Tumor suppressor Lzap regulates cell cycle progression, doming and zebrafish epiboly

PubMed Central

Liu, Dan; Wang, Wen-Der; Melville, David B.; Cha, Yong I.; Yin, Zhirong; Issaeva, Natalia; Knapik, Ela W.; Yarbrough, Wendell G.

2012-01-01

Initial stages of embryonic development rely on rapid, synchronized cell divisions of the fertilized egg followed by a set of morphogenetic movements collectively called epiboly and gastrulation. Lzap is a putative tumor suppressor whose expression is lost in 30% of head and neck squamous cell carcinomas. Lzap activities include regulation of cell cycle progression and response to therapeutic agents. Here we explore developmental roles of the lzap gene during zebrafish morphogenesis. Lzap is highly conserved among vertebrates and is maternally deposited. Expression is initially ubiquitous during gastrulation, and later becomes more prominent in the pharyngeal arches, digestive tract and brain. Antisense morpholino-mediated depletion of Lzap resulted in delayed cell divisions and apoptosis during blastomere formation, resulting in fewer, larger cells. Cell cycle analysis suggested that Lzap loss in early embryonic cells resulted in a G2/M arrest. Furthermore, the Lzap-deficient embryos failed to initiate epiboly – the earliest morphogenetic movement in animal development – which has been shown to be dependent on cell adhesion and migration of epithelial sheets. Our results strongly implicate Lzap in regulation of cell cycle progression, adhesion and migratory activity of epithelial cell sheets during early development. These functions provide further insight into Lzap activity that may contribute not only to development, but also to tumor formation. PMID:21523853

Cell movement is guided by the rigidity of the substrate

NASA Technical Reports Server (NTRS)

Lo, C. M.; Wang, H. B.; Dembo, M.; Wang, Y. L.

2000-01-01

Directional cell locomotion is critical in many physiological processes, including morphogenesis, the immune response, and wound healing. It is well known that in these processes cell movements can be guided by gradients of various chemical signals. In this study, we demonstrate that cell movement can also be guided by purely physical interactions at the cell-substrate interface. We cultured National Institutes of Health 3T3 fibroblasts on flexible polyacrylamide sheets coated with type I collagen. A transition in rigidity was introduced in the central region of the sheet by a discontinuity in the concentration of the bis-acrylamide cross-linker. Cells approaching the transition region from the soft side could easily migrate across the boundary, with a concurrent increase in spreading area and traction forces. In contrast, cells migrating from the stiff side turned around or retracted as they reached the boundary. We call this apparent preference for a stiff substrate "durotaxis." In addition to substrate rigidity, we discovered that cell movement could also be guided by manipulating the flexible substrate to produce mechanical strains in the front or rear of a polarized cell. We conclude that changes in tissue rigidity and strain could play an important controlling role in a number of normal and pathological processes involving cell locomotion.

Rac1/RhoA antagonism defines cell-to-cell heterogeneity during epidermal morphogenesis in nematodes

PubMed Central

Ouellette, Marie-Hélène

2016-01-01

The antagonism between the GTPases Rac1 and RhoA controls cell-to-cell heterogeneity in isogenic populations of cells in vitro and epithelial morphogenesis in vivo. Its involvement in the regulation of cell-to-cell heterogeneity during epidermal morphogenesis has, however, never been addressed. We used a quantitative cell imaging approach to characterize epidermal morphogenesis at a single-cell level during early elongation of Caenorhabditis elegans embryos. This study reveals that a Rac1-like pathway, involving the Rac/Cdc42 guanine-exchange factor β-PIX/PIX-1 and effector PAK1/PAK-1, and a RhoA-like pathway, involving ROCK/LET-502, control the remodeling of apical junctions and the formation of basolateral protrusions in distinct subsets of hypodermal cells. In these contexts, protrusions adopt lamellipodia or an amoeboid morphology. We propose that lamella formation may reduce tension building at cell–cell junctions during morphogenesis. Cell-autonomous antagonism between these pathways enables cells to switch between Rac1- and RhoA-like morphogenetic programs. This study identifies the first case of cell-to-cell heterogeneity controlled by Rac1/RhoA antagonism during epidermal morphogenesis. PMID:27821782

Ras1 interacts with multiple new signaling and cytoskeletal loci in Drosophila eggshell patterning and morphogenesis.

PubMed Central

Schnorr, J D; Holdcraft, R; Chevalier, B; Berg, C A

2001-01-01

Little is known about the genes that interact with Ras signaling pathways to regulate morphogenesis. The synthesis of dorsal eggshell structures in Drosophila melanogaster requires multiple rounds of Ras signaling followed by dramatic epithelial sheet movements. We took advantage of this process to identify genes that link patterning and morphogenesis; we screened lethal mutations on the second chromosome for those that could enhance a weak Ras1 eggshell phenotype. Of 1618 lethal P-element mutations tested, 13 showed significant enhancement, resulting in forked and fused dorsal appendages. Our genetic and molecular analyses together with information from the Berkeley Drosophila Genome Project reveal that 11 of these lines carry mutations in previously characterized genes. Three mutations disrupt the known Ras1 cell signaling components Star, Egfr, and Blistered, while one mutation disrupts Sec61beta, implicated in ligand secretion. Seven lines represent cell signaling and cytoskeletal components that are new to the Ras1 pathway; these are Chickadee (Profilin), Tec29, Dreadlocks, POSH, Peanut, Smt3, and MESK2, a suppressor of dominant-negative Ksr. A twelfth insertion disrupts two genes, Nrk, a "neurospecific" receptor tyrosine kinase, and Tpp, which encodes a neuropeptidase. These results suggest that Ras1 signaling during oogenesis involves novel components that may be intimately associated with additional signaling processes and with the reorganization of the cytoskeleton. To determine whether these Ras1 Enhancers function upstream or downstream of the Egf receptor, four mutations were tested for their ability to suppress an activated Egfr construct (lambdatop) expressed in oogenesis exclusively in the follicle cells. Mutations in Star and l(2)43Bb had no significant effect upon the lambdatop eggshell defect whereas smt3 and dock alleles significantly suppressed the lambdatop phenotype. PMID:11606538

Ras1 interacts with multiple new signaling and cytoskeletal loci in Drosophila eggshell patterning and morphogenesis.

PubMed

Schnorr, J D; Holdcraft, R; Chevalier, B; Berg, C A

2001-10-01

Little is known about the genes that interact with Ras signaling pathways to regulate morphogenesis. The synthesis of dorsal eggshell structures in Drosophila melanogaster requires multiple rounds of Ras signaling followed by dramatic epithelial sheet movements. We took advantage of this process to identify genes that link patterning and morphogenesis; we screened lethal mutations on the second chromosome for those that could enhance a weak Ras1 eggshell phenotype. Of 1618 lethal P-element mutations tested, 13 showed significant enhancement, resulting in forked and fused dorsal appendages. Our genetic and molecular analyses together with information from the Berkeley Drosophila Genome Project reveal that 11 of these lines carry mutations in previously characterized genes. Three mutations disrupt the known Ras1 cell signaling components Star, Egfr, and Blistered, while one mutation disrupts Sec61beta, implicated in ligand secretion. Seven lines represent cell signaling and cytoskeletal components that are new to the Ras1 pathway; these are Chickadee (Profilin), Tec29, Dreadlocks, POSH, Peanut, Smt3, and MESK2, a suppressor of dominant-negative Ksr. A twelfth insertion disrupts two genes, Nrk, a "neurospecific" receptor tyrosine kinase, and Tpp, which encodes a neuropeptidase. These results suggest that Ras1 signaling during oogenesis involves novel components that may be intimately associated with additional signaling processes and with the reorganization of the cytoskeleton. To determine whether these Ras1 Enhancers function upstream or downstream of the Egf receptor, four mutations were tested for their ability to suppress an activated Egfr construct (lambdatop) expressed in oogenesis exclusively in the follicle cells. Mutations in Star and l(2)43Bb had no significant effect upon the lambdatop eggshell defect whereas smt3 and dock alleles significantly suppressed the lambdatop phenotype.

Cell confinement controls centrosome positioning and lumen initiation during epithelial morphogenesis

PubMed Central

Rodríguez-Fraticelli, Alejo E.; Auzan, Muriel; Alonso, Miguel A.; Bornens, Michel

2012-01-01

Epithelial organ morphogenesis involves sequential acquisition of apicobasal polarity by epithelial cells and development of a functional lumen. In vivo, cells perceive signals from components of the extracellular matrix (ECM), such as laminin and collagens, as well as sense physical conditions, such as matrix stiffness and cell confinement. Alteration of the mechanical properties of the ECM has been shown to promote cell migration and invasion in cancer cells, but the effects on epithelial morphogenesis have not been characterized. We analyzed the effects of cell confinement on lumen morphogenesis using a novel, micropatterned, three-dimensional (3D) Madin-Darby canine kidney cell culture method. We show that cell confinement, by controlling cell spreading, limits peripheral actin contractility and promotes centrosome positioning and lumen initiation after the first cell division. In addition, peripheral actin contractility is mediated by master kinase Par-4/LKB1 via the RhoA–Rho kinase–myosin II pathway, and inhibition of this pathway restores lumen initiation in minimally confined cells. We conclude that cell confinement controls nuclear–centrosomal orientation and lumen initiation during 3D epithelial morphogenesis. PMID:22965908

Un(MaSC)ing Stem Cell Dynamics in Mammary Branching Morphogenesis.

PubMed

Greenwood, Erin; Wrenn, Emma D; Cheung, Kevin J

2017-02-27

The properties of stem cells that participate in mammary gland branching morphogenesis remain contested. Reporting in Nature, Scheele et al. (2017) establish a model for post-pubertal mammary branching morphogenesis in which position-dependent, lineage-restricted stem cells undergo cell mixing in order to contribute to long-term growth. Copyright © 2017 Elsevier Inc. All rights reserved.

The cellular and molecular mechanisms of vertebrate lens development

PubMed Central

Cvekl, Aleš; Ashery-Padan, Ruth

2014-01-01

The ocular lens is a model system for understanding important aspects of embryonic development, such as cell specification and the spatiotemporally controlled formation of a three-dimensional structure. The lens, which is characterized by transparency, refraction and elasticity, is composed of a bulk mass of fiber cells attached to a sheet of lens epithelium. Although lens induction has been studied for over 100 years, recent findings have revealed a myriad of extracellular signaling pathways and gene regulatory networks, integrated and executed by the transcription factor Pax6, that are required for lens formation in vertebrates. This Review summarizes recent progress in the field, emphasizing the interplay between the diverse regulatory mechanisms employed to form lens progenitor and precursor cells and highlighting novel opportunities to fill gaps in our understanding of lens tissue morphogenesis. PMID:25406393

Imaging Cell Shape Change in Living Drosophila Embryos

PubMed Central

Figard, Lauren; Sokac, Anna Marie

2011-01-01

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging. Cell shape changes in the fly are analogous to those in higher organisms, and they drive tissue morphogenesis. So, in many cases, their study has direct implications for understanding human disease (Table 1)1-5. On the sub-cellular scale, these cell shape changes are the product of activities ranging from gene expression to signal transduction, cell polarity, cytoskeletal remodeling and membrane trafficking. Thus, the Drosophila embryo provides not only the context to evaluate cell shape changes as they relate to tissue morphogenesis, but also offers a completely physiological environment to study the sub-cellular activities that shape cells. The protocol described here is designed to image a specific cell shape change called cellularization. Cellularization is a process of dramatic plasma membrane growth, and it ultimately converts the syncytial embryo into the cellular blastoderm. That is, at interphase of mitotic cycle 14, the plasma membrane simultaneously invaginates around each of ~6000 cortically anchored nuclei to generate a sheet of primary epithelial cells. Counter to previous suggestions, cellularization is not driven by Myosin-2 contractility6, but is instead fueled largely by exocytosis of membrane from internal stores7. Thus, cellularization is an excellent system for studying membrane trafficking during cell shape changes that require plasma membrane invagination or expansion, such as cytokinesis or transverse-tubule (T-tubule) morphogenesis in muscle. Note that this protocol is easily applied to the imaging of other cell shape changes in the fly embryo, and only requires slight adaptations such as changing the stage of embryo collection, or using "embryo glue" to mount the embryo in a specific orientation (Table 1)8-19. In all cases, the workflow is basically the same (Figure 1). Standard methods for cloning and Drosophila transgenesis are used to prepare stable fly stocks that express a protein of interest, fused to Green Fluorescent Protein (GFP) or its variants, and these flies provide a renewable source of embryos. Alternatively, fluorescent proteins/probes are directly introduced into fly embryos via straightforward micro-injection techniques9-10. Then, depending on the developmental event and cell shape change to be imaged, embryos are collected and staged by morphology on a dissecting microscope, and finally positioned and mounted for time-lapse imaging on a confocal microscope. PMID:21490577

The N- or C-terminal domains of DSH-2 can activate the C. elegans Wnt/β-catenin asymmetry pathway

PubMed Central

King, Ryan S.; Maiden, Stephanie L.; Hawkins, Nancy C.; Kidd, Ambrose R.; Kimble, Judith; Hardin, Jeff; Walston, Timothy D.

2015-01-01

Dishevelleds are modular proteins that lie at the crossroads of divergent Wnt signaling pathways. The DIX domain of dishevelleds modulates a β-catenin destruction complex, and thereby mediates cell fate decisions through differential activation of Tcf transcription factors. The DEP domain of dishevelleds mediates planar polarity of cells within a sheet through regulation of actin modulators. In Caenorhabditis elegans asymmetric cell fate decisions are regulated by asymmetric localization of signaling components in a pathway termed the Wnt/β-catenin asymmetry pathway. Which domain(s) of Disheveled regulate this pathway is unknown. We show that C. elegans embryos from dsh-2(or302) mutant mothers fail to successfully undergo morphogenesis, but transgenes containing either the DIX or the DEP domain of DSH-2 are sufficient to rescue the mutant phenotype. Embryos lacking zygotic function of SYS-1/β-catenin, WRM-1/β-catenin, or POP-1/Tcf show defects similar to dsh-2 mutants, including a loss of asymmetry in some cell fate decisions. Removal of two dishevelleds (dsh-2 and mig-5) leads to a global loss of POP-1 asymmetry, which can be rescued by addition of transgenes containing either the DIX or DEP domain of DSH-2. These results indicate that either the DIX or DEP domain of DSH-2 is capable of activating the Wnt/β-catenin asymmetry pathway and regulating anterior–posterior fate decisions required for proper morphogenesis. PMID:19298786

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

Programming Morphogenesis through Systems and Synthetic Biology.

PubMed

Velazquez, Jeremy J; Su, Emily; Cahan, Patrick; Ebrahimkhani, Mo R

2018-04-01

Mammalian tissue development is an intricate, spatiotemporal process of self-organization that emerges from gene regulatory networks of differentiating stem cells. A major goal in stem cell biology is to gain a sufficient understanding of gene regulatory networks and cell-cell interactions to enable the reliable and robust engineering of morphogenesis. Here, we review advances in synthetic biology, single cell genomics, and multiscale modeling, which, when synthesized, provide a framework to achieve the ambitious goal of programming morphogenesis in complex tissues and organoids. Copyright © 2017 Elsevier Ltd. All rights reserved.

Expression Pattern of the Pro-apoptotic Gene PAR-4 During the Morphogenesis of MCF-10A Human Mammary Epithelial Cells.

PubMed

de Bessa Garcia, Simone A; Pereira, Michelly C; Nagai, Maria A

2010-12-21

The histological organization of the mammary gland involves a spatial interaction of epithelial and myoepithelial cells with the specialized basement membrane (BM), composed of extra-cellular matrix (ECM) proteins, which is disrupted during the tumorigenic process. The interactions between mammary epithelial cells and ECM components play a major role in mammary gland branching morphogenesis. Critical signals for mammary epithelial cell proliferation, differentiation, and survival are provided by the ECM proteins. Three-dimensional (3D) cell culture was developed to establish a system that simulates several features of the breast epithelium in vivo; 3D cell culture of the spontaneously immortalized cell line, MCF10A, is a well-established model system to study breast epithelial cell biology and morphogenesis. Mammary epithelial cells grown in 3D form spheroids, acquire apicobasal polarization, and form lumens that resemble acini structures, processes that involve cell death. Using this system, we evaluated the expression of the pro-apoptotic gene PAWR (PKC apoptosis WT1 regulator; also named PAR-4, prostate apoptosis response-4) by immunofluorescence and quantitative real time PCR (qPCR). A time-dependent increase in PAR-4 mRNA expression was found during the process of MCF10A acinar morphogenesis. Confocal microscopy analysis also showed that PAR-4 protein was highly expressed in the MCF10A cells inside the acini structure. During the morphogenesis of MCF10A cells in 3D cell culture, the cells within the lumen showed caspase-3 activation, indicating apoptotic activity. PAR-4 was only partially co-expressed with activated caspase-3 on these cells. Our results provide evidence, for the first time, that PAR-4 is differentially expressed during the process of MCF10A acinar morphogenesis.

Normal morphogenesis of epithelial tissues and progression of epithelial tumors

PubMed Central

Wang, Chun-Chao; Jamal, Leen; Janes, Kevin A.

2011-01-01

Epithelial cells organize into various tissue architectures that largely maintain their structure throughout the life of an organism. For decades, the morphogenesis of epithelial tissues has fascinated scientists at the interface of cell, developmental, and molecular biology. Systems biology offers ways to combine knowledge from these disciplines by building integrative models that are quantitative and predictive. Can such models be useful for gaining a deeper understanding of epithelial morphogenesis? Here, we take inventory of some recurring themes in epithelial morphogenesis that systems approaches could strive to capture. Predictive understanding of morphogenesis at the systems level would prove especially valuable for diseases such as cancer, where epithelial tissue architecture is profoundly disrupted. PMID:21898857

A System for Modelling Cell–Cell Interactions during Plant Morphogenesis

PubMed Central

Dupuy, Lionel; Mackenzie, Jonathan; Rudge, Tim; Haseloff, Jim

2008-01-01

Background and aims During the development of multicellular organisms, cells are capable of interacting with each other through a range of biological and physical mechanisms. A description of these networks of cell–cell interactions is essential for an understanding of how cellular activity is co-ordinated in regionalized functional entities such as tissues or organs. The difficulty of experimenting on living tissues has been a major limitation to describing such systems, and computer modelling appears particularly helpful to characterize the behaviour of multicellular systems. The experimental difficulties inherent to the multitude of parallel interactions that underlie cellular morphogenesis have led to the need for computer models. Methods A new generic model of plant cellular morphogenesis is described that expresses interactions amongst cellular entities explicitly: the plant is described as a multi-scale structure, and interactions between distinct entities is established through a topological neighbourhood. Tissues are represented as 2D biphasic systems where the cell wall responds to turgor pressure through a viscous yielding of the cell wall. Key Results This principle was used in the development of the CellModeller software, a generic tool dedicated to the analysis and modelling of plant morphogenesis. The system was applied to three contrasting study cases illustrating genetic, hormonal and mechanical factors involved in plant morphogenesis. Conclusions Plant morphogenesis is fundamentally a cellular process and the CellModeller software, through its underlying generic model, provides an advanced research tool to analyse coupled physical and biological morphogenetic mechanisms. PMID:17921524

Direct activation of Shroom3 transcription by Pitx proteins drives epithelial morphogenesis in the developing gut

PubMed Central

Chung, Mei-I; Nascone-Yoder, Nanette M.; Grover, Stephanie A.; Drysdale, Thomas A.; Wallingford, John B.

2010-01-01

Individual cell shape changes are essential for epithelial morphogenesis. A transcriptional network for epithelial cell shape change is emerging in Drosophila, but this area remains largely unexplored in vertebrates. The distinction is important as so far, key downstream effectors of cell shape change in Drosophila appear not to be conserved. Rather, Shroom3 has emerged as a central effector of epithelial morphogenesis in vertebrates, driving both actin- and microtubule-based cell shape changes. To date, the morphogenetic role of Shroom3 has been explored only in the neural epithelium, so the broad expression of this gene raises two important questions: what are the requirements for Shroom3 in non-neural tissues and what factors control Shroom3 transcription? Here, we show in Xenopus that Shroom3 is essential for cell shape changes and morphogenesis in the developing vertebrate gut and that Shroom3 transcription in the gut requires the Pitx1 transcription factor. Moreover, we show that Pitx proteins directly activate Shroom3 transcription, and we identify Pitx-responsive regulatory elements in the genomic DNA upstream of Shroom3. Finally, we show that ectopic expression of Pitx proteins is sufficient to induce Shroom3-dependent cytoskeletal reorganization and epithelial cell shape change. These data demonstrate new breadth to the requirements for Shroom3 in morphogenesis, and they also provide a cell-biological basis for the role of Pitx transcription factors in morphogenesis. More generally, these results provide a foundation for deciphering the transcriptional network that underlies epithelial cell shape change in developing vertebrates. PMID:20332151

Forces Generated by Cell Intercalation Tow Epidermal Sheets in Mammalian Tissue Morphogenesis

PubMed Central

Heller, Evan; Kumar, K. Vijay; Grill, Stephan W.; Fuchs, Elaine

2014-01-01

Summary While gastrulation movements offer mechanistic paradigms for how collective cellular movements shape developing embryos, far less is known about coordinated cellular movements that occur later in development. Studying eyelid closure, we explore a case where an epithelium locally reshapes, expands, and moves over another epithelium. Live imaging, gene targeting and cell cycle inhibitors reveal that closure does not require overlying periderm, proliferation or supracellular actin cable assembly. Laser ablation and quantitative analyses of tissue deformations further distinguish the mechanism from wound-repair and dorsal closure. Rather, cell intercalations parallel to the tissue front locally compress it perpendicularly, pulling the surrounding epidermis along the closure axis. Functional analyses in vivo show that the mechanism requires localized myosin-IIA and α5β1-fibronectin-mediated migration, and E-cadherin downregulation likely stimulated by Wnt signaling. These studies uncover a mode of epithelial closure in which forces generated by cell intercalation are leveraged to tow the surrounding tissue. PMID:24697897

Perithecium morphogenesis in Sordaria macrospora.

PubMed

Lord, Kathryn M; Read, Nick D

2011-04-01

The perithecium of the self-fertile ascomycete Sordaria macrospora provides an excellent model in which to analyse fungal multicellular development. This study provides a detailed analysis of perithecium morphogenesis in the wild type and eight developmental mutants of S. macrospora, using a range of correlative microscopical techniques. Fundamentally, perithecia and other complex multicellular structures produced by fungi arise by hyphal aggregation and adhesion, and these processes are followed by specialization and septation of hyphal compartments within the aggregates. Perithecial morphogenesis can be divided into the ascogonial, protoperithecial, and perithecial stages of development. At least 13 specialized, morphologically distinct cell-types are involved in perithecium morphogenesis, and these fall into three basic classes: hyphae, conglutinate cells and spores. Conglutinate cells arise from hyphal adhesion and certain perithecial hyphae develop from conglutinate cells. Various hypha-conglutinate cell transitions play important roles during the development of the perithecial wall and neck. Copyright © 2010. Published by Elsevier Inc.

Mechanics and morphogenesis of fission yeast cells.

PubMed

Davì, Valeria; Minc, Nicolas

2015-12-01

The integration of biochemical and biomechanical elements is at the heart of morphogenesis. While animal cells are relatively soft objects which shape and mechanics is mostly regulated by cytoskeletal networks, walled cells including those of plants, fungi and bacteria are encased in a rigid cell wall which resist high internal turgor pressure. How these particular mechanical properties may influence basic cellular processes, such as growth, shape and division remains poorly understood. Recent work using the model fungal cell fission yeast, Schizosaccharomyces pombe, highlights important contribution of cell mechanics to various morphogenesis processes. We envision this genetically tractable system to serve as a novel standard for the mechanobiology of walled cell. Copyright © 2015 Elsevier Ltd. All rights reserved.

Morphogenesis of the C. elegans vulva

PubMed Central

Schindler, Adam J

2012-01-01

Understanding how cells move, change shape, and alter cellular behaviors to form organs, a process termed morphogenesis, is one of the great challenges of developmental biology. Formation of the C. elegans vulva is a powerful, simple, and experimentally accessible model for elucidating how morphogenetic processes produce an organ. In the first step of vulval development, three epithelial precursor cells divide and differentiate to generate 22 cells of seven different vulval subtypes. The 22 vulval cells then rearrange from a linear array into a tube, with each of the seven cell types undergoing characteristic morphogenetic behaviours that construct the vulva. Vulval morphogenesis entails many of the same cellular activities that underlie organogenesis and tissue formation across species, including invagination, lumen formation, oriented cell divisions, cell-cell adhesion, cell migration, cell fusion, extracellular matrix remodelling and cell invasion. Studies of vulval development have led to pioneering discoveries in a number of these processes and are beginning to bridge the gap between the pathways that specify cells and their connections to morphogenetic behaviors. The simplicity of the vulva and the experimental tools available in C. elegans will continue to make vulval morphogenesis a powerful paradigm to further our understanding of the largely mysterious mechanisms that build tissues and organs. PMID:23418408

Polycystin-1 Binds Par3/aPKC and Controls Convergent Extension During Renal Tubular Morphogenesis

PubMed Central

Castelli, Maddalena; Boca, Manila; Chiaravalli, Marco; Ramalingam, Harini; Rowe, Isaline; Distefano, Gianfranco; Carroll, Thomas; Boletta, Alessandra

2013-01-01

Several organs, including lungs and kidneys, are formed by epithelial tubes whose proper morphogenesis ensures correct function. This is best exemplified by the kidney, where defective establishment or maintanance of tubular diameter results in polycystic kidney disease, a common genetic disorder. Most polycystic kidney disease cases result from loss-of-function mutations in the PKD1 gene, encoding Polycystin-1 (PC-1), a large receptor of unknown function. Here we demonstrate that PC-1 plays an essential role in establishment of correct tubular diameter during nephron development. PC-1 associates with Par3 favoring the assembly of a pro-polarizing Par3/aPKC complex and it regulates a program of cell polarity important for oriented cell migration and for a convergent extension-like process during tubular morphogenesis. Par3 inactivation in the developing kidney results in defective convergent extension and tubular morphogenesis and in renal cyst formation. Our data define PC-1 as central to cell polarization and to epithelial tube morphogenesis and homeostasis. PMID:24153433

Polycystin-1 binds Par3/aPKC and controls convergent extension during renal tubular morphogenesis

NASA Astrophysics Data System (ADS)

Castelli, Maddalena; Boca, Manila; Chiaravalli, Marco; Ramalingam, Harini; Rowe, Isaline; Distefano, Gianfranco; Carroll, Thomas; Boletta, Alessandra

2013-10-01

Several organs, including the lungs and kidneys, are formed by epithelial tubes whose proper morphogenesis ensures correct function. This is best exemplified by the kidney, where defective establishment or maintenance of tubular diameter results in polycystic kidney disease, a common genetic disorder. Most polycystic kidney disease cases result from loss-of-function mutations in the PKD1 gene, encoding Polycystin-1, a large receptor of unknown function. Here we demonstrate that PC-1 has an essential role in the establishment of correct tubular diameter during nephron development. Polycystin-1 associates with Par3 favouring the assembly of a pro-polarizing Par3/aPKC complex and it regulates a programme of cell polarity important for oriented cell migration and for a convergent extension-like process during tubular morphogenesis. Par3 inactivation in the developing kidney results in defective convergent extension and tubular morphogenesis, and in renal cyst formation. Our data define Polycystin-1 as central to cell polarization and to epithelial tube morphogenesis and homeostasis.

Crk synergizes with epidermal growth factor for epithelial invasion and morphogenesis and is required for the met morphogenic program.

PubMed

Lamorte, Louie; Rodrigues, Sonia; Naujokas, Monica; Park, Morag

2002-10-04

Activation of the Met receptor tyrosine kinase through its ligand, hepatocyte growth factor, stimulates cell spreading, cell dispersal, and the inherent morphogenic program of various epithelial cell lines. Although both hepatocyte growth factor and epidermal growth factor (EGF) can activate downstream signaling pathways in Madin-Darby canine kidney epithelial cells, EGF fails to promote the breakdown of cell-cell junctional complexes and initiate an invasive morphogenic program. We have undertaken a strategy to identify signals that synergize with EGF in this process. We provide evidence that the overexpression of the CrkII adapter protein complements EGF-stimulated pathways to induce cell dispersal in two-dimensional cultures and cell invasion and branching morphogenesis in three-dimensional collagen gels. This finding correlates with the ability of CrkII to promote the breakdown of adherens junctions in stable cell lines and the ability of EGF to stimulate enhanced Rac activity in cells overexpressing CrkII. We have previously shown that the Gab1-docking protein is required for branching morphogenesis downstream of the Met receptor. Consistent with a role for CrkII in promoting EGF-dependent branching morphogenesis, the binding of Gab1 to CrkII is required for the branching morphogenic program downstream of Met. Together, our data support a role for the CrkII adapter protein in epithelial invasion and morphogenesis and underscores the importance of considering the synergistic actions of signaling pathways in cancer progression.

DRhoGEF2 and Diaphanous Regulate Contractile Force during Segmental Groove Morphogenesis in the Drosophila Embryo

PubMed Central

Mulinari, Shai; Barmchi, Mojgan Padash

2008-01-01

Morphogenesis of the Drosophila embryo is associated with dynamic rearrangement of the actin cytoskeleton mediated by small GTPases of the Rho family. These GTPases act as molecular switches that are activated by guanine nucleotide exchange factors. One of these factors, DRhoGEF2, plays an important role in the constriction of actin filaments during pole cell formation, blastoderm cellularization, and invagination of the germ layers. Here, we show that DRhoGEF2 is equally important during morphogenesis of segmental grooves, which become apparent as tissue infoldings during mid-embryogenesis. Examination of DRhoGEF2-mutant embryos indicates a role for DRhoGEF2 in the control of cell shape changes during segmental groove morphogenesis. Overexpression of DRhoGEF2 in the ectoderm recruits myosin II to the cell cortex and induces cell contraction. At groove regression, DRhoGEF2 is enriched in cells posterior to the groove that undergo apical constriction, indicating that groove regression is an active process. We further show that the Formin Diaphanous is required for groove formation and strengthens cell junctions in the epidermis. Morphological analysis suggests that Dia regulates cell shape in a way distinct from DRhoGEF2. We propose that DRhoGEF2 acts through Rho1 to regulate acto-myosin constriction but not Diaphanous-mediated F-actin nucleation during segmental groove morphogenesis. PMID:18287521

Fibronectin Deposition Participates in Extracellular Matrix Assembly and Vascular Morphogenesis

PubMed Central

Hielscher, Abigail; Ellis, Kim; Qiu, Connie; Porterfield, Josh; Gerecht, Sharon

2016-01-01

The extracellular matrix (ECM) has been demonstrated to facilitate angiogenesis. In particular, fibronectin has been documented to activate endothelial cells, resulting in their transition from a quiescent state to an active state in which the cells exhibit enhanced migration and proliferation. The goal of this study is to examine the role of polymerized fibronectin during vascular tubulogenesis using a 3 dimensional (3D) cell-derived de-cellularized matrix. A fibronectin-rich 3D de-cellularized ECM was used as a scaffold to study vascular morphogenesis of endothelial cells (ECs). Confocal analyses of several matrix proteins reveal high intra- and extra-cellular deposition of fibronectin in formed vascular structures. Using a small peptide inhibitor of fibronectin polymerization, we demonstrate that inhibition of fibronectin fibrillogenesis in ECs cultured atop de-cellularized ECM resulted in decreased vascular morphogenesis. Further, immunofluorescence and ultrastructural analyses reveal decreased expression of stromal matrix proteins in the absence of polymerized fibronectin with high co-localization of matrix proteins found in association with polymerized fibronectin. Evaluating vascular kinetics, live cell imaging showed that migration, migration velocity, and mean square displacement, are disrupted in structures grown in the absence of polymerized fibronectin. Additionally, vascular organization failed to occur in the absence of a polymerized fibronectin matrix. Consistent with these observations, we tested vascular morphogenesis following the disruption of EC adhesion to polymerized fibronectin, demonstrating that block of integrins α5β1 and αvβ3, abrogated vascular morphogenesis. Overall, fibronectin deposition in a 3D cell-derived de-cellularized ECM appears to be imperative for matrix assembly and vascular morphogenesis. PMID:26811931

Tissue stiffening coordinates morphogenesis by triggering collective cell migration in vivo.

PubMed

Barriga, Elias H; Franze, Kristian; Charras, Guillaume; Mayor, Roberto

2018-02-22

Collective cell migration is essential for morphogenesis, tissue remodelling and cancer invasion. In vivo, groups of cells move in an orchestrated way through tissues. This movement involves mechanical as well as molecular interactions between cells and their environment. While the role of molecular signals in collective cell migration is comparatively well understood, how tissue mechanics influence collective cell migration in vivo remains unknown. Here we investigated the importance of mechanical cues in the collective migration of the Xenopus laevis neural crest cells, an embryonic cell population whose migratory behaviour has been likened to cancer invasion. We found that, during morphogenesis, the head mesoderm underlying the cephalic neural crest stiffens. This stiffening initiates an epithelial-to-mesenchymal transition in neural crest cells and triggers their collective migration. To detect changes in their mechanical environment, neural crest cells use mechanosensation mediated by the integrin-vinculin-talin complex. By performing mechanical and molecular manipulations, we show that mesoderm stiffening is necessary and sufficient to trigger neural crest migration. Finally, we demonstrate that convergent extension of the mesoderm, which starts during gastrulation, leads to increased mesoderm stiffness by increasing the cell density underneath the neural crest. These results show that convergent extension of the mesoderm has a role as a mechanical coordinator of morphogenesis, and reveal a link between two apparently unconnected processes-gastrulation and neural crest migration-via changes in tissue mechanics. Overall, we demonstrate that changes in substrate stiffness can trigger collective cell migration by promoting epithelial-to-mesenchymal transition in vivo. More broadly, our results raise the idea that tissue mechanics combines with molecular effectors to coordinate morphogenesis.

Differentiated roles for MreB-actin isologues and autolytic enzymes in Bacillus subtilis morphogenesis.

PubMed

Domínguez-Cuevas, Patricia; Porcelli, Ida; Daniel, Richard A; Errington, Jeff

2013-09-01

Cell morphogenesis in most bacteria is governed by spatiotemporal growth regulation of the peptidoglycan cell wall layer. Much is known about peptidoglycan synthesis but regulation of its turnover by hydrolytic enzymes is much less well understood. Bacillus subtilis has a multitude of such enzymes. Two of the best characterized are CwlO and LytE: cells lacking both enzymes have a lethal block in cell elongation. Here we show that activity of CwlO is regulated by an ABC transporter, FtsEX, which is required for cell elongation, unlike cell division as in Escherichia coli. Actin-like MreB proteins are thought to play a key role in orchestrating cell wall morphogenesis. B. subtilis has three MreB isologues with partially differentiated functions. We now show that the three MreB isologues have differential roles in regulation of the CwlO and LytE systems and that autolysins control different aspects of cell morphogenesis. The results add major autolytic activities to the growing list of functions controlled by MreB isologues in bacteria and provide new insights into the different specialized functions of essential cell wall autolysins. © 2013 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.

An early cytoplasmic step of peptidoglycan synthesis is associated to MreB in Bacillus subtilis.

PubMed

Rueff, Anne-Stéphanie; Chastanet, Arnaud; Domínguez-Escobar, Julia; Yao, Zhizhong; Yates, James; Prejean, Maria-Victoria; Delumeau, Olivier; Noirot, Philippe; Wedlich-Söldner, Roland; Filipe, Sergio R; Carballido-López, Rut

2014-01-01

MreB proteins play a major role during morphogenesis of rod-shaped bacteria by organizing biosynthesis of the peptidoglycan cell wall. However, the mechanisms underlying this process are not well understood. In Bacillus subtilis, membrane-associated MreB polymers have been shown to be associated to elongation-specific complexes containing transmembrane morphogenetic factors and extracellular cell wall assembly proteins. We have now found that an early intracellular step of cell wall synthesis is also associated to MreB. We show that the previously uncharacterized protein YkuR (renamed DapI) is required for synthesis of meso-diaminopimelate (m-DAP), an essential constituent of the peptidoglycan precursor, and that it physically interacts with MreB. Highly inclined laminated optical sheet microscopy revealed that YkuR forms uniformly distributed foci that exhibit fast motion in the cytoplasm, and are not detected in cells lacking MreB. We propose a model in which soluble MreB organizes intracellular steps of peptidoglycan synthesis in the cytoplasm to feed the membrane-associated cell wall synthesizing machineries. © 2013 John Wiley & Sons Ltd.

Distinct subsets of Eve-positive pericardial cells stabilise cardiac outflow and contribute to Hox gene-triggered heart morphogenesis in Drosophila.

PubMed

Zmojdzian, Monika; de Joussineau, Svetlana; Da Ponte, Jean Philippe; Jagla, Krzysztof

2018-01-17

The Drosophila heart, composed of discrete subsets of cardioblasts and pericardial cells, undergoes Hox-triggered anterior-posterior morphogenesis, leading to a functional subdivision into heart proper and aorta, with its most anterior part forming a funnel-shaped cardiac outflow. Cardioblasts differentiate into Tin-positive 'working myocytes' and Svp-expressing ostial cells. However, developmental fates and functions of heart-associated pericardial cells remain elusive. Here, we show that the pericardial cells that express the transcription factor Even Skipped adopt distinct fates along the anterior-posterior axis. Among them, the most anterior Antp-Ubx-AbdA - negative cells form a novel cardiac outflow component we call the outflow hanging structure, whereas the Antp-expressing cells differentiate into wing heart precursors. Interestingly, Hox gene expression in the Even Skipped-positive cells not only underlies their antero-posterior diversification, but also influences heart morphogenesis in a non-cell-autonomous way. In brief, we identify a new cardiac outflow component derived from a subset of Even Skipped-expressing cells that stabilises the anterior heart tip, and demonstrate non-cell-autonomous effects of Hox gene expression in the Even Skipped-positive cells on heart morphogenesis. © 2018. Published by The Company of Biologists Ltd.

Morphogenesis of the caenorhabditis elegans vulva.

PubMed

Schindler, Adam J; Sherwood, David R

2013-01-01

Understanding how cells move, change shape, and alter cellular behaviors to form organs, a process termed morphogenesis, is one of the great challenges of developmental biology. Formation of the Caenorhabditis elegans vulva is a powerful, simple, and experimentally accessible model for elucidating how morphogenetic processes produce an organ. In the first step of vulval development, three epithelial precursor cells divide and differentiate to generate 22 cells of 7 different vulval subtypes. The 22 vulval cells then rearrange from a linear array into a tube, with each of the seven cell types undergoing characteristic morphogenetic behaviors that construct the vulva. Vulval morphogenesis entails many of the same cellular activities that underlie organogenesis and tissue formation across species, including invagination, lumen formation, oriented cell divisions, cell–cell adhesion, cell migration, cell fusion, extracellular matrix remodeling, and cell invasion. Studies of vulval development have led to pioneering discoveries in a number of these processes and are beginning to bridge the gap between the pathways that specify cells and their connections to morphogenetic behaviors. The simplicity of the vulva and the experimental tools available in C. elegans will continue to make vulval morphogenesis a powerful paradigm to further our understanding of the largely mysterious mechanisms that build tissues and organs. © 2012 Wiley Periodicals, Inc.

Cell Cycle Dynamics and Quorum Sensing in Candida albicans Chlamydospores Are Distinct from Budding and Hyphal Growth

PubMed Central

Martin, Stephen W.; Douglas, Lois M.; Konopka, James B.

2005-01-01

The regulation of morphogenesis in the human fungal pathogen Candida albicans is under investigation to better understand how the switch between budding and hyphal growth is linked to virulence. Therefore, in this study we examined the ability of C. albicans to undergo a distinct type of morphogenesis to form large thick-walled chlamydospores whose role in infection is unclear, but they act as a resting form in other species. During chlamydospore morphogenesis, cells switch to filamentous growth and then develop elongated suspensor cells that give rise to chlamydospores. These filamentous cells were distinct from true hyphae in that they were wider and were not inhibited by the quorum-sensing factor farnesol. Instead, farnesol increased chlamydospore production, indicating that quorum sensing can also have a positive role. Nuclear division did not occur across the necks of chlamydospores, as it does in budding. Interestingly, nuclei divided within the suspensor cells, and then one daughter nucleus subsequently migrated into the chlamydospore. Septins were not detected near mitotic nuclei but were localized at chlamydospore necks. At later stages, septins localized throughout the chlamydospore plasma membrane and appeared to form long filamentous structures. Deletion of the CDC10 or CDC11 septins caused greater curvature of cells growing in a filamentous manner and morphological defects in suspensor cells and chlamydospores. These studies identify aspects of chlamydospore morphogenesis that are distinct from bud and hyphal morphogenesis. PMID:16002645

Altered tooth morphogenesis after silencing the planar cell polarity core component, Vangl2.

PubMed

Wu, Zhaoming; Epasinghe, Don Jeevanie; He, Jinquan; Li, Liwen; Green, David W; Lee, Min-Jung; Jung, Han-Sung

2016-12-01

Vangl2, one of the core components of the planar cell polarity (PCP) pathway, has an important role in the regulation of morphogenesis in several tissues. Although the expression of Vangl2 has been detected in the developing tooth, its role in tooth morphogenesis is not known. In this study, we show that Vangl2 is expressed in the inner dental epithelium (IDE) and in the secondary enamel knots (SEKs) of bell stage tooth germs. Inhibition of Vangl2 expression by siRNA treatment in in vitro-cultured tooth germs resulted in retarded tooth germ growth with deregulated cell proliferation and apoptosis. After kidney transplantation of Vangl2 siRNA-treated tooth germs, teeth were observed to be small and malformed. We also show that Vangl2 is required to maintain the proper pattern of cell alignment in SEKs, which maybe important for the function of SEKs as signaling centers. These results suggest that Vangl2 plays an important role in the morphogenesis of teeth.

Developmental expression of Hsp90, Hsp70 and HSF during morphogenesis in the vetigastropod Haliotis asinina.

PubMed

Gunter, Helen M; Degnan, Bernard M

2007-08-01

Heat shock proteins (Hsps) have dual functions, participating in both the stress response and a broad range of developmental processes. At physiological temperatures, it has been demonstrated in deuterostomes (vertebrates) and ecdysozoans (insects) that Hsps are expressed in tissues that are undergoing differentiation and morphogenesis. Here we investigate the developmental expression of Hsp70, Hsp90 and their regulatory transcription factor heat shock transcription factor (HSF) in the marine gastropod Haliotis asinina, a representative of the 3rd major lineage of bilaterian animals, the Lophotrochozoa. HasHsp70, HasHsp90 and HasHSF are maternally expressed in H. asinina and are progressively restricted to the micromere lineage during cleavage. During larval morphogenesis, they are expressed in unique and overlapping patterns in the prototroch, foot, and mantle. Hsp expression peaked in these tissues during periods of cell differentiation and morphogenesis, returning to lower levels after morphogenesis was complete. These patterns of Hsp and HSF expression in H. asinina are akin to those observed in ecdysozoans and deuterostomes, with Hsps being activated in cells and tissues undergoing morphogenesis.

Context clues: the importance of stem cell-material interactions

PubMed Central

Murphy, William L.

2014-01-01

Understanding the processes by which stem cells give rise to de novo tissues is an active focus of stem cell biology and bioengineering disciplines. Instructive morphogenic cues surrounding the stem cell during morphogenesis create what is referred to as the stem cell microenvironment. An emerging paradigm in stem cell bioengineering involves “biologically driven assembly,” in which stem cells are encouraged to largely define their own morphogenesis processes. However, even in the case of biologically driven assembly, stem cells do not act alone. The properties of the surrounding microenvironment can be critical regulators of cell fate. Stem cell-material interactions are among the most well-characterized microenvironmental effectors of stem cell fate, and they establish a signaling “context” that can define the mode of influence for morphogenic cues. Here we describe illustrative examples of cell-material interactions that occur during in vitro stem cell studies, with an emphasis on how cell-material interactions create instructive contexts for stem cell differentiation and morphogenesis. PMID:24369691

Ascidian notochord morphogenesis

PubMed Central

Jiang, Di; Smith, William C.

2010-01-01

The development of the notochord involves a complex set of cellular behaviors. While these morphogenic behaviors are common to all chordates, the ascidian provides a particularly attractive experimental model because of its relative simplicity. In particular, all notochord morphogenesis in ascidians takes place with only 40 cells, as opposed to the hundreds of cells in vertebrate models systems. Initial steps in ascidian notochord development convert a monolayer of epithelial-like cells in the pre-gastrula embryo to a cylindrical rod of single-cell diameter. Convergent extension is responsible for the intercalation of notochord cells and some degree of notochord elongation, while a second phase of elongation is observed as the notochord narrows medially and increases in volume. The mechanism by which the volume of the notochord increases differs between ascidian species. Some ascidian species produce extracellular pockets that will eventually coalesce to form a lumen running the length of the notochord, while others appear to make intercellular vacuoles. By either mechanism, the resulting notochord serves as a hydrostatic skeleton allowing for the locomotion of the swimming larva. Several basic cell behaviors, such as cell shape changes, cell rearrangement, establishment of cell polarity, and alteration of extracellular environment, are displayed in the process of notochord morphogenesis. Modern analysis of ascidian notochord morphogenesis promises to contribute to our understanding of these fundamental biological processes. PMID:17497687

Ascidian notochord morphogenesis.

PubMed

Jiang, Di; Smith, William C

2007-07-01

The development of the notochord involves a complex set of cellular behaviors. While these morphogenic behaviors are common to all chordates, the ascidian provides a particularly attractive experimental model because of its relative simplicity. In particular, all notochord morphogenesis in ascidians takes place with only 40 cells, as opposed to the hundreds of cells in vertebrate model systems. Initial steps in ascidian notochord development convert a monolayer of epithelial-like cells in the pregastrula embryo to a cylindrical rod of single-cell diameter. Convergent extension is responsible for the intercalation of notochord cells and some degree of notochord elongation, while a second phase of elongation is observed as the notochord narrows medially and increases in volume. The mechanism by which the volume of the notochord increases differs between ascidian species. Some ascidians produce extracellular pockets that will eventually coalesce to form a lumen running the length of the notochord; whereas others do not. By either mechanism, the resulting notochord serves as a hydrostatic skeleton allowing for the locomotion of the swimming larva. Several basic cell behaviors, such as cell shape changes, cell rearrangement, establishment of cell polarity, and alteration of extracellular environment, are displayed in the process of notochord morphogenesis. Modern analysis of ascidian notochord morphogenesis promises to contribute to our understanding of these fundamental biological processes. Copyright 2007 Wiley-Liss, Inc.

Foregut separation and tracheo-oesophageal malformations: The role of tracheal outgrowth, dorso-ventral patterning and programmed cell death

PubMed Central

Ioannides, Adonis S.; Massa, Valentina; Ferraro, Elisabetta; Cecconi, Francesco; Spitz, Lewis; Henderson, Deborah J.; Copp, Andrew J.

2010-01-01

Foregut division—the separation of dorsal (oesophageal) from ventral (tracheal) foregut components—is a crucial event in gastro-respiratory development, and frequently disturbed in clinical birth defects. Here, we examined three outstanding questions of foregut morphogenesis. The origin of the trachea is suggested to result either from respiratory outgrowth or progressive septation of the foregut tube. We found normal foregut lengthening despite failure of tracheo-oesophageal separation in Adriamycin-treated embryos, whereas active septation was observed only in normal foregut morphogenesis, indicating a primary role for septation. Dorso-ventral patterning of Nkx2.1 (ventral) and Sox2 (dorsal) expression is proposed to be critical for tracheo-oesophageal separation. However, normal dorso-ventral patterning of Nkx2.1 and Sox2 expression occurred in Adriamycin-treated embryos with defective foregut separation. In contrast, Shh expression shifts dynamically, ventral-to-dorsal, solely during normal morphogenesis, particularly implicating Shh in foregut morphogenesis. Dying cells localise to the fusing foregut epithelial ridges, with disturbance of this apoptotic pattern in Adriamycin, Shh and Nkx2.1 models. Strikingly, however, genetic suppression of apoptosis in the Apaf1 mutant did not prevent foregut separation, indicating that apoptosis is not required for tracheo-oesophageal morphogenesis. Epithelial remodelling during septation may cause loss of cell-cell or cell-matrix interactions, resulting in apoptosis (anoikis) as a secondary consequence. PMID:19913007

Tissue architecture, cell traction, deformable scaffolds, and the forces that shape the embryo during morphogenesis.

NASA Astrophysics Data System (ADS)

Davidson, Lance

2005-03-01

Morphogenesis is the process of constucting form and shape. Morphogenesis during early development of the embryo involves orchestrated movements of cells and tissues. These morphogenetic movements establish the body plan and organs of the early embryo. The rates and trajectories of these movements depend on three physical features of the early embryo: 1) the forces generated by cells, 2) the mechanical properties of the tissues, and 3) the architecture of the tissues. These three mechanical features of the embryo are some of the earliest phenotypic features generated by the genome. We are taking an interdisciplinary approach combining biophysical, cell biological, and classical embryological techniques to understand the mechanics of morphogenesis. Using nanoNewton-sensitive force transducers we can apply forces and measure time dependent elastic modulii of tissue fragments 100 micrometers across. Using traction-force microscopy we can measure forces generated by cells on their environment. We use drugs and chimeric proteins to investigate the localization and function of molecular complexes responsible for force generation and the modulus. We use microsurgery to take-apart and construct novel tissues to investigate the role of geometry and architecture in the mechanics of morphogenesis. Together with simulation techniques these quantitative approaches will provide us with a practical nuts-and-bolts understanding of how the genome encodes the shapes and forms of life.

Reverse engineering the mechanical and molecular pathways in stem cell morphogenesis.

PubMed

Lu, Kai; Gordon, Richard; Cao, Tong

2015-03-01

The formation of relevant biological structures poses a challenge for regenerative medicine. During embryogenesis, embryonic cells differentiate into somatic tissues and undergo morphogenesis to produce three-dimensional organs. Using stem cells, we can recapitulate this process and create biological constructs for therapeutic transplantation. However, imperfect imitation of nature sometimes results in in vitro artifacts that fail to recapitulate the function of native organs. It has been hypothesized that developing cells may self-organize into tissue-specific structures given a correct in vitro environment. This proposition is supported by the generation of neo-organoids from stem cells. We suggest that morphogenesis may be reverse engineered to uncover its interacting mechanical pathway and molecular circuitry. By harnessing the latent architecture of stem cells, novel tissue-engineering strategies may be conceptualized for generating self-organizing transplants. Copyright © 2013 John Wiley & Sons, Ltd.

MicroRNA miR-328 Regulates Zonation Morphogenesis by Targeting CD44 Expression

PubMed Central

Wang, Chia-Hui; Lee, Daniel Y.; Deng, Zhaoqun; Jeyapalan, Zina; Lee, Shao-Chen; Kahai, Shireen; Lu, Wei-Yang; Zhang, Yaou; Yang, Burton B.

2008-01-01

Morphogenesis is crucial to initiate physiological development and tumor invasion. Here we show that a microRNA controls zonation morphogenesis by targeting hyaluronan receptor CD44. We have developed a novel system to study microRNA functions by generating constructs expressing pre-miRNAs and mature miRNAs. Using this system, we have demonstrated that expression of miR-328 reduced cell adhesion, aggregation, and migration, and regulated formation of capillary structure. Protein analysis indicated that miR-328 repressed CD44 expression. Activities of luciferase constructs harboring the target site in CD44, but not the one containing mutation, were repressed by miR-328. Zonation morphogenesis appeared in cells transfected by miR-328: miR-328-transfected cells were present on the surface of zonating structures while the control cells stayed in the middle. MiR-328-mediated CD44 actions was validated by anti-CD44 antibody, hyaluronidase, CD44 siRNA, and CD44 expression constructs. In vivo experiments showed that CD44-silencing cells appeared as layers on the surfaces of nodules or zonating structures. Immuno-histochemistry also exhibited CD44-negative cells on the surface layers of normal rat livers and the internal zones of Portal veins. Our results demonstrate that miR-328 targets CD44, which is essential in regulating zonation morphogenesis: silencing of CD44 expression is essential in sealing the zonation structures to facilitate their extension and to inhibit complex expansion. PMID:18560585

MicroRNA miR-328 regulates zonation morphogenesis by targeting CD44 expression.

PubMed

Wang, Chia-Hui; Lee, Daniel Y; Deng, Zhaoqun; Jeyapalan, Zina; Lee, Shao-Chen; Kahai, Shireen; Lu, Wei-Yang; Zhang, Yaou; Yang, Burton B

2008-06-18

Morphogenesis is crucial to initiate physiological development and tumor invasion. Here we show that a microRNA controls zonation morphogenesis by targeting hyaluronan receptor CD44. We have developed a novel system to study microRNA functions by generating constructs expressing pre-miRNAs and mature miRNAs. Using this system, we have demonstrated that expression of miR-328 reduced cell adhesion, aggregation, and migration, and regulated formation of capillary structure. Protein analysis indicated that miR-328 repressed CD44 expression. Activities of luciferase constructs harboring the target site in CD44, but not the one containing mutation, were repressed by miR-328. Zonation morphogenesis appeared in cells transfected by miR-328: miR-328-transfected cells were present on the surface of zonating structures while the control cells stayed in the middle. MiR-328-mediated CD44 actions was validated by anti-CD44 antibody, hyaluronidase, CD44 siRNA, and CD44 expression constructs. In vivo experiments showed that CD44-silencing cells appeared as layers on the surfaces of nodules or zonating structures. Immuno-histochemistry also exhibited CD44-negative cells on the surface layers of normal rat livers and the internal zones of Portal veins. Our results demonstrate that miR-328 targets CD44, which is essential in regulating zonation morphogenesis: silencing of CD44 expression is essential in sealing the zonation structures to facilitate their extension and to inhibit complex expansion.

Chiral cell sliding drives left-right asymmetric organ twisting

PubMed Central

Inaki, Mikiko; Hatori, Ryo; Nakazawa, Naotaka; Okumura, Takashi; Ishibashi, Tomoki; Kikuta, Junichi; Ishii, Masaru

2018-01-01

Polarized epithelial morphogenesis is an essential process in animal development. While this process is mostly attributed to directional cell intercalation, it can also be induced by other mechanisms. Using live-imaging analysis and a three-dimensional vertex model, we identified ‘cell sliding,’ a novel mechanism driving epithelial morphogenesis, in which cells directionally change their position relative to their subjacent (posterior) neighbors by sliding in one direction. In Drosophila embryonic hindgut, an initial left-right (LR) asymmetry of the cell shape (cell chirality in three dimensions), which occurs intrinsically before tissue deformation, is converted through LR asymmetric cell sliding into a directional axial twisting of the epithelial tube. In a Drosophila inversion mutant showing inverted cell chirality and hindgut rotation, cell sliding occurs in the opposite direction to that in wild-type. Unlike directional cell intercalation, cell sliding does not require junctional remodeling. Cell sliding may also be involved in other cases of LR-polarized epithelial morphogenesis. PMID:29891026

Chiral cell sliding drives left-right asymmetric organ twisting.

PubMed

Inaki, Mikiko; Hatori, Ryo; Nakazawa, Naotaka; Okumura, Takashi; Ishibashi, Tomoki; Kikuta, Junichi; Ishii, Masaru; Matsuno, Kenji; Honda, Hisao

2018-06-12

Polarized epithelial morphogenesis is an essential process in animal development. While this process is mostly attributed to directional cell intercalation, it can also be induced by other mechanisms. Using live-imaging analysis and a three-dimensional vertex model, we identified 'cell sliding,' a novel mechanism driving epithelial morphogenesis, in which cells directionally change their position relative to their subjacent (posterior) neighbors by sliding in one direction. In Drosophila embryonic hindgut, an initial left-right (LR) asymmetry of the cell shape (cell chirality in three dimensions), which occurs intrinsically before tissue deformation, is converted through LR asymmetric cell sliding into a directional axial twisting of the epithelial tube. In a Drosophila inversion mutant showing inverted cell chirality and hindgut rotation, cell sliding occurs in the opposite direction to that in wild-type. Unlike directional cell intercalation, cell sliding does not require junctional remodeling. Cell sliding may also be involved in other cases of LR-polarized epithelial morphogenesis. © 2018, Inaki et al.

Lamellipodia-based migrations of larval epithelial cells are required for normal closure of the adult epidermis of Drosophila

PubMed Central

Bischoff, Marcus

2012-01-01

Cell migrations are an important feature of animal development. They are, furthermore, essential to wound healing and tumour progression. Despite recent progress, it is still mysterious how cell migration is spatially and temporally regulated during morphogenesis and how cell migration is coordinated with other cellular behaviours to shape tissues and organs. The formation of the abdominal epithelium of Drosophila during metamorphosis provides an attractive system to study morphogenesis. Here, the diploid adult histoblasts replace the polyploid larval epithelial cells (LECs). Using in vivo 4D microscopy, I show that, besides apical constriction and apoptosis, the LECs undergo extensive coordinated migrations. The migrations follow a transition from a stationary (epithelial) to a migratory mode. The migratory behaviour is stimulated by autocrine Dpp signalling. Directed apical lamellipodia-like protrusions propel the cells. Initially, planar cell polarity determines the orientation of LEC migration. While LECs are migrating they also constrict apically, and changes in activity of the small GTPase Rho1 can favour one behaviour over the other. This study shows that the LECs play a more active role in morphogenesis than previously thought, with their migrations contributing to abdominal closure. It furthermore provides insights into how the migratory behaviour of cells is regulated during morphogenesis. PMID:22230614

Assembly of embryonic and extraembryonic stem cells to mimic embryogenesis in vitro.

PubMed

Harrison, Sarah Ellys; Sozen, Berna; Christodoulou, Neophytos; Kyprianou, Christos; Zernicka-Goetz, Magdalena

2017-04-14

Mammalian embryogenesis requires intricate interactions between embryonic and extraembryonic tissues to orchestrate and coordinate morphogenesis with changes in developmental potential. Here, we combined mouse embryonic stem cells (ESCs) and extraembryonic trophoblast stem cells (TSCs) in a three-dimensional scaffold to generate structures whose morphogenesis is markedly similar to that of natural embryos. By using genetically modified stem cells and specific inhibitors, we show that embryogenesis of ESC- and TSC-derived embryos-ETS-embryos-depends on cross-talk involving Nodal signaling. When ETS-embryos develop, they spontaneously initiate expression of mesoderm and primordial germ cell markers asymmetrically on the embryonic and extraembryonic border, in response to Wnt and BMP signaling. Our study demonstrates the ability of distinct stem cell types to self-assemble in vitro to generate embryos whose morphogenesis, architecture, and constituent cell types resemble those of natural embryos. Copyright © 2017, American Association for the Advancement of Science.

Genetic Interaction of Centrosomin and Bazooka in Apical Domain Regulation in Drosophila Photoreceptor

PubMed Central

Chen, Geng; Rogers, Alicia K.; League, Garrett P.; Nam, Sang-Chul

2011-01-01

Background Cell polarity genes including Crumbs (Crb) and Par complexes are essential for controlling photoreceptor morphogenesis. Among the Crb and Par complexes, Bazooka (Baz, Par-3 homolog) acts as a nodal component for other cell polarity proteins. Therefore, finding other genes interacting with Baz will help us to understand the cell polarity genes' role in photoreceptor morphogenesis. Methodology/Principal Findings Here, we have found a genetic interaction between baz and centrosomin (cnn). Cnn is a core protein for centrosome which is a major microtubule-organizing center. We analyzed the effect of the cnn mutation on developing eyes to determine its role in photoreceptor morphogenesis. We found that Cnn is dispensable for retinal differentiation in eye imaginal discs during the larval stage. However, photoreceptors deficient in Cnn display dramatic morphogenesis defects including the mislocalization of Crumbs (Crb) and Bazooka (Baz) during mid-stage pupal eye development, suggesting that Cnn is specifically required for photoreceptor morphogenesis during pupal eye development. This role of Cnn in apical domain modulation was further supported by Cnn's gain-of-function phenotype. Cnn overexpression in photoreceptors caused the expansion of the apical Crb membrane domain, Baz and adherens junctions (AJs). Conclusions/Significance These results strongly suggest that the interaction of Baz and Cnn is essential for apical domain and AJ modulation during photoreceptor morphogenesis, but not for the initial photoreceptor differentiation in the Drosophila photoreceptor. PMID:21253601

Eph/Ephrin signalling maintains eye field segregation from adjacent neural plate territories during forebrain morphogenesis

PubMed Central

Cavodeassi, Florencia; Ivanovitch, Kenzo; Wilson, Stephen W.

2013-01-01

During forebrain morphogenesis, there is extensive reorganisation of the cells destined to form the eyes, telencephalon and diencephalon. Little is known about the molecular mechanisms that regulate region-specific behaviours and that maintain the coherence of cell populations undergoing specific morphogenetic processes. In this study, we show that the activity of the Eph/Ephrin signalling pathway maintains segregation between the prospective eyes and adjacent regions of the anterior neural plate during the early stages of forebrain morphogenesis in zebrafish. Several Ephrins and Ephs are expressed in complementary domains in the prospective forebrain and combinatorial abrogation of their activity results in incomplete segregation of the eyes and telencephalon and in defective evagination of the optic vesicles. Conversely, expression of exogenous Ephs or Ephrins in regions of the prospective forebrain where they are not usually expressed changes the adhesion properties of the cells, resulting in segregation to the wrong domain without changing their regional fate. The failure of eye morphogenesis in rx3 mutants is accompanied by a loss of complementary expression of Ephs and Ephrins, suggesting that this pathway is activated downstream of the regional fate specification machinery to establish boundaries between domains undergoing different programmes of morphogenesis. PMID:24026122

The polarity protein partitioning-defective 1 (PAR-1) regulates dendritic spine morphogenesis through phosphorylating postsynaptic density protein 95 (PSD-95).

PubMed

Wu, Qian; DiBona, Victoria L; Bernard, Laura P; Zhang, Huaye

2012-08-31

The polarity protein PAR-1 plays an essential role in many cellular contexts, including embryogenesis, asymmetric cell division, directional migration, and epithelial morphogenesis. Despite its known importance in different cellular processes, the role of PAR-1 in neuronal morphogenesis is less well understood. In particular, its role in the morphogenesis of dendritic spines, which are sites of excitatory synaptic inputs, has been unclear. Here, we show that PAR-1 is required for normal spine morphogenesis in hippocampal neurons. We further show that PAR-1 functions through phosphorylating the synaptic scaffolding protein PSD-95 in this process. Phosphorylation at a conserved serine residue in the KXGS motif in PSD-95 regulates spine morphogenesis, and a phosphomimetic mutant of this site can rescue the defects of kinase-dead PAR-1. Together, our findings uncover a role of PAR-1 in spine morphogenesis in hippocampal neurons through phosphorylating PSD-95.

The world of epithelial sheets.

PubMed

Honda, Hisao

2017-06-01

An epithelium is a layer of closely connected cells covering the body or lining a body cavity. In this review, several fundamental questions are addressed regarding the epithelium. (i) While an epithelium functions as barrier against the external environment, how is barrier function maintained during its construction? (ii) What determines the apical and basal sides of epithelial layer? (iii) Is there any relationship between the apical side of the epithelium and the apical membrane of an epithelial cell? (iv) Why are hepatocytes (liver cells) called epithelial, even though they differ completely from column-like shape of typical epithelial cells? Keeping these questions in mind, multiple shapes of epithelia were considered, extracting a few of their elemental processes, and constructing a virtual world of epithelia by combining them. Epithelial cells were also classified into several types based on the number of apical domains of each cell. In addition, an intracellular organelle was introduced within epithelial cells, the vacuolar apical compartment (VAC), which is produced within epithelial cells surrounded by external cell matrix (ECM). The VAC interacts with areas of cell-cell contact of the cell surface membrane and is converted to apical membrane. The properties of VACs enable us to answer the initial questions posed above. Finally, the genetic and molecular mechanisms of epithelial morphogenesis are discussed. © 2017 Japanese Society of Developmental Biologists.

Quantification of shape and cell polarity reveals a novel mechanism underlying malformations resulting from related FGF mutations during facial morphogenesis

PubMed Central

Li, Xin; Young, Nathan M.; Tropp, Stephen; Hu, Diane; Xu, Yanhua; Hallgrímsson, Benedikt; Marcucio, Ralph S.

2013-01-01

Fibroblast growth factor (FGF) signaling mutations are a frequent contributor to craniofacial malformations including midfacial anomalies and craniosynostosis. FGF signaling has been shown to control cellular mechanisms that contribute to facial morphogenesis and growth such as proliferation, survival, migration and differentiation. We hypothesized that FGF signaling not only controls the magnitude of growth during facial morphogenesis but also regulates the direction of growth via cell polarity. To test this idea, we infected migrating neural crest cells of chicken embryos with  replication-competent avian sarcoma virus expressing either FgfR2C278F, a receptor mutation found in Crouzon syndrome or the ligand Fgf8. Treated embryos exhibited craniofacial malformations resembling facial dysmorphologies in craniosynostosis syndrome. Consistent with our hypothesis, ectopic activation of FGF signaling resulted in decreased cell proliferation, increased expression of the Sprouty class of FGF signaling inhibitors, and repressed phosphorylation of ERK/MAPK. Furthermore, quantification of cell polarity in facial mesenchymal cells showed that while orientation of the Golgi body matches the direction of facial prominence outgrowth in normal cells, in FGF-treated embryos this direction is randomized, consistent with aberrant growth that we observed. Together, these data demonstrate that FGF signaling regulates cell proliferation and cell polarity and that these cell processes contribute to facial morphogenesis. PMID:23906837

Luminal mitosis drives epithelial cell dispersal within the branching ureteric bud

PubMed Central

Packard, Adam; Georgas, Kylie; Michos, Odyssé; Riccio, Paul; Cebrian, Cristina; Combes, Alexander N.; Ju, Adler; Ferrer-Vaquer, Anna; Hadjantonakis, Anna-Katerina; Zong, Hui; Little, Melissa H.; Costantini, Frank

2013-01-01

Summary The ureteric bud is an epithelial tube that undergoes branching morphogenesis to form the renal collecting system. Though development of a normal kidney depends on proper ureteric bud morphogenesis, the cellular events underlying this process remain obscure. Here, we used time-lapse microscopy together with several genetic labeling methods to observe ureteric bud cell behaviors in developing mouse kidneys. We observed an unexpected cell behavior in the branching tips of the ureteric bud, which we term “mitosis-associated cell dispersal”. Pre-mitotic ureteric tip cells delaminate from the epithelium and divide within the lumen; while one daughter cell retains a basal process, allowing it to reinsert into the epithelium at the site of origin, the other daughter cell reinserts at a position one to three cell diameters away. Given the high rate of cell division in ureteric tips, this cellular behavior causes extensive epithelial cell rearrangements that may contribute to renal branching morphogenesis. PMID:24183650

Melatonin Inhibits Embryonic Salivary Gland Branching Morphogenesis by Regulating Both Epithelial Cell Adhesion and Morphology

PubMed Central

Miura, Jiro; Sakai, Manabu; Uchida, Hitoshi; Nakamura, Wataru; Nohara, Kanji; Maruyama, Yusuke; Hattori, Atsuhiko; Sakai, Takayoshi

2015-01-01

Many organs, including salivary glands, lung, and kidney, are formed by epithelial branching during embryonic development. Branching morphogenesis occurs via either local outgrowths or the formation of clefts that subdivide epithelia into buds. This process is promoted by various factors, but the mechanism of branching morphogenesis is not fully understood. Here we have defined melatonin as a potential negative regulator or “brake” of branching morphogenesis, shown that the levels of it and its receptors decline when branching morphogenesis begins, and identified the process that it regulates. Melatonin has various physiological functions, including circadian rhythm regulation, free-radical scavenging, and gonadal development. Furthermore, melatonin is present in saliva and may have an important physiological role in the oral cavity. In this study, we found that the melatonin receptor is highly expressed on the acinar epithelium of the embryonic submandibular gland. We also found that exogenous melatonin reduces salivary gland size and inhibits branching morphogenesis. We suggest that this inhibition does not depend on changes in either proliferation or apoptosis, but rather relates to changes in epithelial cell adhesion and morphology. In summary, we have demonstrated a novel function of melatonin in organ formation during embryonic development. PMID:25876057

Purification of fetal mouse hepatoblasts by magnetic beads coated with monoclonal anti-e-cadherin antibodies and their in vitro culture.

PubMed

Nitou, Miho; Sugiyama, Yoshinori; Ishikawa, Katsutoshi; Shiojiri, Nobuyoshi

2002-10-01

A simple, rapid, and reproducible method of fetal hepatoblast purification was established to investigate mechanisms controlling interactions between hepatoblasts and nonparenchymal cells during liver development. Because E-cadherin is exclusively expressed on the cell membrane of hepatoblasts, magnetic beads coated with monoclonal antibodies to an extracellular epitope of its molecule were used to purify hepatoblasts from a cell suspension prepared from 12.5-day fetal mouse livers. The purity and yield in the hepatoblast fraction prepared in our protocol were more than 90% and approximately 30%, respectively. The nonparenchymal fraction rarely contained hepatoblasts; the rate of hepatoblast contamination in this fraction was less than 1%. Separate cultures of these two fractions were compared with cocultures of both fractions. In culture of the hepatoblast fraction, hepatoblasts formed aggregates similar to a bunch of grapes via their loose adhesion, floating in the medium after 24 h, and dissociated into single cells from the aggregates after 120 h of culture. By contrast, in the mixed culture, the majority of hepatoblasts formed multicellular spheroids after 24 h, and these spheroids changed into monolayer cell sheets after 120 h of culture. The cells comprising these monolayer sheets abundantly expressed albumin and carbamoylphosphate synthase I. In the mixed culture, fibroblastic cells also proliferated extensively with spreading on glass slides and surrounded the hepatoblast or hepatocyte colonies. On the other hand, fibroblastic cells spreading on glass slides decreased gradually in cultures of the nonparenchymal cell fraction alone. These findings indicated that the coexistence of hepatoblasts and nonparenchymal cells may be essential for their mutual survival, proliferation, differentiation, and morphogenesis. The conditioned medium of fetal liver cell cultures could partially replace the effects of the nonparenchymal cells on hepatoblasts in vitro. Our isolation protocol for fetal mouse hepatoblasts using immunobeads can greatly facilitate studies on mechanisms of cell-cell interactions during liver development.

klf2a couples mechanotransduction and zebrafish valve morphogenesis through fibronectin synthesis

PubMed Central

Steed, Emily; Faggianelli, Nathalie; Roth, Stéphane; Ramspacher, Caroline; Concordet, Jean-Paul; Vermot, Julien

2016-01-01

The heartbeat and blood flow signal to endocardial cell progenitors through mechanosensitive proteins that modulate the genetic program controlling heart valve morphogenesis. To date, the mechanism by which mechanical forces coordinate tissue morphogenesis is poorly understood. Here we use high-resolution imaging to uncover the coordinated cell behaviours leading to heart valve formation. We find that heart valves originate from progenitors located in the ventricle and atrium that generate the valve leaflets through a coordinated set of endocardial tissue movements. Gene profiling analyses and live imaging reveal that this reorganization is dependent on extracellular matrix proteins, in particular on the expression of fibronectin1b. We show that blood flow and klf2a, a major endocardial flow-responsive gene, control these cell behaviours and fibronectin1b synthesis. Our results uncover a unique multicellular layering process leading to leaflet formation and demonstrate that endocardial mechanotransduction and valve morphogenesis are coupled via cellular rearrangements mediated by fibronectin synthesis. PMID:27221222

Mechanical models for the self-organization of tubular patterns.

PubMed

Guo, Chin-Lin

2013-01-01

Organogenesis, such as long tubule self-organization, requires long-range coordination of cell mechanics to arrange cell positions and to remodel the extracellular matrix. While the current mainstream in the field of tissue morphogenesis focuses primarily on genetics and chemical signaling, the influence of cell mechanics on the programming of patterning cues in tissue morphogenesis has not been adequately addressed. Here, we review experimental evidence and propose quantitative mechanical models by which cells can create tubular patterns.

Solo and keratin filaments regulate epithelial tubule morphology.

PubMed

Nishimura, Ryosuke; Kato, Kagayaki; Fujiwara, Sachiko; Ohashi, Kazumasa; Mizuno, Kensaku

2018-04-28

Epithelial tubules, consisting of the epithelial cell sheet with a central lumen, are the basic structure of many organs. Mechanical forces play an important role in epithelial tubulogenesis; however, little is known about the mechanisms controlling the mechanical forces during epithelial tubule morphogenesis. Solo (also known as ARHGEF40) is a RhoA-targeting guanine-nucleotide exchange factor that is involved in mechanical force-induced RhoA activation and stress fiber formation. Solo binds to keratin-8/keratin-18 (K8/K18) filaments, and this interaction plays a crucial role in mechanotransduction. In this study, we examined the roles of Solo and K8/K18 filaments in epithelial tubulogenesis using MDCK cells cultured in 3D collagen gels. Knockdown of either Solo or K18 resulted in rounder tubules with increased lumen size, indicating that Solo and K8/K18 filaments play critical roles in forming the elongated morphology of epithelial tubules. Moreover, knockdown of Solo or K18 decreased the level of diphosphorylated myosin light chain (a marker of contractile force) at the luminal and outer surfaces of tubules, suggesting that Solo and K8/K18 filaments are involved in the generation of the myosin II-mediated contractile force during epithelial tubule morphogenesis. In addition, K18 filaments were normally oriented along the long axis of the tubule, but knockdown of Solo perturbed their orientation. These results suggest that Solo plays crucial roles in forming the elongated morphology of epithelial tubules and in regulating myosin II activity and K18 filament organization during epithelial tubule formation.

Alteration in the ultrastructural morphology of mycelial hyphae and the dynamics of transcriptional activity of lytic enzyme genes during basidiomycete morphogenesis.

PubMed

Vetchinkina, Elena; Kupryashina, Maria; Gorshkov, Vladimir; Ageeva, Marina; Gogolev, Yuri; Nikitina, Valentina

2017-04-01

The morphogenesis of macromycetes is a complex multilevel process resulting in a set of molecular-genetic, physiological-biochemical, and morphological-ultrastructural changes in the cells. When the xylotrophic basidiomycetes Lentinus edodes, Grifola frondosa, and Ganoderma lucidum were grown on wood waste as the substrate, the ultrastructural morphology of the mycelial hyphal cell walls differed considerably between mycelium and morphostructures. As the macromycetes passed from vegetative to generative development, the expression of the tyr1, tyr2, chi1, chi2, exg1, exg2, and exg3 genes was activated. These genes encode enzymes such as tyrosinase, chitinase, and glucanase, which play essential roles in cell wall growth and morphogenesis.

Polarized protein transport and lumen formation during epithelial tissue morphogenesis.

PubMed

Blasky, Alex J; Mangan, Anthony; Prekeris, Rytis

2015-01-01

One of the major challenges in biology is to explain how complex tissues and organs arise from the collective action of individual polarized cells. The best-studied model of this process is the cross talk between individual epithelial cells during their polarization to form the multicellular epithelial lumen during tissue morphogenesis. Multiple mechanisms of apical lumen formation have been proposed. Some epithelial lumens form from preexisting polarized epithelial structures. However, de novo lumen formation from nonpolarized cells has recently emerged as an important driver of epithelial tissue morphogenesis, especially during the formation of small epithelial tubule networks. In this review, we discuss the latest findings regarding the mechanisms and regulation of de novo lumen formation in vitro and in vivo.

From The Cover: Reconstruction of functionally normal and malignant human breast tissues in mice

NASA Astrophysics Data System (ADS)

Kuperwasser, Charlotte; Chavarria, Tony; Wu, Min; Magrane, Greg; Gray, Joe W.; Carey, Loucinda; Richardson, Andrea; Weinberg, Robert A.

2004-04-01

The study of normal breast epithelial morphogenesis and carcinogenesis in vivo has largely used rodent models. Efforts at studying mammary morphogenesis and cancer with xenotransplanted human epithelial cells have failed to recapitulate the full extent of development seen in the human breast. We have developed an orthotopic xenograft model in which both the stromal and epithelial components of the reconstructed mammary gland are of human origin. Genetic modification of human stromal cells before the implantation of ostensibly normal human mammary epithelial cells resulted in the outgrowth of benign and malignant lesions. This experimental model allows for studies of human epithelial morphogenesis and differentiation in vivo and underscores the critical role of heterotypic interactions in human breast development and carcinogenesis.

Ciona intestinalis notochord as a new model to investigate the cellular and molecular mechanisms of tubulogenesis.

PubMed

Denker, Elsa; Jiang, Di

2012-05-01

Biological tubes are a prevalent structural design across living organisms. They provide essential functions during the development and adult life of an organism. Increasing progress has been made recently in delineating the cellular and molecular mechanisms underlying tubulogenesis. This review aims to introduce ascidian notochord morphogenesis as an interesting model system to study the cell biology of tube formation, to a wider cell and developmental biology community. We present fundamental morphological and cellular events involved in notochord morphogenesis, compare and contrast them with other more established tubulogenesis model systems, and point out some unique features, including bipolarity of the notochord cells, and using cell shape changes and cell rearrangement to connect lumens. We highlight some initial findings in the molecular mechanisms of notochord morphogenesis. Based on these findings, we present intriguing problems and put forth hypotheses that can be addressed in future studies. Copyright © 2012 Elsevier Ltd. All rights reserved.

The heart tube forms and elongates through dynamic cell rearrangement coordinated with foregut extension.

PubMed

Kidokoro, Hinako; Yonei-Tamura, Sayuri; Tamura, Koji; Schoenwolf, Gary C; Saijoh, Yukio

2018-03-29

In the initiation of cardiogenesis, the heart primordia transform from bilateral flat sheets of mesoderm into an elongated midline tube. Here, we discover that this rapid architectural change is driven by actomyosin-based oriented cell rearrangement and resulting dynamic tissue reshaping (convergent extension, CE). By labeling clusters of cells spanning the entire heart primordia, we show that the heart primordia converge toward the midline to form a narrow tube, while extending perpendicularly to rapidly lengthen it. Our data for the first time visualize the process of early heart tube formation from both the medial (second) and lateral (first) heart fields, revealing that both fields form the early heart tube by essentially the same mechanism. Additionally, the adjacent endoderm coordinately forms the foregut through previously unrecognized movements that parallel those of the heart mesoderm and elongates by CE. In conclusion, our data illustrate how initially two-dimensional flat primordia rapidly change their shapes and construct the three-dimensional morphology of emerging organs in coordination with neighboring morphogenesis. © 2018. Published by The Company of Biologists Ltd.

Regulation of polarized morphogenesis by protein kinase C iota in oncogenic epithelial spheroids.

PubMed

Linch, Mark; Sanz-Garcia, Marta; Rosse, Carine; Riou, Philippe; Peel, Nick; Madsen, Chris D; Sahai, Erik; Downward, Julian; Khwaja, Asim; Dillon, Christian; Roffey, Jon; Cameron, Angus J M; Parker, Peter J

2014-02-01

Protein kinase C iota (PKCι), a serine/threonine kinase required for cell polarity, proliferation and migration, is commonly up- or downregulated in cancer. PKCι is a human oncogene but whether this is related to its role in cell polarity and what repertoire of oncogenes acts in concert with PKCι is not known. We developed a panel of candidate oncogene expressing Madin-Darby canine kidney (MDCK) cells and demonstrated that H-Ras, ErbB2 and phosphatidylinositol 3-kinase transformation led to non-polar spheroid morphogenesis (dysplasia), whereas MDCK spheroids expressing c-Raf or v-Src were largely polarized. We show that small interfering RNA (siRNA)-targeting PKCι decreased the size of all spheroids tested and partially reversed the aberrant polarity phenotype in H-Ras and ErbB2 spheroids only. This indicates distinct requirements for PKCι and moreover that different thresholds of PKCι activity are required for these phenotypes. By manipulating PKCι function using mutant constructs, siRNA depletion or chemical inhibition, we have demonstrated that PKCι is required for polarization of parental MDCK epithelial cysts in a 3D matrix and that there is a threshold of PKCι activity above and below which, disorganized epithelial morphogenesis results. Furthermore, treatment with a novel PKCι inhibitor, CRT0066854, was able to restore polarized morphogenesis in the dysplastic H-Ras spheroids. These results show that tightly regulated PKCι is required for normal-polarized morphogenesis in mammalian cells and that H-Ras and ErbB2 cooperate with PKCι for loss of polarization and dysplasia. The identification of a PKCι inhibitor that can restore polarized morphogenesis has implications for the treatment of Ras and ErbB2 driven malignancies.

Unified quantitative characterization of epithelial tissue development

PubMed Central

Guirao, Boris; Rigaud, Stéphane U; Bosveld, Floris; Bailles, Anaïs; López-Gay, Jesús; Ishihara, Shuji; Sugimura, Kaoru

2015-01-01

Understanding the mechanisms regulating development requires a quantitative characterization of cell divisions, rearrangements, cell size and shape changes, and apoptoses. We developed a multiscale formalism that relates the characterizations of each cell process to tissue growth and morphogenesis. Having validated the formalism on computer simulations, we quantified separately all morphogenetic events in the Drosophila dorsal thorax and wing pupal epithelia to obtain comprehensive statistical maps linking cell and tissue scale dynamics. While globally cell shape changes, rearrangements and divisions all significantly participate in tissue morphogenesis, locally, their relative participations display major variations in space and time. By blocking division we analyzed the impact of division on rearrangements, cell shape changes and tissue morphogenesis. Finally, by combining the formalism with mechanical stress measurement, we evidenced unexpected interplays between patterns of tissue elongation, cell division and stress. Our formalism provides a novel and rigorous approach to uncover mechanisms governing tissue development. DOI: http://dx.doi.org/10.7554/eLife.08519.001 PMID:26653285

The Ron Receptor Tyrosine Kinase Negatively Regulates Mammary Gland Branching Morphogenesis

PubMed Central

Meyer, Sara E.; Zinser, Glendon M.; Stuart, William D.; Pathrose, Peterson; Waltz, Susan E.

2009-01-01

The Ron receptor tyrosine kinase is expressed in normal breast tissue and is overexpressed in approximately 50% of human breast cancers. Despite the recent studies on Ron in breast cancer, nothing is known about the importance of this protein during breast development. To investigate the functional significance of Ron in the normal mammary gland, we compared mammary gland development in wild-type mice to mice containing a targeted ablation of the tyrosine kinase (TK) signaling domain of Ron (TK−/−). Mammary glands from RonTK−/− mice exhibited accelerated pubertal development including significantly increased ductal extension and branching morphogenesis. While circulating levels of estrogen, progesterone, and overall rates of epithelial cell turnover were unchanged, significant increases in phosphorylated MAPK, which predominantly localized to the epithelium, were associated with increased branching morphogenesis. Additionally, purified RonTK−/− epithelial cells cultured ex vivo exhibited enhanced branching morphogenesis, which was reduced upon MAPK inhibition. Microarray analysis of pubertal RonTK−/− glands revealed 393 genes temporally impacted by Ron expression with significant changes observed in signaling networks regulating development, morphogenesis, differentiation, cell motility, and adhesion. In total, these studies represent the first evidence of a role for the Ron receptor tyrosine kinase as a critical negative regulator of mammary development. PMID:19576199

The Polarity Protein Partitioning-defective 1 (PAR-1) Regulates Dendritic Spine Morphogenesis through Phosphorylating Postsynaptic Density Protein 95 (PSD-95)*

PubMed Central

Wu, Qian; DiBona, Victoria L.; Bernard, Laura P.; Zhang, Huaye

2012-01-01

The polarity protein PAR-1 plays an essential role in many cellular contexts, including embryogenesis, asymmetric cell division, directional migration, and epithelial morphogenesis. Despite its known importance in different cellular processes, the role of PAR-1 in neuronal morphogenesis is less well understood. In particular, its role in the morphogenesis of dendritic spines, which are sites of excitatory synaptic inputs, has been unclear. Here, we show that PAR-1 is required for normal spine morphogenesis in hippocampal neurons. We further show that PAR-1 functions through phosphorylating the synaptic scaffolding protein PSD-95 in this process. Phosphorylation at a conserved serine residue in the KXGS motif in PSD-95 regulates spine morphogenesis, and a phosphomimetic mutant of this site can rescue the defects of kinase-dead PAR-1. Together, our findings uncover a role of PAR-1 in spine morphogenesis in hippocampal neurons through phosphorylating PSD-95. PMID:22807451

Apico-basal forces exerted by apoptotic cells drive epithelium folding.

PubMed

Monier, Bruno; Gettings, Melanie; Gay, Guillaume; Mangeat, Thomas; Schott, Sonia; Guarner, Ana; Suzanne, Magali

2015-02-12

Epithelium folding is a basic morphogenetic event that is essential in transforming simple two-dimensional epithelial sheets into three-dimensional structures in both vertebrates and invertebrates. Folding has been shown to rely on apical constriction. The resulting cell-shape changes depend either on adherens junction basal shift or on a redistribution of myosin II, which could be driven by mechanical signals. Yet the initial cellular mechanisms that trigger and coordinate cell remodelling remain largely unknown. Here we unravel the active role of apoptotic cells in initiating morphogenesis, thus revealing a novel mechanism of epithelium folding. We show that, in a live developing tissue, apoptotic cells exert a transient pulling force upon the apical surface of the epithelium through a highly dynamic apico-basal myosin II cable. The apoptotic cells then induce a non-autonomous increase in tissue tension together with cortical myosin II apical stabilization in the surrounding tissue, eventually resulting in epithelium folding. Together our results, supported by a theoretical biophysical three-dimensional model, identify an apoptotic myosin-II-dependent signal as the initial signal leading to cell reorganization and tissue folding. This work further reveals that, far from being passively eliminated as generally assumed (for example, during digit individualization), apoptotic cells actively influence their surroundings and trigger tissue remodelling through regulation of tissue tension.

Expression and Functional Role of Sprouty-2 in Breast Morphogenesis

PubMed Central

Hilmarsdottir, Bylgja; Gustafsdottir, Sigrun M.; Franzdottir, Sigridur Rut; Arason, Ari Jon; Steingrimsson, Eirikur; Magnusson, Magnus K.; Gudjonsson, Thorarinn

2013-01-01

Branching morphogenesis is a mechanism used by many species for organogenesis and tissue maintenance. Receptor tyrosine kinases (RTKs), including epidermal growth factor receptor (EGFR) and the sprouty protein family are believed to be critical regulators of branching morphogenesis. The aim of this study was to analyze the expression of Sprouty-2 (SPRY2) in the mammary gland and study its role in branching morphogenesis. Human breast epithelial cells, breast tissue and mouse mammary glands were used for expression studies using immunoblotting, real rime PCR and immunohistochemistry. Knockdown of SPRY2 in the breast epithelial stem cell line D492 was done by lentiviral transduction of shRNA constructs targeting SPRY2. Three dimensional culture of D492 with or without endothelial cells was done in reconstituted basement membrane matrix. We show that in the human breast, SPRY2 is predominantly expressed in the luminal epithelial cells of both ducts and lobuli. In the mouse mammary gland, SPRY2 expression is low or absent in the virgin state, while in the pregnant mammary gland SPRY2 is expressed at branching epithelial buds with increased expression during lactation. This expression pattern is closely associated with the activation of the EGFR pathway. Using D492 which generates branching structures in three-dimensional (3D) culture, we show that SPRY2 expression is low during initiation of branching with subsequent increase throughout the branching process. Immunostaining locates expression of phosphorylated SPRY2 and EGFR at the tip of lobular-like, branching ends. SPRY2 knockdown (KD) resulted in increased migration, increased pERK and larger and more complex branching structures indicating a loss of negative feedback control during branching morphogenesis. In D492 co-cultures with endothelial cells, D492 SPRY2 KD generates spindle-like colonies that bear hallmarks of epithelial to mesenchymal transition. These data indicate that SPRY2 is an important regulator of branching morphogenesis and epithelial to mesenchymal transition in the mammary gland. PMID:23573284

Expression and functional role of sprouty-2 in breast morphogenesis.

PubMed

Sigurdsson, Valgardur; Ingthorsson, Saevar; Hilmarsdottir, Bylgja; Gustafsdottir, Sigrun M; Franzdottir, Sigridur Rut; Arason, Ari Jon; Steingrimsson, Eirikur; Magnusson, Magnus K; Gudjonsson, Thorarinn

2013-01-01

Branching morphogenesis is a mechanism used by many species for organogenesis and tissue maintenance. Receptor tyrosine kinases (RTKs), including epidermal growth factor receptor (EGFR) and the sprouty protein family are believed to be critical regulators of branching morphogenesis. The aim of this study was to analyze the expression of Sprouty-2 (SPRY2) in the mammary gland and study its role in branching morphogenesis. Human breast epithelial cells, breast tissue and mouse mammary glands were used for expression studies using immunoblotting, real rime PCR and immunohistochemistry. Knockdown of SPRY2 in the breast epithelial stem cell line D492 was done by lentiviral transduction of shRNA constructs targeting SPRY2. Three dimensional culture of D492 with or without endothelial cells was done in reconstituted basement membrane matrix. We show that in the human breast, SPRY2 is predominantly expressed in the luminal epithelial cells of both ducts and lobuli. In the mouse mammary gland, SPRY2 expression is low or absent in the virgin state, while in the pregnant mammary gland SPRY2 is expressed at branching epithelial buds with increased expression during lactation. This expression pattern is closely associated with the activation of the EGFR pathway. Using D492 which generates branching structures in three-dimensional (3D) culture, we show that SPRY2 expression is low during initiation of branching with subsequent increase throughout the branching process. Immunostaining locates expression of phosphorylated SPRY2 and EGFR at the tip of lobular-like, branching ends. SPRY2 knockdown (KD) resulted in increased migration, increased pERK and larger and more complex branching structures indicating a loss of negative feedback control during branching morphogenesis. In D492 co-cultures with endothelial cells, D492 SPRY2 KD generates spindle-like colonies that bear hallmarks of epithelial to mesenchymal transition. These data indicate that SPRY2 is an important regulator of branching morphogenesis and epithelial to mesenchymal transition in the mammary gland.

Mechanical influences in bacterial morphogenesis and cell division

NASA Astrophysics Data System (ADS)

Sun, Sean

2010-03-01

Bacterial cells utilize a ring-like organelle (the Z-ring) to accomplish cell division. The Z-ring actively generates a contractile force and influences cell wall growth. We will discuss a general model of bacterial morphogenesis where mechanical forces are coupled to the growth dynamics of the cell wall. The model suggests a physical mechanism that determines the shapes of bacteria cells. The roles of several bacterial cytoskeletal proteins and the Z-ring are discussed. We will also explore molecular mechanisms of force generation by the Z-ring and how cells can generate mechanical forces without molecular motors.

Epithelial organization and cyst lumen expansion require efficient Sec13-Sec31-driven secretion.

PubMed

Townley, Anna K; Schmidt, Katy; Hodgson, Lorna; Stephens, David J

2012-02-01

Epithelial morphogenesis is directed by interactions with the underlying extracellular matrix. Secretion of collagen and other matrix components requires efficient coat complex II (COPII) vesicle formation at the endoplasmic reticulum. Here, we show that suppression of the outer layer COPII component, Sec13, in zebrafish embryos results in a disorganized gut epithelium. In human intestinal epithelial cells (Caco-2), Sec13 depletion causes defective epithelial polarity and organization on permeable supports. Defects are seen in the ability of cells to adhere to the substrate, form a monolayer and form intercellular junctions. When embedded in a three-dimensional matrix, Sec13-depleted Caco-2 cells form cysts but, unlike controls, are defective in lumen expansion. Incorporation of primary fibroblasts within the three-dimensional culture substantially restores normal morphogenesis. We conclude that efficient COPII-dependent secretion, notably assembly of Sec13-Sec31, is required to drive epithelial morphogenesis in both two- and three-dimensional cultures in vitro, as well as in vivo. Our results provide insight into the role of COPII in epithelial morphogenesis and have implications for the interpretation of epithelial polarity and organization assays in cell culture.

Epithelial organization and cyst lumen expansion require efficient Sec13–Sec31-driven secretion

PubMed Central

Townley, Anna K.; Schmidt, Katy; Hodgson, Lorna; Stephens, David J.

2012-01-01

Epithelial morphogenesis is directed by interactions with the underlying extracellular matrix. Secretion of collagen and other matrix components requires efficient coat complex II (COPII) vesicle formation at the endoplasmic reticulum. Here, we show that suppression of the outer layer COPII component, Sec13, in zebrafish embryos results in a disorganized gut epithelium. In human intestinal epithelial cells (Caco-2), Sec13 depletion causes defective epithelial polarity and organization on permeable supports. Defects are seen in the ability of cells to adhere to the substrate, form a monolayer and form intercellular junctions. When embedded in a three-dimensional matrix, Sec13-depleted Caco-2 cells form cysts but, unlike controls, are defective in lumen expansion. Incorporation of primary fibroblasts within the three-dimensional culture substantially restores normal morphogenesis. We conclude that efficient COPII-dependent secretion, notably assembly of Sec13–Sec31, is required to drive epithelial morphogenesis in both two- and three-dimensional cultures in vitro, as well as in vivo. Our results provide insight into the role of COPII in epithelial morphogenesis and have implications for the interpretation of epithelial polarity and organization assays in cell culture. PMID:22331354

Motility and more: the flagellum of Trypanosoma brucei

PubMed Central

Langousis, Gerasimos; Hill, Kent L.

2014-01-01

A central feature of trypanosome cell biology and life cycle is the parasite’s single flagellum, which is an essential and multifunctional organelle involved in cell propulsion, morphogenesis and cytokinesis. The flagellar membrane is also a specialized subdomain of the cell surface that harbors multiple parasite virulence factors with roles in signaling and host-parasite interactions. In this review, we discuss the structure, assembly and function of the trypanosome flagellum, including canonical roles in cell motility as well as novel and emerging roles in cell morphogenesis and host-parasite interaction. PMID:24931043

Extra-embryonic tissue spreading directs early embryo morphogenesis in killifish

PubMed Central

Reig, Germán; Cerda, Mauricio; Sepúlveda, Néstor; Flores, Daniela; Castañeda, Victor; Tada, Masazumi; Härtel, Steffen; Concha, Miguel L.

2017-01-01

The spreading of mesenchymal-like cell layers is critical for embryo morphogenesis and tissue repair, yet we know little of this process in vivo. Here we take advantage of unique developmental features of the non-conventional annual killifish embryo to study the principles underlying tissue spreading in a simple cellular environment, devoid of patterning signals and major morphogenetic cell movements. Using in vivo experimentation and physical modelling we reveal that the extra-embryonic epithelial enveloping cell layer, thought mainly to provide protection to the embryo, directs cell migration and the spreading of embryonic tissue during early development. This function relies on the ability of embryonic cells to couple their autonomous random motility to non-autonomous signals arising from the expansion of the extra-embryonic epithelium, mediated by cell membrane adhesion and tension. Thus, we present a mechanism of extra-embryonic control of embryo morphogenesis that couples the mechanical properties of adjacent tissues in the early killifish embryo. PMID:28580937

The case for applying tissue engineering methodologies to instruct human organoid morphogenesis.

PubMed

Marti-Figueroa, Carlos R; Ashton, Randolph S

2017-05-01

Three-dimensional organoids derived from human pluripotent stem cell (hPSC) derivatives have become widely used in vitro models for studying development and disease. Their ability to recapitulate facets of normal human development during in vitro morphogenesis produces tissue structures with unprecedented biomimicry. Current organoid derivation protocols primarily rely on spontaneous morphogenesis processes to occur within 3-D spherical cell aggregates with minimal to no exogenous control. This yields organoids containing microscale regions of biomimetic tissues, but at the macroscale (i.e. 100's of microns to millimeters), the organoids' morphology, cytoarchitecture, and cellular composition are non-biomimetic and variable. The current lack of control over in vitro organoid morphogenesis at the microscale induces aberrations at the macroscale, which impedes realization of the technology's potential to reproducibly form anatomically correct human tissue units that could serve as optimal human in vitro models and even transplants. Here, we review tissue engineering methodologies that could be used to develop powerful approaches for instructing multiscale, 3-D human organoid morphogenesis. Such technological mergers are critically needed to harness organoid morphogenesis as a tool for engineering functional human tissues with biomimetic anatomy and physiology. Human PSC-derived 3-D organoids are revolutionizing the biomedical sciences. They enable the study of development and disease within patient-specific genetic backgrounds and unprecedented biomimetic tissue microenvironments. However, their uncontrolled, spontaneous morphogenesis at the microscale yields inconsistences in macroscale organoid morphology, cytoarchitecture, and cellular composition that limits their standardization and application. Integration of tissue engineering methods with organoid derivation protocols could allow us to harness their potential by instructing standardized in vitro morphogenesis to generate organoids with biomimicry at all scales. Such advancements would enable the use of organoids as a basis for 'next-generation' tissue engineering of functional, anatomically mimetic human tissues and potentially novel organ transplants. Here, we discuss critical aspects of organoid morphogenesis where application of innovative tissue engineering methodologies would yield significant advancement towards this goal. Copyright © 2017. Published by Elsevier Ltd.

Multicellular Models of Morphogenesis

EPA Science Inventory

EPA’s Virtual Embryo project (v-Embryo™), in collaboration with developers of CompuCell3D, aims to create computer models of morphogenesis that can be used to address the effects of chemical perturbation on embryo development at the cellular level. Such computational (in silico) ...

Mammary collective cell migration involves transient loss of epithelial features and individual cell migration within the epithelium

PubMed Central

Ewald, Andrew J.; Huebner, Robert J.; Palsdottir, Hildur; Lee, Jessie K.; Perez, Melissa J.; Jorgens, Danielle M.; Tauscher, Andrew N.; Cheung, Kevin J.; Werb, Zena; Auer, Manfred

2012-01-01

Normal mammary morphogenesis involves transitions between simple and multilayered epithelial organizations. We used electron microscopy and molecular markers to determine whether intercellular junctions and apico-basal polarity were maintained in the multilayered epithelium. We found that multilayered elongating ducts had polarized apical and basal tissue surfaces both in three-dimensional culture and in vivo. However, individual cells were only polarized on surfaces in contact with the lumen or extracellular matrix. The basolateral marker scribble and the apical marker atypical protein kinase C zeta localized to all interior cell membranes, whereas PAR3 displayed a cytoplasmic localization, suggesting that the apico-basal polarity was incomplete. Despite membrane localization of E-cadherin and β-catenin, we did not observe a defined zonula adherens connecting interior cells. Instead, interior cells were connected through desmosomes and exhibited complex interdigitating membrane protrusions. Single-cell labeling revealed that individual cells were both protrusive and migratory within the epithelial multilayer. Inhibition of Rho kinase (ROCK) further reduced intercellular adhesion on apical and lateral surfaces but did not disrupt basal tissue organization. Following morphogenesis, segregated membrane domains were re-established and junctional complexes re-formed. We observed similar epithelial organization during mammary morphogenesis in organotypic culture and in vivo. We conclude that mammary epithelial morphogenesis involves a reversible, spatially limited, reduction in polarity and intercellular junctions and active individualistic cell migration. Our data suggest that reductions in polarity and adhesion during breast cancer progression might reflect partial recapitulation of a normal developmental program. PMID:22344263

Activation of YUCCA5 by the Transcription Factor TCP4 Integrates Developmental and Environmental Signals to Promote Hypocotyl Elongation in Arabidopsis.

PubMed

Challa, Krishna Reddy; Aggarwal, Pooja; Nath, Utpal

2016-09-05

Cell expansion is an essential process in plant morphogenesis and is regulated by the coordinated action of environmental stimuli and endogenous factors, such as the phytohormones auxin and brassinosteroid. Although the biosynthetic pathways that generate these hormones and their downstream signaling mechanisms have been extensively studied, the upstream transcriptional network that modulates their levels and connects their action to cell morphogenesis is less clear. Here we show that the miR319-regulated TCP (TEOSINTE BRANCHED 1, CYCLODEA, PROLIFERATING CELL FACTORS) transcription factors, notably TCP4, directly activate YUCCA5 transcription and integrate the auxin response to a brassinosteroid-dependent molecular circuit that promotes cell elongation in Arabidopsis hypocotyls. Further, TCP4 modulates the common transcriptional network downstream to auxin-BR signaling, which is also triggered by environmental cues, such as light, to promote cell expansion. Our study links TCP function with the hormone response during cell morphogenesis and shows that developmental and environmental signals converge on a common transcriptional network to promote cell elongation. {copyright, serif} 2016 American Society of Plant Biologists. All rights reserved.

Cell wall accumulation of fluorescent proteins derived from a trans-Golgi cisternal membrane marker and paramural bodies in interdigitated Arabidopsis leaf epidermal cells.

PubMed

Akita, Kae; Kobayashi, Megumi; Sato, Mayuko; Kutsuna, Natsumaro; Ueda, Takashi; Toyooka, Kiminori; Nagata, Noriko; Hasezawa, Seiichiro; Higaki, Takumi

2017-01-01

In most dicotyledonous plants, leaf epidermal pavement cells develop jigsaw puzzle-like shapes during cell expansion. The rapid growth and complicated cell shape of pavement cells is suggested to be achieved by targeted exocytosis that is coordinated with cytoskeletal rearrangement to provide plasma membrane and/or cell wall materials for lobe development during their morphogenesis. Therefore, visualization of membrane trafficking in leaf pavement cells should contribute an understanding of the mechanism of plant cell morphogenesis. To reveal membrane trafficking in pavement cells, we observed monomeric red fluorescent protein-tagged rat sialyl transferases, which are markers of trans-Golgi cisternal membranes, in the leaf epidermis of Arabidopsis thaliana. Quantitative fluorescence imaging techniques and immunoelectron microscopic observations revealed that accumulation of the red fluorescent protein occurred mostly in the curved regions of pavement cell borders and guard cell ends during leaf expansion. Transmission electron microscopy observations revealed that apoplastic vesicular membrane structures called paramural bodies were more frequent beneath the curved cell wall regions of interdigitated pavement cells and guard cell ends in young leaf epidermis. In addition, pharmacological studies showed that perturbations in membrane trafficking resulted in simple cell shapes. These results suggested possible heterogeneity of the curved regions of plasma membranes, implying a relationship with pavement cell morphogenesis.

Identification and Expression of Acetylcholinesterase in Octopus vulgaris Arm Development and Regeneration: a Conserved Role for ACHE?

PubMed

Fossati, Sara Maria; Candiani, Simona; Nödl, Marie-Therese; Maragliano, Luca; Pennuto, Maria; Domingues, Pedro; Benfenati, Fabio; Pestarino, Mario; Zullo, Letizia

2015-08-01

Acetylcholinesterase (ACHE) is a glycoprotein with a key role in terminating synaptic transmission in cholinergic neurons of both vertebrates and invertebrates. ACHE is also involved in the regulation of cell growth and morphogenesis during embryogenesis and regeneration acting through its non-cholinergic sites. The mollusk Octopus vulgaris provides a powerful model for investigating the mechanisms underlying tissue morphogenesis due to its high regenerative power. Here, we performed a comparative investigation of arm morphogenesis during adult arm regeneration and embryonic arm development which may provide insights on the conserved ACHE pathways. In this study, we cloned and characterized O. vulgaris ACHE, finding a single highly conserved ACHE hydrophobic variant, characterized by prototypical catalytic sites and a putative consensus region for a glycosylphosphatidylinositol (GPI)-anchor attachment at the COOH-terminus. We then show that its expression level is correlated to the stage of morphogenesis in both adult and embryonic arm. In particular, ACHE is localized in typical neuronal sites when adult-like arm morphology is established and in differentiating cell locations during the early stages of arm morphogenesis. This possibility is also supported by the presence in the ACHE sequence and model structure of both cholinergic and non-cholinergic sites. This study provides insights into ACHE conserved roles during processes of arm morphogenesis. In addition, our modeling study offers a solid basis for predicting the interaction of the ACHE domains with pharmacological blockers for in vivo investigations. We therefore suggest ACHE as a target for the regulation of tissue morphogenesis.

JunB is required for endothelial cell morphogenesis by regulating core-binding factor β

PubMed Central

Licht, Alexander H.; Pein, Oliver T.; Florin, Lore; Hartenstein, Bettina; Reuter, Hendrik; Arnold, Bernd; Lichter, Peter; Angel, Peter; Schorpp-Kistner, Marina

2006-01-01

The molecular mechanism triggering the organization of endothelial cells (ECs) in multicellular tubules is mechanistically still poorly understood. We demonstrate that cell-autonomous endothelial functions of the AP-1 subunit JunB are required for proper endothelial morphogenesis both in vivo in mouse embryos with endothelial-specific ablation of JunB and in in vitro angiogenesis models. By cDNA microarray analysis, we identified core-binding factor β (CBFβ), which together with the Runx proteins forms the heterodimeric core-binding transcription complex CBF, as a novel JunB target gene. In line with our findings, expression of the CBF target MMP-13 was impaired in JunB-deficient ECs. Reintroduction of CBFβ into JunB-deficient ECs rescued the tube formation defect and MMP-13 expression, indicating an important role for CBFβ in EC morphogenesis. PMID:17158955

Feedback, Lineages and Self-Organizing Morphogenesis

PubMed Central

Calof, Anne L.; Lowengrub, John S.; Lander, Arthur D.

2016-01-01

Feedback regulation of cell lineage progression plays an important role in tissue size homeostasis, but whether such feedback also plays an important role in tissue morphogenesis has yet to be explored. Here we use mathematical modeling to show that a particular feedback architecture in which both positive and negative diffusible signals act on stem and/or progenitor cells leads to the appearance of bistable or bi-modal growth behaviors, ultrasensitivity to external growth cues, local growth-driven budding, self-sustaining elongation, and the triggering of self-organization in the form of lamellar fingers. Such behaviors arise not through regulation of cell cycle speeds, but through the control of stem or progenitor self-renewal. Even though the spatial patterns that arise in this setting are the result of interactions between diffusible factors with antagonistic effects, morphogenesis is not the consequence of Turing-type instabilities. PMID:26989903

Gab1 Mediates Hepatocyte Growth Factor-Stimulated Mitogenicity and Morphogenesis in Multipotent Myeloid Cells

PubMed Central

Felici, Angelina; Giubellino, Alessio; Bottaro, Donald P.

2012-01-01

Hepatocyte growth factor (HGF)-stimulated mitogenesis, motogenesis and morphogenesis in various cell types begins with activation of the Met receptor tyrosine kinase and the recruitment of intracellular adaptors and kinase substrates. The adapter protein Gab1 is a critical effector and substrate of activated Met, mediating morphogenesis, among other activities, in epithelial cells. To define its role downstream of Met in hematopoietic cells, Gab1 was expressed in the HGF-responsive, Gab1-negative murine myeloid cell line 32D. Interestingly, the adhesion and motility of Gab1-expressing cells were significantly greater than parental cells, independent of growth factor treatment. Downstream of activated Met, Gab1 expression was specifically associated with rapid Shp-2 recruitment and activation, increased mitogenic potency, suppression of GATA-1 expression and concomitant upregulation of GATA-2 transcription. In addition to enhanced proliferation, continuous culture of Gab1-expressing 32D cells in HGF resulted in cell attachment, filopodia extension and phenotypic changes suggestive of monocytic differentiation. Our results suggest that in myeloid cells, Gab1 is likely to enhance HGF mitogenicity by coupling Met to Shp-2 and GATA-2 expression, thereby potentially contributing to normal myeloid differentiation as well as oncogenic transformation. PMID:20506405

Tissue-specific activities of the Fat1 cadherin cooperate to control neuromuscular morphogenesis

PubMed Central

2018-01-01

Muscle morphogenesis is tightly coupled with that of motor neurons (MNs). Both MNs and muscle progenitors simultaneously explore the surrounding tissues while exchanging reciprocal signals to tune their behaviors. We previously identified the Fat1 cadherin as a regulator of muscle morphogenesis and showed that it is required in the myogenic lineage to control the polarity of progenitor migration. To expand our knowledge on how Fat1 exerts its tissue-morphogenesis regulator activity, we dissected its functions by tissue-specific genetic ablation. An emblematic example of muscle under such morphogenetic control is the cutaneous maximus (CM) muscle, a flat subcutaneous muscle in which progenitor migration is physically separated from the process of myogenic differentiation but tightly associated with elongating axons of its partner MNs. Here, we show that constitutive Fat1 disruption interferes with expansion and differentiation of the CM muscle, with its motor innervation and with specification of its associated MN pool. Fat1 is expressed in muscle progenitors, in associated mesenchymal cells, and in MN subsets, including the CM-innervating pool. We identify mesenchyme-derived connective tissue (CT) as a cell type in which Fat1 activity is required for the non–cell-autonomous control of CM muscle progenitor spreading, myogenic differentiation, motor innervation, and for motor pool specification. In parallel, Fat1 is required in MNs to promote their axonal growth and specification, indirectly influencing muscle progenitor progression. These results illustrate how Fat1 coordinates the coupling of muscular and neuronal morphogenesis by playing distinct but complementary actions in several cell types. PMID:29768404

Gonadal morphogenesis and gene expression in reptiles with temperature-dependent sex determination.

PubMed

Merchant-Larios, H; Díaz-Hernández, V; Marmolejo-Valencia, A

2010-01-01

In reptiles with temperature-dependent sexual determination, the thermosensitive period (TSP) is the interval in which the sex is defined during gonadal morphogenesis. One-shift experiments in a group of eggs define the onset and the end of the TSP as all and none responses, respectively. Timing for sex-undetermined (UG) and -determined gonads (DG) differs at male- (MPT) or female-producing temperatures (FPT). During the TSP a decreasing number of embryos respond to temperature shifts indicating that in this period embryos with both UG and DG exist. Although most UG correspond to undifferentiated gonads, some embryos extend UG after the onset of histological differentiation. Thus, temperature affects gonadal cells during the process of morphogenesis, but timing of commitment depends on individual embryos. A correlation between gonadal morphogenesis, TSP, and gene expression suggests that determination of the molecular pathways modulated by temperature in epithelial cells (surface epithelium and medullary cords) holds the key for a unifying hypothesis on temperature-dependent sex determination. (c) 2010 S. Karger AG, Basel.

The signalling receptor MCAM coordinates apical-basal polarity and planar cell polarity during morphogenesis

PubMed Central

Gao, Qian; Zhang, Junfeng; Wang, Xiumei; Liu, Ying; He, Rongqiao; Liu, Xingfeng; Wang, Fei; Feng, Jing; Yang, Dongling; Wang, Zhaoqing; Meng, Anming; Yan, Xiyun

2017-01-01

The apical–basal (AB) polarity and planar cell polarity (PCP) provide an animal cell population with different phenotypes during morphogenesis. However, how cells couple these two patterning systems remains unclear. Here we provide in vivo evidence that melanoma cell adhesion molecule (MCAM) coordinates AB polarity-driven lumenogenesis and c-Jun N-terminal kinase (JNK)/PCP-dependent ciliogenesis. We identify that MCAM is an independent receptor of fibroblast growth factor 4 (FGF4), a membrane anchor of phospholipase C-γ (PLC-γ), an immediate upstream receptor of nuclear factor of activated T-cells (NFAT) and a constitutive activator of JNK. We find that MCAM-mediated vesicular trafficking towards FGF4, while generating a priority-grade transcriptional response of NFAT determines lumenogenesis. We demonstrate that MCAM plays indispensable roles in ciliogenesis through activating JNK independently of FGF signals. Furthermore, mcam-deficient zebrafish and Xenopus exhibit a global defect in left-right (LR) asymmetric establishment as a result of morphogenetic failure of their LR organizers. Therefore, MCAM coordination of AB polarity and PCP provides insight into the general mechanisms of morphogenesis. PMID:28589943

Extracellular matrix motion and early morphogenesis

PubMed Central

Loganathan, Rajprasad; Rongish, Brenda J.; Smith, Christopher M.; Filla, Michael B.; Czirok, Andras; Bénazéraf, Bertrand

2016-01-01

For over a century, embryologists who studied cellular motion in early amniotes generally assumed that morphogenetic movement reflected migration relative to a static extracellular matrix (ECM) scaffold. However, as we discuss in this Review, recent investigations reveal that the ECM is also moving during morphogenesis. Time-lapse studies show how convective tissue displacement patterns, as visualized by ECM markers, contribute to morphogenesis and organogenesis. Computational image analysis distinguishes between cell-autonomous (active) displacements and convection caused by large-scale (composite) tissue movements. Modern quantification of large-scale ‘total’ cellular motion and the accompanying ECM motion in the embryo demonstrates that a dynamic ECM is required for generation of the emergent motion patterns that drive amniote morphogenesis. PMID:27302396

Multiple Phosphatidylinositol 3-Kinases Regulate Vaccinia Virus Morphogenesis

PubMed Central

McNulty, Shannon; Bornmann, William; Schriewer, Jill; Werner, Chas; Smith, Scott K.; Olson, Victoria A.; Damon, Inger K.; Buller, R. Mark; Heuser, John; Kalman, Daniel

2010-01-01

Poxvirus morphogenesis is a complex process that involves the successive wrapping of the virus in host cell membranes. We screened by plaque assay a focused library of kinase inhibitors for those that caused a reduction in viral growth and identified several compounds that selectively inhibit phosphatidylinositol 3-kinase (PI3K). Previous studies demonstrated that PI3Ks mediate poxviral entry. Using growth curves and electron microscopy in conjunction with inhibitors, we show that that PI3Ks additionally regulate morphogenesis at two distinct steps: immature to mature virion (IMV) transition, and IMV envelopment to form intracellular enveloped virions (IEV). Cells derived from animals lacking the p85 regulatory subunit of Type I PI3Ks (p85α−/−β−/−) presented phenotypes similar to those observed with PI3K inhibitors. In addition, VV appear to redundantly use PI3Ks, as PI3K inhibitors further reduce plaque size and number in p85α−/−β−/− cells. Together, these data provide evidence for a novel regulatory mechanism for virion morphogenesis involving phosphatidylinositol dynamics and may represent a new therapeutic target to contain poxviruses. PMID:20526370

Mechanical Control of Tissue Morphogenesis

PubMed Central

Patwari, Parth; Lee, Richard T.

2008-01-01

Mechanical forces participate in morphogenesis from the level of individual cells to whole organism patterning. This manuscript reviews recent research that has identified specific roles for mechanical forces in important developmental events. One well-defined example is that dynein-driven cilia create fluid flow that determines left-right patterning in the early mammalian embryo. Fluid flow is also important for vasculogenesis, and evidence suggests that fluid shear stress rather than fluid transport is primarily required for remodeling the early vasculature. Contraction of the actin cytoskeleton, driven by nonmuscle myosins and regulated by the Rho family GTPases, is a recurring mechanism for controlling morphogenesis throughout development, from gastrulation to cardiogenesis. Finally, novel experimental approaches suggest critical roles for the actin cytoskeleton and the mechanical environment in determining differentiation of mesenchymal stem cells. Insights into the mechanisms linking mechanical forces to cell and tissue differentiation pathways are important for understanding many congenital diseases and for developing regenerative medicine strategies. PMID:18669930

Growth patterns for shape-shifting elastic bilayers.

PubMed

van Rees, Wim M; Vouga, Etienne; Mahadevan, L

2017-10-31

Inspired by the differential-growth-driven morphogenesis of leaves, flowers, and other tissues, there is increasing interest in artificial analogs of these shape-shifting thin sheets made of active materials that respond to environmental stimuli such as heat, light, and humidity. But how can we determine the growth patterns to achieve a given shape from another shape? We solve this geometric inverse problem of determining the growth factors and directions (the metric tensors) for a given isotropic elastic bilayer to grow into a target shape by posing and solving an elastic energy minimization problem. A mathematical equivalence between bilayers and curved monolayers simplifies the inverse problem considerably by providing algebraic expressions for the growth metric tensors in terms of those of the final shape. This approach also allows us to prove that we can grow any target surface from any reference surface using orthotropically growing bilayers. We demonstrate this by numerically simulating the growth of a flat sheet into a face, a cylindrical sheet into a flower, and a flat sheet into a complex canyon-like structure.

Growth patterns for shape-shifting elastic bilayers

PubMed Central

van Rees, Wim M.; Vouga, Etienne; Mahadevan, L.

2017-01-01

Inspired by the differential-growth-driven morphogenesis of leaves, flowers, and other tissues, there is increasing interest in artificial analogs of these shape-shifting thin sheets made of active materials that respond to environmental stimuli such as heat, light, and humidity. But how can we determine the growth patterns to achieve a given shape from another shape? We solve this geometric inverse problem of determining the growth factors and directions (the metric tensors) for a given isotropic elastic bilayer to grow into a target shape by posing and solving an elastic energy minimization problem. A mathematical equivalence between bilayers and curved monolayers simplifies the inverse problem considerably by providing algebraic expressions for the growth metric tensors in terms of those of the final shape. This approach also allows us to prove that we can grow any target surface from any reference surface using orthotropically growing bilayers. We demonstrate this by numerically simulating the growth of a flat sheet into a face, a cylindrical sheet into a flower, and a flat sheet into a complex canyon-like structure. PMID:29078336

Tooth-bone morphogenesis during postnatal stages of mouse first molar development

PubMed Central

Lungová, Vlasta; Radlanski, Ralf J; Tucker, Abigail S; Renz, Herbert; Míšek, Ivan; Matalová, Eva

2011-01-01

The first mouse molar (M1) is the most common model for odontogenesis, with research particularly focused on prenatal development. However, the functional dentition forms postnatally, when the histogenesis and morphogenesis of the tooth is completed, the roots form and the tooth physically anchors into the jaw. In this work, M1 was studied from birth to eruption, assessing morphogenesis, proliferation and apoptosis, and correlating these with remodeling of the surrounding bony tissue. The M1 completed crown formation between postnatal (P) days 0–2, and the development of the tooth root was initiated at P4. From P2 until P12, cell proliferation in the dental epithelium reduced and shifted downward to the apical region of the forming root. In contrast, proliferation was maintained or increased in the mesenchymal cells of the dental follicle. At later stages, before tooth eruption (P20), cell proliferation suddenly ceased. This withdrawal from the cell cycle correlated with tooth mineralization and mesenchymal differentiation. Apoptosis was observed during all stages of M1 postnatal morphogenesis, playing a role in the removal of cells such as osteoblasts in the mandibular region and working together with osteoclasts to remodel the bone around the developing tooth. At more advanced developmental stages, apoptotic cells and bodies accumulated in the cell layers above the tooth cusps, in the path of eruption. Three-dimensional reconstruction of the developing postnatal tooth and bone indicates that the alveolar crypts form by resorption underneath the primordia, whereas the ridges form by active bone growth between the teeth and roots to form a functional complex. PMID:21418206

A global sensitivity analysis approach for morphogenesis models.

PubMed

Boas, Sonja E M; Navarro Jimenez, Maria I; Merks, Roeland M H; Blom, Joke G

2015-11-21

Morphogenesis is a developmental process in which cells organize into shapes and patterns. Complex, non-linear and multi-factorial models with images as output are commonly used to study morphogenesis. It is difficult to understand the relation between the uncertainty in the input and the output of such 'black-box' models, giving rise to the need for sensitivity analysis tools. In this paper, we introduce a workflow for a global sensitivity analysis approach to study the impact of single parameters and the interactions between them on the output of morphogenesis models. To demonstrate the workflow, we used a published, well-studied model of vascular morphogenesis. The parameters of this cellular Potts model (CPM) represent cell properties and behaviors that drive the mechanisms of angiogenic sprouting. The global sensitivity analysis correctly identified the dominant parameters in the model, consistent with previous studies. Additionally, the analysis provided information on the relative impact of single parameters and of interactions between them. This is very relevant because interactions of parameters impede the experimental verification of the predicted effect of single parameters. The parameter interactions, although of low impact, provided also new insights in the mechanisms of in silico sprouting. Finally, the analysis indicated that the model could be reduced by one parameter. We propose global sensitivity analysis as an alternative approach to study the mechanisms of morphogenesis. Comparison of the ranking of the impact of the model parameters to knowledge derived from experimental data and from manipulation experiments can help to falsify models and to find the operand mechanisms in morphogenesis. The workflow is applicable to all 'black-box' models, including high-throughput in vitro models in which output measures are affected by a set of experimental perturbations.

Coherent Motion of Monolayer Sheets under Confinement and Its Pathological Implications.

PubMed

Soumya, S S; Gupta, Animesh; Cugno, Andrea; Deseri, Luca; Dayal, Kaushik; Das, Dibyendu; Sen, Shamik; Inamdar, Mandar M

2015-12-01

Coherent angular rotation of epithelial cells is thought to contribute to many vital physiological processes including tissue morphogenesis and glandular formation. However, factors regulating this motion, and the implications of this motion if perturbed, remain incompletely understood. In the current study, we address these questions using a cell-center based model in which cells are polarized, motile, and interact with the neighboring cells via harmonic forces. We demonstrate that, a simple evolution rule in which the polarization of any cell tends to orient with its velocity vector can induce coherent motion in geometrically confined environments. In addition to recapitulating coherent rotational motion observed in experiments, our results also show the presence of radial movements and tissue behavior that can vary between solid-like and fluid-like. We show that the pattern of coherent motion is dictated by the combination of different physical parameters including number density, cell motility, system size, bulk cell stiffness and stiffness of cell-cell adhesions. We further observe that perturbations in the form of cell division can induce a reversal in the direction of motion when cell division occurs synchronously. Moreover, when the confinement is removed, we see that the existing coherent motion leads to cell scattering, with bulk cell stiffness and stiffness of cell-cell contacts dictating the invasion pattern. In summary, our study provides an in-depth understanding of the origin of coherent rotation in confined tissues, and extracts useful insights into the influence of various physical parameters on the pattern of such movements.

Coherent Motion of Monolayer Sheets under Confinement and Its Pathological Implications

PubMed Central

Soumya, S S; Gupta, Animesh; Cugno, Andrea; Deseri, Luca; Dayal, Kaushik; Das, Dibyendu; Sen, Shamik; Inamdar, Mandar M.

2015-01-01

Coherent angular rotation of epithelial cells is thought to contribute to many vital physiological processes including tissue morphogenesis and glandular formation. However, factors regulating this motion, and the implications of this motion if perturbed, remain incompletely understood. In the current study, we address these questions using a cell-center based model in which cells are polarized, motile, and interact with the neighboring cells via harmonic forces. We demonstrate that, a simple evolution rule in which the polarization of any cell tends to orient with its velocity vector can induce coherent motion in geometrically confined environments. In addition to recapitulating coherent rotational motion observed in experiments, our results also show the presence of radial movements and tissue behavior that can vary between solid-like and fluid-like. We show that the pattern of coherent motion is dictated by the combination of different physical parameters including number density, cell motility, system size, bulk cell stiffness and stiffness of cell-cell adhesions. We further observe that perturbations in the form of cell division can induce a reversal in the direction of motion when cell division occurs synchronously. Moreover, when the confinement is removed, we see that the existing coherent motion leads to cell scattering, with bulk cell stiffness and stiffness of cell-cell contacts dictating the invasion pattern. In summary, our study provides an in-depth understanding of the origin of coherent rotation in confined tissues, and extracts useful insights into the influence of various physical parameters on the pattern of such movements. PMID:26691341

Live imaging of heart tube development in mouse reveals alternating phases of cardiac differentiation and morphogenesis

PubMed Central

Ivanovitch, Kenzo; Temiño, Susana

2017-01-01

During vertebrate heart development, two progenitor populations, first and second heart fields (FHF, SHF), sequentially contribute to longitudinal subdivisions of the heart tube (HT), with the FHF contributing the left ventricle and part of the atria, and the SHF the rest of the heart. Here, we study the dynamics of cardiac differentiation and morphogenesis by tracking individual cells in live analysis of mouse embryos. We report that during an initial phase, FHF precursors differentiate rapidly to form a cardiac crescent, while limited morphogenesis takes place. In a second phase, no differentiation occurs while extensive morphogenesis, including splanchnic mesoderm sliding over the endoderm, results in HT formation. In a third phase, cardiac precursor differentiation resumes and contributes to SHF-derived regions and the dorsal closure of the HT. These results reveal tissue-level coordination between morphogenesis and differentiation during HT formation and provide a new framework to understand heart development. PMID:29202929

Ectopic expression of Msx-2 in posterior limb bud mesoderm impairs limb morphogenesis while inducing BMP-4 expression, inhibiting cell proliferation, and promoting apoptosis.

PubMed

Ferrari, D; Lichtler, A C; Pan, Z Z; Dealy, C N; Upholt, W B; Kosher, R A

1998-05-01

During early stages of chick limb development, the homeobox-containing gene Msx-2 is expressed in the mesoderm at the anterior margin of the limb bud and in a discrete group of mesodermal cells at the midproximal posterior margin. These domains of Msx-2 expression roughly demarcate the anterior and posterior boundaries of the progress zone, the highly proliferating posterior mesodermal cells underneath the apical ectodermal ridge (AER) that give rise to the skeletal elements of the limb and associated structures. Later in development as the AER loses its activity, Msx-2 expression expands into the distal mesoderm and subsequently into the interdigital mesenchyme which demarcates the developing digits. The domains of Msx-2 expression exhibit considerably less proliferation than the cells of the progress zone and also encompass several regions of programmed cell death including the anterior and posterior necrotic zones and interdigital mesenchyme. We have thus suggested that Msx-2 may be in a regulatory network that delimits the progress zone by suppressing the morphogenesis of the regions of the limb mesoderm in which it is highly expressed. In the present study we show that ectopic expression of Msx-2 via a retroviral expression vector in the posterior mesoderm of the progress zone from the time of initial formation of the limb bud severely impairs limb morphogenesis. Msx-2-infected limbs are typically very narrow along the anteroposterior axis, are occasionally truncated, and exhibit alterations in the pattern of formation of skeletal elements, indicating that as a consequence of ectopic Msx-2 expression the morphogenesis of large portions of the posterior mesoderm has been suppressed. We further show that Msx-2 impairs limb morphogenesis by reducing cell proliferation and promoting apoptosis in the regions of the posterior mesoderm in which it is ectopically expressed. The domains of ectopic Msx-2 expression in the posterior mesoderm also exhibit ectopic expression of BMP-4, a secreted signaling molecule that is coexpressed with Msx-2 during normal limb development in the anterior limb mesoderm, the posterior necrotic zone, and interdigital mesenchyme. This indicates that Msx-2 regulates BMP-4 expression and that the suppressive effects of Msx-2 on limb morphogenesis might be mediated in part by BMP-4. These studies indicate that during normal limb development Msx-2 is a key component of a regulatory network that delimits the boundaries of the progress zone by suppressing the morphogenesis of the regions of the limb mesoderm in which it is highly expressed, thus restricting the outgrowth and formation of skeletal elements and associated structures to the progress zone. We also report that rather large numbers of apoptotic cells as well as proliferating cells are present throughout the AER during all stages of normal limb development we have examined, indicating that many of the cells of the AER are continuously undergoing programmed cell death at the same time that new AER cells are being generated by cell proliferation. Thus, a balance between cell proliferation and programmed cell death may play a very important role in maintaining the activity of the AER. Copyright 1998 Academic Press.

NFIB regulates embryonic development of submandibular glands.

PubMed

Mellas, R E; Kim, H; Osinski, J; Sadibasic, S; Gronostajski, R M; Cho, M; Baker, O J

2015-02-01

NFIB (nuclear factor I B) is a NFI transcription factor family member, which is essential for the development of a variety of organ systems. Salivary gland development occurs through several stages, including prebud, bud, pseudoglandular, canalicular, and terminal. Although many studies have been done to understand mouse submandibular gland (SMG) branching morphogenesis, little is known about SMG cell differentiation during the terminal stages. The goal of this study was to determine the role of NFIB during SMG development. We analyzed SMGs from wild-type and Nfib-deficient mice (Nfib (-/-)). At embryonic (E) day 18.5, SMGs from wild-type mice showed duct branching morphogenesis and differentiation of tubule ductal cells into tubule secretory cells. In contrast, SMGs from Nfib (-/-) mice at E18.5 failed to differentiate into tubule secretory cells while branching morphogenesis was unaffected. SMGs from wild-type mice at E16.5 displayed well-organized cuboidal inner terminal tubule cells. However, SMGs from Nfib (-/-) at E16.5 displayed disorganized inner terminal tubule cells. SMGs from wild-type mice at E18.5 became fully differentiated, as indicated by a high degree of apicobasal polarization (i.e., presence of apical ZO-1 and basolateral E-cadherin) and columnar shape. Furthermore, SMGs from wild-type mice at E18.5 expressed the protein SMGC, a marker for tubule secretory cells. However, SMGs from Nfib (-/-) mice at E18.5 showed apicobasal polarity, but they were disorganized and lost the ability to secrete SMGC. These findings indicate that the transcription factor NFIB is not required for branching morphogenesis but plays a key role in tubule cell differentiation during mouse SMG development. © International & American Associations for Dental Research 2014.

Extracellular matrix motion and early morphogenesis.

PubMed

Loganathan, Rajprasad; Rongish, Brenda J; Smith, Christopher M; Filla, Michael B; Czirok, Andras; Bénazéraf, Bertrand; Little, Charles D

2016-06-15

For over a century, embryologists who studied cellular motion in early amniotes generally assumed that morphogenetic movement reflected migration relative to a static extracellular matrix (ECM) scaffold. However, as we discuss in this Review, recent investigations reveal that the ECM is also moving during morphogenesis. Time-lapse studies show how convective tissue displacement patterns, as visualized by ECM markers, contribute to morphogenesis and organogenesis. Computational image analysis distinguishes between cell-autonomous (active) displacements and convection caused by large-scale (composite) tissue movements. Modern quantification of large-scale 'total' cellular motion and the accompanying ECM motion in the embryo demonstrates that a dynamic ECM is required for generation of the emergent motion patterns that drive amniote morphogenesis. © 2016. Published by The Company of Biologists Ltd.

Remodeling a tissue: subtraction adds insight.

PubMed

Axelrod, Jeffrey D

2012-11-27

Sculpting a body plan requires both patterning of gene expression and translating that pattern into morphogenesis. Developmental biologists have made remarkable strides in understanding gene expression patterning, but despite a long history of fascination with the mechanics of morphogenesis, knowledge of how patterned gene expression drives the emergence of even simple shapes and forms has grown at a slower pace. The successful merging of approaches from cell biology, developmental biology, imaging, engineering, and mathematical and computational sciences is now accelerating progress toward a fuller and better integrated understanding of the forces shaping morphogenesis.

The TCP4 transcription factor regulates trichome cell differentiation by directly activating GLABROUS INFLORESCENCE STEMS in Arabidopsis thaliana.

PubMed

Vadde, Batthula Vijaya Lakshmi; Challa, Krishna Reddy; Nath, Utpal

2018-01-01

Trichomes are the first cell type to be differentiated during the morphogenesis of leaf epidermis and serve as an ideal model to study cellular differentiation. Many genes involved in the patterning and differentiation of trichome cells have been studied over the past decades, and the majority of these genes encode transcription factors that specifically regulate epidermal cell development. However, the upstream regulators of these genes that link early leaf morphogenesis with cell type differentiation are less studied. The TCP proteins are the plant-specific transcription factors involved in regulating diverse aspects of plant development including lateral organ morphogenesis by modulating cell proliferation and differentiation. Here, we show that the miR319-regulated class II TCP proteins, notably TCP4, suppress trichome branching in Arabidopsis leaves and inflorescence stem by direct transcriptional activation of GLABROUS INFLORESCENCE STEMS (GIS), a known negative regulator of trichome branching. The trichome branch number is increased in plants with reduced TCP activity and decreased in the gain-of-function lines of TCP4. Biochemical analyses show that TCP4 binds to the upstream regulatory region of GIS and activates its expression. Detailed genetic analyses show that GIS and TCP4 work in same pathway and GIS function is required for TCP4-mediated regulation of trichome differentiation. Taken together, these results identify a role for the class II TCP genes in trichome differentiation, thus providing a connection between organ morphogenesis and cellular differentiation. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

Computational Modeling of Tissue Self-Assembly

NASA Astrophysics Data System (ADS)

Neagu, Adrian; Kosztin, Ioan; Jakab, Karoly; Barz, Bogdan; Neagu, Monica; Jamison, Richard; Forgacs, Gabor

As a theoretical framework for understanding the self-assembly of living cells into tissues, Steinberg proposed the differential adhesion hypothesis (DAH) according to which a specific cell type possesses a specific adhesion apparatus that combined with cell motility leads to cell assemblies of various cell types in the lowest adhesive energy state. Experimental and theoretical efforts of four decades turned the DAH into a fundamental principle of developmental biology that has been validated both in vitro and in vivo. Based on computational models of cell sorting, we have developed a DAH-based lattice model for tissues in interaction with their environment and simulated biological self-assembly using the Monte Carlo method. The present brief review highlights results on specific morphogenetic processes with relevance to tissue engineering applications. Our own work is presented on the background of several decades of theoretical efforts aimed to model morphogenesis in living tissues. Simulations of systems involving about 105 cells have been performed on high-end personal computers with CPU times of the order of days. Studied processes include cell sorting, cell sheet formation, and the development of endothelialized tubes from rings made of spheroids of two randomly intermixed cell types, when the medium in the interior of the tube was different from the external one. We conclude by noting that computer simulations based on mathematical models of living tissues yield useful guidelines for laboratory work and can catalyze the emergence of innovative technologies in tissue engineering.

Tracing anti-cancer and cancer-promoting actions of all-trans retinoic acid in breast cancer to a RARα epigenetic mechanism of mammary epithelial cell fate.

PubMed

Rossetti, Stefano; Ren, MingQiang; Visconti, Nicolo; Corlazzoli, Francesca; Gagliostro, Vincenzo; Somenzi, Giulia; Yao, Jin; Sun, Yijun; Sacchi, Nicoletta

2016-12-27

A hallmark of cancer cells is the ability to evade the growth inhibitory/pro-apoptotic action of physiological all-trans retinoic acid (RA) signal, the bioactive derivative of Vitamin A. However, as we and others reported, RA can also promote cancer cell growth and invasion. Here we show that anticancer and cancer-promoting RA actions in breast cancer have roots in a mechanism of mammary epithelial cell morphogenesis that involves both transcriptional (epigenetic) and non-transcriptional RARα (RARA) functions. We found that the mammary epithelial cell-context specific degree of functionality of the RARA transcriptional (epigenetic) component of this mechanism, by tuning the effects of the non-transcriptional RARA component, determines different cell fate decisions during mammary morphogenesis. Indeed, factors that hamper the RARA epigenetic function make physiological RA drive aberrant morphogenesis via non-transcriptional RARA, thus leading to cell transformation. Remarkably, also the cell context-specific degree of functionality of the RARA epigenetic component retained by breast cancer cells is critical to determine cell fate decisions in response to physiological as well as supraphysiological RA variation. Overall this study supports the proof of principle that the epigenetic functional plasticity of the mammary epithelial cell RARA mechanism, which is essential for normal morphogenetic processes, is necessary to deter breast cancer onset/progression consequent to the insidious action of physiological RA.

Cell cycle progression is required for zebrafish somite morphogenesis but not segmentation clock function

PubMed Central

Zhang, Lixia; Kendrick, Christina; Jülich, Dörthe; Holley, Scott A.

2010-01-01

Summary Cell division, differentiation and morphogenesis are coordinated during embryonic development and frequently in disarray in pathologies such as cancer. Here, we present a zebrafish mutant that ceases mitosis at the beginning of gastrulation, but undergoes axis elongation and develops blood, muscle and a beating heart. We identify the mutation as being in early mitotic inhibitor 1 (emi1), a negative regulator of the Anaphase Promoting Complex, and utilize the mutant to examine the role of the cell cycle in somitogenesis. The mutant phenotype indicates that axis elongation during the segmentation period is substantially driven by cell migration. We find that the segmentation clock, which regulates somitogenesis, functions normally in the absence of cell cycle progression and observe that mitosis is a modest source of noise for the clock. Somite morphogenesis involves the epithelialization of the somite border cells around a core of mesenchyme. As in wild-type embryos, somite boundary cells are polarized along a Fibronectin matrix in emi1−/−. The mutants also display evidence of segment polarity. However, in the absence of a normal cell cycle, somites appear to hyper-epithelialize as the internal mesenchymal cells exit the core of the somite after initial boundary formation. Thus, cell cycle progression is not required during the segmentation period for segmentation clock function but is necessary for normal segmental arrangement of epithelial borders and internal mesenchymal cells. PMID:18480162

Molecular mechanisms controlling pavement cell shape in Arabidopsis leaves.

PubMed

Qian, Pingping; Hou, Suiwen; Guo, Guangqin

2009-08-01

Pavement cells have an interlocking jigsaw puzzle-shaped leaf surface pattern. Twenty-three genes involved in the pavement cell morphogenesis were discovered until now. The mutations of these genes through various means lead to pavement cell shape defects, such as loss or lack of interdigitation, the reduction of lobing, gaps between lobe and neck regions in pavement cells, and distorted trichomes. These phenotypes are affected by the organization of microtubules and microfilaments. Microtubule bands are considered corresponding with the neck regions of the cell, while lobe formation depends on patches of microfilaments. The pathway of Rho of plant (ROP) GTPase signaling cascades regulates overall activity of the cytoskeleton in pavement cells. Some other proteins, in addition to the ROPs, SCAR/WAVE, and ARP2/3 complexes, are also involved in the pavement cell morphogenesis.

Synthetic Morphogenesis.

PubMed

Teague, Brian P; Guye, Patrick; Weiss, Ron

2016-09-01

Throughout biology, function is intimately linked with form. Across scales ranging from subcellular to multiorganismal, the identity and organization of a biological structure's subunits dictate its properties. The field of molecular morphogenesis has traditionally been concerned with describing these links, decoding the molecular mechanisms that give rise to the shape and structure of cells, tissues, organs, and organisms. Recent advances in synthetic biology promise unprecedented control over these molecular mechanisms; this opens the path to not just probing morphogenesis but directing it. This review explores several frontiers in the nascent field of synthetic morphogenesis, including programmable tissues and organs, synthetic biomaterials and programmable matter, and engineering complex morphogenic systems de novo. We will discuss each frontier's objectives, current approaches, constraints and challenges, and future potential. Copyright © 2016 Cold Spring Harbor Laboratory Press; all rights reserved.

Case Study: Organotypic human in vitro models of embryonic ...

EPA Pesticide Factsheets

Morphogenetic fusion of tissues is a common event in embryonic development and disruption of fusion is associated with birth defects of the eye, heart, neural tube, phallus, palate, and other organ systems. Embryonic tissue fusion requires precise regulation of cell-cell and cell-matrix interactions that drive proliferation, differentiation, and morphogenesis. Chemical low-dose exposures can disrupt morphogenesis across space and time by interfering with key embryonic fusion events. The Morphogenetic Fusion Task uses computer and in vitro models to elucidate consequences of developmental exposures. The Morphogenetic Fusion Task integrates multiple approaches to model responses to chemicals that leaad to birth defects, including integrative mining on ToxCast DB, ToxRefDB, and chemical structures, advanced computer agent-based models, and human cell-based cultures that model disruption of cellular and molecular behaviors including mechanisms predicted from integrative data mining and agent-based models. The purpose of the poster is to indicate progress on the CSS 17.02 Virtual Tissue Models Morphogenesis Task 1 products for the Board of Scientific Counselors meeting on Nov 16-17.

HGF-induced serine 897 phosphorylation of EphA2 regulates epithelial morphogenesis of MDCK cells in 3D culture.

PubMed

Harada, Kohei; Negishi, Manabu; Katoh, Hironori

2015-05-15

Expression of EphA2 is upregulated in various cancers that are derived from epithelial cells and correlates with the ability of a cancer cell to undergo migration and invasion. Here we have investigated the role of EphA2 in the epithelial morphogenesis of Madin-Darby canine kidney (MDCK) cells in three-dimensional culture. We show that EphA2 is phosphorylated on serine residue 897 through hepatocyte growth factor (HGF) stimulation using a phosphatidylinositol 3-kinase (PI3K)-Akt-dependent mechanism and that this phosphorylation is required for the formation of extensions, the first step of tubulogenesis, in MDCK cysts. By contrast, stimulation using the ligand ephrinA1 dephosphorylates EphA2 on serine residue 897 and suppresses the HGF-induced morphological change. Furthermore, activation of the small GTPase RhoG is involved in the HGF-induced formation of extensions downstream of EphA2. These observations suggest that a ligand-independent activity of EphA2 contributes to epithelial morphogenesis. © 2015. Published by The Company of Biologists Ltd.

In vivo imaging of basement membrane movement: ECM patterning shapes Hydra polyps.

PubMed

Aufschnaiter, Roland; Zamir, Evan A; Little, Charles D; Özbek, Suat; Münder, Sandra; David, Charles N; Li, Li; Sarras, Michael P; Zhang, Xiaoming

2011-12-01

Growth and morphogenesis during embryonic development, asexual reproduction and regeneration require extensive remodeling of the extracellular matrix (ECM). We used the simple metazoan Hydra to examine the fate of ECM during tissue morphogenesis and asexual budding. In growing Hydra, epithelial cells constantly move towards the extremities of the animal and into outgrowing buds. It is not known, whether these tissue movements involve epithelial migration relative to the underlying matrix or whether cells and ECM are displaced as a composite structure. Furthermore, it is unclear, how the ECM is remodeled to adapt to the shape of developing buds and tentacles. To address these questions, we used a new in vivo labeling technique for Hydra collagen-1 and laminin, and tracked the fate of ECM in all body regions of the animal. Our results reveal that Hydra 'tissue movements' are largely displacements of epithelial cells together with associated ECM. By contrast, during the evagination of buds and tentacles, extensive movement of epithelial cells relative to the matrix is observed, together with local ECM remodeling. These findings provide new insights into the nature of growth and morphogenesis in epithelial tissues.

Measurement of cortical elasticity in Drosophila melanogaster embryos using ferrofluids

PubMed Central

Doubrovinski, Konstantin; Swan, Michael; Polyakov, Oleg; Wieschaus, Eric F.

2017-01-01

Many models of morphogenesis are forced to assume specific mechanical properties of cells, because the actual mechanical properties of living tissues are largely unknown. Here, we measure the rheology of epithelial cells in the cellularizing Drosophila embryo by injecting magnetic particles and studying their response to external actuation. We establish that, on timescales relevant to epithelial morphogenesis, the cytoplasm is predominantly viscous, whereas the cellular cortex is elastic. The timescale of elastic stress relaxation has a lower bound of 4 min, which is comparable to the time required for internalization of the ventral furrow during gastrulation. The cytoplasm was measured to be ∼103-fold as viscous as water. We show that elasticity depends on the actin cytoskeleton and conclude by discussing how these results relate to existing mechanical models of morphogenesis. PMID:28096360

MarvelD3 regulates the c-Jun N-terminal kinase pathway during eye development in Xenopus

PubMed Central

Vacca, Barbara; Sanchez-Heras, Elena; Steed, Emily; Balda, Maria S.; Ohnuma, Shin-Ichi; Sasai, Noriaki; Mayor, Roberto

2016-01-01

ABSTRACT Ocular morphogenesis requires several signalling pathways controlling the expression of transcription factors and cell-cycle regulators. However, despite a well-known mechanism, the dialogue between those signals and factors remains to be unveiled. Here, we identify a requirement for MarvelD3, a tight junction transmembrane protein, in eye morphogenesis in Xenopus. MarvelD3 depletion led to an abnormally pigmented eye or even an eye-less phenotype, which was rescued by ectopic MarvelD3 expression. Altering MarvelD3 expression led to deregulated expression of cell-cycle regulators and transcription factors required for eye development. The eye phenotype was rescued by increased c-Jun terminal Kinase activation. Thus, MarvelD3 links tight junctions and modulation of the JNK pathway to eye morphogenesis. PMID:27870636

The cell shape proteins MreB and MreC control cell morphogenesis by positioning cell wall synthetic complexes.

PubMed

Divakaruni, Arun V; Baida, Cyril; White, Courtney L; Gober, James W

2007-10-01

MreB, the bacterial actin homologue, is thought to function in spatially co-ordinating cell morphogenesis in conjunction with MreC, a protein that wraps around the outside of the cell within the periplasmic space. In Caulobacter crescentus, MreC physically associates with penicillin-binding proteins (PBPs) which catalyse the insertion of intracellularly synthesized precursors into the peptidoglycan cell wall. Here we show that MreC is required for the spatial organization of components of the peptidoglycan-synthesizing holoenzyme in the periplasm and MreB directs the localization of a peptidoglycan precursor synthesis protein in the cytosol. Additionally, fluorescent vancomycin (Van-FL) labelling revealed that the bacterial cytoskeletal proteins MreB and FtsZ, as well as MreC and RodA, were required for peptidoglycan synthetic activity. MreB and FtsZ were found to be required for morphogenesis of the polar stalk. FtsZ was required for a cell cycle-regulated burst of peptidoglycan synthesis early in the cell cycle resulting in the synthesis of cross-band structures, whereas MreB was required for lengthening of the stalk. Thus, the bacterial cytoskeleton and cell shape-determining proteins such as MreC, function in concert to orchestrate the localization of cell wall synthetic complexes resulting in spatially co-ordinated and efficient peptidoglycan synthetic activity.

The influence of matrix properties on growth and morphogenesis of human pancreatic ductal epithelial cells in 3D

PubMed Central

Raza, Asad; Ki, Chang Seok; Lin, Chien-Chi

2013-01-01

A highly tunable synthetic biomimetic hydrogel platform was developed to study the growth and morphogenesis of pancreatic ductal epithelial cells (PDEC) under the influence of a myriad of instructive cues. A PDEC line, PANC-1, was used as a model system to illustrate the importance of matrix compositions on cell fate determination. PANC-1 is an immortalized ductal epithelial cell line widely used in the study of pancreatic tumor cell behaviors. PANC-1 cells are also increasingly explored as a potential cell source for endocrine differentiation. Thus far, most studies related to PANC-1, among other PDEC lines, are performed on 2D culture surfaces. Here, we evaluated the effect of matrix compositions on PANC-1 cell growth and morphogenesis in 3D. Specifically, PANC-1 cells were encapsulated in PEG-based hydrogels prepared by step-growth thiol-ene photopolymerization. It was found that thiol-ene hydrogels provided a cytocompatible environment for encapsulation and 3D culture of PANC-1 cells. In contrast to a monolayer morphology on 2D culture surfaces, PANC-1 cells formed clusters in 3D thiol-ene hydrogels within 4 days of culture. After culturing for 10 days, however, the growth and structures of these clusters were significantly impacted by gel matrix properties, including sensitivity of the matrix to proteases, stiffness of the matrix, and ECM-mimetic motifs. The use of matrix metalloproteinase (MMP) sensitive linker or the immobilization of fibronectin-derived RGDS ligand in the matrix promoted PANC-1 cell growth and encouraged them to adopt ductal cyst-like structures. On the other hand, the encapsulated cells formed smaller and more compact aggregates in non-MMP responsive gels. The incorporation of laminin-derived YIGSR peptide did not enhance cell growth and caused the cells to form compact aggregates. Immobilized YIGSR also enhanced the expression of epithelial cell markers including β-catenin and E-cadherin. These studies have established PEG-peptide hydrogels formed by thiol-ene photo-click reaction as a suitable platform for studying and manipulating pancreatic epithelial cell growth and morphogenesis in 3D. PMID:23602364

Regulation of tight junction assembly and epithelial morphogenesis by the heat shock protein Apg-2

PubMed Central

Aijaz, Saima; Sanchez-Heras, Elena; Balda, Maria S; Matter, Karl

2007-01-01

Background Tight junctions are required for epithelial barrier formation and participate in the regulation of signalling mechanisms that control proliferation and differentiation. ZO-1 is a tight junction-associated adaptor protein that regulates gene expression, junction assembly and epithelial morphogenesis. We have previously demonstrated that the heat shock protein Apg-2 binds ZO-1 and thereby regulates its role in cell proliferation. Here, we addressed the question whether Apg-2 is also important for junction formation and epithelial morphogenesis. Results We demonstrate that depletion of Apg-2 by RNAi in MDCK cells did not prevent formation of functional tight junctions. Similar to ZO-1, however, reduced expression of Apg-2 retarded de novo junction assembly if analysed in a Ca-switch model. Formation of functional junctions, as monitored by measuring transepithelial electrical resistance, and recruitment of tight and adherens junction markers were retarded. If cultured in three dimensional extracellular matrix gels, Apg-2 depleted cells, as previously shown for ZO-1 depleted cells, did not form hollow polarised cysts but poorly organised, irregular structures. Conclusion Our data indicate that Apg-2 regulates junction assembly and is required for normal epithelial morphogenesis in a three-dimensional culture system, suggesting that Apg-2 is an important regulator of epithelial differentiation. As the observed phenotypes are similar to those previously described for ZO-1 depleted cells and depletion of Apg-2 retards junctional recruitment of ZO-1, regulation of ZO-1 is likely to be an important functional role for Apg-2 during epithelial differentiation. PMID:18028534

Regulation of tight junction assembly and epithelial morphogenesis by the heat shock protein Apg-2.

PubMed

Aijaz, Saima; Sanchez-Heras, Elena; Balda, Maria S; Matter, Karl

2007-11-20

Tight junctions are required for epithelial barrier formation and participate in the regulation of signalling mechanisms that control proliferation and differentiation. ZO-1 is a tight junction-associated adaptor protein that regulates gene expression, junction assembly and epithelial morphogenesis. We have previously demonstrated that the heat shock protein Apg-2 binds ZO-1 and thereby regulates its role in cell proliferation. Here, we addressed the question whether Apg-2 is also important for junction formation and epithelial morphogenesis. We demonstrate that depletion of Apg-2 by RNAi in MDCK cells did not prevent formation of functional tight junctions. Similar to ZO-1, however, reduced expression of Apg-2 retarded de novo junction assembly if analysed in a Ca-switch model. Formation of functional junctions, as monitored by measuring transepithelial electrical resistance, and recruitment of tight and adherens junction markers were retarded. If cultured in three dimensional extracellular matrix gels, Apg-2 depleted cells, as previously shown for ZO-1 depleted cells, did not form hollow polarised cysts but poorly organised, irregular structures. Our data indicate that Apg-2 regulates junction assembly and is required for normal epithelial morphogenesis in a three-dimensional culture system, suggesting that Apg-2 is an important regulator of epithelial differentiation. As the observed phenotypes are similar to those previously described for ZO-1 depleted cells and depletion of Apg-2 retards junctional recruitment of ZO-1, regulation of ZO-1 is likely to be an important functional role for Apg-2 during epithelial differentiation.

Morphogenesis in Plants: Modeling the Shoot Apical Meristem, and Possible Applications

NASA Technical Reports Server (NTRS)

Mjolsness, Eric; Gor, Victoria; Meyerowitz, Elliot; Mann, Tobias

1998-01-01

A key determinant of overall morphogenesis in flowering plants such as Arabidopsis thaliana is the shoot apical meristem (growing tip of a shoot). Gene regulation networks can be used to model this system. We exhibit a very preliminary two-dimensional model including gene regulation and intercellular signaling, but omitting cell division and dynamical geometry. The model can be trained to have three stable regions of gene expression corresponding to the central zone, peripheral zone, and rib meristem. We also discuss a space-engineering motivation for studying and controlling the morphogenesis of plants using such computational models.

Distinct Recruitment and Function of Gab1 and Gab2 in Met Receptor-mediated Epithelial Morphogenesis

PubMed Central

Lock, Lisa S.; Maroun, Christiane R.; Naujokas, Monica A.; Park, Morag

2002-01-01

The Gab family of docking proteins (Gab1 and Gab2) are phosphorylated in response to various cytokines and growth factors. Gab1 acts to diversify the signal downstream from the Met receptor tyrosine kinase through the recruitment of multiple signaling proteins, and is essential for epithelial morphogenesis. To determine whether Gab1 and Gab2 are functionally redundant, we have examined the role of Gab2 in epithelial cells. Both Gab1 and Gab2 are expressed in epithelial cells and localize to cell-cell junctions. However, whereas overexpression of Gab1 promotes a morphogenic response, the overexpression of Gab2 fails to induce this response. We show that Gab2 recruitment to the Met receptor is dependent on the Grb2 adapter protein. In contrast, Gab1 recruitment to Met is both Grb2 dependent and Grb2 independent. The latter requires a novel amino acid sequence present in the Met-binding domain of Gab1 but not Gab2. Mutation of these residues in Gab1 impairs both association with the Met receptor and the ability of Gab1 to promote a morphogenic response, whereas their insertion into Gab2 increases Gab2 association with Met, but does not confer on Gab2 the ability to promote epithelial morphogenesis. We propose that the Grb2-independent recruitment of Gab proteins to Met is necessary but not sufficient to promote epithelial morphogenesis. PMID:12058075

Connexin 43 Is Necessary for Salivary Gland Branching Morphogenesis and FGF10-induced ERK1/2 Phosphorylation*

PubMed Central

Yamada, Aya; Futagi, Masaharu; Fukumoto, Emiko; Saito, Kan; Yoshizaki, Keigo; Ishikawa, Masaki; Arakaki, Makiko; Hino, Ryoko; Sugawara, Yu; Ishikawa, Momoko; Naruse, Masahiro; Miyazaki, Kanako; Nakamura, Takashi; Fukumoto, Satoshi

2016-01-01

Cell-cell interaction via the gap junction regulates cell growth and differentiation, leading to formation of organs of appropriate size and quality. To determine the role of connexin43 in salivary gland development, we analyzed its expression in developing submandibular glands (SMGs). Connexin43 (Cx43) was found to be expressed in salivary gland epithelium. In ex vivo organ cultures of SMGs, addition of the gap junctional inhibitors 18α-glycyrrhetinic acid (18α-GA) and oleamide inhibited SMG branching morphogenesis, suggesting that gap junctional communication contributes to salivary gland development. In Cx43−/− salivary glands, submandibular and sublingual gland size was reduced as compared with those from heterozygotes. The expression of Pdgfa, Pdgfb, Fgf7, and Fgf10, which induced branching of SMGs in Cx43−/− samples, were not changed as compared with those from heterozygotes. Furthermore, the blocking peptide for the hemichannel and gap junction channel showed inhibition of terminal bud branching. FGF10 induced branching morphogenesis, while it did not rescue the Cx43−/− phenotype, thus Cx43 may regulate FGF10 signaling during salivary gland development. FGF10 is expressed in salivary gland mesenchyme and regulates epithelial proliferation, and was shown to induce ERK1/2 phosphorylation in salivary epithelial cells, while ERK1/2 phosphorylation in HSY cells was dramatically inhibited by 18α-GA, a Cx43 peptide or siRNA. On the other hand, PDGF-AA and PDGF-BB separately induced ERK1/2 phosphorylation in primary cultured salivary mesenchymal cells regardless of the presence of 18α-GA. Together, our results suggest that Cx43 regulates FGF10-induced ERK1/2 phosphorylation in salivary epithelium but not in mesenchyme during the process of SMG branching morphogenesis. PMID:26565022

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

The APC tumor suppressor is required for epithelial cell polarization and three-dimensional morphogenesis

PubMed Central

Lesko, Alyssa C.; Goss, Kathleen H.; Yang, Frank F.; Schwertner, Adam; Hulur, Imge; Onel, Kenan; Prosperi, Jenifer R.

2015-01-01

The Adenomatous Polyposis Coli (APC) tumor suppressor has been previously implicated in the control of apical-basal polarity; yet, the consequence of APC loss-of-function in epithelial polarization and morphogenesis has not been characterized. To test the hypothesis that APC is required for the establishment of normal epithelial polarity and morphogenesis programs, we generated APC-knockdown epithelial cell lines. APC depletion resulted in loss of polarity and multi-layering on permeable supports, and enlarged, filled spheroids with disrupted polarity in 3D culture. Importantly, these effects of APC knockdown were independent of Wnt/β-catenin signaling, but were rescued with either full-length or a carboxy (c)-terminal segment of APC. Moreover, we identified a gene expression signature associated with APC knockdown that points to several candidates known to regulate cell-cell and cell-matrix communication. Analysis of epithelial tissues from mice and humans carrying heterozygous APC mutations further support the importance of APC as a regulator of epithelial behavior and tissue architecture. These data also suggest that the initiation of epithelial-derived tumors as a result of APC mutation or gene silencing may be driven by loss of polarity and dysmorphogenesis. PMID:25578398

The development and geometry of shape change in Arabidopsis thaliana cotyledon pavement cells

PubMed Central

2011-01-01

Background The leaf epidermis is an important architectural control element that influences the growth properties of underlying tissues and the overall form of the organ. In dicots, interdigitated pavement cells are the building blocks of the tissue, and their morphogenesis includes the assembly of specialized cell walls that surround the apical, basal, and lateral (anticlinal) cell surfaces. The microtubule and actin cytoskeletons are highly polarized along the cortex of the anticlinal wall; however, the relationships between these arrays and cell morphogenesis are unclear. Results We developed new quantitative tools to compare population-level growth statistics with time-lapse imaging of cotyledon pavement cells in an intact tissue. The analysis revealed alternating waves of lobe initiation and a phase of lateral isotropic expansion that persisted for days. During lateral isotropic diffuse growth, microtubule organization varied greatly between cell surfaces. Parallel microtubule bundles were distributed unevenly along the anticlinal surface, with subsets marking stable cortical domains at cell indentations and others clearly populating the cortex within convex cell protrusions. Conclusions Pavement cell morphogenesis is discontinuous, and includes punctuated phases of lobe initiation and lateral isotropic expansion. In the epidermis, lateral isotropic growth is independent of pavement cell size and shape. Cortical microtubules along the upper cell surface and stable cortical patches of anticlinal microtubules may coordinate the growth behaviors of orthogonal cell walls. This work illustrates the importance of directly linking protein localization data to the growth behavior of leaf epidermal cells. PMID:21284861

The development and geometry of shape change in Arabidopsis thaliana cotyledon pavement cells.

PubMed

Zhang, Chunhua; Halsey, Leah E; Szymanski, Daniel B

2011-02-01

The leaf epidermis is an important architectural control element that influences the growth properties of underlying tissues and the overall form of the organ. In dicots, interdigitated pavement cells are the building blocks of the tissue, and their morphogenesis includes the assembly of specialized cell walls that surround the apical, basal, and lateral (anticlinal) cell surfaces. The microtubule and actin cytoskeletons are highly polarized along the cortex of the anticlinal wall; however, the relationships between these arrays and cell morphogenesis are unclear. We developed new quantitative tools to compare population-level growth statistics with time-lapse imaging of cotyledon pavement cells in an intact tissue. The analysis revealed alternating waves of lobe initiation and a phase of lateral isotropic expansion that persisted for days. During lateral isotropic diffuse growth, microtubule organization varied greatly between cell surfaces. Parallel microtubule bundles were distributed unevenly along the anticlinal surface, with subsets marking stable cortical domains at cell indentations and others clearly populating the cortex within convex cell protrusions. Pavement cell morphogenesis is discontinuous, and includes punctuated phases of lobe initiation and lateral isotropic expansion. In the epidermis, lateral isotropic growth is independent of pavement cell size and shape. Cortical microtubules along the upper cell surface and stable cortical patches of anticlinal microtubules may coordinate the growth behaviors of orthogonal cell walls. This work illustrates the importance of directly linking protein localization data to the growth behavior of leaf epidermal cells.

Integration of actomyosin contractility with cell-cell adhesion during dorsal closure.

PubMed

Duque, Julia; Gorfinkiel, Nicole

2016-12-15

In this work, we combine genetic perturbation, time-lapse imaging and quantitative image analysis to investigate how pulsatile actomyosin contractility drives cell oscillations, apical cell contraction and tissue closure during morphogenesis of the amnioserosa, the main force-generating tissue during the dorsal closure in Drosophila We show that Myosin activity determines the oscillatory and contractile behaviour of amnioserosa cells. Reducing Myosin activity prevents cell shape oscillations and reduces cell contractility. By contrast, increasing Myosin activity increases the amplitude of cell shape oscillations and the time cells spend in the contracted phase relative to the expanded phase during an oscillatory cycle, promoting cell contractility and tissue closure. Furthermore, we show that in AS cells, Rok controls Myosin foci formation and Mbs regulates not only Myosin phosphorylation but also adhesion dynamics through control of Moesin phosphorylation, showing that Mbs coordinates actomyosin contractility with cell-cell adhesion during amnioserosa morphogenesis. © 2016. Published by The Company of Biologists Ltd.

Asymmetric cell division of stem cells in the lung and other systems

PubMed Central

Berika, Mohamed; Elgayyar, Marwa E.; El-Hashash, Ahmed H. K.

2014-01-01

New insights have been added to identification, behavior and cellular properties of embryonic and tissue-specific stem cells over the last few years. The modes of stem cell division, asymmetric vs. symmetric, are tightly regulated during development and regeneration. The proper choice of a stem cell to divide asymmetrically or symmetrically has great consequences for development and disease because inappropriate asymmetric division disrupts organ morphogenesis, whereas uncontrolled symmetric division induces tumorigenesis. Therefore, understanding the behavior of lung stem cells could identify innovative solutions for restoring normal morphogenesis and/or regeneration of different organs. In this concise review, we describe recent studies in our laboratory about the mode of division of lung epithelial stem cells. We also compare asymmetric cell division (ACD) in the lung stem cells with other tissues in different organisms. PMID:25364740

SILAC-based proteomics of human primary endothelial cell morphogenesis unveils tumor angiogenic markers.

PubMed

Zanivan, Sara; Maione, Federica; Hein, Marco Y; Hernández-Fernaud, Juan Ramon; Ostasiewicz, Pawel; Giraudo, Enrico; Mann, Matthias

2013-12-01

Proteomics has been successfully used for cell culture on dishes, but more complex cellular systems have proven to be challenging and so far poorly approached with proteomics. Because of the complexity of the angiogenic program, we still do not have a complete understanding of the molecular mechanisms involved in this process, and there have been no in depth quantitative proteomic studies. Plating endothelial cells on matrigel recapitulates aspects of vessel growth, and here we investigate this mechanism by using a spike-in SILAC quantitative proteomic approach. By comparing proteomic changes in primary human endothelial cells morphogenesis on matrigel to general adhesion mechanisms in cells spreading on culture dish, we pinpoint pathways and proteins modulated by endothelial cells. The cell-extracellular matrix adhesion proteome depends on the adhesion substrate, and a detailed proteomic profile of the extracellular matrix secreted by endothelial cells identified CLEC14A as a matrix component, which binds to MMRN2. We verify deregulated levels of these proteins during tumor angiogenesis in models of multistage carcinogenesis. This is the most in depth quantitative proteomic study of endothelial cell morphogenesis, which shows the potential of applying high accuracy quantitative proteomics to in vitro models of vessel growth to shed new light on mechanisms that accompany pathological angiogenesis. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium with the data set identifier PXD000359.

Cell death and morphogenesis during early mouse development: Are they interconnected?

PubMed Central

Bedzhov, Ivan; Zernicka-Goetz, Magdalena

2015-01-01

Shortly after implantation the embryonic lineage transforms from a coherent ball of cells into polarized cup shaped epithelium. Recently we elucidated a previously unknown apoptosis-independent morphogenic event that reorganizes the pluripotent lineage. Polarization cues from the surrounding basement membrane rearrange the epiblast into a polarized rosette-like structure, where subsequently a central lumen is established. Thus, we provided a new model revising the current concept of apoptosis-dependent epiblast morphogenesis. Cell death however has to be tightly regulated during embryogenesis to ensure developmental success. Here, we follow the stages of early mouse development and take a glimpse at the critical signaling and morphogenic events that determine cells destiny and reshape the embryonic lineage. PMID:25640415

Using cell deformation and motion to predict forces and collective behavior in morphogenesis.

PubMed

Merkel, Matthias; Manning, M Lisa

2017-07-01

In multi-cellular organisms, morphogenesis translates processes at the cellular scale into tissue deformation at the scale of organs and organisms. To understand how biochemical signaling regulates tissue form and function, we must understand the mechanical forces that shape cells and tissues. Recent progress in developing mechanical models for tissues has led to quantitative predictions for how cell shape changes and polarized cell motility generate forces and collective behavior on the tissue scale. In particular, much insight has been gained by thinking about biological tissues as physical materials composed of cells. Here we review these advances and discuss how they might help shape future experiments in developmental biology. Copyright © 2016 Elsevier Ltd. All rights reserved.

Stromal–epithelial cell interactions and alteration of branching morphogenesis in macromastic mammary glands

PubMed Central

Zhong, Aimei; Wang, Guohua; Yang, Jie; Xu, Qijun; Yuan, Quan; Yang, Yanqing; Xia, Yun; Guo, Ke; Horch, Raymund E; Sun, Jiaming

2014-01-01

True macromastia is a rare but disabling condition characterized by massive breast growth. The aetiology and pathogenic mechanisms for this disorder remain largely unexplored because of the lack of in vivo or in vitro models. Previous studies suggested that regulation of epithelial cell growth and development by oestrogen was dependent on paracrine growth factors from the stroma. In this study, a co-culture model containing epithelial and stromal cells was used to investigate the interactions of these cells in macromastia. Epithelial cell proliferation and branching morphogenesis were measured to assess the effect of macromastic stromal cells on epithelial cells. We analysed the cytokines secreted by stromal cells and identified molecules that were critical for effects on epithelial cells. Our results indicated a significant increase in cell proliferation and branching morphogenesis of macromastic and non-macromastic epithelial cells when co-cultured with macromastic stromal cells or in conditioned medium from macromastic stromal cells. Hepatocyte growth factor (HGF) is a key factor in epithelial–stromal interactions of macromastia-derived cell cultures. Blockade of HGF with neutralizing antibodies dramatically attenuated epithelial cell proliferation in conditioned medium from macromastic stromal cells. The epithelial–stromal cell co-culture model demonstrated reliability for studying interactions of mammary stromal and epithelial cells in macromastia. In this model, HGF secreted by macromastic stromal cells was found to play an important role in modifying the behaviour of co-cultured epithelial cells. This model allows further studies to investigate basic cellular and molecular mechanisms in tissue from patients with true breast hypertrophy. PMID:24720804

Contraction and elongation: Mechanics underlying cell boundary deformations in epithelial tissue.

PubMed

Hara, Yusuke

2017-06-01

The cell-cell boundaries of epithelial cells form cellular frameworks at the apical side of tissues. Deformations in these boundaries, for example, boundary contraction and elongation, and the associated forces form the mechanical basis of epithelial tissue morphogenesis. In this review, using data from recent Drosophila studies on cell boundary contraction and elongation, I provide an overview of the mechanism underlying the bi-directional deformations in the epithelial cell boundary, that are sustained by biased accumulations of junctional and apico-medial non-muscle myosin II. Moreover, how the junctional tensions exist on cell boundaries in different boundary dynamics and morphologies are discussed. Finally, some future perspectives on how recent knowledge about single cell boundary-level mechanics will contribute to our understanding of epithelial tissue morphogenesis are discussed. © 2017 Japanese Society of Developmental Biologists.

Micropatterning of cells reveals chiral morphogenesis

PubMed Central

2013-01-01

Invariant left-right (LR) patterning or chirality is critical for embryonic development. The loss or reversal of LR asymmetry is often associated with malformations and disease. Although several theories have been proposed, the exact mechanism of the initiation of the LR symmetry has not yet been fully elucidated. Recently, chirality has been detected within single cells as well as multicellular structures using several in vitro approaches. These studies demonstrated the universality of cell chirality, its dependence on cell phenotype, and the role of physical boundaries. In this review, we discuss the theories for developmental LR asymmetry, compare various in vitro cell chirality model systems, and highlight possible roles of cell chirality in stem cell differentiation. We emphasize that the in vitro cell chirality systems have great promise for helping unveil the nature of chiral morphogenesis in development. PMID:23672821

Impaired hair follicle morphogenesis and polarized keratinocyte movement upon conditional inactivation of integrin-linked kinase in the epidermis.

PubMed

Nakrieko, Kerry-Ann; Welch, Ian; Dupuis, Holly; Bryce, Dawn; Pajak, Agnieszka; St Arnaud, René; Dedhar, Shoukat; D'Souza, Sudhir J A; Dagnino, Lina

2008-04-01

Integrin-linked kinase (ILK) is key for cell survival, migration, and adhesion, but little is known about its role in epidermal development and homeostasis in vivo. We generated mice with conditional inactivation of the Ilk gene in squamous epithelia. These mice die perinatally and exhibit skin blistering and severe defects in hair follicle morphogenesis, including greatly reduced follicle numbers, failure to progress beyond very early developmental stages, and pronounced defects in follicular keratinocyte proliferation. ILK-deficient epidermis shows abnormalities in adhesion to the basement membrane and in differentiation. ILK-deficient cultured keratinocytes fail to attach and spread efficiently and exhibit multiple abnormalities in actin cytoskeletal organization. Ilk gene inactivation in cultured keratinocytes causes impaired ability to form stable lamellipodia, to directionally migrate, and to polarize. These defects are accompanied by abnormal distribution of active Cdc42 to cell protrusions, as well as reduced activation of Rac1 upon induction of cell migration in scraped keratinocyte monolayers. Significantly, alterations in cell spreading and forward movement in single cells can be rescued by expression of constitutively active Rac1 or RhoG. Our studies underscore a central and distinct role for ILK in hair follicle development and in polarized cell movements, two key aspects of epithelial morphogenesis and function.

Impaired Hair Follicle Morphogenesis and Polarized Keratinocyte Movement upon Conditional Inactivation of Integrin-linked Kinase in the Epidermis

PubMed Central

Nakrieko, Kerry-Ann; Welch, Ian; Dupuis, Holly; Bryce, Dawn; Pajak, Agnieszka; St. Arnaud, René; Dedhar, Shoukat

2008-01-01

Integrin-linked kinase (ILK) is key for cell survival, migration, and adhesion, but little is known about its role in epidermal development and homeostasis in vivo. We generated mice with conditional inactivation of the Ilk gene in squamous epithelia. These mice die perinatally and exhibit skin blistering and severe defects in hair follicle morphogenesis, including greatly reduced follicle numbers, failure to progress beyond very early developmental stages, and pronounced defects in follicular keratinocyte proliferation. ILK-deficient epidermis shows abnormalities in adhesion to the basement membrane and in differentiation. ILK-deficient cultured keratinocytes fail to attach and spread efficiently and exhibit multiple abnormalities in actin cytoskeletal organization. Ilk gene inactivation in cultured keratinocytes causes impaired ability to form stable lamellipodia, to directionally migrate, and to polarize. These defects are accompanied by abnormal distribution of active Cdc42 to cell protrusions, as well as reduced activation of Rac1 upon induction of cell migration in scraped keratinocyte monolayers. Significantly, alterations in cell spreading and forward movement in single cells can be rescued by expression of constitutively active Rac1 or RhoG. Our studies underscore a central and distinct role for ILK in hair follicle development and in polarized cell movements, two key aspects of epithelial morphogenesis and function. PMID:18234842

Retinoic Acid signaling regulates Krt5 and Krt14 independently of stem cell markers in submandibular salivary gland epithelium

PubMed Central

Abashev, Timur M.; Metzler, Melissa A.; Wright, Diana M.; Sandell, Lisa L.

2017-01-01

Background Retinoic Acid (RA), the active metabolite of Vitamin A, has been demonstrated to be important for growth and branching morphogenesis of mammalian embryonic salivary gland epithelium. However, it is not known whether RA functions directly within epithelial cells or in associated tissues that influence morphogenesis of salivary epithelium. Moreover, downstream targets of RA regulation have not been identified. Results Here we show that canonical RA signaling occurs in multiple tissues of embryonic mouse salivary glands, including epithelium, associated parasympathetic ganglion neurons, and non-neuronal mesenchyme. By culturing epithelium explants in isolation from other tissues we demonstrate that RA influences epithelium morphogenesis by direct action in that tissue. Moreover, we demonstrate that inhibition of RA signaling represses cell proliferation and expression of FGF10 signaling targets, and upregulates expression of basal epithelial keratins Krt5 and Krt14. Importantly, we show that the stem cell gene Kit is regulated inversely from Krt5/Krt14 by RA signaling. Conclusions RA regulates Krt5 and Krt14 expression independently of stem cell character in developing salivary epithelium. RA, or chemical inhibitors of RA signaling, could potentially be used for modulating growth and differentiation of epithelial stem cells for the purpose of re-populating damaged glands or generating bioengineered organs. PMID:27884045

Effects of substrate conductivity on cell morphogenesis and proliferation using tailored, atomic layer deposition-grown ZnO thin films

PubMed Central

Choi, Won Jin; Jung, Jongjin; Lee, Sujin; Chung, Yoon Jang; Yang, Cheol-Soo; Lee, Young Kuk; Lee, You-Seop; Park, Joung Kyu; Ko, Hyuk Wan; Lee, Jeong-O

2015-01-01

We demonstrate that ZnO films grown by atomic layer deposition (ALD) can be employed as a substrate to explore the effects of electrical conductivity on cell adhesion, proliferation, and morphogenesis. ZnO substrates with precisely tunable electrical conductivity were fabricated on glass substrates using ALD deposition. The electrical conductivity of the film increased linearly with increasing duration of the ZnO deposition cycle (thickness), whereas other physical characteristics, such as surface energy and roughness, tended to saturate at a certain value. Differences in conductivity dramatically affected the behavior of SF295 glioblastoma cells grown on ZnO films, with high conductivity (thick) ZnO films causing growth arrest and producing SF295 cell morphologies distinct from those cultured on insulating substrates. Based on simple electrostatic calculations, we propose that cells grown on highly conductive substrates may strongly adhere to the substrate without focal-adhesion complex formation, owing to the enhanced electrostatic interaction between cells and the substrate. Thus, the inactivation of focal adhesions leads to cell proliferation arrest. Taken together, the work presented here confirms that substrates with high conductivity disturb the cell-substrate interaction, producing cascading effects on cellular morphogenesis and disrupting proliferation, and suggests that ALD-grown ZnO offers a single-variable method for uniquely tailoring conductivity. PMID:25897486

Reassessing the roles of PIN proteins and anticlinal microtubules during pavement cell morphogenesis

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

Belteton, Samuel; Sawchuk, Megan G.; Donohoe, Bryon S.

The leaf epidermis is a biomechanical shell that influences the size and shape of the organ. Its morphogenesis is a multiscale process in which nanometer-scale cytoskeletal protein complexes, individual cells, and groups of cells pattern growth and define macroscopic leaf traits. Interdigitated growth of neighboring cells is an evolutionarily conserved developmental strategy. Understanding how signaling pathways and cytoskeletal proteins pattern cell walls during this form of tissue morphogenesis is an important research challenge. The cellular and molecular control of a lobed cell morphology is currently thought to involve PIN-FORMED (PIN)-type plasma membrane efflux carriers that generate subcellular auxin gradients. Auxinmore » gradients were proposed to function across cell boundaries to encode stable offset patterns of cortical microtubules and actin filaments between adjacent cells. Many models suggest that long-lived microtubules along the anticlinal cell wall generate local cell wall heterogeneities that restrict local growth and specify the timing and location of lobe formation. Here we used Arabidopsis reverse genetics and multivariate long-term time-lapse imaging to test current cell shape control models. We found that neither PIN proteins nor microtubules along the anticlinal wall predict the patterns of lobe formation. In fields of lobing cells, anticlinal microtubules are not correlated with cell shape and are unstable at the time scales of cell expansion. Our analyses indicate that anticlinal microtubules have multiple functions in pavement cells, and that lobe initiation is likely controlled by complex interactions among cell geometry, cell wall stress patterns, and transient microtubule networks that span the anticlinal and periclinal walls.« less

Reassessing the Roles of PIN Proteins and Anticlinal Microtubules during Pavement Cell Morphogenesis1[OPEN

PubMed Central

Sawchuk, Megan G.; Scarpella, Enrico

2018-01-01

The leaf epidermis is a biomechanical shell that influences the size and shape of the organ. Its morphogenesis is a multiscale process in which nanometer-scale cytoskeletal protein complexes, individual cells, and groups of cells pattern growth and define macroscopic leaf traits. Interdigitated growth of neighboring cells is an evolutionarily conserved developmental strategy. Understanding how signaling pathways and cytoskeletal proteins pattern cell walls during this form of tissue morphogenesis is an important research challenge. The cellular and molecular control of a lobed cell morphology is currently thought to involve PIN-FORMED (PIN)-type plasma membrane efflux carriers that generate subcellular auxin gradients. Auxin gradients were proposed to function across cell boundaries to encode stable offset patterns of cortical microtubules and actin filaments between adjacent cells. Many models suggest that long-lived microtubules along the anticlinal cell wall generate local cell wall heterogeneities that restrict local growth and specify the timing and location of lobe formation. Here, we used Arabidopsis (Arabidopsis thaliana) reverse genetics and multivariate long-term time-lapse imaging to test current cell shape control models. We found that neither PIN proteins nor long-lived microtubules along the anticlinal wall predict the patterns of lobe formation. In fields of lobing cells, anticlinal microtubules are not correlated with cell shape and are unstable at the time scales of cell expansion. Our analyses indicate that anticlinal microtubules have multiple functions in pavement cells and that lobe initiation is likely controlled by complex interactions among cell geometry, cell wall stress patterns, and transient microtubule networks that span the anticlinal and periclinal walls. PMID:29192026

MicroRNAs and the Evolution of Insect Metamorphosis.

PubMed

Belles, Xavier

2017-01-31

MicroRNAs (miRNAs) are involved in the regulation of a number of processes associated with metamorphosis, either in the less modified hemimetabolan mode or in the more modified holometabolan mode. The miR-100/let-7/miR-125 cluster has been studied extensively, especially in relation to wing morphogenesis in both hemimetabolan and holometabolan species. Other miRNAs also participate in wing morphogenesis, as well as in programmed cell and tissue death, neuromaturation, neuromuscular junction formation, and neuron cell fate determination, typically during the pupal stage of holometabolan species. A special case is the control of miR-2 over Kr-h1 transcripts, which determines adult morphogenesis in the hemimetabolan metamorphosis. This is an elegant example of how a single miRNA can control an entire process by acting on a crucial mediator; however, this is a quite exceptional mechanism that was apparently lost during the transition from hemimetaboly to holometaboly.

Engineering Three-dimensional Epithelial Tissues Embedded within Extracellular Matrix.

PubMed

Piotrowski-Daspit, Alexandra S; Nelson, Celeste M

2016-07-10

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, which is regulated by a variety of soluble and physical signals in the microenvironment. Described here is a method created to study the process of branching morphogenesis by forming engineered three-dimensional (3D) epithelial tissues of defined shape and size that are completely embedded within an extracellular matrix (ECM). This method enables the formation of arrays of identical tissues and enables the control of a variety of environmental factors, including tissue geometry, spacing, and ECM composition. This method can also be combined with widely used techniques such as traction force microscopy (TFM) to gain more information about the interactions between cells and their surrounding ECM. The protocol can be used to investigate a variety of cell and tissue processes beyond branching morphogenesis, including cancer invasion.

Computer simulation analysis of normal and abnormal development of the mammalian diaphragm

PubMed Central

Fisher, Jason C; Bodenstein, Lawrence

2006-01-01

Background Congenital diaphragmatic hernia (CDH) is a birth defect with significant morbidity and mortality. Knowledge of diaphragm morphogenesis and the aberrations leading to CDH is limited. Although classical embryologists described the diaphragm as arising from the septum transversum, pleuroperitoneal folds (PPF), esophageal mesentery and body wall, animal studies suggest that the PPF is the major, if not sole, contributor to the muscular diaphragm. Recently, a posterior defect in the PPF has been identified when the teratogen nitrofen is used to induce CDH in fetal rodents. We describe use of a cell-based computer modeling system (Nudge++™) to study diaphragm morphogenesis. Methods and results Key diaphragmatic structures were digitized from transverse serial sections of paraffin-embedded mouse embryos at embryonic days 11.5 and 13. Structure boundaries and simulated cells were combined in the Nudge++™ software. Model cells were assigned putative behavioral programs, and these programs were progressively modified to produce a diaphragm consistent with the observed anatomy in rodents. Homology between our model and recent anatomical observations occurred under the following simulation conditions: (1) cell mitoses are restricted to the edge of growing tissue; (2) cells near the chest wall remain mitotically active; (3) mitotically active non-edge cells migrate toward the chest wall; and (4) movement direction depends on clonal differentiation between anterior and posterior PPF cells. Conclusion With the PPF as the sole source of mitotic cells, an early defect in the PPF evolves into a posteromedial diaphragm defect, similar to that of the rodent nitrofen CDH model. A posterolateral defect, as occurs in human CDH, would be more readily recreated by invoking other cellular contributions. Our results suggest that recent reports of PPF-dominated diaphragm morphogenesis in the rodent may not be strictly applicable to man. The ability to recreate a CDH defect using a combination of experimental data and testable hypotheses gives impetus to simulation modeling as an adjunct to experimental analysis of diaphragm morphogenesis. PMID:16483386

Computer simulation analysis of normal and abnormal development of the mammalian diaphragm.

PubMed

Fisher, Jason C; Bodenstein, Lawrence

2006-02-17

Congenital diaphragmatic hernia (CDH) is a birth defect with significant morbidity and mortality. Knowledge of diaphragm morphogenesis and the aberrations leading to CDH is limited. Although classical embryologists described the diaphragm as arising from the septum transversum, pleuroperitoneal folds (PPF), esophageal mesentery and body wall, animal studies suggest that the PPF is the major, if not sole, contributor to the muscular diaphragm. Recently, a posterior defect in the PPF has been identified when the teratogen nitrofen is used to induce CDH in fetal rodents. We describe use of a cell-based computer modeling system (Nudge++) to study diaphragm morphogenesis. Key diaphragmatic structures were digitized from transverse serial sections of paraffin-embedded mouse embryos at embryonic days 11.5 and 13. Structure boundaries and simulated cells were combined in the Nudge++ software. Model cells were assigned putative behavioral programs, and these programs were progressively modified to produce a diaphragm consistent with the observed anatomy in rodents. Homology between our model and recent anatomical observations occurred under the following simulation conditions: (1) cell mitoses are restricted to the edge of growing tissue; (2) cells near the chest wall remain mitotically active; (3) mitotically active non-edge cells migrate toward the chest wall; and (4) movement direction depends on clonal differentiation between anterior and posterior PPF cells. With the PPF as the sole source of mitotic cells, an early defect in the PPF evolves into a posteromedial diaphragm defect, similar to that of the rodent nitrofen CDH model. A posterolateral defect, as occurs in human CDH, would be more readily recreated by invoking other cellular contributions. Our results suggest that recent reports of PPF-dominated diaphragm morphogenesis in the rodent may not be strictly applicable to man. The ability to recreate a CDH defect using a combination of experimental data and testable hypotheses gives impetus to simulation modeling as an adjunct to experimental analysis of diaphragm morphogenesis.

Sox17 drives functional engraftment of endothelium converted from non-vascular cells.

PubMed

Schachterle, William; Badwe, Chaitanya R; Palikuqi, Brisa; Kunar, Balvir; Ginsberg, Michael; Lis, Raphael; Yokoyama, Masataka; Elemento, Olivier; Scandura, Joseph M; Rafii, Shahin

2017-01-16

Transplanting vascular endothelial cells (ECs) to support metabolism and express regenerative paracrine factors is a strategy to treat vasculopathies and to promote tissue regeneration. However, transplantation strategies have been challenging to develop, because ECs are difficult to culture and little is known about how to direct them to stably integrate into vasculature. Here we show that only amniotic cells could convert to cells that maintain EC gene expression. Even so, these converted cells perform sub-optimally in transplantation studies. Constitutive Akt signalling increases expression of EC morphogenesis genes, including Sox17, shifts the genomic targeting of Fli1 to favour nearby Sox consensus sites and enhances the vascular function of converted cells. Enforced expression of Sox17 increases expression of morphogenesis genes and promotes integration of transplanted converted cells into injured vessels. Thus, Ets transcription factors specify non-vascular, amniotic cells to EC-like cells, whereas Sox17 expression is required to confer EC function.

The large Maf factor Traffic Jam controls gonad morphogenesis in Drosophila.

PubMed

Li, Michelle A; Alls, Jeffrey D; Avancini, Rita M; Koo, Karen; Godt, Dorothea

2003-11-01

Interactions between somatic and germline cells are critical for the normal development of egg and sperm. Here we show that the gene traffic jam (tj) produces a soma-specific factor that controls gonad morphogenesis and is required for female and male fertility. tj encodes the only large Maf factor in Drosophila melanogaster, an orthologue of the atypical basic Leu zipper transcription factors c-Maf and MafB/Kreisler in vertebrates. Expression of tj occurs in somatic gonadal cells that are in direct contact with germline cells throughout development. In tj mutant gonads, somatic cells fail to inter-mingle and properly envelop germline cells, causing an early block in germ cell differentiation. In addition, tj mutant somatic cells show an increase in the level of expression for several adhesion molecules. We propose that tj is a critical modulator of the adhesive properties of somatic cells, facilitating germline-soma interactions that are essential for germ cell differentiation.

Plant Growth and Morphogenesis under Different Gravity Conditions: Relevance to Plant Life in Space.

PubMed

Hoson, Takayuki

2014-05-16

The growth and morphogenesis of plants are entirely dependent on the gravitational acceleration of earth. Under microgravity conditions in space, these processes are greatly modified. Recent space experiments, in combination with ground-based studies, have shown that elongation growth is stimulated and lateral expansion suppressed in various shoot organs and roots under microgravity conditions. Plant organs also show automorphogenesis in space, which consists of altered growth direction and spontaneous curvature in the dorsiventral (back and front) directions. Changes in cell wall properties are responsible for these modifications of growth and morphogenesis under microgravity conditions. Plants live in space with interesting new sizes and forms.

Nrk2b-mediated NAD+ production regulates cell adhesion and is required for muscle morphogenesis in vivo

PubMed Central

Goody, Michelle F.; Kelly, Meghan W.; Lessard, Kevin N.; Khalil, Andre; Henry, Clarissa A.

2010-01-01

Cell-matrix adhesion complexes (CMACs) play fundamental roles during morphogenesis. Given the ubiquitous nature of CMACs and their roles in many cellular processes, one question is how specificity of CMAC function is modulated. The clearly defined cell behaviors that generate segmentally reiterated axial skeletal muscle during zebrafish development comprise an ideal system with which to investigate CMAC function during morphogenesis. We found that Nicotinamide riboside kinase 2b (Nrk2b) cell autonomously modulates the molecular composition of CMACs in vivo. Nrk2b is required for normal Laminin polymerization at the myotendinous junction (MTJ). In Nrk2b-deficient embryos, at MTJ loci where Laminin is not properly polymerized, muscle fibers elongate into adjacent myotomes and are abnormally long. In yeast and human cells, Nrk2 phosphorylates Nicotinamide Riboside and generates NAD+ through an alternative salvage pathway. Exogenous NAD+ treatment rescues MTJ development in Nrk2b-deficient embryos, but not in laminin mutant embryos. Both Nrk2b and Laminin are required for localization of Paxillin, but not β-Dystroglycan, to CMACs at the MTJ. Overexpression of Paxillin in Nrk2b-deficient embryos is sufficient to rescue MTJ integrity. Taken together, these data show that Nrk2b plays a specific role in modulating subcellular localization of discrete CMAC components that in turn play roles in musculoskeletal development. Furthermore, these data suggest that Nrk2b-mediated synthesis of NAD+ is functionally upstream of Laminin adhesion and Paxillin subcellular localization during MTJ development. These results indicate a previously unrecognized complexity to CMAC assembly in vivo and also elucidate a novel role for NAD+ during morphogenesis. PMID:20566368

hmmr mediates anterior neural tube closure and morphogenesis in the frog Xenopus.

PubMed

Prager, Angela; Hagenlocher, Cathrin; Ott, Tim; Schambony, Alexandra; Feistel, Kerstin

2017-10-01

Development of the central nervous system requires orchestration of morphogenetic processes which drive elevation and apposition of the neural folds and their fusion into a neural tube. The newly formed tube gives rise to the brain in anterior regions and continues to develop into the spinal cord posteriorly. Conspicuous differences between the anterior and posterior neural tube become visible already during neural tube closure (NTC). Planar cell polarity (PCP)-mediated convergent extension (CE) movements are restricted to the posterior neural plate, i.e. hindbrain and spinal cord, where they propagate neural fold apposition. The lack of CE in the anterior neural plate correlates with a much slower mode of neural fold apposition anteriorly. The morphogenetic processes driving anterior NTC have not been addressed in detail. Here, we report a novel role for the breast cancer susceptibility gene and microtubule (MT) binding protein Hmmr (Hyaluronan-mediated motility receptor, RHAMM) in anterior neurulation and forebrain development in Xenopus laevis. Loss of hmmr function resulted in a lack of telencephalic hemisphere separation, arising from defective roof plate formation, which in turn was caused by impaired neural tissue narrowing. hmmr regulated polarization of neural cells, a function which was dependent on the MT binding domains. hmmr cooperated with the core PCP component vangl2 in regulating cell polarity and neural morphogenesis. Disrupted cell polarization and elongation in hmmr and vangl2 morphants prevented radial intercalation (RI), a cell behavior essential for neural morphogenesis. Our results pinpoint a novel role of hmmr in anterior neural development and support the notion that RI is a major driving force for anterior neurulation and forebrain morphogenesis. Copyright © 2017 Elsevier Inc. All rights reserved.

The Epithelial Cell Adhesion Molecule EpCAM Is Required for Epithelial Morphogenesis and Integrity during Zebrafish Epiboly and Skin Development

PubMed Central

Slanchev, Krasimir; Carney, Thomas J.; Stemmler, Marc P.; Koschorz, Birgit; Amsterdam, Adam; Schwarz, Heinz; Hammerschmidt, Matthias

2009-01-01

The aberrant expression of the transmembrane protein EpCAM is associated with tumor progression, affecting different cellular processes such as cell–cell adhesion, migration, proliferation, differentiation, signaling, and invasion. However, the in vivo function of EpCAM still remains elusive due to the lack of genetic loss-of-function studies. Here, we describe epcam (tacstd) null mutants in zebrafish. Maternal-zygotic mutants display compromised basal protrusive activity and epithelial morphogenesis in cells of the enveloping layer (EVL) during epiboly. In partial redundancy with E-cadherin (Ecad), EpCAM made by EVL cells is further required for cell–cell adhesion within the EVL and, possibly, for proper attachment of underlying deep cells to the inner surface of the EVL, thereby also affecting deep cell epiboly movements. During later development, EpCAM per se becomes indispensable for epithelial integrity within the periderm of the skin, secondarily leading to disrupted morphology of the underlying basal epidermis and moderate hyper-proliferation of skin cells. On the molecular level, EVL cells of epcam mutant embryos display reduced levels of membranous Ecad, accompanied by an enrichment of tight junction proteins and a basal extension of apical junction complexes (AJCs). Our data suggest that EpCAM acts as a partner of E-cadherin to control adhesiveness and integrity as well as plasticity and morphogenesis within simple epithelia. In addition, EpCAM is required for the interaction of the epithelia with underlying cell layers. PMID:19609345

The fibronectin leucine-rich repeat transmembrane protein Flrt2 is required in the epicardium to promote heart morphogenesis

PubMed Central

Müller, Pari-Sima; Schulz, Ramona; Maretto, Silvia; Costello, Ita; Srinivas, Shankar; Bikoff, Elizabeth; Robertson, Elizabeth

2011-01-01

The epicardium, the outermost tissue layer that envelops the developing heart and provides essential trophic signals for the myocardium, derives from the pro-epicardial organ (PEO). Two of the three members of the Flrt family of transmembrane glycoproteins, Flrt2 and Flrt3, are strongly co-expressed in the PEO. However, beginning at around day 10 of mouse development, following attachment and outgrowth, Flrt3 is selectively downregulated, and only Flrt2 is exclusively expressed in the fully delaminated epicardium. The present gene-targeting experiments demonstrate that mouse embryos lacking Flrt2 expression arrest at mid-gestation owing to cardiac insufficiency. The defects in integrity of the epicardial sheet and disturbed organization of the underlying basement membrane closely resemble those described in Flrt3-deficient embryos that fail to maintain cell-cell contacts in the anterior visceral endoderm (AVE) signalling centre that normally establishes the A-P axis. Using in vitro and in vivo reconstitution assays, we demonstrate that Flrt2 and Flrt3 are functionally interchangeable. When acting alone, either of these proteins is sufficient to rescue functional activities in the AVE and the developing epicardium. PMID:21350012

Tumor endothelial marker 5 expression in endothelial cells during capillary morphogenesis is induced by the small GTPase Rac and mediates contact inhibition of cell proliferation

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

Vallon, Mario, E-mail: m.vallon@arcor.de; Rohde, Franziska; Janssen, Klaus-Peter

2010-02-01

Tumor endothelial marker (TEM) 5 is an adhesion G-protein-coupled receptor upregulated in endothelial cells during tumor and physiologic angiogenesis. So far, the mechanisms leading to upregulation of TEM5 and its function during angiogenesis have not been identified. Here, we report that TEM5 expression in endothelial cells is induced during capillary-like network formation on Matrigel, during capillary morphogenesis in a three-dimensional collagen I matrix, and upon confluence on a two-dimensional matrix. TEM5 expression was not induced by a variety of soluble angiogenic factors, including VEGF and bFGF, in subconfluent endothelial cells. TEM5 upregulation was blocked by toxin B from Clostridium difficile,more » an inhibitor of the small GTPases Rho, Rac, and Cdc42. The Rho inhibitor C3 transferase from Clostridium botulinum did not affect TEM5 expression, whereas the Rac inhibitor NSC23766 suppressed TEM5 upregulation. An excess of the soluble TEM5 extracellular domain or an inhibitory monoclonal TEM5 antibody blocked contact inhibition of endothelial cell proliferation resulting in multilayered islands within the endothelial monolayer and increased vessel density during capillary formation. Based on our results we conclude that TEM5 expression during capillary morphogenesis is induced by the small GTPase Rac and mediates contact inhibition of proliferation in endothelial cells.« less

Notochord Morphogenesis in Mice: Current Understanding & Open Questions

PubMed Central

Balmer, Sophie; Nowotschin, Sonja; Hadjantonakis, Anna-Katerina

2016-01-01

The notochord is the structure which defines chordates. It is a rod-like mesodermal structure that runs the anterior-posterior length of the embryo, adjacent to the ventral neural tube. The notochord plays a critical role in embryonic tissue patterning, for example the dorsal-ventral patterning of the neural tube. The cells that will come to form the notochord are specified at gastrulation. Axial mesodermal cells arising at the anterior primitive streak migrate anteriorly as the precursors of the notochord and populate the notochordal plate. Interestingly, even though a lot of interest has centered on investigating the functional and structural roles of the notochord, we still have a very rudimentary understanding of notochord morphogenesis. The events driving the formation of the notochord are rapid, taking place over the period of approximately a day in mice. In this commentary we provide an overview of our current understanding of mouse notochord morphogenesis, from the initial specification of axial mesendodermal cells at the primitive streak, the emergence of these cells at the midline on the surface of the embryo, to their submergence and organization of the stereotypically positioned notochord. We will also discuss some key open questions. PMID:26845388

In vivo imaging of basement membrane movement: ECM patterning shapes Hydra polyps

PubMed Central

Aufschnaiter, Roland; Zamir, Evan A.; Little, Charles D.; Özbek, Suat; Münder, Sandra; David, Charles N.; Li, Li; Sarras, Michael P.; Zhang, Xiaoming

2011-01-01

Growth and morphogenesis during embryonic development, asexual reproduction and regeneration require extensive remodeling of the extracellular matrix (ECM). We used the simple metazoan Hydra to examine the fate of ECM during tissue morphogenesis and asexual budding. In growing Hydra, epithelial cells constantly move towards the extremities of the animal and into outgrowing buds. It is not known, whether these tissue movements involve epithelial migration relative to the underlying matrix or whether cells and ECM are displaced as a composite structure. Furthermore, it is unclear, how the ECM is remodeled to adapt to the shape of developing buds and tentacles. To address these questions, we used a new in vivo labeling technique for Hydra collagen-1 and laminin, and tracked the fate of ECM in all body regions of the animal. Our results reveal that Hydra ‘tissue movements’ are largely displacements of epithelial cells together with associated ECM. By contrast, during the evagination of buds and tentacles, extensive movement of epithelial cells relative to the matrix is observed, together with local ECM remodeling. These findings provide new insights into the nature of growth and morphogenesis in epithelial tissues. PMID:22194305

Mesenchymal-epithelial interactions during hair follicle morphogenesis and cycling

PubMed Central

Sennett, Rachel; Rendl, Michael

2012-01-01

Embryonic hair follicle induction and formation are regulated by mesenchymal-epithelial interactions between specialized dermal cells and epidermal stem cells that switch to a hair fate. Similarly, during postnatal hair growth, communication between mesenchymal dermal papilla cells and surrounding epithelial matrix cells coordinates hair shaft production. Adult hair follicle regeneration in the hair cycle again is thought to be controlled by activating signals originating from the mesenchymal compartment and acting on hair follicle stem cells. Although many signaling pathways are implicated in hair follicle formation and growth, the precise nature, timing, and intersection of these inductive and regulatory signals remains elusive. The goal of this review is to summarize our current understanding and to discuss recent new insights into mesenchymal-epithelial interactions during hair follicle morphogenesis and cycling. PMID:22960356

Cellular growth in plants requires regulation of cell wall biochemistry.

PubMed

Chebli, Youssef; Geitmann, Anja

2017-02-01

Cell and organ morphogenesis in plants are regulated by the chemical structure and mechanical properties of the extracellular matrix, the cell wall. The two primary load bearing components in the plant cell wall, the pectin matrix and the cellulose/xyloglucan network, are constantly remodelled to generate the morphological changes required during plant development. This remodelling is regulated by a plethora of loosening and stiffening agents such as pectin methyl-esterases, calcium ions, expansins, and glucanases. The tight spatio-temporal regulation of the activities of these agents is a sine qua non condition for proper morphogenesis at cell and tissue levels. The pectin matrix and the cellulose-xyloglucan network operate in concert and their behaviour is mutually dependent on their chemical, structural and mechanical modifications. Copyright © 2017 Elsevier Ltd. All rights reserved.

Cell death and morphogenesis during early mouse development: are they interconnected?

PubMed

Bedzhov, Ivan; Zernicka-Goetz, Magdalena

2015-04-01

Shortly after implantation the embryonic lineage transforms from a coherent ball of cells into polarized cup shaped epithelium. Recently we elucidated a previously unknown apoptosis-independent morphogenic event that reorganizes the pluripotent lineage. Polarization cues from the surrounding basement membrane rearrange the epiblast into a polarized rosette-like structure, where subsequently a central lumen is established. Thus, we provided a new model revising the current concept of apoptosis-dependent epiblast morphogenesis. Cell death however has to be tightly regulated during embryogenesis to ensure developmental success. Here, we follow the stages of early mouse development and take a glimpse at the critical signaling and morphogenic events that determine cells destiny and reshape the embryonic lineage. © 2015 The Authors. Bioessays published by WILEY Periodicals, Inc.

Stomatal Complex Development and F-Actin Organization in Maize Leaf Epidermis Depend on Cellulose Synthesis.

PubMed

Panteris, Emmanuel; Achlati, Theonymphi; Daras, Gerasimos; Rigas, Stamatis

2018-06-06

Cellulose microfibrils reinforce the cell wall for morphogenesis in plants. Herein, we provide evidence on a series of defects regarding stomatal complex development and F-actin organization in Zea mays leaf epidermis, due to inhibition of cellulose synthesis. Formative cell divisions of stomatal complex ontogenesis were delayed or inhibited, resulting in lack of subsidiary cells and frequently in unicellular stomata, with an atypical stomatal pore. Guard cells failed to acquire a dumbbell shape, becoming rounded, while subsidiary cells, whenever present, exhibited aberrant morphogenesis. F-actin organization was also affected, since the stomatal complex-specific arrays were scarcely observed. At late developmental stages, the overall F-actin network was diminished in all epidermal cells, although thick actin bundles persisted. Taken together, stomatal complex development strongly depends on cell wall mechanical properties. Moreover, F-actin organization exhibits a tight relationship with the cell wall.

Dynamics of cell wall elasticity pattern shapes the cell during yeast mating morphogenesis

PubMed Central

Goldenbogen, Björn; Giese, Wolfgang; Hemmen, Marie; Uhlendorf, Jannis; Herrmann, Andreas

2016-01-01

The cell wall defines cell shape and maintains integrity of fungi and plants. When exposed to mating pheromone, Saccharomyces cerevisiae grows a mating projection and alters in morphology from spherical to shmoo form. Although structural and compositional alterations of the cell wall accompany shape transitions, their impact on cell wall elasticity is unknown. In a combined theoretical and experimental approach using finite-element modelling and atomic force microscopy (AFM), we investigated the influence of spatially and temporally varying material properties on mating morphogenesis. Time-resolved elasticity maps of shmooing yeast acquired with AFM in vivo revealed distinct patterns, with soft material at the emerging mating projection and stiff material at the tip. The observed cell wall softening in the protrusion region is necessary for the formation of the characteristic shmoo shape, and results in wider and longer mating projections. The approach is generally applicable to tip-growing fungi and plants cells. PMID:27605377

The C. elegans Intestine As a Model for Intercellular Lumen Morphogenesis and In Vivo Polarized Membrane Biogenesis at the Single-cell Level: Labeling by Antibody Staining, RNAi Loss-of-function Analysis and Imaging.

PubMed

Zhang, Nan; Khan, Liakot A; Membreno, Edward; Jafari, Gholamali; Yan, Siyang; Zhang, Hongjie; Gobel, Verena

2017-10-03

Multicellular tubes, fundamental units of all internal organs, are composed of polarized epithelial or endothelial cells, with apical membranes lining the lumen and basolateral membranes contacting each other and/or the extracellular matrix. How this distinctive membrane asymmetry is established and maintained during organ morphogenesis is still an unresolved question of cell biology. This protocol describes the C. elegans intestine as a model for the analysis of polarized membrane biogenesis during tube morphogenesis, with emphasis on apical membrane and lumen biogenesis. The C. elegans twenty-cell single-layered intestinal epithelium is arranged into a simple bilaterally symmetrical tube, permitting analysis on a single-cell level. Membrane polarization occurs concomitantly with polarized cell division and migration during early embryogenesis, but de novo polarized membrane biogenesis continues throughout larval growth, when cells no longer proliferate and move. The latter setting allows one to separate subcellular changes that simultaneously mediate these different polarizing processes, difficult to distinguish in most polarity models. Apical-, basolateral membrane-, junctional-, cytoskeletal- and endomembrane components can be labeled and tracked throughout development by GFP fusion proteins, or assessed by in situ antibody staining. Together with the organism's genetic versatility, the C. elegans intestine thus provides a unique in vivo model for the visual, developmental, and molecular genetic analysis of polarized membrane and tube biogenesis. The specific methods (all standard) described here include how to: label intestinal subcellular components by antibody staining; analyze genes involved in polarized membrane biogenesis by loss-of-function studies adapted to the typically essential tubulogenesis genes; assess polarity defects during different developmental stages; interpret phenotypes by epifluorescence, differential interference contrast (DIC) and confocal microscopy; quantify visual defects. This protocol can be adapted to analyze any of the often highly conserved molecules involved in epithelial polarity, membrane biogenesis, tube and lumen morphogenesis.

Inhibition of Candida albicans Biofilm Formation by the Synthetic Lactoferricin Derived Peptide hLF1-11

PubMed Central

Morici, Paola; Fais, Roberta; Rizzato, Cosmeri

2016-01-01

The aim of this study was to evaluate the in vitro activity of the synthetic peptide hLF1-11 against biofilm produced by clinical isolates of Candida albicans with different fluconazole susceptibility. The antibiofilm activity of the peptide hLF1-11 was assessed in terms of reduction of biofilm cellular density, metabolic activity and sessile cell viability. The extent of morphogenesis in hLF1-11 treated and untreated biofilms was also investigated microscopically. Transcription levels of genes related to cell adhesion, hyphal development and extracellular matrix production were analysed by qRT-PCR in hLF1-11 treated and untreated biofilms. Exogenous dibutyryl-cAMP (db-cAMP) was used to rescue morphogenesis in cells exposed to the peptide. The results revealed that hLF1-11 exhibited an inhibitory effect on biofilm formation by all C. albicans isolates tested in a dose-dependent manner, regardless of their fluconazole susceptibility. Visual inspection of treated or untreated biofilm cells with an inverted microscope revealed a significant reduction in hyphal formation by hLF1-11 treated cells, as early as 3 hours of incubation. Moreover, hLF1-11 showed a reduced activity on preadherent cells. hLF1-11 induced the down-regulation of biofilm and hyphal-associated genes, which were predominantly regulated via the Ras1-cAMP-Efg1 pathway. Indeed, exogenous db-cAMP restored morphogenesis in hLF1-11 treated cells. The hLF1-11 peptide significantly inhibited biofilm formation by C. albicans mainly at early stages, interfering with biofilm cellular density and metabolic activity, and affected morphogenesis through the Ras1-cAMP-Efg1 pathway. Our findings provide the first evidence that hLF1-11 could represent a potential candidate for the prevention of biofilm formation by C. albicans. PMID:27902776

Inhibition of Candida albicans Biofilm Formation by the Synthetic Lactoferricin Derived Peptide hLF1-11.

PubMed

Morici, Paola; Fais, Roberta; Rizzato, Cosmeri; Tavanti, Arianna; Lupetti, Antonella

2016-01-01

The aim of this study was to evaluate the in vitro activity of the synthetic peptide hLF1-11 against biofilm produced by clinical isolates of Candida albicans with different fluconazole susceptibility. The antibiofilm activity of the peptide hLF1-11 was assessed in terms of reduction of biofilm cellular density, metabolic activity and sessile cell viability. The extent of morphogenesis in hLF1-11 treated and untreated biofilms was also investigated microscopically. Transcription levels of genes related to cell adhesion, hyphal development and extracellular matrix production were analysed by qRT-PCR in hLF1-11 treated and untreated biofilms. Exogenous dibutyryl-cAMP (db-cAMP) was used to rescue morphogenesis in cells exposed to the peptide. The results revealed that hLF1-11 exhibited an inhibitory effect on biofilm formation by all C. albicans isolates tested in a dose-dependent manner, regardless of their fluconazole susceptibility. Visual inspection of treated or untreated biofilm cells with an inverted microscope revealed a significant reduction in hyphal formation by hLF1-11 treated cells, as early as 3 hours of incubation. Moreover, hLF1-11 showed a reduced activity on preadherent cells. hLF1-11 induced the down-regulation of biofilm and hyphal-associated genes, which were predominantly regulated via the Ras1-cAMP-Efg1 pathway. Indeed, exogenous db-cAMP restored morphogenesis in hLF1-11 treated cells. The hLF1-11 peptide significantly inhibited biofilm formation by C. albicans mainly at early stages, interfering with biofilm cellular density and metabolic activity, and affected morphogenesis through the Ras1-cAMP-Efg1 pathway. Our findings provide the first evidence that hLF1-11 could represent a potential candidate for the prevention of biofilm formation by C. albicans.

Cellular Force Microscopy for in Vivo Measurements of Plant Tissue Mechanics1[W][OA

PubMed Central

Routier-Kierzkowska, Anne-Lise; Weber, Alain; Kochova, Petra; Felekis, Dimitris; Nelson, Bradley J.; Kuhlemeier, Cris; Smith, Richard S.

2012-01-01

Although growth and morphogenesis are controlled by genetics, physical shape change in plant tissue results from a balance between cell wall loosening and intracellular pressure. Despite recent work demonstrating a role for mechanical signals in morphogenesis, precise measurement of mechanical properties at the individual cell level remains a technical challenge. To address this challenge, we have developed cellular force microscopy (CFM), which combines the versatility of classical microindentation techniques with the high automation and resolution approaching that of atomic force microscopy. CFM’s large range of forces provides the possibility to map the apparent stiffness of both plasmolyzed and turgid tissue as well as to perform micropuncture of cells using very high stresses. CFM experiments reveal that, within a tissue, local stiffness measurements can vary with the level of turgor pressure in an unexpected way. Altogether, our results highlight the importance of detailed physically based simulations for the interpretation of microindentation results. CFM’s ability to be used both to assess and manipulate tissue mechanics makes it a method of choice to unravel the feedbacks between mechanics, genetics, and morphogenesis. PMID:22353572

SEPT9_v1 Functions in Breast Cancer Cell Division

DTIC Science & Technology

2012-01-01

the regulation and function of septin filaments, and define new mechanisms regulating important cellular functions. BODY: 1). Study the effects...ciliogenesis. However, mechanisms for retaining these proteins and lipids in the primary cilia are not clear. We directly tested the presence of a diffusion...polarity and morphogenesis;  Defined mechanisms involving the roles of septins and microtubules in vesicle trafficking and epithelial morphogenesis

Apical constriction: themes and variations on a cellular mechanism driving morphogenesis

PubMed Central

Martin, Adam C.; Goldstein, Bob

2014-01-01

Apical constriction is a cell shape change that promotes tissue remodeling in a variety of homeostatic and developmental contexts, including gastrulation in many organisms and neural tube formation in vertebrates. In recent years, progress has been made towards understanding how the distinct cell biological processes that together drive apical constriction are coordinated. These processes include the contraction of actin-myosin networks, which generates force, and the attachment of actin networks to cell-cell junctions, which allows forces to be transmitted between cells. Different cell types regulate contractility and adhesion in unique ways, resulting in apical constriction with varying dynamics and subcellular organizations, as well as a variety of resulting tissue shape changes. Understanding both the common themes and the variations in apical constriction mechanisms promises to provide insight into the mechanics that underlie tissue morphogenesis. PMID:24803648

Arabidopsis FH1 Formin Affects Cotyledon Pavement Cell Shape by Modulating Cytoskeleton Dynamics.

PubMed

Rosero, Amparo; Oulehlová, Denisa; Stillerová, Lenka; Schiebertová, Petra; Grunt, Michal; Žárský, Viktor; Cvrčková, Fatima

2016-03-01

Plant cell morphogenesis involves concerted rearrangements of microtubules and actin microfilaments. We previously reported that FH1, the main Arabidopsis thaliana housekeeping Class I membrane-anchored formin, contributes to actin dynamics and microtubule stability in rhizodermis cells. Here we examine the effects of mutations affecting FH1 (At3g25500) on cell morphogenesis and above-ground organ development in seedlings, as well as on cytoskeletal organization and dynamics, using a combination of confocal and variable angle epifluorescence microscopy with a pharmacological approach. Homozygous fh1 mutants exhibited cotyledon epinasty and had larger cotyledon pavement cells with more pronounced lobes than the wild type. The pavement cell shape alterations were enhanced by expression of the fluorescent microtubule marker GFP-microtubule-associated protein 4 (MAP4). Mutant cotyledon pavement cells exhibited reduced density and increased stability of microfilament bundles, as well as enhanced dynamics of microtubules. Analogous results were also obtained upon treatments with the formin inhibitor SMIFH2 (small molecule inhibitor of formin homology 2 domains). Pavement cell shape in wild-type (wt) and fh1 plants in some situations exhibited a differential response towards anti-cytoskeletal drugs, especially the microtubule disruptor oryzalin. Our observations indicate that FH1 participates in the control of microtubule dynamics, possibly via its effects on actin, subsequently influencing cell morphogenesis and macroscopic organ development. © The Author 2016. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

The DCL gene of tomato is required for chloroplast development and palisade cell morphogenesis in leaves.

PubMed

Keddie, J S; Carroll, B; Jones, J D; Gruissem, W

1996-08-15

The defective chloroplasts and leaves-mutable (dcl-m) mutation of tomato was identified in a Ds mutagenesis screen. This unstable mutation affects both chloroplast development and palisade cell morphogenesis in leaves. Mutant plants are clonally variegated as a result of somatic excision of Ds and have albino leaves with green sectors. Leaf midribs and stems are light green with sectors of dark green tissue but fruit and petals are wild-type in appearance. Within dark green sectors of dcl-m leaves, palisade cells are normal, whereas in albino areas of dcl-m leaves, palisade cells do not expand to become their characteristic columnar shape. The development of chloroplasts from proplastids in albino areas is apparently blocked at an early stage. DCL was cloned using Ds as a tag and encodes a novel protein of approximately 25 kDa, containing a chloroplast transit peptide and an acidic alpha-helical region. DCL protein was imported into chloroplasts in vitro and processed to a mature form. Because of the ubiquitous expression of DCL and the proplastid-like appearance of dcl-affected plastids, the DCL protein may regulate a basic and universal function of the plastid. The novel dcl-m phenotype suggests that chloroplast development is required for correct palisade cell morphogenesis during leaf development.

Visualizing morphogenesis in transgenic zebrafish embryos using BODIPY TR methyl ester dye as a vital counterstain for GFP.

PubMed

Cooper, Mark S; Szeto, Daniel P; Sommers-Herivel, Greg; Topczewski, Jacek; Solnica-Krezel, Lila; Kang, Hee-Chol; Johnson, Iain; Kimelman, David

2005-02-01

Green fluorescent protein (GFP) technology is rapidly advancing the study of morphogenesis, by allowing researchers to specifically focus on a subset of labeled cells within the living embryo. However, when imaging GFP-labeled cells using confocal microscopy, it is often essential to simultaneously visualize all of the cells in the embryo using dual-channel fluorescence to provide an embryological context for the cells expressing GFP. Although various counterstains are available, part of their fluorescence overlaps with the GFP emission spectra, making it difficult to clearly identify the cells expressing GFP. In this study, we report that a new fluorophore, BODIPY TR methyl ester dye, serves as a versatile vital counterstain for visualizing the cellular dynamics of morphogenesis within living GFP transgenic zebrafish embryos. The fluorescence of this photostable synthetic dye is spectrally separate from GFP fluorescence, allowing dual-channel, three-dimensional (3D) and four-dimensional (4D) confocal image data sets of living specimens to be easily acquired. These image data sets can be rendered subsequently into uniquely informative 3D and 4D visualizations using computer-assisted visualization software. We discuss a variety of immediate and potential applications of BODIPY TR methyl ester dye as a vital visualization counterstain for GFP in transgenic zebrafish embryos. Copyright 2004 Wiley-Liss, Inc.

Laser capture microdissection to study flower morphogenesis

NASA Astrophysics Data System (ADS)

Pawełkowicz, Magdalena Ewa; Skarzyńska, Agnieszka; Kowalczuk, Cezary; PlÄ der, Wojciech; Przybecki, Zbigniew

2017-08-01

Laser Capture Microdissection (LCM) is a sample preparation microscopic method that enables isolation of an interesting cell or cells population from human, animal or plant tissue. This technique allows for obtaining pure sample from heterogeneous mixture. From isolated cells, it is possible to obtain the appropriate quality material used for genomic research in transcriptomics, proteomics and metabolomics. We used LCM method to study flower morphogenesis and specific bud's organ organization and development. The genes expression level in developing flower buds of male (B10) and female (2gg) lines were analyzed with qPCR. The expression was checked for stamen and carpel primordia obtained with LCM and for whole flower buds at successive stages of growth.

Reassessing the Roles of PIN Proteins and Anticlinal Microtubules during Pavement Cell Morphogenesis.

PubMed

Belteton, Samuel A; Sawchuk, Megan G; Donohoe, Bryon S; Scarpella, Enrico; Szymanski, Daniel B

2018-01-01

The leaf epidermis is a biomechanical shell that influences the size and shape of the organ. Its morphogenesis is a multiscale process in which nanometer-scale cytoskeletal protein complexes, individual cells, and groups of cells pattern growth and define macroscopic leaf traits. Interdigitated growth of neighboring cells is an evolutionarily conserved developmental strategy. Understanding how signaling pathways and cytoskeletal proteins pattern cell walls during this form of tissue morphogenesis is an important research challenge. The cellular and molecular control of a lobed cell morphology is currently thought to involve PIN-FORMED (PIN)-type plasma membrane efflux carriers that generate subcellular auxin gradients. Auxin gradients were proposed to function across cell boundaries to encode stable offset patterns of cortical microtubules and actin filaments between adjacent cells. Many models suggest that long-lived microtubules along the anticlinal cell wall generate local cell wall heterogeneities that restrict local growth and specify the timing and location of lobe formation. Here, we used Arabidopsis ( Arabidopsis thaliana ) reverse genetics and multivariate long-term time-lapse imaging to test current cell shape control models. We found that neither PIN proteins nor long-lived microtubules along the anticlinal wall predict the patterns of lobe formation. In fields of lobing cells, anticlinal microtubules are not correlated with cell shape and are unstable at the time scales of cell expansion. Our analyses indicate that anticlinal microtubules have multiple functions in pavement cells and that lobe initiation is likely controlled by complex interactions among cell geometry, cell wall stress patterns, and transient microtubule networks that span the anticlinal and periclinal walls. © 2018 American Society of Plant Biologists. All Rights Reserved.

Luminal Progenitors Restrict Their Lineage Potential during Mammary Gland Development

PubMed Central

Rodilla, Veronica; Dasti, Alessandro; Huyghe, Mathilde; Lafkas, Daniel; Laurent, Cécile; Reyal, Fabien; Fre, Silvia

2015-01-01

The hierarchical relationships between stem cells and progenitors that guide mammary gland morphogenesis are still poorly defined. While multipotent basal stem cells have been found within the myoepithelial compartment, the in vivo lineage potential of luminal progenitors is unclear. Here we used the expression of the Notch1 receptor, previously implicated in mammary gland development and tumorigenesis, to elucidate the hierarchical organization of mammary stem/progenitor cells by lineage tracing. We found that Notch1 expression identifies multipotent stem cells in the embryonic mammary bud, which progressively restrict their lineage potential during mammary ductal morphogenesis to exclusively generate an ERαneg luminal lineage postnatally. Importantly, our results show that Notch1-labelled cells represent the alveolar progenitors that expand during pregnancy and survive multiple successive involutions. This study reveals that postnatal luminal epithelial cells derive from distinct self-sustained lineages that may represent the cells of origin of different breast cancer subtypes. PMID:25688859

Luminal progenitors restrict their lineage potential during mammary gland development.

PubMed

Rodilla, Veronica; Dasti, Alessandro; Huyghe, Mathilde; Lafkas, Daniel; Laurent, Cécile; Reyal, Fabien; Fre, Silvia

2015-02-01

The hierarchical relationships between stem cells and progenitors that guide mammary gland morphogenesis are still poorly defined. While multipotent basal stem cells have been found within the myoepithelial compartment, the in vivo lineage potential of luminal progenitors is unclear. Here we used the expression of the Notch1 receptor, previously implicated in mammary gland development and tumorigenesis, to elucidate the hierarchical organization of mammary stem/progenitor cells by lineage tracing. We found that Notch1 expression identifies multipotent stem cells in the embryonic mammary bud, which progressively restrict their lineage potential during mammary ductal morphogenesis to exclusively generate an ERαneg luminal lineage postnatally. Importantly, our results show that Notch1-labelled cells represent the alveolar progenitors that expand during pregnancy and survive multiple successive involutions. This study reveals that postnatal luminal epithelial cells derive from distinct self-sustained lineages that may represent the cells of origin of different breast cancer subtypes.

Mammary stem cells and the differentiation hierarchy: current status and perspectives

PubMed Central

Visvader, Jane E.; Stingl, John

2014-01-01

The mammary epithelium is highly responsive to local and systemic signals, which orchestrate morphogenesis of the ductal tree during puberty and pregnancy. Based on transplantation and lineage tracing studies, a hierarchy of stem and progenitor cells has been shown to exist among the mammary epithelium. Lineage tracing has highlighted the existence of bipotent mammary stem cells (MaSCs) in situ as well as long-lived unipotent cells that drive morphogenesis and homeostasis of the ductal tree. Moreover, there is accumulating evidence for a heterogeneous MaSC compartment comprising fetal MaSCs, slow-cycling cells, and both long-term and short-term repopulating cells. In parallel, diverse luminal progenitor subtypes have been identified in mouse and human mammary tissue. Elucidation of the normal cellular hierarchy is an important step toward understanding the “cells of origin” and molecular perturbations that drive breast cancer. PMID:24888586

Interference by 2,3,7,8-tetrachlorodibenzo-p-dioxin with cultured mouse submandibular gland branching morphogenesis involves reduced epidermal growth factor receptor signaling

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

Kiukkonen, Anu; Sahlberg, Carin; Partanen, Anna-Maija

2006-05-01

Toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to mouse embryonic teeth, sharing features of early development with salivary glands in common, involves enhanced apoptosis and depends on the expression of epidermal growth factor (EGF) receptor. EGF receptor signaling, on the other hand, is essential for salivary gland branching morphogenesis. To see if TCDD impairs salivary gland morphogenesis and if the impairment is associated with EGF receptor signaling, we cultured mouse (NMRI) E13 submandibular glands with TCDD or TCDD in combination with EGF or fibronectin (FN), both previously found to enhance branching morphogenesis. Explants were examined stereomicroscopically and processed to paraffin sections. TCDD exposuremore » impaired epithelial branching and cleft formation, resulting in enlarged buds. The glands were smaller than normal. EGF and FN alone concentration-dependently stimulated or inhibited branching morphogenesis but when co-administered with TCDD, failed to compensate for its effect. TCDD induced cytochrome P4501A1 expression in the glandular epithelium, indicating activation of the aryl hydrocarbon receptor. TCDD somewhat increased epithelial apoptosis as observed by terminal deoxynucleotidyl transferase (TdT)-mediated nick end-labeling method but the increase could not be correlated with morphological changes. The frequency of proliferating cells was not altered. Corresponding to the reduced cleft sites in TCDD-exposed explants, FN immunoreactivity in the epithelium was reduced. The results show that TCDD, comparably with EGF and FN at morphogenesis-inhibiting concentrations, impaired salivary gland branching morphogenesis in vitro. Together with the failure of EGF and FN at morphogenesis-stimulating concentrations to compensate for the effect of TCDD this implies that TCDD toxicity to developing salivary gland involves reduced EGF receptor signaling.« less

Deletion of the Vaccinia Virus I2 Protein Interrupts Virion Morphogenesis, Leading to Retention of the Scaffold Protein and Mislocalization of Membrane-Associated Entry Proteins

PubMed Central

Hyun, Seong-In; Weisberg, Andrea

2017-01-01

ABSTRACT The I2L open reading frame of vaccinia virus (VACV) encodes a conserved 72-amino-acid protein with a putative C-terminal transmembrane domain. Previous studies with a tetracycline-inducible mutant demonstrated that I2-deficient virions are defective in cell entry. The purpose of the present study was to determine the step of replication or entry that is affected by loss of the I2 protein. Fluorescence microscopy experiments showed that I2 colocalized with a major membrane protein of immature and mature virions. We generated a cell line that constitutively expressed I2 and allowed construction of the VACV I2L deletion mutant vΔI2. As anticipated, vΔI2 was unable to replicate in cells that did not express I2. Unexpectedly, morphogenesis was interrupted at a stage after immature virion formation, resulting in the accumulation of dense spherical particles instead of brick-shaped mature virions with well-defined core structures. The abnormal particles retained the D13 scaffold protein of immature virions, were severely deficient in the transmembrane proteins that comprise the entry fusion complex (EFC), and had increased amounts of unprocessed membrane and core proteins. Total lysates of cells infected with vΔI2 also had diminished EFC proteins due to instability attributed to their hydrophobicity and failure to be inserted into viral membranes. A similar instability of EFC proteins had previously been found with unrelated mutants blocked earlier in morphogenesis that also accumulated viral membranes retaining the D13 scaffold. We concluded that I2 is required for virion morphogenesis, release of the D13 scaffold, and the association of EFC proteins with viral membranes. IMPORTANCE Poxviruses comprise a large family that infect vertebrates and invertebrates, cause disease in both in humans and in wild and domesticated animals, and are being engineered as vectors for vaccines and cancer therapy. In addition, investigations of poxviruses have provided insights into many aspects of cell biology. The I2 protein is conserved in all poxviruses that infect vertebrates, suggesting an important role. The present study revealed that this protein is essential for vaccinia virus morphogenesis and that its absence results in an accumulation of deformed virus particles retaining the scaffold protein and deficient in surface proteins needed for cell entry. PMID:28490596

Dynamic Analysis of Vascular Morphogenesis Using Transgenic Quail Embryos

PubMed Central

Huss, David; Filla, Michael B.; Czirok, Andras; Rongish, Brenda J.; Little, Charles D.; Fraser, Scott E.; Lansford, Rusty

2010-01-01

Background One of the least understood and most central questions confronting biologists is how initially simple clusters or sheet-like cell collectives can assemble into highly complex three-dimensional functional tissues and organs. Due to the limits of oxygen diffusion, blood vessels are an essential and ubiquitous presence in all amniote tissues and organs. Vasculogenesis, the de novo self-assembly of endothelial cell (EC) precursors into endothelial tubes, is the first step in blood vessel formation [1]. Static imaging and in vitro models are wholly inadequate to capture many aspects of vascular pattern formation in vivo, because vasculogenesis involves dynamic changes of the endothelial cells and of the forming blood vessels, in an embryo that is changing size and shape. Methodology/Principal Findings We have generated Tie1 transgenic quail lines Tg(tie1:H2B-eYFP) that express H2B-eYFP in all of their endothelial cells which permit investigations into early embryonic vascular morphogenesis with unprecedented clarity and insight. By combining the power of molecular genetics with the elegance of dynamic imaging, we follow the precise patterning of endothelial cells in space and time. We show that during vasculogenesis within the vascular plexus, ECs move independently to form the rudiments of blood vessels, all while collectively moving with gastrulating tissues that flow toward the embryo midline. The aortae are a composite of somatic derived ECs forming its dorsal regions and the splanchnic derived ECs forming its ventral region. The ECs in the dorsal regions of the forming aortae exhibit variable mediolateral motions as they move rostrally; those in more ventral regions show significant lateral-to-medial movement as they course rostrally. Conclusions/Significance The present results offer a powerful approach to the major challenge of studying the relative role(s) of the mechanical, molecular, and cellular mechanisms of vascular development. In past studies, the advantages of the molecular genetic tools available in mouse were counterbalanced by the limited experimental accessibility needed for imaging and perturbation studies. Avian embryos provide the needed accessibility, but few genetic resources. The creation of transgenic quail with labeled endothelia builds upon the important roles that avian embryos have played in previous studies of vascular development. PMID:20856866

Counter-rotational cell flows drive morphological and cell fate asymmetries in mammalian hair follicles.

PubMed

Cetera, Maureen; Leybova, Liliya; Joyce, Bradley; Devenport, Danelle

2018-05-01

Organ morphogenesis is a complex process coordinated by cell specification, epithelial-mesenchymal interactions and tissue polarity. A striking example is the pattern of regularly spaced, globally aligned mammalian hair follicles, which emerges through epidermal-dermal signaling and planar polarized morphogenesis. Here, using live-imaging, we discover that developing hair follicles polarize through dramatic cell rearrangements organized in a counter-rotational pattern of cell flows. Upon hair placode induction, Shh signaling specifies a radial pattern of progenitor fates that, together with planar cell polarity, induce counter-rotational rearrangements through myosin and ROCK-dependent polarized neighbour exchanges. Importantly, these cell rearrangements also establish cell fate asymmetry by repositioning radial progenitors along the anterior-posterior axis. These movements concurrently displace associated mesenchymal cells, which then signal asymmetrically to maintain polarized cell fates. Our results demonstrate how spatial patterning and tissue polarity generate an unexpected collective cell behaviour that in turn, establishes both morphological and cell fate asymmetry.

Retinoic acid signaling regulates Krt5 and Krt14 independently of stem cell markers in submandibular salivary gland epithelium.

PubMed

Abashev, Timur M; Metzler, Melissa A; Wright, Diana M; Sandell, Lisa L

2017-02-01

Retinoic acid (RA), the active metabolite of vitamin A, has been demonstrated to be important for growth and branching morphogenesis of mammalian embryonic salivary gland epithelium. However, it is not known whether RA functions directly within epithelial cells or in associated tissues that influence morphogenesis of salivary epithelium. Moreover, downstream targets of RA regulation have not been identified. Here, we show that canonical RA signaling occurs in multiple tissues of embryonic mouse salivary glands, including epithelium, associated parasympathetic ganglion neurons, and nonneuronal mesenchyme. By culturing epithelium explants in isolation from other tissues, we demonstrate that RA influences epithelium morphogenesis by direct action in that tissue. Moreover, we demonstrate that inhibition of RA signaling represses cell proliferation and expression of FGF10 signaling targets, and upregulates expression of basal epithelial keratins Krt5 and Krt14. Importantly, we show that the stem cell gene Kit is regulated inversely from Krt5/Krt14 by RA signaling. RA regulates Krt5 and Krt14 expression independently of stem cell character in developing salivary epithelium. RA, or chemical inhibitors of RA signaling, could potentially be used for modulating growth and differentiation of epithelial stem cells for the purpose of re-populating damaged glands or generating bioengineered organs. Developmental Dynamics 246:135-147, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Feedback control of growth, differentiation, and morphogenesis of pancreatic endocrine progenitors in an epithelial plexus niche

PubMed Central

Bankaitis, Eric D.; Bechard, Matthew E.; Wright, Christopher V.E.

2015-01-01

In the mammalian pancreas, endocrine cells undergo lineage allocation upon emergence from a bipotent duct/endocrine progenitor pool, which resides in the “trunk epithelium.” Major questions remain regarding how niche environments are organized within this epithelium to coordinate endocrine differentiation with programs of epithelial growth, maturation, and morphogenesis. We used EdU pulse-chase and tissue-reconstruction approaches to analyze how endocrine progenitors and their differentiating progeny are assembled within the trunk as it undergoes remodeling from an irregular plexus of tubules to form the eventual mature, branched ductal arbor. The bulk of endocrine progenitors is maintained in an epithelial “plexus state,” which is a transient intermediate during epithelial maturation within which endocrine cell differentiation is continually robust and surprisingly long-lived. Within the plexus, local feedback effects derived from the differentiating and delaminating endocrine cells nonautonomously regulate the flux of endocrine cell birth as well as proliferative growth of the bipotent cell population using Notch-dependent and Notch-independent influences, respectively. These feedback effects in turn maintain the plexus state to ensure prolonged allocation of endocrine cells late into gestation. These findings begin to define a niche-like environment guiding the genesis of the endocrine pancreas and advance current models for how differentiation is coordinated with the growth and morphogenesis of the developing pancreatic epithelium. PMID:26494792

Yeast casein kinase 2 governs morphology, biofilm formation, cell wall integrity, and host cell damage of Candida albicans.

PubMed

Jung, Sook-In; Rodriguez, Natalie; Irrizary, Jihyun; Liboro, Karl; Bogarin, Thania; Macias, Marlene; Eivers, Edward; Porter, Edith; Filler, Scott G; Park, Hyunsook

2017-01-01

The regulatory networks governing morphogenesis of a pleomorphic fungus, Candida albicans are extremely complex and remain to be completely elucidated. This study investigated the function of C. albicans yeast casein kinase 2 (CaYck2p). The yck2Δ/yck2Δ strain displayed constitutive pseudohyphae in both yeast and hyphal growth conditions, and formed enhanced biofilm under non-biofilm inducing condition. This finding was further supported by gene expression analysis of the yck2Δ/yck2Δ strain which showed significant upregulation of UME6, a key transcriptional regulator of hyphal transition and biofilm formation, and cell wall protein genes ALS3, HWP1, and SUN41, all of which are associated with morphogenesis and biofilm architecture. The yck2Δ/yck2Δ strain was hypersensitive to cell wall damaging agents and had increased compensatory chitin deposition in the cell wall accompanied by an upregulation of the expression of the chitin synthase genes, CHS2, CHS3, and CHS8. Absence of CaYck2p also affected fungal-host interaction; the yck2Δ/yck2Δ strain had significantly reduced ability to damage host cells. However, the yck2Δ/yck2Δ strain had wild-type susceptibility to cyclosporine and FK506, suggesting that CaYck2p functions independently from the Ca+/calcineurin pathway. Thus, in C. albicans, Yck2p is a multifunctional kinase that governs morphogenesis, biofilm formation, cell wall integrity, and host cell interactions.

Drosophila glypican Dally-like acts in FGF-receiving cells to modulate FGF signaling during tracheal morphogenesis

PubMed Central

Yan, Dong; Lin, Xinhua

2007-01-01

Summary Previous studies in Drosophila have shown that heparan sulfate proteoglycans (HSPGs) are involved in both breathless (btl)- and heartless (htl)-mediated FGF signaling during embryogenesis. However, the mechanism(s) by which HSPGs control Btl and Htl signaling is unknown. Here we show that dally-like (dlp, a Drosophila glypican) mutant embryos exhibit severe defects in tracheal morphogenesis and show a reduction in btl-mediated FGF signaling activity. However, htl-dependent mesodermal cell migration is not affected in dlp mutant embryos. Furthermore, expression of Dlp, but not other Drosophila HSPGs, can restore effectively the tracheal morphogenesis in dlp embryos. Rescue experiments in dlp embryos demonstrate that Dlp functions only in Bnl/FGF receiving cells in a cell-autonomous manner, but is not essential for Bnl/FGF expression cells. To further dissect the mechanism(s) of Dlp in Btl signaling, we analyzed the role of Dlp in Btl-mediated air sac tracheoblast formation in wing discs. Mosaic analysis experiments show that removal of HSPG activity in FGF-producing or other surrounding cells does not affect tracheoblasts migration, while HSPG mutant tracheoblast cells fail to receive FGF signaling. Together, our results argue strongly that HSPGs regulate Btl signaling exclusively in FGF-receiving cells as co-receptors, but are not essential for the secretion and distribution of the FGF ligand. This mechanism is distinct from HSPG functions in morphogen distribution, and is likely a general paradigm for HSPG functions in FGF signaling in Drosophila. PMID:17959166

Myosin-II controls cellular branching morphogenesis and migration in 3D by minimizing cell surface curvature

PubMed Central

Elliott, Hunter; Fischer, Robert A.; Myers, Kenneth A.; Desai, Ravi A.; Gao, Lin; Chen, Christopher S.; Adelstein, Robert; Waterman, Clare M.; Danuser, Gaudenz

2014-01-01

In many cases cell function is intimately linked to cell shape control. We utilized endothelial cell branching morphogenesis as a model to understand the role of myosin-II in shape control of invasive cells migrating in 3D collagen gels. We applied principles of differential geometry and mathematical morphology to 3D image sets to parameterize cell branch structure and local cell surface curvature. We find that Rho/ROCK-stimulated myosin-II contractility minimizes cell-scale branching by recognizing and minimizing local cell surface curvature. Utilizing micro-fabrication to constrain cell shape identifies a positive feedback mechanism in which low curvature stabilizes myosin-II cortical association, where it acts to maintain minimal curvature. The feedback between myosin-II regulation by and control of curvature drives cycles of localized cortical myosin-II assembly and disassembly. These cycles in turn mediate alternating phases of directionally biased branch initiation and retraction to guide 3D cell migration. PMID:25621949

Autocrine and paracrine Shh signaling are necessary for tooth morphogenesis, but not tooth replacement in snakes and lizards (Squamata).

PubMed

Handrigan, Gregory R; Richman, Joy M

2010-01-01

Here we study the role of Shh signaling in tooth morphogenesis and successional tooth initiation in snakes and lizards (Squamata). By characterizing the expression of Shh pathway receptor Ptc1 in the developing dentitions of three species (Eublepharis macularius, Python regius, and Pogona vitticeps) and by performing gain- and loss-of-function experiments, we demonstrate that Shh signaling is active in the squamate tooth bud and is required for its normal morphogenesis. Shh apparently mediates tooth morphogenesis by separate paracrine- and autocrine-mediated functions. According to this model, paracrine Shh signaling induces cell proliferation in the cervical loop, outer enamel epithelium, and dental papilla. Autocrine signaling within the stellate reticulum instead appears to regulate cell survival. By treating squamate dental explants with Hh antagonist cyclopamine, we induced tooth phenotypes that closely resemble the morphological and differentiation defects of vestigial, first-generation teeth in the bearded dragon P. vitticeps. Our finding that these vestigial teeth are deficient in epithelial Shh signaling further corroborates that Shh is needed for the normal development of teeth in snakes and lizards. Finally, in this study, we definitively refute a role for Shh signaling in successional dental lamina formation and conclude that other pathways regulate tooth replacement in squamates.

Connexin 43 Is Necessary for Salivary Gland Branching Morphogenesis and FGF10-induced ERK1/2 Phosphorylation.

PubMed

Yamada, Aya; Futagi, Masaharu; Fukumoto, Emiko; Saito, Kan; Yoshizaki, Keigo; Ishikawa, Masaki; Arakaki, Makiko; Hino, Ryoko; Sugawara, Yu; Ishikawa, Momoko; Naruse, Masahiro; Miyazaki, Kanako; Nakamura, Takashi; Fukumoto, Satoshi

2016-01-08

Cell-cell interaction via the gap junction regulates cell growth and differentiation, leading to formation of organs of appropriate size and quality. To determine the role of connexin43 in salivary gland development, we analyzed its expression in developing submandibular glands (SMGs). Connexin43 (Cx43) was found to be expressed in salivary gland epithelium. In ex vivo organ cultures of SMGs, addition of the gap junctional inhibitors 18α-glycyrrhetinic acid (18α-GA) and oleamide inhibited SMG branching morphogenesis, suggesting that gap junctional communication contributes to salivary gland development. In Cx43(-/-) salivary glands, submandibular and sublingual gland size was reduced as compared with those from heterozygotes. The expression of Pdgfa, Pdgfb, Fgf7, and Fgf10, which induced branching of SMGs in Cx43(-/-) samples, were not changed as compared with those from heterozygotes. Furthermore, the blocking peptide for the hemichannel and gap junction channel showed inhibition of terminal bud branching. FGF10 induced branching morphogenesis, while it did not rescue the Cx43(-/-) phenotype, thus Cx43 may regulate FGF10 signaling during salivary gland development. FGF10 is expressed in salivary gland mesenchyme and regulates epithelial proliferation, and was shown to induce ERK1/2 phosphorylation in salivary epithelial cells, while ERK1/2 phosphorylation in HSY cells was dramatically inhibited by 18α-GA, a Cx43 peptide or siRNA. On the other hand, PDGF-AA and PDGF-BB separately induced ERK1/2 phosphorylation in primary cultured salivary mesenchymal cells regardless of the presence of 18α-GA. Together, our results suggest that Cx43 regulates FGF10-induced ERK1/2 phosphorylation in salivary epithelium but not in mesenchyme during the process of SMG branching morphogenesis. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

The solid state environment orchestrates embryonic development and tissue remodeling

NASA Technical Reports Server (NTRS)

Damsky, C. H.; Moursi, A.; Zhou, Y.; Fisher, S. J.; Globus, R. K.

1997-01-01

Cell interactions with extracellular matrix and with other cells play critical roles in morphogenesis during development and in tissue homeostasis and remodeling throughout life. Extracellular matrix is information-rich, not only because it is comprised of multifunctional structural ligands for cell surface adhesion receptors, but also because it contains peptide signaling factors, and proteinases and their inhibitors. The functions of these groups of molecules are extensively interrelated. In this review, three primary cell culture models are described that focus on adhesion receptors and their roles in complex aspects of morphogenesis and remodeling: the regulation of proteinase expression by fibronectin and integrins in synovial fibroblasts; the regulation of osteoblast differentiation and survival by fibronectin, and the regulation of trophoblast differentiation and invasion by integrins, cadherins and immunoglobulin family adhesion receptors.

Ylpex5 mutation partially suppresses the defective hyphal growth of a Yarrowia lipolytica ceramide synthase mutant, Yllac1, by recovering lipid raft polarization and vacuole morphogenesis.

PubMed

Bal, Jyotiranjan; Lee, Hye-Jeong; Cheon, Seon Ah; Lee, Kyung Jin; Oh, Doo-Byoung; Kim, Jeong-Yoon

2013-01-01

Sphingolipids are involved in cell differentiation and morphogenesis in eukaryotic cells. In this study, YlLac1p, a ceramide synthase required for glucosylceramide (GlcCer) synthesis, was found to be essential for hyphal growth in Yarrowia lipolytica. Y. lipolytica GlcCer was shown to be composed of a C16:0 fatty acid, which is hydroxylated at C2, and a C18:2 long chain base, which is unsaturated at both C4 and C8 and methylated at C9. Domain swapping analysis revealed that the entire TRAM/Lag1/CLN8 (TLC) domain, not the Lag1 motif, is crucial for the function of YlLac1p. YlDes1p, the C4 desaturase of the ceramide synthesized by YlLac1p, was also required for Y. lipolytica morphogenesis. Both Yllac1Δ and Yldes1Δ mutants neither polarize lipid rafts nor form normal vacuoles. Interestingly, mutation in YlPEX5, which encode a peroxisomal targeting signal receptor, partially suppressed the defective hyphal growth of Yllac1Δ. The Yllac1ΔYlpex5Δ mutant restored the ability to polarize lipid rafts and to form normal vacuoles, although it could not synthesize GlcCer. Taken together, our results suggest that GlcCer or GlcCer derivatives may be involved in hyphal morphogenesis in Y. lipolytica, at least in part, by affecting polarization of lipid rafts and vacuole morphogenesis. Copyright © 2012 Elsevier Inc. All rights reserved.

Foxi3 deficiency compromises hair follicle stem cell specification and activation

PubMed Central

Shirokova, Vera; Biggs, Leah C.; Jussila, Maria; Ohyama, Takahiro; Groves, Andrew K.; Mikkola, Marja L.

2017-01-01

The hair follicle is an ideal system to study stem cell specification and homeostasis due to its well characterized morphogenesis and stereotypic cycles of stem cell activation upon each hair cycle to produce a new hair shaft. The adult hair follicle stem cell niche consists of two distinct populations, the bulge and the more activation-prone secondary hair germ. Hair follicle stem cells are set aside during early stages of morphogenesis. This process is known to depend on the Sox9 transcription factor, but otherwise the establishment of the hair follicle stem cell niche is poorly understood. Here we show that that mutation of Foxi3, a Forkhead family transcription factor mutated in several hairless dog breeds, compromises stem cell specification. Further, loss of Foxi3 impedes hair follicle downgrowth and progression of the hair cycle. Genome-wide profiling revealed a number of downstream effectors of Foxi3 including transcription factors with a recognized function in hair follicle stem cells such as Lhx2, Runx1, and Nfatc1, suggesting that the Foxi3 mutant phenotype results from simultaneous downregulation of several stem cell signature genes. We show that Foxi3 displays a highly dynamic expression pattern during hair morphogenesis and cycling, and identify Foxi3 as a novel secondary hair germ marker. Absence of Foxi3 results in poor hair regeneration upon hair plucking, and a sparse fur phenotype in unperturbed mice that exacerbates with age, caused by impaired secondary hair germ activation leading to progressive depletion of stem cells. Thus, Foxi3 regulates multiple aspects of hair follicle development and homeostasis. PMID:26992132

Axial protocadherin (AXPC) regulates cell fate during notochordal morphogenesis.

PubMed

Yoder, Michael D; Gumbiner, Barry M

2011-11-01

The separation and specification of mesoderm into the notochord and somites involves members of the non-clustered δ-protocadherins. Axial (AXPC) and paraxial (PAPC) protocadherins are expressed in the early dorsal mesoderm and later become refined to the developing notochordal and somitic mesoderm, respectively. The role of PAPC in this process has been studied extensively, but the role of AXPC is poorly understood. Partial knockdown of AXPC causes a specific bent-axis phenotype, while more severe knockdown results in the loss of notochord formation. The inability of these embryos to develop a notochord is not due to a cell-sorting event via changes in cell adhesion during gastrulation, but rather this defect is manifested through the loss of axial mesoderm specification, but not general mesoderm induction. The results presented here show that AXPC functions in notochord morphogenesis by directing cell-fate decisions rather than cell-cell adhesion. Copyright © 2011 Wiley Periodicals, Inc.

An Apical MRCK-driven Morphogenetic Pathway Controls Epithelial Polarity

PubMed Central

Zihni, Ceniz; Vlassaks, Evi; Terry, Stephen; Carlton, Jeremy; Leung, Thomas King Chor; Olson, Michael; Pichaud, Franck; Balda, Maria Susana; Matter, Karl

2017-01-01

Polarized epithelia develop distinct cell surface domains, with the apical membrane acquiring characteristic morphological features such as microvilli. Cell polarization is driven by polarity determinants including the evolutionarily conserved partitioning defective (PAR) proteins that are separated into distinct cortical domains. PAR protein segregation is thought to be a consequence of asymmetric actomyosin contractions. The mechanism of activation of apically polarized actomyosin contractility is unknown. Here we show that the Cdc42 effector MRCK activates Myosin-II at the apical pole to segregate aPKC-Par6 from junctional Par3, defining the apical domain. Apically polarized MRCK-activated actomyosin contractility is reinforced by cooperation with aPKC-Par6 downregulating antagonistic RhoA-driven junctional actomyosin contractility, and drives polarization of cytosolic brush border determinants and apical morphogenesis. MRCK-activated polarized actomyosin contractility is required for apical differentiation and morphogenesis in vertebrate epithelia and Drosophila photoreceptors. Our results identify an apical origin of actomyosin-driven morphogenesis that couples cytoskeletal reorganization to PAR polarity signalling. PMID:28825699

Reprogramming cells and tissue patterning via bioelectrical pathways: molecular mechanisms and biomedical opportunities.

PubMed

Levin, Michael

2013-01-01

Transformative impact in regenerative medicine requires more than the reprogramming of individual cells: advances in repair strategies for birth defects or injuries, tumor normalization, and the construction of bioengineered organs and tissues all require the ability to control large-scale anatomical shape. Much recent work has focused on the transcriptional and biochemical regulation of cell behavior and morphogenesis. However, exciting new data reveal that bioelectrical properties of cells and their microenvironment exert a profound influence on cell differentiation, proliferation, and migration. Ion channels and pumps expressed in all cells, not just excitable nerve and muscle, establish resting potentials that vary across tissues and change with significant developmental events. Most importantly, the spatiotemporal gradients of these endogenous transmembrane voltage potentials (Vmem ) serve as instructive patterning cues for large-scale anatomy, providing organ identity, positional information, and prepattern template cues for morphogenesis. New genetic and pharmacological techniques for molecular modulation of bioelectric gradients in vivo have revealed the ability to initiate complex organogenesis, change tissue identity, and trigger regeneration of whole vertebrate appendages. A large segment of the spatial information processing that orchestrates individual cells' programs toward the anatomical needs of the host organism is electrical; this blurs the line between memory and decision-making in neural networks and morphogenesis in nonneural tissues. Advances in cracking this bioelectric code will enable the rational reprogramming of shape in whole tissues and organs, revolutionizing regenerative medicine, developmental biology, and synthetic bioengineering. Copyright © 2013 Wiley Periodicals, Inc.

Tissue Motion and Assembly During Early Cardiovascular Morphogenesis

NASA Astrophysics Data System (ADS)

Rongish, Brenda

2010-03-01

Conventional dogma in the field of cardiovascular developmental biology suggests that cardiac precursor cells migrate to the embryonic midline to form a tubular heart. These progenitors are believed to move relative to their extracellular matrix (ECM); responding to stimulatory and inhibitory cues in their environment. The tubular heart that is formed by 30 hours post fertilization is comprised of two concentric layers: the muscular myocardium and the endothelial-like endocardium, which are separated by a thick layer of ECM believed to be secreted predominantly by the myocardial cells. Here we describe the origin and motility of fluorescently tagged endocardial precursors in transgenic (Tie1-YFP) quail embryos (R. Lansford, Caltech) using epifluorescence time-lapse imaging. To visualize the environment of migrating endocardial progenitors, we labeled two ECM components, fibronectin and fibrillin-2, via in vivo microinjection of fluorochrome-conjugated monoclonal antibodies. Dynamic imaging was performed at stages encompassing tubular heart assembly and early looping. We established the motion of endocardial precursor cells and presumptive cardiac ECM fibrils using both object tracking and particle image velocimetry (image cross correlation). We determined the relative importance of directed cell autonomous motility versus passive tissue movements in endocardial morphogenesis. The data show presumptive endocardial cells and cardiac ECM fibrils are swept passively into the anterior and posterior poles of the elongating tubular heart. These quantitative data indicate the contribution of cell autonomous motility displayed by endocardial precursors is limited. Thus, tissue motion drives most of the cell displacements during endocardial morphogenesis.

G3139, an Anti-Bcl-2 Antisense Oligomer that Binds Heparin-Binding Growth Factors and Collagen I, Alters In Vitro Endothelial Cell Growth and Tubular Morphogenesis

PubMed Central

Stein, C.A.; Wu, SiJian; Voskresenskiy, Anatoliy M.; Zhou, Jin-Feng; Shin, Joongho; Miller, Paul; Souleimanian, Naira; Benimetskaya, Luba

2009-01-01

Purpose We examined the effects of G3139 on the interaction of heparin-binding proteins (e.g., FGF2 and collagen I) with endothelial cells. G3139 is an 18mer phosphorothioate oligonucleotide targeted to the initiation codon region of the Bcl-2 mRNA. A randomized, prospective global Phase III trial in advanced melanoma (GM301) has evaluted G3139 in combination with dacarbazine. However, the mechanism of action of G3139 is incompletely understood, as it is unlikely that Bcl-2 silencing is the sole mechanism for chemo-sensitization in melanoma cells. Experimental Design The ability of G3139 to interact with and protect heparin-binding proteins was quantitated. The effects of G3139 on the binding of FGF2 to high affinity cell surface receptors, and the induction of cellular mitogenesis and tubular morphogenesis in HMEC-1 and HUVEC cells were determined. Results G3139 binds with picomolar affinity to collagen I. By replacing heparin, the drug can potentiate the binding of FGF2 to FGFR1 IIIc, and it protects FGF from oxidation and from proteolysis. G3139 can increase endothelial cell mitogenesis and tubular morphogenesis of HMEC-1 cells in 3D collagen gels, increases the mitogenesis of HUVEC cells similarly, and induces vessel sprouts in the rat aortic ring model. Conclusions G3139 dramatically affects the behavior of endothelial cells. There may be a correlation between this observation and the treatment interaction with LDH observed clinically. PMID:19351753

The metabolic response of Candida albicans to farnesol under hyphae-inducing conditions.

PubMed

Han, Ting-Li; Cannon, Richard D; Villas-Bôas, Silas G

2012-12-01

Farnesol is a quorum-sensing molecule (QSM) produced, and sensed, by the polymorphic fungus, Candida albicans. This cell-to-cell communication molecule is known to suppress the hyphal formation of C. albicans at high cell density. Despite many studies investigating the signalling mechanisms by which QSMs influence the morphogenesis of C. albicans, the downstream metabolic effect of these signalling pathways in response to farnesol-mediated morphogenesis remains obscure. Here, we have used metabolomics to investigate the metabolic response of C. albicans upon exposure to farnesol under hyphae-inducing conditions. We have found a general up-regulation of central carbon metabolic pathways when hyphal formation was suppressed by farnesol evidenced by a considerably larger number of central carbon metabolic intermediates detected under this condition at an overall lower intracellular level. By combining the metabolic profiles from farnesol-exposed cells with previous metabolomics data for C. albicans undergoing morphogenesis, we have identified several metabolic pathways that are likely to be associated with the morphogenetic process of C. albicans, as well as metabolic pathways such as those involved in lipid metabolism that appeared to be specifically affected by farnesol. Therefore, our results provide important new insights into the metabolic role of farnesol in C. albicans metabolism. © 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

Intersection of FOXO- and RUNX1-mediated gene expression programs in single breast epithelial cells during morphogenesis and tumor progression.

PubMed

Wang, Lixin; Brugge, Joan S; Janes, Kevin A

2011-10-04

Gene expression networks are complicated by the assortment of regulatory factors that bind DNA and modulate transcription combinatorially. Single-cell measurements can reveal biological mechanisms hidden by population averages, but their value has not been fully explored in the context of mRNA regulation. Here, we adapted a single-cell expression profiling technique to examine the gene expression program downstream of Forkhead box O (FOXO) transcription factors during 3D breast epithelial acinar morphogenesis. By analyzing patterns of mRNA fluctuations among individual matrix-attached epithelial cells, we found that a subset of FOXO target genes was jointly regulated by the transcription factor Runt-related transcription factor 1 (RUNX1). Knockdown of RUNX1 causes hyperproliferation and abnormal morphogenesis, both of which require normal FOXO function. Down-regulating RUNX1 and FOXOs simultaneously causes widespread oxidative stress, which arrests proliferation and restores normal acinar morphology. In hormone-negative breast cancers lacking human epidermal growth factor receptor 2 (HER2) amplification, we find that RUNX1 down-regulation is strongly associated with up-regulation of FOXO1, which may be required to support growth of RUNX1-negative tumors. The coordinate function of these two tumor suppressors may provide a failsafe mechanism that inhibits cancer progression.

Notochord morphogenesis in mice: Current understanding & open questions.

PubMed

Balmer, Sophie; Nowotschin, Sonja; Hadjantonakis, Anna-Katerina

2016-05-01

The notochord is a structure common to all chordates, and the feature that the phylum Chordata has been named after. It is a rod-like mesodermal structure that runs the anterior-posterior length of the embryo, adjacent to the ventral neural tube. The notochord plays a critical role in embryonic tissue patterning, for example the dorsal-ventral patterning of the neural tube. The cells that will come to form the notochord are specified at gastrulation. Axial mesodermal cells arising at the anterior primitive streak migrate anteriorly as the precursors of the notochord and populate the notochordal plate. Yet, even though a lot of interest has centered on investigating the functional and structural roles of the notochord, we still have a very rudimentary understanding of notochord morphogenesis. The events driving the formation of the notochord are rapid, taking place over the period of approximately a day in mice. In this commentary, we provide an overview of our current understanding of mouse notochord morphogenesis, from the initial specification of axial mesendodermal cells at the primitive streak, the emergence of these cells at the midline on the surface of the embryo, to their submergence and organization of the stereotypically positioned notochord. We will also discuss some key open questions. Developmental Dynamics 245:547-557, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Emerging pulmonary vasculature lacks fate specification.

PubMed

Schwarz, Margaret A; Caldwell, Lauren; Cafasso, Danielle; Zheng, Haihua

2009-01-01

Lung morphogenesis requires precise coordination between branching morphogenesis and vascularization to generate distal airways capable of supporting respiration at the cell-cell interface. The specific origins and types of blood vessels that initially form in the lung, however, remain obscure. Herein, we definitively show that during the early phases of lung development [i.e., embryonic day (E) 11.5], functional vessels, replete with blood flow, are restricted to the mesenchyme, distal to the epithelium. However, by day E14.5, and in response to epithelial-derived VEGF signals, functional vessels extend from the mesenchyme to the epithelial interface. Moreover, these vessels reside adjacent to multipotent mesenchymal stromal cells that likely play a regulatory role in this process. As well as and distinct from the systemic vasculature, immunostaining for EphrinB2 and EphB4 revealed that arterial and venous identity is not distinguishable in emergent pulmonary vasculature. Collectively, this study provides evidence that lung vascularization initially originates in the mesenchyme, distal to the epithelium, and that arterial-venous specification does not exist in the early lung. At a mechanistic level, we show that basilar epithelial VEGF prompts endothelial cells to move toward the epithelium where they undergo morphogenesis during the proliferative, canalicular stage. Thus our findings challenge existing notions of vascular origin and identity during development.

NuMA-microtubule interactions are critical for spindle orientation and the morphogenesis of diverse epidermal structures

PubMed Central

Seldin, Lindsey; Muroyama, Andrew; Lechler, Terry

2016-01-01

Mitotic spindle orientation is used to generate cell fate diversity and drive proper tissue morphogenesis. A complex of NuMA and dynein/dynactin is required for robust spindle orientation in a number of cell types. Previous research proposed that cortical dynein/dynactin was sufficient to generate forces on astral microtubules (MTs) to orient the spindle, with NuMA acting as a passive tether. In this study, we demonstrate that dynein/dynactin is insufficient for spindle orientation establishment in keratinocytes and that NuMA’s MT-binding domain, which targets MT tips, is also required. Loss of NuMA-MT interactions in skin caused defects in spindle orientation and epidermal differentiation, leading to neonatal lethality. In addition, we show that NuMA-MT interactions are also required in adult mice for hair follicle morphogenesis and spindle orientation within the transit-amplifying cells of the matrix. Loss of spindle orientation in matrix cells results in defective differentiation of matrix-derived lineages. Our results reveal an additional and direct function of NuMA during mitotic spindle positioning, as well as a reiterative use of spindle orientation in the skin to build diverse structures. DOI: http://dx.doi.org/10.7554/eLife.12504.001 PMID:26765568

Sox17 drives functional engraftment of endothelium converted from non-vascular cells

PubMed Central

Schachterle, William; Badwe, Chaitanya R.; Palikuqi, Brisa; Kunar, Balvir; Ginsberg, Michael; Lis, Raphael; Yokoyama, Masataka; Elemento, Olivier; Scandura, Joseph M.; Rafii, Shahin

2017-01-01

Transplanting vascular endothelial cells (ECs) to support metabolism and express regenerative paracrine factors is a strategy to treat vasculopathies and to promote tissue regeneration. However, transplantation strategies have been challenging to develop, because ECs are difficult to culture and little is known about how to direct them to stably integrate into vasculature. Here we show that only amniotic cells could convert to cells that maintain EC gene expression. Even so, these converted cells perform sub-optimally in transplantation studies. Constitutive Akt signalling increases expression of EC morphogenesis genes, including Sox17, shifts the genomic targeting of Fli1 to favour nearby Sox consensus sites and enhances the vascular function of converted cells. Enforced expression of Sox17 increases expression of morphogenesis genes and promotes integration of transplanted converted cells into injured vessels. Thus, Ets transcription factors specify non-vascular, amniotic cells to EC-like cells, whereas Sox17 expression is required to confer EC function. PMID:28091527

Bone morphogenetic protein-4 strongly potentiates growth factor-induced proliferation of mammary epithelial cells

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

Montesano, Roberto; Sarkoezi, Rita; Schramek, Herbert

2008-09-12

Bone morphogenetic proteins (BMPs) are multifunctional cytokines that elicit pleiotropic effects on biological processes such as cell proliferation, cell differentiation and tissue morphogenesis. With respect to cell proliferation, BMPs can exert either mitogenic or anti-mitogenic activities, depending on the target cells and their context. Here, we report that in low-density cultures of immortalized mammary epithelial cells, BMP-4 did not stimulate cell proliferation by itself. However, when added in combination with suboptimal concentrations of fibroblast growth factor (FGF)-2, FGF-7, FGF-10, epidermal growth factor (EGF) or hepatocyte growth factor (HGF), BMP-4 potently enhanced growth factor-induced cell proliferation. These results reveal a hithertomore » unsuspected interplay between BMP-4 and growth factors in the regulation of mammary epithelial cell proliferation. We suggest that the ability of BMP-4 to potentiate the mitogenic activity of multiple growth factors may contribute to mammary gland ductal morphogenesis as well as to breast cancer progression.« less

Cell-cell adhesion in the cnidaria: insights into the evolution of tissue morphogenesis.

PubMed

Magie, Craig R; Martindale, Mark Q

2008-06-01

Cell adhesion is a major aspect of cell biology and one of the fundamental processes involved in the development of a multicellular animal. Adhesive mechanisms, both cell-cell and between cell and extracellular matrix, are intimately involved in assembling cells into the three-dimensional structures of tissues and organs. The modulation of adhesive complexes could therefore be seen as a central component in the molecular control of morphogenesis, translating information encoded within the genome into organismal form. The availability of whole genomes from early-branching metazoa such as cnidarians is providing important insights into the evolution of adhesive processes by allowing for the easy identification of the genes involved in adhesion in these organisms. Discovery of the molecular nature of cell adhesion in the early-branching groups, coupled with comparisons across the metazoa, is revealing the ways evolution has tinkered with this vital cellular process in the generation of the myriad forms seen across the animal kingdom.

WAVE2 is required for directed cell migration and cardiovascular development.

PubMed

Yamazaki, Daisuke; Suetsugu, Shiro; Miki, Hiroaki; Kataoka, Yuki; Nishikawa, Shin-Ichi; Fujiwara, Takashi; Yoshida, Nobuaki; Takenawa, Tadaomi

2003-07-24

WAVE2, a protein related to Wiskott-Aldrich syndrome protein, is crucial for Rac-induced membrane ruffling, which is important in cell motility. Cell movement is essential for morphogenesis, but it is unclear how cell movement is regulated or related to morphogenesis. Here we show the physiological functions of WAVE2 by disruption of the WAVE2 gene in mice. WAVE2 was expressed predominantly in vascular endothelial cells during embryogenesis. WAVE2-/- embryos showed haemorrhages and died at about embryonic day 10. Deficiency in WAVE2 had no significant effect on vasculogenesis, but it decreased sprouting and branching of endothelial cells from existing vessels during angiogenesis. In WAVE2-/- endothelial cells, cell polarity formed in response to vascular endothelial growth factor, but the formation of lamellipodia at leading edges and capillaries was severely impaired. These findings indicate that WAVE2-regulated actin reorganization might be required for proper cell movement and that a lack of functional WAVE2 impairs angiogenesis in vivo.

Scribble is required for normal epithelial cell–cell contacts and lumen morphogenesis in the mammalian lung

PubMed Central

Yates, Laura L.; Schnatwinkel, Carsten; Hazelwood, Lee; Chessum, Lauren; Paudyal, Anju; Hilton, Helen; Romero, M. Rosario; Wilde, Jonathan; Bogani, Debora; Sanderson, Jeremy; Formstone, Caroline; Murdoch, Jennifer N.; Niswander, Lee A.; Greenfield, Andy; Dean, Charlotte H.

2013-01-01

During lung development, proper epithelial cell arrangements are critical for the formation of an arborized network of tubes. Each tube requires a lumen, the diameter of which must be tightly regulated to enable optimal lung function. Lung branching and lumen morphogenesis require close epithelial cell–cell contacts that are maintained as a result of adherens junctions, tight junctions and by intact apical–basal (A/B) polarity. However, the molecular mechanisms that maintain epithelial cohesion and lumen diameter in the mammalian lung are unknown. Here we show that Scribble, a protein implicated in planar cell polarity (PCP) signalling, is necessary for normal lung morphogenesis. Lungs of the Scrib mouse mutant Circletail (Crc) are abnormally shaped with fewer airways, and these airways often lack a visible, ‘open’ lumen. Mechanistically we show that Scrib genetically interacts with the core PCP gene Vangl2 in the developing lung and that the distribution of PCP pathway proteins and Rho mediated cytoskeletal modification is perturbed in ScribCrc/Crc lungs. However A/B polarity, which is disrupted in Drosophila Scrib mutants, is largely unaffected. Notably, we find that Scrib mediates functions not attributed to other PCP proteins in the lung. Specifically, Scrib localises to both adherens and tight junctions of lung epithelia and knockdown of Scrib in lung explants and organotypic cultures leads to reduced cohesion of lung epithelial cells. Live imaging of Scrib knockdown lungs shows that Scrib does not affect bud bifurcation, as previously shown for the PCP protein Celsr1, but is required to maintain epithelial cohesion. To understand the mechanism leading to reduced cell–cell association, we show that Scrib associates with β-catenin in embryonic lung and the sub-cellular distribution of adherens and tight junction proteins is perturbed in mutant lung epithelia. Our data reveal that Scrib is required for normal lung epithelial organisation and lumen morphogenesis by maintaining cell–cell contacts. Thus we reveal novel and important roles for Scrib in lung development operating via the PCP pathway, and in regulating junctional complexes and cell cohesion. PMID:23195221

Rac1 GTPase -deficient mouse lens exhibits defects in shape, suture formation, fiber cell migration and survival

PubMed Central

Maddala, Rupalatha; Chauhan, Bharesh K.; Walker, Christopher; Zheng, Yi; Robinson, Michael L.; Lang, Richard A.; Rao, Ponugoti V.

2011-01-01

Morphogenesis and shape of the ocular lens depend on epithelial cell elongation and differentiation into fiber cells, followed by the symmetric and compact organization of fiber cells within an enclosed extracellular matrix-enriched elastic capsule. The cellular mechanisms orchestrating these different events however, remain obscure. We investigated the role of the Rac1 GTPase in these processes by targeted deletion of expression using the conditional gene knockout (cKO) approach. Rac1 cKO mice were derived from two different Cre (Le-Cre and MLR-10) transgenic mice in which lens-specific Cre expression starts at embryonic day 8.75 and 10.5, respectively, in both the lens epithelium and fiber cells. The Le-Cre/Rac1 cKO mice exhibited an early-onset (E12.5) and severe lens phenotype compared to the MLR-10/Rac1 cKO (E15.5) mice. While the Le-Cre/Rac1 cKO lenses displayed delayed primary fiber cell elongation, lenses from both Rac1 cKO strains were characterized by abnormal shape, impaired secondary fiber cell migration, sutural defects and thinning of the posterior capsule which often led to rupture. Lens fiber cell N-cadherin/β-catenin/Rap1/Nectin-based cell-cell junction formation and WAVE-2/Abi-2/Nap1-regulated actin polymerization were impaired in the Rac1 deficient mice. Additionally, the Rac1 cKO lenses were characterized by a shortened epithelial sheet, reduced levels of extracellular matrix (ECM) proteins and increased apoptosis. Taken together, these data uncover the essential role of Rac1 GTPase activity in establishment and maintenance of lens shape, suture formation and capsule integrity, and in fiber cell migration, adhesion and survival, via regulation of actin cytoskeletal dynamics, cell adhesive interactions and ECM turnover. PMID:21945075

The mechanics of development: models and methods for tissue morphogenesis

PubMed Central

Gjorevski, Nikolce; Nelson, Celeste M.

2011-01-01

Embryonic development is a physical process during which masses of cells are sculpted into functional organs. The mechanical properties of tissues and the forces exerted on them serve as epigenetic regulators of morphogenesis. Understanding these mechanobiological effects in the embryo requires new experimental approaches. Here we focus on branching of the lung airways and bending of the heart tube to describe examples of mechanical and physical cues that guide cell fate decisions and organogenesis. We highlight recent technological advances to measure tissue elasticity and endogenous mechanical stresses in real time during organ development. We also discuss recent progress in manipulating forces in intact embryos. PMID:20860059

Engineering epithelial-stromal interactions in vitro for toxicology assessment.

PubMed

Belair, David G; Abbott, Barbara D

2017-05-01

Crosstalk between epithelial and stromal cells drives the morphogenesis of ectodermal organs during development and promotes normal mature adult epithelial tissue homeostasis. Epithelial-stromal interactions (ESIs) have historically been examined using mammalian models and ex vivo tissue recombination. Although these approaches have elucidated signaling mechanisms underlying embryonic morphogenesis processes and adult mammalian epithelial tissue function, they are limited by the availability of tissue, low throughput, and human developmental or physiological relevance. In this review, we describe how bioengineered ESIs, using either human stem cells or co-cultures of human primary epithelial and stromal cells, have enabled the development of human in vitro epithelial tissue models that recapitulate the architecture, phenotype, and function of adult human epithelial tissues. We discuss how the strategies used to engineer mature epithelial tissue models in vitro could be extrapolated to instruct the design of organotypic culture models that can recapitulate the structure of embryonic ectodermal tissues and enable the in vitro assessment of events critical to organ/tissue morphogenesis. Given the importance of ESIs towards normal epithelial tissue development and function, such models present a unique opportunity for toxicological screening assays to incorporate ESIs to assess the impact of chemicals on mature and developing epidermal tissues. Published by Elsevier B.V.

Engineering epithelial-stromal interactions in vitro for toxicology assessment

PubMed Central

Belair, David G.; Abbott, Barbara D.

2018-01-01

Crosstalk between epithelial and stromal cells drives the morphogenesis of ectodermal organs during development and promotes normal mature adult epithelial tissue homeostasis. Epithelial-stromal interactions (ESIs) have historically been examined using mammalian models and ex vivo tissue recombination. Although these approaches have elucidated signaling mechanisms underlying embryonic morphogenesis processes and adult mammalian epithelial tissue function, they are limited by the availability of tissue, low throughput, and human developmental or physiological relevance. In this review, we describe how bioengineered ESIs, using either human stem cells or co-cultures of human primary epithelial and stromal cells, have enabled the development of human in vitro epithelial tissue models that recapitulate the architecture, phenotype, and function of adult human epithelial tissues. We discuss how the strategies used to engineer mature epithelial tissue models in vitro could be extrapolated to instruct the design of organotypic culture models that can recapitulate the structure of embryonic ectodermal tissues and enable the in vitro assessment of events critical to organ/tissue morphogenesis. Given the importance of ESIs towards normal epithelial tissue development and function, such models present a unique opportunity for toxicological screening assays to incorporate ESIs to assess the impact of chemicals on mature and developing epidermal tissues. PMID:28285100

Solitary waves in morphogenesis: Determination fronts as strain-cued strain transformations among automatous cells

NASA Astrophysics Data System (ADS)

Cox, Brian N.; Landis, Chad M.

2018-02-01

We present a simple theory of a strain pulse propagating as a solitary wave through a continuous two-dimensional population of cells. A critical strain is assumed to trigger a strain transformation, while, simultaneously, cells move as automata to tend to restore a preferred cell density. We consider systems in which the strain transformation is a shape change, a burst of proliferation, or the commencement of growth (which changes the shape of the population sheet), and demonstrate isomorphism among these cases. Numerical and analytical solutions describe a strain pulse whose height does not depend on how the strain disturbance was first launched, or the rate at which the strain transformation is achieved, or the rate constant in the rule for the restorative cell motion. The strain pulse is therefore very stable, surviving the imposition of strong perturbations: it would serve well as a timing signal in development. The automatous wave formulation is simple, with few model parameters. A strong case exists for the presence of a strain pulse during amelogenesis. Quantitative analysis reveals a simple relationship between the velocity of the leading edge of the pulse in amelogenesis and the known speed of migration of ameloblast cells. This result and energy arguments support the depiction of wave motion as an automatous cell response to strain, rather than as a response to an elastic energy gradient. The theory may also contribute to understanding the determination front in somitogenesis, moving fronts of convergent-extension transformation, and mitotic wavefronts in the syncytial drosophila embryo.

Repair Mechanism of Osteochondral Defect Promoted by Bioengineered Chondrocyte Sheet

PubMed Central

Kamei, Naosuke; Adachi, Nobuo; Hamanishi, Michio; Kamei, Goki; Mahmoud, Elhussein Elbadry; Nakano, Tomohiro; Iwata, Takanori; Yamato, Masayuki; Okano, Teruo; Ochi, Mitsuo

2015-01-01

Cell sheet engineering has developed as a remarkable method for cell transplantation. In the field of cartilage regeneration, several studies previously reported that cartilage defects could be regenerated by transplantation of a chondrocyte sheet using cell sheet engineering. However, it remains unclear how such a thin cell sheet could repair a deep cartilage defect. We, therefore, focused on the mechanism of cartilage repair using cell sheet engineering in this study. Chondrocyte sheets and synovial cell sheets were fabricated using cell sheet engineering, and these allogenic cell sheets were transplanted to cover an osteochondral defect in a rat model. Macroscopic and histological evaluation was performed at 4 and 12 weeks after transplantation. Analysis of the gene expression of each cell sheet and of the regenerated tissue at 1 week after transplantation was performed. In addition, green fluorescent protein (GFP) transgenic rats were used as donors (transplanted chondrocyte sheets) or recipients (osteochondral defect models) to identify the cell origin of regenerated cartilage. Cartilage repair was significantly better in the group implanted with a chondrocyte sheet than in that with a synovial cell sheet. The results of gene expression analysis suggest that the possible factor contributing to cartilage repair might be TGFβ1. Cell tracking experiments using GFP transgenic rats showed that the regenerated cartilage was largely composed of cells derived from the transplanted chondrocyte sheets. PMID:25396711

Dynamics of cell wall elasticity pattern shapes the cell during yeast mating morphogenesis.

PubMed

Goldenbogen, Björn; Giese, Wolfgang; Hemmen, Marie; Uhlendorf, Jannis; Herrmann, Andreas; Klipp, Edda

2016-09-01

The cell wall defines cell shape and maintains integrity of fungi and plants. When exposed to mating pheromone, Saccharomyces cerevisiae grows a mating projection and alters in morphology from spherical to shmoo form. Although structural and compositional alterations of the cell wall accompany shape transitions, their impact on cell wall elasticity is unknown. In a combined theoretical and experimental approach using finite-element modelling and atomic force microscopy (AFM), we investigated the influence of spatially and temporally varying material properties on mating morphogenesis. Time-resolved elasticity maps of shmooing yeast acquired with AFM in vivo revealed distinct patterns, with soft material at the emerging mating projection and stiff material at the tip. The observed cell wall softening in the protrusion region is necessary for the formation of the characteristic shmoo shape, and results in wider and longer mating projections. The approach is generally applicable to tip-growing fungi and plants cells. © 2016 The Authors.

Morphogenesis and gravity in a whole amphibian embryo and in isolated blastomeres of sea urchins.

PubMed

Izumi-Kurotani, Akemi; Kiyomoto, Masato

2003-01-01

Fertilization and subsequent embryogenesis of newts occurred normally under microgravity in two Astronewt flight experiments. By accumulation of the results from the amphibian flight experiments including 'Astronewt', it is considered that gravity has rather small effects on the early development of amphibian eggs. However, some temporary abnormalities, which recover in the course of the further developmental process, have been observed. Some regulations may occur in whole embryos. For a thorough knowledge about the role of gravity in morphogenesis, we need to investigate the gravitational effects on a single cell in a whole embryo. We propose a new experimental system with sea urchin embryos and micromeres for further studies at a cellular level of the effects of gravity on morphogenesis.

Microtubules and epithem-cell morphogenesis in hydathodes of Pilea cadierei.

PubMed

Galatis, B

1988-12-01

When cell divisions have ceased, the epithem of the hydathodes of Pilea cadierei Gagnep. et Guill. consists of small polyhedral cells exhibiting a meristematic appearance, and completely lacks intercellular spaces. The cortical microtubules in epithem cells exhibit a unique organization: they are not scattered along the whole wall surface but form groups lying at some distance from each other. In sections, from two to eight groups of microtubules can be observed, each lining a wall region averaging between 0.5 and 1.5 μm in length. These groups represent sections of microtubule bundles girdling a major part or the whole of the cell periphery. They are connected to one another by anastomoses, forming a microtubular reticulum. The assembly of microtubule bundles is followed by the appearance of distinct local thickenings in the adjacent wall areas. The cellulose microfibrils in the thickenings are deposited in parallel to the underlying microtubules. Gradually, the vacuolating epithem cells undergo swelling, except for the areas bounded by the wall thickenings. Since the latter, and actually their constituent bundles of cellulose microfibrils, cannot extend in length the differential cell growth results in schizogenous formation of intercellular spaces between contiguous cell walls at their thickened regions. The spaces then broaden and merge to become an extensive intercellular space system. As a result of the above processes, the epithem cells become constricted and finally deeply lobed. The observations show that (i) the cortical microtubules are intimately involved in the morphogenesis of the epithem cells and (ii) the initiation and development of the epithem intercellular spaces is a phenomenon directly related to cell morphogenesis and therefore to the cortical microtubule cytoskeleton. The sites of initiation of these spaces are highly predictable.

An Organotypic 3D Assay for Primary Human Mammary Epithelial Cells that Recapitulates Branching Morphogenesis.

PubMed

Linnemann, Jelena R; Meixner, Lisa K; Miura, Haruko; Scheel, Christina H

2017-01-01

We have developed a three-dimensional organotypic culture system for primary human mammary epithelial cells (HMECs) in which the cells are cultured in free floating collagen type I gels. In this assay, luminal cells predominantly form multicellular spheres, while basal/myoepithelial cells form complex branched structures resembling terminal ductal lobular units (TDLUs), the functional units of the human mammary gland in situ. The TDLU-like organoids can be cultured for at least 3 weeks and can then be passaged multiple times. Subsequently, collagen gels can be stained with carmine or by immunofluorescence to allow for the analysis of morphology, protein expression and polarization, and to facilitate quantification of structures. In addition, structures can be isolated for gene expression analysis. In summary, this technique is suitable for studying branching morphogenesis, regeneration, and differentiation of HMECs as well as their dependence on the physical environment.

Mechanical Influences on Morphogenesis of the Knee Joint Revealed through Morphological, Molecular and Computational Analysis of Immobilised Embryos

PubMed Central

Roddy, Karen A.; Prendergast, Patrick J.; Murphy, Paula

2011-01-01

Very little is known about the regulation of morphogenesis in synovial joints. Mechanical forces generated from muscle contractions are required for normal development of several aspects of normal skeletogenesis. Here we show that biophysical stimuli generated by muscle contractions impact multiple events during chick knee joint morphogenesis influencing differential growth of the skeletal rudiment epiphyses and patterning of the emerging tissues in the joint interzone. Immobilisation of chick embryos was achieved through treatment with the neuromuscular blocking agent Decamethonium Bromide. The effects on development of the knee joint were examined using a combination of computational modelling to predict alterations in biophysical stimuli, detailed morphometric analysis of 3D digital representations, cell proliferation assays and in situ hybridisation to examine the expression of a selected panel of genes known to regulate joint development. This work revealed the precise changes to shape, particularly in the distal femur, that occur in an altered mechanical environment, corresponding to predicted changes in the spatial and dynamic patterns of mechanical stimuli and region specific changes in cell proliferation rates. In addition, we show altered patterning of the emerging tissues of the joint interzone with the loss of clearly defined and organised cell territories revealed by loss of characteristic interzone gene expression and abnormal expression of cartilage markers. This work shows that local dynamic patterns of biophysical stimuli generated from muscle contractions in the embryo act as a source of positional information guiding patterning and morphogenesis of the developing knee joint. PMID:21386908

Loss of laminin alpha 1 results in multiple structural defects and divergent effects on adhesion during vertebrate optic cup morphogenesis

PubMed Central

Bryan, Chase D.; Chien, Chi-Bin; Kwan, Kristen M.

2016-01-01

The vertebrate eye forms via a complex set of morphogenetic events. The optic vesicle evaginates and undergoes transformative shape changes to form the optic cup, in which neural retina and retinal pigmented epithelium enwrap the lens. It has long been known that a complex, glycoprotein-rich extracellular matrix layer surrounds the developing optic cup throughout the process, yet the functions of the matrix and its specific molecular components have remained unclear. Previous work established a role for laminin extracellular matrix in particular steps of eye development, including optic vesicle evagination, lens differentiation, and retinal ganglion cell polarization, yet it is unknown what role laminin might play in the early process of optic cup formation subsequent to the initial step of optic vesicle evagination. Here, we use the zebrafish lama1 mutant (lama1UW1) to determine the function of laminin during optic cup morphogenesis. Using live imaging, we find, surprisingly, that loss of laminin leads to divergent effects on focal adhesion assembly in a spatiotemporally-specific manner, and that laminin is required for multiple steps of optic cup morphogenesis, including optic stalk constriction, invagination, and formation of a spherical lens. Laminin is not required for single cell behaviors and changes in cell shape. Rather, in lama1UW1 mutants, loss of epithelial polarity and altered adhesion lead to defective tissue architecture and formation of a disorganized retina. These results demonstrate that the laminin extracellular matrix plays multiple critical roles regulating adhesion and polarity to establish and maintain tissue structure during optic cup morphogenesis. PMID:27339294

Yeast casein kinase 2 governs morphology, biofilm formation, cell wall integrity, and host cell damage of Candida albicans

PubMed Central

Irrizary, Jihyun; Liboro, Karl; Bogarin, Thania; Macias, Marlene; Eivers, Edward; Porter, Edith; Filler, Scott G.

2017-01-01

The regulatory networks governing morphogenesis of a pleomorphic fungus, Candida albicans are extremely complex and remain to be completely elucidated. This study investigated the function of C. albicans yeast casein kinase 2 (CaYck2p). The yck2Δ/yck2Δ strain displayed constitutive pseudohyphae in both yeast and hyphal growth conditions, and formed enhanced biofilm under non-biofilm inducing condition. This finding was further supported by gene expression analysis of the yck2Δ/yck2Δ strain which showed significant upregulation of UME6, a key transcriptional regulator of hyphal transition and biofilm formation, and cell wall protein genes ALS3, HWP1, and SUN41, all of which are associated with morphogenesis and biofilm architecture. The yck2Δ/yck2Δ strain was hypersensitive to cell wall damaging agents and had increased compensatory chitin deposition in the cell wall accompanied by an upregulation of the expression of the chitin synthase genes, CHS2, CHS3, and CHS8. Absence of CaYck2p also affected fungal-host interaction; the yck2Δ/yck2Δ strain had significantly reduced ability to damage host cells. However, the yck2Δ/yck2Δ strain had wild-type susceptibility to cyclosporine and FK506, suggesting that CaYck2p functions independently from the Ca+/calcineurin pathway. Thus, in C. albicans, Yck2p is a multifunctional kinase that governs morphogenesis, biofilm formation, cell wall integrity, and host cell interactions. PMID:29107946

Physics of Bacterial Morphogenesis

PubMed Central

Sun, Sean X.; Jiang, Hongyuan

2011-01-01

Summary: Bacterial cells utilize three-dimensional (3D) protein assemblies to perform important cellular functions such as growth, division, chemoreception, and motility. These assemblies are composed of mechanoproteins that can mechanically deform and exert force. Sometimes, small-nucleotide hydrolysis is coupled to mechanical deformations. In this review, we describe the general principle for an understanding of the coupling of mechanics with chemistry in mechanochemical systems. We apply this principle to understand bacterial cell shape and morphogenesis and how mechanical forces can influence peptidoglycan cell wall growth. We review a model that can potentially reconcile the growth dynamics of the cell wall with the role of cytoskeletal proteins such as MreB and crescentin. We also review the application of mechanochemical principles to understand the assembly and constriction of the FtsZ ring. A number of potential mechanisms are proposed, and important questions are discussed. PMID:22126993

Reprogramming cells and tissue patterning via bioelectrical pathways: molecular mechanisms and biomedical opportunities

PubMed Central

Levin, Michael

2013-01-01

Transformative impact in regenerative medicine requires more than the reprogramming of individual cells: advances in repair strategies for birth defects or injuries, tumor normalization, and the construction of bioengineered organs and tissues all require the ability to control large-scale anatomical shape. Much recent work has focused on the transcriptional and biochemical regulation of cell behaviour and morphogenesis. However, exciting new data reveal that bioelectrical properties of cells and their microenvironment exert a profound influence on cell differentiation, proliferation, and migration. Ion channels and pumps expressed in all cells, not just excitable nerve and muscle, establish resting potentials that vary across tissues and change with significant developmental events. Most importantly, the spatio-temporal gradients of these endogenous transmembrane voltage potentials (Vmem) serve as instructive patterning cues for large-scale anatomy, providing organ identity, positional information, and prepattern template cues for morphogenesis. New genetic and pharmacological techniques for molecular modulation of bioelectric gradients in vivo have revealed the ability to initiate complex organogenesis, change tissue identity, and trigger regeneration of whole vertebrate appendages. A large segment of the spatial information processing that orchestrates individual cells’ programs towards the anatomical needs of the host organism is electrical; this blurs the line between memory and decision-making in neural networks and morphogenesis in non-neural tissues. Advances in cracking this bioelectric code will enable the rational reprogramming of shape in whole tissues and organs, revolutionizing regenerative medicine, developmental biology, and synthetic bioengineering. PMID:23897652

A Computational Framework for 3D Mechanical Modeling of Plant Morphogenesis with Cellular Resolution

PubMed Central

Gilles, Benjamin; Hamant, Olivier; Boudaoud, Arezki; Traas, Jan; Godin, Christophe

2015-01-01

The link between genetic regulation and the definition of form and size during morphogenesis remains largely an open question in both plant and animal biology. This is partially due to the complexity of the process, involving extensive molecular networks, multiple feedbacks between different scales of organization and physical forces operating at multiple levels. Here we present a conceptual and modeling framework aimed at generating an integrated understanding of morphogenesis in plants. This framework is based on the biophysical properties of plant cells, which are under high internal turgor pressure, and are prevented from bursting because of the presence of a rigid cell wall. To control cell growth, the underlying molecular networks must interfere locally with the elastic and/or plastic extensibility of this cell wall. We present a model in the form of a three dimensional (3D) virtual tissue, where growth depends on the local modulation of wall mechanical properties and turgor pressure. The model shows how forces generated by turgor-pressure can act both cell autonomously and non-cell autonomously to drive growth in different directions. We use simulations to explore lateral organ formation at the shoot apical meristem. Although different scenarios lead to similar shape changes, they are not equivalent and lead to different, testable predictions regarding the mechanical and geometrical properties of the growing lateral organs. Using flower development as an example, we further show how a limited number of gene activities can explain the complex shape changes that accompany organ outgrowth. PMID:25569615

Solid oxide fuel cell with multi-unit construction and prismatic design

DOEpatents

McPheeters, Charles C.; Dees, Dennis W.; Myles, Kevin M.

1999-01-01

A single cell unit of a solid oxide fuel cell that is individually fabricated and sintered prior to being connected to adjacent cells to form a solid oxide fuel cell. The single cell unit is comprised of a shaped anode sheet positioned between a flat anode sheet and an anode-electrolyte-cathode (A/E/C) sheet, and a shaped cathode sheet positioned between the A/E/C sheet and a cathode-interconnect-anode (C/I/A) sheet. An alternate embodiment comprises a shaped cathode sheet positioned between an A/E/C sheet and a C/I/A sheet. The shaped sheets form channels for conducting reactant gases. Each single cell unit is individually sintered to form a finished sub-assembly. The finished sub-assemblies are connected in electrical series by interposing connective material between the end surfaces of adjacent cells, whereby individual cells may be inspected for defects and interchanged with non-defective single cell units.

Structure of Cellulose Microfibrils in Primary Cell Walls from Collenchyma1[C][W][OA

PubMed Central

Thomas, Lynne H.; Forsyth, V. Trevor; Šturcová, Adriana; Kennedy, Craig J.; May, Roland P.; Altaner, Clemens M.; Apperley, David C.; Wess, Timothy J.; Jarvis, Michael C.

2013-01-01

In the primary walls of growing plant cells, the glucose polymer cellulose is assembled into long microfibrils a few nanometers in diameter. The rigidity and orientation of these microfibrils control cell expansion; therefore, cellulose synthesis is a key factor in the growth and morphogenesis of plants. Celery (Apium graveolens) collenchyma is a useful model system for the study of primary wall microfibril structure because its microfibrils are oriented with unusual uniformity, facilitating spectroscopic and diffraction experiments. Using a combination of x-ray and neutron scattering methods with vibrational and nuclear magnetic resonance spectroscopy, we show that celery collenchyma microfibrils were 2.9 to 3.0 nm in mean diameter, with a most probable structure containing 24 chains in cross section, arranged in eight hydrogen-bonded sheets of three chains, with extensive disorder in lateral packing, conformation, and hydrogen bonding. A similar 18-chain structure, and 24-chain structures of different shape, fitted the data less well. Conformational disorder was largely restricted to the surface chains, but disorder in chain packing was not. That is, in position and orientation, the surface chains conformed to the disordered lattice constituting the core of each microfibril. There was evidence that adjacent microfibrils were noncovalently aggregated together over part of their length, suggesting that the need to disrupt these aggregates might be a constraining factor in growth and in the hydrolysis of cellulose for biofuel production. PMID:23175754

Structure of cellulose microfibrils in primary cell walls from collenchyma.

PubMed

Thomas, Lynne H; Forsyth, V Trevor; Sturcová, Adriana; Kennedy, Craig J; May, Roland P; Altaner, Clemens M; Apperley, David C; Wess, Timothy J; Jarvis, Michael C

2013-01-01

In the primary walls of growing plant cells, the glucose polymer cellulose is assembled into long microfibrils a few nanometers in diameter. The rigidity and orientation of these microfibrils control cell expansion; therefore, cellulose synthesis is a key factor in the growth and morphogenesis of plants. Celery (Apium graveolens) collenchyma is a useful model system for the study of primary wall microfibril structure because its microfibrils are oriented with unusual uniformity, facilitating spectroscopic and diffraction experiments. Using a combination of x-ray and neutron scattering methods with vibrational and nuclear magnetic resonance spectroscopy, we show that celery collenchyma microfibrils were 2.9 to 3.0 nm in mean diameter, with a most probable structure containing 24 chains in cross section, arranged in eight hydrogen-bonded sheets of three chains, with extensive disorder in lateral packing, conformation, and hydrogen bonding. A similar 18-chain structure, and 24-chain structures of different shape, fitted the data less well. Conformational disorder was largely restricted to the surface chains, but disorder in chain packing was not. That is, in position and orientation, the surface chains conformed to the disordered lattice constituting the core of each microfibril. There was evidence that adjacent microfibrils were noncovalently aggregated together over part of their length, suggesting that the need to disrupt these aggregates might be a constraining factor in growth and in the hydrolysis of cellulose for biofuel production.

Zic1 and Zic4 regulate zebrafish roof plate specification and hindbrain ventricle morphogenesis

PubMed Central

Elsen, Gina E.; Choi, Louis; Millen, Kathleen; Grinblat, Yevgenya; Prince, Victoria E.

2008-01-01

During development, the lumen of the neural tube develops into a system of brain cavities or ventricles, which play important roles in normal CNS function. We have established that the formation of the hindbrain (4th) ventricle in zebrafish is dependent upon the pleiotropic functions of the genes implicated in human Dandy Walker Malformation, Zic1 and Zic4. Using morpholino knockdown we show that zebrafish Zic1 and Zic4 are required for normal morphogenesis of the 4th ventricle. In Zic1 and/or Zic4 morphants the ventricle does not open properly, but remains completely or partially fused from the level of rhombomere (r) 2 towards the posterior. In the absence of Zic function early hindbrain regionalization and neural crest development remain unaffected, but dorsal hindbrain progenitor cell proliferation is significantly reduced. Importantly, we find that Zic1 and Zic4 are required for development of the dorsal roof plate. In Zic morphants expression of roof plate markers, including lmx1b.1 and lmx1b.2, is disrupted. We further demonstrate that zebrafish Lmx1b function is required for both hindbrain roof plate development and 4th ventricle morphogenesis, confirming that roof plate formation is a critical component of ventricle development. Finally, we show that dorsal rhombomere boundary signaling centers depend on Zic1 and Zic4 function and on roof plate signals, and provide evidence that these boundary signals are also required for ventricle morphogenesis. In summary, we conclude that Zic1 and Zic4 control zebrafish 4th ventricle morphogenesis by regulating multiple mechanisms including cell proliferation and fate specification in the dorsal hindbrain. PMID:18191121

Patterned cortical tension mediated by N-cadherin controls cell geometric order in the Drosophila eye

PubMed Central

Chan, Eunice HoYee; Chavadimane Shivakumar, Pruthvi; Clément, Raphaël; Laugier, Edith; Lenne, Pierre-François

2017-01-01

Adhesion molecules hold cells together but also couple cell membranes to a contractile actomyosin network, which limits the expansion of cell contacts. Despite their fundamental role in tissue morphogenesis and tissue homeostasis, how adhesion molecules control cell shapes and cell patterns in tissues remains unclear. Here we address this question in vivo using the Drosophila eye. We show that cone cell shapes depend little on adhesion bonds and mostly on contractile forces. However, N-cadherin has an indirect control on cell shape. At homotypic contacts, junctional N-cadherin bonds downregulate Myosin-II contractility. At heterotypic contacts with E-cadherin, unbound N-cadherin induces an asymmetric accumulation of Myosin-II, which leads to a highly contractile cell interface. Such differential regulation of contractility is essential for morphogenesis as loss of N-cadherin disrupts cell rearrangements. Our results establish a quantitative link between adhesion and contractility and reveal an unprecedented role of N-cadherin on cell shapes and cell arrangements. DOI: http://dx.doi.org/10.7554/eLife.22796.001 PMID:28537220

In vitro cementoblast-like differentiation of postmigratory neural crest-derived p75{sup +} stem cells with dental follicle cell conditioned medium

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

Wen, Xiujie; Liu, Luchuan; Deng, Manjing

Cranial neural crest-derived cells (CNCCs) play important role in epithelial–mesenchymal interactions during tooth morphogenesis. However, the heterogeneity of CNCCs and their tendency to spontaneously differentiate along smooth muscle or osteoblast lineages in vitro limit further understanding of their biological properties. We studied the differentiation properties of isolated rat embryonic postmigratory CNCCs, expressing p75 neurotrophin receptor (p75NTR). These p75NTR positive (p75{sup +}) CNCCs, isolated using fluorescence activated cell sorter, exhibited fibroblast-like morphology and characteristics of mesenchymal stem cells. Incubation of p75{sup +} CNCCs in dental follicle cell conditioned medium (DFCCM) combined with dentin non-collagenous proteins (dNCPs), altered their morphological features tomore » cementoblast-like appearance. These cells also showed low proliferative activity, high ALP activity and significantly increased calcified nodule formation. Markers related to mineralization or specific to cementoblast lineage were highly expressed in dNCPs/DFCCM-treated p75{sup +} cells, suggesting their differentiation along cementoblast-like lineage. p75{sup +} stem cells selected from postmigratory CNCCs represent a pure stem cell population and could be used as a stem cell model for in vitro studies due to their intrinsic ability to differentiate to neuronal cells and transform from neuroectoderm to ectomesenchyme. They can provide a potential stem cell resource for tooth engineering studies and help to further investigate mechanisms of epithelial–mesenchymal interactions in tooth morphogenesis. - Highlights: • Cranial neural crest-derived cells (CNCCs) take part in tooth morphogenesis. • positive (p75{sup +}) CNCCs are fibroblast-like and resemble mesenchymal stem cells. • p75{sup +} CNCCs in dental follicle cell medium (DFCCM/dNCP) appear like cementoblasts. • DFCCM/dNCP-treated p75{sup +} cells express cementoblast specific mineralization markers. • p75{sup +} cells are pure stem cells and able to differentiate to neuronal cells.« less

An Antarctic hypotrichous ciliate, Parasterkiella thompsoni (Foissner) nov. gen., nov. comb., recorded in Argentinean peat-bogs: morphology, morphogenesis, and molecular phylogeny.

PubMed

Küppers, Gabriela Cristina; Paiva, Thiago da Silva; Borges, Bárbara do Nascimento; Harada, Maria Lúcia; Garraza, Gabriela González; Mataloni, Gabriela

2011-05-01

The ciliate Parasterkiella thompsoni (Foissner, 1996) nov. gen., nov. comb. was originally described from Antarctica. In the present study, we report the morphology, morphogenesis during cell division, and molecular phylogeny inferred from the 18S-rDNA sequence of a population isolated from the Rancho Hambre peat bog, Tierra del Fuego Province (Argentina). The study is based on live and protargol-impregnated specimens. Molecular phylogeny was inferred from trees constructed by means of the maximum parsimony, neighbor joining, and Bayesian analyses. The interphase morphology matches the original description of the species. During the cell division, stomatogenesis begins with the de novo proliferation of two fields of basal bodies, each one left of the postoral ventral cirri and of transverse cirri, which later unify. Primordia IV-VI of the proter develop from disaggregation of cirrus IV/3, while primordium IV of the opisthe develops from cirrus IV/2 and primordia V and VI from cirrus V/4. Dorsal morphogenesis occurs in the Urosomoida pattern-that is, the fragmentation of kinety 3 is lacking. Three macronuclear nodules are generated before cytokinesis. Phylogenetic analyses consistently placed P. thompsoni within the stylonychines. New data on the morphogenesis of the dorsal ciliature justifies the transference of Sterkiella thompsoni to a new genus Parasterkiella. Copyright © 2011 Elsevier GmbH. All rights reserved.

RodZ links MreB to cell wall synthesis to mediate MreB rotation and robust morphogenesis

PubMed Central

Morgenstein, Randy M.; Bratton, Benjamin P.; Nguyen, Jeffrey P.; Ouzounov, Nikolay; Shaevitz, Joshua W.; Gitai, Zemer

2015-01-01

The rod shape of most bacteria requires the actin homolog, MreB. Whereas MreB was initially thought to statically define rod shape, recent studies found that MreB dynamically rotates around the cell circumference dependent on cell wall synthesis. However, the mechanism by which cytoplasmic MreB is linked to extracytoplasmic cell wall synthesis and the function of this linkage for morphogenesis has remained unclear. Here we demonstrate that the transmembrane protein RodZ mediates MreB rotation by directly or indirectly coupling MreB to cell wall synthesis enzymes. Furthermore, we map the RodZ domains that link MreB to cell wall synthesis and identify mreB mutants that suppress the shape defect of ΔrodZ without restoring rotation, uncoupling rotation from rod-like growth. Surprisingly, MreB rotation is dispensable for rod-like shape determination under standard laboratory conditions but is required for the robustness of rod shape and growth under conditions of cell wall stress. PMID:26396257

RodZ links MreB to cell wall synthesis to mediate MreB rotation and robust morphogenesis.

PubMed

Morgenstein, Randy M; Bratton, Benjamin P; Nguyen, Jeffrey P; Ouzounov, Nikolay; Shaevitz, Joshua W; Gitai, Zemer

2015-10-06

The rod shape of most bacteria requires the actin homolog, MreB. Whereas MreB was initially thought to statically define rod shape, recent studies found that MreB dynamically rotates around the cell circumference dependent on cell wall synthesis. However, the mechanism by which cytoplasmic MreB is linked to extracytoplasmic cell wall synthesis and the function of this linkage for morphogenesis has remained unclear. Here we demonstrate that the transmembrane protein RodZ mediates MreB rotation by directly or indirectly coupling MreB to cell wall synthesis enzymes. Furthermore, we map the RodZ domains that link MreB to cell wall synthesis and identify mreB mutants that suppress the shape defect of ΔrodZ without restoring rotation, uncoupling rotation from rod-like growth. Surprisingly, MreB rotation is dispensable for rod-like shape determination under standard laboratory conditions but is required for the robustness of rod shape and growth under conditions of cell wall stress.

Autonomy and Non-autonomy of Angiogenic Cell Movements Revealed by Experiment-Driven Mathematical Modeling.

PubMed

Sugihara, Kei; Nishiyama, Koichi; Fukuhara, Shigetomo; Uemura, Akiyoshi; Arima, Satoshi; Kobayashi, Ryo; Köhn-Luque, Alvaro; Mochizuki, Naoki; Suda, Toshio; Ogawa, Hisao; Kurihara, Hiroki

2015-12-01

Angiogenesis is a multicellular phenomenon driven by morphogenetic cell movements. We recently reported morphogenetic vascular endothelial cell (EC) behaviors to be dynamic and complex. However, the principal mechanisms orchestrating individual EC movements in angiogenic morphogenesis remain largely unknown. Here we present an experiment-driven mathematical model that enables us to systematically dissect cellular mechanisms in branch elongation. We found that cell-autonomous and coordinated actions governed these multicellular behaviors, and a cell-autonomous process sufficiently illustrated essential features of the morphogenetic EC dynamics at both the single-cell and cell-population levels. Through refining our model and experimental verification, we further identified a coordinated mode of tip EC behaviors regulated via a spatial relationship between tip and follower ECs, which facilitates the forward motility of tip ECs. These findings provide insights that enhance our mechanistic understanding of not only angiogenic morphogenesis, but also other types of multicellular phenomenon. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Deletion of the Vaccinia Virus I2 Protein Interrupts Virion Morphogenesis, Leading to Retention of the Scaffold Protein and Mislocalization of Membrane-Associated Entry Proteins.

PubMed

Hyun, Seong-In; Weisberg, Andrea; Moss, Bernard

2017-08-01

The I2L open reading frame of vaccinia virus (VACV) encodes a conserved 72-amino-acid protein with a putative C-terminal transmembrane domain. Previous studies with a tetracycline-inducible mutant demonstrated that I2-deficient virions are defective in cell entry. The purpose of the present study was to determine the step of replication or entry that is affected by loss of the I2 protein. Fluorescence microscopy experiments showed that I2 colocalized with a major membrane protein of immature and mature virions. We generated a cell line that constitutively expressed I2 and allowed construction of the VACV I2L deletion mutant vΔI2. As anticipated, vΔI2 was unable to replicate in cells that did not express I2. Unexpectedly, morphogenesis was interrupted at a stage after immature virion formation, resulting in the accumulation of dense spherical particles instead of brick-shaped mature virions with well-defined core structures. The abnormal particles retained the D13 scaffold protein of immature virions, were severely deficient in the transmembrane proteins that comprise the entry fusion complex (EFC), and had increased amounts of unprocessed membrane and core proteins. Total lysates of cells infected with vΔI2 also had diminished EFC proteins due to instability attributed to their hydrophobicity and failure to be inserted into viral membranes. A similar instability of EFC proteins had previously been found with unrelated mutants blocked earlier in morphogenesis that also accumulated viral membranes retaining the D13 scaffold. We concluded that I2 is required for virion morphogenesis, release of the D13 scaffold, and the association of EFC proteins with viral membranes. IMPORTANCE Poxviruses comprise a large family that infect vertebrates and invertebrates, cause disease in both in humans and in wild and domesticated animals, and are being engineered as vectors for vaccines and cancer therapy. In addition, investigations of poxviruses have provided insights into many aspects of cell biology. The I2 protein is conserved in all poxviruses that infect vertebrates, suggesting an important role. The present study revealed that this protein is essential for vaccinia virus morphogenesis and that its absence results in an accumulation of deformed virus particles retaining the scaffold protein and deficient in surface proteins needed for cell entry. Copyright © 2017 American Society for Microbiology.

The MARVEL domain protein Nce102 regulates actin organization and invasive growth of Candida albicans.

PubMed

Douglas, Lois M; Wang, Hong X; Konopka, James B

2013-11-26

Invasive growth of the fungal pathogen Candida albicans into tissues promotes disseminated infections in humans. The plasma membrane is essential for pathogenesis because this important barrier mediates morphogenesis and invasive growth, as well as secretion of virulence factors, cell wall synthesis, nutrient import, and other processes. Previous studies showed that the Sur7 tetraspan protein that localizes to MCC (membrane compartment occupied by Can1)/eisosome subdomains of the plasma membrane regulates a broad range of key functions, including cell wall synthesis, morphogenesis, and resistance to copper. Therefore, a distinct tetraspan protein found in MCC/eisosomes, Nce102, was investigated. Nce102 belongs to the MARVEL domain protein family, which is implicated in regulating membrane structure and function. Deletion of NCE102 did not cause the broad defects seen in sur7Δ cells. Instead, the nce102Δ mutant displayed a unique phenotype in that it was defective in forming hyphae and invading low concentrations of agar but could invade well in higher agar concentrations. This phenotype was likely due to a defect in actin organization that was observed by phalloidin staining. In support of this, the invasive growth defect of a bni1Δ mutant that mislocalizes actin due to lack of the Bni1 formin was also reversed at high agar concentrations. This suggests that a denser matrix provides a signal that compensates for the actin defects. The nce102Δ mutant displayed decreased virulence and formed abnormal hyphae in mice. These studies identify novel ways that Nce102 and the physical environment surrounding C. albicans regulate morphogenesis and pathogenesis. The plasma membrane promotes virulence of the human fungal pathogen Candida albicans by acting as a protective barrier around the cell and mediating dynamic activities, such as morphogenesis, cell wall synthesis, secretion of virulence factors, and nutrient uptake. To better understand how the plasma membrane contributes to virulence, we analyzed a set of eight genes encoding MARVEL family proteins that are predicted to function in membrane organization. Interestingly, deletion of one gene, NCE102, caused a strong defect in formation of invasive hyphal growth in vitro and decreased virulence in mice. The nce102Δ mutant cells showed defects in actin organization that underlie the morphogenesis defect, since mutation of a known regulator of actin organization caused a similar defect. These studies identify a novel way in which the plasma membrane regulates the actin cytoskeleton and contributes to pathogenesis.

Tissue morphodynamics shaping the early mouse embryo.

PubMed

Sutherland, Ann E

2016-07-01

Generation of the elongated vertebrate body plan from the initially radially symmetrical embryo requires comprehensive changes to tissue form. These shape changes are generated by specific underlying cell behaviors, coordinated in time and space. Major principles and also specifics are emerging, from studies in many model systems, of the cell and physical biology of how region-specific cell behaviors produce regional tissue morphogenesis, and how these, in turn, are integrated at the level of the embryo. New technical approaches have made it possible more recently, to examine the morphogenesis of the mouse embryo in depth, and to elucidate the underlying cellular mechanisms. This review focuses on recent advances in understanding the cellular basis for the early fundamental events that establish the basic form of the embryo. Copyright © 2016 Elsevier Ltd. All rights reserved.

Tenascin-C in the extracellular matrix promotes the selection of highly proliferative and tubulogenesis-defective endothelial cells

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

Alves, Tercia Rodrigues; Universidade Federal do Rio de Janeiro; Carvalho da Fonseca, Anna Carolina

2011-09-10

The extracellular matrix (ECM) contains important cues for tissue homeostasis and morphogenesis. The matricellular protein tenascin-C (TN-C) is overexpressed in remodeling tissues and cancer. In the present work, we studied the effect of different ECM-which exhibited a significant diversity in their TN-C content-in endothelial survival, proliferation and tubulogenic differentiation: autologous (endothelial) ECM devoid of TN-C, but bearing large amounts of FN; fibroblast ECM, bearing both high TN-C and FN contents; and finally, glioma-derived matrices, usually poor in FN, but very rich in TN-C. HUVECs initially adhered to the immobilized matrix produced by U373 MG glioma cells, but significantly detached andmore » died by anoikis (50 to 80%) after 24 h, as compared with cells incubated with endothelial and fibroblast matrices. Surviving endothelial cells (20 to 50%) became up to 6-fold more proliferative and formed 74-97% less tube-like structures in vitro than cells grown on non-tumoral matrices. An antibody against the EGF-like repeats of tenascin-C (TN-C) partially rescued cells from the tubulogenic defect, indicating that this molecule is responsible for the selection of highly proliferative and tubulogenic defective endothelial cells. Interestingly, by using defined substrata, in conditions that mimic glioma and normal cell ECM composition, we observed that fibronectin (FN) modulates the TN-C-induced selection of endothelial cells. Our data show that TN-C is able to modulate endothelial branching morphogenesis in vitro and, since it is prevalent in matrices of injured and tumor tissues, also suggest a role for this protein in vascular morphogenesis, in these physiological contexts.« less

Unique patterns of organization and migration of FGF-expressing cells during Drosophila morphogenesis.

PubMed

Du, Lijuan; Zhou, Amy; Patel, Akshay; Rao, Mishal; Anderson, Kelsey; Roy, Sougata

2017-07-01

Fibroblast growth factors (FGF) are essential signaling proteins that regulate diverse cellular functions in developmental and metabolic processes. In Drosophila, the FGF homolog, branchless (bnl) is expressed in a dynamic and spatiotemporally restricted pattern to induce branching morphogenesis of the trachea, which expresses the Bnl-receptor, breathless (btl). Here we have developed a new strategy to determine bnl- expressing cells and study their interactions with the btl-expressing cells in the range of tissue patterning during Drosophila development. To enable targeted gene expression specifically in the bnl expressing cells, a new LexA based bnl enhancer trap line was generated using CRISPR/Cas9 based genome editing. Analyses of the spatiotemporal expression of the reporter in various embryonic stages, larval or adult tissues and in metabolic hypoxia, confirmed its target specificity and versatility. With this tool, new bnl expressing cells, their unique organization and functional interactions with the btl-expressing cells were uncovered in a larval tracheoblast niche in the leg imaginal discs, in larval photoreceptors of the developing retina, and in the embryonic central nervous system. The targeted expression system also facilitated live imaging of simultaneously labeled Bnl sources and tracheal cells, which revealed a unique morphogenetic movement of the embryonic bnl- source. Migration of bnl- expressing cells may create a dynamic spatiotemporal pattern of the signal source necessary for the directional growth of the tracheal branch. The genetic tool and the comprehensive profile of expression, organization, and activity of various types of bnl-expressing cells described in this study provided us with an important foundation for future research investigating the mechanisms underlying Bnl signaling in tissue morphogenesis. Copyright © 2017 Elsevier Inc. All rights reserved.

Cell Sheet Stiffness Sensing without taking out from culture liquid.

PubMed

Uchida, Ryohei; Tanaka, Nobuyuki; Higashimori, Mitsuru; Tadakuma, Kenjiro; Kaneko, Makoto; Kondo, Makoto; Yamato, Masayuki

2010-01-01

Stiffness could be an important index for evaluating the vitality of cell sheet. This paper challenges the measurement of stiffness of transparent cell sheet in culture liquid without taking it out from petri dish. The system is composed of a micro air nozzle for supplying an air jet and a regular reflective type laser sensor for measuring the the deformation of transparent cell sheet. This system is called as Cell Sheet Stiffness Sensing system (CS(3) system). When an air jet is given to a cell sheet in culture liquid, it pushes away the liquid toward the outer direction at initial phase and reaches the surface of cell sheet. Without any switching motion, the air jet continuously imparts a force to the surface of cell sheet so that the sensor can measure the stiffness of the cell sheet.

Role of the nuclear migration protein Lis1 in cell morphogenesis in Ustilago maydis

PubMed Central

Valinluck, Michael; Ahlgren, Sara; Sawada, Mizuho; Locken, Kristopher; Banuett, Flora

2010-01-01

Ustilago maydis is a basidiomycete fungus that exhibits a yeast-like and a filamentous form. Growth of the fungus in the host leads to additional morphological transitions. The different morphologies are characterized by distinct nuclear movements. Dynein and α-tubulin are required for nuclear movements and for cell morphogenesis of the yeast-like form. Lis1 is a microtubule plus-end tracking protein (+TIPs) conserved in eukaryotes and required for nuclear migration and spindle positioning. Defects in nuclear migration result in altered cell fate and aberrant development in metazoans, slow growth in fungi and disease in humans (e.g. lissencephaly). Here we investigate the role of the human LIS1 homolog in U. maydis and demonstrate that it is essential for cell viability, not previously seen in other fungi. With a conditional null mutation we show that lis1 is necessary for nuclear migration in the yeast-like cell and during the dimorphic transition. Studies of asynchronous exponentially growing cells and time-lapse microscopy uncovered novel functions of lis1: It is necessary for cell morphogenesis, positioning of the septum and cell wall integrity. lis1-depleted cells exhibit altered axes of growth and loss of cell polarity leading to grossly aberrant cells with clusters of nuclei and morphologically altered buds devoid of nuclei. Altered septum positioning and cell wall deposition contribute to the aberrant morphology. lis1-depleted cells lyse, indicative of altered cell wall properties or composition. We also demonstrate, with indirect immunofluorescence to visualize tubulin, that lis1 is necessary for the normal organization of the microtubule cytoskeleton: lis1-depleted cells contain more and longer microtubules that can form coils perpendicular to the long axis of the cell. We propose that lis1 controls microtubule dynamics and thus the regulated delivery of vesicles to growth sites and other cell domains that govern nuclear movements. PMID:20524583

Solid oxide fuel cell with multi-unit construction and prismatic design

DOEpatents

McPheeters, C.C.; Dees, D.W.; Myles, K.M.

1999-03-16

A single cell unit of a solid oxide fuel cell is described that is individually fabricated and sintered prior to being connected to adjacent cells to form a solid oxide fuel cell. The single cell unit is comprised of a shaped anode sheet positioned between a flat anode sheet and an anode-electrolyte-cathode (A/E/C) sheet, and a shaped cathode sheet positioned between the A/E/C sheet and a cathode-interconnect-anode (C/I/A) sheet. An alternate embodiment comprises a shaped cathode sheet positioned between an A/E/C sheet and a C/I/A sheet. The shaped sheets form channels for conducting reactant gases. Each single cell unit is individually sintered to form a finished sub-assembly. The finished sub-assemblies are connected in electrical series by interposing connective material between the end surfaces of adjacent cells, whereby individual cells may be inspected for defects and interchanged with non-defective single cell units. 7 figs.

Keratinocyte cytoskeletal roles in cell sheet engineering

PubMed Central

2013-01-01

Background There is an increasing need to understand cell-cell interactions for cell and tissue engineering purposes, such as optimizing cell sheet constructs, as well as for examining adhesion defect diseases. For cell-sheet engineering, one major obstacle to sheet function is that cell sheets in suspension are fragile and, over time, will contract. While the role of the cytoskeleton in maintaining the structure and adhesion of cells cultured on a rigid substrate is well-characterized, a systematic examination of the role played by different components of the cytoskeleton in regulating cell sheet contraction and cohesion in the absence of a substrate has been lacking. Results In this study, keratinocytes were cultured until confluent and cell sheets were generated using dispase to remove the influence of the substrate. The effects of disrupting actin, microtubules or intermediate filaments on cell-cell interactions were assessed by measuring cell sheet cohesion and contraction. Keratin intermediate filament disruption caused comparable effects on cell sheet cohesion and contraction, when compared to actin or microtubule disruption. Interfering with actomyosin contraction demonstrated that interfering with cell contraction can also diminish cell cohesion. Conclusions All components of the cytoskeleton are involved in maintaining cell sheet cohesion and contraction, although not to the same extent. These findings demonstrate that substrate-free cell sheet biomechanical properties are dependent on the integrity of the cytoskeleton network. PMID:23442760

Potent blockade of hepatocyte growth factor-stimulated cell motility, matrix invasion and branching morphogenesis by antagonists of Grb2 Src homology 2 domain interactions.

PubMed

Atabey, N; Gao, Y; Yao, Z J; Breckenridge, D; Soon, L; Soriano, J V; Burke, T R; Bottaro, D P

2001-04-27

Hepatocyte growth factor (HGF) stimulates mitogenesis, motogenesis, and morphogenesis in a wide range of cellular targets during development, homeostasis and tissue regeneration. Inappropriate HGF signaling occurs in several human cancers, and the ability of HGF to initiate a program of protease production, cell dissociation, and motility has been shown to promote cellular invasion and is strongly linked to tumor metastasis. Upon HGF binding, several tyrosines within the intracellular domain of its receptor, c-Met, become phosphorylated and mediate the binding of effector proteins, such as Grb2. Grb2 binding through its SH2 domain is thought to link c-Met with downstream mediators of cell proliferation, shape change, and motility. We analyzed the effects of Grb2 SH2 domain antagonists on HGF signaling and observed potent blockade of cell motility, matrix invasion, and branching morphogenesis, with ED(50) values of 30 nm or less, but only modest inhibition of mitogenesis. These compounds are 1000-10,000-fold more potent anti-motility agents than any previously characterized Grb2 SH2 domain antagonists. Our results suggest that SH2 domain-mediated c-Met-Grb2 interaction contributes primarily to the motogenic and morphogenic responses to HGF, and that these compounds may have therapeutic application as anti-metastatic agents for tumors where the HGF signaling pathway is active.

Looking into the sea urchin embryo you can see local cell interactions regulate morphogenesis.

PubMed

Wilt, F H

1997-08-01

The transparent sea urchin embryo provides a laboratory for study of morphogenesis. The calcareous endoskeleton is formed by a syncytium of mesenchyme cells in the blastocoel. The locations of mesenchyme in the blastocoel, the size of the skeleton, and even the branching pattern of the skeletal rods, are governed by interactions with the blastula wall. Now Guss and Ettensohn show that the rate of deposition of CaCO3 in the skeleton is locally controlled in the mesenchymal syncytium, as is the pattern of expression of three genes involved in skeleton formation. They propose that short range signals emanating from the blastula wall regulate many aspects of the biomineralization process.

Mechanical Forces Program the Orientation of Cell Division during Airway Tube Morphogenesis.

PubMed

Tang, Zan; Hu, Yucheng; Wang, Zheng; Jiang, Kewu; Zhan, Cheng; Marshall, Wallace F; Tang, Nan

2018-02-05

Oriented cell division plays a key role in controlling organogenesis. The mechanisms for regulating division orientation at the whole-organ level are only starting to become understood. By combining 3D time-lapse imaging, mouse genetics, and mathematical modeling, we find that global orientation of cell division is the result of a combination of two types of spindles with distinct spindle dynamic behaviors in the developing airway epithelium. Fixed spindles follow the classic long-axis rule and establish their division orientation before metaphase. In contrast, rotating spindles do not strictly follow the long-axis rule and determine their division orientation during metaphase. By using both a cell-based mechanical model and stretching-lung-explant experiments, we showed that mechanical force can function as a regulatory signal in maintaining the stable ratio between fixed spindles and rotating spindles. Our findings demonstrate that mechanical forces, cell geometry, and oriented cell division function together in a highly coordinated manner to ensure normal airway tube morphogenesis. Copyright © 2017 Elsevier Inc. All rights reserved.

Apolar and polar transitions drive the conversion between amoeboid and mesenchymal shapes in melanoma cells

PubMed Central

Cooper, Sam; Sadok, Amine; Bousgouni, Vicky; Bakal, Chris

2015-01-01

Melanoma cells can adopt two functionally distinct forms, amoeboid and mesenchymal, which facilitates their ability to invade and colonize diverse environments during the metastatic process. Using quantitative imaging of single living tumor cells invading three-dimensional collagen matrices, in tandem with unsupervised computational analysis, we found that melanoma cells can switch between amoeboid and mesenchymal forms via two different routes in shape space—an apolar and polar route. We show that whereas particular Rho-family GTPases are required for the morphogenesis of amoeboid and mesenchymal forms, others are required for transitions via the apolar or polar route and not amoeboid or mesenchymal morphogenesis per se. Altering the transition rates between particular routes by depleting Rho-family GTPases can change the morphological heterogeneity of cell populations. The apolar and polar routes may have evolved in order to facilitate conversion between amoeboid and mesenchymal forms, as cells are either searching for, or attracted to, particular migratory cues, respectively. PMID:26310441

Mechanisms of bacterial morphogenesis: evolutionary cell biology approaches provide new insights.

PubMed

Jiang, Chao; Caccamo, Paul D; Brun, Yves V

2015-04-01

How Darwin's "endless forms most beautiful" have evolved remains one of the most exciting questions in biology. The significant variety of bacterial shapes is most likely due to the specific advantages they confer with respect to the diverse environments they occupy. While our understanding of the mechanisms generating relatively simple shapes has improved tremendously in the last few years, the molecular mechanisms underlying the generation of complex shapes and the evolution of shape diversity are largely unknown. The emerging field of bacterial evolutionary cell biology provides a novel strategy to answer this question in a comparative phylogenetic framework. This relatively novel approach provides hypotheses and insights into cell biological mechanisms, such as morphogenesis, and their evolution that would have been difficult to obtain by studying only model organisms. We discuss the necessary steps, challenges, and impact of integrating "evolutionary thinking" into bacterial cell biology in the genomic era. © 2015 WILEY Periodicals, Inc.

Cytoskeletal self-organization in neuromorphogenesis.

PubMed

Dehmelt, Leif

2014-01-01

Self-organization of dynamic microtubules via interactions with associated motors plays a critical role in spindle formation. The microtubule-based mechanisms underlying other aspects of cellular morphogenesis, such as the formation and development of protrusions from neuronal cells is less well understood. In a recent study, we investigated the molecular mechanism that underlies the massive reorganization of microtubules induced in non-neuronal cells by expression of the neuronal microtubule stabilizer MAP2c. In that study we directly observed cortical dynein complexes and how they affect the dynamic behavior of motile microtubules in living cells. We found that stationary dynein complexes transiently associate with motile microtubules near the cell cortex and that their rapid turnover facilitates efficient microtubule transport. Here, we discuss our findings in the larger context of cellular morphogenesis with specific focus on self-organizing principles from which cellular shape patterns such as the thin protrusions of neurons can emerge.

Development and Function of the Drosophila Tracheal System.

PubMed

Hayashi, Shigeo; Kondo, Takefumi

2018-06-01

The tracheal system of insects is a network of epithelial tubules that functions as a respiratory organ to supply oxygen to various target organs. Target-derived signaling inputs regulate stereotyped modes of cell specification, branching morphogenesis, and collective cell migration in the embryonic stage. In the postembryonic stages, the same set of signaling pathways controls highly plastic regulation of size increase and pattern elaboration during larval stages, and cell proliferation and reprograming during metamorphosis. Tracheal tube morphogenesis is also regulated by physicochemical interaction of the cell and apical extracellular matrix to regulate optimal geometry suitable for air flow. The trachea system senses both the external oxygen level and the metabolic activity of internal organs, and helps organismal adaptation to changes in environmental oxygen level. Cellular and molecular mechanisms underlying the high plasticity of tracheal development and physiology uncovered through research on Drosophila are discussed. Copyright © 2018 by the Genetics Society of America.

Cytoskeletal self-organization in neuromorphogenesis

PubMed Central

Dehmelt, Leif

2014-01-01

Self-organization of dynamic microtubules via interactions with associated motors plays a critical role in spindle formation. The microtubule-based mechanisms underlying other aspects of cellular morphogenesis, such as the formation and development of protrusions from neuronal cells is less well understood. In a recent study, we investigated the molecular mechanism that underlies the massive reorganization of microtubules induced in non-neuronal cells by expression of the neuronal microtubule stabilizer MAP2c. In that study we directly observed cortical dynein complexes and how they affect the dynamic behavior of motile microtubules in living cells. We found that stationary dynein complexes transiently associate with motile microtubules near the cell cortex and that their rapid turnover facilitates efficient microtubule transport. Here, we discuss our findings in the larger context of cellular morphogenesis with specific focus on self-organizing principles from which cellular shape patterns such as the thin protrusions of neurons can emerge. PMID:24847718

Quantitative semi-automated analysis of morphogenesis with single-cell resolution in complex embryos.

PubMed

Giurumescu, Claudiu A; Kang, Sukryool; Planchon, Thomas A; Betzig, Eric; Bloomekatz, Joshua; Yelon, Deborah; Cosman, Pamela; Chisholm, Andrew D

2012-11-01

A quantitative understanding of tissue morphogenesis requires description of the movements of individual cells in space and over time. In transparent embryos, such as C. elegans, fluorescently labeled nuclei can be imaged in three-dimensional time-lapse (4D) movies and automatically tracked through early cleavage divisions up to ~350 nuclei. A similar analysis of later stages of C. elegans development has been challenging owing to the increased error rates of automated tracking of large numbers of densely packed nuclei. We present Nucleitracker4D, a freely available software solution for tracking nuclei in complex embryos that integrates automated tracking of nuclei in local searches with manual curation. Using these methods, we have been able to track >99% of all nuclei generated in the C. elegans embryo. Our analysis reveals that ventral enclosure of the epidermis is accompanied by complex coordinated migration of the neuronal substrate. We can efficiently track large numbers of migrating nuclei in 4D movies of zebrafish cardiac morphogenesis, suggesting that this approach is generally useful in situations in which the number, packing or dynamics of nuclei present challenges for automated tracking.

Importance of MAP Kinases during Protoperithecial Morphogenesis in Neurospora crassa

PubMed Central

Jeffree, Chris E.; Oborny, Radek; Boonyarungsrit, Patid; Read, Nick D.

2012-01-01

In order to produce multicellular structures filamentous fungi combine various morphogenetic programs that are fundamentally different from those used by plants and animals. The perithecium, the female sexual fruitbody of Neurospora crassa, differentiates from the vegetative mycelium in distinct morphological stages, and represents one of the more complex multicellular structures produced by fungi. In this study we defined the stages of protoperithecial morphogenesis in the N. crassa wild type in greater detail than has previously been described; compared protoperithecial morphogenesis in gene-deletion mutants of all nine mitogen-activated protein (MAP) kinases conserved in N. crassa; confirmed that all three MAP kinase cascades are required for sexual development; and showed that the three different cascades each have distinctly different functions during this process. However, only MAP kinases equivalent to the budding yeast pheromone response and cell wall integrity pathways, but not the osmoregulatory pathway, were essential for vegetative cell fusion. Evidence was obtained for MAP kinase signaling cascades performing roles in extracellular matrix deposition, hyphal adhesion, and envelopment during the construction of fertilizable protoperithecia. PMID:22900028

A novel closed cell culture device for fabrication of corneal epithelial cell sheets.

PubMed

Nakajima, Ryota; Kobayashi, Toyoshige; Moriya, Noboru; Mizutani, Manabu; Kan, Kazutoshi; Nozaki, Takayuki; Saitoh, Kazuo; Yamato, Masayuki; Okano, Teruo; Takeda, Shizu

2015-11-01

Automation technology for cell sheet-based tissue engineering would need to optimize the cell sheet fabrication process, stabilize cell sheet quality and reduce biological contamination risks. Biological contamination must be avoided in clinical settings. A closed culture system provides a solution for this. In the present study, we developed a closed culture device called a cell cartridge, to be used in a closed cell culture system for fabricating corneal epithelial cell sheets. Rabbit limbal epithelial cells were cultured on the surface of a porous membrane with 3T3 feeder cells, which are separate from the epithelial cells in the cell cartridges and in the cell-culture inserts as a control. To fabricate the stratified cell sheets, five different thicknesses of the membranes which were welded to the cell cartridge, were examined. Multilayered corneal epithelial cell sheets were fabricated in cell cartridges that were welded to a 25 µm-thick gas-permeable membrane, which was similar to the results with the cell-culture inserts. However, stratification of corneal epithelial cell sheets did not occur with cell cartridges that were welded to 100-300 µm-thick gas-permeable membranes. The fabricated cell sheets were evaluated by histological analyses to examine the expression of corneal epithelial-specific markers. Immunohistochemical analyses showed that a putative stem cell marker, p63, a corneal epithelial differentiation maker, CK3, and a barrier function marker, Claudin-1, were expressed in the appropriate position in the cell sheets. These results suggest that the cell cartridge is effective for fabricating corneal epithelial cell sheets. Copyright © 2012 John Wiley & Sons, Ltd.

bullwinkle and shark regulate dorsal-appendage morphogenesis in Drosophila oogenesis.

PubMed

Tran, David H; Berg, Celeste A

2003-12-01

bullwinkle (bwk) regulates embryonic anteroposterior patterning and, through a novel germline-to-soma signal, morphogenesis of the eggshell dorsal appendages. We screened for dominant modifiers of the bullwinkle mooseantler eggshell phenotype and identified shark, which encodes an SH2-domain, ankyrin-repeat tyrosine kinase. At the onset of dorsal-appendage formation, shark is expressed in a punctate pattern in the squamous stretch cells overlying the nurse cells. Confocal microscopy with cell-type-specific markers demonstrates that the stretch cells act as a substrate for the migrating dorsal-appendage-forming cells and extend cellular projections towards them. Mosaic analyses reveal that shark is required in follicle cells for cell migration and chorion deposition. Proper shark RNA expression in the stretch cells requires bwk activity, while restoration of shark expression in the stretch cells suppresses the bwk dorsal-appendage phenotype. These results suggest that shark plays an important downstream role in the bwk-signaling pathway. Candidate testing implicates Src42A in a similar role, suggesting conservation with a vertebrate signaling pathway involving non-receptor tyrosine kinases.

Supplying osteogenesis to dead bone using an osteogenic matrix cell sheet.

PubMed

Uchihara, Yoshinobu; Akahane, Manabu; Okuda, Akinori; Shimizu, Takamasa; Masuda, Keisuke; Kira, Tsutomu; Kawate, Kenji; Tanaka, Yasuhito

2018-02-22

To evaluate whether osteogenic matrix cell sheets can supply osteogenesis to dead bone. Femur bone fragments (5 mm in length) were obtained from Fisher 344 rats and irradiated by a single exposure of 60 Gy to produce bones that were no longer viable. Osteogenic matrix cell sheets were created from rat bone marrow-derived stromal cells (BMSCs). After wrapping the dead bone with an osteogenic matrix cell sheet, it was subcutaneously transplanted into the back of a rat and harvested after 4 weeks. Bone formation around the dead bone was evaluated by X-ray imaging and histology. Alkaline phosphatase (ALP) and osteocalcin (OC) mRNA expression levels were measured to confirm osteogenesis of the transplanted bone. The contribution of donor cells to bone formation was assessed using the Sry gene and PKH26. After the cell sheet was transplanted together with dead bone, X-ray images showed abundant calcification around the dead bone. In contrast, no newly formed bone was seen in samples that were transplanted without the cell sheet. Histological sections also showed newly formed bone around dead bone in samples transplanted with the cell sheet, whereas many empty lacunae and no newly formed bone were observed in samples transplanted without the cell sheet. ALP and OC mRNA expression levels were significantly higher in dead bones transplanted with cell sheets than in those without a cell sheet (P < 0.01). Sry gene expression and cells derived from cell sheets labeled with PKH26 were detected in samples transplanted with a cell sheet, indicating survival of donor cells after transplantation. Our study indicates that osteogenic matrix cell sheet transplantation can supply osteogenesis to dead bone. Copyright © 2018. Published by Elsevier B.V.

CPK3-phosphorylated RhoGDI1 is essential in the development of Arabidopsis seedlings and leaf epidermal cells

PubMed Central

Wu, Yan

2013-01-01

The regulation of Rho of plants (ROP) in morphogenesis of leaf epidermal cells has been well studied, but the roles concerning regulators of ROPs such as RhoGDIs are poorly understood. This study reports that AtRhoGDI1 (GDI1) acts as a versatile regulator to modulate development of seedlings and leaf pavement cells. In mutant gdi1, leaf pavement cells showed shorter lobes in comparison with those in wild type. In GDI1-14 seedlings (GDI1-overexpression line) the growth of lobes in pavement cells was severely suppressed and the development of seedlings was altered. These results indicate that GDI1 plays an essential role in morphogenesis of epidermal pavement cells through modulating the ROP signalling pathways. The interaction between GDI1 and ROP2 or ROP6 was detected in the leaf pavement cells using FRET analysis. Dominant negative, not constitutively active, DN-rop6 could weaken the effect caused by overexpression of GDI1; because the pleiotropic phenotype of GDI1-14 plants was eliminated in the hybrid line GDI1-14 DN-rop6. GDI1 could be phosphorylated by CPK3. Three conserved Ser/Thr residues in GDI1 were determined as targeted amino acids for CPK3. Overexpression of GDI1(3D), not GDI1(3A), could rescue the abnormal growth phenotypes of gdi1-1 seedlings, demonstrating the impact of GDI1 phosphorylation in the development of Arabidopsis. In summary, these results suggest that GDI1 regulation in morphogenesis of seedlings and leaf pavement cells could be undergone through modulating the ROP signalling pathways and the phosphorylation of GDI1 by CPK3 was required for the developmental modulation in Arabidopsis. PMID:23846874

The C. elegans Excretory Canal as a Model for Intracellular Lumen Morphogenesis and In Vivo Polarized Membrane Biogenesis in a Single Cell: labeling by GFP-fusions, RNAi Interaction Screen and Imaging.

PubMed

Zhang, Nan; Membreno, Edward; Raj, Susan; Zhang, Hongjie; Khan, Liakot A; Gobel, Verena

2017-10-03

The four C. elegans excretory canals are narrow tubes extended through the length of the animal from a single cell, with almost equally far extended intracellular endotubes that build and stabilize the lumen with a membrane and submembraneous cytoskeleton of apical character. The excretory cell expands its length approximately 2,000 times to generate these canals, making this model unique for the in vivo assessment of de novo polarized membrane biogenesis, intracellular lumen morphogenesis and unicellular tubulogenesis. The protocol presented here shows how to combine standard labeling, gain- and loss-of-function genetic or RNA interference (RNAi)-, and microscopic approaches to use this model to visually dissect and functionally analyze these processes on a molecular level. As an example of a labeling approach, the protocol outlines the generation of transgenic animals with fluorescent fusion proteins for live analysis of tubulogenesis. As an example of a genetic approach, it highlights key points of a visual RNAi-based interaction screen designed to modify a gain-of-function cystic canal phenotype. The specific methods described are how to: label and visualize the canals by expressing fluorescent proteins; construct a targeted RNAi library and strategize RNAi screening for the molecular analysis of canal morphogenesis; visually assess modifications of canal phenotypes; score them by dissecting fluorescence microscopy; characterize subcellular canal components at higher resolution by confocal microscopy; and quantify visual parameters. The approach is useful for the investigator who is interested in taking advantage of the C. elegans excretory canal for identifying and characterizing genes involved in the phylogenetically conserved processes of intracellular lumen and unicellular tube morphogenesis.

Sea Urchin Morphogenesis.

PubMed

McClay, David R

2016-01-01

In the sea urchin morphogenesis follows extensive molecular specification. The specification controls the many morphogenetic events and these, in turn, precede patterning steps that establish the larval body plan. To understand how the embryo is built it was necessary to understand those series of molecular steps. Here an example of the historical sequence of those discoveries is presented as it unfolded over the last 50 years, the years during which major progress in understanding development of many animals and plants was documented by CTDB. In sea urchin development a rich series of experimental studies first established many of the phenomenological components of skeletal morphogenesis and patterning without knowledge of the molecular components. The many discoveries of transcription factors, signals, and structural proteins that contribute to the shape of the endoskeleton of the sea urchin larva then followed as molecular tools became available. A number of transcription factors and signals were discovered that were necessary for specification, morphogenesis, and patterning. Perturbation of the transcription factors and signals provided the means for assembling models of the gene regulatory networks used for specification and controlled the subsequent morphogenetic events. The earlier experimental information informed perturbation experiments that asked how patterning worked. As a consequence it was learned that ectoderm provides a series of patterning signals to the skeletogenic cells and as a consequence the skeletogenic cells secrete a highly patterned skeleton based on their ability to genotypically decode the localized reception of several signals. We still do not understand the complexity of the signals received by the skeletogenic cells, nor do we understand in detail how the genotypic information shapes the secreted skeletal biomineral, but the current knowledge at least outlines the sequence of events and provides a useful template for future discoveries. © 2016 Elsevier Inc. All rights reserved.

In vivo imaging of emerging endocrine cells reveals a requirement for PI3K-regulated motility in pancreatic islet morphogenesis

PubMed Central

Freudenblum, Julia; Iglesias, José A.; Hermann, Martin; Walsen, Tanja; Wilfinger, Armin; Meyer, Dirk

2018-01-01

ABSTRACT The three-dimensional architecture of the pancreatic islet is integral to beta cell function, but the process of islet formation remains poorly understood due to the difficulties of imaging internal organs with cellular resolution. Within transparent zebrafish larvae, the developing pancreas is relatively superficial and thus amenable to live imaging approaches. We performed in vivo time-lapse and longitudinal imaging studies to follow islet development, visualizing both naturally occurring islet cells and cells arising with an accelerated timecourse following an induction approach. These studies revealed previously unappreciated fine dynamic protrusions projecting between neighboring and distant endocrine cells. Using pharmacological compound and toxin interference approaches, and single-cell analysis of morphology and cell dynamics, we determined that endocrine cell motility is regulated by phosphoinositide 3-kinase (PI3K) and G-protein-coupled receptor (GPCR) signaling. Linking cell dynamics to islet formation, perturbation of protrusion formation disrupted endocrine cell coalescence, and correlated with decreased islet cell differentiation. These studies identified novel cell behaviors contributing to islet morphogenesis, and suggest a model in which dynamic exploratory filopodia establish cell-cell contacts that subsequently promote cell clustering. PMID:29386244

Early local differentiation of the cell wall matrix defines the contact sites in lobed mesophyll cells of Zea mays.

PubMed

Giannoutsou, E; Sotiriou, P; Apostolakos, P; Galatis, B

2013-10-01

The morphogenesis of lobed mesophyll cells (MCs) is highly controlled and coupled with intercellular space formation. Cortical microtubule rings define the number and the position of MC isthmi. This work investigated early events of MC morphogenesis, especially the mechanism defining the position of contacts between MCs. The distributions of plasmodesmata, the hemicelluloses callose and (1 → 3,1 → 4)-β-d-glucans (MLGs) and the pectin epitopes recognized by the 2F4, JIM5, JIM7 and LM6 antibodies were studied in the cell walls of Zea mays MCs. Matrix cell wall polysaccharides were immunolocalized in hand-made sections and in sections of material embedded in LR White resin. Callose was also localized using aniline blue in hand-made sections. Plasmodesmata distribution was examined by transmission electron microscopy. Before reorganization of the dispersed cortical microtubules into microtubule rings, particular bands of the longitudinal MC walls, where the MC contacts will form, locally differentiate by selective (1) deposition of callose and the pectin epitopes recognized by the 2F4, LM6, JIM5 and JIM7 antibodies, (2) degradation of MLGs and (3) formation of secondary plasmodesmata clusterings. This cell wall matrix differentiation persists in cell contacts of mature MCs. Simultaneously, the wall bands between those of future cell contacts differentiate with (1) deposition of local cell wall thickenings including cellulose microfibrils, (2) preferential presence of MLGs, (3) absence of callose and (4) transient presence of the pectins identified by the JIM5 and JIM7 antibodies. The wall areas between cell contacts expand determinately to form the cell isthmi and the cell lobes. The morphogenesis of lobed MCs is characterized by the early patterned differentiation of two distinct cell wall subdomains, defining the sites of the future MC contacts and of the future MC isthmi respectively. This patterned cell wall differentiation precedes cortical microtubule reorganization and may define microtubule ring disposition.

Bone Marrow Stromal Cells Stimulate an Angiogenic Program that Requires Endothelial MT1-MMP

PubMed Central

Kachgal, Suraj; Carrion, Bita; Janson, Isaac A.; Putnam, Andrew J.

2012-01-01

Bone marrow-derived stromal/stem cells (BMSCs) have recently been characterized as mediators of tissue regeneration after injury. In addition to preventing fibrosis at the wound site, BMSCs elicit an angiogenic response within the fibrin matrix. The mechanistic interactions between BMSCs and invading endothelial cells (ECs) during this process are not fully understood. Using a three-dimensional, fibrin-based angiogenesis model, we sought to investigate the proteolytic mechanisms by which BMSCs promote vessel morphogenesis. We find that BMSC-mediated vessel formation depends on the proteolytic ability of membrane type 1-matrix metalloproteinase (MT1-MMP). Knockdown of the protease results in a small network of vessels with enlarged lumens. Contrastingly, vessel morphogenesis is unaffected by the knockdown of MMP-2 and MMP-9. Furthermore, we find that BMSC-mediated vessel morphogenesis in vivo follows mechanisms similar to what we observe in vitro. Subcutaneous, cellular fibrin implants in C.B-17/SCID mice form aberrant vasculature when MMPs are inhibited with a broad spectrum chemical inhibitor, and a very minimal amount of vessels when MT1-MMP proteolytic activity is interrupted in ECs. Other studies have debated the necessity of MT1-MMP in the context of vessel invasion in fibrin, but this study clearly demonstrates its requirement in BMSC-mediated angiogenesis. PMID:22262018

Muscle contraction controls skeletal morphogenesis through regulation of chondrocyte convergent extension.

PubMed

Shwartz, Yulia; Farkas, Zsuzsanna; Stern, Tomer; Aszódi, Attila; Zelzer, Elazar

2012-10-01

Convergent extension driven by mediolateral intercalation of chondrocytes is a key process that contributes to skeletal growth and morphogenesis. While progress has been made in deciphering the molecular mechanism that underlies this process, the involvement of mechanical load exerted by muscle contraction in its regulation has not been studied. Using the zebrafish as a model system, we found abnormal pharyngeal cartilage morphology in both chemically and genetically paralyzed embryos, demonstrating the importance of muscle contraction for zebrafish skeletal development. The shortening of skeletal elements was accompanied by prominent changes in cell morphology and organization. While in control the cells were elongated, chondrocytes in paralyzed zebrafish were smaller and exhibited a more rounded shape, confirmed by a reduction in their length-to-width ratio. The typical columnar organization of cells was affected too, as chondrocytes in various skeletal elements exhibited abnormal stacking patterns, indicating aberrant intercalation. Finally, we demonstrate impaired chondrocyte intercalation in growth plates of muscle-less Sp(d) mouse embryos, implying the evolutionary conservation of muscle force regulation of this essential morphogenetic process.Our findings provide a new perspective on the regulatory interaction between muscle contraction and skeletal morphogenesis by uncovering the role of muscle-induced mechanical loads in regulating chondrocyte intercalation in two different vertebrate models. Copyright © 2012 Elsevier Inc. All rights reserved.

3D stromal tissue equivalent affects intestinal epithelium morphogenesis in vitro.

PubMed

De Gregorio, Vincenza; Imparato, Giorgia; Urciuolo, Francesco; Netti, Paolo A

2018-04-01

Current in vitro models of human intestine commonly fail to mimic the complex intestinal functions and features required for drug development and disease research. Here, we deeply investigate the interaction existing between epithelium and the underneath stroma, and its role in the epithelium morphogenesis. We cultured human intestinal subepithelial myofibroblasts (ISEMFs) in two different 3D configurations: 3D-collagen gel equivalent (3D-CGE) and 3D cell-synthetized stromal equivalent (3D-CSSE). The 3D-CGEs were obtained by means of the traditional collagen-based cell technique and the 3D-CSSE were obtained by bottom-up tissue engineering strategy. The biophysical properties of both 3D models with regard to cell growth and composition (via histological analysis, immunofluorescence, and multiphoton imaging) were assessed. Then, human colorectal adenocarcinoma cell line (CaCo-2) was cultured on both the 3D constructs in order to produce the intestinal model. We identified higher levels of matrix-associated proteins from ISEMFs cultured in 3D-CSSE compared to 3D-CGE. Furthermore, multiphoton investigation revealed differences in the collagen network architecture in both models. At last, the more physiologically relevant stromal environment of the 3D-CSSE drove the CaCo-2 cell differentiation toward the four different type of intestinal epithelial cells (absorptive, mucus-secretory, enteroendocrine, and Paneth) phenotype and promotes, in contrast to the 3D-CGE, the production of the basement membrane. Taken together, these results highlight a fundamental role of the 3D stromal environment in addressing a correct epithelium morphogenesis as well as epithelial-stromal interface establishment. © 2017 Wiley Periodicals, Inc.

Modulation of Morphogenesis in Candida albicans by Various Small Molecules ▿

PubMed Central

Shareck, Julie; Belhumeur, Pierre

2011-01-01

The pathogenic yeast Candida albicans, a member of the mucosal microbiota, is responsible for a large spectrum of infections, ranging from benign thrush and vulvovaginitis in both healthy and immunocompromised individuals to severe, life-threatening infections in immunocompromised patients. A striking feature of C. albicans is its ability to grow as budding yeast and as filamentous forms, including hyphae and pseudohyphae. The yeast-to-hypha transition contributes to the overall virulence of C. albicans and may even constitute a target for the development of antifungal drugs. Indeed, impairing morphogenesis in C. albicans has been shown to be a means to treat candidiasis. Additionally, a large number of small molecules such as farnesol, fatty acids, rapamycin, geldanamycin, histone deacetylase inhibitors, and cell cycle inhibitors have been reported to modulate the yeast-to-hypha transition in C. albicans. In this minireview, we take a look at molecules that modulate morphogenesis in this pathogenic yeast. When possible, we address experimental findings regarding their mechanisms of action and their therapeutic potential. We discuss whether or not modulating morphogenesis constitutes a strategy to treat Candida infections. PMID:21642508

Growth and morphogenesis of embryonic mouse organs on non-coated and extracellular matrix-coated Biopore membrane

NASA Technical Reports Server (NTRS)

Hardman, P.; Klement, B. J.; Spooner, B. S.

1993-01-01

Embryonic mouse salivary glands, pancreata, and kidneys were isolated from embryos of appropriate gestational age by microdissection, and were cultured on Biopore membrane either non-coated or coated with type I collagen or Matrigel. As expected, use of Biopore membrane allowed high quality photomicroscopy of the living organs. In all organs extensive mesenchymal spreading was observed in the presence of type I collagen or Matrigel. However, differences were noted in the effects of extracellular matrix (ECM) coatings on epithelial growth and morphogenesis: salivary glands were minimally affected, pancreas morphogenesis was adversely affected, and kidney growth and branching apparently was enhanced. It is suggested that these differences in behaviour reflect differences in the strength of interactions between the mesenchymal cells and their surrounding endogenous matrix, compared to the exogenous ECM macromolecules. This method will be useful for culture of these and other embryonic organs. In particular, culture of kidney rudiments on ECM-coated Biopore offers a great improvement over previously used methods which do not allow morphogenesis to be followed in vitro.

Huntingtin Is Required for Epithelial Polarity through RAB11A-Mediated Apical Trafficking of PAR3-aPKC

PubMed Central

Elias, Salah; McGuire, John Russel; Yu, Hua; Humbert, Sandrine

2015-01-01

The establishment of apical-basolateral polarity is important for both normal development and disease, for example, during tumorigenesis and metastasis. During this process, polarity complexes are targeted to the apical surface by a RAB11A-dependent mechanism. Huntingtin (HTT), the protein that is mutated in Huntington disease, acts as a scaffold for molecular motors and promotes microtubule-based dynamics. Here, we investigated the role of HTT in apical polarity during the morphogenesis of the mouse mammary epithelium. We found that the depletion of HTT from luminal cells in vivo alters mouse ductal morphogenesis and lumen formation. HTT is required for the apical localization of PAR3-aPKC during epithelial morphogenesis in virgin, pregnant, and lactating mice. We show that HTT forms a complex with PAR3, aPKC, and RAB11A and ensures the microtubule-dependent apical vesicular translocation of PAR3-aPKC through RAB11A. We thus propose that HTT regulates polarized vesicular transport, lumen formation and mammary epithelial morphogenesis. PMID:25942483

Effect of Cell Sheet Manipulation Techniques on the Expression of Collagen Type II and Stress Fiber Formation in Human Chondrocyte Sheets.

PubMed

Wongin, Sopita; Waikakul, Saranatra; Chotiyarnwong, Pojchong; Siriwatwechakul, Wanwipa; Viravaidya-Pasuwat, Kwanchanok

2018-03-01

Cell sheet technology is applied to human articular chondrocytes to construct a tissue-like structure as an alternative treatment for cartilage defect. The effect of a gelatin manipulator, as a cell sheet transfer system, on the quality of the chondrocyte sheets was investigated. The changes of important chondrogenic markers and stress fibers, resulting from the cell sheet manipulation, were also studied. The chondrocyte cell sheets were constructed with patient-derived chondrocytes using a temperature-responsive polymer and a gelatin manipulator as a transfer carrier. The properties of the cell sheets, including sizes, expression levels of collagen type II and I, and the localization of the stress fibers, were assessed and compared with those of the cell sheets harvested without the gelatin manipulator. Using the gelatin manipulator, the original size of the chondrocyte cell sheets was retained with abundant stress fibers, but with a decrease in the expression of collagen type II. Without the gelatin manipulator, although the cell shrinkage occurred, the cell sheet with suppressed stress fiber formation showed significantly higher levels of collagen type II. These results support our observations that stress fiber formation in chondrocyte cell sheets affected the production of chondrogenic markers. These densely packed tissue-like structures possessed a good chondrogenic activity, indicating their potential for use in autologous chondrocyte implantation to treat cartilage defects.

Endothelial Cell Hyperproliferation and Stratification in Uteroplacental Blood Vessels of the Black Mastiff Bat, Molossus rufus

PubMed Central

Rasweiler IV, J.J.; Badwaik, N.K.; Salame, G.; Abulafia, O.

2011-01-01

Placentation was studied histologically and immunocytochemically in black mastiff bats obtained at frequent intervals throughout pregnancy. These were bred in a captive colony or collected from a reproductively-synchronized wild population. During late pregnancy, the single fetus was largely sustained by a discoidal, hemochorial placenta located at the cranial end of the right uterine horn. This invariant positioning was determined by a vascular tuft that developed there both during early pregnancy and non-pregnant cycles. This provided a scaffold for early placental morphogenesis. As development proceeded, small arterioles and venules serving the tuft were converted to large uteroplacental vessels. Within the base of the placenta, these became lined by an unusual vascular epithelium composed of intermingled patches of multilayered endothelial cells and cytotrophoblast. Initially, the endothelium became multilayered by hypertrophy, proliferation, and infolding of its basal lamina. These created endothelial bilayers usually insinuated between basal laminae. The development of temporary gaps in the laminae then permitted further enlargement of the vessels and proliferation of the endothelial cells as monolayer sheets or chains. The latter were interconnected, forming a complex, stratified, cellular network associated with a prominent meshwork of basal laminae. Throughout much of pregnancy, these endothelial cells were cuboidal to columnar and possessed an abundance of basal glycoprotein granules presumably containing basal lamina precursors. The cells also expressed vimentin and frequently von Willebrand factor, but not cytokeratins or desmin. Pronounced thickening of the endothelia and amplification of their basal laminae likely evolved to greatly strengthen the walls of the uteroplacental vessels. PMID:21764447

Fabrication of corneal epithelial cell sheets maintaining colony-forming cells without feeder cells by oxygen-controlled method.

PubMed

Nakajima, Ryota; Takeda, Shizu

2014-01-01

The use of murine 3T3 feeder cells needs to be avoided when fabricating corneal epithelial cell sheets for use in treating ocular surface diseases. However, the expression level of the epithelial stem/progenitor cell marker, p63, is down-regulated in feeder-free culture systems. In this study, in order to fabricate corneal epithelial cell sheets that maintain colony-forming cells without using any feeder cells, we investigated the use of an oxygen-controlled method that was developed previously to fabricate cell sheets efficiently. Rabbit limbal epithelial cells were cultured under hypoxia (1-10% O2) and under normoxia during stratification after reaching confluence. Multilayered corneal epithelial cell sheets were fabricated using an oxygen-controlled method, and immunofluorescence analysis showed that cytokeratin 3 and p63 was expressed in appropriate localization in the cell sheets. The colony-forming efficiency of the cell sheets fabricated by the oxygen-controlled method without feeder cells was significantly higher than that of cell sheets fabricated under 20% O2 without feeder cells. These results indicate that the oxygen-controlled method has the potential to achieve a feeder-free culture system for fabricating corneal epithelial cell sheets for corneal regeneration. Copyright © 2013 Elsevier Ltd. All rights reserved.

Connecting Teratogen-Induced Congenital Heart Defects to Neural Crest Cells and Their Effect on Cardiac Function

PubMed Central

Karunamuni, Ganga H.; Ma, Pei; Gu, Shi; Rollins, Andrew M.; Jenkins, Michael W.; Watanabe, Michiko

2014-01-01

Neural crest cells play many key roles in embryonic development, as demonstrated by the abnormalities that result from their specific absence or dysfunction. Unfortunately, these key cells are particularly sensitive to abnormalities in various intrinsic and extrinsic factors, such as genetic deletions or ethanol-exposure that lead to morbidity and mortality for organisms. This review discusses the role identified for a segment of neural crest is in regulating the morphogenesis of the heart and associated great vessels. The paradox is that their derivatives constitute a small proportion of cells to the cardiovascular system. Findings supporting that these cells impact early cardiac function raises the interesting possibility that they indirectly control cardiovascular development at least partially through regulating function. Making connections between insults to the neural crest, cardiac function, and morphogenesis is more approachable with technological advances. Expanding our understanding of early functional consequences could be useful in improving diagnosis and testing therapies. PMID:25220155

Bidirectional Interplay between Vimentin Intermediate Filaments and Contractile Actin Stress Fibers.

PubMed

Jiu, Yaming; Lehtimäki, Jaakko; Tojkander, Sari; Cheng, Fang; Jäälinoja, Harri; Liu, Xiaonan; Varjosalo, Markku; Eriksson, John E; Lappalainen, Pekka

2015-06-16

The actin cytoskeleton and cytoplasmic intermediate filaments contribute to cell migration and morphogenesis, but the interplay between these two central cytoskeletal elements has remained elusive. Here, we find that specific actin stress fiber structures, transverse arcs, interact with vimentin intermediate filaments and promote their retrograde flow. Consequently, myosin-II-containing arcs are important for perinuclear localization of the vimentin network in cells. The vimentin network reciprocally restricts retrograde movement of arcs and hence controls the width of flat lamellum at the leading edge of the cell. Depletion of plectin recapitulates the vimentin organization phenotype of arc-deficient cells without affecting the integrity of vimentin filaments or stress fibers, demonstrating that this cytoskeletal cross-linker is required for productive interactions between vimentin and arcs. Collectively, our results reveal that plectin-mediated interplay between contractile actomyosin arcs and vimentin intermediate filaments controls the localization and dynamics of these two cytoskeletal systems and is consequently important for cell morphogenesis. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

YAP is essential for mechanical force production and epithelial cell proliferation during lung branching morphogenesis

PubMed Central

Lin, Chuwen; Yao, Erica; Zhang, Kuan; Jiang, Xuan; Croll, Stacey; Thompson-Peer, Katherine; Chuang, Pao-Tien

2017-01-01

Branching morphogenesis is a fundamental program for tissue patterning. We show that active YAP, a key mediator of Hippo signaling, is distributed throughout the murine lung epithelium and loss of epithelial YAP severely disrupts branching. Failure to branch is restricted to regions where YAP activity is removed. This suggests that YAP controls local epithelial cell properties. In support of this model, mechanical force production is compromised and cell proliferation is reduced in Yap mutant lungs. We propose that defective force generation and insufficient epithelial cell number underlie the branching defects. Through genomic analysis, we also uncovered a feedback control of pMLC levels, which is critical for mechanical force production, likely through the direct induction of multiple regulators by YAP. Our work provides a molecular pathway that could control epithelial cell properties required for proper morphogenetic movement and pattern formation. DOI: http://dx.doi.org/10.7554/eLife.21130.001 PMID:28323616

Cell wall matrix polysaccharide distribution and cortical microtubule organization: two factors controlling mesophyll cell morphogenesis in land plants

PubMed Central

Sotiriou, P.; Giannoutsou, E.; Panteris, E.; Apostolakos, P.; Galatis, B.

2016-01-01

Background and aims This work investigates the involvement of local differentiation of cell wall matrix polysaccharides and the role of microtubules in the morphogenesis of mesophyll cells (MCs) of three types (lobed, branched and palisade) in the dicotyledon Vigna sinensis and the fern Asplenium nidus. Methods Homogalacturonan (HGA) epitopes recognized by the 2F4, JIM5 and JIM7 antibodies and callose were immunolocalized in hand-made leaf sections. Callose was also stained with aniline blue. We studied microtubule organization by tubulin immunofluorescence and transmission electron microscopy. Results In both plants, the matrix cell wall polysaccharide distribution underwent definite changes during MC differentiation. Callose constantly defined the sites of MC contacts. The 2F4 HGA epitope in V. sinensis first appeared in MC contacts but gradually moved towards the cell wall regions facing the intercellular spaces, while in A. nidus it was initially localized at the cell walls delimiting the intercellular spaces, but finally shifted to MC contacts. In V. sinensis, the JIM5 and JIM7 HGA epitopes initially marked the cell walls delimiting the intercellular spaces and gradually shifted in MC contacts, while in A. nidus they constantly enriched MC contacts. In all MC types examined, the cortical microtubules played a crucial role in their morphogenesis. In particular, in palisade MCs, cortical microtubule helices, by controlling cellulose microfibril orientation, forced these MCs to acquire a truncated cone-like shape. Unexpectedly in V. sinensis, the differentiation of colchicine-affected MCs deviated completely, since they developed a cell wall ingrowth labyrinth, becoming transfer-like cells. Conclusions The results of this work and previous studies on Zea mays (Giannoutsou et al., Annals of Botany 2013; 112: 1067–1081) revealed highly controlled local cell wall matrix differentiation in MCs of species belonging to different plant groups. This, in coordination with microtubule-dependent cellulose microfibril alignment, spatially controlled cell wall expansion, allowing MCs to acquire their particular shape. PMID:26802013

Mechanical feedback coordinates cell wall expansion and assembly in yeast mating morphogenesis

PubMed Central

2018-01-01

The shaping of individual cells requires a tight coordination of cell mechanics and growth. However, it is unclear how information about the mechanical state of the wall is relayed to the molecular processes building it, thereby enabling the coordination of cell wall expansion and assembly during morphogenesis. Combining theoretical and experimental approaches, we show that a mechanical feedback coordinating cell wall assembly and expansion is essential to sustain mating projection growth in budding yeast (Saccharomyces cerevisiae). Our theoretical results indicate that the mechanical feedback provided by the Cell Wall Integrity pathway, with cell wall stress sensors Wsc1 and Mid2 increasingly activating membrane-localized cell wall synthases Fks1/2 upon faster cell wall expansion, stabilizes mating projection growth without affecting cell shape. Experimental perturbation of the osmotic pressure and cell wall mechanics, as well as compromising the mechanical feedback through genetic deletion of the stress sensors, leads to cellular phenotypes that support the theoretical predictions. Our results indicate that while the existence of mechanical feedback is essential to stabilize mating projection growth, the shape and size of the cell are insensitive to the feedback. PMID:29346368

Rapid fabricating technique for multi-layered human hepatic cell sheets by forceful contraction of the fibroblast monolayer.

PubMed

Sakai, Yusuke; Koike, Makiko; Hasegawa, Hideko; Yamanouchi, Kosho; Soyama, Akihiko; Takatsuki, Mitsuhisa; Kuroki, Tamotsu; Ohashi, Kazuo; Okano, Teruo; Eguchi, Susumu

2013-01-01

Cell sheet engineering is attracting attention from investigators in various fields, from basic research scientists to clinicians focused on regenerative medicine. However, hepatocytes have a limited proliferation potential in vitro, and it generally takes a several days to form a sheet morphology and multi-layered sheets. We herein report our rapid and efficient technique for generating multi-layered human hepatic cell (HepaRG® cell) sheets using pre-cultured fibroblast monolayers derived from human skin (TIG-118 cells) as a feeder layer on a temperature-responsive culture dish. Multi-layered TIG-118/HepaRG cell sheets with a thick morphology were harvested on day 4 of culturing HepaRG cells by forceful contraction of the TIG-118 cells, and the resulting sheet could be easily handled. In addition, the human albumin and alpha 1-antitrypsin synthesis activities of TIG-118/HepaRG cells were approximately 1.2 and 1.3 times higher than those of HepaRG cells, respectively. Therefore, this technique is considered to be a promising modality for rapidly fabricating multi-layered human hepatocyte sheets from cells with limited proliferation potential, and the engineered cell sheet could be used for cell transplantation with highly specific functions.

Actin capping protein CAPZB regulates cell morphology, differentiation, and neural crest migration in craniofacial morphogenesis†

PubMed Central

Mukherjee, Kusumika; Ishii, Kana; Pillalamarri, Vamsee; Kammin, Tammy; Atkin, Joan F.; Hickey, Scott E.; Xi, Qiongchao J.; Zepeda, Cinthya J.; Gusella, James F.; Talkowski, Michael E.; Morton, Cynthia C.; Maas, Richard L.; Liao, Eric C.

2016-01-01

CAPZB is an actin-capping protein that caps the growing end of F-actin and modulates the cytoskeleton and tethers actin filaments to the Z-line of the sarcomere in muscles. Whole-genome sequencing was performed on a subject with micrognathia, cleft palate and hypotonia that harbored a de novo, balanced chromosomal translocation that disrupts the CAPZB gene. The function of capzb was analyzed in the zebrafish model. capzb−/− mutants exhibit both craniofacial and muscle defects that recapitulate the phenotypes observed in the human subject. Loss of capzb affects cell morphology, differentiation and neural crest migration. Differentiation of both myogenic stem cells and neural crest cells requires capzb. During palate morphogenesis, defective cranial neural crest cell migration in capzb−/− mutants results in loss of the median cell population, creating a cleft phenotype. capzb is also required for trunk neural crest migration, as evident from melanophores disorganization in capzb−/− mutants. In addition, capzb over-expression results in embryonic lethality. Therefore, proper capzb dosage is important during embryogenesis, and regulates both cell behavior and tissue morphogenesis. PMID:26758871

Cell chirality: emergence of asymmetry from cell culture.

PubMed

Wan, Leo Q; Chin, Amanda S; Worley, Kathryn E; Ray, Poulomi

2016-12-19

Increasing evidence suggests that intrinsic cell chirality significantly contributes to the left-right (LR) asymmetry in embryonic development, which is a well-conserved characteristic of living organisms. With animal embryos, several theories have been established, but there are still controversies regarding mechanisms associated with embryonic LR symmetry breaking and the formation of asymmetric internal organs. Recently, in vitro systems have been developed to determine cell chirality and to recapitulate multicellular chiral morphogenesis on a chip. These studies demonstrate that chirality is indeed a universal property of the cell that can be observed with well-controlled experiments such as micropatterning. In this paper, we discuss the possible benefits of these in vitro systems to research in LR asymmetry, categorize available platforms for single-cell chirality and multicellular chiral morphogenesis, and review mathematical models used for in vitro cell chirality and its applications in in vivo embryonic development. These recent developments enable the interrogation of the intracellular machinery in LR axis establishment and accelerate research in birth defects in laterality.This article is part of the themed issue 'Provocative questions in left-right asymmetry'. © 2016 The Author(s).

Cell chirality: emergence of asymmetry from cell culture

PubMed Central

Wan, Leo Q.; Chin, Amanda S.; Worley, Kathryn E.; Ray, Poulomi

2016-01-01

Increasing evidence suggests that intrinsic cell chirality significantly contributes to the left–right (LR) asymmetry in embryonic development, which is a well-conserved characteristic of living organisms. With animal embryos, several theories have been established, but there are still controversies regarding mechanisms associated with embryonic LR symmetry breaking and the formation of asymmetric internal organs. Recently, in vitro systems have been developed to determine cell chirality and to recapitulate multicellular chiral morphogenesis on a chip. These studies demonstrate that chirality is indeed a universal property of the cell that can be observed with well-controlled experiments such as micropatterning. In this paper, we discuss the possible benefits of these in vitro systems to research in LR asymmetry, categorize available platforms for single-cell chirality and multicellular chiral morphogenesis, and review mathematical models used for in vitro cell chirality and its applications in in vivo embryonic development. These recent developments enable the interrogation of the intracellular machinery in LR axis establishment and accelerate research in birth defects in laterality. This article is part of the themed issue ‘Provocative questions in left–right asymmetry’. PMID:27821525

Morphogenesis of the vulva and the vulval-uterine connection.

PubMed Central

Gupta, Bhagwati P; Hanna-Rose, Wendy; Sternberg, Paul W

2012-01-01

The C. elegans hermaphrodite vulva is an established model system to study mechanisms of cell fate specification and tissue morphogenesis. The adult vulva is a tubular shaped organ composed of seven concentric toroids that arise from selective fusion between differentiated vulval progeny. The dorsal end of the vulval tubule is connected to the uterus via a multinucleate syncytium utse (uterine-seam) cell. The vulval tubule and utse are formed as a result of changes in morphogenetic processes such as cell polarity, adhesion, and invagination. A number of genes controlling these processes are conserved all the way up to human and function in similar developmental contexts. This makes it possible to extend the findings to other metazoan systems. Gene expression studies in the vulval and uterine cells have revealed the presence of regulatory networks specifying distinct cell fates. Thus, these two cell types serve as a good system to understand how gene networks confer unique cell identities both experimentally and computationally. This chapter focuses on morphogenetic processes during the formation of the vulva and its connection to uterus. PMID:23208727

Morphogenesis of the vulva and the vulval-uterine connection.

PubMed

Gupta, Bhagwati P; Hanna-Rose, Wendy; Sternberg, Paul W

2012-11-30

The C. elegans hermaphrodite vulva is an established model system to study mechanisms of cell fate specification and tissue morphogenesis. The adult vulva is a tubular shaped organ composed of seven concentric toroids that arise from selective fusion between differentiated vulval progeny. The dorsal end of the vulval tubule is connected to the uterus via a multinucleate syncytium utse (uterine-seam) cell. The vulval tubule and utse are formed as a result of changes in morphogenetic processes such as cell polarity, adhesion, and invagination. A number of genes controlling these processes are conserved all the way up to human and function in similar developmental contexts. This makes it possible to extend the findings to other metazoan systems. Gene expression studies in the vulval and uterine cells have revealed the presence of regulatory networks specifying distinct cell fates. Thus, these two cell types serve as a good system to understand how gene networks confer unique cell identities both experimentally and computationally. This chapter focuses on morphogenetic processes during the formation of the vulva and its connection to uterus.

New robust algorithm for tracking cells in videos of Drosophila morphogenesis based on finding an ideal path in segmented spatio-temporal cellular structures.

PubMed

Bellaïche, Yohanns; Bosveld, Floris; Graner, François; Mikula, Karol; Remesíková, Mariana; Smísek, Michal

2011-01-01

In this paper, we present a novel algorithm for tracking cells in time lapse confocal microscopy movie of a Drosophila epithelial tissue during pupal morphogenesis. We consider a 2D + time video as a 3D static image, where frames are stacked atop each other, and using a spatio-temporal segmentation algorithm we obtain information about spatio-temporal 3D tubes representing evolutions of cells. The main idea for tracking is the usage of two distance functions--first one from the cells in the initial frame and second one from segmented boundaries. We track the cells backwards in time. The first distance function attracts the subsequently constructed cell trajectories to the cells in the initial frame and the second one forces them to be close to centerlines of the segmented tubular structures. This makes our tracking algorithm robust against noise and missing spatio-temporal boundaries. This approach can be generalized to a 3D + time video analysis, where spatio-temporal tubes are 4D objects.

WNTLESS IS REQUIRED FOR PERIPHERAL LUNG DIFFERENTIATION AND PULMONARY VASCULAR DEVELOPMENT

PubMed Central

Cornett, Bridget; Snowball, John; Varisco, Brian M.; Lang, Richard; Whitsett, Jeffrey; Sinner, Debora

2013-01-01

Wntless (Wls), a gene highly conserved across the animal kingdom, encodes for a transmembrane protein that mediates Wnt ligand secretion. Wls is expressed in developing lung, wherein Wnt signaling is necessary for pulmonary morphogenesis. We hypothesize that Wls plays a critical role in modulating Wnt signaling during lung development and therefore affects processes critical for pulmonary morphogenesis. We generated conditional Wls mutant mice utilizing Shh-Cre and Dermo1-Cre mice to delete Wls in the embryonic respiratory epithelium and mesenchyme, respectively. Epithelial deletion of Wls disrupted lung branching morphogenesis, peripheral lung development and pulmonary endothelial differentiation. Epithelial Wls mutant mice died at birth due to respiratory failure caused by lung hypoplasia and pulmonary hemorrhage. In the lungs of these mice, VEGF and Tie2-angiopoietin signaling pathways, which mediate vascular development, were downregulated from early stages of development. In contrast, deletion of Wls in mesenchymal cells of the developing lung did not alter branching morphogenesis or early mesenchymal differentiation. In vitro assays support the concept that Wls acts in part via Wnt5a to regulate pulmonary vascular development. We conclude that epithelial Wls modulates Wnt ligand activities critical for pulmonary vascular differentiation and peripheral lung morphogenesis. These studies provide a new framework for understanding the molecular mechanisms underlying normal pulmonary vasculature formation and the dysmorphic pulmonary vasculature development associated with congenital lung disease. PMID:23523683

Wntless is required for peripheral lung differentiation and pulmonary vascular development.

PubMed

Cornett, Bridget; Snowball, John; Varisco, Brian M; Lang, Richard; Whitsett, Jeffrey; Sinner, Debora

2013-07-01

Wntless (Wls), a gene highly conserved across the animal kingdom, encodes for a transmembrane protein that mediates Wnt ligand secretion. Wls is expressed in developing lung, wherein Wnt signaling is necessary for pulmonary morphogenesis. We hypothesize that Wls plays a critical role in modulating Wnt signaling during lung development and therefore affects processes critical for pulmonary morphogenesis. We generated conditional Wls mutant mice utilizing Shh-Cre and Dermo1-Cre mice to delete Wls in the embryonic respiratory epithelium and mesenchyme, respectively. Epithelial deletion of Wls disrupted lung branching morphogenesis, peripheral lung development and pulmonary endothelial differentiation. Epithelial Wls mutant mice died at birth due to respiratory failure caused by lung hypoplasia and pulmonary hemorrhage. In the lungs of these mice, VEGF and Tie2-angiopoietin signaling pathways, which mediate vascular development, were downregulated from early stages of development. In contrast, deletion of Wls in mesenchymal cells of the developing lung did not alter branching morphogenesis or early mesenchymal differentiation. In vitro assays support the concept that Wls acts in part via Wnt5a to regulate pulmonary vascular development. We conclude that epithelial Wls modulates Wnt ligand activities critical for pulmonary vascular differentiation and peripheral lung morphogenesis. These studies provide a new framework for understanding the molecular mechanisms underlying normal pulmonary vasculature formation and the dysmorphic pulmonary vasculature development associated with congenital lung disease. Copyright © 2013 Elsevier Inc. All rights reserved.

Epithelial tricellular junctions act as interphase cell shape sensors to orient mitosis

PubMed Central

Bosveld, Floris; Markova, Olga; Guirao, Boris; Martin, Charlotte; Wang, Zhimin; Pierre, Anaëlle; Balakireva, Maria; Gaugue, Isabelle; Ainslie, Anna; Christophorou, Nicolas; Lubensky, David K.; Minc, Nicolas; Bellaïche, Yohanns

2017-01-01

The orientation of cell division along the interphase cell long-axis, the century old Hertwig’s rule, has profound roles in tissue proliferation, morphogenesis, architecture and mechanics1,2. In epithelial tissues, the shape of the interphase cell is influenced by cell adhesion, mechanical stress, neighbour topology, and planar polarity pathways3–12. At mitosis, epithelial cells usually round up to ensure faithful chromosome segregation and to promote morphogenesis1. The mechanisms underlying interphase cell shape sensing in tissues are therefore unknown. We found that in Drosophila epithelia, tricellular junctions (TCJ) localize microtubule force generators, orienting cell division via the Dynein associated protein Mud independently of the classical Pins/Gαi pathway. Moreover, as cells round up during mitosis, TCJs serve as spatial landmarks, encoding information about interphase cell shape anisotropy to orient division in the rounded mitotic cell. Finally, experimental and simulation data show that shape and mechanical strain sensing by the TCJ emerge from a general geometric property of TCJ distributions in epithelial tissues. Thus, in addition to their function as epithelial barrier structures, TCJs serve as polarity cues promoting geometry and mechanical sensing in epithelial tissues. PMID:26886796

Stem/Progenitor Cell Proteoglycans Decorated with 7-D-4, 4-C-3 and 3-B-3(-) Chondroitin Sulphate Motifs Are Morphogenetic Markers Of Tissue Development.

PubMed

Hayes, Anthony J; Smith, Susan M; Caterson, Bruce; Melrose, James

2018-06-11

This study reviewed the occurrence of chondroitin sulphate (CS) motifs 4-C-3, 7-D-4 and 3-B-3(-) which are expressed by progenitor cells in tissues undergoing morphogenesis. These motifs have a transient early expression pattern during tissue development and also appear in mature tissues during pathological remodeling and attempted repair processes by activated adult stem cells. The CS motifs are information and recognition modules, which may regulate cellular behavior and delineate stem cell niches in developmental tissues. One of the difficulties in determining the precise role of stem cells in tissue development and repair processes is their short engraftment period and the lack of specific markers, which differentiate the activated stem cell lineages from the resident cells. The CS sulphation motifs 7-D-4, 4-C-3 and 3-B-3 (-) decorate cell surface proteoglycans on activated stem/progenitor cells and appear to identify these cells in transitional areas of tissue development and in tissue repair and may be applicable to determining a more precise role for stem cells in tissue morphogenesis. This article is protected by copyright. All rights reserved. © 2018 AlphaMed Press.

Construction of osteochondral-like tissue graft combining β-tricalcium phosphate block and scaffold-free centrifuged chondrocyte cell sheet.

PubMed

Niyama, Kouhei; Ide, Naoto; Onoue, Kaori; Okabe, Takahiro; Wakitani, Shigeyuki; Takagi, Mutsumi

2011-09-01

The combination of a β-tricalcium phosphate (βTCP) block with a scaffold-free chondrocyte sheet formed by the centrifugation of chondrocytes in a well was investigated with the aim of constructing an osteochondral-like structure. Human and porcine articular cartilage chondrocytes were respectively centrifuged in a 96-well plate or cell culture insert (0.32 cm(2)) that was set in a 24-well plate, cultivated in the respective vessel for 3 weeks, and the cell sheets were harvested. In some cases, a cylindrical βTCP block (diameter 5 mm, height 3 mm) was placed on the sheet on days 1-7. The sheet size, cell number, and sulfated glycosaminoglycan accumulation were determined. The use of a 96-well plate for not suspension but adhesion culture and the initial centrifugation of a well containing cells were crucial to obtaining a uniformly thick cell sheet. The glycosaminoglycan density of the harvested cell sheet was comparable to that of the pellet culture. An inoculum cell number of more than 31 × 10(5) cells tended to result in a curved cell sheet. Culture involving 18.6 × 10(5) cells and the 96-well plate for adhesion culture showed no curving of the cell sheet (thickness of 0.85 mm), and these were found to be the best of the culture conditions tested. The timing of the addition of a βTCP block to the cell sheet (1-7 days) markedly affected the balance between the thickness of cell sheet parts on and in the βTCP block. Centrifugation and subsequent cultivation of chondrocytes (18.6 × 10(5) cells) in a 96-well plate for adhesion culture led to the production of a scaffold-free cartilage-like cell sheet with a thickness of 0.85 mm. A combined osteochondral-like structure was produced by putting a βTCP block on the cell sheet. The thickness of the cell sheet on the βTCP block and the binding strength between the cell sheet and the βTCP block could be optimized by adjusting the inoculum cell number and timing of βTCP block addition.

Expression of genes responsible for cell morphogenesis involved in differentiation in porcine buccal pouch mucosal cells during long-term primary culture and real-time proliferation in vitro.

PubMed

Dyszkiewicz-Konwińska, M; Bryja, A; Jopek, K; Budna, J; Khozmi, R; Jeseta, M; Bukowska, D; Antosik, P; Bruska, M; Nowicki, M; Zabel, M; Kempisty, B

2017-01-01

Recently, using experimental animal model, we demonstrated that porcine buccal pouch mucosal cells reflect increased proliferation capability during primary cultivation in vitro. Although the histological structure and morphogenesis in oral cavity is well recognized, the molecular mechanisms which regulate this process still need further investigation. This study was aimed to analyze the molecular marker expression profile involved in morphogenesis and differentiation capacity of porcine buccal pouch mucosal cells during their long-term primary cultivation in vitro. The experiment was performed on buccal pouch mucosal cells isolated from 80 pubertal crossbred Landrace gilts. After collection, the cells were treated enzymatically and transferred into a primary in vitro culture (IVC) system and cultured for 30 days. The cells were collected for RNA isolation after 7, 15 and 30 days of IVC and were checked for their real-time proliferative status using the RTCA system. We found an increased expression of FN1 and SOX9 genes when calculated against ACTB after 7, and 30 days of IVC, (P less than 0.01, P less than 0.001, respectively). The CXCL12 mRNA was down-regulated after 7, 15 and 30 days of IVC, but not statistically significant. Similar expression profile was observed when calculated against HPRT, however, DAB2 was found to be higher expressed at day 15 of IVC, (P less than 0.05). The cell index measured during real-time cell proliferation was substantially increased between 96 h and 147h of IVC and reached the log phase. Since FN1 and SOX9 revealed significant increase of expression after long-term culture in vitro, it is suggested that expression of these differentiation and stemness genes is accompanied by cell proliferation. Moreover, FN1 and SOX9 might be recognized as new markers of buccal pouch mucosal cell proliferation and differentiation in pigs in in vitro primary culture model.

Simple rules for a "simple" nervous system? Molecular and biomathematical approaches to enteric nervous system formation and malformation.

PubMed

Newgreen, Donald F; Dufour, Sylvie; Howard, Marthe J; Landman, Kerry A

2013-10-01

We review morphogenesis of the enteric nervous system from migratory neural crest cells, and defects of this process such as Hirschsprung disease, centering on cell motility and assembly, and cell adhesion and extracellular matrix molecules, along with cell proliferation and growth factors. We then review continuum and agent-based (cellular automata) models with rules of cell movement and logistical proliferation. Both movement and proliferation at the individual cell level are modeled with stochastic components from which stereotyped outcomes emerge at the population level. These models reproduced the wave-like colonization of the intestine by enteric neural crest cells, and several new properties emerged, such as colonization by frontal expansion, which were later confirmed biologically. These models predict a surprising level of clonal heterogeneity both in terms of number and distribution of daughter cells. Biologically, migrating cells form stable chains made up of unstable cells, but this is not seen in the initial model. We outline additional rules for cell differentiation into neurons, axon extension, cell-axon and cell-cell adhesions, chemotaxis and repulsion which can reproduce chain migration. After the migration stage, the cells re-arrange as a network of ganglia. Changes in cell adhesion molecules parallel this, and we describe additional rules based on Steinberg's Differential Adhesion Hypothesis, reflecting changing levels of adhesion in neural crest cells and neurons. This was able to reproduce enteric ganglionation in a model. Mouse mutants with disturbances of enteric nervous system morphogenesis are discussed, and these suggest future refinement of the models. The modeling suggests a relatively simple set of cell behavioral rules could account for complex patterns of morphogenesis. The model has allowed the proposal that Hirschsprung disease is mostly an enteric neural crest cell proliferation defect, not a defect of cell migration. In addition, the model suggests an explanations for zonal and skip segment variants of Hirschsprung disease, and also gives a novel stochastic explanation for the observed discordancy of Hirschsprung disease in identical twins. © 2013 Elsevier Inc. All rights reserved.

Role of cranial neural crest cells in visceral arch muscle positioning and morphogenesis in the Mexican axolotl, Ambystoma mexicanum.

PubMed

Ericsson, Rolf; Cerny, Robert; Falck, Pierre; Olsson, Lennart

2004-10-01

The role of cranial neural crest cells in the formation of visceral arch musculature was investigated in the Mexican axolotl, Ambystoma mexicanum. DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine, perchlorate) labeling and green fluorescent protein (GFP) mRNA injections combined with unilateral transplantations of neural folds showed that neural crest cells contribute to the connective tissues but not the myofibers of developing visceral arch muscles in the mandibular, hyoid, and branchial arches. Extirpations of individual cranial neural crest streams demonstrated that neural crest cells are necessary for correct morphogenesis of visceral arch muscles. These do, however, initially develop in their proper positions also in the absence of cranial neural crest. Visceral arch muscles forming in the absence of neural crest cells start to differentiate at their origins but fail to extend toward their insertions and may have a frayed appearance. Our data indicate that visceral arch muscle positioning is controlled by factors that do not have a neural crest origin. We suggest that the cranial neural crest-derived connective tissues provide directional guidance important for the proper extension of the cranial muscles and the subsequent attachment to the insertion on the correct cartilage. In a comparative context, our data from the Mexican axolotl support the view that the cranial neural crest plays a fundamental role in the development of not only the skeleton of the vertebrate head but also in the morphogenesis of the cranial muscles and that this might be a primitive feature of cranial development in vertebrates. 2004 Wiley-Liss, Inc.

Membrane Targeting of Grb2-associated Binder-1 (Gab1) Scaffolding Protein through Src Myristoylation Sequence Substitutes for Gab1 Pleckstrin Homology Domain and Switches an Epidermal Growth Factor Response to an Invasive Morphogenic Program

PubMed Central

Maroun, Christiane R.; Naujokas, Monica A.; Park, Morag

2003-01-01

The hepatocyte growth factor receptor tyrosine kinase Met promotes cell dissociation and the inherent morphogenic program of epithelial cells. In a search for substrates downstream from Met, we have previously identified the Grb2-associated binder-1 (Gab1) as critical for the morphogenic program. Gab1 is a scaffold protein that acts to diversify the signal downstream from the Met receptor through its ability to couple with multiple signal transduction pathways. Gab1 contains a pleckstrin homology (PH) domain with specificity for phosphatidylinositol 3,4,5-trisphosphate. The phospholipid binding capacity of the Gab1 PH domain is required for the localization of Gab1 at sites of cell-cell contact in colonies of epithelial cells and for epithelial morphogenesis, suggesting that PH domain-dependent subcellular localization of Gab1 is a prerequisite for function. We have investigated the requirement for membrane localization of Gab1 for biological activity. We show that substitution of the Gab1 PH domain with the myristoylation signal from the c-Src protein is sufficient to replace the Gab1 PH domain for epithelial morphogenesis. The membrane targeting of Gab1 enhances Rac activity in the absence of stimulation and switches a nonmorphogenic noninvasive response to epidermal growth factor to a morphogenic invasive program. These results suggest that the subcellular localization of Gab1 is a critical determinant for epithelial morphogenesis and invasiveness. PMID:12686619

Inverse tissue mechanics of cell monolayer expansion.

PubMed

Kondo, Yohei; Aoki, Kazuhiro; Ishii, Shin

2018-03-01

Living tissues undergo deformation during morphogenesis. In this process, cells generate mechanical forces that drive the coordinated cell motion and shape changes. Recent advances in experimental and theoretical techniques have enabled in situ measurement of the mechanical forces, but the characterization of mechanical properties that determine how these forces quantitatively affect tissue deformation remains challenging, and this represents a major obstacle for the complete understanding of morphogenesis. Here, we proposed a non-invasive reverse-engineering approach for the estimation of the mechanical properties, by combining tissue mechanics modeling and statistical machine learning. Our strategy is to model the tissue as a continuum mechanical system and to use passive observations of spontaneous tissue deformation and force fields to statistically estimate the model parameters. This method was applied to the analysis of the collective migration of Madin-Darby canine kidney cells, and the tissue flow and force were simultaneously observed by the phase contrast imaging and traction force microscopy. We found that our monolayer elastic model, whose elastic moduli were reverse-engineered, enabled a long-term forecast of the traction force fields when given the tissue flow fields, indicating that the elasticity contributes to the evolution of the tissue stress. Furthermore, we investigated the tissues in which myosin was inhibited by blebbistatin treatment, and observed a several-fold reduction in the elastic moduli. The obtained results validate our framework, which paves the way to the estimation of mechanical properties of living tissues during morphogenesis.

Feather regeneration as a model for organogenesis

PubMed Central

Lin, Sung-Jan; Wideliz, Randall B; Yue, Zhicao; Li, Ang; Wu, Xiaoshan; Jiang, Ting-Xin; Wu, Ping; Chuong, Cheng-Ming

2013-01-01

In the process of organogenesis, different cell types form organized tissues and tissues are integrated into an organ. Most organs form in the developmental stage, but new organs can also form in physiological states or following injuries during adulthood. Feathers are a good model to study post-natal organogenesis because they regenerate episodically under physiological conditions and in response to injuries such as plucking. Epidermal stem cells in the collar can respond to activation signals. Dermal papilla located at the follicle base controls the regenerative process. Adhesion molecules (e.g., NCAM, tenascin), morphogens (e.g., Wnt3a, sprouty, FGF10), and differentiation markers (e.g., keratins) are expressed dynamically in initiation, growth and resting phases of the feather cycle. Epidermal cells are shaped into different feather morphologies based on the molecular micro-environment at the moment of morphogenesis. Chicken feather variants provide a rich resource for us to identify genetic determinants involved in feather regeneration and morphogenesis. An example of using genome-wide SNP analysis to identify alpha keratin 75 as the mutation in frizzled chickens is demonstrated. Due to its accessibility to experimental manipulation and observation, results of regeneration can be analyzed in a comprehensive way. The layout of time dimension along the distal (formed earlier) - proximal (formed later) feather axis makes the morphological analyses easier. Therefore feather regeneration can be a unique model for understanding organogenesis: from activation of stems cell under various physiological conditions to serving as the Rosetta stone for deciphering the language of morphogenesis. PMID:23294361

Morphogenesis and calcification of the statoconia in the chick (Gallus domesticus) embryo - Implications for future studies

NASA Technical Reports Server (NTRS)

Fermin, C. D.; Igarashi, M.

1985-01-01

The morphogenesis of the statoconia in the chick, Gallus domesticus, injected with a carbon anhydrase inhibitor is studied. The preparation of the embryo specimens for analysis is described. The early, middle, and late stages of embryonic development are examined. The data reveal that acetozolamide inhibits statoconia formation in the middle stage of development and the calcification process follows statoconia formation. The spatial relationship between the development of type 1 and type 2 hair cells and the appearance and maturation of the statoconia is investigated.

Latrunculin B-induced plant dwarfism: Plant cell elongation is F-actin-dependent.

PubMed

Baluska, F; Jasik, J; Edelmann, H G; Salajová, T; Volkmann, D

2001-03-01

Marine macrolides latrunculins are highly specific toxins which effectively depolymerize actin filaments (generally F-actin) in all eukaryotic cells. We show that latrunculin B is effective on diverse cell types in higher plants and describe the use of this drug in probing F-actin-dependent growth and in plant development-related processes. In contrast to other eukaryotic organisms, cell divisions occurs in plant cells devoid of all actin filaments. However, the alignment of the division planes is often distorted. In addition to cell division, postembryonic development and morphogenesis also continue in the absence of F-actin. These experimental data suggest that F-actin is of little importance in the morphogenesis of higher plants, and that plants can develop more or less normally without F-actin. In contrast, F-actin turns out to be essential for cell elongation. When latrunculin B was added during germination, morphologically normal Arabidopsis and rye seedlings developed but, as a result of the absence of cell elongation, these were stunted, resembling either genetic dwarfs or environmental bonsai plants. In conclusion, F-actin is essential for the plant cell elongation, while this F-actin-dependent cell elongation is not an essential feature of plant-specific developmental programs.

Canonical TGF-β Signaling Negatively Regulates Neuronal Morphogenesis through TGIF/Smad Complex-Mediated CRMP2 Suppression.

PubMed

Nakashima, Hideyuki; Tsujimura, Keita; Irie, Koichiro; Ishizu, Masataka; Pan, Miao; Kameda, Tomonori; Nakashima, Kinichi

2018-05-16

Functional neuronal connectivity requires proper neuronal morphogenesis and its dysregulation causes neurodevelopmental diseases. Transforming growth factor-β (TGF-β) family cytokines play pivotal roles in development, but little is known about their contribution to morphological development of neurons. Here we show that the Smad-dependent canonical signaling of TGF-β family cytokines negatively regulates neuronal morphogenesis during brain development. Mechanistically, activated Smads form a complex with transcriptional repressor TG-interacting factor (TGIF), and downregulate the expression of a neuronal polarity regulator, collapsin response mediator protein 2. We also demonstrate that TGF-β family signaling inhibits neurite elongation of human induced pluripotent stem cell-derived neurons. Furthermore, the expression of TGF-β receptor 1, Smad4, or TGIF, which have mutations found in patients with neurodevelopmental disorders, disrupted neuronal morphogenesis in both mouse (male and female) and human (female) neurons. Together, these findings suggest that the regulation of neuronal morphogenesis by an evolutionarily conserved function of TGF-β signaling is involved in the pathogenesis of neurodevelopmental diseases. SIGNIFICANCE STATEMENT Canonical transforming growth factor-β (TGF-β) signaling plays a crucial role in multiple organ development, including brain, and mutations in components of the signaling pathway associated with several human developmental disorders. In this study, we found that Smads/TG-interacting factor-dependent canonical TGF-β signaling regulates neuronal morphogenesis through the suppression of collapsin response mediator protein-2 (CRMP2) expression during brain development, and that function of this signaling is evolutionarily conserved in the mammalian brain. Mutations in canonical TGF-β signaling factors identified in patients with neurodevelopmental disorders disrupt the morphological development of neurons. Thus, our results suggest that proper control of TGF-β/Smads/CRMP2 signaling pathways is critical for the precise execution of neuronal morphogenesis, whose impairment eventually results in neurodevelopmental disorders. Copyright © 2018 the authors 0270-6474/18/384791-20$15.00/0.

The Nematode Caenorhabditis Elegans.

ERIC Educational Resources Information Center

Kenyon, Cynthia

1988-01-01

Discusses advantages of nematode use for studying patterns of cell division, differentiation, and morphogenesis. Describes nematode development. Cites experimental approaches available for genetic studies. Reviews the topics of control of cell division and differentiation, the nervous system, and muscle assembly and function of the organism. (RT)

Rapid Fabricating Technique for Multi-Layered Human Hepatic Cell Sheets by Forceful Contraction of the Fibroblast Monolayer

PubMed Central

Sakai, Yusuke; Koike, Makiko; Hasegawa, Hideko; Yamanouchi, Kosho; Soyama, Akihiko; Takatsuki, Mitsuhisa; Kuroki, Tamotsu; Ohashi, Kazuo; Okano, Teruo; Eguchi, Susumu

2013-01-01

Cell sheet engineering is attracting attention from investigators in various fields, from basic research scientists to clinicians focused on regenerative medicine. However, hepatocytes have a limited proliferation potential in vitro, and it generally takes a several days to form a sheet morphology and multi-layered sheets. We herein report our rapid and efficient technique for generating multi-layered human hepatic cell (HepaRG® cell) sheets using pre-cultured fibroblast monolayers derived from human skin (TIG-118 cells) as a feeder layer on a temperature-responsive culture dish. Multi-layered TIG-118/HepaRG cell sheets with a thick morphology were harvested on day 4 of culturing HepaRG cells by forceful contraction of the TIG-118 cells, and the resulting sheet could be easily handled. In addition, the human albumin and alpha 1-antitrypsin synthesis activities of TIG-118/HepaRG cells were approximately 1.2 and 1.3 times higher than those of HepaRG cells, respectively. Therefore, this technique is considered to be a promising modality for rapidly fabricating multi-layered human hepatocyte sheets from cells with limited proliferation potential, and the engineered cell sheet could be used for cell transplantation with highly specific functions. PMID:23923035

A novel gelatin hydrogel carrier sheet for corneal endothelial transplantation.

PubMed

Watanabe, Ryou; Hayashi, Ryuhei; Kimura, Yu; Tanaka, Yuji; Kageyama, Tomofumi; Hara, Susumu; Tabata, Yasuhiko; Nishida, Kohji

2011-09-01

We examined the feasibility of using gelatin hydrogels as carrier sheets for the transplantation of cultivated corneal endothelial cells. The mechanical properties, transparency, and permeability of gelatin hydrogel sheets were compared with those of atelocollagen sheets. Immunohistochemistry (ZO-1, Na(+)/K(+)-ATPase, and N-cadherin), hematoxylin and eosin staining, and scanning electron microscopy were performed to assess the integrity of corneal endothelial cells that were cultured on gelatin hydrogel sheets. The gelatin hydrogel sheets displayed greater transparency, elastic modulus, and albumin permeability compared to those of atelocollagen sheets. The corneal endothelial cells on gelatin hydrogel sheets showed normal expression levels of ZO-1, Na(+)/K(+)-ATPase, and N-cadherin. Hematoxylin and eosin staining revealed the formation of a continuous monolayer of cells attached to the gelatin hydrogel sheet. Scanning electron microscopy observations showed that the corneal endothelial cells were arranged in a regular, mosaic, and polygonal pattern with normal cilia. These results indicate that the gelatin hydrogel sheet is a promising material to transport corneal endothelial cells during transplantation.

Preparation of Caco-2 cell sheets using plasma polymerised acrylic acid as a weak boundary layer.

PubMed

Majani, Ruby; Zelzer, Mischa; Gadegaard, Nikolaj; Rose, Felicity R; Alexander, Morgan R

2010-09-01

The use of cell sheets for tissue engineering applications has considerable advantages over single cell seeding techniques. So far, only thermoresponsive surfaces have been used to manufacture cell sheets without chemically disrupting the cell-surface interactions. Here, we present a new and facile technique to prepare sheets of epithelial cells using plasma polymerised acrylic acid films. The cell sheets are harvested by gentle agitation of the media without the need of any additional external stimulus. We demonstrate that the plasma polymer deposition conditions affect the viability and metabolic activity of the cells in the sheet and relate these effects to the different surface properties of the plasma polymerised acrylic acid films. Based on surface analysis data, a first attempt is made to explain the mechanism behind the cell sheet formation. The advantage of the epithelial cell sheets generated here over single cell suspensions to seed a PLGA scaffold is presented. The scaffold itself, prepared using a mould fabricated via photolithography, exhibits a unique architecture that mimics closely the dimensions of the native tissue (mouse intestine). Copyright 2010 Elsevier Ltd. All rights reserved.

Collective epithelial cell sheet adhesion and migration on polyelectrolyte multilayers with uniform and gradients of compliance

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

Martinez, Jessica S.; Schlenoff, Joseph B.; Keller, Thomas C.S., E-mail: tkeller@bio.fsu.edu

Polyelectrolyte multilayers (PEMUs) are tunable thin films that could serve as coatings for biomedical implants. PEMUs built layer by layer with the polyanion poly(acrylic acid) (PAA) modified with a photosensitive 4-(2-hydroxyethoxy) benzophenone (PAABp) group and the polycation poly(allylamine hydrochloride) (PAH) are mechanically tunable by UV irradiation, which forms covalent bonds between the layers and increases PEMU stiffness. PAH-terminated PEMUs (PAH-PEMUs) that were uncrosslinked, UV-crosslinked to a uniform stiffness, or UV-crosslinked with an edge mask or through a neutral density optical gradient filter to form continuous compliance gradients were used to investigate how differences in PEMU stiffness affect the adhesion andmore » migration of epithelial cell sheets from scales of the fish Poecilia sphenops (Black Molly) and Carassius auratus (Comet Goldfish). During the progressive collective cell migration, the edge cells (also known as ‘leader’ cells) in the sheets on softer uncrosslinked PEMUs and less crosslinked regions of the gradient formed more actin filaments and vinculin-containing adherens junctions and focal adhesions than formed in the sheet cells on stiffer PEMUs or glass. During sheet migration, the ratio of edge cell to internal cell (also known as ‘follower’ cells) motilities were greater on the softer PEMUs than on the stiffer PEMUs or glass, causing tension to develop across the sheet and periods of retraction, during which the edge cells lost adhesion to the substrate and regions of the sheet retracted toward the more adherent internal cell region. These retraction events were inhibited by the myosin II inhibitor Blebbistatin, which reduced the motility velocity ratios to those for sheets on the stiffer PEMUs. Blebbistatin also caused disassembly of actin filaments, reorganization of focal adhesions, increased cell spreading at the leading edge, as well as loss of edge cell-cell connections in epithelial cell sheets on all surfaces. Interestingly, cells throughout the interior region of the sheets on uncrosslinked PEMUs retained their actin and vinculin organization at adherens junctions after treatment with Blebbistatin. Like Blebbistatin, a Rho-kinase (ROCK) inhibitor, Y27632, promoted loss of cell-cell connections between edge cells, whereas a Rac1 inhibitor, NSC23766, primarily altered the lamellipodial protrusion in edge cells. Compliance gradient PAH-PEMUs promoted durotaxis of the cell sheets but not of individual keratocytes, demonstrating durotaxis, like plithotaxis, is an emergent property of cell sheet organization. - Highlights: • Fish scale cell sheets migrate on PAH-PAABp polyelectrolyte multilayers. • Sheets migrating on softer PEMUs periodically retract. • Sheets durotax on modulus gradients. • Myosin II inhibitors inhibit sheet integrity and migration.« less

The F-BAR domains from srGAP1, srGAP2 and srGAP3 regulate membrane deformation differently

PubMed Central

Coutinho-Budd, Jaeda; Ghukasyan, Vladimir; Zylka, Mark J.; Polleux, Franck

2012-01-01

Summary Coordination of membrane deformation and cytoskeletal dynamics lies at the heart of many biological processes critical for cell polarity, motility and morphogenesis. We have recently shown that Slit-Robo GTPase-activating protein 2 (srGAP2) regulates neuronal morphogenesis through the ability of its F-BAR domain to regulate membrane deformation and induce filopodia formation. Here, we demonstrate that the F-BAR domains of two closely related family members, srGAP1 and srGAP3 [designated F-BAR(1) and F-BAR(3), respectively] display significantly different membrane deformation properties in non-neuronal COS7 cells and in cortical neurons. F-BAR(3) induces filopodia in both cell types, though less potently than F-BAR(2), whereas F-BAR(1) prevents filopodia formation in cortical neurons and reduces plasma membrane dynamics. These three F-BAR domains can heterodimerize, and they act synergistically towards filopodia induction in COS7 cells. As measured by fluorescence recovery after photobleaching, F-BAR(2) displays faster molecular dynamics than F-BAR(3) and F-BAR(1) at the plasma membrane, which correlates well with its increased potency to induce filopodia. We also show that the molecular dynamic properties of F-BAR(2) at the membrane are partially dependent on F-Actin. Interestingly, acute phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] depletion in cells does not interfere with plasma membrane localization of F-BAR(2), which is compatible with our result showing that F-BAR(2) binds to a broad range of negatively-charged phospholipids present at the plasma membrane, including phosphatidylserine (PtdSer). Overall, our results provide novel insights into the functional diversity of the membrane deformation properties of this subclass of F-BAR-domains required for cell morphogenesis. PMID:22467852

Tissue organization by cadherin adhesion molecules: dynamic molecular and cellular mechanisms of morphogenetic regulation

PubMed Central

Niessen, Carien M.; Leckband, Deborah; Yap, Alpha S.

2013-01-01

This review addresses the cellular and molecular mechanisms of cadherin-based tissue morphogenesis. Tissue physiology is profoundly influenced by the distinctive organizations of cells in organs and tissues. In metazoa, adhesion receptors of the classical cadherin family play important roles in establishing and maintaining such tissue organization. Indeed, it is apparent that cadherins participate in a range of morphogenetic events that range from support of tissue integrity to dynamic cellular rearrangements. A comprehensive understanding of cadherin-based morphogenesis must then define the molecular and cellular mechanisms that support these distinct cadherin biologies. Here we focus on four key mechanistic elements: the molecular basis for adhesion through cadherin ectodomains; the regulation of cadherin expression at the cell surface; cooperation between cadherins and the actin cytoskeleton; and regulation by cell signaling. We discuss current progress and outline issues for further research in these fields. PMID:21527735

Regulation of Facial Morphogenesis by Endothelin Signaling: Insights from Mice and Fish

PubMed Central

Clouthier, David E.; Garcia, Elvin; Schilling, Thomas F.

2010-01-01

Craniofacial morphogenesis is accomplished through a complex set of developmental events, most of which are initiated in neural crest cells within the pharyngeal arches. Local patterning cues from the surrounding environment induce gene expression within neural crest cells, leading to formation of a diverse set of skeletal elements. Endothelin-1 (Edn1) is one of the primary signals that establish the identities of neural crest cells within the mandibular portion of the first pharyngeal arch. Signaling through its cognate receptor, the endothelin-A receptor, is critical for patterning the ventral/distal portion of the arch (lower jaw) and also participates with Hox genes in patterning more posterior arches. Edn1/Ednra signaling is highly conserved between mouse and zebrafish, and genetic analyses in these two species have provided complementary insights into the patterning cues responsible for establishing the craniofacial complex as well as the genetic basis of facial birth defect syndromes. PMID:20684004

Microfabricated tissues for investigating traction forces involved in cell migration and tissue morphogenesis

PubMed Central

Nerger, Bryan A.; Siedlik, Michael J.; Nelson, Celeste M.

2016-01-01

Cell-generated forces drive an array of biological processes ranging from wound healing to tumor metastasis. Whereas experimental techniques such as traction force microscopy are capable of quantifying traction forces in multidimensional systems, the physical mechanisms by which these forces induce changes in tissue form remain to be elucidated. Understanding these mechanisms will ultimately require techniques that are capable of quantifying traction forces with high precision and accuracy in vivo or in systems that recapitulate in vivo conditions, such as microfabricated tissues and engineered substrata. To that end, here we review the fundamentals of traction forces, their quantification, and the use of microfabricated tissues designed to study these forces during cell migration and tissue morphogenesis. We emphasize the differences between traction forces in two- and three-dimensional systems, and highlight recently developed techniques for quantifying traction forces. PMID:28008471

The use of platelet-rich fibrin combined with periodontal ligament and jaw bone mesenchymal stem cell sheets for periodontal tissue engineering.

PubMed

Wang, Zhong-Shan; Feng, Zhi-Hong; Wu, Guo-Feng; Bai, Shi-Zhu; Dong, Yan; Chen, Fa-Ming; Zhao, Yi-Min

2016-06-21

Periodontal regeneration involves the restoration of at least three unique tissues: cementum, periodontal ligament tissue (PDL) and alveolar bone tissue. Here, we first isolated human PDL stem cells (PDLSCs) and jaw bone mesenchymal stem cells (JBMSCs). These cells were then induced to form cell sheets using an ascorbic acid-rich approach, and the cell sheet properties, including morphology, thickness and gene expression profile, were compared. Platelet-rich fibrin (PRF) derived from human venous blood was then fabricated into bioabsorbable fibrin scaffolds containing various growth factors. Finally, the in vivo potential of a cell-material construct based on PDLSC sheets, PRF scaffolds and JBMSC sheets to form periodontal tissue was assessed in a nude mouse model. In this model, PDLSC sheet/PRF/JBMSC sheet composites were placed in a simulated periodontal space comprising human treated dentin matrix (TDM) and hydroxyapatite (HA)/tricalcium phosphate (TCP) frameworks. Eight weeks after implantation, the PDLSC sheets tended to develop into PDL-like tissues, while the JBMSC sheets tended to produce predominantly bone-like tissues. In addition, the PDLSC sheet/PRF/JBMSC sheet composites generated periodontal tissue-like structures containing PDL- and bone-like tissues. Further improvements in this cell transplantation design may have the potential to provide an effective approach for future periodontal tissue regeneration.

Quantitative semi-automated analysis of morphogenesis with single-cell resolution in complex embryos

PubMed Central

Giurumescu, Claudiu A.; Kang, Sukryool; Planchon, Thomas A.; Betzig, Eric; Bloomekatz, Joshua; Yelon, Deborah; Cosman, Pamela; Chisholm, Andrew D.

2012-01-01

A quantitative understanding of tissue morphogenesis requires description of the movements of individual cells in space and over time. In transparent embryos, such as C. elegans, fluorescently labeled nuclei can be imaged in three-dimensional time-lapse (4D) movies and automatically tracked through early cleavage divisions up to ~350 nuclei. A similar analysis of later stages of C. elegans development has been challenging owing to the increased error rates of automated tracking of large numbers of densely packed nuclei. We present Nucleitracker4D, a freely available software solution for tracking nuclei in complex embryos that integrates automated tracking of nuclei in local searches with manual curation. Using these methods, we have been able to track >99% of all nuclei generated in the C. elegans embryo. Our analysis reveals that ventral enclosure of the epidermis is accompanied by complex coordinated migration of the neuronal substrate. We can efficiently track large numbers of migrating nuclei in 4D movies of zebrafish cardiac morphogenesis, suggesting that this approach is generally useful in situations in which the number, packing or dynamics of nuclei present challenges for automated tracking. PMID:23052905

Functional studies on the role of Notch signaling in Hydractinia development.

PubMed

Gahan, James M; Schnitzler, Christine E; DuBuc, Timothy Q; Doonan, Liam B; Kanska, Justyna; Gornik, Sebastian G; Barreira, Sofia; Thompson, Kerry; Schiffer, Philipp; Baxevanis, Andreas D; Frank, Uri

2017-08-01

The function of Notch signaling was previously studied in two cnidarians, Hydra and Nematostella, representing the lineages Hydrozoa and Anthozoa, respectively. Using pharmacological inhibition in Hydra and a combination of pharmacological and genetic approaches in Nematostella, it was shown in both animals that Notch is required for tentacle morphogenesis and for late stages of stinging cell maturation. Surprisingly, a role for Notch in neural development, which is well documented in bilaterians, was evident in embryonic Nematostella but not in adult Hydra. Adult neurogenesis in the latter seemed to be unaffected by DAPT, a drug that inhibits Notch signaling. To address this apparent discrepancy, we studied the role of Notch in Hydractinia echinata, an additional hydrozoan, in all life stages. Using CRISPR-Cas9 mediated mutagenesis, transgenesis, and pharmacological interference we show that Notch is dispensable for Hydractinia normal neurogenesis in all life stages but is required for the maturation of stinging cells and for tentacle morphogenesis. Our results are consistent with a conserved role for Notch in morphogenesis and nematogenesis across Cnidaria, and a lineage-specific loss of Notch dependence in neurogenesis in hydrozoans. Copyright © 2017 Elsevier Inc. All rights reserved.

A Transient Exposure to Symbiosis-Competent Bacteria Induces Light Organ Morphogenesis in the Host Squid.

PubMed

Doino, J A; McFall-Ngai, M J

1995-12-01

Recent studies of the symbiotic association between the Hawaiian sepiolid squid Euprymna scolopes and the luminous bacterium Vibrio fischeri have shown that colonization of juvenile squid with symbiosis-competent bacteria induces morphogenetic changes of the light organ. These changes occur over a 4-day period and include cell death and tissue regression of the external ciliated epithelium. In the absence of bacterial colonization, morphogenesis does not occur. To determine whether the bacteria must be present throughout the morphogenetic process, we used the antibiotic chloramphenicol to clear the light organ of bacteria at various times during the initial colonization. We provide evidence in this study that a transient, 12-hour exposure to symbiosis-competent bacteria is necessary and sufficient to induce tissue regression in the light organ over the next several days. Further, we show that successful entrance into the light organ is necessary to induce morphogenesis, suggesting that induction results from bacterial interaction with internal crypt cells and not with the external ciliated epithelium. Finally, no difference in development was observed when the light organ was colonized by a mutant strain of V. fischeri that did not produce autoinducer, a potential light organ morphogen.

The role of integrin α8β1 in fetal lung morphogenesis and injury

PubMed Central

Benjamin, John T.; Gaston, David C.; Halloran, Brian A.; Schnapp, Lynn M.; Zent, Roy; Prince, Lawrence S.

2009-01-01

Prenatal inflammation prevents normal lung morphogenesis and leads to bronchopulmonary dysplasia (BPD), a common complication of preterm birth. We previously demonstrated in a bacterial endotoxin mouse model of BPD that disrupting fibronectin localization in the fetal lung mesenchyme causes arrested saccular airway branching. In this study we show that expression of the fibronectin receptor, integrin α8β1, is decreased in the lung mesenchyme in the same inflammation model suggesting it is required for normal lung development. We verified a role for integrin α8β1 in lung development using integrin α8-null mice, which develop fusion of the medial and caudal lobes as well as abnormalities in airway division. We further show in vivo and vitro that α8-null fetal lung mesenchymal cells fail to form stable adhesions and have increased migration. Thus we propose that integrin α8β1 plays a critical role in lung morphogenesis by regulating mesenchymal cell adhesion and migration. Furthermore, our data suggests that disruption of the interactions between extracellular matrix and integrin α8β1 may contribute to the pathogenesis of BPD. PMID:19769957

Proinsulin in development: New roles for an ancient prohormone.

PubMed

Hernández-Sánchez, C; Mansilla, A; de la Rosa, E J; de Pablo, F

2006-06-01

In postnatal organisms, insulin is well known as an essential anabolic hormone responsible for maintaining glucose homeostasis. Its biosynthesis by the pancreatic beta cell has been considered a model of tissue-specific gene expression. However, proinsulin mRNA and protein have been found in embryonic stages before the formation of the pancreatic primordium, and later, in extrapancreatic tissues including the nervous system. Phylogenetic studies have also confirmed that production of insulin-like peptides antecedes the morphogenesis of a pancreas, and that these peptides contribute to normal development. In recent years, other roles for insulin distinct from its metabolic function have emerged also in vertebrates. During embryonic development, insulin acts as a survival factor and is involved in early morphogenesis. These findings are consistent with the observation that, at these stages, the proinsulin gene product remains as the precursor form, proinsulin. Independent of its low metabolic activity, proinsulin stimulates proliferation in developing neuroretina, as well as cell survival and cardiogenesis in early embryos. Insulin/proinsulin levels are finely regulated during development, since an excess of the protein interferes with correct morphogenesis and is deleterious for the embryo. This fine-tuned regulation is achieved by the expression of alternative embryonic proinsulin transcripts that have diminished translational activity.

Epithelial junction formation requires confinement of Cdc42 activity by a novel SH3BP1 complex

PubMed Central

Elbediwy, Ahmed; Zihni, Ceniz; Terry, Stephen J.; Clark, Peter

2012-01-01

Epithelial cell–cell adhesion and morphogenesis require dynamic control of actin-driven membrane remodeling. The Rho guanosine triphosphatase (GTPase) Cdc42 regulates sequential molecular processes during cell–cell junction formation; hence, mechanisms must exist that inactivate Cdc42 in a temporally and spatially controlled manner. In this paper, we identify SH3BP1, a GTPase-activating protein for Cdc42 and Rac, as a regulator of junction assembly and epithelial morphogenesis using a functional small interfering ribonucleic acid screen. Depletion of SH3BP1 resulted in loss of spatial control of Cdc42 activity, stalled membrane remodeling, and enhanced growth of filopodia. SH3BP1 formed a complex with JACOP/paracingulin, a junctional adaptor, and CD2AP, a scaffolding protein; both were required for normal Cdc42 signaling and junction formation. The filamentous actin–capping protein CapZ also associated with the SH3BP1 complex and was required for control of actin remodeling. Epithelial junction formation and morphogenesis thus require a dual activity complex, containing SH3BP1 and CapZ, that is recruited to sites of active membrane remodeling to guide Cdc42 signaling and cytoskeletal dynamics. PMID:22891260

Epithelial stratification and placode invagination are separable functions in early morphogenesis of the molar tooth

PubMed Central

Li, Jingjing; Chatzeli, Lemonia; Panousopoulou, Eleni; Tucker, Abigail S.; Green, Jeremy B. A.

2016-01-01

Ectodermal organs, which include teeth, hair follicles, mammary ducts, and glands such as sweat, mucous and sebaceous glands, are initiated in development as placodes, which are epithelial thickenings that invaginate and bud into the underlying mesenchyme. These placodes are stratified into a basal and several suprabasal layers of cells. The mechanisms driving stratification and invagination are poorly understood. Using the mouse molar tooth as a model for ectodermal organ morphogenesis, we show here that vertical, stratifying cell divisions are enriched in the forming placode and that stratification is cell division dependent. Using inhibitor and gain-of-function experiments, we show that FGF signalling is necessary and sufficient for stratification but not invagination as such. We show that, instead, Shh signalling is necessary for, and promotes, invagination once suprabasal tissue is generated. Shh-dependent suprabasal cell shape suggests convergent migration and intercalation, potentially accounting for post-stratification placode invagination to bud stage. We present a model in which FGF generates suprabasal tissue by asymmetric cell division, while Shh triggers cell rearrangement in this tissue to drive invagination all the way to bud formation. PMID:26755699

Whole-mount Confocal Microscopy for Adult Ear Skin: A Model System to Study Neuro-vascular Branching Morphogenesis and Immune Cell Distribution.

PubMed

Yamazaki, Tomoko; Li, Wenling; Mukouyama, Yoh-Suke

2018-03-29

Here, we present a protocol of a whole-mount adult ear skin imaging technique to study comprehensive three-dimensional neuro-vascular branching morphogenesis and patterning, as well as immune cell distribution at a cellular level. The analysis of peripheral nerve and blood vessel anatomical structures in adult tissues provides some insights into the understanding of functional neuro-vascular wiring and neuro-vascular degeneration in pathological conditions such as wound healing. As a highly informative model system, we have focused our studies on adult ear skin, which is readily accessible for dissection. Our simple and reproducible protocol provides an accurate depiction of the cellular components in the entire skin, such as peripheral nerves (sensory axons, sympathetic axons, and Schwann cells), blood vessels (endothelial cells and vascular smooth muscle cells), and inflammatory cells. We believe this protocol will pave the way to investigate morphological abnormalities in peripheral nerves and blood vessels as well as the inflammation in the adult ear skin under different pathological conditions.

Spatial control of translation repression and polarized growth by conserved NDR kinase Orb6 and RNA-binding protein Sts5.

PubMed

Nuñez, Illyce; Rodriguez Pino, Marbelys; Wiley, David J; Das, Maitreyi E; Chen, Chuan; Goshima, Tetsuya; Kume, Kazunori; Hirata, Dai; Toda, Takashi; Verde, Fulvia

2016-07-30

RNA-binding proteins contribute to the formation of ribonucleoprotein (RNP) granules by phase transition, but regulatory mechanisms are not fully understood. Conserved fission yeast NDR (Nuclear Dbf2-Related) kinase Orb6 governs cell morphogenesis in part by spatially controlling Cdc42 GTPase. Here we describe a novel, independent function for Orb6 kinase in negatively regulating the recruitment of RNA-binding protein Sts5 into RNPs to promote polarized cell growth. We find that Orb6 kinase inhibits Sts5 recruitment into granules, its association with processing (P) bodies, and degradation of Sts5-bound mRNAs by promoting Sts5 interaction with 14-3-3 protein Rad24. Many Sts5-bound mRNAs encode essential factors for polarized cell growth, and Orb6 kinase spatially and temporally controls the extent of Sts5 granule formation. Disruption of this control system affects cell morphology and alters the pattern of polarized cell growth, revealing a role for Orb6 kinase in the spatial control of translational repression that enables normal cell morphogenesis.

Intrinsic properties of limb bud cells can be differentially reset.

PubMed

Saiz-Lopez, Patricia; Chinnaiya, Kavitha; Towers, Matthew; Ros, Maria A

2017-02-01

An intrinsic timing mechanism specifies the positional values of the zeugopod (i.e. radius/ulna) and then autopod (i.e. wrist/digits) segments during limb development. Here, we have addressed whether this timing mechanism ensures that patterning events occur only once by grafting GFP-expressing autopod progenitor cells to the earlier host signalling environment of zeugopod progenitor cells. We show by detecting Hoxa13 expression that early and late autopod progenitors fated for the wrist and phalanges, respectively, both contribute to the entire host autopod, indicating that the autopod positional value is irreversibly determined. We provide evidence that Hoxa13 provides an autopod-specific positional value that correctly allocates cells into the autopod, most likely through the control of cell-surface properties as shown by cell-cell sorting analyses. However, we demonstrate that only the earlier autopod cells can adopt the host proliferation rate to permit normal morphogenesis. Therefore, our findings reveal that the ability of embryonic cells to differentially reset their intrinsic behaviours confers robustness to limb morphogenesis. We speculate that this plasticity could be maintained beyond embryogenesis in limbs with regenerative capacity. © 2017. Published by The Company of Biologists Ltd.

Adenosine kinase modulates root gravitropism and cap morphogenesis in Arabidopsis.

PubMed

Young, Li-Sen; Harrison, Benjamin R; Narayana Murthy, U M; Moffatt, Barbara A; Gilroy, Simon; Masson, Patrick H

2006-10-01

Adenosine kinase (ADK) is a key enzyme that regulates intra- and extracellular levels of adenosine, thereby modulating methyltransferase reactions, production of polyamines and secondary compounds, and cell signaling in animals. Unfortunately, little is known about ADK's contribution to the regulation of plant growth and development. Here, we show that ADK is a modulator of root cap morphogenesis and gravitropism. Upon gravistimulation, soluble ADK levels and activity increase in the root tip. Mutation in one of two Arabidopsis (Arabidopsis thaliana) ADK genes, ADK1, results in cap morphogenesis defects, along with alterations in root sensitivity to gravistimulation and slower kinetics of root gravitropic curvature. The kinetics defect can be partially rescued by adding spermine to the growth medium, whereas the defects in cap morphogenesis and gravitropic sensitivity cannot. The root morphogenesis and gravitropism defects of adk1-1 are accompanied by altered expression of the PIN3 auxin efflux facilitator in the cap and decreased expression of the auxin-responsive DR5-GUS reporter. Furthermore, PIN3 fails to relocalize to the bottom membrane of statocytes upon gravistimulation. Consequently, adk1-1 roots cannot develop a lateral auxin gradient across the cap, necessary for the curvature response. Interestingly, adk1-1 does not affect gravity-induced cytoplasmic alkalinization of the root statocytes, suggesting either that ADK1 functions between cytoplasmic alkalinization and PIN3 relocalization in a linear pathway or that the pH and PIN3-relocalization responses to gravistimulation belong to distinct branches of the pathway. Our data are consistent with a role for ADK and the S-adenosyl-L-methionine pathway in the control of root gravitropism and cap morphogenesis.

Adenosine Kinase Modulates Root Gravitropism and Cap Morphogenesis in Arabidopsis1[W][OA

PubMed Central

Young, Li-Sen; Harrison, Benjamin R.; U.M., Narayana Murthy; Moffatt, Barbara A.; Gilroy, Simon; Masson, Patrick H.

2006-01-01

Adenosine kinase (ADK) is a key enzyme that regulates intra- and extracellular levels of adenosine, thereby modulating methyltransferase reactions, production of polyamines and secondary compounds, and cell signaling in animals. Unfortunately, little is known about ADK's contribution to the regulation of plant growth and development. Here, we show that ADK is a modulator of root cap morphogenesis and gravitropism. Upon gravistimulation, soluble ADK levels and activity increase in the root tip. Mutation in one of two Arabidopsis (Arabidopsis thaliana) ADK genes, ADK1, results in cap morphogenesis defects, along with alterations in root sensitivity to gravistimulation and slower kinetics of root gravitropic curvature. The kinetics defect can be partially rescued by adding spermine to the growth medium, whereas the defects in cap morphogenesis and gravitropic sensitivity cannot. The root morphogenesis and gravitropism defects of adk1-1 are accompanied by altered expression of the PIN3 auxin efflux facilitator in the cap and decreased expression of the auxin-responsive DR5-GUS reporter. Furthermore, PIN3 fails to relocalize to the bottom membrane of statocytes upon gravistimulation. Consequently, adk1-1 roots cannot develop a lateral auxin gradient across the cap, necessary for the curvature response. Interestingly, adk1-1 does not affect gravity-induced cytoplasmic alkalinization of the root statocytes, suggesting either that ADK1 functions between cytoplasmic alkalinization and PIN3 relocalization in a linear pathway or that the pH and PIN3-relocalization responses to gravistimulation belong to distinct branches of the pathway. Our data are consistent with a role for ADK and the S-adenosyl-l-methionine pathway in the control of root gravitropism and cap morphogenesis. PMID:16891550

Role of an expansin-like molecule in Dictyostelium morphogenesis and regulation of its gene expression by the signal transducer and activator of transcription protein Dd-STATa.

PubMed

Ogasawara, Shun; Shimada, Nao; Kawata, Takefumi

2009-02-01

Expansins are proteins involved in plant morphogenesis, exerting their effects on cellulose to extend cell walls. Dictyostelium is an organism that possesses expansin-like molecules, but their functions are not known. In this study, we analyzed the expL7 (expansin-like 7) gene, which has been identified as a putative target of Dd-STATa, a Dictyostelium homolog of the metazoan signal transducer and activator of transcription (STAT) proteins. Promoter fragments of the expL7 were fused to a lacZ reporter and the expression patterns determined. As expected from the behavior of the endogenous expL7 gene, the expL7/lacZ fusion gene was downregulated in Dd-STATa null slugs. In the parental strain, the expL7 promoter was activated in the anterior tip region. Mutational analysis of the promoter identified a sequence that was necessary for expression in tip cells. In addition, an activator sequence for pstAB cells was identified. These sequences act in combination with the repressor region to prevent ectopic expL7 expression in the prespore and prestalk regions of the slug and culminant. Although the expL7 null mutant showed no phenotypic change, the expL7 overexpressor showed aberrant stalk formation. These results indicate that the expansin-like molecule is important for morphogenesis in Dictyostelium.

Identifying the cellular mechanisms of symbiont-induced epithelial morphogenesis in the squid-vibrio association

PubMed Central

Koropatnick, Tanya; Goodson, Michael S.; Heath-Heckman, Elizabeth A. C.; McFall-Ngai, Margaret

2014-01-01

The symbiotic association between the Hawaiian bobtail squid Euprymna scolopes and the luminous marine bacterium Vibrio fischeri provides a unique opportunity to study epithelial morphogenesis. Shortly after hatching, the squid host harvests bacteria from the seawater using currents created by two elaborate fields of ciliated epithelia on the surface of the juvenile light organ. After light organ colonization, the symbiont population signals the gradual loss of the ciliated epithelia through apoptosis of the cells, which culminates in the complete regression of these tissues. Whereas aspects of this process have been studied at the morphological, biochemical and molecular levels, no in-depth analysis of the cellular events has been reported. Here we describe the cellular structure of the epithelial field and present evidence that the symbiosis-induced regression occurs in two steps. Using confocal microscopic analyses, we observed an initial epithelial remodeling, which serves to disable the function of the harvesting apparatus, followed by a protracted regression involving actin rearrangements and epithelial cell extrusion. We identified a metal-dependent gelatinolytic activity in the symbiont-induced morphogenic epithelial fields, suggesting the involvement of Zn-dependent matrix metalloproteinase(s) (MMP) in light organ morphogenesis. These data show that the bacterial symbionts not only induce apoptosis of the field, but also change the form, function and biochemistry of the cells as part of the morphogenic program. PMID:24648207

Epithelial Markers aSMA, Krt14, and Krt19 Unveil Elements of Murine Lacrimal Gland Morphogenesis and Maturation.

PubMed

Kuony, Alison; Michon, Frederic

2017-01-01

As an element of the lacrimal apparatus, the lacrimal gland (LG) produces the aqueous part of the tear film, which protects the eye surface. Therefore, a defective LG can lead to serious eyesight impairment. Up to now, little is known about LG morphogenesis and subsequent maturation. In this study, we delineated elements of the cellular and molecular events involved in LG formation by using three epithelial markers, namely aSMA, Krt14, and Krt19. While aSMA marked a restricted epithelial population of the terminal end buds (TEBs) in the forming LG, Krt14 was found in the whole embryonic LG epithelial basal cell layer. Interestingly, Krt19 specifically labeled the presumptive ductal domain and subsequently, the luminal cell layer. By combining these markers, the Fucci reporter mouse strain and genetic fate mapping of the Krt14 + population, we demonstrated that LG epithelium expansion is fuelled by a patterned cell proliferation, and to a lesser extent by epithelial reorganization and possible mesenchymal-to-epithelial transition. We pointed out that this epithelial reorganization, which is associated with apoptosis, regulated the lumen formation. Finally, we showed that the inhibition of Notch signaling prevented the ductal identity from setting, and led to a LG covered by ectopic TEBs. Taken together our results bring a deeper understanding on LG morphogenesis, epithelial domain identity, and organ expansion.

Epithelial Markers aSMA, Krt14, and Krt19 Unveil Elements of Murine Lacrimal Gland Morphogenesis and Maturation

PubMed Central

Kuony, Alison; Michon, Frederic

2017-01-01

As an element of the lacrimal apparatus, the lacrimal gland (LG) produces the aqueous part of the tear film, which protects the eye surface. Therefore, a defective LG can lead to serious eyesight impairment. Up to now, little is known about LG morphogenesis and subsequent maturation. In this study, we delineated elements of the cellular and molecular events involved in LG formation by using three epithelial markers, namely aSMA, Krt14, and Krt19. While aSMA marked a restricted epithelial population of the terminal end buds (TEBs) in the forming LG, Krt14 was found in the whole embryonic LG epithelial basal cell layer. Interestingly, Krt19 specifically labeled the presumptive ductal domain and subsequently, the luminal cell layer. By combining these markers, the Fucci reporter mouse strain and genetic fate mapping of the Krt14+ population, we demonstrated that LG epithelium expansion is fuelled by a patterned cell proliferation, and to a lesser extent by epithelial reorganization and possible mesenchymal-to-epithelial transition. We pointed out that this epithelial reorganization, which is associated with apoptosis, regulated the lumen formation. Finally, we showed that the inhibition of Notch signaling prevented the ductal identity from setting, and led to a LG covered by ectopic TEBs. Taken together our results bring a deeper understanding on LG morphogenesis, epithelial domain identity, and organ expansion. PMID:29033846

Identifying the cellular mechanisms of symbiont-induced epithelial morphogenesis in the squid-Vibrio association.

PubMed

Koropatnick, Tanya; Goodson, Michael S; Heath-Heckman, Elizabeth A C; McFall-Ngai, Margaret

2014-02-01

The symbiotic association between the Hawaiian bobtail squid Euprymna scolopes and the luminous marine bacterium Vibrio fischeri provides a unique opportunity to study epithelial morphogenesis. Shortly after hatching, the squid host harvests bacteria from the seawater using currents created by two elaborate fields of ciliated epithelia on the surface of the juvenile light organ. After light organ colonization, the symbiont population signals the gradual loss of the ciliated epithelia through apoptosis of the cells, which culminates in the complete regression of these tissues. Whereas aspects of this process have been studied at the morphological, biochemical, and molecular levels, no in-depth analysis of the cellular events has been reported. Here we describe the cellular structure of the epithelial field and present evidence that the symbiosis-induced regression occurs in two steps. Using confocal microscopic analyses, we observed an initial epithelial remodeling, which serves to disable the function of the harvesting apparatus, followed by a protracted regression involving actin rearrangements and epithelial cell extrusion. We identified a metal-dependent gelatinolytic activity in the symbiont-induced morphogenic epithelial fields, suggesting the involvement of Zn-dependent matrix metalloproteinase(s) (MMP) in light organ morphogenesis. These data show that the bacterial symbionts not only induce apoptosis of the field, but also change the form, function, and biochemistry of the cells as part of the morphogenic program.

Bacterial Community Morphogenesis Is Intimately Linked to the Intracellular Redox State

PubMed Central

Okegbe, Chinweike; Price-Whelan, Alexa; Sakhtah, Hassan; Hunter, Ryan C.; Newman, Dianne K.

2013-01-01

Many microbial species form multicellular structures comprising elaborate wrinkles and concentric rings, yet the rules governing their architecture are poorly understood. The opportunistic pathogen Pseudomonas aeruginosa produces phenazines, small molecules that act as alternate electron acceptors to oxygen and nitrate to oxidize the intracellular redox state and that influence biofilm morphogenesis. Here, we show that the depth occupied by cells within colony biofilms correlates well with electron acceptor availability. Perturbations in the environmental provision, endogenous production, and utilization of electron acceptors affect colony development in a manner consistent with redox control. Intracellular NADH levels peak before the induction of colony wrinkling. These results suggest that redox imbalance is a major factor driving the morphogenesis of P. aeruginosa biofilms and that wrinkling itself is an adaptation that maximizes oxygen accessibility and thereby supports metabolic homeostasis. This type of redox-driven morphological change is reminiscent of developmental processes that occur in metazoans. PMID:23292774

Vitamin C treatment promotes mesenchymal stem cell sheet formation and tissue regeneration by elevating telomerase activity.

PubMed

Wei, Fulan; Qu, Cunye; Song, Tieli; Ding, Gang; Fan, Zhipeng; Liu, Dayong; Liu, Yi; Zhang, Chunmei; Shi, Songtao; Wang, Songlin

2012-09-01

Cell sheet engineering has been developed as an alternative approach to improve mesenchymal stem cell-mediated tissue regeneration. In this study, we found that vitamin C (Vc) was capable of inducing telomerase activity in periodontal ligament stem cells (PDLSCs), leading to the up-regulated expression of extracellular matrix type I collagen, fibronectin, and integrin β1, stem cell markers Oct4, Sox2, and Nanog as well as osteogenic markers RUNX2, ALP, OCN. Under Vc treatment, PDLSCs can form cell sheet structures because of increased cell matrix production. Interestingly, PDLSC sheets demonstrated a significant improvement in tissue regeneration compared with untreated control dissociated PDLSCs and offered an effective treatment for periodontal defects in a swine model. In addition, bone marrow mesenchymal stem cell sheets and umbilical cord mesenchymal stem cell sheets were also well constructed using this method. The development of Vc-mediated mesenchymal stem cell sheets may provide an easy and practical approach for cell-based tissue regeneration. Copyright © 2011 Wiley Periodicals, Inc.

Lateral dimension-dependent antibacterial activity of graphene oxide sheets.

PubMed

Liu, Shaobin; Hu, Ming; Zeng, Tingying Helen; Wu, Ran; Jiang, Rongrong; Wei, Jun; Wang, Liang; Kong, Jing; Chen, Yuan

2012-08-21

Graphene oxide (GO) is a promising precursor to produce graphene-family nanomaterials for various applications. Their potential health and environmental impacts need a good understanding of their cellular interactions. Many factors may influence their biological interactions with cells, and the lateral dimension of GO sheets is one of the most relevant material properties. In this study, a model bacterium, Escherichia coli ( E. coli ), was used to evaluate the antibacterial activity of well-dispersed GO sheets, whose lateral size differs by more than 100 times. Our results show that the antibacterial activity of GO sheets toward E. coli cells is lateral size dependent. Larger GO sheets show stronger antibacterial activity than do smaller ones, and they have different time- and concentration-dependent antibacterial activities. Large GO sheets lead to most cell loss after 1 h incubation, and their concentration strongly influences antibacterial activity at relative low concentration (<10 μg/mL). In contrast, when incubating with small GO sheets up to 4 h, the inactivation rate of E. coli cells continues increasing. The increase of small GO sheet concentration also results in persistent increases in their antibacterial activity. In this study, GO sheets with different lateral sizes are all well dispersed, and their oxidation capacity toward glutathione is similar, consistent with X-ray photoelectron spectroscopy and ultraviolet-visible absorption spectroscopy results. This suggests the lateral size-dependent antibacterial activity of GO sheets is caused by neither their aggregation states, nor oxidation capacity. Atomic force microscope analysis of GO sheets and cells shows that GO sheets interact strongly with cells. Large GO sheets more easily cover cells, and cells cannot proliferate once fully covered, resulting in the cell viability loss observed in the followed colony counting test. In contrast, small GO sheets adhere to the bacterial surfaces, which cannot effectively isolate cells from environment. This study highlights the importance of tailoring the lateral dimension of GO sheets to optimize the application potential with minimal risks for environmental health and safety.

Generation of Functional Thyroid Tissue Using 3D-Based Culture of Embryonic Stem Cells.

PubMed

Antonica, Francesco; Kasprzyk, Dominika Figini; Schiavo, Andrea Alex; Romitti, Mírian; Costagliola, Sabine

2017-01-01

During the last decade three-dimensional (3D) cultures of pluripotent stem cells have been intensively used to understand morphogenesis and molecular signaling important for the embryonic development of many tissues. In addition, pluripotent stem cells have been shown to be a valid tool for the in vitro modeling of several congenital or chronic human diseases, opening new possibilities to study their physiopathology without using animal models. Even more interestingly, 3D culture has proved to be a powerful and versatile tool to successfully generate functional tissues ex vivo. Using similar approaches, we here describe a protocol for the generation of functional thyroid tissue using mouse embryonic stem cells and give all the details and references for its characterization and analysis both in vitro and in vivo. This model is a valid approach to study the expression and the function of genes involved in the correct morphogenesis of thyroid gland, to elucidate the mechanisms of production and secretion of thyroid hormones and to test anti-thyroid drugs.

Cell sheet-based tissue engineering for fabricating 3-dimensional heart tissues.

PubMed

Shimizu, Tatsuya

2014-01-01

In addition to stem cell biology, tissue engineering is an essential research field for regenerative medicine. In contrast to cell injection, bioengineered tissue transplantation minimizes cell loss and has the potential to repair tissue defects. A popular approach is scaffold-based tissue engineering, which utilizes a biodegradable polymer scaffold for seeding cells; however, new techniques of cell sheet-based tissue engineering have been developed. Cell sheets are harvested from temperature-responsive culture dishes by simply lowering the temperature. Monolayer or stacked cell sheets are transplantable directly onto damaged tissues and cell sheet transplantation has already been clinically applied. Cardiac cell sheet stacking produces pulsatile heart tissue; however, lack of vasculature limits the viable tissue thickness to 3 layers. Multistep transplantation of triple-layer cardiac cell sheets cocultured with endothelial cells has been used to form thick vascularized cardiac tissue in vivo. Furthermore, in vitro functional blood vessel formation within 3-dimensional (3D) tissues has been realized by successfully imitating in vivo conditions. Triple-layer cardiac cell sheets containing endothelial cells were layered on vascular beds and the constructs were media-perfused using novel bioreactor systems. Interestingly, cocultured endothelial cells migrate into the vascular beds and form perfusable blood vessels. An in vitro multistep procedure has also enabled the fabrication of thick, vascularized heart tissues. Cell sheet-based tissue engineering has revealed great potential to fabricate 3D cardiac tissues and should contribute to future treatment of severe heart diseases and human tissue model production.

Msx-2 expression and glucocorticoid-induced overexpression in embryonic mouse submandibular glands.

PubMed

Jaskoll, T; Luo, W; Snead, M L

1998-01-01

It is well known that the process of branching morphogenesis requires epithelial-mesenchymal interactions. One outstanding model for the study of tissue interactions during branching morphogenesis is the embryonic mouse submandibular gland (SMG). Although it has been clearly demonstrated that the branching pattern is dependent on interactions between the epithelium and the surrounding mesenchyme, little is known about the molecular mechanism underlying the branching process. One group of transcription factors that likely participates in the control of epithelial-mesenchymal inductive interactions are the Msx-class of homeodomain-containing proteins. In this paper, we focus on Msx-2 because its developmental expression is correlated with inductive interactions, suggesting that Msx-2 may play a functional role during cell-cell interactions. We demonstrate the expression of Msx-2 mRNA and protein to be primarily in the branching epithelia with progressive embryonic (E13 to E15) SMG development and, to a lesser extent, in the mesenchyme. We also show that Msx-2 is expressed by embryonic SMG primordia cultured under defined conditions. In addition, to begin to delineate a functional role for Msx-2, we employed an experimental strategy by using exogenous glucocorticoid (CORT) treatment of embryonic SMGs in vitro and in vivo to significantly enhance branching morphogenesis and evaluate the effect of CORT treatment on embryonic SMG Msx-2 expression. A marked increase in Msx-2 transcripts and protein is detected with in vitro and in vivo CORT treatment. Our studies indicate that one mechanism of CORT regulation of salivary gland morphogenesis is likely through the modulation of Msx-2 gene expression.

Regulation of lung branching morphogenesis by bombesin-like peptides and neutral endopeptidase.

PubMed

Aguayo, S M; Schuyler, W E; Murtagh, J J; Roman, J

1994-06-01

The expression of bombesin-like peptides (BLPs) by pulmonary neuroendocrine cells is transiently upregulated during lung development. A functional role for BLPs is supported by their ability to stimulate lung growth and maturation both in vitro and in vivo during the late stages of lung development. In addition, the cell membrane-associated enzyme CD10/neutral endopeptidase 24.11 (CD10/NEP), which inactivates BLPs and other regulatory peptides, is also expressed by developing lungs and modulates the stimulatory effects of BLPs on lung growth and maturation. We hypothesized that, in addition to expressing BLPs and CD10/NEP, embryonic lungs must express BLP receptors, and that BLPs may also regulate processes that occur during early lung development such as branching morphogenesis. Using reverse transcriptase-polymerase chain reaction and oligonucleotide primers designed for amplifying a BLP receptor originally isolated from Swiss 3T3 mouse fibroblasts, we found that embryonic mouse lungs express a similar BLP receptor mRNA during the pseudoglandular stage of lung development when branching morphogenesis take place. Subsequently, we evaluated the effects of ligands for this BLP receptor using embryonic mouse lungs in an in vitro model of lung branching morphogenesis. We found that, in comparison with control lungs, treatment with bombesin (1 to 100 nM) resulted in a modest increase in clefts or branching points. In contrast, embryonic mouse lungs treated with the BLP analog [Leu13-psi(CH2NH)Leu14]bombesin (1 microM), which also binds to this BLP receptor but has predominantly antagonistic effects, demonstrated fewer branching points.(ABSTRACT TRUNCATED AT 250 WORDS)

Rotator cuff repair using cell sheets derived from human rotator cuff in a rat model.

PubMed

Harada, Yoshifumi; Mifune, Yutaka; Inui, Atsuyuki; Sakata, Ryosuke; Muto, Tomoyuki; Takase, Fumiaki; Ueda, Yasuhiro; Kataoka, Takeshi; Kokubu, Takeshi; Kuroda, Ryosuke; Kurosaka, Masahiro

2017-02-01

To achieve biological regeneration of tendon-bone junctions, cell sheets of human rotator-cuff derived cells were used in a rat rotator cuff injury model. Human rotator-cuff derived cells were isolated, and cell sheets were made using temperature-responsive culture plates. Infraspinatus tendons in immunodeficient rats were resected bilaterally at the enthesis. In right shoulders, infraspinatus tendons were repaired by the transosseous method and covered with the cell sheet (sheet group), whereas the left infraspinatus tendons were repaired in the same way without the cell sheet (control group). Histological examinations (safranin-O and fast green staining, isolectin B4, type II collagen, and human-specific CD31) and mRNA expression (vascular endothelial growth factor; VEGF, type II collagen; Col2, and tenomodulin; TeM) were analyzed 4 weeks after surgery. Biomechanical tests were performed at 8 weeks. In the sheet group, proteoglycan at the enthesis with more type II collagen and isolectin B4 positive cells were seen compared with in the control group. Human specific CD31-positive cells were detected only in the sheet group. VEGF and Col2 gene expressions were higher and TeM gene expression was lower in the sheet group than in the control group. In mechanical testing, the sheet group showed a significantly higher ultimate failure load than the control group at 8 weeks. Our results indicated that the rotator-cuff derived cell sheet could promote cartilage regeneration and angiogenesis at the enthesis, with superior mechanical strength compared with the control. Treatment for rotator cuff injury using cell sheets could be a promising strategy for enthesis of tendon tissue engineering. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:289-296, 2017. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

Geometric control of capillary architecture via cell-matrix mechanical interactions.

PubMed

Sun, Jian; Jamilpour, Nima; Wang, Fei-Yue; Wong, Pak Kin

2014-03-01

Capillary morphogenesis is a multistage, multicellular activity that plays a pivotal role in various developmental and pathological situations. In-depth understanding of the regulatory mechanism along with the capability of controlling the morphogenic process will have direct implications on tissue engineering and therapeutic angiogenesis. Extensive research has been devoted to elucidate the biochemical factors that regulate capillary morphogenesis. The roles of geometric confinement and cell-matrix mechanical interactions on the capillary architecture, nevertheless, remain largely unknown. Here, we show geometric control of endothelial network topology by creating physical confinements with microfabricated fences and wells. Decreasing the thickness of the matrix also results in comparable modulation of the network architecture, supporting the boundary effect is mediated mechanically. The regulatory role of cell-matrix mechanical interaction on the network topology is further supported by alternating the matrix stiffness by a cell-inert PEG-dextran hydrogel. Furthermore, reducing the cell traction force with a Rho-associated protein kinase inhibitor diminishes the boundary effect. Computational biomechanical analysis delineates the relationship between geometric confinement and cell-matrix mechanical interaction. Collectively, these results reveal a mechanoregulation scheme of endothelial cells to regulate the capillary network architecture via cell-matrix mechanical interactions. Copyright © 2014 Elsevier Ltd. All rights reserved.

Sequoia, a tramtrack-related zinc finger protein, functions as a pan-neural regulator for dendrite and axon morphogenesis in Drosophila.

PubMed

Brenman, J E; Gao, F B; Jan, L Y; Jan, Y N

2001-11-01

Morphological complexity of neurons contributes to their functional complexity. How neurons generate different dendritic patterns is not known. We identified the sequoia mutant from a previous screen for dendrite mutants. Here we report that Sequoia is a pan-neural nuclear protein containing two putative zinc fingers homologous to the DNA binding domain of Tramtrack. sequoia mutants affect the cell fate decision of a small subset of neurons but have global effects on axon and dendrite morphologies of most and possibly all neurons. In support of sequoia as a specific regulator of neuronal morphogenesis, microarray experiments indicate that sequoia may regulate downstream genes that are important for executing neurite development rather than altering a variety of molecules that specify cell fates.

Msx-1 and Msx-2 in mammary gland development.

PubMed

Satoh, Kennichi; Ginsburg, Erika; Vonderhaar, Barbara K

2004-04-01

Homeobox genes do not generally function alone to determine cell fate and morphogenesis. Rather it is the distinct combination of various members of the homeobox family of genes and their spatiotemporal patterns of expression that determine cell identity and function. Functional redundancy often makes it difficult to clearly discern the role of any one given homeobox gene. The roles that Msx1 and Msx2 play in branching morphogenesis of the mammary gland are only now becoming more evident. Many signaling pathways and transcription factors are implicated in how these homeobox genes correctly determine the morphological development of the gland. Overexpression of Msx1 and Msx2 may also be involved in tumorigenesis. Additional studies are needed to elucidate the roles of these genes in both breast development and cancer.

Partial regeneration of uterine horns in rats through adipose-derived stem cell sheets.

PubMed

Sun, Huijun; Lu, Jie; Li, Bo; Chen, Shuqiang; Xiao, Xifeng; Wang, Jun; Wang, Jingjing; Wang, Xiaohong

2018-06-20

Severe uterine damage and infection lead to intrauterine adhesions, which result in hypomenorrhea, amenorrhea and infertility. Cell sheet engineering has shown great promise in clinical applications. Adipose-derived stem cells (ADSCs) are emerging as an alternative source of stem cells for cell-based therapies. In the present study, we investigated the feasibility of applying ADSCs as seed cells to form scaffold-free cell sheet. Data showed that ADSC sheets expressed higher levels of FGF, Col I, TGFβ and VEGF than ADSCs in suspension, while increased expression of this gene set was associated with stemness, including Nanog, Oct4 and Sox2. We then investigated the therapeutic effects of 3D ADSCs sheet on regeneration in a rat model. We found that ADSCs were mainly detected in the basal layer of the regenerating endometrium in the cell sheet group at 21 days after transplantation. Additionally, some ADSCs differentiated into stromal-like cells. Moreover, ADSC sheets transplanted into partially excised uteri promoted regeneration of the endometrium cells, muscle cells and stimulated angiogenesis, and also resulted in better pregnancy outcomes. Therefore, ADSC sheet therapy shows considerable promise as a new treatment for severe uterine damage.

Culturing bone marrow cells with dexamethasone and ascorbic acid improves osteogenic cell sheet structure.

PubMed

Akahane, M; Shimizu, T; Kira, T; Onishi, T; Uchihara, Y; Imamura, T; Tanaka, Y

2016-11-01

To assess the structure and extracellular matrix molecule expression of osteogenic cell sheets created via culture in medium with both dexamethasone (Dex) and ascorbic acid phosphate (AscP) compared either Dex or AscP alone. Osteogenic cell sheets were prepared by culturing rat bone marrow stromal cells in a minimal essential medium (MEM), MEM with AscP, MEM with Dex, and MEM with Dex and AscP (Dex/AscP). The cell number and messenger (m)RNA expression were assessed in vitro, and the appearance of the cell sheets was observed after mechanical retrieval using a scraper. β-tricalcium phosphate (β-TCP) was then wrapped with the cell sheets from the four different groups and subcutaneously implanted into rats. After mechanical retrieval, the osteogenic cell sheets from the MEM, MEM with AscP, and MEM with Dex groups appeared to be fragmented or incomplete structures. The cell sheets cultured with Dex/AscP remained intact after mechanical retrieval, without any identifiable tears. Culture with Dex/AscP increased the mRNA and protein expression of extracellular matrix proteins and cell number compared with those of the other three groups. More bridging bone formation was observed after transplantation of the β-TCP scaffold wrapped with cell sheets cultured with Dex/AscP, than in the other groups. These results suggest that culture with Dex/AscP improves the mechanical integrity of the osteogenic cell sheets, allowing retrieval of the confluent cells in a single cell sheet structure. This method may be beneficial when applied in cases of difficult tissue reconstruction, such as nonunion, bone defects, and osteonecrosis.Cite this article: M. Akahane, T. Shimizu, T. Kira, T. Onishi, Y. Uchihara, T. Imamura, Y. Tanaka. Culturing bone marrow cells with dexamethasone and ascorbic acid improves osteogenic cell sheet structure. Bone Joint Res 2016;5:569-576. DOI: 10.1302/2046-3758.511.BJR-2016-0013.R1. © 2016 Akahane et al.

The use of platelet-rich fibrin combined with periodontal ligament and jaw bone mesenchymal stem cell sheets for periodontal tissue engineering

PubMed Central

Wang, Zhong-Shan; Feng, Zhi-Hong; Wu, Guo-Feng; Bai, Shi-Zhu; Dong, Yan; Chen, Fa-Ming; Zhao, Yi-Min

2016-01-01

Periodontal regeneration involves the restoration of at least three unique tissues: cementum, periodontal ligament tissue (PDL) and alveolar bone tissue. Here, we first isolated human PDL stem cells (PDLSCs) and jaw bone mesenchymal stem cells (JBMSCs). These cells were then induced to form cell sheets using an ascorbic acid-rich approach, and the cell sheet properties, including morphology, thickness and gene expression profile, were compared. Platelet-rich fibrin (PRF) derived from human venous blood was then fabricated into bioabsorbable fibrin scaffolds containing various growth factors. Finally, the in vivo potential of a cell-material construct based on PDLSC sheets, PRF scaffolds and JBMSC sheets to form periodontal tissue was assessed in a nude mouse model. In this model, PDLSC sheet/PRF/JBMSC sheet composites were placed in a simulated periodontal space comprising human treated dentin matrix (TDM) and hydroxyapatite (HA)/tricalcium phosphate (TCP) frameworks. Eight weeks after implantation, the PDLSC sheets tended to develop into PDL-like tissues, while the JBMSC sheets tended to produce predominantly bone-like tissues. In addition, the PDLSC sheet/PRF/JBMSC sheet composites generated periodontal tissue-like structures containing PDL- and bone-like tissues. Further improvements in this cell transplantation design may have the potential to provide an effective approach for future periodontal tissue regeneration. PMID:27324079

Activation of PPARbeta/delta induces endothelial cell proliferation and angiogenesis.

PubMed

Piqueras, Laura; Reynolds, Andrew R; Hodivala-Dilke, Kairbaan M; Alfranca, Arántzazu; Redondo, Juan M; Hatae, Toshihisa; Tanabe, Tadashi; Warner, Timothy D; Bishop-Bailey, David

2007-01-01

The role of the nuclear receptor peroxisome-proliferator activated receptor (PPAR)-beta/delta in endothelial cells remains unclear. Interestingly, the selective PPARbeta/delta ligand GW501516 is in phase II clinical trials for dyslipidemia. Here, using GW501516, we have assessed the involvement of PPARbeta/delta in endothelial cell proliferation and angiogenesis. Western blot analysis indicated PPARbeta/delta was expressed in primary human umbilical and aortic endothelial cells, and in the endothelial cell line, EAHy926. Treatment with GW501516 increased human endothelial cell proliferation and morphogenesis in cultures in vitro, endothelial cell outgrowth from murine aortic vessels in vitro, and angiogenesis in a murine matrigel plug assay in vivo. GW501516 induced vascular endothelial cell growth factor mRNA and peptide release, as well as adipose differentiation-related protein (ADRP), a PPARbeta/delta target gene. GW501516-induced proliferation, morphogenesis, vascular endothelial growth factor (VEGF), and ADRP were absent in endothelial cells transfected with dominant-negative PPARbeta/delta. Furthermore, treatment of cells with cyclo-VEGFI, a VEGF receptor1/2 antagonist, abolished GW501516-induced endothelial cell proliferation and tube formation. PPARbeta/delta is a novel regulator of endothelial cell proliferation and angiogenesis through VEGF. The use of GW501516 to treat dyslipidemia may need to be carefully monitored in patients susceptible to angiogenic disorders.

MEMBRANOUS LABYRINTH IN BACULOVIRUS-INFECTED CRUSTRACEAN CELLS: POSSIBLE ROLES IN VIRAL REPRODUCTION

EPA Science Inventory

The origins and morphogenesis of the membranous labyrinth (ML) in Baculovirus penaei (BP) infected cells of penaeid shrimps (Crustacea:Decapoda) are described. t is hypothesized that, because of the close parallel and concurrent development of the ML and virus reproduction, and o...

A Mathematical Model to study the Dynamics of Epithelial Cellular Networks

PubMed Central

Abate, Alessandro; Vincent, Stéphane; Dobbe, Roel; Silletti, Alberto; Master, Neal; Axelrod, Jeffrey D.; Tomlin, Claire J.

2013-01-01

Epithelia are sheets of connected cells that are essential across the animal kingdom. Experimental observations suggest that the dynamical behavior of many single-layered epithelial tissues has strong analogies with that of specific mechanical systems, namely large networks consisting of point masses connected through spring-damper elements and undergoing the influence of active and dissipating forces. Based on this analogy, this work develops a modeling framework to enable the study of the mechanical properties and of the dynamic behavior of large epithelial cellular networks. The model is built first by creating a network topology that is extracted from the actual cellular geometry as obtained from experiments, then by associating a mechanical structure and dynamics to the network via spring-damper elements. This scalable approach enables running simulations of large network dynamics: the derived modeling framework in particular is predisposed to be tailored to study general dynamics (for example, morphogenesis) of various classes of single-layered epithelial cellular networks. In this contribution we test the model on a case study of the dorsal epithelium of the Drosophila melanogaster embryo during early dorsal closure (and, less conspicuously, germband retraction). PMID:23221083

Development of a Cell Sheet Transportation Technique for Regenerative Medicine

PubMed Central

Oie, Yoshinori; Nozaki, Takayuki; Takayanagi, Hiroshi; Hara, Susumu; Hayashi, Ryuhei; Takeda, Shizu; Mori, Keisuke; Moriya, Noboru; Soma, Takeshi; Tsujikawa, Motokazu; Saito, Kazuo

2014-01-01

Purpose: A transportation technique for cell sheets is necessary to standardize regenerative medicine. The aim of this article is to develop and evaluate a new transportation technique for cell sheets. Material and Methods: We developed a transportation container with three basic functions: the maintenance of interior temperature, air pressure, and sterility. The interior temperature and air pressure were monitored by a recorder. Human oral mucosal epithelial cells obtained from two healthy volunteers were cultured on temperature-responsive culture dishes. The epithelial cell sheets were transported via an airplane between the Osaka University and Tohoku University using the developed cell transportation container. Histological and immunohistochemical analyses and flow cytometric analyses for cell viability and cell purity were performed for the cell sheets before and 12 h after transportation to assess the influence of transportation on the cell sheets. Sterility tests and screening for endotoxin and mycoplasma in the cell sheets were performed before and after transportation. Results: During transportation via an airplane, the temperature inside the container was maintained above 32°C, and the changes in air pressure remained within 10 hPa. The cell sheets were well stratified and successfully harvested before and after transportation. The expression patterns of keratin 3/76, p63, and MUC16 were equivalent before and after transportation. However, the expression of ZO-1 in the cell sheet after transportation was slightly weaker than that before transportation. The cell viability was 72.0% before transportation and 77.3% after transportation. The epithelial purity was 94.6% before transportation and 87.9% after transportation. Sterility tests and screening for endotoxin and mycoplasma were negative for all cell sheets. Conclusion: The newly developed transportation technique for air travel is essential technology for regenerative medicine and promotes the standardization and spread of regenerative therapies. PMID:24044382

Development of a cell sheet transportation technique for regenerative medicine.

PubMed

Oie, Yoshinori; Nozaki, Takayuki; Takayanagi, Hiroshi; Hara, Susumu; Hayashi, Ryuhei; Takeda, Shizu; Mori, Keisuke; Moriya, Noboru; Soma, Takeshi; Tsujikawa, Motokazu; Saito, Kazuo; Nishida, Kohji

2014-05-01

A transportation technique for cell sheets is necessary to standardize regenerative medicine. The aim of this article is to develop and evaluate a new transportation technique for cell sheets. We developed a transportation container with three basic functions: the maintenance of interior temperature, air pressure, and sterility. The interior temperature and air pressure were monitored by a recorder. Human oral mucosal epithelial cells obtained from two healthy volunteers were cultured on temperature-responsive culture dishes. The epithelial cell sheets were transported via an airplane between the Osaka University and Tohoku University using the developed cell transportation container. Histological and immunohistochemical analyses and flow cytometric analyses for cell viability and cell purity were performed for the cell sheets before and 12 h after transportation to assess the influence of transportation on the cell sheets. Sterility tests and screening for endotoxin and mycoplasma in the cell sheets were performed before and after transportation. During transportation via an airplane, the temperature inside the container was maintained above 32°C, and the changes in air pressure remained within 10 hPa. The cell sheets were well stratified and successfully harvested before and after transportation. The expression patterns of keratin 3/76, p63, and MUC16 were equivalent before and after transportation. However, the expression of ZO-1 in the cell sheet after transportation was slightly weaker than that before transportation. The cell viability was 72.0% before transportation and 77.3% after transportation. The epithelial purity was 94.6% before transportation and 87.9% after transportation. Sterility tests and screening for endotoxin and mycoplasma were negative for all cell sheets. The newly developed transportation technique for air travel is essential technology for regenerative medicine and promotes the standardization and spread of regenerative therapies.

Elimination of remaining undifferentiated induced pluripotent stem cells in the process of human cardiac cell sheet fabrication using a methionine-free culture condition.

PubMed

Matsuura, Katsuhisa; Kodama, Fumiko; Sugiyama, Kasumi; Shimizu, Tatsuya; Hagiwara, Nobuhisa; Okano, Teruo

2015-03-01

Cardiac tissue engineering is a promising method for regenerative medicine. Although we have developed human cardiac cell sheets by integration of cell sheet-based tissue engineering and scalable bioreactor culture, the risk of contamination by induced pluripotent stem (iPS) cells in cardiac cell sheets remains unresolved. In the present study, we established a novel culture method to fabricate human cardiac cell sheets with a decreased risk of iPS cell contamination while maintaining viabilities of iPS cell-derived cells, including cardiomyocytes and fibroblasts, using a methionine-free culture condition. When cultured in the methionine-free condition, human iPS cells did not survive without feeder cells and could not proliferate or form colonies on feeder cells or in coculture with cells for cardiac cell sheet fabrication. When iPS cell-derived cells after the cardiac differentiation were transiently cultured in the methionine-free condition, gene expression of OCT3/4 and NANOG was downregulated significantly compared with that in the standard culture condition. Furthermore, in fabricated cardiac cell sheets, spontaneous and synchronous beating was observed in the whole area while maintaining or upregulating the expression of various cardiac and extracellular matrix genes. These findings suggest that human iPS cells are methionine dependent and a methionine-free culture condition for cardiac cell sheet fabrication might reduce the risk of iPS cell contamination.

Chondrocyte Differentiation of Human Endometrial Gland-Derived MSCs in Layered Cell Sheets

PubMed Central

Shimizu, Tatsuya; Yamato, Masayuki; Umezawa, Akihiro; Okano, Teruo

2013-01-01

Recently, regenerative medicine using engineered three-dimensional (3D) tissues has been focused. In the fields of cell therapy and regenerative medicine, mesenchymal stem cells (MSCs) are attractive autologous cell sources. While, in bioengineered tissues, a 3D environment may affect the differentiation of the stem cells, little is known regarding the effect of 3D environment on cellular differentiation. In this study, MSC differentiation in in vitro 3D tissue models was assessed by human endometrial gland-derived MSCs (hEMSCs) and cell sheet technology. hEMSC sheets were layered into cell-dense 3D tissues and were cultured on porous membranes. The tissue sections revealed that chondrocyte-like cells were found within the multilayered cell sheets even at 24 h after layering. Immunostainings of chondrospecific markers were positive within those cell sheet constructs. In addition, sulfated glycosaminoglycan accumulation within the tissues increased in proportion to the numbers of layered cell sheets. The findings suggested that a high cell density and hypoxic environment in 3D tissues by layering cell sheets might accelerate a rapid differentiation of hEMSCs into chondrocytes without the help of chondro-differentiation reagents. These tissue models using cell sheets would give new insights to stem cell differentiation in 3D environment and contribute to the future application of stem cells to cartilage regenerative therapy. PMID:24348153

Fabrication method for cores of structural sandwich materials including star shaped core cells

DOEpatents

Christensen, Richard M.

1997-01-01

A method for fabricating structural sandwich materials having a core pattern which utilizes star and non-star shaped cells. The sheets of material are bonded together or a single folded sheet is used, and bonded or welded at specific locations, into a flat configuration, and are then mechanically pulled or expanded normal to the plane of the sheets which expand to form the cells. This method can be utilized to fabricate other geometric cell arrangements than the star/non-star shaped cells. Four sheets of material (either a pair of bonded sheets or a single folded sheet) are bonded so as to define an area therebetween, which forms the star shaped cell when expanded.

Effect of Levonorgestrel (NORPLANT) on the Immune Regulation of Bone Morphogenesis in Calvarial Cultures from the Laboratory Mouse (Mus muscularis).

DTIC Science & Technology

1995-10-01

continually releases a synthetic progestin, levonorgestrel , for five years. In order to assess the impact of levonorgestrel on bone cells, murine calvarial...cell cultures were harvested, grown to confluence and treated with levonorgestrel , progesterone and estrogen. The majority of the cells grown in these

The microRNA-200 family coordinately regulates cell adhesion and proliferation in hair morphogenesis.

PubMed

Hoefert, Jaimee E; Bjerke, Glen A; Wang, Dongmei; Yi, Rui

2018-06-04

The microRNA (miRNA)-200 (miR-200) family is highly expressed in epithelial cells and frequently lost in metastatic cancer. Despite intensive studies into their roles in cancer, their targets and functions in normal epithelial tissues remain unclear. Importantly, it remains unclear how the two subfamilies of the five-miRNA family, distinguished by a single nucleotide within the seed region, regulate their targets. By directly ligating miRNAs to their targeted mRNA regions, we identify numerous miR-200 targets involved in the regulation of focal adhesion, actin cytoskeleton, cell cycle, and Hippo/Yap signaling. The two subfamilies bind to largely distinct target sites, but many genes are coordinately regulated by both subfamilies. Using inducible and knockout mouse models, we show that the miR-200 family regulates cell adhesion and orientation in the hair germ, contributing to precise cell fate specification and hair morphogenesis. Our findings demonstrate that combinatorial targeting of many genes is critical for miRNA function and provide new insights into miR-200's functions. © 2018 Hoefert et al.

Actin cables and the exocyst form two independent morphogenesis pathways in the fission yeast

PubMed Central

Bendezú, Felipe O.; Martin, Sophie G.

2011-01-01

Cell morphogenesis depends on polarized exocytosis. One widely held model posits that long-range transport and exocyst-dependent tethering of exocytic vesicles at the plasma membrane sequentially drive this process. Here, we describe that disruption of either actin-based long-range transport and microtubules or the exocyst did not abolish polarized growth in rod-shaped fission yeast cells. However, disruption of both actin cables and exocyst led to isotropic growth. Exocytic vesicles localized to cell tips in single mutants but were dispersed in double mutants. In contrast, a marker for active Cdc42, a major polarity landmark, localized to discreet cortical sites even in double mutants. Localization and photobleaching studies show that the exocyst subunits Sec6 and Sec8 localize to cell tips largely independently of the actin cytoskeleton, but in a cdc42 and phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2)–dependent manner. Thus in fission yeast long-range cytoskeletal transport and PIP2-dependent exocyst represent parallel morphogenetic modules downstream of Cdc42, raising the possibility of similar mechanisms in other cell types. PMID:21148300

MpWIP regulates air pore complex development in the liverwort Marchantia polymorpha

PubMed Central

Jones, Victor A. S.

2017-01-01

The colonisation of the land by plants was accompanied by the evolution of complex tissues and multicellular structures comprising different cell types as morphological adaptations to the terrestrial environment. Here, we show that the single WIP protein in the early-diverging land plant Marchantia polymorpha L. is required for the development of the multicellular gas exchange structure: the air pore complex. This 16-cell barrel-shaped structure surrounds an opening between epidermal cells that facilitates the exchange of gases between the chamber containing the photosynthetic cells inside the plant and the air outside. MpWIP is expressed in cells of the developing air pore complex and the morphogenesis of the complex is defective in plants with reduced MpWIP function. The role of WIP proteins in the control of different multicellular structures in M. polymorpha and the flowering plant Arabidopsis thaliana suggests that these proteins controlled the development of multicellular structures in the common ancestor of land plants. We hypothesise that WIP genes were subsequently co-opted in the control of morphogenesis of novel multicellular structures that evolved during the diversification of land plants. PMID:28174248

Combinatorial effect of substratum properties on mesenchymal stem cell sheet engineering and subsequent multi-lineage differentiation.

PubMed

Chuah, Yon Jin; Zhang, Ying; Wu, Yingnan; Menon, Nishanth V; Goh, Ghim Hian; Lee, Ann Charlene; Chan, Vincent; Zhang, Yilei; Kang, Yuejun

2015-09-01

Cell sheet engineering has been exploited as an alternative approach in tissue regeneration and the use of stem cells to generate cell sheets has further showed its potential in stem cell-mediated tissue regeneration. There exist vast interests in developing strategies to enhance the formation of stem cell sheets for downstream applications. It has been proved that stem cells are sensitive to the biophysical cues of the microenvironment. Therefore we hypothesized that the combinatorial substratum properties could be tailored to modulate the development of cell sheet formation and further influence its multipotency. For validation, polydimethylsiloxane (PDMS) of different combinatorial substratum properties (including stiffness, roughness and wettability) were created, on which the human bone marrow derived mesenchymal stem cells (BMSCs) were cultured to form cell sheets with their multipotency evaluated after induced differentiation. The results showed that different combinatorial effects of these substratum properties were able to influence BMSC behavior such as adhesion, spreading and proliferation during cell sheet development. Collagen formation within the cell sheet was enhanced on substrates with lower stiffness, higher hydrophobicity and roughness, which further assisted the induced chondrogenesis and osteogenesis, respectively. These findings suggested that combinatorial substratum properties had profound effects on BMSC cell sheet integrity and multipotency, which had significant implications for future biomaterials and scaffold designs in the field of BMSC-mediated tissue regeneration. Copyright © 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

3D tissue formation by stacking detachable cell sheets formed on nanofiber mesh.

PubMed

Kim, Min Sung; Lee, Byungjun; Kim, Hong Nam; Bang, Seokyoung; Yang, Hee Seok; Kang, Seong Min; Suh, Kahp-Yang; Park, Suk-Hee; Jeon, Noo Li

2017-03-23

We present a novel approach for assembling 3D tissue by layer-by-layer stacking of cell sheets formed on aligned nanofiber mesh. A rigid frame was used to repeatedly collect aligned electrospun PCL (polycaprolactone) nanofiber to form a mesh structure with average distance between fibers 6.4 µm. When human umbilical vein endothelial cells (HUVECs), human foreskin dermal fibroblasts, and skeletal muscle cells (C2C12) were cultured on the nanofiber mesh, they formed confluent monolayers and could be handled as continuous cell sheets with areas 3 × 3 cm 2 or larger. Thicker 3D tissues have been formed by stacking multiple cell sheets collected on frames that can be nested (i.e. Matryoshka dolls) without any special tools. When cultured on the nanofiber mesh, skeletal muscle, C2C12 cells oriented along the direction of the nanofibers and differentiated into uniaxially aligned multinucleated myotube. Myotube cell sheets were stacked (upto 3 layers) in alternating or aligned directions to form thicker tissue with ∼50 µm thickness. Sandwiching HUVEC cell sheets with two dermal fibroblast cell sheets resulted in vascularized 3D tissue. HUVECs formed extensive networks and expressed CD31, a marker of endothelial cells. Cell sheets formed on nanofiber mesh have a number of advantages, including manipulation and stacking of multiple cell sheets for constructing 3D tissue and may find applications in a variety of tissue engineering applications.

Ecdysone signaling induces two phases of cell cycle exit in Drosophila cells

PubMed Central

Guo, Yongfeng; Flegel, Kerry; Kumar, Jayashree; McKay, Daniel J.

2016-01-01

ABSTRACT During development, cell proliferation and differentiation must be tightly coordinated to ensure proper tissue morphogenesis. Because steroid hormones are central regulators of developmental timing, understanding the links between steroid hormone signaling and cell proliferation is crucial to understanding the molecular basis of morphogenesis. Here we examined the mechanism by which the steroid hormone ecdysone regulates the cell cycle in Drosophila. We find that a cell cycle arrest induced by ecdysone in Drosophila cell culture is analogous to a G2 cell cycle arrest observed in the early pupa wing. We show that in the wing, ecdysone signaling at the larva-to-puparium transition induces Broad which in turn represses the cdc25c phosphatase String. The repression of String generates a temporary G2 arrest that synchronizes the cell cycle in the wing epithelium during early pupa wing elongation and flattening. As ecdysone levels decline after the larva-to-puparium pulse during early metamorphosis, Broad expression plummets, allowing String to become re-activated, which promotes rapid G2/M progression and a subsequent synchronized final cell cycle in the wing. In this manner, pulses of ecdysone can both synchronize the final cell cycle and promote the coordinated acquisition of terminal differentiation characteristics in the wing. PMID:27737823

Transcription Factors Runx1 to 3 Are Expressed in the Lacrimal Gland Epithelium and Are Involved in Regulation of Gland Morphogenesis and Regeneration

PubMed Central

Voronov, Dmitry; Gromova, Anastasia; Liu, Daren; Zoukhri, Driss; Medvinsky, Alexander; Meech, Robyn; Makarenkova, Helen P.

2013-01-01

Purpose. Lacrimal gland (LG) morphogenesis and repair are regulated by a complex interplay of intrinsic factors (e.g., transcription factors) and extrinsic signals (e.g., soluble growth/signaling factors). Many of these interconnections remain poorly characterized. Runt-related (Runx) factors belong to a small family of heterodimeric transcription factors known to regulate lineage-specific proliferation and differentiation of stem cells. The purpose of this study was to define the expression pattern and the role of Runx proteins in LG development and regeneration. Methods. Expression of epithelial-restricted transcription factors in murine LG was examined using immunostaining, qRT-PCR, and RT2Profiler PCR microarrays. The role of Runx transcription factors in LG morphogenesis was studied using siRNA and ex vivo LG cultures. Expression of Runx transcription factors during LG regeneration was assessed using in vivo model of LG regeneration. Results. We found that Runx factors are expressed in the epithelial compartment of the LG; in particular, Runx1 was restricted to the epithelium with highest level of expression in ductal and centroacinar cells. Downregulation of Runx1 to 3 expression using Runx-specific siRNAs abolished LG growth and branching and our data suggest that Runx1, 2, and 3 are partially redundant in LG development. In siRNA-treated LG, reduction of branching correlated with reduction of epithelial proliferation, as well as expression of cyclin D1 and the putative epithelial progenitor cell marker cytokeratin-5. Runx1, Runx3, and cytokeratin-5 expression increased significantly in regenerating LG and there was modest increase in Runx2 expression during LG differentiation. Conclusions. Runx1 and 2 are new markers of the LG epithelial lineage and Runx factors are important for normal LG morphogenesis and regeneration. PMID:23532528

Transfer of fibroblast sheets cultured on thermoresponsive dishes with membranes.

PubMed

Kawecki, Marek; Kraut, Małgorzata; Klama-Baryła, Agnieszka; Łabuś, Wojciech; Kitala, Diana; Nowak, Mariusz; Glik, Justyna; Sieroń, Aleksander L; Utrata-Wesołek, Alicja; Trzebicka, Barbara; Dworak, Andrzej; Szweda, Dawid

2016-06-01

In cell or tissue engineering, it is essential to develop a support for cell-to-cell adhesion, which leads to the generation of cell sheets connected by extracellular matrix. Such supports must be hydrophobic and should result in a detachable cell sheet. A thermoresponsive support that enables the cultured cell sheet to detach using only a change in temperature could be an interesting alternative in regenerative medicine. The aim of this study was to evaluate plates covered with thermoresponsive polymers as supports for the formation of fibroblast sheets and to develop a damage-free procedure for cell sheet transfer with the use of membranes as transfer tools. Human skin fibroblasts were seeded on supports coated with a thermoresponsive polymer: commercial UpCell™ dishes (NUNC™) coated with thermoresponsive poly(N-isopropylacrylamide) (PNIPAM) and dishes coated with thermoresponsive poly(tri(ethylene glycol) monoethyl ether methacrylate) (P(TEGMA-EE)). Confluent fibroblast sheets were effectively cultured and harvested from both commercial PNIPAM-coated dishes and laboratory P(TEGMA-EE)-coated dishes. To transfer a detached cell sheet, two membranes, Immobilon-P(®) and SUPRATHEL(®), were examined. The use of SUPRATHEL for relocating the cell sheets opens a new possibility for the clinical treatment of wounds. This study established the background for implementing thermoresponsive supports for transplanting in vitro cultured fibroblasts.

Cartilage engineering using chondrocyte cell sheets and its application in reconstruction of microtia.

PubMed

Zhou, Libin; Ding, Ruiying; Li, Baowei; Han, Haolun; Wang, Hongnan; Wang, Gang; Xu, Bingxin; Zhai, Suoqiang; Wu, Wei

2015-01-01

The imperfections of scaffold materials have hindered the clinical application of cartilage tissue engineering. The recently developed cell-sheet technique is adopted to engineer tissues without scaffold materials, thus is considered being potentially able to overcome the problems concerning the scaffold imperfections. This study constructed monolayer and bilayer chondrocyte cell sheets and harvested the sheets with cell scraper instead of temperature-responsive culture dishes. The properties of the cultured chondrocyte cell sheets and the feasibility of cartilage engineering using the chondrocyte cell sheets was further investigated via in vitro and in vivo study. Primary extracellular matrix (ECM) formation and type II collagen expression was detected in the cell sheets during in vitro culture. After implanted into nude mice for 8 weeks, mature cartilage discs were harvested. The morphology of newly formed cartilage was similar in the constructs originated from monolayer and bilayer chondrocyte cell sheet. The chondrocytes were located within evenly distributed ovoid lacunae. Robust ECM formation and intense expression of type II collagen was observed surrounding the evenly distributed chondrocytes in the neocartilages. Biochemical analysis showed that the DNA contents of the neocartilages were higher than native human costal cartilage; while the contents of the main component of ECM, glycosaminoglycan and hydroxyproline, were similar to native human costal cartilage. In conclusion, the chondrocyte cell sheet constructed using the simple and low-cost technique is basically the same with the cell sheet cultured and harvested in temperature-responsive culture dishes, and can be used for cartilage tissue engineering.

Rsr1 Focuses Cdc42 Activity at Hyphal Tips and Promotes Maintenance of Hyphal Development in Candida albicans

PubMed Central

Pulver, Rebecca; Heisel, Timothy; Gonia, Sara; Robins, Robert; Norton, Jennifer; Haynes, Paula

2013-01-01

The extremely elongated morphology of fungal hyphae is dependent on the cell's ability to assemble and maintain polarized growth machinery over multiple cell cycles. The different morphologies of the fungus Candida albicans make it an excellent model organism in which to study the spatiotemporal requirements for constitutive polarized growth and the generation of different cell shapes. In C. albicans, deletion of the landmark protein Rsr1 causes defects in morphogenesis that are not predicted from study of the orthologous protein in the related yeast Saccharomyces cerevisiae, thus suggesting that Rsr1 has expanded functions during polarized growth in C. albicans. Here, we show that Rsr1 activity localizes to hyphal tips by the differential localization of the Rsr1 GTPase-activating protein (GAP), Bud2, and guanine nucleotide exchange factor (GEF), Bud5. In addition, we find that Rsr1 is needed to maintain the focused localization of hyphal polarity structures and proteins, including Bem1, a marker of the active GTP-bound form of the Rho GTPase, Cdc42. Further, our results indicate that tip-localized Cdc42 clusters are associated with the cell's ability to express a hyphal transcriptional program and that the ability to generate a focused Cdc42 cluster in early hyphae (germ tubes) is needed to maintain hyphal morphogenesis over time. We propose that in C. albicans, Rsr1 “fine-tunes” the distribution of Cdc42 activity and that self-organizing (Rsr1-independent) mechanisms of polarized growth are not sufficient to generate narrow cell shapes or to provide feedback to the transcriptional program during hyphal morphogenesis. PMID:23223038

Rho-associated kinase activity is required for proper morphogenesis of the inner cell mass in the mouse blastocyst.

PubMed

Laeno, Arlene May A; Tamashiro, Dana Ann A; Alarcon, Vernadeth B

2013-11-01

The blastocyst consists of the outer layer of trophectoderm and pluripotent inner cell mass (ICM), the precursor of the placenta and fetus, respectively. During blastocyst expansion, the ICM adopts a compact, ovoidal shape, whose proper morphology is crucial for normal embryogenesis. Rho-associated kinase (ROCK), an effector of small GTPase RHO signaling, mediates the diverse cellular processes of morphogenesis, but its role in ICM morphogenesis is unclear. Here, we demonstrate that ROCK is required for cohesion of ICM cells and formation of segregated tissues called primitive endoderm (PrE) and epiblast (Epi) in the ICM of the mouse blastocyst. Blastocyst treatment with ROCK inhibitors Y-27632 and Fasudil caused widening or spreading of the ICM, and intermingling of PrE and Epi. Widening of ICM was independent of trophectoderm because isolated ICMs as well as colonies of mouse embryonic stem cells (mESC) also spread upon Y-27632 treatment. PrE, Epi, and trophectoderm cell numbers were similar between control and treated blastocysts, suggesting that ROCK inhibition affected ICM morphology but not lineage differentiation. Rock1 and Rock2 knockdown via RNA interference in mESC also induced spreading, supporting the conclusion that morphological defects caused by the pharmacological inhibitors were due to ROCK inactivation. When blastocysts were transferred into surrogates, implantation efficiencies were unaffected by ROCK inhibition, but treated blastocysts yielded greater fetal loss. These results show that proper ICM morphology is dependent on ROCK activity and is crucial for fetal development. Our studies have wider implication for improving efficiencies of human assisted reproductive technologies that diminish pregnancy loss and promote successful births.

Fungal Morphogenesis, from the Polarized Growth of Hyphae to Complex Reproduction and Infection Structures.

PubMed

Riquelme, Meritxell; Aguirre, Jesús; Bartnicki-García, Salomon; Braus, Gerhard H; Feldbrügge, Michael; Fleig, Ursula; Hansberg, Wilhelm; Herrera-Estrella, Alfredo; Kämper, Jörg; Kück, Ulrich; Mouriño-Pérez, Rosa R; Takeshita, Norio; Fischer, Reinhard

2018-06-01

Filamentous fungi constitute a large group of eukaryotic microorganisms that grow by forming simple tube-like hyphae that are capable of differentiating into more-complex morphological structures and distinct cell types. Hyphae form filamentous networks by extending at their tips while branching in subapical regions. Rapid tip elongation requires massive membrane insertion and extension of the rigid chitin-containing cell wall. This process is sustained by a continuous flow of secretory vesicles that depends on the coordinated action of the microtubule and actin cytoskeletons and the corresponding motors and associated proteins. Vesicles transport cell wall-synthesizing enzymes and accumulate in a special structure, the Spitzenkörper, before traveling further and fusing with the tip membrane. The place of vesicle fusion and growth direction are enabled and defined by the position of the Spitzenkörper, the so-called cell end markers, and other proteins involved in the exocytic process. Also important for tip extension is membrane recycling by endocytosis via early endosomes, which function as multipurpose transport vehicles for mRNA, septins, ribosomes, and peroxisomes. Cell integrity, hyphal branching, and morphogenesis are all processes that are largely dependent on vesicle and cytoskeleton dynamics. When hyphae differentiate structures for asexual or sexual reproduction or to mediate interspecies interactions, the hyphal basic cellular machinery may be reprogrammed through the synthesis of new proteins and/or the modification of protein activity. Although some transcriptional networks involved in such reprogramming of hyphae are well studied in several model filamentous fungi, clear connections between these networks and known determinants of hyphal morphogenesis are yet to be established. Copyright © 2018 American Society for Microbiology.

Concise review: can the intrinsic power of branching morphogenesis be used for engineering epithelial tissues and organs?

PubMed

Nigam, Sanjay K

2013-12-01

Branching morphogenesis is critical to the development of organs such as kidney, lung, mammary gland, prostate, pancreas, and salivary gland. Essentially, an epithelial bud becomes an iterative tip-stalk generator (ITSG) able to form a tree of branching ducts and/or tubules. In different organs, branching morphogenesis is governed by similar sets of genes. Epithelial branching has been recapitulated in vitro (or ex vivo) using three-dimensional cell culture and partial organ culture systems, and several such systems relevant to kidney tissue engineering are discussed here. By adapting systems like these it may be possible to harness the power inherent in the ITSG program to propagate and engineer epithelial tissues and organs. It is also possible to conceive of a universal ITSG capable of propagation that may, by recombination with organ-specific mesenchymal cells, be used for engineering many organ-like tissues similar to the organ from which the mesenchyme cells were derived, or toward which they are differentiated (from stem cells). The three-dimensional (3D) branched epithelial structure could act as a dynamic branching cellular scaffold to establish the architecture for the rest of the tissue. Another strategy-that of recombining propagated organ-specific ITSGs in 3D culture with undifferentiated mesenchymal stem cells-is also worth exploring. If feasible, such engineered tissues may be useful for the ex vivo study of drug toxicity, developmental biology, and physiology in the laboratory. Over the long term, they have potential clinical applications in the general fields of transplantation, regenerative medicine, and bioartificial medical devices to aid in the treatment of chronic kidney disease, diabetes, and other diseases.

8-Oxoguanine DNA glycosylase 1 (ogg1) maintains the function of cardiac progenitor cells during heart formation in zebrafish

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

Yan, Lifeng; Key Laboratory of Modern Toxicology of Ministry of Education, School of Public Health, Nanjing Medical University, Nanjing 210029; Zhou, Yong

Genomic damage may devastate the potential of progenitor cells and consequently impair early organogenesis. We found that ogg1, a key enzyme initiating the base-excision repair, was enriched in the embryonic heart in zebrafish. So far, little is known about DNA repair in cardiogenesis. Here, we addressed the critical role of ogg1 in cardiogenesis for the first time. ogg1 mainly expressed in the anterior lateral plate mesoderm (ALPM), the primary heart tube, and subsequently the embryonic myocardium by in situ hybridisation. Loss of ogg1 resulted in severe cardiac morphogenesis and functional abnormalities, including the short heart length, arrhythmia, decreased cardiomyocytes andmore » nkx2.5{sup +} cardiac progenitor cells. Moreover, the increased apoptosis and repressed proliferation of progenitor cells caused by ogg1 deficiency might contribute to the heart phenotype. The microarray analysis showed that the expression of genes involved in embryonic heart tube morphogenesis and heart structure were significantly changed due to the lack of ogg1. Among those, foxh1 is an important partner of ogg1 in the cardiac development in response to DNA damage. Our work demonstrates the requirement of ogg1 in cardiac progenitors and heart development in zebrafish. These findings may be helpful for understanding the aetiology of congenital cardiac deficits. - Highlights: • A key DNA repair enzyme ogg1 is expressed in the embryonic heart in zebrafish. • We found that ogg1 is essential for normal cardiac morphogenesis in zebrafish. • The production of embryonic cardiomyocytes requires appropriate ogg1 expression. • Ogg1 critically regulated proliferation of cardiac progenitor cells in zebrafish. • foxh1 is a partner of ogg1 in the cardiac development in response to DNA damage.« less

The Cotton Kinesin-Like Calmodulin-Binding Protein Associates with Cortical Microtubles in Cotton Fibers

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

Preuss, Mary L.; Delmar, Deborah P.; Liu, Bo

Microtubules in interphase plant cells form a cortical array, which is critical for plant cell morphogenesis. Genetic studies imply that the minus end-directed microtubule motor kinesin-like calmodulin-binding protein (KCBP) plays a role in trichome morphogenesis in Arabidopsis. However, it was not clear whether this motor interacted with interphase microtubules. In cotton (Gossypium hirsutum) fibers, cortical microtubules undergo dramatic reorganization during fiber development. In this study, cDNA clones of the cotton KCBP homolog GhKCBP were isolated from a cotton fiber-specific cDNA library. During cotton fiber development from 10 to 21 DPA, the GhKCBP protein level gradually decreases. By immunofluorescence, GhKCBP wasmore » detected as puncta along cortical microtubules in fiber cells of different developmental stages. Thus the results provide evidence that GhKCBP plays a role in interphase cell growth likely by interacting with cortical microtubules. In contrast to fibers, in dividing cells of cotton, GhKCBP localized to the nucleus, the microtubule preprophase band, mitotic spindle, and the phragmoplast. Therefore KCBP likely exerts multiple roles in cell division and cell growth in flowering plants.« less

On the genetic control of planar growth during tissue morphogenesis in plants.

PubMed

Enugutti, Balaji; Kirchhelle, Charlotte; Schneitz, Kay

2013-06-01

Tissue morphogenesis requires extensive intercellular communication. Plant organs are composites of distinct radial cell layers. A typical layer, such as the epidermis, is propagated by stereotypic anticlinal cell divisions. It is presently unclear what mechanisms coordinate cell divisions relative to the plane of a layer, resulting in planar growth and maintenance of the layer structure. Failure in the regulation of coordinated growth across a tissue may result in spatially restricted abnormal growth and the formation of a tumor-like protrusion. Therefore, one way to approach planar growth control is to look for genetic mutants that exhibit localized tumor-like outgrowths. Interestingly, plants appear to have evolved quite robust genetic mechanisms that govern these aspects of tissue morphogenesis. Here we provide a short summary of the current knowledge about the genetics of tumor formation in plants and relate it to the known control of coordinated cell behavior within a tissue layer. We further portray the integuments of Arabidopsis thaliana as an excellent model system to study the regulation of planar growth. The value of examining this process in integuments was established by the recent identification of the Arabidopsis AGC VIII kinase UNICORN as a novel growth suppressor involved in the regulation of planar growth and the inhibition of localized ectopic growth in integuments and other floral organs. An emerging insight is that misregulation of central determinants of adaxial-abaxial tissue polarity can lead to the formation of spatially restricted multicellular outgrowths in several tissues. Thus, there may exist a link between the mechanisms regulating adaxial-abaxial tissue polarity and planar growth in plants.

An Engineered Organoid Culture Model That Incorporates Human Mesenchymal and Epithelial Cells to Model Palatal Fusion.

EPA Science Inventory

During embryonic development, fusion events are critical to morphogenesis of organs and tissues, including the iris, urethra, heart, neural tube, and secondary palate. Modeling this process in vitro is challenging as the interactions of mesenchymal and epithelial cells can be cr...

Fabrication method for cores of structural sandwich materials including star shaped core cells

DOEpatents

Christensen, R.M.

1997-07-15

A method for fabricating structural sandwich materials having a core pattern which utilizes star and non-star shaped cells is disclosed. The sheets of material are bonded together or a single folded sheet is used, and bonded or welded at specific locations, into a flat configuration, and are then mechanically pulled or expanded normal to the plane of the sheets which expand to form the cells. This method can be utilized to fabricate other geometric cell arrangements than the star/non-star shaped cells. Four sheets of material (either a pair of bonded sheets or a single folded sheet) are bonded so as to define an area therebetween, which forms the star shaped cell when expanded. 3 figs.

Cell wall matrix polysaccharide distribution and cortical microtubule organization: two factors controlling mesophyll cell morphogenesis in land plants.

PubMed

Sotiriou, P; Giannoutsou, E; Panteris, E; Apostolakos, P; Galatis, B

2016-03-01

This work investigates the involvement of local differentiation of cell wall matrix polysaccharides and the role of microtubules in the morphogenesis of mesophyll cells (MCs) of three types (lobed, branched and palisade) in the dicotyledon Vigna sinensis and the fern Asplenium nidus. Homogalacturonan (HGA) epitopes recognized by the 2F4, JIM5 and JIM7 antibodies and callose were immunolocalized in hand-made leaf sections. Callose was also stained with aniline blue. We studied microtubule organization by tubulin immunofluorescence and transmission electron microscopy. In both plants, the matrix cell wall polysaccharide distribution underwent definite changes during MC differentiation. Callose constantly defined the sites of MC contacts. The 2F4 HGA epitope in V. sinensis first appeared in MC contacts but gradually moved towards the cell wall regions facing the intercellular spaces, while in A. nidus it was initially localized at the cell walls delimiting the intercellular spaces, but finally shifted to MC contacts. In V. sinensis, the JIM5 and JIM7 HGA epitopes initially marked the cell walls delimiting the intercellular spaces and gradually shifted in MC contacts, while in A. nidus they constantly enriched MC contacts. In all MC types examined, the cortical microtubules played a crucial role in their morphogenesis. In particular, in palisade MCs, cortical microtubule helices, by controlling cellulose microfibril orientation, forced these MCs to acquire a truncated cone-like shape. Unexpectedly in V. sinensis, the differentiation of colchicine-affected MCs deviated completely, since they developed a cell wall ingrowth labyrinth, becoming transfer-like cells. The results of this work and previous studies on Zea mays (Giannoutsou et al., Annals of Botany 2013; 112: : 1067-1081) revealed highly controlled local cell wall matrix differentiation in MCs of species belonging to different plant groups. This, in coordination with microtubule-dependent cellulose microfibril alignment, spatially controlled cell wall expansion, allowing MCs to acquire their particular shape. © The Author 2016. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Human platelet lysate supports the formation of robust human periodontal ligament cell sheets.

PubMed

Tian, Bei-Min; Wu, Rui-Xin; Bi, Chun-Sheng; He, Xiao-Tao; Yin, Yuan; Chen, Fa-Ming

2018-04-01

The use of stem cell-derived sheets has become increasingly common in a wide variety of biomedical applications. Although substantial evidence has demonstrated that human platelet lysate (PL) can be used for therapeutic cell expansion, either as a substitute for or as a supplement to xenogeneic fetal bovine serum (FBS), its impact on cell sheet production remains largely unexplored. In this study, we manufactured periodontal ligament stem cell (PDLSC) sheets in vitro by incubating PDLSCs in sheet-induction media supplemented with various ratios of PL and FBS, i.e. 10% PL without FBS, 7.5% PL + 2.5% FBS, 5% PL + 5% FBS, 2.5% PL + 7.5% FBS or 10% FBS without PL. Cultures with the addition of all the designed supplements led to successful cell sheet production. In addition, all the resultant cellular materials exhibited similar expression profiles of matrix-related genes and proteins, such as collagen I, fibronectin and integrin β1. Interestingly, the cell components within sheets generated by media containing both PL and FBS exhibited improved osteogenic potential. Following in vivo transplantation, all sheets supported significant new bone formation. Our data suggest that robust PDLSC sheets can be produced by applying PL as either an alternative or an adjuvant to FBS. Further examination of the relevant influences of human PL that benefit cell behaviour and matrix production will pave the way towards optimized and standardized conditions for cell sheet production. Copyright © 2017 John Wiley & Sons, Ltd.

Infused polymers for cell sheet release

NASA Astrophysics Data System (ADS)

Juthani, Nidhi; Howell, Caitlin; Ledoux, Haylea; Sotiri, Irini; Kelso, Susan; Kovalenko, Yevgen; Tajik, Amanda; Vu, Thy L.; Lin, Jennifer J.; Sutton, Amy; Aizenberg, Joanna

2016-05-01

Tissue engineering using whole, intact cell sheets has shown promise in many cell-based therapies. However, current systems for the growth and release of these sheets can be expensive to purchase or difficult to fabricate, hindering their widespread use. Here, we describe a new approach to cell sheet release surfaces based on silicone oil-infused polydimethylsiloxane. By coating the surfaces with a layer of fibronectin (FN), we were able to grow mesenchymal stem cells to densities comparable to those of tissue culture polystyrene controls (TCPS). Simple introduction of oil underneath an edge of the sheet caused it to separate from the substrate. Characterization of sheets post-transfer showed that they retain their FN layer and morphology, remain highly viable, and are able to grow and proliferate normally after transfer. We expect that this method of cell sheet growth and detachment may be useful for low-cost, flexible, and customizable production of cellular layers for tissue engineering.

Infused polymers for cell sheet release

PubMed Central

Juthani, Nidhi; Howell, Caitlin; Ledoux, Haylea; Sotiri, Irini; Kelso, Susan; Kovalenko, Yevgen; Tajik, Amanda; Vu, Thy L.; Lin, Jennifer J.; Sutton, Amy; Aizenberg, Joanna

2016-01-01

Tissue engineering using whole, intact cell sheets has shown promise in many cell-based therapies. However, current systems for the growth and release of these sheets can be expensive to purchase or difficult to fabricate, hindering their widespread use. Here, we describe a new approach to cell sheet release surfaces based on silicone oil-infused polydimethylsiloxane. By coating the surfaces with a layer of fibronectin (FN), we were able to grow mesenchymal stem cells to densities comparable to those of tissue culture polystyrene controls (TCPS). Simple introduction of oil underneath an edge of the sheet caused it to separate from the substrate. Characterization of sheets post-transfer showed that they retain their FN layer and morphology, remain highly viable, and are able to grow and proliferate normally after transfer. We expect that this method of cell sheet growth and detachment may be useful for low-cost, flexible, and customizable production of cellular layers for tissue engineering. PMID:27189419

Infused polymers for cell sheet release.

PubMed

Juthani, Nidhi; Howell, Caitlin; Ledoux, Haylea; Sotiri, Irini; Kelso, Susan; Kovalenko, Yevgen; Tajik, Amanda; Vu, Thy L; Lin, Jennifer J; Sutton, Amy; Aizenberg, Joanna

2016-05-18

Tissue engineering using whole, intact cell sheets has shown promise in many cell-based therapies. However, current systems for the growth and release of these sheets can be expensive to purchase or difficult to fabricate, hindering their widespread use. Here, we describe a new approach to cell sheet release surfaces based on silicone oil-infused polydimethylsiloxane. By coating the surfaces with a layer of fibronectin (FN), we were able to grow mesenchymal stem cells to densities comparable to those of tissue culture polystyrene controls (TCPS). Simple introduction of oil underneath an edge of the sheet caused it to separate from the substrate. Characterization of sheets post-transfer showed that they retain their FN layer and morphology, remain highly viable, and are able to grow and proliferate normally after transfer. We expect that this method of cell sheet growth and detachment may be useful for low-cost, flexible, and customizable production of cellular layers for tissue engineering.

Rapid fabrication of detachable three-dimensional tissues by layering of cell sheets with heating centrifuge.

PubMed

Haraguchi, Yuji; Kagawa, Yuki; Hasegawa, Akiyuki; Kubo, Hirotsugu; Shimizu, Tatsuya

2018-01-18

Confluent cultured cells on a temperature-responsive culture dish can be harvested as an intact cell sheet by decreasing temperature below 32°C. A three-dimensional (3-D) tissue can be fabricated by the layering of cell sheets. A resulting 3-D multilayered cell sheet-tissue on a temperature-responsive culture dish can be also harvested without any damage by only temperature decreasing. For shortening the fabrication time of the 3-D multilayered constructs, we attempted to layer cell sheets on a temperature-responsive culture dish with centrifugation. However, when a cell sheet was attached to the culture surface with a conventional centrifuge at 22-23°C, the cell sheet hardly adhere to the surface due to its noncell adhesiveness. Therefore, in this study, we have developed a heating centrifuge. In centrifugation (55g) at 36-37°C, the cell sheet adhered tightly within 5 min to the dish without significant cell damage. Additionally, centrifugation accelerated the cell sheet-layering process. The heating centrifugation shortened the fabrication time by one-fifth compared to a multilayer tissue fabrication without centrifugation. Furthermore, the multilayered constructs were finally detached from the dishes by decreasing temperature. This rapid tissue-fabrication method will be used as a valuable tool in the field of tissue engineering and regenerative therapy. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 2018. © 2018 American Institute of Chemical Engineers.

Rotation is the primary motion of paired human epidermal keratinocytes.

PubMed

Tate, Sota; Imai, Matome; Matsushita, Natsuki; Nishimura, Emi K; Higashiyama, Shigeki; Nanba, Daisuke

2015-09-01

Collective motion of keratinocytes is involved in morphogenesis, homeostasis, and wound healing of the epidermis. Yet how the collective motion of keratinocytes emerges from the behavior of individual cells is still largely unknown. The aim of this study was to find the cellular behavior that links single and collective motion of keratinocytes. We investigated the behavior of two-cell colonies of HaCaT keratinocytes by a combination of time-lapse imaging and image processing. The two-cell colonies of HaCaT cells were formed as a contacted pair of keratinocyte clones. Image analysis and cell culture experiments revealed that the rotational speed of two-cell colonies was positively associated with their proliferative capacity. α6 integrin was required for the rotational motion of two-cell keratinocyte colonies. We also confirmed that two-cell colonies of keratinocytes predominantly exhibited the rotational, but not translational, motion, two modes of motion in a contact pair of rotating objects. The rotational motion is the primary motion of two-cell keratinocyte colonies and its speed is positively associated with their proliferative capacity. This study suggests that the assembly of rotating keratinocytes generates the collective motion of proliferative keratinocytes during morphogenesis and wound healing of the epidermis. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

ROP GTPase-mediated auxin signaling regulates pavement cell interdigitation in Arabidopsis thaliana.

PubMed

Lin, Deshu; Ren, Huibo; Fu, Ying

2015-01-01

In multicellular plant organs, cell shape formation depends on molecular switches to transduce developmental or environmental signals and to coordinate cell-to-cell communication. Plants have a specific subfamily of the Rho GTPase family, usually called Rho of Plants (ROP), which serve as a critical signal transducer involved in many cellular processes. In the last decade, important advances in the ROP-mediated regulation of plant cell morphogenesis have been made by using Arabidopsis thaliana leaf and cotyledon pavement cells. Especially, the auxin-ROP signaling networks have been demonstrated to control interdigitated growth of pavement cells to form jigsaw-puzzle shapes. Here, we review findings related to the discovery of this novel auxin-signaling mechanism at the cell surface. This signaling pathway is to a large extent independent of the well-known Transport Inhibitor Response (TIR)-Auxin Signaling F-Box (AFB) pathway, and instead requires Auxin Binding Protein 1 (ABP1) interaction with the plasma membrane-localized, transmembrane kinase (TMK) receptor-like kinase to regulate ROP proteins. Once activated, ROP influences cytoskeletal organization and inhibits endocytosis of the auxin transporter PIN1. The present review focuses on ROP signaling and its self-organizing feature allowing ROP proteins to serve as a bustling signal decoder and integrator for plant cell morphogenesis. © 2014 Institute of Botany, Chinese Academy of Sciences.

Virtual Tissue Models in Developmental Toxicity Research

EPA Science Inventory

Prenatal exposure to drugs and chemicals may perturb, directly or indirectly, core developmental processes in the embryo (patterning, morphogenesis, proliferation and apoptosis, and cell differentiation), leading to adverse developmental outcomes. Because embryogenesis entails a...

Development and characterization of hybrid tubular structure of PLCL porous scaffold with hMSCs/ECs cell sheet.

PubMed

Pangesty, Azizah Intan; Arahira, Takaaki; Todo, Mitsugu

2017-09-15

Tissue engineering offers an alternate approach to providing vascular graft with potential to grow similar with native tissue by seeding autologous cells into biodegradable scaffold. In this study, we developed a combining technique by layering a sheet of cells onto a porous tubular scaffold. The cell sheet prepared from co-culturing human mesenchymal stem cells (hMSCs) and endothelial cells (ECs) were able to infiltrate through porous structure of the tubular poly (lactide-co-caprolactone) (PLCL) scaffold and further proliferated on luminal wall within a week of culture. Moreover, the co-culture cell sheet within the tubular scaffold has demonstrated a faster proliferation rate than the monoculture cell sheet composed of MSCs only. We also found that the co-culture cell sheet expressed a strong angiogenic marker, including vascular endothelial growth factor (VEGF) and its receptor (VEGFR), as compared with the monoculture cell sheet within 2 weeks of culture, indicating that the co-culture system could induce differentiation into endothelial cell lineage. This combined technique would provide cellularization and maturation of vascular construct in relatively short period with a strong expression of angiogenic properties.

Spatially Assembled Bilayer Cell Sheets of Stem Cells and Endothelial Cells Using Thermosensitive Hydrogels for Therapeutic Angiogenesis.

PubMed

Jun, Indong; Ahmad, Taufiq; Bak, Seongwoo; Lee, Joong-Yup; Kim, Eun Mi; Lee, Jinkyu; Lee, Yu Bin; Jeong, Hongsoo; Jeon, Hojeong; Shin, Heungsoo

2017-05-01

Although the coculture of multiple cell types has been widely employed in regenerative medicine, in vivo transplantation of cocultured cells while maintaining the hierarchical structure remains challenging. Here, a spatially assembled bilayer cell sheet of human mesenchymal stem cells and human umbilical vein endothelial cells on a thermally expandable hydrogel containing fibronectin is prepared and its effect on in vitro proangiogenic functions and in vivo ischemic injury is investigated. The expansion of hydrogels in response to a temperature change from 37 to 4 °C allows rapid harvest and delivery of the bilayer cell sheet to two different targets (an in vitro model glass surface and in vivo tissue). The in vitro study confirms that the bilayer sheet significantly increases proangiogenic functions such as the release of nitric oxide and expression of vascular endothelial cell genes. In addition, transplantation of the cell sheet from the hydrogels into a hindlimb ischemia mice model demonstrates significant retardation of necrosis particularly in the group transplated with the bilayer sheet. Collectively, the bilayer cell sheet is readily transferrable from the thermally expandable hydrogel and represents an alternative approach for recovery from ischemic injury, potentially via improved cell-cell communication. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Plant development. Arabidopsis NAC45/86 direct sieve element morphogenesis culminating in enucleation.

PubMed

Furuta, Kaori Miyashima; Yadav, Shri Ram; Lehesranta, Satu; Belevich, Ilya; Miyashima, Shunsuke; Heo, Jung-ok; Vatén, Anne; Lindgren, Ove; De Rybel, Bert; Van Isterdael, Gert; Somervuo, Panu; Lichtenberger, Raffael; Rocha, Raquel; Thitamadee, Siripong; Tähtiharju, Sari; Auvinen, Petri; Beeckman, Tom; Jokitalo, Eija; Helariutta, Ykä

2014-08-22

Photoassimilates such as sugars are transported through phloem sieve element cells in plants. Adapted for effective transport, sieve elements develop as enucleated living cells. We used electron microscope imaging and three-dimensional reconstruction to follow sieve element morphogenesis in Arabidopsis. We show that sieve element differentiation involves enucleation, in which the nuclear contents are released and degraded in the cytoplasm at the same time as other organelles are rearranged and the cytosol is degraded. These cellular reorganizations are orchestrated by the genetically redundant NAC domain-containing transcription factors, NAC45 and NAC86 (NAC45/86). Among the NAC45/86 targets, we identified a family of genes required for enucleation that encode proteins with nuclease domains. Thus, sieve elements differentiate through a specialized autolysis mechanism. Copyright © 2014, American Association for the Advancement of Science.

Dynamin 2 regulation of integrin endocytosis, but not VEGF signaling, is crucial for developmental angiogenesis

PubMed Central

Lee, Monica Y.; Skoura, Athanasia; Park, Eon Joo; Landskroner-Eiger, Shira; Jozsef, Levente; Luciano, Amelia K.; Murata, Takahisa; Pasula, Satish; Dong, Yunzhou; Bouaouina, Mohamed; Calderwood, David A.; Ferguson, Shawn M.; De Camilli, Pietro; Sessa, William C.

2014-01-01

Here we show that dynamin 2 (Dnm2) is essential for angiogenesis in vitro and in vivo. In cultured endothelial cells lacking Dnm2, vascular endothelial growth factor (VEGF) signaling and receptor levels are augmented whereas cell migration and morphogenesis are impaired. Mechanistically, the loss of Dnm2 increases focal adhesion size and the surface levels of multiple integrins and reduces the activation state of β1 integrin. In vivo, the constitutive or inducible loss of Dnm2 in endothelium impairs branching morphogenesis and promotes the accumulation of β1 integrin at sites of failed angiogenic sprouting. Collectively, our data show that Dnm2 uncouples VEGF signaling from function and coordinates the endocytic turnover of integrins in a manner that is crucially important for angiogenesis in vitro and in vivo. PMID:24598168

Using In Vivo and Tissue and Cell Explant Approaches to Study the Morphogenesis and Pathogenesis of the Embryonic and Perinatal Aorta.

PubMed

Misra, Ashish; Feng, Zhonghui; Zhang, Jiasheng; Lou, Zhi-Yin; Greif, Daniel M

2017-09-12

The aorta is the largest artery in the body. The aortic wall is composed of an inner layer of endothelial cells, a middle layer of alternating elastic lamellae and smooth muscle cells (SMCs), and an outer layer of fibroblasts and extracellular matrix. In contrast to the widespread study of pathological models (e.g., atherosclerosis) in the adult aorta, much less is known about the embryonic and perinatal aorta. Here, we focus on SMCs and provide protocols for the analysis of the morphogenesis and pathogenesis of embryonic and perinatal aortic SMCs in normal development and disease. Specifically, the four protocols included are: i) in vivo embryonic fate mapping and clonal analysis; ii) explant embryonic aorta culture; iii) SMC isolation from the perinatal aorta; and iv) subcutaneous osmotic mini-pump placement in pregnant (or non-pregnant) mice. Thus, these approaches facilitate the investigation of the origin(s), fate, and clonal architecture of SMCs in the aorta in vivo. They allow for modulating embryonic aorta morphogenesis in utero by continuous exposure to pharmacological agents. In addition, isolated aortic tissue explants or aortic SMCs can be used to gain insights into the role of specific gene targets during fundamental processes such as muscularization, proliferation, and migration. These hypothesis-generating experiments on isolated SMCs and the explanted aorta can then be assessed in the in vivo context through pharmacological and genetic approaches.

ΔNp63 is an ectodermal gatekeeper of epidermal morphogenesis

PubMed Central

Shalom-Feuerstein, R; Lena, A M; Zhou, H; De La Forest Divonne, S; Van Bokhoven, H; Candi, E; Melino, G; Aberdam, D

2011-01-01

p63, a member of p53 family, has a significant role in the development and maintenance of stratified epithelia. However, a persistent dispute remained over the last decade concerning the interpretation of the severe failure of p63-null embryos to develop stratified epithelia. In this study, by investigating both p63-deficient strains, we demonstrated that p63-deficient epithelia failed to develop beyond ectodermal stage as they remained a monolayer of non-proliferating cells expressing K8/K18. Importantly, in the absence of p63, corneal-epithelial commitment (which occurs at embryonic day 12.5 of mouse embryogenesis) was hampered 3 weeks before corneal stem cell renewal (that begins at P14). Taken together, these data illustrate the significant role of p63 in epithelial embryogenesis, before and independently of other functions of p63 in adult stem cells regulation. Transcriptome analysis of laser captured-embryonic tissues confirmed the latter hypothesis, demonstrating that a battery of epidermal genes that were activated in wild-type epidermis remained silent in p63-null tissues. Furthermore, we defined a subset of novel bona fide p63-induced genes orchestrating first epidermal stratification and a subset of p63-repressed mesodermal-specific genes. These data highlight the earliest recognized action of ΔNp63 in the induction epidermal morphogenesis at E11.5. In the absence of p63, a mesodermal program is activated while epidermal morphogenesis does not initiate. PMID:21127502

Virus-induced gene silencing unravels multiple transcription factors involved in floral growth and development in Phalaenopsis orchids.

PubMed

Hsieh, Ming-Hsien; Pan, Zhao-Jun; Lai, Pei-Han; Lu, Hsiang-Chia; Yeh, Hsin-Hung; Hsu, Chia-Chi; Wu, Wan-Lin; Chung, Mei-Chu; Wang, Shyh-Shyan; Chen, Wen-Huei; Chen, Hong-Hwa

2013-09-01

Orchidaceae, one of the largest angiosperm families, has significant commercial value. Isolation of genes involved in orchid floral development and morphogenesis, scent production, and colouration will advance knowledge of orchid flower formation and facilitate breeding new varieties to increase the commercial value. With high-throughput virus-induced gene silencing (VIGS), this study identified five transcription factors involved in various aspects of flower morphogenesis in the orchid Phalaenopsis equestris. These genes are PeMADS1, PeMADS7, PeHB, PebHLH, and PeZIP. Silencing PeMADS1 and PebHLH resulted in reduced flower size together with a pelaloid column containing petal-like epidermal cells and alterations of epidermal cell arrangement in lip lateral lobes, respectively. Silencing PeMADS7, PeHB, and PeZIP alone resulted in abortion of the first three fully developed flower buds of an inflorescence, which indicates the roles of the genes in late flower development. Furthermore, double silencing PeMADS1 and PeMADS6, C- and B-class MADS-box genes, respectively, produced a combinatorial phenotype with two genes cloned in separate vectors. Both PeMADS1 and PeMADS6 are required to ensure the normal development of the lip and column as well as the cuticle formation on the floral epidermal cell surface. Thus, VIGS allows for unravelling the interaction between two classes of MADS transcription factors for dictating orchid floral morphogenesis.

Virus-induced gene silencing unravels multiple transcription factors involved in floral growth and development in Phalaenopsis orchids

PubMed Central

Hsieh, Ming-Hsien; Pan, Zhao-Jun; Lai, Pei-Han; Lu, Hsiang-Chia; Yeh, Hsin-Hung; Hsu, Chia-Chi; Wu, Wan-Lin; Chung, Mei-Chu; Wang, Shyh-Shyan; Chen, Wen-Huei; Chen, Hong-Hwa

2013-01-01

Orchidaceae, one of the largest angiosperm families, has significant commercial value. Isolation of genes involved in orchid floral development and morphogenesis, scent production, and colouration will advance knowledge of orchid flower formation and facilitate breeding new varieties to increase the commercial value. With high-throughput virus-induced gene silencing (VIGS), this study identified five transcription factors involved in various aspects of flower morphogenesis in the orchid Phalaenopsis equestris. These genes are PeMADS1, PeMADS7, PeHB, PebHLH, and PeZIP. Silencing PeMADS1 and PebHLH resulted in reduced flower size together with a pelaloid column containing petal-like epidermal cells and alterations of epidermal cell arrangement in lip lateral lobes, respectively. Silencing PeMADS7, PeHB, and PeZIP alone resulted in abortion of the first three fully developed flower buds of an inflorescence, which indicates the roles of the genes in late flower development. Furthermore, double silencing PeMADS1 and PeMADS6, C- and B-class MADS-box genes, respectively, produced a combinatorial phenotype with two genes cloned in separate vectors. Both PeMADS1 and PeMADS6 are required to ensure the normal development of the lip and column as well as the cuticle formation on the floral epidermal cell surface. Thus, VIGS allows for unravelling the interaction between two classes of MADS transcription factors for dictating orchid floral morphogenesis. PMID:23956416

Nonlinear Chemical Dynamics and Synchronization

NASA Astrophysics Data System (ADS)

Li, Ning

Alan Turing's work on morphogenesis, more than half a century ago, continues to motivate and inspire theoretical and experimental biologists even today. That said, there are very few experimental systems for which Turing's theory is applicable. In this thesis we present an experimental reaction-diffusion system ideally suited for testing Turing's ideas in synthetic "cells" consisting of microfluidically produced surfactant-stabilized emulsions in which droplets containing the Belousov-Zhabotinsky (BZ) oscillatory chemical reactants are dispersed in oil. The BZ reaction has become the prototype of nonlinear dynamics in chemistry and a preferred system for exploring the behavior of coupled nonlinear oscillators. Our system consists of a surfactant stabilized monodisperse emulsion of drops of aqueous BZ solution dispersed in a continuous phase of oil. In contrast to biology, here the chemistry is understood, rate constants are measured and interdrop coupling is purely diffusive. We explore a large set of parameters through control of rate constants, drop size, spacing, and spatial arrangement of the drops in lines and rings in one-dimension (1D) and hexagonal arrays in two-dimensions (2D). The Turing model is regarded as a metaphor for morphogenesis in biology but not for prediction. Here, we develop a quantitative and falsifiable reaction-diffusion model that we experimentally test with synthetic cells. We quantitatively establish the extent to which the Turing model in 1D describes both stationary pattern formation and temporal synchronization of chemical oscillators via reaction-diffusion and in 2D demonstrate that chemical morphogenesis drives physical differentiation in synthetic cells.

Whole-Mount Adult Ear Skin Imaging Reveals Defective Neuro-Vascular Branching Morphogenesis in Obese and Type 2 Diabetic Mouse Models.

PubMed

Yamazaki, Tomoko; Li, Wenling; Yang, Ling; Li, Ping; Cao, Haiming; Motegi, Sei-Ichiro; Udey, Mark C; Bernhard, Elise; Nakamura, Takahisa; Mukouyama, Yoh-Suke

2018-01-11

Obesity and type 2 diabetes are frequently associated with peripheral neuropathy. Though there are multiple methods for diagnosis and analysis of morphological changes of peripheral nerves and blood vessels, three-dimensional high-resolution imaging is necessary to appreciate the pathogenesis with an anatomically recognizable branching morphogenesis and patterning. Here we established a novel technique for whole-mount imaging of adult mouse ear skin to visualize branching morphogenesis and patterning of peripheral nerves and blood vessels. Whole-mount immunostaining of adult mouse ear skin showed that peripheral sensory and sympathetic nerves align with large-diameter blood vessels. Diet-induced obesity (DIO) mice exhibit defective vascular smooth muscle cells (VSMCs) coverage, while there is no significant change in the amount of peripheral nerves. The leptin receptor-deficient db/db mice, a severe obese and type 2 diabetic mouse model, exhibit defective VSMC coverage and a large increase in the amount of smaller-diameter nerve bundles with myelin sheath and unmyelinated nerve fibers. Interestingly, an increase in the amount of myeloid immune cells was observed in the DIO but not db/db mouse skin. These data suggest that our whole-mount imaging method enables us to investigate the neuro-vascular and neuro-immune phenotypes in the animal models of obesity and diabetes.

Gonad morphogenesis defects drive hybrid male sterility in asymmetric hybrid breakdown of Caenorhabditis nematodes

PubMed Central

Dey, Alivia; Jin, Qi; Chen, Yen-Chu; Cutter, Asher D.

2014-01-01

Determining the causes and evolution of reproductive barriers to gene flow between populations, speciation, is the key to understanding the origin of diversity in nature. Many species manifest hybrid breakdown when they intercross, characterized by increasingly exacerbated problems in later generations of hybrids. Recently, Caenorhabditis nematodes have emerged as a genetic model for studying speciation, and here we investigate the nature and causes of hybrid breakdown between C. remanei and C. latens. We quantify partial F1 hybrid inviability and extensive F2 hybrid inviability; the ~75% F2 embryonic arrest occurs primarily during gastrulation or embryonic elongation. Moreover, F1 hybrid males exhibit Haldane’s rule asymmetrically for both sterility and inviability, being strongest when C. remanei serves as maternal parent. We show that the mechanism by which sterile hybrid males are incapable of transferring sperm or a copulatory plug involves defective gonad morphogenesis, which we hypothesize results from linker cell defects in migration and/or cell death during development. This first documented case of partial hybrid male sterility in Caenorhabditis follows expectations of Darwin’s corollary to Haldane’s rule for asymmetric male fitness, providing a powerful foundation for molecular dissection of intrinsic reproductive barriers and divergence of genetic pathways controlling organ morphogenesis. PMID:25196892

Gonad morphogenesis defects drive hybrid male sterility in asymmetric hybrid breakdown of Caenorhabditis nematodes.

PubMed

Dey, Alivia; Jin, Qi; Chen, Yen-Chu; Cutter, Asher D

2014-01-01

Determining the causes and evolution of reproductive barriers to gene flow between populations, speciation, is the key to understanding the origin of diversity in nature. Many species manifest hybrid breakdown when they intercross, characterized by increasingly exacerbated problems in later generations of hybrids. Recently, Caenorhabditis nematodes have emerged as a genetic model for studying speciation, and here we investigate the nature and causes of hybrid breakdown between Caenorhabditis remanei and C. latens. We quantify partial F1 hybrid inviability and extensive F2 hybrid inviability; the ~75% F2 embryonic arrest occurs primarily during gastrulation or embryonic elongation. Moreover, F1 hybrid males exhibit Haldane's rule asymmetrically for both sterility and inviability, being strongest when C. remanei serves as maternal parent. We show that the mechanism by which sterile hybrid males are incapable of transferring sperm or a copulatory plug involves defective gonad morphogenesis, which we hypothesize results from linker cell defects in migration and/or cell death during development. This first documented case of partial hybrid male sterility in Caenorhabditis follows expectations of Darwin's corollary to Haldane's rule for asymmetric male fitness, providing a powerful foundation for molecular dissection of intrinsic reproductive barriers and divergence of genetic pathways controlling organ morphogenesis. © 2014 Wiley Periodicals, Inc.

Mesodermal expression of integrin α5β1 regulates neural crest development and cardiovascular morphogenesis

PubMed Central

Liang, Dong; Wang, Xia; Mittal, Ashok; Dhiman, Sonam; Hou, Shuan-Yu; Degenhardt, Karl; Astrof, Sophie

2014-01-01

Integrin α5-null embryos die in mid-gestation from severe defects in cardiovascular morphogenesis, which stem from defective development of the neural crest, heart and vasculature. To investigate the role of integrin α5β1 in cardiovascular development, we used the Mesp1Cre knock-in strain of mice to ablate integrin α5 in the anterior mesoderm, which gives rise to all of the cardiac and many of the vascular and muscle lineages in the anterior portion of the embryo. Surprisingly, we found that mutant embryos displayed numerous defects related to the abnormal development of the neural crest such as cleft palate, ventricular septal defect, abnormal development of hypoglossal nerves, and defective remodeling of the aortic arch arteries. We found that defects in arch artery remodeling stem from the role of mesodermal integrin α5β1 in neural crest proliferation and differentiation into vascular smooth muscle cells, while proliferation of pharyngeal mesoderm and differentiation of mesodermal derivatives into vascular smooth muscle cells was not defective. Taken together our studies demonstrate a requisite role for mesodermal integrin α5β1 in signaling between the mesoderm and the neural crest, thereby regulating neural crest-dependent morphogenesis of essential embryonic structures. PMID:25242040

Fucus as a Model System to Study the Role of Auxin Transport and the Actin Cytoskeleton in Gravity Response

NASA Technical Reports Server (NTRS)

Muday, Gloria K.

2003-01-01

The overarching goal of this proposal was to examine the mechanisms for the cellular asymmetry in auxin transport proteins. As auxin transport polarity changes in response to reorientation of algal and plant cells relative to the gravity vector, it was critical to ask how auxin transport polarity is established and how this transport polarity may change in response to gravity stimulation. The experiments conducted with this NASA grant fell into two categories. The first area of experimentation was to explore the biochemical interactions between an auxin transport protein and the actin cytoskeleton. These experiments used biochemical techniques, including actin affinity chromatography, to demonstrate that one auxin transport protein interacts with the actin cytoskeleton. The second line of experiments examined whether in the initially symmetrical single celled embryos of Fucus distichus, whether auxin regulates development and whether gravity is a cue to control the morphogenesis of these embryos and whether gravi-morphogenesis is auxin dependent. Results in these two areas are summarized separately below. As a result of this funding, in combination with results from other investigators, we have strong evidence for an important role for the actin cytoskeleton in both establishing and change auxin transport polarity. It is also clear that Fucus distichus embryos are auxin responsive and gravity controls their morphogenesis.

The Bio-Logic and machinery of plant morphogenesis.

PubMed

Niklas, Karl J

2003-04-01

Morphogenesis (the development of organic form) requires signal-trafficking and cross-talking across all levels of organization to coordinate the operation of metabolic and genomic networked systems. Many biologists are currently converging on the pictorial conventions of computer scientists to render biological signaling as logic circuits supervising the operation of one or more signal-activated metabolic or gene networks. This approach can redact and simplify complex morphogenetic phenomena and allows for their aggregation into diagrams of larger, more "global" networked systems. This conceptualization is discussed in terms of how logic circuits and signal-activated subsystems work, and it is illustrated for examples of increasingly more complex morphogenetic phenomena, e.g., auxin-mediated cell expansion, entry into the mitotic cell cycle phases, and polar/lateral intercellular auxin transport. For each of these phenomena, a posited circuit/subsystem diagram draws rapid attention to missing components, either in the logic circuit or in the subsystem it supervises. These components must be identified experimentally if each of these basic phenomena is to be fully understood. Importantly, the power of the circuit/subsystem approach to modeling developmental phenomena resides not in its pictorial appeal but in the mathematical tools that are sufficiently strong to reveal and quantify the synergistics of networked systems and thus foster a better understanding of morphogenesis.

Compensatory branching morphogenesis of stalk cells in the Drosophila trachea.

PubMed

Francis, Deanne; Ghabrial, Amin S

2015-06-01

Tubes are essential for nutrient transport and gas exchange in multicellular eukaryotes, but how connections between different tube types are maintained over time is unknown. In the Drosophila tracheal system, mutations in oak gall (okg) and conjoined (cnj) confer identical defects, including late onset blockage near the terminal cell-stalk cell junction and the ectopic extension of autocellular, seamed tubes into the terminal cell. We determined that okg and cnj encode the E and G subunits of the vacuolar ATPase (vATPase) and showed that both the V0 and V1 domains are required for terminal cell morphogenesis. Remarkably, the ectopic seamed tubes running along vATPase-deficient terminal cells belonged to the neighboring stalk cells. All vATPase-deficient tracheal cells had reduced apical domains and terminal cells displayed mislocalized apical proteins. Consistent with recent reports that the mTOR and vATPase pathways intersect, we found that mTOR pathway mutants phenocopied okg and cnj. Furthermore, terminal cells depleted for the apical determinants Par6 or aPKC had identical ectopic seamed tube defects. We thus identify a novel mechanism of compensatory branching in which stalk cells extend autocellular tubes into neighboring terminal cells with undersized apical domains. This compensatory branching also occurs in response to injury, with damaged terminal cells being rapidly invaded by their stalk cell neighbor. © 2015. Published by The Company of Biologists Ltd.

Spm1, a stress-activated MAP kinase that regulates morphogenesis in S.pombe.

PubMed Central

Zaitsevskaya-Carter, T; Cooper, J A

1997-01-01

A gene encoding a novel MAP kinase family member, Spm1, was isolated from the fission yeast Schizosaccharomyces pombe. Overproduction of Spm1 inhibits proliferation. Disruption of the spm1+ gene interferes with cell separation and morphogenesis. Under conditions of nutrient limitation, hypertonic stress or elevated temperature, spm1 delta cells grow as short branched filaments in which the cell walls and septa are thickened, suggesting defects in polarized growth and cell wall remodeling. At high osmolarity, spm1 delta cells fail to form colonies. The Spm1 protein is tyrosine phosphorylated and activated in response to osmotic and heat stress, consistent with a role for Spm1 in adaptation to these conditions. Two other S.pombe MAP kinases are known, Spk1, required for sexual differentiation and sporulation, and Spc1/Sty1/Phh1, which is activated in hypertonic conditions. However, the distinctive features of the spm1 delta mutant phenotype and direct biochemical assays suggest that Spm1 does not lie on other known MAP kinase pathways. Our results demonstrate the existence of a new MAP kinase pathway that regulates cell wall remodeling and cytokinesis in response to environmental stresses. PMID:9135147

Glycosylphosphatidylinositol-anchored proteins are required for cell wall synthesis and morphogenesis in Arabidopsis.

PubMed

Gillmor, C Stewart; Lukowitz, Wolfgang; Brininstool, Ginger; Sedbrook, John C; Hamann, Thorsten; Poindexter, Patricia; Somerville, Chris

2005-04-01

Mutations at five loci named PEANUT1-5 (PNT) were identified in a genetic screen for radially swollen embryo mutants. pnt1 cell walls showed decreased crystalline cellulose, increased pectins, and irregular and ectopic deposition of pectins, xyloglucans, and callose. Furthermore, pnt1 pollen is less viable than the wild type, and pnt1 embryos were delayed in morphogenesis and showed defects in shoot and root meristems. The PNT1 gene encodes the Arabidopsis thaliana homolog of mammalian PIG-M, an endoplasmic reticulum-localized mannosyltransferase that is required for synthesis of the glycosylphosphatidylinositol (GPI) anchor. All five pnt mutants showed strongly reduced accumulation of GPI-anchored proteins, suggesting that they all have defects in GPI anchor synthesis. Although the mutants are seedling lethal, pnt1 cells are able to proliferate for a limited time as undifferentiated callus and do not show the massive deposition of ectopic cell wall material seen in pnt1 embryos. The different phenotype of pnt1 cells in embryos and callus suggest a differential requirement for GPI-anchored proteins in cell wall synthesis in these two tissues and points to the importance of GPI anchoring in coordinated multicellular growth.

Latest status of the clinical and industrial applications of cell sheet engineering and regenerative medicine.

PubMed

Egami, Mime; Haraguchi, Yuji; Shimizu, Tatsuya; Yamato, Masayuki; Okano, Teruo

2014-01-01

Cell sheet engineering, which allows tissue engineering to be realized without the use of biodegradable scaffolds as an original approach, using a temperature-responsive intelligent surface, has been applied in regenerative medicine for various tissues, and a number of clinical studies have been already performed for life-threatening diseases. By using the results and findings obtained from the initial clinical studies, additional investigative clinical studies in several tissues with cell sheet engineering are currently in preparation stage. For treating many patients effectively by cell sheet engineering, an automated system integrating cell culture, cell-sheet fabrication, and layering is essential, and the system should include an advanced three-dimensional suspension cell culture system and an in vitro bioreactor system to scale up the production of cultured cells and fabricate thicker vascularized tissues. In this paper, cell sheet engineering, its clinical application, and further the authors' challenge to develop innovative cell culture systems under newly legislated regulatory platform in Japan are summarized and discussed.

The anatomical placode in reptile scale morphogenesis indicates shared ancestry among skin appendages in amniotes

PubMed Central

Di-Poï, Nicolas; Milinkovitch, Michel C.

2016-01-01

Most mammals, birds, and reptiles are readily recognized by their hairs, feathers, and scales, respectively. However, the lack of fossil intermediate forms between scales and hairs and substantial differences in their morphogenesis and protein composition have fueled the controversy pertaining to their potential common ancestry for decades. Central to this debate is the apparent lack of an “anatomical placode” (that is, a local epidermal thickening characteristic of feathers’ and hairs’ early morphogenesis) in reptile scale development. Hence, scenarios have been proposed for the independent development of the anatomical placode in birds and mammals and parallel co-option of similar signaling pathways for their morphogenesis. Using histological and molecular techniques on developmental series of crocodiles and snakes, as well as of unique wild-type and EDA (ectodysplasin A)–deficient scaleless mutant lizards, we show for the first time that reptiles, including crocodiles and squamates, develop all the characteristics of an anatomical placode: columnar cells with reduced proliferation rate, as well as canonical spatial expression of placode and underlying dermal molecular markers. These results reveal a new evolutionary scenario where hairs, feathers, and scales of extant species are homologous structures inherited, with modification, from their shared reptilian ancestor’s skin appendages already characterized by an anatomical placode and associated signaling molecules. PMID:28439533

The anatomical placode in reptile scale morphogenesis indicates shared ancestry among skin appendages in amniotes.

PubMed

Di-Poï, Nicolas; Milinkovitch, Michel C

2016-06-01

Most mammals, birds, and reptiles are readily recognized by their hairs, feathers, and scales, respectively. However, the lack of fossil intermediate forms between scales and hairs and substantial differences in their morphogenesis and protein composition have fueled the controversy pertaining to their potential common ancestry for decades. Central to this debate is the apparent lack of an "anatomical placode" (that is, a local epidermal thickening characteristic of feathers' and hairs' early morphogenesis) in reptile scale development. Hence, scenarios have been proposed for the independent development of the anatomical placode in birds and mammals and parallel co-option of similar signaling pathways for their morphogenesis. Using histological and molecular techniques on developmental series of crocodiles and snakes, as well as of unique wild-type and EDA (ectodysplasin A)-deficient scaleless mutant lizards, we show for the first time that reptiles, including crocodiles and squamates, develop all the characteristics of an anatomical placode: columnar cells with reduced proliferation rate, as well as canonical spatial expression of placode and underlying dermal molecular markers. These results reveal a new evolutionary scenario where hairs, feathers, and scales of extant species are homologous structures inherited, with modification, from their shared reptilian ancestor's skin appendages already characterized by an anatomical placode and associated signaling molecules.

Neurovascular Cell Sheet Transplantation in a Canine Model of Intracranial Hemorrhage

PubMed Central

Lee, Woo-Jin; Lee, Jong Young; Jung, Keun-Hwa; Lee, Soon-Tae; Kim, Hyo Yeol; Park, Dong-Kyu; Yu, Jung-Suk; Kim, So-Yun; Jeon, Daejong; Kim, Manho; Lee, Sang Kun; Roh, Jae-Kyu; Chu, Kon

2017-01-01

Cell-based therapy for intracerebral hemorrhage (ICH) has a great therapeutic potential. However, methods to effectively induce direct regeneration of the damaged neural tissue after cell transplantation have not been established, which, if done, would improve the efficacy of cell-based therapy. In this study, we aimed to develop a cell sheet with neurovasculogenic potential and evaluate its usefulness in a canine ICH model. We designed a composite cell sheet made of neural progenitors derived from human olfactory neuroepithelium and vascular progenitors from human adipose tissue-derived stromal cells. We also generated a physiologic canine ICH model by manually injecting and then infusing autologous blood under arterial pressure. We transplanted the sheet cells (cell sheet group) or saline (control group) at the cortex over the hematoma at subacute stages (2 weeks from ICH induction). At 4 weeks from the cell transplantation, cell survival, migration, and differentiation were evaluated. Hemispheric atrophy and neurobehavioral recovery were also compared between the groups. As a result, the cell sheet was rich in extracellular matrices and expressed neurotrophic factors as well as the markers for neuronal development. After transplantation, the cells successfully survived for 4 weeks, and a large portion of those migrated to the perihematomal site and differentiated into neurons and pericytes (20% and 30% of migrated stem cells, respectively). Transplantation of cell sheets alleviated hemorrhage-related hemispheric atrophy (p = 0.042) and showed tendency for improving functional recovery (p = 0.062). Therefore, we concluded that the cell sheet transplantation technique might induce direct regeneration of neural tissue and might improve outcomes of intracerebral hemorrhage. PMID:28713638

A flexible thermoresponsive cell culture substrate for direct transfer of keratinocyte cell sheets.

PubMed

Praveen, Wulligundam; Madathil, Bernadette K; Sajin Raj, R S; Kumary, T V; Anil Kumar, P R

2017-10-25

Most cell sheet engineering systems require a support or carrier to handle the harvested cell sheets. In this study, polyethylene terephthalate-based overhead projection transparency sheets (OHPS) were subjected to surface hydrolysis by alkali treatment to increase pliability and hydrophilicity and enable poly(N-isopropylacrylamide-co-glycidylmethacrylate) copolymer (NGMA) coating to impart thermoresponsiveness. NGMA was applied on the modified OHPS by the technique of spin coating using an indigenously designed spin coater. The spin coating had the advantage of using low volumes of the polymer and a reduced coating time. The surface chemistry and thermoresponsive coating was analyzed by Fourier transform infrared spectroscopy and water contact angle. Human keratinocyte cells were cultured on the spin coated surface and scaffold-free cell sheets were successfully harvested by simple variation of temperature. These cell sheets were found to be viable, exhibited epithelial characteristic and cell-cell contact as confirmed by positive immunostaining for ZO-1. The integrity and morphology of the cell sheet was confirmed by stereomicroscopy and E-SEM. These results highlight the potential of the NGMA spin coated modified OHPS to serve as a thermoresponsive culture surface-cum-flexible transfer tool.

MorphoGraphX: A platform for quantifying morphogenesis in 4D.

PubMed

Barbier de Reuille, Pierre; Routier-Kierzkowska, Anne-Lise; Kierzkowski, Daniel; Bassel, George W; Schüpbach, Thierry; Tauriello, Gerardo; Bajpai, Namrata; Strauss, Sören; Weber, Alain; Kiss, Annamaria; Burian, Agata; Hofhuis, Hugo; Sapala, Aleksandra; Lipowczan, Marcin; Heimlicher, Maria B; Robinson, Sarah; Bayer, Emmanuelle M; Basler, Konrad; Koumoutsakos, Petros; Roeder, Adrienne H K; Aegerter-Wilmsen, Tinri; Nakayama, Naomi; Tsiantis, Miltos; Hay, Angela; Kwiatkowska, Dorota; Xenarios, Ioannis; Kuhlemeier, Cris; Smith, Richard S

2015-05-06

Morphogenesis emerges from complex multiscale interactions between genetic and mechanical processes. To understand these processes, the evolution of cell shape, proliferation and gene expression must be quantified. This quantification is usually performed either in full 3D, which is computationally expensive and technically challenging, or on 2D planar projections, which introduces geometrical artifacts on highly curved organs. Here we present MorphoGraphX ( www.MorphoGraphX.org), a software that bridges this gap by working directly with curved surface images extracted from 3D data. In addition to traditional 3D image analysis, we have developed algorithms to operate on curved surfaces, such as cell segmentation, lineage tracking and fluorescence signal quantification. The software's modular design makes it easy to include existing libraries, or to implement new algorithms. Cell geometries extracted with MorphoGraphX can be exported and used as templates for simulation models, providing a powerful platform to investigate the interactions between shape, genes and growth.

An FGF autocrine loop initiated in second heart field mesoderm regulates morphogenesis at the arterial pole of the heart

PubMed Central

Park, Eon Joo; Watanabe, Yusuke; Smyth, Graham; Miyagawa-Tomita, Sachiko; Meyers, Erik; Klingensmith, John; Camenisch, Todd; Buckingham, Margaret; Moon, Anne M.

2009-01-01

In order to understand how secreted signals regulate complex morphogenetic events, it is crucial to identify their cellular targets. By conditional inactivation of Fgfr1 and Fgfr2 and overexpression of the FGF antagonist sprouty 2 in different cell types, we have dissected the role of FGF signaling during heart outflow tract development in mouse. Contrary to expectation, cardiac neural crest and endothelial cells are not primary paracrine targets. FGF signaling within second heart field mesoderm is required for remodeling of the outflow tract: when disrupted, outflow myocardium fails to produce extracellular matrix and TGFβ and BMP signals essential for endothelial cell transformation and invasion of cardiac neural crest. We conclude that an autocrine regulatory loop, initiated by the reception of FGF signals by the mesoderm, regulates correct morphogenesis at the arterial pole of the heart. These findings provide new insight into how FGF signaling regulates context-dependent cellular responses during development. PMID:18832392

Cell delivery in regenerative medicine: the cell sheet engineering approach.

PubMed

Yang, Joseph; Yamato, Masayuki; Nishida, Kohji; Ohki, Takeshi; Kanzaki, Masato; Sekine, Hidekazu; Shimizu, Tatsuya; Okano, Teruo

2006-11-28

Recently, cell-based therapies have developed as a foundation for regenerative medicine. General approaches for cell delivery have thus far involved the use of direct injection of single cell suspensions into the target tissues. Additionally, tissue engineering with the general paradigm of seeding cells into biodegradable scaffolds has also evolved as a method for the reconstruction of various tissues and organs. With success in clinical trials, regenerative therapies using these approaches have therefore garnered significant interest and attention. As a novel alternative, we have developed cell sheet engineering using temperature-responsive culture dishes, which allows for the non-invasive harvest of cultured cells as intact sheets along with their deposited extracellular matrix. Using this approach, cell sheets can be directly transplanted to host tissues without the use of scaffolding or carrier materials, or used to create in vitro tissue constructs via the layering of individual cell sheets. In addition to simple transplantation, cell sheet engineered constructs have also been applied for alternative therapies such as endoscopic transplantation, combinatorial tissue reconstruction, and polysurgery to overcome limitations of regenerative therapies and cell delivery using conventional approaches.

Pituitary gland morphogenesis and ontogeny of adenohypophyseal cells of Salminus brasiliensis (Teleostei, Characiformes).

PubMed

de Jesus, Lázaro Wender Oliveira; Chehade, Chayrra; Costa, Fabiano Gonçalves; Borella, Maria Inês

2014-06-01

In this study, we describe for the first time the details of the pituitary gland morphogenesis and the ontogeny of adenohypophyseal cells of a South American Characiform species with great importance for Brazilian Aquaculture, Salminus brasiliensis (Characiformes, Characidae), from hatching to 25 days after hatching (dah), by histochemical and immunocytochemical methods. The pituitary placode was first detected at hatching (0 dah), and the pituitary anlage became more defined at 0.5 dah. The neurohypophysis (NH) development started at 3 dah, and the early formation of its stalk at 12.5 dah. An increase in adenohypophyseal and NH tissues was also observed, and in juveniles at 25 dah, the pituitary displayed similar morphology to that found in adults of this species, displaying the main features of the teleost pituitary. PRL cells were detected at 0.5 dah, together with ACTH and α-MSH cells, followed by GH and SL cells at 1.5 dah. β-FSH cells were detected at 25 dah, while β-LH cells at 5 dah. The pituitary development in this species comprises a dynamic process similar to other teleosts. Our findings in S. brasiliensis corroborate the heterogeneity in the ontogeny of adenohypophyseal cells in teleosts and suggest a role for adenohypophyseal hormones in the early development of this species.

Stochasticity and stereotypy in the Ciona notochord.

PubMed

Carlson, Maia; Reeves, Wendy; Veeman, Michael

2015-01-15

Fate mapping with single cell resolution has typically been confined to embryos with completely stereotyped development. The lineages giving rise to the 40 cells of the Ciona notochord are invariant, but the intercalation of those cells into a single-file column is not. Here we use genetic labeling methods to fate map the Ciona notochord with both high resolution and large sample sizes. We find that the ordering of notochord cells into a single column is not random, but instead shows a distinctive signature characteristic of mediolaterally-biased intercalation. We find that patterns of cell intercalation in the notochord are somewhat stochastic but far more stereotyped than previously believed. Cell behaviors vary by lineage, with the secondary notochord lineage being much more constrained than the primary lineage. Within the primary lineage, patterns of intercalation reflect the geometry of the intercalating tissue. We identify the latest point at which notochord morphogenesis is largely stereotyped, which is shortly before the onset of mediolateral intercalation and immediately after the final cell divisions in the primary lineage. These divisions are consistently oriented along the AP axis. Our results indicate that the interplay between stereotyped and stochastic cell behaviors in morphogenesis can only be assessed by fate mapping experiments that have both cellular resolution and large sample sizes. Copyright © 2014 Elsevier Inc. All rights reserved.

Stochasticity and Stereotypy in the Ciona Notochord

PubMed Central

Carlson, Maia; Reeves, Wendy; Veeman, Michael

2015-01-01

Fate mapping with single cell resolution has typically been confined to embryos with completely stereotyped development. The lineages giving rise to the 40 cells of the Ciona notochord are invariant, but the intercalation of those cells into a single-file column is not. Here we use genetic labeling methods to fate map the Ciona notochord with both high resolution and large sample sizes. We find that the ordering of notochord cells into a single column is not random, but instead shows a distinctive signature characteristic of mediolaterally-biased intercalation. We find that patterns of cell intercalation in the notochord are somewhat stochastic but far more stereotyped than previously believed. Cell behaviors vary by lineage, with the secondary notochord lineage being much more constrained than the primary lineage. Within the primary lineage, patterns of intercalation reflect the geometry of the intercalating tissue. We identify the latest point at which notochord morphogenesis is largely stereotyped, which is shortly before the onset of mediolateral intercalation and immediately after the final cell divisions in the primary lineage. These divisions are consistently oriented along the AP axis. Our results indicate that the interplay between stereotyped and stochastic cell behaviors in morphogenesis can only be assessed by fate mapping experiments that have both cellular resolution and large sample sizes. PMID:25459659

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

Exogenous Cellulase Switches Cell Interdigitation to Cell Elongation in an RIC1-dependent Manner in Arabidopsis thaliana Cotyledon Pavement Cells.

PubMed

Higaki, Takumi; Takigawa-Imamura, Hisako; Akita, Kae; Kutsuna, Natsumaro; Kobayashi, Ryo; Hasezawa, Seiichiro; Miura, Takashi

2017-01-01

Pavement cells in cotyledons and true leaves exhibit a jigsaw puzzle-like morphology in most dicotyledonous plants. Among the molecular mechanisms mediating cell morphogenesis, two antagonistic Rho-like GTPases regulate local cell outgrowth via cytoskeletal rearrangements. Analyses of several cell wall-related mutants suggest the importance of cell wall mechanics in the formation of interdigitated patterns. However, how these factors are integrated is unknown. In this study, we observed that the application of exogenous cellulase to hydroponically grown Arabidopsis thaliana cotyledons switched the interdigitation of pavement cells to the production of smoothly elongated cells. The cellulase-induced inhibition of cell interdigitation was not observed in a RIC1 knockout mutant. This gene encodes a Rho-like GTPase-interacting protein important for localized cell growth suppression via microtubule bundling on concave cell interfaces. Additionally, to characterize pavement cell morphologies, we developed a mathematical model that considers the balance between cell and cell wall growth, restricted global cell growth orientation, and regulation of local cell outgrowth mediated by a Rho-like GTPase-cytoskeleton system. Our computational simulations fully support our experimental observations, and suggest that interdigitated patterns form because of mechanical buckling in the absence of Rho-like GTPase-dependent regulation of local cell outgrowth. Our model clarifies the cell wall mechanics influencing pavement cell morphogenesis. © The Author 2016. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Cellular Basis of Mechanotransduction

NASA Technical Reports Server (NTRS)

Ingber, Donald E.

1996-01-01

Physical forces, such as those due to gravity are fundamental regulators of tissue development. To influence morphogenesis, mechanical forces must alter growth and function. Yet little is known about how cells convert mechanical signals into a chemical response. This presentation attempts to place the potential molecular mediators of mechanotransduction within the context of the structural complexity of living cells.

Differences in mushroom bodies morphogenesis in workers, queens and drones of Apis mellifera: neuroblasts proliferation and death.

PubMed

Roat, Thaisa Cristina; da Cruz Landim, Carminda

2010-06-01

Apis mellifera is an interesting model to neurobiological studies. It has a relatively small brain that commands the complex learning and memory tasks demanded by the social organization. An A. mellifera colony is made up of a queen, thousands of workers and a varying number of drones. The latter are males, whereas the former are the two female castes. These three phenotypes differ in morphology, physiology and behavior, correlated with their respective functions in the society. Such differences include the morphology and architecture of their brains. To understand the processes generating such polymorphic brains we characterized the cell division and cell death dynamics which underlie the morphogenesis of the mushroom bodies, through several methods suitable for evidence the time and place of occurrence. Cell death was detected in mushroom bodies of last larval instar and mainly in black-eyed pupae. Cell division was observed in mushroom bodies, primarily at the start of metamorphosis, exhibiting temporal differences among workers, queens and males. Copyright 2010 Elsevier Ltd. All rights reserved.

Cdc42 regulates junctional actin but not cell polarization in the Caenorhabditis elegans epidermis

PubMed Central

Zilberman, Yuliya; Abrams, Joshua; Anderson, Dorian C.

2017-01-01

During morphogenesis, adherens junctions (AJs) remodel to allow changes in cell shape and position while preserving adhesion. Here, we examine the function of Rho guanosine triphosphatase CDC-42 in AJ formation and regulation during Caenorhabditis elegans embryo elongation, a process driven by asymmetric epidermal cell shape changes. cdc-42 mutant embryos arrest during elongation with epidermal ruptures. Unexpectedly, we find using time-lapse fluorescence imaging that cdc-42 is not required for epidermal cell polarization or junction assembly, but rather is needed for proper junctional actin regulation during elongation. We show that the RhoGAP PAC-1/ARHGAP21 inhibits CDC-42 activity at AJs, and loss of PAC-1 or the interacting linker protein PICC-1/CCDC85A-C blocks elongation in embryos with compromised AJ function. pac-1 embryos exhibit dynamic accumulations of junctional F-actin and an increase in AJ protein levels. Our findings identify a previously unrecognized molecular mechanism for inhibiting junctional CDC-42 to control actin organization and AJ protein levels during epithelial morphogenesis. PMID:28903999

BAF200 is required for heart morphogenesis and coronary artery development.

PubMed

He, Lingjuan; Tian, Xueying; Zhang, Hui; Hu, Tianyuan; Huang, Xiuzhen; Zhang, Libo; Wang, Zhong; Zhou, Bin

2014-01-01

ATP-dependent SWI/SNF chromatin remodeling complexes utilize ATP hydrolysis to non-covalently change nucleosome-DNA interactions and are essential in stem cell development, organogenesis, and tumorigenesis. Biochemical studies show that SWI/SNF in mammalian cells can be divided into two subcomplexes BAF and PBAF based on the subunit composition. ARID2 or BAF200 has been defined as an intrinsic subunit of PBAF complex. However, the function of BAF200 in vivo is not clear. To dissect the possible role of BAF200 in regulating embryogenesis and organ development, we generated BAF200 mutant mice and found they were embryonic lethal. BAF200 mutant embryos exhibited multiple cardiac defects including thin myocardium, ventricular septum defect, common atrioventricular valve, and double outlet right ventricle around E14.5. Moreover, we also detected reduced intramyocardial coronary arteries in BAF200 mutants, suggesting that BAF200 is required for proper migration and differentiation of subepicardial venous cells into arterial endothelial cells. Our work revealed that PBAF complex plays a critical role in heart morphogenesis and coronary artery angiogenesis.

The mechanics behind plant development.

PubMed

Hamant, Olivier; Traas, Jan

2010-01-01

Morphogenesis in living organisms relies on the integration of both biochemical and mechanical signals. During the last decade, attention has been mainly focused on the role of biochemical signals in patterning and morphogenesis, leaving the contribution of mechanics largely unexplored. Fortunately, the development of new tools and approaches has made it possible to re-examine these processes. In plants, shape is defined by two local variables: growth rate and growth direction. At the level of the cell, these variables depend on both the cell wall and turgor pressure. Multidisciplinary approaches have been used to understand how these cellular processes are integrated in the growing tissues. These include quantitative live imaging to measure growth rate and direction in tissues with cellular resolution. In parallel, stress patterns have been artificially modified and their impact on strain and cell behavior been analysed. Importantly, computational models based on analogies with continuum mechanics systems have been useful in interpreting the results. In this review, we will discuss these issues focusing on the shoot apical meristem, a population of stem cells that is responsible for the initiation of the aerial organs of the plant.

Wound Repair: Toward Understanding and Integration of Single-Cell and Multicellular Wound Responses

PubMed Central

Sonnemann, Kevin J.; Bement, William M.

2016-01-01

The importance of wound healing to medicine and biology has long been evident, and consequently, wound healing has been the subject of intense investigation for many years. However, several relatively recent developments have added new impetus to wound repair research: the increasing application of model systems; the growing recognition that single cells have a robust, complex, and medically relevant wound healing response; and the emerging recognition that different modes of wound repair bear an uncanny resemblance to other basic biological processes such as morphogenesis and cytokinesis. In this review, each of these developments is described, and their significance for wound healing research is considered. In addition, overlapping mechanisms of single-cell and multicellular wound healing are highlighted, and it is argued that they are more similar than is often recognized. Based on this and other information, a simple model to explain the evolutionary relationships of cytokinesis, single-cell wound repair, multicellular wound repair, and developmental morphogenesis is proposed. Finally, a series of important, but as yet unanswered, questions is posed. PMID:21721944

Oligomerization-Dependent Regulation of Motility and Morphogenesis by the Collagen Xviii Nc1/Endostatin Domain

PubMed Central

Kuo, Calvin J.; LaMontagne, Kenneth R.; Garcia-Cardeña, Guillermo; Ackley, Brian D.; Kalman, Daniel; Park, Susan; Christofferson, Rolf; Kamihara, Junne; Ding, Yuan-Hua; Lo, Kin-Ming; Gillies, Stephen; Folkman, Judah; Mulligan, Richard C.; Javaherian, Kashi

2001-01-01

Collagen XVIII (c18) is a triple helical endothelial/epithelial basement membrane protein whose noncollagenous (NC)1 region trimerizes a COOH-terminal endostatin (ES) domain conserved in vertebrates, Caenorhabditis elegans and Drosophila. Here, the c18 NC1 domain functioned as a motility-inducing factor regulating the extracellular matrix (ECM)-dependent morphogenesis of endothelial and other cell types. This motogenic activity required ES domain oligomerization, was dependent on rac, cdc42, and mitogen-activated protein kinase, and exhibited functional distinction from the archetypal motogenic scatter factors hepatocyte growth factor and macrophage stimulatory protein. The motility-inducing and mitogen-activated protein kinase–stimulating activities of c18 NC1 were blocked by its physiologic cleavage product ES monomer, consistent with a proteolysis-dependent negative feedback mechanism. These data indicate that the collagen XVIII NC1 region encodes a motogen strictly requiring ES domain oligomerization and suggest a previously unsuspected mechanism for ECM regulation of motility and morphogenesis. PMID:11257123

Electronically transparent graphene replicas of diatoms: a new technique for the investigation of frustule morphology

PubMed Central

Pan, Zhengwei; Lerch, Sarah J. L.; Xu, Liang; Li, Xufan; Chuang, Yen-Jun; Howe, Jane Y.; Mahurin, Shannon M.; Dai, Sheng; Hildebrand, Mark

2014-01-01

The morphogenesis of the silica cell walls (called frustules) of unicellular algae known as diatoms is one of the most intriguing mysteries of the diatoms. To study frustule morphogenesis, optical, electron and atomic force microscopy has been extensively used to reveal the frustule morphology. However, since silica frustules are opaque, past observations were limited to outer and fracture surfaces, restricting observations of interior structures. Here we show that opaque silica frustules can be converted into electronically transparent graphene replicas, fabricated using chemical vapor deposition of methane. Chemical vapor deposition creates a continuous graphene coating preserving the frustule's shape and fine, complicated internal features. Subsequent dissolution of the silica with hydrofluoric acid yields a free-standing replica of the internal and external native frustule morphologies. Electron microscopy renders these graphene replicas highly transparent, revealing previously unobserved, complex, three-dimensional, interior frustule structures, which lend new insights into the investigation of frustule morphogenesis. PMID:25135739

Cell Sheet-Based Tissue Engineering for Organizing Anisotropic Tissue Constructs Produced Using Microfabricated Thermoresponsive Substrates.

PubMed

Takahashi, Hironobu; Okano, Teruo

2015-11-18

In some native tissues, appropriate microstructures, including orientation of the cell/extracellular matrix, provide specific mechanical and biological functions. For example, skeletal muscle is made of oriented myofibers that is responsible for the mechanical function. Native artery and myocardial tissues are organized three-dimensionally by stacking sheet-like tissues of aligned cells. Therefore, to construct any kind of complex tissue, the microstructures of cells such as myotubes, smooth muscle cells, and cardiomyocytes also need to be organized three-dimensionally just as in the native tissues of the body. Cell sheet-based tissue engineering allows the production of scaffold-free engineered tissues through a layer-by-layer construction technique. Recently, using microfabricated thermoresponsive substrates, aligned cells are being harvested as single continuous cell sheets. The cell sheets act as anisotropic tissue units to build three-dimensional tissue constructs with the appropriate anisotropy. This cell sheet-based technology is straightforward and has the potential to engineer a wide variety of complex tissues. In addition, due to the scaffold-free cell-dense environment, the physical and biological cell-cell interactions of these cell sheet constructs exhibit unique cell behaviors. These advantages will provide important clues to enable the production of well-organized tissues that closely mimic the structure and function of native tissues, required for the future of tissue engineering. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Crystal growth for high-efficiency silicon solar cells workshop: Summary

NASA Technical Reports Server (NTRS)

Dumas, K. A.

1985-01-01

The state of the art in the growth of silicon crystals for high-efficiency solar cells are reviewed, sheet requirements are defined, and furture areas of research are identified. Silicon sheet material characteristics that limit cell efficiencies and yields were described as well as the criteria for the ideal sheet-growth method. The device engineers wish list to the material engineer included: silicon sheet with long minority carrier lifetime that is uniform throughout the sheet, and which doesn't change during processing; and sheet material that stays flat throughout device processing, has uniform good mechanical strength, and is low cost. Impurities in silicon solar cells depreciate cell performance by reducing diffusion length and degrading junctions. The impurity behavior, degradation mechanisms, and variations in degradation threshold with diffusion length for silicon solar cells were described.

Morphogenesis by symbiogenesis

NASA Technical Reports Server (NTRS)

Chapman, M. J.; Margulis, L.

1998-01-01

Here we review cases where initiation of morphogenesis, including the differentiation of specialized cells and tissues, has clearly evolved due to cyclical symbiont integration. For reasons of space, our examples are drawn chiefly from the plant, fungal and bacterial kingdoms. Partners live in symbioses and show unique morphological specializations that result when they directly and cyclically interact. We include here brief citations to relevant literature where plant, bacterial or fungal partners alternate independent with entirely integrated living. The independent, or at least physically unassociated stages, are correlated with the appearance of distinctive morphologies that can be traced to the simultaneous presence and strong interaction of the plant with individuals that represent different taxa.

Periodontal healing by periodontal ligament cell sheets in a teeth replantation model.

PubMed

Zhou, Yefang; Li, Yusheng; Mao, Ling; Peng, Hao

2012-02-01

Successful transplantation of avulsed teeth is to restore the attachment and regenerate the periodontal support. Different strategies have been applied in treatment from modification of teeth storage, antibiotic usage to peridontium tissue replacement. We developed a novel periodontal ligament cell-sheet delivery system to apply on delayed replanted teeth in promoting periodontal healing in a canine model. Autologous periodontal ligament (PDL) fibroblasts were isolated from extracted premolars of beagle dog. The cell-sheets were fabricated using normal culture dish after stimulation of extracellular matrix formation. Teeth were surgically extracted and attached soft tissues were removed. After root canal treatment, the root of teeth were wrapped by the PDL cell-sheets and replanted back to prior socket accordingly whilst teeth without cell sheets as a control. Eight weeks after surgery, the animals were sacrificed and decalcified specimens were prepared. Regeneration of periodontal tissue was evaluated through histology assay. Multi-layered PDL cell-sheet could be attached on tooth root and most cells on sheet-tooth constructs were viable before replantation. Minimum clinical signs of inflammation were observed in experiment. PDL cell-sheets group show significant higher occurrence of favourable healing (88.4%) than control group with low healing (5.3%). Periodontal ligament and cememtum tissue regeneration was observed in the experimental group, and the regenerated tissues showed high collagen type III, type I and fibronectin expression. The periodontal ligament cell-sheets fabricated through normal cell culture dish has a potential for regeneration of periodontal ligament and may become a novel therapy for avulsed teeth replantation. Copyright © 2011 Elsevier Ltd. All rights reserved.

MicroRNA-200c-141 and ∆Np63 are required for breast epithelial differentiation and branching morphogenesis.

PubMed

Hilmarsdóttir, Bylgja; Briem, Eirikur; Sigurdsson, Valgardur; Franzdóttir, Sigrídur Rut; Ringnér, Markus; Arason, Ari Jon; Bergthorsson, Jon Thor; Magnusson, Magnus Karl; Gudjonsson, Thorarinn

2015-07-15

The epithelial compartment of the breast contains two lineages, the luminal- and the myoepithelial cells. D492 is a breast epithelial cell line with stem cell properties that forms branching epithelial structures in 3D culture with both luminal- and myoepithelial differentiation. We have recently shown that D492 undergo epithelial to mesenchymal transition (EMT) when co-cultured with endothelial cells. This 3D co-culture model allows critical analysis of breast epithelial lineage development and EMT. In this study, we compared the microRNA (miR) expression profiles for D492 and its mesenchymal-derivative D492M. Suppression of the miR-200 family in D492M was among the most profound changes observed. Exogenous expression of miR-200c-141 in D492M reversed the EMT phenotype resulting in gain of luminal but not myoepithelial differentiation. In contrast, forced expression of ∆Np63 in D492M restored the myoepithelial phenotype only. Co-expression of miR-200c-141 and ∆Np63 in D492M restored the branching morphogenesis in 3D culture underlining the requirement for both luminal and myoepithelial elements for obtaining full branching morphogenesis in breast epithelium. Introduction of a miR-200c-141 construct in both D492 and D492M resulted in resistance to endothelial induced EMT. In conclusion, our data suggests that expression of miR-200c-141 and ∆Np63 in D492M can reverse EMT resulting in luminal- and myoepithelial differentiation, respectively, demonstrating the importance of these molecules in epithelial integrity in the human breast. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Influence of nanotopography on periodontal ligament stem cell functions and cell sheet based periodontal regeneration

PubMed Central

Gao, Hui; Li, Bei; Zhao, Lingzhou; Jin, Yan

2015-01-01

Periodontal regeneration is an important part of regenerative medicine, with great clinical significance; however, the effects of nanotopography on the functions of periodontal ligament (PDL) stem cells (PDLSCs) and on PDLSC sheet based periodontal regeneration have never been explored. Titania nanotubes (NTs) layered on titanium (Ti) provide a good platform to study this. In the current study, the influence of NTs of different tube size on the functions of PDLSCs was observed. Afterward, an ectopic implantation model using a Ti/cell sheets/hydroxyapatite (HA) complex was applied to study the effect of the NTs on cell sheet based periodontal regeneration. The NTs were able to enhance the initial PDLSC adhesion and spread, as well as collagen secretion. With the Ti/cell sheets/HA complex model, it was demonstrated that the PDLSC sheets were capable of regenerating the PDL tissue, when combined with bone marrow mesenchymal stem cell (BMSC) sheets and HA, without the need for extra soluble chemical cues. Simultaneously, the NTs improved the periodontal regeneration result of the ectopically implanted Ti/cell sheets/HA complex, giving rise to functionally aligned collagen fiber bundles. Specifically, much denser collagen fibers, with abundant blood vessels as well as cementum-like tissue on the Ti surface, which well-resembled the structure of natural PDL, were observed in the NT5 and NT10 sample groups. Our study provides the first evidence that the nanotopographical cues obviously influence the functions of PDLSCs and improve the PDLSC sheet based periodontal regeneration size dependently, which provides new insight to the periodontal regeneration. The Ti/cell sheets/HA complex may constitute a good model to predict the effect of biomaterials on periodontal regeneration. PMID:26150714

Influence of nanotopography on periodontal ligament stem cell functions and cell sheet based periodontal regeneration.

PubMed

Gao, Hui; Li, Bei; Zhao, Lingzhou; Jin, Yan

2015-01-01

Periodontal regeneration is an important part of regenerative medicine, with great clinical significance; however, the effects of nanotopography on the functions of periodontal ligament (PDL) stem cells (PDLSCs) and on PDLSC sheet based periodontal regeneration have never been explored. Titania nanotubes (NTs) layered on titanium (Ti) provide a good platform to study this. In the current study, the influence of NTs of different tube size on the functions of PDLSCs was observed. Afterward, an ectopic implantation model using a Ti/cell sheets/hydroxyapatite (HA) complex was applied to study the effect of the NTs on cell sheet based periodontal regeneration. The NTs were able to enhance the initial PDLSC adhesion and spread, as well as collagen secretion. With the Ti/cell sheets/HA complex model, it was demonstrated that the PDLSC sheets were capable of regenerating the PDL tissue, when combined with bone marrow mesenchymal stem cell (BMSC) sheets and HA, without the need for extra soluble chemical cues. Simultaneously, the NTs improved the periodontal regeneration result of the ectopically implanted Ti/cell sheets/HA complex, giving rise to functionally aligned collagen fiber bundles. Specifically, much denser collagen fibers, with abundant blood vessels as well as cementum-like tissue on the Ti surface, which well-resembled the structure of natural PDL, were observed in the NT5 and NT10 sample groups. Our study provides the first evidence that the nanotopographical cues obviously influence the functions of PDLSCs and improve the PDLSC sheet based periodontal regeneration size dependently, which provides new insight to the periodontal regeneration. The Ti/cell sheets/HA complex may constitute a good model to predict the effect of biomaterials on periodontal regeneration.

Rapid Genetic Analysis of Epithelial-Mesenchymal Signaling During Hair Regeneration

PubMed Central

Zhen, Hanson H.; Oro, Anthony E.

2013-01-01

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an attractive model system to study organ development and tissue-specific signaling. Although hair follicle development is genetically tractable, fast and reproducible analysis of factors essential for this process remains a challenge. Here we describe a procedure to generate targeted overexpression or shRNA-mediated knockdown of factors using lentivirus in a tissue-specific manner. Using a modified version of a hair regeneration model 5, 6, 11, we can achieve robust gain- or loss-of-function analysis in primary mouse keratinocytes or dermal cells to facilitate study of epithelial-mesenchymal signaling pathways that lead to hair follicle morphogenesis. We describe how to isolate fresh primary mouse keratinocytes and dermal cells, which contain dermal papilla cells and their precursors, deliver lentivirus containing either shRNA or cDNA to one of the cell populations, and combine the cells to generate fully formed hair follicles on the backs of nude mice. This approach allows analysis of tissue-specific factors required to generate hair follicles within three weeks and provides a fast and convenient companion to existing genetic models. PMID:23486463

Spore formation in Myxococcus xanthus is tied to cytoskeleton functions and polysaccharide spore coat deposition

PubMed Central

Müller, Frank D.; Schink, Christian W.; Hoiczyk, Egbert; Cserti, Emöke; Higgs, Penelope I.

2011-01-01

Summary Myxococcus xanthus is a Gram-negative bacterium that differentiates into environmentally resistant spores. Spore differentiation involves septation-independent remodelling of the rod-shaped vegetative cell into a spherical spore and deposition of a thick and compact spore coat outside of the outer membrane. Our analyses suggest that spore coat polysaccharides are exported to the cell surface by the Exo outer membrane polysaccharide export/polysaccharide co-polymerase 2a (OPX/PCP-2a) machinery. Conversion of the capsule-like polysaccharide layer into a compact spore coat layer requires the Nfs proteins which likely form a complex in the cell envelope. Mutants in either nfs, exo, or two other genetic loci encoding homologs of polysaccharide synthesis enzymes, fail to complete morphogenesis from rods to spherical spores and instead produce a transient state of deformed cell morphology before reversion into typical rods. We additionally provide evidence that the cell cytoskeletal protein, MreB, plays an important role in rod to spore morphogenesis and for spore outgrowth. These studies provide evidence that this novel gram-negative differentiation process is tied to cytoskeleton functions and polysaccharide spore coat deposition. PMID:22188356

MpWIP regulates air pore complex development in the liverwort Marchantia polymorpha.

PubMed

Jones, Victor A S; Dolan, Liam

2017-04-15

The colonisation of the land by plants was accompanied by the evolution of complex tissues and multicellular structures comprising different cell types as morphological adaptations to the terrestrial environment. Here, we show that the single WIP protein in the early-diverging land plant Marchantia polymorpha L. is required for the development of the multicellular gas exchange structure: the air pore complex. This 16-cell barrel-shaped structure surrounds an opening between epidermal cells that facilitates the exchange of gases between the chamber containing the photosynthetic cells inside the plant and the air outside. Mp WIP is expressed in cells of the developing air pore complex and the morphogenesis of the complex is defective in plants with reduced Mp WIP function. The role of WIP proteins in the control of different multicellular structures in M. polymorpha and the flowering plant Arabidopsis thaliana suggests that these proteins controlled the development of multicellular structures in the common ancestor of land plants. We hypothesise that WIP genes were subsequently co-opted in the control of morphogenesis of novel multicellular structures that evolved during the diversification of land plants. © 2017. Published by The Company of Biologists Ltd.

Coordinating cell and tissue behavior during zebrafish neural tube morphogenesis.

PubMed

Araya, Claudio; Ward, Laura C; Girdler, Gemma C; Miranda, Miguel

2016-03-01

The development of a vertebrate neural epithelium with well-organized apico-basal polarity and a central lumen is essential for its proper function. However, how this polarity is established during embryonic development and the potential influence of surrounding signals and tissues on such organization has remained less understood. In recent years the combined superior transparency and genetics of the zebrafish embryo has allowed for in vivo visualization and quantification of the cellular and molecular dynamics that govern neural tube structure. Here, we discuss recent studies revealing how co-ordinated cell-cell interactions coupled with adjacent tissue dynamics are critical to regulate final neural tissue architecture. Furthermore, new findings show how the spatial regulation and timing of orientated cell division is key in defining precise lumen formation at the tissue midline. In addition, we compare zebrafish neurulation with that of amniotes and amphibians in an attempt to understand the conserved cellular mechanisms driving neurulation and resolve the apparent differences among animals. Zebrafish neurulation not only offers fundamental insights into early vertebrate brain development but also the opportunity to explore in vivo cell and tissue dynamics during complex three-dimensional animal morphogenesis. © 2015 Wiley Periodicals, Inc.

Cellular context–mediated Akt dynamics regulates MAP kinase signaling thresholds during angiogenesis

PubMed Central

Hellesøy, Monica; Lorens, James B.

2015-01-01

The formation of new blood vessels by sprouting angiogenesis is tightly regulated by contextual cues that affect angiogeneic growth factor signaling. Both constitutive activation and loss of Akt kinase activity in endothelial cells impair angiogenesis, suggesting that Akt dynamics mediates contextual microenvironmental regulation. We explored the temporal regulation of Akt in endothelial cells during formation of capillary-like networks induced by cell–cell contact with vascular smooth muscle cells (vSMCs) and vSMC-associated VEGF. Expression of constitutively active Akt1 strongly inhibited network formation, whereas hemiphosphorylated Akt1 epi-alleles with reduced kinase activity had an intermediate inhibitory effect. Conversely, inhibition of Akt signaling did not affect endothelial cell migration or morphogenesis in vSMC cocultures that generate capillary-like structures. We found that endothelial Akt activity is transiently blocked by proteasomal degradation in the presence of SMCs during the initial phase of capillary-like structure formation. Suppressed Akt activity corresponded to the increased endothelial MAP kinase signaling that was required for angiogenic endothelial morphogenesis. These results reveal a regulatory principle by which cellular context regulates Akt protein dynamics, which determines MAP kinase signaling thresholds necessary drive a morphogenetic program during angiogenesis. PMID:26023089

Partial functional redundancy of MreB isoforms, MreB, Mbl and MreBH, in cell morphogenesis of Bacillus subtilis.

PubMed

Kawai, Yoshikazu; Asai, Kei; Errington, Jeffery

2009-08-01

MreB proteins are bacterial actin homologues thought to have a role in cell shape determination by positioning the cell wall synthetic machinery. Many bacteria, particularly Gram-positives, have more than one MreB isoform. Bacillus subtilis has three, MreB, Mbl and MreBH, which colocalize in a single helical structure. We now show that the helical pattern of peptidoglycan (PG) synthesis in the cylindrical part of the rod-shaped cell is governed by the redundant action of the three MreB isoforms. Single mutants for any one of mreB isoforms can still incorporate PG in a helical pattern and generate a rod shape. However, after depletion of MreB in an mbl mutant (or depletion of all three isoforms) lateral wall PG synthesis was impaired and the cells became spherical and lytic. Overexpression of any one of the MreB isoforms overcame the lethality as well as the defects in lateral PG synthesis and cell shape. Furthermore, MreB and Mbl can associate with the peptidoglycan biosynthetic machinery independently. However, no single MreB isoform was able to support normal growth under various stress conditions, suggesting that the multiple isoforms are used to allow cells to maintain proper growth and morphogenesis under changing and sometimes adverse conditions.

Epimorphin regulates bile duct formation via effects on mitosis orientation in rat liver epithelial stem-like cells.

PubMed

Zhou, Junnian; Zhao, Lei; Qin, Lipeng; Wang, Jing; Jia, Yali; Yao, Hailei; Sang, Chen; Hu, Qinghua; Shi, Shuangshuang; Nan, Xue; Yue, Wen; Zhuang, Fengyuan; Yang, Chun; Wang, Yunfang; Pei, Xuetao

2010-03-17

Understanding how hepatic precursor cells can generate differentiated bile ducts is crucial for studies on epithelial morphogenesis and for development of cell therapies for hepatobiliary diseases. Epimorphin (EPM) is a key morphogen for duct morphogenesis in various epithelial organs. The role of EPM in bile duct formation (DF) from hepatic precursor cells, however, is not known. To address this issue, we used WB-F344 rat epithelial stem-like cells as model for bile duct formation. A micropattern and a uniaxial static stretch device was used to investigate the effects of EPM and stress fiber bundles on the mitosis orientation (MO) of WB cells. Immunohistochemistry of liver tissue sections demonstrated high EPM expression around bile ducts in vivo. In vitro, recombinant EPM selectively induced DF through upregulation of CK19 expression and suppression of HNF3alpha and HNF6, with no effects on other hepatocytic genes investigated. Our data provide evidence that EPM guides MO of WB-F344 cells via effects on stress fiber bundles and focal adhesion assembly, as supported by blockade EPM, beta1 integrin, and F-actin assembly. These blockers can also inhibit EPM-induced DF. These results demonstrate a new biophysical action of EPM in bile duct formation, during which determination of MO plays a crucial role.

Apolar and polar transitions drive the conversion between amoeboid and mesenchymal shapes in melanoma cells.

PubMed

Cooper, Sam; Sadok, Amine; Bousgouni, Vicky; Bakal, Chris

2015-11-05

Melanoma cells can adopt two functionally distinct forms, amoeboid and mesenchymal, which facilitates their ability to invade and colonize diverse environments during the metastatic process. Using quantitative imaging of single living tumor cells invading three-dimensional collagen matrices, in tandem with unsupervised computational analysis, we found that melanoma cells can switch between amoeboid and mesenchymal forms via two different routes in shape space--an apolar and polar route. We show that whereas particular Rho-family GTPases are required for the morphogenesis of amoeboid and mesenchymal forms, others are required for transitions via the apolar or polar route and not amoeboid or mesenchymal morphogenesis per se. Altering the transition rates between particular routes by depleting Rho-family GTPases can change the morphological heterogeneity of cell populations. The apolar and polar routes may have evolved in order to facilitate conversion between amoeboid and mesenchymal forms, as cells are either searching for, or attracted to, particular migratory cues, respectively. © 2015 Cooper et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Endogenous Sheet-Averaged Tension Within a Large Epithelial Cell Colony.

PubMed

Dumbali, Sandeep P; Mei, Lanju; Qian, Shizhi; Maruthamuthu, Venkat

2017-10-01

Epithelial cells form quasi-two-dimensional sheets that function as contractile media to effect tissue shape changes during development and homeostasis. Endogenously generated intrasheet tension is a driver of such changes, but has predominantly been measured in the presence of directional migration. The nature of epithelial cell-generated forces transmitted over supracellular distances, in the absence of directional migration, is thus largely unclear. In this report, we consider large epithelial cell colonies which are archetypical multicell collectives with extensive cell-cell contacts but with a symmetric (circular) boundary. Using the traction force imbalance method (TFIM) (traction force microscopy combined with physical force balance), we first show that one can determine the colony-level endogenous sheet forces exerted at the midline by one half of the colony on the other half with no prior assumptions on the uniformity of the mechanical properties of the cell sheet. Importantly, we find that this colony-level sheet force exhibits large variations with orientation-the difference between the maximum and minimum sheet force is comparable to the average sheet force itself. Furthermore, the sheet force at the colony midline is largely tensile but the shear component exhibits significantly more variation with orientation. We thus show that even an unperturbed epithelial colony with a symmetric boundary shows significant directional variation in the endogenous sheet tension and shear forces that subsist at the colony level.

Composite cell sheet for periodontal regeneration: crosstalk between different types of MSCs in cell sheet facilitates complex periodontal-like tissue regeneration.

PubMed

Zhang, Hao; Liu, Shiyu; Zhu, Bin; Xu, Qiu; Ding, Yin; Jin, Yan

2016-11-14

Tissue-engineering strategies based on mesenchymal stem cells (MSCs) and cell sheets have been widely used for periodontal tissue regeneration. However, given the complexity in periodontal structure, the regeneration methods using a single species of MSC could not fulfill the requirement for periodontal regeneration. We researched the interaction between the periodontal ligament stem cells (PDLSCs) and jaw bone marrow-derived mesenchymal stem cells (JBMMSCs), and constructed a composite cell sheet comprising both of the above MSCs to regenerate complex periodontium-like structures in nude mice. Our results show that by co-culturing PDLSCs and JBMMSCs, the expressions of bone and extracellular matrix (ECM)-related genes and proteins were significantly improved in both MSCs. Further investigations showed that, compared to the cell sheet using PDLSCs or JBMMSCs, the composite stem cell sheet (CSCS), which comprises these two MSCs, expressed higher levels of bone- and ECM-related genes and proteins, and generated a composite structure more similar to the native periodontal tissue physiologically in vivo. In conclusion, our results demonstrate that the crosstalk between PDLSCs and JBMMSCs in cell sheets facilitate regeneration of complex periodontium-like structures, providing a promising new strategy for physiological and functional regeneration of periodontal tissue.

Engineering epithelial-stromal interactions in vitro for toxicology assessment

EPA Science Inventory

Background: Crosstalk between epithelial and stromal cells drives the morphogenesis of ectodermal organs during development and promotes normal mature adult epithelial tissue function. Epithelial-mesenchymal interactions (EMIs) have been examined using mammalian models, ex vivo t...

Co-assembly, spatiotemporal control and morphogenesis of a hybrid protein-peptide system.

PubMed

Inostroza-Brito, Karla E; Collin, Estelle; Siton-Mendelson, Orit; Smith, Katherine H; Monge-Marcet, Amàlia; Ferreira, Daniela S; Rodríguez, Raúl Pérez; Alonso, Matilde; Rodríguez-Cabello, José Carlos; Reis, Rui L; Sagués, Francesc; Botto, Lorenzo; Bitton, Ronit; Azevedo, Helena S; Mata, Alvaro

2015-11-01

Controlling molecular interactions between bioinspired molecules can enable the development of new materials with higher complexity and innovative properties. Here we report on a dynamic system that emerges from the conformational modification of an elastin-like protein by peptide amphiphiles and with the capacity to access, and be maintained in, non-equilibrium for substantial periods of time. The system enables the formation of a robust membrane that displays controlled assembly and disassembly capabilities, adhesion and sealing to surfaces, self-healing and the capability to undergo morphogenesis into tubular structures with high spatiotemporal control. We use advanced microscopy along with turbidity and spectroscopic measurements to investigate the mechanism of assembly and its relation to the distinctive membrane architecture and the resulting dynamic properties. Using cell-culture experiments with endothelial and adipose-derived stem cells, we demonstrate the potential of this system to generate complex bioactive scaffolds for applications such as tissue engineering.

Co-assembly, spatiotemporal control and morphogenesis of a hybrid protein-peptide system

NASA Astrophysics Data System (ADS)

Inostroza-Brito, Karla E.; Collin, Estelle; Siton-Mendelson, Orit; Smith, Katherine H.; Monge-Marcet, Amàlia; Ferreira, Daniela S.; Rodríguez, Raúl Pérez; Alonso, Matilde; Rodríguez-Cabello, José Carlos; Reis, Rui L.; Sagués, Francesc; Botto, Lorenzo; Bitton, Ronit; Azevedo, Helena S.; Mata, Alvaro

2015-11-01

Controlling molecular interactions between bioinspired molecules can enable the development of new materials with higher complexity and innovative properties. Here we report on a dynamic system that emerges from the conformational modification of an elastin-like protein by peptide amphiphiles and with the capacity to access, and be maintained in, non-equilibrium for substantial periods of time. The system enables the formation of a robust membrane that displays controlled assembly and disassembly capabilities, adhesion and sealing to surfaces, self-healing and the capability to undergo morphogenesis into tubular structures with high spatiotemporal control. We use advanced microscopy along with turbidity and spectroscopic measurements to investigate the mechanism of assembly and its relation to the distinctive membrane architecture and the resulting dynamic properties. Using cell-culture experiments with endothelial and adipose-derived stem cells, we demonstrate the potential of this system to generate complex bioactive scaffolds for applications such as tissue engineering.

Brown Mycelial Mat as an Essential Morphological Structure of the Shiitake Medicinal Mushroom Lentinus edodes (Agaricomycetes).

PubMed

Vetchinkina, Elena; Gorshkov, Vladimir; Ageeva, Marina; Gogolev, Yuri; Nikitina, Valentina E

2017-01-01

We show here, to our knowledge for the first time, that the brown mycelial mat of the xylotrophic shiitake medicinal mushroom, Lentinus edodes, not only performs a protective function owing to significant changes in the ultrastructure (thickening of the cell wall, increased density, and pigmentation of the fungal hyphae) but also is a metabolically active stage in the development of the mushroom. The cells of this morphological structure exhibit repeated activation of expression of the genes lcc4, tir, exp1, chi, and exg1, coding for laccase, tyrosinase, a specific transcription factor, chitinase, and glucanase, which are required for fungal growth and morphogenesis. This study revealed the maximum activity of functionally important proteins with phenol oxidase and lectin activities, and the emergence of additional laccases, tyrosinases, and lectins, which are typical of only this stage of morphogenesis and have a regulatory function in the development and formation of fruiting bodies.

Inversin relays Frizzled-8 signals to promote proximal pronephros development

PubMed Central

Lienkamp, Soeren; Ganner, Athina; Boehlke, Christopher; Schmidt, Thorsten; Arnold, Sebastian J.; Schäfer, Tobias; Romaker, Daniel; Schuler, Julia; Hoff, Sylvia; Powelske, Christian; Eifler, Annekathrin; Krönig, Corinna; Bullerkotte, Axel; Nitschke, Roland; Kuehn, E. Wolfgang; Kim, Emily; Burkhardt, Hans; Brox, Thomas; Ronneberger, Olaf; Gloy, Joachim; Walz, Gerd

2010-01-01

Mutations of inversin cause type II nephronophthisis, an infantile autosomal recessive disease characterized by cystic kidney disease and developmental defects. Inversin regulates Wnt signaling and is required for convergent extension movements during early embryogenesis. We now show that Inversin is essential for Xenopus pronephros formation, involving two distinct and opposing forms of cell movements. Knockdown of Inversin abrogated both proximal pronephros extension and distal tubule differentiation, phenotypes similar to that of Xenopus deficient in Frizzled-8. Exogenous Inversin rescued the pronephric defects caused by lack of Frizzled-8, indicating that Inversin acts downstream of Frizzled-8 in pronephros morphogenesis. Depletion of Inversin prevents the recruitment of Dishevelled in response to Frizzled-8 and impeded the accumulation of Dishevelled at the apical membrane of tubular epithelial cells in vivo. Thus, defective tubule morphogenesis seems to contribute to the renal pathology observed in patients with nephronophthisis type II. PMID:21059920

Bile system morphogenesis defects and liver dysfunction upon targeted deletion of HNF1beta.

PubMed

Coffinier, Catherine; Gresh, Lionel; Fiette, Laurence; Tronche, François; Schütz, Günther; Babinet, Charles; Pontoglio, Marco; Yaniv, Moshe; Barra, Jacqueline

2002-04-01

The inactivation of the Hnf1beta gene identified an essential role in epithelial differentiation of the visceral endoderm and resulted in early embryonic death. In the present study, we have specifically inactivated this gene in hepatocytes and bile duct cells using the Cre/loxP system. Mutant animals exhibited severe jaundice caused by abnormalities of the gallbladder and intrahepatic bile ducts (IHBD). The paucity of small IHBD was linked to a failure in the organization of duct structures during liver organogenesis, suggesting an essential function of Hnf1b in bile duct morphogenesis. Mutant mice also lacked interlobular arteries. As HNF1beta is not expressed in these cells, it further emphasizes the link between arterial and biliary formation. Hepatocyte metabolism was also affected and we identified hepatocyte-specific HNF1beta target genes involved in bile acids sensing and in fatty acid oxidation.

Stochastic model of cell rearrangements in convergent extension of ascidian notochord

NASA Astrophysics Data System (ADS)

Lubkin, Sharon; Backes, Tracy; Latterman, Russell; Small, Stephen

2007-03-01

We present a discrete stochastic cell based model of convergent extension of the ascidian notochord. Our work derives from research that clarifies the coupling of invagination and convergent extension in ascidian notochord morphogenesis (Odell and Munro, 2002). We have tested the roles of cell-cell adhesion, cell-extracellular matrix adhesion, random motion, and extension of individual cells, as well as the presence or absence of various tissue types, and determined which factors are necessary and/or sufficient for convergent extension.

Microenvironments and Signaling Pathways Regulating Early Dissemination, Dormancy, and Metastasis

DTIC Science & Technology

2016-09-01

regulators of branching morphogenesis during mammary gland development 17,18, arguing that normal mammary epithelial cells cooperate with these innate ...CD45+CD11b+F4/80+ cells lacking lymphoid and granulocytic markers (Supplementary Fig.3B). viSNE plots 30 of myelo- monocytic cells (Fig.5A) showed that...cancer cells and how the microenvironment in these primary sites named P-TMEM (Primary Tumor Microenvironment of Metastases) contribute to early

Cell-cell and cell-extracellular matrix adhesions cooperate to organize actomyosin networks and maintain force transmission during dorsal closure.

PubMed

Goodwin, Katharine; Lostchuck, Emily E; Cramb, Kaitlyn M L; Zulueta-Coarasa, Teresa; Fernandez-Gonzalez, Rodrigo; Tanentzapf, Guy

2017-05-15

Tissue morphogenesis relies on the coordinated action of actin networks, cell-cell adhesions, and cell-extracellular matrix (ECM) adhesions. Such coordination can be achieved through cross-talk between cell-cell and cell-ECM adhesions. Drosophila dorsal closure (DC), a morphogenetic process in which an extraembryonic tissue called the amnioserosa contracts and ingresses to close a discontinuity in the dorsal epidermis of the embryo, requires both cell-cell and cell-ECM adhesions. However, whether the functions of these two types of adhesions are coordinated during DC is not known. Here we analyzed possible interdependence between cell-cell and cell-ECM adhesions during DC and its effect on the actomyosin network. We find that loss of cell-ECM adhesion results in aberrant distributions of cadherin-mediated adhesions and actin networks in the amnioserosa and subsequent disruption of myosin recruitment and dynamics. Moreover, loss of cell-cell adhesion caused up-regulation of cell-ECM adhesion, leading to reduced cell deformation and force transmission across amnioserosa cells. Our results show how interdependence between cell-cell and cell-ECM adhesions is important in regulating cell behaviors, force generation, and force transmission critical for tissue morphogenesis. © 2017 Goodwin, Lostchuck, et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Successful transplantation of in vitro expanded human cadaver corneal endothelial precursor cells on to a cadaver bovine's eye using a nanocomposite gel sheet.

PubMed

Parikumar, Periyasamy; Haraguchi, Kazutoshi; Ohbayashi, Akira; Senthilkumar, Rajappa; Abraham, Samuel J K

2014-05-01

In vitro expansion of human corneal endothelial precursor (HCEP) cells has been reported via production of cell aggregated spheres. However, to translate this procedure in human patients warrants maintaining the position of the eyeballs facing down for 36 h, which is not feasible. In this study, we report a method using a nanocomposite (NC) gel sheet to accomplish the integration of HCEP cells to the endothelium of cadaver bovine's eyes. HCEP cells were isolated from the corneal endothelium of a cadaver human eye and then expanded using a thermoreversible gelation polymer (TGP) as reported earlier. For the study, three cadaver bovine eyes were used. The NC gel sheets were inserted into the bovine eyes', aligned and suture-fixed in position under the host endothelium. HCEP cells previously expanded in the TGP were harvested and injected using a 26-gauge syringe between the endothelium and the NC gel sheet. The eyes were left undisturbed for three hours following which the NC gel sheets were gently removed. The corneas were harvested and subjected to histopathological studies. Histopathological studies showed that all the three corneas used for NC gel sheet implantation showed the presence of engrafted HCEP cells, seen as multi-layered cells over the native endothelium of the bovine cornea. Examination of the NC gel sheets used for implantation showed that only very few corneal endothelial cells remained on the sheets amounting to what could be considered negligible. The use of the NC gel sheet makes HCEP cell transplantation feasible for human patients. Further in vitro basic studies followed by translational studies are necessary to bring this method for clinical application in appropriate indications.

Mesenchymal stem cell sheets exert anti-stenotic effects in a rat arterial injury model.

PubMed

Homma, Jun; Sekine, Hidekazu; Matsuura, Katsuhisa; Kobayashi, Eiji; Shimizu, Tatsuya

2018-05-04

Restenosis after catheter or surgical intervention substantially affects the prognosis of arterial occlusive disease. Mesenchymal stem cells (MSCs) may have anti-stenotic effects on injured arteries. MSC transplantation from the adventitial side of an artery is safer than endovascular transplantation but has not been extensively examined. In this study, a rat model of femoral artery injury was used to compare the anti-stenotic effects of transplanted cell sheets and transplanted cell suspensions. Rat adipose-derived stem cells (ASCs) were used as the source of MSCs. For both cell sheets and suspensions, 6×106 MSCs were transplanted on the day of arterial injury. MSC sheets attenuated neointimal hyperplasia more than MSC suspensions (intima-to-media ratio in haematoxylin/eosin-stained sections: 0.55±0.13 vs. 1.14±0.12; P<0.05). Cell engraftment (assessed by immunohistochemistry or bioluminescence imaging of luciferase-expressing cells), arterial re-endothelialisation (evaluated by immunohistochemical staining for rat endothelial cell antigen-1) and restriction of vascular smooth muscle cell proliferation in the neointima (double-staining of alpha-smooth muscle actin and phospho-histone H3) were greater when MSC sheets were applied than when MSC suspensions were used. In conclusion, MSC sheets exhibited better anti-stenotic and cell engraftment properties than MSC suspensions. MSC sheet transplantation from the adventitial side is a promising therapy for prevention of arterial restenosis.

3D Printing of Thermo-Responsive Methylcellulose Hydrogels for Cell-Sheet Engineering.

PubMed

Cochis, Andrea; Bonetti, Lorenzo; Sorrentino, Rita; Contessi Negrini, Nicola; Grassi, Federico; Leigheb, Massimiliano; Rimondini, Lia; Farè, Silvia

2018-04-10

A possible strategy in regenerative medicine is cell-sheet engineering (CSE), i.e., developing smart cell culture surfaces from which to obtain intact cell sheets (CS). The main goal of this study was to develop 3D printing via extrusion-based bioprinting of methylcellulose (MC)-based hydrogels. Hydrogels were prepared by mixing MC powder in saline solutions (Na₂SO₄ and PBS). MC-based hydrogels were analyzed to investigate the rheological behavior and thus optimize the printing process parameters. Cells were tested in vitro on ring-shaped printed hydrogels; bulk MC hydrogels were used for comparison. In vitro tests used murine embryonic fibroblasts (NIH/3T3) and endothelial murine cells (MS1), and the resulting cell sheets were characterized analyzing cell viability and immunofluorescence. In terms of CS preparation, 3D printing proved to be an optimal approach to obtain ring-shaped CS. Cell orientation was observed for the ring-shaped CS and was confirmed by the degree of circularity of their nuclei: cell nuclei in ring-shaped CS were more elongated than those in sheets detached from bulk hydrogels. The 3D printing process appears adequate for the preparation of cell sheets of different shapes for the regeneration of complex tissues.

Cultures of Schwann-like cells differentiated from adipose-derived stem cells on PDMS/MWNT sheets as a scaffold for peripheral nerve regeneration.

PubMed

Han, In Ho; Sun, Fangfang; Choi, Yoon Ji; Zou, Fengming; Nam, Kyoung Hyup; Cho, Won Ho; Choi, Byung Kwan; Song, Geun Sung; Koh, Kwangnak; Lee, Jaebeom

2015-11-01

Carbon nanotubes (CNTs) are promising candidates as novel scaffolds for peripheral nerve regeneration. Schwann cells (SCs) are attractive therapeutic targets due to their pivotal role in peripheral nerve regeneration, but primary SCs have limitations for clinical application. However, adipose-derived stem cells (ASCs) may differentiate into Schwann-like cells. The present study assesses the potential applicability of multiwall CNTs (MWNTs) composited with polydimethylsiloxane (PDMS), which were then seeded with differentiated adipose-derived stem cells (dASCs) to promote neuronal differentiation and growth. Aqueous MWNT dispersion was filtered, and the PDMS/MWNT sheets were prepared using a simple printing-transfer method. Characterization of PDMS/MWNT sheets indicated their unique physical properties, such as superior mechanical strength and electroconductivity, compared with bare PDMS sheets. ASCs were differentiated into Schwann-like cells using a mixture of glial growth factors. Dorsal root ganglion (DRG) neurons were co-cultured with SCs and dASCs on PDMS/MWNTs sheets or noncoated dishes. An alamar blue proliferation assay of dASC and SCs showed significantly more dASC and SCs cultured on PDMS/MWNT sheets at 48 h and 72 h than when cultured on noncoated dishes (p < 0.05). Additionally, when DRG were cultured on PDMS/MWNT sheets seeded with dASCs, the proliferation of DRG neurons and the longest neurite outgrowth length per neuron were significantly greater than when DRG were cultured on PDMS/MWNT sheets alone or on noncoated dishes seeded with SCs or dASCs (p < 0.05). Overall, PDMS/MWNT sheets exhibited excellent biocompatibility for culturing Schwann-like cells differentiated from ASCs. Seeding the dASCs on PDMS/MWNT sheets may produce synergistic effects in peripheral nerve regeneration, similarly to SCs. © 2015 Wiley Periodicals, Inc.

The use of cell-sheet technique eliminates arrhythmogenicity of skeletal myoblast-based therapy to the heart with enhanced therapeutic effects.

PubMed

Narita, Takuya; Shintani, Yasunori; Ikebe, Chiho; Kaneko, Masahiro; Harada, Narumi; Tshuma, Nomathamsanqa; Takahashi, Kunihiko; Campbell, Niall G; Coppen, Steven R; Yashiro, Kenta; Sawa, Yoshiki; Suzuki, Ken

2013-09-20

Clinical application of skeletal myoblast transplantation has been curtailed due to arrhythmogenicity and inconsistent therapeutic benefits observed in previous studies. However, these issues may be solved by the use of a new cell-delivery mode. It is now possible to generate "cell-sheets" using temperature-responsive dishes without artificial scaffolds. This study aimed to validate the safety and efficacy of epicardial placement of myoblast-sheets (myoblast-sheet therapy) in treating heart failure. After coronary artery ligation in rats, the same numbers of syngeneic myoblasts were transplanted by intramyocardial injection or cell-sheet placement. Continuous radio-telemetry monitoring detected increased ventricular arrhythmias, including ventricular tachycardia, after intramyocardial injection compared to the sham-control, while these were abolished in myoblast-sheet therapy. This effect was conjunct with avoidance of islet-like cell-cluster formation that disrupts electrical conduction, and with prevention of increased arrhythmogenic substrates due to exaggerated inflammation. Persistent ectopic donor cells were found in the lung only after intramyocardial injection, strengthening the improved safety of myoblast-sheet therapy. In addition, myoblast-sheet therapy enhanced cardiac function, corresponding to a 9.2-fold increase in donor cell survival, compared to intramyocardial injection. Both methods achieved reduced infarct size, decreased fibrosis, attenuated cardiomyocyte hypertrophy, and increased neovascular formation, in association with myocardial upregulation of a group of relevant molecules. The pattern of these beneficial changes was similar between two methods, but the degree was more substantial after myoblast-sheet therapy. The cell-sheet technique enhanced safety and therapeutic efficacy of myoblast-based therapy, compared to the current method, thereby paving the way for clinical application. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

Tissue-engineered thyroid cell sheet rescued hypothyroidism in rat models after receiving total thyroidectomy comparing with nontransplantation models.

PubMed

Arauchi, Ayumi; Shimizu, Tatsuya; Yamato, Masayuki; Obara, Takao; Okano, Teruo

2009-12-01

For hormonal deficiency caused by endocrine organ diseases, continuous oral hormone administration is indispensable to supplement the shortage of hormones. In this study, as a more effective therapy, we have tried to reconstruct the three-dimensional thyroid tissue by the cell sheet technology, a novel tissue engineering approach. The cell suspension obtained from rat thyroid gland was cultured on temperature-responsive culture dishes, from which confluent cells detach as a cell sheet simply by reducing temperature without any enzymatic treatment. The 8-week-old Lewis rats were exposed to total thyroidectomy as hypothyroidism models and received thyroid cell sheet transplantation 1 week after total thyroidectomy. Serum levels of free triiodothyronine (fT(3)) and free thyroxine (fT(4)) significantly decreased 1 week after total thyroidectomy. On the other hand, transplantation of the thyroid cell sheets was able to restore the thyroid function 1 week after the cell sheet transplantation, and improvement was maintained for 4 weeks. Moreover, morphological analyses showed typical thyroid follicle organization, and anti-thyroid-transcription-factor-1 antibody staining demonstrated the presence of follicle epithelial cells. The presence of functional microvessels was also detected within the engineered thyroid tissues. In conclusion, our results indicate that thyroid cell sheets transplanted in a model of total thyroidectomy can reorganize histologically to resemble a typical thyroid gland and restore thyroid function in vivo. In this study, we are the first to confirm that engineered thyroid tissue can repair hypothyroidism models in rats and, therefore, cell sheet transplantation of endocrine organs may be suitable for the therapy of hormonal deficiency.

Morphogenesis checkpoint kinase Swe1 is the executor of lipolysis-dependent cell-cycle progression

PubMed Central

Chauhan, Neha; Visram, Myriam; Cristobal-Sarramian, Alvaro; Sarkleti, Florian

2015-01-01

Cell growth and division requires the precise duplication of cellular DNA content but also of membranes and organelles. Knowledge about the cell-cycle–dependent regulation of membrane and storage lipid homeostasis is only rudimentary. Previous work from our laboratory has shown that the breakdown of triacylglycerols (TGs) is regulated in a cell-cycle–dependent manner, by activation of the Tgl4 lipase by the major cyclin-dependent kinase Cdc28. The lipases Tgl3 and Tgl4 are required for efficient cell-cycle progression during the G1/S (Gap1/replication phase) transition, at the onset of bud formation, and their absence leads to a cell-cycle delay. We now show that defective lipolysis activates the Swe1 morphogenesis checkpoint kinase that halts cell-cycle progression by phosphorylation of Cdc28 at tyrosine residue 19. Saturated long-chain fatty acids and phytosphingosine supplementation rescue the cell-cycle delay in the Tgl3/Tgl4 lipase-deficient strain, suggesting that Swe1 activity responds to imbalanced sphingolipid metabolism, in the absence of TG degradation. We propose a model by which TG-derived sphingolipids are required to activate the protein phosphatase 2A (PP2ACdc55) to attenuate Swe1 phosphorylation and its inhibitory effect on Cdc28 at the G1/S transition of the cell cycle. PMID:25713391

Morphogenesis checkpoint kinase Swe1 is the executor of lipolysis-dependent cell-cycle progression.

PubMed

Chauhan, Neha; Visram, Myriam; Cristobal-Sarramian, Alvaro; Sarkleti, Florian; Kohlwein, Sepp D

2015-03-10

Cell growth and division requires the precise duplication of cellular DNA content but also of membranes and organelles. Knowledge about the cell-cycle-dependent regulation of membrane and storage lipid homeostasis is only rudimentary. Previous work from our laboratory has shown that the breakdown of triacylglycerols (TGs) is regulated in a cell-cycle-dependent manner, by activation of the Tgl4 lipase by the major cyclin-dependent kinase Cdc28. The lipases Tgl3 and Tgl4 are required for efficient cell-cycle progression during the G1/S (Gap1/replication phase) transition, at the onset of bud formation, and their absence leads to a cell-cycle delay. We now show that defective lipolysis activates the Swe1 morphogenesis checkpoint kinase that halts cell-cycle progression by phosphorylation of Cdc28 at tyrosine residue 19. Saturated long-chain fatty acids and phytosphingosine supplementation rescue the cell-cycle delay in the Tgl3/Tgl4 lipase-deficient strain, suggesting that Swe1 activity responds to imbalanced sphingolipid metabolism, in the absence of TG degradation. We propose a model by which TG-derived sphingolipids are required to activate the protein phosphatase 2A (PP2A(Cdc55)) to attenuate Swe1 phosphorylation and its inhibitory effect on Cdc28 at the G1/S transition of the cell cycle.

Rap1 GTPase is required for mouse lens epithelial maintenance and morphogenesis

PubMed Central

Maddala, Rupalatha; Nagendran, Tharkika; Lang, Richard A.; Morozov, Alexei; Rao, Ponugoti V.

2015-01-01

Rap1, a Ras-like small GTPase, plays a crucial role in cell-matrix adhesive interactions, cell-cell junction formation, cell polarity and migration. The role of Rap1 in vertebrate organ development and tissue architecture, however, remains elusive. We addressed this question in a mouse lens model system using a conditional gene targeting approach. While individual germline deficiency of either Rap1a or Rap1b did not cause overt defects in mouse lens, conditional double deficiency (Rap1 cKO) prior to lens placode formation led to an ocular phenotype including microphthalmia and lens opacification in embryonic mice. The embryonic Rap1 cKO mouse lens exhibited striking defects including loss of E-cadherin- and ZO-1-based cell-cell junctions, disruption of paxillin and β1-integrin-based cell adhesive interactions along with abnormalities in cell shape and apical-basal polarity of epithelium. These epithelial changes were accompanied by increased levels of α-smooth muscle actin, vimentin and N-cadherin, and expression of transcriptional suppressors of E-cadherin (Snai1, Slug and Zeb2), and a mesenchymal metabolic protein (Dihydropyrimidine dehydrogenase). Additionally, while lens differentiation was not overtly affected, increased apoptosis and dysregulated cell cycle progression were noted in epithelium and fibers in Rap1 cKO mice. Collectively these observations uncover a requirement for Rap1 in maintenance of lens epithelial phenotype and morphogenesis. PMID:26212757

Endothelial-monocyte activating polypeptide II disrupts alveolar epithelial type II to type I cell transdifferentiation

PubMed Central

2012-01-01

Background Distal alveolar morphogenesis is marked by differentiation of alveolar type (AT)-II to AT-I cells that give rise to the primary site of gas exchange, the alveolar/vascular interface. Endothelial-Monocyte Activating Polypeptide (EMAP) II, an endogenous protein with anti-angiogenic properties, profoundly disrupts distal lung neovascularization and alveolar formation during lung morphogenesis, and is robustly expressed in the dysplastic alveolar regions of infants with Bronchopulmonary dysplasia. Determination as to whether EMAP II has a direct or indirect affect on ATII→ATI trans-differentiation has not been explored. Method In a controlled nonvascular environment, an in vitro model of ATII→ATI cell trans-differentiation was utilized to demonstrate the contribution that one vascular mediator has on distal epithelial cell differentiation. Results Here, we show that EMAP II significantly blocked ATII→ATI cell transdifferentiation by increasing cellular apoptosis and inhibiting expression of ATI markers. Moreover, EMAP II-treated ATII cells displayed myofibroblast characteristics, including elevated cellular proliferation, increased actin cytoskeleton stress fibers and Rho-GTPase activity, and increased nuclear:cytoplasmic volume. However, EMAP II-treated cells did not express the myofibroblast markers desmin or αSMA. Conclusion Our findings demonstrate that EMAP II interferes with ATII → ATI transdifferentiation resulting in a proliferating non-myofibroblast cell. These data identify the transdifferentiating alveolar cell as a possible target for EMAP II's induction of alveolar dysplasia. PMID:22214516

Alpha-catenin-dependent recruitment of the centrosomal protein CAP350 to adherens junctions allows epithelial cells to acquire a columnar shape.

PubMed

Gavilan, Maria P; Arjona, Marina; Zurbano, Angel; Formstecher, Etienne; Martinez-Morales, Juan R; Bornens, Michel; Rios, Rosa M

2015-03-01

Epithelial morphogenesis involves a dramatic reorganisation of the microtubule cytoskeleton. How this complex process is controlled at the molecular level is still largely unknown. Here, we report that the centrosomal microtubule (MT)-binding protein CAP350 localises at adherens junctions in epithelial cells. By two-hybrid screening, we identified a direct interaction of CAP350 with the adhesion protein α-catenin that was further confirmed by co-immunoprecipitation experiments. Block of epithelial cadherin (E-cadherin)-mediated cell-cell adhesion or α-catenin depletion prevented CAP350 localisation at cell-cell junctions. Knocking down junction-located CAP350 inhibited the establishment of an apico-basal array of microtubules and impaired the acquisition of columnar shape in Madin-Darby canine kidney II (MDCKII) cells grown as polarised epithelia. Furthermore, MDCKII cystogenesis was also defective in junctional CAP350-depleted cells. CAP350-depleted MDCKII cysts were smaller and contained either multiple lumens or no lumen. Membrane polarity was not affected, but cortical microtubule bundles did not properly form. Our results indicate that CAP350 may act as an adaptor between adherens junctions and microtubules, thus regulating epithelial differentiation and contributing to the definition of cell architecture. We also uncover a central role of α-catenin in global cytoskeleton remodelling, in which it acts not only on actin but also on MT reorganisation during epithelial morphogenesis.

The Rap GTPase Activator Drosophila PDZ-GEF Regulates Cell Shape in Epithelial Migration and Morphogenesis▿

PubMed Central

Boettner, Benjamin; Van Aelst, Linda

2007-01-01

Epithelial morphogenesis is characterized by an exquisite control of cell shape and position. Progression through dorsal closure in Drosophila gastrulation depends on the ability of Rap1 GTPase to signal through the adherens junctional multidomain protein Canoe. Here, we provide genetic evidence that epithelial Rap activation and Canoe effector usage are conferred by the Drosophila PDZ-GEF (dPDZ-GEF) exchange factor. We demonstrate that dPDZ-GEF/Rap/Canoe signaling modulates cell shape and apicolateral cell constriction in embryonic and wing disc epithelia. In dPDZ-GEF mutant embryos with strong dorsal closure defects, cells in the lateral ectoderm fail to properly elongate. Postembryonic dPDZ-GEF mutant cells generated in mosaic tissue display a striking extension of lateral cell perimeters in the proximity of junctional complexes, suggesting a loss of normal cell contractility. Furthermore, our data indicate that dPDZ-GEF signaling is linked to myosin II function. Both dPDZ-GEF and cno show strong genetic interactions with the myosin II-encoding gene, and myosin II distribution is severely perturbed in epithelia of both mutants. These findings provide the first insight into the molecular machinery targeted by Rap signaling to modulate epithelial plasticity. We propose that dPDZ-GEF-dependent signaling functions as a rheostat linking Rap activity to the regulation of cell shape in epithelial morphogenesis at different developmental stages. PMID:17846121

Distinct roles of cell wall biogenesis in yeast morphogenesis as revealed by multivariate analysis of high-dimensional morphometric data

PubMed Central

Okada, Hiroki; Ohnuki, Shinsuke; Roncero, Cesar; Konopka, James B.; Ohya, Yoshikazu

2014-01-01

The cell wall of budding yeast is a rigid structure composed of multiple components. To thoroughly understand its involvement in morphogenesis, we used the image analysis software CalMorph to quantitatively analyze cell morphology after treatment with drugs that inhibit different processes during cell wall synthesis. Cells treated with cell wall–affecting drugs exhibited broader necks and increased morphological variation. Tunicamycin, which inhibits the initial step of N-glycosylation of cell wall mannoproteins, induced morphologies similar to those of strains defective in α-mannosylation. The chitin synthase inhibitor nikkomycin Z induced morphological changes similar to those of mutants defective in chitin transglycosylase, possibly due to the critical role of chitin in anchoring the β-glucan network. To define the mode of action of echinocandin B, a 1,3-β-glucan synthase inhibitor, we compared the morphology it induced with mutants of Fks1 that contains the catalytic domain for 1,3-β-glucan synthesis. Echinocandin B exerted morphological effects similar to those observed in some fks1 mutants, with defects in cell polarity and reduced glucan synthesis activity, suggesting that echinocandin B affects not only 1,3-β-glucan synthesis, but also another functional domain. Thus our multivariate analyses reveal discrete functions of cell wall components and increase our understanding of the pharmacology of antifungal drugs. PMID:24258022

Alpha-catenin-Dependent Recruitment of the Centrosomal Protein CAP350 to Adherens Junctions Allows Epithelial Cells to Acquire a Columnar Shape

PubMed Central

Zurbano, Angel; Formstecher, Etienne; Martinez-Morales, Juan R.; Bornens, Michel; Rios, Rosa M.

2015-01-01

Epithelial morphogenesis involves a dramatic reorganisation of the microtubule cytoskeleton. How this complex process is controlled at the molecular level is still largely unknown. Here, we report that the centrosomal microtubule (MT)-binding protein CAP350 localises at adherens junctions in epithelial cells. By two-hybrid screening, we identified a direct interaction of CAP350 with the adhesion protein α-catenin that was further confirmed by co-immunoprecipitation experiments. Block of epithelial cadherin (E-cadherin)-mediated cell-cell adhesion or α-catenin depletion prevented CAP350 localisation at cell-cell junctions. Knocking down junction-located CAP350 inhibited the establishment of an apico-basal array of microtubules and impaired the acquisition of columnar shape in Madin-Darby canine kidney II (MDCKII) cells grown as polarised epithelia. Furthermore, MDCKII cystogenesis was also defective in junctional CAP350-depleted cells. CAP350-depleted MDCKII cysts were smaller and contained either multiple lumens or no lumen. Membrane polarity was not affected, but cortical microtubule bundles did not properly form. Our results indicate that CAP350 may act as an adaptor between adherens junctions and microtubules, thus regulating epithelial differentiation and contributing to the definition of cell architecture. We also uncover a central role of α-catenin in global cytoskeleton remodelling, in which it acts not only on actin but also on MT reorganisation during epithelial morphogenesis. PMID:25764135

Overexpression of Robo2 causes defects in the recruitment of metanephric mesenchymal cells and ureteric bud branching morphogenesis

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

Ji, Jiayao; Medical College of NanKai University, Tianjin; Li, Qinggang

2012-05-11

Highlights: Black-Right-Pointing-Pointer Overexpression of Robo2 caused reduced UB branching and glomerular number. Black-Right-Pointing-Pointer Fewer MM cells surrounding the UB after overexpression of Robo2 in vitro. Black-Right-Pointing-Pointer No abnormal Epithelial Morphology of UB or apoptosis of mm cells in the kidney. Black-Right-Pointing-Pointer Overexpression of Robo2 affected MM cells migration and caused UB deficit. Black-Right-Pointing-Pointer The reduced glomerular number can also be caused by fewer MM cells. -- Abstract: Roundabout 2 (Robo2) is a member of the membrane protein receptor family. The chemorepulsive effect of Slit2-Robo2 signaling plays vital roles in nervous system development and neuron migration. Slit2-Robo2 signaling is also importantmore » for maintaining the normal morphogenesis of the kidney and urinary collecting system, especially for the branching of the ureteric bud (UB) at the proper site. Slit2 or Robo2 mouse mutants exhibit multilobular kidneys, multiple ureters, and dilatation of the ureter, renal pelvis, and collecting duct system, which lead to vesicoureteral reflux. To understand the effect of Robo2 on kidney development, we used microinjection and electroporation to overexpress GFP-Robo2 in an in vitro embryonic kidney model. Our results show reduced UB branching and decreased glomerular number after in vitro Robo2 overexpression in the embryonic kidneys. We found fewer metanephric mesenchymal (MM) cells surrounding the UB but no abnormal morphology in the branching epithelial UB. Meanwhile, no significant change in MM proliferation or apoptosis was observed. These findings indicate that Robo2 is involved in the development of embryonic kidneys and that the normal expression of Robo2 can help maintain proper UB branching and glomerular morphogenesis. Overexpression of Robo2 leads to reduced UB branching caused by fewer surrounding MM cells, but MM cell apoptosis is not involved in this effect. Our study demonstrates that overexpression of Robo2 by microinjection in embryonic kidneys is an effective approach to study the function of Robo2.« less

Silk Film Topography Directs Collective Epithelial Cell Migration

PubMed Central

Rosenblatt, Mark I.

2012-01-01

The following study provides new insight into how surface topography dictates directed collective epithelial cell sheet growth through the guidance of individual cell movement. Collective cell behavior of migrating human corneal limbal-epithelial cell sheets were studied on highly biocompatible flat and micro-patterned silk film surfaces. The silk film edge topography guided the migratory direction of individual cells making up the collective epithelial sheet, which resulted in a 75% increase in total culture elongation. This was due to a 3-fold decrease in cell sheet migration rate efficiency for movement perpendicular to the topography edge. Individual cell migration direction is preferred in the parallel approach to the edge topography where localization of cytoskeletal proteins to the topography’s edge region is reduced, which results in the directed growth of the collective epithelial sheet. Findings indicate customized biomaterial surfaces may be created to direct both the migration rate and direction of tissue epithelialization. PMID:23185573

In Silico Dynamics: computer simulation in a Virtual Embryo (SOT)

EPA Science Inventory

Abstract: Utilizing cell biological information to predict higher order biological processes is a significant challenge in predictive toxicology. This is especially true for highly dynamical systems such as the embryo where morphogenesis, growth and differentiation require preci...

Inhibition of breast tumor growth and angiogenesis by a medicinal herb: Ocimum sanctum

PubMed Central

Nangia-Makker, Pratima; Tait, Larry; Hogan, Victor; Shekhar, Malathy P.V.; Funasaka, Tatsuyoshi; Raz, Avraham

2013-01-01

Ocimum sanctum (OS) is a traditionally used medicinal herb, which shows anti-oxidant, anti-carcinogenic, radio-protective and free radical scavenging properties. So far no detailed studies have been reported on its effects on human cancers. Thus, we analyzed its effects on human breast cancer utilizing in vitro and in vivo methodologies. Aqueous extracts were prepared from the mature leaves of Ocimum sanctum cultivated devoid of pesticides. Tumor progression and angiogenesis related processes like chemotaxis, proliferation, apoptosis, 3-dimensional growth and morphogenesis, angiogenesis, and tumor growth were studied in the presence or absence of the extract and in some experiments a comparison was made with purified commercially available eugenol, apigenin and ursolic acid. Aqueous OS leaf extract inhibits proliferation, migration, anchorage independent growth, three dimensional growth and morphogenesis, and induction of COX-2 protein in breast cancer cells. A comparative analysis with eugenol, apigenin and ursolic acid showed that the inhibitory effects on chemotaxis and three dimensional morphogenesis of breast cancer cells were specific to OS extract. In addition, OS extracts also reduced tumor size and neoangiogenesis in a MCF10 DCIS.com xenograft model of human DCIS. This is the first detailed report showing that OS leaf extract may be of value as a breast cancer preventive and therapeutic agent and might be considered as additional additive in the arsenal of components aiming at combating breast cancer progression and metastasis. PMID:17437270

3D bioprinting matrices with controlled pore structure and release function guide in vitro self-organization of sweat gland.

PubMed

Liu, Nanbo; Huang, Sha; Yao, Bin; Xie, Jiangfan; Wu, Xu; Fu, Xiaobing

2016-10-03

3D bioprinting matrices are novel platforms for tissue regeneration. Tissue self-organization is a critical process during regeneration that implies the features of organogenesis. However, it is not clear from the current evidences whether 3D printed construct plays a role in guiding tissue self-organization in vitro. Based on our previous study, we bioprinted a 3D matrix as the restrictive niche for direct sweat gland differentiation of epidermal progenitors by different pore structure (300-μm or 400-μm nozzle diameters printed) and reported a long-term gradual transition of differentiated cells into glandular morphogenesis occurs within the 3D construct in vitro. At the initial 14-day culture, an accelerated cell differentiation was achieved with inductive cues released along with gelatin reduction. After protein release completed, the 3D construct guide the self-organized formation of sweat gland tissues, which is similar to that of the natural developmental process. However, glandular morphogenesis was only observed in 300-μm-printed constructs. In the absence of 3D architectural support, glandular morphogenesis was not occurred. This striking finding made us to identify a previously unknown role of the 3D-printed structure in glandular tissue regeneration, and this self-organizing strategy can be applied to forming other tissues in vitro.

Role of Wnt5a-Ror2 Signaling in Morphogenesis of the Metanephric Mesenchyme during Ureteric Budding

PubMed Central

Qiao, Sen; Miyamoto, Mari; Okinaka, Yuka; Yamada, Makiko; Hashimoto, Ryuju; Iijima, Kazumoto; Otani, Hiroki; Hartmann, Christine; Nishinakamura, Ryuichi

2014-01-01

Development of the metanephric kidney begins with the induction of a single ureteric bud (UB) on the caudal Wolffian duct (WD) in response to GDNF (glial cell line-derived neurotrophic factor) produced by the adjacent metanephric mesenchyme (MM). Mutual interaction between the UB and MM maintains expression of GDNF in the MM, thereby supporting further outgrowth and branching morphogenesis of the UB, while the MM also grows and aggregates around the branched tips of the UB. Ror2, a member of the Ror family of receptor tyrosine kinases, has been shown to act as a receptor for Wnt5a to mediate noncanonical Wnt signaling. We show that Ror2 is predominantly expressed in the MM during UB induction and that Ror2- and Wnt5a-deficient mice exhibit duplicated ureters and kidneys due to ectopic UB induction. During initial UB formation, these mutant embryos show dysregulated positioning of the MM, resulting in spatiotemporally aberrant interaction between the MM and WD, which provides the WD with inappropriate GDNF signaling. Furthermore, the numbers of proliferating cells in the mutant MM are markedly reduced compared to the wild-type MM. These results indicate an important role of Wnt5a-Ror2 signaling in morphogenesis of the MM to ensure proper epithelial tubular formation of the UB required for kidney development. PMID:24891614

Calcium signals act through histone deacetylase to mediate pronephric kidney morphogenesis.

PubMed

Rothschild, Sarah C; Lee, Hunter J; Ingram, Sarah R; Mohammadi, Daniel K; Walsh, Gregory S; Tombes, Robert M

2018-06-01

Autosomal dominant polycystic kidney disease is the most common monogenetic kidney disorder and is linked to mutations in PKD1 and PKD2. PKD2, a Ca 2+ -conducting TRP channel enriched in ciliated cells and gated by extracellular signals, is necessary to activate the multifunctional Ca 2+/ calmodulin-dependent protein kinase type 2 (CaMK-II), enabling kidney morphogenesis and cilia stability. In this study, antisense morpholino oligonucleotides and pharmacological compounds were employed to investigate the roles of class II HDAC family members (HDAC 4, 5, and 6) in Zebrafish kidney development. While all three class II HDAC genes were expressed throughout the embryo during early development, HDAC5-morphant embryos exhibited anterior cysts and destabilized cloacal cilia, similar to PKD2 and CaMK-II morphants. In contrast, HDAC4-morphant embryos exhibited elongated cloacal cilia and lacked anterior kidney defects. Suppression of HDAC4 partially reversed the cilia shortening and anterior convolution defects caused by CaMK-II deficiency, whereas HDAC5 loss exacerbated these defects. EGFP-HDAC4, but not EGFP-HDAC5, translocated into the nucleus upon CaMK-II suppression in pronephric kidney cells. These results support a model by which activated CaMK-II sequesters HDAC4 in the cytosol to enable primary cilia formation and kidney morphogenesis. Developmental Dynamics 247:807-817, 2018. © 2018 Wiley Periodicals, Inc. © 2018 Wiley Periodicals, Inc.

Morphogenetic Studies of the Drosophila DA1 Ventral Olfactory Projection Neuron

PubMed Central

Yu, Hung-Hsiang

2016-01-01

In the Drosophila olfactory system, odorant information is sensed by olfactory sensory neurons and relayed from the primary olfactory center, the antennal lobe (AL), to higher olfactory centers via olfactory projection neurons (PNs). A major portion of the AL is constituted with dendrites of four groups of PNs, anterodorsal PNs (adPNs), lateral PNs (lPNs), lateroventral PNs (lvPNs) and ventral PNs (vPNs). Previous studies have been focused on the development and function of adPNs and lPNs, while the investigation on those of lvPNs and vPNs received less attention. Here, we study the molecular and cellular mechanisms underlying the morphogenesis of a putative male-pheromone responding vPN, the DA1 vPN. Using an intersection strategy to remove background neurons labeled within a DA1 vPN-containing GAL4 line, we depicted morphological changes of the DA1 vPN that occurs at the pupal stage. We then conducted a pilot screen using RNA interference knock-down approach to identify cell surface molecules, including Down syndrome cell adhesion molecule 1 and Semaphorin-1a, that might play essential roles for the DA1 vPN morphogenesis. Taken together, by revealing molecular and cellular basis of the DA1 vPN morphogenesis, we should provide insights into future comprehension of how vPNs are assembled into the olfactory neural circuitry. PMID:27163287

Morphogenetic Studies of the Drosophila DA1 Ventral Olfactory Projection Neuron.

PubMed

Shen, Hung-Chang; Wei, Jia-Yi; Chu, Sao-Yu; Chung, Pei-Chi; Hsu, Tsai-Chi; Yu, Hung-Hsiang

2016-01-01

In the Drosophila olfactory system, odorant information is sensed by olfactory sensory neurons and relayed from the primary olfactory center, the antennal lobe (AL), to higher olfactory centers via olfactory projection neurons (PNs). A major portion of the AL is constituted with dendrites of four groups of PNs, anterodorsal PNs (adPNs), lateral PNs (lPNs), lateroventral PNs (lvPNs) and ventral PNs (vPNs). Previous studies have been focused on the development and function of adPNs and lPNs, while the investigation on those of lvPNs and vPNs received less attention. Here, we study the molecular and cellular mechanisms underlying the morphogenesis of a putative male-pheromone responding vPN, the DA1 vPN. Using an intersection strategy to remove background neurons labeled within a DA1 vPN-containing GAL4 line, we depicted morphological changes of the DA1 vPN that occurs at the pupal stage. We then conducted a pilot screen using RNA interference knock-down approach to identify cell surface molecules, including Down syndrome cell adhesion molecule 1 and Semaphorin-1a, that might play essential roles for the DA1 vPN morphogenesis. Taken together, by revealing molecular and cellular basis of the DA1 vPN morphogenesis, we should provide insights into future comprehension of how vPNs are assembled into the olfactory neural circuitry.

Functional Role of the microRNA-200 Family in Breast Morphogenesis and Neoplasia

PubMed Central

Hilmarsdottir, Bylgja; Briem, Eirikur; Bergthorsson, Jon Thor; Magnusson, Magnus Karl; Gudjonsson, Thorarinn

2014-01-01

Branching epithelial morphogenesis is closely linked to epithelial-to-mesenchymal transition (EMT), a process important in normal development and cancer progression. The miR-200 family regulates epithelial morphogenesis and EMT through a negative feedback loop with the ZEB1 and ZEB2 transcription factors. miR-200 inhibits expression of ZEB1/2 mRNA, which in turn can down-regulate the miR-200 family that further results in down-regulation of E-cadherin and induction of a mesenchymal phenotype. Recent studies show that the expression of miR-200 genes is high during late pregnancy and lactation, thereby indicating that these miRs are important for breast epithelial morphogenesis and differentiation. miR-200 genes have been studied intensively in relation to breast cancer progression and metastasis, where it has been shown that miR-200 members are down-regulated in basal-like breast cancer where the EMT phenotype is prominent. There is growing evidence that the miR-200 family is up-regulated in distal breast metastasis indicating that these miRs are important for colonization of metastatic breast cancer cells through induction of mesenchymal to epithelial transition. The dual role of miR-200 in primary and metastatic breast cancer is of interest for future therapeutic interventions, making it important to understand its role and interacting partners in more detail. PMID:25216122

Characterization and role of p53 family members in the symbiont-induced morphogenesis of the Euprymna scolopes light organ.

PubMed

Goodson, Michael S; Crookes-Goodson, Wendy J; Kimbell, Jennifer R; McFall-Ngai, Margaret J

2006-08-01

Within hours of hatching, the squid Euprymna scolopes forms a specific light organ symbiosis with the marine luminous bacterium Vibrio fischeri. Interactions with the symbiont result in the loss of a complex ciliated epithelium dedicated to promoting colonization of host tissue, and some or all of this loss is due to widespread, symbiont-induced apoptosis. Members of the p53 family, including p53, p63, and p73, are conserved across broad phyletic lines and p63 is thought to be the ancestral gene. These proteins have been shown to induce apoptosis and developmental morphogenesis. In this study, we characterized p63-like transcripts from mRNA isolated from the symbiotic tissues of E. scolopes and described their role in symbiont-induced morphogenesis. Using degenerate RT-PCR and RACE PCR, we identified two p63-like transcripts encoding proteins of 431 and 567 amino acids. These transcripts shared identical nucleotides where they overlapped, suggesting that they are splice variants of the same gene. Immunocytochemistry and Western blots using an antibody specific for E. scolopes suggested that the p53 family members are activated in cells of the symbiont-harvesting structures of the symbiotic light organ. We propose that once the symbiosis is initiated, a symbiont-induced signal activates p53 family members, inducing apoptosis and developmental morphogenesis of the light organ.

Stacked endoplasmic reticulum sheets are connected by helicoidal membrane motifs

PubMed Central

Terasaki, Mark; Shemesh, Tom; Kasthuri, Narayanan; Klemm, Robin W.; Schalek, Richard; Hayworth, Kenneth J.; Hand, Arthur R.; Yankova, Maya; Huber, Greg; Lichtman, Jeff W.; Rapoport, Tom A.; Kozlov, Michael M.

2013-01-01

The endoplasmic reticulum (ER) often forms stacked membrane sheets, an arrangement that is likely required to accommodate a maximum of membrane-bound polysomes for secretory protein synthesis. How sheets are stacked is unknown. Here, we used novel staining and automated ultra-thin sectioning electron microscopy methods to analyze stacked ER sheets in neuronal cells and secretory salivary gland cells of mice. Our results show that stacked ER sheets form a continuous membrane system in which the sheets are connected by twisted membrane surfaces with helical edges of left- or right-handedness. The three-dimensional structure of tightly stacked ER sheets resembles a parking garage, in which the different levels are connected by helicoidal ramps. A theoretical model explains the experimental observations and indicates that the structure corresponds to a minimum of elastic energy of sheet edges and surfaces. The structure allows the dense packing of ER sheets in the restricted space of a cell. PMID:23870120

Retinal pigment epithelium expansion around the neural retina occurs in two separate phases with distinct mechanisms.

PubMed

Cechmanek, Paula Bernice; McFarlane, Sarah

2017-08-01

The retinal pigment epithelium (RPE) is a specialized monolayer of epithelial cells that forms a tight barrier surrounding the neural retina. RPE cells are indispensable for mature photoreceptor renewal and survival, yet how the initial RPE cell population expands around the neural retina during eye development is poorly understood. Here we characterize the differentiation, proliferation, and movements of RPE progenitors in the Zebrafish embryo over the period of optic cup morphogenesis. RPE progenitors are present in the dorsomedial eye vesicle shortly after eye vesicle evagination. We define two separate phases that allow for full RPE expansion. The first phase involves a previously uncharacterized antero-wards expansion of the RPE progenitor domain in the inner eye vesicle leaflet, driven largely by an increase in cell number. During this phase, RPE progenitors start to express differentiation markers. In the second phase, the progenitor domain stretches in the dorsoventral and posterior axes, involving cell movements and shape changes, and coinciding with optic cup morphogenesis. Significantly, cell division is not required for RPE expansion. RPE development to produce the monolayer epithelium that covers the back of the neural retina occurs in two distinct phases driven by distinct mechanisms. Developmental Dynamics 246:598-609, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Regulation of cell wall morphogenesis in Bacillus subtilis by recruitment of PBP1 to the MreB helix.

PubMed

Kawai, Yoshikazu; Daniel, Richard A; Errington, Jeffery

2009-03-01

The bacterial actin homologue MreB plays a key role in cell morphogenesis. In Bacillus subtilis MreB is essential under normal growth conditions and mreB mutants are defective in the control of cell diameter. However, the precise role of MreB is still unclear. Analysis of the lethal phenotypic consequences of mreB disruption revealed an unusual bulging phenotype that precedes cell death. A similar phenotype was seen in wild-type cells at very low Mg(2+) concentrations. We found that inactivation of the major bi-functional penicillin-binding protein (PBP) PBP1 of B. subtilis restored the viability of an mreB null mutant as well as preventing bulging in both mutant and wild-type backgrounds. Bulging was associated with delocalization of PBP1. We show that the normal pattern of localization of PBP1 is dependent on MreB and that the proteins can physically interact using in vivo pull-down and bacterial two-hybrid approaches. Interactions between MreB and several other PBPs were also detected. Our results suggest that MreB filaments associate directly with the peptidoglycan biosynthetic machinery in B. subtilis as part of the mechanism that brings about controlled cell elongation.

Rules and Self-Organizing Properties of Post-embryonic Plant Organ Cell Division Patterns.

PubMed

von Wangenheim, Daniel; Fangerau, Jens; Schmitz, Alexander; Smith, Richard S; Leitte, Heike; Stelzer, Ernst H K; Maizel, Alexis

2016-02-22

Plants form new organs with patterned tissue organization throughout their lifespan. It is unknown whether this robust post-embryonic organ formation results from stereotypic dynamic processes, in which the arrangement of cells follows rigid rules. Here, we combine modeling with empirical observations of whole-organ development to identify the principles governing lateral root formation in Arabidopsis. Lateral roots derive from a small pool of founder cells in which some take a dominant role as seen by lineage tracing. The first division of the founders is asymmetric, tightly regulated, and determines the formation of a layered structure. Whereas the pattern of subsequent cell divisions is not stereotypic between different samples, it is characterized by a regular switch in division plane orientation. This switch is also necessary for the appearance of patterned layers as a result of the apical growth of the primordium. Our data suggest that lateral root morphogenesis is based on a limited set of rules. They determine cell growth and division orientation. The organ-level coupling of the cell behavior ensures the emergence of the lateral root's characteristic features. We propose that self-organizing, non-deterministic modes of development account for the robustness of plant organ morphogenesis. Copyright © 2016 Elsevier Ltd. All rights reserved.

A cell-body groove housing the new flagellum tip suggests an adaptation of cellular morphogenesis for parasitism in the bloodstream form of Trypanosoma brucei.

PubMed

Hughes, Louise; Towers, Katie; Starborg, Tobias; Gull, Keith; Vaughan, Sue

2013-12-15

Flagella are highly conserved organelles present in a wide variety of species. In Trypanosoma brucei the single flagellum is necessary for morphogenesis, cell motility and pathogenesis, and is attached along the cell body. A new flagellum is formed alongside the old during the cell division cycle. In the (insect) procyclic form, the flagella connector (FC) attaches the tip of the new flagellum to the side of the old flagellum, ensuring faithful replication of cell architecture. The FC is not present in the bloodstream form of the parasite. We show here, using new imaging techniques including serial block-face scanning electron microscopy (SBF-SEM), that the distal tip of the new flagellum in the bloodstream form is embedded within an invagination in the cell body plasma membrane, named the groove. We suggest that the groove has a similar function to the flagella connector. The groove is a mobile junction located alongside the microtubule quartet (MtQ) and occurred within a gap in the subpellicular microtubule corset, causing significant modification of microtubules during elongation of the new flagellum. It appears likely that this novel form of morphogenetic structure has evolved to withstand the hostile immune response in the mammalian blood.

A Robust Method to Generate Mechanically Anisotropic Vascular Smooth Muscle Cell Sheets for Vascular Tissue Engineering.

PubMed

Backman, Daniel E; LeSavage, Bauer L; Shah, Shivem B; Wong, Joyce Y

2017-06-01

In arterial tissue engineering, mimicking native structure and mechanical properties is essential because compliance mismatch can lead to graft failure and further disease. With bottom-up tissue engineering approaches, designing tissue components with proper microscale mechanical properties is crucial to achieve the necessary macroscale properties in the final implant. This study develops a thermoresponsive cell culture platform for growing aligned vascular smooth muscle cell (VSMC) sheets by photografting N-isopropylacrylamide (NIPAAm) onto micropatterned poly(dimethysiloxane) (PDMS). The grafting process is experimentally and computationally optimized to produce PNIPAAm-PDMS substrates optimal for VSMC attachment. To allow long-term VSMC sheet culture and increase the rate of VSMC sheet formation, PNIPAAm-PDMS surfaces were further modified with 3-aminopropyltriethoxysilane yielding a robust, thermoresponsive cell culture platform for culturing VSMC sheets. VSMC cell sheets cultured on patterned thermoresponsive substrates exhibit cellular and collagen alignment in the direction of the micropattern. Mechanical characterization of patterned, single-layer VSMC sheets reveals increased stiffness in the aligned direction compared to the perpendicular direction whereas nonpatterned cell sheets exhibit no directional dependence. Structural and mechanical anisotropy of aligned, single-layer VSMC sheets makes this platform an attractive microstructural building block for engineering a vascular graft to match the in vivo mechanical properties of native arterial tissue. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Effect of cryopreservation on proliferation and differentiation of periodontal ligament stem cell sheets.

PubMed

Li, Mengying; Feng, Cheng; Gu, Xiuge; He, Qin; Wei, Fulan

2017-04-17

Cryopreservation has been extensively applied to the long-term storage of a diverse range of biological materials. However, no comprehensive study is currently available on the cryopreservation of periodontal ligament stem cell (PDLSC) sheets which have been suggested as excellent transplant materials for periodontal tissue regeneration. The aim of this study is to investigate the effect of cryopreservation on the structural integrity and functional viability of PDLSC sheets. PDLSC sheets prepared from extracted human molars were divided into two groups: the cryopreservation group (cPDLSC sheets) and the freshly prepared control group (fPDLSC sheets). The cPDLSC sheets were cryopreserved in a solution consisting of 90% fetal bovine serum and 10% dimethyl sulfoxide for 3 months. Cell viability and cell proliferation rates of PDLSCs in both groups were evaluated by cell viability assay and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, respectively. The multilineage differentiation potentials of the cells were assessed by von Kossa staining and Oil Red O staining. The chromosomal stability was examined by karyotype analysis. Moreover, the cell sheets in each group were transplanted subcutaneously into the dorsal site of nude mice, after which Sirius Red staining was performed to analyze the efficiency of tissue regeneration. The PDLSCs derived from both groups of cell sheets showed no significant difference in their viability, proliferative capacities, and multilineage differentiation potentials, as well as chromosomal stability. Furthermore, transplantation experiments based on a mouse model demonstrated that the cPDLSC sheets were equally effective in generating viable osteoid tissues in vivo as their freshly prepared counterparts. In both cases, the regenerated tissues showed similar network patterns of bone-like matrix. Our results offer convincing evidence that cryopreservation does not alter the biological properties of PDLSC sheets and could enhance their clinical utility in tissue regeneration.

Combining cell sheet technology and electrospun scaffolding for engineered tubular, aligned, and contractile blood vessels.

PubMed

Rayatpisheh, Shahrzad; Heath, Daniel E; Shakouri, Amir; Rujitanaroj, Pim-On; Chew, Sing Yian; Chan-Park, Mary B

2014-03-01

Herein we combine cell sheet technology and electrospun scaffolding to rapidly generate circumferentially aligned tubular constructs of human aortic smooth muscles cells with contractile gene expression for use as tissue engineered blood vessel media. Smooth muscle cells cultured on micropatterned and N-isopropylacrylamide-grafted (pNIPAm) polydimethylsiloxane (PDMS), a small portion of which was covered by aligned electrospun scaffolding, resulted in a single sheet of unidirectionally aligned cells. Upon cooling to room temperature, the scaffold, its adherent cells, and the remaining cell sheet detached and were collected on a mandrel to generating tubular constructs with circumferentially aligned smooth muscle cells which possess contractile gene expression and a single layer of electrospun scaffold as an analogue to a small diameter blood vessel's internal elastic lamina (IEL). This method improves cell sheet handling, results in rapid circumferential alignment of smooth muscle cells which immediately express contractile genes, and introduction of an analogue to small diameter blood vessel IEL. Copyright © 2013 Elsevier Ltd. All rights reserved.

Mechanisms of Regulating Tissue Elongation in Drosophila Wing: Impact of Oriented Cell Divisions, Oriented Mechanical Forces, and Reduced Cell Size

PubMed Central

Li, Yingzi; Naveed, Hammad; Kachalo, Sema; Xu, Lisa X.; Liang, Jie

2014-01-01

Regulation of cell growth and cell division plays fundamental roles in tissue morphogenesis. However, the mechanisms of regulating tissue elongation through cell growth and cell division are still not well understood. The wing imaginal disc of Drosophila provides a model system that has been widely used to study tissue morphogenesis. Here we use a recently developed two-dimensional cellular model to study the mechanisms of regulating tissue elongation in Drosophila wing. We simulate the effects of directional cues on tissue elongation. We also computationally analyze the role of reduced cell size. Our simulation results indicate that oriented cell divisions, oriented mechanical forces, and reduced cell size can all mediate tissue elongation, but they function differently. We show that oriented cell divisions and oriented mechanical forces act as directional cues during tissue elongation. Between these two directional cues, oriented mechanical forces have a stronger influence than oriented cell divisions. In addition, we raise the novel hypothesis that reduced cell size may significantly promote tissue elongation. We find that reduced cell size alone cannot drive tissue elongation. However, when combined with directional cues, such as oriented cell divisions or oriented mechanical forces, reduced cell size can significantly enhance tissue elongation in Drosophila wing. Furthermore, our simulation results suggest that reduced cell size has a short-term effect on cell topology by decreasing the frequency of hexagonal cells, which is consistent with experimental observations. Our simulation results suggest that cell divisions without cell growth play essential roles in tissue elongation. PMID:24504016

A Comparison of 2,4-Dichlorophenoxyacetic Acid Metabolism in Cultured Soybean Cells and in Embryogenic Carrot Cells

PubMed Central

Montague, Michael J.; Enns, Russell K.; Siegel, Ned R.; Jaworski, Ernest G.

1981-01-01

The removal or reduction in concentration of auxin is often a successful method for obtaining morphogenesis in cell cultures of higher plants, such as carrot, but not for soybean. For this reason, the metabolism of one auxin, 2,4-dichlorophenoxyacetic acid (2,4-D), was compared in both carrot and soybean cells. Whereas soybean cells conjugated a high percentage of their 2,4-D to amino acids, carrot cells contained primarily free 2,4-D. Moreover, after long-term exposure to 2,4-D, carrot cells released much more 2,4-D upon transfer to 2,4-D-free (embryogenic) medium than did soybean cells. It appears that the retention of 2,4-D by soybean cells might interfere with subsequent morphogenesis. Because no impairment of 2,4-D efflux was found with short-term exposure to radiolabeled 2,4-D, it was concluded that 2,4-D retention in soybean cells might be due to a time-dependent, metabolic process. The conjugation of 2,4-D to amino acids was shown to be one such time-dependent process. Additionally, the release of 2,4-D from the cells was shown to be due primarily to a loss of free 2,4-D and not 2,4-D-amino acid conjugates. It seems that the greater retention of 2,4-D by soybean cells upon transfer to 2,4-D-free medium is due to greater formation of 2,4-D-amino acid conjugates. PMID:16661722

Epithelial Patterning, Morphogenesis, and Evolution: Drosophila Eggshell as a Model.

PubMed

Osterfield, Miriam; Berg, Celeste A; Shvartsman, Stanislav Y

2017-05-22

Understanding the mechanisms driving tissue and organ formation requires knowledge across scales. How do signaling pathways specify distinct tissue types? How does the patterning system control morphogenesis? How do these processes evolve? The Drosophila egg chamber, where EGF and BMP signaling intersect to specify unique cell types that construct epithelial tubes for specialized eggshell structures, has provided a tractable system to ask these questions. Work there has elucidated connections between scales of development, including across evolutionary scales, and fostered the development of quantitative modeling tools. These tools and general principles can be applied to the understanding of other developmental processes across organisms. Copyright © 2017 Elsevier Inc. All rights reserved.

Involvement of actin filaments in rhizoid morphogenesis of Spirogyra.

PubMed

Yoshida, Katsuhisa; Shimmen, Teruo

2009-01-01

The role of actin filaments in rhizoid morphogenesis was studied in Spirogyra. When the algal filaments were severed, new terminal cells started tip growth and finally formed rhizoids. Actin inhibitors, latrunculin B and cytochalasin D, reversibly inhibited the process. A mesh-like structure of actin filaments (AFs) was formed at the tip region. Gd(3+) inhibited tip growth and decreased AFs in the tip region. Either a decrease in turgor pressure or lowering of the external Ca(2+) concentration also induced similar results. It was suggested that the mesh-like AF structure is indispensable for the elongation of rhizoids. A possible organization mechanism of the mesh-like AF structure was discussed.

Hedgehog Is a Positive Regulator of FGF Signalling during Embryonic Tracheal Cell Migration

PubMed Central

Butí, Elisenda; Mesquita, Duarte; Araújo, Sofia J.

2014-01-01

Cell migration is a widespread and complex process that is crucial for morphogenesis and for the underlying invasion and metastasis of human cancers. During migration, cells are steered toward target sites by guidance molecules that induce cell direction and movement through complex intracellular mechanisms. The spatio-temporal regulation of the expression of these guidance molecules is of extreme importance for both normal morphogenesis and human disease. One way to achieve this precise regulation is by combinatorial inputs of different transcription factors. Here we used Drosophila melanogaster mutants with migration defects in the ganglionic branches of the tracheal system to further clarify guidance regulation during cell migration. By studying the cellular consequences of overactivated Hh signalling, using ptc mutants, we found that Hh positively regulates Bnl/FGF levels during embryonic stages. Our results show that Hh modulates cell migration non-autonomously in the tissues surrounding the action of its activity. We further demonstrate that the Hh signalling pathway regulates bnl expression via Stripe (Sr), a zinc-finger transcription factor with homology to the Early Growth Response (EGR) family of vertebrate transcription factors. We propose that Hh modulates embryonic cell migration by participating in the spatio-temporal regulation of bnl expression in a permissive mode. By doing so, we provide a molecular link between the activation of Hh signalling and increased chemotactic responses during cell migration. PMID:24651658

Hedgehog is a positive regulator of FGF signalling during embryonic tracheal cell migration.

PubMed

Butí, Elisenda; Mesquita, Duarte; Araújo, Sofia J

2014-01-01

Cell migration is a widespread and complex process that is crucial for morphogenesis and for the underlying invasion and metastasis of human cancers. During migration, cells are steered toward target sites by guidance molecules that induce cell direction and movement through complex intracellular mechanisms. The spatio-temporal regulation of the expression of these guidance molecules is of extreme importance for both normal morphogenesis and human disease. One way to achieve this precise regulation is by combinatorial inputs of different transcription factors. Here we used Drosophila melanogaster mutants with migration defects in the ganglionic branches of the tracheal system to further clarify guidance regulation during cell migration. By studying the cellular consequences of overactivated Hh signalling, using ptc mutants, we found that Hh positively regulates Bnl/FGF levels during embryonic stages. Our results show that Hh modulates cell migration non-autonomously in the tissues surrounding the action of its activity. We further demonstrate that the Hh signalling pathway regulates bnl expression via Stripe (Sr), a zinc-finger transcription factor with homology to the Early Growth Response (EGR) family of vertebrate transcription factors. We propose that Hh modulates embryonic cell migration by participating in the spatio-temporal regulation of bnl expression in a permissive mode. By doing so, we provide a molecular link between the activation of Hh signalling and increased chemotactic responses during cell migration.

Flat-plate solar array project. Volume 3: Silicon sheet: Wafers and ribbons

NASA Technical Reports Server (NTRS)

Briglio, A.; Dumas, K.; Leipold, M.; Morrison, A.

1986-01-01

The primary objective of the Silicon Sheet Task of the Flat-Plate Solar Array (FSA) Project was the development of one or more low cost technologies for producing silicon sheet suitable for processing into cost-competitive solar cells. Silicon sheet refers to high purity crystalline silicon of size and thickness for fabrication into solar cells. Areas covered in the project were ingot growth and casting, wafering, ribbon growth, and other sheet technologies. The task made and fostered significant improvements in silicon sheet including processing of both ingot and ribbon technologies. An additional important outcome was the vastly improved understanding of the characteristics associated with high quality sheet, and the control of the parameters required for higher efficiency solar cells. Although significant sheet cost reductions were made, the technology advancements required to meet the task cost goals were not achieved.

CNT Sheet Air Electrode for the Development of Ultra-High Cell Capacity in Lithium-Air Batteries

PubMed Central

Nomura, Akihiro; Ito, Kimihiko; Kubo, Yoshimi

2017-01-01

Lithium-air batteries (LABs) are expected to provide a cell with a much higher capacity than ever attained before, but their prototype cells present a limited areal cell capacity of no more than 10 mAh cm−2, mainly due to the limitation of their air electrodes. Here, we demonstrate the use of flexible carbon nanotube (CNT) sheets as a promising air electrode for developing ultra-high capacity in LAB cells, achieving areal cell capacities of up to 30 mAh cm−2, which is approximately 15 times higher than the capacity of cells with lithium-ion battery (LiB) technology (~2 mAh cm−2). During discharge, the CNT sheet electrode experienced enormous swelling to a thickness of a few millimeters because of the discharge product deposition of lithium peroxide (Li2O2), but the sheet was fully recovered after being fully charged. This behavior results from the CNT sheet characteristics of the flexible and fibrous conductive network and suggests that the CNT sheet is an effective air electrode material for developing a commercially available LAB cell with an ultra-high cell capacity. PMID:28378746

In Vitro Engineering of Vascularized Tissue Surrogates

PubMed Central

Sakaguchi, Katsuhisa; Shimizu, Tatsuya; Horaguchi, Shigeto; Sekine, Hidekazu; Yamato, Masayuki; Umezu, Mitsuo; Okano, Teruo

2013-01-01

In vitro scaling up of bioengineered tissues is known to be limited by diffusion issues, specifically a lack of vasculature. Here, we report a new strategy for preserving cell viability in three-dimensional tissues using cell sheet technology and a perfusion bioreactor having collagen-based microchannels. When triple-layer cardiac cell sheets are incubated within this bioreactor, endothelial cells in the cell sheets migrate to vascularize in the collagen gel, and finally connect with the microchannels. Medium readily flows into the cell sheets through the microchannels and the newly developed capillaries, while the cardiac construct shows simultaneous beating. When additional triple-layer cell sheets are repeatedly layered, new multi-layer construct spontaneously integrates and the resulting construct becomes a vascularized thick tissue. These results confirmed our method to fabricate in vitro vascularized tissue surrogates that overcomes engineered-tissue thickness limitations. The surrogates promise new therapies for damaged organs as well as new in vitro tissue models. PMID:23419835

Cell sheet engineering using the stromal vascular fraction of adipose tissue as a vascularization strategy.

PubMed

Costa, Marina; Cerqueira, Mariana T; Santos, Tírcia C; Sampaio-Marques, Belém; Ludovico, Paula; Marques, Alexandra P; Pirraco, Rogério P; Reis, Rui L

2017-06-01

Current vascularization strategies for Tissue Engineering constructs, in particular cell sheet-based, are limited by time-consuming and expensive endothelial cell isolation and/or by the complexity of using extrinsic growth factors. Herein, we propose an alternative strategy using angiogenic cell sheets (CS) obtained from the stromal vascular fraction (SVF) of adipose tissue that can be incorporated into more complex constructs. Cells from the SVF were cultured in normoxic and hypoxic conditions for up to 8days in the absence of extrinsic growth factors. Immunocytochemistry against CD31 and CD146 revealed spontaneous organization in capillary-like structures, more complex after hypoxic conditioning. Inhibition of HIF-1α pathway hindered capillary-like structure formation in SVF cells cultured in hypoxia, suggesting a role of HIF-1α. Moreover, hypoxic SVF cells showed a trend for increased secretion of angiogenic factors, which was reflected in increased network formation by endothelial cells cultured on matrigel using that conditioned medium. In vivo implantation of SVF CS in a mouse hind limb ischemia model revealed that hypoxia-conditioned CS led to improved restoration of blood flow. Both in vitro and in vivo data suggest that SVF CS can be used as simple and cost-efficient tools to promote functional vascularization of TE constructs. Neovascularization after implantation is a major obstacle for producing clinically viable cell sheet-based tissue engineered constructs. Strategies using endothelial cells and extrinsic angiogenic growth factors are expensive and time consuming and may raise concerns of tumorigenicity. In this manuscript, we describe a simplified approach using angiogenic cell sheets fabricated from the stromal vascular fraction of adipose tissue. The strong angiogenic behavior of these cell sheets, achieved without the use of external growth factors, was further stimulated by low oxygen culture. When implanted in an in vivo model of hind limb ischemia, the angiogenic cell sheets contributed to blood flux recovery. These cell sheets can therefore be used as a straightforward tool to increase the neovascularization of cell sheet-based thick constructs. Copyright © 2017. Published by Elsevier Ltd.

Periodontal regeneration in swine after cell injection and cell sheet transplantation of human dental pulp stem cells following good manufacturing practice.

PubMed

Hu, Jingchao; Cao, Yu; Xie, Yilin; Wang, Hua; Fan, Zhipeng; Wang, Jinsong; Zhang, Chunmei; Wang, Jinsong; Wu, Chu-Tse; Wang, Songlin

2016-09-09

Periodontitis, one of the most prevalent infectious diseases in humans, results in the destruction of tooth-supporting tissues. The purpose of the present study is to evaluate the effect of cell injection and cell sheet transplantation on periodontal regeneration in a swine model. In the present study, human dental pulp stem cells (hDPSCs) were transplanted into a swine model for periodontal regeneration. Twelve miniature pigs were used to generate periodontitis with bone defects of 5 mm in width, 7 mm in length, and 3 mm in depth. hDPSCs were obtained for bone regeneration using cell injection or cell sheet transplantation. After 12 weeks, clinical, radiological, and histological assessments of regenerated periodontal tissues were performed to compare periodontal regeneration treated with xenogeneic cell injection and cell sheet implantation. Our study showed that translating hDPSCs into this large animal model could significantly improve periodontal bone regeneration and soft tissue healing. After 12 weeks, both the hDPSC sheet treatment and hDPSC injection significantly improved periodontal tissue healing clinically in comparison with the control group. The volume of regenerative bone in the hDPSC sheet group (52.7 ± 4.1 mm(3)) was significantly larger than in the hDPSC injection group (32.4 ± 5.1 mm(3)) (P < 0.05). The percentage of bone in the periodontium in the hDPSC injection group was 12.8 ± 4.4 %, while it was 17.4 ± 5.3 % in the hDPSC sheet group (P < 0.05). Both hDPSC injection and cell sheet transplantation significantly regenerated periodontal bone in swine. The hDPSC sheet had more bone regeneration capacity compared with hDPSC injection.

Polycrystalline silicon sheets for solar cells by the spinning method

NASA Astrophysics Data System (ADS)

Maeda, Y.; Yokoyama, T.; Hide, I.

1984-03-01

A new method has been developed in which polycrystalline silicon sheets are formed directly from molten silicon on a spinning wheel. The sheet is 5 cm x 5 cm, 0.1-0.5 mm thick, and made at a rate of four sheets per 15 s; power conversion rate of a solar cell assembled with these silicon sheets is more than 10 percent.

3D Printing of Thermo-Responsive Methylcellulose Hydrogels for Cell-Sheet Engineering

PubMed Central

Cochis, Andrea; Sorrentino, Rita; Grassi, Federico; Leigheb, Massimiliano; Farè, Silvia

2018-01-01

A possible strategy in regenerative medicine is cell-sheet engineering (CSE), i.e., developing smart cell culture surfaces from which to obtain intact cell sheets (CS). The main goal of this study was to develop 3D printing via extrusion-based bioprinting of methylcellulose (MC)-based hydrogels. Hydrogels were prepared by mixing MC powder in saline solutions (Na2SO4 and PBS). MC-based hydrogels were analyzed to investigate the rheological behavior and thus optimize the printing process parameters. Cells were tested in vitro on ring-shaped printed hydrogels; bulk MC hydrogels were used for comparison. In vitro tests used murine embryonic fibroblasts (NIH/3T3) and endothelial murine cells (MS1), and the resulting cell sheets were characterized analyzing cell viability and immunofluorescence. In terms of CS preparation, 3D printing proved to be an optimal approach to obtain ring-shaped CS. Cell orientation was observed for the ring-shaped CS and was confirmed by the degree of circularity of their nuclei: cell nuclei in ring-shaped CS were more elongated than those in sheets detached from bulk hydrogels. The 3D printing process appears adequate for the preparation of cell sheets of different shapes for the regeneration of complex tissues. PMID:29642573

Exogenous ROS-induced cell sheet transfer based on hematoporphyrin-polyketone film via a one-step process.

PubMed

Koo, Min-Ah; Lee, Mi Hee; Kwon, Byeong-Ju; Seon, Gyeung Mi; Kim, Min Sung; Kim, Dohyun; Nam, Ki Chang; Park, Jong-Chul

2018-04-01

To date, most of invasive cell sheet harvesting methods have used culture surface property variations, such as wettability, pH, electricity, and magnetism, to induce cell detachment. These methods that rely on surface property changes are effective when cell detachment prior to application is necessary, but of limited use when used for cell sheet transfer to target regions. The study reports a new reactive oxygen species (ROS)-induced strategy based on hematoporphyrin-incorporated polyketone film (Hp-PK film) to transfer cell sheets directly to target areas without an intermediate harvesting process. After green LED (510 nm) irradiation, production of exogenous ROS from the Hp-PK films induces cell sheet detachment and transfer. The study suggests that ROS-induced cell detachment property of the Hp-PK film is closely related to conformational changes of extracellular matrix (ECM) proteins. Also, this strategy with the Hp-PK film can be applied by regulating production rate of exogenous ROS in various types of cells, including fibroblasts, mesenchymal stem cells and keratinocytes. In conclusion, ROS-induced method using the Hp-PK film can be used for one-step cell sheet transplantation and has potential in biomedical applications. Copyright © 2018 Elsevier Ltd. All rights reserved.

Periodontal regeneration with multi-layered periodontal ligament-derived cell sheets in a canine model.

PubMed

Iwata, Takanori; Yamato, Masayuki; Tsuchioka, Hiroaki; Takagi, Ryo; Mukobata, Shigeki; Washio, Kaoru; Okano, Teruo; Ishikawa, Isao

2009-05-01

Periodontal regeneration has been challenged with chemical reagents and/or biological approaches, however, there is still no sufficient technique that can regenerate complete periodontium, including alveolar bone, cementum, and well-oriented collagen fibers. The purpose of this study was to examine multi-layered sheets of periodontal ligament (PDL)-derived cells for periodontal regeneration. Canine PDL cells were isolated enzymatically and expanded in vitro. The cell population contained cells capable of making single cell-derived colonies at an approximately 20% frequency. Expression of mRNA of periodontal marker genes, S100 calcium binding protein A4 and periostin, was observed. Alkaline phosphatase activity and gene expression of both osteoblastic/cementoblastic and periodontal markers were upregulated by osteoinductive medium. Then, three-layered PDL cell sheets supported with woven polyglycolic acid were transplanted to dental root surfaces having three-wall periodontal defects in an autologous manner, and bone defects were filled with porous beta-tricalcium phosphate. Cell sheet transplantation regenerated both new bone and cementum connecting with well-oriented collagen fibers, while only limited bone regeneration was observed in control group where cell sheet transplantation was eliminated. These results suggest that PDL cells have multiple differentiation properties to regenerate periodontal tissues comprising hard and soft tissues. PDL cell sheet transplantation should prove useful for periodontal regeneration in clinical settings.

Mammalian aPKC/Par polarity complex mediated regulation of epithelial division orientation and cell fate

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

Vorhagen, Susanne; Niessen, Carien M., E-mail: carien.niessen@uni-koeln.de

2014-11-01

Oriented cell division is a key regulator of tissue architecture and crucial for morphogenesis and homeostasis. Balanced regulation of proliferation and differentiation is an essential property of tissues not only to drive morphogenesis but also to maintain and restore homeostasis. In many tissues orientation of cell division is coupled to the regulation of differentiation producing daughters with similar (symmetric cell division, SCD) or differential fate (asymmetric cell division, ACD). This allows the organism to generate cell lineage diversity from a small pool of stem and progenitor cells. Division orientation and/or the ratio of ACD/SCD need to be tightly controlled. Lossmore » of orientation or an altered ratio can promote overgrowth, alter tissue architecture and induce aberrant differentiation, and have been linked to morphogenetic diseases, cancer and aging. A key requirement for oriented division is the presence of a polarity axis, which can be established through cell intrinsic and/or extrinsic signals. Polarity proteins translate such internal and external cues to drive polarization. In this review we will focus on the role of the polarity complex aPKC/Par3/Par6 in the regulation of division orientation and cell fate in different mammalian epithelia. We will compare the conserved function of this complex in mitotic spindle orientation and distribution of cell fate determinants and highlight common and differential mechanisms in which this complex is used by tissues to adapt division orientation and cell fate to the specific properties of the epithelium.« less

Arabidopsis thaliana plants lacking the ARP2/3 complex show defects in cell wall assembly and auxin distribution.

PubMed

Pratap Sahi, Vaidurya; Cifrová, Petra; García-González, Judith; Kotannal Baby, Innu; Mouillé, Gregory; Gineau, Emilie; Müller, Karel; Baluška, František; Soukup, Aleš; Petrášek, Jan; Schwarzerová, Katerina

2017-12-25

The cytoskeleton plays an important role in the synthesis of plant cell walls. Both microtubules and actin cytoskeleton are known to be involved in the morphogenesis of plant cells through their role in cell wall building. The role of ARP2/3-nucleated actin cytoskeleton in the morphogenesis of cotyledon pavement cells has been described before. Seedlings of Arabidopsis mutants lacking a functional ARP2/3 complex display specific cell wall-associated defects. In three independent Arabidopsis mutant lines lacking subunits of the ARP2/3 complex, phenotypes associated with the loss of the complex were analysed throughout plant development. Organ size and anatomy, cell wall composition, and auxin distribution were investigated. ARP2/3-related phenotype is associated with changes in cell wall composition, and the phenotype is manifested especially in mature tissues. Cell walls of mature plants contain less cellulose and a higher amount of homogalacturonan, and display changes in cell wall lignification. Vascular bundles of mutant inflorescence stems show a changed pattern of AUX1-YFP expression. Plants lacking a functional ARP2/3 complex have decreased basipetal auxin transport. The results suggest that the ARP2/3 complex has a morphogenetic function related to cell wall synthesis and auxin transport. © The Author(s) 2017. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Geometry can provide long-range mechanical guidance for embryogenesis

PubMed Central

Dicko, Mahamar; Saramito, Pierre

2017-01-01

Downstream of gene expression, effectors such as the actomyosin contractile machinery drive embryo morphogenesis. During Drosophila embryonic axis extension, actomyosin has a specific planar-polarised organisation, which is responsible for oriented cell intercalation. In addition to these cell rearrangements, cell shape changes also contribute to tissue deformation. While cell-autonomous dynamics are well described, understanding the tissue-scale behaviour challenges us to solve the corresponding mechanical problem at the scale of the whole embryo, since mechanical resistance of all neighbouring epithelia will feedback on individual cells. Here we propose a novel numerical approach to compute the whole-embryo dynamics of the actomyosin-rich apical epithelial surface. We input in the model specific patterns of actomyosin contractility, such as the planar-polarisation of actomyosin in defined ventro-lateral regions of the embryo. Tissue strain rates and displacements are then predicted over the whole embryo surface according to the global balance of stresses and the material behaviour of the epithelium. Epithelia are modelled using a rheological law that relates the rate of deformation to the local stresses and actomyosin anisotropic contractility. Predicted flow patterns are consistent with the cell flows observed when imaging Drosophila axis extension in toto, using light sheet microscopy. The agreement between model and experimental data indicates that the anisotropic contractility of planar-polarised actomyosin in the ventro-lateral germband tissue can directly cause the tissue-scale deformations of the whole embryo. The three-dimensional mechanical balance is dependent on the geometry of the embryo, whose curved surface is taken into account in the simulations. Importantly, we find that to reproduce experimental flows, the model requires the presence of the cephalic furrow, a fold located anteriorly of the extending tissues. The presence of this geometric feature, through the global mechanical balance, guides the flow and orients extension towards the posterior end. PMID:28346461

Polyamine and ethylene biosynthesis in relation to somatic embryogenesis in carrot (Daucus carota L.) cell cultures

Treesearch

Subhash C. Minocha; Cheryl A. Robie; Akhtar J. Khan; Nancy S. Papa; Andrew I. Samuelsen; Rakesh Minocha

1990-01-01

Carrot cell cultures provide a model experimental system for the analysis of biochemical and molecular events associated with morphogenesis in plants (3, 4, 5, 14). Among the biochemical changes accompanying somatic embryogenesis in this tissue is an increased biosynthesis ofpolyamines (1, 2, 7, 10, 11, 13). A variety of inhibitors of polyamine biosynthetic enzymes...

Ontogeny of plants under various gravity condition

NASA Astrophysics Data System (ADS)

Laurinavičius, R.; Švegždienṡ, D.; Raklevičienė, D.; Kenstavičienė, P.

2001-01-01

The results of experiments performed under conditions of microgravity (MG) or under its simulation on the horizontal clinostat (HC) with the callus, seedlings of various species and embryogenic structures have revealed a definite role of gravity as an ecological factor in the processes of cytomorphogenesis, growth, and development. The transformation of differentiated somatic cells of arabidopsis seed into undifferentiated callus was not inhibited under MG, though modifications of the whole callus morphology and of mean cell and nucleus size were observed. The morphogenesis of polar structures such as root-hair bearing cells of Lactuca primary root has been shown to be modified in the course of differentiation under mass acceleration diminished below 0.1 g. Seed germination and seedling morphogenesis under MG follow their normal course, but a significant stimulation of shoot growth with no effect on primary root growth has been determined. A successful in vitro regeneration of Nicotiana tabacum plantlets from leaf cells and subsequent formation of shoots and roots on a continuously rotating HC as well as the formation of viable seeds during seed-to-seed growth of Arabidopsis plants under MG have indicated that gravity plays but a limited role in the processes of embryogenesis and organogenesis.