manganese (Mn), followed by validation in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and Mn mouse models. Morphological analysis highlighted loss of neuronal processes in the MPP + and not the Mn model. Cellular network dynamics of dopaminergic neurons characterized by spike frequency and inter-spike intervals indicated major neuronal population (~ 93%) with slow discharge rates (0-5 Hz). While MPP + exposure suppressed the firing of these neurons, Mn neither suppressed nor elevated the neuronal activity. High-throughput transcriptomic analysis revealed up-regulation of 694 and 603 genes and down-regulation of 428 and 255 genes in the MPP + and Mn models respectively. Many differentially expressed genes were unique to either models and contributed to neuroinflammation, metabolic/mitochondrial function, apoptosis and nuclear function, synaptic plasticity, neurotransmission and cytoskeleton. Analysis of the Janus kinase-signal transducer and activator of transcription pathway with implications for neuritogenesis and neuronal proliferation revealed contrasting profile in both models. Genome-wide DNA methylomics revealed differences between both models and substantiated the epigenetic basis of the difference in the Janus kinase-signal transducer and activator of transcription pathway. We conclude that idiopathic Parkinson's disease and atypical parkinsonism have divergent neurotoxicological manifestation at the dopaminergic neuronal level with implications for pathobiology and evolution of novel therapeutics. Cover Image for this issue: doi. 10.1111/jnc.13821. © 2017 International Society for Neurochemistry.

Harnessing 3D models of mammary epithelial morphogenesis. An off the beaten path approach to identify candidate biomarkers of early stage breast cancer

PubMed Central

Rossetti, Stefano; Bshara, Wiam; Reiners, Johanna A.; Corlazzoli, Francesca; Miller, Austin; Sacchi, Nicoletta

2016-01-01

Regardless of the etiological factor, an aberrant morphology is the common hallmark of ductal carcinoma in situ (DCIS), which is a highly heterogeneous disease. To test if critical core morphogenetic mechanisms are compromised by different mutations, we performed proteomics analysis of five mammary epithelial HME1 mutant lines that develop a DCIS-like morphology in three dimensional (3D) culture. Here we show first, that all HME1 mutant lines share a common protein signature highlighting an inverse deregulation of two annexins, ANXA2 and ANXA8. Either ANXA2 downregulation or ANXA8 upregulation in the HME1 cell context are per se sufficient to confer a 3D DCIS-like morphology. Seemingly, different mutations impinged on a common mechanism that differentially regulates the two annexins. Second, we show that ANXA8 expression is significantly higher in DCIS tissue samples versus normal breast tissue and atypical ductal hyperplasia (ADH). Apparently, ANXA8 expression is significantly more upregulated in ER- negative versus ER-positive cases, and significantly correlates with tumor stage, grade and positive lymph node. Based on our study, 3D mammary morphogenesis models can be an alternate/complementary strategy for unraveling new DCIS mechanisms and biomarkers. PMID:27422542

The role of insulin-like-growth factor binding protein 2 (IGFBP2) and phosphatase and tensin homologue (PTEN) in the regulation of myoblast differentiation and hypertrophy.

PubMed

Sharples, Adam P; Al-Shanti, Nasser; Hughes, David C; Lewis, Mark P; Stewart, Claire E

2013-06-01

The complex actions of the insulin-like-growth factor binding proteins (IGFBPs) in skeletal muscle are becoming apparent, with IGFBP2 being implicated in skeletal muscle cell proliferation and differentiation (Ernst et al., 1992; Sharples et al., 2010). Furthermore, PTEN signalling has been linked to IGFBP2 action in other cell types by co-ordinating downstream Akt signalling, a known modulator of myoblast differentiation. The present study therefore aimed to determine the interaction between IGFBP2 and PTEN on myoblast differentiation. It has previously been established that C2C12 cells have high IGFBP2 gene expression upon transfer to low serum media, and that expression reduces rapidly as cells differentiate over 72 h [1]. Wishing to establish a potential role for IGFBP2 in this model, a neutralising IGFBP2 antibody was administered to C2C12 myoblasts upon initiation of differentiation. Myoblasts subsequently displayed reduced morphological differentiation (myotube number), biochemical differentiation (creatine kinase) and myotube hypertrophy (myotube area) with an early reduction in Akt phosphorylation. Knock-down of phosphatase and tensin homologue (PTEN) using siRNA in the absence of the neutralising antibody did not improve differentiation or hypertrophy vs. control conditions, however, in the presence of the neutralising IGFBP2 antibody, differentiation was restored and importantly hypertrophy exceeded that of control levels. Overall, these data suggest that; 1) reduced early availability of IGFBP2 can inhibit myoblast differentiation at later time points, 2) knock-down of PTEN levels can restore myoblast differentiation in the presence of neutralising IGFBP2 antibody, and 3) PTEN inhibition acts as a potent inducer of myotube hypertrophy when the availability of IGFBP2 is reduced in C2C12 myoblasts. Copyright © 2013 Elsevier Ltd. All rights reserved.

Phenotypic Diagnosis of Lineage and Differentiation During Sake Yeast Breeding

PubMed Central

Ohnuki, Shinsuke; Okada, Hiroki; Friedrich, Anne; Kanno, Yoichiro; Goshima, Tetsuya; Hasuda, Hirokazu; Inahashi, Masaaki; Okazaki, Naoto; Tamura, Hiroyasu; Nakamura, Ryo; Hirata, Dai; Fukuda, Hisashi; Shimoi, Hitoshi; Kitamoto, Katsuhiko; Watanabe, Daisuke; Schacherer, Joseph; Akao, Takeshi; Ohya, Yoshikazu

2017-01-01

Sake yeast was developed exclusively in Japan. Its diversification during breeding remains largely uncharacterized. To evaluate the breeding processes of the sake lineage, we thoroughly investigated the phenotypes and differentiation of 27 sake yeast strains using high-dimensional, single-cell, morphological phenotyping. Although the genetic diversity of the sake yeast lineage is relatively low, its morphological diversity has expanded substantially compared to that of the Saccharomyces cerevisiae species as a whole. Evaluation of the different types of breeding processes showed that the generation of hybrids (crossbreeding) has more profound effects on cell morphology than the isolation of mutants (mutation breeding). Analysis of phenotypic robustness revealed that some sake yeast strains are more morphologically heterogeneous, possibly due to impairment of cellular network hubs. This study provides a new perspective for studying yeast breeding genetics and micro-organism breeding strategies. PMID:28642365

When Can Clades Be Potentially Resolved with Morphology?

PubMed Central

Bapst, David W.

2013-01-01

Morphology-based phylogenetic analyses are the only option for reconstructing relationships among extinct lineages, but often find support for conflicting hypotheses of relationships. The resulting lack of phylogenetic resolution is generally explained in terms of data quality and methodological issues, such as character selection. A previous suggestion is that sampling ancestral morphotaxa or sampling multiple taxa descended from a long-lived, unchanging lineage can also yield clades which have no opportunity to share synapomorphies. This lack of character information leads to a lack of ‘intrinsic’ resolution, an issue that cannot be solved with additional morphological data. It is unclear how often we should expect clades to be intrinsically resolvable in realistic circumstances, as intrinsic resolution must increase as taxonomic sampling decreases. Using branching simulations, I quantify intrinsic resolution across several models of morphological differentiation and taxonomic sampling. Intrinsically unresolvable clades are found to be relatively frequent in simulations of both extinct and living taxa under realistic sampling scenarios, implying that intrinsic resolution is an issue for morphology-based analyses of phylogeny. Simulations which vary the rates of sampling and differentiation were tested for their agreement to observed distributions of durations from well-sampled fossil records and also having high intrinsic resolution. This combination only occurs in those datasets when differentiation and sampling rates are both unrealistically high relative to branching and extinction rates. Thus, the poor phylogenetic resolution occasionally observed in morphological phylogenetics may result from a lack of intrinsic resolvability within groups. PMID:23638034

MicroRNA-378 regulates neural stem cell proliferation and differentiation in vitro by modulating Tailless expression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Huang, Yanxia; Department of Rehabilitation, Xi'an Children's Hospital, Xi'an 710003; Liu, Xiaoguai

Previous studies have suggested that microRNAs (miRNAs) play an important role in regulating neural stem cell (NSC) proliferation and differentiation. However, the precise role of miRNAs in NSC remains largely unexplored. In this study, we showed that miR-378 can target Tailless (TLX), a critical regulator of NSC, to regulate NSC proliferation and differentiation. By bioinformatic algorithms, miR-378 was found to have a predicted target site in the 3′-untranslated region of TLX, which was verified by a dual-luciferase reporter assay. The expression of miR-378 was increased during NSC differentiation and inversely correlated with TLX expression. qPCR and Western blot analysis alsomore » showed that miR-378 negatively regulated TLX mRNA and protein expression in neural stem cells (NSCs). Intriguingly, overexpression of miR-378 increased NSC differentiation and reduced NSC proliferation, whereas suppression of miR-378 led to decreased NSC differentiation and increased NSC proliferation. Moreover, the downstream targets of TLX, including p21, PTEN and Wnt/β-catenin were also found to be regulated by miR-378. Additionally, overexpression of TLX rescued the NSC proliferation deficiency induced by miR-378 overexpression and abolished miR-378-promoted NSC differentiation. Taken together, our data suggest that miR-378 is a novel miRNA that regulates NSC proliferation and differentiation via targeting TLX. Therefore, manipulating miR-378 in NSCs could be a novel strategy to develop novel interventions for the treatment of relevant neurological disorders. - Highlights: • miR-378 targeted and regulated TLX. • miR-378 was increased during NSC differentiation. • miR-378 regulated NSC proliferation and differentiation. • miR-378 regulated NSC self-renew through TLX.« less

Growth inhibition of N1E-115 mouse neuroblastoma cells by c-myc or N-myc antisense oligodeoxynucleotides causes limited differentiation but is not coupled to neurite formation.

PubMed

Larcher, J C; Basseville, M; Vayssiere, J L; Cordeau-Lossouarn, L; Croizat, B; Gros, F

1992-06-30

Antisense oligodeoxynucleotides were found to be stable in the culture medium containing fetal calf serum (heat-inactivated 30 minutes at 65 degrees C) and in cells. Antisense oligomer treatment causes cessation of mitoses, but does not lead to morphological differentiation. Under antisense conditions, we have observed an increase in the amount of two neurospecific protein, namely peripherin and gamma-enolase. Comparison of the results obtained with chemical inducers and antisense oligodeoxynucleotides allows us to postulate three phases in N1E-115 differentiation: the first correspond to the arrest of mitosis, the second to the expression of a limited neuronal program, and the third to the morphological and electrophysiological differentiation.

Nicotine induces cell proliferation in association with cyclin D1 up-regulation and inhibits cell differentiation in association with p53 regulation in a murine pre-osteoblastic cell line

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sato, Tsuyoshi; Abe, Takahiro; Nakamoto, Norimichi

Recent studies have suggested that nicotine critically affects bone metabolism. Many studies have examined the effects of nicotine on proliferation and differentiation, but the underlying molecular mechanisms remain unclear. We examined cell cycle regulators involved in the proliferation and differentiation of MC3T3-E1 cells. Nicotine induced cell proliferation in association with p53 down-regulation and cyclin D1 up-regulation. In differentiated cells, nicotine reduced alkaline phosphatase activity and mineralized nodule formation in dose-dependent manners. Furthermore, p53 expression was sustained in nicotine-treated cells during differentiation. These findings indicate that nicotine promotes the cell cycle and inhibits differentiation in association with p53 regulation in pre-osteoblasticmore » cells.« less

Down-regulated non-coding RNA (lncRNA-ANCR) promotes osteogenic differentiation of periodontal ligament stem cells.

PubMed

Jia, Qian; Jiang, Wenkai; Ni, Longxing

2015-02-01

Our studies aimed to figure out how anti-differentiation noncoding RNA (ANCR) regulates the proliferation and osteogenic differentiation of periodontal ligament stem cells (PDLSCs). In this study, we used lentivirus infection to down-regulate the expression of ANCR in PDLSCs. Then we compared the proliferation of control cells and PDLSC/ANCR-RNAi cells by Cell Counting Kit-8. And the osteogenic differentiation of control cells and PDLSC/ANCR-RNAi cells were evaluated by Alkaline phosphatase (ALP) activity quantification and Alizarin red staining. WNT inhibitor was used to analyze the relationship between ANCR and canonical WNT signalling pathway. The expression of osteogenic differentiation marker mRNAs, DKK1, GSK3-β and β-catenin were evaluated by qRT-PCR. The results showed that down-regulated ANCR promoted proliferation of PDLSCs. Down-regulated ANCR also promoted osteogenic differentiation of PDLSCs by up-regulating osteogenic differentiation marker genes. After the inhibition of canonical WNT signalling pathway, the osteogenic differentiation of PDLSC/ANCR-RNAi cells was inhibited too. qRT-PCR results also demonstrated that canonical WNT signalling pathway was activated for ANCR-RNAi on PDLSCs during the procedure of proliferation and osteogenic induction. These results indicated that ANCR was a key regulator of the proliferation and osteogenic differentiation of PDLSCs, and its regulating effects was associated with the canonical WNT signalling pathway, thus offering a new target for oral stem cell differentiation studies that could also facilitate oral tissue engineering. Copyright © 2014. Published by Elsevier Ltd.

The Spore Differentiation Pathway in the Enteric Pathogen Clostridium difficile

PubMed Central

Pereira, Fátima C.; Saujet, Laure; Tomé, Ana R.; Serrano, Mónica; Monot, Marc; Couture-Tosi, Evelyne; Martin-Verstraete, Isabelle; Dupuy, Bruno; Henriques, Adriano O.

2013-01-01

Endosporulation is an ancient bacterial developmental program that culminates with the differentiation of a highly resistant endospore. In the model organism Bacillus subtilis, gene expression in the forespore and in the mother cell, the two cells that participate in endospore development, is governed by cell type-specific RNA polymerase sigma subunits. σF in the forespore, and σE in the mother cell control early stages of development and are replaced, at later stages, by σG and σK, respectively. Starting with σF, the activation of the sigma factors is sequential, requires the preceding factor, and involves cell-cell signaling pathways that operate at key morphological stages. Here, we have studied the function and regulation of the sporulation sigma factors in the intestinal pathogen Clostridium difficile, an obligate anaerobe in which the endospores are central to the infectious cycle. The morphological characterization of mutants for the sporulation sigma factors, in parallel with use of a fluorescence reporter for single cell analysis of gene expression, unraveled important deviations from the B. subtilis paradigm. While the main periods of activity of the sigma factors are conserved, we show that the activity of σE is partially independent of σF, that σG activity is not dependent on σE, and that the activity of σK does not require σG. We also show that σK is not strictly required for heat resistant spore formation. In all, our results indicate reduced temporal segregation between the activities of the early and late sigma factors, and reduced requirement for the σF-to-σE, σE-to-σG, and σG-to-σK cell-cell signaling pathways. Nevertheless, our results support the view that the top level of the endosporulation network is conserved in evolution, with the sigma factors acting as the key regulators of the pathway, established some 2.5 billion years ago upon its emergence at the base of the Firmicutes Phylum. PMID:24098139

Influence of organophosphate poisoning on human dendritic cells.

PubMed

Schäfer, Marina; Koppe, Franziska; Stenger, Bernhard; Brochhausen, Christoph; Schmidt, Annette; Steinritz, Dirk; Thiermann, Horst; Kirkpatrick, Charles James; Pohl, Christine

2013-12-05

Organophosphourus compounds (OPC, including nerve agents and pesticides) exhibit acute toxicity by inhibition of acetylcholinesterase. Lung affections are frequent complications and a risk factor for death. In addition, epidemiological studies reported immunological alterations after OPC exposure. In our experiments we investigated the effects of organophosphourus pesticides dimethoate and chlorpyrifos on dendritic cells (DC) that are essential for the initial immune response, especially in the pulmonary system. DC, differentiated from the monocyte cell line THP-1 by using various cytokines (IL-4, GM-CSF, TNF-α, Ionomycin), were exposed to organophosphourus compounds at different concentrations for a 24h time period. DC were characterized by flow cytometry and immunofluorescence using typical dendritic cell markers (e.g., CD11c, CD209 and CD83). After OPC exposure we investigated cell death, the secretion profile of inflammatory mediators, changes of DC morphology, and the effect on protein kinase signalling pathways. Our results revealed a successful differentiation of THP-1 into DC. OPC exposure caused a significant concentration-dependent influence on DC: Dendrites of the DC were shortened and damaged, DC-specific cell surface markers (i.e., CD83and CD209) decreased dramatically after chlorpyrifos exposure. Interestingly, the effects caused by dimethoate were in general less pronounced. The organophosphourus compounds affected the release of inflammatory cytokines, such as IL-1ß and IL-8. The anti-inflammatory cytokine IL-10 was significantly down regulated. Protein kinases like the Akt family or ERK, which are essential for cell survival and proliferation, were inhibited by both OPC. These findings indicate that the tested organophosphourus compounds induced significant changes in cell morphology, inhibited anti-inflammatory cytokines and influenced important protein signalling pathways which are involved in regulation of apoptosis. Thus our results highlight novel aspects -apparently independent of AChE inhibition- of OPC poisoning with regard to lung toxicity. Our findings contribute to the basic understanding of pulmonary complications caused by OPC poisoning. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Subsurface Grain Morphology Reconstruction by Differential Aperture X-ray Microscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Eisenlohr, Philip; Shanthraj, Pratheek; Vande Kieft, Brendan R.

A multistep, non-destructive grain morphology reconstruction methodology that is applicable to near-surface volumes is developed and tested on synthetic grain structures. This approach probes the subsurface crystal orientation using differential aperture x-ray microscopy on a sparse grid across the microstructure volume of interest. Resulting orientation data are clustered according to proximity in physical and orientation space and used as seed points for an initial Voronoi tessellation to (crudely) approximate the grain morphology. Curvature-driven grain boundary relaxation, simulated by means of the Voronoi implicit interface method, progressively improves the reconstruction accuracy. The similarity between bulk and readily accessible surface reconstruction errormore » provides an objective termination criterion for boundary relaxation.« less

Fragility of foot process morphology in kidney podocytes arises from chaotic spatial propagation of cytoskeletal instability

PubMed Central

Deerinck, Thomas J.; Chen, Yibang; He, John C.; Ellisman, Mark H.; Iyengar, Ravi

2017-01-01

Kidney podocytes’ function depends on fingerlike projections (foot processes) that interdigitate with those from neighboring cells to form the glomerular filtration barrier. The integrity of the barrier depends on spatial control of dynamics of actin cytoskeleton in the foot processes. We determined how imbalances in regulation of actin cytoskeletal dynamics could result in pathological morphology. We obtained 3-D electron microscopy images of podocytes and used quantitative features to build dynamical models to investigate how regulation of actin dynamics within foot processes controls local morphology. We find that imbalances in regulation of actin bundling lead to chaotic spatial patterns that could impair the foot process morphology. Simulation results are consistent with experimental observations for cytoskeletal reconfiguration through dysregulated RhoA or Rac1, and they predict compensatory mechanisms for biochemical stability. We conclude that podocyte morphology, optimized for filtration, is intrinsically fragile, whereby local transient biochemical imbalances may lead to permanent morphological changes associated with pathophysiology. PMID:28301477

JAG1-Mediated Notch Signaling Regulates Secretory Cell Differentiation of the Human Airway Epithelium.

PubMed

Gomi, Kazunori; Staudt, Michelle R; Salit, Jacqueline; Kaner, Robert J; Heldrich, Jonna; Rogalski, Allison M; Arbelaez, Vanessa; Crystal, Ronald G; Walters, Matthew S

2016-08-01

Basal cells (BC) are the stem/progenitor cells of the human airway epithelium capable of differentiating into secretory and ciliated cells. Notch signaling activation increases BC differentiation into secretory cells, but the role of individual Notch ligands in regulating this process in the human airway epithelium is largely unknown. The objective of this study was to define the role of the Notch ligand JAG1 in regulating human BC differentiation. JAG1 over-expression in BC increased secretory cell differentiation, with no effect on ciliated cell differentiation. Conversely, knockdown of JAG1 decreased expression of secretory cell genes. These data demonstrate JAG1-mediated Notch signaling regulates differentiation of BC into secretory cells.

Adaptation of a Simple Microfluidic Platform for High-Dimensional Quantitative Morphological Analysis of Human Mesenchymal Stromal Cells on Polystyrene-Based Substrates.

PubMed

Lam, Johnny; Marklein, Ross A; Jimenez-Torres, Jose A; Beebe, David J; Bauer, Steven R; Sung, Kyung E

2017-12-01

Multipotent stromal cells (MSCs, often called mesenchymal stem cells) have garnered significant attention within the field of regenerative medicine because of their purported ability to differentiate down musculoskeletal lineages. Given the inherent heterogeneity of MSC populations, recent studies have suggested that cell morphology may be indicative of MSC differentiation potential. Toward improving current methods and developing simple yet effective approaches for the morphological evaluation of MSCs, we combined passive pumping microfluidic technology with high-dimensional morphological characterization to produce robust tools for standardized high-throughput analysis. Using ultraviolet (UV) light as a modality for reproducible polystyrene substrate modification, we show that MSCs seeded on microfluidic straight channel devices incorporating UV-exposed substrates exhibited morphological changes that responded accordingly to the degree of substrate modification. Substrate modification also effected greater morphological changes in MSCs seeded at a lower rather than higher density within microfluidic channels. Despite largely comparable trends in morphology, MSCs seeded in microscale as opposed to traditional macroscale platforms displayed much higher sensitivity to changes in substrate properties. In summary, we adapted and qualified microfluidic cell culture platforms comprising simple straight channel arrays as a viable and robust tool for high-throughput quantitative morphological analysis to study cell-material interactions.

Towards a morphogenetic perspective on cancer.

PubMed

Aranda-Anzaldo, Armando

2002-01-01

The purpose of this paper is to present a critique of the current view that reduces cancer to a cellular problem caused by specific gene mutations and to propose, instead, that such a problem might become more intelligible, if it is understood as a phenomenon which results from the breakdown of the morphological plan or Gestalt of the organism. Such an organism, in Aristotelian terms, is characterised for presenting a specific morpho or logos (form) and for having a telos (end) to fulfill. A malignant tumour represents an entity separated from both, the organic logos and the organic telos. According to the basic postulates of Semiophysics--a blend of Aristotelian physics and Catastrophe Theory developed by René Thom--an organism is a source (original) form individuated by a dominant pregnancy which corresponds to its morphogenetic field. Here it is suggested that cancer in aged individuals may result from the progressive exhaustion of the developmental constraints that regulate the process of ontogeny, that is expected to go from the fertilised non-differentiated zygote to the mature fully-developed organism, because there is no further point ahead in the developmental pathway past the reproductive age. Cancer in young individuals (before their reproductive maturity) may then be consequence of premature derangement of such fundamental developmental constraints. In all cases the result is the loss of morphological coherence within the organism. Thus representing a conflict between an organised morphology (the organism) and a part of such a morphology that drifts towards an amorphous state (the tumour).

Chronic intermittent ethanol exposure and withdrawal leads to adaptations in nucleus accumbens core postsynaptic density proteome and dendritic spines.

PubMed

Uys, Joachim D; McGuier, Natalie S; Gass, Justin T; Griffin, William C; Ball, Lauren E; Mulholland, Patrick J

2016-05-01

Alcohol use disorder is a chronic relapsing brain disease characterized by the loss of ability to control alcohol (ethanol) intake despite knowledge of detrimental health or personal consequences. Clinical and pre-clinical models provide strong evidence for chronic ethanol-associated alterations in glutamatergic signaling and impaired synaptic plasticity in the nucleus accumbens (NAc). However, the neural mechanisms that contribute to aberrant glutamatergic signaling in ethanol-dependent individuals in this critical brain structure remain unknown. Using an unbiased proteomic approach, we investigated the effects of chronic intermittent ethanol (CIE) exposure on neuroadaptations in postsynaptic density (PSD)-enriched proteins in the NAc of ethanol-dependent mice. Compared with controls, CIE exposure significantly changed expression levels of 50 proteins in the PSD-enriched fraction. Systems biology and functional annotation analyses demonstrated that the dysregulated proteins are expressed at tetrapartite synapses and critically regulate cellular morphology. To confirm this latter finding, the density and morphology of dendritic spines were examined in the NAc core of ethanol-dependent mice. We found that CIE exposure and withdrawal differentially altered dendrite diameter and dendritic spine density and morphology. Through the use of quantitative proteomics and functional annotation, these series of experiments demonstrate that ethanol dependence produces neuroadaptations in proteins that modify dendritic spine morphology. In addition, these studies identified novel PSD-related proteins that contribute to the neurobiological mechanisms of ethanol dependence that drive maladaptive structural plasticity of NAc neurons. © 2015 Society for the Study of Addiction.

SACE_5599, a putative regulatory protein, is involved in morphological differentiation and erythromycin production in Saccharopolyspora erythraea.

PubMed

Kirm, Benjamin; Magdevska, Vasilka; Tome, Miha; Horvat, Marinka; Karničar, Katarina; Petek, Marko; Vidmar, Robert; Baebler, Spela; Jamnik, Polona; Fujs, Štefan; Horvat, Jaka; Fonovič, Marko; Turk, Boris; Gruden, Kristina; Petković, Hrvoje; Kosec, Gregor

2013-12-17

Erythromycin is a medically important antibiotic, biosynthesized by the actinomycete Saccharopolyspora erythraea. Genes encoding erythromycin biosynthesis are organized in a gene cluster, spanning over 60 kbp of DNA. Most often, gene clusters encoding biosynthesis of secondary metabolites contain regulatory genes. In contrast, the erythromycin gene cluster does not contain regulatory genes and regulation of its biosynthesis has therefore remained poorly understood, which has for a long time limited genetic engineering approaches for erythromycin yield improvement. We used a comparative proteomic approach to screen for potential regulatory proteins involved in erythromycin biosynthesis. We have identified a putative regulatory protein SACE_5599 which shows significantly higher levels of expression in an erythromycin high-producing strain, compared to the wild type S. erythraea strain. SACE_5599 is a member of an uncharacterized family of putative regulatory genes, located in several actinomycete biosynthetic gene clusters. Importantly, increased expression of SACE_5599 was observed in the complex fermentation medium and at controlled bioprocess conditions, simulating a high-yield industrial fermentation process in the bioreactor. Inactivation of SACE_5599 in the high-producing strain significantly reduced erythromycin yield, in addition to drastically decreasing sporulation intensity of the SACE_5599-inactivated strains when cultivated on ABSM4 agar medium. In contrast, constitutive overexpression of SACE_5599 in the wild type NRRL23338 strain resulted in an increase of erythromycin yield by 32%. Similar yield increase was also observed when we overexpressed the bldD gene, a previously identified regulator of erythromycin biosynthesis, thereby for the first time revealing its potential for improving erythromycin biosynthesis. SACE_5599 is the second putative regulatory gene to be identified in S. erythraea which has positive influence on erythromycin yield. Like bldD, SACE_5599 is involved in morphological development of S. erythraea, suggesting a very close relationship between secondary metabolite biosynthesis and morphological differentiation in this organism. While the mode of action of SACE_5599 remains to be elucidated, the manipulation of this gene clearly shows potential for improvement of erythromycin production in S. erythraea in industrial setting. We have also demonstrated the applicability of the comparative proteomics approach for identifying new regulatory elements involved in biosynthesis of secondary metabolites in industrial conditions.

SACE_5599, a putative regulatory protein, is involved in morphological differentiation and erythromycin production in Saccharopolyspora erythraea

PubMed Central

2013-01-01

Background Erythromycin is a medically important antibiotic, biosynthesized by the actinomycete Saccharopolyspora erythraea. Genes encoding erythromycin biosynthesis are organized in a gene cluster, spanning over 60 kbp of DNA. Most often, gene clusters encoding biosynthesis of secondary metabolites contain regulatory genes. In contrast, the erythromycin gene cluster does not contain regulatory genes and regulation of its biosynthesis has therefore remained poorly understood, which has for a long time limited genetic engineering approaches for erythromycin yield improvement. Results We used a comparative proteomic approach to screen for potential regulatory proteins involved in erythromycin biosynthesis. We have identified a putative regulatory protein SACE_5599 which shows significantly higher levels of expression in an erythromycin high-producing strain, compared to the wild type S. erythraea strain. SACE_5599 is a member of an uncharacterized family of putative regulatory genes, located in several actinomycete biosynthetic gene clusters. Importantly, increased expression of SACE_5599 was observed in the complex fermentation medium and at controlled bioprocess conditions, simulating a high-yield industrial fermentation process in the bioreactor. Inactivation of SACE_5599 in the high-producing strain significantly reduced erythromycin yield, in addition to drastically decreasing sporulation intensity of the SACE_5599-inactivated strains when cultivated on ABSM4 agar medium. In contrast, constitutive overexpression of SACE_5599 in the wild type NRRL23338 strain resulted in an increase of erythromycin yield by 32%. Similar yield increase was also observed when we overexpressed the bldD gene, a previously identified regulator of erythromycin biosynthesis, thereby for the first time revealing its potential for improving erythromycin biosynthesis. Conclusions SACE_5599 is the second putative regulatory gene to be identified in S. erythraea which has positive influence on erythromycin yield. Like bldD, SACE_5599 is involved in morphological development of S. erythraea, suggesting a very close relationship between secondary metabolite biosynthesis and morphological differentiation in this organism. While the mode of action of SACE_5599 remains to be elucidated, the manipulation of this gene clearly shows potential for improvement of erythromycin production in S. erythraea in industrial setting. We have also demonstrated the applicability of the comparative proteomics approach for identifying new regulatory elements involved in biosynthesis of secondary metabolites in industrial conditions. PMID:24341557

Knockdown of SLC41A1 magnesium transporter promotes mineralization and attenuates magnesium inhibition during osteogenesis of mesenchymal stromal cells.

PubMed

Tsao, Yu-Tzu; Shih, Ya-Yi; Liu, Yu-An; Liu, Yi-Shiuan; Lee, Oscar K

2017-02-21

Magnesium is essential for numerous physiological functions. Magnesium exists mostly in bone and the amount is dynamically regulated by skeletal remodeling. Accelerating bone mass loss occurs when magnesium intake is insufficient; whereas high magnesium could lead to mineralization defects. However, the underlying magnesium regulatory mechanisms remain elusive. In the present study, we investigated the effects of high extracellular magnesium concentration on osteogenic differentiation of mesenchymal stromal/stem cells (MSCs) and the role of magnesium transporter SLC41A1 in the mineralization process. Murine MSCs derived from the bone marrow of BALB/c mouse or commercially purchased human MSCs were treated with osteogenic induction medium containing 5.8 mM magnesium chloride and the osteogenic differentiation efficiency was compared with that of MSCs in normal differentiation medium containing 0.8 mM magnesium chloride by cell morphology, gene expression profile of osteogenic markers, and Alizarin Red staining. Slc41a1 gene knockdown in MSCs was performed by siRNA transfection using Lipofectamine RNAiMAX, and the differentiation efficiency of siRNA-treated MSCs was also assessed. High concentration of extracellular magnesium ion inhibited mineralization during osteogenic differentiation of MSCs. Early osteogenic marker genes including osterix, alkaline phosphatase, and type I collagen were significantly downregulated in MSCs under high concentration of magnesium, whereas late marker genes such as osteopontin, osteocalcin, and bone morphogenetic protein 2 were upregulated with statistical significance compared with those in normal differentiation medium containing 0.8 mM magnesium. siRNA treatment targeting SLC41A1 magnesium transporter, a member of the solute carrier family with a predominant Mg 2+ efflux system, accelerated the mineralization process and ameliorated the inhibition of mineralization caused by high concentration of magnesium. High concentration of magnesium significantly upregulated Dkk1 gene expression and the upregulation was attenuated after the Slc41a1 gene was knocked down. Immunofluorescent staining showed that Slc41a1 gene knockdown promoted the translocation of phosphorylated β-catenin into nuclei. In addition, secreted MGP protein was elevated after Slc41a1 was knocked down. High concentration of extracellular magnesium modulates gene expression of MSCs during osteogenic differentiation and inhibits the mineralization process. Additionally, we identified magnesium transporter SLC41A1 that regulates the interaction of magnesium and MSCs during osteogenic differentiation. Wnt signaling is suggested to be involved in SLC41A1-mediated regulation. Tissue-specific SLC41A1 could be a potential treatment for bone mass loss; in addition, caution should be taken regarding the role of magnesium in osteoporosis and the design of magnesium alloys for implantation.

Tetraspanin 7 regulates sealing zone formation and the bone-resorbing activity of osteoclasts

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kwon, Jun-Oh; Lee, Yong Deok; Kim, Haemin

Tetraspanin family proteins regulate morphology, motility, fusion, and signaling in various cell types. We investigated the role of the tetraspanin 7 (Tspan7) isoform in the differentiation and function of osteoclasts. Tspan7 was up-regulated during osteoclastogenesis. When Tspan7 expression was reduced in primary precursor cells by siRNA-mediated gene knock-down, the generation of multinuclear osteoclasts was not affected. However, a striking cytoskeletal abnormality was observed: the formation of the podosome belt structure was inhibited and the microtubular network were disrupted by Tspan7 knock-down. Decreases in acetylated microtubules and levels of phosphorylated Src and Pyk2 in Tspan7 knock-down cells supported the involvement ofmore » Tspan7 in cytoskeletal rearrangement signaling in osteoclasts. This cytoskeletal defect interfered with sealing zone formation and subsequently the bone-resorbing activity of mature osteoclasts on dentin surfaces. Our results suggest that Tspan7 plays an important role in cytoskeletal organization required for the bone-resorbing function of osteoclasts by regulating signaling to Src, Pyk2, and microtubules. - Highlights: • Tspan7 expression is up-regulated during osteoclastogenesis. • Tspan7 regulates podosome belt organization in osteoclasts. • Tspan7 is crucial for sealing zone formation and bone-resorption by osteoclasts. • Src and Pyk2 phosphorylation and microtubule acetylation mediate Tspan7 function.« less

The Extracellular Environment's Effect on Cellular Processes: An In Vitro Study of Mechanical and Chemical Cues on Human Mesenchymal Stem Cells and C17.2 Neural Stem Cells

NASA Astrophysics Data System (ADS)

Casey, Meghan E.

Stem cells are widely used in the area of tissue engineering. The ability of cells to interact with materials on the nano- and micro- level is important in the success of the biomaterial. It is well-known that cells respond to their micro- and nano-environments through a process termed chemo-mechanotransduction. It is important to establish standard protocols for cellular experiments, as chemical modifications to maintenance environments can alter long-term research results. In this work, the effects of different media compositions on human mesenchymal stem cells (hMSCs) throughout normal in vitro maintenance are investigated. Changes in RNA regulation, protein expression and proliferation are studied via quantitative polymerase chain reaction (qPCR), immunocytochemistry (ICC) and cell counts, respectively. Morphological differences are also observed throughout the experiment. Results of this study illustrate the dynamic response of hMSC maintenance to differences in growth medium and passage number. These experiments highlight the effect growth medium has on in vitro experiments and the need of consistent protocols in hMSC research. A substantial opportunity exists in neuronal research to develop a material platform that allows for both the proliferation and differentiation of stem cells into neurons and the ability to quantify the secretome of neuronal cells. Anodic aluminum oxide (AAO) membranes are fabricated in a two-step anodization procedure where voltage is varied to control the pore size and morphology of the membranes. C17.2 neural stem cells are differentiated on the membranes via serum-withdrawal. Cellular growth is characterized by scanning electron microscopy (SEM), ICC and qPCR. ImageJ software is used to obtain phenotypic cell counts and neurite outgrowth lengths. Results indicate a highly tunable correlation between AAO nanopore sizes and differentiated cell populations. By selecting AAO membranes with specific pore size ranges, control of neuronal network density and neurite outgrowth length is achievable. To understand differentiation marker expressions in C17.2 NSCs and how material stiffness affects differentiation, cells are cultured on substrates of varying stiffness. qPCR is used to analyze neural stem cell, neural progenitor cell, neuron-restricted progenitor and differentiated post-mitotic neuronal cell RNA expression. Results suggest a relationship between material stiffness and neuronal development in C17.2 neural stem cells.

Mutation of the murC and murB Genes Impairs Heterocyst Differentiation in Anabaena sp. Strain PCC 7120

PubMed Central

Videau, Patrick; Rivers, Orion S.; Ushijima, Blake; Oshiro, Reid T.; Kim, Min Joo; Philmus, Benjamin

2016-01-01

ABSTRACT To stabilize cellular integrity in the face of environmental perturbations, most bacteria, including cyanobacteria, synthesize and maintain a strong, flexible, three-dimensional peptidoglycan lattice. Anabaena sp. strain PCC 7120 is a filamentous cyanobacterium capable of differentiating morphologically distinct nitrogen-fixing heterocyst cells in a periodic pattern. While heterocyst development has been shown to require proper peptidoglycan remodeling, the role of peptidoglycan synthesis has remained unclear. Here we report the identification of two peptidoglycan synthesis genes, murC (alr5065) and murB (alr5066), as required for heterocyst development. The murC and murB genes are predicted to encode a UDP-N-acetylmuramate:l-alanine ligase and a UDP-N-acetylenolpyruvoylglucosamine reductase, respectively, and we confirm enzymatic function through complementation of Escherichia coli strains deficient for these enzymes. Cells depleted of either murC or murB expression failed to differentiate heterocysts under normally inducing conditions and displayed decreased filament integrity. To identify the stage(s) of development affected by murC or murB depletion, the spatial distribution of expression of the patterning marker gene, patS, was examined. Whereas murB depletion did not affect the pattern of patS expression, murC depletion led to aberrant expression of patS in all cells of the filament. Finally, expression of gfp controlled by the region of DNA immediately upstream of murC was enriched in differentiating cells and was repressed by the transcription factor NtcA. Collectively, the data in this work provide evidence for a direct link between peptidoglycan synthesis and the maintenance of a biological pattern in a multicellular organism. IMPORTANCE Multicellular organisms that differentiate specialized cells must regulate morphological changes such that both cellular integrity and the dissemination of developmental signals are preserved. Here we show that the multicellular bacterium Anabaena, which differentiates a periodic pattern of specialized heterocyst cells, requires peptidoglycan synthesis by the murine ligase genes murC (alr5065) and murB (alr5066) for maintenance of patterned gene expression, filament integrity, and overall development. This work highlights the significant influence that intracellular structure and intercellular connections can have on the execution of a developmental program. PMID:26811320

Mutation of the murC and murB Genes Impairs Heterocyst Differentiation in Anabaena sp. Strain PCC 7120.

PubMed

Videau, Patrick; Rivers, Orion S; Ushijima, Blake; Oshiro, Reid T; Kim, Min Joo; Philmus, Benjamin; Cozy, Loralyn M

2016-04-01

To stabilize cellular integrity in the face of environmental perturbations, most bacteria, including cyanobacteria, synthesize and maintain a strong, flexible, three-dimensional peptidoglycan lattice. Anabaena sp. strain PCC 7120 is a filamentous cyanobacterium capable of differentiating morphologically distinct nitrogen-fixing heterocyst cells in a periodic pattern. While heterocyst development has been shown to require proper peptidoglycan remodeling, the role of peptidoglycan synthesis has remained unclear. Here we report the identification of two peptidoglycan synthesis genes, murC (alr5065) and murB (alr5066), as required for heterocyst development. The murC and murB genes are predicted to encode a UDP-N-acetylmuramate:L-alanine ligase and a UDP-N-acetylenolpyruvoylglucosamine reductase, respectively, and we confirm enzymatic function through complementation of Escherichia coli strains deficient for these enzymes. Cells depleted of either murC or murB expression failed to differentiate heterocysts under normally inducing conditions and displayed decreased filament integrity. To identify the stage(s) of development affected by murC or murB depletion, the spatial distribution of expression of the patterning marker gene, patS, was examined. Whereas murB depletion did not affect the pattern of patS expression, murC depletion led to aberrant expression of patS in all cells of the filament. Finally, expression of gfp controlled by the region of DNA immediately upstream of murC was enriched in differentiating cells and was repressed by the transcription factor NtcA. Collectively, the data in this work provide evidence for a direct link between peptidoglycan synthesis and the maintenance of a biological pattern in a multicellular organism. Multicellular organisms that differentiate specialized cells must regulate morphological changes such that both cellular integrity and the dissemination of developmental signals are preserved. Here we show that the multicellular bacterium Anabaena, which differentiates a periodic pattern of specialized heterocyst cells, requires peptidoglycan synthesis by the murine ligase genes murC (alr5065) and murB (alr5066) for maintenance of patterned gene expression, filament integrity, and overall development. This work highlights the significant influence that intracellular structure and intercellular connections can have on the execution of a developmental program. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Genetic, morphological, and acoustic evidence reveals lack of diversification in the colonization process in an island bird.

PubMed

Illera, Juan Carlos; Palmero, Ana M; Laiolo, Paola; Rodríguez, Felipe; Moreno, Ángel C; Navascués, Miguel

2014-08-01

Songbirds with recently (i.e., early Holocene) founded populations are suitable models for studying incipient differentiation in oceanic islands. On such systems each colonization event represents a different evolutionary episode that can be studied by addressing sets of diverging phenotypic and genetic traits. We investigate the process of early differentiation in the spectacled warbler (Sylvia conspicillata) in 14 populations separated by sea barriers from three Atlantic archipelagos and from continental regions spanning from tropical to temperate latitudes. Our approach involved the study of sexual acoustic signals, morphology, and genetic data. Mitochondrial DNA did not provide clear population structure. However, microsatellites analyses consistently identified two genetic groups, albeit without correspondence to subspecies classification and little correspondence to geography. Coalescent analyses showed significant evidence for gene flow between the two genetic groups. Discriminant analyses could not correctly assign morphological or acoustic traits to source populations. Therefore, although theory predicting that in isolated populations genetic, morphological, or acoustic traits can lead to radiation, we have strikingly failed to document differentiation on these attributes in a resident passerine throughout three oceanic archipelagos. © 2014 The Author(s). Evolution © 2014 The Society for the Study of Evolution.

Genetic and Morphological Differentiation of the Semiterrestrial Crab Armases angustipes (Brachyura: Sesarmidae) along the Brazilian Coast.

PubMed

Marochi, Murilo Zanetti; Masunari, Setuko; Schubart, Christoph D

2017-02-01

The genetic and morphometric population structures of the semiterrestrial crab Armases angustipes from along the Brazilian coast were examined. The influence of the Central South Equatorial Current on larval dispersal of A. angustipes also was evaluated. Six populations were sampled from estuarine areas in São Luis do Maranhão, Maranhão; Natal, Rio Grande do Norte; Maceió, Alagoas; Ilhéus, Bahia; Aracruz, Espírito Santo; and Guaratuba, Paraná. Patterns of genetic differentiation were assessed using DNA sequence data corresponding to parts of the mitochondrial cytochrome c oxidase subunit 1. Geometric morphometric techniques were used to evaluate morphological variation in shape and size of the carapace and right cheliped propodus. Our results revealed low genetic variability and lack of phylogeographic structure; geometric morphometrics showed statistically significant morphological differentiation and geographic structuring. Our data indicate the absence of possible barriers to gene flow for this mobile species, and no clear correlation of morphological or genetic variation with ocean currents and/or geographic distance. Our results also suggest that historical geological and climatological events and/or possible bottleneck effects influenced the current low genetic variability among the populations of A. angustipes.

Increasing morphological complexity in multiple parallel lineages of the Crustacea

PubMed Central

Adamowicz, Sarah J.; Purvis, Andy; Wills, Matthew A.

2008-01-01

The prospect of finding macroevolutionary trends and rules in the history of life is tremendously appealing, but very few pervasive trends have been found. Here, we demonstrate a parallel increase in the morphological complexity of most of the deep lineages within a major clade. We focus on the Crustacea, measuring the morphological differentiation of limbs. First, we show a clear trend of increasing complexity among 66 free-living, ordinal-level taxa from the Phanerozoic fossil record. We next demonstrate that this trend is pervasive, occurring in 10 or 11 of 12 matched-pair comparisons (across five morphological diversity indices) between extinct Paleozoic and related Recent taxa. This clearly differentiates the pattern from the effects of lineage sorting. Furthermore, newly appearing taxa tend to have had more types of limbs and a higher degree of limb differentiation than the contemporaneous average, whereas those going extinct showed higher-than-average limb redundancy. Patterns of contemporary species diversity partially reflect the paleontological trend. These results provide a rare demonstration of a large-scale and probably driven trend occurring across multiple independent lineages and influencing both the form and number of species through deep time and in the present day. PMID:18347335

Effects of keratinocyte growth factor on skin epithelial differentiation of human amnion epithelial cells.

PubMed

Fatimah, Simat Siti; Tan, Geok Chin; Chua, Kienhui; Tan, Ay Eeng; Nur Azurah, Abdul Ghani; Hayati, Abdul Rahman

2013-08-01

The aim of the present study was to determine the effects of KGF on the differentiation of cultured human amnion epithelial cells (HAECs) towards skin keratinocyte. HAECs at passage 1 were cultured in medium HAM's F12: Dulbecco's Modified Eagles Medium (1:1) supplemented with different concentrations of KGF (0, 5, 10, 20, 30 and 50 ng/ml KGF). Dose-response of KGF on HAECs was determined by morphological assessment; growth kinetic evaluation; immunocytochemical analysis; stemness and epithelial gene expression quantification with two step real time RT-PCR. KGF promotes the proliferation of HAECs with maximal effect observed at 10 ng/ml KGF. However, KGF decreased the stemness genes expression: Oct-3/4, Sox-2, Nanog3, Rex-1, FGF-4, FZD-9 and BST-1. KGF also down-regulates epithelial genes expression: CK3, CK18, CK19, Integrin-β1, p63 and involucrin in cultured HAECs. No significant difference on the gene expression was detected for each Nestin, ABCG-2, CK1 and CK14 in KGF-treated HAECs. Immunocytochemical analysis for both control and KGF-treated HAECs demonstrated positive staining against CK14 and CK18 but negative staining against involucrin. The results suggested that KGF stimulates an early differentiation of HAECs towards epidermal cells. Differentiation of KGF-treated HAECs to corneal lineage is unfavourable. Therefore, further studies are needed to elucidate the roles of KGF in the differentiation of HAECs towards skin keratinocytes. Copyright © 2012 Elsevier Ltd and ISBI. All rights reserved.

Gene expression profiling in multipotent DFAT cells derived from mature adipocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ono, Hiromasa; Database Center for Life Science; Oki, Yoshinao

2011-04-15

Highlights: {yields} Adipocyte dedifferentiation is evident in a significant decrease in typical genes. {yields} Cell proliferation is strongly related to adipocyte dedifferentiation. {yields} Dedifferentiated adipocytes express several lineage-specific genes. {yields} Comparative analyses using publicly available datasets boost the interpretation. -- Abstract: Cellular dedifferentiation signifies the withdrawal of cells from a specific differentiated state to a stem cell-like undifferentiated state. However, the mechanism of dedifferentiation remains obscure. Here we performed comparative transcriptome analyses during dedifferentiation in mature adipocytes (MAs) to identify the transcriptional signatures of multipotent dedifferentiated fat (DFAT) cells derived from MAs. Using microarray systems, we explored similarly expressed asmore » well as significantly differentially expressed genes in MAs during dedifferentiation. This analysis revealed significant changes in gene expression during this process, including a significant reduction in expression of genes for lipid metabolism concomitantly with a significant increase in expression of genes for cell movement, cell migration, tissue developmental processes, cell growth, cell proliferation, cell morphogenesis, altered cell shape, and cell differentiation. Our observations indicate that the transcriptional signatures of DFAT cells derived from MAs are summarized in terms of a significant decrease in functional phenotype-related genes and a parallel increase in cell proliferation, altered cell morphology, and regulation of the differentiation of related genes. A better understanding of the mechanisms involved in dedifferentiation may enable scientists to control and possibly alter the plasticity of the differentiated state, which may lead to benefits not only in stem cell research but also in regenerative medicine.« less

Tunable Microfibers Suppress Fibrotic Encapsulation via Inhibition of TGFβ Signaling

PubMed Central

Allen, Jessica; Ryu, Jubin; Maggi, Alessandro; Flores, Bianca; Greer, Julia R.

2016-01-01

Fibrotic encapsulation limits the efficacy and lifetime of implantable biomedical devices. Microtopography has shown promise in the regulation of myofibroblast differentiation, a key driver of fibrotic encapsulation. However, existing studies have not systematically isolated the requisite geometric parameters for suppression of myofibroblast differentiation via microtopography, and there has not been in vivo validation of this technology to date. To address these issues, a novel lamination method was developed to afford more control over topography dimensions. Specifically, in this study we focus on fiber length and its effect on myofibroblast differentiation. Fibroblasts cultured on films with microfibers exceeding 16 μm in length lost the characteristic morphology associated with myofibroblast differentiation, while shorter microfibers of 6 μm length failed to produce this phenotype. This increase in length corresponded to a 50% decrease in fiber stiffness, which acts as a mechanical cue to influence myofibroblast differentiation. Longer microfiber films suppressed expression of myofibroblast-specific genes (αSMA, Col1α2, and Col3α1) and TGFβ signaling components (TGFβ1, TβR2, and Smad3). About 16 μm long microfiber films subcutaneously implanted in a mouse wound-healing model generated a substantially thinner fibrotic capsule and less deposition of collagen in the wound bed. Together, these results identify a critical feature length threshold for microscale topography-mediated repression of fibrotic encapsulation. This study also demonstrates a simple and powerful strategy to improve surface biocompatibility and reduce fibrotic encapsulation around implanted materials. PMID:26507808

Overexpressed Calponin3 by Subsonic Vibration Induces Neural Differentiation of hUC-MSCs by Regulating the Ionotropic Glutamate Receptor.

PubMed

Kim, Hyun-Jung; Kim, Jin-Hee; Song, Yeo-Ju; Seo, Young-Kwon; Park, Jung-Keug; Kim, Chan-Wha

2015-09-01

In this study, we used proteomics to investigate the effects of sonic vibration (SV) on mesenchymal stem cells derived from human umbilical cords (hUC-MSCs) during neural differentiation to understand how SV enhances neural differentiation of hUC-MSCs. We investigated the levels of gene and protein related to neural differentiation after 3 or 5 days in a group treated with 40-Hz SV. In addition, protein expression patterns were compared between the control and the 40-Hz SV-treated hUC-MSC groups via a proteomic approach. Among these proteins, calponin3 (CNN3) was confirmed to have 299 % higher expression in the 40-Hz SV stimulated hUC-MSCs group than that in the control by Western blotting. Notably, overexpression of CNN3-GFP in Chinese hamster ovary (CHO)-K1 cells had positive effects on the stability and reorganization of F-actin compared with that in GFP-transfected cells. Moreover, CNN3 changed the morphology of the cells by making a neurite-like form. After being subjected to SV, messenger RNA (mRNA) levels of glutamate receptors such as PSD95, GluR1, and NR1 as well as intracellular calcium levels were upregulated. These results suggest that the activity of glutamate receptors increased because of CNN3 characteristics. Taken together, these results demonstrate that overexpressed CNN3 during SV increases expression of glutamate receptors and promotes functional neural differentiation of hUC-MSCs.

A TCP domain transcription factor controls flower type specification along the radial axis of the Gerbera (Asteraceae) inflorescence.

PubMed

Broholm, Suvi K; Tähtiharju, Sari; Laitinen, Roosa A E; Albert, Victor A; Teeri, Teemu H; Elomaa, Paula

2008-07-01

Several key processes in plant development are regulated by TCP transcription factors. CYCLOIDEA-like (CYC-like) TCP domain proteins have been shown to control flower symmetry in distantly related plant lineages. Gerbera hybrida, a member of one of the largest clades of angiosperms, the sunflower family (Asteraceae), is an interesting model for developmental studies because its elaborate inflorescence comprises different types of flowers that have specialized structures and functions. The morphological differentiation of flower types involves gradual changes in flower size and symmetry that follow the radial organization of the densely packed inflorescence. Differences in the degree of petal fusion further define the distinct shapes of the Gerbera flower types. To study the role of TCP transcription factors during specification of this complex inflorescence organization, we characterized the CYC-like homolog GhCYC2 from Gerbera. The expression of GhCYC2 follows a gradient along the radial axis of the inflorescence. GhCYC2 is expressed in the marginal, bilaterally symmetrical ray flowers but not in the centermost disk flowers, which are nearly radially symmetrical and have significantly less fused petals. Overexpression of GhCYC2 causes disk flowers to obtain morphologies similar to ray flowers. Both expression patterns and transgenic phenotypes suggest that GhCYC2 is involved in differentiation among Gerbera flower types, providing the first molecular evidence that CYC-like TCP factors take part in defining the complex inflorescence structure of the Asteraceae, a major determinant of the family's evolutionary success.

Human induced pluripotent stem cell-derived beating cardiac tissues on paper.

PubMed

Wang, Li; Xu, Cong; Zhu, Yujuan; Yu, Yue; Sun, Ning; Zhang, Xiaoqing; Feng, Ke; Qin, Jianhua

2015-11-21

There is a growing interest in using paper as a biomaterial scaffold for cell-based applications. In this study, we made the first attempt to fabricate a paper-based array for the culture, proliferation, and direct differentiation of human induced pluripotent stem cells (hiPSCs) into functional beating cardiac tissues and create "a beating heart on paper." This array was simply constructed by binding a cured multi-well polydimethylsiloxane (PDMS) mold with common, commercially available paper substrates. Three types of paper material (print paper, chromatography paper and nitrocellulose membrane) were tested for adhesion, proliferation and differentiation of human-derived iPSCs. We found that hiPSCs grew well on these paper substrates, presenting a three-dimensional (3D)-like morphology with a pluripotent property. The direct differentiation of human iPSCs into functional cardiac tissues on paper was also achieved using our modified differentiation approach. The cardiac tissue retained its functional activities on the coated print paper and chromatography paper with a beating frequency of 40-70 beats per min for up to three months. Interestingly, human iPSCs could be differentiated into retinal pigment epithelium on nitrocellulose membrane under the conditions of cardiac-specific induction, indicating the potential roles of material properties and mechanical cues that are involved in regulating stem cell differentiation. Taken together, these results suggest that different grades of paper could offer great opportunities as bioactive, low-cost, and 3D in vitro platforms for stem cell-based high-throughput drug testing at the tissue/organ level and for tissue engineering applications.

Epigenetic regulation of osteogenesis: human embryonic palatal mesenchymal cells.

PubMed

Barkhordarian, Andre; Sison, Jay; Cayabyab, Riana; Mahanian, Nicole; Chiappelli, Francesco

2011-01-06

Mesenchymal stem cells (MSCs) provide an appropriate model to study epigenetic changes during osteogenesis and bone regeneration due to their differentiation potential. Since there are no unique markers for MSCs, methods of identification are limited. The complex morphology of human embryonic palatal mesenchyme stem cell (HEPM) requires analysis of fractal dimensions to provide an objective quantification of self-similarity, a statistical transformation of cellular shape and border complexity. We propose the hypothesis of a study to compare and contrast sequential steps of osteogenic differentiation in HEPMs both phenotypically using immunocytochemistry, and morphometrically using fractal analysis from undifferentiated passage 1 (P1) to passage 7 (P7) cells. The proof-of-concept is provided by results we present here that identify and compare the modulation of expression of certain epigenetic biomarkers (alkaline phosphatase, ALP; stromal interaction molecule-1, STRO-1; runt-related transcription factor-2, RUNX2), which are established markers of osteogenesis in bone marrow studies, of osteoblastic/skeletal morphogenesis, and of osteoblast maturation. We show that Osteoinductive medium (OIM) modulates the rate of differentiation of HEPM into Run-2+ cells, the most differentiated subpopulation, followed by ALP+ and STRO-1+ cells. Taken together, our phenotypical and morphometric data demonstrate the feasibility of using HEPM to assess osteogenic differentiation from an early undifferentiated to a differentiated stage. This research model may lay the foundation for future studies aimed at characterizing the epigenetic characteristics of osteoimmunological disorders and dysfunctions (e.g., osteoarthritis, temporomandibular joint disorders), so that proteomic profiling can aid the diagnosis and monitor the prognosis of these and other osteoimmunopathologies.

Differential gene expression in response to juvenile hormone analog treatment in the damp-wood termite Hodotermopsis sjostedti (Isoptera, Archotermopsidae).

PubMed

Cornette, Richard; Hayashi, Yoshinobu; Koshikawa, Shigeyuki; Miura, Toru

2013-04-01

Termite societies are characterized by a highly organized division of labor among conspicuous castes, groups of individuals with various morphological specializations. Termite caste differentiation is under control of juvenile hormone (JH), but the molecular mechanism underlying the response to JH and early events triggering caste differentiation are still poorly understood. In order to profile candidate gene expression during early soldier caste differentiation of the damp-wood termite, Hodotermopsis sjostedti, we treated pseudergates (workers) with a juvenile hormone analog (JHA) to induce soldier caste differentiation. We then used Suppressive Subtractive Hybridization to create two cDNA libraries enriched for transcripts that were either up- or downregulated at 24h after treatment. Finally, we used quantitative PCR to confirm temporal expression patterns. Hexamerins represent a large proportion of the genes upregulated following JHA treatment and have an expression pattern that shows roughly an inverse correlation to intrinsic JH titers. This data is consistent with the role of a JH "sink", which was demonstrated for hexamerins in another termite, Reticulitermes flavipes. A putative nuclear protein was also upregulated a few hours after JHA treatment, which suggests a role in the early response to JH and subsequent regulation of transcriptional events associated with soldier caste differentiation. Some digestive enzymes, such as endogenous beta-endoglucanase and chymotrypsin, as well as a protein associated to digestion were identified among genes downregulated after JHA treatment. This suggests that JH may directly influence the pseudergate-specific digestive system. Copyright © 2013 Elsevier Ltd. All rights reserved.

Sexually dimorphic differentiation of a C. elegans hub neuron is cell-autonomously controlled by a conserved transcription factor

PubMed Central

Serrano-Saiz, Esther; Oren-Suissa, Meital; Bayer, Emily A.; Hobert, Oliver

2018-01-01

SUMMARY Functional and anatomical sexual dimorphisms in the brain are either the result of cells that are generated only in one sex, or a manifestation of sex-specific differentiation of neurons present in both sexes. The PHC neurons of the nematode C. elegans differentiate in a strikingly sex-specific manner. While in hermaphrodites the PHC neurons display a canonical pattern of synaptic connectivity similar to that of other sensory neurons, PHC differentiates into a densely connected hub sensory/interneuron in males, integrating a large number of male-specific synaptic inputs and conveying them to both male-specific and sex-shared circuitry. We show that the differentiation into such a hub neuron involves the sex-specific scaling of several components of the synaptic vesicle machinery, including the vesicular glutamate transporter eat-4/VGLUT, induction of neuropeptide expression, changes in axonal projection morphology and a switch in neuronal function. We demonstrate that these molecular and anatomical remodeling events are controlled cell-autonomously by the phylogenetically conserved Doublesex homolog dmd-3, which is both required and sufficient for sex-specific PHC differentiation. Cellular specificity of dmd-3 action is ensured by its collaboration with non-sex specific terminal selector-type transcription factors whereas sex-specificity of dmd-3 action is ensured by the hermaphrodite-specific, transcriptional master regulator of hermaphroditic cell identity, tra-1, which represses transcription of dmd-3 in hermaphrodite PHC. Taken together, our studies provide mechanistic insights into how neurons are specified in a sexually dimorphic manner. PMID:28065609

6-gingerol inhibits rosiglitazone-induced adipogenesis in 3T3-L1 adipocytes.

PubMed

Tzeng, Thing-Fong; Chang, Chia Ju; Liu, I-Min

2014-02-01

We investigated the effects of 6-gingerol ((S)-5-hydroxy-1-(4-hydroxy-3-methoxyphenyl)-3-decanone) on the inhibition of rosiglitazone (RGZ)-induced adipogenesis in 3T3-L1 cells. The morphological changes were photographed based on staining lipid accumulation by Oil-Red O in RGZ (1 µmol/l)-treated 3T3-L1 cells without or with various concentrations of 6-gingerol on differentiation day 8. Quantitation of triglycerides content was performed in cells on day 8 after differentiation induction. Differentiated cells were lysed to detect mRNA and protein levels of adipocyte-specific transcription factors by real-time reverse transcription-polymerase chain reaction and Western blot analysis, respectively. 6-gingerol (50 µmol/l) effectively suppressed oil droplet accumulation and reduced the sizes of the droplets in RGZ-induced adipocyte differentiation in 3T3-L1 cells. The triglyceride accumulation induced by RGZ in differentiated 3T3-L1 cells was also reduced by 6-gingerol (50 µmol/l). Treatment of differentiated 3T3-L1 cells with 6-gingerol (50 µmol/l) antagonized RGZ-induced gene expression of peroxisome proliferator-activated receptor (PPAR)γ and CCAAT/enhancer-binding protein α. Additionally, the increased levels of mRNA and protein in adipocyte-specific fatty acid binding protein 4 and fatty acid synthase induced by RGZ in 3T3-L1 cells were decreased upon treatment with 6-gingerol. Our data suggests that 6-gingerol may be beneficial in obesity, by reducing adipogenesis partly through the down-regulating PPARγ activity. Copyright © 2013 John Wiley & Sons, Ltd.

Diversity and population structure of a dominant deciduous tree based on morphological and genetic data

PubMed Central

Zhang, Qin-di; Jia, Rui-Zhi; Meng, Chao; Ti, Chao-Wen; Wang, Yi-Ling

2015-01-01

Knowledge of the genetic diversity and structure of tree species across their geographic ranges is essential for sustainable use and management of forest ecosystems. Acer grosseri Pax., an economically and ecologically important maple species, is mainly distributed in North China. In this study, the genetic diversity and population differentiation of 24 natural populations of this species were evaluated using sequence-related amplified polymorphism markers and morphological characters. The results show that highly significant differences occurred in 32 morphological traits. The coefficient of variation of 34 characters was 18.19 %. Principal component analysis indicated that 18 of 34 traits explained 60.20 % of the total variance. The phenotypic differentiation coefficient (VST) was 36.06 % for all morphological traits. The Shannon–Wiener index of 34 morphological characters was 6.09, while at the population level, it was 1.77. The percentage of polymorphic bands of all studied A. grosseri populations was 82.14 %. Nei's gene diversity (He) and Shannon's information index (I) were 0.35 and 0.50, respectively. Less genetic differentiation was detected among the natural populations (GST = 0.20, ΦST = 0.10). Twenty-four populations of A. grosseri formed two main clusters, which is consistent with morphological cluster analysis. Principal coordinates analysis and STRUCTURE analysis supported the UPGMA-cluster dendrogram. There was no significant correlation between genetic and geographical distances among populations. Both molecular and morphological data suggested that A. grosseri is rich in genetic diversity. The high level of genetic variation within populations could be affected by the biological characters, mating system and lifespan of A. grosseri, whereas the lower genetic diversity among populations could be caused by effective gene exchange, selective pressure from environmental heterogeneity and the species' geographical range. PMID:26311734

Differentiation between diverticulitis and colorectal cancer: quantitative CT perfusion measurements versus morphologic criteria--initial experience.

PubMed

Goh, Vicky; Halligan, Steve; Taylor, Stuart A; Burling, David; Bassett, Paul; Bartram, Clive I

2007-02-01

To determine whether computed tomographic (CT) perfusion measurements in prospectively recruited patients can be used to differentiate between diverticulitis and colorectal cancer and to compare this discrimination with that of standard morphologic criteria. After institutional review board approval and written informed consent were obtained, 60 patients (24 men, 36 women; mean age, 69 years; range, 33.5-90.4 years; 20 patients with cancer, 20 with diverticulitis, and 20 with inactive diverticular disease) underwent CT perfusion imaging at the level of the colonic abnormality, and perfusion parameters were calculated. Analysis of variance was used to investigate any differences in perfusion between the patient groups. Two independent observers also analyzed an abdominopelvic CT study obtained immediately after the CT perfusion study and noted standard morphologic criteria for differential diagnosis. The sensitivity and specificity of CT perfusion measurements for determining the diagnostic category were compared with morphologic criteria by means of multivariate analysis to identify the most discriminatory criteria. Mean blood volume, blood flow, transit time, and permeability were significantly different between patients with cancer and those with diverticulitis (P < .0001); patients with cancer had the highest blood volume, blood flow, and permeability and the shortest transit time. The most discriminatory criteria for determining diagnostic category were blood volume, transit time, permeability, and presence of pericolonic nodes (P = .05, .02, .04, and .02, respectively). Blood volume and blood flow each had a sensitivity of 80% and had specificity of 70% and 75%, respectively, for cancer in comparison with standard morphologic criteria: less than 5 cm of bowel involvement (45% sensitivity, 95% specificity), presence of a mass (85% sensitivity, 90% specificity), pericolonic inflammation (75% sensitivity, 5% specificity), and pericolonic nodes (90% sensitivity, 45% specificity). CT perfusion measurements enable differentiation and better discrimination, in comparison with morphologic criteria, between cancer and diverticulitis. (c) RSNA, 2007.

Population delimitation across contrasting evolutionary clines in deer mice (Peromyscus maniculatus)

PubMed Central

Yang, D-S; Kenagy, G

2011-01-01

Despite current interest in population genetics, a concrete definition of a “population” remains elusive. Multiple ecologically and evolutionarily based definitions of population are in current use, which focus, respectively, on demographic and genetic interactions. Accurate population delimitation is crucial for not only evolutionary and ecological population biology, but also for conservation of threatened populations. Along the Pacific Coast of North America, two contrasting patterns of geographic variation in deer mice (Peromyscus maniculatus) converge within the state of Oregon. Populations of these mice diverge morphologically across an east–west axis, and they diverge in mitochondrial DNA haplotypes across a north–south axis. In this study, we investigate these geographically contrasting patterns of differentiation in the context of ecological and evolutionary definitions (paradigms) of populations. We investigate these patterns using a new and geographically expansive sample that integrates data on morphology, mitochondrial DNA, and nuclear DNA. We found no evidence of nuclear genetic differentiation between the morphologically and mitochondrially distinct populations, thus indicating the occurrence of gene flow across Oregon. Under the evolutionary paradigm, Oregon populations can be considered a single population, whereas morphological and mitochondrial differentiations do not indicate distinct populations. In contrast, under the ecological paradigm morphological differentiation indicates distinct populations based on the low likelihood of demographic interactions between geographically distant individuals. The two sympatric but mitochondrially distinct haplogroups form a single population under the ecological paradigm. Hence, we find that the difference between evolutionary and ecological paradigms is the time-scale of interest, and we believe that the more chronologically inclusive evolutionary paradigm may be preferable except in cases where only a single generation is of interest. PMID:22393480

Gender Differentiations of Some Anthropological Characteristics of Karate Players--Cadets.

PubMed

Jukić, Josefina

2015-07-01

The research was conducted with the aim of determining gender differentiations of some anthropological characteristics of male and female cadet karate players. To achieve this aim, a group of 10 morphological variables, ten motor tests, five situation motor test and six technical performance evaluation tests was applied. Two hundred and six competing players were measured, of which 105 male and 101 female. The examinees were divided according to weight categories (lower and higher weight categories). Based on the variance analysis and canon discrimination analysis a conclusion can be made: gender differentiations are most strongly expressed in morphological and basic motor area, and significantly less in the area of specific motor abilities and motor knowledge (technical efficiency).

PKCalpha-mediated ERK, JNK and p38 activation regulates the myogenic program in human rhabdomyosarcoma cells.

PubMed

Mauro, Annunziata; Ciccarelli, Carmela; De Cesaris, Paola; Scoglio, Arianna; Bouché, Marina; Molinaro, Mario; Aquino, Angelo; Zani, Bianca Maria

2002-09-15

We have previously suggested that PKCalpha has a role in 12-O-Tetradecanoylphorbol-13-acetate (TPA)-mediated growth arrest and myogenic differentiation in human embryonal rhabdomyosarcoma cells (RD). Here, by monitoring the signalling pathways triggered by TPA, we demonstrate that PKCalpha mediates these effects by inducing transient activation of c-Jun N-terminal protein kinases (JNKs) and sustained activation of both p38 kinase and extracellular signal-regulated kinases (ERKs) (all referred to as MAPKs). Activation of MAPKs following ectopic expression of constitutively active PKCalpha, but not its dominant-negative form, is also demonstrated. We investigated the selective contribution of MAPKs to growth arrest and myogenic differentiation by monitoring the activation of MAPK pathways, as well as by dissecting MAPK pathways using MEK1/2 inhibitor (UO126), p38 inhibitor (SB203580) and JNK and p38 agonist (anisomycin) treatments. Growth-arresting signals are triggered either by transient and sustained JNK activation (by TPA and anisomycin, respectively) or by preventing both ERK and JNK activation (UO126) and are maintained, rather than induced, by p38. We therefore suggest a key role for JNK in controlling ERK-mediated mitogenic activity. Notably, sarcomeric myosin expression is induced by both TPA and UO126 but is abrogated by the p38 inhibitor. This finding indicates a pivotal role for p38 in controlling the myogenic program. Anisomycin persistently activates p38 and JNKs but prevents myosin expression induced by TPA. In accordance with this negative role, reactivation of JNKs by anisomycin, in UO126-pre-treated cells, also prevents myosin expression. This indicates that, unlike the transient JNK activation that occurs in the TPA-mediated myogenic process, long-lasting JNK activation supports the growth-arrest state but antagonises p38-mediated myosin expression. Lastly, our results with the MEK inhibitor suggest a key role of the ERK pathway in regulating myogenic-related morphology in differentiated RD cells.

Calcium signaling mediates five types of cell morphological changes to form neural rosettes.

PubMed

Hříbková, Hana; Grabiec, Marta; Klemová, Dobromila; Slaninová, Iva; Sun, Yuh-Man

2018-02-12

Neural rosette formation is a critical morphogenetic process during neural development, whereby neural stem cells are enclosed in rosette niches to equipoise proliferation and differentiation. How neural rosettes form and provide a regulatory micro-environment remains to be elucidated. We employed the human embryonic stem cell-based neural rosette system to investigate the structural development and function of neural rosettes. Our study shows that neural rosette formation consists of five types of morphological change: intercalation, constriction, polarization, elongation and lumen formation. Ca 2+ signaling plays a pivotal role in the five steps by regulating the actions of the cytoskeletal complexes, actin, myosin II and tubulin during intercalation, constriction and elongation. These, in turn, control the polarizing elements, ZO-1, PARD3 and β-catenin during polarization and lumen production for neural rosette formation. We further demonstrate that the dismantlement of neural rosettes, mediated by the destruction of cytoskeletal elements, promotes neurogenesis and astrogenesis prematurely, indicating that an intact rosette structure is essential for orderly neural development. © 2018. Published by The Company of Biologists Ltd.

Mitochondrial control by DRP1 in brain tumor initiating cells.

PubMed

Xie, Qi; Wu, Qiulian; Horbinski, Craig M; Flavahan, William A; Yang, Kailin; Zhou, Wenchao; Dombrowski, Stephen M; Huang, Zhi; Fang, Xiaoguang; Shi, Yu; Ferguson, Ashley N; Kashatus, David F; Bao, Shideng; Rich, Jeremy N

2015-04-01

Brain tumor initiating cells (BTICs) co-opt the neuronal high affinity glucose transporter, GLUT3, to withstand metabolic stress. We investigated another mechanism critical to brain metabolism, mitochondrial morphology, in BTICs. BTIC mitochondria were fragmented relative to non-BTIC tumor cell mitochondria, suggesting that BTICs increase mitochondrial fission. The essential mediator of mitochondrial fission, dynamin-related protein 1 (DRP1), showed activating phosphorylation in BTICs and inhibitory phosphorylation in non-BTIC tumor cells. Targeting DRP1 using RNA interference or pharmacologic inhibition induced BTIC apoptosis and inhibited tumor growth. Downstream, DRP1 activity regulated the essential metabolic stress sensor, AMP-activated protein kinase (AMPK), and targeting AMPK rescued the effects of DRP1 disruption. Cyclin-dependent kinase 5 (CDK5) phosphorylated DRP1 to increase its activity in BTICs, whereas Ca(2+)-calmodulin-dependent protein kinase 2 (CAMK2) inhibited DRP1 in non-BTIC tumor cells, suggesting that tumor cell differentiation induces a regulatory switch in mitochondrial morphology. DRP1 activation correlated with poor prognosis in glioblastoma, suggesting that mitochondrial dynamics may represent a therapeutic target for BTICs.

Altered expression of miRNAs in the uterus from a letrozole-induced rat PCOS model.

PubMed

Li, Chunjin; Chen, Lu; Zhao, Yun; Chen, Shuxiong; Fu, Lulu; Jiang, Yanwen; Gao, Shan; Liu, Zhuo; Wang, Fengge; Zhu, Xiaoling; Rao, Jiahui; Zhang, Jing; Zhou, Xu

2017-01-20

Polycystic ovary syndrome (PCOS) causes female subfertility with ovarian disorders and may be associated with increased rate of early-pregnancy failure. Rat PCOS models were established using letrozole to understand the uterine pathogenesis of PCOS. The differential expression of microRNAs (miRNAs) was observed in rat uterus with PCOS. After estrous cycles were disrupted, significantly abnormal ovarian morphology and hormone level were observed in rats with PCOS. A total of 148 miRNAs differentially expressed were identified in the uterus from the letrozole-induced rat model compared with the control. These miRNAs included 111 upregulated miRNAs and 37 downregulated miRNAs. The differential expression of miR-484, miR-375-3p, miR-324-5p, and miR-223-3p was further confirmed by quantitative reverse transcription polymerase chain reaction. Bioinformatic analysis showed that these four miRNAs were predicted to regulate a large number of genes with different functions. Pathway analysis supported that target genes of miRNAs were involved in insulin secretion and signaling pathways, such as wnt, AMPK, PI3K-Akt, and Ras. These data indicated that miRNAs differentially expressed in rat uterus with PCOS may be associated with PCOS pathogenesis in the uterus. Our findings can help clarify the mechanism of uterine defects in PCOS. Copyright © 2016. Published by Elsevier B.V.

Pathophysiological role of prostaglandin E2-induced up-regulation of the EP2 receptor in motor neuron-like NSC-34 cells and lumbar motor neurons in ALS model mice.

PubMed

Kosuge, Yasuhiro; Miyagishi, Hiroko; Yoneoka, Yuki; Yoneda, Keiko; Nango, Hiroshi; Ishige, Kumiko; Ito, Yoshihisa

2017-07-04

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by selective degeneration of motor neurons. The primary triggers for motor neuronal death are still unknown, but inflammation is considered to be an important factor contributing to the pathophysiology of ALS both clinically and in ALS models. Prostaglandin E2 (PGE2) and its corresponding four E-prostanoid receptors play a pivotal role in the degeneration of motor neurons in human and transgenic models of ALS. It has also been shown that PGE2-EP2 signaling in glial cells (astrocytes or microglia) promotes motor neuronal death in G93A mice. The present study was designed to investigate the levels of expression of EP receptors in the spinal motor neurons of ALS model mice and to examine whether PGE2 alters the expression of EP receptors in differentiated NSC-34 cells, a motor neuron-like cell line. Immunohistochemical staining demonstrated that EP2 and EP3 immunoreactivity was localized in NeuN-positive large cells showing the typical morphology of motor neurons in mice. Semi-quantitative analysis showed that the immunoreactivity of EP2 in motor neurons was significantly increased in the early symptomatic stage in ALS model mice. In contrast, the level of EP3 expression remained constant, irrespective of age. In differentiated NSC-34 cells, bath application of PGE2 resulted in a concentration-dependent decrease of MTT reduction. Although PGE2 had no effect on cell survival at concentrations of less than 10 μM, pretreatment with 10 μM PGE2 significantly up-regulated EP2 and concomitantly potentiated cell death induced by 30 μM PGE2. These results suggest that PGE2 is an important effector for induction of the EP2 subtype in differentiated NSC-34 cells, and that not only EP2 up-regulation in glial cells but also EP2 up-regulation in motor neurons plays a pivotal role in the vulnerability of motor neurons in ALS model mice. Copyright © 2017 Elsevier Ltd. All rights reserved.

Dynamics of DNA methylation and Histone H4 acetylation during floral bud differentiation in azalea

PubMed Central

2010-01-01

Background The ability to control the timing of flowering is a key strategy for planning production in ornamental species such as azalea, however it requires a thorough understanding of floral transition. Floral transition is achieved through a complex genetic network and regulated by multiple environmental and endogenous cues. Dynamic changes between chromatin states facilitating or inhibiting DNA transcription regulate the expression of floral induction pathways in response to environmental and developmental signals. DNA methylation and histone modifications are involved in controlling the functional state of chromatin and gene expression. Results The results of this work indicate that epigenetic mechanisms such as DNA methylation and histone H4 acetylation have opposite and particular dynamics during the transition from vegetative to reproductive development in the apical shoots of azalea. Global levels of DNA methylation and histone H4 acetylation as well as immunodetection of 5-mdC and acetylated H4, in addition to a morphological study have permitted the delimitation of four basic phases in the development of the azalea bud and allowed the identification of a stage of epigenetic reprogramming which showed a sharp decrease of whole DNA methylation similar to that is defined in other developmental processes in plants and in mammals. Conclusion The epigenetic control and reorganization of chromatin seem to be decisive for coordinating floral development in azalea. DNA methylation and H4 deacetylation act simultaneously and co-ordinately, restructuring the chromatin and regulating the gene expression during soot apical meristem development and floral differentiation. PMID:20067625

Mammalian knock out cells reveal prominent roles for atlastin GTPases in ER network morphology

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhao, Guohua; Zhu, Peng-Peng; Renvoisé, Benoît

Atlastins are large, membrane-bound GTPases that participate in the fusion of endoplasmic reticulum (ER) tubules to generate the polygonal ER network in eukaryotes. They also regulate lipid droplet size and inhibit bone morphogenetic protein (BMP) signaling, though mechanisms remain unclear. Humans have three atlastins (ATL1, ATL2, and ATL3), and ATL1 and ATL3 are mutated in autosomal dominant hereditary spastic paraplegia and hereditary sensory neuropathies. Cellular investigations of atlastin orthologs in most yeast, plants, flies and worms are facilitated by the presence of a single or predominant isoform, but loss-of-function studies in mammalian cells are complicated by multiple, broadly-expressed paralogs. Wemore » have generated mouse NIH-3T3 cells lacking all three mammalian atlastins (Atl1/2/3) using CRISPR/Cas9-mediated gene knockout (KO). ER morphology is markedly disrupted in these triple KO cells, with prominent impairment in formation of three-way ER tubule junctions. This phenotype can be rescued by expression of distant orthologs from Saccharomyces cerevisiae (Sey1p) and Arabidopsis (ROOT HAIR DEFECTIVE3) as well as any one of the three human atlastins. Minimal, if any, changes are observed in the morphology of mitochondria and the Golgi apparatus. Alterations in BMP signaling and increased sensitivity to ER stress are also noted, though effects appear more modest. Finally, atlastins appear required for the proper differentiation of NIH-3T3 cells into an adipocyte-like phenotype. These findings have important implications for the pathogenesis of hereditary spastic paraplegias and sensory neuropathies associated with atlastin mutations. - Highlights: • NIH-3T3 cells lacking all three atlastin paralogs were generated using CRISPR/Cas9. • Cells lacking all atlastin GTPases exhibit far fewer 3-way ER tubule junctions. • ER morphology defects in atlastin knockout cells are rescued by distant plant and yeast orthologs. • Atlastin knock out cells also exhibit differences in ER stress and BMP signaling. • Atlastins appear important for adipocyte-like differentiation of NIH-3T3 cells.« less

Regenerative effects of human embryonic stem cell-derived neural crest cells for treatment of peripheral nerve injury.

PubMed

Jones, Iwan; Novikova, Liudmila N; Novikov, Lev N; Renardy, Monika; Ullrich, Andreas; Wiberg, Mikael; Carlsson, Leif; Kingham, Paul J

2018-04-01

Surgical intervention is the current gold standard treatment following peripheral nerve injury. However, this approach has limitations, and full recovery of both motor and sensory modalities often remains incomplete. The development of artificial nerve grafts that either complement or replace current surgical procedures is therefore of paramount importance. An essential component of artificial grafts is biodegradable conduits and transplanted cells that provide trophic support during the regenerative process. Neural crest cells are promising support cell candidates because they are the parent population to many peripheral nervous system lineages. In this study, neural crest cells were differentiated from human embryonic stem cells. The differentiated cells exhibited typical stellate morphology and protein expression signatures that were comparable with native neural crest. Conditioned media harvested from the differentiated cells contained a range of biologically active trophic factors and was able to stimulate in vitro neurite outgrowth. Differentiated neural crest cells were seeded into a biodegradable nerve conduit, and their regeneration potential was assessed in a rat sciatic nerve injury model. A robust regeneration front was observed across the entire width of the conduit seeded with the differentiated neural crest cells. Moreover, the up-regulation of several regeneration-related genes was observed within the dorsal root ganglion and spinal cord segments harvested from transplanted animals. Our results demonstrate that the differentiated neural crest cells are biologically active and provide trophic support to stimulate peripheral nerve regeneration. Differentiated neural crest cells are therefore promising supporting cell candidates to aid in peripheral nerve repair. © 2018 The Authors. Journal of Tissue Engineering and Regenerative Medicine published by John Wiley & Sons, Ltd.

Phylogeography and alpha taxonomy of the common dolphin (Delphinus sp.).

PubMed

Natoli, A; Cañadas, A; Peddemors, V M; Aguilar, A; Vaquero, C; Fernández-Piqueras, P; Hoelzel, A R

2006-05-01

The resolution of taxonomic classifications for delphinid cetaceans has been problematic, especially for species in the genera Delphinus, Tursiops and Stenella. The frequent lack of correspondence between morphological and genetic differentiation in these species raises questions about the mechanisms responsible for their evolution. In this study we focus on the genus Delphinus, and use molecular markers to address questions about speciation and the evolution of population structure. Delphinus species have a worldwide distribution and show a high degree of morphological variation. Two distinct morphotypes, long-beaked and short-beaked, have been considered different species named D. capensis and D. delphis, respectively. However, genetic differentiation between these two forms has only been demonstrated in the Pacific. We analysed samples from eight different geographical regions, including two morphologically defined long-beaked form populations, and compared these with the eastern North Pacific populations. We found high differentiation among the populations described as long-beaked instead of the expected monophyly, suggesting that these populations may have evolved from independent events converging on the same morphotype. We observed low genetic differentiation among the short-beaked populations across a large geographical scale. We interpret these phylogeographical patterns in the context of life history and population structure in related species.

Prevalence of disruptive selection predicts extent of species differentiation in Lake Victoria cichlids.

PubMed

van Rijssel, Jacco C; Moser, Florian N; Frei, David; Seehausen, Ole

2018-01-31

Theory suggests that speciation with gene flow is most likely when both sexual and ecological selection are divergent or disruptive. Divergent sexual and natural selection on the visual system have been demonstrated before in sympatric, morphologically similar sister species of Lake Victoria cichlids, but this does not explain the subtle morphological differences between them. To investigate the significance of natural selection on morphology during speciation, we here ask whether the prevalence of disruptive ecological selection differs between sympatric sister species that are at different stages of speciation. Some of our species pairs do ( Pundamilia ) and others do not ( Neochromis ) differ distinctively in sexually selected male nuptial coloration. We find that (i) evidence for disruptive selection, and for evolutionary response to it, is prevalent in traits that are differentiated between sister species; (ii) prevalence of both predicts the extent of genetic differentiation; and (iii) genetic differentiation is weaker in species pairs with conserved male nuptial coloration. Our results speak to the existence of two different mechanisms of speciation with gene flow: speciation mainly by sexual selection tightly followed by ecological character displacement in some cases and speciation mainly by divergent ecological selection in others. © 2018 The Author(s).

TRIM67 Protein Negatively Regulates Ras Activity through Degradation of 80K-H and Induces Neuritogenesis*

PubMed Central

Yaguchi, Hiroaki; Okumura, Fumihiko; Takahashi, Hidehisa; Kano, Takahiro; Kameda, Hiroyuki; Uchigashima, Motokazu; Tanaka, Shinya; Watanabe, Masahiko; Sasaki, Hidenao; Hatakeyama, Shigetsugu

2012-01-01

Tripartite motif (TRIM)-containing proteins, which are defined by the presence of a common domain structure composed of a RING finger, one or two B-box motifs and a coiled-coil motif, are involved in many biological processes including innate immunity, viral infection, carcinogenesis, and development. Here we show that TRIM67, which has a TRIM motif, an FN3 domain and a SPRY domain, is highly expressed in the cerebellum and that TRIM67 interacts with PRG-1 and 80K-H, which is involved in the Ras-mediated signaling pathway. Ectopic expression of TRIM67 results in degradation of endogenous 80K-H and attenuation of cell proliferation and enhances neuritogenesis in the neuroblastoma cell line N1E-115. Furthermore, morphological and biological changes caused by knockdown of 80K-H are similar to those observed by overexpression of TRIM67. These findings suggest that TRIM67 regulates Ras signaling via degradation of 80K-H, leading to neural differentiation including neuritogenesis. PMID:22337885

TRIM67 protein negatively regulates Ras activity through degradation of 80K-H and induces neuritogenesis.

PubMed

Yaguchi, Hiroaki; Okumura, Fumihiko; Takahashi, Hidehisa; Kano, Takahiro; Kameda, Hiroyuki; Uchigashima, Motokazu; Tanaka, Shinya; Watanabe, Masahiko; Sasaki, Hidenao; Hatakeyama, Shigetsugu

2012-04-06

Tripartite motif (TRIM)-containing proteins, which are defined by the presence of a common domain structure composed of a RING finger, one or two B-box motifs and a coiled-coil motif, are involved in many biological processes including innate immunity, viral infection, carcinogenesis, and development. Here we show that TRIM67, which has a TRIM motif, an FN3 domain and a SPRY domain, is highly expressed in the cerebellum and that TRIM67 interacts with PRG-1 and 80K-H, which is involved in the Ras-mediated signaling pathway. Ectopic expression of TRIM67 results in degradation of endogenous 80K-H and attenuation of cell proliferation and enhances neuritogenesis in the neuroblastoma cell line N1E-115. Furthermore, morphological and biological changes caused by knockdown of 80K-H are similar to those observed by overexpression of TRIM67. These findings suggest that TRIM67 regulates Ras signaling via degradation of 80K-H, leading to neural differentiation including neuritogenesis.

Genomic and transcriptomic analysis of NDM-1 Klebsiella pneumoniae in spaceflight reveal mechanisms underlying environmental adaptability

PubMed Central

Li, Jia; Liu, Fei; Wang, Qi; Ge, Pupu; Woo, Patrick C. Y.; Yan, Jinghua; Zhao, Yanlin; Gao, George F.; Liu, Cui Hua; Liu, Changting

2014-01-01

The emergence and rapid spread of New Delhi Metallo-beta-lactamase-1 (NDM-1)-producing Klebsiella pneumoniae strains has caused a great concern worldwide. To better understand the mechanisms underlying environmental adaptation of those highly drug-resistant K. pneumoniae strains, we took advantage of the China's Shenzhou 10 spacecraft mission to conduct comparative genomic and transcriptomic analysis of a NDM-1 K. pneumoniae strain (ATCC BAA-2146) being cultivated under different conditions. The samples were recovered from semisolid medium placed on the ground (D strain), in simulated space condition (M strain), or in Shenzhou 10 spacecraft (T strain) for analysis. Our data revealed multiple variations underlying pathogen adaptation into different environments in terms of changes in morphology, H2O2 tolerance and biofilm formation ability, genomic stability and regulation of metabolic pathways. Additionally, we found a few non-coding RNAs to be differentially regulated. The results are helpful for better understanding the adaptive mechanisms of drug-resistant bacterial pathogens. PMID:25163721

DNA barcodes for bio-surveillance: regulated and economically important arthropod plant pests.

PubMed

Ashfaq, Muhammad; Hebert, Paul D N

2016-11-01

Many of the arthropod species that are important pests of agriculture and forestry are impossible to discriminate morphologically throughout all of their life stages. Some cannot be differentiated at any life stage. Over the past decade, DNA barcoding has gained increasing adoption as a tool to both identify known species and to reveal cryptic taxa. Although there has not been a focused effort to develop a barcode library for them, reference sequences are now available for 77% of the 409 species of arthropods documented on major pest databases. Aside from developing the reference library needed to guide specimen identifications, past barcode studies have revealed that a significant fraction of arthropod pests are a complex of allied taxa. Because of their importance as pests and disease vectors impacting global agriculture and forestry, DNA barcode results on these arthropods have significant implications for quarantine detection, regulation, and management. The current review discusses these implications in light of the presence of cryptic species in plant pests exposed by DNA barcoding.

PAK4 kinase is essential for embryonic viability and for proper neuronal development.

PubMed

Qu, Jian; Li, Xiaofan; Novitch, Bennet G; Zheng, Ye; Kohn, Matthew; Xie, Jian-Ming; Kozinn, Spencer; Bronson, Roderick; Beg, Amer A; Minden, Audrey

2003-10-01

The serine/threonine kinase PAK4 is a target for the Rho GTPase Cdc42 and has been shown to regulate cell morphology and cytoskeletal organization in mammalian cells. To examine the physiological and developmental functions of PAK4, we have disrupted the PAK4 gene in mice. The absence of PAK4 led to lethality by embryonic day 11.5, a result most likely due to a defect in the fetal heart. Striking abnormalities were also evident in the nervous systems of PAK4-deficient embryos. These embryos had dramatic defects in neuronal development and axonal outgrowth. In particular, spinal cord motor neurons and interneurons failed to differentiate and migrate to their proper positions. This is probably related to the role for PAK4 in the regulation of cytoskeletal organization and cell and/or extracellular matrix adhesion. PAK4-null embryos also had defects in proper folding of the caudal portion of the neural tube, suggesting an important role for PAK4 in neural tube development.

APC/C-Cdh1 coordinates neurogenesis and cortical size during development

NASA Astrophysics Data System (ADS)

Delgado-Esteban, Maria; García-Higuera, Irene; Maestre, Carolina; Moreno, Sergio; Almeida, Angeles

2013-12-01

The morphology of the adult brain is the result of a delicate balance between neural progenitor proliferation and the initiation of neurogenesis in the embryonic period. Here we assessed whether the anaphase-promoting complex/cyclosome (APC/C) cofactor, Cdh1—which regulates mitosis exit and G1-phase length in dividing cells—regulates neurogenesis in vivo. We use an embryo-restricted Cdh1 knockout mouse model and show that functional APC/C-Cdh1 ubiquitin ligase activity is required for both terminal differentiation of cortical neurons in vitro and neurogenesis in vivo. Further, genetic ablation of Cdh1 impairs the ability of APC/C to promote neurogenesis by delaying the exit of the progenitor cells from the cell cycle. This causes replicative stress and p53-mediated apoptotic death resulting in decreased number of cortical neurons and cortex size. These results demonstrate that APC/C-Cdh1 coordinates cortical neurogenesis and size, thus posing Cdh1 in the molecular pathogenesis of congenital neurodevelopmental disorders, such as microcephaly.

Morphological adaptation with no mitochondrial DNA differentiation in the coastal plain swamp sparrow

USGS Publications Warehouse

Greenberg, R.; Cordero, P.J.; Droege, S.; Fleischer, R.C.

1998-01-01

We estimated genetic differentiation between morphologically distinct tidal marsh populations of Swamp Sparrows (Melospiza georgiana nigrescens) and the more widespread inland populations (M. g. georgiana and M. g. ericrypta). The tidal marsh populations are consistently grayer with more extensive black markings (particularly in the crown), and their bills are larger. These differences are variously shared with other species of salt marsh birds and small mammals. We analyzed mitochondrial DNA sequences (5′ end of control region, COII/t-lys/ATPase8, and ND2) of Swamp Sparrows and found low levels of genetic variation and no evidence of geographic structure. These results suggest a rapid and recent geographic expansion of Swamp Sparrows from restricted Pleistocene populations. Morphological differentiation has occurred without long-term genetic isolation, suggesting that selection on the divergent traits is intense. The grayer and more melanistic plumage is probably cryptic coloration for foraging on tidal mud, which tends to be grayish as a result of the formation of iron sulfides, rather than iron oxides, under anaerobic conditions.

[Monoclonal antibodies in diagnosis of acute leukemias].

PubMed

Krawczyńska, A; Robak, T

1996-01-01

Immunophenotyping has become an essential component for the study of acute myeloblastic (AML) and lymphoblastic (ALL) leukaemias. The recent development of highly specific monoclonal antibodies (Mc Ab) to differentiation antigens (CD) of haematopoetic cells have made it readily available to clinical laboratories in most major hospitals. Immunophenotyping complements standard morphology by providing information on lineage, stage of differentiation and clonality. In addition some of the flow cytometry findings have independent prognostic significance. Monoclonal antibodies useful in defining lineage (B-cell versus T-cell) and stages of differentiation of ALL. It can be also used in identifying characteristic feature of AML and aiding in lineage determination in acute leukaemias that are morphologically undifferentiated. Surface immunophenotyping is especially helpful for recognizing mixed lineage acute leukaemia and diagnosing certain rare entities such as erythroleukaemia (M6), acute megakaryocytic leukaemia (M7) and minimally differentiation acute myeloid leukaemia.

Morphology of the external genitalia of the adult male and female mice as an endpoint of sex differentiation

PubMed Central

Weiss, Dana A.; Rodriguez, Esequiel; Cunha, Tristan; Menshenina, Julia; Barcellos, Dale; Chan, Lok Yun; Risbridger, Gail; Baskin, Laurence; Cunha, Gerald

2013-01-01

Adult external genitalia (ExG) are the endpoints of normal sex differentiation. Detailed morphometric analysis and comparison of adult mouse ExG has revealed 10 homologous features distinguishing the penis and clitoris that define masculine vs. feminine sex differentiation. These features have enabled the construction of a simple metric to evaluate various intersex conditions in mutant or hormonally manipulated mice. This review focuses on the morphology of the adult mouse penis and clitoris through detailed analysis of histologic sections, scanning electron microscopy, and three-dimensional reconstruction. We also present previous results from evaluation of “non-traditional” mammals, such as the spotted hyena and wallaby to demonstrate the complex process of sex differentiation that involves not only androgen-dependent processes, but also estrogen-dependent and hormone-independent mechanisms. PMID:21893161

Genome-wide analysis of miRNA and mRNA transcriptomes during amelogenesis.

PubMed

Yin, Kaifeng; Hacia, Joseph G; Zhong, Zhe; Paine, Michael L

2014-11-19

In the rodent incisor during amelogenesis, as ameloblast cells transition from secretory stage to maturation stage, their morphology and transcriptome profiles change dramatically. Prior whole genome transcriptome analysis has given a broad picture of the molecular activities dominating both stages of amelogenesis, but this type of analysis has not included miRNA transcript profiling. In this study, we set out to document which miRNAs and corresponding target genes change significantly as ameloblasts transition from secretory- to maturation-stage amelogenesis. Total RNA samples from both secretory- and maturation-stage rat enamel organs were subjected to genome-wide miRNA and mRNA transcript profiling. We identified 59 miRNAs that were differentially expressed at the maturation stage relative to the secretory stage of enamel development (False Discovery Rate (FDR)<0.05, fold change (FC)≥1.8). In parallel, transcriptome profiling experiments identified 1,729 mRNA transcripts that were differentially expressed in the maturation stage compared to the secretory stage (FDR<0.05, FC≥1.8). Based on bioinformatics analyses, 5.8% (629 total) of these differentially expressed genes (DEGS) were highlighted as being the potential targets of 59 miRNAs that were differentially expressed in the opposite direction, in the same tissue samples. Although the number of predicted target DEGs was not higher than baseline expectations generated by examination of stably expressed miRNAs, Gene Ontology (GO) analysis showed that these 629 DEGS were enriched for ion transport, pH regulation, calcium handling, endocytotic, and apoptotic activities. Seven differentially expressed miRNAs (miR-21, miR-31, miR-488, miR-153, miR-135b, miR-135a and miR298) in secretory- and/or maturation-stage enamel organs were confirmed by in situ hybridization. Further, we used luciferase reporter assays to provide evidence that two of these differentially expressed miRNAs, miR-153 and miR-31, are potential regulators for their predicated target mRNAs, Lamp1 (miR-153) and Tfrc (miR-31). In conclusion, these data indicate that miRNAs exhibit a dynamic expression pattern during the transition from secretory-stage to maturation-stage tooth enamel formation. Although they represent only one of numerous mechanisms influencing gene activities, miRNAs specific to the maturation stage could be involved in regulating several key processes of enamel maturation by influencing mRNA stability and translation.

Differentiating Human Multipotent Mesenchymal Stromal Cells Regulate microRNAs: Prediction of microRNA Regulation by PDGF During Osteogenesis

PubMed Central

Goff, Loyal A.; Boucher, Shayne; Ricupero, Christopher L.; Fenstermacher, Sara; Swerdel, Mavis; Chase, Lucas; Adams, Christopher; Chesnut, Jonathan; Lakshmipathy, Uma; Hart, Ronald P.

2009-01-01

Objective Human multipotent mesenchymal stromal cells (MSC) have the potential to differentiate into multiple cell types, although little is known about factors that control their fate. Differentiation-specific microRNAs may play a key role in stem cell self renewal and differentiation. We propose that specific intracellular signalling pathways modulate gene expression during differentiation by regulating microRNA expression. Methods Illumina mRNA and NCode microRNA expression analyses were performed on MSC and their differentiated progeny. A combination of bioinformatic prediction and pathway inhibition was used to identify microRNAs associated with PDGF signalling. Results The pattern of microRNA expression in MSC is distinct from that in pluripotent stem cells such as human embryonic stem cells. Specific populations of microRNAs are regulated in MSC during differentiation targeted towards specific cell types. Complementary mRNA expression analysis increases the pool of markers characteristic of MSC or differentiated progeny. To identify microRNA expression patterns affected by signalling pathways, we examined the PDGF pathway found to be regulated during osteogenesis by microarray studies. A set of microRNAs bioinformatically predicted to respond to PDGF signalling was experimentally confirmed by direct PDGF inhibition. Conclusion Our results demonstrate that a subset of microRNAs regulated during osteogenic differentiation of MSCs is responsive to perturbation of the PDGF pathway. This approach not only identifies characteristic classes of differentiation-specific mRNAs and microRNAs, but begins to link regulated molecules with specific cellular pathways. PMID:18657893

Merging mythology and morphology: the multifaceted lifestyle of Proteus mirabilis.

PubMed

Armbruster, Chelsie E; Mobley, Harry L T

2012-11-01

Proteus mirabilis, named for the Greek god who changed shape to avoid capture, has fascinated microbiologists for more than a century with its unique swarming differentiation, Dienes line formation and potent urease activity. Transcriptome profiling during both host infection and swarming motility, coupled with the availability of the complete genome sequence for P. mirabilis, has revealed the occurrence of interbacterial competition and killing through a type VI secretion system, and the reciprocal regulation of adhesion and motility, as well as the intimate connections between metabolism, swarming and virulence. This Review addresses some of the unique and recently described aspects of P. mirabilis biology and pathogenesis, and emphasizes the potential role of this bacterium in single-species and polymicrobial urinary tract infections.

Merging mythology and morphology: the multifaceted lifestyle of Proteus mirabilis

PubMed Central

Armbruster, Chelsie E.; Mobley, Harry L. T.

2013-01-01

Proteus mirabilis, named for the Greek god who changed shape to avoid capture, has fascinated microbiologists for more than a century with its unique swarming differentiation, Dienes line formation and potent urease activity. Transcriptome profiling during both host infection and swarming motility, coupled with the availability of the complete genome sequence for P. mirabilis, has revealed the occurrence of interbacterial competition and killing through a type VI secretion system, and the reciprocal regulation of adhesion and motility, as well as the intimate connections between metabolism, swarming and virulence. This Review addresses some of the unique and recently described aspects of P. mirabilis biology and pathogenesis, and emphasizes the potential role of this bacterium in single- species and polymicrobial urinary tract infections. PMID:23042564

Morphological Similarity and Ecological Overlap in Two Rotifer Species

PubMed Central

Gabaldón, Carmen; Montero-Pau, Javier; Serra, Manuel; Carmona, María José

2013-01-01

Co-occurrence of cryptic species raises theoretically relevant questions regarding their coexistence and ecological similarity. Given their great morphological similitude and close phylogenetic relationship (i.e., niche retention), these species will have similar ecological requirements and are expected to have strong competitive interactions. This raises the problem of finding the mechanisms that may explain the coexistence of cryptic species and challenges the conventional view of coexistence based on niche differentiation. The cryptic species complex of the rotifer Brachionus plicatilis is an excellent model to study these questions and to test hypotheses regarding ecological differentiation. Rotifer species within this complex are filtering zooplankters commonly found inhabiting the same ponds across the Iberian Peninsula and exhibit an extremely similar morphology—some of them being even virtually identical. Here, we explore whether subtle differences in body size and morphology translate into ecological differentiation by comparing two extremely morphologically similar species belonging to this complex: B. plicatilis and B. manjavacas. We focus on three key ecological features related to body size: (1) functional response, expressed by clearance rates; (2) tolerance to starvation, measured by growth and reproduction; and (3) vulnerability to copepod predation, measured by the number of preyed upon neonates. No major differences between B. plicatilis and B. manjavacas were found in the response to these features. Our results demonstrate the existence of a substantial niche overlap, suggesting that the subtle size differences between these two cryptic species are not sufficient to explain their coexistence. This lack of evidence for ecological differentiation in the studied biotic niche features is in agreement with the phylogenetic limiting similarity hypothesis but requires a mechanistic explanation of the coexistence of these species not based on differentiation related to biotic niche axes. PMID:23451154

Regulation of DNA replication during development

PubMed Central

Nordman, Jared; Orr-Weaver, Terry L.

2012-01-01

As development unfolds, DNA replication is not only coordinated with cell proliferation, but is regulated uniquely in specific cell types and organs. This differential regulation of DNA synthesis requires crosstalk between DNA replication and differentiation. This dynamic aspect of DNA replication is highlighted by the finding that the distribution of replication origins varies between differentiated cell types and changes with differentiation. Moreover, differential DNA replication in some cell types can lead to increases or decreases in gene copy number along chromosomes. This review highlights the recent advances and technologies that have provided us with new insights into the developmental regulation of DNA replication. PMID:22223677

Isolated Polynucleotides and Methods of Promoting a Morphology in a Fungus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lasure, Linda L; Dai, Ziyu

2008-10-21

The invention includes isolated polynucleotide molecules that are differentially expressed in a native fungus exhibiting a first morphology relative to the native fungus exhibiting a second morphology. The invention includes a method of enhancing a bioprocess utilizing a fungus. A transformed fungus is produced by transforming a fungus with a recombinant polynucleotide molecule. The recombinant polynucleotide molecule contains an isolated polynucleotide sequence linked operably to a promoter. The polynucleotide sequence is expressed to promote a first morphology. The first morphology of the transformed fungus enhances a bioprocess relative to the bioprocess utilizing a second morphology.

Differential Effects of Tissue Culture Coating Substrates on Prostate Cancer Cell Adherence, Morphology and Behavior

PubMed Central

Liberio, Michelle S.; Sadowski, Martin C.; Soekmadji, Carolina; Davis, Rohan A.; Nelson, Colleen C.

2014-01-01

Weak cell-surface adhesion of cell lines to tissue culture surfaces is a common problem and presents technical limitations to the design of experiments. To overcome this problem, various surface coating protocols have been developed. However, a comparative and precise real-time measurement of their impact on cell behavior has not been conducted. The prostate cancer cell line LNCaP, derived from a patient lymph node metastasis, is a commonly used model system in prostate cancer research. However, the cells’ characteristically weak attachment to the surface of tissue culture vessels and cover slips has impeded their manipulation and analysis and use in high throughput screening. To improve the adherence of LNCaP cells to the culture surface, we compared different coating reagents (poly-l-lysine, poly-l-ornithine, collagen type IV, fibronectin, and laminin) and culturing conditions and analyzed their impact on cell proliferation, adhesion, morphology, mobility and gene expression using real-time technologies. The results showed that fibronectin, poly-l-lysine and poly-l-ornithine improved LNCaP cells adherence and provoked cell morphology alterations, such as increase of nuclear and cellular area. These coating reagents also induced a higher expression of F-actin and reduced cell mobility. In contrast, laminin and collagen type IV did not improve adherence but promoted cell aggregation and affected cell morphology. Cells cultured in the presence of laminin displayed higher mobility than control cells. All the coating conditions significantly affected cell viability; however, they did not affect the expression of androgen receptor-regulated genes. Our comparative findings provide important insight for the selection of the ideal coating reagent and culture conditions for the cancer cell lines with respect to their effect on proliferation rate, attachment, morphology, migration, transcriptional response and cellular cytoskeleton arrangement. PMID:25375165

Sex differences of gray matter morphology in cortico-limbic-striatal neural system in major depressive disorder.

PubMed

Kong, Lingtao; Chen, Kaiyuan; Womer, Fay; Jiang, Wenyan; Luo, Xingguang; Driesen, Naomi; Liu, Jie; Blumberg, Hilary; Tang, Yanqing; Xu, Ke; Wang, Fei

2013-06-01

Sex differences are observed in both epidemiological and clinical aspects of major depressive disorder (MDD). The cortico-limbic-striatal neural system, including the prefrontal cortex, amygdala, hippocampus, and striatum, have shown sexually dimorphic morphological features and have been implicated in the dysfunctional regulation of mood and emotion in MDD. In this study, we utilized a whole-brain, voxel-based approach to examine sex differences in the regional distribution of gray matter (GM) morphological abnormalities in medication-naïve participants with MDD. Participants included 29 medication-naïve individuals with MDD (16 females and 13 males) and 33 healthy controls (HC) (17 females and 16 males). Gray matter morphology of the cortico-limbic-striatal neural system was examined using voxel-based morphometry analyzes of high-resolution structural magnetic resonance imaging scans. The main effect of diagnosis and interaction effect of diagnosis by sex on GM morphology were statistically significant (p < 0.05, corrected) in the left ventral prefrontal cortex, right amygdala, right hippocampus and bilateral caudate when comparing the MDD and HC groups. Posthoc analyzes showed that females with MDD had significant GM decreases in limbic regions (p < 0.05, corrected), compared to female HC; while males with MDD demonstrated significant GM reduction in striatal regions, (p < 0.05, corrected), compared to HC males. The observed sex-related patterns of abnormalities within the cortico-limbic-strial neural system, such as predominant prefrontal-limbic abnormalities in MDD females vs. predominant prefrontal-striatal abnormalities in MDD males, suggest differences in neural circuitry that may mediate sex differences in the clinical presentation of MDD and potential targets for sex-differentiated treatment of the disorder. Copyright © 2013 Elsevier Ltd. All rights reserved.

Biochemical and morphological differentiation of acetylcholinesterase-positive efferent fibers in the mouse cochlea.

PubMed

Emmerling, M R; Sobkowicz, H M; Levenick, C V; Scott, G L; Slapnick, S M; Rose, J E

1990-06-01

We have compared the biochemical expression of cholinergic enzymes with the morphological differentiation of efferent nerve fibers and endings in the cochlea of the postnatally developing mouse. Choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) are present in the newborn cochlea at specific activities 63% and 25%, respectively, of their mature levels. The relative increases in ChAT, in AChE, and in its molecular forms over the newborn values start about day 4 and reach maturity by about day 10. The biochemical results correlate well with the massive presence of nerve fibers stained immunocytochemically for ChAT and AChE or enzymatically for AChE in the inner and outer hair cell regions. Ultrastructral studies, however, indicate the presence of only few vesiculated fibers and endings in the inner and outer hair cell regions. The appearance of large, cytologically mature endings occurs only toward the end of the third postnatal week. The discrepancy may be resolved in the electron microscopy using the enzymatic staining for AChE. Labeling is seen on many nonvesiculated fibers and endings in the hair cell regions, suggesting that the majority of the efferent fibers in the perinatal organ may be biochemically differentiated but morphologically immature. The results may imply that the efferents to inner and outer hair cells develop earlier than indicated by previous ultrastructral studies. Moreover, the pattern of development suggests that in the cochlea, as in other tissues, the biochemical differentiation of the efferent innervation may precede the morphological maturation.

The value of whole lesion ADC histogram profiling to differentiate between morphologically indistinguishable ring enhancing lesions-comparison of glioblastomas and brain abscesses.

PubMed

Horvath-Rizea, Diana; Surov, Alexey; Hoffmann, Karl-Titus; Garnov, Nikita; Vörkel, Cathrin; Kohlhof-Meinecke, Patricia; Ganslandt, Oliver; Bäzner, Hansjörg; Gihr, Georg Alexander; Kalman, Marcell; Henkes, Elina; Henkes, Hans; Schob, Stefan

2018-04-06

Morphologically similar appearing ring enhancing lesions in the brain parenchyma can be caused by a number of distinct pathologies, however, they consistently represent life-threatening conditions. The two most frequently encountered diseases manifesting as such are glioblastoma multiforme (GBM) and brain abscess (BA), each requiring disparate therapeutical approaches. As a result of their morphological resemblance, essential treatment might be significantly delayed or even ommited, in case results of conventional imaging remain inconclusive. Therefore, our study aimed to investigate, whether ADC histogram profiling reliably can distinguish between both entities, thus enhancing the differential diagnostic process and preventing treatment failure in this highly critical context. 103 patients (51 BA, 52 GBM) with histopathologically confirmed diagnosis were enrolled. Pretreatment diffusion weighted imaging (DWI) was obtained in a 1.5T system using b values of 0, 500, and 1000 s/mm 2 . Whole lesion ADC volumes were analyzed using a histogram-based approach. Statistical analysis was performed using SPSS version 23. All investigated parameters were statistically different in comparison of both groups. Most importantly, ADCp10 was able to differentiate reliably between BA and GBM with excellent accuracy (0.948) using a cutpoint value of 70 × 10 -5 mm 2 × s -1 . ADC whole lesion histogram profiling provides a valuable tool to differentiate between morphologically indistinguishable mass lesions. Among the investigated parameters, the 10th percentile of the ADC volume distinguished best between GBM and BA.

Prohibitin (PHB) inhibits apoptosis in rat granulosa cells (GCs) through the extracellular signal-regulated kinase 1/2 (ERK1/2) and the Bcl family of proteins.

PubMed

Chowdhury, Indrajit; Thompson, Winston E; Welch, Crystal; Thomas, Kelwyn; Matthews, Roland

2013-12-01

Mammalian ovarian follicular development is tightly regulated by crosstalk between cell death and survival signals, which include both endocrine and intra-ovarian regulators. Whether the follicle ultimately ovulates or undergoes atresia is dependent on the expression and actions of factors promoting follicular cell proliferation, differentiation or apoptosis. Prohibitin (PHB) is a highly conserved, ubiquitous protein that is abundantly expressed in granulosa cells (GCs) and associated with GC differentiation and apoptosis. The current study was designed to characterize the regulation of anti-apoptotic and pro-apoptotic factors in undifferentiated rat GCs (gonadotropin independent phase) governed by PHB. Microarray technology was initially employed to identify potential apoptosis-related genes, whose expression levels within GCs were altered by either staurosporine (STS) alone or STS in presence of ectopically over-expressed PHB. Next, immunoblot studies were performed to examine the expression patterns of selective Bcl-2 family members identified by the microarray analysis, which are commonly regulated in the intrinsic-apoptotic pathway. These studies were designed to measure protein levels of Bcl2 family in relation to expression of the acidic isoform (phosphorylated) PHB and the components of MEK-Erk1/2 pathway. These studies indicated that over-expression of PHB in undifferentiated GCs inhibit apoptosis which concomitantly results in an increased level of the anti-apoptotic proteins Bcl2 and Bclxl, reduced release of cytochrome c from mitochondria and inhibition of caspase-3 activity. In contrast, silencing of PHB expression resulted in change of mitochondrial morphology from the regular reticular network to a fragmented form, which enhanced sensitization of these GCs to the induction of apoptosis. Collectively, these studies have provided new insights on the PHB-mediated anti-apoptotic mechanism, which occurs in undifferentiated GCs through a PHB → Mek-Erk1/2 → Bcl/Bcl-xL pathway and may have important clinical implications.

Subsurface Grain Morphology Reconstruction by Differential Aperture X-ray Microscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Eisenlohr, Philip; Shanthraj, Pratheek; Vande Kieft, Brendan R.

A multistep, non-destructive grain morphology reconstruction methodology that is applicable to near-surface volumes is developed and tested on synthetic grain structures. This approach probes the subsurface crystal orientation using differential aperture X-ray microscopy (DAXM) on a sparse grid across the microstructure volume of interest. Resulting orientation data is clustered according to proximity in physical and orientation space and used as seed points for an initial Voronoi tessellation to (crudely) approximate the grain morphology. Curvature-driven grain boundary relaxation, simulated by means of the Voronoi Implicit Interface Method (VIIM), progressively improves the reconstruction accuracy. The similarity between bulk and readily accessible surfacemore » reconstruction error provides an objective termination criterion for boundary relaxation.« less

Mitochondrial morphology transitions and functions: implications for retrograde signaling?

PubMed Central

Picard, Martin; Shirihai, Orian S.; Gentil, Benoit J.

2013-01-01

In response to cellular and environmental stresses, mitochondria undergo morphology transitions regulated by dynamic processes of membrane fusion and fission. These events of mitochondrial dynamics are central regulators of cellular activity, but the mechanisms linking mitochondrial shape to cell function remain unclear. One possibility evaluated in this review is that mitochondrial morphological transitions (from elongated to fragmented, and vice-versa) directly modify canonical aspects of the organelle's function, including susceptibility to mitochondrial permeability transition, respiratory properties of the electron transport chain, and reactive oxygen species production. Because outputs derived from mitochondrial metabolism are linked to defined cellular signaling pathways, fusion/fission morphology transitions could regulate mitochondrial function and retrograde signaling. This is hypothesized to provide a dynamic interface between the cell, its genome, and the fluctuating metabolic environment. PMID:23364527

Drone and Worker Brood Microclimates Are Regulated Differentially in Honey Bees, Apis mellifera.

PubMed

Li, Zhiyong; Huang, Zachary Y; Sharma, Dhruv B; Xue, Yunbo; Wang, Zhi; Ren, Bingzhong

2016-01-01

Honey bee (Apis mellifera) drones and workers show differences in morphology, physiology, and behavior. Because the functions of drones are more related to colony reproduction, and those of workers relate to both survival and reproduction, we hypothesize that the microclimate for worker brood is more precisely regulated than that of drone brood. We assessed temperature and relative humidity (RH) inside honey bee colonies for both drone and worker brood throughout the three-stage development period, using digital HOBO® Data Loggers. The major findings of this study are that 1) both drone and worker castes show the highest temperature for eggs, followed by larvae and then pupae; 2) temperature in drones are maintained at higher precision (smaller variance) in drone eggs and larvae, but at a lower precision in pupae than the corresponding stages of workers; 3) RH regulation showed higher variance in drone than workers across all brood stages; and 4) RH regulation seems largely due to regulation by workers, as the contribution from empty honey combs are much smaller compared to that from adult workers. We conclude that honey bee colonies maintain both temperature and humidity actively; that the microclimate for sealed drone brood is less precisely regulated than worker brood; and that combs with honey contribute very little to the increase of RH in honey bee colonies. These findings increase our understanding of microclimate regulation in honey bees and may have implications for beekeeping practices.

Drone and Worker Brood Microclimates Are Regulated Differentially in Honey Bees, Apis mellifera

PubMed Central

Li, Zhiyong; Huang, Zachary Y.; Sharma, Dhruv B.; Xue, Yunbo; Wang, Zhi; Ren, Bingzhong

2016-01-01

Background Honey bee (Apis mellifera) drones and workers show differences in morphology, physiology, and behavior. Because the functions of drones are more related to colony reproduction, and those of workers relate to both survival and reproduction, we hypothesize that the microclimate for worker brood is more precisely regulated than that of drone brood. Methodology/Principal Findings We assessed temperature and relative humidity (RH) inside honey bee colonies for both drone and worker brood throughout the three-stage development period, using digital HOBO® Data Loggers. The major findings of this study are that 1) both drone and worker castes show the highest temperature for eggs, followed by larvae and then pupae; 2) temperature in drones are maintained at higher precision (smaller variance) in drone eggs and larvae, but at a lower precision in pupae than the corresponding stages of workers; 3) RH regulation showed higher variance in drone than workers across all brood stages; and 4) RH regulation seems largely due to regulation by workers, as the contribution from empty honey combs are much smaller compared to that from adult workers. Conclusions/Significance We conclude that honey bee colonies maintain both temperature and humidity actively; that the microclimate for sealed drone brood is less precisely regulated than worker brood; and that combs with honey contribute very little to the increase of RH in honey bee colonies. These findings increase our understanding of microclimate regulation in honey bees and may have implications for beekeeping practices. PMID:26882104

Regulation of cAMP and GSK3 signaling pathways contributes to the neuronal conversion of glioma

PubMed Central

Kim, Yongbo; Che, Lihua; Kim, Jeong Beom; Chang, Gyeong Eon; Cheong, Eunji; Kang, Seok-Gu; Ha, Yoon

2017-01-01

Glioma is the most malignant type of primary central nervous system tumors, and has an extremely poor prognosis. One potential therapeutic approach is to induce the terminal differentiation of glioma through the forced expression of pro-neural factors. Our goal is to show the proof of concept of the neuronal conversion of C6 glioma through the combined action of small molecules. We investigated the various changes in gene expression, cell-specific marker expression, signaling pathways, physiological characteristics, and morphology in glioma after combination treatment with two small molecules (CHIR99021, a glycogen synthase kinase 3 [GSK3] inhibitor and forskolin, a cyclic adenosine monophosphate [cAMP] activator). Here, we show that the combined action of CHIR99021 and forskolin converted malignant glioma into fully differentiated neurons with no malignant characteristics; inhibited the proliferation of malignant glioma; and significantly down-regulated gene ontology and gene expression profiles related to cell division, gliogenesis, and angiogenesis in small molecule–induced neurons. In vivo, the combined action of CHIR99021 and forskolin markedly delayed neurological deficits and significantly reduced the tumor volume. We suggest that reprogramming technology may be a potential treatment strategy replacing the therapeutic paradigm of traditional treatment of malignant glioma, and a combination molecule comprising a GSK3 inhibitor and a cAMP inducer could be the next generation of anticancer drugs. PMID:29161257

Identification of differentially expressed genes in human lung squamous cell carcinoma using suppression subtractive hybridization.

PubMed

Sun, Wenyue; Zhang, Kaitai; Zhang, Xinyu; Lei, Wendong; Xiao, Ting; Ma, Jinfang; Guo, Suping; Shao, Shujuan; Zhang, Husheng; Liu, Yan; Yuan, Jinsong; Hu, Zhi; Ma, Ying; Feng, Xiaoli; Hu, Songnian; Zhou, Jun; Cheng, Shujun; Gao, Yanning

2004-08-20

Lung cancer is one of the major causes of cancer-related deaths. Over the past decade, much has been known about the molecular changes associated with lung carcinogenesis; however, our understanding to lung tumorigenesis is still incomplete. To identify genes that are differentially expressed in squamous cell carcinoma (SCC) of the lung, we compared the expression profiles between primarily cultured SCC tumor cells and bronchial epithelial cells derived from morphologically normal bronchial epithelium of the same patient. Using suppression subtractive hybridization (SSH), two cDNA libraries containing up- and down-regulated genes in the tumor cells were constructed, named as LCTP and LCBP. The two libraries comprise 258 known genes and 133 unknown genes in total. The known up-regulated genes in the library LCTP represented a variety of functional groups; including metabolism-, cell adhesion and migration-, signal transduction-, and anti-apoptosis-related genes. Using semi-quantitative reverse transcription-polymerase chain reaction, seven genes chosen randomly from the LCTP were analyzed in the tumor tissue paired with its corresponding adjacent normal lung tissue derived from 16 cases of the SCC. Among them, the IQGAP1, RAP1GDS1, PAICS, MLF1, and MARK1 genes showed a consistent expression pattern with that of the SSH analysis. Identification and further characterization of these genes may allow a better understanding of lung carcinogenesis.

Genome-Wide Analysis of Long Non-Coding RNAs in Potato and Their Potential Role in Tuber Sprouting Process

PubMed Central

Hou, Xiaodong; Du, Yongmei; Liu, Xinmin; Zhang, Hongbo; Liu, Yanhua; Yan, Ning; Zhang, Zhongfeng

2017-01-01

Sprouting is a key factor affecting the quality of potato tubers. The present study aimed to compare the differential expression of long non-coding RNAs (lncRNAs) in the apical meristem during the dormancy release and sprouting stages by using lncRNA sequencing. Microscopic observations and Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses revealed the changes in the morphology and expression of lncRNAs in potato tubers during sprouting. Meristematic cells of potato tuber apical buds divided continuously and exhibited vegetative cone bulging and vascularisation. In all, 3175 lncRNAs were identified from the apical buds of potato tubers, among which 383 lncRNAs were up-regulated and 340 were down-regulated during sprouting. The GO enrichment analysis revealed that sprouting mainly influenced the expression of lncRNAs related to the cellular components of potato apical buds (e.g., cytoplasm and organelles) and cellular metabolic processes. The KEGG enrichment analysis also showed significant enrichment of specific metabolic pathways. In addition, 386 differentially expressed lncRNAs during sprouting were identified as putative targets of 235 potato miRNAs. Quantitative real-time polymerase chain reaction results agreed with the sequencing data. Our study provides the first systematic study of numerous lncRNAs involved in the potato tuber sprouting process and lays the foundation for further studies to elucidate their precise functions. PMID:29286332

Global analysis of differential gene expression related to long-term sperm storage in oviduct of Chinese Soft-Shelled Turtle Pelodiscus sinensis

PubMed Central

Liu, Tengfei; Yang, Ping; Chen, Hong; Huang, Yufei; Liu, Yi; Waqas, Yasir; Ahmed, Nisar; Chu, Xiaoya; Chen, Qiusheng

2016-01-01

Important evolutionary and ecological consequences arise from the ability of female turtles to store viable spermatozoa for an extended period. Although previous morphological studies have observed the localization of spermatozoa in Pelodiscus sinensis oviduct, no systematic study on the identification of genes that are involved in long-term sperm storage has been performed. In this study, the oviduct of P. sinensis at different phases (reproductive and hibernation seasons) was prepared for RNA-Seq and gene expression profiling. In total, 2,662 differentially expressed genes (DEGs) including 1,224 up- and 1,438 down-regulated genes were identified from two cDNA libraries. Functional enrichment analysis indicated that many genes were predominantly involved in the immune response, apoptosis pathway and regulation of autophagy. RT-qPCR, ELISA, western blot and IHC analyses showed that the expression profiles of mRNA and protein in selected DEGs were in consistent with results from RNA-Seq analysis. Remarkably, TUNEL analysis revealed the reduced number of apoptotic cells during sperm storage. IHC and TEM analyses found that autophagy occurred in the oviduct epithelial cells, where the spermatozoa were closely attached. The outcomes of this study provide fundamental insights into the complex sperm storage regulatory process and facilitate elucidating the mechanism of sperm storage in P. sinensis. PMID:27628424

The regulation of tooth morphogenesis is associated with epithelial cell proliferation and the expression of Sonic hedgehog through epithelial-mesenchymal interactions

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ishida, Kentaro; Murofushi, Mayumi; Nakao, Kazuhisa

2011-02-18

Research highlights: {yields} Bioengineered teeth regulated the contact area of epithelium and mesenchyme. {yields} The crown width is regulated by the contact area of the epithelium and mesenchyme. {yields} This regulation is associated with cell proliferation and Sonic hedgehog expression. {yields} The cusp number is correlated with the crown width of the bioengineered tooth. {yields} Cell proliferation and Shh expression areas regulate the tooth morphogenesis. -- Abstract: Ectodermal organs, such as the tooth, salivary gland, hair, and mammary gland, develop through reciprocal epithelial-mesenchymal interactions. Tooth morphologies are defined by the crown width and tooth length (macro-morphologies), and by the numbermore » and locations of the cusp and roots (micro-morphologies). In our current study, we report that the crown width of a bioengineered molar tooth, which was reconstructed using dissociated epithelial and mesenchymal cells via an organ germ method, can be regulated by the contact area between epithelial and mesenchymal cell layers. We further show that this is associated with cell proliferation and Sonic hedgehog (Shh) expression in the inner enamel epithelium after the germ stage has formed a secondary enamel knot. We also demonstrate that the cusp number is significantly correlated with the crown width of the bioengineered tooth. These findings suggest that the tooth micro-morphology, i.e. the cusp formation, is regulated after the tooth width, or macro-morphology, is determined. These findings also suggest that the spatiotemporal patterning of cell proliferation and the Shh expression areas in the epithelium regulate the crown width and cusp formation of the developing tooth.« less

Effects of let-7b and TLX on the proliferation and differentiation of retinal progenitor cells in vitro

PubMed Central

Ni, Ni; Zhang, Dandan; Xie, Qing; Chen, Junzhao; Wang, Zi; Deng, Yuan; Wen, Xuyang; Zhu, Mengyu; Ji, Jing; Fan, Xianqun; Luo, Min; Gu, Ping

2014-01-01

MicroRNAs manifest significant functions in brain neural stem cell (NSC) self-renewal and differentiation through the post-transcriptional regulation of neurogenesis genes. Let-7b is expressed in the mammalian brain and regulates NSC proliferation and differentiation by targeting the nuclear receptor TLX, which is an essential regulator of NSC self-renewal. Whether let-7b and TLX act as important regulators in retinal progenitor cell (RPC) proliferation and differentiation remains unknown. Here, our data show that let-7b and TLX play important roles in controlling RPC fate determination in vitro. Let-7b suppresses TLX expression to negatively regulate RPC proliferation and accelerate the neuronal and glial differentiation of RPCs. The overexpression of let-7b downregulates TLX levels in RPCs, leading to reduced RPC proliferation and increased neuronal and glial differentiation, whereas antisense knockdown of let-7b produces robust TLX expression,enhanced RPC proliferation and decreased differentiation. Moreover, the inhibition of endogenous TLX by small interfering RNA suppresses RPC proliferation and promotes RPC differentiation. Furthermore, overexpression of TLX rescues let-7b-induced proliferation deficiency and weakens the RPC differentiation enhancement caused by let-7b alone. These results suggest that let-7b, by forming a negative feedback loop with TLX, provides a novel model to regulate the proliferation and differentiation of retinal progenitors in vitro. PMID:25327364

Effects of let-7b and TLX on the proliferation and differentiation of retinal progenitor cells in vitro.

PubMed

Ni, Ni; Zhang, Dandan; Xie, Qing; Chen, Junzhao; Wang, Zi; Deng, Yuan; Wen, Xuyang; Zhu, Mengyu; Ji, Jing; Fan, Xianqun; Luo, Min; Gu, Ping

2014-10-20

MicroRNAs manifest significant functions in brain neural stem cell (NSC) self-renewal and differentiation through the post-transcriptional regulation of neurogenesis genes. Let-7b is expressed in the mammalian brain and regulates NSC proliferation and differentiation by targeting the nuclear receptor TLX, which is an essential regulator of NSC self-renewal. Whether let-7b and TLX act as important regulators in retinal progenitor cell (RPC) proliferation and differentiation remains unknown. Here, our data show that let-7b and TLX play important roles in controlling RPC fate determination in vitro. Let-7b suppresses TLX expression to negatively regulate RPC proliferation and accelerate the neuronal and glial differentiation of RPCs. The overexpression of let-7b downregulates TLX levels in RPCs, leading to reduced RPC proliferation and increased neuronal and glial differentiation, whereas antisense knockdown of let-7b produces robust TLX expression,enhanced RPC proliferation and decreased differentiation. Moreover, the inhibition of endogenous TLX by small interfering RNA suppresses RPC proliferation and promotes RPC differentiation. Furthermore, overexpression of TLX rescues let-7b-induced proliferation deficiency and weakens the RPC differentiation enhancement caused by let-7b alone. These results suggest that let-7b, by forming a negative feedback loop with TLX, provides a novel model to regulate the proliferation and differentiation of retinal progenitors in vitro.

Geographic in wing morphology relate to colonization history in New World but not Old World populations of yellow dung flies.

PubMed

Schäfer, Martin A; Berger, David; Rohner, Patrick T; Kjaersgaard, Anders; Bauerfeind, Stephanie S; Guillaume, Frédéric; Fox, Charles W; Blanckenhorn, Wolf U

2018-06-17

Geographic clines offer insights about putative targets and agents of natural selection as well as tempo and mode of adaptation. However, demographic processes can lead to clines that are indistinguishable from adaptive divergence. Using the widespread yellow dung fly Scathophaga stercoraria (Diptera: Scathophagidae), we examine quantitative genetic differentiation (Q ST ) of wing shape across North America, Europe and Japan, and compare this differentiation with that of ten microsatellites (F ST ). Morphometric analyses of 28 populations reared at three temperatures revealed significant thermal plasticity, sexual dimorphism and geographic differentiation in wing shape. In North America morphological differentiation followed the decline in microsatellite variability along the presumed route of recent colonization from the southeast to the northwest. Across Europe, where S. stercoraria presumably existed for much longer time and where no molecular pattern of isolation by distance was evident, clinal variation was less pronounced despite significant morphological differentiation (Q ST >F ST ). Shape vector comparisons further indicate that thermal plasticity (hot-to-cold) does not mirror patterns of latitudinal divergence (south-to-north), as might have been expected under a scenario with temperature as the major agent of selection. Our findings illustrate the importance of detailed phylogeographic information when interpreting geographic clines of dispersal traits in an adaptive evolutionary framework. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Investigating the molecular underpinnings underlying morphology and changes in carbon partitioning during tension wood formation in Eucalyptus.

PubMed

Mizrachi, Eshchar; Maloney, Victoria J; Silberbauer, Janine; Hefer, Charles A; Berger, Dave K; Mansfield, Shawn D; Myburg, Alexander A

2015-06-01

Tension wood has distinct physical and chemical properties, including altered fibre properties, cell wall composition and ultrastructure. It serves as a good system for investigating the genetic regulation of secondary cell wall biosynthesis and wood formation. The reference genome sequence for Eucalyptus grandis allows investigation of the global transcriptional reprogramming that accompanies tension wood formation in this global wood fibre crop. We report the first comprehensive analysis of physicochemical wood property changes in tension wood of Eucalyptus measured in a hybrid (E. grandis × Eucalyptus urophylla) clone, as well as genome-wide gene expression changes in xylem tissues 3 wk post-induction using RNA sequencing. We found that Eucalyptus tension wood in field-grown trees is characterized by an increase in cellulose, a reduction in lignin, xylose and mannose, and a marked increase in galactose. Gene expression profiling in tension wood-forming tissue showed corresponding down-regulation of monolignol biosynthetic genes, and differential expression of several carbohydrate active enzymes. We conclude that alterations of cell wall traits induced by tension wood formation in Eucalyptus are a consequence of a combination of down-regulation of lignin biosynthesis and hemicellulose remodelling, rather than the often proposed up-regulation of the cellulose biosynthetic pathway. © 2014 University of Pretoria New Phytologist © 2014 New Phytologist Trust.

Myostatin acts as an autocrine/paracrine negative regulator in myoblast differentiation from human induced pluripotent stem cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gao, Fei; Kishida, Tsunao; Ejima, Akika

Highlights: ► iPS-derived cells express myostatin and its receptor upon myoblast differentiation. ► Myostatin inhibits myoblast differentiation by inhibiting MyoD and Myo5a induction. ► Silencing of myostatin promotes differentiation of human iPS cells into myoblasts. -- Abstract: Myostatin, also known as growth differentiation factor (GDF-8), regulates proliferation of muscle satellite cells, and suppresses differentiation of myoblasts into myotubes via down-regulation of key myogenic differentiation factors including MyoD. Recent advances in stem cell biology have enabled generation of myoblasts from pluripotent stem cells, but it remains to be clarified whether myostatin is also involved in regulation of artificial differentiation of myoblastsmore » from pluripotent stem cells. Here we show that the human induced pluripotent stem (iPS) cell-derived cells that were induced to differentiate into myoblasts expressed myostatin and its receptor during the differentiation. An addition of recombinant human myostatin (rhMyostatin) suppressed induction of MyoD and Myo5a, resulting in significant suppression of myoblast differentiation. The rhMyostatin treatment also inhibited proliferation of the cells at a later phase of differentiation. RNAi-mediated silencing of myostatin promoted differentiation of human iPS-derived embryoid body (EB) cells into myoblasts. These results strongly suggest that myostatin plays an important role in regulation of myoblast differentiation from iPS cells of human origin. The present findings also have significant implications for potential regenerative medicine for muscular diseases.« less

Morphological Integration of Soft-Tissue Facial Morphology in Down Syndrome and Siblings

PubMed Central

Starbuck, John; Reeves, Roger H.; Richtsmeier, Joan

2011-01-01

Down syndrome (DS), resulting from trisomy of chromosome 21, is the most common live-born human aneuploidy. The phenotypic expression of trisomy 21 produces variable, though characteristic, facial morphology. Although certain facial features have been documented quantitatively and qualitatively as characteristic of DS (e.g., epicanthic folds, macroglossia, and hypertelorism), all of these traits occur in other craniofacial conditions with an underlying genetic cause. We hypothesize that the typical DS face is integrated differently than the face of non-DS siblings, and that the pattern of morphological integration unique to individuals with DS will yield information about underlying developmental associations between facial regions. We statistically compared morphological integration patterns of immature DS faces (N = 53) with those of non-DS siblings (N = 54), aged 6–12 years using 31 distances estimated from 3D coordinate data representing 17 anthropometric landmarks recorded on 3D digital photographic images. Facial features are affected differentially in DS, as evidenced by statistically significant differences in integration both within and between facial regions. Our results suggest a differential affect of trisomy on facial prominences during craniofacial development. PMID:21996933

Morphological integration of soft-tissue facial morphology in Down Syndrome and siblings.

PubMed

Starbuck, John; Reeves, Roger H; Richtsmeier, Joan

2011-12-01

Down syndrome (DS), resulting from trisomy of chromosome 21, is the most common live-born human aneuploidy. The phenotypic expression of trisomy 21 produces variable, though characteristic, facial morphology. Although certain facial features have been documented quantitatively and qualitatively as characteristic of DS (e.g., epicanthic folds, macroglossia, and hypertelorism), all of these traits occur in other craniofacial conditions with an underlying genetic cause. We hypothesize that the typical DS face is integrated differently than the face of non-DS siblings, and that the pattern of morphological integration unique to individuals with DS will yield information about underlying developmental associations between facial regions. We statistically compared morphological integration patterns of immature DS faces (N = 53) with those of non-DS siblings (N = 54), aged 6-12 years using 31 distances estimated from 3D coordinate data representing 17 anthropometric landmarks recorded on 3D digital photographic images. Facial features are affected differentially in DS, as evidenced by statistically significant differences in integration both within and between facial regions. Our results suggest a differential affect of trisomy on facial prominences during craniofacial development. 2011 Wiley Periodicals, Inc.

Differential staining of bacteria: gram stain.

PubMed

Moyes, Rita B; Reynolds, Jackie; Breakwell, Donald P

2009-11-01

In 1884, Hans Christian Gram, a Danish doctor, developed a differential staining technique that is still the cornerstone of bacterial identification and taxonomic division. This multistep, sequential staining protocol separates bacteria into four groups based on cell morphology and cell wall structure: Gram-positive cocci, Gram-negative cocci, Gram-positive rods, and Gram-negative rods. The Gram stain is useful for assessing bacterial contamination of tissue culture samples or for examining the Gram stain status and morphological features of bacteria isolated from mixed or isolated bacterial cultures. (c) 2009 by John Wiley & Sons, Inc.

Molecular differentiation of Entamoeba spp. in a rural community of Loja province, South Ecuador.

PubMed

Levecke, B; Dreesen, L; Barrionuevo-Samaniego, M; Ortiz, W Benitez; Praet, N; Brandt, J; Dorny, P

2011-12-01

Although previous epidemiological surveys in Ecuador indicate the presence of Entamoeba histolytica, prevalence data of this parasite remain scarce. Most of the studies were based on microscopic examination, which does not allow a morphological differentiation from the non-pathogenic Ent. dispar and Ent. moshkovskii. In the present study, 674 stool samples from a South Ecuadorian rural community were screened for Entamoeba spp. Subsequently, molecular identification was performed on 101 samples containing Ent. histolytica/Ent. dispar/Ent. moshkovskii cysts. The study indicated the absence of Ent. histolytica in this South Ecuadorian community and confirmed the difficulty of differentiating Entamoeba spp. based on morphological features. Copyright © 2011 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd. All rights reserved.

Labelling and targeted ablation of specific bipolar cell types in the zebrafish retina

PubMed Central

2009-01-01

Background Development of a functional retina depends on regulated differentiation of several types of neurons and generation of a highly complex network between the different types of neurons. In addition, each type of retinal neuron includes several distinct morphological types. Very little is known about the mechanisms responsible for generating this diversity of retinal neurons, which may also display specific patterns of regional distribution. Results In a screen in zebrafish, using a trapping vector carrying an engineered yeast Gal4 transcription activator and a UAS:eGFP reporter cassette, we have identified two transgenic lines of zebrafish co-expressing eGFP and Gal4 in specific subsets of retinal bipolar cells. The eGFP-labelling facilitated analysis of axon terminals within the inner plexiform layer of the adult retina and showed that the fluorescent bipolar cells correspond to previously defined morphological types. Strong regional restriction of eGFP-positive bipolar cells to the central part of the retina surrounding the optic nerve was observed in adult zebrafish. Furthermore, we achieved specific ablation of the labelled bipolar cells in 5 days old larvae, using a bacterial nitroreductase gene under Gal4-UAS control in combination with the prodrug metronidazole. Following prodrug treatment, nitroreductase expressing bipolar cells were efficiently ablated without affecting surrounding retina architecture, and recovery occurred within a few days due to increased generation of new bipolar cells. Conclusion This report shows that enhancer trapping can be applied to label distinct morphological types of bipolar cells in the zebrafish retina. The genetic labelling of these cells yielded co-expression of a modified Gal4 transcription activator and the fluorescent marker eGFP. Our work also demonstrates the potential utility of the Gal4-UAS system for induction of other transgenes, including a bacterial nitroreductase fusion gene, which can facilitate analysis of bipolar cell differentiation and how the retina recovers from specific ablation of these cells. PMID:19712466

Behavioral stress alters corticolimbic microglia in a sex- and brain region-specific manner.

PubMed

Bollinger, Justin L; Collins, Kaitlyn E; Patel, Rushi; Wellman, Cara L

2017-01-01

Women are more susceptible to numerous stress-linked psychological disorders (e.g., depression) characterized by dysfunction of corticolimbic brain regions critical for emotion regulation and cognitive function. Although sparsely investigated, a number of studies indicate sex differences in stress effects on neuronal structure, function, and behaviors associated with these regions. We recently demonstrated a basal sex difference in- and differential effects of stress on- microglial activation in medial prefrontal cortex (mPFC). The resident immune cells of the brain, microglia are implicated in synaptic and dendritic plasticity, and cognitive-behavioral function. Here, we examined the effects of acute (3h/day, 1 day) and chronic (3h/day, 10 days) restraint stress on microglial density and morphology, as well as immune factor expression in orbitofrontal cortex (OFC), basolateral amygdala (BLA), and dorsal hippocampus (DHC) in male and female rats. Microglia were visualized, classified based on their morphology, and stereologically counted. Microglia-associated transcripts (CD40, iNOS, Arg1, CX3CL1, CX3CR1, CD200, and CD200R) were assessed in brain punches from each region. Expression of genes linked with cellular stress, neuroimmune state, and neuron-microglia communication varied between unstressed male and female rats in a region-specific manner. In OFC, chronic stress upregulated a wider variety of immune factors in females than in males. Acute stress increased microglia-associated transcripts in BLA in males, whereas chronic stress altered immune factor expression in BLA more broadly in females. In DHC, chronic stress increased immune factor expression in males but not females. Moreover, acute and chronic stress differentially affected microglial morphological activation state in male and female rats across all brain regions investigated. In males, chronic stress altered microglial activation in a pattern consistent with microglial involvement in stress-induced dendritic remodeling across OFC, BLA, and DHC. Together, these data suggest the potential for microglia-mediated sex differences in stress effects on neural structure, function, and behavior.

Behavioral stress alters corticolimbic microglia in a sex- and brain region-specific manner

PubMed Central

Bollinger, Justin L.; Collins, Kaitlyn E.; Patel, Rushi

2017-01-01

Women are more susceptible to numerous stress-linked psychological disorders (e.g., depression) characterized by dysfunction of corticolimbic brain regions critical for emotion regulation and cognitive function. Although sparsely investigated, a number of studies indicate sex differences in stress effects on neuronal structure, function, and behaviors associated with these regions. We recently demonstrated a basal sex difference in- and differential effects of stress on- microglial activation in medial prefrontal cortex (mPFC). The resident immune cells of the brain, microglia are implicated in synaptic and dendritic plasticity, and cognitive-behavioral function. Here, we examined the effects of acute (3h/day, 1 day) and chronic (3h/day, 10 days) restraint stress on microglial density and morphology, as well as immune factor expression in orbitofrontal cortex (OFC), basolateral amygdala (BLA), and dorsal hippocampus (DHC) in male and female rats. Microglia were visualized, classified based on their morphology, and stereologically counted. Microglia-associated transcripts (CD40, iNOS, Arg1, CX3CL1, CX3CR1, CD200, and CD200R) were assessed in brain punches from each region. Expression of genes linked with cellular stress, neuroimmune state, and neuron-microglia communication varied between unstressed male and female rats in a region-specific manner. In OFC, chronic stress upregulated a wider variety of immune factors in females than in males. Acute stress increased microglia-associated transcripts in BLA in males, whereas chronic stress altered immune factor expression in BLA more broadly in females. In DHC, chronic stress increased immune factor expression in males but not females. Moreover, acute and chronic stress differentially affected microglial morphological activation state in male and female rats across all brain regions investigated. In males, chronic stress altered microglial activation in a pattern consistent with microglial involvement in stress-induced dendritic remodeling across OFC, BLA, and DHC. Together, these data suggest the potential for microglia-mediated sex differences in stress effects on neural structure, function, and behavior. PMID:29194444

New insights into redox regulation of stem cell self-renewal and differentiation.

PubMed

Ren, Fenglian; Wang, Kui; Zhang, Tao; Jiang, Jingwen; Nice, Edouard Collins; Huang, Canhua

2015-08-01

Reactive oxygen species (ROS), the natural byproducts of aerobic metabolism, are precisely orchestrated to evoke diverse signaling pathways. To date, studies have focused mainly on the detrimental effects of ROS in stem cells. Recently, accumulating evidence has suggested that ROS also function as second messengers that modulate stem cell self-renewal and differentiation by regulating intricate signaling networks. Although many efforts have been made to clarify the general effects of ROS on signal transduction in stem cells, less is known about the initial and direct executors of ROS signaling, which are known as 'redox sensors'. Modifications of cysteine residues in redox sensors are of significant importance in the modulation of protein function in response to different redox conditions. Intriguingly, most key molecules in ROS signaling and cell cycle regulation (including transcriptional factors and kinases) that are crucial in the regulation of stem cell self-renewal and differentiation have the potential to be redox sensors. We highlight herein the importance of redox regulation of these key regulators in stem cell self-renewal and differentiation. Understanding the mechanisms of redox regulation in stem cell self-renewal and differentiation will open exciting new perspectives for stem cell biology. This article is part of a Special Issue entitled Redox regulation of differentiation and de-differentiation. Copyright © 2015 Elsevier B.V. All rights reserved.

Induction of neurite extension and survival in pheochromocytoma cells by the Rit GTPase.

PubMed

Spencer, Michael L; Shao, Haipeng; Andres, Douglas A

2002-06-07

The Rit, Rin, and Ric proteins comprise a distinct and evolutionarily conserved subfamily of the Ras-like small G-proteins. Although these proteins share the majority of core effector domain residues with Ras, recent studies suggest that Rit uses novel effector pathways to regulate NIH3T3 cell proliferation and transformation, while the functions of Rin and Ric remain largely unknown. Since we demonstrate that Rit is expressed in neurons, we investigated the role of Rit signaling in promoting the differentiation and survival of pheochromocytoma cells. In this study, we show that expression of constitutively active Rit (RitL79) in PC6 cells results in neuronal differentiation, characterized by the elaboration of an extensive network of neurite-like processes that are morphologically distinct from those mediated by the expression of oncogenic Ras. Although activated Rit fails to stimulate mitogen-activated protein kinase/extracellular-signal-regulated kinase (MAPK/ERK) signaling pathways in COS cells, RitL79 induced the phosphorylation of ERK1/2 in PC6 cells. We also find that Rit-mediated effects on neurite outgrowth can be blocked by co-expression of dominant-negative mutants of C-Raf1 or mitogen-activated protein kinase kinase 1 (MEK1). Moreover, expression of dominant-negative Rit is sufficient to inhibit NGF-induced neurite outgrowth. Expression of active Rit inhibits growth factor-withdrawal mediated apoptosis of PC6 cells, but does not induce phosphorylation of Akt/protein kinase B, suggesting that survival does not utilize the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. Instead, pharmacological inhibitors of MEK block Rit-stimulated cell survival. Taken together, these studies suggest that Rit represents a distinct regulatory protein, capable of mediating differentiation and cell survival in PC6 cells using a MEK-dependent signaling pathway to achieve its effects.

The flavonoid apigenin from Croton betulaster Mull inhibits proliferation, induces differentiation and regulates the inflammatory profile of glioma cells.

PubMed

Coelho, Paulo L C; Oliveira, Mona N; da Silva, Alessandra B; Pitanga, Bruno P S; Silva, Victor D A; Faria, Giselle P; Sampaio, Geraldo P; Costa, Maria de Fatima D; Braga-de-Souza, Suzana; Costa, Silvia L

2016-11-01

This study aimed to investigate the antitumor and immunomodulatory properties of the flavonoid apigenin (5,7,4'-trihydroxyflavone), which was extracted from Croton betulaster Mull, in glioma cell culture using the high-proliferative rat C6 glioma cell line as a model. Apigenin was found to have the ability to reduce the viability and proliferation of C6 cells in a time-dependent and dose-dependent manner, with an IC50 of 22.8 µmol/l, 40 times lower than that of temozolomide (1000 µmol/l), after 72 h of apigenin treatment. Even after C6 cells were treated with apigenin for 48 h, high proportions of C6 cells entered apoptosis (39.56%) and autophagy (22%) as shown by flow cytometry using annexin V/propidium iodide and acridine orange staining, respectively. In addition, the flavonoid apigenin induced cell accumulation in the G0/G1 phase of the cell cycle and inhibited glioma cell migration efficiently. Moreover, apigenin induced astroglial differentiation and morphological changes in C6 cells, characterized by increased expression of glial fibrillary acidic protein and decreased expression of nestin protein, a typical marker of neuronal precursors. The immunomodulating effects of apigenin were also characterized by a change in the inflammatory profile as evidenced by a significant decrease in interleukin-10 and tumor necrosis factor production and increased nitric oxide levels. Because apigenin can induce differentiation, apoptosis, and autophagy, can alter the profile of cytokines involved in regulating the immune response, and can reduce the survival, growth, proliferation, and migration of C6 cells, this flavonoid may be considered a potential antitumor drug for the adjuvant treatment of malignant gliomas.

Global gene expression profiling of brown to white adipose tissue transformation in sheep reveals novel transcriptional components linked to adipose remodeling.

PubMed

Basse, Astrid L; Dixen, Karen; Yadav, Rachita; Tygesen, Malin P; Qvortrup, Klaus; Kristiansen, Karsten; Quistorff, Bjørn; Gupta, Ramneek; Wang, Jun; Hansen, Jacob B

2015-03-19

Large mammals are capable of thermoregulation shortly after birth due to the presence of brown adipose tissue (BAT). The majority of BAT disappears after birth and is replaced by white adipose tissue (WAT). We analyzed the postnatal transformation of adipose in sheep with a time course study of the perirenal adipose depot. We observed changes in tissue morphology, gene expression and metabolism within the first two weeks of postnatal life consistent with the expected transition from BAT to WAT. The transformation was characterized by massively decreased mitochondrial abundance and down-regulation of gene expression related to mitochondrial function and oxidative phosphorylation. Global gene expression profiling demonstrated that the time points grouped into three phases: a brown adipose phase, a transition phase and a white adipose phase. Between the brown adipose and the transition phase 170 genes were differentially expressed, and 717 genes were differentially expressed between the transition and the white adipose phase. Thirty-eight genes were shared among the two sets of differentially expressed genes. We identified a number of regulated transcription factors, including NR1H3, MYC, KLF4, ESR1, RELA and BCL6, which were linked to the overall changes in gene expression during the adipose tissue remodeling. Finally, the perirenal adipose tissue expressed both brown and brite/beige adipocyte marker genes at birth, the expression of which changed substantially over time. Using global gene expression profiling of the postnatal BAT to WAT transformation in sheep, we provide novel insight into adipose tissue plasticity in a large mammal, including identification of novel transcriptional components linked to adipose tissue remodeling. Moreover, our data set provides a useful resource for further studies in adipose tissue plasticity.

The effect of pro-inflammatory cytokines on immunophenotype, differentiation capacity and immunomodulatory functions of human mesenchymal stem cells.

PubMed

Pourgholaminejad, Arash; Aghdami, Nasser; Baharvand, Hossein; Moazzeni, Seyed Mohammad

2016-09-01

Mesenchymal stem cells (MSCs), as cells with potential clinical utilities, have demonstrated preferential incorporation into inflammation sites. Immunophenotype and immunomodulatory functions of MSCs could alter by inflamed-microenvironments due to the local pro-inflammatory cytokine milieu. A major cellular mediator with specific function in promoting inflammation and pathogenicity of autoimmunity are IL-17-producing T helper 17 (Th17) cells that polarize in inflamed sites in the presence of pro-inflammatory cytokines such as Interleukin-1β (IL-1β), IL-6 and IL-23. Since MSCs are promising candidate for cell-based therapeutic strategies in inflammatory and autoimmune diseases, Th17 cell polarizing factors may alter MSCs phenotype and function. In this study, human bone-marrow-derived MSCs (BM-MSC) and adipose tissue-derived MSCs (AD-MSC) were cultured with or without IL-1β, IL-6 and IL-23 as pro-inflammatory cytokines. The surface markers and their differentiation capacity were measured in cytokine-untreated and cytokine-treated MSCs. MSCs-mediated immunomodulation was analyzed by their regulatory effects on mixed lymphocyte reaction (MLR) and the level of IL-10, TGF-β, IL-4, IFN-γ and TNF-α production as immunomodulatory cytokines. Pro-inflammatory cytokines showed no effect on MSCs morphology, immunophenotype and co-stimulatory molecules except up-regulation of CD45. Adipogenic and osteogenic differentiation capacity increased in CD45+ MSCs. Moreover, cytokine-treated MSCs preserved the suppressive ability of allogeneic T cell proliferation and produced higher level of TGF-β and lower level of IL-4. We concluded pro-inflammatory cytokines up-regulate the efficacy of MSCs in cell-based therapy of degenerative, inflammatory and autoimmune disorders. Copyright © 2016. Published by Elsevier Ltd.

Magnesium modification of a calcium phosphate cement alters bone marrow stromal cell behavior via an integrin-mediated mechanism.

PubMed

Zhang, Jing; Ma, Xiaoyu; Lin, Dan; Shi, Hengsong; Yuan, Yuan; Tang, Wei; Zhou, Huanjun; Guo, Han; Qian, Jiangchao; Liu, Changsheng

2015-06-01

The chemical composition, structure and surface characteristics of biomaterials/scaffold can affect the adsorption of proteins, and this in turn influences the subsequent cellular response and tissue regeneration. With magnesium/calcium phosphate cements (MCPC) as model, the effects of magnesium (Mg) on the initial adhesion and osteogenic differentiation of bone marrow stromal cells (BMSCs) as well as the underlying mechanism were investigated. A series of MCPCs with different magnesium phosphate cement (MPC) content (0∼20%) in calcium phosphate cement (CPC) were synthesized. MCPCs with moderate proportion of MPC (5% and 10%, referred to as 5MCPC and 10MCPC) were found to effectively modulate the orientation of the adsorbed fibronectin (Fn) to exhibit enhanced receptor binding affinity, and to up-regulate integrin α5β1 expression of BMSCs, especially for 5MCPC. As a result, the attachment, morphology, focal adhesion formation, actin filaments assembly and osteogenic differentiation of BMSCs on 5MCPC were strongly enhanced. Further in vivo experiments confirmed that 5MCPC induced promoted osteogenesis in comparison to ot her CPC/MCPCs. Our results also suggested that the Mg on the underlying substrates but not the dissolved Mg ions was the main contributor to the above positive effects. Based on these results, it can be inferred that the specific interaction of Fn and integrin α5β1 had predominant effect on the MCPC-induced enhanced cellular response of BMSCs. These results provide a new strategy to regulate BMSCs adhesion and osteogenic differentiation by adjusting the Mg/Ca content and distribution in CPC, guiding the development of osteoinductive scaffolds for bone tissue regeneration. Copyright © 2015 Elsevier Ltd. All rights reserved.

The effects of increased testicular temperature on testis-specific isoform of Na+/K+ -ATPase in sperm and its role in spermatogenesis and sperm function.

PubMed

Thundathil, J C; Rajamanickam, G D; Kastelic, J P; Newton, L D

2012-08-01

Impaired testicular thermoregulation is commonly implicated in abnormal spermatogenesis and impaired sperm function in animals and humans, with outcomes ranging from subclinical infertility to sterility. Bovine testes must be maintained 4-5 °C below body-core temperature for normal spermatogenesis. The effects of elevated testicular temperature have been extensively studied in cattle using a scrotal insulation model, which results in abnormal spermatogenesis and impaired sperm morphology and function. Using this model and proteomic approaches, we compared normal and abnormal sperm (from the same bulls) to elucidate the molecular basis of impaired function. We identified a cohort of sperm functional proteins differentially expressed between normal vs abnormal sperm, including a testis-specific isoform of Na(+) /K(+) -ATPase. In addition to its role as a sodium pump regulating sperm motility, Na(+) /K(+) -ATPase is also involved as a signalling molecule during sperm capacitation. In conclusion, because of its involvement in regulation of sperm function, this protein has potential as a fertility marker. Furthermore, comparing normal vs abnormal sperm (induced by scrotal insulation) is a useful model for identifying proteins regulating sperm function. © 2012 Blackwell Verlag GmbH.

Target molecules in 3T3-L1 adipocytes differentiation are regulated by maslinic acid, a natural triterpene from Olea europaea.

PubMed

Pérez-Jiménez, Amalia; Rufino-Palomares, Eva E; Fernández-Gallego, Nieves; Ortuño-Costela, M Carmen; Reyes-Zurita, Fernando J; Peragón, Juan; García-Salguero, Leticia; Mokhtari, Khalida; Medina, Pedro P; Lupiáñez, José A

2016-11-15

Metabolic syndrome is a set of pathologies among which stand out the obesity, which is related to the lipid droplet accumulation and changes to cellular morphology regulated by several molecules and transcription factors. Maslinic acid (MA) is a natural product with demonstrated pharmacological functions including anti-inflammation, anti-tumor and anti-oxidation, among others. Here we report the effects of MA on the adipogenesis process in 3T3-L1 cells. Cell viability, glucose uptake, cytoplasmic triglyceride droplets, triglycerides quantification, gene transcription factors such as peroxisome proliferator-activated receptor γ (PPARγ) and adipocyte fatty acid-binding protein (aP2) and intracellular Ca 2+ levels were determined in pre-adipocytes and adipocytes of 3T3-L1 cells. MA increased glucose uptake. MA also decreased lipid droplets and triglyceride levels, which is in concordance with the down-regulation of PPARγ and aP2. Finally, MA increased the intracellular Ca 2+ concentration, which could also be involved in the demonstrated antiadipogenic effect of this triterpene. MA has been demonstrated as potential antiadipogenic compound in 3T3-L1 cells. Copyright © 2016 Elsevier GmbH. All rights reserved.

Toward an understanding of the pathophysiology of clear cell carcinoma of the ovary (Review)

PubMed Central

UEKURI, CHIHARU; SHIGETOMI, HIROSHI; ONO, SUMIRE; SASAKI, YOSHIKAZU; MATSUURA, MIYUKI; KOBAYASHI, HIROSHI

2013-01-01

Endometriosis-associated ovarian cancers demonstrate substantial morphological and genetic diversity. The transcription factor, hepatocyte nuclear factor (HNF)-1β, may be one of several key genes involved in the identity of ovarian clear cell carcinoma (CCC). The present study reviews a considerably expanded set of HNF-1β-associated genes and proteins that determine the pathophysiology of CCC. The current literature was reviewed by searching MEDLINE/PubMed. Functional interpretations of gene expression profiling in CCC are provided. Several important CCC-related genes overlap with those known to be regulated by the upregulation of HNF-1β expression, along with a lack of estrogen receptor (ER) expression. Furthermore, the genetic expression pattern in CCC resembles that of the Arias-Stella reaction, decidualization and placentation. HNF-1β regulates a subset of progesterone target genes. HNF-1β may also act as a modulator of female reproduction, playing a role in endometrial regeneration, differentiation, decidualization, glycogen synthesis, detoxification, cell cycle regulation, implantation, uterine receptivity and a successful pregnancy. In conclusion, the present study focused on reviewing the aberrant expression of CCC-specific genes and provided an update on the pathological implications and molecular functions of well-characterized CCC-specific genes. PMID:24179489

Testis development, fertility, and survival in Ethanolamine kinase 2-deficient mice.

PubMed

Gustin, Sonja E; Western, Patrick S; McClive, Peter J; Harley, Vincent R; Koopman, Peter A; Sinclair, Andrew H

2008-12-01

Ethanolamine kinase 2 (Eki2) was previously isolated from a differential expression screen designed to identify candidate genes involved in testis development and differentiation. In mouse, Eki2 is specifically up-regulated in Sertoli cells of the developing testis at the time of sex determination. Based on this expression profile, Eki2 was considered a good candidate testis-determining gene. To investigate a possible role of Eki2 in testis development, we have generated a mouse with targeted disruption of the Eki2 gene by using an EGFP replacement strategy. No abnormalities were detected in the Eki2-deficient mice with regard to embryonic and adult testis morphology, differentiation, function, or fertility. Furthermore, no significant differences were observed in litter sizes, pup mortality rates, or distribution of the sexes among the offspring. Ethanolamine kinases are involved in the biosynthesis of phosphatidylethanolamine, a major membrane phospholipid. Expression analysis indicates that the absence of an apparent phenotype in the Eki2-deficient mice may be due to compensation by Eki2-family members or the activation of an alternative pathway to generate phosphatidylethanolamine. Expression of EGFP in this mouse model enabled the isolation of gonad cell populations, providing a useful resource from which to obtain relatively pure early steroidogenic cells for further studies.

Methylglyoxal synthase regulates cell elongation via alterations of cellular methylglyoxal and spermidine content in Bacillus subtilis.

PubMed

Shin, Sang-Min; Song, Sung-Hyun; Lee, Jin-Woo; Kwak, Min-Kyu; Kang, Sa-Ouk

2017-10-01

Methylglyoxal regulates cell division and differentiation through its interaction with polyamines. Loss of their biosynthesizing enzyme causes physiological impairment and cell elongation in eukaryotes. However, the reciprocal effects of methylglyoxal and polyamine production and its regulatory metabolic switches on morphological changes in prokaryotes have not been addressed. Here, Bacillus subtilis methylglyoxal synthase (mgsA) and polyamine biosynthesizing genes encoding arginine decarboxylase (SpeA), agmatinase (SpeB), and spermidine synthase (SpeE), were disrupted or overexpressed. Treatment of 0.2mM methylglyoxal and 1mM spermidine led to the elongation and shortening of B. subtilis wild-type cells to 12.38±3.21μm (P<0.05) and 3.24±0.73μm (P<0.01), respectively, compared to untreated cells (5.72±0.68μm). mgsA-deficient (mgsA - ) and -overexpressing (mgsA OE ) mutants also demonstrated cell shortening and elongation, similar to speB- and speE-deficient (speB - and speE - ) and -overexpressing (speB OE and speE OE ) mutants. Importantly, both mgsA-depleted speB OE and speE OE mutants (speB OE /mgsA - and speE OE /mgsA - ) were drastically shortened to 24.5% and 23.8% of parental speB OE and speE OE mutants, respectively. These phenotypes were associated with reciprocal alterations of mgsA and polyamine transcripts governed by the contents of methylglyoxal and spermidine, which are involved in enzymatic or genetic metabolite-control mechanisms. Additionally, biophysically detected methylglyoxal-spermidine Schiff bases did not affect morphogenesis. Taken together, the findings indicate that methylglyoxal triggers cell elongation. Furthermore, cells with methylglyoxal accumulation commonly exhibit an elongated rod-shaped morphology through upregulation of mgsA, polyamine genes, and the global regulator spx, as well as repression of the cell division and shape regulator, FtsZ. Copyright © 2017 Elsevier Ltd. All rights reserved.

Hepatic differentiation capability of rat bone marrow-derived mesenchymal stem cells and hematopoietic stem cells.

PubMed

Shu, Sai-Nan; Wei, Lai; Wang, Jiang-Hua; Zhan, Yu-Tao; Chen, Hong-Song; Wang, Yu

2004-10-01

To investigate the different effects of mesenchymal stem cells (MSCs) and hematopoietic stem cells (HSCs) on hepatic differentiation. MSCs from rat bone marrow were isolated and cultured by standard methods. HSCs from rat bone marrow were isolated and purified by magnetic activated cell sorting. Both cell subsets were induced. Morphology, RT-PCR and immunocytochemistry were used to identify the hepatic differentiation grade. MSCs exhibited round in shape after differentiation, instead of fibroblast-like morphology before differentiation. Albumin mRNA and protein were expressed positively in MSCs, without detection of alpha-fetoprotein (AFP). HSCs were polygonal in shape after differentiation. The expression of albumin signal decreased and AFP signal increased. The expression of CK18 was continuous in MSCs and HSCs both before and after induction. Both MSCs and HSCs have hepatic differentiation capabilities. However, their capabilities are not the same. MSCs can differentiate into mature hepatocyte-like cells, never expressing early hepatic specific genes, while Thy-1.1(+) cells are inclined to differentiate into hepatic stem cell-like cells, with an increasing AFP expression and a decreasing albumin signal. CK18 mRNA is positive in Thy-1.1(+) cells and MSCs, negative in Thy-1.1(-) cells. It seems that CK18 has some relationship with Thy-1.1 antigen, and CK18 may be a predictive marker of hepatic differentiation capability.

Usual and unusual development of the dicot leaf: involvement of transcription factors and hormones.

PubMed

Fambrini, Marco; Pugliesi, Claudio

2013-06-01

Morphological diversity exhibited by higher plants is essentially related to the tremendous variation of leaf shape. With few exceptions, leaf primordia are initiated postembryonically at the flanks of a group of undifferentiated and proliferative cells within the shoot apical meristem (SAM) in characteristic position for the species and in a regular phyllotactic sequence. Auxin is critical for this process, because genes involved in auxin biosynthesis, transport, and signaling are required for leaf initiation. Down-regulation of transcription factors (TFs) and cytokinins are also involved in the light-dependent leaf initiation pathway. Furthermore, mechanical stresses in SAM determine the direction of cell division and profoundly influence leaf initiation suggesting a link between physical forces, gene regulatory networks and biochemical gradients. After the leaf is initiated, its further growth depends on cell division and cell expansion. Temporal and spatial regulation of these processes determines the size and the shape of the leaf, as well as the internal structure. A complex array of intrinsic signals, including phytohormones and TFs control the appropriate cell proliferation and differentiation to elaborate the final shape and complexity of the leaf. Here, we highlight the main determinants involved in leaf initiation, epidermal patterning, and elaboration of lamina shape to generate small marginal serrations, more deep lobes or a dissected compound leaf. We also outline recent advances in our knowledge of regulatory networks involved with the unusual pattern of leaf development in epiphyllous plants as well as leaf morphology aberrations, such as galls after pathogenic attacks of pests.

A novel function of the cell polarity-regulating kinase PAR-1/MARK in dendritic spines

PubMed Central

Hayashi, Kenji; Suzuki, Atsushi; Ohno, Shigeo

2011-01-01

Dendritic spines are postsynaptic structures that receive excitatory synaptic signals from presynaptic terminals in neurons. Because the morphology of spines has been considered to be a crucial factor for the efficiency of synaptic transmission, understanding the mechanisms regulating their morphology is important for neuroscience. Actin filaments and their regulatory proteins are known to actively maintain spine morphology; recent studies have also shown an essential role of microtubules (MTs). Live imaging of the plus-ends of MTs in mature neurons revealed that MTs stochastically enter spines and mediate accumulation of p140Cap, which regulates reorganization of actin filaments. However, the molecular mechanism by which MT dynamics is controlled has remained largely unknown. A cell polarity-regulating serine/threonine kinase, partitioning-defective 1 (PAR-1), phosphorylates classical MAPs and inhibits their binding to MTs. Because the interaction of MAPs with MTs can decrease MT dynamic instability, PAR-1 is supposed to activate MT dynamics through its MAP/MT affinity-regulating kinase (MARK) activity, although there is not yet any direct evidence for this. Here, we review recent findings on the localization of PAR-1b in the dendrites of mouse hippocampal neurons, and its novel function in the maintenance of mature spine morphology by regulating MT dynamics. PMID:22545177

A novel function of the cell polarity-regulating kinase PAR-1/MARK in dendritic spines.

PubMed

Hayashi, Kenji; Suzuki, Atsushi; Ohno, Shigeo

2011-11-01

Dendritic spines are postsynaptic structures that receive excitatory synaptic signals from presynaptic terminals in neurons. Because the morphology of spines has been considered to be a crucial factor for the efficiency of synaptic transmission, understanding the mechanisms regulating their morphology is important for neuroscience. Actin filaments and their regulatory proteins are known to actively maintain spine morphology; recent studies have also shown an essential role of microtubules (MTs). Live imaging of the plus-ends of MTs in mature neurons revealed that MTs stochastically enter spines and mediate accumulation of p140Cap, which regulates reorganization of actin filaments. However, the molecular mechanism by which MT dynamics is controlled has remained largely unknown. A cell polarity-regulating serine/threonine kinase, partitioning-defective 1 (PAR-1), phosphorylates classical MAPs and inhibits their binding to MTs. Because the interaction of MAPs with MTs can decrease MT dynamic instability, PAR-1 is supposed to activate MT dynamics through its MAP/MT affinity-regulating kinase (MARK) activity, although there is not yet any direct evidence for this. Here, we review recent findings on the localization of PAR-1b in the dendrites of mouse hippocampal neurons, and its novel function in the maintenance of mature spine morphology by regulating MT dynamics.

The role of selection and historical factors in driving population differentiation along an elevational gradient in an island bird.

PubMed

Bertrand, J A M; Delahaie, B; Bourgeois, Y X C; Duval, T; García-Jiménez, R; Cornuault, J; Pujol, B; Thébaud, C; Milá, B

2016-04-01

Adaptation to local environmental conditions and the range dynamics of populations can influence evolutionary divergence along environmental gradients. Thus, it is important to investigate patterns of both phenotypic and genetic variations among populations to reveal the respective roles of these two types of factors in driving population differentiation. Here, we test for evidence of phenotypic and genetic structure across populations of a passerine bird (Zosterops borbonicus) distributed along a steep elevational gradient on the island of Réunion. Using 11 microsatellite loci screened in 401 individuals from 18 localities distributed along the gradient, we found that genetic differentiation occurred at two spatial levels: (i) between two main population groups corresponding to highland and lowland areas, respectively, and (ii) within each of these two groups. In contrast, several morphological traits varied gradually along the gradient. Comparison of neutral genetic differentiation (FST ) and phenotypic differentiation (PST ) showed that PST largely exceeds FST at several morphological traits, which is consistent with a role for local adaptation in driving morphological divergence along the gradient. Overall, our results revealed an area of secondary contact midway up the gradient between two major, cryptic, population groups likely diverged in allopatry. Remarkably, local adaptation has shaped phenotypic differentiation irrespective of population history, resulting in different patterns of variation along the elevational gradient. Our findings underscore the importance of understanding both historical and selective factors when trying to explain variation along environmental gradients. © 2016 European Society For Evolutionary Biology. Journal of Evolutionary Biology © 2016 European Society For Evolutionary Biology.

Chondrogenesis, osteogenesis and adipogenesis of canine mesenchymal stem cells: a biochemical, morphological and ultrastructural study.

PubMed

Csaki, C; Matis, U; Mobasheri, A; Ye, H; Shakibaei, M

2007-12-01

Musculoskeletal diseases with osteochondrotic articular cartilage defects, such as osteoarthritis, are an increasing problem for humans and companion animals which necessitates the development of novel and improved therapeutic strategies. Canine mesenchymal stem cells (cMSCs) offer significant promise as a multipotent source for cell-based therapies and could form the basis for the differentiation and cultivation of tissue grafts to replace damaged tissue. However, no comprehensive analysis has been undertaken to characterize the ultrastructure of in vitro differentiated cMSCs. The main goal of this paper was to focus on cMSCs and to analyse their differentiation capacity. To achieve this aim, bone marrow cMSCs from three canine patients were isolated, expanded in monolayer culture and characterized with respect to their ability for osteogenic, adipogenic and chondrogenic differentiation capacities. cMSCs showed proliferative potential and were capable of osteogenic, adipogenic and chondrogenic differentiation. cMSCs treated with the osteogenic induction medium differentiated into osteoblasts, produced typical bone matrix components, beta1-integrins and upregulated the osteogenic specific transcription factor Cbfa-1. cMSCs treated with the adipogenic induction medium showed typical adipocyte morphology, produced adiponectin, collagen type I and beta1-integrins, and upregulated the adipogenic specific transcription factor PPAR-gamma. cMSCs treated with the chondrogenic induction medium exhibited a round to oval shape, produced a cartilage-specific extracellular matrix, beta1-integrins and upregulated the chondrogenic specific transcription factor Sox9. These results demonstrate, at the biochemical, morphological and ultrastructural levels, the multipotency of cMSCs and thus highlight their potential therapeutic value for cell-based tissue engineering.