Evasion of cell senescence in SHH medulloblastoma

PubMed Central

Tamayo-Orrego, Lukas; Swikert, Shannon M.; Charron, Frédéric

2016-01-01

ABSTRACT The mechanisms leading to brain tumor formation are poorly understood. Using Ptch1+/− mice as a medulloblastoma model, sequential mutations were found to shape tumor evolution. Initially, medulloblastoma preneoplastic lesions display loss of heterozygosity of the Ptch1 wild-type allele, an event associated with cell senescence in preneoplasia. Subsequently, p53 mutations lead to senescence evasion and progression from preneoplasia to medulloblastoma. These findings are consistent with a model where high levels of Hedgehog signaling caused by the loss of the tumor suppressor Ptch1 lead to oncogene-induced senescence and drive p53 mutations. Thus, cell senescence is an important characteristic of a subset of SHH medulloblastoma and might explain the acquisition of somatic TP53 mutations in human medulloblastoma. This mode of medulloblastoma formation contrasts with the one characterizing Li-Fraumeni patients with medulloblastoma, where TP53 germ-line mutations cause chromothriptic genomic instability and lead to mutations in Hedgehog signaling genes, which drive medulloblastoma growth. Here we discuss in detail these 2 alternative mechanisms leading to medulloblastoma tumorigenesis. PMID:27229128

Initiation and patterning of the snake dentition are dependent on Sonic hedgehog signaling.

PubMed

Buchtová, Marcela; Handrigan, Gregory R; Tucker, Abigail S; Lozanoff, Scott; Town, Liam; Fu, Katherine; Diewert, Virginia M; Wicking, Carol; Richman, Joy M

2008-07-01

Here we take the first look at cellular dynamics and molecular signaling in the developing snake dentition. We found that tooth formation differs from rodents in several respects. The majority of snake teeth bud off of a deep, ribbon-like dental lamina rather than as separate tooth germs. Prior to and after dental lamina ingrowth, we observe asymmetries in cell proliferation and extracellular matrix distribution suggesting that localized signaling by a secreted protein is involved. We cloned Sonic hedgehog from the African rock python Python sebae and traced its expression in the species as well as in two other snakes, the closely-related Python regius and the more derived corn snake Elaphe guttata (Colubridae). We found that expression of Shh is first confined to the odontogenic band and defines the position of the future dental lamina. Shh transcripts in pythons are progressively restricted to the oral epithelium on one side of the dental lamina and remain in this position throughout the prehatching period. Shh is expressed in the inner enamel epithelium and the stellate reticulum of the tooth anlagen, but is absent from the outer enamel epithelium and its derivative, the successional lamina. This suggests that signals other than Shh are responsible for replacement tooth formation. Functional studies using cyclopamine to block Hh signaling during odontogenesis prevented initiation and extension of the dental lamina into the mesenchyme, and also affected the directionality of this process. Further, blocking Hh signaling led to disruptions of the inner enamel epithelium. To explore the role of Shh in lamina extension, we looked at its expression in the premaxillary teeth, which form closer to the oral surface than elsewhere in the mouth. Oral ectodermal Shh expression in premaxillary teeth is lost soon after the teeth form reinforcing the idea that Shh is controlling the depth of the dental lamina. In summary, we have found diverse roles for Shh in patterning the snake dentition but, have excluded the participation of this signal in replacement tooth formation.

A dynamic Shh expression pattern, regulated by SHH and BMP signaling, coordinates fusion of primordia in the amniote face

PubMed Central

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

2015-01-01

The mechanisms of morphogenesis are not well understood, yet shaping structures during development is essential for establishing correct organismal form and function. Here, we examine mechanisms that help to shape the developing face during the crucial period of facial primordia fusion. This period of development is a time when the faces of amniote embryos exhibit the greatest degree of similarity, and it probably results from the necessity for fusion to occur to establish the primary palate. Our results show that hierarchical induction mechanisms, consisting of iterative signaling by Sonic hedgehog (SHH) followed by Bone morphogenetic proteins (BMPs), regulate a dynamic expression pattern of Shh in the ectoderm covering the frontonasal (FNP) and maxillary (MxP) processes. Furthermore, this Shh expression domain contributes to the morphogenetic processes that drive the directional growth of the globular process of the FNP toward the lateral nasal process and MxP, in part by regulating cell proliferation in the facial mesenchyme. The nature of the induction mechanism that we discovered suggests that the process of fusion of the facial primordia is intrinsically buffered against producing maladaptive morphologies, such as clefts of the primary palate, because there appears to be little opportunity for variation to occur during expansion of the Shh expression domain in the ectoderm of the facial primordia. Ultimately, these results might explain why this period of development constitutes a phylotypic stage of facial development among amniotes. PMID:25605783

Multivalency of Sonic hedgehog conjugated to linear polymer chains modulates protein potency.

PubMed

Wall, Samuel T; Saha, Krishanu; Ashton, Randolph S; Kam, Kimberly R; Schaffer, David V; Healy, Kevin E

2008-04-01

A potently active multivalent form of the protein Sonic hedgehog (Shh) was produced by bioconjugation of a modified recombinant form of Shh to the linear polymers poly(acrylic acid) (pAAc) and hyaluronic acid (HyA) via a two-step reaction exploiting carboimiide and maleimide chemistry. Efficiency of the conjugation was approximately 75% even at stoichiometric ratios of 30 Shh molecules per linear HyA chain (i.e., 30:1 Shh/HyA). Bioactivity of the conjugates was tested via a cellular assay across a range of stoichiometric ratios of Shh molecules to HyA linear chains, which was varied from 0.6:1 Shh/HyA to 22:1 Shh/HyA. Results indicate that low conjugation ratios decrease Shh bioactivity and high ratios increase this activity beyond the potency of monomeric Shh, with approximately equal activity between monomeric soluble Shh and conjugated Shh at 7:1 Shh/HyA. In addition, high-ratio constructs increased angiogenesis determined by the in vivo chick chorioallantoic membrane (CAM) assay. These results are captured by a kinetic model of multiple interactions between the Shh/HyA conjugates and cell surface receptors resulting in higher cell signaling at lower bulk Shh concentrations.

Sonic hedgehog (SHH) and glioblastoma-2 (Gli-2) expressions are associated with poor jaundice-free survival in biliary atresia.

PubMed

Jung, Hae Yoen; Jing, Jin; Lee, Kyoung Bun; Jang, Ja-June

2015-03-01

Biliary atresia (BA) causes biliary obstruction in neonates. Although the Kasai operation can successfully treat certain BA cases, many patients exhibit recurrent jaundice and secondary biliary cirrhosis requiring liver transplantation. Consequently, studies of the prognostic factors of the Kasai operation are needed. Accordingly, sonic hedgehog (SHH) pathway expression at the extrahepatic bile duct (EHBD), an important bile duct repair mechanism, will be investigated via immunohistochemistry in patients with BA to examine the association with post-Kasai operation prognosis. Fifty-seven EHBD specimens were obtained during Kasai operations from 1992 to 2009. The SHH, patched (PTCH), and glioblastoma-2 (Gli-2) immunohistochemical staining results were analyzed quantitatively. Overall, 57.9% of patients had bile flow normalization after the Kasai operation; 43.1% did not. High preoperative serum total bilirubin, direct bilirubin, and aspartate aminotransferase levels were associated with sustained jaundice post-Kasai operation, as was an age ≥65days at the time of surgery (all p<0.05). High Gli-2 and SHH expression rates were significantly associated with early post-Kasai operation jaundice relapse. Strong Gli-2 and SHH expression in the EHBD might be a poor prognostic factor in Kasai operation-treated patients with BA. Copyright © 2015 Elsevier Inc. All rights reserved.

Single-nucleotide variants in two Hedgehog genes, SHH and HHIP, as genetic cause of combined pituitary hormone deficiency.

PubMed

Gorbenko del Blanco, Darya; de Graaff, Laura C G; Visser, Theo J; Hokken-Koelega, Anita C S

2013-03-01

Combined pituitary hormone deficiency (CPHD) is characterized by deficiencies of two or more anterior pituitary hormones. Its genetic cause is unknown in the majority of cases. The Hedgehog (Hh) signalling pathway has been implicated in disorders associated with pituitary development. Mutations in Sonic Hedgehog (SHH) have been described in patients with holoprosencephaly (with or without pituitary involvement). Hedgehog interacting protein (HHIP) has been associated with variations in adult height in genome wide association studies. We investigated whether mutations in these two genes of the Hh pathway, SHH and HHIP, could result in 'idiopathic' CPHD. We directly sequenced the coding regions and exon - intron boundaries of SHH and HHIP in 93 CPHD patients of the Dutch HYPOPIT study in whom mutations in the classical CPHD genes PROP1, POU1F1, HESX1, LHX3 and LHX4 had been ruled out. We compared the expression of Hh genes in Hep3B transfected cells between wild-type proteins and mutants. We identified three single-nucleotide variants (p.Ala226Thr, c.1078C>T and c.*8G>T) in SHH. The function of the latter was severely affected in our in vitro assay. In HHIP, we detected a new activating variant c.-1G>C, which increases HHIP's inhibiting function on the Hh pathway. Our results suggest involvement of the Hedgehog pathway in CPHD. We suggest that both SHH and HHIP are investigated as a second screening in CPHD, after mutations in the classical CPHD genes have been ruled out. © 2012 Blackwell Publishing Ltd.

Neural Influences on Sonic Hedgehog and Apoptosis in the Rat Penis1

PubMed Central

Bond, Christopher; Tang, Yi; Podlasek, Carol A.

2010-01-01

The role of sonic hedgehog (SHH) in maintaining corpora cavernosal morphology in the adult penis has been established; however, the mechanism of how SHH itself is regulated remains unclear. Since decreased SHH protein is a cause of smooth muscle apoptosis and erectile dysfunction (ED) in the penis, and SHH treatment can suppress cavernous nerve (CN) injury-induced apoptosis, the question of how SHH signaling is regulated is significant. It is likely that neural input is involved in this process since two models of neuropathy-induced ED exhibit decreased SHH protein and increased apoptosis in the penis. We propose the hypothesis that SHH abundance in the corpora cavernosa is regulated by SHH signaling in the pelvic ganglia, neural activity, or neural transport of a trophic factor from the pelvic ganglia to the corpora. We have examined each of these potential mechanisms. SHH inhibition in the penis shows a 12-fold increase in smooth muscle apoptosis. SHH inhibition in the pelvic ganglia causes significantly increased apoptosis (1.3-fold) and decreased SHH protein (1.1-fold) in the corpora cavernosa. SHH protein is not transported by the CN. Colchicine treatment of the CN resulted in significantly increased smooth muscle apoptosis (1.2-fold) and decreased SHH protein (1.3-fold) in the penis. Lidocaine treatment of the CN caused a similar increase in apoptosis (1.6-fold) and decrease in SHH protein (1.3-fold) in the penis. These results show that neural activity and a trophic factor from the pelvic ganglia/CN are necessary to regulate SHH protein and smooth muscle abundance in the penis. PMID:18256331

Plasma Shh levels reduced in pancreatic cancer patients

PubMed Central

El-Zaatari, Mohamad; Daignault, Stephanie; Tessier, Art; Kelsey, Gail; Travnikar, Lisa A.; Cantu, Esperanza F.; Lee, Jamie; Plonka, Caitlyn M.; Simeone, Diane M.; Anderson, Michelle A.; Merchant, Juanita L.

2012-01-01

Objectives Normally, sonic hedgehog (Shh) is expressed in the pancreas during fetal development and transiently after tissue injury. Although pancreatic cancers express Shh, it is not known if the protein is secreted into the blood and whether its plasma levels change with pancreatic transformation. The goal of this study was to develop an ELISA to detect human Shh in blood, and determine the levels in subjects with and without pancreatic cancer. Methods A human Shh ELISA assay was developed, and plasma Shh levels were measured in blood samples from normal volunteers and subjects with pancreatitis or pancreatic cancer. The biological activity of plasma Shh was tested using NIH-3T3 cells. Results The average levels of Shh in human blood were lower in pancreatitis and pancreatic cancer patients than in normal individuals. Hematopoietic cells did not express Shh suggesting that Shh is secreted into the bloodstream. Plasma fractions enriched for Shh did not induce Gli-1 mRNA suggesting that the protein was not biologically active. Conclusions Shh is secreted from tissues and organs into the circulation but its activity is blocked by plasma proteins. Reduced plasma levels were found in pancreatic cancer patients, but alone were not sufficient to predict pancreatic cancer. PMID:22513293

Plasma Shh levels reduced in pancreatic cancer patients.

PubMed

El-Zaatari, Mohamad; Daignault, Stephanie; Tessier, Art; Kelsey, Gail; Travnikar, Lisa A; Cantu, Esperanza F; Lee, Jamie; Plonka, Caitlyn M; Simeone, Diane M; Anderson, Michelle A; Merchant, Juanita L

2012-10-01

Normally, sonic hedgehog (Shh) is expressed in the pancreas during fetal development and transiently after tissue injury. Although pancreatic cancers express Shh, it is not known if the protein is secreted into the blood and whether its plasma levels change with pancreatic transformation. The goal of this study was to develop an enzyme-linked immunosorbent assay to detect human Shh in blood and determine its levels in subjects with and without pancreatic cancer. A human Shh enzyme-linked immunosorbent assay was developed, and plasma Shh levels were measured in blood samples from healthy subjects and patients with pancreatitis or pancreatic cancer. The biological activity of plasma Shh was tested using NIH-3T3 cells. The mean levels of Shh in human blood were lower in patients with pancreatitis and pancreatic cancer than in healthy subjects. Hematopoietic cells did not express Shh, suggesting that Shh is secreted into the bloodstream. Plasma fractions enriched with Shh did not induce Gli-1 messenger RNA, suggesting that the protein was not biologically active. Shh is secreted from tissues and organs into the circulation, but its activity is blocked by plasma proteins. Reduced plasma levels were found in pancreatic cancer patients, but alone were not sufficient to predict pancreatic cancer.

Direct and indirect requirements of Shh/Gli signaling in early pituitary development.

PubMed

Wang, Yiwei; Martin, James F; Bai, C Brian

2010-12-15

Induction of early pituitary progenitors is achieved through combined activities of signals from adjacent embryonic tissues. Previous studies have identified a requirement for oral ectoderm derived Sonic Hedgehog (Shh) in specification and/or proliferation of early pituitary progenitors, however how different Gli genes mediate Shh signaling to control pituitary progenitor development has not yet been determined. Here we show that Gli2, which encodes a major Gli activator, is required for proliferation of specific groups of pituitary progenitors but not for initial dorsoventral patterning. We further show that the action of Gli2 occurs prior to the closure of Rathke' pouch. Lastly, we show that Shh/Gli2 signaling controls the diencephalic expression of Bone morphogenetic protein 4 (Bmp4) and Fibroblast growth factor 8 (Fgf8), two genes that are known to play critical roles in patterning and growth of Rathke's pouch. Our results therefore suggest both cell-autonomous and non-cell-autonomous requirements for Gli2 in regulation of pituitary progenitor specification, proliferation and differentiation. Copyright © 2010 Elsevier Inc. All rights reserved.

Temporally Distinct Six2-Positive Second Heart Field Progenitors Regulate Mammalian Heart Development and Disease.

PubMed

Zhou, Zhengfang; Wang, Jingying; Guo, Chaoshe; Chang, Weiting; Zhuang, Jian; Zhu, Ping; Li, Xue

2017-01-24

The embryonic process of forming a complex structure such as the heart remains poorly understood. Here, we show that Six2 marks a dynamic subset of second heart field progenitors. Six2-positive (Six2 + ) progenitors are rapidly recruited and assigned, and their descendants are allocated successively to regions of the heart from the right ventricle (RV) to the pulmonary trunk. Global ablation of Six2 + progenitors resulted in RV hypoplasia and pulmonary atresia. An early stage-specific ablation of a small subset of Six2 + progenitors did not cause any apparent structural defect at birth but rather resulted in adult-onset cardiac hypertrophy and dysfunction. Furthermore, Six2 expression depends in part on Shh signaling, and Shh deletion resulted in severe deficiency of Six2 + progenitors. Collectively, these findings unveil the chronological features of cardiogenesis, in which the mammalian heart is built sequentially by temporally distinct populations of cardiac progenitors, and provide insights into late-onset congenital heart disease. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Injury-stimulated Sonic hedgehog expression in microglia contributes to neuroinflammatory response in the MPTP model of Parkinson's disease

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

Lee, Jeong Hwi; Department of Biochemistry & Molecular Biology, School of Medicine Kyung Hee University, Seoul 130-701; Chung, Young Cheul

Parkinson's disease (PD) is a progressive neurodegenerative disorder in which dopamine (DA) neurons in the substantia nigra pars compacta (SNpc) region are selectively destroyed. Sonic hedgehog (Shh) has been well known to play a key role in a variety of processes such as embryogenesis, cell proliferation and protection, and tissue repair during inflammation. However, the evidences for the innate role of Shh in adult brain injury are presently lacking and studies have been needed to unveil the importance of Shh in the process of neurodegeneration. Here, we investigated the role of Shh in the pathologic progress of Parkinson's disease inmore » MPTP-induced animal model system. Interestingly, we observed that Shh expression was gradually increased in MPTP affected SNpc region. Activated microglia exclusively expressed SHH in vivo and we could recapitulate Shh induction in activated cultured primary microglia cells. Using the SHH responsive Cre-loxP binary genetic reporter transgenic mouse system, we also found that most of the cell types except for oligodendrocyte in the SNpc region reacted to the SHH by MPTP injection. Taken together, activated microglia induced Shh expression and most neural cells except oligodendrocyte responded to microglia-derived SHH in MPTP-treated SN. These results suggest that SHH in activated microglia by MPTP-injection might be involved in the innate processes of recovery from neurotoxin induced injury in the PD animal model system. - Highlights: • Sonic hedgehog (Shh) was induced by MPTP neurotoxin at the Substantia Nigra (SN) in vivo. • Activated microglia are major cell type for SHH expression in vivo and in vitro. • Different types of cells in the brain, except oligodendrocyte, respond to microglia-derived SHH in SN region.« less

The Acid-Secreting Parietal Cell as an Endocrine Source of Sonic Hedgehog During Gastric Repair

PubMed Central

Engevik, Amy C.; Feng, Rui; Yang, Li

2013-01-01

Sonic Hedgehog (Shh) has been shown to regulate wound healing in various tissues. Despite its known function in tissue regeneration, the role of Shh secreted from the gastric epithelium during tissue repair in the stomach remains unknown. Here we tested the hypothesis that Shh secreted from the acid-secreting parietal cell is a fundamental circulating factor that drives gastric repair. A mouse model expressing a parietal cell-specific deletion of Shh (PC-ShhKO) was generated using animals bearing loxP sites flanking exon 2 of the Shh gene (Shhflx/flx) and mice expressing a Cre transgene under the control of the H+,K+-ATPase β-subunit promoter. Shhflx/flx, the H+,K+-ATPase β-subunit promoter, and C57BL/6 mice served as controls. Ulcers were induced via acetic acid injury. At 1, 2, 3, 4, 5, and 7 days after the ulcer induction, gastric tissue and blood samples were collected. Parabiosis experiments were used to establish the effect of circulating Shh on ulcer repair. Control mice exhibited an increased expression of Shh in the gastric tissue and plasma that correlated with the repair of injury within 7 days after surgery. PC-ShhKO mice showed a loss of ulcer repair and reduced Shh tissue and plasma concentrations. In a parabiosis experiment whereby a control mouse was paired with a PC-ShhKO littermate and both animals subjected to gastric injury, a significant increase in the circulating Shh was measured in both parabionts. Elevated circulating Shh concentrations correlated with the repair of gastric ulcers in the PC-ShhKO parabionts. Therefore, the acid-secreting parietal cell within the stomach acts as an endocrine source of Shh during repair. PMID:24092639

Adult-type myogenesis of the frog Xenopus laevis specifically suppressed by notochord cells but promoted by spinal cord cells in vitro.

PubMed

Yamane, Hitomi; Ihara, Setsunosuke; Kuroda, Masaaki; Nishikawa, Akio

2011-08-01

Larval-to-adult myogenic conversion occurs in the dorsal muscle but not in the tail muscle during Xenopus laevis metamorphosis. To know the mechanism for tail-specific suppression of adult myogenesis, response character was compared between adult myogenic cells (Ad-cells) and larval tail myogenic cells (La-cells) to a Sonic hedgehog (Shh) inhibitor, notochord (Nc) cells, and spinal cord (SC) cells in vitro. Cyclopamine, an Shh inhibitor, suppressed the differentiation of cultured Ad (but not La) cells, suggesting the significance of Shh signaling in promoting adult myogenesis. To test the possibility that Shh-producing axial elements (notochord and spinal cord) regulate adult myogenesis, Ad-cells or La-cells were co-cultured with Nc or SC cells. The results showed that differentiation of Ad-cells were strongly inhibited by Nc cells but promoted by SC cells. If Ad-cells were "separately" co-cultured with Nc cells without direct cell-cell interactions, adult differentiation was not inhibited but rather promoted, suggesting that Nc cells have two roles, one is a short-range suppression and another is a long-range promotion for adult myogenesis. Immunohistochemical analysis showed both notochord and spinal cord express the N-terminal Shh fragment throughout metamorphosis. The "spinal cord-promotion" and long-range effect by Nc cells on adult myogenesis is thus involved in Shh signaling, while the signaling concerning the short-range "Nc suppression" will be determined by future studies. Interestingly, these effects, "Nc suppression" and "SC promotion" were not observed for La-cells. Situation where the spinal cord/notochord cross-sectional ratio is quite larger in tadpole trunk than in the tail seems to contribute to trunk-specific promotion and tail-specific suppression of adult myogenesis during Xenopus metamorphosis.

Melatonin influences the sonic hedgehog signaling pathway in porcine cumulus oocyte complexes.

PubMed

Lee, Sanghoon; Jin, Jun-Xue; Taweechaipaisankul, Anukul; Kim, Geon A; Ahn, Curie; Lee, Byeong Chun

2017-10-01

Melatonin, which is synthesized in the pineal gland and peripheral reproductive organs, has antioxidant properties and regulates physiological processes. It is well known that melatonin affects in vitro maturation (IVM) of oocytes and embryonic development in many species. However, beneficial effects of melatonin on IVM have been explained mainly by indirect antioxidant effects and little information is available on the underlying mechanism by which melatonin directly acts on porcine cumulus oocyte complexes (COCs). Sonic hedgehog (Shh) signaling is important for follicle development, oocyte maturation, and embryo development, and there may be a relationship between melatonin and Shh signaling. To examine this, we designed three groups: (i) control, (ii) melatonin (10 -9  mol/L), and (iii) melatonin with cyclopamine (2 μmol/L; Shh signaling inhibitor). The aim of this study was to investigate the effects of these agents on cumulus expansion, oocyte maturation, embryo development after parthenogenetic activation (PA), gene expression in cumulus cells, oocytes and blastocysts, and protein expression in COCs. Melatonin significantly increased the proportion of COCs exhibiting complete cumulus expansion (degree 4), PA blastocyst formation rates, and total cell numbers, which were inhibited by addition of cyclopamine. Simultaneously, the expression of cumulus expansion-related genes (Ptgs1, Ptgs2, and Has2) and Shh signaling-related genes (Shh, Pthc1, Smo, and Gli1) and proteins (Ptch1, Smo, and Gli1) in cumulus cells was upregulated in the melatonin-treated group, and these effects were also inhibited by cyclopamine. In conclusion, our results suggest that Shh signaling mediates effects of melatonin to improve porcine cumulus expansion and subsequent embryo development. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

β-Catenin signaling regulates temporally discrete phases of anterior taste bud development

PubMed Central

Thirumangalathu, Shoba; Barlow, Linda A.

2015-01-01

The sense of taste is mediated by multicellular taste buds located within taste papillae on the tongue. In mice, individual taste buds reside in fungiform papillae, which develop at mid-gestation as epithelial placodes in the anterior tongue. Taste placodes comprise taste bud precursor cells, which express the secreted factor sonic hedgehog (Shh) and give rise to taste bud cells that differentiate around birth. We showed previously that epithelial activation of β-catenin is the primary inductive signal for taste placode formation, followed by taste papilla morphogenesis and taste bud differentiation, but the degree to which these later elements were direct or indirect consequences of β-catenin signaling was not explored. Here, we define discrete spatiotemporal functions of β-catenin in fungiform taste bud development. Specifically, we show that early epithelial activation of β-catenin, before taste placodes form, diverts lingual epithelial cells from a taste bud fate. By contrast, β-catenin activation a day later within Shh+ placodes, expands taste bud precursors directly, but enlarges papillae indirectly. Further, placodal activation of β-catenin drives precocious differentiation of Type I glial-like taste cells, but not other taste cell types. Later activation of β-catenin within Shh+ precursors during papilla morphogenesis also expands taste bud precursors and accelerates Type I cell differentiation, but papilla size is no longer enhanced. Finally, although Shh regulates taste placode patterning, we find that it is dispensable for the accelerated Type I cell differentiation induced by β-catenin. PMID:26525674

Sonic hedgehog, Apoptosis and the Penis

PubMed Central

Podlasek, Carol A.

2009-01-01

Introduction Smooth muscle apoptosis in the penis is common in prostatectomy patients and animal models of erectile dysfunction (ED). A critical regulator of smooth muscle apoptosis in the penis is the secreted protein Sonic hedgehog (SHH). Since SHH protein treatment of the penis prevents cavernous nerve (CN) injury induced apoptosis, SHH has the potential to treat post-prostatectomy apoptosis. However little is known about how SHH signaling is regulated in the adult penis. Aim The goal of this review is to examine what is known about SHH signaling in the penis, to offer insight as to how SHH inhibition induces apoptosis in penile smooth muscle, and to define the role of the SHH pathway in maintaining CN integrity. Methods Information presented in this review was derived from a literature search using the National Library of Medicine PubMed Services. Search terms included SHH, apoptosis, smooth muscle, penis, ED, pelvic ganglia, corpora cavernosa, CN, regeneration, Schwann cell, neural activity and transport. Results In this review we have discussed the role of the CN in regulation of SHH abundance and apoptosis induction in the penis and have examined the function and localization of SHH signaling in the CN. Conclusion There is substantial potential to develop SHH for delivery to the penis of prostatectomy patients at the time of surgery in order to prevent apoptosis induction and long term ED development. Studies are in progress which will identify if SHH may be used as a regenerative therapy to speed CN regeneration. PMID:19267857

A Cascade of Wnt, Eda, and Shh Signaling Is Essential for Touch Dome Merkel Cell Development.

PubMed

Xiao, Ying; Thoresen, Daniel T; Miao, Lingling; Williams, Jonathan S; Wang, Chaochen; Atit, Radhika P; Wong, Sunny Y; Brownell, Isaac

2016-07-01

The Sonic hedgehog (Shh) signaling pathway regulates developmental, homeostatic, and repair processes throughout the body. In the skin, touch domes develop in tandem with primary hair follicles and contain sensory Merkel cells. The developmental signaling requirements for touch dome specification are largely unknown. We found dermal Wnt signaling and subsequent epidermal Eda/Edar signaling promoted Merkel cell morphogenesis by inducing Shh expression in early follicles. Lineage-specific gene deletions revealed intraepithelial Shh signaling was necessary for Merkel cell specification. Additionally, a Shh signaling agonist was sufficient to rescue Merkel cell differentiation in Edar-deficient skin. Moreover, Merkel cells formed in Fgf20 mutant skin where primary hair formation was defective but Shh production was preserved. Although developmentally associated with hair follicles, fate mapping demonstrated Merkel cells primarily originated outside the hair follicle lineage. These findings suggest that touch dome development requires Wnt-dependent mesenchymal signals to establish reciprocal signaling within the developing ectoderm, including Eda signaling to primary hair placodes and ultimately Shh signaling from primary follicles to extrafollicular Merkel cell progenitors. Shh signaling often demonstrates pleiotropic effects within a structure over time. In postnatal skin, Shh is known to regulate the self-renewal, but not the differentiation, of touch dome stem cells. Our findings relate the varied effects of Shh in the touch dome to the ligand source, with locally produced Shh acting as a morphogen essential for lineage specification during development and neural Shh regulating postnatal touch dome stem cell maintenance.

Dual role of Brg chromatin remodeling factor in Sonic hedgehog signaling during neural development.

PubMed

Zhan, Xiaoming; Shi, Xuanming; Zhang, Zilai; Chen, Yu; Wu, Jiang I

2011-08-02

Sonic hedgehog (Shh) signaling plays diverse roles during animal development and adult tissue homeostasis through differential regulation of Gli family transcription factors. Dysregulated Shh signaling activities have been linked to birth defects and tumorigenesis. Here we report that Brg, an ATP-dependent chromatin remodeling factor, has dual functions in regulating Shh target gene expression. Using a Brg conditional deletion in Shh-responding neural progenitors and fibroblasts, we demonstrate that Brg is required both for repression of the basal expression and for the activation of signal-induced transcription of Shh target genes. In developing telencephalons deficient for Brg, Shh target genes were derepressed, whereas Brg-deleted cerebellar granule neuron precursors failed to respond to Shh to increase their proliferation. The repressor function of Brg was mediated through Gli3 and both the repressor and activator functions of Brg appeared to be independent of its ATPase activity. Furthermore, Brg facilitates Gli coactivator histone deacetylase (HDAC) binding to the regulatory regions of Shh target genes, providing a possible mechanism for its positive role in Shh signaling. Our results thus reveal that a complex chromatin regulation mechanism underlies the precise transcription outcomes of Shh signaling and its diverse roles during development.

Shh mediates PDGF-induced contractile-to-synthetic phenotypic modulation in vascular smooth muscle cells through regulation of KLF4

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

Zeng, Qiu; Wei, Bin; Zhao, Yu

Platelet-derived growth factor (PDGF) is known to induce phenotypic switching of vascular smooth muscle cells (VSMCs) from contractile to a pathological synthetic state, which played an essential role in proliferation of VSMCs. Sonic hedgehog (Shh) contributes to the proliferation of VSMCs when induced by PDGF. Here, we investigated the probable role of Shh in PDGF-induced VSMC dedifferentiation and its underlying mechanisms. We found that PDGF stimulated Shh expression in VSMCs, which was mediated by activation of PDGFRβ/ERK1/2 cell signaling pathway. Further, we found PDGF-induced VSMC phenotypic modulation was accompanied by up-regulation of Shh/Gli family zinc finger 2 (Gli2) signaling andmore » Krüppel-like factor 4 (KLF4). When inhibited Shh in the presence of PDGF, the expressions of KLF4 and VSMC dedifferentiation markers were down-regulated and the effect of PDGF in inducing VSMC dedifferentiation was blocked. In the absence of PDGF, Shh signaling activation increased the expression of KLF4 and promoted VSMC dedifferentiation. The results indicate Shh participated in the regulation of PDGF-induced VSMC dedifferentiation. Finally, we found that KLF4 was closely involved in this process. On inhibition of KLF4, PDGF induced VSMC dedifferentiation was abrogated, even in the presence of Shh. Taken together, the results provide critical insights into the newly discovered role of Shh in phenotypic modulation of VSMCs which depends on KLF4. - Highlights: • Shh as a downstream effector of PDGF participates in PDGF-induced VSMC phenotypic modulation. • Shh can promote VSMC phenotypic switching from contractile to synthetic state. • Shh mediates VSMC phenotypic modulation through regulation of KLF4.« less

Modulation of Sonic hedgehog-induced mouse embryonic stem cell behaviors through E-cadherin expression and Integrin β1-dependent F-actin formation.

PubMed

Oh, Ji Young; Suh, Han Na; Choi, Gee Euhn; Lee, Hyun Jik; Jung, Young Hyun; Ko, So Hee; Kim, Jun Sung; Chae, Chang Woo; Lee, Chang-Kyu; Han, Ho Jae

2018-06-22

Sonic hedgehog pathway (Shh) plays a central role in maintaining stem cell function and behavior in various processes related to self-renewal and tissue regeneration. However, the therapeutic effect of Shh on mouse embryonic stem cells (mESCs) has not yet been clearly described. Thus, we investigated the effect of Shh on the regulation of mESC behaviors as well as the effect of Shh-pretreated mESCs in skin wound healing. The present study investigated the underlying mechanisms of Shh signaling pathway in growth and motility of mESCs using western blot analysis, cell proliferation assay, and cell migration assay. In addition, the effect of Shh-pretreated mESCs in skin wound healing was determined using mouse excisional wound splinting model. Shh induced adherens junction disruption through proteolysis by activating matrix metallopeptidases. In addition, the release of β-catenin from adherens junctions mediated by Shh led to cell cycle-dependent mESC proliferation. Shh-mediated Gli1 expression led to integrin β1 upregulation, followed by FAK and Src phosphorylation. Furthermore, among the Rho-GTPases, Rac1 and Cdc42 were activated in a Shh-dependent manner while F-actin expression was suppressed by Rac1 and Cdc42 siRNA transfection. Consistent with the in vitro results, skin wound healing assay revealed that Shh-treated mESCs induced angiogenesis and skin wound repair compared to that in Shh-treated mESCs transfected with integrin β1 siRNA in vivo. Our results imply that Shh induces adherens junction disruption and integrin β1-dependent F-actin formation involving FAK/Src and Rac1/Cdc42 signaling pathways in mESCs. This article is protected by copyright. All rights reserved.

Cooperatively transcriptional and epigenetic regulation of sonic hedgehog overexpression drives malignant potential of breast cancer

PubMed Central

Duan, Zhao-Heng; Wang, Hao-Chuan; Zhao, Dong-Mei; Ji, Xiao-Xin; Song, Min; Yang, Xiao-Jun; Cui, Wei

2015-01-01

Sonic hedgehog (Shh), a ligand of Hedgehog signaling pathway, is considered an important oncogene and an exciting potential therapeutic target in several cancers. Comprehensive understanding of the regulation mechanism of Shh in cancer cells is necessary to find an effective approach to selectively block its tumorigenic function. We and others previously demonstrated that nuclear factor-kappa B (NF-κB) activation and promoter hypomethylation contributed to the overexpression of Shh. However, the relationship between transcriptional and epigenetic regulation of Shh, and their roles in the malignant phenotype of cancer cells are still not clearly elucidated. In the present study, our data showed that the level of Shh was higher in breast cancer tissues with positive NF-κB nuclear staining and promoter hypomethylation. In addition, survival analysis revealed that Shh overexpression, but not hypomethylation and NF-κB nuclear staining, was a poor prognosis indicator for breast cancers. Moreover, in vitro data demonstrated that both NF-κB activation and hypomethylation in promoter region were positively associated with the overexpression of Shh. Mechanistically, the hypomethylation in Shh promoter could facilitate NF-κB binding to its site, and subsequently cooperate to induce transcription of Shh. Furthermore, the biological function data indicated that overexpressed Shh enhanced the self-renewal capacity and migration ability of breast cancer cells, which could be augmented by promoter demethylation and NF-κB activation. Overall, our findings reveal multiple and cooperative mechanisms of Shh upregulation in cancer cells, and the roles of Shh in tumor malignant behavior, thus suggesting a new strategy for therapeutic interventions to reduce Shh in tumors and improve patients’ prognosis. PMID:25990213

Sonic Hedgehog Guides Axons via Zipcode Binding Protein 1-Mediated Local Translation.

PubMed

Lepelletier, Léa; Langlois, Sébastien D; Kent, Christopher B; Welshhans, Kristy; Morin, Steves; Bassell, Gary J; Yam, Patricia T; Charron, Frédéric

2017-02-15

Sonic hedgehog (Shh) attracts spinal cord commissural axons toward the floorplate. How Shh elicits changes in the growth cone cytoskeleton that drive growth cone turning is unknown. We find that the turning of rat commissural axons up a Shh gradient requires protein synthesis. In particular, Shh stimulation increases β-actin protein at the growth cone even when the cell bodies have been removed. Therefore, Shh induces the local translation of β-actin at the growth cone. We hypothesized that this requires zipcode binding protein 1 (ZBP1), an mRNA-binding protein that transports β-actin mRNA and releases it for local translation upon phosphorylation. We found that Shh stimulation increases phospho-ZBP1 levels in the growth cone. Disruption of ZBP1 phosphorylation in vitro abolished the turning of commissural axons toward a Shh gradient. Disruption of ZBP1 function in vivo in mouse and chick resulted in commissural axon guidance errors. Therefore, ZBP1 is required for Shh to guide commissural axons. This identifies ZBP1 as a new mediator of noncanonical Shh signaling in axon guidance. SIGNIFICANCE STATEMENT Sonic hedgehog (Shh) guides axons via a noncanonical signaling pathway that is distinct from the canonical Hedgehog signaling pathway that specifies cell fate and morphogenesis. Axon guidance is driven by changes in the growth cone in response to gradients of guidance molecules. Little is known about the molecular mechanism of how Shh orchestrates changes in the growth cone cytoskeleton that are required for growth cone turning. Here, we show that the guidance of axons by Shh requires protein synthesis. Zipcode binding protein 1 (ZBP1) is an mRNA-binding protein that regulates the local translation of proteins, including actin, in the growth cone. We demonstrate that ZBP1 is required for Shh-mediated axon guidance, identifying a new member of the noncanonical Shh signaling pathway. Copyright © 2017 the authors 0270-6474/17/371685-11$15.00/0.

Analysis of testosterone effects on sonic hedgehog signaling in juvenile, adolescent and adult sprague dawley rat penis.

PubMed

Bond, Christopher W; Angeloni, Nicholas L; Podlasek, Carol A

2010-03-01

Smooth muscle apoptosis is a major contributing factor to erectile dysfunction (ED) development in prostatectomy and diabetic patients and animal models. A critical regulator of penile smooth muscle and apoptosis is Sonic hedgehog (SHH). The SHH protein is decreased in ED models and SHH treatment of cavernous nerve (CN) injured rats prevents smooth muscle apoptosis. A close association between androgen deficiency and ED has been suggested in the literature, but few studies have examined the molecular effects on penile smooth muscle and on known signaling mechanisms that regulate morphology. Aim. Examine testosterone and SHH interaction in eugonadal adult, adolescent and juvenile rats by performing castration studies and treatment with supraphysiological testosterone. The eugonadal adult Sprague Dawley rats were either treated with testosterone for 7 or 14 days (N = 14) or were castrated for 4 or 7 days (N = 12). The juvenile rats were treated with testosterone for 8 days (N = 7). The adolescent rats were castrated and sacrificed at P88 (N = 8). The control rats had empty vehicle (N = 22) or sham surgery (N = 20). The active form of SHH protein and mRNA were quantified by semi-quantitative immunohistochemical analysis and real-time reverse transcriptase polymerase chain reaction (RT-PCR). Testosterone treatment did not alter SHH signaling in juvenile rats. Shh mRNA increased 3.2-fold and SHH protein increased 1.2-fold in rats castrated during puberty. In adult rats, castration decreased Shh mRNA 3.2-fold but did not alter SHH protein. Testosterone supplement in adult rats increased Shh mRNA 2.3-fold and decreased SHH protein 1.3-fold. SHH signaling is independent of testosterone in normal juvenile rats and is sensitive to testosterone during adolescence, while testosterone supplement in the adult adversely impacts SHH signaling in a very similar manner to that observed with CN injury.

Hedgehog-mediated regulation of PPARγ controls metabolic patterns in neural precursors and shh-driven medulloblastoma.

PubMed

Bhatia, Bobby; Potts, Chad R; Guldal, Cemile; Choi, SunPhil; Korshunov, Andrey; Pfister, Stefan; Kenney, Anna M; Nahlé, Zaher A

2012-04-01

Sonic hedgehog (Shh) signaling is critical during development and its aberration is common across the spectrum of human malignancies. In the cerebellum, excessive activity of the Shh signaling pathway is associated with the devastating pediatric brain tumor medulloblastoma. We previously demonstrated that exaggerated de novo lipid synthesis is a hallmark of Shh-driven medulloblastoma and that hedgehog signaling inactivates the Rb/E2F tumor suppressor complex to promote lipogenesis. Indeed, such Shh-mediated metabolic reprogramming fuels tumor progression, in an E2F1- and FASN-dependent manner. Here, we show that the nutrient sensor PPARγ is a key component of the Shh metabolic network, particularly its regulation of glycolysis. Our data show that in primary cerebellar granule neural precursors (CGNPs), proposed medulloblastoma cells-of-origin, Shh stimulation elicits a marked induction of PPARγ alongside major glycolytic markers. This is also documented in the actively proliferating Shh-responsive CGNPs in the developing cerebellum, and PPARγ expression is strikingly elevated in Shh-driven medulloblastoma in vivo. Importantly, pharmacological blockade of PPARγ and/or Rb inactivation inhibits CGNP proliferation, drives medulloblastoma cell death and extends survival of medulloblastoma-bearing animals in vivo. This coupling of mitogenic Shh signaling to a major nutrient sensor and metabolic transcriptional regulator define a novel mechanism through which Shh signaling engages the nutrient sensing machinery in brain cancer, controls the cell cycle, and regulates the glycolytic index. This also reveals a dominant role of Shh in the etiology of glucose metabolism in medulloblastoma and underscores the function of the Shh → E2F1 → PPARγ axis in altering substrate utilization patterns in brain cancers in favor of tumor growth. These findings emphasize the value of PPARγ downstream of Shh as a global therapeutic target in hedgehog-dependent and/or Rb-inactivated tumors.

Is sonic Hedgehog involved in human fracture healing? --a prospective study on local and systemic concentrations of SHH.

PubMed

Eipeldauer, Stefan; Thomas, Anita; Hoechtl-Lee, Leonard; Kecht, Mathias; Binder, Harald; Koettstorfer, Julia; Gregori, Markus; Sarahrudi, Kambiz

2014-01-01

Sonic Hedgehog (SHH) is a new signalling pathway in bone repair. Evidence exist that SHH pathway plays a significant role in vasculogenesis and limb development during embryogenesis. Some in vitro and animal studies has already proven its potential for bone regeneration. However, no data on the role of SHH in the human fracture healing have been published so far. Seventy-five patients with long bone fractures were included into the study and divided in 2 groups. First group contained 69 patients with normal fracture healing. Four patients with impaired fracture healing formed the second group. 34 volunteers donated blood samples as control. Serum samples were collected over a period of 1 year following a standardized time schedule. In addition, SHH levels were measured in fracture haematoma and serum of 16 patients with bone fractures. Fracture haematoma and patients serum both contained lower SHH concentrations compared to control serum. The comparison between the patients' serum SHH level and the control serum revealed lower levels for the patients at all measurement time points. Significantly lower concentrations were observed at weeks 1 and 2 after fracture. SHH levels were slightly decreased in patients with impaired fracture healing without statistical significance. This is the first study to report local and systemic concentration of SHH in human fracture healing and SHH serum levels in healthy adults. A significant reduction of the SHH levels during the inflammatory phase of fracture healing was found. SHH concentrations in fracture haematoma and serum were lower than the concentration in control serum for the rest of the healing period. Our findings indicate that there is no relevant involvement of SHH in human fracture healing. Fracture repair process seem to reduce the SHH level in human. Further studies are definitely needed to clarify the underlying mechanisms.

Sonic Hedgehog Protein Is Decreased and Penile Morphology Is Altered in Prostatectomy and Diabetic Patients

PubMed Central

Angeloni, Nicholas L.; Bond, Christopher W.; McVary, Kevin T.; Podlasek, Carol A.

2013-01-01

Erectile dysfunction (ED) is a debilitating medical condition and current treatments are ineffective in patients with cavernous nerve (CN) injury, due to penile remodeling and apoptosis. A critical regulator of penile smooth muscle and apoptosis is the secreted protein sonic hedgehog (SHH). SHH protein is decreased in rat prostatectomy and diabetic ED models, SHH inhibition in the penis induces apoptosis and ED, and SHH treatment at the time of CN injury suppresses smooth muscle apoptosis and promotes regeneration of erectile function. Thus SHH treatment has significant translational potential as an ED therapy if similar mechanisms underlie ED development in patients. In this study we quantify SHH protein and morphological changes in corpora cavernosal tissue of control, prostatectomy and diabetic patients and hypothesize that decreased SHH protein is an underlying cause of ED development in prostatectomy and diabetic patients. Our results show significantly decreased SHH protein in prostatectomy and diabetic penis. Morphological remodelling of the penis, including significantly increased apoptotic index and decreased smooth muscle/collagen ratio, accompanies declining SHH. SHH signaling is active in human penis and is altered in a parallel manner to previous observations in the rat. These results suggest that SHH has significant potential to be developed as an ED therapy in prostatectomy and diabetic patients. The increased apoptotic index long after initial injury is suggestive of ongoing remodeling that may be clinically manipulatable. PMID:23967143

Sonic Hedgehog promotes the survival of neural crest cells by limiting apoptosis induced by the dependence receptor CDON during branchial arch development.

PubMed

Delloye-Bourgeois, Céline; Rama, Nicolas; Brito, José; Le Douarin, Nicole; Mehlen, Patrick

2014-09-26

Cell-adhesion molecule-related/Downregulated by Oncogenes (CDO or CDON) was identified as a receptor for the classic morphogen Sonic Hedgehog (SHH). It has been shown that, in cell culture, CDO also behaves as a SHH dependence receptor: CDO actively triggers apoptosis in absence of SHH via a proteolytic cleavage in CDO intracellular domain. We present evidence that CDO is also pro-apoptotic in the developing neural tube where SHH is known to act as a survival factor. SHH, produced by the ventral foregut endoderm, was shown to promote survival of facial neural crest cells (NCCs) that colonize the first branchial arch (BA1). We show here that the survival activity of SHH on neural crest cells is due to SHH-mediated inhibition of CDO pro-apoptotic activity. Silencing of CDO rescued NCCs from apoptosis observed upon SHH inhibition in the ventral foregut endoderm. Thus, the pair SHH/dependence receptor CDO may play an important role in neural crest cell survival during the formation of the first branchial arch. Copyright © 2014 Elsevier Inc. All rights reserved.

Sonic hedgehog-expressing basal cells are general post-mitotic precursors of functional taste receptor cells

PubMed Central

Miura, Hirohito; Scott, Jennifer K.; Harada, Shuitsu; Barlow, Linda A.

2014-01-01

Background Taste buds contain ~60 elongate cells and several basal cells. Elongate cells comprise three functional taste cell types: I - glial cells, II - bitter/sweet/umami receptor cells, and III - sour detectors. Although taste cells are continuously renewed, lineage relationships among cell types are ill-defined. Basal cells have been proposed as taste bud stem cells, a subset of which express Sonic hedgehog (Shh). However, Shh+ basal cells turnover rapidly suggesting that Shh+ cells are precursors of some or all taste cell types. Results To fate map Shh-expressing cells, mice carrying ShhCreERT2 and a high (CAG-CAT-EGFP) or low (R26RLacZ) efficiency reporter allele were given tamoxifen to activate Cre in Shh+ cells. Using R26RLacZ, lineage-labeled cells occur singly within buds, supporting a post-mitotic state for Shh+ cells. Using either reporter, we show that Shh+ cells differentiate into all three taste cell types, in proportions reflecting cell type ratios in taste buds (I > II > III). Conclusions Shh+ cells are not stem cells, but are post-mitotic, immediate precursors of taste cells. Shh+ cells differentiate into each of the three taste cell types, and the choice of a specific taste cell fate is regulated to maintain the proper ratio within buds. PMID:24590958

Sonic hedgehog in the notochord is sufficient for patterning of the intervertebral discs

PubMed Central

Choi, Kyung-Suk; Lee, Chanmi; Harfe, Brian D.

2012-01-01

The intervertebral discs, located between adjacent vertebrae, are required for stability of the spine and distributing mechanical load throughout the vertebral column. All cell types located in thes middle regions of the discs, called nuclei pulposi, are derived from the embryonic notochord. Recently, it was shown that the hedgehog signaling pathway plays an essential role during formation of nuclei pulposi. However, during the time that nuclei pulposi are forming, Shh is expressed in both the notochord and the nearby floor plate. To determine the source of SHH protein sufficient for formation of nuclei pulposi we removed Shh from either the floor plate or the notochord using tamoxifen-inducible Cre alleles. Removal of Shh from the floor plate resulted in phenotypically normal intervertebral discs, indicating that Shh expression in this tissue is not required for disc patterning. In addition, embryos that lacked Shh in the floor plate had normal vertebral columns, demonstrating that Shh expression in the notochord is sufficient for pattering the entire vertebral column. Removal of Shh from the notochord resulted in the absence of Shh in the floor plate, loss of intervertebral discs and vertebral structures. These data indicate that Shh expression in the notochord is sufficient for patterning of the intervertebral discs and the vertebral column. PMID:22841806

Sonic hedgehog in the notochord is sufficient for patterning of the intervertebral discs.

PubMed

Choi, Kyung-Suk; Lee, Chanmi; Harfe, Brian D

2012-01-01

The intervertebral discs, located between adjacent vertebrae, are required for stability of the spine and distributing mechanical load throughout the vertebral column. All cell types located in the middle regions of the discs, called nuclei pulposi, are derived from the embryonic notochord. Recently, it was shown that the hedgehog signaling pathway plays an essential role during formation of nuclei pulposi. However, during the time that nuclei pulposi are forming, Shh is expressed in both the notochord and the nearby floor plate. To determine the source of SHH protein sufficient for formation of nuclei pulposi we removed Shh from either the floor plate or the notochord using tamoxifen-inducible Cre alleles. Removal of Shh from the floor plate resulted in phenotypically normal intervertebral discs, indicating that Shh expression in this tissue is not required for disc patterning. In addition, embryos that lacked Shh in the floor plate had normal vertebral columns, demonstrating that Shh expression in the notochord is sufficient for pattering the entire vertebral column. Removal of Shh from the notochord resulted in the absence of Shh in the floor plate, loss of intervertebral discs and vertebral structures. These data indicate that Shh expression in the notochord is sufficient for patterning of the intervertebral discs and the vertebral column. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

Recombinant Human Sonic Hedgehog Protein Regulates the Expression of ZO-1 and Occludin by Activating Angiopoietin-1 in Stroke Damage

PubMed Central

Chen, Sheng-cai; Huang, Ming; Wang, Yong; Gao, Yuan; Huang, Yan; Wang, Meng-die; Mao, Ling; Hu, Bo

2013-01-01

This study examines the regulating effect of Sonic Hedgehog (Shh) on the permeability of the blood-brain barrier (BBB) in cerebral ischemia. By employing permanent middle cerebral artery occlusion (pMCAO) model, we find that Shh significantly decreases brain edema and preserves BBB permeability. Moreover, Shh increases zonula occludens-1 (ZO-1), occludin and angiopiotetin-1 (Ang-1) expression in the ischemic penumbra. Blockage of Shh with cyclopamine abolishes the effects of Shh on brain edema, BBB permeability and ZO-1, occludin, Ang-1 expression. Primary brain microvessel endothelial cells (BMECs) and astrocytes were pre-treated with Shh, cyclopamine, Ang-1-neutralizing antibody, and subjected to oxygen-glucose deprivation (OGD). Results show that the Ang-1 protein level in the culture medium of Shh-treated astrocytes is significantly higher. Shh also increased ZO-1, occludin and Ang-1 expression in BMECs, while cyclopamine and Ang-1-neutralizing antibody inhibited the effects of Shh on the ZO-1 and occludin expression, respectively. This study suggests that, under ischemic insults, Shh triggers Ang-1 production predominantly in astrocytes, and the secreted Ang-1 acts on BMECs, thereby upregulating ZO-1 and occludin to repair the tight junction and ameliorate the brain edema and BBB leakage. PMID:23894369

β-Catenin signaling regulates temporally discrete phases of anterior taste bud development.

PubMed

Thirumangalathu, Shoba; Barlow, Linda A

2015-12-15

The sense of taste is mediated by multicellular taste buds located within taste papillae on the tongue. In mice, individual taste buds reside in fungiform papillae, which develop at mid-gestation as epithelial placodes in the anterior tongue. Taste placodes comprise taste bud precursor cells, which express the secreted factor sonic hedgehog (Shh) and give rise to taste bud cells that differentiate around birth. We showed previously that epithelial activation of β-catenin is the primary inductive signal for taste placode formation, followed by taste papilla morphogenesis and taste bud differentiation, but the degree to which these later elements were direct or indirect consequences of β-catenin signaling was not explored. Here, we define discrete spatiotemporal functions of β-catenin in fungiform taste bud development. Specifically, we show that early epithelial activation of β-catenin, before taste placodes form, diverts lingual epithelial cells from a taste bud fate. By contrast, β-catenin activation a day later within Shh(+) placodes, expands taste bud precursors directly, but enlarges papillae indirectly. Further, placodal activation of β-catenin drives precocious differentiation of Type I glial-like taste cells, but not other taste cell types. Later activation of β-catenin within Shh(+) precursors during papilla morphogenesis also expands taste bud precursors and accelerates Type I cell differentiation, but papilla size is no longer enhanced. Finally, although Shh regulates taste placode patterning, we find that it is dispensable for the accelerated Type I cell differentiation induced by β-catenin. © 2015. Published by The Company of Biologists Ltd.

Sonic hedgehog signaling in spinal cord contributes to morphine-induced hyperalgesia and tolerance through upregulating brain-derived neurotrophic factor expression

PubMed Central

Song, Zhi-Jing; Miao, Shuai; Zhao, Ye; Wang, Xiu-Li; Liu, Yue-Peng

2018-01-01

Purpose Preventing opioid-induced hyperalgesia and tolerance continues to be a major clinical challenge, and the underlying mechanisms of hyperalgesia and tolerance remain elusive. Here, we investigated the role of sonic hedgehog (Shh) signaling in opioid-induced hyperalgesia and tolerance. Methods Shh signaling expression, behavioral changes, and neurochemical alterations induced by morphine were analyzed in male adult CD-1 mice with repeated administration of morphine. To investigate the contribution of Shh to morphine-induced hyperalgesia (MIH) and tolerance, Shh signaling inhibitor cyclopamine and Shh small interfering RNA (siRNA) were used. To explore the mechanisms of Shh signaling in MIH and tolerance, brain-derived neurotrophic factor (BDNF) inhibitor K252 and anti-BDNF antibody were used. Results Repeated administration of morphine produced obvious hyperalgesia and tolerance. The behavioral changes were correlated with the upregulation and activation of morphine treatment-induced Shh signaling. Pharmacologic and genetic inhibition of Shh signaling significantly delayed the generation of MIH and tolerance and associated neurochemical changes. Chronic morphine administration also induced upregulation of BDNF. Inhibiting BDNF effectively delayed the generation of MIH and tolerance. The upregulation of BDNF induced by morphine was significantly suppressed by inhibiting Shh signaling. In naïve mice, exogenous activation of Shh signaling caused a rapid increase of BDNF expression, as well as thermal hyperalgesia. Inhibiting BDNF significantly suppressed smoothened agonist-induced hyperalgesia. Conclusion These findings suggest that Shh signaling may be a critical mediator for MIH and tolerance by regulating BDNF expression. Inhibiting Shh signaling, especially during the early phase, may effectively delay or suppress MIH and tolerance. PMID:29662325

Mesenchymal stem cells induce epithelial proliferation within the inflamed stomach.

PubMed

Donnelly, Jessica M; Engevik, Amy; Feng, Rui; Xiao, Chang; Boivin, Gregory P; Li, Jing; Houghton, JeanMarie; Zavros, Yana

2014-06-15

Bone marrow-derived mesenchymal stem cells (MSCs) sustain cancer cells by creating a microenvironment favorable for tumor growth. In particular, MSCs have been implicated in gastric cancer development. There is extensive evidence suggesting that Hedgehog signaling regulates tumor growth. However, very little is known regarding the precise roles of Hedgehog signaling and MSCs in tumor development within the stomach. The current study tests that hypothesis that Sonic Hedgehog (Shh), secreted from MSCs, provides a proliferative stimulus for the gastric epithelium in the presence of inflammation. Red fluorescent protein-expressing MSCs transformed in vitro (stMSCs) were transduced with lentiviral constructs containing a vector control (stMSC(vect)) or short hairpin RNA (shRNA) targeting the Shh gene (stMSC(ShhKO)). Gastric submucosal transplantation of wild-type MSCs (wtMSCs), wild-type MSCs overexpressing Shh (wtMSC(Shh)), stMSC(vect), or stMSC(ShhKO) cells in C57BL/6 control (BL/6) or gastrin-deficient (GKO) mice was performed and mice analyzed 30 and 60 days posttransplantation. Compared with BL/6 mice transplanted with wtMSC(Shh) and stMSC(vect) cells, inflamed GKO mice developed aggressive gastric tumors. Tumor development was not observed in mouse stomachs transplanted with wtMSC or stMSC(ShhKO) cells. Compared with stMSC(ShhKO)-transplanted mice, within the inflamed GKO mouse stomach, Shh-expressing stMSC(vect)- and wtMSC(Shh)-induced proliferation of CD44-positive cells. CD44-positive cells clustered in gland-like structures within the tumor stroma and were positive for Patched (Ptch) expression. We conclude that Shh, secreted from MSCs, provides a proliferative stimulus for the gastric epithelium that is associated with tumor development, a response that is sustained by chronic inflammation. Copyright © 2014 the American Physiological Society.

Endogenous Sonic Hedgehog limits inflammation and angiogenesis in the ischaemic skeletal muscle of mice.

PubMed

Caradu, Caroline; Guy, Alexandre; James, Chloé; Reynaud, Annabel; Gadeau, Alain-Pierre; Renault, Marie-Ange

2018-04-01

Hedgehog (Hh) signalling has been shown to be re-activated in ischaemic tissues and participate in ischaemia-induced angiogenesis. Sonic Hedgehog (Shh) is upregulated by more than 80-fold in the ischaemic skeletal muscle, however its specific role in ischaemia-induced angiogenesis has not yet been fully investigated. The purpose of the present study was to investigate the role of endogenous Shh in ischaemia-induced angiogenesis. To this aim, we used inducible Shh knock-out (KO) mice and unexpectedly found that capillary density was significantly increased in re-generating muscle of Shh deficient mice 5 days after hind limb ischaemia was induced, demonstrating that endogenous Shh does not promote angiogenesis but more likely limits it. Myosin and MyoD expression were equivalent in Shh deficient mice and control mice, indicating that endogenous Shh is not required for ischaemia-induced myogenesis. Additionally, we observed a significant increase in macrophage infiltration in the ischaemic muscle of Shh deficient mice. Our data indicate that this was due to an increase in chemokine expression by myoblasts in the setting of impaired Hh signalling, using tissue specific Smoothened conditional KO mice. The increased macrophage infiltration in mice deficient for Hh signalling in myocytes was associated with increased VEGFA expression and a transiently increased angiogenesis, demonstrating that Shh limits inflammation and angiogenesis indirectly by signalling to myocytes. Although ectopic administration of Shh has previously been shown to promote ischaemia-induced angiogenesis, the present study reveals that endogenous Shh does not promote ischaemia-induced angiogenesis. On the contrary, the absence of Shh leads to aberrant ischaemic tissue inflammation and a transiently increased angiogenesis.

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

Ge, Xin; Lyu, Pengwei; Gu, Yuanting

Sonic hesgehog (Shh) signaling has been reported to play an essential role in cancer progression. The mechanism of Shh involved in breast cancer carcinogenesis remains unclear. The present study sought to explore whether Shh signaling could regulate the glycolytic metabolism in breast cancers. Overexpression of the smoothed (Smo) and Gli-1 was found in human primary breast cancers. The expressions of Shh and Gli-1 correlated significantly with tumor size and tumor stage. In vitro, human recombinant Shh (rShh) triggered Smo and Gli-1 expression, promoted glucose utilization and lactate production, and accelerated cell proliferation in MCF-7 and MDA-MB-231 cells. Notably, rShh did notmore » alter 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3) expression but augmented PFKFB3 phosphorylation on ser{sup 461}, along with elevated fructose-2,6-bisphosphate (F2,6BP) generation by MCF-7 and MDA-MB-231 cells. This effect could be dampened by Smo siRNA but not by Gli-1 siRNA. In addition, our data showed the upregulated expressions of MAPK by rShh and elevatory PFKFB3 phosphorylation by p38/MAPK activated kinase (MK2). In conclusion, our study characterized a novel role of Shh in promoting glycolysis and proliferation of breast cancer cells via PFKFB3 phosphorylation, which was mediated by Smo and p38/MK2. - Highlights: • Overexpression of Smo and Gli-1 was found in human primary breast cancers. • Shh promoted glucose utilization, lactate production, and cell proliferation. • Shh did not alter PFKFB3 expression but augmented PFKFB3 phosphorylation on ser461. • Shh acts on PFKFB3 phosphorylation via Smo and p38 MAPK/MK2.« less

Evidence for an expansion-based temporal Shh gradient in specifying vertebrate digit identities.

PubMed

Harfe, Brian D; Scherz, Paul J; Nissim, Sahar; Tian, Hua; McMahon, Andrew P; Tabin, Clifford J

2004-08-20

The zone of polarizing activity (ZPA) in the posterior limb bud produces Sonic Hedgehog (Shh) protein, which plays a critical role in establishing distinct fates along the anterior-posterior axis. This activity has been modeled as a concentration-dependent response to a diffusible morphogen. Using recombinase base mapping in the mouse, we determine the ultimate fate of the Shh-producing cells. Strikingly, the descendants of the Shh-producing cells encompass all cells in the two most posterior digits and also contribute to the middle digit. Our analysis suggests that, while specification of the anterior digits depends upon differential concentrations of Shh, the length of time of exposure to Shh is critical in the specification of the differences between the most posterior digits. Genetic studies of the effects of limiting accessibility of Shh within the limb support this model, in which the effect of the Shh morphogen is dictated by a temporal as well as a spatial gradient.

Resveratrol Downregulates Interleukin-6-Stimulated Sonic Hedgehog Signaling in Human Acute Myeloid Leukemia

PubMed Central

Su, Yu-Chieh; Li, Szu-Chin; Wu, Yin-Chi; Wang, Li-Min; Chao, K. S. Clifford; Liao, Hui-Fen

2013-01-01

IL-6 and sonic hedgehog (Shh) signaling molecules are considered to maintain the growth of cancer stem cells (CSCs). Resveratrol, an important integrant in traditional Chinese medicine, possesses certain antitumor effects. However, the mechanisms on regulating acute myeloid leukemia (AML) are unclear. This study first used human subjects to demonstrate that the plasma levels of IL-6 and IL-1β in AML patients were higher and lower, respectively, than healthy donors. The expression of Shh preproproteins, and C- and N-terminal Shh peptides increased in bone marrow and peripheral blood mononuclear cells isolated from AML patients, and the plasma N-Shh secretion was greater. To further clarify the effect of IL-6 and resveratrol in Shh signaling, human AML HL-60 cells were tested. IL-6 upregulated Shh and Gli-1 expression and was accompanied by an increase of cell viability. Resveratrol significantly decreased CSC-related Shh expression, Gli-1 nuclear translocation, and cell viability in IL-6-treated HL-60 cells and had synergistic effect with Shh inhibitor cyclopamine on inhibiting cell growth. Conclusions. IL-6 stimulated the growth of AML cells through Shh signaling, and this effect might be blocked by resveratrol. Further investigations of Shh as a prognostic marker and resveratrol as a therapeutic drug target to CSCs in AML are surely warranted. PMID:23533494

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.

An inversion involving the mouse Shh locus results in brachydactyly through dysregulation of Shh expression.

PubMed

Niedermaier, Michael; Schwabe, Georg C; Fees, Stephan; Helmrich, Anne; Brieske, Norbert; Seemann, Petra; Hecht, Jochen; Seitz, Volkhard; Stricker, Sigmar; Leschik, Gundula; Schrock, Evelin; Selby, Paul B; Mundlos, Stefan

2005-04-01

Short digits (Dsh) is a radiation-induced mouse mutant. Homozygous mice are characterized by multiple defects strongly resembling those resulting from Sonic hedgehog (Shh) inactivation. Heterozygous mice show a limb reduction phenotype with fusion and shortening of the proximal and middle phalanges in all digits, similar to human brachydactyly type A1, a condition caused by mutations in Indian hedgehog (IHH). We mapped Dsh to chromosome 5 in a region containing Shh and were able to demonstrate an inversion comprising 11.7 Mb. The distal breakpoint is 13.298 kb upstream of Shh, separating the coding sequence from several putative regulatory elements identified by interspecies comparison. The inversion results in almost complete downregulation of Shh expression during E9.5-E12.5, explaining the homozygous phenotype. At E13.5 and E14.5, however, Shh is upregulated in the phalangeal anlagen of Dsh/+ mice, at a time point and in a region where WT Shh is never expressed. The dysregulation of Shh expression causes the local upregulation of hedgehog target genes such as Gli1-3, patched, and Pthlh, as well as the downregulation of Ihh and Gdf5. This results in shortening of the digits through an arrest of chondrocyte differentiation and the disruption of joint development.

An inversion involving the mouse Shh locus results in brachydactyly through dysregulation of Shh expression

PubMed Central

Niedermaier, Michael; Schwabe, Georg C.; Fees, Stephan; Helmrich, Anne; Brieske, Norbert; Seemann, Petra; Hecht, Jochen; Seitz, Volkhard; Stricker, Sigmar; Leschik, Gundula; Schrock, Evelin; Selby, Paul B.; Mundlos, Stefan

2005-01-01

Short digits (Dsh) is a radiation-induced mouse mutant. Homozygous mice are characterized by multiple defects strongly resembling those resulting from Sonic hedgehog (Shh) inactivation. Heterozygous mice show a limb reduction phenotype with fusion and shortening of the proximal and middle phalanges in all digits, similar to human brachydactyly type A1, a condition caused by mutations in Indian hedgehog (IHH). We mapped Dsh to chromosome 5 in a region containing Shh and were able to demonstrate an inversion comprising 11.7 Mb. The distal breakpoint is 13.298 kb upstream of Shh, separating the coding sequence from several putative regulatory elements identified by interspecies comparison. The inversion results in almost complete downregulation of Shh expression during E9.5–E12.5, explaining the homozygous phenotype. At E13.5 and E14.5, however, Shh is upregulated in the phalangeal anlagen of Dsh/+ mice, at a time point and in a region where WT Shh is never expressed. The dysregulation of Shh expression causes the local upregulation of hedgehog target genes such as Gli1-3, patched, and Pthlh, as well as the downregulation of Ihh and Gdf5. This results in shortening of the digits through an arrest of chondrocyte differentiation and the disruption of joint development. PMID:15841179

Cell fate specification in the lingual epithelium is controlled by antagonistic activities of Sonic hedgehog and retinoic acid.

PubMed

El Shahawy, Maha; Reibring, Claes-Göran; Neben, Cynthia L; Hallberg, Kristina; Marangoni, Pauline; Harfe, Brian D; Klein, Ophir D; Linde, Anders; Gritli-Linde, Amel

2017-07-01

The interaction between signaling pathways is a central question in the study of organogenesis. Using the developing murine tongue as a model, we uncovered unknown relationships between Sonic hedgehog (SHH) and retinoic acid (RA) signaling. Genetic loss of SHH signaling leads to enhanced RA activity subsequent to loss of SHH-dependent expression of Cyp26a1 and Cyp26c1. This causes a cell identity switch, prompting the epithelium of the tongue to form heterotopic minor salivary glands and to overproduce oversized taste buds. At developmental stages during which Wnt10b expression normally ceases and Shh becomes confined to taste bud cells, loss of SHH inputs causes the lingual epithelium to undergo an ectopic and anachronic expression of Shh and Wnt10b in the basal layer, specifying de novo taste placode induction. Surprisingly, in the absence of SHH signaling, lingual epithelial cells adopted a Merkel cell fate, but this was not caused by enhanced RA signaling. We show that RA promotes, whereas SHH, acting strictly within the lingual epithelium, inhibits taste placode and lingual gland formation by thwarting RA activity. These findings reveal key functions for SHH and RA in cell fate specification in the lingual epithelium and aid in deciphering the molecular mechanisms that assign cell identity.

Integration of shallow gradients of Shh and Netrin-1 guides commissural axons.

PubMed

Sloan, Tyler F W; Qasaimeh, Mohammad A; Juncker, David; Yam, Patricia T; Charron, Frédéric

2015-03-01

During nervous system development, gradients of Sonic Hedgehog (Shh) and Netrin-1 attract growth cones of commissural axons toward the floor plate of the embryonic spinal cord. Mice defective for either Shh or Netrin-1 signaling have commissural axon guidance defects, suggesting that both Shh and Netrin-1 are required for correct axon guidance. However, how Shh and Netrin-1 collaborate to guide axons is not known. We first quantified the steepness of the Shh gradient in the spinal cord and found that it is mostly very shallow. We then developed an in vitro microfluidic guidance assay to simulate these shallow gradients. We found that axons of dissociated commissural neurons respond to steep but not shallow gradients of Shh or Netrin-1. However, when we presented axons with combined Shh and Netrin-1 gradients, they had heightened sensitivity to the guidance cues, turning in response to shallower gradients that were unable to guide axons when only one cue was present. Furthermore, these shallow gradients polarized growth cone Src-family kinase (SFK) activity only when Shh and Netrin-1 were combined, indicating that SFKs can integrate the two guidance cues. Together, our results indicate that Shh and Netrin-1 synergize to enable growth cones to sense shallow gradients in regions of the spinal cord where the steepness of a single guidance cue is insufficient to guide axons, and we identify a novel type of synergy that occurs when the steepness (and not the concentration) of a guidance cue is limiting.

Integration of Shallow Gradients of Shh and Netrin-1 Guides Commissural Axons

PubMed Central

Sloan, Tyler F. W.; Qasaimeh, Mohammad A.; Juncker, David; Yam, Patricia T.; Charron, Frédéric

2015-01-01

During nervous system development, gradients of Sonic Hedgehog (Shh) and Netrin-1 attract growth cones of commissural axons toward the floor plate of the embryonic spinal cord. Mice defective for either Shh or Netrin-1 signaling have commissural axon guidance defects, suggesting that both Shh and Netrin-1 are required for correct axon guidance. However, how Shh and Netrin-1 collaborate to guide axons is not known. We first quantified the steepness of the Shh gradient in the spinal cord and found that it is mostly very shallow. We then developed an in vitro microfluidic guidance assay to simulate these shallow gradients. We found that axons of dissociated commissural neurons respond to steep but not shallow gradients of Shh or Netrin-1. However, when we presented axons with combined Shh and Netrin-1 gradients, they had heightened sensitivity to the guidance cues, turning in response to shallower gradients that were unable to guide axons when only one cue was present. Furthermore, these shallow gradients polarized growth cone Src-family kinase (SFK) activity only when Shh and Netrin-1 were combined, indicating that SFKs can integrate the two guidance cues. Together, our results indicate that Shh and Netrin-1 synergize to enable growth cones to sense shallow gradients in regions of the spinal cord where the steepness of a single guidance cue is insufficient to guide axons, and we identify a novel type of synergy that occurs when the steepness (and not the concentration) of a guidance cue is limiting. PMID:25826604

Sonic hedgehog: restricted expression and limb dysmorphologies

PubMed Central

Hill, Robert E; Heaney, Simon JH; Lettice, Laura A

2003-01-01

Sonic hedgehog, SHH, is required for patterning the limb. The array of skeletal elements that compose the hands and feet, and the ordered arrangement of these bones to form the pattern of fingers and toes are dependent on SHH. The mechanism of action of SHH in the limb is not fully understood; however, an aspect that appears to be important is the localized, asymmetric expression of Shh. Shh is expressed in the posterior margin of the limb bud in a region defined as the zone of polarizing activity (ZPA). Analysis of mouse mutants which have polydactyly (extra toes) shows that asymmetric expression of Shh is lost due to the appearance of an ectopic domain of expression in the anterior limb margin. One such polydactylous mouse mutant, sasquatch (Ssq), maps to the corresponding chromosomal region of the human condition pre-axial polydactyly (PPD) and thus represents a model for this condition. The mutation responsible for Ssq is located 1 Mb away from the Shh gene; however, the mutation disrupts a long-range cis-acting regulator of Shh expression. By inference, human pre-axial polydactyly results from a similar disruption of Shh expression. Other human congenital abnormalities also map near the pre-axial polydactyly locus, suggesting a major chromosomal region for limb dysmorphologies. The distinct phenotypes range from loss of all bones of the hands and feet to syndactyly of the soft tissue and fusion of the digits. We discuss the role played by Shh expression in mouse mutant phenotypes and the human limb dysmorphologies. PMID:12587915

Pleiotropic functions of embryonic sonic hedgehog expression link jaw and taste bud amplification with eye loss during cavefish evolution.

PubMed

Yamamoto, Yoshiyuki; Byerly, Mardi S; Jackman, William R; Jeffery, William R

2009-06-01

This study addresses the role of sonic hedgehog (shh) in increasing oral-pharyngeal constructive traits (jaws and taste buds) at the expense of eyes in the blind cavefish Astyanax mexicanus. In cavefish embryos, eye primordia degenerate under the influence of hyperactive Shh signaling. In concert, cavefish show amplified jaw size and taste bud numbers as part of a change in feeding behavior. To determine whether pleiotropic effects of hyperactive Shh signaling link these regressive and constructive traits, shh expression was compared during late development of the surface-dwelling (surface fish) and cave-dwelling (cavefish) forms of Astyanax. After an initial expansion along the midline of early embryos, shh was elevated in the oral-pharyngeal region in cavefish and later was confined to taste buds. The results of shh inhibition and overexpression experiments indicate that Shh signaling has an important role in oral and taste bud development. Conditional overexpression of an injected shh transgene at specific times in development showed that taste bud amplification and eye degeneration are sensitive to shh overexpression during the same early developmental period, although taste buds are not formed until much later. Genetic crosses between cavefish and surface fish revealed an inverse relationship between eye size and jaw size/taste bud number, supporting a link between oral-pharyngeal constructive traits and eye degeneration. The results suggest that hyperactive Shh signaling increases oral and taste bud amplification in cavefish at the expense of eyes. Therefore, selection for constructive oral-pharyngeal traits may be responsible for eye loss during cavefish evolution via pleiotropic function of the Shh signaling pathway.

Long-range sclerotome induction by sonic hedgehog: direct role of the amino-terminal cleavage product and modulation by the cyclic AMP signaling pathway.

PubMed

Fan, C M; Porter, J A; Chiang, C; Chang, D T; Beachy, P A; Tessier-Lavigne, M

1995-05-05

A long-range signal encoded by the Sonic hedgehog (Shh) gene has been implicated as the ventral patterning influence from the notochord that induces sclerotome and represses dermomyotome in somite differentiation. Long-range effects of hedgehog (hh) signaling have been suggested to result either from local induction of a secondary diffusible signal or from the direct action of the highly diffusible carboxy-terminal product of HH autoproteolytic cleavage. Here we provide evidence that the long-range somite patterning effects of SHH are instead mediated by a direct action of the amino-terminal cleavage product. We also show that pharmacological manipulations to increase the activity of cyclic AMP-dependent protein kinase A can selectively antagonize the effects of the amino-terminal cleavage product. Our results support the operation of a single evolutionarily conserved signaling pathway for both local and direct long-range inductive actions of HH family members.

Vismodegib, an antagonist of hedgehog signaling, directly alters taste molecular signaling in taste buds.

PubMed

Yang, Hyekyung; Cong, Wei-Na; Yoon, Jeong Seon; Egan, Josephine M

2015-02-01

Vismodegib, a highly selective inhibitor of hedgehog (Hh) pathway, is an approved treatment for basal-cell carcinoma. Patients on treatment with vismodegib often report profound alterations in taste sensation. The cellular mechanisms underlying the alterations have not been studied. Sonic Hh (Shh) signaling is required for cell growth and differentiation. In taste buds, Shh is exclusively expressed in type IV taste cells, which are undifferentiated basal cells and the precursors of the three types of taste sensing cells. Thus, we investigated if vismodegib has an inhibitory effect on taste cell turnover because of its known effects on Hh signaling. We gavaged C57BL/6J male mice daily with either vehicle or 30 mg/kg vismodegib for 15 weeks. The gustatory behavior and immunohistochemical profile of taste cells were examined. Vismodegib-treated mice showed decreased growth rate and behavioral responsivity to sweet and bitter stimuli, compared to vehicle-treated mice. We found that vismodegib-treated mice had significant reductions in taste bud size and numbers of taste cells per taste bud. Additionally, vismodegib treatment resulted in decreased numbers of Ki67- and Shh-expressing cells in taste buds. The numbers of phospholipase Cβ2- and α-gustducin-expressing cells, which contain biochemical machinery for sweet and bitter sensing, were reduced in vismodegib-treated mice. Furthermore, vismodegib treatment resulted in reduction in numbers of T1R3, glucagon-like peptide-1, and glucagon-expressing cells, which are known to modulate sweet taste sensitivity. These results suggest that inhibition of Shh signaling by vismodegib treatment directly results in alteration of taste due to local effects in taste buds. Published 2014. This article is a U.S. Government work and is in the public domain in the USA.

Vismodegib, an antagonist of hedgehog signaling, directly alters taste molecular signaling in taste buds

PubMed Central

Yang, Hyekyung; Cong, Wei-na; Yoon, Jeong Seon; Egan, Josephine M

2015-01-01

Vismodegib, a highly selective inhibitor of hedgehog (Hh) pathway, is an approved treatment for basal-cell carcinoma. Patients on treatment with vismodegib often report profound alterations in taste sensation. The cellular mechanisms underlying the alterations have not been studied. Sonic Hh (Shh) signaling is required for cell growth and differentiation. In taste buds, Shh is exclusively expressed in type IV taste cells, which are undifferentiated basal cells and the precursors of the three types of taste sensing cells. Thus, we investigated if vismodegib has an inhibitory effect on taste cell turnover because of its known effects on Hh signaling. We gavaged C57BL/6J male mice daily with either vehicle or 30 mg/kg vismodegib for 15 weeks. The gustatory behavior and immunohistochemical profile of taste cells were examined. Vismodegib-treated mice showed decreased growth rate and behavioral responsivity to sweet and bitter stimuli, compared to vehicle-treated mice. We found that vismodegib-treated mice had significant reductions in taste bud size and numbers of taste cells per taste bud. Additionally, vismodegib treatment resulted in decreased numbers of Ki67- and Shh-expressing cells in taste buds. The numbers of phospholipase Cβ2- and α-gustducin-expressing cells, which contain biochemical machinery for sweet and bitter sensing, were reduced in vismodegib-treated mice. Furthermore, vismodegib treatment resulted in reduction in numbers of T1R3, glucagon-like peptide-1, and glucagon-expressing cells, which are known to modulate sweet taste sensitivity. These results suggest that inhibition of Shh signaling by vismodegib treatment directly results in alteration of taste due to local effects in taste buds. PMID:25354792

Floor plate-derived sonic hedgehog regulates glial and ependymal cell fates in the developing spinal cord.

PubMed

Yu, Kwanha; McGlynn, Sean; Matise, Michael P

2013-04-01

Cell fate specification in the CNS is controlled by the secreted morphogen sonic hedgehog (Shh). At spinal cord levels, Shh produced by both the notochord and floor plate (FP) diffuses dorsally to organize patterned gene expression in dividing neural and glial progenitors. Despite the fact that two discrete sources of Shh are involved in this process, the individual contribution of the FP, the only intrinsic source of Shh throughout both neurogenesis and gliogenesis, has not been clearly defined. Here, we have used conditional mutagenesis approaches in mice to selectively inactivate Shh in the FP (Shh(FP)) while allowing expression to persist in the notochord, which underlies the neural tube during neurogenesis but not gliogenesis. We also inactivated Smo, the common Hh receptor, in neural tube progenitors. Our findings confirm and extend prior studies suggesting an important requirement for Shh(FP) in specifying oligodendrocyte cell fates via repression of Gli3 in progenitors. Our studies also uncover a connection between embryonic Shh signaling and astrocyte-mediated reactive gliosis in adults, raising the possibility that this pathway is involved in the development of the most common cell type in the CNS. Finally, we find that intrinsic spinal cord Shh signaling is required for the proper formation of the ependymal zone, the epithelial cell lining of the central canal that is also an adult stem cell niche. Together, our studies identify a crucial late embryonic role for Shh(FP) in regulating the specification and differentiation of glial and epithelial cells in the mouse spinal cord.

Administration of sonic hedgehog protein induces angiogenesis and has therapeutic effects after stroke in rats.

PubMed

Chen, Sheng-Cai; Huang, Ming; He, Quan-Wei; Zhang, Yan; Opoku, Elvis Nana; Yang, Hang; Jin, Hui-Juan; Xia, Yuan-Peng; Hu, Bo

2017-06-03

The Sonic hedgehog (Shh) signaling pathway is recapitulated in response to ischemic injury. Here, we investigated the clinical implications of Shh protein in the ischemic stroke and explored the underlying mechanism. Intracerebroventricular injection of Shh, Cyclopamine, or anti-vascular endothelial growth factor (VEGF) was performed immediately after permanent middle cerebral artery occlusion (pMCAO) surgery and lasted for 7days (d). Phosphate-buffered saline (PBS) was used as control. Neurological deficits and infarct volume were examined 7d after pMCAO. Microvascular density with fluorescein-iso-thiocyanate (FITC) assay and double staining with CD31 and Ki-67 was measured at 7d. To observe in vitro angiogenesis, rat brain microvascular endothelial cells (RBMECs) were incubated under oxygen glucose deprivation (OGD) for 6h (h) and treated with Shh/anti-VEGF. We found that (1) Shh improved neurological scores and reduced infarct volume, which was blocked by Cyclopamine, (2) Shh improved the microvascular density and promoted angiogenesis and neuron survival in the ischemic boundary zone, (3) Shh enhanced VEGF expression and VEGF antibody could reverse angiogenic and protective effect of Shh in vivo and in vitro. These data demonstrate that the administration of Shh protein could protect brain from ischemic injury, in part by promoting angiogenic repair. Copyright © 2017 IBRO. Published by Elsevier Ltd. All rights reserved.

Cell fate specification in the lingual epithelium is controlled by antagonistic activities of Sonic hedgehog and retinoic acid

PubMed Central

Neben, Cynthia L.; Harfe, Brian D.; Linde, Anders

2017-01-01

The interaction between signaling pathways is a central question in the study of organogenesis. Using the developing murine tongue as a model, we uncovered unknown relationships between Sonic hedgehog (SHH) and retinoic acid (RA) signaling. Genetic loss of SHH signaling leads to enhanced RA activity subsequent to loss of SHH-dependent expression of Cyp26a1 and Cyp26c1. This causes a cell identity switch, prompting the epithelium of the tongue to form heterotopic minor salivary glands and to overproduce oversized taste buds. At developmental stages during which Wnt10b expression normally ceases and Shh becomes confined to taste bud cells, loss of SHH inputs causes the lingual epithelium to undergo an ectopic and anachronic expression of Shh and Wnt10b in the basal layer, specifying de novo taste placode induction. Surprisingly, in the absence of SHH signaling, lingual epithelial cells adopted a Merkel cell fate, but this was not caused by enhanced RA signaling. We show that RA promotes, whereas SHH, acting strictly within the lingual epithelium, inhibits taste placode and lingual gland formation by thwarting RA activity. These findings reveal key functions for SHH and RA in cell fate specification in the lingual epithelium and aid in deciphering the molecular mechanisms that assign cell identity. PMID:28715412

Sonic hedgehog controls growth of external genitalia by regulating cell cycle kinetics

PubMed Central

Seifert, Ashley W.; Zheng, Zhengui; Ormerod, Brandi K.; Cohn, Martin J.

2010-01-01

During embryonic development, cells are instructed which position to occupy, they interpret these cues as differentiation programmes, and expand these patterns by growth. Sonic hedgehog (Shh) specifies positional identity in many organs; however, its role in growth is not well understood. In this study, we show that inactivation of Shh in external genitalia extends the cell cycle from 8.5 to 14.4 h, and genital growth is reduced by ∼75%. Transient Shh signalling establishes pattern in the genital tubercle; however, transcriptional levels of G1 cell cycle regulators are reduced. Consequently, G1 length is extended, leading to fewer progenitor cells entering S-phase. Cell cycle genes responded similarly to Shh inactivation in genitalia and limbs, suggesting that Shh may regulate growth by similar mechanisms in different organ systems. The finding that Shh regulates cell number by controlling the length of specific cell cycle phases identifies a novel mechanism by which Shh elaborates pattern during appendage development. PMID:20975695

ATP oscillations mediate inductive action of FGF and Shh signalling on prechondrogenic condensation.

PubMed

Kwon, Hyuck Joon

2013-01-01

Skeletal patterns are prefigured by prechondrogenic condensation. Morphogens such as fibroblast growth factor (FGF) and sonic hedgehog (Shh) specify the skeletal patterns in limb development. However, how morphogens regulate prechondrogenic condensation has remained unclear. Recently, it was demonstrated that synchronized Adenosine triphosphate (ATP) oscillations play a critical role in prechondrogenic condensation. Thus, the present study has focused on whether ATP oscillations mediate the actions of major developmental morphogens such as FGF and Shh on prechondrogenic condensation. It has been shown that both FGF and Shh signalling promoted cellular condensation but not chondrogenic differentiation and also induced ATP oscillations. In addition, blockage of FGF and Shh signalling prevented both ATP oscillations and prechondrogenic condensation. Furthermore, it was found that inhibition of ATP oscillations suppressed FGF/Shh-induced prechondrogenic condensation. These results indicate that ATP oscillations mediate the actions of FGF and Shh signalling on prechondrogenic condensation. This study proposes that morphogens organize skeletal patterns via ATP oscillations. Copyright © 2012 John Wiley & Sons, Ltd.

Msx genes are important apoptosis effectors downstream of the Shh/Gli3 pathway in the limb.

PubMed

Lallemand, Yvan; Bensoussan, Vardina; Cloment, Cécile Saint; Robert, Benoît

2009-07-15

In tetrapods, the anteroposterior (AP) patterning of the limb is under the control of the antagonistic activities of the secreted factor Sonic hedgehog (Shh) and Gli3R, the truncated repressor form of the transcription factor Gli3. In this report, we show that Msx1 and Msx2 are targets and downstream effectors of Gli3R. Consequently, in Shh null mutants, Msx genes are overexpressed and, furthermore, partially responsible for the limb phenotype. This is exemplified by the fact that reducing Msx activity in Shh mutants partially restores a normal limb development. Finally, we show that the main action of the Msx genes, in both normal and Shh(-/-) limb development, is to control cell death in the mesenchyme. We propose that, in the limb, Msx genes act downstream of the Shh/Gli3 pathway by transducing BMP signaling and that, in the absence of Shh signaling, their deregulation contributes to the extensive apoptosis that impairs limb development.

Development of five digits is controlled by a bipartite long-range cis-regulator.

PubMed

Lettice, Laura A; Williamson, Iain; Devenney, Paul S; Kilanowski, Fiona; Dorin, Julia; Hill, Robert E

2014-04-01

Conservation within intergenic DNA often highlights regulatory elements that control gene expression from a long range. How conservation within a single element relates to regulatory information and how internal composition relates to function is unknown. Here, we examine the structural features of the highly conserved ZRS (also called MFCS1) cis-regulator responsible for the spatiotemporal control of Shh in the limb bud. By systematically dissecting the ZRS, both in transgenic assays and within in the endogenous locus, we show that the ZRS is, in effect, composed of two distinct domains of activity: one domain directs spatiotemporal activity but functions predominantly from a short range, whereas a second domain is required to promote long-range activity. We show further that these two domains encode activities that are highly integrated and that the second domain is crucial in promoting the chromosomal conformational changes correlated with gene activity. During limb bud development, these activities encoded by the ZRS are interpreted differently by the fore limbs and the hind limbs; in the absence of the second domain there is no Shh activity in the fore limb, and in the hind limb low levels of Shh lead to a variant digit pattern ranging from two to four digits. Hence, in the embryo, the second domain stabilises the developmental programme providing a buffer for SHH morphogen activity and this ensures that five digits form in both sets of limbs.

Sonic hedgehog promotes stem-cell potential of Mueller glia in the mammalian retina

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

Wan Jin; Zheng Hua; Xiao Honglei

2007-11-16

Mueller glia have been demonstrated to display stem-cell properties after retinal damage. Here, we report this potential can be regulated by Sonic hedgehog (Shh) signaling. Shh can stimulate proliferation of Mueller glia through its receptor and target gene expressed on them, furthermore, Shh-treated Mueller glia are induced to dedifferentiate by expressing progenitor-specific markers, and then adopt cell fate of rod photoreceptor. Inhibition of signaling by cyclopamine inhibits proliferation and dedifferentiation. Intraocular injection of Shh promotes Mueller glia activation in the photoreceptor-damaged retina, Shh also enhances neurogenic potential by producing more rhodopsin-positive photoreceptors from Mueller glia-derived cells. Together, these results providemore » evidences that Mueller glia act as potential stem cells in mammalian retina, Shh may have therapeutic effects on these cells for promoting the regeneration of retinal neurons.« less

Induction of ectopic taste buds by SHH reveals the competency and plasticity of adult lingual epithelium

PubMed Central

Castillo, David; Seidel, Kerstin; Salcedo, Ernesto; Ahn, Christina; de Sauvage, Frederic J.; Klein, Ophir D.; Barlow, Linda A.

2014-01-01

Taste buds are assemblies of elongated epithelial cells, which are innervated by gustatory nerves that transmit taste information to the brain stem. Taste cells are continuously renewed throughout life via proliferation of epithelial progenitors, but the molecular regulation of this process remains unknown. During embryogenesis, sonic hedgehog (SHH) negatively regulates taste bud patterning, such that inhibition of SHH causes the formation of more and larger taste bud primordia, including in regions of the tongue normally devoid of taste buds. Here, using a Cre-lox system to drive constitutive expression of SHH, we identify the effects of SHH on the lingual epithelium of adult mice. We show that misexpression of SHH transforms lingual epithelial cell fate, such that daughter cells of lingual epithelial progenitors form cell type-replete, onion-shaped taste buds, rather than non-taste, pseudostratified epithelium. These SHH-induced ectopic taste buds are found in regions of the adult tongue previously thought incapable of generating taste organs. The ectopic buds are composed of all taste cell types, including support cells and detectors of sweet, bitter, umami, salt and sour, and recapitulate the molecular differentiation process of endogenous taste buds. In contrast to the well-established nerve dependence of endogenous taste buds, however, ectopic taste buds form independently of both gustatory and somatosensory innervation. As innervation is required for SHH expression by endogenous taste buds, our data suggest that SHH can replace the need for innervation to drive the entire program of taste bud differentiation. PMID:24993944

Preclinical characterization of therapeutic antibodies targeted at the carboxy-terminus of Sonic hedgehog

PubMed Central

Tolani, Bhairavi; Hoang, Ngoc T.; Acevedo, Luis A.; Leprieur, Etienne Giroux; Li, Hui; He, Biao; Jablons, David M.

2018-01-01

The Sonic Hedgehog (Shh) signaling pathway has been implicated in the development and tumor progression of a number of human cancers. Using synthetic peptide mimics to mount an immune response, we generated a mouse mAb to the carboxy (C)-terminus of the Shh protein and characterized its preclinical antitumor effects. In vitro screening guided selection of the best candidate for mAb scale-up production and therapeutic development. C-term anti-Shh, Ab 1C11-2G4 was selected based on ELISA screens, Western blotting, and flow cytometric analyses. Purified Ab 1C11-2G4 was shown to recognize and bind both Shh peptide mimics and cell surface Shh. Administration of Ab 1C11-2G4 not only reduced cell viability in 7 cancer cell lines but also significantly inhibitted tumor growth in a xenograft model of A549 lung cancer cells. Ex vivo analyses of xenograft tumors revealed a reduction in Shh signal transduction and apoptosis in 2G4-treated mice. Collectively, our results provide early demonstration of the antitumor utility of antibodies specific for the C-terminal region of Shh, and support continued development to evaluate their potential efficacy in cancers in which Shh activity is elevated. PMID:29581846

Not so Fast: Co-Requirements for Sonic Hedgehog Induced Brain Tumorigenesis.

PubMed

Ward, Stacey A; Rubin, Joshua B

2015-08-06

The Sonic hedgehog (Shh) pathway plays an integral role in cellular proliferation during normal brain development and also drives growth in a variety of cancers including brain cancer. Clinical trials of Shh pathway inhibitors for brain tumors have yielded disappointing results, indicating a more nuanced role for Shh signaling. We postulate that Shh signaling does not work alone but requires co-activation of other signaling pathways for tumorigenesis and stem cell maintenance. This review will focus on the interplay between the Shh pathway and these pathways to promote tumor growth in brain tumors, presenting opportunities for the study of combinatorial therapies.

Sonic hedgehog protein promotes proliferation and chondrogenic differentiation of bone marrow-derived mesenchymal stem cells in vitro.

PubMed

Warzecha, Jörg; Göttig, Stephan; Brüning, Christian; Lindhorst, Elmar; Arabmothlagh, Mohammad; Kurth, Andreas

2006-10-01

Sonic hedgehog (Shh) protein is known to be an important signaling protein in early embryonic development. Also, Shh is involved in the induction of early cartilaginous differentiation of mesenchymal cells in the limb and in the spine. The impact of Shh on adult stem cells, human bone marrow-derived mesenchymal stem cells (MSCs), was tested. The MSCs were treated either with recombinant Sonic hedgehog protein (r-Shh) or with transforming growth factor-beta 1 (TGF-beta(1)) as a positive control in vitro for 3 weeks. The effects on cartilaginous differentiation and proliferation were assayed. MSCs when treated with either Shh or TGF-beta(1) showed expression of cartilage markers aggrecan, Sox9, CEP-68, and collagen type II and X within 3 weeks. Only r-Shh-treated cells showed a very strong cell proliferation and much higher BrdU incorporation in cell assay systems. These are the first data that indicate an important role of Shh for the induction of cartilage production by MSCs in vitro.

The disruption of a novel limb cis-regulatory element of SHH is associated with autosomal dominant preaxial polydactyly-hypertrichosis

PubMed Central

Petit, Florence; Jourdain, Anne-Sophie; Holder-Espinasse, Muriel; Keren, Boris; Andrieux, Joris; Duterque-Coquillaud, Martine; Porchet, Nicole; Manouvrier-Hanu, Sylvie; Escande, Fabienne

2016-01-01

The expression gradient of the morphogen Sonic Hedgehog (SHH) is crucial in establishing the number and the identity of the digits during anteroposterior patterning of the limb. Its anterior ectopic expression is responsible for preaxial polydactyly (PPD). Most of these malformations are due to the gain-of-function of the Zone of Polarizing Activity Regulatory Sequence, the only limb-specific enhancer of SHH known to date. We report a family affected with a novel condition associating PPD and hypertrichosis of the upper back, following an autosomal dominant mode of inheritance. This phenotype is consistent with deregulation of SHH expression during limb and follicle development. In affected members, we identified a 2 kb deletion located ~240 kb upstream from the SHH promoter. The deleted sequence is capable of repressing the transcriptional activity of the SHH promoter in vitro, consistent with a silencer activity. We hypothesize that the deletion of this silencer could be responsible for SHH deregulation during development, leading to a PPD-hypertrichosis phenotype. PMID:25782671

Heterochronic shift in Hox-mediated activation of sonic hedgehog leads to morphological changes during fin development.

PubMed

Sakamoto, Koji; Onimaru, Koh; Munakata, Keijiro; Suda, Natsuno; Tamura, Mika; Ochi, Haruki; Tanaka, Mikiko

2009-01-01

We explored the molecular mechanisms of morphological transformations of vertebrate paired fin/limb evolution by comparative gene expression profiling and functional analyses. In this study, we focused on the temporal differences of the onset of Sonic hedgehog (Shh) expression in paired appendages among different vertebrates. In limb buds of chick and mouse, Shh expression is activated as soon as there is a morphological bud, concomitant with Hoxd10 expression. In dogfish (Scyliorhinus canicula), however, we found that Shh was transcribed late in fin development, concomitant with Hoxd13 expression. We utilized zebrafish as a model to determine whether quantitative changes in hox expression alter the timing of shh expression in pectoral fins of zebrafish embryos. We found that the temporal shift of Shh activity altered the size of endoskeletal elements in paired fins of zebrafish and dogfish. Thus, a threshold level of hox expression determines the onset of shh expression, and the subsequent heterochronic shift of Shh activity can affect the size of the fin endoskeleton. This process may have facilitated major morphological changes in paired appendages during vertebrate limb evolution.

Identification of Sonic Hedgehog-Induced Stromal Factors That Stimulate Prostate Tumor Growth

DTIC Science & Technology

2006-11-01

LN -Shh xenograft tumors is unabated after castration of the host mouse. However, castration of mice bearing LNCaP + Gli3-/- UGSM bi-clonal...canonical xenograft undergoes involution and growth arrest, growth of LN -Shh xenograft tumors is unabated after castration. As we have shown...signalingindependent of Shh ligand in tumor stroma accelerates tumor growth. We have identified potential stromal Shh target genes in xenograft tumors and have begun

Sonic hedgehog pathway activation increases mitochondrial abundance and activity in hippocampal neurons

PubMed Central

Yao, Pamela J.; Manor, Uri; Petralia, Ronald S.; Brose, Rebecca D.; Wu, Ryan T. Y.; Ott, Carolyn; Wang, Ya-Xian; Charnoff, Ari; Lippincott-Schwartz, Jennifer; Mattson, Mark P.

2017-01-01

Mitochondria are essential organelles whose biogenesis, structure, and function are regulated by many signaling pathways. We present evidence that, in hippocampal neurons, activation of the Sonic hedgehog (Shh) signaling pathway affects multiple aspects of mitochondria. Mitochondrial mass was increased significantly in neurons treated with Shh. Using biochemical and fluorescence imaging analyses, we show that Shh signaling activity reduces mitochondrial fission and promotes mitochondrial elongation, at least in part, via suppression of the mitochondrial fission protein dynamin-like GTPase Drp1. Mitochondria from Shh-treated neurons were more electron-dense, as revealed by electron microscopy, and had higher membrane potential and respiratory activity. We further show that Shh protects neurons against a variety of stresses, including the mitochondrial poison rotenone, amyloid β-peptide, hydrogen peroxide, and high levels of glutamate. Collectively our data suggest a link between Shh pathway activity and the physiological properties of mitochondria in hippocampal neurons. PMID:27932496

Induction of ectopic taste buds by SHH reveals the competency and plasticity of adult lingual epithelium.

PubMed

Castillo, David; Seidel, Kerstin; Salcedo, Ernesto; Ahn, Christina; de Sauvage, Frederic J; Klein, Ophir D; Barlow, Linda A

2014-08-01

Taste buds are assemblies of elongated epithelial cells, which are innervated by gustatory nerves that transmit taste information to the brain stem. Taste cells are continuously renewed throughout life via proliferation of epithelial progenitors, but the molecular regulation of this process remains unknown. During embryogenesis, sonic hedgehog (SHH) negatively regulates taste bud patterning, such that inhibition of SHH causes the formation of more and larger taste bud primordia, including in regions of the tongue normally devoid of taste buds. Here, using a Cre-lox system to drive constitutive expression of SHH, we identify the effects of SHH on the lingual epithelium of adult mice. We show that misexpression of SHH transforms lingual epithelial cell fate, such that daughter cells of lingual epithelial progenitors form cell type-replete, onion-shaped taste buds, rather than non-taste, pseudostratified epithelium. These SHH-induced ectopic taste buds are found in regions of the adult tongue previously thought incapable of generating taste organs. The ectopic buds are composed of all taste cell types, including support cells and detectors of sweet, bitter, umami, salt and sour, and recapitulate the molecular differentiation process of endogenous taste buds. In contrast to the well-established nerve dependence of endogenous taste buds, however, ectopic taste buds form independently of both gustatory and somatosensory innervation. As innervation is required for SHH expression by endogenous taste buds, our data suggest that SHH can replace the need for innervation to drive the entire program of taste bud differentiation. © 2014. Published by The Company of Biologists Ltd.

ESCRT-II/Vps25 constrains digit number by endosome-mediated selective modulation of FGF-SHH signaling.

PubMed

Handschuh, Karen; Feenstra, Jennifer; Koss, Matthew; Ferretti, Elisabetta; Risolino, Maurizio; Zewdu, Rediet; Sahai, Michelle A; Bénazet, Jean-Denis; Peng, Xiao P; Depew, Michael J; Quintana, Laura; Sharpe, James; Wang, Baolin; Alcorn, Heather; Rivi, Roberta; Butcher, Stephen; Manak, J Robert; Vaccari, Thomas; Weinstein, Harel; Anderson, Kathryn V; Lacy, Elizabeth; Selleri, Licia

2014-10-23

Sorting and degradation of receptors and associated signaling molecules maintain homeostasis of conserved signaling pathways during cell specification and tissue development. Yet, whether machineries that sort signaling proteins act preferentially on different receptors and ligands in different contexts remains mysterious. Here, we show that Vacuolar protein sorting 25, Vps25, a component of ESCRT-II (Endosomal Sorting Complex Required for Transport II), directs preferential endosome-mediated modulation of FGF signaling in limbs. By ENU-induced mutagenesis, we isolated a polydactylous mouse line carrying a hypomorphic mutation of Vps25 (Vps25(ENU)). Unlike Vps25-null embryos we generated, Vps25(ENU/ENU) mutants survive until late gestation. Their limbs display FGF signaling enhancement and consequent hyperactivation of the FGF-SHH feedback loop causing polydactyly, whereas WNT and BMP signaling remain unperturbed. Notably, Vps25(ENU/ENU) Mouse Embryonic Fibroblasts exhibit aberrant FGFR trafficking and degradation; however, SHH signaling is unperturbed. These studies establish that the ESCRT-II machinery selectively limits FGF signaling in vertebrate skeletal patterning.

SMARCA4/Brg1 coordinates genetic and epigenetic networks underlying Shh-type medulloblastoma development.

PubMed

Shi, X; Wang, Q; Gu, J; Xuan, Z; Wu, J I

2016-11-03

Recent large-scale genomic studies have classified medulloblastoma into four subtypes: Wnt, Shh, Group 3 and Group 4. Each is characterized by specific mutations and distinct epigenetic states. Previously, we showed that a chromatin regulator SMARCA4/Brg1 is required for Gli-mediated transcription activation in Sonic hedgehog (Shh) signaling. We report here that Brg1 controls a transcriptional program that specifically regulates Shh-type medulloblastoma growth. Using a mouse model of Shh-type medulloblastoma, we deleted Brg1 in precancerous progenitors and primary or transplanted tumors. Brg1 deletion significantly inhibited tumor formation and progression. Genome-wide expression analyses and binding experiments indicate that Brg1 specifically coordinates with key transcription factors including Gli1, Atoh1 and REST to regulate the expression of both oncogenes and tumor suppressors that are required for medulloblastoma identity and proliferation. Shh-type medulloblastoma displays distinct H3K27me3 properties. We demonstrate that Brg1 modulates activities of H3K27me3 modifiers to regulate the expression of medulloblastoma genes. Brg1-regulated pathways are conserved in human Shh-type medulloblastoma, and Brg1 is important for the growth of a human medulloblastoma cell line. Thus, Brg1 coordinates a genetic and epigenetic network that regulates the transcriptional program underlying the Shh-type medulloblastoma development.

Multiple Shh signaling centers participate in fungiform papilla and taste bud formation and maintenance

PubMed Central

Liu, H-X; Ermilov, A; Grachtchouk, M; Li, L; Gumucio, DL; Dlugosz, AA; Mistretta, CM

2014-01-01

The adult fungiform taste papilla is a complex of specialized cell types residing in the stratified squamous tongue epithelium. This unique sensory organ includes taste buds, papilla epithelium and lateral walls that extend into underlying connective tissue to surround a core of lamina propria cells. Fungiform papillae must contain long-lived, sustaining or stem cells and short-lived, maintaining or transit amplifying cells that support the papilla and specialized taste buds. Shh signaling has established roles in supporting fungiform induction, development and patterning. However, for a full understanding of how Shh transduced signals act in tongue, papilla and taste bud formation and maintenance, it is necessary to know where and when the Shh ligand and pathway components are positioned. We used immunostaining, in situ hybridization and mouse reporter strains for Shh, Ptch1, Gli1 and Gli2-expression and proliferation markers to identify cells that participate in hedgehog signaling. Whereas there is a progressive restriction in location of Shh ligand-expressing cells, from placode and apical papilla cells to taste bud cells only, a surrounding population of Ptch1 and Gli1 responding cells is maintained in signaling centers throughout papilla and taste bud development and differentiation. The Shh signaling targets are in regions of active cell proliferation. Using genetic-inducible lineage tracing for Gli1-expression, we found that Shh-responding cells contribute not only to maintenance of filiform and fungiform papillae, but also to taste buds. A requirement for normal Shh signaling in fungiform papilla, taste bud and filiform papilla maintenance was shown by Gli2 constitutive activation. We identified proliferation niches where Shh signaling is active and suggest that epithelial and mesenchymal compartments harbor potential stem and/or progenitor cell zones. In all, we report a set of hedgehog signaling centers that regulate development and maintenance of taste organs, the fungiform papilla and taste bud, and surrounding lingual cells. Shh signaling has roles in forming and maintaining fungiform papillae and taste buds, most likely via stage-specific autocrine and/or paracrine mechanisms, and by engaging epithelial/mesenchymal interactions. PMID:23916850

Multiple Shh signaling centers participate in fungiform papilla and taste bud formation and maintenance.

PubMed

Liu, Hong Xiang; Ermilov, Alexandre; Grachtchouk, Marina; Li, Libo; Gumucio, Deborah L; Dlugosz, Andrzej A; Mistretta, Charalotte M

2013-10-01

The adult fungiform taste papilla is a complex of specialized cell types residing in the stratified squamous tongue epithelium. This unique sensory organ includes taste buds, papilla epithelium and lateral walls that extend into underlying connective tissue to surround a core of lamina propria cells. Fungiform papillae must contain long-lived, sustaining or stem cells and short-lived, maintaining or transit amplifying cells that support the papilla and specialized taste buds. Shh signaling has established roles in supporting fungiform induction, development and patterning. However, for a full understanding of how Shh transduced signals act in tongue, papilla and taste bud formation and maintenance, it is necessary to know where and when the Shh ligand and pathway components are positioned. We used immunostaining, in situ hybridization and mouse reporter strains for Shh, Ptch1, Gli1 and Gli2-expression and proliferation markers to identify cells that participate in hedgehog signaling. Whereas there is a progressive restriction in location of Shh ligand-expressing cells, from placode and apical papilla cells to taste bud cells only, a surrounding population of Ptch1 and Gli1 responding cells is maintained in signaling centers throughout papilla and taste bud development and differentiation. The Shh signaling targets are in regions of active cell proliferation. Using genetic-inducible lineage tracing for Gli1-expression, we found that Shh-responding cells contribute not only to maintenance of filiform and fungiform papillae, but also to taste buds. A requirement for normal Shh signaling in fungiform papilla, taste bud and filiform papilla maintenance was shown by Gli2 constitutive activation. We identified proliferation niches where Shh signaling is active and suggest that epithelial and mesenchymal compartments harbor potential stem and/or progenitor cell zones. In all, we report a set of hedgehog signaling centers that regulate development and maintenance of taste organs, the fungiform papilla and taste bud, and surrounding lingual cells. Shh signaling has roles in forming and maintaining fungiform papillae and taste buds, most likely via stage-specific autocrine and/or paracrine mechanisms, and by engaging epithelial/mesenchymal interactions. © 2013 Elsevier Inc. All rights reserved.

Shh pathway in wounds in non-diabetic Shh-Cre-eGFP/Ptch1-LacZ mice treated with MAA beads.

PubMed

Lisovsky, Alexandra; Sefton, Michael V

2016-09-01

Previously, poly(methacrylic acid-co-methyl methacrylate) (MAA) beads were shown to improve vessel formation with a concomitant increase in the expression of the sonic hedgehog (Shh) gene, a pleiotropic factor implicated in vascularization. The aim of this study was to follow up on this observation in the absence of the confounding factors of diabetes in non-diabetic Shh-Cre-eGFP/Ptch1-LacZ mice; in this mouse, expression of GFP and β-Gal is consistent with the transcription patterns of Shh and its receptor patched 1 (Ptch1), respectively. In agreement with studies in diabetic males, MAA beads improved vascularization in large (15 mm × 15 mm) wounds in non-diabetic males at day 7. Shh pathway activation was suggested, as the numbers of GFP+ (Shh) and β-Gal+ (Ptch1, a target of the pathway) cells increased in the granulation tissue. Shh signaling pathway modulation was also suggested in the healthy skin surrounding the wound bed, as evidenced by an increase in the number of GFP+ and β-Gal+ cells in males at day 4. Gene expression analysis of the wounds confirmed increase in Ptch1 and showed the upregulation of a downstream transcription factor Gli3, involved in the vascular effect of the Shh pathway, implicating the pathway in the effect of MAA beads. The efficacy of MAA beads was also investigated in females; MAA beads modulated the Shh pathway within granulation tissue similarly as in males, but had no enhancement effect on the healthy skin and on vascularization. We believe that understanding the molecular and cellular mechanisms of MAA-based biomaterials and testing the efficacy of therapeutics in both sexes will inform the development of novel therapeutic biomaterials. Copyright © 2016 Elsevier Ltd. All rights reserved.

Vismodegib Exerts Targeted Efficacy Against Recurrent Sonic Hedgehog–Subgroup Medulloblastoma: Results From Phase II Pediatric Brain Tumor Consortium Studies PBTC-025B and PBTC-032

PubMed Central

Robinson, Giles W.; Orr, Brent A.; Wu, Gang; Gururangan, Sridharan; Lin, Tong; Qaddoumi, Ibrahim; Packer, Roger J.; Goldman, Stewart; Prados, Michael D.; Desjardins, Annick; Chintagumpala, Murali; Takebe, Naoko; Kaste, Sue C.; Rusch, Michael; Allen, Sariah J.; Onar-Thomas, Arzu; Stewart, Clinton F.; Fouladi, Maryam; Boyett, James M.; Gilbertson, Richard J.; Curran, Tom; Ellison, David W.; Gajjar, Amar

2015-01-01

Purpose Two phase II studies assessed the efficacy of vismodegib, a sonic hedgehog (SHH) pathway inhibitor that binds smoothened (SMO), in pediatric and adult recurrent medulloblastoma (MB). Patients and Methods Adult patients enrolled onto PBTC-025B and pediatric patients enrolled onto PBTC-032 were treated with vismodegib (150 to 300 mg/d). Protocol-defined response, which had to be sustained for 8 weeks, was confirmed by central neuroimaging review. Molecular tests to identify patterns of response and insensitivity were performed when tissue was available. Results A total of 31 patients were enrolled onto PBTC-025B, and 12 were enrolled onto PBTC-032. Three patients in PBTC-025B and one in PBTC-032, all with SHH-subgroup MB (SHH-MB), exhibited protocol-defined responses. Progression-free survival (PFS) was longer in those with SHH-MB than in those with non-SHH–MB, and prolonged disease stabilization occurred in 41% of patient cases of SHH-MB. Among those with SHH-MB, loss of heterozygosity of PTCH1 was associated with prolonged PFS, and diffuse staining of P53 was associated with reduced PFS. Whole-exome sequencing identified mutations in SHH genes downstream from SMO in four of four tissue samples from nonresponders and upstream of SMO in two of four patients with favorable responses. Conclusion Vismodegib exhibits activity against adult recurrent SHH-MB but not against recurrent non-SHH–MB. Inadequate accrual of pediatric patients precluded conclusions in this population. Molecular analyses support the hypothesis that SMO inhibitor activity depends on the genomic aberrations within the tumor. Such inhibitors should be advanced in SHH-MB studies; however, molecular and genomic work remains imperative to identify target populations that will truly benefit. PMID:26169613

Proliferation of murine midbrain neural stem cells depends upon an endogenous sonic hedgehog (Shh) source.

PubMed

Martínez, Constanza; Cornejo, Víctor Hugo; Lois, Pablo; Ellis, Tammy; Solis, Natalia P; Wainwright, Brandon J; Palma, Verónica

2013-01-01

The Sonic Hedgehog (Shh) pathway is responsible for critical patterning events early in development and for regulating the delicate balance between proliferation and differentiation in the developing and adult vertebrate brain. Currently, our knowledge of the potential role of Shh in regulating neural stem cells (NSC) is largely derived from analyses of the mammalian forebrain, but for dorsal midbrain development it is mostly unknown. For a detailed understanding of the role of Shh pathway for midbrain development in vivo, we took advantage of mouse embryos with cell autonomously activated Hedgehog (Hh) signaling in a conditional Patched 1 (Ptc1) mutant mouse model. This animal model shows an extensive embryonic tectal hypertrophy as a result of Hh pathway activation. In order to reveal the cellular and molecular origin of this in vivo phenotype, we established a novel culture system to evaluate neurospheres (nsps) viability, proliferation and differentiation. By recreating the three-dimensional (3-D) microenvironment we highlight the pivotal role of endogenous Shh in maintaining the stem cell potential of tectal radial glial cells (RGC) and progenitors by modulating their Ptc1 expression. We demonstrate that during late embryogenesis Shh enhances proliferation of NSC, whereas blockage of endogenous Shh signaling using cyclopamine, a potent Hh pathway inhibitor, produces the opposite effect. We propose that canonical Shh signaling plays a central role in the control of NSC behavior in the developing dorsal midbrain by acting as a niche factor by partially mediating the response of NSC to epidermal growth factor (EGF) and fibroblast growth factor (FGF) signaling. We conclude that endogenous Shh signaling is a critical mechanism regulating the proliferation of stem cell lineages in the embryonic dorsal tissue.

Mechanical stimulation promote the osteogenic differentiation of bone marrow stromal cells through epigenetic regulation of Sonic Hedgehog.

PubMed

Wang, Chuandong; Shan, Shengzhou; Wang, Chenglong; Wang, Jing; Li, Jiao; Hu, Guoli; Dai, Kerong; Li, Qingfeng; Zhang, Xiaoling

2017-03-15

Mechanical unloading leads to bone loss and disuse osteoporosis partly due to impaired osteoblastogenesis of bone marrow stromal cells (BMSCs). However, the underlying molecular mechanisms of this phenomenon are not fully understood. In this study, we demonstrated that cyclic mechanical stretch (CMS) promotes osteoblastogenesis of BMSCs both in vivo and in vitro. Besides, we found that Hedgehog (Hh) signaling pathway was activated in this process. Inhibition of which by either knockdown of Sonic hedgehog (Shh) or treating BMSCs with Hh inhibitors attenuated the osteogenic effect of CMS on BMSCs, suggesting that Hh signaling pathway acts as an endogenous mediator of mechanical stimuli on BMSCs. Furthermore, we demonstrated that Shh expression level was regulated by DNA methylation mechanism. Chromatin Immunoprecipitation (ChIP) assay showed that DNA methyltransferase 3b (Dnmt3b) binds to Shh gene promoter, leading to DNA hypermethylation in mechanical unloading BMSCs. However, mechanical stimulation down-regulates the protein level of Dnmt3b, results in DNA demethylation and Shh expression. More importantly, we found that inhibition of Dnmt3b partly rescued bone loss in HU mice by mechanical unloading. Our results demonstrate, for the first time, that mechanical stimulation regulates osteoblastic genes expression via direct regulation of Dnmt3b, and the therapeutic inhibition of Dnmt3b may be an efficient strategy for enhancing bone formation under mechanical unloading. Copyright © 2017 Elsevier Inc. All rights reserved.

Medulloblastomas derived from Cxcr6 mutant mice respond to treatment with a smoothened inhibitor.

PubMed

Sasai, Ken; Romer, Justyna T; Kimura, Hiromichi; Eberhart, Derek E; Rice, Dennis S; Curran, Tom

2007-04-15

The sonic hedgehog (Shh) pathway is activated in approximately 30% of human medulloblastoma resulting in increased expression of downstream target genes. In about half of these cases, this has been shown to be a consequence of mutations in regulatory genes within the pathway, including Ptc1, Smo, and Sufu. However, for some tumors, no mutations have been detected in known pathway genes. This suggests that either mutations in other genes promote tumorigenesis or that epigenetic alterations increase pathway activity in these tumors. Here, we report that 3% to 4% of mice lacking either one or both functional copies of Cxcr6 develop medulloblastoma. Although CXCR6 is not known to be involved in Shh signaling, tumors derived from Cxcr6 mutant mice expressed Shh pathway target genes including Gli1, Gli2, Ptc2, and Sfrp1, indicating elevated pathway activity. Interestingly, the level of Ptc1 expression was decreased in tumor cells although two normal copies of Ptc1 were retained. This implies that reduced CXCR6 function leads to suppression of Ptc1 thereby increasing Smoothened function and promoting tumorigenesis. We used a direct transplant model to test the sensitivity of medulloblastoma arising in Cxcr6 mutant mice to a small-molecule inhibitor of Smoothened (HhAntag). We found that transplanted tumors were dramatically inhibited in mice treated for only 4 days with HhAntag. These findings suggest that HhAntag may be effective against tumors lacking mutations in known Shh pathway genes.

Development of five digits is controlled by a bipartite long-range cis-regulator

PubMed Central

Lettice, Laura A.; Williamson, Iain; Devenney, Paul S.; Kilanowski, Fiona; Dorin, Julia; Hill, Robert E.

2014-01-01

Conservation within intergenic DNA often highlights regulatory elements that control gene expression from a long range. How conservation within a single element relates to regulatory information and how internal composition relates to function is unknown. Here, we examine the structural features of the highly conserved ZRS (also called MFCS1) cis-regulator responsible for the spatiotemporal control of Shh in the limb bud. By systematically dissecting the ZRS, both in transgenic assays and within in the endogenous locus, we show that the ZRS is, in effect, composed of two distinct domains of activity: one domain directs spatiotemporal activity but functions predominantly from a short range, whereas a second domain is required to promote long-range activity. We show further that these two domains encode activities that are highly integrated and that the second domain is crucial in promoting the chromosomal conformational changes correlated with gene activity. During limb bud development, these activities encoded by the ZRS are interpreted differently by the fore limbs and the hind limbs; in the absence of the second domain there is no Shh activity in the fore limb, and in the hind limb low levels of Shh lead to a variant digit pattern ranging from two to four digits. Hence, in the embryo, the second domain stabilises the developmental programme providing a buffer for SHH morphogen activity and this ensures that five digits form in both sets of limbs. PMID:24715461

Reprogramming Medulloblastoma-Propagating Cells by a Combined Antagonism of Sonic Hedgehog and CXCR4.

PubMed

Ward, Stacey A; Warrington, Nicole M; Taylor, Sara; Kfoury, Najla; Luo, Jingqin; Rubin, Joshua B

2017-03-15

The CXCR4 chemokine and Sonic Hedgehog (SHH) morphogen pathways are well-validated therapeutic targets in cancer, including medulloblastoma. However, single-agent treatments with SHH or CXCR4 antagonists have not proven efficacious in clinical trials to date. Here, we discovered that dual inhibition of the SHH and CXCR4 pathways in a murine model of SHH-subtype medulloblastoma exerts potent antitumor effects. This therapeutic synergy resulted in the suppression of tumor-propagating cell function and correlated with increased histone H3 lysine 27 trimethylation within the promoters of stem cell genes, resulting in their decreased expression. These results demonstrate that CXCR4 contributes to the epigenetic regulation of a tumor-propagating cell phenotype. Moreover, they provide a mechanistic rationale to evaluate the combination of SHH and CXCR4 inhibitors in clinical trials for the treatment of medulloblastoma, as well as other cancers driven by SHH that coexpress high levels of CXCR4. Cancer Res; 77(6); 1416-26. ©2016 AACR . ©2016 American Association for Cancer Research.

Murine models of VACTERL syndrome: Role of sonic hedgehog signaling pathway.

PubMed

Kim, P C; Mo, R; Hui Cc, C

2001-02-01

VACTERL syndrome is a common surgical condition affecting the development of many midaxial organs. The etiology, embryology, and pathogenesis of the VACTERL syndrome are not known. The authors report here new mouse models of VACTERL syndrome involving the Sonic hedgehog (Shh) signaling pathway. Mutant mice involving Shh signaling, the Shh transcription factors Gli2-/- and Gli3-/-, Gli2-/-;Gli3+/- double heterozygotes, and Shh-/- were analyzed. In addition to reported vertebral, anal, tracheoesophageal, and limb anomalies, mutant mice display cardiac, renal, and associated anomalies, namely congenital diaphragmatic hernia and omphalocele, known to be associated in VACTERL syndrome. The Shh transcription factors Gli2 and Gli3 have specific and overlapping roles in the induction of VACTERL phenotypes in a gene-dose dependent manner in these mutants. To the authors' knowledge, these mutant mice represent the first animal model that mimics the human VACTERL syndrome, and suggests that aberrations in Shh signaling might be involved in the VACTERL syndrome.

The fickle finger of fate

PubMed Central

de la Fuente, Luis; Helms, Jill A.

2005-01-01

In this issue of the JCI, Niedermaier and colleagues demonstrate that a chromosomal inversion in mice results in dysregulation of Sonic hedgehog (Shh), such that Shh is ectopically expressed in a skeletogenic domain typically occupied by Indian hedgehog (Ihh). This molecular reversal eliminates phalangeal joint spaces, and consequently, Short digits (Dsh) heterozygotes (Dsh/+) have brachydactyly (shortened digits). Ihh is normally downregulated in regions that will become the joint space, but in Dsh/+ mice, Shh bypasses this regulatory control and persists; accordingly, cells maintain their chondrogenic fate and the developed digits are shorter than normal. The significance of these data extends far beyond the field of skeletal biology: they hint at the very real possibility that the endogenous Shh regulatory region contains a repressor designed to segregate the activity of Shh from Ihh. The existence of such a repressor provides a window into the distant past, revealing that Shh and Ihh must once have shared responsibilities in establishing tissue boundaries and orchestrating vertebrate tissue morphogenesis. PMID:15841172

Peptide amphiphile nanofiber hydrogel delivery of sonic hedgehog protein to the cavernous nerve to promote regeneration and prevent erectile dysfunction.

PubMed

Choe, Shawn; Bond, Christopher W; Harrington, Daniel A; Stupp, Samuel I; McVary, Kevin T; Podlasek, Carol A

2017-01-01

Erectile dysfunction (ED) has high impact on quality of life in prostatectomy, diabetic and aging patients. An underlying mechanism is cavernous nerve (CN) injury, which causes ED in up to 80% of prostatectomy patients. We examine how sonic hedgehog (SHH) treatment with innovative peptide amphiphile nanofiber hydrogels (PA), promotes CN regeneration after injury. SHH and its receptors patched (PTCH1) and smoothened (SMO) are localized in PG neurons and glia. SMO undergoes anterograde transport to signal to downstream targets. With crush injury, PG neurons degenerate and undergo apoptosis. SHH protein decreases, SMO localization changes to the neuronal cell surface, and anterograde transport stops. With SHH treatment SHH is taken up at the injury site and undergoes retrograde transport to PG neurons, allowing SMO transport to occur, and neurons remain intact. SHH treatment prevents neuronal degeneration, maintains neuronal, glial and downstream target signaling, and is significant as a regenerative therapy. Published by Elsevier Inc.

Sonic hedgehog-Dependent Induction of MicroRNA 31 and MicroRNA 150 Regulates Mycobacterium bovis BCG-Driven Toll-Like Receptor 2 Signaling

PubMed Central

Ghorpade, Devram Sampat; Holla, Sahana; Kaveri, Srini V.; Bayry, Jagadeesh; Patil, Shripad A.

2013-01-01

Hedgehog (HH) signaling is a significant regulator of cell fate decisions during embryogenesis, development, and perpetuation of various disease conditions. Testing whether pathogen-specific HH signaling promotes unique innate recognition of intracellular bacteria, we demonstrate that among diverse Gram-positive or Gram-negative microbes, Mycobacterium bovis BCG, a vaccine strain, elicits a robust activation of Sonic HH (SHH) signaling in macrophages. Interestingly, sustained tumor necrosis factor alpha (TNF-α) secretion by macrophages was essential for robust SHH activation, as TNF-α−/− macrophages exhibited compromised ability to activate SHH signaling. Neutralization of TNF-α or blockade of TNF-α receptor signaling significantly reduced the infection-induced SHH signaling activation both in vitro and in vivo. Intriguingly, activated SHH signaling downregulated M. bovis BCG-mediated Toll-like receptor 2 (TLR2) signaling events to regulate a battery of genes associated with divergent functions of M1/M2 macrophages. Genome-wide expression profiling as well as conventional gain-of-function or loss-of-function analysis showed that SHH signaling-responsive microRNA 31 (miR-31) and miR-150 target MyD88, an adaptor protein of TLR2 signaling, thus leading to suppression of TLR2 responses. SHH signaling signatures could be detected in vivo in tuberculosis patients and M. bovis BCG-challenged mice. Collectively, these investigations identify SHH signaling to be what we believe is one of the significant regulators of host-pathogen interactions. PMID:23166298

Sonic hedgehog promotes neurite outgrowth of cortical neurons under oxidative stress: Involving of mitochondria and energy metabolism.

PubMed

He, Weiliang; Cui, Lili; Zhang, Cong; Zhang, Xiangjian; He, Junna; Xie, Yanzhao; Chen, Yanxia

2017-01-01

Oxidative stress has been demonstrated to be involved in the etiology of several neurobiological disorders. Sonic hedgehog (Shh), a secreted glycoprotein factor, has been implicated in promoting several aspects of brain remodeling process. Mitochondria may play an important role in controlling fundamental processes in neuroplasticity. However, little evidence is available about the effect and the potential mechanism of Shh on neurite outgrowth in primary cortical neurons under oxidative stress. Here, we revealed that Shh treatment significantly increased the viability of cortical neurons in a dose-dependent manner, which was damaged by hydrogen peroxide (H 2 O 2 ). Shh alleviated the apoptosis rate of H 2 O 2 -induced neurons. Shh also increased neuritogenesis injuried by H 2 O 2 in primary cortical neurons. Moreover, Shh reduced the generation of reactive oxygen species (ROS), increased the activities of SOD and and decreased the productions of MDA. In addition, Shh protected mitochondrial functions, elevated the cellular ATP levels and amelioratesd the impairment of mitochondrial complex II activities of cortical neurons induced by H 2 O 2 . In conclusion, all these results suggest that Shh acts as a prosurvival factor playing an essential role to neurite outgrowth of cortical neuron under H 2 O 2 -induced oxidative stress, possibly through counteracting ROS release and preventing mitochondrial dysfunction and ATP as well as mitochondrial complex II activities against oxidative stress. Copyright © 2016 Elsevier Inc. All rights reserved.

In smokers, Sonic hedgehog modulates pulmonary endothelial function through vascular endothelial growth factor.

PubMed

Henno, Priscilla; Grassin-Delyle, Stanislas; Belle, Emeline; Brollo, Marion; Naline, Emmanuel; Sage, Edouard; Devillier, Philippe; Israël-Biet, Dominique

2017-05-23

Tobacco-induced pulmonary vascular disease is partly driven by endothelial dysfunction. The Sonic hedgehog (SHH) pathway is involved in vascular physiology. We sought to establish whether the SHH pathway has a role in pulmonary endothelial dysfunction in smokers. The ex vivo endothelium-dependent relaxation of pulmonary artery rings in response to acetylcholine (Ach) was compared in 34 current or ex-smokers and 8 never-smokers. The results were expressed as a percentage of the contraction with phenylephrine. We tested the effects of SHH inhibitors (GANT61 and cyclopamine), an SHH activator (SAG) and recombinant VEGF on the Ach-induced relaxation. The level of VEGF protein in the pulmonary artery ring was measured in an ELISA. SHH pathway gene expression was quantified in reverse transcriptase-quantitative polymerase chain reactions. Ach-induced relaxation was much less intense in smokers than in never-smokers (respectively 24 ± 6% and 50 ± 7% with 10 -4 M Ach; p = 0.028). All SHH pathway genes were expressed in pulmonary artery rings from smokers. SHH inhibition by GANT61 reduced Ach-induced relaxation and VEGF gene expression in the pulmonary artery ring. Recombinant VEGF restored the ring's endothelial function. VEGF gene and protein expression levels in the pulmonary artery rings were positively correlated with the degree of Ach-induced relaxation and negatively correlated with the number of pack-years. SHH pathway genes and proteins are expressed in pulmonary artery rings from smokers, where they modulate endothelial function through VEGF.

Holocephalan embryos provide evidence for gill arch appendage reduction and opercular evolution in cartilaginous fishes

PubMed Central

Gillis, J. Andrew; Rawlinson, Kate A.; Bell, Justin; Lyon, Warrick S.; Baker, Clare V. H.; Shubin, Neil H.

2011-01-01

Chondrichthyans possess endoskeletal appendages called branchial rays that extend laterally from their hyoid and gill-bearing (branchial) arches. Branchial ray outgrowth, like tetrapod limb outgrowth, is maintained by Sonic hedgehog (Shh) signaling. In limbs, distal endoskeletal elements fail to form in the absence of normal Shh signaling, whereas shortened duration of Shh expression correlates with distal endoskeletal reduction in naturally variable populations. Chondrichthyans also exhibit natural variation with respect to branchial ray distribution—elasmobranchs (sharks and batoids) possess a series of ray-supported septa on their hyoid and gill arches, whereas holocephalans (chimaeras) possess a single hyoid arch ray-supported operculum. Here we show that the elongate hyoid rays of the holocephalan Callorhinchus milii grow in association with sustained Shh expression within an opercular epithelial fold, whereas Shh is only transiently expressed in the gill arches. Coincident with this transient Shh expression, branchial ray outgrowth is initiated in C. milii but is not maintained, yielding previously unrecognized vestigial gill arch branchial rays. This is in contrast to the condition seen in sharks, where sustained Shh expression corresponds to the presence of fully formed branchial rays on the hyoid and gill arches. Considered in light of current hypotheses of chondrichthyan phylogeny, our data suggest that the holocephalan operculum evolved in concert with gill arch appendage reduction by attenuation of Shh-mediated branchial ray outgrowth, and that chondrichthyan branchial rays and tetrapod limbs exhibit parallel developmental mechanisms of evolutionary reduction. PMID:21220324

Mapping the Shh long-range regulatory domain

PubMed Central

Anderson, Eve; Devenney, Paul S.; Hill, Robert E.; Lettice, Laura A.

2014-01-01

Coordinated gene expression controlled by long-distance enhancers is orchestrated by DNA regulatory sequences involving transcription factors and layers of control mechanisms. The Shh gene and well-established regulators are an example of genomic composition in which enhancers reside in a large desert extending into neighbouring genes to control the spatiotemporal pattern of expression. Exploiting the local hopping activity of the Sleeping Beauty transposon, the lacZ reporter gene was dispersed throughout the Shh region to systematically map the genomic features responsible for expression activity. We found that enhancer activities are retained inside a genomic region that corresponds to the topological associated domain (TAD) defined by Hi-C. This domain of approximately 900 kb is in an open conformation over its length and is generally susceptible to all Shh enhancers. Similar to the distal enhancers, an enhancer residing within the Shh second intron activates the reporter gene located at distances of hundreds of kilobases away, suggesting that both proximal and distal enhancers have the capacity to survey the Shh topological domain to recognise potential promoters. The widely expressed Rnf32 gene lying within the Shh domain evades enhancer activities by a process that may be common among other housekeeping genes that reside in large regulatory domains. Finally, the boundaries of the Shh TAD do not represent the absolute expression limits of enhancer activity, as expression activity is lost stepwise at a number of genomic positions at the verges of these domains. PMID:25252942

Defining the molecular pathologies in cloaca malformation: similarities between mouse and human

PubMed Central

Runck, Laura A.; Method, Anna; Bischoff, Andrea; Levitt, Marc; Peña, Alberto; Collins, Margaret H.; Gupta, Anita; Shanmukhappa, Shiva; Wells, James M.; Guasch, Géraldine

2014-01-01

Anorectal malformations are congenital anomalies that form a spectrum of disorders, from the most benign type with excellent functional prognosis, to very complex, such as cloaca malformation in females in which the rectum, vagina and urethra fail to develop separately and instead drain via a single common channel into the perineum. The severity of this phenotype suggests that the defect occurs in the early stages of embryonic development of the organs derived from the cloaca. Owing to the inability to directly investigate human embryonic cloaca development, current research has relied on the use of mouse models of anorectal malformations. However, even studies of mouse embryos lack analysis of the earliest stages of cloaca patterning and morphogenesis. Here we compared human and mouse cloaca development and retrospectively identified that early mis-patterning of the embryonic cloaca might underlie the most severe forms of anorectal malformation in humans. In mouse, we identified that defective sonic hedgehog (Shh) signaling results in early dorsal-ventral epithelial abnormalities prior to the reported defects in septation. This is manifested by the absence of Sox2 and aberrant expression of keratins in the embryonic cloaca of Shh knockout mice. Shh knockout embryos additionally develop a hypervascular stroma, which is defective in BMP signaling. These epithelial and stromal defects persist later, creating an indeterminate epithelium with molecular alterations in the common channel. We then used these animals to perform a broad comparison with patients with mild-to-severe forms of anorectal malformations including cloaca malformation. We found striking parallels with the Shh mouse model, including nearly identical defective molecular identity of the epithelium and surrounding stroma. Our work strongly suggests that early embryonic cloacal epithelial differentiation defects might be the underlying cause of severe forms of anorectal malformations in humans. Moreover, deranged Shh and BMP signaling is correlated with severe anorectal malformations in both mouse and humans. PMID:24524909

Wnt signaling activates Shh signaling in early postnatal intervertebral discs, and re-activates Shh signaling in old discs in the mouse.

PubMed

Winkler, Tamara; Mahoney, Eric J; Sinner, Debora; Wylie, Christopher C; Dahia, Chitra Lekha

2014-01-01

Intervertebral discs (IVDs) are strong fibrocartilaginous joints that connect adjacent vertebrae of the spine. As discs age they become prone to failure, with neurological consequences that are often severe. Surgical repair of discs treats the result of the disease, which affects as many as one in seven people, rather than its cause. An ideal solution would be to repair degenerating discs using the mechanisms of their normal differentiation. However, these mechanisms are poorly understood. Using the mouse as a model, we previously showed that Shh signaling produced by nucleus pulposus cells activates the expression of differentiation markers, and cell proliferation, in the postnatal IVD. In the present study, we show that canonical Wnt signaling is required for the expression of Shh signaling targets in the IVD. We also show that Shh and canonical Wnt signaling pathways are down-regulated in adult IVDs. Furthermore, this down-regulation is reversible, since re-activation of the Wnt or Shh pathways in older discs can re-activate molecular markers of the IVD that are lost with age. These data suggest that biological treatments targeting Wnt and Shh signaling pathways may be feasible as a therapeutic for degenerative disc disease.

Identification of antibiotic resistance genes in the multidrug-resistant Acinetobacter baumannii strain, MDR-SHH02, using whole-genome sequencing.

PubMed

Wang, Hualiang; Wang, Jinghua; Yu, Peijuan; Ge, Ping; Jiang, Yanqun; Xu, Rong; Chen, Rong; Liu, Xuejie

2017-02-01

This study aimed to investigate antibiotic resistance genes in the multidrug-resistant (MDR) Acinetobacter baumannii (A. baumanii) strain, MDR-SHH02, using whole‑genome sequencing (WGS). The antibiotic resistance of MDR-SHH02 isolated from a patient with breast cancer to 19 types of antibiotics was determined using the Kirby‑Bauer method. WGS of MDR-SHH02 was then performed. Following quality control and transcriptome assembly, functional annotation of genes was conducted, and the phylogenetic tree of MDR-SHH02, along with another 5 A. baumanii species and 2 Acinetobacter species, was constructed using PHYLIP 3.695 and FigTree v1.4.2. Furthermore, pathogenicity islands (PAIs) were predicted by the pathogenicity island database. Potential antibiotic resistance genes in MDR-SHH02 were predicted based on the information in the Antibiotic Resistance Genes Database (ARDB). MDR-SHH02 was found to be resistant to all of the tested antibiotics. The total draft genome length of MDR-SHH02 was 4,003,808 bp. There were 74.25% of coding sequences to be annotated into 21 of the Clusters of Orthologous Groups (COGs) of protein terms, such as 'transcription' and 'amino acid transport and metabolism'. Furthermore, there were 45 PAIs homologous to the sequence MDRSHH02000806. Additionally, a total of 12 gene sequences in MDR-SHH02 were highly similar to the sequences of antibiotic resistance genes in ARDB, including genes encoding aminoglycoside‑modifying enzymes [e.g., aac(3)-Ia, ant(2'')‑Ia, aph33ib and aph(3')-Ia], β-lactamase genes (bl2b_tem and bl2b_tem1), sulfonamide-resistant dihydropteroate synthase genes (sul1 and sul2), catb3 and tetb. These results suggest that numerous genes mediate resistance to various antibiotics in MDR-SHH02, and provide a clinical guidance for the personalized therapy of A. baumannii-infected patients.

The Subjective Health Horizon Questionnaire (SHH-Q): Assessing Future Time Perspectives for Facets of an Active Lifestyle.

PubMed

Düzel, Sandra; Voelkle, Manuel C; Düzel, Emrah; Gerstorf, Denis; Drewelies, Johanna; Steinhagen-Thiessen, Elisabeth; Demuth, Ilja; Lindenberger, Ulman

2016-01-01

A wider subjective time horizon is assumed to be positively associated with longevity and vitality. In particular, a lifestyle with exposure to novel and varied information is considered beneficial for healthy cognitive aging. At present, measures that specifically assess individuals' perceived temporal extension to engage in active lifestyles in the future are not available. We introduce and validate a new self-report measure, the Subjective Health Horizon Questionnaire (SHH-Q). The SHH-Q assesses individuals' future time perspectives in relation to four interrelated but distinct lifestyle dimensions: (1) novelty-oriented exploration (Novelty), (2) bodily fitness (Body), (3) work goals (Work), and (4) goals in life (Life Goals). The present study aims at: (a) validating the hypothesized factor structure of the SHH-Q, according to which the SHH-Q consists of four interrelated but distinct subscales, and (b) testing the hypothesis that the Novelty and Body subscales of the SHH-Q show positive and selective associations with markers of cognition and somatic health, respectively. Using structural equation modeling, we analyzed data from 1,371 healthy individuals (51% women) with a mean age of 70.1 years (SD = 3.6) who participated in the Berlin Aging Study II (BASE-II) and completed the SHH-Q. As predicted, the SHH-Q formed four correlated but distinct subscales: (1) Novelty, (2) Body, (3) Work, and (4) Life Goals. Greater self-reported future novelty orientation was associated with higher current memory performance, and greater future expectations regarding bodily fitness with better current metabolic status. The SHH-Q reliably assesses individual differences in four distinct dimensions of future time perspective. Two of these dimensions, Novelty and Body, show differential associations with cognitive status and somatic health. The SHH-Q may serve as a tool to assess how different facets of future time perspective relate to somatic health, cognition, motivation, and affect, and may help to identify the socioeconomic and individual antecedents, correlates, and consequences of an active lifestyle. © 2016 S. Karger AG, Basel.

Genotypic and phenotypic analysis of 396 individuals with mutations in Sonic Hedgehog.

PubMed

Solomon, Benjamin D; Bear, Kelly A; Wyllie, Adrian; Keaton, Amelia A; Dubourg, Christele; David, Veronique; Mercier, Sandra; Odent, Sylvie; Hehr, Ute; Paulussen, Aimee; Clegg, Nancy J; Delgado, Mauricio R; Bale, Sherri J; Lacbawan, Felicitas; Ardinger, Holly H; Aylsworth, Arthur S; Bhengu, Ntombenhle Louisa; Braddock, Stephen; Brookhyser, Karen; Burton, Barbara; Gaspar, Harald; Grix, Art; Horovitz, Dafne; Kanetzke, Erin; Kayserili, Hulya; Lev, Dorit; Nikkel, Sarah M; Norton, Mary; Roberts, Richard; Saal, Howard; Schaefer, G B; Schneider, Adele; Smith, Erika K; Sowry, Ellen; Spence, M Anne; Shalev, Stavit A; Steiner, Carlos E; Thompson, Elizabeth M; Winder, Thomas L; Balog, Joan Z; Hadley, Donald W; Zhou, Nan; Pineda-Alvarez, Daniel E; Roessler, Erich; Muenke, Maximilian

2012-07-01

Holoprosencephaly (HPE), the most common malformation of the human forebrain, may result from mutations in over 12 genes. Sonic Hedgehog (SHH) was the first such gene discovered; mutations in SHH remain the most common cause of non-chromosomal HPE. The severity spectrum is wide, ranging from incompatibility with extrauterine life to isolated midline facial differences. To characterise genetic and clinical findings in individuals with SHH mutations. Through the National Institutes of Health and collaborating centres, DNA from approximately 2000 individuals with HPE spectrum disorders were analysed for SHH variations. Clinical details were examined and combined with published cases. This study describes 396 individuals, representing 157 unrelated kindreds, with SHH mutations; 141 (36%) have not been previously reported. SHH mutations more commonly resulted in non-HPE (64%) than frank HPE (36%), and non-HPE was significantly more common in patients with SHH than in those with mutations in the other common HPE related genes (p<0.0001 compared to ZIC2 or SIX3). Individuals with truncating mutations were significantly more likely to have frank HPE than those with non-truncating mutations (49% vs 35%, respectively; p=0.012). While mutations were significantly more common in the N-terminus than in the C-terminus (including accounting for the relative size of the coding regions, p=0.00010), no specific genotype-phenotype correlations could be established regarding mutation location. SHH mutations overall result in milder disease than mutations in other common HPE related genes. HPE is more frequent in individuals with truncating mutations, but clinical predictions at the individual level remain elusive.

Shh-ushing Midline Crossing through Remote Protein Transport.

PubMed

Herrera, Eloísa; Sitko, Austen A; Bovolenta, Paola

2018-01-17

Shh contributes to neural circuit formation with different mechanisms. In this issue, Peng and colleagues (2018) identify a novel trans-axonal mechanism by which Shh derived from contralateral projecting retinal ganglion cells prevents midline crossing of Boc-expressing ipsilateral axons at the optic chiasm. Copyright © 2018 Elsevier Inc. All rights reserved.

Lentivirus-mediated delivery of sonic hedgehog into the striatum stimulates neuroregeneration in a rat model of Parkinson disease.

PubMed

Zhang, Yi; Dong, Weiren; Guo, Suiqun; Zhao, Shu; He, Suifen; Zhang, Lihua; Tang, Yinjuan; Wang, Haihong

2014-12-01

Parkinson disease (PD) is a progressive neurodegenerative disorder in which the nigrostriatal pathway, consisting of dopaminergic neuronal projections from the substantia nigra to the striatum, degenerates. Viral transduction is currently the most promising in vivo strategy for delivery of therapeutic proteins into the brain for treatment of PD. Sonic hedgehog (Shh) is necessary for cell proliferation, differentiation and neuroprotection in the central nervous system. In this study, we investigated the effects of overexpressed N-terminal product of SHH (SHH-N) in a PD model rat. A lentiviral vector containing SHH-N was stereotactically injected into the striatum 24 h after a striatal 6-OHDA lesion. We found that overexpressed SHH-N attenuated behavioral deficits and reduced the loss of dopamine neurons in the substantia nigra and the loss of dopamine fibers in the striatum. In addition, fluoro-ruby-labeled nigrostriatal projections were also repaired. Together, our results demonstrate the feasibility and efficacy of using the strategy of lentivirus-mediated Shh-N delivery to delay nigrostriatal pathway degeneration. This strategy holds the potential for therapeutic application in the treatment of PD.

Sonic hedgehog signaling in kidney fibrosis: a master communicator.

PubMed

Zhou, Dong; Tan, Roderick J; Liu, Youhua

2016-09-01

The hedgehog signaling cascade is an evolutionarily conserved pathway that regulates multiple aspects of embryonic development and plays a decisive role in tissue homeostasis. As the best studied member of three hedgehog ligands, sonic hedgehog (Shh) is known to be associated with kidney development and tissue repair after various insults. Recent studies uncover an intrinsic link between dysregulated Shh signaling and renal fibrogenesis. In various types of chronic kidney disease (CKD), Shh is upregulated specifically in renal tubular epithelium but targets interstitial fibroblasts, thereby mediating a dynamic epithelial- mesenchymal communication (EMC). Tubule-derived Shh acts as a growth factor for interstitial fibroblasts and controls a hierarchy of fibrosis-related genes, which lead to the excessive deposition of extracellular matrix in renal interstitium. In this review, we recapitulate the principle of Shh signaling, its activation and regulation in a variety of kidney diseases. We also discuss the potential mechanisms by which Shh promotes renal fibrosis and assess the efficacy of blocking this signaling in preclinical settings. Continuing these lines of investigations will provide novel opportunities for designing effective therapies to improve CKD prognosis in patients.

Sonic hedgehog signaling in kidney fibrosis: a master communicator

PubMed Central

Zhou, Dong; Tan, Roderick J.; Liu, Youhua

2017-01-01

The hedgehog signaling cascade is an evolutionarily conserved pathway that regulates multiple aspects of embryonic development and plays a decisive role in tissue homeostasis. As the best studied member of three hedgehog ligands, sonic hedgehog (Shh) is known to be associated with kidney development and tissue repair after various insults. Recent studies uncover an intrinsic link between dysregulated Shh signaling and renal fibrogenesis. In various types of chronic kidney disease (CKD), Shh is upregulated specifically in renal tubular epithelium but targets interstitial fibroblasts, thereby mediating a dynamic epithelial-mesenchymal communication (EMC). Tubule-derived Shh acts as a growth factor for interstitial fibroblasts and controls a hierarchy of fibrosis-related genes, which lead to the excessive deposition of extracellular matrix in renal interstitium. In this review, we recapitulate the principle of Shh signaling, its activation and regulation in a variety of kidney diseases. We also discuss the potential mechanisms by which Shh promotes renal fibrosis and assess the efficacy of blocking this signaling in preclinical settings. Continuing these lines of investigations will provide novel opportunities for designing effective therapies to improve CKD prognosis in patients. PMID:27333788

Intraflagellar transport protein 122 antagonizes Sonic Hedgehog signaling and controls ciliary localization of pathway components.

PubMed

Qin, Jian; Lin, Yulian; Norman, Ryan X; Ko, Hyuk W; Eggenschwiler, Jonathan T

2011-01-25

Primary cilia are required for proper Sonic Hedgehog (Shh) signaling in mammals. However, their role in the signal transduction process remains unclear. We have identified sister of open brain (sopb), a null allele of mouse Intraflagellar transport protein 122 (Ift122). IFT122 negatively regulates the Shh pathway in the cilium at a step downstream of the Shh ligand and the transmembrane protein Smoothened, but upstream of the Gli2 transcription factor. Ift122(sopb) mutants generate primary cilia, but they show features of defective retrograde intraflagellar transport. IFT122 controls the ciliary localization of Shh pathway regulators in different ways. Disruption of IFT122 leads to accumulation of Gli2 and Gli3 at cilia tips while blocking the ciliary localization of the antagonist TULP3. Suppressor of Fused and Smoothened localize to the cilium through an IFT122-independent mechanism. We propose that the balance between positive and negative regulators of the Shh pathway at the cilium tip controls the output of the pathway and that Shh signaling regulates this balance through intraflagellar transport.

Epithelial heparan sulfate regulates Sonic Hedgehog signaling in lung development.

PubMed

He, Hua; Huang, Meina; Sun, Shenfei; Wu, Yihui; Lin, Xinhua

2017-08-01

The tree-like structure of the mammalian lung is generated from branching morphogenesis, a reiterative process that is precisely regulated by numerous factors. How the cell surface and extra cellular matrix (ECM) molecules regulate this process is still poorly understood. Herein, we show that epithelial deletion of Heparan Sulfate (HS) synthetase Ext1 resulted in expanded branching tips and reduced branching number, associated with several mesenchymal developmental defects. We further demonstrate an expanded Fgf10 expression and increased FGF signaling activity in Ext1 mutant lungs, suggesting a cell non-autonomous mechanism. Consistent with this, we observed reduced levels of SHH signaling which is responsible for suppressing Fgf10 expression. Moreover, reactivating SHH signaling in mutant lungs rescued the tip dilation phenotype and attenuated FGF signaling. Importantly, the reduced SHH signaling activity did not appear to be caused by decreased Shh expression or protein stability; instead, biologically active form of SHH proteins were reduced in both the Ext1 mutant epithelium and surrounding wild type mesenchymal cells. Together, our study highlights the epithelial HS as a key player for dictating SHH signaling critical for lung morphogenesis.

Sonic hedgehog from both nerves and epithelium is a key trophic factor for taste bud maintenance.

PubMed

Castillo-Azofeifa, David; Losacco, Justin T; Salcedo, Ernesto; Golden, Erin J; Finger, Thomas E; Barlow, Linda A

2017-09-01

The integrity of taste buds is intimately dependent on an intact gustatory innervation, yet the molecular nature of this dependency is unknown. Here, we show that differentiation of new taste bud cells, but not progenitor proliferation, is interrupted in mice treated with a hedgehog (Hh) pathway inhibitor (HPI), and that gustatory nerves are a source of sonic hedgehog (Shh) for taste bud renewal. Additionally, epithelial taste precursor cells express Shh transiently, and provide a local supply of Hh ligand that supports taste cell renewal. Taste buds are minimally affected when Shh is lost from either tissue source. However, when both the epithelial and neural supply of Shh are removed, taste buds largely disappear. We conclude Shh supplied by taste nerves and local taste epithelium act in concert to support continued taste bud differentiation. However, although neurally derived Shh is in part responsible for the dependence of taste cell renewal on gustatory innervation, neurotrophic support of taste buds likely involves a complex set of factors. © 2017. Published by The Company of Biologists Ltd.

BMP7 and SHH regulate Pax2 in mouse retinal astrocytes by relieving TLX repression.

PubMed

Sehgal, Rachna; Sheibani, Nader; Rhodes, Simon J; Belecky Adams, Teri L

2009-08-15

Pax2 is essential for development of the neural tube, urogenital system, optic vesicle, optic cup and optic tract. In the eye, Pax2 deficiency is associated with coloboma, a loss of astrocytes in the optic nerve and retina, and abnormal axonal pathfinding of the ganglion cell axons at the optic chiasm. Thus, appropriate expression of Pax2 is essential for astrocyte determination and differentiation. Although BMP7 and SHH have been shown to regulate Pax2 expression, the molecular mechanism by which this regulation occurs is not well understood. In this study, we determined that BMP7 and SHH activate Pax2 expression in mouse retinal astrocyte precursors in vitro. SHH appeared to play a dual role in Pax2 regulation; 1) SHH may regulate BMP7 expression, and 2) the SHH pathway cooperates with the BMP pathway to regulate Pax2 expression. BMP and SHH pathway members can interact separately or together with TLX, a repressor protein in the tailless transcription factor family. Here we show that the interaction of both pathways with TLX relieves the repression of Pax2 expression in mouse retinal astrocytes. Together these data reveal a new mechanism for the cooperative actions of signaling pathways in astrocyte determination and differentiation and suggest interactions of regulatory pathways that are applicable to other developmental programs.

Neural Progenitors Adopt Specific Identities by Directly Repressing All Alternative Progenitor Transcriptional Programs.

PubMed

Kutejova, Eva; Sasai, Noriaki; Shah, Ankita; Gouti, Mina; Briscoe, James

2016-03-21

In the vertebrate neural tube, a morphogen-induced transcriptional network produces multiple molecularly distinct progenitor domains, each generating different neuronal subtypes. Using an in vitro differentiation system, we defined gene expression signatures of distinct progenitor populations and identified direct gene-regulatory inputs corresponding to locations of specific transcription factor binding. Combined with targeted perturbations of the network, this revealed a mechanism in which a progenitor identity is installed by active repression of the entire transcriptional programs of other neural progenitor fates. In the ventral neural tube, sonic hedgehog (Shh) signaling, together with broadly expressed transcriptional activators, concurrently activates the gene expression programs of several domains. The specific outcome is selected by repressive input provided by Shh-induced transcription factors that act as the key nodes in the network, enabling progenitors to adopt a single definitive identity from several initially permitted options. Together, the data suggest design principles relevant to many developing tissues. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

FOREBRAIN AND HINDBRAIN DEVELOPMENT IN ZEBRAFISH IS SENSITIVE TO ETHANOL EXPOSURE INVOLVING AGRIN, FGF AND SONIC HEDGEHOG FUNCTION

PubMed Central

Zhang, Chengjin; Ojiaku, Princess; Cole, Gregory J.

2014-01-01

BACKGROUND Ethanol is a teratogen that affects numerous developmental processes in the nervous system, which includes development and survival of GABAergic and glutamatergic neurons. Possible molecular mechanisms accounting for ethanol’s effects on nervous system development include perturbed fibroblast growth factor (Fgf) and Sonic hedgehog (Shh) signaling. In zebrafish, forebrain GABAergic neuron development is dependent on Fgf19 and Shh signaling. The present study was conducted to test the hypothesis that ethanol affects GABAergic and glutamatergic neuron development by disrupting Fgf, Shh, and agrin function. METHODS Zebrafish embryos were exposed to varying concentrations of ethanol during a range of developmental stages, in the absence or presence of morpholino oligonucleotides (MOs) that disrupt agrin or Shh function. In situ hybridization was employed to analyze glutamic acid decarboxylase (GAD1) gene expression, as well as markers of glutamatergic neurons. RESULTS Acute ethanol exposure results in marked reduction in GAD1 gene expression in forebrain and hindbrain, and reduction of glutamatergic neuronal markers in hindbrain. Subthreshold ethanol exposure, combined with agrin or Shh MO treatment, produces a similar diminution in expression of markers for GABAergic and glutamatergic neurons. Consistent with the ethanol effects on Fgf and Shh pathways, Fgf19, Fgf8 or Shh mRNA overexpression rescues ethanol-induced decreases in GAD1 and atonal1a gene expression. CONCLUSIONS These studies demonstrate that GABAergic and glutamatergic neuron development in zebrafish forebrain or cerebellum is sensitive to ethanol exposure, and provides additional evidence that a signaling pathway involving agrin, Fgfs and Shh may be a critical target of ethanol exposure during zebrafish embryogenesis. PMID:23184466

Sonic Hedgehog Signaling in Limb Development

PubMed Central

Tickle, Cheryll; Towers, Matthew

2017-01-01

The gene encoding the secreted protein Sonic hedgehog (Shh) is expressed in the polarizing region (or zone of polarizing activity), a small group of mesenchyme cells at the posterior margin of the vertebrate limb bud. Detailed analyses have revealed that Shh has the properties of the long sought after polarizing region morphogen that specifies positional values across the antero-posterior axis (e.g., thumb to little finger axis) of the limb. Shh has also been shown to control the width of the limb bud by stimulating mesenchyme cell proliferation and by regulating the antero-posterior length of the apical ectodermal ridge, the signaling region required for limb bud outgrowth and the laying down of structures along the proximo-distal axis (e.g., shoulder to digits axis) of the limb. It has been shown that Shh signaling can specify antero-posterior positional values in limb buds in both a concentration- (paracrine) and time-dependent (autocrine) fashion. Currently there are several models for how Shh specifies positional values over time in the limb buds of chick and mouse embryos and how this is integrated with growth. Extensive work has elucidated downstream transcriptional targets of Shh signaling. Nevertheless, it remains unclear how antero-posterior positional values are encoded and then interpreted to give the particular structure appropriate to that position, for example, the type of digit. A distant cis-regulatory enhancer controls limb-bud-specific expression of Shh and the discovery of increasing numbers of interacting transcription factors indicate complex spatiotemporal regulation. Altered Shh signaling is implicated in clinical conditions with congenital limb defects and in the evolution of the morphological diversity of vertebrate limbs. PMID:28293554

Transcriptional repressor foxl1 regulates central nervous system development by suppressing shh expression in zebra fish.

PubMed

Nakada, Chisako; Satoh, Shinya; Tabata, Yoko; Arai, Ken-ichi; Watanabe, Sumiko

2006-10-01

We identified zebra fish forkhead transcription factor l1 (zfoxl1) as a gene strongly expressed in neural tissues such as midbrain, hindbrain, and the otic vesicle at the early embryonic stage. Loss of the function of zfoxl1 effected by morpholino antisense oligonucleotide resulted in defects in midbrain and eye development, and in that of formation of the pectoral fins. Interestingly, ectopic expression of shh in the midbrain and elevated pax2a expression in the optic stalk were observed in foxl1 MO-injected embryos. In contrast, expression of pax6a, which is negatively regulated by shh, was suppressed in the thalamus and pretectum regions, supporting the idea of augmentation of the shh signaling pathway by suppression of foxl1. Expression of zfoxl1-EnR (repressing) rather than zfoxl1-VP16 (activating) resulted in a phenotype similar to that induced by foxl1-mRNA, suggesting that foxl1 may act as a transcriptional repressor of shh in zebra fish embryos. Supporting this notion, foxl1 suppressed isolated 2.7-kb shh promoter activity in PC12 cells, and the minimal region of foxl1 required for its transcriptional repressor activity showed strong homology with the groucho binding motif, which is found in genes encoding various homeodomain proteins. In view of all of our data taken together, we propose zfoxl1 to be a novel regulator of neural development that acts by suppressing shh expression.

Transcriptional Repressor foxl1 Regulates Central Nervous System Development by Suppressing shh Expression in Zebra Fish†

PubMed Central

Nakada, Chisako; Satoh, Shinya; Tabata, Yoko; Arai, Ken-ichi; Watanabe, Sumiko

2006-01-01

We identified zebra fish forkhead transcription factor l1 (zfoxl1) as a gene strongly expressed in neural tissues such as midbrain, hindbrain, and the otic vesicle at the early embryonic stage. Loss of the function of zfoxl1 effected by morpholino antisense oligonucleotide resulted in defects in midbrain and eye development, and in that of formation of the pectoral fins. Interestingly, ectopic expression of shh in the midbrain and elevated pax2a expression in the optic stalk were observed in foxl1 MO-injected embryos. In contrast, expression of pax6a, which is negatively regulated by shh, was suppressed in the thalamus and pretectum regions, supporting the idea of augmentation of the shh signaling pathway by suppression of foxl1. Expression of zfoxl1-EnR (repressing) rather than zfoxl1-VP16 (activating) resulted in a phenotype similar to that induced by foxl1-mRNA, suggesting that foxl1 may act as a transcriptional repressor of shh in zebra fish embryos. Supporting this notion, foxl1 suppressed isolated 2.7-kb shh promoter activity in PC12 cells, and the minimal region of foxl1 required for its transcriptional repressor activity showed strong homology with the groucho binding motif, which is found in genes encoding various homeodomain proteins. In view of all of our data taken together, we propose zfoxl1 to be a novel regulator of neural development that acts by suppressing shh expression. PMID:16980626

YB-1 is elevated in medulloblastoma and drives proliferation in Sonic hedgehog-dependent cerebellar granule neuron progenitor cells and medulloblastoma cells.

PubMed

Dey, A; Robitaille, M; Remke, M; Maier, C; Malhotra, A; Gregorieff, A; Wrana, J L; Taylor, M D; Angers, S; Kenney, A M

2016-08-11

Postnatal proliferation of cerebellar granule neuron precursors (CGNPs), proposed cells of origin for the SHH-associated subgroup of medulloblastoma, is driven by Sonic hedgehog (Shh) and insulin-like growth factor (IGF) in the developing cerebellum. Shh induces the oncogene Yes-associated protein (YAP), which drives IGF2 expression in CGNPs and mouse Shh-associated medulloblastomas. To determine how IGF2 expression is regulated downstream of YAP, we carried out an unbiased screen for transcriptional regulators bound to IGF2 promoters. We report that Y-box binding protein-1 (YB-1), an onco-protein regulating transcription and translation, binds to IGF2 promoter P3. We observed that YB-1 is upregulated across human medulloblastoma subclasses as well as in other varieties of pediatric brain tumors. Utilizing the cerebellar progenitor model for the Shh subgroup of medulloblastoma in mice, we show for the first time that YB-1 is induced by Shh in CGNPs. Its expression is YAP-dependent and it is required for IGF2 expression in CGNPs. Finally, both gain-of function and loss-of-function experiments reveal that YB-1 activity is required for sustaining CGNP and medulloblastoma cell (MBC) proliferation. Collectively, our findings describe a novel role for YB-1 in driving proliferation in the developing cerebellum and MBCs and they identify the SHH:YAP:YB1:IGF2 axis as a powerful target for therapeutic intervention in medulloblastomas.

Sonic Hedgehog, VACTERL, and Fanconi anemia: Pathogenetic connections and therapeutic implications.

PubMed

Lubinsky, Mark

2015-11-01

Three systems with VACTERL association findings- mutations of the Sonic Hedgehog (SHH) signaling pathway in mice, murine adriamycin teratogenicity, and human Fanconi anemia (FA) pathway mutations, may all involve a similar mechanism. SHH is up-regulated in irradiated cells, and DNA breaks common with radiation damage in the adriamycin and FA systems are plausible signals for such effects, which would affect development. Since FA related DNA breakage occurs throughout life, SHH disturbances may account for later FA related findings involving hematopoietic and malignancy issues. In support, androgen, a standard treatment for FA hematologic failure, down-regulates SHH, and common FA malignancies such as squamous cell carcinomas and acute myeloid leukemia have been linked to enhanced SHH function. This suggests that interventions lowering SHH levels may be useful therapeutically. Also supporting a connection between pre- and post- natal findings, the frequency and number of VACTERL anomalies with FA correlate with the severity and onset of hematopoietic and malignancy issues. In FA, radial anomalies are the most common of these defects, followed by renal findings, while vertebral and gastrointestinal anomalies are relatively uncommon, a pattern that differs from observations of the VACTERL association. Genes with more severe effects also show a greatly increased incidence of brain abnormalities, and a paucity of such findings with other FA genes suggests that brain development is relatively refractory to SHH related effects, accounting for the rarity of such findings with the association. © 2015 Wiley Periodicals, Inc.

Sonic hedgehog initiates cochlear hair cell regeneration through downregulation of retinoblastoma protein

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

Lu, Na; Department of Otolaryngology and Program in Neuroscience, Harvard Medical School and Eaton Peabody Laboratory, Massachusetts Eye and Ear Infirmary, Boston, MA 02114; Chen, Yan

Highlights: Black-Right-Pointing-Pointer Shh activation in neonatal cochleae enhances sensory cell proliferation. Black-Right-Pointing-Pointer Proliferating supporting cells can transdifferentiate into hair cells. Black-Right-Pointing-Pointer Shh promotes proliferation by transiently modulating pRb activity. Black-Right-Pointing-Pointer Shh inhibits pRb by inhibiting transcription and increasing phosphorylation of pRb. -- Abstract: Cell cycle re-entry by cochlear supporting cells and/or hair cells is considered one of the best approaches for restoring hearing loss as a result of hair cell damage. To identify mechanisms that can be modulated to initiate cell cycle re-entry and hair cell regeneration, we studied the effect of activating the sonic hedgehog (Shh) pathway. We showmore » that Shh signaling in postnatal rat cochleae damaged by neomycin leads to renewed proliferation of supporting cells and hair cells. Further, proliferating supporting cells are likely to transdifferentiate into hair cells. Shh treatment leads to inhibition of retinoblastoma protein (pRb) by increasing phosphorylated pRb and reducing retinoblastoma gene transcription. This results in upregulation of cyclins B1, D2, and D3, and CDK1. These results suggest that Shh signaling induces cell cycle re-entry in cochlear sensory epithelium and the production of new hair cells, in part by attenuating pRb function. This study provides an additional route to modulate pRb function with important implications in mammalian hair cell regeneration.« less

Sonic hedgehog multimerization: a self-organizing event driven by post-translational modifications?

PubMed

Koleva, Mirella V; Rothery, Stephen; Spitaler, Martin; Neil, Mark A A; Magee, Anthony I

2015-01-01

Sonic hedgehog (Shh) is a morphogen active during vertebrate development and tissue homeostasis in adulthood. Dysregulation of the Shh signalling pathway is known to incite carcinogenesis. Due to the highly lipophilic nature of this protein imparted by two post-translational modifications, Shh's method of transit through the aqueous extracellular milieu has been a long-standing conundrum, prompting the proposition of numerous hypotheses to explain the manner of its displacement from the surface of the producing cell. Detection of high molecular-weight complexes of Shh in the intercellular environment has indicated that the protein achieves this by accumulating into multimeric structures prior to release from producing cells. The mechanism of assembly of the multimers, however, has hitherto remained mysterious and contentious. Here, with the aid of high-resolution optical imaging and post-translational modification mutants of Shh, we show that the C-terminal cholesterol and the N-terminal palmitate adducts contribute to the assembly of large multimers and regulate their shape. Moreover, we show that small Shh multimers are produced in the absence of any lipid modifications. Based on an assessment of the distribution of various dimensional characteristics of individual Shh clusters, in parallel with deductions about the kinetics of release of the protein from the producing cells, we conclude that multimerization is driven by self-assembly underpinned by the law of mass action. We speculate that the lipid modifications augment the size of the multimolecular complexes through prolonging their association with the exoplasmic membrane.

Rubinstein-Taybi syndrome predisposing to non-WNT, non-SHH, group 3 medulloblastoma.

PubMed

Bourdeaut, Franck; Miquel, Catherine; Richer, Wilfrid; Grill, Jacques; Zerah, Michel; Grison, Camille; Pierron, Gaelle; Amiel, Jeanne; Krucker, Clementine; Radvanyi, Francois; Brugieres, Laurence; Delattre, Olivier

2014-02-01

Medulloblastomas (MB) are classified in four subgroups: the well defined WNT and Sonic Hedgehog (SHH) subgroups, and the less defined groups 3 and 4. They occasionally occur in the context of a cancer predisposition syndrome. While germline APC mutations predispose to WNT MB, germline mutations in SUFU, PTCH1, and TP53 predispose to SHH tumors. We report on a child with a Rubinstein-Taybi syndrome (RTS) due to a germline deletion in CREBBP, who developed a MB. Biological profilings demonstrate that this tumor belongs to the group 3. RTS may therefore be the first predisposition syndrome identified for non-WNT/non-SHH MB. © 2013 Wiley Periodicals, Inc.

Opposing Effects of CREBBP Mutations Govern the Phenotype of Rubinstein-Taybi Syndrome and Adult SHH Medulloblastoma.

PubMed

Merk, Daniel J; Ohli, Jasmin; Merk, Natalie D; Thatikonda, Venu; Morrissy, Sorana; Schoof, Melanie; Schmid, Susanne N; Harrison, Luke; Filser, Severin; Ahlfeld, Julia; Erkek, Serap; Raithatha, Kaamini; Andreska, Thomas; Weißhaar, Marc; Launspach, Michael; Neumann, Julia E; Shakarami, Mehdi; Plenker, Dennis; Marra, Marco A; Li, Yisu; Mungall, Andrew J; Moore, Richard A; Ma, Yussanne; Jones, Steven J M; Lutz, Beat; Ertl-Wagner, Birgit; Rossi, Andrea; Wagener, Rabea; Siebert, Reiner; Jung, Andreas; Eberhart, Charles G; Lach, Boleslaw; Sendtner, Michael; Pfister, Stefan M; Taylor, Michael D; Chavez, Lukas; Kool, Marcel; Schüller, Ulrich

2018-03-26

Recurrent mutations in chromatin modifiers are specifically prevalent in adolescent or adult patients with Sonic hedgehog-associated medulloblastoma (SHH MB). Here, we report that mutations in the acetyltransferase CREBBP have opposing effects during the development of the cerebellum, the primary site of origin of SHH MB. Our data reveal that loss of Crebbp in cerebellar granule neuron progenitors (GNPs) during embryonic development of mice compromises GNP development, in part by downregulation of brain-derived neurotrophic factor (Bdnf). Interestingly, concomitant cerebellar hypoplasia was also observed in patients with Rubinstein-Taybi syndrome, a congenital disorder caused by germline mutations of CREBBP. By contrast, loss of Crebbp in GNPs during postnatal development synergizes with oncogenic activation of SHH signaling to drive MB growth, thereby explaining the enrichment of somatic CREBBP mutations in SHH MB of adult patients. Together, our data provide insights into time-sensitive consequences of CREBBP mutations and corresponding associations with human diseases. Copyright © 2018 Elsevier Inc. All rights reserved.

Sonic hedgehog regulates its own receptor on postcrossing commissural axons in a glypican1-dependent manner.

PubMed

Wilson, Nicole H; Stoeckli, Esther T

2013-08-07

Upon reaching their intermediate target, the floorplate, commissural axons acquire responsiveness to repulsive guidance cues, allowing the axons to exit the midline and adopt a contralateral, longitudinal trajectory. The molecular mechanisms that regulate this switch from attraction to repulsion remain poorly defined. Here, we show that the heparan sulfate proteoglycan Glypican1 (GPC1) is required as a coreceptor for the Shh-dependent induction of Hedgehog-interacting protein (Hhip) in commissural neurons. In turn, Hhip is required for postcrossing axons to respond to a repulsive anteroposterior Shh gradient. Thus, Shh is a cue with dual function. In precrossing axons it acts as an attractive guidance molecule in a transcription-independent manner. At the same time, Shh binds to GPC1 to induce the expression of its own receptor, Hhip, which mediates the repulsive response of postcrossing axons to Shh. Our study characterizes a molecular mechanism by which navigating axons switch their responsiveness at intermediate targets. Copyright © 2013 Elsevier Inc. All rights reserved.

Correlation Between Hedgehog (Hh) Protein Family and Brain-Derived Neurotrophic Factor (BDNF) in Autism Spectrum Disorder (ASD).

PubMed

Halepoto, Dost Muhammad; Bashir, Shahid; Zeina, Rana; Al-Ayadhi, Laila Y

2015-12-01

To determine the correlation of Sonic Hedgehog (SHH), Indian Hedgehog (IHH), and Brain-Derived Neurotrophic Factor (BDNF) in children with Autism Spectrum Disorder (ASD). An observational, comparative study. Autism Research and Treatment Center, Al-Amodi Autism Research Chair, Department of Physiology, Faculty of Medicine, King Khalid University Hospital, King Saud University, Riyadh, Saudi Arabia, from October 2011 to May 2012. Serum levels of SHH, IHH and BDNF were determined in recently diagnosed autistic patients and age-matched healthy children (n=25), using the Enzyme-Linked Immunosorbent Assay (ELISA). Childhood Autism Rating Scale (CARS) was used for the assessment of autistic severity. Spearman correlation co-efficient 'r' was determined. The serum levels of IHH and SHH were significantly higher in autistic subjects than those of control subjects. There was significant correlation between age and IHH (r = 0.176, p = 0.03), BDNF and severe IHH (r = 0.1763, p = 0.003), and severe BDNF and severe SHH (r = 0.143, p < 0.001). However, there were no significant relationships among the serum levels of SHH, IHH and BDNF and the CARS score, age or gender. The findings support a correlation between SHH, IHH and BDNF in autistic children, suggesting their pathological role in autism.

Zic2-associated holoprosencephaly is caused by a transient defect in the organizer region during gastrulation.

PubMed

Warr, Nicholas; Powles-Glover, Nicola; Chappell, Anna; Robson, Joan; Norris, Dominic; Arkell, Ruth M

2008-10-01

The putative transcription factor ZIC2 is associated with a defect of forebrain development, known as Holoprosencephaly (HPE), in humans and mouse, yet the mechanism by which aberrant ZIC2 function causes classical HPE is unexplained. The zinc finger domain of all mammalian Zic genes is highly homologous with that of the Gli genes, which are transcriptional mediators of Shh signalling. Mutations in Shh and many other Hh pathway members cause HPE and it has been proposed that Zic2 acts within the Shh pathway to cause HPE. We have investigated the embryological cause of Zic2-associated HPE and the relationship between Zic2 and the Shh pathway using mouse genetics. We show that Zic2 does not interact with Shh to produce HPE. Moreover, molecular defects that are able to account for the HPE phenotype are present in Zic2 mutants before the onset of Shh signalling. Mutation of Zic2 causes HPE via a transient defect in the function of the organizer region at mid-gastrulation which causes an arrest in the development of the prechordal plate (PCP), a structure required for forebrain midline morphogenesis. The analysis provides genetic evidence that Zic2 functions during organizer formation and that the PCP develops via a multi-step process.

Sonic Hedgehog Signaling in Thyroid Cancer

PubMed Central

Xu, Xiulong; Lu, Yurong; Li, Yi; Prinz, Richard A.

2017-01-01

Thyroid cancer is the most common malignancy of the endocrine system. The initiation of thyroid cancer is often triggered by a genetic mutation in the phosphortidylinositol-3 kinase (PI3K) or mitogen-activated protein kinase (MAPK) pathway, such as RAS and BRAF, or by the rearrangement of growth factor receptor tyrosine kinase genes such as RET/PTC. The sonic hedgehog (Shh) pathway is evolutionarily conserved and plays an important role in the embryonic development of normal tissues and organs. Gene mutations in the Shh pathway are involved in basal cell carcinomas (BCC). Activation of the Shh pathway due to overexpression of the genes encoding the components of this pathway stimulates the growth and spread of a wide range of cancer types. The Shh pathway also plays an important role in cancer stem cell (CSC) self-renewal. GDC-0449 and LDE-225, two inhibitors of this pathway, have been approved for treating BCC and are being tested as a single agent or in combination with other drugs for treating various other cancers. Here, we review the recent findings on activation of the Shh pathway in thyroid cancer and its role in maintaining thyroid CSC self-renewal. We also summarize the recent developments on crosstalk of the Shh pathway with the MAPK and PI3K oncogenic pathways, and its implications for combination therapy. PMID:29163356

Rescue Effects and Underlying Mechanisms of Intragland Shh Gene Delivery on Irradiation-Induced Hyposalivation.

PubMed

Hai, Bo; Zhao, Qingguo; Qin, Lizheng; Rangaraj, Dharanipathy; Gutti, Veera R; Liu, Fei

2016-05-01

Irreversible hypofunction of salivary glands is common in head and neck cancer survivors treated with radiotherapy and can only be temporarily relieved with current treatments. We found in an inducible sonic hedgehog (Shh) transgenic mouse model that transient activation of the Hedgehog pathway after irradiation rescued salivary gland function in males by preserving salivary stem/progenitor cells and parasympathetic innervation. To translate these findings into feasible clinical application, we evaluated the effects of Shh gene transfer to salivary glands of wild-type mice on irradiation-induced hyposalivation. Shh or control GFP gene was delivered by noninvasive retrograde ductal instillation of corresponding adenoviral vectors. In both male and female mice, Shh gene delivery efficiently activated Hedgehog/Gli signaling, and significantly improved stimulated saliva secretion and preserved saliva-producing acinar cells after irradiation. In addition to preserving parasympathetic innervation through induction of neurotrophic factors, Shh gene delivery also alleviated the irradiation damage of the microvasculature, likely via inducing angiogenic factors, but did not expand the progeny of cells responsive to Hedgehog/Gli signaling. These data indicate that transient activation of the Hedgehog pathway by gene delivery is promising to rescue salivary function after irradiation in both sexes, and the Hedgehog/Gli pathway may function mainly in cell nonautonomous manners to achieve the rescue effect.

Medulloblastoma, WNT-activated/SHH-activated: clinical impact of molecular analysis and histogenetic evaluation.

PubMed

Cambruzzi, Eduardo

2018-05-01

Medulloblastoma (MDB) is a small cell poorly differentiated embryonal tumor of the cerebellum, which more frequently compromises children. Overall prognosis is favorable, but dependent of stage, histopathological pattern and molecular group. Approximately 30% of the affected patients will die from the disease. WHO 2016 Classification of Tumors of the Central Nervous System (CNS) has been classified MDB into four principal groups: WNT-activated MDB, SHH-activated MDB, group 3 MDB, and group 4 MDB. WNT-activated MDB is associated to monosomy 6, CTNNB1, DDX3X and TP53 mutations, beta-catenin nuclear immunoexpression, and a better prognosis than SHH-activated MDB. WNT-activated tumors account approximately for 10% of cases of MDBs, and are thought to arise from cells in the dorsal brain stem/lower rhombic lip progenitor cells. SHH-activated MDB more frequently arises in the lateral hemispheres of the cerebellum, and clinical outcome in this group is variable. TP53-mutant SHHactivated MDB usually shows the large cell/anaplastic pattern, and can be related to MYCN amplification, GLI2 amplification and 17p loss. TP53-wildtype SHH-activated MDB is more commonly of desmoplastic/nodular morphology, and can be related to PTCH1 deletion and 10q loss. Gene expression and methylation profiling is the gold standard for defining molecular groups of MDB. In immunohistochemistry assays, anti-GAB1 antibody expression is positive in tumors showing SHH pathway activation or PTCH mutation, while positive immunoexpression for YAP1 antibody can be only found in WNT-activated and SHH-activated MDB.

SHH desmoplastic/nodular medulloblastoma and Gorlin syndrome in the setting of Down syndrome: case report, molecular profiling, and review of the literature.

PubMed

Mangum, Ross; Varga, Elizabeth; Boué, Daniel R; Capper, David; Benesch, Martin; Leonard, Jeffrey; Osorio, Diana S; Pierson, Christopher R; Zumberge, Nicholas; Sahm, Felix; Schrimpf, Daniel; Pfister, Stefan M; Finlay, Jonathan L

2016-12-01

Individuals with Down syndrome (DS) have an increased risk of acute leukemia compared to a markedly decreased incidence of solid tumors. Medulloblastoma, the most common malignant brain tumor of childhood, is particularly rare in the DS population, with only one published case. As demonstrated in a mouse model, DS is associated with cerebellar hypoplasia and a decreased number of cerebellar granule neuron progenitor cells (CGNPs) in the external granule cell layer (EGL). Treatment of these mice with sonic hedgehog signaling pathway (Shh) agonists promote normalization of CGNPs and improved cognitive functioning. We describe a 21-month-old male with DS and concurrent desmoplastic/nodular medulloblastoma (DNMB)-a tumor derived from Shh dysregulation and over-activation of CGNPs. Molecular profiling further classified the tumor into the new consensus SHH molecular subgroup. Additional testing revealed a de novo heterozygous germ line mutation in the PTCH1 gene encoding a tumor suppressor protein in the Shh pathway. The developmental failure of CGNPs in DS patients offers a plausible explanation for the rarity of medulloblastoma in this population. Conversely, patients with PTCH1 germline mutations experience Shh overstimulation resulting in Gorlin (Nevoid Basal Cell Carcinoma) syndrome and an increased incidence of malignant transformation of CGNPs leading to medulloblastoma formation. This represents the first documented report of an individual with DS simultaneously carrying PTCH1 germline mutation. We have observed a highly unusual circumstance in which the PTCH1 mutation appears to "trump" the effects of DS in causation of Shh-activated medulloblastoma.

Learning Induces Sonic Hedgehog Signaling in the Amygdala which Promotes Neurogenesis and Long-Term Memory Formation

PubMed Central

Hung, Hui-Chi; Hsiao, Ya-Hsin

2015-01-01

Background: It is known that neurogenesis occurs throughout the life mostly in the subgranular zone of the hippocampus and the subventricular zone of the lateral ventricle. We investigated whether neurogenesis occurred in the amygdala and its function in fear memory formation. Methods: For detection of newborn neurons, mice were injected intraperitoneally with 5-bromo-2’-deoxyuridine (BrdU) 2h before receiving 15 tone–footshock pairings, and newborn neurons were analyzed 14 and 42 days after training. To determine the relationship between neurogenesis and memory formation, mice were given a proliferation inhibitor methylazoxymethanol (MAM) or a DNA synthesis inhibitor cytosine arabinoside (Ara-C). To test whether sonic hedgehog (Shh) signaling was required for neurogenesis, Shh-small hairpin–interfering RNA (shRNA) was inserted into a retroviral vector (Retro-Shh-shRNA). Results: The number of BrdU+/Neuronal nuclei (NeuN)+ cells was significantly higher in the conditioned mice, suggesting that association of tone with footshock induced neurogenesis. MAM and Ara-C markedly reduced neurogenesis and impaired fear memory formation. Shh, its receptor patched 1 (Ptc1), and transcription factor Gli1 protein levels increased at 1 day and returned to baseline at 7 days after fear conditioning. Retro-Shh-shRNA, which knocked down Shh specifically in the mitotic neurons, reduced the number of BrdU+/NeuN+ cells and decreased freezing responses. Conclusions: These results suggest that fear learning induces Shh signaling activation in the amygdala, which promotes neurogenesis and fear memory formation. PMID:25522410

Multiple developmental programs are altered by loss of Zic1 and Zic4 to cause Dandy-Walker malformation cerebellar pathogenesis

PubMed Central

Blank, Marissa C.; Grinberg, Inessa; Aryee, Emmanuel; Laliberte, Christine; Chizhikov, Victor V.; Henkelman, R. Mark; Millen, Kathleen J.

2011-01-01

Heterozygous deletions encompassing the ZIC1;ZIC4 locus have been identified in a subset of individuals with the common cerebellar birth defect Dandy-Walker malformation (DWM). Deletion of Zic1 and Zic4 in mice produces both cerebellar size and foliation defects similar to human DWM, confirming a requirement for these genes in cerebellar development and providing a model to delineate the developmental basis of this clinically important congenital malformation. Here, we show that reduced cerebellar size in Zic1 and Zic4 mutants results from decreased postnatal granule cell progenitor proliferation. Through genetic and molecular analyses, we show that Zic1 and Zic4 have Shh-dependent function promoting proliferation of granule cell progenitors. Expression of the Shh-downstream genes Ptch1, Gli1 and Mycn was downregulated in Zic1/4 mutants, although Shh production and Purkinje cell gene expression were normal. Reduction of Shh dose on the Zic1+/−;Zic4+/− background also resulted in cerebellar size reductions and gene expression changes comparable with those observed in Zic1−/−;Zic4−/− mice. Zic1 and Zic4 are additionally required to pattern anterior vermis foliation. Zic mutant folial patterning abnormalities correlated with disrupted cerebellar anlage gene expression and Purkinje cell topography during late embryonic stages; however, this phenotype was Shh independent. In Zic1+/−;Zic4+/−;Shh+/−, we observed normal cerebellar anlage patterning and foliation. Furthermore, cerebellar patterning was normal in both Gli2-cko and Smo-cko mutant mice, where all Shh function was removed from the developing cerebellum. Thus, our data demonstrate that Zic1 and Zic4 have both Shh-dependent and -independent roles during cerebellar development and that multiple developmental disruptions underlie Zic1/4-related DWM. PMID:21307096

Arsenic inhibits hedgehog signaling during P19 cell differentiation

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

Liu, Jui Tung; Bain, Lisa J., E-mail: lbain@clemson.edu; Department of Biological Sciences, Clemson University, 132 Long Hall, Clemson, SC 29634

Arsenic is a toxicant found in ground water around the world, and human exposure mainly comes from drinking water or from crops grown in areas containing arsenic in soils or water. Epidemiological studies have shown that arsenic exposure during development decreased intellectual function, reduced birth weight, and altered locomotor activity, while in vitro studies have shown that arsenite decreased muscle and neuronal cell differentiation. The sonic hedgehog (Shh) signaling pathway plays an important role during the differentiation of both neurons and skeletal muscle. The purpose of this study was to investigate whether arsenic can disrupt Shh signaling in P19 mousemore » embryonic stem cells, leading to changes muscle and neuronal cell differentiation. P19 embryonic stem cells were exposed to 0, 0.25, or 0.5 μM of sodium arsenite for up to 9 days during cell differentiation. We found that arsenite exposure significantly reduced transcript levels of genes in the Shh pathway in both a time and dose-dependent manner. This included the Shh ligand, which was decreased 2- to 3-fold, the Gli2 transcription factor, which was decreased 2- to 3-fold, and its downstream target gene Ascl1, which was decreased 5-fold. GLI2 protein levels and transcriptional activity were also reduced. However, arsenic did not alter GLI2 primary cilium accumulation or nuclear translocation. Moreover, additional extracellular SHH rescued the inhibitory effects of arsenic on cellular differentiation due to an increase in GLI binding activity. Taken together, we conclude that arsenic exposure affected Shh signaling, ultimately decreasing the expression of the Gli2 transcription factor. These results suggest a mechanism by which arsenic disrupts cell differentiation. - Highlights: • Arsenic exposure decreases sonic hedgehog pathway-related gene expression. • Arsenic decreases GLI2 protein levels and transcriptional activity in P19 cells. • Arsenic exposure does not alter the levels of SHH expression and GLI2 primary cilum accumulation. • Induction of the Shh pathway rescues arsenic's inhibitory effects on cell differentiation.« less

A compartmentalized phosphoinositide signaling axis at cilia is regulated by INPP5E to maintain cilia and promote Sonic Hedgehog medulloblastoma.

PubMed

Conduit, S E; Ramaswamy, V; Remke, M; Watkins, D N; Wainwright, B J; Taylor, M D; Mitchell, C A; Dyson, J M

2017-10-26

Sonic Hedgehog (SHH) signaling at primary cilia drives the proliferation and progression of a subset of medulloblastomas, the most common malignant paediatric brain tumor. Severe side effects associated with conventional treatments and resistance to targeted therapies has led to the need for new strategies. SHH signaling is dependent on primary cilia for signal transduction suggesting the potential for cilia destabilizing mechanisms as a therapeutic target. INPP5E is an inositol polyphosphate 5-phosphatase that hydrolyses PtdIns(4,5)P 2 and more potently, the phosphoinositide (PI) 3-kinase product PtdIns(3,4,5)P 3 . INPP5E promotes SHH signaling during embryonic development via PtdIns(4,5)P 2 hydrolysis at cilia, that in turn regulates the cilia recruitment of the SHH suppressor GPR161. However, the role INPP5E plays in cancer is unknown and the contribution of PI3-kinase signaling to cilia function is little characterized. Here, we reveal INPP5E promotes SHH signaling in SHH medulloblastoma by negatively regulating a cilia-compartmentalized PI3-kinase signaling axis that maintains primary cilia on tumor cells. Conditional deletion of Inpp5e in a murine model of constitutively active Smoothened-driven medulloblastoma slowed tumor progression, suppressed cell proliferation, reduced SHH signaling and promoted tumor cell cilia loss. PtdIns(3,4,5)P 3 , its effector pAKT and the target pGSK3β, which when non-phosphorylated promotes cilia assembly/stability, localized to tumor cell cilia. The number of PtdIns(3,4,5)P 3 /pAKT/pGSK3β-positive cilia was increased in cultured Inpp5e-null tumor cells relative to controls. PI3-kinase inhibition or expression of wild-type, but not catalytically inactive HA-INPP5E partially rescued cilia loss in Inpp5e-null tumor cells in vitro. INPP5E mRNA and copy number were reduced in human SHH medulloblastoma compared to other molecular subtypes and consistent with the murine model, reduced INPP5E was associated with improved overall survival. Therefore our study identifies a compartmentalized PtdIns(3,4,5)P 3 /AKT/GSK3β signaling axis at cilia in SHH-dependent medulloblastoma that is regulated by INPP5E to maintain tumor cell cilia, promote SHH signaling and thereby medulloblastoma progression.

A Study of Failure Characteristics in Thermoplastic Composite Material.

DTIC Science & Technology

1988-03-01

plastic, such as epoxy, or a metal, such as aluminium . These advanced composites have two major advantages among many others: (i) improved strength-to...8010 85 2.095 21.823 85.0 C2-SH-H-11 0.09632 8011 85 2.102 21.823 85.0 C3-SH-H-13 0.09744 8013 95% 2.377 24.394 94.3 C3-SH-H-14 0.09728 8014 95 2.373

Hypomorphic Recessive Variants in SUFU Impair the Sonic Hedgehog Pathway and Cause Joubert Syndrome with Cranio-facial and Skeletal Defects.

PubMed

De Mori, Roberta; Romani, Marta; D'Arrigo, Stefano; Zaki, Maha S; Lorefice, Elisa; Tardivo, Silvia; Biagini, Tommaso; Stanley, Valentina; Musaev, Damir; Fluss, Joel; Micalizzi, Alessia; Nuovo, Sara; Illi, Barbara; Chiapparini, Luisa; Di Marcotullio, Lucia; Issa, Mahmoud Y; Anello, Danila; Casella, Antonella; Ginevrino, Monia; Leggins, Autumn Sa'na; Roosing, Susanne; Alfonsi, Romina; Rosati, Jessica; Schot, Rachel; Mancini, Grazia Maria Simonetta; Bertini, Enrico; Dobyns, William B; Mazza, Tommaso; Gleeson, Joseph G; Valente, Enza Maria

2017-10-05

The Sonic Hedgehog (SHH) pathway is a key signaling pathway orchestrating embryonic development, mainly of the CNS and limbs. In vertebrates, SHH signaling is mediated by the primary cilium, and genetic defects affecting either SHH pathway members or ciliary proteins cause a spectrum of developmental disorders. SUFU is the main negative regulator of the SHH pathway and is essential during development. Indeed, Sufu knock-out is lethal in mice, and recessive pathogenic variants of this gene have never been reported in humans. Through whole-exome sequencing in subjects with Joubert syndrome, we identified four children from two unrelated families carrying homozygous missense variants in SUFU. The children presented congenital ataxia and cerebellar vermis hypoplasia with elongated superior cerebellar peduncles (mild "molar tooth sign"), typical cranio-facial dysmorphisms (hypertelorism, depressed nasal bridge, frontal bossing), and postaxial polydactyly. Two siblings also showed polymicrogyria. Molecular dynamics simulation predicted random movements of the mutated residues, with loss of the native enveloping movement of the binding site around its ligand GLI3. Functional studies on cellular models and fibroblasts showed that both variants significantly reduced SUFU stability and its capacity to bind GLI3 and promote its cleavage into the repressor form GLI3R. In turn, this impaired SUFU-mediated repression of the SHH pathway, as shown by altered expression levels of several target genes. We demonstrate that germline hypomorphic variants of SUFU cause deregulation of SHH signaling, resulting in recessive developmental defects of the CNS and limbs which share features with both SHH-related disorders and ciliopathies. Copyright © 2017 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

Downregulation of the Sonic Hedgehog/Gli pathway transcriptional target Neogenin-1 is associated with basal cell carcinoma aggressiveness.

PubMed

Casas, Bárbara S; Adolphe, Christelle; Lois, Pablo; Navarrete, Nelson; Solís, Natalia; Bustamante, Eva; Gac, Patricio; Cabané, Patricio; Gallegos, Ivan; Wainwright, Brandon J; Palma, Verónica

2017-10-13

Basal Cell Carcinoma (BCC) is one of the most diagnosed cancers worldwide. It develops due to an unrestrained Sonic Hedgehog (SHH) signaling activity in basal cells of the skin. Certain subtypes of BCC are more aggressive than others, although the molecular basis of this phenomenon remains unknown. We have previously reported that Neogenin-1 (NEO1) is a downstream target gene of the SHH/GLI pathway in neural tissue. Given that SHH participates in epidermal homeostasis, here we analyzed the epidermal expression of NEO1 in order to identify whether it plays a role in adult epidermis or BCC. We describe the mRNA and protein expression profile of NEO1 and its ligands (Netrin-1 and RGMA) in human and mouse control epidermis and in a broad range of human BCCs. We identify in human BCC a significant positive correlation in the levels of NEO1 receptor, NTN-1 and RGMA ligands with respect to GLI1 , the main target gene of the canonical SHH pathway. Moreover, we show via cyclopamine inhibition of the SHH/GLI pathway of ex vivo cultures that NEO1 likely functions as a downstream target of SHH/GLI signaling in the skin. We also show how Neo1 expression decreases throughout BCC progression in the K14-Cre:Ptch1 lox/lox mouse model and that aggressive subtypes of human BCC exhibit lower levels of NEO1 than non-aggressive BCC samples. Taken together, these data suggest that NEO1 is a SHH/GLI target in epidermis. We propose that NEO1 may be important in tumor onset and is then down-regulated in advanced BCC or aggressive subtypes.

Downregulation of the Sonic Hedgehog/Gli pathway transcriptional target Neogenin-1 is associated with basal cell carcinoma aggressiveness

PubMed Central

Casas, Bárbara S.; Adolphe, Christelle; Lois, Pablo; Navarrete, Nelson; Solís, Natalia; Bustamante, Eva; Gac, Patricio; Cabané, Patricio; Gallegos, Ivan; Wainwright, Brandon J.; Palma, Verónica

2017-01-01

Basal Cell Carcinoma (BCC) is one of the most diagnosed cancers worldwide. It develops due to an unrestrained Sonic Hedgehog (SHH) signaling activity in basal cells of the skin. Certain subtypes of BCC are more aggressive than others, although the molecular basis of this phenomenon remains unknown. We have previously reported that Neogenin-1 (NEO1) is a downstream target gene of the SHH/GLI pathway in neural tissue. Given that SHH participates in epidermal homeostasis, here we analyzed the epidermal expression of NEO1 in order to identify whether it plays a role in adult epidermis or BCC. We describe the mRNA and protein expression profile of NEO1 and its ligands (Netrin-1 and RGMA) in human and mouse control epidermis and in a broad range of human BCCs. We identify in human BCC a significant positive correlation in the levels of NEO1 receptor, NTN-1 and RGMA ligands with respect to GLI1, the main target gene of the canonical SHH pathway. Moreover, we show via cyclopamine inhibition of the SHH/GLI pathway of ex vivo cultures that NEO1 likely functions as a downstream target of SHH/GLI signaling in the skin. We also show how Neo1 expression decreases throughout BCC progression in the K14-Cre:Ptch1lox/lox mouse model and that aggressive subtypes of human BCC exhibit lower levels of NEO1 than non-aggressive BCC samples. Taken together, these data suggest that NEO1 is a SHH/GLI target in epidermis. We propose that NEO1 may be important in tumor onset and is then down-regulated in advanced BCC or aggressive subtypes. PMID:29137400

CD271+ cells are diagnostic and prognostic and exhibit elevated MAPK activity in SHH medulloblastoma.

PubMed

Liang, Lisa; Morrison, Ludivine Coudière; Tatari, Nazanin; Stromecki, Margaret; Fresnoza, Agnes; Porter, Christopher J; Del Bigio, Marc R; Hawkins, Cynthia; Chan, Jennifer A; Ryken, Timothy C; Taylor, Michael D; Ramaswamy, Vijay; Werbowetski-Ogilvie, Tamra E

2018-06-21

The extensive heterogeneity both between and within the medulloblastoma (MB) subgroups underscores a critical need for variant-specific biomarkers and therapeutic strategies. We previously identified a role for the CD271/p75 neurotrophin receptor (p75NTR) in regulating stem/progenitor cells in the SHH MB subgroup. Here we demonstrate the utility of CD271 as a novel diagnostic and prognostic marker for SHH MB using immunohistochemical analysis and transcriptome data across 763 primary tumors. RNA sequencing of CD271+ and CD271- cells revealed molecularly distinct, co-existing cellular subsets both in vitro and in vivo. MAPK/ERK signaling was upregulated in the CD271+ population, and inhibiting this pathway reduced endogenous CD271 levels, stem/progenitor cell proliferation, and cell survival as well as cell migration in vitro. Treatment with the MEK inhibitor selumetinib extended survival and reduced CD271 levels in vivo; whereas, treatment with vismodegib, a well-known smoothened (SMO) inhibitor currently in clinical trials for the treatment of recurrent SHH medulloblastoma, had no significant effect in our models. Our study demonstrates the clinical utility of CD271 as both a diagnostic and prognostic tool for SHH MB tumors and reveals a novel role for MEK inhibitors in targeting CD271+ SHH MB cells. Copyright ©2018, American Association for Cancer Research.

Molecular mechanisms of Sonic hedgehog mutant effects in holoprosencephaly.

PubMed

Maity, Tapan; Fuse, Naoyuki; Beachy, Philip A

2005-11-22

Holoprosencephaly (HPE), a human developmental brain defect, usually is also associated with varying degrees of midline facial dysmorphism. Heterozygous mutations in the Sonic hedgehog (SHH) gene are the most common genetic lesions associated with HPE, and loss of Shh function in the mouse produces cyclopia and alobar forebrain development. The N-terminal domain (ShhNp) of Sonic hedgehog protein, generated by cholesterol-dependent autoprocessing and modification at the C terminus and by palmitate addition at the N terminus, is the active ligand in the Shh signal transduction pathway. Here, we analyze seven reported missense mutations (G31R, D88V, Q100H, N115K, W117G, W117R, and E188Q) that alter the N-terminal signaling domain of Shh protein, and show that two of these mutations (Q100H and E188Q), which are questionably linked to HPE, produce no detectable effects on function. The remaining five alterations affect normal processing, Ptc binding, and signaling to varying degrees. These effects include introduction of a recognition site for furin-like proteases by the G31R alteration, resulting in cleavage of 11 amino acid residues from the N terminus of ShhNp and consequent reduced signaling potency. Two other alterations, W117G and W117R, cause temperature-dependent misfolding and retention in the sterol-poor endoplasmic reticulum, thus disrupting cholesterol-dependent autoprocessing.

Aspirin inhibits the SHH/GLI1 signaling pathway and sensitizes malignant glioma cells to temozolomide therapy

PubMed Central

Li, Ziwei; Lin, Lin; Meng, Xiangqi; Han, Bo; Wang, Ruijia; Wu, Pengfei; Li, Jianlong; Cai, Jinquan; Jiang, Chuanlu

2017-01-01

Aberrant activation of sonic hedgehog (SHH)/glioma-associated oncogene homolog 1 (GLI1) pathway plays an important role in the tumorigenicity of malignant glioma cells and resistance to temozolomide (TMZ). Here we investigated the aspirin's antineoplastic molecular route by targeting SHH/GLI1 pathway and examined the feasibility of aspirin combined with TMZ therapy. Western blot and quantitative real-time polymerase chain reaction (qRT-PCR) revealed that the activity of the SHH/GLI1 pathway was strongly inhibited by aspirin. Aspirin acted as the glioma growth-inhibitory and pro-apoptosis roles by inhibiting the SHH/GLI1 pathway and reprogramming the epithelial to mesenchymal transition (EMT). The immunofluorescence assay showed aspirin could prevent the nuclear translocation of GLI1 to inhibit its transcriptional regulation. The stable lentiviral overexpression of GLI1 reversed the DNA double strand breaks (DSBs) caused by the GANT61 and TMZ. Furthermore, aspirin combined with TMZ enhanced chemosensitivity and GLI1-induced chemoprotection was partly blocked by aspirin in vitro and in vivo. Collectively, aspirin has a therapeutic potential for SHH/GLI1 targeted therapy against glioma cells. Acquired activation of GLI1 protects glioma cells against TMZ therapy. Impairment of DNA DSBs repair activity might be involved in the route of aspirin-induced chemosensitivity. Combined aspirin with TMZ may be a promising strategy against malignant glioma. PMID:28446712

Aspirin inhibits the SHH/GLI1 signaling pathway and sensitizes malignant glioma cells to temozolomide therapy.

PubMed

Ming, Jianguang; Sun, Bo; Li, Ziwei; Lin, Lin; Meng, Xiangqi; Han, Bo; Wang, Ruijia; Wu, Pengfei; Li, Jianlong; Cai, Jinquan; Jiang, Chuanlu

2017-04-01

Aberrant activation of sonic hedgehog (SHH)/glioma-associated oncogene homolog 1 (GLI1) pathway plays an important role in the tumorigenicity of malignant glioma cells and resistance to temozolomide (TMZ). Here we investigated the aspirin's antineoplastic molecular route by targeting SHH/GLI1 pathway and examined the feasibility of aspirin combined with TMZ therapy. Western blot and quantitative real-time polymerase chain reaction (qRT-PCR) revealed that the activity of the SHH/GLI1 pathway was strongly inhibited by aspirin. Aspirin acted as the glioma growth-inhibitory and pro-apoptosis roles by inhibiting the SHH/GLI1 pathway and reprogramming the epithelial to mesenchymal transition (EMT). The immunofluorescence assay showed aspirin could prevent the nuclear translocation of GLI1 to inhibit its transcriptional regulation. The stable lentiviral overexpression of GLI1 reversed the DNA double strand breaks (DSBs) caused by the GANT61 and TMZ. Furthermore, aspirin combined with TMZ enhanced chemosensitivity and GLI1-induced chemoprotection was partly blocked by aspirin in vitro and in vivo . Collectively, aspirin has a therapeutic potential for SHH/GLI1 targeted therapy against glioma cells. Acquired activation of GLI1 protects glioma cells against TMZ therapy. Impairment of DNA DSBs repair activity might be involved in the route of aspirin-induced chemosensitivity. Combined aspirin with TMZ may be a promising strategy against malignant glioma.

Reflection does not undermine self-interested prosociality

PubMed Central

Rand, David G.; Kraft-Todd, Gordon T.

2014-01-01

The cognitive basis of prosocial behavior has received considerable recent attention. Previous work using economic games has found that in social dilemmas, intuitive decisions are more prosocial on average. The Social Heuristics Hypothesis (SHH) explains this result by contending that strategies which are successful in daily life become automatized as intuitions. Deliberation then causes participants to adjust to the self-interested strategy in the specific setting at hand. Here we provide further evidence for the SHH by confirming several predictions regarding when and for whom time pressure/delay will and will not alter contributions in a Public Goods Game (PGG). First, we replicate and extend previous results showing that (as predicted by the SHH) trust of daily-life interaction partners and previous experience with economic games moderate the effect of time pressure/delay in social dilemmas. We then confirm a novel prediction of the SHH: that deliberation should not undermine the decision to benefit others when doing so is also individually payoff-maximizing. Our results lend further support to the SHH, and shed light on the role that deliberation plays in social dilemmas. PMID:25232309

Reflection does not undermine self-interested prosociality.

PubMed

Rand, David G; Kraft-Todd, Gordon T

2014-01-01

The cognitive basis of prosocial behavior has received considerable recent attention. Previous work using economic games has found that in social dilemmas, intuitive decisions are more prosocial on average. The Social Heuristics Hypothesis (SHH) explains this result by contending that strategies which are successful in daily life become automatized as intuitions. Deliberation then causes participants to adjust to the self-interested strategy in the specific setting at hand. Here we provide further evidence for the SHH by confirming several predictions regarding when and for whom time pressure/delay will and will not alter contributions in a Public Goods Game (PGG). First, we replicate and extend previous results showing that (as predicted by the SHH) trust of daily-life interaction partners and previous experience with economic games moderate the effect of time pressure/delay in social dilemmas. We then confirm a novel prediction of the SHH: that deliberation should not undermine the decision to benefit others when doing so is also individually payoff-maximizing. Our results lend further support to the SHH, and shed light on the role that deliberation plays in social dilemmas.

Hedgehog Signaling in the Stomach

PubMed Central

Konstantinou, Daniel; Bertaux-Skeirik, Nina; Zavros, Yana

2016-01-01

The Hedgehog (Hh) signaling pathway not only plays a key part in controlling embryonic development, but in the adult stomach governs important cellular events such as epithelial cell differentiation, proliferation, gastric disease and regeneration. In particular, Sonic Hedgehog (Shh) signaling has been well studied for its role in gastric physiology and pathophysiology. Shh is secreted from the gastric parietal cells and contributes to the regeneration of the epithelium in response to injury, or the development of gastritis during Helicobacter pylori infection. Dysregulation of the Shh signaling pathway leads to the disruption of gastric differentiation, loss of gastric acid secretion and the development of cancer. In this chapter, we will review the most recent findings that reveal the role of Shh as a regulator of gastric physiology, regeneration and disease. PMID:27750091

Hedgehog signaling in the stomach.

PubMed

Konstantinou, Daniel; Bertaux-Skeirik, Nina; Zavros, Yana

2016-12-01

The Hedgehog (Hh) signaling pathway not only plays a key part in controlling embryonic development, but in the adult stomach governs important cellular events such as epithelial cell differentiation, proliferation, gastric disease, and regeneration. In particular, Sonic Hedgehog (Shh) signaling has been well studied for its role in gastric physiology and pathophysiology. Shh is secreted from the gastric parietal cells and contributes to the regeneration of the epithelium in response to injury, or the development of gastritis during Helicobacter pylori infection. Dysregulation of the Shh signaling pathway leads to the disruption of gastric differentiation, loss of gastric acid secretion and the development of cancer. In this chapter, we will review the most recent findings that reveal the role of Shh as a regulator of gastric physiology, regeneration, and disease. Copyright © 2016 Elsevier Ltd. All rights reserved.

Endogenous stem cell proliferation induced by intravenous hedgehog agonist administration after contusion in the adult rat spinal cord.

PubMed

Bambakidis, Nicholas C; Horn, Eric M; Nakaji, Peter; Theodore, Nicholas; Bless, Elizabeth; Dellovade, Tammy; Ma, Chiyuan; Wang, Xukui; Preul, Mark C; Coons, Stephen W; Spetzler, Robert F; Sonntag, Volker K H

2009-02-01

Sonic hedgehog (Shh) is a glycoprotein molecule that upregulates the transcription factor Gli1. The Shh protein plays a critical role in the proliferation of endogenous neural precursor cells when directly injected into the spinal cord after a spinal cord injury in adult rodents. Small-molecule agonists of the hedgehog (Hh) pathway were used in an attempt to reproduce these findings through intravenous administration. The expression of Gli1 was measured in rat spinal cord after the intravenous administration of an Hh agonist. Ten adult rats received a moderate contusion and were treated with either an Hh agonist (10 mg/kg, intravenously) or vehicle (5 rodents per group) 1 hour and 4 days after injury. The rats were killed 5 days postinjury. Tissue samples were immediately placed in fixative. Samples were immunohistochemically stained for neural precursor cells, and these cells were counted. Systemic dosing with an Hh agonist significantly upregulated Gli1 expression in the spinal cord (p < 0.005). After spinal contusion, animals treated with the Hh agonist had significantly more nestin-positive neural precursor cells around the rim of the lesion cavity than in vehicle-treated controls (means +/- SDs, 46.9 +/- 12.9 vs 20.9 +/- 8.3 cells/hpf, respectively, p < 0.005). There was no significant difference in the area of white matter injury between the groups. An intravenous Hh agonist at doses that upregulate spinal cord Gli1 transcription also increases the population of neural precursor cells after spinal cord injury in adult rats. These data support previous findings based on injections of Shh protein directly into the spinal cord.

Medulloblastoma in China: Clinicopathologic Analyses of SHH, WNT, and Non-SHH/WNT Molecular Subgroups Reveal Different Therapeutic Responses to Adjuvant Chemotherapy

PubMed Central

Ren, Yong; Yao, Yu; Li, Kay Ka-Wai; Ng, Ho-Keung; Mao, Ying; Zhou, Liang-Fu; Zhong, Ping

2014-01-01

Medulloblastoma (MB) is one of the most common primary central nervous system tumors in children. Data is lacking of a large cohort of medulloblastoma patients in China. Also, our knowledge on the sensitivity of different molecular subgroups of MB to adjuvant radiation therapy (RT) or chemotherapy (CHT) is still limited. The authors performed a retrospective study of 173 medulloblastoma patients treated at two institutions from 2002 to 2011. Formalin-fixed paraffin embedded (FFPE) tissues were available in all the cases and sections were stained to classify histological and molecular subgroups. Univariate and multivariate analyses were used to investigate prognostic factors. Of 173 patients, there were 118 children and 55 adults, 112 males and 61 females. Estimated 5-year overall survival (OS) rates for all patients, children and adults were 52%, 48% and 63%, respectively. After multivariate analysis, postoperative primary radiation therapy (RT) and chemotherapy (CHT) were revealed as favorable prognostic factors influencing OS and EFS. Postoperative primary chemotherapy (CHT) was found significantly improving the survival of children (p<0.001) while it was not a significant prognostic factor for adult patients. Moreover, patients in WNT subtype had better OS (p = 0.028) than others (SHH and Non-SHH/WNT subtypes) given postoperative adjuvant therapies. Postoperative primary RT was found to be a strong prognostic factor influencing the survival in all histological and molecular subgroups (p<0.001). Postoperative primary CHT was found significantly to influence the survival of classic medulloblastoma (CMB) (OS p<0.001, EFS p<0.001), SHH subgroup (OS p = 0.020, EFS p = 0.049) and WNT subgroup (OS p = 0.003, EFS p = 0.016) but not in desmoplastic/nodular medulloblastoma (DMB) (OS p = 0.361, EFS p = 0.834) and Non-SHH/WNT subgroup (OS p = 0.127, EFS p = 0.055). Our study showed postoperative primary CHT significantly influence the survival of CMB, SHH subgroup and WNT subgroup but not in DMB and Non-SHH/WNT subgroup of MB. PMID:24932704

Sonic hedgehog (Shh)/Gli modulates the spatial organization of neuroepithelial cell proliferation in the developing chick optic tectum.

PubMed

Rapacioli, Melina; Botelho, Joao; Cerda, Gustavo; Duarte, Santiago; Elliot, Matías; Palma, Verónica; Flores, Vladimir

2012-10-02

Sonic hedgehog (Shh)/Gli pathway plays an important regulatory role on the neuroepithelial cells (NEc) proliferation in the dorsal regions of the developing vertebrate Central Nervous System. The aim of this paper was to analyze the effect of the Shh/Gli signaling pathway activation on the proliferation dynamics and/or the spatial organization of the NEc proliferation activity during early stages of the developing chick optic tectum (OT). In ovo pharmacological gain and loss of hedgehog function approaches were complemented with in vivo electroporation experiments in order to create ectopic sources of either Shh or Gli activator (GliA) proteins in the OT. NEc proliferating activity was analyzed at ED 4/4.5 by recording the spatial co-ordinates of the entire population of mitotic NEc (mNEc) located along OT dorsal-ventral sections. Several space signals (numerical sequences) were derived from the mNEc spatial co-ordinate records and analyzed by different standardized non-linear methods of signal analysis. In ovo pharmacologic treatment with cyclopamine resulted in dramatic failure in the OT expansion while the agonist purmorphamine produced the opposite result, a huge expansion of the OT vesicle. Besides, GliA and Shh misexpressions interfere with the formation of the intertectal fissure located along the dorsal midline. This morphogenetic alteration is accompanied by an increase in the mNEc density. There is a gradient in the response of NEcs to Shh and GliA: the increase in mNEc density is maximal near the dorsal regions and decrease towards the OT-tegmental boundary. Biomathematical analyses of the signals derived from the mNEc records show that both Shh and GliA electroporations change the proliferation dynamics and the spatial organization of the mNEc as revealed by the changes in the scaling index estimated by these methods. The present results show that the Shh/Gli signaling pathway plays a critical role in the OT expansion and modelling. This effect is probably mediated by a differential mitogenic effect that increases the NEc proliferation and modulates the spatial organization of the NEc proliferation activity.

Argentine references for the assessment of body proportions from birth to 17 years of age.

PubMed

Del Pino, Mariana; Orden, Alicia B; Arenas, María A; Fano, Virginia

2017-06-01

Abnormal body proportions may indicate skeletal disorders; therefore, their detection has great clinical significance. To estimate centiles for head circumference/height (HC/H) and sitting height/height (SH/H) ratios, and assess their diagnostic usefulness among a group of children with skeletal dysplasia. Centiles 3, 10, 25, 50, 75, 90 and 97 for HC/H and SH/H ratios were estimated with the LMS method using Box-Cox transformation to normalize data distribution for each age. Q-Q plot tests were applied to evaluate normality of residuals and the Q test to calculate goodness-of-fit. The sample included 4818 girls and4803 boys, all healthy, between 0-17 years old. The median of the SH/H ratio for each age decreased from 0.67 at birth to 0.57 at age 4. At 12 years of age, values reached 0.52 and 0.53 for males and females, respectively, remaining unchanged until age 17. The median of the HC/H ratio decreased from 0.45 at 6 years old to 0.34 at 17 years old for both sexes. Z-scores for SH/H among 20 children diagnosed with hypochondroplasia were better at showing abnormal proportions than the SH/H ratio not adjusted by age. Estimated centiles for HC/H and SH/H ratios show that the most dramatic changes in body proportions occur in the prepubertal period. These references allow an earlier detection of abnormal body proportions in children with skeletal dysplasia.

The anti-neoplastic activity of Vandetanib against high-risk medulloblastoma variants is profoundly enhanced by additional PI3K inhibition

PubMed Central

Velz, Julia; Olschewski, Martin; Goetz, Barbara; Pietsch, Torsten; Dilloo, Dagmar

2017-01-01

Medulloblastoma is comprised of at least four molecular subgroups with distinct clinical outcome (WHO classification 2016). SHH-TP53-mutated as well as MYC-amplified Non-WNT/Non-SHH medulloblastoma show the worst prognosis. Here we present evidence that single application of the multi-kinase inhibitor Vandetanib displays anti-neoplastic efficacy against cell lines derived from high-risk SHH-TP53-mutated and MYC-amplified Non-WNT/Non-SHH medulloblastoma. The narrow target spectrum of Vandetanib along with a favourable toxicity profile renders this drug ideal for multimodal treatment approaches. In this context our investigation documents that Vandetanib in combination with the clinically available PI3K inhibitor GDC-0941 leads to enhanced cytotoxicity against MYC-amplified and SHH-TP53-mutated medulloblastoma. In line with these findings we show for MYC-amplified medulloblastoma a profound reduction in activity of the oncogenes STAT3 and AKT. Furthermore, we document that Vandetanib and the standard chemotherapeutic Etoposide display additive anti-neoplastic efficacy in the investigated medulloblastoma cell lines that could be further enhanced by PI3K inhibition. Of note, the combination of Vandetanib, GDC-0941 and Etoposide results in MYC-amplified and SHH-TP53-mutated cell lines in complete loss of cell viability. Our findings therefore provide a rational to further evaluate Vandetanib in combination with PI3K inhibitors as well as standard chemotherapeutics in vivo for the treatment of most aggressive medulloblastoma variants. PMID:28159923

The anti-neoplastic activity of Vandetanib against high-risk medulloblastoma variants is profoundly enhanced by additional PI3K inhibition.

PubMed

Craveiro, Rogerio B; Ehrhardt, Michael; Velz, Julia; Olschewski, Martin; Goetz, Barbara; Pietsch, Torsten; Dilloo, Dagmar

2017-07-18

Medulloblastoma is comprised of at least four molecular subgroups with distinct clinical outcome (WHO classification 2016). SHH-TP53-mutated as well as MYC-amplified Non-WNT/Non-SHH medulloblastoma show the worst prognosis.Here we present evidence that single application of the multi-kinase inhibitor Vandetanib displays anti-neoplastic efficacy against cell lines derived from high-risk SHH-TP53-mutated and MYC-amplified Non-WNT/Non-SHH medulloblastoma. The narrow target spectrum of Vandetanib along with a favourable toxicity profile renders this drug ideal for multimodal treatment approaches. In this context our investigation documents that Vandetanib in combination with the clinically available PI3K inhibitor GDC-0941 leads to enhanced cytotoxicity against MYC-amplified and SHH-TP53-mutated medulloblastoma. In line with these findings we show for MYC-amplified medulloblastoma a profound reduction in activity of the oncogenes STAT3 and AKT. Furthermore, we document that Vandetanib and the standard chemotherapeutic Etoposide display additive anti-neoplastic efficacy in the investigated medulloblastoma cell lines that could be further enhanced by PI3K inhibition. Of note, the combination of Vandetanib, GDC-0941 and Etoposide results in MYC-amplified and SHH-TP53-mutated cell lines in complete loss of cell viability. Our findings therefore provide a rational to further evaluate Vandetanib in combination with PI3K inhibitors as well as standard chemotherapeutics in vivo for the treatment of most aggressive medulloblastoma variants.

Characterization of the human oncogene SCL/TAL1 interrupting locus (Stil) mediated Sonic hedgehog (Shh) signaling transduction in proliferating mammalian dopaminergic neurons

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

Sun, Lei; Department of Physiology, Nankai University School of Medicine, Tianjin 300071; Carr, Aprell L.

2014-07-11

Highlights: • Stil is a human oncogene that is conserved in vertebrate species. • Stil functions in the Shh pathway in mammalian cells. • The expression of Stil is required for mammalian dopaminergic cell proliferation. - Abstract: The human oncogene SCL/TAL1 interrupting locus (Stil) is highly conserved in all vertebrate species. In humans, the expression of Stil is involved in cancer cell survival, apoptosis and proliferation. In this research, we investigated the roles of Stil expression in cell proliferation of mammalian dopaminergic (DA) PC12 cells. Stil functions through the Sonic hedgehog (Shh) signal transduction pathway. Co-immunoprecipitation tests revealed that STILmore » interacts with Shh downstream components, which include SUFU and GLI1. By examining the expression of Stil, Gli1, CyclinD2 (cell-cycle marker) and PCNA (proliferating cell nuclear antigen), we found that up-regulation of Stil expression (transfection with overexpression plasmids) increased Shh signaling transduction and PC12 cell proliferation, whereas down-regulation of Stil expression (by shRNA) inhibited Shh signaling transduction, and thereby decreased PC12 cell proliferation. Transient transfection of PC12 cells with Stil knockdown or overexpression plasmids did not affect PC12 cell neural differentiation, further indicating the specific roles of Stil in cell proliferation. The results from this research suggest that Stil may serve as a bio-marker for neurological diseases involved in DA neurons, such as Parkinson’s disease.« less

Differential requirements for Gli2 and Gli3 in the regional specification of the mouse hypothalamus

PubMed Central

Haddad-Tóvolli, Roberta; Paul, Fabian A.; Zhang, Yuanfeng; Zhou, Xunlei; Theil, Thomas; Puelles, Luis; Blaess, Sandra; Alvarez-Bolado, Gonzalo

2015-01-01

Secreted protein Sonic hedgehog (Shh) ventralizes the neural tube by modulating the crucial balance between activating and repressing functions (GliA, GliR) of transcription factors Gli2 and Gli3. This balance—the Shh-Gli code—is species- and context-dependent and has been elucidated for the mouse spinal cord. The hypothalamus, a forebrain region regulating vital functions like homeostasis and hormone secretion, shows dynamic and intricate Shh expression as well as complex regional differentiation. Here we asked if particular combinations of Gli2 and Gli3 and of GliA and GliR functions contribute to the variety of hypothalamic regions, i.e., we wanted to approach the question of a possible hypothalamic version of the Shh-Gli code. Based on mouse mutant analysis, we show that: (1) hypothalamic regional heterogeneity is based in part on differentially stringent requirements for Gli2 or Gli3; (2) another source of diversity are differential requirements for Shh of neural vs. non-neural origin; (3) the medial progenitor domain known to depend on Gli2 for its development generates several essential hypothalamic nuclei plus the pituitary and median eminence; (4) the suppression of Gli3R by neural and non-neural Shh is essential for hypothalamic specification. Finally, we have mapped our results on a recent model which considers the hypothalamus as a transverse region with alar and basal portions. Our data confirm the model and are explained by it. PMID:25859185

Combination of Cyclopamine and Tamoxifen Promotes Survival and Migration of MCF-7 Breast Cancer Cells – Interaction of Hedgehog-Gli and Estrogen Receptor Signaling Pathways

PubMed Central

Uzarevic, Zvonimir; Ozretic, Petar; Musani, Vesna; Rafaj, Maja; Cindric, Mario; Levanat, Sonja

2014-01-01

Hedgehog-Gli (Hh-Gli) signaling pathway is one of the new molecular targets found upregulated in breast tumors. Estrogen receptor alpha (ERα) signaling has a key role in the development of hormone-dependent breast cancer. We aimed to investigate the effects of inhibiting both pathways simultaneously on breast cancer cell survival and the potential interactions between these two signaling pathways. ER-positive MCF-7 cells show decreased viability after treatment with cyclopamine, a Hh-Gli pathway inhibitor, as well as after tamoxifen (an ERα inhibitor) treatment. Simultaneous treatment with cyclopamine and tamoxifen on the other hand, causes short-term survival of cells, and increased migration. We found upregulated Hh-Gli signaling under these conditions and protein profiling revealed increased expression of proteins involved in cell proliferation and migration. Therefore, even though Hh-Gli signaling seems to be a good potential target for breast cancer therapy, caution must be advised, especially when combining therapies. In addition, we also show a potential direct interaction between the Shh protein and ERα in MCF-7 cells. Our data suggest that the Shh protein is able to activate ERα independently of the canonical Hh-Gli signaling pathway. Therefore, this may present an additional boost for ER-positive cells that express Shh, even in the absence of estrogen. PMID:25503972

M1 macrophage recruitment correlates with worse outcome in SHH Medulloblastomas.

PubMed

Lee, Chanhee; Lee, Joongyub; Choi, Seung Ah; Kim, Seung-Ki; Wang, Kyu-Chang; Park, Sung-Hye; Kim, Se Hoon; Lee, Ji Yeoun; Phi, Ji Hoon

2018-05-08

Recent progress in molecular analysis has advanced the understanding of medulloblastoma (MB) and is anticipated to facilitate management of the disease. MB is composed of 4 molecular subgroups: WNT, SHH, Group 3, and Group 4. Macrophages play a crucial role in the tumor microenvironment; however, the functional role of their activated phenotype (M1/M2) remains controversial. Herein, we investigate the correlation between tumor-associated macrophage (TAM) recruitment within the MB subgroups and prognosis. Molecular subgrouping was performed by a nanoString-based RNA assay on retrieved snap-frozen tissue samples. Immunohistochemistry (IHC) and immunofluorescence (IF) assays were performed on subgroup identified samples, and the number of polarized macrophages was quantified from IHC. Survival analyses were conducted on collected clinical data and quantified macrophage data. TAM (M1/M2) recruitment in SHH MB was significantly higher compared to that in other subgroups. A Kaplan-Meier survival curve and multivariate Cox regression demonstrated that high M1 expressers showed worse overall survival (OS) and progression-free survival (PFS) than low M1 expressers in SHH MB, with relative risk (RR) values of 11.918 and 6.022, respectively. M1 rather than M2 correlates more strongly with worse outcome in SHH medulloblastoma.

Targeting of Cancer Stem Cells and Their Microenvironment in Early-Stage MutantK-ras Lung Cancer

DTIC Science & Technology

2015-10-01

a combination of SHH/ALDH/ NOTCH3 as potential stem cell markers. We will also test the hypothesis that SHH+ cell population may be a quiescent stem...and SHH are expressed within the same cell population. We will also test NOTCH3 in combination with 5E1-647 alone or in combination with 5E1-647 and...Aldefluor assays. Anti- NOTCH3 antibody will be labeled with Alexa Fluor 594 (red color spectrum) in an analogous manner to 5E1-594. As ALDH+ and

Association of expression of the hedgehog signal with Merkel cell polyomavirus infection and prognosis of Merkel cell carcinoma.

PubMed

Kuromi, Teruyuki; Matsushita, Michiko; Iwasaki, Takeshi; Nonaka, Daisuke; Kuwamoto, Satoshi; Nagata, Keiko; Kato, Masako; Akizuki, Gen; Kitamura, Yukisato; Hayashi, Kazuhiko

2017-11-01

Merkel cell carcinoma (MCC) is an aggressive neuroendocrine skin cancer that mostly occurs in the elderly. Merkel cell polyomavirus (MCPyV) is detected in approximately 80% of MCCs and is associated with carcinogenesis. Hedgehog signaling pathway plays a role in human embryogenesis and organogenesis. In addition, reactivation of this pathway later in life can cause tumors. Twenty-nineMCPyV-positive and 21 MCPyV-negative MCCs were immunohistochemically stained with primary antibodies for hedgehog signaling (SHH, IHH, PTCH1, SMO, GLI1, GLI2, and GLI3) and evaluated using H-score. Polymerase chain reaction and sequence analysis for SHH and GLI1 exons were also performed. Expression of SHH was higher in MCPyV-positive MCCs than in MCPyV-negative MCCs (P<.001). Higher expression of GLI1, MCPyV infection, male sex, and Japanese ethnicity were associated with better overall survival (P=.034, P=.001, P=.042, and P=.036, respectively). Higher expression of SHH and MCPyV infection were associated with improved MCC-specific survival (P=.037 and P=.002, respectively). The mutation analysis of prognosis-related GLI1 and SHH genes in our study revealed a low frequency of mutations in the 10 exons examined, except GLI1 exon 5 (18/22 cases), all having the same silent mutation of c.576G>A. Only 2 mutations with amino acid changes were detected in MCPyV-negative MCCs only: 1 missense mutation in GLI1 exon 4 and 1 nonsense mutation in SHH-3B. Expression of SHH and GLI1 may be useful prognostic markers of MCC because increased expression was associated with better prognosis. The high rate of c.576G>A silent mutation in GLI1 exon 5 was a feature of MCC. Copyright © 2017 Elsevier Inc. All rights reserved.

The SHH/Gli axis regulates CD90-mediated liver cancer stem cell function by activating the IL6/JAK2 pathway.

PubMed

Zhang, Ketao; Che, Siyao; Pan, Chuzhi; Su, Zheng; Zheng, Shangyou; Yang, Shanglin; Zhang, Huayao; Li, Wenda; Wang, Weidong; Liu, Jianping

2018-05-02

The cell surface antigen CD90 has recently been established as a promising marker for liver cancer stem cells. This study aimed to investigate potential implications of SHH/Gli signalling in CD90+ liver cancer stem cells. Correlation of the expression of SHH signalling components and CD90 in liver cancer cells and clinical tissues, as well as in enriched CD90+ liver cancer stem cells and the TCGA database, were analysed by quantitative RT-PCR, Western blotting and flow cytometry. Functional analysis was conducted by siRNA-mediated CD90, Gli1 and Gli3 gene knockdown, SHH treatment and application of the JAK2 inhibitor AZD1480 and IL6 neutralizing antibody in CD90+ liver cancer stem cells, followed by cell proliferation, migration, sphere formation and tumorigenicity assays. CD90 expression exhibited a high positive correlation with Gli1 and Gli3 in multiple liver cancer cell lines and human cancerous liver tissues, both of which showed a significant increase in liver cancer. Analysis of TCGA data revealed an association of CD90, Gli1 and Gli3 with a short overall survival and positive correlation between CD90 expression and Gli3 expression level. The stem cell potentials of CD90+ 97L liver cancer cells were greatly impaired by Gli1/3 knockdown with siRNA but enhanced by SHH treatment. Application of the JAK2 inhibitor AZD1480 and IL6 neutralizing antibody showed the CD90 and SHH/Gli-regulated liver cancer stem cell functions were mediated by the IL6/JAK2/STAT3 pathway. The stem cell properties of CD90+ liver cancer cells are regulated by the downstream SHH/Gli and IL6/JAK2/STAT3 signalling pathways. © 2018 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

A novel role for FOXA2 and SHH in organizing midbrain signaling centers.

PubMed

Bayly, Roy D; Brown, Charmaine Y; Agarwala, Seema

2012-09-01

The floor plate (FP) is a midline signaling center, known to direct ventral cell fates and axon guidance in the neural tube. The recent identification of midbrain FP as a source of dopaminergic neurons has renewed interest in its specification and organization, which remain poorly understood. In this study, we have examined the chick midbrain and spinal FP and show that both can be partitioned into medial (MFP) and lateral (LFP) subdivisions. Although Hedgehog (HH) signaling is necessary and sufficient for LFP specification, it is not sufficient for MFP induction. By contrast, the transcription factor FOXA2 can execute the full midbrain and spinal cord FP program via HH-independent and dependent mechanisms. Interestingly, although HH-independent FOXA2 activity is necessary and sufficient for inducing MFP-specific gene expression (e.g., LMX1B, BMP7), it cannot confer ventral identity to midline cells without also turning on Sonic hedgehog (SHH). We also note that the signaling centers of the midbrain, the FP, roof plate (RP) and the midbrain-hindbrain boundary (MHB) are physically contiguous, with each expressing LMX1B and BMP7. Possibly as a result, SHH or FOXA2 misexpression can transform the MHB into FP and also suppress RP induction. Conversely, HH or FOXA2 knockdown expands the endogenous RP and transforms the MFP into a RP and/or MHB fate. Finally, combined HH blockade and FOXA2 misexpression in ventral midbrain induces LMX1B expression, which triggers the specification of the RP, rather than the MFP. Thus we identify HH-independent and dependent roles for FOXA2 in specifying the FP. In addition, we elucidate for the first time, a novel role for SHH in determining whether a midbrain signaling center will become the FP, MHB or RP. Copyright © 2012 Elsevier Inc. All rights reserved.

Molecular differences between mature and immature dental pulp cells: Bioinformatics and preliminary results.

PubMed

Chen, Long; Jiang, Yifeng; Du, Zhen

2018-04-01

Although previous studies have demonstrated that dental pulp stem cells (DPSCs) from mature and immature teeth exhibit potential for multi-directional differentiation, the molecular and biological difference between the DPSCs from mature and immature permanent teeth has not been fully investigated. In the present study, 500 differentially expressed genes from dental pulp cells (DPCs) in mature and immature permanent teeth were obtained from the Gene Expression Omnibus online database. Based on bioinformatics analysis using the Database for Annotation, Visualization and Integrated Discovery, these genes were divided into a number of subgroups associated with immunity, inflammation and cell signaling. The results of the present study suggest that immune features, response to infection and cell signaling may be different in DPCs from mature and immature permanent teeth; furthermore, DPCs from immature permanent teeth may be more suitable for use in tissue engineering or stem cell therapy. The Online Mendelian Inheritance in Man database stated that Sonic Hedgehog (SHH), a differentially expressed gene in DPCs from mature and immature permanent teeth, serves a crucial role in the development of craniofacial tissues, including teeth, which further confirmed that SHH may cause DPCs from mature and immature permanent teeth to exhibit different biological characteristics. The Search Tool for the Retrieval of Interacting Genes/Proteins database revealed that SHH has functional protein associations with a number of other proteins, including Glioma-associated oncogene (GLI)1, GLI2, growth arrest-specific protein 1, bone morphogenetic protein (BMP)2 and BMP4, in mice and humans. It was also demonstrated that SHH may interact with other genes to regulate the biological characteristics of DPCs. The results of the present study may provide a useful reference basis for selecting suitable DPSCs and molecules for the treatment of these cells to optimize features for tissue engineering or stem cell therapy. Quantitative polymerase chain reaction should be performed to confirm the differential expression of these genes prior to the beginning of a functional study.

Sox2 is required for embryonic development of the ventral telencephalon through the activation of the ventral determinants Nkx2.1 and Shh.

PubMed

Ferri, Anna; Favaro, Rebecca; Beccari, Leonardo; Bertolini, Jessica; Mercurio, Sara; Nieto-Lopez, Francisco; Verzeroli, Cristina; La Regina, Federico; De Pietri Tonelli, Davide; Ottolenghi, Sergio; Bovolenta, Paola; Nicolis, Silvia K

2013-03-01

The Sox2 transcription factor is active in stem/progenitor cells throughout the developing vertebrate central nervous system. However, its conditional deletion at E12.5 in mouse causes few brain developmental problems, with the exception of the postnatal loss of the hippocampal radial glia stem cells and the dentate gyrus. We deleted Sox2 at E9.5 in the telencephalon, using a Bf1-Cre transgene. We observed embryonic brain defects that were particularly severe in the ventral, as opposed to the dorsal, telencephalon. Important tissue loss, including the medial ganglionic eminence (MGE), was detected at E12.5, causing the subsequent impairment of MGE-derived neurons. The defect was preceded by loss of expression of the essential ventral determinants Nkx2.1 and Shh, and accompanied by ventral spread of dorsal markers. This phenotype is reminiscent of that of mice mutant for the transcription factor Nkx2.1 or for the Shh receptor Smo. Nkx2.1 is known to mediate the initial activation of ventral telencephalic Shh expression. A partial rescue of the normal phenotype at E14.5 was obtained by administration of a Shh agonist. Experiments in Medaka fish indicate that expression of Nkx2.1 is regulated by Sox2 in this species also. We propose that Sox2 contributes to Nkx2.1 expression in early mouse development, thus participating in the region-specific activation of Shh, thereby mediating ventral telencephalic patterning induction.

Lycium barbarum Polysaccharides Protect against Trimethyltin Chloride-Induced Apoptosis via Sonic Hedgehog and PI3K/Akt Signaling Pathways in Mouse Neuro-2a Cells.

PubMed

Zhao, Wanyun; Pan, Xiaoqi; Li, Tao; Zhang, Changchun; Shi, Nian

2016-01-01

Trimethyltin chloride (TMT) is a classic neurotoxicant that can cause severe neurodegenerative diseases. Some signaling pathways involving cell death play pivotal roles in the central nervous system. In this study, the role of Sonic Hedgehog (Shh) and PI3K/Akt pathways in TMT-induced apoptosis and protective effect of Lycium barbarum polysaccharides (LBP) on mouse neuro-2a (N2a) cells were investigated. Results showed that TMT treatment significantly enhanced apoptosis, upregulated proapoptotic Bax, downregulated antiapoptotic Bcl-2 expression, and increased caspase-3 activity in a dose-dependent manner in N2a cells. TMT induced oxidative stress in cells, performing reactive oxygen species (ROS) and malondialdehyde (MDA) excessive generation, and superoxide dismutase (SOD) activity reduction. TMT significantly decreased phosphorylated glycogen synthase kinase-3β (GSK-3β) and inhibited Shh and PI3K/Akt pathways. However, the addition of LBP upregulated GSK-3β phosphorylation, activated Shh and PI3K/Akt pathways, and eventually reduced apoptosis and oxidative stress caused by TMT. The interaction between Shh and PI3K/Akt pathways was clarified by specific PI3K inhibitor LY294002 or Shh inhibitor GDC-0449. Moreover, LY294002 and GDC-0449 pretreatment both induced phosphorylated GSK-3β downregulation and significantly promoted apoptosis induced by TMT. These results suggest that LBP could reduce TMT-induced N2a cells apoptosis by regulating GSK-3β phosphorylation, Shh, and PI3K/Akt signaling pathways.

Lycium barbarum Polysaccharides Protect against Trimethyltin Chloride-Induced Apoptosis via Sonic Hedgehog and PI3K/Akt Signaling Pathways in Mouse Neuro-2a Cells

PubMed Central

Zhao, Wanyun; Pan, Xiaoqi; Li, Tao; Zhang, Changchun; Shi, Nian

2016-01-01

Trimethyltin chloride (TMT) is a classic neurotoxicant that can cause severe neurodegenerative diseases. Some signaling pathways involving cell death play pivotal roles in the central nervous system. In this study, the role of Sonic Hedgehog (Shh) and PI3K/Akt pathways in TMT-induced apoptosis and protective effect of Lycium barbarum polysaccharides (LBP) on mouse neuro-2a (N2a) cells were investigated. Results showed that TMT treatment significantly enhanced apoptosis, upregulated proapoptotic Bax, downregulated antiapoptotic Bcl-2 expression, and increased caspase-3 activity in a dose-dependent manner in N2a cells. TMT induced oxidative stress in cells, performing reactive oxygen species (ROS) and malondialdehyde (MDA) excessive generation, and superoxide dismutase (SOD) activity reduction. TMT significantly decreased phosphorylated glycogen synthase kinase-3β (GSK-3β) and inhibited Shh and PI3K/Akt pathways. However, the addition of LBP upregulated GSK-3β phosphorylation, activated Shh and PI3K/Akt pathways, and eventually reduced apoptosis and oxidative stress caused by TMT. The interaction between Shh and PI3K/Akt pathways was clarified by specific PI3K inhibitor LY294002 or Shh inhibitor GDC-0449. Moreover, LY294002 and GDC-0449 pretreatment both induced phosphorylated GSK-3β downregulation and significantly promoted apoptosis induced by TMT. These results suggest that LBP could reduce TMT-induced N2a cells apoptosis by regulating GSK-3β phosphorylation, Shh, and PI3K/Akt signaling pathways. PMID:27143997

Evolutionary analyses of hedgehog and Hoxd-10 genes in fish species closely related to the zebrafish

PubMed Central

Zardoya, Rafael; Abouheif, Ehab; Meyer, Axel

1996-01-01

The study of development has relied primarily on the isolation of mutations in genes with specific functions in development and on the comparison of their expression patterns in normal and mutant phenotypes. Comparative evolutionary analyses can complement these approaches. Phylogenetic analyses of Sonic hedgehog (Shh) and Hoxd-10 genes from 18 cyprinid fish species closely related to the zebrafish provide novel insights into the functional constraints acting on Shh. Our results confirm and extend those gained from expression and crystalline structure analyses of this gene. Unexpectedly, exon 1 of Shh is found to be almost invariant even in third codon positions among these morphologically divergent species suggesting that this exon encodes for a functionally important domain of the hedgehog protein. This is surprising because the main functional domain of Shh had been thought to be that encoded by exon 2. Comparisons of Shh and Hoxd-10 gene sequences and of resulting gene trees document higher evolutionary constraints on the former than on the latter. This might be indicative of more general evolutionary patterns in networks of developmental regulatory genes interacting in a hierarchical fashion. The presence of four members of the hedgehog gene family in cyprinid fishes was documented and their homologies to known hedgehog genes in other vertebrates were established. PMID:8917540

Evolutionary analyses of hedgehog and Hoxd-10 genes in fish species closely related to the zebrafish.

PubMed

Zardoya, R; Abouheif, E; Meyer, A

1996-11-12

The study of development has relied primarily on the isolation of mutations in genes with specific functions in development and on the comparison of their expression patterns in normal and mutant phenotypes. Comparative evolutionary analyses can complement these approaches. Phylogenetic analyses of Sonic hedgehog (Shh) and Hoxd-10 genes from 18 cyprinid fish species closely related to the zebrafish provide novel insights into the functional constraints acting on Shh. Our results confirm and extend those gained from expression and crystalline structure analyses of this gene. Unexpectedly, exon 1 of Shh is found to be almost invariant even in third codon positions among these morphologically divergent species suggesting that this exon encodes for a functionally important domain of the hedgehog protein. This is surprising because the main functional domain of Shh had been thought to be that encoded by exon 2. Comparisons of Shh and Hoxd-10 gene sequences and of resulting gene trees document higher evolutionary constraints on the former than on the latter. This might be indicative of more general evolutionary patterns in networks of developmental regulatory genes interacting in a hierarchical fashion. The presence of four members of the hedgehog gene family in cyprinid fishes was documented and their homologies to known hedgehog genes in other vertebrates were established.

Formation of intestinal atresias in the Fgfr2IIIb-/- mice is not associated with defects in notochord development or alterations in Shh expression.

PubMed

Reeder, Amy L; Botham, Robert A; Franco, Marta; Zaremba, Krzysztof M; Nichol, Peter F

2012-09-01

The etiology of intestinal atresia remains elusive but has been ascribed to a number of possible events including in utero vascular accidents, failure of recanalization of the intestinal lumen, and mechanical compression. Another such event that has been postulated to be a cause in atresia formation is disruption in notochord development. This hypothesis arose from clinical observations of notochord abnormalities in patients with intestinal atresias as well as abnormal notochord development observed in a pharmacologic animal model of intestinal atresia. Atresias in this model result from in utero exposure to Adriamycin, wherein notochord defects were noted in up to 80% of embryos that manifested intestinal atresias. Embryos with notochord abnormalities were observed to have ectopic expression of Sonic Hedgehog (Shh), which in turn was postulated to be causative in atresia formation. We were interested in determining whether disruptions in notochord development or Shh expression occurred in an established genetic model of intestinal atresia and used the fibroblast growth factor receptor 2IIIb homozygous mutant (Fgfr2IIIb-/-) mouse model. These embryos develop colonic atresias (100% penetrance) and duodenal atresias (42% penetrance). Wild-type and Fgfr2IIIb-/- mouse embryos were harvested at embryonic day (E) 10.5, E11.5, E12.5, and E13.5. Whole-mount in situ hybridization was performed on E10.5 embryos for Shh. Embryos at each time point were harvested and sectioned for hematoxylin-eosin staining. Sections were photographed specifically for the notochord and resulting images reconstructed in 3-D using Amira software. Colons were isolated from wild-type and Fgfr2IIIb-/- embryos at E10.5, then cultured for 48 hours in Matrigel with FGF10 in the presence or absence of exogenous Shh protein. Explants were harvested, fixed in formalin, and photographed. Fgfr2IIIb-/- mouse embryos exhibit no disruptions in Shh expression at E10.5, when the first events in atresia formation are known to occur. Three-dimensional reconstructions failed to demonstrate any anatomical disruptions in the notochord by discontinuity or excessive branching. Culture of wild-type intestines in the presence of Shh failed to induce atresia formation in either the duodenum or colon. Cultured Fgfr2IIIb-/- intestines developed atresias of the colon in either the presence or absence of Shh protein. Although disruptions in notochord development can be associated with intestinal atresia formation, in the Fgfr2IIIb-/- genetic animal model neither disruptions in notochord development nor the presence of exogenous Shh protein are causative in the formation of these defects. Copyright © 2012 Elsevier Inc. All rights reserved.

Formation of Intestinal Atresias in the Fgfr2IIIb−/− Mice is not Associated with Defects in Notochord Development or Alterations in Shh Expression

PubMed Central

Reader, Amy L.; Botham, Robert A.; Franco, Marta; Zaremba, Krzysztof M.; Nichol, Peter F.

2012-01-01

Purpose The etiology of intestinal atresia remains elusive but has been ascribed to a number of possible events including in utero vascular accidents, failure of recanalization of the intestinal lumen and mechanical compression. Another such event that has been postulated to be a cause in atresia formation is disruption in notochord development. This hypothesis arose from clinical observations of notochord abnormalities in patients with intestinal atresias as well as abnormal notochord development observed in a pharmacological animal model of intestinal atresia. Atresias in this model result from in utero exposure to Adriamycin, wherein notochord defects were noted in up to 80% of embryos that manifested intestinal atresias. Embryos with notochord abnormalities were observed to have ectopic expression of Sonic Hedgehog (Shh) which in turn was postulated to be causative in atresia formation. We were interested in determining whether disruptions in notochord development or Shh expression occurred in an established genetic model of intestinal atresia and utilized the Fibroblast Growth Factor Receptor 2IIIb homozygous mutant (Fgfr2IIIb−/−) mouse model. These embryos develop colonic atresias (100% penetrance) and duodenal atresias (42% penetrance). Methods Wild-type and Fgfr2IIIb−/− mouse embryos were harvested at E10.5, E11.5, E12.5 and E13.5. Whole mount in situ hybridization was performed on E10.5 embryos for Shh. Embryos at each time point were harvested and sectioned for H&E staining. Sections were photographed specifically for the notochord and resulting images reconstructed in 3-D using Amira software. Colons were isolated from wild-type and Fgfr2IIIb−/− embryos at E10.5, then cultured for 48 hours in matrigel with FGF10 in the presence or absence of exogenous SHH protein. Explants were harvested, fixed in formalin and photographed. Results Fgfr2IIIb−/− mouse embryos exhibit no disruptions in Shh expression at E10.5, when the first events in atresia formation are known to occur. Three-dimensional reconstructions failed to demonstrate any anatomical disruptions in the notochord by discontinuity or excessive branching. Culture of wild-type intestines in the presence of Shh failed to induce atresia formation in either the duodenum or colon. Cultured Fgfr2IIIb−/− intestines developed atresias of the colon in either the presence, or absence, of Shh protein. Conclusions Although disruptions in notochord development can be associated with intestinal atresia formation, in the Fgfr2IIIb−/− genetic animal model neither disruptions in notochord development nor the presence of exogenous Shh protein are causative in the formation of these defects. PMID:22572615

Dynamic Bayesian Network Modeling of the Interplay between EGFR and Hedgehog Signaling.

PubMed

Fröhlich, Holger; Bahamondez, Gloria; Götschel, Frank; Korf, Ulrike

2015-01-01

Aberrant activation of sonic Hegdehog (SHH) signaling has been found to disrupt cellular differentiation in many human cancers and to increase proliferation. The SHH pathway is known to cross-talk with EGFR dependent signaling. Recent studies experimentally addressed this interplay in Daoy cells, which are presumable a model system for medulloblastoma, a highly malignant brain tumor that predominately occurs in children. Currently ongoing are several clinical trials for different solid cancers, which are designed to validate the clinical benefits of targeting the SHH in combination with other pathways. This has motivated us to investigate interactions between EGFR and SHH dependent signaling in greater depth. To our knowledge, there is no mathematical model describing the interplay between EGFR and SHH dependent signaling in medulloblastoma so far. Here we come up with a fully probabilistic approach using Dynamic Bayesian Networks (DBNs). To build our model, we made use of literature based knowledge describing SHH and EGFR signaling and integrated gene expression (Illumina) and cellular location dependent time series protein expression data (Reverse Phase Protein Arrays). We validated our model by sub-sampling training data and making Bayesian predictions on the left out test data. Our predictions focusing on key transcription factors and p70S6K, showed a high level of concordance with experimental data. Furthermore, the stability of our model was tested by a parametric bootstrap approach. Stable network features were in agreement with published data. Altogether we believe that our model improved our understanding of the interplay between two highly oncogenic signaling pathways in Daoy cells. This may open new perspectives for the future therapy of Hedghog/EGF-dependent solid tumors.

The immunoprofile of odontogenic keratocyst (keratocystic odontogenic tumor) that includes expression of PTCH, SMO, GLI-1 and bcl-2 is similar to ameloblastoma but different from odontogenic cysts.

PubMed

Vered, M; Peleg, O; Taicher, S; Buchner, A

2009-08-01

The aggressive biological behavior of odontogenic keratocysts (OKCs), unlike that of other odontogenic cysts, has argued for its recent re-classification as a neoplasm, 'keratocystic odontogenic tumor'. Identification of mutations in the PTCH gene in some of the OKCs that were expected to produce truncated proteins, resulting in loss of control of the cell cycle, provided additional support for OKCs having a neoplastic nature. We investigated the immunohistochemical expression of the sonic hedgehog (SHH) signaling pathway-related proteins, PTCH, smoothened (SMO) and GLI-1, and of the SHH-induced bcl-2 oncoprotein in a series of primary OKC (pOKC), recurrent OKC (rOKC) and nevoid basal cell carcinoma syndrome-associated OKCs (NBCCS-OKCs), and compared them to solid ameloblastomas (SAMs), unicystic ameloblastomas (UAMs), 'orthokeratinized' OKCs (oOKCs), dentigerous cysts (DCs) and radicular cysts (RCs). All studied lesions expressed the SHH pathway-related proteins in a similar pattern. The expression of bcl-2 in OKCs (pOKCs and NBCCS-OKCs) and SAMs was significantly higher than in oOKCs, DCs and RCs (P < 0.001). The present results of the immunoprofile of OKCs (that includes the expression of the SHH-related proteins and the SHH-induced bcl-2 oncoprotein) further support the notion of OKC having a neoplastic nature. As OKCs vary considerably in their biologic behavior, it is suggested that the quality and quantity of interactions between the SHH and other cell cycle regulatory pathways are likely to work synergistically to define the individual phenotype and corresponding biological behavior of this lesion.

Protein kinase inhibitor SU6668 attenuates positive regulation of Gli proteins in cancer and multipotent progenitor cells.

PubMed

Piirsoo, Alla; Kasak, Lagle; Kauts, Mari-Liis; Loog, Mart; Tints, Kairit; Uusen, Piia; Neuman, Toomas; Piirsoo, Marko

2014-04-01

Observations that Glioma-associated transcription factors Gli1 and Gli2 (Gli1/2), executers of the Sonic Hedgehog (Shh) signaling pathway and targets of the Transforming Growth Factor β (TGF-β) signaling axis, are involved in numerous developmental and pathological processes unveil them as attractive pharmaceutical targets. Unc-51-like serine/threonine kinase Ulk3 has been suggested to play kinase activity dependent and independent roles in the control of Gli proteins in the context of the Shh signaling pathway. This study aimed at investigating whether the mechanism of generation of Gli1/2 transcriptional activators has similarities regardless of the signaling cascade evoking their activation. We also elucidate further the role of Ulk3 kinase in regulation of Gli1/2 proteins and examine SU6668 as an inhibitor of Ulk3 catalytic activity and a compound targeting Gli1/2 proteins in different cell-based experimental models. Here we demonstrate that Ulk3 is required not only for maintenance of basal levels of Gli1/2 proteins but also for TGF-β or Shh dependent activation of endogenous Gli1/2 proteins in human adipose tissue derived multipotent stromal cells (ASCs) and mouse immortalized progenitor cells, respectively. We show that cultured ASCs possess the functional Shh signaling axis and differentiate towards osteoblasts in response to Shh. Also, we demonstrate that similarly to Ulk3 RNAi, SU6668 prevents de novo expression of Gli1/2 proteins and antagonizes the Gli-dependent activation of the gene expression programs induced by either Shh or TGF-β. Our data suggest SU6668 as an efficient inhibitor of Ulk3 kinase allowing manipulation of the Gli-dependent transcriptional outcome. Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.

A review of hedgehog signaling in cranial bone development

PubMed Central

Pan, Angel; Chang, Le; Nguyen, Alan; James, Aaron W.

2013-01-01

During craniofacial development, the Hedgehog (HH) signaling pathway is essential for mesodermal tissue patterning and differentiation. The HH family consists of three protein ligands: Sonic Hedgehog (SHH), Indian Hedgehog (IHH), and Desert Hedgehog (DHH), of which two are expressed in the craniofacial complex (IHH and SHH). Dysregulations in HH signaling are well documented to result in a wide range of craniofacial abnormalities, including holoprosencephaly (HPE), hypotelorism, and cleft lip/palate. Furthermore, mutations in HH effectors, co-receptors, and ciliary proteins result in skeletal and craniofacial deformities. Cranial suture morphogenesis is a delicate developmental process that requires control of cell commitment, proliferation and differentiation. This review focuses on both what is known and what remains unknown regarding HH signaling in cranial suture morphogenesis and intramembranous ossification. As demonstrated from murine studies, expression of both SHH and IHH is critical to the formation and fusion of the cranial sutures and calvarial ossification. SHH expression has been observed in the cranial suture mesenchyme and its precise function is not fully defined, although some postulate SHH to delay cranial suture fusion. IHH expression is mainly found on the osteogenic fronts of the calvarial bones, and functions to induce cell proliferation and differentiation. Unfortunately, neonatal lethality of IHH deficient mice precludes a detailed examination of their postnatal calvarial phenotype. In summary, a number of basic questions are yet to be answered regarding domains of expression, developmental role, and functional overlap of HH morphogens in the calvaria. Nevertheless, SHH and IHH ligands are integral to cranial suture development and regulation of calvarial ossification. When HH signaling goes awry, the resultant suite of morphologic abnormalities highlights the important roles of HH signaling in cranial development. PMID:23565096

Activation of Sonic Hedgehog Signaling Is Associated with Human Osteosarcoma Cells Radioresistance Characterized by Increased Proliferation, Migration, and Invasion.

PubMed

Qu, Wei; Li, Dichen; Wang, Yufei; Wu, Qining; Hao, Dingjun

2018-06-04

BACKGROUND Radioresistance restricts the application of radiotherapy in human osteosarcoma (OS). This study investigated the molecular mechanism of radioresistance in OS, which may provide clues to finding ideal targets for genetic therapy. MATERIAL AND METHODS The human OS cell line MG63 was employed as parent cells. After repeat low-dose X-ray irradiation of MG63, the radioresistant OS cell line MG63R was produced. Colony formation assay was used to assess the radioresistance. Cell viability was evaluated by CCK-8 assay. Flow cytometry was used to detect cell apoptosis, and wound healing assay was used to evaluate invasive capacity. The nuclear translocation was evaluated by fluorescent immunohistochemistry. Protein expression levels were assessed by Western blotting. Specific siRNA against Shh was used to silence Shh. RESULTS More survival colony formation, elevated cell viability, less cell apoptosis, and increased wound closure were found in MG63R than in MG63 cells exposed to irradiation. The nuclear translocation of Gli, expression levels of Shh, Smo, Ptch1, Bcl2, active MMP2, and active MMP9 were increased in MG63R cells compared with MG63 cells. Transfection of Shh-siRNA suppressed expression levels of Shh, Smo, Ptch1, Bcl2, active MMP2, and active MMP9, as well as the nuclear translocation of Gli in MG63R cells. The cell viability, survival colony formation, and wound closure were impaired, whereas cell apoptosis was increased, in siRNA-transfected MG63R cells than in control MG63R cells exposed to irradiation. CONCLUSIONS Activation of Shh signaling was involved in radioresistance of OS cells. Blocking this signaling can impair the radioresistance capacity of OS cells.

Polycomb-Mediated Repression and Sonic Hedgehog Signaling Interact to Regulate Merkel Cell Specification during Skin Development

PubMed Central

Bar, Carmit; Tsai, Pai-Chi; Valdes, Victor J.; Cohen, Idan; Santoriello, Francis J.; Zhao, Dejian; Hsu, Ya-Chieh; Ezhkova, Elena

2016-01-01

An increasing amount of evidence indicates that developmental programs are tightly regulated by the complex interplay between signaling pathways, as well as transcriptional and epigenetic processes. Here, we have uncovered coordination between transcriptional and morphogen cues to specify Merkel cells, poorly understood skin cells that mediate light touch sensations. In murine dorsal skin, Merkel cells are part of touch domes, which are skin structures consisting of specialized keratinocytes, Merkel cells, and afferent neurons, and are located exclusively around primary hair follicles. We show that the developing primary hair follicle functions as a niche required for Merkel cell specification. We find that intraepidermal Sonic hedgehog (Shh) signaling, initiated by the production of Shh ligand in the developing hair follicles, is required for Merkel cell specification. The importance of Shh for Merkel cell formation is further reinforced by the fact that Shh overexpression in embryonic epidermal progenitors leads to ectopic Merkel cells. Interestingly, Shh signaling is common to primary, secondary, and tertiary hair follicles, raising the possibility that there are restrictive mechanisms that regulate Merkel cell specification exclusively around primary hair follicles. Indeed, we find that loss of Polycomb repressive complex 2 (PRC2) in the epidermis results in the formation of ectopic Merkel cells that are associated with all hair types. We show that PRC2 loss expands the field of epidermal cells competent to differentiate into Merkel cells through the upregulation of key Merkel-differentiation genes, which are known PRC2 targets. Importantly, PRC2-mediated repression of the Merkel cell differentiation program requires inductive Shh signaling to form mature Merkel cells. Our study exemplifies how the interplay between epigenetic and morphogen cues regulates the complex patterning and formation of the mammalian skin structures. PMID:27414999

Polycomb-Mediated Repression and Sonic Hedgehog Signaling Interact to Regulate Merkel Cell Specification during Skin Development.

PubMed

Perdigoto, Carolina N; Dauber, Katherine L; Bar, Carmit; Tsai, Pai-Chi; Valdes, Victor J; Cohen, Idan; Santoriello, Francis J; Zhao, Dejian; Zheng, Deyou; Hsu, Ya-Chieh; Ezhkova, Elena

2016-07-01

An increasing amount of evidence indicates that developmental programs are tightly regulated by the complex interplay between signaling pathways, as well as transcriptional and epigenetic processes. Here, we have uncovered coordination between transcriptional and morphogen cues to specify Merkel cells, poorly understood skin cells that mediate light touch sensations. In murine dorsal skin, Merkel cells are part of touch domes, which are skin structures consisting of specialized keratinocytes, Merkel cells, and afferent neurons, and are located exclusively around primary hair follicles. We show that the developing primary hair follicle functions as a niche required for Merkel cell specification. We find that intraepidermal Sonic hedgehog (Shh) signaling, initiated by the production of Shh ligand in the developing hair follicles, is required for Merkel cell specification. The importance of Shh for Merkel cell formation is further reinforced by the fact that Shh overexpression in embryonic epidermal progenitors leads to ectopic Merkel cells. Interestingly, Shh signaling is common to primary, secondary, and tertiary hair follicles, raising the possibility that there are restrictive mechanisms that regulate Merkel cell specification exclusively around primary hair follicles. Indeed, we find that loss of Polycomb repressive complex 2 (PRC2) in the epidermis results in the formation of ectopic Merkel cells that are associated with all hair types. We show that PRC2 loss expands the field of epidermal cells competent to differentiate into Merkel cells through the upregulation of key Merkel-differentiation genes, which are known PRC2 targets. Importantly, PRC2-mediated repression of the Merkel cell differentiation program requires inductive Shh signaling to form mature Merkel cells. Our study exemplifies how the interplay between epigenetic and morphogen cues regulates the complex patterning and formation of the mammalian skin structures.

Identification of a neuronal transcription factor network involved in medulloblastoma development.

PubMed

Lastowska, Maria; Al-Afghani, Hani; Al-Balool, Haya H; Sheth, Harsh; Mercer, Emma; Coxhead, Jonathan M; Redfern, Chris P F; Peters, Heiko; Burt, Alastair D; Santibanez-Koref, Mauro; Bacon, Chris M; Chesler, Louis; Rust, Alistair G; Adams, David J; Williamson, Daniel; Clifford, Steven C; Jackson, Michael S

2013-07-11

Medulloblastomas, the most frequent malignant brain tumours affecting children, comprise at least 4 distinct clinicogenetic subgroups. Aberrant sonic hedgehog (SHH) signalling is observed in approximately 25% of tumours and defines one subgroup. Although alterations in SHH pathway genes (e.g. PTCH1, SUFU) are observed in many of these tumours, high throughput genomic analyses have identified few other recurring mutations. Here, we have mutagenised the Ptch+/- murine tumour model using the Sleeping Beauty transposon system to identify additional genes and pathways involved in SHH subgroup medulloblastoma development. Mutagenesis significantly increased medulloblastoma frequency and identified 17 candidate cancer genes, including orthologs of genes somatically mutated (PTEN, CREBBP) or associated with poor outcome (PTEN, MYT1L) in the human disease. Strikingly, these candidate genes were enriched for transcription factors (p=2x10-5), the majority of which (6/7; Crebbp, Myt1L, Nfia, Nfib, Tead1 and Tgif2) were linked within a single regulatory network enriched for genes associated with a differentiated neuronal phenotype. Furthermore, activity of this network varied significantly between the human subgroups, was associated with metastatic disease, and predicted poor survival specifically within the SHH subgroup of tumours. Igf2, previously implicated in medulloblastoma, was the most differentially expressed gene in murine tumours with network perturbation, and network activity in both mouse and human tumours was characterised by enrichment for multiple gene-sets indicating increased cell proliferation, IGF signalling, MYC target upregulation, and decreased neuronal differentiation. Collectively, our data support a model of medulloblastoma development in SB-mutagenised Ptch+/- mice which involves disruption of a novel transcription factor network leading to Igf2 upregulation, proliferation of GNPs, and tumour formation. Moreover, our results identify rational therapeutic targets for SHH subgroup tumours, alongside prognostic biomarkers for the identification of poor-risk SHH patients.

Transcriptional profiles of SHH pathway genes in keratocystic odontogenic tumor and ameloblastoma.

PubMed

Gurgel, Clarissa Araújo Silva; Buim, Marcilei Eliza Cavichiolli; Carvalho, Kátia Cândido; Sales, Caroline Brandi Schlaepfer; Reis, Mitermayer Galvão; de Souza, Renata Oliveira; de Faro Valverde, Ludmila; de Azevedo, Roberto Almeida; Dos Santos, Jean Nunes; Soares, Fernando Augusto; Ramos, Eduardo Antônio Gonçalves

2014-09-01

Sonic hedgehog (SHH) pathway activation has been identified as a key factor in the development of many types of tumors, including odontogenic tumors. Our study examined the expression of genes in the SHH pathway to characterize their roles in the pathogenesis of keratocystic odontogenic tumors (KOT) and ameloblastomas (AB). We quantified the expression of SHH, SMO, PTCH1, SUFU, GLI1, CCND1, and BCL2 genes by qPCR in a total of 23 KOT, 11 AB, and three non-neoplastic oral mucosa (NNM). We also measured the expression of proteins related to this pathway (CCND1 and BCL2) by immunohistochemistry. We observed overexpression of SMO, PTCH1, GLI1, and CCND1 genes in both KOT (23/23) and AB (11/11). However, we did not detect expression of the SHH gene in 21/23 KOT and 10/11 AB tumors. Low levels of the SUFU gene were expressed in KOT (P = 0.0199) and AB (P = 0.0127) relative to the NNM. Recurrent KOT exhibited high levels of SMO (P = 0.035), PTCH1 (P = 0.048), CCND1 (P = 0.048), and BCL2 (P = 0.045) transcripts. Using immunolabeling of CCND1, we observed no statistical difference between primary and recurrent KOT (P = 0.8815), sporadic and NBCCS-KOT (P = 0.7688), and unicystic and solid AB (P = 0.7521). Overexpression of upstream (PTCH1 and SMO) and downstream (GLI1, CCND1 and BCL2) genes in the SHH pathway leads to the constitutive activation of this pathway in KOT and AB and may suggest a mechanism for the development of these types of tumors. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Identification of conserved regions and residues within Hedgehog acyltransferase critical for palmitoylation of Sonic Hedgehog.

PubMed

Buglino, John A; Resh, Marilyn D

2010-06-23

Sonic hedgehog (Shh) is a palmitoylated protein that plays key roles in mammalian development and human cancers. Palmitoylation of Shh is required for effective long and short range Shh-mediated signaling. Attachment of palmitate to Shh is catalyzed by Hedgehog acyltransferase (Hhat), a member of the membrane bound O-acyl transferase (MBOAT) family of multipass membrane proteins. The extremely hydrophobic composition of MBOAT proteins has limited their biochemical characterization. Except for mutagenesis of two conserved residues, there has been no structure-function analysis of Hhat, and the regions of the protein required for Shh palmitoylation are unknown. Here we undertake a systematic approach to identify residues within Hhat that are required for protein stability and/or enzymatic activity. We also identify a second, novel MBOAT homology region (residues 196-234) that is required for Hhat activity. In total, ten deletion mutants and eleven point mutants were generated and analyzed. Truncations at the N- and C-termini of Hhat yielded inactive proteins with reduced stability. Four Hhat mutants with deletions within predicted loop regions and five point mutants retained stability but lost palmitoylation activity. We purified two point mutants, W378A and H379A, with defective Hhat activity. Kinetic analyses revealed alterations in apparent K(m) and V(max) for Shh and/or palmitoyl CoA, changes that likely explain the catalytic defects observed for these mutants. This study has pinpointed specific regions and multiple residues that regulate Hhat stability and catalysis. Our findings should be applicable to other MBOAT proteins that mediate lipid modification of Wnt proteins and ghrelin, and should serve as a model for understanding how secreted morphogens are modified by palmitoyl acyltransferases.

Novel insights into the pathways regulating the canine hair cycle and their deregulation in alopecia X.

PubMed

Brunner, Magdalena A T; Jagannathan, Vidhya; Waluk, Dominik P; Roosje, Petra; Linek, Monika; Panakova, Lucia; Leeb, Tosso; Wiener, Dominique J; Welle, Monika M

2017-01-01

Alopecia X is a hair cycle arrest disorder in Pomeranians. Histologically, kenogen and telogen hair follicles predominate, whereas anagen follicles are sparse. The induction of anagen relies on the activation of hair follicle stem cells and their subsequent proliferation and differentiation. Stem cell function depends on finely tuned interactions of signaling molecules and transcription factors, which are not well defined in dogs. We performed transcriptome profiling on skin biopsies to analyze altered molecular pathways in alopecia X. Biopsies from five affected and four non-affected Pomeranians were investigated. Differential gene expression revealed a downregulation of key regulator genes of the Wnt (CTNNB1, LEF1, TCF3, WNT10B) and Shh (SHH, GLI1, SMO, PTCH2) pathways. In mice it has been shown that Wnt and Shh signaling results in stem cell activation and differentiation Thus our findings are in line with the lack of anagen hair follicles in dogs with Alopecia X. We also observed a significant downregulation of the stem cell markers SOX9, LHX2, LGR5, TCF7L1 and GLI1 whereas NFATc1, a quiescence marker, was upregulated in alopecia X. Moreover, genes coding for enzymes directly involved in the sex hormone metabolism (CYP1A1, CYP1B1, HSD17B14) were differentially regulated in alopecia X. These findings are in agreement with the so far proposed but not yet proven deregulation of the sex hormone metabolism in this disease.

Signaling by Bone Morphogenetic Proteins directs formation of an ectodermal signaling center that regulates craniofacial development

PubMed Central

Foppiano, Silvia; Hu, Diane; Marcucio, Ralph S.

2008-01-01

We previously described a signaling center, the Frontonasal Ectodermal Zone (FEZ) that regulates growth and patterning of the frontonasal process (FNP). The FEZ is comprised of FNP ectoderm flanking a boundary between Sonic hedgehog (Shh) and Fibroblast growth factor 8 (Fgf8) expression domains. Our objective was to examine BMP signaling during formation of the FEZ. We blocked BMP signaling throughout the FNP prior to FEZ formation by infecting chick embryos at stage 10 (HH10) with a replication competent avian retrovirus encoding the BMP antagonist Noggin. We assessed gene expression patterns in the FNP 72 hours after infection (~HH22) and observed that Shh expression was reduced or absent. In the mesenchyme we observed that Bmp2 transcripts were absent while the Bmp4 expression domain was expanded proximally. In addition to the molecular changes, infected embryos also exhibited facial malformations at 72 and 96 hours after infection suggesting that the FEZ did not form. Our data indicate that reduced cell proliferation, but not apoptosis, in the mesenchyme contributed to the phenotype that we observed. Additionally, adding exogenous SHH into the mesenchyme of RCAS-Noggin infected embryos did not restore Bmp2 and Bmp4 to a normal pattern of expression. These data indicate that BMP signaling mediates interactions between tissues in the FNP that regulate FEZ formation; and that the correct pattern of Bmp2 and Bmp4, but not Bmp7, expression in the FNP mesenchyme requires signaling by the BMP pathway. PMID:18028903

Complementary Gli activity mediates early patterning of the mouse visual system.

PubMed

Furimsky, Marosh; Wallace, Valerie A

2006-03-01

The Sonic hedgehog (Shh) signaling pathway plays a key role in the development of the vertebrate central nervous system, including the eye. This pathway is mediated by the Gli transcription factors (Gli1, Gli2, and Gli3) that differentially activate and repress the expression of specific downstream target genes. In this study, we investigated the roles of the three vertebrate Glis in mediating midline Shh signaling in early ocular development. We examined the ocular phenotypes of Shh and Gli combination mutant mouse embryos and monitored proximodistal and dorsoventral patterning by the expression of specific eye development regulatory genes using in situ hybridization. We show that midline Shh signaling relieves the repressor activity of Gli3 adjacent to the midline and then promotes eye pattern formation through the nonredundant activities of all three Gli proteins. Gli3, in particular, is required to specify the dorsal optic stalk and to define the boundary between the optic stalk and the optic cup.

Formation of the sacrum requires down-regulation of sonic hedgehog signaling in the sacral intervertebral discs.

PubMed

Bonavita, Raffaella; Vincent, Kathleen; Pinelli, Robert; Dahia, Chitra Lekha

2018-05-21

In humans, the sacrum forms an important component of the pelvic arch, and it transfers the weight of the body to the lower limbs. The sacrum is formed by collapse of the intervertebral discs (IVDs) between the five sacral vertebrae during childhood, and their fusion to form a single bone. We show that collapse of the sacral discs in the mouse is associated with the down-regulation of sonic hedgehog (SHH) signaling in the nucleus pulposus (NP) of the disc, and many aspects of this phenotype can be reversed by experimental postnatal activation of HH signaling. We have previously shown that SHH signaling is essential for the normal postnatal growth and differentiation of intervertebral discs elsewhere in the spine, and that loss of SHH signaling leads to pathological disc degeneration, a very common disorder of aging. Thus, loss of SHH is pathological in one region of the spine but part of normal development in another. © 2018. Published by The Company of Biologists Ltd.

Sonic Hedgehog promotes proliferation of Notch-dependent monociliated choroid plexus tumour cells

PubMed Central

Li, Li; Grausam, Katie B.; Wang, Jun; Lun, Melody P.; Ohli, Jasmin; Lidov, Hart G. W.; Calicchio, Monica L.; Zeng, Erliang; Salisbury, Jeffrey L.; Wechsler-Reya, Robert J.; Lehtinen, Maria K.; Schüller, Ulrich; Zhao, Haotian

2016-01-01

Aberrant Notch signaling has been linked to many cancers including choroid plexus (CP) tumours, a group of rare and predominantly pediatric brain neoplasms. We developed animal models of CP tumours by inducing sustained expression of Notch1 that recapitulate properties of human CP tumours with aberrant NOTCH signaling. Whole transcriptome and functional analyses showed that tumour cell proliferation is associated with Sonic Hedgehog (Shh) in the tumour microenvironment. Unlike CP epithelial cells, which have multiple primary cilia, tumour cells possess a solitary primary cilium as a result of Notch-mediated suppression of multiciliate diffferentiation. A Shh-driven signaling cascade in the primary cilium occurs in tumour cells but not in epithelial cells. Lineage studies show that CP tumours arise from mono-ciliated progenitors in the roof plate characterized by elevated Notch signaling. Abnormal SHH signaling and distinct ciliogenesis are detected in human CP tumours, suggesting SHH pathway and cilia differentiation as potential therapeutic avenues. PMID:26999738

Sonic Hedgehog modulates EGFR dependent proliferation of neural stem cells during late mouse embryogenesis through EGFR transactivation

PubMed Central

Reinchisi, Gisela; Parada, Margarita; Lois, Pablo; Oyanadel, Claudia; Shaughnessy, Ronan; Gonzalez, Alfonso; Palma, Verónica

2013-01-01

Sonic Hedgehog (Shh/GLI) and EGFR signaling pathways modulate Neural Stem Cell (NSC) proliferation. How these signals cooperate is therefore critical for understanding normal brain development and function. Here we report a novel acute effect of Shh signaling on EGFR function. We show that during late neocortex development, Shh mediates the activation of the ERK1/2 signaling pathway in Radial Glial cells (RGC) through EGFR transactivation. This process is dependent on metalloprotease activity and accounts for almost 50% of the EGFR-dependent mitogenic response of late NSCs. Furthermore, in HeLa cancer cells, a well-known model for studying the EGFR receptor function, Shh also induces cell proliferation involving EGFR activation, as reflected by EGFR internalization and ERK1/2 phosphorylation. These findings may have important implications for understanding the mechanisms that regulate NSC proliferation during neurogenesis and may lead to novel approaches to the treatment of tumors. PMID:24133411

Motile cilia of human airway epithelia contain hedgehog signaling components that mediate noncanonical hedgehog signaling.

PubMed

Mao, Suifang; Shah, Alok S; Moninger, Thomas O; Ostedgaard, Lynda S; Lu, Lin; Tang, Xiao Xiao; Thornell, Ian M; Reznikov, Leah R; Ernst, Sarah E; Karp, Philip H; Tan, Ping; Keshavjee, Shaf; Abou Alaiwa, Mahmoud H; Welsh, Michael J

2018-02-06

Differentiated airway epithelia produce sonic hedgehog (SHH), which is found in the thin layer of liquid covering the airway surface. Although previous studies showed that vertebrate HH signaling requires primary cilia, as airway epithelia mature, the cells lose primary cilia and produce hundreds of motile cilia. Thus, whether airway epithelia have apical receptors for SHH has remained unknown. We discovered that motile cilia on airway epithelial cells have HH signaling proteins, including patched and smoothened. These cilia also have proteins affecting cAMP-dependent signaling, including Gα i and adenylyl cyclase 5/6. Apical SHH decreases intracellular levels of cAMP, which reduces ciliary beat frequency and pH in airway surface liquid. These results suggest that apical SHH may mediate noncanonical HH signaling through motile cilia to dampen respiratory defenses at the contact point between the environment and the lung, perhaps counterbalancing processes that stimulate airway defenses. Copyright © 2018 the Author(s). Published by PNAS.

The sonic hedgehog signaling pathway maintains the cancer stem cell self-renewal of anaplastic thyroid cancer by inducing snail expression.

PubMed

Heiden, Katherine B; Williamson, Ashley J; Doscas, Michelle E; Ye, Jin; Wang, Yimin; Liu, Dingxie; Xing, Mingzhao; Prinz, Richard A; Xu, Xiulong

2014-11-01

Cancer stem cells (CSCs) have been recently identified in thyroid neoplasm. Anaplastic thyroid cancer (ATC) contains a higher percentage of CSCs than well-differentiated thyroid cancer. The signaling pathways and the transcription factors that regulate thyroid CSC self-renewal remain poorly understood. The objective of this study is to use two ATC cell lines (KAT-18 and SW1736) as a model to study the role of the sonic hedgehog (Shh) pathway in maintaining thyroid CSC self-renewal and to understand its underlying molecular mechanisms. The expression and activity of aldehyde dehydrogenase (ALDH), a marker for thyroid CSCs, was analyzed by Western blot and ALDEFLUOR assay, respectively. The effect of three Shh pathway inhibitors (cyclopamine, HhAntag, GANT61), Shh, Gli1, Snail knockdown, and Gli1 overexpression on thyroid CSC self-renewal was analyzed by ALDEFLUOR assay and thyrosphere formation. The sensitivity of transfected KAT-18 cells to radiation was evaluated by a colony survival assay. Western blot analysis revealed that ALDH protein levels in five thyroid cancer cell lines (WRO82, a follicular thyroid cancer cell line; BCPAP and TPC1, two papillary thyroid cancer cell lines; KAT-18 and SW1736, two ATC cell lines) correlated with the percentage of the ALDH(High) cells as well as Gli1 and Snail expression. The Shh pathway inhibitors, Shh and Gli1 knockdown, in KAT-18 cells decreased thyroid CSC self-renewal and increased radiation sensitivity. In contrast, Gli1 overexpression led to increased thyrosphere formation, an increased percentage of ALDH(High) cells, and increased radiation resistance in KAT-18 cells. Inhibition of the Shh pathway by three specific inhibitors led to decreased Snail expression and a decreased number of ALDH(High) cells in KAT-18 and SW1736. Snail gene knockdown decreased the number of ALDH(High) cells in KAT-18 and SW1736 cells. The Shh pathway promotes the CSC self-renewal in ATC cell lines by Gli1-induced Snail expression.

Left cardiac isomerism in the Sonic hedgehog null mouse.

PubMed

Hildreth, Victoria; Webb, Sandra; Chaudhry, Bill; Peat, Jonathan D; Phillips, Helen M; Brown, Nigel; Anderson, Robert H; Henderson, Deborah J

2009-06-01

Sonic hedgehog (Shh) is a secreted morphogen necessary for the production of sidedness in the developing embryo. In this study, we describe the morphology of the atrial chambers and atrioventricular junctions of the Shh null mouse heart. We demonstrate that the essential phenotypic feature is isomerism of the left atrial appendages, in combination with an atrioventricular septal defect and a common atrioventricular junction. These malformations are known to be frequent in humans with left isomerism. To confirm the presence of left isomerism, we show that Pitx2c, a recognized determinant of morphological leftness, is expressed in the Shh null mutants on both the right and left sides of the inflow region, and on both sides of the solitary arterial trunk exiting from the heart. It has been established that derivatives of the second heart field expressing Isl1 are asymmetrically distributed in the developing normal heart. We now show that this population is reduced in the hearts from the Shh null mutants, likely contributing to the defects. To distinguish the consequences of reduced contributions from the second heart field from those of left-right patterning disturbance, we disrupted the movement of second heart field cells into the heart by expressing dominant-negative Rho kinase in the population of cells expressing Isl1. This resulted in absence of the vestibular spine, and presence of atrioventricular septal defects closely resembling those seen in the hearts from the Shh null mutants. The primary atrial septum, however, was well formed, and there was no evidence of isomerism of the atrial appendages, suggesting that these features do not relate to disruption of the contributions made by the second heart field. We demonstrate, therefore, that the Shh null mouse is a model of isomerism of the left atrial appendages, and show that the recognized associated malformations found at the venous pole of the heart in the setting of left isomerism are likely to arise from the loss of the effects of Shh in the establishment of laterality, combined with a reduced contribution made by cells derived from the second heart field.

Strong sonic hedgehog signaling in the mouse ventral spinal cord is not required for oligodendrocyte precursor cell (OPC) generation but is necessary for correct timing of its generation.

PubMed

Hashimoto, Hirokazu; Jiang, Wen; Yoshimura, Takeshi; Moon, Kyeong-Hye; Bok, Jinwoong; Ikenaka, Kazuhiro

2017-11-06

In the mouse neural tube, sonic hedgehog (Shh) secreted from the floor plate (FP) and the notochord (NC) regulates ventral patterning of the neural tube, and later is essential for the generation of oligodendrocyte precursor cells (OPCs). During early development, the NC is adjacent to the neural tube and induces ventral domains in it, including the FP. In the later stage of development, during gliogenesis in the spinal cord, the pMN domain receives strong Shh signaling input. While this is considered to be essential for the generation of OPCs, the actual role of this strong input in OPC generation remains unclear. Here we studied OPC generation in bromi mutant mice which show abnormal ciliary structure. Shh signaling occurs within cilia and has been reported to be weak in bromi mutants. At E11.5, accumulation of Patched1 mRNA, a Shh signaling reporter, is observed in the pMN domain of wild type but not bromi mutants, whereas expression of Gli1 mRNA, another Shh reporter, disappeared. Thus, Shh signaling input to the pMN domain at E12.5 was reduced in bromi mutant mice. In these mutants, induction of the FP structure was delayed and its size was reduced compared to wild type mice. Furthermore, while the p3 and pMN domains were induced, the length of the Nkx2.2-positive region and the number of Olig2-positive cells decreased. The number of OPCs was also significantly decreased in the E12.5 and E14.5 bromi mutant spinal cord. In contrast, motor neuron (MN) production, detected by HB9 expression, significantly increased. It is likely that the transition from MN production to OPC generation in the pMN domain is impaired in bromi mutant mice. These results suggest that strong Shh input to the pMN domain is not required for OPC generation but is essential for producing a sufficient number of OPCs. Copyright © 2017 Elsevier Ltd. All rights reserved.

Functional diversification of maize RNA polymerase IV and V subtypes via alternative catalytic subunits

DOE PAGES

Haag, Jeremy R.; Brower-Toland, Brent; Krieger, Elysia K.; ...

2014-10-02

Unlike nuclear multisubunit RNA polymerases I, II, and III, whose subunit compositions are conserved throughout eukaryotes, plant RNA polymerases IV and V are nonessential, Pol II-related enzymes whose subunit compositions are still evolving. Whereas Arabidopsis Pols IV and V differ from Pol II in four or five of their 12 subunits, respectively, and differ from one another in three subunits, proteomic analyses show that maize Pols IV and V differ from Pol II in six subunits but differ from each other only in their largest subunits. Use of alternative catalytic second subunits, which are nonredundant for development and paramutation, yieldsmore » at least two sub-types of Pol IV and three subtypes of Pol V in maize. Pol IV/Pol V associations with MOP1, RMR1, AGO121, Zm_DRD1/CHR127, SHH2a, and SHH2b extend parallels between paramutation in maize and the RNA-directed DNA methylation pathway in Arabidopsis.« less

Functional diversification of maize RNA polymerase IV and V subtypes via alternative catalytic subunits

PubMed Central

Haag, Jeremy R.; Brower-Toland, Brent; Krieger, Elysia K.; Sidorenko, Lyudmila; Nicora, Carrie D.; Norbeck, Angela D.; Irsigler, Andre; LaRue, Huachun; Brzeski, Jan; McGinnis, Karen; Ivashuta, Sergey; Pasa-Tolic, Ljiljana; Chandler, Vicki L.; Pikaard, Craig S.

2014-01-01

Summary Unlike nuclear multisubunit RNA polymerases I, II and III, whose subunit compositions are conserved throughout eukaryotes, plant RNA Polymerases IV and V are non-essential, Pol II-related enzymes whose subunit compositions are still evolving. Whereas Arabidopsis Pols IV and V differ from Pol II in four or five of their twelve subunits, respectively, and differ from one another in three subunits, proteomic analyses show that maize Pols IV and V differ from Pol II in six subunits, but differ from each other only in their largest subunits. Use of alternative catalytic second-subunits, which are non-redundant for development and paramutation, yields at least two subtypes of Pol IV, and three subtypes of Pol V in maize. Pol IV/V associations with MOP1, RMR1, AGO121, Zm_DRD1/CHR127, SHH2a and SHH2b extend parallels between paramutation in maize and the RNA-directed DNA methylation pathway in Arabidopsis. PMID:25284785

Gene expression analysis of murine cells producing amphotropic mouse leukaemia virus at a cultivation temperature of 32 and 37 degrees C.

PubMed

Beer, Christiane; Buhr, Petra; Hahn, Heidi; Laubner, Daniela; Wirth, Manfred

2003-07-01

Cultivation of retrovirus packaging cells at 32 degrees C represents a common procedure to achieve high titres in mouse retrovirus production. Gene expression profiling of mouse NIH 3T3 cells producing amphotropic mouse leukaemia virus 4070A revealed that 10 % of the 1176 cellular genes investigated were regulated by temperature shift (37/32 degrees C), while 5 % were affected by retrovirus infection. Strikingly, retrovirus production at 32 degrees C activated the cholesterol biosynthesis/transport pathway and caused an increase in plasma membrane cholesterol levels. Furthermore, these conditions resulted in transcriptional activation of smoothened (smo), patched (ptc) and gli-1; Smo, Ptc and Gli-1, as well as cholesterol, are components of the Sonic hedgehog (Shh) signalling pathway, which directs pattern formation, diversification and tumourigenesis in mammalian cells. These findings suggest a link between cultivation at 32 degrees C, production of MLV-A and the Shh signalling pathway.

Characterization of novel biomarkers in selecting for subtype specific medulloblastoma phenotypes.

PubMed

Liang, Lisa; Aiken, Christopher; McClelland, Robyn; Morrison, Ludivine Coudière; Tatari, Nazanin; Remke, Marc; Ramaswamy, Vijay; Issaivanan, Magimairajan; Ryken, Timothy; Del Bigio, Marc R; Taylor, Michael D; Werbowetski-Ogilvie, Tamra E

2015-11-17

Major research efforts have focused on defining cell surface marker profiles for characterization and selection of brain tumor stem/progenitor cells. Medulloblastoma is the most common primary malignant pediatric brain cancer and consists of 4 molecular subgroups: WNT, SHH, Group 3 and Group 4. Given the heterogeneity within and between medulloblastoma variants, surface marker profiles may be subtype-specific. Here, we employed a high throughput flow cytometry screen to identify differentially expressed cell surface markers in self-renewing vs. non-self-renewing SHH medulloblastoma cells. The top 25 markers were reduced to 4, CD271/p75NTR/NGFR, CD106/VCAM1, EGFR and CD171/NCAM-L1, by evaluating transcript levels in SHH tumors relative to samples representing the other variants. However, only CD271/p75NTR/NGFR and CD171/NCAM-L1 maintain differential expression between variants at the protein level. Functional characterization of CD271, a low affinity neurotrophin receptor, in cell lines and primary cultures suggested that CD271 selects for lower self-renewing progenitors or stem cells. Moreover, CD271 levels were negatively correlated with expression of SHH pathway genes. Our study reveals a novel role for CD271 in SHH medulloblastoma and suggests that targeting CD271 pathways could lead to the design of more selective therapies that lessen the broad impact of current treatments on developing nervous systems.

Active Sonic Hedgehog Signaling between Androgen Independent Human Prostate Cancer Cells and Normal/Benign but Not Cancer-Associated Prostate Stromal Cells

PubMed Central

Shigemura, Katsumi; Huang, Wen-Chin; Li, Xiangyan; Zhau, Haiyen E.; Zhu, Guodong; Gotoh, Akinobu; Fujisawa, Masato; Xie, Jingwu; Marshall, Fray F.; Chung, Leland W. K.

2012-01-01

BACKGROUND Sonic hedgehog (Shh) signaling plays a pivotal role in stromal-epithelial interaction during normal development but its role in tumor-stromal interaction during carcinogenic progression is less well defined. Since hormone refractory prostate cancer with bone metastasis is difficult to treat, it is crucial to investigate how androgen independent (AI) human prostate cancer cells communicate with their associated stroma. METHODS Shh and its target transcription factor, Gli1 mRNA, were assessed by RT-PCR and/or quantitative RT-PCR in co-cultured cell recombinants comprised of AI C4-2 either with NPF (prostate fibroblasts from normal/benign prostate gland) or CPF cancer-associated stromal fibroblasts) under Shh/cyclopamine (a hedgehog signaling inhibitor) treatment. Human bone marrow stromal (HS27A) cells were used as controls. In vivo investigation was performed by checking serum PSA and immunohistochemical staining for the apoptosis-associated M30 gene in mice bearing chimeric C4-2/NPF tumors. RESULTS CONCLUSIONS Based on co-culture and chimeric tumor models, active Shh-mediated signaling was demonstrated between AI prostate cancer and NPF in a paracrine- and tumor progression-dependent manner. Our study suggests that drugs like cyclopamine that interfere with Shh signaling could be beneficial in preventing AI progression in prostate cancer cells. PMID:21520153

The ADAMTS5 Metzincin Regulates Zebrafish Somite Differentiation

PubMed Central

Dancevic, Carolyn M.; Gibert, Yann; Smith, Adam D.; Ward, Alister C.; McCulloch, Daniel R.

2018-01-01

The ADAMTS5 metzincin, a secreted zinc-dependent metalloproteinase, modulates the extracellular matrix (ECM) during limb morphogenesis and other developmental processes. Here, the role of ADAMTS5 was investigated by knockdown of zebrafish adamts5 during embryogenesis. This revealed impaired Sonic Hedgehog (Shh) signaling during somite patterning and early myogenesis. Notably, synergistic regulation of myod expression by ADAMTS5 and Shh during somite differentiation was observed. These roles were not dependent upon the catalytic activity of ADAMTS5. These data identify a non-enzymatic function for ADAMTS5 in regulating an important cell signaling pathway that impacts on muscle development, with implications for musculoskeletal diseases in which ADAMTS5 and Shh have been associated. PMID:29518972

Sonic Hedgehog Initiates Cochlear Hair Cell Regeneration through Downregulation of Retinoblastoma Protein

PubMed Central

Lu, Na; Chen, Yan; Wang, Zhengmin; Chen, Guoling; Lin, Qin; Chen, Zheng-Yi; Li, Huawei

2013-01-01

Cell cycle re-entry by cochlear supporting cells and/or hair cells is considered one of the best approaches for restoring hearing loss as a result of hair cell damage. To identify mechanisms that can be modulated to initiate cell cycle re-entry and hair cell regeneration, we studied the effect of activating the sonic hedgehog (Shh) pathway. We show that Shh signaling in postnatal rat cochleae damaged by neomycin leads to renewed proliferation of supporting cells and hair cells. Further, proliferating supporting cells are likely to transdifferentiate into hair cells. Shh treatment leads to inhibition of retinoblastoma protein (pRb) by increasing phosphorylated pRb and reducing retinoblastoma gene transcription. This results in upregulation of cyclins B1, D2, and D3, and CDK1. These results suggest that Shh signaling induces cell cycle re-entry in cochlear sensory epithelium and the production of new hair cells, in part by attenuating pRb function. This study provides an additional route to modulate pRb function with important implications in mammalian hair cell regeneration. PMID:23211596

Transient Overexpression of Sonic Hedgehog Alters the Architecture and Mechanical Properties of Trabecular Bone

PubMed Central

Kiuru, Maija; Solomon, Jason; Ghali, Bassem; van der Meulen, Marjolein; Crystal, Ronald G; Hidaka, Chisa

2009-01-01

Bone formation and remodeling involve coordinated interactions between osteoblasts and osteoclasts through signaling networks involving a variety of molecular pathways. We hypothesized that overexpression of Sonic hedgehog (Shh), a morphogen with a crucial role in skeletal development, would stimulate osteoblastogenesis and bone formation in adult animals in vivo. Systemic administration of adenovirus expressing the N-terminal form of Shh into adult mice resulted in a primary increase in osteoblasts and their precursors. Surprisingly, however, this was associated with altered trabecular morphology, decreased bone volume, and decreased compressive strength in the vertebrae. Whereas no change was detected in the number of osteoclast precursors, bone marrow stromal cells from Shh-treated mice showed enhanced osteoclastogenic potential in vitro. These effects were mediated by the PTH/PTH-related protein (PTHrP) pathway as evidenced by increased sensitivity to PTH stimulation and upregulation of the PTH/PTHrP receptor (PPR). Together, these data show that Shh has stimulatory effects on osteoprogenitors and osteoblasts in adult animals in vivo, which results in bone remodeling and reduced bone strength because of a secondary increase in osteoclastogenesis. PMID:19338448

A new role for Hedgehogs in juxtacrine signaling.

PubMed

Pettigrew, Christopher A; Asp, Eva; Emerson, Charles P

2014-02-01

The Hedgehog pathway plays important roles in embryonic development, adult stem cell maintenance and tumorigenesis. In mammals these effects are mediated by Sonic, Desert and Indian Hedgehog (Shh, Dhh and Ihh). Shh undergoes autocatalytic cleavage and dual lipidation prior to secretion and forming a response gradient. Post-translational processing and secretion of Dhh and Ihh ligands has not previously been investigated. This study reports on the synthesis, processing, secretion and signaling activities of SHH, IHH and DHH preproteins expressed in cultured cells, providing unexpected evidence that DHH does not undergo substantial autoprocessing or secretion, and does not function in paracrine signaling. Rather, DHH functions as a juxtacrine signaling ligand to activate a cell contact-mediated HH signaling response, consistent with its localised signaling in vivo. Further, the LnCAP prostate cancer cell, when induced to express endogenous DHH and SHH, is active only in juxtacrine signaling. Domain swap studies reveal that the C-terminal domain of HH regulates its processing and secretion. These findings establish a new regulatory role for HHs in cell-mediated juxtacrine signaling in development and cancer. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

HIF-1 maintains a functional relationship between pancreatic cancer cells and stromal fibroblasts by upregulating expression and secretion of Sonic hedgehog

PubMed Central

Katagiri, Tomohiro; Kobayashi, Minoru; Yoshimura, Michio; Morinibu, Akiyo; Itasaka, Satoshi; Hiraoka, Masahiro; Harada, Hiroshi

2018-01-01

Hypoxic and stroma-rich microenvironments, characteristic features of pancreatic cancers, are strongly associated with a poor prognosis. However, whether and how hypoxia increases stromal compartments remain largely unknown. Here, we investigated the potential importance of a master regulator of the cellular adaptive response to hypoxia, hypoxia-inducible factor-1 (HIF-1), in the formation of stroma-rich microenvironments of pancreatic tumors. We found that pancreatic cancer cells secreted more Sonic hedgehog protein (SHH) under hypoxia by upregulating its expression and efficiency of secretion in a HIF-1-dependent manner. Recombinant SHH, which was confirmed to activate the hedgehog signaling pathway, accelerated the growth of fibroblasts in a dose-dependent manner. The SHH protein secreted from pancreatic cancer cells under hypoxic conditions promoted the growth of fibroblasts by stimulating their Sonic hedgehog signaling pathway. These results suggest that the increased secretion of SHH by HIF-1 is potentially responsible for the formation of detrimental and stroma-rich microenvironments in pancreatic cancers, therefore providing a rational basis to target it in cancer therapy. PMID:29535824

Constitutive Activation of Smoothened in the Renal Collecting Ducts Leads to Renal Hypoplasia, Hydronephrosis, and Hydroureter.

PubMed

Gupta, Deepak Prasad; Hwang, Jae-Won; Cho, Eui-Sic; Kim, Won; Song, Chang Ho; Chai, Ok Hee

2017-01-01

Sonic Hedgehog (Shh) signaling plays a major role in and is essential for regulation, patterning, and proliferation during renal development. Smoothened (Smo) plays a pivot role in transducing the Shh-glioma-associated oncogene Kruppel family member. However, the cellular and molecular mechanism underlying the role of sustained Smo activation in postnatal kidney development is still not clearly understood. Using a conditional knockin mouse model that expresses a constitutively activated form of Smo (SmoM2) upon Homeobox-B7-mediated recombination (Hoxb7-Cre), the effects of Shh signaling were determined in postnatal kidney development. SmoM2;Hoxb7-Cre mutant mice showed growth retardation with a reduction of body weight. Constitutive activation of Smo in the renal collecting ducts caused renal hypoplasia, hydronephrosis, and hydroureter. The parenchymal area and glomerular numbers were reduced, but the glomerular density was increased in SmoM2;Hoxb7-Cre mutant mice. The expression of Patched 1, the receptor of Shh and a downstream target gene of the Shh signaling pathway, was highly restricted and it was upregulated in the inner medullary collecting ducts of the kidney. The proliferative cells in the mesenchyme and collecting ducts were decreased in SmoM2;Hoxb7-Cre mutant mice. This study showed for the first time that sustained Smo inhibits postnatal kidney development by suppressing the proliferation of the mesenchyme and medullary collecting ducts in mice. © 2017 S. Karger AG, Basel.

Usp7 promotes medulloblastoma cell survival and metastasis by activating Shh pathway

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

Zhan, Meixiao; Zhuhai Precision Medicine Center, Zhuhai People's Hospital, Jinan University, Zhuhai; Sun, Xiaohan

The ubiquitin-specific protease Usp7 plays roles in multiple cellular processes through deubiquitinating and stabilizing numerous substrates, including P53, Pten and Gli. Aberrant Usp7 activity has been implicated in many disorders and tumorigenesis, making it as a potential target for therapeutic intervention. Although it is clear that Usp7 is involved in many types of cancer, its role in regulating medulloblastoma (MB) is still unknown. In this study, we show that knockdown of Usp7 inhibits the proliferation and migration of MB cells, while Usp7 overexpression exerts an opposite effect. Furthermore, we establish Usp7 knockout MB cell line using the CRISPR/Cas9 system andmore » further confirm that Usp7 knockout also blocks MB cell proliferation and metastasis. In addition, we reveal that knockdown of Usp7 compromises Shh pathway activity and decrease Gli protein levels, while P53 level and P53 target gene expression have no obvious changes. Finally, we find that Usp7 inhibitors apparently inhibit MB cell viability and migration. Taken together, our findings suggest that Usp7 is important for MB cell proliferation and metastasis by activating Shh pathway, and is a putative therapeutic target for MBs. - Highlights: • Loss of usp7 blocks the proliferation and metastasis of MB cells. • Usp7 regulates MB cell growth and migration through stimulating Shh pathway. • Usp7 inhibitors hamper MB cell proliferation and migration. • Usp7 inhibitors could attenuate Shh pathway activity.« less

An early role for sonic hedgehog from foregut endoderm in jaw development: ensuring neural crest cell survival.

PubMed

Brito, José M; Teillet, Marie-Aimée; Le Douarin, Nicole M

2006-08-01

We have investigated the role of Sonic hedgehog (Shh) in the development of facial structures by depriving chicken embryos of the most anterior sources of this morphogen, including the prechordal plate and the anterior ventral endoderm of the foregut, before the onset of neural crest cell (NCC) migration to the first branchial arch (BA1). The entire forehead, including the foregut endoderm, was removed at 5- to 10-somite stage (ss), which led to the absence of the lower jaw when the operation was performed before 7-ss. If the embryos were deprived of their forehead at 8- to 10-ss, they were later on endowed with a lower beak. In embryos that were operated on early, the NCCs migrated normally to BA1 but were subjected to massive apoptosis a few hours later. Cell death did not occur when forehead excision was performed at a later stage. In this case, onward expression of Shh in the ventral foregut endoderm extended caudally over the excision limit, and we hypothesized that absence of Shh production by the endoderm in embryos that were operated on early could be responsible for the NCC apoptosis and the failure of BA1 development. We thus provided exogenous Shh to the embryos that were operated on before 7-ss. In this case, the development of the lower jaw was rescued. Therefore, Shh derived from the ventral foregut endoderm ensures the survival of NCCs at a critical stage of BA1 development.

SHOX mutations in idiopathic short stature and Leri-Weill dyschondrosteosis: frequency and phenotypic variability.

PubMed

Jorge, Alexander A L; Souza, Silvia C; Nishi, Miriam Y; Billerbeck, Ana E; Libório, Débora C C; Kim, Chong A; Arnhold, Ivo J P; Mendonca, Berenice B

2007-01-01

The frequency of SHOX mutations in children with idiopathic short stature (ISS) has been found to be variable. We analysed the SHOX gene in children with ISS and Leri-Weill dyschondrosteosis (LWD) and evaluated the phenotypic variability in patients harbouring SHOX mutations. Sixty-three ISS, nine LWD children and 21 affected relatives. SHOX gene deletion was evaluated by fluorescence in situ hybridization (FISH), Southern blotting and segregation study of polymorphic marker. Point mutations were assessed by direct DNA sequencing. None of the ISS patients presented SHOX deletions, but two (3.2%) presented heterozygous point mutations, including the novel R147H mutation. However, when ISS patients were selected by sitting height : height ratio (SH/H) for age > 2 SD, mutation frequency detection increased to 22%. Eight (89%) LWD patients had SHOX deletions, but none had point mutations. Analysis of the other relatives in the families carrying SHOX mutations identified 14 children and 17 adult patients. A broad phenotypic variability was observed in all families regarding short stature severity and Madelung deformities. However, the presence of disproportional height, assessed by SH/H, was observed in all children and 82% of adult patients, being the most common feature in our patients with SHOX mutations. Patients with SHOX mutations present a broad phenotypic variability. SHOX mutations are very frequent in LWD (89%), in opposition to ISS (3.2%) in our cohort. The use of SH/H SDS as a selection criterion increases the frequency of SHOX mutation detection to 22% and should be used for selecting ISS children to undergo SHOX mutation molecular studies.

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

PubMed Central

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

2010-01-01

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

Identification of a neuronal transcription factor network involved in medulloblastoma development

PubMed Central

2013-01-01

Background Medulloblastomas, the most frequent malignant brain tumours affecting children, comprise at least 4 distinct clinicogenetic subgroups. Aberrant sonic hedgehog (SHH) signalling is observed in approximately 25% of tumours and defines one subgroup. Although alterations in SHH pathway genes (e.g. PTCH1, SUFU) are observed in many of these tumours, high throughput genomic analyses have identified few other recurring mutations. Here, we have mutagenised the Ptch+/- murine tumour model using the Sleeping Beauty transposon system to identify additional genes and pathways involved in SHH subgroup medulloblastoma development. Results Mutagenesis significantly increased medulloblastoma frequency and identified 17 candidate cancer genes, including orthologs of genes somatically mutated (PTEN, CREBBP) or associated with poor outcome (PTEN, MYT1L) in the human disease. Strikingly, these candidate genes were enriched for transcription factors (p=2x10-5), the majority of which (6/7; Crebbp, Myt1L, Nfia, Nfib, Tead1 and Tgif2) were linked within a single regulatory network enriched for genes associated with a differentiated neuronal phenotype. Furthermore, activity of this network varied significantly between the human subgroups, was associated with metastatic disease, and predicted poor survival specifically within the SHH subgroup of tumours. Igf2, previously implicated in medulloblastoma, was the most differentially expressed gene in murine tumours with network perturbation, and network activity in both mouse and human tumours was characterised by enrichment for multiple gene-sets indicating increased cell proliferation, IGF signalling, MYC target upregulation, and decreased neuronal differentiation. Conclusions Collectively, our data support a model of medulloblastoma development in SB-mutagenised Ptch+/- mice which involves disruption of a novel transcription factor network leading to Igf2 upregulation, proliferation of GNPs, and tumour formation. Moreover, our results identify rational therapeutic targets for SHH subgroup tumours, alongside prognostic biomarkers for the identification of poor-risk SHH patients. PMID:24252690

Directed midbrain and spinal cord neurogenesis from pluripotent stem cells to model development and disease in a dish

PubMed Central

Allodi, Ilary; Hedlund, Eva

2014-01-01

Induction of specific neuronal fates is restricted in time and space in the developing CNS through integration of extrinsic morphogen signals and intrinsic determinants. Morphogens impose regional characteristics on neural progenitors and establish distinct progenitor domains. Such domains are defined by unique expression patterns of fate determining transcription factors. These processes of neuronal fate specification can be recapitulated in vitro using pluripotent stem cells. In this review, we focus on the generation of dopamine neurons and motor neurons, which are induced at ventral positions of the neural tube through Sonic hedgehog (Shh) signaling, and defined at anteroposterior positions by fibroblast growth factor (Fgf) 8, Wnt1, and retinoic acid (RA). In vitro utilization of these morphogenic signals typically results in the generation of multiple neuronal cell types, which are defined at the intersection of these signals. If the purpose of in vitro neurogenesis is to generate one cell type only, further lineage restriction can be accomplished by forced expression of specific transcription factors in a permissive environment. Alternatively, cell-sorting strategies allow for selection of neuronal progenitors or mature neurons. However, modeling development, disease and prospective therapies in a dish could benefit from structured heterogeneity, where desired neurons are appropriately synaptically connected and thus better reflect the three-dimensional structure of that region. By modulating the extrinsic environment to direct sequential generation of neural progenitors within a domain, followed by self-organization and synaptic establishment, a reductionist model of that brain region could be created. Here we review recent advances in neuronal fate induction in vitro, with a focus on the interplay between cell intrinsic and extrinsic factors, and discuss the implications for studying development and disease in a dish. PMID:24904255

Directed midbrain and spinal cord neurogenesis from pluripotent stem cells to model development and disease in a dish.

PubMed

Allodi, Ilary; Hedlund, Eva

2014-01-01

Induction of specific neuronal fates is restricted in time and space in the developing CNS through integration of extrinsic morphogen signals and intrinsic determinants. Morphogens impose regional characteristics on neural progenitors and establish distinct progenitor domains. Such domains are defined by unique expression patterns of fate determining transcription factors. These processes of neuronal fate specification can be recapitulated in vitro using pluripotent stem cells. In this review, we focus on the generation of dopamine neurons and motor neurons, which are induced at ventral positions of the neural tube through Sonic hedgehog (Shh) signaling, and defined at anteroposterior positions by fibroblast growth factor (Fgf) 8, Wnt1, and retinoic acid (RA). In vitro utilization of these morphogenic signals typically results in the generation of multiple neuronal cell types, which are defined at the intersection of these signals. If the purpose of in vitro neurogenesis is to generate one cell type only, further lineage restriction can be accomplished by forced expression of specific transcription factors in a permissive environment. Alternatively, cell-sorting strategies allow for selection of neuronal progenitors or mature neurons. However, modeling development, disease and prospective therapies in a dish could benefit from structured heterogeneity, where desired neurons are appropriately synaptically connected and thus better reflect the three-dimensional structure of that region. By modulating the extrinsic environment to direct sequential generation of neural progenitors within a domain, followed by self-organization and synaptic establishment, a reductionist model of that brain region could be created. Here we review recent advances in neuronal fate induction in vitro, with a focus on the interplay between cell intrinsic and extrinsic factors, and discuss the implications for studying development and disease in a dish.

The transcription factor Foxg1 regulates the competence of telencephalic cells to adopt subpallial fates in mice

PubMed Central

Manuel, Martine; Martynoga, Ben; Yu, Tian; West, John D.; Mason, John O.; Price, David J.

2010-01-01

Summary Foxg1 is required for development of the ventral telencephalon in the embryonic mammalian forebrain. Although one existing hypothesis suggests that failed ventral telencephalic development in the absence of Foxg1 is due to reduced production of the morphogens sonic hedgehog (Shh) and fibroblast growth factor 8 (Fgf8), the possibility that telencephalic cells lacking Foxg1 are intrinsically incompetent to generate the ventral telencephalon has remained untested. We examined the ability of Foxg1−/− telencephalic cells to respond to Shh and Fgf8 by examining the expression of genes whose activation requires Shh or Fgf8 in vivo and by testing their responses to Shh and Fgf8 in culture. We found that many elements of the Shh and Fgf8 signalling pathways continue to function in the absence of Foxg1 but, nevertheless, we were unable to elicit normal responses of key ventral telencephalic marker genes in Foxg1−/− telencephalic tissue following a range of in vivo and in vitro manipulations. We explored the development of Foxg1−/− cells in Foxg1−/− Foxg1+/+ chimeric embryos that contained ventral telencephalon created by normally patterned wild-type cells. We found that Foxg1−/− cells contributed to the chimeric ventral telencephalon, but that they retained abnormal specification, expressing dorsal rather than ventral telencephalic markers. These findings indicate that, in addition to regulating the production of ventralising signals, Foxg1 acts cell-autonomously in the telencephalon to ensure that cells develop the competence to adopt ventral identities. PMID:20081193

Targeting placental growth factor/neuropilin 1 pathway inhibits growth and spread of medulloblastoma

PubMed Central

Snuderl, Matija; Batista, Ana; Kirkpatrick, Nathaniel D.; de Almodovar, Carmen Ruiz; Riedemann, Lars; Walsh, Elisa C.; Anolik, Rachel; Huang, Yuhui; Martin, John D.; Kamoun, Walid; Knevels, Ellen; Schmidt, Thomas; Farrar, Christian T.; Vakoc, Benjamin J.; Mohan, Nishant; Chung, Euiheon; Roberge, Sylvie; Peterson, Teresa; Bais, Carlos; Zhelyazkova, Boryana H.; Yip, Stephen; Hasselblatt, Martin; Rossig, Claudia; Niemeyer, Elisabeth; Ferrara, Napoleone; Klagsbrun, Michael; Duda, Dan G.; Fukumura, Dai; Xu, Lei; Carmeliet, Peter; Jain, Rakesh K.

2013-01-01

SUMMARY Medulloblastoma is the most common pediatric malignant brain tumor. Although current therapies improve survival, these regimens are highly toxic and associated with significant morbidity. Here, we report that placental growth factor (PlGF) is expressed in the majority of medulloblastomas independent of their subtype. Moreover, high expression of PlGF receptor neuropilin 1 (Nrp1) correlates with poor overall survival in patients. We demonstrate that PlGF and Nrp1 are required for the growth and spread of medulloblastoma: PlGF/Nrp1 blockade results in direct antitumor effects in vivo, resulting in medulloblastoma regression, decreased metastases, and increased mouse survival. We reveal that PlGF is produced in the cerebellar stroma via tumor-derived Sonic hedgehog (Shh) and show that PlGF acts through Nrp1—and not vascular endothelial growth factor receptor 1 (VEGFR1)—to promote tumor cell survival. This critical tumor-stroma interaction—mediated by Shh, PlGF, and Nrp1 across medulloblastoma subtypes—supports the development of therapies targeting PlGF/Nrp1 pathway. PMID:23452854

Regulator of G protein signaling 5 (RGS5) inhibits sonic hedgehog function in mouse cortical neurons.

PubMed

Liu, Chuanliang; Hu, Qiongqiong; Jing, Jia; Zhang, Yun; Jin, Jing; Zhang, Liulei; Mu, Lili; Liu, Yumei; Sun, Bo; Zhang, Tongshuai; Kong, Qingfei; Wang, Guangyou; Wang, Dandan; Zhang, Yao; Liu, Xijun; Zhao, Wei; Wang, Jinghua; Feng, Tao; Li, Hulun

2017-09-01

Regulator of G protein signaling 5 (RGS5) acts as a GTPase-activating protein (GAP) for the Gαi subunit and negatively regulates G protein-coupled receptor signaling. However, its presence and function in postmitotic differentiated primary neurons remains largely uncharacterized. During neural development, sonic hedgehog (Shh) signaling is involved in cell signaling pathways via Gαi activity. In particular, Shh signaling is essential for embryonic neural tube patterning, which has been implicated in neuronal polarization involving neurite outgrowth. Here, we examined whether RGS5 regulates Shh signaling in neurons. RGS5 transcripts were found to be expressed in cortical neurons and their expression gradually declined in a time-dependent manner in culture system. When an adenovirus expressing RGS5 was introduced into an in vitro cell culture model of cortical neurons, RGS5 overexpression significantly reduced neurite outgrowth and FM4-64 uptake, while cAMP-PKA signaling was also affected. These findings suggest that RGS5 inhibits Shh function during neurite outgrowth and the presynaptic terminals of primary cortical neurons mature via modulation of cAMP. Copyright © 2017 Elsevier Inc. All rights reserved.

Two domains in vertebral development: antagonistic regulation by SHH and BMP4 proteins.

PubMed

Watanabe, Y; Duprez, D; Monsoro-Burq, A H; Vincent, C; Le Douarin, N M

1998-07-01

It has previously been shown that the notochord grafted laterally to the neural tube enhances the differentiation of the vertebral cartilage at the expense of the derivatives of the dermomyotome. In contrast, the dorsomedial graft of a notochord inhibits cartilage differentiation of the dorsal part of the vertebra carrying the spinous process. Cartilage differentiation is preceded by the expression of transcription factors of the Pax family (Pax1/Pax9) in the ventrolateral domain and of the Msx family in the dorsal domain. The proliferation and differentiation of Msx-expressing cells in the dorsal precartilaginous domain of the vertebra are stimulated by BMP4, which acts upstream of Msx genes. It has previously been shown that the SHH protein arising from the notochord (and floor plate) is necessary for the survival and further development of Pax1/Pax9-expressing sclerotomal cells. We show here that SHH acts antagonistically to BMP4. SHH-producing cells grafted dorsally to the neural tube at E2 inhibit expression of Bmp4 and Msx genes and also inhibits the differentiation of the spinous process. We present a model that accounts for cartilage differentiation in the vertebra.

SoxB1-driven transcriptional network underlies neural-specific interpretation of morphogen signals.

PubMed

Oosterveen, Tony; Kurdija, Sanja; Ensterö, Mats; Uhde, Christopher W; Bergsland, Maria; Sandberg, Magnus; Sandberg, Rickard; Muhr, Jonas; Ericson, Johan

2013-04-30

The reiterative deployment of a small cadre of morphogen signals underlies patterning and growth of most tissues during embyogenesis, but how such inductive events result in tissue-specific responses remains poorly understood. By characterizing cis-regulatory modules (CRMs) associated with genes regulated by Sonic hedgehog (Shh), retinoids, or bone morphogenetic proteins in the CNS, we provide evidence that the neural-specific interpretation of morphogen signaling reflects a direct integration of these pathways with SoxB1 proteins at the CRM level. Moreover, expression of SoxB1 proteins in the limb bud confers on mesodermal cells the potential to activate neural-specific target genes upon Shh, retinoid, or bone morphogenetic protein signaling, and the collocation of binding sites for SoxB1 and morphogen-mediatory transcription factors in CRMs faithfully predicts neural-specific gene activity. Thus, an unexpectedly simple transcriptional paradigm appears to conceptually explain the neural-specific interpretation of pleiotropic signaling during vertebrate development. Importantly, genes induced in a SoxB1-dependent manner appear to constitute repressive gene regulatory networks that are directly interlinked at the CRM level to constrain the regional expression of patterning genes. Accordingly, not only does the topology of SoxB1-driven gene regulatory networks provide a tissue-specific mode of gene activation, but it also determines the spatial expression pattern of target genes within the developing neural tube.

Melittin induces PTCH1 expression by down-regulating MeCP2 in human hepatocellular carcinoma SMMC-7721 cells

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

Wu, Xiaoqin; Zhao, Bin; Cheng, Yahui

Hepatocellular carcinoma (HCC) has a high mortality rate worldwide and still remains to be a noticeable public health problem. Therefore, new remedies are urgently needed. Melittin, a major component of bee venom, is known to suppress cell growth in various cancers including HCC. However, the mechanism of the anticancer effect of melittin on HCC has not been fully elucidated. It has been reported that Methyl-CpG binding protein 2 (MeCP2) plays a key role in tumor proliferation, apoptosis, migration and invasion. In the present study, we found the high expression of MeCP2 in human HCC tissues and in the SMMC-7721 cellmore » line. MeCP2 silencing inhibited cell proliferation, while over-expression of MeCP2 promoted cell growth in SMMC-7721 cells. It indicates that MeCP2 may be an attractive target for human HCC. We further found that melittin could inhibit cell proliferation by reducing MeCP2 expression in vitro. Interestingly, the inhibitory effect of melittin on cell proliferation was due to a delay in G{sub 0}/G{sub 1} cell cycle progression, without influencing cell apoptosis. Next, we investigated the potential molecular mechanisms and found that MeCP2 could modulate Shh signaling in SMMC-7721 cells. Further study indicates that melittin may induce the demethylation of PTCH1 promoter, resulting in the increased expression of PTCH1. Furthermore, the expression of Shh and GLI1 was significantly lowered upon treatment of melittin. These results suggest that melittin can block Shh signaling in vitro. In short, these results indicate that melittin inhibits cell proliferation by down-regulating MeCP2 through Shh signaling in SMMC-7721 cells. - Highlights: • MeCP2 plays a key role in the proliferation of human HCC cells. • Melittin reduces MeCP2 expression in vitro. • Melittin induces G{sub 0}/G{sub 1} cell cycle arrest in SMMC-7721 cells. • MeCP2 modulates the Shh signaling pathway in SMMC-7721 cells. • Melittin blocks the Shh signaling pathway in SMMC-7721 cells.« less

Molecular subgroups of medulloblastoma identification using noninvasive magnetic resonance spectroscopy.

PubMed

Blüml, Stefan; Margol, Ashley S; Sposto, Richard; Kennedy, Rebekah J; Robison, Nathan J; Vali, Marzieh; Hung, Long T; Muthugounder, Sakunthala; Finlay, Jonathan L; Erdreich-Epstein, Anat; Gilles, Floyd H; Judkins, Alexander R; Krieger, Mark D; Dhall, Girish; Nelson, Marvin D; Asgharzadeh, Shahab

2016-01-01

Medulloblastomas in children can be categorized into 4 molecular subgroups with differing clinical characteristics, such that subgroup determination aids in prognostication and risk-adaptive treatment strategies. Magnetic resonance spectroscopy (MRS) is a widely available, noninvasive tool that is used to determine the metabolic characteristics of tumors and provide diagnostic information without the need for tumor tissue. In this study, we investigated the hypothesis that metabolite concentrations measured by MRS would differ between molecular subgroups of medulloblastoma and allow accurate subgroup determination. MRS was used to measure metabolites in medulloblastomas across molecular subgroups (SHH = 12, Groups 3/4 = 17, WNT = 1). Levels of 14 metabolites were analyzed to determine those that were the most discriminant for medulloblastoma subgroups in order to construct a multivariable classifier for distinguishing between combined Group 3/4 and SHH tumors. Medulloblastomas across molecular subgroups revealed distinct spectral features. Group 3 and Group 4 tumors demonstrated metabolic profiles with readily detectable taurine, lower levels of lipids, and high levels of creatine. SHH tumors showed prominent choline and lipid with low levels of creatine and little or no evidence of taurine. A 5-metabolite subgroup classifier inclusive of creatine, myo-inositol, taurine, aspartate, and lipid 13a was developed that could discriminate between Group 3/4 and SHH medulloblastomas with excellent accuracy (cross-validated area under the curve [AUC] = 0.88). The data show that medulloblastomas of Group 3/4 differ metabolically as measured using MRS when compared with SHH molecular subgroups. MRS is a useful and accurate tool to determine medulloblastoma molecular subgroups. © The Author(s) 2015. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Prenatal cadmium exposure dysregulates sonic hedgehog and Wnt/beta-catenin signaling in the thymus resulting in altered thymocyte development

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

Hanson, Miranda L.; Brundage, Kathleen M.; Schafer, Rosana

2010-01-15

Cadmium (Cd) is both an environmental pollutant and a component of cigarette smoke. Although evidence demonstrates that adult exposure to Cd causes changes in the immune system, there are limited reports in the literature of immunomodulatory effects of prenatal exposure to Cd. The sonic hedgehog (Shh) and Wnt/beta-catenin pathways are required for thymocyte maturation. Several studies have demonstrated that Cd exposure affects these pathways in different organ systems. This study was designed to investigate the effect of prenatal Cd exposure on thymocyte development, and to determine if these effects were linked to dysregulation of Shh and Wnt/beta-catenin pathways. Pregnant C57Bl/6more » mice were exposed to an environmentally relevant dose (10 ppm) of Cd throughout pregnancy and effects on the thymus were assessed on the day of birth. Thymocyte phenotype was determined by flow cytometry. A Gli:luciferase reporter cell line was used to measure Shh signaling. Transcription of target genes and translation of key components of both signaling pathways were assessed using real-time RT-PCR and western blot, respectively. Prenatal Cd exposure increased the number of CD4{sup +} cells and a subpopulation of double-negative cells (DN; CD4{sup -}CD8{sup -}), DN4 (CD44{sup -}CD25{sup -}). Shh and Wnt/beta-catenin signaling were both decreased in the thymus. Target genes of Shh (Patched1 and Gli1) and Wnt/beta-catenin (c-fos, and c-myc) were affected differentially among thymocyte subpopulations. These findings suggest that prenatal exposure to Cd dysregulates two signaling pathways in the thymus, resulting in altered thymocyte development.« less

Mesenchymal stem cells induce epithelial proliferation within the inflamed stomach

PubMed Central

Donnelly, Jessica M.; Engevik, Amy; Feng, Rui; Xiao, Chang; Boivin, Gregory P.; Li, Jing; Houghton, JeanMarie

2014-01-01

Bone marrow-derived mesenchymal stem cells (MSCs) sustain cancer cells by creating a microenvironment favorable for tumor growth. In particular, MSCs have been implicated in gastric cancer development. There is extensive evidence suggesting that Hedgehog signaling regulates tumor growth. However, very little is known regarding the precise roles of Hedgehog signaling and MSCs in tumor development within the stomach. The current study tests that hypothesis that Sonic Hedgehog (Shh), secreted from MSCs, provides a proliferative stimulus for the gastric epithelium in the presence of inflammation. Red fluorescent protein-expressing MSCs transformed in vitro (stMSCs) were transduced with lentiviral constructs containing a vector control (stMSCvect) or short hairpin RNA (shRNA) targeting the Shh gene (stMSCShhKO). Gastric submucosal transplantation of wild-type MSCs (wtMSCs), wild-type MSCs overexpressing Shh (wtMSCShh), stMSCvect, or stMSCShhKO cells in C57BL/6 control (BL/6) or gastrin-deficient (GKO) mice was performed and mice analyzed 30 and 60 days posttransplantation. Compared with BL/6 mice transplanted with wtMSCShh and stMSCvect cells, inflamed GKO mice developed aggressive gastric tumors. Tumor development was not observed in mouse stomachs transplanted with wtMSC or stMSCShhKO cells. Compared with stMSCShhKO-transplanted mice, within the inflamed GKO mouse stomach, Shh-expressing stMSCvect- and wtMSCShh-induced proliferation of CD44-positive cells. CD44-positive cells clustered in gland-like structures within the tumor stroma and were positive for Patched (Ptch) expression. We conclude that Shh, secreted from MSCs, provides a proliferative stimulus for the gastric epithelium that is associated with tumor development, a response that is sustained by chronic inflammation. PMID:24789207

Clinical and experimental evidence suggest a link between KIF7 and C5orf42-related ciliopathies through Sonic Hedgehog signaling.

PubMed

Asadollahi, Reza; Strauss, Justin E; Zenker, Martin; Beuing, Oliver; Edvardson, Simon; Elpeleg, Orly; Strom, Tim M; Joset, Pascal; Niedrist, Dunja; Otte, Christine; Oneda, Beatrice; Boonsawat, Paranchai; Azzarello-Burri, Silvia; Bartholdi, Deborah; Papik, Michael; Zweier, Markus; Haas, Cordula; Ekici, Arif B; Baumer, Alessandra; Boltshauser, Eugen; Steindl, Katharina; Nothnagel, Michael; Schinzel, Albert; Stoeckli, Esther T; Rauch, Anita

2018-02-01

Acrocallosal syndrome (ACLS) is an autosomal recessive neurodevelopmental disorder caused by KIF7 defects and belongs to the heterogeneous group of ciliopathies related to Joubert syndrome (JBTS). While ACLS is characterized by macrocephaly, prominent forehead, depressed nasal bridge, and hypertelorism, facial dysmorphism has not been emphasized in JBTS cohorts with molecular diagnosis. To evaluate the specificity and etiology of ACLS craniofacial features, we performed whole exome or targeted Sanger sequencing in patients with the aforementioned overlapping craniofacial appearance but variable additional ciliopathy features followed by functional studies. We found (likely) pathogenic variants of KIF7 in 5 out of 9 families, including the original ACLS patients, and delineated 1000 to 4000-year-old Swiss founder alleles. Three of the remaining families had (likely) pathogenic variants in the JBTS gene C5orf42, and one patient had a novel de novo frameshift variant in SHH known to cause autosomal dominant holoprosencephaly. In accordance with the patients' craniofacial anomalies, we showed facial midline widening after silencing of C5orf42 in chicken embryos. We further supported the link between KIF7, SHH, and C5orf42 by demonstrating abnormal primary cilia and diminished response to a SHH agonist in fibroblasts of C5orf42-mutated patients, as well as axonal pathfinding errors in C5orf42-silenced chicken embryos similar to those observed after perturbation of Shh signaling. Our findings, therefore, suggest that beside the neurodevelopmental features, macrocephaly and facial widening are likely more general signs of disturbed SHH signaling. Nevertheless, long-term follow-up revealed that C5orf42-mutated patients showed catch-up development and fainting of facial features contrary to KIF7-mutated patients.

Functional analysis of the zebrafish ortholog of HMGCS1 reveals independent functions for cholesterol and isoprenoids in craniofacial development

PubMed Central

Hernandez, Jose A.; Gonzalez, Cesar G.

2017-01-01

There are 8 different human syndromes caused by mutations in the cholesterol synthesis pathway. A subset of these disorders such as Smith-Lemli-Opitz disorder, are associated with facial dysmorphia. However, the molecular and cellular mechanisms underlying such facial deficits are not fully understood, primarily because of the diverse functions associated with the cholesterol synthesis pathway. Recent evidence has demonstrated that mutation of the zebrafish ortholog of HMGCR results in orofacial clefts. Here we sought to expand upon these data, by deciphering the cholesterol dependent functions of the cholesterol synthesis pathway from the cholesterol independent functions. Moreover, we utilized loss of function analysis and pharmacological inhibition to determine the extent of sonic hedgehog (Shh) signaling in animals with aberrant cholesterol and/or isoprenoid synthesis. Our analysis confirmed that mutation of hmgcs1, which encodes the first enzyme in the cholesterol synthesis pathway, results in craniofacial abnormalities via defects in cranial neural crest cell differentiation. Furthermore targeted pharmacological inhibition of the cholesterol synthesis pathway revealed a novel function for isoprenoid synthesis during vertebrate craniofacial development. Mutation of hmgcs1 had no effect on Shh signaling at 2 and 3 days post fertilization (dpf), but did result in a decrease in the expression of gli1, a known Shh target gene, at 4 dpf, after morphological deficits in craniofacial development and chondrocyte differentiation were observed in hmgcs1 mutants. These data raise the possibility that deficiencies in cholesterol modulate chondrocyte differentiation by a combination of Shh independent and Shh dependent mechanisms. Moreover, our results describe a novel function for isoprenoids in facial development and collectively suggest that cholesterol regulates craniofacial development through versatile mechanisms. PMID:28686747

Role of HERP and a HERP-related protein in HRD1-dependent protein degradation at the endoplasmic reticulum.

PubMed

Huang, Chih-Hsiang; Chu, Yue-Ru; Ye, Yihong; Chen, Xin

2014-02-14

Misfolded proteins of the endoplasmic reticulum (ER) are retrotranslocated to the cytosol and degraded by the proteasome via a process termed ER-associated degradation (ERAD). The precise mechanism of retrotranslocation is unclear. Here, we use several lumenal ERAD substrates targeted for degradation by the ubiquitin ligase HRD1 including SHH (sonic hedgehog) and NHK (null Hong Kong α1-antitrypsin) to study the geometry, organization, and regulation of the HRD1-containing ERAD machinery. We report a new HRD1-associated membrane protein named HERP2, which is homologous to the previously identified HRD1 partner HERP1. Despite sequence homology, HERP2 is constitutively expressed in cells, whereas HERP1 is highly induced by ER stress. We find that these proteins are required for efficient degradation of both glycosylated and nonglycosylated SHH proteins as well as NHK. In cells depleted of HERPs, SHH proteins are largely trapped inside the ER with a fraction of the stabilized SHH protein bound to the HRD1-SEL1L ligase complex. Ubiquitination of SHH is significantly attenuated in the absence of HERPs, suggesting a defect in retrotranslocation. Both HERP proteins interact with HRD1 through a region located in the cytosol. However, unlike its homolog in Saccharomyces cerevisiae, HERPs do not regulate HRD1 stability or oligomerization status. Instead, they help recruit DERL2 to the HRD1-SEL1L complex. Additionally, the UBL domain of HERP1 also seems to have a function independent of DERL2 recruitment in ERAD. Our studies have revealed a critical scaffolding function for mammalian HERP proteins that is required for forming an active retrotranslocation complex containing HRD1, SEL1L, and DERL2.

Low-intensity pulsed ultrasound promotes spinal fusion and enhances migration and proliferation of MG63s through sonic hedgehog signaling pathway.

PubMed

Zhou, Xiao-Yi; Xu, Xi-Ming; Wu, Sui-Yi; Zhang, Zi-Cheng; Wang, Fei; Yang, Yi-Lin; Li, Ming; Wei, Xian-Zhao

2018-05-01

Low-intensity pulsed ultrasound (LIPUS) has been found to accelerate the healing process of spinal fusion via a process closely related to osteoblast differentiation and migration. Sonic hedgehog (Shh) signaling plays an important role in development and homeostasis, including a critical function in bone formation. However, its role in spinal fusion during LIPUS treatment is still unknown. This study showed that LIPUS treatment after spinal fusion surgery increased bone formation. The increased bone mass under LIPUS treatment appeared to result from the increased migration and proliferation of osteoblasts, resulting from upregulation of the Shh signaling pathway. In contrast, inhibition of Shh reduced the migratory and proliferative ability of osteoblast-like MG63 cells and blocked the efficacy of LIPUS treatment. Copyright © 2018 Elsevier Inc. All rights reserved.

Inhibition of Shh signalling in the chick wing gives insights into digit patterning and evolution.

PubMed

Pickering, Joseph; Towers, Matthew

2016-10-01

In an influential model of pattern formation, a gradient of Sonic hedgehog (Shh) signalling in the chick wing bud specifies cells with three antero-posterior positional values, which give rise to three morphologically different digits by a self-organizing mechanism with Turing-like properties. However, as four of the five digits of the mouse limb are morphologically similar in terms of phalangeal pattern, it has been suggested that self-organization alone could be sufficient. Here, we show that inhibition of Shh signalling at a specific stage of chick wing development results in a pattern of four digits, three of which can have the same number of phalanges. These patterning changes are dependent on a posterior extension of the apical ectodermal ridge, and this also allows the additional digit to arise from the Shh-producing cells of the polarizing region - an ability lost in ancestral theropod dinosaurs. Our analyses reveal that, if the specification of antero-posterior positional values is curtailed, self-organization can then produce several digits with the same number of phalanges. We present a model that may give important insights into how the number of digits and phalanges has diverged during the evolution of avian and mammalian limbs. © 2016. Published by The Company of Biologists Ltd.

SJDAWN: St. Jude Children's Research Hospital Phase 1 Study Evaluating Molecularly-Driven Doublet Therapies for Children and Young Adults With Recurrent Brain Tumors

ClinicalTrials.gov

2018-04-09

Anaplastic Astrocytoma; Anaplastic Ependymoma; Anaplastic Ganglioglioma; Anaplastic Meningioma; Anaplastic Oligodendroglioma; Pleomorphic Xanthoastrocytoma, Anaplastic; Atypical Teratoid/Rhabdoid Tumor; Brain Cancer; Brain Tumor; Central Nervous System Neoplasms; Choroid Plexus Carcinoma; CNS Embryonal Tumor With Rhabdoid Features; Ganglioneuroblastoma of Central Nervous System; CNS Tumor; Embryonal Tumor of CNS; Ependymoma; Glioblastoma; Glioma; Glioma, Malignant; Medulloblastoma; Medulloblastoma; Unspecified Site; Medulloepithelioma; Neuroepithelial Tumor; Neoplasms; Neoplasms, Neuroepithelial; Papillary Tumor of the Pineal Region (High-grade Only); Pediatric Brain Tumor; Pineal Parenchymal Tumor of Intermediate Differentiation (High-grade Only); Pineoblastoma; Primitive Neuroectodermal Tumor; Recurrent Medulloblastoma; Refractory Brain Tumor; Neuroblastoma. CNS; Glioblastoma, IDH-mutant; Glioblastoma, IDH-wildtype; Medulloblastoma, Group 3; Medulloblastoma, Group 4; Glioma, High Grade; Neuroepithelial Tumor, High Grade; Medulloblastoma, SHH-activated and TP53 Mutant; Medulloblastoma, SHH-activated and TP53 Wildtype; Medulloblastoma, Chromosome 9q Loss; Medulloblastoma, Non-WNT Non-SHH, NOS; Medulloblastoma, Non-WNT/Non-SHH; Medulloblastoma, PTCH1 Mutation; Medulloblastoma, WNT-activated; Ependymoma, Recurrent; Glioma, Recurrent High Grade; Glioma, Recurrent Malignant; Embryonal Tumor, NOS; Glioma, Diffuse Midline, H3K27M-mutant; Embryonal Tumor With Multilayered Rosettes (ETMR); Ependymoma, NOS, WHO Grade III; Ependymoma, NOS, WHO Grade II; Medulloblastoma, G3/G4; Ependymoma, RELA Fusion Positive

Angiogenesis within the developing mouse neural tube is dependent on sonic hedgehog signaling: possible roles of motor neurons.

PubMed

Nagase, Takashi; Nagase, Miki; Yoshimura, Kotaro; Fujita, Toshiro; Koshima, Isao

2005-06-01

Embryonic morphogenesis of vascular and nervous systems is tightly coordinated, and recent studies revealed that some neurogenetic factors such as Sonic hedgehog (Shh) also exhibit angiogenetic potential. Vascularization within the developing mouse neural tube depends on vessel sprouting from the surrounding vascular plexus. Previous studies implicated possible roles of VEGF/Flk-1 and Angiopoietin-1(Ang-1)/Tie-2 signaling as candidate molecules functioning in this process. Examining gene expressions of these factors at embryonic day (E) 9.5 and 10.5, we unexpectedly found that both VEGF and Ang-1 were expressed in the motor neurons in the ventral neural tube. The motor neurons were indeed located in the close vicinity of the infiltrating vessels, suggesting involvement of motor neurons in the sprouting. To substantiate this possibility, we inhibited induction of the motor neurons in the cultured mouse embryos by cyclopamine, a Shh signaling blocker. The vessel sprouting was dramatically impaired by inhibition of Shh signaling, together with nearly complete loss of the motor neurons. Expression of Ang-1, but not VEGF, within the neural tube was remarkably reduced in the cyclopamine treated embryos. These results suggest that the neural tube angiogenesis is dependent on Shh signaling, and mediated, at least in part, by the Ang-1 positive motor neurons.

Semiotics and semiology of Nursing: evaluation of undergraduate students' knowledge on procedures.

PubMed

Melo, Gabriela de Sousa Martins; Tibúrcio, Manuela Pinto; Freitas, Camylla Cavalcante Soares de; Vasconcelos, Quinídia Lúcia Duarte de Almeida Quithé de; Costa, Isabel Karolyne Fernandes; Torres, Gilson de Vasconcelos

2017-04-01

to assess the knowledge of scholars on Nursing regarding simple hands hygiene (SHH), blood pressure measurement (BP), peripheral venipuncture (PV) with venous catheter and male urethral catheterization delay (UCD) procedures. quantitative study carried out between February and May 2014, with 186 undergraduate Nursing students from 5th to 9th period of a public university of Rio Grande do Norte, with application of four questionnaires. One carried out descriptive and analytic analysis. the students presented low average percentage of right answers, especially in blood pressure measurement (55.5%); SHH's average was higher than 70%. The average of correct answers was the highest in SHH (8.6), followed by UCD (7.8), PV (7.4) and BP (6.7). The questions regarding the topic "concepts" showed less correct answers when comparing it to the topic "technique steps". it is necessary to establish knowledge monitoring strategies, in order to stimulate the constant improvement.

Distinct Neural Stem Cell Populations Give Rise to Disparate Brain Tumors in Response to N-MYC

PubMed Central

Swartling, Fredrik J.; Savov, Vasil; Persson, Anders I.; Chen, Justin; Hackett, Christopher S.; Northcott, Paul A.; Grimmer, Matthew R.; Lau, Jasmine; Chesler, Louis; Perry, Arie; Phillips, Joanna J.; Taylor, Michael D.; Weiss, William A.

2012-01-01

SUMMARY The proto-oncogene MYCN is mis-expressed in various types of human brain tumors. To clarify how developmental and regional differences influence transformation, we transduced wild-type or mutationally-stabilized murine N-mycT58A into neural stem cells (NSCs) from perinatal murine cerebellum, brain stem and forebrain. Transplantation of N-mycWT NSCs was insufficient for tumor formation. N-mycT58A cerebellar and brain stem NSCs generated medulloblastoma/primitive neuroectodermal tumors, whereas forebrain NSCs developed diffuse glioma. Expression analyses distinguished tumors generated from these different regions, with tumors from embryonic versus postnatal cerebellar NSCs demonstrating SHH-dependence and SHH-independence, respectively. These differences were regulated in-part by the transcription factor SOX9, activated in the SHH subclass of human medulloblastoma. Our results demonstrate context-dependent transformation of NSCs in response to a common oncogenic signal. PMID:22624711

Cross-regulatory interactions between Fgf8 and Shh in the avian frontonasal prominence.

PubMed

Abzhanov, Arhat; Cordero, Dwight R; Sen, Jonaki; Tabin, Clifford J; Helms, Jill A

2007-12-01

The frontonasal prominence of the developing avian embryo contains an organizing center, defined by juxtaposition of the Sonic hedgehog (Shh) and Fibroblast growth factor 8 (Fgf8) expression domains. This molecular interface presages any detectable growth of the frontonasal prominence, and experiments involving transplantation of this boundary epithelium have demonstrated it is a source of dorsal-ventral and rostral-caudal patterning information for the neural crest-derived mesenchyme of the upper beak. We explored the ontogeny of this organizing center by mapping the expression domains of both genes and their receptors and downstream targets. We tested the extent to which Shh and Fgf8 regulate each other's expression in this frontonasal organizer by either blocking or ectopically activating these pathways. Our experiments revealed mutual antagonism between the two molecules, which aids in establishing and maintaining a molecular boundary that subsequently influences patterning and growth of the middle and upper face.

Holoprosencephaly: from Homer to Hedgehog.

PubMed

Ming, J E; Muenke, M

1998-03-01

Holoprosencephaly (HPE), a common developmental defect affecting the forebrain and face, is etiologically heterogeneous and exhibits wide phenotypic variation. Graded degrees of severity of the brain malformation are also reflected in the highly variable craniofacial malformations associated with HPE. In addition, individuals with microforms of HPE, who usually have normal cognition and normal brain imaging, are at risk for having children with HPE. Some obligate carriers for HPE may not have any phenotypic abnormalities. Recurrent chromosomal rearrangements in individuals with HPE suggest loci containing genes important for brain development, and abnormalities in these genes may result in HPE. Recently, Sonic Hedgehog (SHH) was the first gene identified as causing HPE in humans. Proper function of SHH depends on cholesterol modification. Other candidate genes that may be involved in HPE include components of the SHH pathway, elements involved in cholesterol metabolism, and genes expressed in the developing forebrain.

Spatiotemporal antagonism in mesenchymal-epithelial signaling in sweat versus hair fate decision.

PubMed

Lu, Catherine P; Polak, Lisa; Keyes, Brice E; Fuchs, Elaine

2016-12-23

The gain of eccrine sweat glands in hairy body skin has empowered humans to run marathons and tolerate temperature extremes. Epithelial-mesenchymal cross-talk is integral to the diverse patterning of skin appendages, but the molecular events underlying their specification remain largely unknown. Using genome-wide analyses and functional studies, we show that sweat glands are specified by mesenchymal-derived bone morphogenetic proteins (BMPs) and fibroblast growth factors that signal to epithelial buds and suppress epithelial-derived sonic hedgehog (SHH) production. Conversely, hair follicles are specified when mesenchymal BMP signaling is blocked, permitting SHH production. Fate determination is confined to a critical developmental window and is regionally specified in mice. In contrast, a shift from hair to gland fates is achieved in humans when a spike in BMP silences SHH during the final embryonic wave(s) of bud morphogenesis. Copyright © 2016, American Association for the Advancement of Science.

Glioma-Associated Oncogene Homolog Inhibitors Have the Potential of Suppressing Cancer Stem Cells of Breast Cancer.

PubMed

Jeng, Kuo-Shyang; Jeng, Chi-Juei; Sheen, I-Shyan; Wu, Szu-Hua; Lu, Ssu-Jung; Wang, Chih-Hsuan; Chang, Chiung-Fang

2018-05-05

Overexpression of Sonic Hedgehog signaling (Shh) pathway molecules is associated with invasiveness and recurrence in breast carcinoma. Therefore, inhibition of the Shh pathway downstream molecule Glioma-associated Oncogene Homolog (Gli) was investigated for its ability to reduce progression and invasiveness of patient-derived breast cancer cells and cell lines. Human primary breast cancer T2 cells with high expression of Shh signaling pathway molecules were compared with breast cancer line MDA-MB-231 cells. The therapeutic effects of Gli inhibitors were examined in terms of the cell proliferation, apoptosis, cancer stem cells, cell migration and gene expression. Blockade of the Shh signaling pathway could reduce cell proliferation and migration only in MDA-MB-231 cells. Hh pathway inhibitor-1 (HPI-1) increased the percentages of late apoptotic cells in MDA-MB-231 cells and early apoptotic cells in T2 cells. It reduced Bcl2 expression for cell proliferation and increased Bim expression for apoptosis. In addition, Gli inhibitor HPI-1 decreased significantly the percentages of cancer stem cells in T2 cells. HPI-1 worked more effectively than GANT-58 against breast carcinoma cells. In conclusion, HPI-1 could inhibit cell proliferation, reduce cell invasion and decrease cancer stem cell population in breast cancer cells. To target Gli-1 could be a potential strategy to suppress breast cancer stem cells.

GLI inhibitor GANT-61 diminishes embryonal and alveolar rhabdomyosarcoma growth by inhibiting Shh/AKT-mTOR axis

PubMed Central

Srivastava, Ritesh K.; Kaylani, Samer Zaid; Edrees, Nayf; Li, Changzhao; Talwelkar, Sarang S.; Xu, Jianmin; Palle, Komaraiah; Pressey, Joseph G.; Athar, Mohammad

2014-01-01

Rhabdomyosarcoma (RMS) typically arises from skeletal muscle. Currently, RMS in patients with recurrent and metastatic disease have no successful treatment. The molecular pathogenesis of RMS varies based on cancer sub-types. Some embryonal RMS but not other sub-types are driven by sonic hedgehog (Shh) signaling pathway. However, Shh pathway inhibitors particularly smoothened inhibitors are not highly effective in animals. Here, we show that Shh pathway effectors GLI1 and/or GLI2 are over-expressed in the majority of RMS cells and that GANT-61, a specific GLI1/2 inhibitor dampens the proliferation of both embryonal and alveolar RMS cells-derived xenograft tumors thereby blocking their growth. As compared to vehicle-treated control, about 50% tumor growth inhibition occurs in mice receiving GANT-61 treatment. The proliferation inhibition was associated with slowing of cell cycle progression which was mediated by the reduced expression of cyclins D1/2/3 & E and the concomitant induction of p21. GANT-61 not only reduced expression of GLI1/2 in these RMS but also significantly diminished AKT/mTOR signaling. The therapeutic action of GANT-61 was significantly augmented when combined with chemotherapeutic agents employed for RMS therapy such as temsirolimus or vincristine. Finally, reduced expression of proteins driving epithelial mesenchymal transition (EMT) characterized the residual tumors. PMID:25432075

Sonic hedgehog signaling regulates amygdalar neurogenesis and extinction of fear memory.

PubMed

Hung, Hui-Chi; Hsiao, Ya-Hsin; Gean, Po-Wu

2015-10-01

It is now recognized that neurogenesis occurs throughout life predominantly in the subgranular zone (SGZ) of the hippocampus and the subventricular zone (SVZ) of the lateral ventricle. In the present study, we investigated the relationship between neurogenesis in the amygdala and extinction of fear memory. Mice received 15 tone-footshock pairings. Twenty-four hours after training, the mice were given 15 tone-alone trials (extinction training) once per day for 7 days. Two hours before extinction training, the mice were injected intraperitoneally with 5-bromo-3-deoxyuridine (BrdU). BrdU-positive and NeuN-positive cells were analyzed 52 days after the training. A group of mice that received tone-footshock pairings but no extinction training served as controls (FC+No-Ext). The number of BrdU(+)/NeuN(+) cells was significantly higher in the extinction (FC+Ext) than in the FC+No-Ext mice. Proliferation inhibitor methylazoxymethanol acetate (MAM) or DNA synthesis inhibitor cytosine arabinoside (Ara-C) reduced neurogenesis and retarded extinction. Silencing Sonic hedgehog (Shh) gene with short hairpin interfering RNA (shRNA) by means of a retrovirus expression system to knockdown Shh specifically in the mitotic neurons reduced neurogenesis and retarded extinction. By contrast, over-expression of Shh increased neurogenesis and facilitated extinction. These results suggest that amygdala neurogenesis and Shh signaling are involved in the extinction of fear memory. Copyright © 2015 Elsevier B.V. and ECNP. All rights reserved.

Disruption of sonic hedgehog signaling in Ellis-van Creveld dwarfism confers protection against bipolar affective disorder.

PubMed

Ginns, E I; Galdzicka, M; Elston, R C; Song, Y E; Paul, S M; Egeland, J A

2015-10-01

Ellis-van Creveld syndrome, an autosomal recessively inherited chondrodysplastic dwarfism, is frequent among Old Order Amish of Pennsylvania. Decades of longitudinal research on bipolar affective disorder (BPAD) revealed cosegregation of high numbers of EvC and Bipolar I (BPI) cases in several large Amish families descending from the same pioneer. Despite the high prevalence of both disorders in these families, no EvC individual has ever been reported with BPI. The proximity of the EVC gene to our previously reported chromosome 4p16 BPAD locus with protective alleles, coupled with detailed clinical observations that EvC and BPI do not occur in the same individuals, led us to hypothesize that the genetic defect causing EvC in the Amish confers protection from BPI. This hypothesis is supported by a significant negative association of these two disorders when contrasted with absence of disease (P=0.029, Fisher's exact test, two-sided, verified by permutation to estimate the null distribution of the test statistic). As homozygous Amish EVC mutations causing EvC dwarfism do so by disrupting sonic hedgehog (Shh) signaling, our data implicate Shh signaling in the underlying pathophysiology of BPAD. Understanding how disrupted Shh signaling protects against BPI could uncover variants in the Shh pathway that cause or increase risk for this and related mood disorders.

LKB1 Regulates Cerebellar Development by Controlling Sonic Hedgehog-mediated Granule Cell Precursor Proliferation and Granule Cell Migration.

PubMed

Men, Yuqin; Zhang, Aizhen; Li, Haixiang; Jin, Yecheng; Sun, Xiaoyang; Li, Huashun; Gao, Jiangang

2015-11-09

The Liver Kinase B1 (LKB1) gene plays crucial roles in cell differentiation, proliferation and the establishment of cell polarity. We created LKB1 conditional knockout mice (LKB1(Atoh1) CKO) to investigate the function of LKB1 in cerebellar development. The LKB1(Atoh1) CKO mice displayed motor dysfunction. In the LKB1(Atoh1) CKO cerebellum, the overall structure had a larger volume and more lobules. LKB1 inactivation led to an increased proliferation of granule cell precursors (GCPs), aberrant granule cell migration and overproduction of unipolar brush cells. To investigate the mechanism underlying the abnormal foliation, we examined sonic hedgehog signalling (Shh) by testing its transcriptional mediators, the Gli proteins, which regulate the GCPs proliferation and cerebellar foliation during cerebellar development. The expression levels of Gli genes were significantly increased in the mutant cerebellum. In vitro assays showed that the proliferation of cultured GCPs from mutant cerebellum significantly increased, whereas the proliferation of mutant GCPs significantly decreased in the presence of a Shh inhibitor GDC-0049. Thus, LKB1 deficiency in the LKB1(Atoh1) CKO mice enhanced Shh signalling, leading to the excessive GCP proliferation and the formation of extra lobules. We proposed that LKB1 regulates cerebellar development by controlling GCPs proliferation through Shh signalling during cerebellar development.

LKB1 Regulates Cerebellar Development by Controlling Sonic Hedgehog-mediated Granule Cell Precursor Proliferation and Granule Cell Migration

PubMed Central

Men, Yuqin; Zhang, Aizhen; Li, Haixiang; Jin, Yecheng; Sun, Xiaoyang; Li, Huashun; Gao, Jiangang

2015-01-01

The Liver Kinase B1 (LKB1) gene plays crucial roles in cell differentiation, proliferation and the establishment of cell polarity. We created LKB1 conditional knockout mice (LKB1Atoh1 CKO) to investigate the function of LKB1 in cerebellar development. The LKB1Atoh1 CKO mice displayed motor dysfunction. In the LKB1Atoh1 CKO cerebellum, the overall structure had a larger volume and morelobules. LKB1 inactivationled to an increased proliferation of granule cell precursors (GCPs), aberrant granule cell migration and overproduction of unipolar brush cells. To investigate the mechanism underlying the abnormal foliation, we examined sonic hedgehog signalling (Shh) by testing its transcriptional mediators, the Gli proteins, which regulate the GCPs proliferation and cerebellar foliation during cerebellar development. The expression levels of Gli genes were significantly increased in the mutant cerebellum. In vitro assays showed that the proliferation of cultured GCPs from mutant cerebellum significantly increased, whereas the proliferation of mutant GCPs significantly decreased in the presence of a Shh inhibitor GDC-0049. Thus, LKB1 deficiency in the LKB1Atoh1 CKO mice enhanced Shh signalling, leading to the excessive GCP proliferation and the formation of extra lobules. We proposed that LKB1 regulates cerebellar development by controlling GCPs proliferation through Shh signalling during cerebellar development. PMID:26549569

The role of the sonic hedgehog signalling pathway in patients with midline defects and congenital hypopituitarism.

PubMed

Gregory, L C; Gaston-Massuet, C; Andoniadou, C L; Carreno, G; Webb, E A; Kelberman, D; McCabe, M J; Panagiotakopoulos, L; Saldanha, J W; Spoudeas, H A; Torpiano, J; Rossi, M; Raine, J; Canham, N; Martinez-Barbera, J P; Dattani, M T

2015-05-01

The Gli family of zinc finger (GLI) transcription factors mediates the sonic hedgehog signalling pathway (HH) essential for CNS, early pituitary and ventral forebrain development in mice. Human mutations in this pathway have been described in patients with holoprosencephaly (HPE), isolated congenital hypopituitarism (CH) and cranial/midline facial abnormalities. Mutations in Sonic hedgehog (SHH) have been associated with HPE but not CH, despite murine studies indicating involvement in pituitary development. We aimed to establish the role of the HH pathway in the aetiology of hypothalamo-pituitary disorders by screening our cohort of patients with midline defects and/or CH for mutations in SHH, GLI2, Shh brain enhancer 2 (SBE2) and growth-arrest specific 1 (GAS1). Two variants and a deletion of GLI2 were identified in three patients. A novel variant at a highly conserved residue in the zinc finger DNA-binding domain, c.1552G > A [pE518K], was identified in a patient with growth hormone deficiency and low normal free T4. A nonsynonymous variant, c.2159G > A [p.R720H], was identified in a patient with a short neck, cleft palate and hypogonadotrophic hypogonadism. A 26·6 Mb deletion, 2q12·3-q21·3, encompassing GLI2 and 77 other genes, was identified in a patient with short stature and impaired growth. Human embryonic expression studies and molecular characterisation of the GLI2 mutant p.E518K support the potential pathogenicity of GLI2 mutations. No mutations were identified in GAS1 or SBE2. A novel SHH variant, c.1295T>A [p.I432N], was identified in two siblings with variable midline defects but normal pituitary function. Our data suggest that mutations in SHH, GAS1 and SBE2 are not associated with hypopituitarism, although GLI2 is an important candidate for CH. © 2014 John Wiley & Sons Ltd.

Human platelet lysate versus minoxidil stimulates hair growth by activating anagen promoting signaling pathways.

PubMed

Dastan, Maryam; Najafzadeh, Nowruz; Abedelahi, Ali; Sarvi, Mohammadreza; Niapour, Ali

2016-12-01

Minoxidil and human platelet lysate (HPL) are commonly used to treat patients with hair loss. However, the roles of HPL versus minoxidil in hair follicle biology largely remain unknown. Here, we hypothesized that bulge and dermal papilla (DP) cells may express specific genes, including Kras, Erk, Akt, Shh and β-catenin after exposure to minoxidil or HPL. The mouse hair follicles were isolated on day 10 after depilation and bulge or DP regions were dissected. The bulge and DP cells were cultured for 14days in DMEM/F12 medium. Then, the cells were treated with 100μM minoxidil and 10% HPL for 10 days. Nuclear morphology was identified using DAPi staining. Reverse transcriptase and real-time polymerase chain reaction (PCR) analysis were also performed to examine the expression of Kras, Erk, Akt, Shh and β-catenin mRNA levels in the treated bulge and DP regions after organ culture. Here, we found that minoxidil influences bulge and DP cell survival (P<0.05). Apoptosis in DP cells was also meaningfully decreased by HPL treatment (P=0.014). In addition, Kras, Akt, Erk, Shh and β-catenin mRNA levels were changed in response to minoxidil treatment in both bulge and DP cells. HPL mediated Erk upregulation in both bulge and DP cells (P<0.05), but Kras and Akt mRNA levels were not considerably different in the HPL-treated cells. β-catenin mRNA level was also significantly increased in the bulge region by HPL. We also found that Shh mRNA level was considerably higher in HPL-treated bulge cells than in minoxidil-treated bulge cells. In contrast, the expression of β-cateinin and Shh in the DP cells was not meaningfully increased after treatment with HPL. Our results suggest that minoxidil and HPL can promote hair growth by activating the main anagen inducing signaling pathways. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

YAP regulates neuronal differentiation through Sonic hedgehog signaling pathway

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

Lin, Yi-Ting; Ding, Jing-Ya; Li, Ming-Yang

2012-09-10

Tight regulation of cell numbers by controlling cell proliferation and apoptosis is important during development. Recently, the Hippo pathway has been shown to regulate tissue growth and organ size in Drosophila. In mammalian cells, it also affects cell proliferation and differentiation in various tissues, including the nervous system. Interplay of several signaling cascades, such as Notch, Wnt, and Sonic Hedgehog (Shh) pathways, control cell proliferation during neuronal differentiation. However, it remains unclear whether the Hippo pathway coordinates with other signaling cascades in regulating neuronal differentiation. Here, we used P19 cells, a mouse embryonic carcinoma cell line, as a model tomore » study roles of YAP, a core component of the Hippo pathway, in neuronal differentiation. P19 cells can be induced to differentiate into neurons by expressing a neural bHLH transcription factor gene Ascl1. Our results showed that YAP promoted cell proliferation and inhibited neuronal differentiation. Expression of Yap activated Shh but not Wnt or Notch signaling activity during neuronal differentiation. Furthermore, expression of Yap increased the expression of Patched homolog 1 (Ptch1), a downstream target of the Shh signaling. Knockdown of Gli2, a transcription factor of the Shh pathway, promoted neuronal differentiation even when Yap was over-expressed. We further demonstrated that over-expression of Yap inhibited neuronal differentiation in primary mouse cortical progenitors and Gli2 knockdown rescued the differentiation defect in Yap over-expressing cells. In conclusion, our study reveals that Shh signaling acts downstream of YAP in regulating neuronal differentiation. -- Highlights: Black-Right-Pointing-Pointer YAP promotes cell proliferation and inhibits neuronal differentiation in P19 cells. Black-Right-Pointing-Pointer YAP promotes Sonic hedgehog signaling activity during neuronal differentiation. Black-Right-Pointing-Pointer Knockdown of Gli2 rescues the Yap-overexpression phenotype in P19 cells. Black-Right-Pointing-Pointer Knockdown of Gli2 rescues the Yap-overexpression phenotype in cortical progenitors.« less

Mycobacterium bovis BCG promotes tumor cell survival from tumor necrosis factor-α-induced apoptosis.

PubMed

Holla, Sahana; Ghorpade, Devram Sampat; Singh, Vikas; Bansal, Kushagra; Balaji, Kithiganahalli Narayanaswamy

2014-09-11

Increased incidence of lung cancer among pulmonary tuberculosis patients suggests mycobacteria-induced tumorigenic response in the host. The alveolar epithelial cells, candidate cells that form lung adenocarcinoma, constitute a niche for mycobacterial replication and infection. We thus explored the possible mechanism of M. bovis Bacillus Calmette-Guérin (BCG)-assisted tumorigenicity in type II epithelial cells, human lung adenocarcinoma A549 and other cancer cells. Cancer cell lines originating from lung, colon, bladder, liver, breast, skin and cervix were treated with tumor necrosis factor (TNF)-α in presence or absence of BCG infection. p53, COP1 and sonic hedgehog (SHH) signaling markers were determined by immunoblotting and luciferase assays, and quantitative real time PCR was done for p53-responsive pro-apoptotic genes and SHH signaling markers. MTT assays and Annexin V staining were utilized to study apoptosis. Gain- and loss-of-function approaches were used to investigate the role for SHH and COP1 signaling during apoptosis. A549 xenografted mice were used to validate the contribution of BCG during TNF-α treatment. Here, we show that BCG inhibits TNF-α-mediated apoptosis in A549 cells via downregulation of p53 expression. Substantiating this observation, BCG rescued A549 xenografts from TNF-α-mediated tumor clearance in nude mice. Furthermore, activation of SHH signaling by BCG induced the expression of an E3 ubiquitin ligase, COP1. SHH-driven COP1 targeted p53, thereby facilitating downregulation of p53-responsive pro-apoptotic genes and inhibition of apoptosis. Similar effects of BCG could be shown for HCT116, T24, MNT-1, HepG2 and HELA cells but not for HCT116 p53(-/-) and MDA-MB-231 cells. Our results not only highlight possible explanations for the coexistence of pulmonary tuberculosis and lung cancer but also address probable reasons for failure of BCG immunotherapy of cancers.

The relationship between sitting height, sitting height to height ratio with blood pressure among Polokwane private school children aged 6-13 years.

PubMed

Ramoshaba, Nthai E; Monyeki, Kotsedi D; Mpya, Joyce; Monyeki, Mafolwa S

2018-01-04

It is notable that sitting height (SH) correlates with blood pressure (BP) in children and adolescents of developed countries. However, little is known about the relationships between SH and SH to height ratio (SH/H) with BP in South African children from middle and upper socio-economic groups. The purpose of this study was to compare SH and SH/H of private school attending children in the Polokwane area with National Health and Nutrition Examination Survey III (NHANES III) reference population and to determine the relationship between SH, SH/H with BP among private school attending children. A total of 1665 children (846 boys and 819 girls) aged between 6 and 13 years attending three private schools in Polokwane, underwent anthropometric and BP measurements using standard procedures. Linear regression was used to determine the relationship between height, SH, SH/H with BP among these children. Polokwane private school attending boys from age 7 to 13 years displayed a lower mean SH compared to the NHANES III whereas NHANHES III girls from age 10 to 13 years had a higher mean SH compared to those in private school. In the simple regression analysis, SH was positively associated with SBP (β =1.318; 95% CI = 1.217-1.418) and DBP (β = 0.641; 95% Cl = 0.555-0.727). The findings remains statistically significant only for SH with both SBP (β = 1.025; 95% Cl = 0.844-1.201) and DBP (β = 0.585; 95% Cl 0.434-0.736) after adjusting for age, gender and BMI among these children. In South African children, SH and SH/H were lower compared to the NHANES III children. There was a significant positive association between DBP and SBP together with the components of height among Polokwane private school children.

TRAIL, Wnt, Sonic Hedgehog, TGFβ, and miRNA Signalings Are Potential Targets for Oral Cancer Therapy

PubMed Central

Farooqi, Ammad Ahmad; Shu, Chih-Wen; Huang, Hurng-Wern; Wang, Hui-Ru; Chang, Yung-Ting; Fayyaz, Sundas; Yuan, Shyng-Shiou F.; Tang, Jen-Yang

2017-01-01

Clinical studies and cancer cell models emphasize the importance of targeting therapies for oral cancer. The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is highly expressed in cancer, and is a selective killing ligand for oral cancer. Signaling proteins in the wingless-type mouse mammary tumor virus (MMTV) integration site family (Wnt), Sonic hedgehog (SHH), and transforming growth factor β (TGFβ) pathways may regulate cell proliferation, migration, and apoptosis. Accordingly, the genes encoding these signaling proteins are potential targets for oral cancer therapy. In this review, we focus on recent advances in targeting therapies for oral cancer and discuss the gene targets within TRAIL, Wnt, SHH, and TGFβ signaling for oral cancer therapies. Oncogenic microRNAs (miRNAs) and tumor suppressor miRNAs targeting the genes encoding these signaling proteins are summarized, and the interactions between Wnt, SHH, TGFβ, and miRNAs are interpreted. With suitable combination treatments, synergistic effects are expected to improve targeting therapies for oral cancer. PMID:28708091

Cellular origin of bladder neoplasia and tissue dynamics of its progression to invasive carcinoma

PubMed Central

Shin, Kunyoo; Lim, Agnes; Odegaard, Justin I.; Honeycutt, Jared D.; Kawano, Sally; Hsieh, Michael H.; Beachy, Philip A.

2014-01-01

Understanding how malignancies arise within normal tissues requires identification of the cancer cell of origin and knowledge of the cellular and tissue dynamics of tumor progression. Here we examine bladder cancer in a chemical carcinogenesis model that mimics muscle-invasive human bladder cancer. With no prior bias regarding genetic pathways or cell types, we prospectively mark or ablate cells to show that muscle-invasive bladder carcinomas arise exclusively from Sonic hedgehog (Shh)-expressing stem cells in basal urothelium. These carcinomas arise clonally from a single cell whose progeny aggressively colonize a major portion of the urothelium to generate a lesion with histological features identical to human carcinoma-in-situ. Shh-expressing basal cells within this precursor lesion become tumor-initiating cells, although Shh expression is lost in subsequent carcinomas. We thus find that invasive carcinoma is initiated from basal urothelial stem cells but that tumor cell phenotype can diverge significantly from that of the cancer cell-of-origin. PMID:24747439

Subcellular Localization of Patched and Smoothened, the Receptors for Sonic Hedgehog Signaling, in the Hippocampal Neuron

PubMed Central

Petralia, Ronald S.; Schwartz, Catherine M.; Wang, Ya-Xian; Mattson, Mark P.; Yao, Pamela J.

2011-01-01

Cumulative evidence suggests that, aside from patterning the embryonic neural tube, Sonic hedgehog (Shh) signaling plays important roles in the mature nervous system. In this study, we investigate the expression and localization of the Shh signaling receptors, Patched (Ptch) and Smoothened (Smo), in the hippocampal neurons of young and mature rats. Reverse transcriptase-polymerase chain reaction and immunoblotting analyses show that the expression of Ptch and Smo remains at a moderate level in young postnatal and adult brains. By using immunofluorescence light microscopy and immunoelectron microscopy, we examine the spatial distribution of Ptch and Smo within the hippocampal neurons. In young developing neurons, Ptch and Smo are present in the processes and are clustered at their growth cones. In mature neurons, Ptch and Smo are concentrated in dendrites, spines, and postsynaptic sites. Synaptic Ptch and Smo often co-exist with unusual structures—synaptic spinules and autophagosomes. Our results reveal the anatomical organization of the Shh receptors within both the young and the mature hippocampal neurons. PMID:21618238

TRAIL, Wnt, Sonic Hedgehog, TGFβ, and miRNA Signalings Are Potential Targets for Oral Cancer Therapy.

PubMed

Farooqi, Ammad Ahmad; Shu, Chih-Wen; Huang, Hurng-Wern; Wang, Hui-Ru; Chang, Yung-Ting; Fayyaz, Sundas; Yuan, Shyng-Shiou F; Tang, Jen-Yang; Chang, Hsueh-Wei

2017-07-14

Clinical studies and cancer cell models emphasize the importance of targeting therapies for oral cancer. The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is highly expressed in cancer, and is a selective killing ligand for oral cancer. Signaling proteins in the wingless-type mouse mammary tumor virus (MMTV) integration site family (Wnt), Sonic hedgehog (SHH), and transforming growth factor β (TGFβ) pathways may regulate cell proliferation, migration, and apoptosis. Accordingly, the genes encoding these signaling proteins are potential targets for oral cancer therapy. In this review, we focus on recent advances in targeting therapies for oral cancer and discuss the gene targets within TRAIL, Wnt, SHH, and TGFβ signaling for oral cancer therapies. Oncogenic microRNAs (miRNAs) and tumor suppressor miRNAs targeting the genes encoding these signaling proteins are summarized, and the interactions between Wnt, SHH, TGFβ, and miRNAs are interpreted. With suitable combination treatments, synergistic effects are expected to improve targeting therapies for oral cancer.

Attenuated sensing of SHH by Ptch1 underlies evolution of bovine limbs.

PubMed

Lopez-Rios, Javier; Duchesne, Amandine; Speziale, Dario; Andrey, Guillaume; Peterson, Kevin A; Germann, Philipp; Unal, Erkan; Liu, Jing; Floriot, Sandrine; Barbey, Sarah; Gallard, Yves; Müller-Gerbl, Magdalena; Courtney, Andrew D; Klopp, Christophe; Rodriguez, Sabrina; Ivanek, Robert; Beisel, Christian; Wicking, Carol; Iber, Dagmar; Robert, Benoit; McMahon, Andrew P; Duboule, Denis; Zeller, Rolf

2014-07-03

The large spectrum of limb morphologies reflects the wide evolutionary diversification of the basic pentadactyl pattern in tetrapods. In even-toed ungulates (artiodactyls, including cattle), limbs are adapted for running as a consequence of progressive reduction of their distal skeleton to symmetrical and elongated middle digits with hoofed phalanges. Here we analyse bovine embryos to establish that polarized gene expression is progressively lost during limb development in comparison to the mouse. Notably, the transcriptional upregulation of the Ptch1 gene, which encodes a Sonic hedgehog (SHH) receptor, is disrupted specifically in the bovine limb bud mesenchyme. This is due to evolutionary alteration of a Ptch1 cis-regulatory module, which no longer responds to graded SHH signalling during bovine handplate development. Our study provides a molecular explanation for the loss of digit asymmetry in bovine limb buds and suggests that modifications affecting the Ptch1 cis-regulatory landscape have contributed to evolutionary diversification of artiodactyl limbs.

PM2.5 promotes human bronchial smooth muscle cell migration via the sonic hedgehog signaling pathway.

PubMed

Ye, Xiuqin; Hong, Wei; Hao, Binwei; Peng, Gongyong; Huang, Lingmei; Zhao, Zhuxiang; Zhou, Yumin; Zheng, Mengning; Li, Chenglong; Liang, Chunxiao; Yi, Erkang; Pu, Jinding; Li, Bing; Ran, Pixin

2018-03-02

The contribution of airway remodeling in chronic obstructive pulmonary disease (COPD) has been well documented, with airway smooth muscle cell proliferation and migration playing a role in the remodeling process. Here, we aimed to verify the effects of fine particulate matter (PM2.5) on human bronchial smooth muscle cell (HBSMC) migration and to explore the underlying signaling pathways. HBSMC apoptosis, proliferation and migration were measured using flow cytometry, cell counting and transwell migration assays, respectively. The role of the hedgehog pathway in cell migration was assessed by western blotting to measure the expression of Sonic hedgehog (Shh), Gli1 and Snail. Furthermore, siRNA was used to knock down Gli1 or Snail expression. PM2.5 induced HBSMC apoptosis in a dose-dependent manner, although certain concentrations of PM2.5 did not induce HBSMC proliferation or apoptosis. Interestingly, cell migration was stimulated by PM2.5 doses far below those that induced apoptosis. Additional experiments revealed that these PM2.5 doses enhanced the expression of Shh, Gli1 and Snail in HBSMCs. Furthermore, PM2.5-induced cell migration and protein expression were enhanced by recombinant Shh and attenuated by cyclopamine. Similar results were obtained by knocking down Gli1 or Snail. These findings suggest that PM2.5, which may exert its effects through the Shh signaling pathway, is necessary for the migration of HBSMCs. These data define a novel role for PM2.5 in airway remodeling in COPD.

Prospective evaluation of surgical management of sliding hiatal hernia and gastroesophageal reflux in dogs.

PubMed

Mayhew, Philipp D; Marks, Stanley L; Pollard, Rachel; Culp, William T N; Kass, Philip H

2017-11-01

To evaluate response to surgical management of sliding hiatal hernia (SHH) and gastroesophageal reflux (GER) in dogs using standardized clinical scoring, videofluoroscopic swallow studies, and impedance planimetry. Prospective clinical trial. A total of 17 client-owned dogs. Dogs were included if they had clinical signs and videofluoroscopic evidence of SHH and/or GER. Owners were asked to complete a standardized canine dysphagia assessment tool (CDAT) preoperatively and postoperatively. Conscious videofluoroscopic swallowing studies and impedance planimetry (IP) were used to evaluate esophageal function and lower esophageal sphincter location and geometry preoperatively and in a subsection of dogs postoperatively. Preoperatively, 13/17 dogs included in the study had a history of regurgitation, and 4/17 had radiographic evidence of aspiration pneumonia. Postprandial regurgitation improved in 8/10 dogs with preoperative regurgitation, and for which completed preoperative and postoperative CDAT questionnaires were available (P < .01). The hiatal hernia severity score improved postoperatively (P = .046) in dogs with preoperative and postoperative videofluoroscopic swallowing studies (n = 12). However, hernia frequency score (P = .2) and IP parameters did not differ significantly between time points. Clinical signs of SHH generally improved with surgery but did not consistently resolve. Videofluoroscopic studies provide evidence that GER and SHH can persist postoperatively in some patients. Based on IP findings, clinical improvement may be attributed to a mechanism independent of lower esophageal sphincter attenuation. © 2017 The American College of Veterinary Surgeons.

Dental developmental abnormalities in a patient with subtelomeric 7q36 deletion syndrome may confirm a novel role for the SHH gene☆

PubMed Central

Linhares, Natália D.; Svartman, Marta; Salgado, Mauro Ivan; Rodrigues, Tatiane C.; da Costa, Silvia S.; Rosenberg, Carla; Valadares, Eugênia R.

2013-01-01

Studies in mice demonstrated that the Shh gene is crucial for normal development of both incisors and molars, causing a severe retardation in tooth growth, which leads to abnormal placement of the tooth in the jaw and disrupted tooth morphogenesis. In humans the SHH gene is located on chromosome 7q36. Defects in its protein or signaling pathway may cause holoprosencephaly spectrum, a disorder in which the developing forebrain fails to correctly separate into right and left hemispheres and that can be manifested in microforms such as single maxillary central incisor. A novel role for this gene in the developing human primary dentition was recently demonstrated. We report a 12-year old boy with a de novo 7q36.1-qter deletion characterized by high-resolution karyotyping, oligonucleotide aCGH and FISH. His phenotype includes intellectual disability, non-verbal communication, hypospadia, partial sacral agenesis and absence of coccyx, which are distinctive features of the syndrome and mainly correlated with the MNX1, HTR5A and EN2 genes. No microforms of holoprosencephaly spectrum were observed; but the patient had diastema and dental developmental abnormalities, such as conical, asymmetric and tapered inferior central incisors. The dental anomalies are reported herein for the first time in subtelomeric 7q36 deletion syndrome and may confirm clinically a novel role for the SHH gene in dental development. PMID:25606385

The pleiotropic mouse phenotype extra-toes spotting is caused by translation initiation factor Eif3c mutations and is associated with disrupted sonic hedgehog signaling.

PubMed

Gildea, Derek E; Luetkemeier, Erin S; Bao, Xiaozhong; Loftus, Stacie K; Mackem, Susan; Yang, Yingzi; Pavan, William J; Biesecker, Leslie G

2011-05-01

Polydactyly is a common malformation and can be an isolated anomaly or part of a pleiotropic syndrome. The elucidation of the mutated genes that cause polydactyly provides insight into limb development pathways. The extra-toes spotting (Xs) mouse phenotype manifests anterior polydactyly, predominantly in the forelimbs, with ventral hypopigmenation. The mapping of Xs(J) to chromosome 7 was confirmed, and the interval was narrowed to 322 kb using intersubspecific crosses. Two mutations were identified in eukaryotic translation initiation factor 3 subunit C (Eif3c). An Eif3c c.907C>T mutation (p.Arg303X) was identified in Xs(J), and a c.1702_1758del mutation (p.Leu568_Leu586del) was identified in extra-toes spotting-like (Xsl), an allele of Xs(J). The effect of the Xs(J) mutation on the SHH/GLI3 pathway was analyzed by in situ hybridization analysis, and we show that Xs mouse embryos have ectopic Shh and Ptch1 expression in the anterior limb. In addition, anterior limb buds show aberrant Gli3 processing, consistent with perturbed SHH/GLI3 signaling. Based on the occurrence of Eif3c mutations in 2 Xs lines and haploinsufficiency of the Xs(J) allele, we conclude that the Xs phenotype is caused by a mutation in Eif3c, a component of the translation initiation complex, and that the phenotype is associated with aberrant SHH/GLI3 signaling.

Human germline hedgehog pathway mutations predispose to fatty liver.

PubMed

Guillen-Sacoto, Maria J; Martinez, Ariel F; Abe, Yu; Kruszka, Paul; Weiss, Karin; Everson, Joshua L; Bataller, Ramon; Kleiner, David E; Ward, Jerrold M; Sulik, Kathleen K; Lipinski, Robert J; Solomon, Benjamin D; Muenke, Maximilian

2017-10-01

Non-alcoholic fatty liver disease (NAFLD) is the most common form of liver disease. Activation of hedgehog (Hh) signaling has been implicated in the progression of NAFLD and proposed as a therapeutic target; however, the effects of Hh signaling inhibition have not been studied in humans with germline mutations that affect this pathway. Patients with holoprosencephaly (HPE), a disorder associated with germline mutations disrupting Sonic hedgehog (SHH) signaling, were clinically evaluated for NAFLD. A combined mouse model of Hh signaling attenuation (Gli2 heterozygous null: Gli2 +/- ) and diet-induced NAFLD was used to examine aspects of NAFLD and hepatic gene expression profiles, including molecular markers of hepatic fibrosis and inflammation. Patients with HPE had a higher prevalence of liver steatosis compared to the general population, independent of obesity. Exposure of Gli2 +/- mice to fatty liver-inducing diets resulted in increased liver steatosis compared to wild-type mice. Similar to humans, this effect was independent of obesity in the mutant mice and was associated with decreased expression of pro-fibrotic and pro-inflammatory genes, and increased expression of PPARγ, a potent anti-fibrogenic and anti-inflammatory regulator. Interestingly, tumor suppressors p53 and p16INK4 were found to be downregulated in the Gli2 +/- mice exposed to a high-fat diet. Our results indicate that germline mutations disrupting Hh signaling promotes liver steatosis, independent of obesity, with reduced fibrosis. While Hh signaling inhibition has been associated with a better NAFLD prognosis, further studies are required to evaluate the long-term effects of mutations affecting this pathway. Lay summary: Non-alcoholic fatty liver disease (NAFLD) is characterized by excess fat deposition in the liver predominantly due to high calorie intake and a sedentary lifestyle. NAFLD progression is usually accompanied by activation of the Sonic hedgehog (SHH) pathway leading to fibrous buildup (scar tissue) and inflammation of the liver tissue. For the first time patients with holoprosencephaly, a disease caused by SHH signaling mutations, are shown to have increased liver steatosis independent of obesity. This observation was recapitulated in a mouse model of attenuated SHH signaling that also showed increased liver steatosis but with decreased fibrosis and inflammation. While SHH inhibition is associated with a good NAFLD prognosis, this increase in liver fat accumulation in the context of SHH signaling inhibition must be studied prospectively to evaluate its long-term effects, especially in individuals with Western-type dietary habits. Published by Elsevier B.V.

The Role of Polycomb Group Gene BMI-1 in the Development of Prostate Cancer

DTIC Science & Technology

2013-07-01

cyclin D1 (Wnt target) and Bcl-2 (Sonic Hedgehog -SHH target). The novel finding in presented in the 2nd annual report was that regulation of Bcl-2... hedgehog (SHH) pathway that is very well know to regulate Bcl-2. For this purpose we determined the expression level of Bcl-2 in BMI1- overexpressing and...2006; 15: 217-27. 16. Hegde GV, Munger CM, Emanuel K, Joshi AD, Greiner TC, Weisenburger DD, Vose JM, et al. Targeting of sonic hedgehog -GLI signaling

Potential evolution of neurosurgical treatment paradigms for craniopharyngioma based on genomic and transcriptomic characteristics.

PubMed

Robinson, Leslie C; Santagata, Sandro; Hankinson, Todd C

2016-12-01

The recent genomic and transcriptomic characterization of human craniopharyngiomas has provided important insights into the pathogenesis of these tumors and supports that these tumor types are distinct entities. Critically, the insights provided by these data offer the potential for the introduction of novel therapies and surgical treatment paradigms for these tumors, which are associated with high morbidity rates and morbid conditions. Mutations in the CTNNB1 gene are primary drivers of adamantinomatous craniopharyngioma (ACP) and lead to the accumulation of β-catenin protein in a subset of the nuclei within the neoplastic epithelium of these tumors. Dysregulation of epidermal growth factor receptor (EGFR) and of sonic hedgehog (SHH) signaling in ACP suggest that paracrine oncogenic mechanisms may underlie ACP growth and implicate these signaling pathways as potential targets for therapeutic intervention using directed therapies. Recent work shows that ACP cells have primary cilia, further supporting the potential importance of SHH signaling in the pathogenesis of these tumors. While further preclinical data are needed, directed therapies could defer, or replace, the need for radiation therapy and/or allow for less aggressive surgical interventions. Furthermore, the prospect for reliable control of cystic disease without the need for surgery now exists. Studies of papillary craniopharyngioma (PCP) are more clinically advanced than those for ACP. The vast majority of PCPs harbor the BRAF v600e mutation. There are now 2 reports of patients with PCP that had dramatic therapeutic responses to targeted agents. Ongoing clinical and research studies promise to not only advance our understanding of these challenging tumors but to offer new approaches for patient management.

Diarylheptanoids suppress proliferation of pancreatic cancer PANC-1 cells through modulating shh-Gli-FoxM1 pathway.

PubMed

Dong, Guang-Zhi; Jeong, Ji Hye; Lee, Yu-Ih; Lee, So Yoon; Zhao, Hui-Yuan; Jeon, Raok; Lee, Hwa Jin; Ryu, Jae-Ha

2017-04-01

Pancreatic cancer is one of the leading causes of cancer, and it has the lowest 5-year survival rates. It is necessary to develop more potent anti-pancreatic cancer drugs to overcome the fast metastasis and resistance to surgery, radiotherapy, chemotherapy, and combinations of these. We have identified several diarylheptanoids as anti-pancreatic cancer agents from Alpinia officinarum (lesser galangal) and Alnus japonica. These diarylheptanoids suppressed cell proliferation and induced the cell cycle arrest of pancreatic cancer cells (PANC-1). Among them, the most potent compounds 1 and 7 inhibited the shh-Gli-FoxM1 pathway and their target gene expression in PANC-1 cells. Furthermore, they suppressed the expression of the cell cycle associated genes that were rescued by the overexpression of exogenous FoxM1. Taken together, (E)-7-(4-hydroxy-3-methoxyphenyl)-1-phenylhept-4-en-3-one (1) from Alpinia officinarum (lesser galangal) and platyphyllenone (7) from Alnus japonica inhibit PANC-1 cell proliferation by suppressing the shh-Gli-FoxM1 pathway, and they can be potential candidates for anti-pancreatic cancer drug development.

AMP-activated Protein Kinase Stimulates Warburg-like Glycolysis and Activation of Satellite Cells during Muscle Regeneration*

PubMed Central

Fu, Xing; Zhu, Mei-Jun; Dodson, Mike V.; Du, Min

2015-01-01

Satellite cells are the major myogenic stem cells residing inside skeletal muscle and are indispensable for muscle regeneration. Satellite cells remain largely quiescent but are rapidly activated in response to muscle injury, and the derived myogenic cells then fuse to repair damaged muscle fibers or form new muscle fibers. However, mechanisms eliciting metabolic activation, an inseparable step for satellite cell activation following muscle injury, have not been defined. We found that a noncanonical Sonic Hedgehog (Shh) pathway is rapidly activated in response to muscle injury, which activates AMPK and induces a Warburg-like glycolysis in satellite cells. AMPKα1 is the dominant AMPKα isoform expressed in satellite cells, and AMPKα1 deficiency in satellite cells impairs their activation and myogenic differentiation during muscle regeneration. Drugs activating noncanonical Shh promote proliferation of satellite cells, which is abolished because of satellite cell-specific AMPKα1 knock-out. Taken together, AMPKα1 is a critical mediator linking noncanonical Shh pathway to Warburg-like glycolysis in satellite cells, which is required for satellite activation and muscle regeneration. PMID:26370082

E2F4 is required for early eye patterning.

PubMed

Ruzhynsky, Vladimir A; Furimsky, Marosh; Park, David S; Wallace, Valerie A; Slack, Ruth S

2009-01-01

Increasingly, studies reveal novel functions for cell cycle proteins during development. Here, we investigated the role of E2F4 in eye development. E2F4-deficient mouse embryos exhibit severe early eye patterning defects, which are evident from embryonic day 11.5 and characterized by aberrant shape of the optic cup, coloboma as well as abnormal eye pigmentation. Loss of E2F4 is associated with proximal-distal patterning defects in the optic vesicle. These defects are characterized by the expansion of optic stalk marker gene expression to the optic cup and reduced expression of ventral optic cup markers. These defects are associated with a split of Shh expression domain at the ventral midline of the forebrain and expansion of the Shh activity into the ventral optic cup. Despite these patterning defects, early neuronal differentiation and Shh expression in the retina are not affected by E2F4 deletion. Overall, the results of our studies show a novel role of E2F4 in the early eye development. 2009 S. Karger AG, Basel.

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

Cross cultural research in palliative care.

PubMed

Field, Annette; Maher, Paul; Webb, David

2002-01-01

Hospices within Australia, such as the Sacred Heart Hospice (SHH) at Darlinghurst, have over many years proven to be highly successful in meeting the needs of persons who require palliative care and addressing the concerns raised by their families. However, health professionals have increasingly recognised that caring for persons who are from a Non-English Speaking Background (NESB) and for their families requires giving them special consideration because of their different cultural needs. This project involved reviewing all inpatient files of the SHH over a three year period (1 October 1994 to 30 September 1997) and quantitatively reviewing all inpatient files of NESB inpatients during this period. Following this review, the researchers coordinated a focus group that comprised various health professionals from SHH and other health and community services and representatives of four non-English speaking countries--Greece, Italy, the former USSR, and China. These countries had the highest representation of inpatients during the three-year term covered by the research project. The project identified a number of specific strategies aimed at providing a more culturally sensitive health care service to NESB inpatients.

Subcellular localization of Patched and Smoothened, the receptors for Sonic hedgehog signaling, in the hippocampal neuron.

PubMed

Petralia, Ronald S; Schwartz, Catherine M; Wang, Ya-Xian; Mattson, Mark P; Yao, Pamela J

2011-12-15

Cumulative evidence suggests that, aside from patterning the embryonic neural tube, Sonic hedgehog (Shh) signaling plays important roles in the mature nervous system. In this study, we investigate the expression and localization of the Shh signaling receptors, Patched (Ptch) and Smoothened (Smo), in the hippocampal neurons of young and mature rats. Reverse transcriptase-polymerase chain reaction and immunoblotting analyses show that the expression of Ptch and Smo remains at a moderate level in young postnatal and adult brains. By using immunofluorescence light microscopy and immunoelectron microscopy, we examine the spatial distribution of Ptch and Smo within the hippocampal neurons. In young developing neurons, Ptch and Smo are present in the processes and are clustered at their growth cones. In mature neurons, Ptch and Smo are concentrated in dendrites, spines, and postsynaptic sites. Synaptic Ptch and Smo often co-exist with unusual structures-synaptic spinules and autophagosomes. Our results reveal the anatomical organization of the Shh receptors within both the young and the mature hippocampal neurons. Copyright © 2011 Wiley-Liss, Inc.

Zika Virus Can Strongly Infect and Disrupt Secondary Organizers in the Ventricular Zone of the Embryonic Chicken Brain.

PubMed

Thawani, Ankita; Sirohi, Devika; Kuhn, Richard J; Fekete, Donna M

2018-04-17

Zika virus (ZIKV) is associated with severe neurodevelopmental impairments in human fetuses, including microencephaly. Previous reports examining neural progenitor tropism of ZIKV in organoid and animal models did not address whether the virus infects all neural progenitors uniformly. To explore this, ZIKV was injected into the neural tube of 2-day-old chicken embryos, resulting in nonuniform periventricular infection 3 days later. Recurrent foci of intense infection were present at specific signaling centers that influence neuroepithelial patterning at a distance through secretion of morphogens. ZIKV infection reduced transcript levels for 3 morphogens, SHH, BMP7, and FGF8 expressed at the midbrain basal plate, hypothalamic floor plate, and isthmus, respectively. Levels of Patched1, a SHH-pathway downstream gene, were also reduced, and a SHH-dependent cell population in the ventral midbrain was shifted in position. Thus, the diminishment of signaling centers through ZIKV-mediated apoptosis may yield broader, non-cell-autonomous changes in brain patterning. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Mouse Mesenchyme forkhead 2 (Mf2): expression, DNA binding and induction by sonic hedgehog during somitogenesis.

PubMed

Wu, S C; Grindley, J; Winnier, G E; Hargett, L; Hogan, B L

1998-01-01

Cloning and sequencing of mouse Mf2 (mesoderm/mesenchyme forkhead 2) cDNAs revealed an open reading frame encoding a putative protein of 492 amino acids which, after in vitro translation, binds to a DNA consensus sequence. Mf2 is expressed at high levels in the ventral region of newly formed somites, in sclerotomal derivatives, in lateral plate and cephalic mesoderm and in the first and second branchial arches. Other regions of mesodermal expression include the developing tongue, meninges, nose, whiskers, kidney, genital tubercule and limb joints. In the nervous system Mf2 is transcribed in restricted regions of the mid- and forebrain. In several tissues, including the early somite, Mf2 is expressed in cell populations adjacent to regions expressing sonic hedgehog (Shh) and in explant cultures of presomitic mesoderm Mf2 is induced by Shh secreted by COS cells. These results suggest that Mf2, like other murine forkhead genes, has multiple roles in embryogenesis, possibly mediating the response of cells to signaling molecules such as SHH.

Novel molecular subgroups for clinical classification and outcome prediction in childhood medulloblastoma: a cohort study.

PubMed

Schwalbe, Edward C; Lindsey, Janet C; Nakjang, Sirintra; Crosier, Stephen; Smith, Amanda J; Hicks, Debbie; Rafiee, Gholamreza; Hill, Rebecca M; Iliasova, Alice; Stone, Thomas; Pizer, Barry; Michalski, Antony; Joshi, Abhijit; Wharton, Stephen B; Jacques, Thomas S; Bailey, Simon; Williamson, Daniel; Clifford, Steven C

2017-07-01

International consensus recognises four medulloblastoma molecular subgroups: WNT (MB WNT ), SHH (MB SHH ), group 3 (MB Grp3 ), and group 4 (MB Grp4 ), each defined by their characteristic genome-wide transcriptomic and DNA methylomic profiles. These subgroups have distinct clinicopathological and molecular features, and underpin current disease subclassification and initial subgroup-directed therapies that are underway in clinical trials. However, substantial biological heterogeneity and differences in survival are apparent within each subgroup, which remain to be resolved. We aimed to investigate whether additional molecular subgroups exist within childhood medulloblastoma and whether these could be used to improve disease subclassification and prognosis predictions. In this retrospective cohort study, we assessed 428 primary medulloblastoma samples collected from UK Children's Cancer and Leukaemia Group (CCLG) treatment centres (UK), collaborating European institutions, and the UKCCSG-SIOP-PNET3 European clinical trial. An independent validation cohort (n=276) of archival tumour samples was also analysed. We analysed samples from patients with childhood medulloblastoma who were aged 0-16 years at diagnosis, and had central review of pathology and comprehensive clinical data. We did comprehensive molecular profiling, including DNA methylation microarray analysis, and did unsupervised class discovery of test and validation cohorts to identify consensus primary molecular subgroups and characterise their clinical and biological significance. We modelled survival of patients aged 3-16 years in patients (n=215) who had craniospinal irradiation and had been treated with a curative intent. Seven robust and reproducible primary molecular subgroups of childhood medulloblastoma were identified. MB WNT remained unchanged and each remaining consensus subgroup was split in two. MB SHH was split into age-dependent subgroups corresponding to infant (<4·3 years; MB SHH-Infant ; n=65) and childhood patients (≥4·3 years; MB SHH-Child ; n=38). MB Grp3 and MB Grp4 were each split into high-risk (MB Grp3-HR [n=65] and MB Grp4-HR [n=85]) and low-risk (MB Grp3-LR [n=50] and MB Grp4-LR [n=73]) subgroups. These biological subgroups were validated in the independent cohort. We identified features of the seven subgroups that were predictive of outcome. Cross-validated subgroup-dependent survival models, incorporating these novel subgroups along with secondary clinicopathological and molecular features and established disease risk-factors, outperformed existing disease risk-stratification schemes. These subgroup-dependent models stratified patients into four clinical risk groups for 5-year progression-free survival: favourable risk (54 [25%] of 215 patients; 91% survival [95% CI 82-100]); standard risk (50 [23%] patients; 81% survival [70-94]); high-risk (82 [38%] patients; 42% survival [31-56]); and very high-risk (29 [13%] patients; 28% survival [14-56]). The discovery of seven novel, clinically significant subgroups improves disease risk-stratification and could inform treatment decisions. These data provide a new foundation for future research and clinical investigations. Cancer Research UK, The Tom Grahame Trust, Star for Harris, Action Medical Research, SPARKS, The JGW Patterson Foundation, The INSTINCT network (co-funded by The Brain Tumour Charity, Great Ormond Street Children's Charity, and Children with Cancer UK). Copyright © 2017 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY 4.0 license. Published by Elsevier Ltd.. All rights reserved.

Inflammatory PAF Receptor Signaling Initiates Hedgehog Signaling and Kidney Fibrogenesis During Ethanol Consumption

PubMed Central

Latchoumycandane, Calivarathan; Hanouneh, Mohamad; Nagy, Laura E.; McIntyre, Thomas M.

2015-01-01

Acute inflammation either resolves or proceeds to fibrotic repair that replaces functional tissue. Pro-fibrotic hedgehog signaling and induction of its Gli transcription factor in pericytes induces fibrosis in kidney, but molecular instructions connecting inflammation to fibrosis are opaque. We show acute kidney inflammation resulting from chronic ingestion of the common xenobiotic ethanol initiates Gli1 transcription and hedgehog synthesis in kidney pericytes, and promotes renal fibrosis. Ethanol ingestion stimulated transcription of TGF-ß, collagens I and IV, and alpha-smooth muscle actin with accumulation of these proteins. This was accompanied by deposition of extracellular fibrils. Ethanol catabolism by CYP2E1 in kidney generates local reactive oxygen species that oxidize cellular phospholipids to phospholipid products that activate the Platelet-activating Factor receptor (PTAFR) for inflammatory phospholipids. Genetically deleting this ptafr locus abolished accumulation of mRNA for TGF-ß, collagen IV, and α-smooth muscle actin. Loss of PTAFR also abolished ethanol-stimulated Sonic (Shh) and Indian hedgehog (Ihh) expression, and abolished transcription and accumulation of Gli1. Shh induced in pericytes and Ihh in tubules escaped to urine of ethanol-fed mice. Neutrophil myeloperoxidase (MPO) is required for ethanol-induced kidney inflammation, and Shh was not present in kidney or urine of mpo -/- mice. Shh also was present in urine of patients with acute kidney injury, but not in normal individuals or those with fibrotic liver cirrhosis We conclude neither endogenous PTAFR signaling nor CYP2E1-generated radicals alone are sufficient to initiate hedgehog signaling, but instead PTAFR-dependent neutrophil infiltration with myeloperoxidase activation is necessary to initiate ethanol-induced fibrosis in kidney. We also show fibrogenic mediators escape to urine, defining a new class of urinary mechanistic biomarkers of fibrogenesis for an organ not commonly biopsied. PMID:26720402

Hedgehog signaling in the murine melanoma microenvironment.

PubMed

Geng, Ling; Cuneo, Kyle C; Cooper, Michael K; Wang, Hong; Sekhar, Konjeti; Fu, Allie; Hallahan, Dennis E

2007-01-01

The Hedgehog intercellular signaling pathway regulates cell proliferation and differentiation. This pathway has been implicated to play a role in the pathogenesis of cancer and in embryonic blood vessel development. In the current study, Hedgehog signaling in tumor related vasculature and microenvironment was examined using human umbilical vein endothelial cells and B16F0 (murine melanoma) tumors models. Use of exogenous Sonic hedgehog (Shh) peptide significantly increased BrdU incorporation in endothelial cells in vitro by a factor of 2 (P < 0.001). The Hedgehog pathway antagonist cyclopamine effectively reduced Shh-induced proliferation to control levels. To study Hedgehog signaling in vivo a hind limb tumor model with the B16F0 cell line was used. Treatment with 25 mg/kg cyclopamine significantly attenuated BrdU incorporation in tumor cells threefold (P < 0.001), in tumor related endothelial cells threefold (P = 0.004), and delayed tumor growth by 4 days. Immunohistochemistry revealed that the Hedgehog receptor Patched was localized to the tumor stroma and that B16F0 cells expressed Shh peptide. Furthermore, mouse embryonic fibroblasts required the presence of B16F0 cells to express Patched in a co-culture assay system. These studies indicate that Shh peptide produced by melanoma cells induces Patched expression in fibroblasts. To study tumor related angiogenesis a vascular window model was used to monitor tumor vascularity. Treatment with cyclopamine significantly attenuated vascular formation by a factor of 2.5 (P < 0.001) and altered vascular morphology. Furthermore, cyclopamine reduced tumor blood vessel permeability to FITC labeled dextran while having no effect on normal blood vessels. These studies suggest that Hedgehog signaling regulates melanoma related vascular formation and function.

The involvement of immunoglobulin E isotype switch in scleroderma skin tissue.

PubMed

Ohtsuka, Tsutomu; Yamazaki, Soji

2005-08-01

The involvement of mast cell, which is activated by immunoglobulin E (IgE), has been reported in the formation of systemic sclerosis (SSc) abnormality. IgE is generated with isotype switch. During isotype switch, switch circles resulting from direct mu to epsilon, or from sequential mu to gamma via epsilon switching will be created. We studied whether switching occurs in SSc. We used nested polymerase chain reaction to analyze the S fragments from switch circles. Fifty-two patients with SSc, and 62 healthy women were studied. Neither of 62 normal skin tissues showed direct switch, nor sequential switch. Neither of seven normal whole blood cells showed direct switch, nor sequential switch. In 52SSc skin tissues, three (5.8%) showed direct switch, and two (3.8%) showed sequential switch. As a result, five (9.6%) of SSc skin tissue showed immunogobulin E class switch. These results were confirmed by DNA sequencing. These results demonstrated that isotype switch to the epsilon locus achieved by direct and/or sequential switch are involved in SSc skin.

AAV-mediated gene therapy in Dystrophin-Dp71 deficient mouse leads to blood-retinal barrier restoration and oedema reabsorption.

PubMed

Vacca, Ophélie; Charles-Messance, Hugo; El Mathari, Brahim; Sene, Abdoulaye; Barbe, Peggy; Fouquet, Stéphane; Aragón, Jorge; Darche, Marie; Giocanti-Aurégan, Audrey; Paques, Michel; Sahel, José-Alain; Tadayoni, Ramin; Montañez, Cecilia; Dalkara, Deniz; Rendon, Alvaro

2016-07-15

Dystrophin-Dp71 being a key membrane cytoskeletal protein, expressed mainly in Müller cells that provide a mechanical link at the Müller cell membrane by direct binding to actin and a transmembrane protein complex. Its absence has been related to blood-retinal barrier (BRB) permeability through delocalization and down-regulation of the AQP4 and Kir4.1 channels (1). We have previously shown that the adeno-associated virus (AAV) variant, ShH10, transduces Müller cells in the Dp71-null mouse retina efficiently and specifically (2,3). Here, we use ShH10 to restore Dp71 expression in Müller cells of Dp71 deficient mouse to study molecular and functional effects of this restoration in an adult mouse displaying retinal permeability. We show that strong and specific expression of exogenous Dp71 in Müller cells leads to correct localization of Dp71 protein restoring all protein interactions in order to re-establish a proper functional BRB and retina homeostasis thus preventing retina from oedema. This study is the basis for the development of new therapeutic strategies in dealing with diseases with BRB breakdown and macular oedema such as diabetic retinopathy (DR). © The Author 2016. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Cell cycle regulator E2F4 is essential for the development of the ventral telencephalon.

PubMed

Ruzhynsky, Vladimir A; McClellan, Kelly A; Vanderluit, Jacqueline L; Jeong, Yongsu; Furimsky, Marosh; Park, David S; Epstein, Douglas J; Wallace, Valerie A; Slack, Ruth S

2007-05-30

Early forebrain development is characterized by extensive proliferation of neural precursors coupled with complex structural transformations; however, little is known regarding the mechanisms by which these processes are integrated. Here, we show that deficiency of the cell cycle regulatory protein, E2F4, results in the loss of ventral telencephalic structures and impaired self-renewal of neural precursor cells. The mechanism underlying aberrant ventral patterning lies in a dramatic loss of Sonic hedgehog (Shh) expression specifically in this region. The E2F4-deficient phenotype can be recapitulated by interbreeding mice heterozygous for E2F4 with those lacking one allele of Shh, suggesting a genetic interaction between these pathways. Treatment of E2F4-deficient cells with a Hh agonist rescues stem cell self-renewal and cells expressing the homeodomain proteins that specify the ventral telencephalic structures. Finally, we show that E2F4 deficiency results in impaired activity of Shh forebrain-specific enhancers. In conclusion, these studies establish a novel requirement for the cell cycle regulatory protein, E2F4, in the development of the ventral telencephalon.

Update on the integrated histopathological and genetic classification of medulloblastoma – a practical diagnostic guideline

PubMed Central

Pietsch, Torsten; Haberler, Christine

2016-01-01

The revised WHO classification of tumors of the CNS 2016 has introduced the concept of the integrated diagnosis. The definition of medulloblastoma entities now requires a combination of the traditional histological information with additional molecular/genetic features. For definition of the histopathological component of the medulloblastoma diagnosis, the tumors should be assigned to one of the four entities classic, desmoplastic/nodular (DNMB), extensive nodular (MBEN), or large cell/anaplastic (LC/A) medulloblastoma. The genetically defined component comprises the four entities WNT-activated, SHH-activated and TP53 wildtype, SHH-activated and TP53 mutant, or non-WNT/non-SHH medulloblastoma. Robust and validated methods are available to allow a precise diagnosis of these medulloblastoma entities according to the updated WHO classification, and for differential diagnostic purposes. A combination of immunohistochemical markers including β-catenin, Yap1, p75-NGFR, Otx2, and p53, in combination with targeted sequencing and copy number assessment such as FISH analysis for MYC genes allows a precise assignment of patients for risk-adapted stratification. It also allows comparison to results of study cohorts in the past and provides a robust basis for further treatment refinement. PMID:27781424

Update on the integrated histopathological and genetic classification of medulloblastoma - a practical diagnostic guideline.

PubMed

Pietsch, Torsten; Haberler, Christine

The revised WHO classification of tumors of the CNS 2016 has introduced the concept of the integrated diagnosis. The definition of medulloblastoma entities now requires a combination of the traditional histological information with additional molecular/genetic features. For definition of the histopathological component of the medulloblastoma diagnosis, the tumors should be assigned to one of the four entities classic, desmoplastic/nodular (DNMB), extensive nodular (MBEN), or large cell/anaplastic (LC/A) medulloblastoma. The genetically defined component comprises the four entities WNT-activated, SHH-activated and TP53 wildtype, SHH-activated and TP53 mutant, or non-WNT/non-SHH medulloblastoma. Robust and validated methods are available to allow a precise diagnosis of these medulloblastoma entities according to the updated WHO classification, and for differential diagnostic purposes. A combination of immunohistochemical markers including β-catenin, Yap1, p75-NGFR, Otx2, and p53, in combination with targeted sequencing and copy number assessment such as FISH analysis for MYC genes allows a precise assignment of patients for risk-adapted stratification. It also allows comparison to results of study cohorts in the past and provides a robust basis for further treatment refinement.
.

Transplantation of oligodendrocyte precursors and sonic hedgehog results in improved function and white matter sparing in the spinal cords of adult rats after contusion.

PubMed

Bambakidis, Nicholas C; Miller, Robert H

2004-01-01

A substantial cause of neurological disability in spinal cord injury is oligodendrocyte death leading to demyelination and axonal degeneration. Rescuing oligodendrocytes and preserving myelin is expected to result in significant improvement in functional outcome after spinal cord injury. Although previous investigators have used cellular transplantation of xenografted pluripotent embryonic stem cells and observed improved functional outcome, these transplants have required steroid administration and only a minority of these cells develop into oligodendrocytes. The objective of the present study was to determine whether allografts of oligodendrocyte precursors transplanted into an area of incomplete spinal cord contusion would improve behavioral and electrophysiological measures of spinal cord function. Additional treatment incorporated the use of the glycoprotein molecule Sonic hedgehog (Shh), which has been shown to play a critical role in oligodendroglial development and induce proliferation of endogenous neural precursors after spinal cord injury. Laboratory study. Moderate spinal cord contusion injury was produced in 39 adult rats at T9-T10. Ten animals died during the course of the study. Nine rats served as contusion controls (Group 1). Six rats were treated with oligodendrocyte precursor transplantation 5 days after injury (Group 2). The transplanted cells were isolated from newborn rat pups using immunopanning techniques. Another eight rats received an injection of recombinant Shh along with the oligodendrocyte precursors (Group 3), while six more rats were treated with Shh alone (Group 4). Eight additional rats received only T9 laminectomies to serve as noninjured controls (Group 0). Animals were followed for 28 days. After an initial complete hindlimb paralysis, rats of all groups receiving a contusive injury recovered substantial function within 1 week. By 28 days, rats in Groups 2 and 3 scored 4.7 and 5.8 points better on the Basso, Beattie, Bresnahan (BBB) open field locomotor score than rats in group 1 (Groups 2 and 3=18.2 and 19.4 points, respectively, after 28 days vs. Group 1=13.6 points; p=.015). Rats in Group 4 scored no better than those in Group 1 (BBB=16.4). Motor evoked potential (MEP) recordings revealed a strong trend towards significant improvement in latency measurements in all treatment groups compared with controls at 28 days, although three animals in Group 1 and two animals in Group 3 were not recordable. Histological examination demonstrated significantly more spared white matter in the same groups that correlated with the improvements in BBB scores and MEP latencies. Immunohistochemical analysis showed the survival, proliferation and migration of the transplanted cells, as well as the induction of proliferating endogenous neural precursor cells in animals treated with Shh. These findings suggest that the transplantation of oligodendrocyte precursors may improve axonal conduction and spinal cord function in the injured spinal cord. The benefits seem more pronounced with the addition of Shh, and the addition of Shh alone results in the proliferation of an endogenous population of neural precursor cells.

Risk-adapted therapy for young children with medulloblastoma (SJYC07): therapeutic and molecular outcomes from a multicentre, phase 2 trial.

PubMed

Robinson, Giles W; Rudneva, Vasilisa A; Buchhalter, Ivo; Billups, Catherine A; Waszak, Sebastian M; Smith, Kyle S; Bowers, Daniel C; Bendel, Anne; Fisher, Paul G; Partap, Sonia; Crawford, John R; Hassall, Tim; Indelicato, Daniel J; Boop, Frederick; Klimo, Paul; Sabin, Noah D; Patay, Zoltan; Merchant, Thomas E; Stewart, Clinton F; Orr, Brent A; Korbel, Jan O; Jones, David T W; Sharma, Tanvi; Lichter, Peter; Kool, Marcel; Korshunov, Andrey; Pfister, Stefan M; Gilbertson, Richard J; Sanders, Robert P; Onar-Thomas, Arzu; Ellison, David W; Gajjar, Amar; Northcott, Paul A

2018-05-16

Young children with medulloblastoma have a poor overall survival compared with older children, due to use of radiation-sparing therapy in young children. Radiotherapy is omitted or reduced in these young patients to spare them from debilitating long-term side-effects. We aimed to estimate event-free survival and define the molecular characteristics associated with progression-free survival in young patients with medulloblastoma using a risk-stratified treatment strategy designed to defer, reduce, or delay radiation exposure. In this multicentre, phase 2 trial, we enrolled children younger than 3 years with newly diagnosed medulloblastoma at six centres in the USA and Australia. Children aged 3-5 years with newly diagnosed, non-metastatic medulloblastoma without any high-risk features were also eligible. Eligible patients were required to start therapy within 31 days from definitive surgery, had a Lansky performance score of at least 30, and did not receive previous radiotherapy or chemotherapy. Patients were stratified postoperatively by clinical and histological criteria into low-risk, intermediate-risk, and high-risk treatment groups. All patients received identical induction chemotherapy (methotrexate, vincristine, cisplatin, and cyclophosphamide), with high-risk patients also receiving an additional five doses of vinblastine. Induction was followed by risk-adapted consolidation therapy: low-risk patients received cyclophosphamide (1500 mg/m 2 on day 1), etoposide (100 mg/m 2 on days 1 and 2), and carboplatin (area under the curve 5 mg/mL per min on day 2) for two 4-week cycles; intermediate-risk patients received focal radiation therapy (54 Gy with a clinical target volume of 5 mm over 6 weeks) to the tumour bed; and high-risk patients received chemotherapy with targeted intravenous topotecan (area under the curve 120-160 ng-h/mL intravenously on days 1-5) and cyclophosphamide (600 mg/m 2 intravenously on days 1-5). After consolidation, all patients received maintenance chemotherapy with cyclophosphamide, topotecan, and erlotinib. The coprimary endpoints were event-free survival and patterns of methylation profiling associated with progression-free survival. Outcome and safety analyses were per protocol (all patients who received at least one dose of induction chemotherapy); biological analyses included all patients with tissue available for methylation profiling. This trial is registered with ClinicalTrials.gov, number NCT00602667, and was closed to accrual on April 19, 2017. Between Nov 27, 2007, and April 19, 2017, we enrolled 81 patients with histologically confirmed medulloblastoma. Accrual to the low-risk group was suspended after an interim analysis on Dec 2, 2015, when the 1-year event-free survival was estimated to be below the stopping rule boundary. After a median follow-up of 5·5 years (IQR 2·7-7·3), 5-year event-free survival was 31·3% (95% CI 19·3-43·3) for the whole cohort, 55·3% (95% CI 33·3-77·3) in the low-risk cohort (n=23) versus 24·6% (3·6-45·6) in the intermediate-risk cohort (n=32; hazard ratio 2·50, 95% CI 1·19-5·27; p=0·016) and 16·7% (3·4-30·0) in the high-risk cohort (n=26; 3·55, 1·66-7·59; p=0·0011; overall p=0·0021). 5-year progression-free survival by methylation subgroup was 51·1% (95% CI 34·6-67·6) in the sonic hedgehog (SHH) subgroup (n=42), 8·3% (95% CI 0·0-24·0%) in the group 3 subgroup (n=24), and 13·3% (95% CI 0·0-37·6%) in the group 4 subgroup (n=10). Within the SHH subgroup, two distinct methylation subtypes were identified and named iSHH-I and iSHH-II. 5-year progression-free survival was 27·8% (95% CI 9·0-46·6; n=21) for iSHH-I and 75·4% (55·0-95·8; n=21) for iSHH-II. The most common adverse events were grade 3-4 febrile neutropenia (48 patients [59%]), neutropenia (21 [26%]), infection with neutropenia (20 [25%]), leucopenia (15 [19%]), vomiting (15 [19%]), and anorexia (13 [16%]). No treatment-related deaths occurred. The risk-adapted approach did not improve event-free survival in young children with medulloblastoma. However, the methylation subgroup analyses showed that the SHH subgroup had improved progression-free survival compared with the group 3 subgroup. Moreover, within the SHH subgroup, the iSHH-II subtype had improved progression-free survival in the absence of radiation, intraventricular chemotherapy, or high-dose chemotherapy compared with the iSHH-I subtype. These findings support the development of a molecularly driven, risk-adapted, treatment approach in future trials in young children with medulloblastoma. American Lebanese Syrian Associated Charities, St Jude Children's Research Hospital, NCI Cancer Center, Alexander and Margaret Stewart Trust, Sontag Foundation, and American Association for Cancer Research. Copyright © 2018 Elsevier Ltd. All rights reserved.

Devaluation and sequential decisions: linking goal-directed and model-based behavior

PubMed Central

Friedel, Eva; Koch, Stefan P.; Wendt, Jean; Heinz, Andreas; Deserno, Lorenz; Schlagenhauf, Florian

2014-01-01

In experimental psychology different experiments have been developed to assess goal–directed as compared to habitual control over instrumental decisions. Similar to animal studies selective devaluation procedures have been used. More recently sequential decision-making tasks have been designed to assess the degree of goal-directed vs. habitual choice behavior in terms of an influential computational theory of model-based compared to model-free behavioral control. As recently suggested, different measurements are thought to reflect the same construct. Yet, there has been no attempt to directly assess the construct validity of these different measurements. In the present study, we used a devaluation paradigm and a sequential decision-making task to address this question of construct validity in a sample of 18 healthy male human participants. Correlational analysis revealed a positive association between model-based choices during sequential decisions and goal-directed behavior after devaluation suggesting a single framework underlying both operationalizations and speaking in favor of construct validity of both measurement approaches. Up to now, this has been merely assumed but never been directly tested in humans. PMID:25136310

Inhibition of Wnt signaling by Wise (Sostdc1) and negative feedback from Shh controls tooth number and patterning.

PubMed

Ahn, Youngwook; Sanderson, Brian W; Klein, Ophir D; Krumlauf, Robb

2010-10-01

Mice carrying mutations in Wise (Sostdc1) display defects in many aspects of tooth development, including tooth number, size and cusp pattern. To understand the basis of these defects, we have investigated the pathways modulated by Wise in tooth development. We present evidence that, in tooth development, Wise suppresses survival of the diastema or incisor vestigial buds by serving as an inhibitor of Lrp5- and Lrp6-dependent Wnt signaling. Reducing the dosage of the Wnt co-receptor genes Lrp5 and Lrp6 rescues the Wise-null tooth phenotypes. Inactivation of Wise leads to elevated Wnt signaling and, as a consequence, vestigial tooth buds in the normally toothless diastema region display increased proliferation and continuous development to form supernumerary teeth. Conversely, gain-of-function studies show that ectopic Wise reduces Wnt signaling and tooth number. Our analyses demonstrate that the Fgf and Shh pathways are major downstream targets of Wise-regulated Wnt signaling. Furthermore, our experiments revealed that Shh acts as a negative-feedback regulator of Wnt signaling and thus determines the fate of the vestigial buds and later tooth patterning. These data provide insight into the mechanisms that control Wnt signaling in tooth development and into how crosstalk among signaling pathways controls tooth number and morphogenesis.

Opposing Shh and Fgf signals initiate nasotemporal patterning of the zebrafish retina.

PubMed

Hernández-Bejarano, María; Gestri, Gaia; Spawls, Lana; Nieto-López, Francisco; Picker, Alexander; Tada, Masazumi; Brand, Michael; Bovolenta, Paola; Wilson, Stephen W; Cavodeassi, Florencia

2015-11-15

The earliest known determinants of retinal nasotemporal identity are the transcriptional regulators Foxg1, which is expressed in the prospective nasal optic vesicle, and Foxd1, which is expressed in the prospective temporal optic vesicle. Previous work has shown that, in zebrafish, Fgf signals from the dorsal forebrain and olfactory primordia are required to specify nasal identity in the dorsal, prospective nasal, optic vesicle. Here, we show that Hh signalling from the ventral forebrain is required for specification of temporal identity in the ventral optic vesicle and is sufficient to induce temporal character when activated in the prospective nasal retina. Consequently, the evaginating optic vesicles become partitioned into prospective nasal and temporal domains by the opposing actions of Fgfs and Shh emanating from dorsal and ventral domains of the forebrain primordium. In absence of Fgf activity, foxd1 expression is established irrespective of levels of Hh signalling, indicating that the role of Shh in promoting foxd1 expression is only required in the presence of Fgf activity. Once the spatially complementary expression of foxd1 and foxg1 is established, the boundary between expression domains is maintained by mutual repression between Foxd1 and Foxg1. © 2015. Published by The Company of Biologists Ltd.

Hyperinnervation improves Xenopus laevis limb regeneration.

PubMed

Mitogawa, Kazumasa; Makanae, Aki; Satoh, Akira

2018-01-15

Xenopus laevis (an anuran amphibian) shows limb regeneration ability between that of urodele amphibians and that of amniotes. Xenopus frogs can initiate limb regeneration but fail to form patterned limbs. Regenerated limbs mainly consist of cone-shaped cartilage without any joints or branches. These pattern defects are thought to be caused by loss of proper expressions of patterning-related genes. This study shows that hyperinnervation surgery resulted in the induction of a branching regenerate. The hyperinnervated blastema allows the identification and functional analysis of the molecules controlling this patterning of limb regeneration. This paper focuses on the nerve affects to improve Xenopus limb patterning ability during regeneration. The nerve molecules, which regulate limb patterning, were also investigated. Blastemas grown in a hyperinnervated forelimb upregulate limb patterning-related genes (shh, lmx1b, and hoxa13). Nerves projecting their axons to limbs express some growth factors (bmp7, fgf2, fgf8, and shh). Inputs of these factors to a blastema upregulated some limb patterning-related genes and resulted in changes in the cartilage patterns in the regenerates. These results indicate that additional nerve factors enhance Xenopus limb patterning-related gene expressions and limb regeneration ability, and that bmp, fgf, and shh are candidate nerve substitute factors. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Effect of abnormal notochord delamination on hindgut development in the Adriamycin mouse model.

PubMed

Sato, Hideaki; Hajduk, Piotr; Furuta, Shigeyuki; Wakisaka, Munechika; Murphy, Paula; Puri, Prem; Kitagawa, Hiroaki

2013-11-01

Adriamycin mouse model (AMM) is a model of VACTERL anomalies. Sonic hedgehog (Shh) pathway, sourced by the notochord, is implicated of anorectal malformations. We hypothesized hindgut anomalies observed in the AMM are the result of abnormal effect of the notochord. Time-mated CBA/Ca mice received two intraperitoneal injections of Adriamycin (6 mg/kg) or saline as control on embryonic day (E) 7 and 8. Fetuses were harvested from E9 to E11, stained following whole mount in situ hybridization with labeled RNA probes to detect Shh and Fork head box F1(Foxf1) transcripts. Immunolocalization with endoderm marker Hnf3β was used to visualize morphology. Embryos were scanned by OPT to obtain 3D representations of expressions. In AMM, the notochord was abnormally displaced ventrally with attachment to the hindgut endoderm in 71 % of the specimens. In 32 % of the treated embryos abnormal hindgut ended blindly in a cystic structure, and both of types were remarked in 29 % of treated embryos. Endodermal Shh and mesenchymal Foxf1 genes expression were preserved around the hindgut cystic malformation. The delamination of the developing notochord in the AMM is disrupted, which may influence signaling mechanisms from the notochord to the hindgut resulting in abnormal patterning of the hindgut.

Genetic interactions between the hedgehog co-receptors Gas1 and Boc regulate cell proliferation during murine palatogenesis

PubMed Central

Xavier, Guilherme M.; Seppala, Maisa; Papageorgiou, Spyridon N.; Fan, Chen-Ming; Cobourne, Martyn T.

2016-01-01

Abnormal regulation of Sonic hedgehog (Shh) signaling has been described in a variety of human cancers and developmental anomalies, which highlights the essential role of this signaling molecule in cell cycle regulation and embryonic development. Gas1 and Boc are membrane co-receptors for Shh, which demonstrate overlapping domains of expression in the early face. This study aims to investigate potential interactions between these co-receptors during formation of the secondary palate. Mice with targeted mutation in Gas1 and Boc were used to generate Gas1; Boc compound mutants. The expression of key Hedgehog signaling family members was examined in detail during palatogenesis via radioactive in situ hybridization. Morphometric analysis involved computational quantification of BrdU-labeling and cell packing; whilst TUNEL staining was used to assay cell death. Ablation of Boc in a Gas1 mutant background leads to reduced Shh activity in the palatal shelves and an increase in the penetrance and severity of cleft palate, associated with failed elevation, increased proliferation and reduced cell death. Our findings suggest a dual requirement for Boc and Gas1 during early development of the palate, mediating cell cycle regulation during growth and subsequent fusion of the palatal shelves. PMID:27811357

Loss and Re-emergence of Legs in Snakes by Modular Evolution of Sonic hedgehog and HOXD Enhancers.

PubMed

Leal, Francisca; Cohn, Martin J

2016-11-07

Limb reduction and loss are hallmarks of snake evolution. Although advanced snakes are completely limbless, basal and intermediate snakes retain pelvic girdles and small rudiments of the femur. Moreover, legs may have re-emerged in extinct snake lineages [1-5], suggesting that the mechanisms of limb development were not completely lost in snakes. Here we report that hindlimb development arrests in python embryos as a result of mutations that abolish essential transcription factor binding sites in the limb-specific enhancer of Sonic hedgehog (SHH). Consequently, SHH transcription is weak and transient in python hindlimb buds, leading to early termination of a genetic circuit that drives limb outgrowth. Our results suggest that degenerate evolution of the SHH limb enhancer played a role in reduction of hindlimbs during snake evolution. By contrast, HOXD digit enhancers are conserved in pythons, and HOXD gene expression in the hindlimb buds progresses to the distal phase, forming an autopodial (digit) domain. Python hindlimb buds then develop transitory pre-chondrogenic condensations of the tibia, fibula, and footplate, raising the possibility that re-emergence of hindlimbs during snake evolution did not require de novo re-evolution of lost structures but instead could have resulted from persistence of embryonic legs. VIDEO ABSTRACT. Copyright © 2016 Elsevier Ltd. All rights reserved.

Hedgehog signaling plays roles in epithelial cell proliferation in neonatal mouse uterus and vagina.

PubMed

Nakajima, Tadaaki; Iguchi, Taisen; Sato, Tomomi

2012-04-01

Both the uterus and vagina develop from the Müllerian duct but are quite distinct in morphology and function. To investigate factors controlling epithelial differentiation and cell proliferation in neonatal uterus and vagina, we focused on Hedgehog (HH) signaling. In neonatal mice, Sonic hh (Shh) was localized in the vaginal epithelium and Indian hh (Ihh) was slightly expressed in the uterus and vagina, whereas all Glioma-associated oncogene homolog (Gli) genes were mainly expressed in the stroma. The expression of target genes of HH signaling was high in the neonatal vagina and in the uterus, it increased with growth. Thus, in neonatal mice, Shh in the vaginal epithelium and Ihh in the uterus and vagina activated HH signaling in the stroma. Tissue recombinants showed that vaginal Shh expression was inhibited by the vaginal stroma and uterine Ihh expression was stimulated by the uterine stroma. Addition of a HH signaling inhibitor decreased epithelial cell proliferation in organ-cultured uterus and vagina and increased stromal cell proliferation in organ-cultured uterus. However, it did not affect epithelial differentiation or the expression of growth factors in organ-cultured uterus and vagina. Thus, activated HH signaling stimulates epithelial cell proliferation in neonatal uterus and vagina but inhibits stromal cell proliferation in neonatal uterus.

Basal Cell Carcinoma in Gorlin's Patients: a Matter of Fibroblasts-Led Protumoral Microenvironment?

PubMed

Gache, Yannick; Brellier, Florence; Rouanet, Sophie; Al-Qaraghuli, Sahar; Goncalves-Maia, Maria; Burty-Valin, Elodie; Barnay, Stéphanie; Scarzello, Sabine; Ruat, Martial; Sevenet, Nicolas; Avril, Marie-Françoise; Magnaldo, Thierry

2015-01-01

Basal cell carcinoma (BCC) is the commonest tumor in human. About 70% sporadic BCCs bear somatic mutations in the PATCHED1 tumor suppressor gene which encodes the receptor for the Sonic Hedgehog morphogen (SHH). PATCHED1 germinal mutations are associated with the dominant Nevoid Basal Cell Carcinoma Syndrome (NBCCS), a major hallmark of which is a high susceptibility to BCCs. Although the vast majority of sporadic BCCs arises exclusively in sun exposed skin areas, 40 to 50% BCCs from NBCCS patients develop in non photo-exposed skin. Since overwhelming evidences indicate that microenvironment may both be modified by- and influence the- epithelial tumor, we hypothesized that NBCCS fibroblasts could contribute to BCCs in NBCCS patients, notably those developing in non photo-exposed skin areas. The functional impact of NBCCS fibroblasts was then assessed in organotypic skin cultures with control keratinocytes. Onset of epidermal differentiation was delayed in the presence of primary NBCCS fibroblasts. Unexpectedly, keratinocyte proliferation was severely reduced and showed high levels of nuclear P53 in both organotypic skin cultures and in fibroblast-led conditioning experiments. However, in spite of increased levels of senescence associated β-galactosidase activity in keratinocytes cultured in the presence of medium conditioned by NBCCS fibroblasts, we failed to observe activation of P16 and P21 and then of bona fide features of senescence. Constitutive extinction of P53 in WT keratinocytes resulted in an invasive phenotype in the presence of NBCCS fibroblasts. Finally, we found that expression of SHH was limited to fibroblasts but was dependent on the presence of keratinocytes. Inhibition of SHH binding resulted in improved epidermal morphogenesis. Altogether, these data suggest that the repertoire of diffusible factors (including SHH) expressed by primary NBCCS fibroblasts generate a stress affecting keratinocytes behavior and epidermal homeostasis. Our findings suggest that defects in dermo/epidermal interactions could contribute to BCC susceptibility in NBCCS patients.

Imaging Biomarkers for Adult Medulloblastomas: Genetic Entities May Be Identified by Their MR Imaging Radiophenotype.

PubMed

Keil, V C; Warmuth-Metz, M; Reh, C; Enkirch, S J; Reinert, C; Beier, D; Jones, D T W; Pietsch, T; Schild, H H; Hattingen, E; Hau, P

2017-10-01

The occurrence of medulloblastomas in adults is rare; nevertheless, these tumors can be subdivided into genetic and histologic entities each having distinct prognoses. This study aimed to identify MR imaging biomarkers to classify these entities and to uncover differences in MR imaging biomarkers identified in pediatric medulloblastomas. Eligible preoperative MRIs from 28 patients (11 women; 22-53 years of age) of the Multicenter Pilot-study for the Therapy of Medulloblastoma of Adults (NOA-7) cohort were assessed by 3 experienced neuroradiologists. Lesions and perifocal edema were volumetrized and multiparametrically evaluated for classic morphologic characteristics, location, hydrocephalus, and Chang criteria. To identify MR imaging biomarkers, we correlated genetic entities sonic hedgehog ( SHH ) TP53 wild type, wingless ( WNT ), and non -WNT/ non -SHH medulloblastomas (in adults, Group 4), and histologic entities were correlated with the imaging criteria. These MR imaging biomarkers were compared with corresponding data from a pediatric study. There were 19 SHH TP53 wild type (69%), 4 WNT -activated (14%), and 5 Group 4 (17%) medulloblastomas. Six potential MR imaging biomarkers were identified, 3 of which, hydrocephalus ( P = .03), intraventricular macrometastases ( P = .02), and hemorrhage ( P = .04), when combined, could identify WNT medulloblastoma with 100% sensitivity and 88.3% specificity (95% CI, 39.8%-100.0% and 62.6%-95.3%). WNT -activated nuclear β-catenin accumulating medulloblastomas were smaller than the other entities (95% CI, 5.2-22.3 cm 3 versus 35.1-47.6 cm 3 ; P = .03). Hemorrhage was exclusively present in non -WNT/ non -SHH medulloblastomas ( P = .04; n = 2/5). MR imaging biomarkers were all discordant from those identified in the pediatric cohort. Desmoplastic/nodular medulloblastomas were more rarely in contact with the fourth ventricle (4/15 versus 7/13; P = .04). MR imaging biomarkers can help distinguish histologic and genetic medulloblastoma entities in adults and appear to be different from those identified in children. © 2017 by American Journal of Neuroradiology.

Histogenesis of retinal dysplasia in trisomy 13

PubMed Central

Chan, Ada; Lakshminrusimha, Satyan; Heffner, Reid; Gonzalez-Fernandez, Federico

2007-01-01

Background Although often associated with holoprosencephaly, little detail of the histopathology of cyclopia is available. Here, we describe the ocular findings in a case of trisomy 13 to better understand the histogenesis of the rosettes, or tubules, characteristic of the retinal dysplasia associated with this condition. Methods A full pediatric autopsy was performed of a near term infant who died shortly after birth from multiple congenital anomalies including fused facial-midline structures. A detailed histopathological study of the ocular structures was performed. The expression of interphotoreceptor retinoid-binding protein (IRBP), cellular retinal-binding protein (CRALBP), rod opsin, and Sonic Hedgehog (Shh) were studied by immunohistochemistry. Results Holoprosencephaly, and a spectrum of anatomical findings characteristic of Patau's syndrome, were found. Cytogenetic studies demonstrated trisomy 13 [47, XY, +13]. The eyes were fused but contained two developed separate lenses. In contrast, the cornea, and angle structures were hypoplastic, and the anterior chamber had failed to form. The retina showed areas of normally laminated neural retina, whereas in other areas it was replaced by numerous neuronal rosettes. Histological and immunohistochemical studies revealed that the rosettes were composed of differentiated retinal neurons and Müller cell glia. In normally laminated retina, Shh expression was restricted to retinal-ganglion cells, and to a population of neurons in the inner zone of the outer nuclear layer. In contrast, Shh could not be detected in the dysplastic rosettes. Conclusion The histopathology of cyclopia appears to be more complex than what may have been previously appreciated. In fact, the terms "cyclopia" and "synophthalmia" are misnomers as the underlying mechanism is a failure of the eyes to form separately during development. The rosettes found in the dysplastic retina are fundamentally different than those of retinoblastoma, being composed of a variety of differentiated cell types. The dysplastic rosettes are essentially laminated retina failing to establish a polarized orientation, resulting in the formation of tubules. Finally, our findings suggest that defective ganglion cell Shh expression may contribute to the ocular pathology of cyclopia. PMID:18088410

Lentivirus-mediated silencing of the PTC1 and PTC2 genes promotes recovery from spinal cord injury by activating the Hedgehog signaling pathway in a rat model.

PubMed

Zhang, Ya-Dong; Zhu, Zhong-Sheng; Zhang, Dong; Zhang, Zhen; Ma, Bin; Zhao, Shi-Chang; Xue, Feng

2017-12-15

This study aimed to investigate the effect of Patched-1 (PTC1) and PTC2 silencing in a rat model, on Hedgehog (Hh) pathway-mediated recovery from spinal cord injury (SCI). An analytical emphasis on the relationship between the sonic hedgehog (Shh) pathway and nerve regeneration was explored. A total of 126 rats were divided into normal, sham, SCI, negative control (NC), PTC1-RNAi, PTC2-RNAi and PTC1/PTC2-RNAi groups. The Basso, Beattie and Bresnahan (BBB) scale was employed to assess hind limb motor function. Quantitative real-time polymerase chain reaction and western blotting were performed to examine the mRNA and protein levels of PTC1, PTC2, Shh, glioma-associated oncogene homolog 1 (Gli-1), Smo and Nestin. Tissue morphology was analyzed using immunohistochemistry, and immunofluorescent staining was conducted to detect neurofilament protein 200 (NF-200) and glial fibrillary acidic protein (GFAP). The PTC1/PTC2-RNAi group displayed higher BBB scores than the SCI and NC groups. Shh, Gli-1, Smo and Nestin expression levels were elevated in the PTC1/PTC2-RNAi group. PTC1 and PTC2 mRNA and protein expression was lower in the PTC1/PTC2-RNAi group than in the normal, sham and SCI groups. Among the seven groups, the PTC1/PTC2-RNAi group had the largest positive area of NF-200 staining, whereas the SCI group exhibited a larger GFAP-positive area than both the normal and the sham groups. The Shh pathway may provide new insights into therapeutic indications and regenerative recovery tools for the treatment of SCI. Activation of the Hh signaling pathway by silencing PTC1 and PTC2 may reduce inflammation and may ultimately promote SCI recovery.

Cellular Functions of the Autism Risk Factor PTCHD1 in Mice.

PubMed

Tora, David; Gomez, Andrea M; Michaud, Jean-Francois; Yam, Patricia T; Charron, Frédéric; Scheiffele, Peter

2017-12-06

The gene patched domain containing 1 ( PTCHD1 ) is mutated in patients with autism spectrum disorders and intellectual disabilities and has been hypothesized to contribute to Sonic hedgehog (Shh) signaling and synapse formation. We identify a panel of Ptchd1-interacting proteins that include postsynaptic density proteins and the retromer complex, revealing a link to critical regulators of dendritic and postsynaptic trafficking. Ptchd1 knock-out (KO) male mice exhibit cognitive alterations, including defects in a novel object recognition task. To test whether Ptchd1 is required for Shh-dependent signaling, we examined two Shh-dependent cell populations that express high levels of Ptchd1 mRNA: cerebellar granule cell precursors and dentate granule cells in the hippocampus. We found that proliferation of these neuronal precursors was not altered significantly in Ptchd1 KO male mice. We used whole-cell electrophysiology and anatomical methods to assess synaptic function in Ptchd1-deficient dentate granule cells. In the absence of Ptchd1, we observed profound disruption in excitatory/inhibitory balance despite normal dendritic spine density on dentate granule cells. These findings support a critical role of the Ptchd1 protein in the dentate gyrus, but indicate that it is not required for structural synapse formation in dentate granule cells or for Shh-dependent neuronal precursor proliferation. SIGNIFICANCE STATEMENT The mechanisms underlying neuronal and cellular alterations resulting from patched domain containing 1 ( Ptchd1 ) gene mutations are unknown. The results from this study support an association with dendritic trafficking complexes of Ptchd1. Loss-of-function experiments do not support a role in sonic hedgehog-dependent signaling, but reveal a disruption of synaptic transmission in the mouse dentate gyrus. The findings will help to guide ongoing efforts to understand the etiology of neurodevelopmental disorders arising from Ptchd1 deficiency. Copyright © 2017 the authors 0270-6474/17/3711993-13$15.00/0.

Hedgehog signaling pathway in neuroblastoma differentiation.

PubMed

Souzaki, Ryota; Tajiri, Tatsuro; Souzaki, Masae; Kinoshita, Yoshiaki; Tanaka, Sakura; Kohashi, Kenichi; Oda, Yoshinao; Katano, Mitsuo; Taguchi, Tomoaki

2010-12-01

The hedgehog (Hh) signaling pathway is activated in some adult cancers. On the other hand, the Hh signaling pathway plays an important role in the development of the neural crest in embryos. The aim of this study is to show the activation of Hh signaling pathway in neuroblastoma (NB), a pediatric malignancy arising from neural crest cells, and to reveal the meaning of the Hh signaling pathway in NB development. This study analyzed the expression of Sonic hedgehog (Shh), GLI1, and Patched 1 (Ptch1), transactivators of Hh signaling pathway, by immunohistochemistry in 82 NB and 10 ganglioneuroblastoma cases. All 92 cases were evaluated for the status of MYCN amplification. Of the 92 cases, 67 (73%) were positive for Shh, 62 cases (67%) were positive for GLI1, and 73 cases (79%) were positive for Ptch1. Only 2 (10%) of the 20 cases with MYCN amplification were positive for Shh and GLI1, and 4 cases (20%) were positive for Ptch1 (MYCN amplification vs no MYCN amplification, P ≦ .01). The percentage of GLI1-positive cells in the cases with INSS stage 1 without MYCN amplification was significantly higher than that with INSS stage 4. Of 72 cases without MYCN amplification, 60 were GLI1-positive. Twelve cases were GLI1-negative, and the prognosis of the GLI1-positive cases was significantly better than that of the GLI1-negative cases (P = .015). Most of NBs without MYCN amplification were positive for Shh, GLI1, and Ptch1. In the cases without MYCN amplification, the high expression of GLI1 was significantly associated with early clinical stage and a good prognosis of the patients. In contrast to adult cancers, the activation of the Hh signaling pathway in NB may be associated with the differentiation of the NB. Copyright © 2010 Elsevier Inc. All rights reserved.

Involvement of PI3K/Akt pathway in the inhibition of hepatocarcinoma cell invasion and metastasis induced by SASH1 through downregulating Shh-Gli1 signaling.

PubMed

Sun, Changyu; Zhang, Zhihao; He, Ping; Zhou, Yan; Xie, Xuhua

2017-08-01

The SASH1 gene is discovered as a tumor suppressor recently. However, the molecular mechanisms of SASH1 in hepatocarcinoma (HCC) remain unclear. In present studies, we investigated the molecular mechanisms of SASH1 on cell invasion and metastasis of hepatocarcinoma in vivo and in vitro. In this study, SASH1 overexpression HCC cell lines were treated with purmorphamine (0, 0.5, 1, 2μmol/l). Western blot and qRT-PCR were used to examine the related gene expression of EMT markers and the Shh-Gli1 and PI3K/Akt-dependent pathway. Cell migration and invasion were assessed by Transwell assay. In addition, a mice SASH1 overexpression HCC orthotopic xenograft model was established and treated with purmorphamine or 740Y-P or PDGF. Tumor volume was assessed, and H&E staining was applied to histopathologic analysis. The results showed that purmorphamine exposure significantly increased the mRNA and protein expression levels of Shh and Gli1 in a dose-dependent manner in the SASH1 overexpression HepG2 and HCCLM3 cells. Besides, purmorphamine promoted the migration and invasion of SASH1 overexpression HCC cells, as well as the EMT progress. Moreover, purmorphamine significantly increased the synthesis of PI3K and pAkt in a dose-dependent manner. Furthermore, the invasion and migration abilities were also improved by treatment with 740Y-P or PDGF in the SASH1 overexpression HCC cells. Additionally, the agonists promoted tumor growth and intrahepatic and pulmonary metastasis of the orthotopic transplantation tumors grown from SASH1 overexpression HCC cells in vivo. In conclusion, SASH1 may inhibit hepatocarcinoma cell invasion and metastasis through down-regulating the Shh-Gli1 and PI3K-AKT pathways in vivo and in vitro. Copyright © 2017. Published by Elsevier Ltd.

Development of hemipenes in the ball python snake Python regius.

PubMed

Leal, Francisca; Cohn, Martin J

2015-01-01

Within amniotes, external copulatory organs have undergone extensive morphological diversification. One of the most extreme examples is squamate (lizards and snakes) hemipenes, which are paired copulatory organs that extend from the lateral margins of the cloaca. Here, we describe the development of hemipenes in a basal snake, the ball python (Python regius). Snake hemipenes arise as a pair of lateral swellings on either side of the caudal part of the cloaca, and these paired outgrowths persist to form the left and right hemipenes. In non-squamate amniotes, external genitalia form from paired swellings that arise on the anterior side of the cloaca, which then fuse medially to form a single genital tubercle, the anlagen of the penis or clitoris. Whereas in non-squamate amniotes, Sonic hedgehog (Shh)-expressing cells of the cloacal endoderm form the urethral or sulcus epithelium and are required for phallus outgrowth, the hemipenes of squamates lack an endodermal contribution, and the sulcus does not express Shh. Thus, snake hemipenes differ from the genital tubercles of non-squamate amniotes both in their embryonic origins and in at least part of patterning mechanisms, which raises the possibility that hemipenes may not be direct homologs of the unpaired amniote penis. Nonetheless, we find that some developmental genes show similar expression patterns in snake hemipenes buds and non-squamate genital tubercles, suggesting that homologous developmental mechanisms are involved in aspects of external genital development across amniotes, even when these structures may have different developmental origins and may have arisen independently during evolution.

Neural patterning of human induced pluripotent stem cells in 3-D cultures for studying biomolecule-directed differential cellular responses.

PubMed

Yan, Yuanwei; Bejoy, Julie; Xia, Junfei; Guan, Jingjiao; Zhou, Yi; Li, Yan

2016-09-15

Appropriate neural patterning of human induced pluripotent stem cells (hiPSCs) is critical to generate specific neural cells/tissues and even mini-brains that are physiologically relevant to model neurological diseases. However, the capacity of signaling factors that regulate 3-D neural tissue patterning in vitro and differential responses of the resulting neural populations to various biomolecules have not yet been fully understood. By tuning neural patterning of hiPSCs with small molecules targeting sonic hedgehog (SHH) signaling, this study generated different 3-D neuronal cultures that were mainly comprised of either cortical glutamatergic neurons or motor neurons. Abundant glutamatergic neurons were observed following the treatment with an antagonist of SHH signaling, cyclopamine, while Islet-1 and HB9-expressing motor neurons were enriched by an SHH agonist, purmorphamine. In neurons derived with different neural patterning factors, whole-cell patch clamp recordings showed similar voltage-gated Na(+)/K(+) currents, depolarization-evoked action potentials and spontaneous excitatory post-synaptic currents. Moreover, these different neuronal populations exhibited differential responses to three classes of biomolecules, including (1) matrix metalloproteinase inhibitors that affect extracellular matrix remodeling; (2) N-methyl-d-aspartate that induces general neurotoxicity; and (3) amyloid β (1-42) oligomers that cause neuronal subtype-specific neurotoxicity. This study should advance our understanding of hiPSC self-organization and neural tissue development and provide a transformative approach to establish 3-D models for neurological disease modeling and drug discovery. Appropriate neural patterning of human induced pluripotent stem cells (hiPSCs) is critical to generate specific neural cells, tissues and even mini-brains that are physiologically relevant to model neurological diseases. However, the capability of sonic hedgehog-related small molecules to tune different neuronal subtypes in 3-D differentiation from hiPSCs and the differential cellular responses of region-specific neuronal subtypes to various biomolecules have not been fully investigated. By tuning neural patterning of hiPSCs with small molecules targeting sonic hedgehog signaling, this study provides knowledge on the differential susceptibility of region-specific neuronal subtypes derived from hiPSCs to different biomolecules in extracellular matrix remodeling and neurotoxicity. The findings are significant for understanding 3-D neural patterning of hiPSCs for the applications in brain organoid formation, neurological disease modeling, and drug discovery. Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Social dilemma cooperation (unlike Dictator Game giving) is intuitive for men as well as women.

PubMed

Rand, David G

2017-11-01

Does intuition favor prosociality, or does prosocial behavior require deliberative self-control? The Social Heuristics Hypothesis (SHH) stipulates that intuition favors typically advantageous behavior - but which behavior is typically advantageous depends on both the individual and the context. For example, non-zero-sum cooperation (e.g. in social dilemmas like the Prisoner's Dilemma) typically pays off because of the opportunity for reciprocity. Conversely, reciprocity does not promote zero-sum cash transfers (e.g. in the Dictator Game, DG). Instead, DG giving can be long-run advantageous because of reputation concerns: social norms often require such behavior of women but not men. Thus, the SHH predicts that intuition will favor social dilemma cooperation regardless of gender, but only favor DG giving among women. Here I present meta-analytic evidence in support of this prediction. In 31 studies examining social dilemma cooperation (N=13,447), I find that promoting intuition increases cooperation to a similar extent for both men and women. This stands in contrast to the results from 22 DG studies (analyzed in Rand et al., 2016) where intuition promotes giving among women but not men. Furthermore, I show using meta-regression that the interaction between gender and intuition is significantly larger in the DG compared to the cooperation games. Thus, I find clear evidence that the role of intuition and deliberation varies across both setting and individual as predicted by the SHH.

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

Risk factors for the prognosis of pediatric medulloblastoma: a retrospective analysis of 40 cases.

PubMed

Yu, Jianzhong; Zhao, Rui; Shi, Wei; Li, Hao

2017-05-01

In this study, we evaluated the association of molecular subtypes, clinical characteristics and pathological types with the prognosis of patients with medulloblastoma. We analyzed forty patients with medulloblastoma who underwent surgical resection at our center between January 2004 and June 2014. Risk factors associated with survival, disease progression and recurrence were analyzed with a univariate Cox regression analysis, and the identified significant risk factors were further analyzed by Kaplan-Meier survival curves. Factors associated with overall survival included M stage (p=0.014), calcification (p=0.012), postoperative treatment, postoperative Karnofsky Performance Scale (KPS) score (p=0.015), and molecular subtype (p=0.005 for WNT and p=0.008 for SHH). Number of symptoms (p=0.029), M stage (p<0.001), and postoperative radiotherapy (p=0.033) were associated with disease progression. Patients with the WNT or SHH subtype had better survival outcomes than patients with non-WNT/SHH subtypes. Risk factors for disease progression-free survival were symptoms >2 and ≥M1 stage without postoperative radiotherapy. The risk of recurrence increased with advanced M stage. Protective factors for recurrence included M0 stage and a combination of chemotherapy and radiotherapy. We identified the risk factors associated with survival, disease progression and recurrence of medulloblastoma patients. This information is helpful for understanding the prognostic factors related to medulloblastoma.

Hedgehog signaling regulates FOXA2 in esophageal embryogenesis and Barrett’s metaplasia

PubMed Central

Wang, David H.; Tiwari, Anjana; Kim, Monica E.; Clemons, Nicholas J.; Regmi, Nanda L.; Hodges, William A.; Berman, David M.; Montgomery, Elizabeth A.; Watkins, D. Neil; Zhang, Xi; Zhang, Qiuyang; Jie, Chunfa; Spechler, Stuart J.; Souza, Rhonda F.

2014-01-01

Metaplasia can result when injury reactivates latent developmental signaling pathways that determine cell phenotype. Barrett’s esophagus is a squamous-to-columnar epithelial metaplasia caused by reflux esophagitis. Hedgehog (Hh) signaling is active in columnar-lined, embryonic esophagus and inactive in squamous-lined, adult esophagus. We showed previously that Hh signaling is reactivated in Barrett’s metaplasia and overexpression of Sonic hedgehog (SHH) in mouse esophageal squamous epithelium leads to a columnar phenotype. Here, our objective was to identify Hh target genes involved in Barrett’s pathogenesis. By microarray analysis, we found that the transcription factor Foxa2 is more highly expressed in murine embryonic esophagus compared with postnatal esophagus. Conditional activation of Shh in mouse esophageal epithelium induced FOXA2, while FOXA2 expression was reduced in Shh knockout embryos, establishing Foxa2 as an esophageal Hh target gene. Evaluation of patient samples revealed FOXA2 expression in Barrett’s metaplasia, dysplasia, and adenocarcinoma but not in esophageal squamous epithelium or squamous cell carcinoma. In esophageal squamous cell lines, Hh signaling upregulated FOXA2, which induced expression of MUC2, an intestinal mucin found in Barrett’s esophagus, and the MUC2-processing protein AGR2. Together, these data indicate that Hh signaling induces expression of genes that determine an intestinal phenotype in esophageal squamous epithelial cells and may contribute to the development of Barrett’s metaplasia. PMID:25083987

Suppressor of Fused Chaperones Gli Proteins To Generate Transcriptional Responses to Sonic Hedgehog Signaling

PubMed Central

Zhang, Ziyu; Shen, Longyan; Law, Kelvin; Zhang, Zengdi; Liu, Xiaotong; Hua, Hu; Li, Sanen; Huang, Huijie; Yue, Shen; Hui, Chi-chung

2016-01-01

ABSTRACT Cellular responses to the graded Sonic Hedgehog (Shh) morphogenic signal are orchestrated by three Gli genes that give rise to both transcription activators and repressors. An essential downstream regulator of the pathway, encoded by the tumor suppressor gene Suppressor of fused (Sufu), plays critical roles in the production, trafficking, and function of Gli proteins, but the mechanism remains controversial. Here, we show that Sufu is upregulated in active Shh responding tissues and accompanies Gli activators translocating into and Gli repressors out of the nucleus. Trafficking of Sufu to the primary cilium, potentiated by Gli activators but not repressors, was found to be coupled to its nuclear import. We have identified a nuclear export signal (NES) motif of Sufu in juxtaposition to the protein kinase A (PKA) and glycogen synthase kinase 3 (GSK3) dual phosphorylation sites and show that Sufu binds the chromatin with both Gli1 and Gli3. Close comparison of neural tube development among individual Ptch1−/−, Sufu−/−, and Ptch1−/−; Sufu−/− double mutant embryos indicates that Sufu is critical for the maximal activation of Shh signaling essential to the specification of the most-ventral neurons. These data define Sufu as a novel class of molecular chaperone required for every aspect of Gli regulation and function. PMID:27849569

BMP signaling in the development of the mouse esophagus and forestomach

PubMed Central

Rodriguez, Pavel; Da Silva, Susana; Oxburgh, Leif; Wang, Fan; Hogan, Brigid L. M.; Que, Jianwen

2010-01-01

The stratification and differentiation of the epidermis are known to involve the precise control of multiple signaling pathways. By contrast, little is known about the development of the mouse esophagus and forestomach, which are composed of a stratified squamous epithelium. Based on prior work in the skin, we hypothesized that bone morphogenetic protein (BMP) signaling is a central player. To test this hypothesis, we first used a BMP reporter mouse line harboring a BRE-lacZ allele, along with in situ hybridization to localize transcripts for BMP signaling components, including various antagonists. We then exploited a Shh-Cre allele that drives recombination in the embryonic foregut epithelium to generate gain- or loss-of-function models for the Bmpr1a (Alk3) receptor. In gain-of-function (Shh-Cre;Rosa26CAG-loxpstoploxp-caBmprIa) embryos, high levels of ectopic BMP signaling stall the transition from simple columnar to multilayered undifferentiated epithelium in the esophagus and forestomach. In loss-of-function experiments, conditional deletion of the BMP receptor in Shh-Cre;Bmpr1aflox/flox embryos allows the formation of a multilayered squamous epithelium but this fails to differentiate, as shown by the absence of expression of the suprabasal markers loricrin and involucrin. Together, these findings suggest multiple roles for BMP signaling in the developing esophagus and forestomach. PMID:21068065

Three-body effects in the Hoyle-state decay

NASA Astrophysics Data System (ADS)

Refsgaard, J.; Fynbo, H. O. U.; Kirsebom, O. S.; Riisager, K.

2018-04-01

We use a sequential R-matrix model to describe the breakup of the Hoyle state into three α particles via the ground state of 8Be. It is shown that even in a sequential picture, features resembling a direct breakup branch appear in the phase-space distribution of the α particles. We construct a toy model to describe the Coulomb interaction in the three-body final state and its effects on the decay spectrum are investigated. The framework is also used to predict the phase-space distribution of the α particles emitted in a direct breakup of the Hoyle state and the possibility of interference between a direct and sequential branch is discussed. Our numerical results are compared to the current upper limit on the direct decay branch determined in recent experiments.

Krebs cycle metabolon formation: metabolite concentration gradient enhanced compartmentation of sequential enzymes.

PubMed

Wu, Fei; Pelster, Lindsey N; Minteer, Shelley D

2015-01-25

Dynamics of metabolon formation in mitochondria was probed by studying diffusional motion of two sequential Krebs cycle enzymes in a microfluidic channel. Enhanced directional co-diffusion of both enzymes against a substrate concentration gradient was observed in the presence of intermediate generation. This reveals a metabolite directed compartmentation of metabolic pathways.

Dental Cell Sheet Biomimetic Tooth Bud Model

PubMed Central

Monteiro, Nelson; Smith, Elizabeth E.; Angstadt, Shantel; Zhang, Weibo; Khademhosseini, Ali

2016-01-01

Tissue engineering and regenerative medicine technologies offer promising therapies for both medicine and dentistry. Our long-term goal is to create functional biomimetic tooth buds for eventual tooth replacement in humans. Here, our objective was to create a biomimetic 3D tooth bud model consisting of dental epithelial (DE) – dental mesenchymal (DM) cell sheets (CSs) combined with biomimetic enamel organ and pulp organ layers created using GelMA hydrogels. Pig DE or DM cells seeded on temperature-responsive plates at various cell densities (0.02, 0.114 and 0.228 cells 106/cm2) and cultured for 7, 14 and 21 days were used to generate DE and DM cell sheets, respectively. Dental CSs were combined with GelMA encapsulated DE and DM cell layers to form bioengineered 3D tooth buds. Biomimetic 3D tooth bud constructs were cultured in vitro, or implanted in vivo for 3 weeks. Analyses were performed using micro-CT, H&E staining, polarized light (Pol) microscopy, immunofluorescent (IF) and immunohistochemical (IHC) analyses. H&E, IHC and IF analyses showed that in vitro cultured multilayered DE-DM CSs expressed appropriate tooth marker expression patterns including SHH, BMP2, RUNX2, tenascin and syndecan, which normally direct DE-DM interactions, DM cell condensation, and dental cell differentiation. In vivo implanted 3D tooth bud constructs exhibited mineralized tissue formation of specified size and shape, and SHH, BMP2 and RUNX2and dental cell differentiation marker expression. We propose our biomimetic 3D tooth buds as models to study optimized DE-DM cell interactions leading to functional biomimetic replacement tooth formation. PMID:27565550

Characterising the developmental profile of human embryonic stem cell-derived medium spiny neuron progenitors and assessing mature neuron function using a CRISPR-generated human DARPP-32WT/eGFP-AMP reporter line.

PubMed

Hunt, C P J; Pouton, C W; Haynes, J M

2017-06-01

In the developing ventral telencephalon, cells of the lateral ganglionic eminence (LGE) give rise to all medium spiny neurons (MSNs). This development occurs in response to a highly orchestrated series of morphogenetic stimuli that pattern the resultant neurons as they develop. Striatal MSNs are characterised by expression of dopamine receptors, dopamine-and cyclic AMP-regulated phosphoprotein (DARPP32) and the neurotransmitter GABA. In this study, we demonstrate that fine tuning Wnt and hedgehog (SHH) signaling early in human embryonic stem cell differentiation can induce a subpallial progenitor molecular profile. Stimulation of TGFβ signaling pathway by activin-A further supports patterning of progenitors to striatal precursors which adopt an LGE-specific gene signature. Moreover, we report that these MSNs also express markers associated with mature neuron function (cannabinoid, adenosine and dopamine receptors). To facilitate live-cell identification we generated a human embryonic stem cell line using CRISPR-mediated gene editing at the DARPP32 locus (DARPP32 WT/eGFP-AMP-LacZ ). The addition of dopamine to MSNs either increased, decreased or had no effect on intracellular calcium, indicating the presence of multiple dopamine receptor subtypes. In summary, we demonstrate greater control over early fate decisions using activin-A, Wnt and SHH to direct differentiation into MSNs. We also generate a DARPP32 reporter line that enables deeper pharmacological profiling and interrogation of complex receptor interactions in human MSNs. Copyright © 2017 Elsevier Ltd. All rights reserved.

The buccohypophyseal canal is an ancestral vertebrate trait maintained by modulation in sonic hedgehog signaling

PubMed Central

2013-01-01

Background The pituitary gland is formed by the juxtaposition of two tissues: neuroectoderm arising from the basal diencephalon, and oral epithelium, which invaginates towards the central nervous system from the roof of the mouth. The oral invagination that reaches the brain from the mouth is referred to as Rathke’s pouch, with the tip forming the adenohypophysis and the stalk disappearing after the earliest stages of development. In tetrapods, formation of the cranial base establishes a definitive barrier between the pituitary and oral cavity; however, numerous extinct and extant vertebrate species retain an open buccohypophyseal canal in adulthood, a vestige of the stalk of Rathke’s pouch. Little is currently known about the formation and function of this structure. Here we have investigated molecular mechanisms driving the formation of the buccohypophyseal canal and their evolutionary significance. Results We show that Rathke’s pouch is located at a boundary region delineated by endoderm, neural crest-derived oral mesenchyme and the anterior limit of the notochord, using CD1, R26R-Sox17-Cre and R26R-Wnt1-Cre mouse lines. As revealed by synchrotron X-ray microtomography after iodine staining in mouse embryos, the pouch has a lobulated three-dimensional structure that embraces the descending diencephalon during pituitary formation. Polarisfl/fl; Wnt1-Cre, Ofd1-/- and Kif3a-/- primary cilia mouse mutants have abnormal sonic hedgehog (Shh) signaling and all present with malformations of the anterior pituitary gland and midline structures of the anterior cranial base. Changes in the expressions of Shh downstream genes are confirmed in Gas1-/- mice. From an evolutionary perspective, persistence of the buccohypophyseal canal is a basal character for all vertebrates and its maintenance in several groups is related to a specific morphology of the midline that can be related to modulation in Shh signaling. Conclusion These results provide insight into a poorly understood ancestral vertebrate structure. It appears that the opening of the buccohypophyseal canal depends upon Shh signaling and that modulation in this pathway most probably accounts for its persistence in phylogeny. PMID:23537390

Risk stratification of childhood medulloblastoma in the molecular era: the current consensus.

PubMed

Ramaswamy, Vijay; Remke, Marc; Bouffet, Eric; Bailey, Simon; Clifford, Steven C; Doz, Francois; Kool, Marcel; Dufour, Christelle; Vassal, Gilles; Milde, Till; Witt, Olaf; von Hoff, Katja; Pietsch, Torsten; Northcott, Paul A; Gajjar, Amar; Robinson, Giles W; Padovani, Laetitia; André, Nicolas; Massimino, Maura; Pizer, Barry; Packer, Roger; Rutkowski, Stefan; Pfister, Stefan M; Taylor, Michael D; Pomeroy, Scott L

2016-06-01

Historical risk stratification criteria for medulloblastoma rely primarily on clinicopathological variables pertaining to age, presence of metastases, extent of resection, histological subtypes and in some instances individual genetic aberrations such as MYC and MYCN amplification. In 2010, an international panel of experts established consensus defining four main subgroups of medulloblastoma (WNT, SHH, Group 3 and Group 4) delineated by transcriptional profiling. This has led to the current generation of biomarker-driven clinical trials assigning WNT tumors to a favorable prognosis group in addition to clinicopathological criteria including MYC and MYCN gene amplifications. However, outcome prediction of non-WNT subgroups is a challenge due to inconsistent survival reports. In 2015, a consensus conference was convened in Heidelberg with the objective to further refine the risk stratification in the context of subgroups and agree on a definition of risk groups of non-infant, childhood medulloblastoma (ages 3-17). Published and unpublished data over the past 5 years were reviewed, and a consensus was reached regarding the level of evidence for currently available biomarkers. The following risk groups were defined based on current survival rates: low risk (>90 % survival), average (standard) risk (75-90 % survival), high risk (50-75 % survival) and very high risk (<50 % survival) disease. The WNT subgroup and non-metastatic Group 4 tumors with whole chromosome 11 loss or whole chromosome 17 gain were recognized as low-risk tumors that may qualify for reduced therapy. High-risk strata were defined as patients with metastatic SHH or Group 4 tumors, or MYCN-amplified SHH medulloblastomas. Very high-risk patients are Group 3 with metastases or SHH with TP53 mutation. In addition, a number of consensus points were reached that should be standardized across future clinical trials. Although we anticipate new data will emerge from currently ongoing and recently completed clinical trials, this consensus can serve as an outline for prioritization of certain molecular subsets of tumors to define and validate risk groups as a basis for future clinical trials.

Risk stratification of childhood medulloblastoma in the molecular era: The Current Consensus

PubMed Central

Ramaswamy, Vijay; Remke, Marc; Bouffet, Eric; Bailey, Simon; Clifford, Steven C.; Doz, Francois; Kool, Marcel; Dufour, Christelle; Vassal, Gilles; Milde, Till; Witt, Olaf; von Hoff, Katja; Pietsch, Torsten; Northcott, Paul A.; Gajjar, Amar; Robinson, Giles W.; Padovani, Laetitia; André, Nicolas; Massimino, Maura; Pizer, Barry; Packer, Roger; Rutkowski, Stefan; Pfister, Stefan M.; Taylor, Michael D.; Pomeroy, Scott L.

2016-01-01

Historical risk stratification criteria for medulloblastoma rely primarily on clinicopathological variables pertaining to age, presence of metastases, extent of resection, histological subtypes and in some instances individual genetic aberrations such as MYC and MYCN amplification. In 2010, an international panel of experts established consensus defining four main subgroups of medulloblastoma (WNT, SHH, Group 3 and Group 4) delineated by transcriptional profiling. This has led to the current generation of biomarker-driven clinical trials assigning WNT tumors to a favorable prognosis group in addition to clinicopathological criteria including MYC and MYCN gene amplifications. However, outcome prediction of non-WNT subgroups is a challenge due to inconsistent survival reports. In 2015, a consensus conference was convened in Heidelberg with the objective to further refine the risk stratification in the context of subgroups and agree on a definition of risk groups of non-infant, childhood medulloblastoma (ages 3–17). Published and unpublished data over the past five years were reviewed, and a consensus was reached regarding the level of evidence for currently available biomarkers. The following risk groups were defined based on current survival rates: low risk (>90% survival), average (standard) risk (75–90% survival), high risk (50–75% survival) and very high risk (<50% survival) disease. The WNT subgroup and non-metastatic Group 4 tumors with whole chromosome 11 loss or whole chromosome 17 gain were recognized as low risk tumors that may qualify for reduced therapy. High-risk strata were defined as patients with metastatic SHH or Group 4 tumors, or MYCN amplified SHH medulloblastomas. Very high-risk patients are Group 3 with metastases or SHH with TP53 mutation. In addition, a number of consensus points were reached that should be standardized across future clinical trials. Although we anticipate new data will emerge from currently ongoing and recently completed clinical trials, this consensus can serve as an outline for prioritization of certain molecular subsets of tumors to define and validate risk groups as a basis for future clinical trials. PMID:27040285

The buccohypophyseal canal is an ancestral vertebrate trait maintained by modulation in sonic hedgehog signaling.

PubMed

Khonsari, Roman H; Seppala, Maisa; Pradel, Alan; Dutel, Hugo; Clément, Gaël; Lebedev, Oleg; Ghafoor, Sarah; Rothova, Michaela; Tucker, Abigael; Maisey, John G; Fan, Chen-Ming; Kawasaki, Maiko; Ohazama, Atsushi; Tafforeau, Paul; Franco, Brunella; Helms, Jill; Haycraft, Courtney J; David, Albert; Janvier, Philippe; Cobourne, Martyn T; Sharpe, Paul T

2013-03-28

The pituitary gland is formed by the juxtaposition of two tissues: neuroectoderm arising from the basal diencephalon, and oral epithelium, which invaginates towards the central nervous system from the roof of the mouth. The oral invagination that reaches the brain from the mouth is referred to as Rathke's pouch, with the tip forming the adenohypophysis and the stalk disappearing after the earliest stages of development. In tetrapods, formation of the cranial base establishes a definitive barrier between the pituitary and oral cavity; however, numerous extinct and extant vertebrate species retain an open buccohypophyseal canal in adulthood, a vestige of the stalk of Rathke's pouch. Little is currently known about the formation and function of this structure. Here we have investigated molecular mechanisms driving the formation of the buccohypophyseal canal and their evolutionary significance. We show that Rathke's pouch is located at a boundary region delineated by endoderm, neural crest-derived oral mesenchyme and the anterior limit of the notochord, using CD1, R26R-Sox17-Cre and R26R-Wnt1-Cre mouse lines. As revealed by synchrotron X-ray microtomography after iodine staining in mouse embryos, the pouch has a lobulated three-dimensional structure that embraces the descending diencephalon during pituitary formation. Polaris(fl/fl); Wnt1-Cre, Ofd1(-/-) and Kif3a(-/-) primary cilia mouse mutants have abnormal sonic hedgehog (Shh) signaling and all present with malformations of the anterior pituitary gland and midline structures of the anterior cranial base. Changes in the expressions of Shh downstream genes are confirmed in Gas1(-/-) mice. From an evolutionary perspective, persistence of the buccohypophyseal canal is a basal character for all vertebrates and its maintenance in several groups is related to a specific morphology of the midline that can be related to modulation in Shh signaling. These results provide insight into a poorly understood ancestral vertebrate structure. It appears that the opening of the buccohypophyseal canal depends upon Shh signaling and that modulation in this pathway most probably accounts for its persistence in phylogeny.

The Role of Sonic Hedgehog in Craniofacial Patterning, Morphogenesis and Cranial Neural Crest Survival

PubMed Central

Dworkin, Sebastian; Boglev, Yeliz; Owens, Harley; Goldie, Stephen J.

2016-01-01

Craniofacial defects (CFD) are a significant healthcare problem worldwide. Understanding both the morphogenetic movements which underpin normal facial development, as well as the molecular factors which regulate these processes, forms the cornerstone of future diagnostic, and ultimately, preventative therapies. The soluble morphogen Sonic hedgehog (Shh), a vertebrate orthologue of Drosophila hedgehog, is a key signalling factor in the regulation of craniofacial skeleton development in vertebrates, operating within numerous tissue types in the craniofacial primordia to spatiotemporally regulate the formation of the face and jaws. This review will provide an overview of normal craniofacial skeleton development, and focus specifically on the known roles of Shh in regulating the development and progression of the first pharyngeal arch, which in turn gives rise to both the upper jaw (maxilla) and lower jaw (mandible). PMID:29615588

The Role of Sonic Hedgehog in Craniofacial Patterning, Morphogenesis and Cranial Neural Crest Survival.

PubMed

Dworkin, Sebastian; Boglev, Yeliz; Owens, Harley; Goldie, Stephen J

2016-08-03

Craniofacial defects (CFD) are a significant healthcare problem worldwide. Understanding both the morphogenetic movements which underpin normal facial development, as well as the molecular factors which regulate these processes, forms the cornerstone of future diagnostic, and ultimately, preventative therapies. The soluble morphogen Sonic hedgehog ( Shh ), a vertebrate orthologue of Drosophila hedgehog , is a key signalling factor in the regulation of craniofacial skeleton development in vertebrates, operating within numerous tissue types in the craniofacial primordia to spatiotemporally regulate the formation of the face and jaws. This review will provide an overview of normal craniofacial skeleton development, and focus specifically on the known roles of Shh in regulating the development and progression of the first pharyngeal arch, which in turn gives rise to both the upper jaw (maxilla) and lower jaw (mandible).

Patterning of mammalian somites by surface ectoderm and notochord: evidence for sclerotome induction by a hedgehog homolog.

PubMed

Fan, C M; Tessier-Lavigne, M

1994-12-30

An early step in the development of vertebrae, ribs, muscle, and dermis is the differentiation of the somitic mesoderm into dermomyotome dorsally and sclerotome ventrally. To analyze this process, we have developed an in vitro assay for somitic mesoderm differentiation. We show that sclerotomal markers can be induced by a diffusible factor secreted by notochord and floor plate and that heterologous cells expressing Sonic hedgehog (shh/vhh-1) mimic this effect. In contrast, expression of dermomyotomal markers can be caused by a contact-dependent signal from surface ectoderm and a diffusible signal from dorsal neural tube. Our results extend previous studies by suggesting that dorsoventral patterning of somites involves the coordinate action of multiple dorsalizing and ventralizing signals and that a diffusible form of Shh/Vhh-1 mediates sclerotome induction.

Prognostic value of hedgehog signaling pathway in digestive system cancers: A systematic review and meta-analysis.

PubMed

Wang, Yihan; Peng, Qian; Jia, Hongyuan; Du, Xiao

2016-01-01

The Hedgehog (Hh) signaling pathway has recently been reported to be associated with the prognosis of digestive system cancers. However, the results are inconsistent. This study aimed to investigate the association between Hh pathway components and survival outcomes in patients with digestive system cancers. We conducted a comprehensive retrieval in PubMed, EMBASE and Cochrane library for relevant literatures until May 1st, 2015. The pooled hazard ratios (HRs) for overall survival (OS) and disease-free survival (DFS) with 95% confidence intervals (CIs) were calculated to clarify the prognostic value of Hh pathway components, including Shh, Gli1, Gli2, Smo and Ptch1. A total of 16 eligible articles with 3222 patients were included in the meta-analysis. Pooled HR suggested that over-expression of Shh and Gli1 were both associated with poor OS (HR = 1.87, 95% CI: 1.14-3.07 and HR = 1.96, 95% CI: 1.66-2.32, respectively) and DFS (HR = 2.37, 95% CI: 1.19-4.72 and HR = 2.18, 95% CI: 1.61-2.96, respectively). In addition, over-expression of Smo was associated with poor DFS (HR = 1.38, 95% CI: 1.08-1.75). This study reveals that over-expressed Hh pathway components, including Shh, Gli1 and Smo, are associated with poor prognosis in digestive system cancer patients. Hh signaling pathway may become a potential therapeutic target in digestive system cancers.

Temporal and spatial expression patterns of Hedgehog receptors in the developing inner and middle ear.

PubMed

Shin, Jeong-Oh; Ankamreddy, Harinarayana; Jakka, Naga Mahesh; Lee, Seokwon; Kim, Un-Kyung; Bok, Jinwoong

2017-01-01

The mammalian inner ear is a complex organ responsible for balance and hearing. Sonic hedgehog (Shh), a member of the Hedgehog (Hh) family of secreted proteins, has been shown to play important roles in several aspects of inner ear development, including dorsoventral axial specification, cochlear elongation, tonotopic patterning, and hair cell differentiation. Hh proteins initiate a downstream signaling cascade by binding to the Patched 1 (Ptch1) receptor. Recent studies have revealed that other types of co-receptors can also mediate Hh signaling, including growth arrest-specific 1 (Gas1), cell-adhesion molecules-related/down-regulated by oncogenes (Cdon), and biregional Cdon binding protein (Boc). However, little is known about the role of these Hh co-receptors in inner ear development. In this study, we examined the expression patterns of Gas1, Cdon, and Boc, as well as that of Ptch1, in the developing mouse inner ear from otocyst (embryonic day (E) 9.5) until birth and in the developing middle ear at E15.5. Ptch1, a readout of Hh signaling, was expressed in a graded pattern in response to Shh signaling throughout development. Expression patterns of Gas1, Cdon, and Boc differed from that of Ptch1, and each Hh co-receptor was expressed in specific cells and domains in the developing inner and middle ear. These unique and differential expression patterns of Hh co-receptors suggest their roles in mediating various time- and space-specific functions of Shh during ear development.

CXCL14 is a candidate biomarker for Hedgehog signalling in idiopathic pulmonary fibrosis.

PubMed

Jia, Guiquan; Chandriani, Sanjay; Abbas, Alexander R; DePianto, Daryle J; N'Diaye, Elsa N; Yaylaoglu, Murat B; Moore, Heather M; Peng, Ivan; DeVoss, Jason; Collard, Harold R; Wolters, Paul J; Egen, Jackson G; Arron, Joseph R

2017-09-01

Idiopathic pulmonary fibrosis (IPF) is associated with aberrant expression of developmental pathways, including Hedgehog (Hh). As Hh signalling contributes to multiple pro-fibrotic processes, Hh inhibition may represent a therapeutic option for IPF. However, no non-invasive biomarkers are available to monitor lung Hh activity. We assessed gene and protein expression in IPF and control lung biopsies, mouse lung, fibroblasts stimulated in vitro with sonic hedgehog (SHh), and plasma in IPF patients versus controls, and cancer patients before and after treatment with vismodegib, a Hh inhibitor. Lung tissue from IPF patients exhibited significantly greater expression of Hh-related genes versus controls. The gene most significantly upregulated in both IPF lung biopsies and fibroblasts stimulated in vitro with SHh was CXCL14 , which encodes a soluble secreted chemokine whose expression is inhibited in vitro by the addition of vismodegib. CXCL14 expression was induced by SHh overexpression in mouse lung. Circulating CXCL14 protein levels were significantly higher in plasma from IPF patients than controls. In cancer patients, circulating CXCL14 levels were significantly reduced upon vismodegib treatment. CXCL14 is a systemic biomarker that could be used to identify IPF patients with increased Hh pathway activity and monitor the pharmacodynamic effects of Hh antagonist therapy in IPF. Post-results, NCT00968981. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.

Retinoic acid is required for specification of the ventral eye field and for Rathke's pouch in the avian embryo.

PubMed

Maden, Malcolm; Blentic, Aida; Reijntjes, Susan; Seguin, Sophie; Gale, Emily; Graham, Anthony

2007-01-01

We have investigated the role of retinoic acid (RA) in eye development using the vitamin A deficient quail model system, which overcomes problems of retinoic acid synthesising enzyme redundancy in the embryo. In the absence of retinoic acid, the ventral optic stalk and ventral retina are missing, whereas the dorsal optic stalk and dorsal retina develop appropriately. Other ocular abnormalities observed were a thinner retina and the lack of differentiation of the lens. In an attempt to explain this, we studied the expression of various dorsally and ventrally expressed genes such as Pax2, Pax6, Tbx6, Vax2, Raldh1 and Raldh3 and noted that they were unchanged in their expression patterns. In contrast, the RA catabolising enzymes Cyp26A1 and Cyp26B1 which are known to be RA-responsive were not expressed at all in the developing eye. At much earlier stages, the expression domain of Shh in the prechordal plate was reduced, as was Nkx2.1 and we suggest a model whereby the eye field is specified according to the concentration of SHH protein that is present. We also describe another organ, Rathke's pouch which fails to develop in the absence of retinoic acid. We attribute this to the down-regulation of Bmp2, Shh and Fgf8 which are known to be involved in the induction of this structure.

Microduplication of 7q36.3 encompassing the SHH long-range regulator (ZRS) in a patient with triphalangeal thumb-polysyndactyly syndrome and congenital heart disease

PubMed Central

Liu, Zhenghua; Yin, Ni; Gong, Lianghui; Tan, Zhiping; Yin, Bangliang; Yang, Yifeng; Luo, Cheng

2017-01-01

Triphalangeal thumb-polysyndactyly syndrome (TPT-PS) is an autosomal dominant disorder with complete penetrance and a variable expression consisting of opposable triphalangeal thumbs, duplication of the distal thumb phalanx, pre-axial polydactyly and duplication of the big toes (hallux). The causative gene of TPT-PS has been mapped to 7q36.3. Sonic hedgehog (SHH) expressed in the zone of polarizing activity (ZPA) has an important role in defining the anterior-posterior axis and numbers of digits in limb bud development. Point mutation or duplication in the ZPA regulatory sequence (ZRS), a cis-regulator of SHH, will lead to TPT-PS. The present study describes a 1-year-old female congenital heart disease (CHD) patient with TPT-PS phenotype. In this Han Chinese family with TPT-PS, high resolution single nucleotide polymorphism array technology identified a novel 0.29 Mb duplication comprising ZRS at 7q36.3 where LMBR1 is located. Additionally, a novel deletion of 22q11.21 was detected in the proband with Tetralogy of Fallot. However, the parents and other relatives of the patient did not harbor this genomic lesion nor CHD. The findings supported the hypothesis that an increased copy number variation of ZRS is the genetic mechanism underlying the phenotype of TPT-PS, and corroborated that 22q11.21 deletion is a genetic cause of CHD. PMID:28035386

Umbilical cord blood cells regulate endogenous neural stem cell proliferation via hedgehog signaling in hypoxic ischemic neonatal rats.

PubMed

Wang, Xiao-Li; Zhao, Yan-Song; Hu, Ming-Ying; Sun, Ye-Quan; Chen, Yu-Xi; Bi, Xue-Hui

2013-06-26

Umbilical cord blood mononuclear cells (UCBMC) transplantation may improve hypoxia-induced brain injury in neonatal rats, but the mechanism is unclear. This study examines whether UCBMC promote neural stem cell (NSC) proliferation via the Sonic hedgehog (Shh) signaling pathway. The rats underwent left carotid ligation followed by hypoxic stress. UCBMC were transplanted 24h after hypoxia ischemia (HI), and immunohistochemistry, immmunoblotting, and morphology analyses were performed at different time points after transplantation. Increased numbers of NSCs were observed in the subventrical zone (SVZ) of the HI+UCBMC group, but these increases were attenuated by cyclopamine treatment. There were significant increases in Shh and Gli1 protein levels after transplantation in the HI group treated with UCBMC compared to HI rats treated with phosphate-buffered solution (PBS). Significantly more Gli1(+)DAPI(+) cells were observed in the SVZ of the HI+UCBMC group compared to the HI+PBS and N+UCBMC groups, but few Gli1(+)DAPI(+) cells were found in the SVZ of the HI+cyclopamine+UCBMC group. The HI+UCBMC group had significantly less neuronal loss in the cortex and CA1 sector of the hippocampus compared to the HI+PBS group, but more neuron loss was observed in the HI+cyclopamine+UCBMC group compared to HI+UCBMC. These results indicate that UCBMC may promote NSC proliferation and alleviate brain injury in HI neonatal rats via Shh signaling. Copyright © 2013 Elsevier B.V. All rights reserved.

Multiuser signal detection using sequential decoding

NASA Astrophysics Data System (ADS)

Xie, Zhenhua; Rushforth, Craig K.; Short, Robert T.

1990-05-01

The application of sequential decoding to the detection of data transmitted over the additive white Gaussian noise channel by K asynchronous transmitters using direct-sequence spread-spectrum multiple access is considered. A modification of Fano's (1963) sequential-decoding metric, allowing the messages from a given user to be safely decoded if its Eb/N0 exceeds -1.6 dB, is presented. Computer simulation is used to evaluate the performance of a sequential decoder that uses this metric in conjunction with the stack algorithm. In many circumstances, the sequential decoder achieves results comparable to those obtained using the much more complicated optimal receiver.

C-learning: A new classification framework to estimate optimal dynamic treatment regimes.

PubMed

Zhang, Baqun; Zhang, Min

2017-12-11

A dynamic treatment regime is a sequence of decision rules, each corresponding to a decision point, that determine that next treatment based on each individual's own available characteristics and treatment history up to that point. We show that identifying the optimal dynamic treatment regime can be recast as a sequential optimization problem and propose a direct sequential optimization method to estimate the optimal treatment regimes. In particular, at each decision point, the optimization is equivalent to sequentially minimizing a weighted expected misclassification error. Based on this classification perspective, we propose a powerful and flexible C-learning algorithm to learn the optimal dynamic treatment regimes backward sequentially from the last stage until the first stage. C-learning is a direct optimization method that directly targets optimizing decision rules by exploiting powerful optimization/classification techniques and it allows incorporation of patient's characteristics and treatment history to improve performance, hence enjoying advantages of both the traditional outcome regression-based methods (Q- and A-learning) and the more recent direct optimization methods. The superior performance and flexibility of the proposed methods are illustrated through extensive simulation studies. © 2017, The International Biometric Society.

Spectrum and prevalence of genetic predisposition in medulloblastoma: a retrospective genetic study and prospective validation in a clinical trial cohort.

PubMed

Waszak, Sebastian M; Northcott, Paul A; Buchhalter, Ivo; Robinson, Giles W; Sutter, Christian; Groebner, Susanne; Grund, Kerstin B; Brugières, Laurence; Jones, David T W; Pajtler, Kristian W; Morrissy, A Sorana; Kool, Marcel; Sturm, Dominik; Chavez, Lukas; Ernst, Aurelie; Brabetz, Sebastian; Hain, Michael; Zichner, Thomas; Segura-Wang, Maia; Weischenfeldt, Joachim; Rausch, Tobias; Mardin, Balca R; Zhou, Xin; Baciu, Cristina; Lawerenz, Christian; Chan, Jennifer A; Varlet, Pascale; Guerrini-Rousseau, Lea; Fults, Daniel W; Grajkowska, Wiesława; Hauser, Peter; Jabado, Nada; Ra, Young-Shin; Zitterbart, Karel; Shringarpure, Suyash S; De La Vega, Francisco M; Bustamante, Carlos D; Ng, Ho-Keung; Perry, Arie; MacDonald, Tobey J; Hernáiz Driever, Pablo; Bendel, Anne E; Bowers, Daniel C; McCowage, Geoffrey; Chintagumpala, Murali M; Cohn, Richard; Hassall, Timothy; Fleischhack, Gudrun; Eggen, Tone; Wesenberg, Finn; Feychting, Maria; Lannering, Birgitta; Schüz, Joachim; Johansen, Christoffer; Andersen, Tina V; Röösli, Martin; Kuehni, Claudia E; Grotzer, Michael; Kjaerheim, Kristina; Monoranu, Camelia M; Archer, Tenley C; Duke, Elizabeth; Pomeroy, Scott L; Shelagh, Redmond; Frank, Stephan; Sumerauer, David; Scheurlen, Wolfram; Ryzhova, Marina V; Milde, Till; Kratz, Christian P; Samuel, David; Zhang, Jinghui; Solomon, David A; Marra, Marco; Eils, Roland; Bartram, Claus R; von Hoff, Katja; Rutkowski, Stefan; Ramaswamy, Vijay; Gilbertson, Richard J; Korshunov, Andrey; Taylor, Michael D; Lichter, Peter; Malkin, David; Gajjar, Amar; Korbel, Jan O; Pfister, Stefan M

2018-06-01

Medulloblastoma is associated with rare hereditary cancer predisposition syndromes; however, consensus medulloblastoma predisposition genes have not been defined and screening guidelines for genetic counselling and testing for paediatric patients are not available. We aimed to assess and define these genes to provide evidence for future screening guidelines. In this international, multicentre study, we analysed patients with medulloblastoma from retrospective cohorts (International Cancer Genome Consortium [ICGC] PedBrain, Medulloblastoma Advanced Genomics International Consortium [MAGIC], and the CEFALO series) and from prospective cohorts from four clinical studies (SJMB03, SJMB12, SJYC07, and I-HIT-MED). Whole-genome sequences and exome sequences from blood and tumour samples were analysed for rare damaging germline mutations in cancer predisposition genes. DNA methylation profiling was done to determine consensus molecular subgroups: WNT (MB WNT ), SHH (MB SHH ), group 3 (MB Group3 ), and group 4 (MB Group4 ). Medulloblastoma predisposition genes were predicted on the basis of rare variant burden tests against controls without a cancer diagnosis from the Exome Aggregation Consortium (ExAC). Previously defined somatic mutational signatures were used to further classify medulloblastoma genomes into two groups, a clock-like group (signatures 1 and 5) and a homologous recombination repair deficiency-like group (signatures 3 and 8), and chromothripsis was investigated using previously established criteria. Progression-free survival and overall survival were modelled for patients with a genetic predisposition to medulloblastoma. We included a total of 1022 patients with medulloblastoma from the retrospective cohorts (n=673) and the four prospective studies (n=349), from whom blood samples (n=1022) and tumour samples (n=800) were analysed for germline mutations in 110 cancer predisposition genes. In our rare variant burden analysis, we compared these against 53 105 sequenced controls from ExAC and identified APC, BRCA2, PALB2, PTCH1, SUFU, and TP53 as consensus medulloblastoma predisposition genes according to our rare variant burden analysis and estimated that germline mutations accounted for 6% of medulloblastoma diagnoses in the retrospective cohort. The prevalence of genetic predispositions differed between molecular subgroups in the retrospective cohort and was highest for patients in the MB SHH subgroup (20% in the retrospective cohort). These estimates were replicated in the prospective clinical cohort (germline mutations accounted for 5% of medulloblastoma diagnoses, with the highest prevalence [14%] in the MB SHH subgroup). Patients with germline APC mutations developed MB WNT and accounted for most (five [71%] of seven) cases of MB WNT that had no somatic CTNNB1 exon 3 mutations. Patients with germline mutations in SUFU and PTCH1 mostly developed infant MB SHH . Germline TP53 mutations presented only in childhood patients in the MB SHH subgroup and explained more than half (eight [57%] of 14) of all chromothripsis events in this subgroup. Germline mutations in PALB2 and BRCA2 were observed across the MB SHH , MB Group3 , and MB Group4 molecular subgroups and were associated with mutational signatures typical of homologous recombination repair deficiency. In patients with a genetic predisposition to medulloblastoma, 5-year progression-free survival was 52% (95% CI 40-69) and 5-year overall survival was 65% (95% CI 52-81); these survival estimates differed significantly across patients with germline mutations in different medulloblastoma predisposition genes. Genetic counselling and testing should be used as a standard-of-care procedure in patients with MB WNT and MB SHH because these patients have the highest prevalence of damaging germline mutations in known cancer predisposition genes. We propose criteria for routine genetic screening for patients with medulloblastoma based on clinical and molecular tumour characteristics. German Cancer Aid; German Federal Ministry of Education and Research; German Childhood Cancer Foundation (Deutsche Kinderkrebsstiftung); European Research Council; National Institutes of Health; Canadian Institutes for Health Research; German Cancer Research Center; St Jude Comprehensive Cancer Center; American Lebanese Syrian Associated Charities; Swiss National Science Foundation; European Molecular Biology Organization; Cancer Research UK; Hertie Foundation; Alexander and Margaret Stewart Trust; V Foundation for Cancer Research; Sontag Foundation; Musicians Against Childhood Cancer; BC Cancer Foundation; Swedish Council for Health, Working Life and Welfare; Swedish Research Council; Swedish Cancer Society; the Swedish Radiation Protection Authority; Danish Strategic Research Council; Swiss Federal Office of Public Health; Swiss Research Foundation on Mobile Communication; Masaryk University; Ministry of Health of the Czech Republic; Research Council of Norway; Genome Canada; Genome BC; Terry Fox Research Institute; Ontario Institute for Cancer Research; Pediatric Oncology Group of Ontario; The Family of Kathleen Lorette and the Clark H Smith Brain Tumour Centre; Montreal Children's Hospital Foundation; The Hospital for Sick Children: Sonia and Arthur Labatt Brain Tumour Research Centre, Chief of Research Fund, Cancer Genetics Program, Garron Family Cancer Centre, MDT's Garron Family Endowment; BC Childhood Cancer Parents Association; Cure Search Foundation; Pediatric Brain Tumor Foundation; Brainchild; and the Government of Ontario. Copyright © 2018 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY-NC-ND 4.0 license. Published by Elsevier Ltd.. All rights reserved.

Eccrine sweat gland development and sweat secretion

PubMed Central

Cui, Chang-Yi; Schlessinger, David

2017-01-01

Eccrine sweat glands help to maintain homoeostasis, primarily by stabilizing body temperature. Derived from embryonic ectoderm, millions of eccrine glands are distributed across human skin and secrete litres of sweat per day. Their easy accessibility has facilitated the start of analyses of their development and function. Mouse genetic models find sweat gland development regulated sequentially by Wnt, Eda and Shh pathways, although precise subpathways and additional regulators require further elucidation. Mature glands have two secretory cell types, clear and dark cells, whose comparative development and functional interactions remain largely unknown. Clear cells have long been known as the major secretory cells, but recent studies suggest that dark cells are also indispensable for sweat secretion. Dark cell-specific Foxa1 expression was shown to regulate a Ca2+-dependent Best2 anion channel that is the candidate driver for the required ion currents. Overall, it was shown that cholinergic impulses trigger sweat secretion in mature glands through second messengers – for example InsP3 and Ca2+ – and downstream ion channels/transporters in the framework of a Na+-K+-Cl− cotransporter model. Notably, the microenvironment surrounding secretory cells, including acid–base balance, was implicated to be important for proper sweat secretion, which requires further clarification. Furthermore, multiple ion channels have been shown to be expressed in clear and dark cells, but the degree to which various ion channels function redundantly or indispensably also remains to be determined. PMID:26014472

β-catenin-mediated Wnt signaling regulates neurogenesis in the ventral telencephalon

PubMed Central

Gulacsi, Alexandra A.; Anderson, Stewart A.

2009-01-01

Development of the telencephalon involves the coordinated growth of diversely patterned brain structures. Previous studies have demonstrated the importance of β-catenin-mediated Wnt signaling in proliferation and fate determination during cerebral cortical development. In this paper, we present novel evidence that β-catenin-mediated Wnt signaling also critically maintains progenitor proliferation in the subcortical (pallidal) telencephalon of mice. Targeted deletion of β-catenin severely impairs proliferation in the medial ganglionic eminence without grossly altering differentiated fate. Several lines of evidence suggest that this phenotype is primarily due to loss of canonical Wnt signaling. As previous studies have suggested that the ventral patterning factor Shh also stimulates dorsal telencephalic proliferation, we propose a model whereby Wnt and Shh signaling promote distinct dorsal-ventral patterning, while also having broader effects on proliferation that serve to coordinate the growth of telencephalic subregions. PMID:18997789

Functional expression of SCL/TAL1 interrupting locus (Stil) protects retinal dopaminergic cells from neurotoxin-induced degeneration.

PubMed

Li, Jingling; Li, Ping; Carr, Aprell; Wang, Xiaokai; DeLaPaz, April; Sun, Lei; Lee, Eric; Tomei, Erika; Li, Lei

2013-01-11

We previously isolated a dominant mutation, night blindness b (nbb), which causes a late onset of retinal dopaminergic cell degeneration in zebrafish. In this study, we cloned the zebrafish nbb locus. Sequencing results revealed that nbb is a homolog of the vertebrate SCL/TAL1 interrupting locus (Stil). The Stil gene has been shown to play important roles in the regulation of vertebrate embryonic neural development and human cancer cell proliferation. In this study, we demonstrate that functional expression of Stil is also required for neural survival. In zebrafish, decreased expression of Stil resulted in increased toxic susceptibility of retinal dopaminergic cells to 6-hydroxydopamine. Increases in Stil-mediated Shh signaling transduction (i.e. by knocking down the Shh repressor Sufu) prevented dopaminergic cell death induced by neurotoxic insult. The data suggest that the oncogene Stil also plays important roles in neural protection.

Exclusion of the Sonic Hedgehog gene as responsible for Currarino syndrome and anorectal malformations with sacral hypodevelopment.

PubMed

Seri, M; Martucciello, G; Paleari, L; Bolino, A; Priolo, M; Salemi, G; Forabosco, P; Caroli, F; Cusano, R; Tocco, T; Lerone, M; Cama, A; Torre, M; Guys, J M; Romeo, G; Jasonni, V

1999-01-01

Anorectal malformations (ARMs) are common congenital anomalies that account for 1:4 digestive malformations. ARM patients show different degrees of sacral hypodevelopment while the hemisacrum is characteristic of the Currarino syndrome (CS). Cases of CS present an association of ARM, hemisacrum and presacral mass. A gene responsible for CS has recently been mapped in 7q36. Among the genes localized in this critical region, sonic hedgehog (SHH) was thought to represent a candidate gene for CS as well as for ARM with different levels of sacral hypodevelopment according to its role in the differentiation of midline mesoderm. By linkage analysis we confirmed the critical region in one large family with recurrence of CS. In addition, the screening of SHH in 7 CS and in 15 sporadic ARM patients with sacral hypodevelopment allowed us to exclude its role in the pathogenesis of these disorders.

Sonic Hedgehog switches on Wnt/planar cell polarity signaling in commissural axon growth cones by reducing levels of Shisa2

PubMed Central

Onishi, Keisuke

2017-01-01

Commissural axons switch on responsiveness to Wnt attraction during midline crossing and turn anteriorly only after exiting the floor plate. We report here that Sonic Hedgehog (Shh)-Smoothened signaling downregulates Shisa2, which inhibits the glycosylation and cell surface presentation of Frizzled3 in rodent commissural axon growth cones. Constitutive Shisa2 expression causes randomized turning of post-crossing commissural axons along the anterior–posterior (A–P) axis. Loss of Shisa2 led to precocious anterior turning of commissural axons before or during midline crossing. Post-crossing commissural axon turning is completely randomized along the A–P axis when Wntless, which is essential for Wnt secretion, is conditionally knocked out in the floor plate. This regulatory link between Shh and planar cell polarity (PCP) signaling may also occur in other developmental processes. PMID:28885142

The epidemiology of supernumerary teeth and the associated molecular mechanism

PubMed Central

Lu, Xi; Yu, Fang; Liu, Junjun; Cai, Wenping; Zhao, Yumei; Zhao, Shouliang; Liu, Shangfeng

2017-01-01

ABSTRACT Supernumerary teeth are common clinical dental anomalies. Although various studies have provided abundant information regarding genes and signaling pathways involved in tooth morphogenesis, which include Wnt, FGF, BMP, and Shh, the molecular mechanism of tooth formation, especially for supernumerary teeth, is still unclear. In the population, some cases of supernumerary teeth are sporadic, while others are syndrome-related with familial hereditary. The prompt and accurate diagnosis of syndrome related supernumerary teeth is quite important for some distinctive disorders. Mice are the most commonly used model system for investigating supernumerary teeth. The upregulation of Wnt and Shh signaling in the dental epithelium results in the formation of multiple supernumerary teeth in mice. Understanding the molecular mechanism of supernumerary teeth is also a component of understanding tooth formation in general and provides clinical guidance for early diagnosis and treatment in the future. PMID:28598258

Targeted Reduction of Vascular Msx1 and Msx2 Mitigates Arteriosclerotic Calcification and Aortic Stiffness in LDLR-Deficient Mice Fed Diabetogenic Diets

PubMed Central

Cheng, Su-Li; Behrmann, Abraham; Shao, Jian-Su; Ramachandran, Bindu; Krchma, Karen; Bello Arredondo, Yoanna; Kovacs, Attila; Mead, Megan; Maxson, Robert

2014-01-01

When fed high-fat diets, male LDLR−/− mice develop obesity, hyperlipidemia, hyperglycemia, and arteriosclerotic calcification. An osteogenic Msx-Wnt regulatory program is concomitantly upregulated in the vasculature. To better understand the mechanisms of diabetic arteriosclerosis, we generated SM22-Cre;Msx1(fl/fl);Msx2(fl/fl);LDLR−/− mice, assessing the impact of Msx1+Msx2 gene deletion in vascular myofibroblast and smooth muscle cells. Aortic Msx2 and Msx1 were decreased by 95% and 34% in SM22-Cre;Msx1(fl/fl);Msx2(fl/fl);LDLR−/− animals versus Msx1(fl/fl);Msx2(fl/fl);LDLR−/− controls, respectively. Aortic calcium was reduced by 31%, and pulse wave velocity, an index of stiffness, was decreased in SM22-Cre;Msx1(fl/fl);Msx2(fl/fl);LDLR−/− mice vs. controls. Fasting blood glucose and lipids did not differ, yet SM22-Cre;Msx1(fl/fl);Msx2(fl/fl);LDLR−/− siblings became more obese. Aortic adventitial myofibroblasts from SM22-Cre;Msx1(fl/fl);Msx2(fl/fl);LDLR−/− mice exhibited reduced osteogenic gene expression and mineralizing potential with concomitant reduction in multiple Wnt genes. Sonic hedgehog (Shh) and Sca1, markers of aortic osteogenic progenitors, were also reduced, paralleling a 78% reduction in alkaline phosphatase (TNAP)–positive adventitial myofibroblasts. RNA interference revealed that although Msx1+Msx2 supports TNAP and Wnt7b expression, Msx1 selectively maintains Shh and Msx2 sustains Wnt2, Wnt5a, and Sca1 expression in aortic adventitial myofibroblast cultures. Thus, Msx1 and Msx2 support vascular mineralization by directing the osteogenic programming of aortic progenitors in diabetic arteriosclerosis. PMID:25056439

Multi-Level Sequential Pattern Mining Based on Prime Encoding

NASA Astrophysics Data System (ADS)

Lianglei, Sun; Yun, Li; Jiang, Yin

Encoding is not only to express the hierarchical relationship, but also to facilitate the identification of the relationship between different levels, which will directly affect the efficiency of the algorithm in the area of mining the multi-level sequential pattern. In this paper, we prove that one step of division operation can decide the parent-child relationship between different levels by using prime encoding and present PMSM algorithm and CROSS-PMSM algorithm which are based on prime encoding for mining multi-level sequential pattern and cross-level sequential pattern respectively. Experimental results show that the algorithm can effectively extract multi-level and cross-level sequential pattern from the sequence database.

Long-term behavioral change as a result of acute ethanol exposure in zebrafish: Evidence for a role for sonic hedgehog but not retinoic acid signaling.

PubMed

Burton, Derek F; Zhang, Chengjin; Boa-Amponsem, Oswald; Mackinnon, Shanta; Cole, Gregory J

2017-05-01

Developmental exposure to ethanol is recognized to produce long-term neurobehavioral impairment in multiple animal models. However, the molecular mechanisms underlying these deficits remain poorly understood. The present study was undertaken to ascertain whether two well-characterized targets of prenatal alcohol exposure, sonic hedgehog (Shh) and retinoic acid (RA), that induce the hallmark morphological phenotypes of fetal alcohol spectrum disorders (FASD), are involved in the generation of behavioral alterations as a result of alcohol exposure. Zebrafish embryos were exposed to ethanol (0%, 1%, 3%) at either 8-10 or 24-27h post-fertilization (hpf) and then evaluated during adolescence in the novel tank dive test to assess anxiety and risk-taking behavior. Overt signs of dysmorphogenesis were also scored and behavioral and morphological changes were compared for embryos treated with alcohol alone or in combination with subthreshold doses of shh or alhh1a3 morpholinos (MOs). Ethanol treated fish displayed altered tank diving behavior that was not exacerbated by combined MO treatment. While treatment of embryos with either shha mRNA or RA prior to ethanol exposure only ameliorated the altered tank diving response in the case of shha mRNA overexpression, dysmorphogenesis was rescued by both treatments. These results suggest that the effects of ethanol exposure on changes in anxiety and risk-taking behavior in adolescent zebrafish is manifested by a blunting of Shh, but not RA, signaling during early development. Copyright © 2017 Elsevier Inc. All rights reserved.

Wnt/β-Catenin Regulates the Activity of Epiprofin/Sp6, SHH, FGF, and BMP to Coordinate the Stages of Odontogenesis

PubMed Central

Aurrekoetxea, Maitane; Irastorza, Igor; García-Gallastegui, Patricia; Jiménez-Rojo, Lucia; Nakamura, Takashi; Yamada, Yoshihiko; Ibarretxe, Gaskon; Unda, Fernando J.

2016-01-01

Background: We used an in vitro tooth development model to investigate the effects of overactivation of the Wnt/β-catenin pathway during odontogenesis by bromoindirubin oxime reagent (BIO), a specific inhibitor of GSK-3 activity. Results: Overactivating the Wnt/β-catenin pathway at tooth initiation upregulated and ectopically expressed the epithelial markers Sonic Hedgehog (Shh), Epiprofin (Epfn), and Fibroblast growth factor8 (Fgf8), which are involved in the delimitation of odontogenic fields in the oral ectoderm. This result indicated an ectopic extension of the odontogenic potential. During tooth morphogenesis, Fibroblast growth factor4 (Fgf4), Fibroblast growth factor10 (Fgf10), Muscle segment homeobox 1 (Msx-1), Bone Morphogenetic protein 4 (Bmp4), and Dickkopf WNT signaling pathway inhibitor 1 (Dkk-1) were overexpressed in first molars cultured with BIO. Conversely, the expression levels of Wingless integration site 10b (Wnt-10b) and Shh were reduced. Additionally, the odontoblast differentiation markers Nestin and Epfn showed ectopic overexpression in the dental mesenchyme of BIO-treated molars. Moreover, alkaline phosphatase activity increased in the dental mesenchyme, again suggesting aberrant, ectopic mesenchymal cell differentiation. Finally, Bmp4 downregulated Epfn expression during dental morphogenesis. Conclusions: We suggest the presence of a positive feedback loop wherein Epfn and β-catenin activate each other. The balance of the expression of these two molecules is essential for proper tooth development. We propose a possible link between Wnt, Bmp, and Epfn that would critically determine the correct patterning of dental cusps and the differentiation of odontoblasts and ameloblasts. PMID:27066482

Continuous light affects mineralization and delays osteoid incorporation in vertebral bone of Atlantic salmon (Salmo salar L.).

PubMed

Wargelius, Anna; Fjelldal, Per Gunnar; Nordgarden, Ulla; Hansen, Tom

2009-03-01

In order to study the effects of photoperiod on fish bone, Atlantic salmon (Salmo salar L.) were exposed to two light regimes (natural and continuous light) from January until June. During the experimental period, several parameters related to the inorganic (minerals) and organic (osteoid) phases were measured. Changes in the organic phase were related to mechanical strength (yield-load) and the expression of the genes sonic hedgehog (shh) and collagen type I alpha 2 (col I). Co-variation between yield-load and the expression of both shh and col I were detected in both groups. It was also shown that fish on the continuous light regime had delayed activation of osteoid incorporation. Mineralization properties were measured with stiffness, mineral incorporation per day and expression of alkaline phosphatase (alp) and matrix Gla protein (mgp). Stiffness, mineral incorporation and gene expression followed the same trend in both light groups in late spring, whereas an increase in the expression of mgp and alp was detected in April, followed by significantly higher stiffness at last sampling in both light groups. These results indicate that constant light affects mineralization and delays osteoid incorporation in Atlantic salmon during the spring. However, in this experiment light treatment did not promote the development of vertebral deformities. Our results also suggest that shh can be used as a marker of osteoblast proliferation and col I a marker of osteoid incorporation, and that both alp and mgp expression could be associated with a rapid increase in mineralization in Atlantic salmon vertebrae.

Genome-wide analysis of alternative splicing in medulloblastoma identifies splicing patterns characteristic of normal cerebellar development

PubMed Central

Menghi, Francesca; Jacques, Thomas S.; Barenco, Martino; Schwalbe, Ed C.; Clifford, Steven C.; Hubank, Mike; Ham, Jonathan

2011-01-01

Alternative splicing is an important mechanism for the generation of protein diversity at a post-transcriptional level. Modifications in the splicing patterns of several genes have been shown to contribute to the malignant transformation of different tissue types. In this study, we used the Affymetrix Exon arrays to investigate patterns of differential splicing between paediatric medulloblastomas and normal cerebellum on a genome-wide scale. Of the 1262 genes identified as potentially generating tumour-associated splice forms, we selected 14 examples of differential splicing of known cassette exons and successfully validated 11 of them by RT-PCR. The pattern of differential splicing of three validated events was characteristic for the molecular subset of Sonic Hedgehog (Shh)-driven medulloblastomas, suggesting that their unique gene signature includes the expression of distinctive transcript variants. Generally, we observed that tumour and normal fetal cerebellar samples shared significantly lower exon inclusion rates compared to normal adult cerebellum. We investigated whether tumour-associated splice forms were expressed in primary cultures of Shh-dependent mouse cerebellar granule cell precursors (GCPs) and found that Shh caused a decrease in the cassette exon inclusion rate of five out of the seven tested genes. Furthermore, we observed a significant increase in exon inclusion between post-natal days 7 and 14 of mouse cerebellar development, at the time when GCPs mature into post-mitotic neurons. We conclude that inappropriate splicing frequently occurs in human medulloblastomas and may be linked to the activation of developmental signalling pathways and a failure of cerebellar precursor cells to differentiate. PMID:21248070

Intestinal cell kinase, a protein associated with endocrine-cerebro-osteodysplasia syndrome, is a key regulator of cilia length and Hedgehog signaling.

PubMed

Moon, Heejung; Song, Jieun; Shin, Jeong-Oh; Lee, Hankyu; Kim, Hong-Kyung; Eggenschwiller, Jonathan T; Bok, Jinwoong; Ko, Hyuk Wan

2014-06-10

Endocrine-cerebro-osteodysplasia (ECO) syndrome is a recessive genetic disorder associated with multiple congenital defects in endocrine, cerebral, and skeletal systems that is caused by a missense mutation in the mitogen-activated protein kinase-like intestinal cell kinase (ICK) gene. In algae and invertebrates, ICK homologs are involved in flagellar formation and ciliogenesis, respectively. However, it is not clear whether this role of ICK is conserved in mammals and how a lack of functional ICK results in the characteristic phenotypes of human ECO syndrome. Here, we generated Ick knockout mice to elucidate the precise role of ICK in mammalian development and to examine the pathological mechanisms of ECO syndrome. Ick null mouse embryos displayed cleft palate, hydrocephalus, polydactyly, and delayed skeletal development, closely resembling ECO syndrome phenotypes. In cultured cells, down-regulation of Ick or overexpression of kinase-dead or ECO syndrome mutant ICK resulted in an elongation of primary cilia and abnormal Sonic hedgehog (Shh) signaling. Wild-type ICK proteins were generally localized in the proximal region of cilia near the basal bodies, whereas kinase-dead ICK mutant proteins accumulated in the distal part of bulged ciliary tips. Consistent with these observations in cultured cells, Ick knockout mouse embryos displayed elongated cilia and reduced Shh signaling during limb digit patterning. Taken together, these results indicate that ICK plays a crucial role in controlling ciliary length and that ciliary defects caused by a lack of functional ICK leads to abnormal Shh signaling, resulting in congenital disorders such as ECO syndrome.

Curcumin inhibits bladder cancer stem cells by suppressing Sonic Hedgehog pathway.

PubMed

Wang, Dengdian; Kong, Xiaochuan; Li, Yuan; Qian, Weiwei; Ma, Jiaxing; Wang, Daming; Yu, Dexin; Zhong, Caiyun

2017-11-04

Cancer stem cells (CSCs) is responsible for the recurrence of human cancers. Thus, targeting CSCs is considered to be a valid way for human cancer treatment. Curcumin is a major component of phytochemicals that exerts potent anticancer activities. However, the effect of curcumin on bladder cancer stem cells (BCSCs) remains to be elucidated. In this study, we investigated the mechanism of curcumin suppressing bladder cancer stem cells. In this study, UM-UC-3 and EJ cells were cultured in serum-free medium (SFM) to form cell spheres that was characterized as BCSCs. Then cell spheres were separately treated with different concentrations of curcumin and purmorphamine. Cell cycle analysis were used to determine the percentage of cells in different phases. Western blot and quantitative real-time PCR analysis were used to detect the expression of relative molecules. Immunofluorescence staining analysis were also utilized to measure the protein level of CD44. We found that CSC markers, including CD44, CD133, ALDH1-A1, OCT-4 and Nanog, were obviously highly expressed in cell spheres. Moreover, we observed that curcumin reduced the cell spheres formation, decreased the expression of CSC markers, suppressed cell proliferation and induced cell apoptosis. We also found that curcumin inhibited the activation of Shh pathway, while the inhibitory effects of curcumin on BCSCs could be weakened by upregulation of Sonic Hedgehog (Shh) pathway. Altogether, these data suggested that curcumin inhibited the activities of BCSCs through suppressing Shh pathway, which might be an effective chemopreventive agent for bladder cancer intervention. Copyright © 2017. Published by Elsevier Inc.

Defective ciliogenesis, embryonic lethality and severe impairment of the Sonic Hedgehog pathway caused by inactivation of the mouse complex A intraflagellar transport gene Ift122/Wdr10, partially overlapping with the DNA repair gene Med1/Mbd4

PubMed Central

Cortellino, Salvatore; Wang, Chengbing; Wang, Baolin; Bassi, Maria Rosaria; Caretti, Elena; Champeval, Delphine; Calmont, Amelie; Jarnik, Michal; Burch, John; Zaret, Kenneth; Larue, Lionel; Bellacosa, Alfonso

2009-01-01

Primary cilia are assembled and maintained by evolutionarily conserved intraflagellar transport (IFT) proteins that are involved in the coordinated movement of macromolecular cargo from the basal body to the cilium tip and back. The IFT machinery is organized in two structural complexes named complex A and complex B. Recently, inactivation in the mouse germline of Ift genes belonging to complex B revealed a requirement of ciliogenesis, or proteins involved in ciliogenesis, for Sonic Hedgehog (Shh) signaling in mammals. Here we report on a complex A mutant mouse, defective for the Ift122 gene. Ift122-null embryos show multiple developmental defects (exencephaly, situs viscerum inversus, delay in turning, hemorrhage and defects in limb development) that result in lethality. In the node, primary cilia were absent or malformed in homozygous mutant and heterozygous embryos, respectively. Impairment of the Shh pathway was apparent in both neural tube patterning (expansion of motoneurons and rostro-caudal level-dependent contraction or expansion of the dorso-lateral interneurons), and limb patterning (ectrosyndactyly). These phenotypes are distinct from both complex B IFT mutant embryos and embryos defective for the ciliary protein hennin/Arl13b, and suggest reduced levels of both Gli2/Gli3 activator and Gli3 repressor functions. We conclude that complex A and complex B factors play similar but distinct roles in ciliogenesis and Shh/Gli3 signaling. PMID:19000668

Auctions with Dynamic Populations: Efficiency and Revenue Maximization

NASA Astrophysics Data System (ADS)

Said, Maher

We study a stochastic sequential allocation problem with a dynamic population of privately-informed buyers. We characterize the set of efficient allocation rules and show that a dynamic VCG mechanism is both efficient and periodic ex post incentive compatible; we also show that the revenue-maximizing direct mechanism is a pivot mechanism with a reserve price. We then consider sequential ascending auctions in this setting, both with and without a reserve price. We construct equilibrium bidding strategies in this indirect mechanism where bidders reveal their private information in every period, yielding the same outcomes as the direct mechanisms. Thus, the sequential ascending auction is a natural institution for achieving either efficient or optimal outcomes.

MUC4 stabilizes HER2 expression and maintains the cancer stem cell population in ovarian cancer cells.

PubMed

Ponnusamy, Moorthy P; Seshacharyulu, Parthasarathy; Vaz, Arokiapriyanka; Dey, Parama; Batra, Surinder K

2011-04-26

Recent evidence has suggested that the capability of cancer to grow, propagate and relapse after therapy is dependent on a small subset of the cell population within the tumor, called cancer stem cells. Therefore, this subpopulation of cells needs to be targeted with different approaches by identification of unique stem-cell specific target antigens. One of the well known tumor antigens is the epithelial cell mucin MUC4, which is aberrantly expressed in ovarian cancer as compared to the normal ovary and plays a pivotal role in the aggressiveness and metastasis of ovarian cancer cells. In the present study, we aimed to analyze the cancer stem cell population in MUC4 overexpressed ovarian cancer cells. MUC4 was ectopically overexpressed in SKOV3 ovarian cancer cells. Western blot analysis was performed for MUC4, HER2, CD133, ALDH1 and Shh expression in MUC4 overexpressed cells. Confocal analysis of MUC4, HER2 and CD133 was also done in the MUC4 overexpressed cells. CD133 and Hoechst33342 dye staining was used to analyze the cancer stem cell population via FACS method in SKOV3-MUC4 cells. MUC4 overexpressed SKOV3 cells showed an increased expression of HER2 compared to control cells. MUC4 overexpression leads to increased (0.1%) side population (SP) and CD133-positive cancer stem cells compared to the control cells. Interestingly, the tumor sphere type circular colony formation was observed only in the MUC4 overexpressed ovarian cancer cells. Furthermore, the cancer stem cell marker CD133 was expressed along with MUC4 in the isolated circular colonies as analyzed by both confocal and western blot analysis. HER2 and cancer stem cell specific marker ALDH1 along with Shh, a self-renewal marker, showed increased expression in the isolated circular colonies compared to MUC4-transfected cells. These studies demonstrate that MUC4 overexpression leads to an enriched ovarian cancer stem cell population either directly or indirectly through HER2. In future, this study would be helpful for MUC4-directed therapy for the ovarian cancer stem cell population.

MUC4 stabilizes HER2 expression and maintains the cancer stem cell population in ovarian cancer cells

PubMed Central

2011-01-01

Background Recent evidence has suggested that the capability of cancer to grow, propagate and relapse after therapy is dependent on a small subset of the cell population within the tumor, called cancer stem cells. Therefore, this subpopulation of cells needs to be targeted with different approaches by identification of unique stem-cell specific target antigens. One of the well known tumor antigens is the epithelial cell mucin MUC4, which is aberrantly expressed in ovarian cancer as compared to the normal ovary and plays a pivotal role in the aggressiveness and metastasis of ovarian cancer cells. In the present study, we aimed to analyze the cancer stem cell population in MUC4 overexpressed ovarian cancer cells. Methods MUC4 was ectopically overexpressed in SKOV3 ovarian cancer cells. Western blot analysis was performed for MUC4, HER2, CD133, ALDH1 and Shh expression in MUC4 overexpressed cells. Confocal analysis of MUC4, HER2 and CD133 was also done in the MUC4 overexpressed cells. CD133 and Hoechst33342 dye staining was used to analyze the cancer stem cell population via FACS method in SKOV3-MUC4 cells. Results MUC4 overexpressed SKOV3 cells showed an increased expression of HER2 compared to control cells. MUC4 overexpression leads to increased (0.1%) side population (SP) and CD133-positive cancer stem cells compared to the control cells. Interestingly, the tumor sphere type circular colony formation was observed only in the MUC4 overexpressed ovarian cancer cells. Furthermore, the cancer stem cell marker CD133 was expressed along with MUC4 in the isolated circular colonies as analyzed by both confocal and western blot analysis. HER2 and cancer stem cell specific marker ALDH1 along with Shh, a self-renewal marker, showed increased expression in the isolated circular colonies compared to MUC4-transfected cells. Conclusion These studies demonstrate that MUC4 overexpression leads to an enriched ovarian cancer stem cell population either directly or indirectly through HER2. In future, this study would be helpful for MUC4-directed therapy for the ovarian cancer stem cell population. PMID:21521521

Direct Associations or Internal Transformations? Exploring the Mechanisms Underlying Sequential Learning Behavior

PubMed Central

Gureckis, Todd M.; Love, Bradley C.

2009-01-01

We evaluate two broad classes of cognitive mechanisms that might support the learning of sequential patterns. According to the first, learning is based on the gradual accumulation of direct associations between events based on simple conditioning principles. The other view describes learning as the process of inducing the transformational structure that defines the material. Each of these learning mechanisms predict differences in the rate of acquisition for differently organized sequences. Across a set of empirical studies, we compare the predictions of each class of model with the behavior of human subjects. We find that learning mechanisms based on transformations of an internal state, such as recurrent network architectures (e.g., Elman, 1990), have difficulty accounting for the pattern of human results relative to a simpler (but more limited) learning mechanism based on learning direct associations. Our results suggest new constraints on the cognitive mechanisms supporting sequential learning behavior. PMID:20396653

Development of a standardized sequential extraction protocol for simultaneous extraction of multiple actinide elements

DOE PAGES

Faye, Sherry A.; Richards, Jason M.; Gallardo, Athena M.; ...

2017-02-07

Sequential extraction is a useful technique for assessing the potential to leach actinides from soils; however, current literature lacks uniformity in experimental details, making direct comparison of results impossible. This work continued development toward a standardized five-step sequential extraction protocol by analyzing extraction behaviors of 232Th, 238U, 239,240Pu and 241Am from lake and ocean sediment reference materials. Results produced a standardized procedure after creating more defined reaction conditions to improve method repeatability. A NaOH fusion procedure is recommended following sequential leaching for the complete dissolution of insoluble species.

Sequential EMT-MET induces neuronal conversion through Sox2

PubMed Central

He, Songwei; Chen, Jinlong; Zhang, Yixin; Zhang, Mengdan; Yang, Xiao; Li, Yuan; Sun, Hao; Lin, Lilong; Fan, Ke; Liang, Lining; Feng, Chengqian; Wang, Fuhui; Zhang, Xiao; Guo, Yiping; Pei, Duanqing; Zheng, Hui

2017-01-01

Direct neuronal conversion can be achieved with combinations of small-molecule compounds and growth factors. Here, by studying the first or induction phase of the neuronal conversion induced by defined 5C medium, we show that the Sox2-mediated switch from early epithelial–mesenchymal transition (EMT) to late mesenchymal–epithelial transition (MET) within a high proliferation context is essential and sufficient for the conversion from mouse embryonic fibroblasts (MEFs) to TuJ+ cells. At the early stage, insulin and basic fibroblast growth factor (bFGF)-induced cell proliferation, early EMT, the up-regulation of Stat3 and Sox2, and the subsequent activation of neuron projection. Up-regulated Sox2 then induced MET and directed cells towards a neuronal fate at the late stage. Inhibiting either stage of this sequential EMT-MET impaired the conversion. In addition, Sox2 could replace sequential EMT-MET to induce a similar conversion within a high proliferation context, and its functions were confirmed with other neuronal conversion protocols and MEFs reprogramming. Therefore, the critical roles of the sequential EMT-MET were implicated in direct cell fate conversion in addition to reprogramming, embryonic development and cancer progression. PMID:28580167

IS SONIC HEDGEHOG (SHH) A CANDIDATE GENE FOR SPINA BIFIDA? (R828292)

EPA Science Inventory

The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

Expression of Wise in chick embryos.

PubMed

Shigetani, Y; Itasaki, N

2007-08-01

We have performed in situ hybridization to study the expression of Wise in early chick embryos. Wise expression is first detectable in the ectoderm at posterior levels of late neurula. As development proceeds, Wise expression is seen in specific patterns in the ectoderm of the trunk region, pharyngeal arches, limb buds, and feather buds. In addition to these areas, particular cartilages such as the ones in the maxillary process and limbs start to express Wise at the late pharyngula stage, and the expression in these cartilages becomes stronger than that in epidermal components at later stages. Importantly, Wise is expressed in regions where other signaling molecules such as Wnt, Bmp, and Shh are known to function in morphogenesis and differentiation. Direct comparisons of the expression of Wise and these genes are also demonstrated. (c) 2007 Wiley-Liss, Inc.

Regulation of Egr-1, VIP, and Shh mRNA and Egr-1 protein in the mouse retina by light and image quality.

PubMed

Brand, Christine; Burkhardt, Eva; Schaeffel, Frank; Choi, Jeong Won; Feldkaemper, Marita Pauline

2005-04-28

To analyze mRNA expression changes of Egr-1, VIP, and Shh under different light and treatment conditions in mice. The mRNA expression levels of the three genes and additionally the Egr-1 protein expression were compared in form deprived eyes and eyes with normal vision. Moreover, the influence of dark to light and light to dark transitions and of changes in retinal illumination on mRNA levels was investigated. Form deprivation of mice was induced by fitting frosted diffusers over one eye and an attentuation matched neutral density (ND) filter over the other eye. To measure the effects of retinal illumination changes on mRNA expression, animals were bilaterally fitted with different ND filters. Semiquantitative real-time RT-PCR was used to measure the mRNA levels and immunohistochemistry was applied to localize and detect Egr-1 protein. The expression levels of both Egr-1 mRNA and protein were reduced in form deprived eyes compared to their fellow eyes after 30 min and 1 h, respectively. Egr-1 mRNA was strikingly upregulated both after dark to light and light to dark transitions, whereas minor changes in retinal illumination by covering the eyes with neutral density filters did not alter Egr-1 mRNA expression. In mice, the mRNA levels of VIP and Shh were not affected by form deprivation, but they were found to be regulated depending on the time of day. Both Egr-1 mRNA and protein expression levels were strongly regulated by light, especially by transitions between light and darkness. Image contrast may exert an additional influence on mRNA and protein expression of Egr-1, particularly in the cells in the ganglion cell layer and in bipolar cells.

Inhibition of Hepatocellular Carcinoma by Total Alkaloids of Rubus alceifolius Poir Involves Suppression of Hedgehog Signaling.

PubMed

Zhao, Jinyan; Liu, Liya; Wan, Yun; Zhang, Yuchen; Zhuang, Qunchuan; Zhong, Xiaoyong; Hong, Zhenfeng; Peng, Jun

2015-07-01

We evaluated the effects of total alkaloids of Rubus alceifolius Poir (TARAP) on the migration and invasion of hepatocellular carcinoma (HCC) and furthermore investigated the possible molecular mechanisms mediating its anticancer activity. We implanted nude mice with human HCC HepG2 cells and fed them with vehicle (physiological saline) or 3 g/kg/day dose of TARAP 5 days per week for 21 days. We determined the in vitro effect of TARAP on the migration and invasion of HepG2 cells by transwell assay. We evaluated SHH signaling components' (SHH, PTCH, SMO, and Gli1) expression levels by reverse transcriptase-polymerase chain reaction and immunohistochemistry. Activity of the matrix metalloproteinases (MMPs) in supernatants was analyzed by zymography. The expression of the MMPs and their specific tissue inhibitor (tissue inhibitor of matrix metalloproteinases, TIMP-1, 2) in HCC tissues was detected by immunohistochemistry. We discovered that TARAP inhibited hepatocellular migration and invasion in a dose-dependent manner in vitro. In addition, TARAP decreased the expression of SHH, PTCH, SMO, and Gli1 in HCC mouse tumors at both transcriptional and translational levels. Moreover, TARAP inhibited the activity of MMP2 and MMP9. We found that TARAP reduced the expression of MMP2 and MMP9, as well as the tissue inhibitor of MMPs. Our study showed that TARAP inhibits HCC migration and invasion likely through suppression of the hedgehog pathway. This may, in part, explain its anticancer properties. These results suggest that total alkaloids in Rubus alceifolius may have potential as a novel antimetastasis drug in the treatment of HCC. © The Author(s) 2015.

Stromal adipocyte enhancer-binding protein (AEBP1) promotes mammary epithelial cell hyperplasia via proinflammatory and hedgehog signaling.

PubMed

Holloway, Ryan W; Bogachev, Oleg; Bharadwaj, Alamelu G; McCluskey, Greg D; Majdalawieh, Amin F; Zhang, Lei; Ro, Hyo-Sung

2012-11-09

Disruption of mammary stromal-epithelial communication leads to aberrant mammary gland development and induces mammary tumorigenesis. Macrophages have been implicated in carcinogenesis primarily by creating an inflammatory microenvironment, which promotes growth of the adjacent epithelial cells. Adipocyte enhancer-binding protein 1 (AEBP1), a novel proinflammatory mediator, promotes macrophage inflammatory responsiveness by inducing NF-κB activity, which has been implicated in tumor cell growth and survival by aberrant sonic hedgehog (Shh) expression. Here, we show that stromal macrophage AEBP1 overexpression results in precocious alveologenesis in the virgin AEBP1 transgenic (AEBP1(TG)) mice, and the onset of ductal hyperplasia was accelerated in AEBP1(TG) mice fed a high fat diet, which induces endogenous AEBP1 expression. Transplantation of AEBP1(TG) bone marrow cells into non-transgenic (AEBP1(NT)) mice resulted in alveolar hyperplasia with up-regulation of NF-κB activity and TNFα expression as displayed in the AEBP1(TG) mammary macrophages and epithelium. Shh expression was induced in AEBP1(TG) macrophages and RAW264.7 macrophages overexpressing AEBP1. The Shh target genes Gli1 and Bmi1 expression was induced in the AEBP1(TG) mammary epithelium and HC11 mammary epithelial cells co-cultured with AEBP1(TG) peritoneal macrophages. The conditioned AEBP1(TG) macrophage culture media promoted NF-κB activity and survival signal, Akt activation, in HC11 cells, whereas such effects were abolished by TNFα neutralizing antibody treatment. Furthermore, HC11 cells displayed enhanced proliferation in response to AEBP1(TG) macrophages and their conditioned media. Our findings highlight the role of AEBP1 in the signaling pathways regulating the cross-talk between mammary epithelium and stroma that could predispose the mammary tissue to tumorigenesis.

Stromal Adipocyte Enhancer-binding Protein (AEBP1) Promotes Mammary Epithelial Cell Hyperplasia via Proinflammatory and Hedgehog Signaling*

PubMed Central

Holloway, Ryan W.; Bogachev, Oleg; Bharadwaj, Alamelu G.; McCluskey, Greg D.; Majdalawieh, Amin F.; Zhang, Lei; Ro, Hyo-Sung

2012-01-01

Disruption of mammary stromal-epithelial communication leads to aberrant mammary gland development and induces mammary tumorigenesis. Macrophages have been implicated in carcinogenesis primarily by creating an inflammatory microenvironment, which promotes growth of the adjacent epithelial cells. Adipocyte enhancer-binding protein 1 (AEBP1), a novel proinflammatory mediator, promotes macrophage inflammatory responsiveness by inducing NF-κB activity, which has been implicated in tumor cell growth and survival by aberrant sonic hedgehog (Shh) expression. Here, we show that stromal macrophage AEBP1 overexpression results in precocious alveologenesis in the virgin AEBP1 transgenic (AEBP1TG) mice, and the onset of ductal hyperplasia was accelerated in AEBP1TG mice fed a high fat diet, which induces endogenous AEBP1 expression. Transplantation of AEBP1TG bone marrow cells into non-transgenic (AEBP1NT) mice resulted in alveolar hyperplasia with up-regulation of NF-κB activity and TNFα expression as displayed in the AEBP1TG mammary macrophages and epithelium. Shh expression was induced in AEBP1TG macrophages and RAW264.7 macrophages overexpressing AEBP1. The Shh target genes Gli1 and Bmi1 expression was induced in the AEBP1TG mammary epithelium and HC11 mammary epithelial cells co-cultured with AEBP1TG peritoneal macrophages. The conditioned AEBP1TG macrophage culture media promoted NF-κB activity and survival signal, Akt activation, in HC11 cells, whereas such effects were abolished by TNFα neutralizing antibody treatment. Furthermore, HC11 cells displayed enhanced proliferation in response to AEBP1TG macrophages and their conditioned media. Our findings highlight the role of AEBP1 in the signaling pathways regulating the cross-talk between mammary epithelium and stroma that could predispose the mammary tissue to tumorigenesis. PMID:22995915

Nicotine induces self-renewal of pancreatic cancer stem cells via neurotransmitter-driven activation of sonic hedgehog signalling.

PubMed

Al-Wadei, Mohammed H; Banerjee, Jheelam; Al-Wadei, Hussein A N; Schuller, Hildegard M

2016-01-01

A small subpopulation of pancreatic cancer cells with characteristics of stem cells drive tumour initiation, progression and metastasis. A better understanding of the regulation of cancer stem cells may lead to more effective cancer prevention and therapy. We have shown that the proliferation and migration of pancreatic cancer cell lines is activated by the nicotinic receptor-mediated release of stress neurotransmitters, responses reversed by γ-aminobutyric acid (GABA). However, the observed cancer inhibiting effects of GABA will only succeed clinically if GABA inhibits pancreatic cancer stem cells (PCSCs) in addition to the more differentiated cancer cells that comprise the majority of cancer tissues and cell lines. Using PCSCs isolated from two pancreatic cancer patients by cell sorting and by spheroid formation assay from pancreatic cancer cell line Panc-1, we tested the hypothesis that nicotine induces the self-renewal of PCSCs. Nicotinic acetylcholine receptors (nAChRs) α3, α4, α5 and α7 were expressed and chronic exposure to nicotine increased the protein expression of these receptors. Immunoassays showed that PCSCs produced the stress neurotransmitters epinephrine and norepinephrine and the inhibitory neurotransmitter GABA. Chronic nicotine significantly increased the production of stress neurotransmitters and sonic hedgehog (SHH) while inducing Gli1 protein and decreasing GABA. GABA treatment inhibited the induction of SHH and Gli1. Spheroid formation and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazoliumbromide assays showed significant nicotine-induced increases in self renewal and cell proliferation, responses blocked by GABA. Our data suggest that nicotine increases the SHH-mediated malignant potential of PCSCs and that GABA prevents these effects. Copyright © 2015 Elsevier Ltd. All rights reserved.

FoxG1 interacts with Bmi1 to regulate self-renewal and tumorigenicity of medulloblastoma stem cells.

PubMed

Manoranjan, Branavan; Wang, Xin; Hallett, Robin M; Venugopal, Chitra; Mack, Stephen C; McFarlane, Nicole; Nolte, Sara M; Scheinemann, Katrin; Gunnarsson, Thorsteinn; Hassell, John A; Taylor, Michael D; Lee, Cathy; Triscott, Joanna; Foster, Colleen M; Dunham, Christopher; Hawkins, Cynthia; Dunn, Sandra E; Singh, Sheila K

2013-07-01

Brain tumors represent the leading cause of childhood cancer mortality, of which medulloblastoma (MB) is the most frequent malignant tumor. Recent studies have demonstrated the presence of several MB molecular subgroups, each distinct in terms of prognosis and predicted therapeutic response. Groups 1 and 2 are characterized by relatively good clinical outcomes and activation of the Wnt and Shh pathways, respectively. In contrast, groups 3 and 4 ("non-Shh/Wnt MBs") are distinguished by metastatic disease, poor patient outcome, and lack a molecular pathway phenotype. Current gene expression platforms have not detected brain tumor-initiating cell (BTIC) self-renewal genes in groups 3 and 4 MBs as BTICs typically comprise a minority of tumor cells and may therefore go undetected on bulk tumor analyses. Since increasing BTIC frequency has been associated with increasing tumor aggressiveness and poor patient outcome, we investigated the subgroup-specific gene expression profile of candidate stem cell genes within 251 primary human MBs from four nonoverlapping MB transcriptional databases (Amsterdam, Memphis, Toronto, Boston) and 74 NanoString-subgrouped MBs (Vancouver). We assessed the functional relevance of two genes, FoxG1 and Bmi1, which were significantly enriched in non-Shh/Wnt MBs and showed these genes to mediate MB stem cell self-renewal and tumor initiation in mice. We also identified their transcriptional regulation through reciprocal promoter occupancy in CD15+ MB stem cells. Our work demonstrates the application of stem cell data gathered from genomic platforms to guide functional BTIC assays, which may then be used to develop novel BTIC self-renewal mechanisms amenable to therapeutic targeting. Copyright © 2013 AlphaMed Press.

Abrogation of Gli3 expression suppresses the growth of colon cancer cells via activation of p53

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

Kang, Han Na; Oh, Sang Cheul; Kim, Jun Suk

2012-03-10

p53, the major human tumor suppressor, appears to be related to sonic hedgehog (Shh)-Gli-mediated tumorigenesis. However, the role of p53 in tumor progression by the Shh-Gli signaling pathway is poorly understood. Herein we investigated the critical regulation of Gli3-p53 in tumorigenesis of colon cancer cells and the molecular mechanisms underlying these effects. RT-PCR analysis indicated that the mRNA level of Shh and Gli3 in colon tumor tissues was significantly higher than corresponding normal tissues (P < 0.001). The inhibition of Gli3 by treatment with Gli3 siRNA resulted in a clear decrease in cell proliferation and enhanced the level of expressionmore » of p53 proteins compared to treatment with control siRNA. The half-life of p53 was dramatically increased by treatment with Gli3 siRNA. In addition, treatment with MG132 blocked MDM2-mediated p53 ubiquitination and degradation, and led to accumulation of p53 in Gli3 siRNA-overexpressing cells. Importantly, ectopic expression of p53 siRNA reduced the ability of Gli3 siRNA to suppress proliferation of those cells compared with the cells treated with Gli3 siRNA alone. Moreover, Gli3 siRNA sensitized colon cancer cells to treatment with anti-cancer agents (5-FU and bevacizumab). Taken together, our studies demonstrate that loss of Gli3 signaling leads to disruption of the MDM2-p53 interaction and strongly potentiate p53-dependent cell growth inhibition in colon cancer cells, indicating a basis for the rational use of Gli3 antagonists as a novel treatment option for colon cancer.« less

Differential Immune Microenvironments and Response to Immune Checkpoint Blockade among Molecular Subtypes of Murine Medulloblastoma.

PubMed

Pham, Christina D; Flores, Catherine; Yang, Changlin; Pinheiro, Elaine M; Yearley, Jennifer H; Sayour, Elias J; Pei, Yanxin; Moore, Colin; McLendon, Roger E; Huang, Jianping; Sampson, John H; Wechsler-Reya, Robert; Mitchell, Duane A

2016-02-01

Despite significant strides in the identification and characterization of potential therapeutic targets for medulloblastoma, the role of the immune system and its interplay with the tumor microenvironment within these tumors are poorly understood. To address this, we adapted two syngeneic animal models of human Sonic Hedgehog (SHH)-driven and group 3 medulloblastoma for preclinical evaluation in immunocompetent C57BL/6 mice. Multicolor flow cytometric analyses were used to phenotype and characterize immune infiltrating cells within established cerebellar tumors. We observed significantly higher percentages of dendritic cells, infiltrating lymphocytes, myeloid-derived suppressor cells, and tumor-associated macrophages in murine SHH model tumors compared with group 3 tumors. However, murine group 3 tumors had higher percentages of CD8(+) PD-1(+) T cells within the CD3 population. PD-1 blockade conferred superior antitumor efficacy in animals bearing intracranial group 3 tumors compared with SHH group tumors, indicating that immunologic differences within the tumor microenvironment can be leveraged as potential targets to mediate antitumor efficacy. Further analysis of anti-PD-1 monoclonal antibody localization revealed binding to PD-1(+) peripheral T cells, but not tumor infiltrating lymphocytes within the brain tumor microenvironment. Peripheral PD-1 blockade additionally resulted in a marked increase in CD3(+) T cells within the tumor microenvironment. This is the first immunologic characterization of preclinical models of molecular subtypes of medulloblastoma and demonstration that response to immune checkpoint blockade differs across subtype classification. Our findings also suggest that effective anti-PD-1 blockade does not require that systemically administered antibodies penetrate the brain tumor microenvironment. ©2015 American Association for Cancer Research.

Subtypes of medulloblastoma have distinct developmental origins

PubMed Central

Gibson, Paul; Tong, Yiai; Robinson, Giles; Thompson, Margaret C.; Currle, D. Spencer; Eden, Christopher; Kranenburg, Tanya A.; Hogg, Twala; Poppleton, Helen; Martin, Julie; Finkelstein, David; Pounds, Stanley; Weiss, Aaron; Patay, Zoltan; Scoggins, Matthew; Ogg, Robert; Pei, Yanxin; Yang, Zeng-Jie; Brun, Sonja; Lee, Youngsoo; Zindy, Frederique; Lindsey, Janet C.; Taketo, Makoto M.; Boop, Frederick A.; Sanford, Robert A.; Gajjar, Amar; Clifford, Steven C.; Roussel, Martine F.; McKinnon, Peter J.; Gutmann, David H.; Ellison, David W.; Wechsler-Reya, Robert; Gilbertson, Richard J.

2010-01-01

Medulloblastoma encompasses a collection of clinically and molecularly diverse tumor subtypes that together comprise the most common malignant childhood brain tumor1–4. These tumors are thought to arise within the cerebellum, with approximately 25% originating from granule neuron precursor cells (GNPCs) following aberrant activation of the Sonic Hedgehog pathway (hereafter, SHH-subtype)3–8. The pathological processes that drive heterogeneity among the other medulloblastoma subtypes are not known, hindering the development of much needed new therapies. Here, we provide evidence that a discrete subtype of medulloblastoma that contains activating mutations in the WNT pathway effector CTNNB1 (hereafter, WNT-subtype)1,3,4, arises outside the cerebellum from cells of the dorsal brainstem. We found that genes marking human WNT-subtype medulloblastomas are more frequently expressed in the lower rhombic lip (LRL) and embryonic dorsal brainstem than in the upper rhombic lip (URL) and developing cerebellum. Magnetic resonance imaging (MRI) and intra-operative reports showed that human WNT-subtype tumors infiltrate the dorsal brainstem, while SHH-subtype tumors are located within the cerebellar hemispheres. Activating mutations in Ctnnb1 had little impact on progenitor cell populations in the cerebellum, but caused the abnormal accumulation of cells on the embryonic dorsal brainstem that included aberrantly proliferating Zic1+ precursor cells. These lesions persisted in all mutant adult mice and in 15% of cases in which Tp53 was concurrently deleted, progressed to form medulloblastomas that recapitulated the anatomy and gene expression profiles of human WNT-subtype medulloblastoma. We provide the first evidence that subtypes of medulloblastoma have distinct cellular origins. Our data provide an explanation for the marked molecular and clinical differences between SHH and WNT-subtype medulloblastomas and have profound implications for future research and treatment of this important childhood cancer. PMID:21150899

Spontaneous Recovery of Cavernous Nerve Crush Injury

PubMed Central

Kim, Hyo Jong; Kim, Ha Young; Kim, Sung Young; Lee, Seong Ho; Lee, Won Ki

2011-01-01

Purpose To investigate pathophysiological consequences and spontaneous recovery after cavernous nerve crush injury (CNCI) in a rat model. Materials and Methods Twenty 4-week-old male Sprague-Dawley rats were divided into the following groups: sham-operated group (n=10) and bilateral CNCI groups (n=10) for two different durations (12 and 24 weeks). At both time points, CN electrical stimulation was used to assess erectile function by measuring the intracavernous pressure. The expression of hypoxia inducible factor (HIF)-1α and sonic hedgehog (SHH) was examined in penile tissue. Immunohistochemical staining was performed for nerve growth factor (NGF), endothelial nitric oxide synthase (eNOS), neuronal nitric oxide synthase (nNOS), and smooth muscle α-actin. Results CNCI significantly decreased erectile function at 12 weeks (51.7% vs. 71.9%, mean ICP/BP ratio, p<0.05) and increased the expression of HIF-1α and decreased the expression of eNOS, nNOS, and SHH. At 24 weeks, erectile function in the CNCI group was improved with no significant difference versus the sham group (70.5% vs. 63.3%, mean ICP/BP ratio, p<0.05) or the CN group at 12 weeks (51.7% vs. 63.3%, mean ICP/BP ratio, p<0.05). By RT-PCR, the increase in HIF-1α and decrease in SHH mRNA was restored at 24 weeks. By immunohistochemistry, the expression of eNOS and nNOS was increased at 24 weeks. Conclusions CN injury induces significantly impaired erectile function and altered gene and protein expression, which suggests that local hypoxic and inflammatory processes may contribute to this change. Significant spontaneous recovery of erectile function was observed at 6 months after CN crush injury. PMID:21927704

Expression analysis of some genes regulated by retinoic acid in controls and triadimefon-exposed embryos: is the amphibian Xenopus laevis a suitable model for gene-based comparative teratology?

PubMed

Di Renzo, Francesca; Rossi, Federica; Bacchetta, Renato; Prati, Mariangela; Giavini, Erminio; Menegola, Elena

2011-06-01

The use of nonmammal models in teratological studies is a matter of debate and seems to be justified if the embryotoxic mechanism involves conserved processes. Published data on mammals and Xenopus laevis suggest that azoles are teratogenic by altering the endogenous concentration of retinoic acid (RA). The expression of some genes (Shh, Ptch-1, Gsc, and Msx2) controlled by retinoic acid is downregulated in rat embryos exposed at the phylotypic stage to the triazole triadimefon (FON). In order to propose X. laevis as a model for gene-based comparative teratology, this work evaluates the expression of Shh, Ptch-1, Gsc, and Msx2 in FON-exposed X. laevis embryos. Embryos, exposed to a high concentration level (500 µM) of FON from stage 13 till 17, were examined at stages 17, 27, and 47. Stage 17 and 27 embryos were processed to perform quantitative RT-PCR. The developmental rate was never affected by FON at any considered stage. FON-exposed stage 47 larvae showed the typical craniofacial malformations. A significant downregulation of Gsc was observed in FON-exposed stage 17 embryos. Shh, Ptch-1, Msx2 showed a high fluctuation of expression both in control and in FON-exposed samples both at stages 17 and 27. The downregulation of Gsc mimics the effects of FON on rat embryos, showing for this gene a common effect of FON in the two vertebrate classes. The high fluctuation observed in the gene expression of the other genes, however, suggests that X. laevis at this stage has limited utility for gene-based comparative teratology. © 2011 Wiley-Liss, Inc.

Sall4-Gli3 system in early limb progenitors is essential for the development of limb skeletal elements.

PubMed

Akiyama, Ryutaro; Kawakami, Hiroko; Wong, Julia; Oishi, Isao; Nishinakamura, Ryuichi; Kawakami, Yasuhiko

2015-04-21

Limb skeletal elements originate from the limb progenitor cells, which undergo expansion and patterning to develop each skeletal element. Posterior-distal skeletal elements, such as the ulna/fibula and posterior digits develop in a Sonic hedgehog (Shh)-dependent manner. However, it is poorly understood how anterior-proximal elements, such as the humerus/femur, the radius/tibia and the anterior digits, are developed. Here we show that the zinc finger factors Sall4 and Gli3 cooperate for proper development of the anterior-proximal skeletal elements and also function upstream of Shh-dependent posterior skeletal element development. Conditional inactivation of Sall4 in the mesoderm before limb outgrowth caused severe defects in the anterior-proximal skeletal elements in the hindlimb. We found that Gli3 expression is reduced in Sall4 mutant hindlimbs, but not in forelimbs. This reduction caused posteriorization of nascent hindlimb buds, which is correlated with a loss of anterior digits. In proximal development, Sall4 integrates Gli3 and the Plzf-Hox system, in addition to proliferative expansion of cells in the mesenchymal core of nascent hindlimb buds. Whereas forelimbs developed normally in Sall4 mutants, further genetic analysis identified that the Sall4-Gli3 system is a common regulator of the early limb progenitor cells in both forelimbs and hindlimbs. The Sall4-Gli3 system also functions upstream of the Shh-expressing ZPA and the Fgf8-expressing AER in fore- and hindlimbs. Therefore, our study identified a critical role of the Sall4-Gli3 system at the early steps of limb development for proper development of the appendicular skeletal elements.

Acute upregulation of hedgehog signaling in mice causes differential effects on cranial morphology.

PubMed

Singh, Nandini; Dutka, Tara; Devenney, Benjamin M; Kawasaki, Kazuhiko; Reeves, Roger H; Richtsmeier, Joan T

2015-03-01

Hedgehog (HH) signaling, and particularly signaling by sonic hedgehog (SHH), is implicated in several essential activities during morphogenesis, and its misexpression causes a number of developmental disorders in humans. In particular, a reduced mitogenic response of cerebellar granule cell precursors to SHH signaling in a mouse model for Down syndrome (DS), Ts65Dn, is substantially responsible for reduced cerebellar size. A single treatment of newborn trisomic mice with an agonist of the SHH pathway (SAG) normalizes cerebellar morphology and restores some cognitive deficits, suggesting a possible therapeutic application of SAG for treating the cognitive impairments of DS. Although the beneficial effects on the cerebellum are compelling, inappropriate activation of the HH pathway causes anomalies elsewhere in the head, particularly in the formation and patterning of the craniofacial skeleton. To determine whether an acute treatment of SAG has an effect on craniofacial morphology, we quantitatively analyzed the cranial form of adult euploid and Ts65Dn mice that were injected with either SAG or vehicle at birth. We found significant deformation of adult craniofacial shape in some animals that had received SAG at birth. The most pronounced differences between the treated and untreated mice were in the midline structures of the facial skeleton. The SAG-driven craniofacial dysmorphogenesis was dose-dependent and possibly incompletely penetrant at lower concentrations. Our findings illustrate that activation of HH signaling, even with an acute postnatal stimulation, can lead to localized dysmorphology of the skull by generating modular shape changes in the facial skeleton. These observations have important implications for translating HH-agonist-based treatments for DS. © 2015. Published by The Company of Biologists Ltd.

G-protein coupled receptor expression patterns delineate medulloblastoma subgroups

PubMed Central

2013-01-01

Background Medulloblastoma is the most common malignant brain tumor in children. Genetic profiling has identified four principle tumor subgroups; each subgroup is characterized by different initiating mutations, genetic and clinical profiles, and prognoses. The two most well-defined subgroups are caused by overactive signaling in the WNT and SHH mitogenic pathways; less is understood about Groups 3 and 4 medulloblastoma. Identification of tumor subgroup using molecular classification is set to become an important component of medulloblastoma diagnosis and staging, and will likely guide therapeutic options. However, thus far, few druggable targets have emerged. G-protein coupled receptors (GPCRs) possess characteristics that make them ideal targets for molecular imaging and therapeutics; drugs targeting GPCRs account for 30-40% of all current pharmaceuticals. While expression patterns of many proteins in human medulloblastoma subgroups have been discerned, the expression pattern of GPCRs in medulloblastoma has not been investigated. We hypothesized that analysis of GPCR expression would identify clear subsets of medulloblastoma and suggest distinct GPCRs that might serve as molecular targets for both imaging and therapy. Results Our study found that medulloblastoma tumors fall into distinct clusters based solely on GPCR expression patterns. Normal cerebellum clustered separately from the tumor samples. Further, two of the tumor clusters correspond with high fidelity to the WNT and SHH subgroups of medulloblastoma. Distinct over-expressed GPCRs emerge; for example, LGR5 and GPR64 are significantly and uniquely over-expressed in the WNT subgroup of tumors, while PTGER4 is over-expressed in the SHH subgroup. Uniquely under-expressed GPCRs were also observed. Our key findings were independently validated using a large international dataset. Conclusions Our results identify GPCRs with potential to act as imaging and therapeutic targets. Elucidating tumorigenic pathways is a secondary benefit to identifying differential GPCR expression patterns in medulloblastoma tumors. PMID:24252460

The regulation of tooth morphogenesis is associated with epithelial cell proliferation and the expression of Sonic hedgehog through epithelial-mesenchymal interactions

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

Ishida, Kentaro; Murofushi, Mayumi; Nakao, Kazuhisa

2011-02-18

Research highlights: {yields} Bioengineered teeth regulated the contact area of epithelium and mesenchyme. {yields} The crown width is regulated by the contact area of the epithelium and mesenchyme. {yields} This regulation is associated with cell proliferation and Sonic hedgehog expression. {yields} The cusp number is correlated with the crown width of the bioengineered tooth. {yields} Cell proliferation and Shh expression areas regulate the tooth morphogenesis. -- Abstract: Ectodermal organs, such as the tooth, salivary gland, hair, and mammary gland, develop through reciprocal epithelial-mesenchymal interactions. Tooth morphologies are defined by the crown width and tooth length (macro-morphologies), and by the numbermore » and locations of the cusp and roots (micro-morphologies). In our current study, we report that the crown width of a bioengineered molar tooth, which was reconstructed using dissociated epithelial and mesenchymal cells via an organ germ method, can be regulated by the contact area between epithelial and mesenchymal cell layers. We further show that this is associated with cell proliferation and Sonic hedgehog (Shh) expression in the inner enamel epithelium after the germ stage has formed a secondary enamel knot. We also demonstrate that the cusp number is significantly correlated with the crown width of the bioengineered tooth. These findings suggest that the tooth micro-morphology, i.e. the cusp formation, is regulated after the tooth width, or macro-morphology, is determined. These findings also suggest that the spatiotemporal patterning of cell proliferation and the Shh expression areas in the epithelium regulate the crown width and cusp formation of the developing tooth.« less

Differential immune microenvironments and response to immune checkpoint blockade amongst molecular subtypes of murine medulloblastoma

PubMed Central

Pham, Christina D.; Flores, Catherine; Yang, Changlin; Pinheiro, Elaine M.; Yearley, Jennifer H.; Sayour, Elias J.; Pei, Yanxin; Moore, Colin; McLendon, Roger E.; Huang, Jianping; Sampson, John H.; Wechsler-Reya, Robert; Mitchell, Duane A.

2016-01-01

PURPOSE Despite significant strides in the identification and characterization of potential therapeutic targets for medulloblastoma (MB), the role of the immune system and its interplay with the tumor microenvironment within these tumors are poorly understood. To address this, we adapted two syngeneic animal models of human Sonic Hedgehog (SHH)-driven and Group 3 MB for preclinical evaluation in immunocompetent C57BL/6 mice. METHODS AND RESULTS Multicolor flow cytometric analyses were used to phenotype and characterize immune infiltrating cells within established cerebellar tumors. We observed significantly higher percentages of dendritic cells, infiltrating lymphocytes, myeloid derived suppressor cells and tumor-associated macrophages in murine SHH model tumors compared with Group 3 tumors. However, murine Group 3 tumors had higher percentages of CD8+ PD-1+ T cells within the CD3 population. PD-1 blockade conferred superior antitumor efficacy in animals bearing intracranial Group 3 tumors compared to SHH group tumors, indicating that immunologic differences within the tumor microenvironment can be leveraged as potential targets to mediate antitumor efficacy. Further analysis of anti-PD-1 monoclonal antibody localization revealed binding to PD-1+ peripheral T cells, but not tumor infiltrating lymphocytes within the brain tumor microenvironment. Peripheral PD-1 blockade additionally resulted in a marked increase in CD3+ T cells within the tumor microenvironment. CONCLUSIONS This is the first immunologic characterization of preclinical models of molecular subtypes of MB and demonstration that response to immune checkpoint blockade differs across subtype classification. Our findings also suggest that effective anti-PD-1 blockade does not require that systemically administered antibodies penetrate the brain tumor microenvironment. PMID:26405194

Fibroblast Growth Factors Stimulate Hair Growth through β-Catenin and Shh Expression in C57BL/6 Mice

PubMed Central

Lin, Wei-hong; Xiang, Li-Jun; Shi, Hong-Xue; Zhang, Jian; Jiang, Li-ping; Cai, Ping-tao; Lin, Zhen-Lang; Lin, Bei-Bei; Huang, Yan; Zhang, Hai-Lin; Fu, Xiao-Bing; Guo, Ding-Jiong; Li, Xiao-Kun; Wang, Xiao-Jie; Xiao, Jian

2015-01-01

Growth factors are involved in the regulation of hair morphogenesis and cycle hair growth. The present study sought to investigate the hair growth promoting activities of three approved growth factor drugs, fibroblast growth factor 10 (FGF-10), acidic fibroblast growth factor (FGF-1), and basic fibroblast growth factor (FGF-2), and the mechanism of action. We observed that FGFs promoted hair growth by inducing the anagen phase in telogenic C57BL/6 mice. Specifically, the histomorphometric analysis data indicates that topical application of FGFs induced an earlier anagen phase and prolonged the mature anagen phase, in contrast to the control group. Moreover, the immunohistochemical analysis reveals earlier induction of β-catenin and Sonic hedgehog (Shh) in hair follicles of the FGFs-treated group. These results suggest that FGFs promote hair growth by inducing the anagen phase in resting hair follicles and might be a potential hair growth-promoting agent. PMID:25685806

Fgf16 is essential for pectoral fin bud formation in zebrafish

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

Nomura, Ryohei; Kamei, Eriko; Hotta, Yuuhei

2006-08-18

Zebrafish pectoral fin bud formation is an excellent model for studying morphogenesis. Fibroblast growth factors (Fgfs) and sonic hedgehog (shh) are essential for pectoral fin bud formation. We found that Fgf16 was expressed in the apical ectodermal ridge (AER) of fin buds. A knockdown of Fgf16 function resulted in no fin bud outgrowth. Fgf16 is required for cell proliferation and differentiation in the mesenchyme and the AER of the fin buds, respectively. Fgf16 functions downstream of Fgf10, a mesenchymal factor, signaling to induce the expression of Fgf4 and Fgf8 in the AER. Fgf16 in the AER and shh in themore » zone of polarizing activity (ZPA) interact to induce and/or maintain each other's expression. These findings have revealed that Fgf16, a newly identified AER factor, plays a crucial role in pectoral fin bud outgrowth by mediating the interactions of AER-mesenchyme and AER-ZPA.« less

Analyzing notochord segmentation and intervertebral disc formation using the twhh:gfp transgenic zebrafish model.

PubMed

Haga, Yutaka; Dominique, Vincent J; Du, Shao Jun

2009-10-01

To characterize the process of vertebral segmentation and disc formation in living animals, we analyzed tiggy-winkle hedgehog (twhh):green fluorescent protein (gfp) and sonic hedgehog (shh):gfp transgenic zebrafish models that display notochord-specific GFP expression. We found that they showed distinct patterns of expression in the intervertebral discs of late stage fish larvae and adult zebrafish. A segmented pattern of GFP expression was detected in the intervertebral disc of twhh:gfp transgenic fish. In contrast, little GFP expression was found in the intervertebral disc of shh:gfp transgenic fish. Treating twhh:gfp transgenic zebrafish larvae with exogenous retinoic acid (RA), a teratogenic factor on normal development, resulted in disruption of notochord segmentation and formation of oversized vertebrae. Histological analysis revealed that the oversized vertebrae are likely due to vertebral fusion. These studies demonstrate that the twhh:gfp transgenic zebrafish is a useful model for studying vertebral segmentation and disc formation, and moreover, that RA signaling may play a role in this process.

Disturbed MEK/ERK signaling increases osteoclast activity via the Hedgehog-Gli pathway in postmenopausal osteoporosis.

PubMed

Li, Xiaojie; Jie, Qiang; Zhang, Hongyang; Zhao, Yantao; Lin, Yangjing; Du, Junjie; Shi, Jun; Wang, Long; Guo, Kai; Li, Yong; Wang, Chunhui; Gao, Bo; Huang, Qiang; Liu, Jian; Yang, Liu; Luo, Zhuojing

2016-11-01

Postmenopausal osteoporosis is a worldwide health problem and is characterized by increased and activated osteoclasts. However, the mechanism by which osteoclasts are dysregulated in postmenopausal osteoporosis is not fully understood. In this study, we found that the Hedgehog-Gli pathway was upregulated in postmenopausal osteoporotic osteoclasts and that 17β-estradiol both inhibited osteoclastogenesis and induced osteoclast apoptosis by downregulating Hedgehog-Gli signaling. Furthermore, we demonstrated that the Hedgehog-Gli pathway was negatively regulated by MEK/ERK signaling and that this effect was Sonic Hedgehog (SHH)-dependent and was partially blocked by an anti-SHH antibody. Moreover, we found that the stimulatory effect of Hedgehog signaling on osteoclastogenesis and the inhibitory effect on osteoclast apoptosis were dependent on the Gli family of transcription factors. The pathways and molecules that contribute to the regulation of osteoclastogenesis and apoptosis represent potential new strategies for designing molecular drugs for the treatment of postmenopausal osteoporosis. Copyright © 2016 Elsevier Ltd. All rights reserved.

Hippi is essential for node cilia assembly and Sonic hedgehog signaling

PubMed Central

Houde, Caroline; Dickinson, Robin J.; Houtzager, Vicky M.; Cullum, Rebecca; Montpetit, Rachel; Metzler, Martina; Simpson, Elizabeth M.; Roy, Sophie; Hayden, Michael R.; Hoodless, Pamela A.; Nicholson, Donald W.

2016-01-01

Hippi functions as an adapter protein that mediates pro-apoptotic signaling from poly-glutamine-expanded huntingtin, an established cause of Huntington disease, to the extrinsic cell death pathway. To explore other functions of Hippi we generated Hippi knock-out mice. This deletion causes randomization of the embryo turning process and heart looping, which are hallmarks of defective left–right (LR) axis patterning. We report that motile monocilia normally present at the surface of the embryonic node, and proposed to initiate the break in LR symmetry, are absent on Hippi−/− embryos. Furthermore, defects in central nervous system development are observed. The Sonic hedgehog (Shh) pathway is downregulated in the neural tube in the absence of Hippi, which results in failure to establish ventral neural cell fate. Together, these findings demonstrate a dual role for Hippi in cilia assembly and Shh signaling during development, in addition to its proposed role in apoptosis signal transduction in the adult brain under pathogenically stressful conditions. PMID:17027958

Performance Modeling and Measurement of Parallelized Code for Distributed Shared Memory Multiprocessors

NASA Technical Reports Server (NTRS)

Waheed, Abdul; Yan, Jerry

1998-01-01

This paper presents a model to evaluate the performance and overhead of parallelizing sequential code using compiler directives for multiprocessing on distributed shared memory (DSM) systems. With increasing popularity of shared address space architectures, it is essential to understand their performance impact on programs that benefit from shared memory multiprocessing. We present a simple model to characterize the performance of programs that are parallelized using compiler directives for shared memory multiprocessing. We parallelized the sequential implementation of NAS benchmarks using native Fortran77 compiler directives for an Origin2000, which is a DSM system based on a cache-coherent Non Uniform Memory Access (ccNUMA) architecture. We report measurement based performance of these parallelized benchmarks from four perspectives: efficacy of parallelization process; scalability; parallelization overhead; and comparison with hand-parallelized and -optimized version of the same benchmarks. Our results indicate that sequential programs can conveniently be parallelized for DSM systems using compiler directives but realizing performance gains as predicted by the performance model depends primarily on minimizing architecture-specific data locality overhead.

Eccrine sweat gland development and sweat secretion.

PubMed

Cui, Chang-Yi; Schlessinger, David

2015-09-01

Eccrine sweat glands help to maintain homoeostasis, primarily by stabilizing body temperature. Derived from embryonic ectoderm, millions of eccrine glands are distributed across human skin and secrete litres of sweat per day. Their easy accessibility has facilitated the start of analyses of their development and function. Mouse genetic models find sweat gland development regulated sequentially by Wnt, Eda and Shh pathways, although precise subpathways and additional regulators require further elucidation. Mature glands have two secretory cell types, clear and dark cells, whose comparative development and functional interactions remain largely unknown. Clear cells have long been known as the major secretory cells, but recent studies suggest that dark cells are also indispensable for sweat secretion. Dark cell-specific Foxa1 expression was shown to regulate a Ca(2+) -dependent Best2 anion channel that is the candidate driver for the required ion currents. Overall, it was shown that cholinergic impulses trigger sweat secretion in mature glands through second messengers - for example InsP3 and Ca(2+) - and downstream ion channels/transporters in the framework of a Na(+) -K(+) -Cl(-) cotransporter model. Notably, the microenvironment surrounding secretory cells, including acid-base balance, was implicated to be important for proper sweat secretion, which requires further clarification. Furthermore, multiple ion channels have been shown to be expressed in clear and dark cells, but the degree to which various ion channels function redundantly or indispensably also remains to be determined. Published 2015. This article is a U.S. Government work and is in the public domain in the USA.

Parallelization of sequential Gaussian, indicator and direct simulation algorithms

NASA Astrophysics Data System (ADS)

Nunes, Ruben; Almeida, José A.

2010-08-01

Improving the performance and robustness of algorithms on new high-performance parallel computing architectures is a key issue in efficiently performing 2D and 3D studies with large amount of data. In geostatistics, sequential simulation algorithms are good candidates for parallelization. When compared with other computational applications in geosciences (such as fluid flow simulators), sequential simulation software is not extremely computationally intensive, but parallelization can make it more efficient and creates alternatives for its integration in inverse modelling approaches. This paper describes the implementation and benchmarking of a parallel version of the three classic sequential simulation algorithms: direct sequential simulation (DSS), sequential indicator simulation (SIS) and sequential Gaussian simulation (SGS). For this purpose, the source used was GSLIB, but the entire code was extensively modified to take into account the parallelization approach and was also rewritten in the C programming language. The paper also explains in detail the parallelization strategy and the main modifications. Regarding the integration of secondary information, the DSS algorithm is able to perform simple kriging with local means, kriging with an external drift and collocated cokriging with both local and global correlations. SIS includes a local correction of probabilities. Finally, a brief comparison is presented of simulation results using one, two and four processors. All performance tests were carried out on 2D soil data samples. The source code is completely open source and easy to read. It should be noted that the code is only fully compatible with Microsoft Visual C and should be adapted for other systems/compilers.

Direct quantum process tomography via measuring sequential weak values of incompatible observables.

PubMed

Kim, Yosep; Kim, Yong-Su; Lee, Sang-Yun; Han, Sang-Wook; Moon, Sung; Kim, Yoon-Ho; Cho, Young-Wook

2018-01-15

The weak value concept has enabled fundamental studies of quantum measurement and, recently, found potential applications in quantum and classical metrology. However, most weak value experiments reported to date do not require quantum mechanical descriptions, as they only exploit the classical wave nature of the physical systems. In this work, we demonstrate measurement of the sequential weak value of two incompatible observables by making use of two-photon quantum interference so that the results can only be explained quantum physically. We then demonstrate that the sequential weak value measurement can be used to perform direct quantum process tomography of a qubit channel. Our work not only demonstrates the quantum nature of weak values but also presents potential new applications of weak values in analyzing quantum channels and operations.

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

Faye, Sherry A.; Richards, Jason M.; Gallardo, Athena M.

Sequential extraction is a useful technique for assessing the potential to leach actinides from soils; however, current literature lacks uniformity in experimental details, making direct comparison of results impossible. This work continued development toward a standardized five-step sequential extraction protocol by analyzing extraction behaviors of 232Th, 238U, 239,240Pu and 241Am from lake and ocean sediment reference materials. Results produced a standardized procedure after creating more defined reaction conditions to improve method repeatability. A NaOH fusion procedure is recommended following sequential leaching for the complete dissolution of insoluble species.

Parallelization of NAS Benchmarks for Shared Memory Multiprocessors

NASA Technical Reports Server (NTRS)

Waheed, Abdul; Yan, Jerry C.; Saini, Subhash (Technical Monitor)

1998-01-01

This paper presents our experiences of parallelizing the sequential implementation of NAS benchmarks using compiler directives on SGI Origin2000 distributed shared memory (DSM) system. Porting existing applications to new high performance parallel and distributed computing platforms is a challenging task. Ideally, a user develops a sequential version of the application, leaving the task of porting to new generations of high performance computing systems to parallelization tools and compilers. Due to the simplicity of programming shared-memory multiprocessors, compiler developers have provided various facilities to allow the users to exploit parallelism. Native compilers on SGI Origin2000 support multiprocessing directives to allow users to exploit loop-level parallelism in their programs. Additionally, supporting tools can accomplish this process automatically and present the results of parallelization to the users. We experimented with these compiler directives and supporting tools by parallelizing sequential implementation of NAS benchmarks. Results reported in this paper indicate that with minimal effort, the performance gain is comparable with the hand-parallelized, carefully optimized, message-passing implementations of the same benchmarks.

Mechanical System Reliability and Cost Integration Using a Sequential Linear Approximation Method

NASA Technical Reports Server (NTRS)

Kowal, Michael T.

1997-01-01

The development of new products is dependent on product designs that incorporate high levels of reliability along with a design that meets predetermined levels of system cost. Additional constraints on the product include explicit and implicit performance requirements. Existing reliability and cost prediction methods result in no direct linkage between variables affecting these two dominant product attributes. A methodology to integrate reliability and cost estimates using a sequential linear approximation method is proposed. The sequential linear approximation method utilizes probability of failure sensitivities determined from probabilistic reliability methods as well a manufacturing cost sensitivities. The application of the sequential linear approximation method to a mechanical system is demonstrated.

Sequential lineups: shift in criterion or decision strategy?

PubMed

Gronlund, Scott D

2004-04-01

R. C. L. Lindsay and G. L. Wells (1985) argued that a sequential lineup enhanced discriminability because it elicited use of an absolute decision strategy. E. B. Ebbesen and H. D. Flowe (2002) argued that a sequential lineup led witnesses to adopt a more conservative response criterion, thereby affecting bias, not discriminability. Height was encoded as absolute (e.g., 6 ft [1.83 m] tall) or relative (e.g., taller than). If a sequential lineup elicited an absolute decision strategy, the principle of transfer-appropriate processing predicted that performance should be best when height was encoded absolutely. Conversely, if a simultaneous lineup elicited a relative decision strategy, performance should be best when height was encoded relatively. The predicted interaction was observed, providing direct evidence for the decision strategies explanation of what happens when witnesses view a sequential lineup.

Cost-effectiveness of simultaneous versus sequential surgery in head and neck reconstruction.

PubMed

Wong, Kevin K; Enepekides, Danny J; Higgins, Kevin M

2011-02-01

To determine whether simultaneous (ablation and reconstruction overlaps by two teams) head and neck reconstruction is cost effective compared to sequentially (ablation followed by reconstruction) performed surgery. Case-controlled study. Tertiary care hospital. Oncology patients undergoing free flap reconstruction of the head and neck. A match paired comparison study was performed with a retrospective chart review examining the total time of surgery for sequential and simultaneous surgery. Nine patients were selected for both the sequential and simultaneous groups. Sequential head and neck reconstruction patients were pair matched with patients who had undergone similar oncologic ablative or reconstructive procedures performed in a simultaneous fashion. A detailed cost analysis using the microcosting method was then undertaken looking at the direct costs of the surgeons, anesthesiologist, operating room, and nursing. On average, simultaneous surgery required 3 hours 15 minutes less operating time, leading to a cost savings of approximately $1200/case when compared to sequential surgery. This represents approximately a 15% reduction in the cost of the entire operation. Simultaneous head and neck reconstruction is more cost effective when compared to sequential surgery.

Efficient differentiation of mouse embryonic stem cells into motor neurons.

PubMed

Wu, Chia-Yen; Whye, Dosh; Mason, Robert W; Wang, Wenlan

2012-06-09

Direct differentiation of embryonic stem (ES) cells into functional motor neurons represents a promising resource to study disease mechanisms, to screen new drug compounds, and to develop new therapies for motor neuron diseases such as spinal muscular atrophy (SMA) and amyotrophic lateral sclerosis (ALS). Many current protocols use a combination of retinoic acid (RA) and sonic hedgehog (Shh) to differentiate mouse embryonic stem (mES) cells into motor neurons. However, the differentiation efficiency of mES cells into motor neurons has only met with moderate success. We have developed a two-step differentiation protocol that significantly improves the differentiation efficiency compared with currently established protocols. The first step is to enhance the neuralization process by adding Noggin and fibroblast growth factors (FGFs). Noggin is a bone morphogenetic protein (BMP) antagonist and is implicated in neural induction according to the default model of neurogenesis and results in the formation of anterior neural patterning. FGF signaling acts synergistically with Noggin in inducing neural tissue formation by promoting a posterior neural identity. In this step, mES cells were primed with Noggin, bFGF, and FGF-8 for two days to promote differentiation towards neural lineages. The second step is to induce motor neuron specification. Noggin/FGFs exposed mES cells were incubated with RA and a Shh agonist, Smoothened agonist (SAG), for another 5 days to facilitate motor neuron generation. To monitor the differentiation of mESs into motor neurons, we used an ES cell line derived from a transgenic mouse expressing eGFP under the control of the motor neuron specific promoter Hb9. Using this robust protocol, we achieved 51 ± 0.8% of differentiation efficiency (n = 3; p < 0.01, Student's t-test). Results from immunofluorescent staining showed that GFP+ cells express the motor neuron specific markers, Islet-1 and choline acetyltransferase (ChAT). Our two-step differentiation protocol provides an efficient way to differentiate mES cells into spinal motor neurons.

Targeted reduction of vascular Msx1 and Msx2 mitigates arteriosclerotic calcification and aortic stiffness in LDLR-deficient mice fed diabetogenic diets.

PubMed

Cheng, Su-Li; Behrmann, Abraham; Shao, Jian-Su; Ramachandran, Bindu; Krchma, Karen; Bello Arredondo, Yoanna; Kovacs, Attila; Mead, Megan; Maxson, Robert; Towler, Dwight A

2014-12-01

When fed high-fat diets, male LDLR(-/-) mice develop obesity, hyperlipidemia, hyperglycemia, and arteriosclerotic calcification. An osteogenic Msx-Wnt regulatory program is concomitantly upregulated in the vasculature. To better understand the mechanisms of diabetic arteriosclerosis, we generated SM22-Cre;Msx1(fl/fl);Msx2(fl/fl);LDLR(-/-) mice, assessing the impact of Msx1+Msx2 gene deletion in vascular myofibroblast and smooth muscle cells. Aortic Msx2 and Msx1 were decreased by 95% and 34% in SM22-Cre;Msx1(fl/fl);Msx2(fl/fl);LDLR(-/-) animals versus Msx1(fl/fl);Msx2(fl/fl);LDLR(-/-) controls, respectively. Aortic calcium was reduced by 31%, and pulse wave velocity, an index of stiffness, was decreased in SM22-Cre;Msx1(fl/fl);Msx2(fl/fl);LDLR(-/-) mice vs. controls. Fasting blood glucose and lipids did not differ, yet SM22-Cre;Msx1(fl/fl);Msx2(fl/fl);LDLR(-/-) siblings became more obese. Aortic adventitial myofibroblasts from SM22-Cre;Msx1(fl/fl);Msx2(fl/fl);LDLR(-/-) mice exhibited reduced osteogenic gene expression and mineralizing potential with concomitant reduction in multiple Wnt genes. Sonic hedgehog (Shh) and Sca1, markers of aortic osteogenic progenitors, were also reduced, paralleling a 78% reduction in alkaline phosphatase (TNAP)-positive adventitial myofibroblasts. RNA interference revealed that although Msx1+Msx2 supports TNAP and Wnt7b expression, Msx1 selectively maintains Shh and Msx2 sustains Wnt2, Wnt5a, and Sca1 expression in aortic adventitial myofibroblast cultures. Thus, Msx1 and Msx2 support vascular mineralization by directing the osteogenic programming of aortic progenitors in diabetic arteriosclerosis. © 2014 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.

U.S. EPA, Pesticide Product Label, GARRATT-CALLAHAN FORMULA 315, 09/08/1989

EPA Pesticide Factsheets

2011-04-21

... 'Up.it II II , Sillt.,~ 1 .. ,,,11 • • r " .t'tf ,rluhttS .",.·d 1:1, shh Ifl' hul .. tt..tltus h..STI( COIT.IIDS T'I,h tlUt

Subgroup Specific Alternative Splicing in Medulloblastoma

PubMed Central

Kloosterhof, Nanne K; Northcott, Paul A; Yu, Emily PY; Shih, David; Peacock, John; Grajkowska, Wieslawa; van Meter, Timothy; Eberhart, Charles G; Pfister, Stefan; Marra, Marco A; Weiss, William A; Scherer, Stephen W; Rutka, James T; French, Pim J; Taylor, Michael D

2014-01-01

Medulloblastoma is comprised of four distinct molecular variants: WNT, SHH, Group 3, and Group 4. We analyzed alternative splicing usage in 14 normal cerebellar samples and 103 medulloblastomas of known subgroup. Medulloblastoma samples have a statistically significant increase in alternative splicing as compared to normal fetal cerebella (2.3-times; P<6.47E-8). Splicing patterns are distinct and specific between molecular subgroups. Unsupervised hierarchical clustering of alternative splicing events accurately assigns medulloblastomas to their correct subgroup. Subgroup-specific splicing and alternative promoter usage was most prevalent in Group 3 (19.4%) and SHH (16.2%) medulloblastomas, while observed less frequently in WNT (3.2%), and Group 4 (9.3%) tumors. Functional annotation of alternatively spliced genes reveals over-representation of genes important for neuronal development. Alternative splicing events in medulloblastoma may be regulated in part by the correlative expression of antisense transcripts, suggesting a possible mechanism affecting subgroup specific alternative splicing. Our results identify additional candidate markers for medulloblastoma subgroup affiliation, further support the existence of distinct subgroups of the disease, and demonstrate an additional level of transcriptional heterogeneity between medulloblastoma subgroups. PMID:22358458

Overexpression of SASH1 Inhibits the Proliferation, Invasion, and EMT in Hepatocarcinoma Cells.

PubMed

He, Ping; Zhang, Hong-Xia; Sun, Chang-Yu; Chen, Chun-Yong; Jiang, He-Qing

2016-01-01

The SASH1 (SAM- and SH3-domain containing 1) gene, a member of the SLY (SH3 domain containing expressed in lymphocytes) family of signal adapter proteins, has been implicated in tumorigenesis of many types of cancers. However, the role and mechanism of SASH1 in the invasion and metastasis of hepatocarcinoma are largely unknown. In this study, we investigated the role and mechanism of SASH1 in the invasion and metastasis of hepatocarcinoma. Our results showed that SASH1 was lowly expressed in hepatocarcinoma cell lines. The in vitro experiments showed that overexpression of SASH1 inhibited the proliferation and migration/invasion of hepatocarcinoma cells, as well as the epithelial-mesenchymal transition (EMT) progress. Furthermore, overexpression of SASH1 suppressed the expression of Shh as well as Smo, Ptc, and Gli-1 in hepatocarcinoma cells. Taken together, these results suggest that overexpression of SASH1 inhibited the proliferation and invasion of hepatocarcinoma cells through the inactivation of Shh signaling pathway. Therefore, these findings reveal that SASH1 may be a potential therapeutic target for the treatment of hepatocarcinoma.

Reversible logic gates on Physarum Polycephalum

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

Schumann, Andrew

2015-03-10

In this paper, we consider possibilities how to implement asynchronous sequential logic gates and quantum-style reversible logic gates on Physarum polycephalum motions. We show that in asynchronous sequential logic gates we can erase information because of uncertainty in the direction of plasmodium propagation. Therefore quantum-style reversible logic gates are more preferable for designing logic circuits on Physarum polycephalum.

Simultaneous and Sequential Feature Negative Discriminations: Elemental Learning and Occasion Setting in Human Pavlovian Conditioning

ERIC Educational Resources Information Center

Baeyens, Frank; Vervliet, Bram; Vansteenwegen, Debora; Beckers, Tom; Hermans, Dirk; Eelen, Paul

2004-01-01

Using a conditioned suppression task, we investigated simultaneous (XA-/A+) vs. sequential (X [right arrow] A-/A+) Feature Negative (FN) discrimination learning in humans. We expected the simultaneous discrimination to result in X (or alternatively the XA configuration) becoming an inhibitor acting directly on the US, and the sequential…

A Robust Real Time Direction-of-Arrival Estimation Method for Sequential Movement Events of Vehicles.

PubMed

Liu, Huawei; Li, Baoqing; Yuan, Xiaobing; Zhou, Qianwei; Huang, Jingchang

2018-03-27

Parameters estimation of sequential movement events of vehicles is facing the challenges of noise interferences and the demands of portable implementation. In this paper, we propose a robust direction-of-arrival (DOA) estimation method for the sequential movement events of vehicles based on a small Micro-Electro-Mechanical System (MEMS) microphone array system. Inspired by the incoherent signal-subspace method (ISM), the method that is proposed in this work employs multiple sub-bands, which are selected from the wideband signals with high magnitude-squared coherence to track moving vehicles in the presence of wind noise. The field test results demonstrate that the proposed method has a better performance in emulating the DOA of a moving vehicle even in the case of severe wind interference than the narrowband multiple signal classification (MUSIC) method, the sub-band DOA estimation method, and the classical two-sided correlation transformation (TCT) method.

High glucose alters the expression of genes involved in proliferation and cell-fate specification of embryonic neural stem cells.

PubMed

Fu, J; Tay, S S W; Ling, E A; Dheen, S T

2006-05-01

Maternal diabetes induces neural tube defects during embryogenesis. Since the neural tube is derived from neural stem cells (NSCs), it is hypothesised that in diabetic pregnancy neural tube defects result from altered expression of developmental control genes, leading to abnormal proliferation and cell-fate choice of NSCs. Cell viability, proliferation index and apoptosis of NSCs and differentiated cells from mice exposed to physiological or high glucose concentration medium were examined by a tetrazolium salt assay, 5-bromo-2'-deoxyuridine incorporation, terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling and immunocytochemistry. Expression of developmental genes, including sonic hedgehog (Shh), bone morphogenetic protein 4 (Bmp4), neurogenin 1/2 (Neurog1/2), achaete-scute complex-like 1 (Ascl1), oligodendrocyte transcription factor 1 (Olig1), oligodendrocyte lineage transcription factor 2 (Olig2), hairy and enhancer of split 1/5 (Hes1/5) and delta-like 1 (Dll1), was analysed by real-time RT-PCR. Proliferation index and neuronal specification in the forebrain of embryos at embryonic day 11.5 were examined histologically. High glucose decreased the proliferation of NSCs and differentiated cells. The incidence of apoptosis was increased in NSCs treated with high glucose, but not in the differentiated cells. High glucose also accelerated neuronal and glial differentiation from NSCs. The decreased proliferation index and early differentiation of neurons were evident in the telencephalon of embryos derived from diabetic mice. Exposure to high glucose altered the mRNA expression levels of Shh, Bmp4, Neurog1/2, Ascl1, Hes1, Dll1 and Olig1 in NSCs and Shh, Dll1, Neurog1/2 and Hes5 in differentiated cells. The changes in proliferation and differentiation of NSCs exposed to high glucose are associated with altered expression of genes that are involved in cell-cycle progression and cell-fate specification during neurulation. These changes may form the basis for the defective neural tube patterning observed in embryos of diabetic pregnancies.

The Hedgehog Signal Induced Modulation of Bone Morphogenetic Protein Signaling: An Essential Signaling Relay for Urinary Tract Morphogenesis

PubMed Central

Nakagata, Naomi; Miyagawa, Shinichi; Suzuki, Kentaro; Kitazawa, Sohei; Yamada, Gen

2012-01-01

Background Congenital diseases of the urinary tract are frequently observed in infants. Such diseases present a number of developmental anomalies such as hydroureter and hydronephrosis. Although some genetically-modified mouse models of growth factor signaling genes reproduce urinary phenotypes, the pathogenic mechanisms remain obscure. Previous studies suggest that a portion of the cells in the external genitalia and bladder are derived from peri-cloacal mesenchymal cells that receive Hedgehog (Hh) signaling in the early developmental stages. We hypothesized that defects in such progenitor cells, which give rise to urinary tract tissues, may be a cause of such diseases. Methodology/Principal Findings To elucidate the pathogenic mechanisms of upper urinary tract malformations, we analyzed a series of Sonic hedgehog (Shh) deficient mice. Shh−/− displayed hydroureter and hydronephrosis phenotypes and reduced expression of several developmental markers. In addition, we suggested that Shh modulation at an early embryonic stage is responsible for such phenotypes by analyzing the Shh conditional mutants. Tissue contribution assays of Hh-responsive cells revealed that peri-cloacal mesenchymal cells, which received Hh signal secreted from cloacal epithelium, could contribute to the ureteral mesenchyme. Gain- and loss-of-functional mutants for Hh signaling revealed a correlation between Hh signaling and Bone morphogenetic protein (Bmp) signaling. Finally, a conditional ablation of Bmp receptor type IA (BmprIA) gene was examined in Hh-responsive cell lineages. This system thus made it possible to analyze the primary functions of the growth factor signaling relay. The defective Hh-to-Bmp signaling relay resulted in severe urinary tract phenotypes with a decrease in the number of Hh-responsive cells. Conclusions/Significance This study identified the essential embryonic stages for the pathogenesis of urinary tract phenotypes. These results suggested that Hh-responsive mesenchymal Bmp signaling maintains the population of peri-cloacal mesenchyme cells, which is essential for the development of the ureter and the upper urinary tract. PMID:22860096

OTX2 exhibits cell-context-dependent effects on cellular and molecular properties of human embryonic neural precursors and medulloblastoma cells

PubMed Central

Kaur, Ravinder; Aiken, Christopher; Morrison, Ludivine Coudière; Rao, Radhika; Del Bigio, Marc R.; Rampalli, Shravanti; Werbowetski-Ogilvie, Tamra

2015-01-01

ABSTRACT Medulloblastoma (MB) is the most common malignant primary pediatric brain tumor and is currently divided into four subtypes based on different genomic alterations, gene expression profiles and response to treatment: WNT, Sonic Hedgehog (SHH), Group 3 and Group 4. This extensive heterogeneity has made it difficult to assess the functional relevance of genes to malignant progression. For example, expression of the transcription factor Orthodenticle homeobox2 (OTX2) is frequently dysregulated in multiple MB variants; however, its role may be subtype specific. We recently demonstrated that neural precursors derived from transformed human embryonic stem cells (trans-hENs), but not their normal counterparts (hENs), resemble Groups 3 and 4 MB in vitro and in vivo. Here, we tested the utility of this model system as a means of dissecting the role of OTX2 in MB using gain- and loss-of-function studies in hENs and trans-hENs, respectively. Parallel experiments with MB cells revealed that OTX2 exerts inhibitory effects on hEN and SHH MB cells by regulating growth, self-renewal and migration in vitro and tumor growth in vivo. This was accompanied by decreased expression of pluripotent genes, such as SOX2, and was supported by overexpression of SOX2 in OTX2+ SHH MB and hENs that resulted in significant rescue of self-renewal and cell migration. By contrast, OTX2 is oncogenic and promotes self-renewal of trans-hENs and Groups 3 and 4 MB independent of pluripotent gene expression. Our results demonstrate a novel role for OTX2 in self-renewal and migration of hENs and MB cells and reveal a cell-context-dependent link between OTX2 and pluripotent genes. Our study underscores the value of human embryonic stem cell derivatives as alternatives to cell lines and heterogeneous patient samples for investigating the contribution of key developmental regulators to MB progression. PMID:26398939

A SHH-FOXF1-BMP4 signaling axis regulating growth and differentiation of epithelial and mesenchymal tissues in ureter development.

PubMed

Bohnenpoll, Tobias; Wittern, Anna B; Mamo, Tamrat M; Weiss, Anna-Carina; Rudat, Carsten; Kleppa, Marc-Jens; Schuster-Gossler, Karin; Wojahn, Irina; Lüdtke, Timo H-W; Trowe, Mark-Oliver; Kispert, Andreas

2017-08-01

The differentiated cell types of the epithelial and mesenchymal tissue compartments of the mature ureter of the mouse arise in a precise temporal and spatial sequence from uncommitted precursor cells of the distal ureteric bud epithelium and its surrounding mesenchyme. Previous genetic efforts identified a member of the Hedgehog (HH) family of secreted proteins, Sonic hedgehog (SHH) as a crucial epithelial signal for growth and differentiation of the ureteric mesenchyme. Here, we used conditional loss- and gain-of-function experiments of the unique HH signal transducer Smoothened (SMO) to further characterize the cellular functions and unravel the effector genes of HH signaling in ureter development. We showed that HH signaling is not only required for proliferation and SMC differentiation of cells of the inner mesenchymal region but also for survival of cells of the outer mesenchymal region, and for epithelial proliferation and differentiation. We identified the Forkhead transcription factor gene Foxf1 as a target of HH signaling in the ureteric mesenchyme. Expression of a repressor version of FOXF1 in this tissue completely recapitulated the mesenchymal and epithelial proliferation and differentiation defects associated with loss of HH signaling while re-expression of a wildtype version of FOXF1 in the inner mesenchymal layer restored these cellular programs when HH signaling was inhibited. We further showed that expression of Bmp4 in the ureteric mesenchyme depends on HH signaling and Foxf1, and that exogenous BMP4 rescued cell proliferation and epithelial differentiation in ureters with abrogated HH signaling or FOXF1 function. We conclude that SHH uses a FOXF1-BMP4 module to coordinate the cellular programs for ureter elongation and differentiation, and suggest that deregulation of this signaling axis occurs in human congenital anomalies of the kidney and urinary tract (CAKUT).

BarTeL, a Genetically Versatile, Bioluminescent and Granule Neuron Precursor-Targeted Mouse Model for Medulloblastoma

PubMed Central

Mahdi, Min Y.; Gonzalez-Gomez, Ignacio; Asgharzadeh, Shahab; D’Apuzzo, Massimo; Erdreich-Epstein, Anat; Moats, Rex A.

2016-01-01

Medulloblastomas are the most common malignant pediatric brain tumor and have been divided into four major molecular subgroups. Animal models that mimic the principal molecular aberrations of these subgroups will be important tools for preclinical studies and allow greater understanding of medulloblastoma biology. We report a new transgenic model of medulloblastoma that possesses a unique combination of desirable characteristics including, among others, the ability to incorporate multiple and variable genes of choice and to produce bioluminescent tumors from a limited number of somatic cells within a normal cellular environment. This model, termed BarTeL, utilizes a Barhl1 homeobox gene promoter to target expression of a bicistronic transgene encoding both the avian retroviral receptor TVA and an eGFP-Luciferase fusion protein to neonatal cerebellar granule neuron precursor (cGNP) cells, which are cells of origin for the sonic hedgehog (SHH) subgroup of human medulloblastomas. The Barhl1 promoter-driven transgene is expressed strongly in mammalian cGNPs and weakly or not at all in mature granule neurons. We efficiently induced bioluminescent medulloblastomas expressing eGFP-luciferase in BarTeL mice by infection of a limited number of somatic cGNPs with avian retroviral vectors encoding the active N-terminal fragment of SHH and a stabilized MYCN mutant. Detection and quantification of the increasing bioluminescence of growing tumors in young BarTeL mice was facilitated by the declining bioluminescence of their uninfected maturing cGNPs. Inclusion of eGFP in the transgene allowed enriched sorting of cGNPs from neonatal cerebella. Use of a single bicistronic avian vector simultaneously expressing both Shh and Mycn oncogenes increased the medulloblastoma incidence and aggressiveness compared to mixed virus infections. Bioluminescent tumors could also be produced by ex vivo transduction of neonatal BarTeL cerebellar cells by avian retroviruses and subsequent implantation into nontransgenic cerebella. Thus, BarTeL mice provide a versatile model with opportunities for use in medulloblastoma biology and therapeutics. PMID:27310018

SHH-dependent knockout of HIF-1 alpha accelerates the degenerative process in mouse intervertebral disc.

PubMed

Wu, W J; Zhang, X K; Zheng, X F; Yang, Y H; Jiang, S D; Jiang, L S

2013-01-01

Hypoxia-inducible factor-1alpha (HIF-1 alpha) has been reported to have an important role in the metabolism and synthesis of extracellular matrix of the nucleus pulposus cells (NPCs) and was assumed to be involved in the process of intervertebral disc degeneration. The objective of this study was to investigate the role of HIF-1alpha in disc degeneration in vivo using a conditional HIF-1alpha knockout (KO) mouse model. ShhCre transgenic mice were mated with HIF-1 alpha fl/fl mice to generate conditional HIF-1alpha KO mice (HIF-1alpha fl/fl-ShhCre+). Three mice of each genotype (Wide-type and HIF-1alpha KO) at the age of 3 days, 6, and 12 weeks were sacrificed after genotyping. Five lumbar disc samples were harvested from each mouse, with a total of 45 disc samples for each genotype. In situ hybridization and immunohistochemical analysis were used to check the efficacy of HIF-1alpha knockout. Histological grading of the disc degeneration was performed according to the classification system proposed by Boos et al. Picro-sirius red staining, Safranine O/fast green staining and immunohistochemical study were used to evaluate the expression of aggrecan, type-II collagen and vascular endothelial growth factor (VEGF). Histologic analysis revealed more NPC deaths and signs of degeneration in HIF-1alpha KO mice and the degeneration scores of HIF-1alpha KO mice were significantly higher than those of the Wide-type mice at the age of 6 weeks and 12 weeks. There were less expressions of aggrecan, type-II collagen and VEGF in the intervertebral discs of HIF1-alpha KO mice than in those of wild-type mice. Taken together, the results of our study indicated that HIF-1alpha is a pivotal contributor to NPC survival and the homeotasis of extracellular matrix through the HIF-1alpha/VEGF signaling pathway, and plays an important role in the development of disc degeneration.

Distinct Effects of the mesenchymal dysplasia Gene Variant of Murine Patched-1 Protein on Canonical and Non-canonical Hedgehog Signaling Pathways*

PubMed Central

Harvey, Malcolm C.; Fleet, Andrew; Okolowsky, Nadia; Hamel, Paul A.

2014-01-01

Hedgehog (Hh) signaling requires regulation of the receptor Patched-1 (Ptch1), which, in turn, regulates Smoothened activity (canonical Hh signaling) as well as other non-canonical signaling pathways. The mutant Ptch1 allele mesenchymal dysplasia (mes), which truncates the Ptch1 C terminus, produces a limited spectrum of developmental defects in mice as well as deregulation of canonical Hh signaling in some, but not all, affected tissues. Paradoxically, mes suppresses canonical Hh signaling and binds to Hh ligands with an affinity similar to wild-type mouse Ptch1 (mPtch1). We characterized the distinct activities of the mes variant of mPtch1 mediating Hh signaling through both canonical and non-canonical pathways. We demonstrated that mPtch1 bound c-src in an Hh-regulated manner. Stimulation with Sonic Hedgehog (Shh) of primary mammary mesenchymal cells from wild-type and mes animals activated Erk1/2. Although Shh activated c-src in wild-type cells, c-src was constitutively activated in mes mesenchymal cells. Transient assays showed that wild-type mPtch1, mes, or mPtch1 lacking the C terminus repressed Hh signaling in Ptch1-deficient mouse embryo fibroblasts and that repression was reversed by Shh, revealing that the C terminus was dispensable for mPtch1-dependent regulation of canonical Hh signaling. In contrast to these transient assays, constitutively high levels of mGli1 but not mPtch1 were present in primary mammary mesenchymal cells from mes mice, whereas the expression of mPtch1 was similarly induced in both mes and wild-type cells. These data define a novel signal transduction pathway involving c-src that is activated by the Hh ligands and reveals the requirement for the C terminus of Ptch in regulation of canonical and non-canonical Hh signaling pathways. PMID:24570001

A Prospective Sequential Analysis of the Relation between Physical Aggression and Peer Rejection Acts in a High-Risk Preschool Sample

ERIC Educational Resources Information Center

Chen, Chin-Chih; McComas, Jennifer J.; Hartman, Ellie; Symons, Frank J.

2011-01-01

Research Findings: In early childhood education, the social ecology of the child is considered critical for healthy behavioral development. There is, however, relatively little information based on directly observing what children do that describes the moment-by-moment (i.e., sequential) relation between physical aggression and peer rejection acts…

Article and method of forming an article

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

Lacy, Benjamin Paul; Kottilingam, Srikanth Chandrudu; Dutta, Sandip

Provided are an article and a method of forming an article. The method includes providing a metallic powder, heating the metallic powder to a temperature sufficient to joint at least a portion of the metallic powder to form an initial layer, sequentially forming additional layers in a build direction by providing a distributed layer of the metallic powder over the initial layer and heating the distributed layer of the metallic powder, repeating the steps of sequentially forming the additional layers in the build direction to form a portion of the article having a hollow space formed in the build direction,more » and forming an overhang feature extending into the hollow space. The article includes an article formed by the method described herein.« less

Sequential accelerated tests: Improving the correlation of accelerated tests to module performance in the field

NASA Astrophysics Data System (ADS)

Felder, Thomas; Gambogi, William; Stika, Katherine; Yu, Bao-Ling; Bradley, Alex; Hu, Hongjie; Garreau-Iles, Lucie; Trout, T. John

2016-09-01

DuPont has been working steadily to develop accelerated backsheet tests that correlate with solar panels observations in the field. This report updates efforts in sequential testing. Single exposure tests are more commonly used and can be completed more quickly, and certain tests provide helpful predictions of certain backsheet failure modes. DuPont recommendations for single exposure tests are based on 25-year exposure levels for UV and humidity/temperature, and form a good basis for sequential test development. We recommend a sequential exposure of damp heat followed by UV then repetitions of thermal cycling and UVA. This sequence preserves 25-year exposure levels for humidity/temperature and UV, and correlates well with a large body of field observations. Measurements can be taken at intervals in the test, although the full test runs 10 months. A second, shorter sequential test based on damp heat and thermal cycling tests mechanical durability and correlates with loss of mechanical properties seen in the field. Ongoing work is directed toward shorter sequential tests that preserve good correlation to field data.

The Role of the Sonic Hedgehog Pathway for Prostate Cancer Progression

DTIC Science & Technology

2005-02-01

GY, Qi YP, Gysin S, Fernandez-Del Castillo C, Yalnik V, Antoniu and real-time PCR analyses. Sumnin Chi contributed to B, McMahon M, Warshaw AL, Hebrok... Romer JT, Kimura H, Magdaleno Set al:. 391(6662), 90-92 (1998). in a mouse model of prostate carcinogenesis. Suppression of the Shh pathway using a 10󈧘

Preclinical Studies of Induced Pluripotent Stem Cell-Derived Astrocyte Transplantation in ALS

DTIC Science & Technology

2013-10-01

followed by caudalization and ventralization using retinoic acid and sonic hedgehog , respectively (Fig. 1A). By day 15 of differentiation, neural...μg/ml, Sigma-Aldrich), were added. At day 7, rhBDNF (10 ng/ml, R&D) and sonic hedgehog (SHH-C, 200 ng/ml, Invitrogen) were added. At day 10

Comparative efficacy of simultaneous versus sequential multiple health behavior change interventions among adults: A systematic review of randomised trials.

PubMed

James, Erica; Freund, Megan; Booth, Angela; Duncan, Mitch J; Johnson, Natalie; Short, Camille E; Wolfenden, Luke; Stacey, Fiona G; Kay-Lambkin, Frances; Vandelanotte, Corneel

2016-08-01

Growing evidence points to the benefits of addressing multiple health behaviors rather than single behaviors. This review evaluates the relative effectiveness of simultaneous and sequentially delivered multiple health behavior change (MHBC) interventions. Secondary aims were to identify: a) the most effective spacing of sequentially delivered components; b) differences in efficacy of MHBC interventions for adoption/cessation behaviors and lifestyle/addictive behaviors, and; c) differences in trial retention between simultaneously and sequentially delivered interventions. MHBC intervention trials published up to October 2015 were identified through a systematic search. Eligible trials were randomised controlled trials that directly compared simultaneous and sequential delivery of a MHBC intervention. A narrative synthesis was undertaken. Six trials met the inclusion criteria and across these trials the behaviors targeted were smoking, diet, physical activity, and alcohol consumption. Three trials reported a difference in intervention effect between a sequential and simultaneous approach in at least one behavioral outcome. Of these, two trials favoured a sequential approach on smoking. One trial favoured a simultaneous approach on fat intake. There was no difference in retention between sequential and simultaneous approaches. There is limited evidence regarding the relative effectiveness of sequential and simultaneous approaches. Given only three of the six trials observed a difference in intervention effectiveness for one health behavior outcome, and the relatively consistent finding that the sequential and simultaneous approaches were more effective than a usual/minimal care control condition, it appears that both approaches should be considered equally efficacious. PROSPERO registration number: CRD42015027876. Copyright © 2016 Elsevier Inc. All rights reserved.

Dissociating hippocampal and striatal contributions to sequential prediction learning

PubMed Central

Bornstein, Aaron M.; Daw, Nathaniel D.

2011-01-01

Behavior may be generated on the basis of many different kinds of learned contingencies. For instance, responses could be guided by the direct association between a stimulus and response, or by sequential stimulus-stimulus relationships (as in model-based reinforcement learning or goal-directed actions). However, the neural architecture underlying sequential predictive learning is not well-understood, in part because it is difficult to isolate its effect on choice behavior. To track such learning more directly, we examined reaction times (RTs) in a probabilistic sequential picture identification task. We used computational learning models to isolate trial-by-trial effects of two distinct learning processes in behavior, and used these as signatures to analyze the separate neural substrates of each process. RTs were best explained via the combination of two delta rule learning processes with different learning rates. To examine neural manifestations of these learning processes, we used functional magnetic resonance imaging to seek correlates of timeseries related to expectancy or surprise. We observed such correlates in two regions, hippocampus and striatum. By estimating the learning rates best explaining each signal, we verified that they were uniquely associated with one of the two distinct processes identified behaviorally. These differential correlates suggest that complementary anticipatory functions drive each region's effect on behavior. Our results provide novel insights as to the quantitative computational distinctions between medial temporal and basal ganglia learning networks and enable experiments that exploit trial-by-trial measurement of the unique contributions of both hippocampus and striatum to response behavior. PMID:22487032

Undermining plastic surgery as a possible option for treating basal cell carcinoma of the forehead.

PubMed

Tchernev, Georgi; Pidakev, Ivan; Lozev, Ilia; Lotti, Torello; Cardoso, Jose Carlos; Patterson, James W

2017-04-01

Basal cell carcinoma (BCC) is the most common cutaneous cancer. Although most cases can be cured with simple surgical procedures and are associated with a good prognosis, a minority of BCCs may pose significant therapeutic challenges. This occurs mostly in cases of so-called advanced BCC, which a loosely defined term that encompasses locally advanced lesions and tumors with metastatic spread. Treatment of these cases is often complex and sometimes may need combinations of therapeutic modalities, including surgery, radiotherapy and/or targeted therapy directed towards sonic hedgehog (SHH) signaling pathways, such as vismodegib. We herein present the case of a 74-year-old man presenting with a large basal cell of the forehead evolving for more than 7 years. The patient underwent excision of the lesion with clear surgical margins. Reconstruction of the defect was performed after extensive undermining of the skin allowing subsequent direct closure with a simple suture, which resulted in an acceptable cosmetic outcome. We discuss the potential advantages, disadvantages, and applicability of this relatively simple surgical maneuver in the reconstruction of defects resulting from excision of considerably large cutaneous tumors.

Enantioselective Cobalt-Catalyzed Sequential Nazarov Cyclization/Electrophilic Fluorination: Access to Chiral α-Fluorocyclopentenones.

PubMed

Zhang, Heyi; Cheng, Biao; Lu, Zhan

2018-06-20

A newly designed thiazoline iminopyridine ligand for enantioselective cobalt-catalyzed sequential Nazarov cyclization/electrophilic fluorination was developed. Various chiral α-fluorocyclopentenones were prepared with good yields and diastereo- and enantioselectivities. Further derivatizations could be easily carried out to provide chiral cyclopentenols with three contiguous stereocenters. Furthermore, a direct deesterification of fluorinated products could afford chiral α-single fluorine-substituted cyclopentenones.

Decay modes of the Hoyle state in 12C

NASA Astrophysics Data System (ADS)

Zheng, H.; Bonasera, A.; Huang, M.; Zhang, S.

2018-04-01

Recent experimental results give an upper limit less than 0.043% (95% C.L.) to the direct decay of the Hoyle state into 3α respect to the sequential decay into 8Be + α. We performed one and two-dimensional tunneling calculations to estimate such a ratio and found it to be more than one order of magnitude smaller than experiment depending on the range of the nuclear force. This is within high statistics experimental capabilities. Our results can also be tested by measuring the decay modes of high excitation energy states of 12C where the ratio of direct to sequential decay might reach 10% at E*(12C) = 10.3 MeV. The link between a Bose Einstein Condensate (BEC) and the direct decay of the Hoyle state is also addressed. We discuss a hypothetical 'Efimov state' at E*(12C) = 7.458 MeV, which would mainly sequentially decay with 3α of equal energies: a counterintuitive result of tunneling. Such a state, if it would exist, is at least 8 orders of magnitude less probable than the Hoyle's, thus below the sensitivity of recent and past experiments.

Chemical Suppression of the Reactivated Androgen Signaling Pathway in Androgen-Independent Prostate Cancer

DTIC Science & Technology

2011-07-01

when it is ingested during pregnancy [20,21]. Aside from its role in development, Hh signaling also supports stem cells in adult tissues [22-24]. However...For the mo.~t commonly uti - lized human prostate cancer cell lines (LNCaP and derivatives, DUI45, PC3 or CWR22rvl) grown in culture, Shh, Glil/2 and

Computational Modeling and Simulation of Genital Tubercle ...

EPA Pesticide Factsheets

Hypospadias is a developmental defect of urethral tube closure that has a complex etiology. Here, we describe a multicellular agent-based model of genital tubercle development that simulates urethrogenesis from the urethral plate stage to urethral tube closure in differentiating male embryos. The model, constructed in CompuCell3D, implemented spatially dynamic signals from SHH, FGF10, and androgen signaling pathways. These signals modulated stochastic cell behaviors, such as differential adhesion, cell motility, proliferation, and apoptosis. Urethral tube closure was an emergent property of the model that was quantitatively dependent on SHH and FGF10 induced effects on mesenchymal proliferation and endodermal apoptosis, ultimately linked to androgen signaling. In the absence of androgenization, simulated genital tubercle development defaulted to the female condition. Intermediate phenotypes associated with partial androgen deficiency resulted in incomplete closure. Using this computer model, complex relationships between urethral tube closure defects and disruption of underlying signaling pathways could be probed theoretically in multiplex disturbance scenarios and modeled into probabilistic predictions for individual risk for hypospadias and potentially other developmental defects of the male genital tubercle. We identify the minimal molecular network that determines the outcome of male genital tubercle development in mice.

Low-intensity pulsed ultrasound stimulation promotes osteoblast differentiation through hedgehog signaling.

PubMed

Matsumoto, Kenichi; Shimo, Tsuyoshi; Kurio, Naito; Okui, Tatsuo; Ibaragi, Soichiro; Kunisada, Yuki; Obata, Kyoichi; Masui, Masanori; Pai, Pang; Horikiri, Yuu; Yamanaka, Nobuyuki; Takigawa, Masaharu; Sasaki, Akira

2018-06-01

Low-intensity pulsed ultrasound (LIPUS) has been used as an adjunct to fracture healing therapies, but the mechanisms underlying its action are not known. We reported that sonic hedgehog (SHH) signaling was activated in osteoblasts at the dynamic remodeling site of a bone fracture. Mechanical stimulation is a crucial factor in bone remodeling, and it is related to the primary cilia as a sensor of hedgehog signaling. Here we observed that LIPUS promoted callus formation in accord with Gli2-positive cells after 14 days at the mouse femur fractured site compared with a control group. An immunofluorescence analysis showed that the numbers of primary cilia and cilia/osterix double-positive osteoblasts were increased at the fracture site by LIPUS. LIPUS stimulated not only the number and the length of primary cilia, but also the levels of ciliated protein, Ift88 mRNA, and SHH, Gli1, and Gli2 in MC3T3-E1 cells. Further experiments revealed that LIPUS stimulated osteogenic differentiation in the presence of smoothened agonist (SAG) treatment. These results indicate that LIPUS stimulates osteogenic differentiation and the maturation of osteoblasts by a primary cilium-mediated activation of hedgehog signaling. © 2017 Wiley Periodicals, Inc.

Computational Modeling and Simulation of Genital Tubercle Development

EPA Pesticide Factsheets

Hypospadias is a developmental defect of urethral tube closure that has a complex etiology involving genetic and environmental factors, including anti-androgenic and estrogenic disrupting chemicals; however, little is known about the morphoregulatory consequences of androgen/estrogen balance during genital tubercle (GT) development. Computer models that predictively model sexual dimorphism of the GT may provide a useful resource to translate chemical-target bipartite networks and their developmental consequences across the human-relevant chemical universe. Here, we describe a multicellular agent-based model of genital tubercle (GT) development that simulates urethrogenesis from the sexually-indifferent urethral plate stage to urethral tube closure. The prototype model, constructed in CompuCell3D, recapitulates key aspects of GT morphogenesis controlled by SHH, FGF10, and androgen pathways through modulation of stochastic cell behaviors, including differential adhesion, motility, proliferation, and apoptosis. Proper urethral tube closure in the model was shown to depend quantitatively on SHH- and FGF10-induced effects on mesenchymal proliferation and epithelial apoptosis??both ultimately linked to androgen signaling. In the absence of androgen, GT development was feminized and with partial androgen deficiency, the model resolved with incomplete urethral tube closure, thereby providing an in silico platform for probabilistic prediction of hypospadias risk across c

High expression of DNA methyltransferases in primary human medulloblastoma.

PubMed

Pócza, T; Krenács, T; Turányi, E; Csáthy, J; Jakab, Z; Hauser, P

2016-01-01

Epigenetic alterations have been implicated in cancer development. DNA methylation modulates gene expression, which is catalyzed by DNA methyltransferases (DNMTs). The objective of our study was to evaluate expression of DNMTs in medulloblastoma and analyze its correlation with clinical features. Nuclear expression of DNMT1, DNMT3A and DNMT3B was analyzed in human primary medulloblastoma of 44 patients using immunohistochemistry. Correlation of expression of DNMT levels with classical histological subtypes, novel molecular subgroups and survival of patients was analyzed. Elevated expression of DNMT1, DNMT3A and DNMT3B was observed in 63.64%, 68.18% and 72.73% of all cases, respectively. None of them showed a correlation with classical histology or survival. Concerning molecular subtypes, significantly higher expression of DNMT1 was observed in the SHH group compared to non-SHH samples (p = 0.02), but without significant difference in DNMT3A or DNMT3B levels between any subtypes. In conclusion, DNMT1, DNMT3A and DNMT3B are highly expressed in human medulloblastoma samples, suggesting that promoter hypermethylation may play a role in medulloblastoma development. Demethylation of tumor suppressor gene promoters may be considered as a possible future target in therapy of medulloblastoma.

Aspm sustains postnatal cerebellar neurogenesis and medulloblastoma growth in mice

PubMed Central

Williams, Scott E.; Garcia, Idoia; Crowther, Andrew J.; Li, Shiyi; Stewart, Alyssa; Liu, Hedi; Lough, Kendall J.; O'Neill, Sean; Veleta, Katherine; Oyarzabal, Esteban A.; Merrill, Joseph R.; Shih, Yen-Yu Ian; Gershon, Timothy R.

2015-01-01

Alterations in genes that regulate brain size may contribute to both microcephaly and brain tumor formation. Here, we report that Aspm, a gene that is mutated in familial microcephaly, regulates postnatal neurogenesis in the cerebellum and supports the growth of medulloblastoma, the most common malignant pediatric brain tumor. Cerebellar granule neuron progenitors (CGNPs) express Aspm when maintained in a proliferative state by sonic hedgehog (Shh) signaling, and Aspm is expressed in Shh-driven medulloblastoma in mice. Genetic deletion of Aspm reduces cerebellar growth, while paradoxically increasing the mitotic rate of CGNPs. Aspm-deficient CGNPs show impaired mitotic progression, altered patterns of division orientation and differentiation, and increased DNA damage, which causes progenitor attrition through apoptosis. Deletion of Aspm in mice with Smo-induced medulloblastoma reduces tumor growth and increases DNA damage. Co-deletion of Aspm and either of the apoptosis regulators Bax or Trp53 (also known as p53) rescues the survival of neural progenitors and reduces the growth restriction imposed by Aspm deletion. Our data show that Aspm functions to regulate mitosis and to mitigate DNA damage during CGNP cell division, causes microcephaly through progenitor apoptosis when mutated, and sustains tumor growth in medulloblastoma. PMID:26450969

Heterochrony in the regulation of the developing marsupial limb.

PubMed

Chew, Keng Yih; Shaw, Geoffrey; Yu, Hongshi; Pask, Andrew J; Renfree, Marilyn B

2014-02-01

At birth, marsupial neonates have precociously developed forelimbs. The development of the tammar wallaby (Macropus eugenii) hindlimbs lags significantly behind that of the forelimbs. This differs from the grey short-tailed opossum, Monodelphis domestica, which has relatively similar fore- and hindlimbs at birth. This study examines the expression of the key patterning genes TBX4, TBX5, PITX1, FGF8, and SHH in developing limb buds in the tammar wallaby. All genes examined were highly conserved with orthologues from opossum and mouse. TBX4 expression appeared earlier in development than in the mouse, but later than in the opossum. SHH expression is restricted to the zone of polarising activity, while TBX5 (forelimb) and PITX1 (hindlimb) showed diffuse mRNA expression. FGF8 is specifically localised to the apical ectodermal ridge, which is more prominent than in the opossum. The most marked divergence in limb size in marsupials occurs in the kangaroos and wallabies. The faster development of the fore limb compared to that of the hind limb correlates with the early timing of the expression of the key patterning genes in these limbs. Copyright © 2013 Wiley Periodicals, Inc.

Learning to Monitor Machine Health with Convolutional Bi-Directional LSTM Networks

PubMed Central

Zhao, Rui; Yan, Ruqiang; Wang, Jinjiang; Mao, Kezhi

2017-01-01

In modern manufacturing systems and industries, more and more research efforts have been made in developing effective machine health monitoring systems. Among various machine health monitoring approaches, data-driven methods are gaining in popularity due to the development of advanced sensing and data analytic techniques. However, considering the noise, varying length and irregular sampling behind sensory data, this kind of sequential data cannot be fed into classification and regression models directly. Therefore, previous work focuses on feature extraction/fusion methods requiring expensive human labor and high quality expert knowledge. With the development of deep learning methods in the last few years, which redefine representation learning from raw data, a deep neural network structure named Convolutional Bi-directional Long Short-Term Memory networks (CBLSTM) has been designed here to address raw sensory data. CBLSTM firstly uses CNN to extract local features that are robust and informative from the sequential input. Then, bi-directional LSTM is introduced to encode temporal information. Long Short-Term Memory networks (LSTMs) are able to capture long-term dependencies and model sequential data, and the bi-directional structure enables the capture of past and future contexts. Stacked, fully-connected layers and the linear regression layer are built on top of bi-directional LSTMs to predict the target value. Here, a real-life tool wear test is introduced, and our proposed CBLSTM is able to predict the actual tool wear based on raw sensory data. The experimental results have shown that our model is able to outperform several state-of-the-art baseline methods. PMID:28146106

Learning to Monitor Machine Health with Convolutional Bi-Directional LSTM Networks.

PubMed

Zhao, Rui; Yan, Ruqiang; Wang, Jinjiang; Mao, Kezhi

2017-01-30

In modern manufacturing systems and industries, more and more research efforts have been made in developing effective machine health monitoring systems. Among various machine health monitoring approaches, data-driven methods are gaining in popularity due to the development of advanced sensing and data analytic techniques. However, considering the noise, varying length and irregular sampling behind sensory data, this kind of sequential data cannot be fed into classification and regression models directly. Therefore, previous work focuses on feature extraction/fusion methods requiring expensive human labor and high quality expert knowledge. With the development of deep learning methods in the last few years, which redefine representation learning from raw data, a deep neural network structure named Convolutional Bi-directional Long Short-Term Memory networks (CBLSTM) has been designed here to address raw sensory data. CBLSTM firstly uses CNN to extract local features that are robust and informative from the sequential input. Then, bi-directional LSTM is introduced to encode temporal information. Long Short-Term Memory networks(LSTMs) are able to capture long-term dependencies and model sequential data, and the bi-directional structure enables the capture of past and future contexts. Stacked, fully-connected layers and the linear regression layer are built on top of bi-directional LSTMs to predict the target value. Here, a real-life tool wear test is introduced, and our proposed CBLSTM is able to predict the actual tool wear based on raw sensory data. The experimental results have shown that our model is able to outperform several state-of-the-art baseline methods.

Effects of sequential and discrete rapid naming on reading in Japanese children with reading difficulty.

PubMed

Wakamiya, Eiji; Okumura, Tomohito; Nakanishi, Makoto; Takeshita, Takashi; Mizuta, Mekumi; Kurimoto, Naoko; Tamai, Hiroshi

2011-06-01

To clarify whether rapid naming ability itself is a main underpinning factor of rapid automatized naming tests (RAN) and how deep an influence the discrete decoding process has on reading, we performed discrete naming tasks and discrete hiragana reading tasks as well as sequential naming tasks and sequential hiragana reading tasks with 38 Japanese schoolchildren with reading difficulty. There were high correlations between both discrete and sequential hiragana reading and sentence reading, suggesting that some mechanism which automatizes hiragana reading makes sentence reading fluent. In object and color tasks, there were moderate correlations between sentence reading and sequential naming, and between sequential naming and discrete naming. But no correlation was found between reading tasks and discrete naming tasks. The influence of rapid naming ability of objects and colors upon reading seemed relatively small, and multi-item processing may work in relation to these. In contrast, in the digit naming task there was moderate correlation between sentence reading and discrete naming, while no correlation was seen between sequential naming and discrete naming. There was moderate correlation between reading tasks and sequential digit naming tasks. Digit rapid naming ability has more direct effect on reading while its effect on RAN is relatively limited. The ratio of how rapid naming ability influences RAN and reading seems to vary according to kind of the stimuli used. An assumption about components in RAN which influence reading is discussed in the context of both sequential processing and discrete naming speed. Copyright © 2010 The Japanese Society of Child Neurology. Published by Elsevier B.V. All rights reserved.

Disentangling beat perception from sequential learning and examining the influence of attention and musical abilities on ERP responses to rhythm.

PubMed

Bouwer, Fleur L; Werner, Carola M; Knetemann, Myrthe; Honing, Henkjan

2016-05-01

Beat perception is the ability to perceive temporal regularity in musical rhythm. When a beat is perceived, predictions about upcoming events can be generated. These predictions can influence processing of subsequent rhythmic events. However, statistical learning of the order of sounds in a sequence can also affect processing of rhythmic events and must be differentiated from beat perception. In the current study, using EEG, we examined the effects of attention and musical abilities on beat perception. To ensure we measured beat perception and not absolute perception of temporal intervals, we used alternating loud and soft tones to create a rhythm with two hierarchical metrical levels. To control for sequential learning of the order of the different sounds, we used temporally regular (isochronous) and jittered rhythmic sequences. The order of sounds was identical in both conditions, but only the regular condition allowed for the perception of a beat. Unexpected intensity decrements were introduced on the beat and offbeat. In the regular condition, both beat perception and sequential learning were expected to enhance detection of these deviants on the beat. In the jittered condition, only sequential learning was expected to affect processing of the deviants. ERP responses to deviants were larger on the beat than offbeat in both conditions. Importantly, this difference was larger in the regular condition than in the jittered condition, suggesting that beat perception influenced responses to rhythmic events in addition to sequential learning. The influence of beat perception was present both with and without attention directed at the rhythm. Moreover, beat perception as measured with ERPs correlated with musical abilities, but only when attention was directed at the stimuli. Our study shows that beat perception is possible when attention is not directed at a rhythm. In addition, our results suggest that attention may mediate the influence of musical abilities on beat perception. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

Probing finite coarse-grained virtual Feynman histories with sequential weak values

NASA Astrophysics Data System (ADS)

Georgiev, Danko; Cohen, Eliahu

2018-05-01

Feynman's sum-over-histories formulation of quantum mechanics has been considered a useful calculational tool in which virtual Feynman histories entering into a coherent quantum superposition cannot be individually measured. Here we show that sequential weak values, inferred by consecutive weak measurements of projectors, allow direct experimental probing of individual virtual Feynman histories, thereby revealing the exact nature of quantum interference of coherently superposed histories. Because the total sum of sequential weak values of multitime projection operators for a complete set of orthogonal quantum histories is unity, complete sets of weak values could be interpreted in agreement with the standard quantum mechanical picture. We also elucidate the relationship between sequential weak values of quantum histories with different coarse graining in time and establish the incompatibility of weak values for nonorthogonal quantum histories in history Hilbert space. Bridging theory and experiment, the presented results may enhance our understanding of both weak values and quantum histories.

An adaptive two-stage sequential design for sampling rare and clustered populations

USGS Publications Warehouse

Brown, J.A.; Salehi, M.M.; Moradi, M.; Bell, G.; Smith, D.R.

2008-01-01

How to design an efficient large-area survey continues to be an interesting question for ecologists. In sampling large areas, as is common in environmental studies, adaptive sampling can be efficient because it ensures survey effort is targeted to subareas of high interest. In two-stage sampling, higher density primary sample units are usually of more interest than lower density primary units when populations are rare and clustered. Two-stage sequential sampling has been suggested as a method for allocating second stage sample effort among primary units. Here, we suggest a modification: adaptive two-stage sequential sampling. In this method, the adaptive part of the allocation process means the design is more flexible in how much extra effort can be directed to higher-abundance primary units. We discuss how best to design an adaptive two-stage sequential sample. ?? 2008 The Society of Population Ecology and Springer.

Sequential causal inference: Application to randomized trials of adaptive treatment strategies

PubMed Central

Dawson, Ree; Lavori, Philip W.

2009-01-01

SUMMARY Clinical trials that randomize subjects to decision algorithms, which adapt treatments over time according to individual response, have gained considerable interest as investigators seek designs that directly inform clinical decision making. We consider designs in which subjects are randomized sequentially at decision points, among adaptive treatment options under evaluation. We present a sequential method to estimate the comparative effects of the randomized adaptive treatments, which are formalized as adaptive treatment strategies. Our causal estimators are derived using Bayesian predictive inference. We use analytical and empirical calculations to compare the predictive estimators to (i) the ‘standard’ approach that allocates the sequentially obtained data to separate strategy-specific groups as would arise from randomizing subjects at baseline; (ii) the semi-parametric approach of marginal mean models that, under appropriate experimental conditions, provides the same sequential estimator of causal differences as the proposed approach. Simulation studies demonstrate that sequential causal inference offers substantial efficiency gains over the standard approach to comparing treatments, because the predictive estimators can take advantage of the monotone structure of shared data among adaptive strategies. We further demonstrate that the semi-parametric asymptotic variances, which are marginal ‘one-step’ estimators, may exhibit significant bias, in contrast to the predictive variances. We show that the conditions under which the sequential method is attractive relative to the other two approaches are those most likely to occur in real studies. PMID:17914714

The Role of NG2 Glial Cells in ALS Pathogenesis

DTIC Science & Technology

2013-10-01

line of OPC differentiation from iPS cells. SHH, sonic hedgehog ; RA, retinoitic acid; bFGF, basic FGF; PDGF, platelet-derived growth factor; IGF...University School of Medicine, Baltimore, Maryland, USA. 3Department of Anatomy , Kitasato University School of Medicine, Sagamihara, Japan. 4Brain Science...6Present address: Shriners Hospital Pediatric Research Center, Department of Anatomy and Cell Biology, Temple University School of Medicine

Function of Brg1 Chromatin Remodeling Factor in Sonic Hedgehog-Dependent Medulloblastoma Initiation and Maintenance

DTIC Science & Technology

2014-10-01

Remodeling Factor in Sonic Hedgehog -Dependent Medulloblastoma Initiation and Maintenance PRINCIPAL INVESTIGATOR: Xuanming Shi CONTRACTING...Function of Brg1 Chromatin Remodeling Factor in Sonic Hedgehog -Dependent 5b. GRANT NUMBER W81XWH-12-1-0527 Medulloblastoma Initiation and Maintenance...medulloblastoma. 15. SUBJECT TERMS Medulloblastoma, Sonic Hedgehog , Chromatin remodeling, BAF complex, Brg1, mouse model of shh-subtype medulloblastoma

Gorlin syndrome-derived induced pluripotent stem cells are hypersensitive to hedgehog-mediated osteogenic induction.

PubMed

Hasegawa, Daigo; Ochiai-Shino, Hiromi; Onodera, Shoko; Nakamura, Takashi; Saito, Akiko; Onda, Takeshi; Watanabe, Katsuhito; Nishimura, Ken; Ohtaka, Manami; Nakanishi, Mahito; Kosaki, Kenjiro; Yamaguchi, Akira; Shibahara, Takahiko; Azuma, Toshifumi

2017-01-01

Gorlin syndrome is an autosomal dominant inherited syndrome that predisposes a patient to the formation of basal cell carcinomas, odontogenic keratocysts, and skeletal anomalies. Causative mutations in several genes associated with the sonic hedgehog (SHH) signaling pathway, including PTCH1, have been identified in Gorlin syndrome patients. However, no definitive genotype-phenotype correlations are evident in these patients, and their clinical presentation varies greatly, often leading to delayed diagnosis and treatment. We generated iPSCs from four unrelated Gorlin syndrome patients with loss-of-function mutations in PTCH1 using the Sendai virus vector (SeVdp(KOSM)302). The patient-derived iPSCs exhibited basic iPSC features, including stem cell marker expression, totipotency, and the ability to form teratomas. GLI1 expression levels were greater in fibroblasts and patient-derived iPSCs than in the corresponding control cells. Patient-derived iPSCs expressed lower basal levels than control iPSCs of the genes encoding the Hh ligands Indian Hedgehog (IHH) and SHH, the Hh acetyltransferase HHAT, Wnt proteins, BMP4, and BMP6. Most of these genes were upregulated in patient-derived iPSCs grown in osteoblast differentiation medium (OBM) and downregulated in control iPSCs cultured in OBM. The expression of GLI1 and GLI2 substantially decreased in both control and patient-derived iPSCs cultured in OBM, whereas GLI3, SHH, and IHH were upregulated in patient-derived iPSCs and downregulated in control iPSCs grown in OBM. Activation of Smoothened by SAG in cells grown in OBM significantly enhanced alkaline phosphatase activity in patient-derived iPSCs compared with control iPSC lines. In summary, patient-derived iPSCs expressed lower basal levels than the control iPSCs of the genes encoding Hh, Wnt, and bone morphogenetic proteins, but their expression of these genes strongly increased under osteogenic conditions. These findings indicate that patient-derived iPSCs are hypersensitive to osteogenic induction. We propose that Hh signaling is constituently active in iPSCs from Gorlin syndrome patients, enhancing their response to osteogenic induction and contributing to disease-associated abnormalities.

Long-term effect of neonatal inhibition of APP gamma-secretase on hippocampal development in the Ts65Dn mouse model of Down syndrome.

PubMed

Stagni, Fiorenza; Raspanti, Alessandra; Giacomini, Andrea; Guidi, Sandra; Emili, Marco; Ciani, Elisabetta; Giuliani, Alessandro; Bighinati, Andrea; Calzà, Laura; Magistretti, Jacopo; Bartesaghi, Renata

2017-07-01

Neurogenesis impairment is considered a major determinant of the intellectual disability that characterizes Down syndrome (DS), a genetic condition caused by triplication of chromosome 21. Previous evidence obtained in the Ts65Dn mouse model of DS showed that the triplicated gene APP (amyloid precursor protein) is critically involved in neurogenesis alterations. In particular, excessive levels of AICD (amyloid precursor protein intracellular domain) resulting from APP cleavage by gamma-secretase increase the transcription of Ptch1, a Sonic Hedgehog (Shh) receptor that keeps the mitogenic Shh pathway repressed. Previous evidence showed that neonatal treatment with ELND006, an inhibitor of gamma-secretase, reinstates the Shh pathway and fully restores neurogenesis in Ts65Dn pups. In the framework of potential therapies for DS, it is extremely important to establish whether the positive effects of early intervention are retained after treatment cessation. Therefore, the goal of the current study was to establish whether early treatment with ELND006 leaves an enduring trace in the brain of Ts65Dn mice. Ts65Dn and euploid pups were treated with ELND006 in the postnatal period P3-P15 and the outcome of treatment was examined at ~one month after treatment cessation. We found that in treated Ts65Dn mice the pool of proliferating cells in the hippocampal dentate gyrus (DG) and total number of granule neurons were still restored as was the number of pre- and postsynaptic terminals in the stratum lucidum of CA3, the site of termination of the mossy fibers from the DG. Accordingly, patch-clamp recording from field CA3 showed functional normalization of the input to CA3. Unlike in field CA3, the number of pre- and postsynaptic terminals in the DG of treated Ts65Dn mice was no longer fully restored. The finding that many of the positive effects of neonatal treatment were retained after treatment cessation provides proof of principle demonstration of the efficacy of early inhibition of gamma-secretase for the improvement of brain development in DS. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Evolution of an adaptive behavior and its sensory receptors promotes eye regression in blind cavefish.

PubMed

Yoshizawa, Masato; Yamamoto, Yoshiyuki; O'Quin, Kelly E; Jeffery, William R

2012-12-27

How and why animals lose eyesight during adaptation to the dark and food-limited cave environment has puzzled biologists since the time of Darwin. More recently, several different adaptive hypotheses have been proposed to explain eye degeneration based on studies in the teleost Astyanax mexicanus, which consists of blind cave-dwelling (cavefish) and sighted surface-dwelling (surface fish) forms. One of these hypotheses is that eye regression is the result of indirect selection for constructive characters that are negatively linked to eye development through the pleiotropic effects of Sonic Hedgehog (SHH) signaling. However, subsequent genetic analyses suggested that other mechanisms also contribute to eye regression in Astyanax cavefish. Here, we introduce a new approach to this problem by investigating the phenotypic and genetic relationships between a suite of non-visual constructive traits and eye regression. Using quantitative genetic analysis of crosses between surface fish, the Pachón cavefish population and their hybrid progeny, we show that the adaptive vibration attraction behavior (VAB) and its sensory receptors, superficial neuromasts (SN) specifically found within the cavefish eye orbit (EO), are genetically correlated with reduced eye size. The quantitative trait loci (QTL) for these three traits form two clusters of congruent or overlapping QTL on Astyanax linkage groups (LG) 2 and 17, but not at the shh locus on LG 13. Ablation of EO SN in cavefish demonstrated a major role for these sensory receptors in VAB expression. Furthermore, experimental induction of eye regression in surface fish via shh overexpression showed that the absence of eyes was insufficient to promote the appearance of VAB or EO SN. We conclude that natural selection for the enhancement of VAB and EO SN indirectly promotes eye regression in the Pachón cavefish population through an antagonistic relationship involving genetic linkage or pleiotropy among the genetic factors underlying these traits. This study demonstrates a trade-off between the evolution of a non-visual sensory system and eye regression during the adaptive evolution of Astyanax to the cave environment.

Sequential Polarity-Reversing Circuit

NASA Technical Reports Server (NTRS)

Labaw, Clayton C.

1994-01-01

Proposed circuit reverses polarity of electric power supplied to bidirectional dc motor, reversible electro-mechanical actuator, or other device operating in direction depending on polarity. Circuit reverses polarity each time power turned on, without need for additional polarity-reversing or direction signals and circuitry to process them.

A temperature-sensitive mutation in the nodal-related gene cyclops reveals that the floor plate is induced during gastrulation in zebrafish.

PubMed

Tian, Jing; Yam, Caleb; Balasundaram, Gayathri; Wang, Hui; Gore, Aniket; Sampath, Karuna

2003-07-01

The floor plate, a specialized group of cells in the ventral midline of the neural tube of vertebrates, plays crucial roles in patterning the central nervous system. Recent work from zebrafish, chick, chick-quail chimeras and mice to investigate the development of the floor plate have led to several models of floor-plate induction. One model suggests that the floor plate is formed by inductive signalling from the notochord to the overlying neural tube. The induction is thought to be mediated by notochord-derived Sonic hedgehog (Shh), a secreted protein, and requires direct cellular contact between the notochord and the neural tube. Another model proposes a role for the organizer in generating midline precursor cells that produce floor plate cells independent of notochord specification, and proposes that floor plate specification occurs early, during gastrulation. We describe a temperature-sensitive mutation that affects the zebrafish Nodal-related secreted signalling factor, Cyclops, and use it to address the issue of when the floor plate is induced in zebrafish. Zebrafish cyclops regulates the expression of shh in the ventral neural tube. Although null mutations in cyclops result in the lack of the medial floor plate, embryos homozygous for the temperature-sensitive mutation have floor plate cells at the permissive temperature and lack floor plate cells at the restrictive temperature. We use this mutant allele in temperature shift-up and shift-down experiments to answer a central question pertaining to the timing of vertebrate floor plate induction. Abrogation of Cyc/Nodal signalling in the temperature-sensitive mutant embryos at various stages indicates that the floor plate in zebrafish is induced early in development, during gastrulation. In addition, continuous Cyclops signalling is required through gastrulation for a complete ventral neural tube throughout the length of the neuraxis. Finally, by modulation of Nodal signalling levels in mutants and in ectopic overexpression experiments, we show that, similar to the requirements for prechordal plate mesendoderm fates, uninterrupted and high levels of Cyclops signalling are required for induction and specification of a complete ventral neural tube.

Structural profiling of individual glycosphingolipids in a single thin-layer chromatogram by multiple sequential immunodetection matched with Direct IR-MALDI-o-TOF mass spectrometry.

PubMed

Souady, Jamal; Soltwisch, Jens; Dreisewerd, Klaus; Haier, Jörg; Peter-Katalinić, Jasna; Müthing, Johannes

2009-11-15

The thin-layer chromatography (TLC) immunoenzyme overlay assay is a widely used tool for antibody-mediated identification of glycosphingolipids (GSLs) in mixtures. However, because the majority of GSLs is left unexamined in a chromatogram of a single assay, we developed a novel method that permits detection of various GSLs by sequential multiple immunostaining combined with individual coloring of GSLs in the same chromatogram. Specific staining was achieved by means of primary anti-GSL antibodies, directed against lactosylceramide, globotriaosylceramide, and globotetraosylceramide, in conjunction with alkaline phosphatase (AP)- or horseradish peroxidase (HRP)-conjugated secondary antibodies together with the appropriate chromogenic substrates. Triple coloring with 5-bromo-4-chloro-3-indolyl phosphate (BCIP)-AP, Fast Red-AP, and 3,3'-diaminobenzidine (DAB)-HRP resulted in blue, red, and black precipitates, respectively, following three sequential immunostaining rounds. Structures of antibody-detected GSLs were determined by direct coupling of TLC with infrared matrix-assisted laser desorption/ionization orthogonal time-of-flight mass spectrometry. This combinatorial technique was used to demonstrate structural GSL profiling of crude lipid extracts from human hepatocellular cancer. This powerful technology allows efficient structural characterization of GSLs in small tissue samples and marks a further step forward in the emerging field of glycosphingolipidomics.

Mineralization of the vertebral bodies in Atlantic salmon (Salmo salar L.) is initiated segmentally in the form of hydroxyapatite crystal accretions in the notochord sheath

PubMed Central

Wang, Shou; Kryvi, Harald; Grotmol, Sindre; Wargelius, Anna; Krossøy, Christel; Epple, Mattias; Neues, Frank; Furmanek, Tomasz; Totland, Geir K

2013-01-01

We performed a sequential morphological and molecular biological study of the development of the vertebral bodies in Atlantic salmon (Salmo salar L.). Mineralization starts in separate bony elements which fuse to form complete segmental rings within the notochord sheath. The nucleation and growth of hydroxyapatite crystals in both the lamellar type II collagen matrix of the notochord sheath and the lamellar type I collagen matrix derived from the sclerotome, were highly similar. In both matrices the hydroxyapatite crystals nucleate and accrete on the surface of the collagen fibrils rather than inside the fibrils, a process that may be controlled by a template imposed by the collagen fibrils. Apatite crystal growth starts with the formation of small plate-like structures, about 5 nm thick, that gradually grow and aggregate to form extensive multi-branched crystal arborizations, resembling dendritic growth. The hydroxyapatite crystals are always oriented parallel to the long axis of the collagen fibrils, and the lamellar collagen matrices provide oriented support for crystal growth. We demonstrate here for the first time by means of synchroton radiation based on X-ray diffraction that the chordacentra contain hydroxyapatite. We employed quantitative real-time PCR to study the expression of key signalling molecule transcripts expressed in the cellular core of the notochord. The results indicate that the notochord not only produces and maintains the notochord sheath but also expresses factors known to regulate skeletogenesis: sonic hedgehog (shh), indian hedgehog homolog b (ihhb), parathyroid hormone 1 receptor (pth1r) and transforming growth factor beta 1 (tgfb1). In conclusion, our study provides evidence for the process of vertebral body development in teleost fishes, which is initially orchestrated by the notochord. PMID:23711083

An Abbreviated Protocol for In Vitro Generation of Functional Human Embryonic Stem Cell-Derived Beta-Like Cells

PubMed Central

Massumi, Mohammad; Pourasgari, Farzaneh; Nalla, Amarnadh; Batchuluun, Battsetseg; Nagy, Kristina; Neely, Eric; Gull, Rida; Nagy, Andras; Wheeler, Michael B.

2016-01-01

The ability to yield glucose-responsive pancreatic beta-cells from human pluripotent stem cells in vitro will facilitate the development of the cell replacement therapies for the treatment of Type 1 Diabetes. Here, through the sequential in vitro targeting of selected signaling pathways, we have developed an abbreviated five-stage protocol (25–30 days) to generate human Embryonic Stem Cell-Derived Beta-like Cells (ES-DBCs). We showed that Geltrex, as an extracellular matrix, could support the generation of ES-DBCs more efficiently than that of the previously described culture systems. The activation of FGF and Retinoic Acid along with the inhibition of BMP, SHH and TGF-beta led to the generation of 75% NKX6.1+/NGN3+ Endocrine Progenitors. The inhibition of Notch and tyrosine kinase receptor AXL, and the treatment with Exendin-4 and T3 in the final stage resulted in 35% mono-hormonal insulin positive cells, 1% insulin and glucagon positive cells and 30% insulin and NKX6.1 co-expressing cells. Functionally, ES-DBCs were responsive to high glucose in static incubation and perifusion studies, and could secrete insulin in response to successive glucose stimulations. Mitochondrial metabolic flux analyses using Seahorse demonstrated that the ES-DBCs could efficiently metabolize glucose and generate intracellular signals to trigger insulin secretion. In conclusion, targeting selected signaling pathways for 25–30 days was sufficient to generate ES-DBCs in vitro. The ability of ES-DBCs to secrete insulin in response to glucose renders them a promising model for the in vitro screening of drugs, small molecules or genes that may have potential to influence beta-cell function. PMID:27755557

Mineralization of the vertebral bodies in Atlantic salmon (Salmo salar L.) is initiated segmentally in the form of hydroxyapatite crystal accretions in the notochord sheath.

PubMed

Wang, Shou; Kryvi, Harald; Grotmol, Sindre; Wargelius, Anna; Krossøy, Christel; Epple, Mattias; Neues, Frank; Furmanek, Tomasz; Totland, Geir K

2013-08-01

We performed a sequential morphological and molecular biological study of the development of the vertebral bodies in Atlantic salmon (Salmo salar L.). Mineralization starts in separate bony elements which fuse to form complete segmental rings within the notochord sheath. The nucleation and growth of hydroxyapatite crystals in both the lamellar type II collagen matrix of the notochord sheath and the lamellar type I collagen matrix derived from the sclerotome, were highly similar. In both matrices the hydroxyapatite crystals nucleate and accrete on the surface of the collagen fibrils rather than inside the fibrils, a process that may be controlled by a template imposed by the collagen fibrils. Apatite crystal growth starts with the formation of small plate-like structures, about 5 nm thick, that gradually grow and aggregate to form extensive multi-branched crystal arborizations, resembling dendritic growth. The hydroxyapatite crystals are always oriented parallel to the long axis of the collagen fibrils, and the lamellar collagen matrices provide oriented support for crystal growth. We demonstrate here for the first time by means of synchroton radiation based on X-ray diffraction that the chordacentra contain hydroxyapatite. We employed quantitative real-time PCR to study the expression of key signalling molecule transcripts expressed in the cellular core of the notochord. The results indicate that the notochord not only produces and maintains the notochord sheath but also expresses factors known to regulate skeletogenesis: sonic hedgehog (shh), indian hedgehog homolog b (ihhb), parathyroid hormone 1 receptor (pth1r) and transforming growth factor beta 1 (tgfb1). In conclusion, our study provides evidence for the process of vertebral body development in teleost fishes, which is initially orchestrated by the notochord. © 2013 Anatomical Society.

A posteriori model validation for the temporal order of directed functional connectivity maps.

PubMed

Beltz, Adriene M; Molenaar, Peter C M

2015-01-01

A posteriori model validation for the temporal order of neural directed functional connectivity maps is rare. This is striking because models that require sequential independence among residuals are regularly implemented. The aim of the current study was (a) to apply to directed functional connectivity maps of functional magnetic resonance imaging data an a posteriori model validation procedure (i.e., white noise tests of one-step-ahead prediction errors combined with decision criteria for revising the maps based upon Lagrange Multiplier tests), and (b) to demonstrate how the procedure applies to single-subject simulated, single-subject task-related, and multi-subject resting state data. Directed functional connectivity was determined by the unified structural equation model family of approaches in order to map contemporaneous and first order lagged connections among brain regions at the group- and individual-levels while incorporating external input, then white noise tests were run. Findings revealed that the validation procedure successfully detected unmodeled sequential dependencies among residuals and recovered higher order (greater than one) simulated connections, and that the procedure can accommodate task-related input. Findings also revealed that lags greater than one were present in resting state data: With a group-level network that contained only contemporaneous and first order connections, 44% of subjects required second order, individual-level connections in order to obtain maps with white noise residuals. Results have broad methodological relevance (e.g., temporal validation is necessary after directed functional connectivity analyses because the presence of unmodeled higher order sequential dependencies may bias parameter estimates) and substantive implications (e.g., higher order lags may be common in resting state data).

Sequential and simultaneous choices: testing the diet selection and sequential choice models.

PubMed

Freidin, Esteban; Aw, Justine; Kacelnik, Alex

2009-03-01

We investigate simultaneous and sequential choices in starlings, using Charnov's Diet Choice Model (DCM) and Shapiro, Siller and Kacelnik's Sequential Choice Model (SCM) to integrate function and mechanism. During a training phase, starlings encountered one food-related option per trial (A, B or R) in random sequence and with equal probability. A and B delivered food rewards after programmed delays (shorter for A), while R ('rejection') moved directly to the next trial without reward. In this phase we measured latencies to respond. In a later, choice, phase, birds encountered the pairs A-B, A-R and B-R, the first implementing a simultaneous choice and the second and third sequential choices. The DCM predicts when R should be chosen to maximize intake rate, and SCM uses latencies of the training phase to predict choices between any pair of options in the choice phase. The predictions of both models coincided, and both successfully predicted the birds' preferences. The DCM does not deal with partial preferences, while the SCM does, and experimental results were strongly correlated to this model's predictions. We believe that the SCM may expose a very general mechanism of animal choice, and that its wider domain of success reflects the greater ecological significance of sequential over simultaneous choices.

Breast conserving treatment for breast cancer: dosimetric comparison of sequential versus simultaneous integrated photon boost.

PubMed

Van Parijs, Hilde; Reynders, Truus; Heuninckx, Karina; Verellen, Dirk; Storme, Guy; De Ridder, Mark

2014-01-01

Breast conserving surgery followed by whole breast irradiation is widely accepted as standard of care for early breast cancer. Addition of a boost dose to the initial tumor area further reduces local recurrences. We investigated the dosimetric benefits of a simultaneously integrated boost (SIB) compared to a sequential boost to hypofractionate the boost volume, while maintaining normofractionation on the breast. For 10 patients 4 treatment plans were deployed, 1 with a sequential photon boost, and 3 with different SIB techniques: on a conventional linear accelerator, helical TomoTherapy, and static TomoDirect. Dosimetric comparison was performed. PTV-coverage was good in all techniques. Conformity was better with all SIB techniques compared to sequential boost (P = 0.0001). There was less dose spilling to the ipsilateral breast outside the PTVboost (P = 0.04). The dose to the organs at risk (OAR) was not influenced by SIB compared to sequential boost. Helical TomoTherapy showed a higher mean dose to the contralateral breast, but less than 5 Gy for each patient. SIB showed less dose spilling within the breast and equal dose to OAR compared to sequential boost. Both helical TomoTherapy and the conventional technique delivered acceptable dosimetry. SIB seems a safe alternative and can be implemented in clinical routine.

Breast Conserving Treatment for Breast Cancer: Dosimetric Comparison of Sequential versus Simultaneous Integrated Photon Boost

PubMed Central

Reynders, Truus; Heuninckx, Karina; Verellen, Dirk; Storme, Guy; De Ridder, Mark

2014-01-01

Background. Breast conserving surgery followed by whole breast irradiation is widely accepted as standard of care for early breast cancer. Addition of a boost dose to the initial tumor area further reduces local recurrences. We investigated the dosimetric benefits of a simultaneously integrated boost (SIB) compared to a sequential boost to hypofractionate the boost volume, while maintaining normofractionation on the breast. Methods. For 10 patients 4 treatment plans were deployed, 1 with a sequential photon boost, and 3 with different SIB techniques: on a conventional linear accelerator, helical TomoTherapy, and static TomoDirect. Dosimetric comparison was performed. Results. PTV-coverage was good in all techniques. Conformity was better with all SIB techniques compared to sequential boost (P = 0.0001). There was less dose spilling to the ipsilateral breast outside the PTVboost (P = 0.04). The dose to the organs at risk (OAR) was not influenced by SIB compared to sequential boost. Helical TomoTherapy showed a higher mean dose to the contralateral breast, but less than 5 Gy for each patient. Conclusions. SIB showed less dose spilling within the breast and equal dose to OAR compared to sequential boost. Both helical TomoTherapy and the conventional technique delivered acceptable dosimetry. SIB seems a safe alternative and can be implemented in clinical routine. PMID:25162031

Two-IMU FDI performance of the sequential probability ratio test during shuttle entry

NASA Technical Reports Server (NTRS)

Rich, T. M.

1976-01-01

Performance data for the sequential probability ratio test (SPRT) during shuttle entry are presented. Current modeling constants and failure thresholds are included for the full mission 3B from entry through landing trajectory. Minimum 100 percent detection/isolation failure levels and a discussion of the effects of failure direction are presented. Finally, a limited comparison of failures introduced at trajectory initiation shows that the SPRT algorithm performs slightly worse than the data tracking test.

REML/BLUP and sequential path analysis in estimating genotypic values and interrelationships among simple maize grain yield-related traits.

PubMed

Olivoto, T; Nardino, M; Carvalho, I R; Follmann, D N; Ferrari, M; Szareski, V J; de Pelegrin, A J; de Souza, V Q

2017-03-22

Methodologies using restricted maximum likelihood/best linear unbiased prediction (REML/BLUP) in combination with sequential path analysis in maize are still limited in the literature. Therefore, the aims of this study were: i) to use REML/BLUP-based procedures in order to estimate variance components, genetic parameters, and genotypic values of simple maize hybrids, and ii) to fit stepwise regressions considering genotypic values to form a path diagram with multi-order predictors and minimum multicollinearity that explains the relationships of cause and effect among grain yield-related traits. Fifteen commercial simple maize hybrids were evaluated in multi-environment trials in a randomized complete block design with four replications. The environmental variance (78.80%) and genotype-vs-environment variance (20.83%) accounted for more than 99% of the phenotypic variance of grain yield, which difficult the direct selection of breeders for this trait. The sequential path analysis model allowed the selection of traits with high explanatory power and minimum multicollinearity, resulting in models with elevated fit (R 2 > 0.9 and ε < 0.3). The number of kernels per ear (NKE) and thousand-kernel weight (TKW) are the traits with the largest direct effects on grain yield (r = 0.66 and 0.73, respectively). The high accuracy of selection (0.86 and 0.89) associated with the high heritability of the average (0.732 and 0.794) for NKE and TKW, respectively, indicated good reliability and prospects of success in the indirect selection of hybrids with high-yield potential through these traits. The negative direct effect of NKE on TKW (r = -0.856), however, must be considered. The joint use of mixed models and sequential path analysis is effective in the evaluation of maize-breeding trials.

Movement plans for posture selection do not transfer across hands

PubMed Central

Schütz, Christoph; Schack, Thomas

2015-01-01

In a sequential task, the grasp postures people select depend on their movement history. This motor hysteresis effect results from the reuse of former movement plans and reduces the cognitive cost of movement planning. Movement plans for hand trajectories not only transfer across successive trials, but also across hands. We therefore asked whether such a transfer would also be found in movement plans for hand postures. To this end, we designed a sequential, continuous posture selection task. Participants had to open a column of drawers with cylindrical knobs in ascending and descending sequences. A hand switch was required in each sequence. Hand pro/supination was analyzed directly before and after the hand switch. Results showed that hysteresis effects were present directly before, but absent directly after the hand switch. This indicates that, in the current study, movement plans for hand postures only transfer across trials, but not across hands. PMID:26441734

Function of Brg1 Chromatin Remodeling Factor in Sonic Hedgehog-Dependent Medulloblastoma Initiation and Maintenance

DTIC Science & Technology

2015-12-01

tumor suppressors and REST-targeted neuronal genes. Brg1 deletion led to the inhibition of Shh-type medulloblastoma growth by deregulation of the...China University of Rostock & Research Institute for the Biology of Farm Animals, Germany University of Texas Southwestern Medical Center at...of Rostock & Research Institute for the Biology of Farm Animals, Germany . Mentor: Prof. Dr. Hans-Martin Seyfert 2010- 2014 Postdoctoral

Evolution of antero‐posterior patterning of the limb: Insights from the chick

PubMed Central

2017-01-01

Summary The developing limbs of chicken embryos have served as pioneering models for understanding pattern formation for over a century. The ease with which chick wing and leg buds can be experimentally manipulated, while the embryo is still in the egg, has resulted in the discovery of important developmental organisers, and subsequently, the signals that they produce. Sonic hedgehog (Shh) is produced by mesenchyme cells of the polarizing region at the posterior margin of the limb bud and specifies positional values across the antero‐posterior axis (the axis running from the thumb to the little finger). Detailed experimental embryology has revealed the fundamental parameters required to specify antero‐posterior positional values in response to Shh signaling in chick wing and leg buds. In this review, the evolution of the avian wing and leg will be discussed in the broad context of tetrapod paleontology, and more specifically, ancestral theropod dinosaur paleontology. How the parameters that dictate antero‐posterior patterning could have been modulated to produce the avian wing and leg digit patterns will be considered. Finally, broader speculations will be made regarding what the antero‐posterior patterning of chick limbs can tell us about the evolution of other digit patterns, including those that were found in the limbs of the earliest tetrapods. PMID:28734068

Molecular signaling in intervertebral disk development.

PubMed

DiPaola, Christian P; Farmer, James C; Manova, Katia; Niswander, Lee A

2005-09-01

The purpose of this investigation is to identify and study the expression pattern of pertinent molecular factors involved in the differentiation of the intervertebral disk (IVD). It is likely that hedgehog genes and the BMP inhibitors are key factors involved in spinal joint formation. Radioactive in situ hybridization with mRNA probes for pax-1, SHH, IHH and Noggin gene was performed on mouse embryo and adult tissue. Immunohistochemistry was performed to localize hedgehog receptor, "patched" (ptc). From 14.5 dpc until birth pax-1 mRNA was expressed in the developing anulus fibrosus (AF). During the same developmental period Noggin mRNA is highly expressed throughout the spine, in the developing AF, while ptc protein and SHH mRNA were expressed in the developing nucleus pulposus (NP). IHH mRNA was expressed by condensing chondrocytes of the vertebral bodies and later becomes confined to the vertebral endplate. We show for the first time that pax-1 is expressed in the adult intervertebral disk. Ptc expression in the NP is an indicator of hedgehog protein signaling in the developing IVD. The expression pattern of the BMP inhibitor Noggin appears to be important for the normal formation of the IVD and may prove to play a role in its segmental pattern formation.

Dual odontogenic origins develop at the early stage of rat maxillary incisor development.

PubMed

Kriangkrai, Rungarun; Iseki, Sachiko; Eto, Kazuhiro; Chareonvit, Suconta

2006-03-01

Developmental process of rat maxillary incisor has been studied through histological analysis and investigation of tooth-related gene expression patterns at initial tooth development. The tooth-related genes studied here are fibroblast growth factor-8 (Fgf-8), pituitary homeobox gene-2 (Pitx-2), sonic hedgehog (Shh), muscle segment homeobox-1 (Msx-1), paired box-9 (Pax-9) and bone morphogenetic protein-4 (Bmp-4). The genes are expressed in oral epithelium and/or ectomesenchyme at the stage of epithelial thickening to the early bud stage of tooth development. Both the histological observation and tooth-related gene expression patterns during early stage of maxillary incisor development demonstrate that dual odontogenic origins aligned medio-laterally in the medial nasal process develop, subsequently only single functional maxillary incisor dental placode forms. The cascade of tooth-related gene expression patterns in rat maxillary incisor studied here is quite similar to those of the previous studies in mouse mandibular molar, even though the origins of oral epithelium and ectomesenchyme involved in development of maxillary incisor and mandibular molar are different. Thus, we conclude that maxillary incisor and mandibular molar share a similar signaling control of Fgf-8, Pitx-2, Shh, Msx-1, Pax-9 and Bmp-4 genes at the stage of oral epithelial thickening to the early bud stage of tooth development.

Knockdown of connexin43-mediated regulation of the zone of polarizing activity in the developing chick limb leads to digit truncation.

PubMed

Law, Lee Yong; Lin, Jun Sheng; Becker, David L; Green, Colin R

2002-12-01

In the developing chick wing, the use of antisense oligodeoxynucleotides to transiently knock down the expression of the gap junction protein, connexin43 (Cx43), results in limb patterning defects, including deletion of the anterior digits. To understand more about how such defects arise, the effects of transient Cx43 knockdown on the expression patterns of several genes known to play pivotal roles in limb formation were examined. Sonic hedgehog (Shh), which is normally expressed in the zone of polarizing activity (ZPA) and is required to maintain both the ZPA and the apical ectodermal ridge (AER), was found to be downregulated in treated limbs within 30 h. Bone morphogenetic protein-2 (Bmp-2), a gene downstream of Shh, was similarly downregulated. Fibroblast growth factor-8 expression, however, was unaltered 30 h after treatment but was greatly reduced at 48 h post-treatment, when the AER begins to regress. Expressions of Bmp-4 and Muscle segment homeobox-like gene (Msx-1) were not affected at any of the time points examined. Cx43 expression is therefore involved in some, but not all patterning cascades, and appears to play a role in the regulation of ZPA activity.

Mechanisms for the development of esophageal atresia.

PubMed

Orford, J; Manglick, P; Cass, D T; Tam, P P

2001-07-01

There is no universally accepted theory to explain esophageal embryology and the abnormal development that produces esophageal atresia. The impact of Adriamycin administration on the pathogenesis of esophageal atresia was studied in the rat model of VATER association, from embryonic day (ED) 10 to ED 13. Tissues in the ED10 Adriamycin-exposed embryos displayed less cell proliferation as shown by the reduced population of MIB-5-labelled cells. Cell apoptosis that is characteristic of the normal ED 12 lateral epithelial folds of the foregut (the prospective site of tracheoesophageal septation) was absent in the foregut of the Adriamycin-exposed embryo. Histologic examination of the ED 11-exposed embryo showed the presence of abnormal notochord that was stretched, split, or tethered to the foregut. This contrasts with the normal embryo in which the notochord was localized in close vicinity of the ventral part of the neural tube and separated from the foregut by ample amount of mesenchyme. The abnormal localization of the notochord was accompanied by the lack of down-regulation of the sonic hedgehog (Shh) activity in the prospective site of future tracheoesophageal separation in the exposed ED 12 embryo. The authors proposed that the ectopic location of the notochord leads to the disruption in Shh signalling that may underpin the development of esophageal atresia. Copyright 2001 by W.B. Saunders Company.

Biologically active and biomimetic dual gelatin scaffolds for tissue engineering.

PubMed

Sánchez, P; Pedraz, J L; Orive, G

2017-05-01

We have designed, developed and optimized Genipin cross-linked 3D gelatin scaffolds that were biologically active and biomimetic, show a dual activity both for growth factor and cell delivery. Type B gelatin powder was dissolved in DI water. 100mg of genipin was dissolved in 10ml of DI water. Three genipin concentrations were prepared: 0.1%, 0.2% and 0.3% (w/v). Solutions were mixed at 40°C and under stirring and then left crosslinking for 72h. Scaffolds were obtained by punching 8 mm-cylinders into ethanol 70% solution for 10min and then freeze-drying. Scaffolds were biologically, biomechanically and morphologically evaluated. Cell adhesion and morphology of D1-Mesenchymal stem cells (MSCs) and L-929 fibroblast was studied. Vascular endothelial grwoth factor (VEGF) and Sonic hedgehog (SHH) were used as model proteins. Swelling ratio increased and younǵs module decreased along with the concentration of genipin. All scaffolds were biocompatible according to the toxicity test. MSC and L-929 cell adhesion improved in 0.2% of genipin, obtaining better results with MSCs. VEGF and SHH were released from the gels. This preliminary study suggest that the biologically active and dual gelatin scaffolds may be used for tissue engineering approaches like bone regeneration. Copyright © 2017 Elsevier B.V. All rights reserved.

Molecular analysis of holoprosencephaly in South America

PubMed Central

Savastano, Clarice Pagani; El-Jaick, Kênia Balbi; Costa-Lima, Marcelo Aguiar; Abath, Cristina Maria Batista; Bianca, Sebastiano; Cavalcanti, Denise Pontes; Félix, Têmis Maria; Scarano, Gioacchino; Llerena, Juan Clinton; Vargas, Fernando Regla; Moreira, Miguel Ângelo Martins; Seuánez, Hector N.; Castilla, Eduardo Enrique; Orioli, Iêda Maria

2014-01-01

Holoprosencephaly (HPE) is a spectrum of brain and facial malformations primarily reflecting genetic factors, such as chromosomal abnormalities and gene mutations. Here, we present a clinical and molecular analysis of 195 probands with HPE or microforms; approximately 72% of the patients were derived from the Latin American Collaborative Study of Congenital Malformations (ECLAMC), and 82% of the patients were newborns. Alobar HPE was the predominant brain defect in almost all facial defect categories, except for patients without oral cleft and median or lateral oral clefts. Ethmocephaly, cebocephaly, and premaxillary agenesis were primarily observed among female patients. Premaxillary agenesis occurred in six of the nine diabetic mothers. Recurrence of HPE or microform was approximately 19%. The frequency of microdeletions, detected using Multiplex Ligation-dependant Probe Amplification (MLPA) was 17% in patients with a normal karyotype. Cytogenetics or QF-PCR analyses revealed chromosomal anomalies in 27% of the probands. Mutational analyses in genes SHH, ZIC2, SIX3 and TGIF were performed in 119 patients, revealing eight mutations in SHH, two mutations in SIX3 and two mutations in ZIC2. Thus, a detailed clinical description of new HPE cases with identified genetic anomalies might establish genotypic and phenotypic correlations and contribute to the development of additional strategies for the analysis of new cases. PMID:24764759

The regulation of tooth morphogenesis is associated with epithelial cell proliferation and the expression of Sonic hedgehog through epithelial-mesenchymal interactions.

PubMed

Ishida, Kentaro; Murofushi, Mayumi; Nakao, Kazuhisa; Morita, Ritsuko; Ogawa, Miho; Tsuji, Takashi

2011-02-18

Ectodermal organs, such as the tooth, salivary gland, hair, and mammary gland, develop through reciprocal epithelial-mesenchymal interactions. Tooth morphologies are defined by the crown width and tooth length (macro-morphologies), and by the number and locations of the cusp and roots (micro-morphologies). In our current study, we report that the crown width of a bioengineered molar tooth, which was reconstructed using dissociated epithelial and mesenchymal cells via an organ germ method, can be regulated by the contact area between epithelial and mesenchymal cell layers. We further show that this is associated with cell proliferation and Sonic hedgehog (Shh) expression in the inner enamel epithelium after the germ stage has formed a secondary enamel knot. We also demonstrate that the cusp number is significantly correlated with the crown width of the bioengineered tooth. These findings suggest that the tooth micro-morphology, i.e. the cusp formation, is regulated after the tooth width, or macro-morphology, is determined. These findings also suggest that the spatiotemporal patterning of cell proliferation and the Shh expression areas in the epithelium regulate the crown width and cusp formation of the developing tooth. Copyright © 2011 Elsevier Inc. All rights reserved.

Dark sequential Z ' portal: Collider and direct detection experiments

NASA Astrophysics Data System (ADS)

Arcadi, Giorgio; Campos, Miguel D.; Lindner, Manfred; Masiero, Antonio; Queiroz, Farinaldo S.

2018-02-01

We revisit the status of a Majorana fermion as a dark matter candidate when a sequential Z' gauge boson dictates the dark matter phenomenology. Direct dark matter detection signatures rise from dark matter-nucleus scatterings at bubble chamber and liquid xenon detectors, and from the flux of neutrinos from the Sun measured by the IceCube experiment, which is governed by the spin-dependent dark matter-nucleus scattering. On the collider side, LHC searches for dilepton and monojet + missing energy signals play an important role. The relic density and perturbativity requirements are also addressed. By exploiting the dark matter complementarity we outline the region of parameter space where one can successfully have a Majorana dark matter particle in light of current and planned experimental sensitivities.

Modeling of a Sequential Two-Stage Combustor

NASA Technical Reports Server (NTRS)

Hendricks, R. C.; Liu, N.-S.; Gallagher, J. R.; Ryder, R. C.; Brankovic, A.; Hendricks, J. A.

2005-01-01

A sequential two-stage, natural gas fueled power generation combustion system is modeled to examine the fundamental aerodynamic and combustion characteristics of the system. The modeling methodology includes CAD-based geometry definition, and combustion computational fluid dynamics analysis. Graphical analysis is used to examine the complex vortical patterns in each component, identifying sources of pressure loss. The simulations demonstrate the importance of including the rotating high-pressure turbine blades in the computation, as this results in direct computation of combustion within the first turbine stage, and accurate simulation of the flow in the second combustion stage. The direct computation of hot-streaks through the rotating high-pressure turbine stage leads to improved understanding of the aerodynamic relationships between the primary and secondary combustors and the turbomachinery.

The use of sequential extraction to evaluate the remediation potential of heavy metals from contaminated harbour sediment

NASA Astrophysics Data System (ADS)

Nystrøm, G. M.; Ottosen, L. M.; Villumsen, A.

2003-05-01

In this work sequential extraction is performed with harbour sediment in order to evaluate the electrodialytic remediation potential for harbour sediments. Sequential extraction was performed on a sample of Norwegian harbour sediment; with the original sediment and after the sediment was treated with acid. The results from the sequential extraction show that 75% Zn and Pb and about 50% Cu are found in the most mobile phases in the original sediment and more than 90% Zn and Pb and 75% Cu are found in the most mobile phase in the sediment treated with acid. Electrodialytic remediation experiments were made. The method uses a low direct current as cleaning agent, removing the heavy metals towards the anode and cathode according to the charge of the heavy metals in the electric field. The electrodialytic experiments show that up to 50% Cu, 85% Zn and 60% Pb can be removed after 20 days. Thus, there is still a potential for a higher removal, with some changes in the experimental set-up and longer remediation time. The experiments show that thc use of sequential extraction can be used to predict the electrodialytic remediation potential for harbour sediments.

A posteriori model validation for the temporal order of directed functional connectivity maps

PubMed Central

Beltz, Adriene M.; Molenaar, Peter C. M.

2015-01-01

A posteriori model validation for the temporal order of neural directed functional connectivity maps is rare. This is striking because models that require sequential independence among residuals are regularly implemented. The aim of the current study was (a) to apply to directed functional connectivity maps of functional magnetic resonance imaging data an a posteriori model validation procedure (i.e., white noise tests of one-step-ahead prediction errors combined with decision criteria for revising the maps based upon Lagrange Multiplier tests), and (b) to demonstrate how the procedure applies to single-subject simulated, single-subject task-related, and multi-subject resting state data. Directed functional connectivity was determined by the unified structural equation model family of approaches in order to map contemporaneous and first order lagged connections among brain regions at the group- and individual-levels while incorporating external input, then white noise tests were run. Findings revealed that the validation procedure successfully detected unmodeled sequential dependencies among residuals and recovered higher order (greater than one) simulated connections, and that the procedure can accommodate task-related input. Findings also revealed that lags greater than one were present in resting state data: With a group-level network that contained only contemporaneous and first order connections, 44% of subjects required second order, individual-level connections in order to obtain maps with white noise residuals. Results have broad methodological relevance (e.g., temporal validation is necessary after directed functional connectivity analyses because the presence of unmodeled higher order sequential dependencies may bias parameter estimates) and substantive implications (e.g., higher order lags may be common in resting state data). PMID:26379489

An Embryonic Growth Pathway is Reactivated in Human Prostate Cancer

DTIC Science & Technology

2005-06-01

and 13 weeks in the prostatic urothelium and nascent prostatic buds. Staining was slightly diminished at 16.5, further diminished at 18 to 20 and...in the human fetal prostate is contemporaneous with the fetal testosterone surge and with ductal budding of the prostatic urothelium . SHH expression...expression in the human fetal the fetal mouse.8 There was intense staining of all layers of prostate. the prostatic urothelium at 11.5 weeks

Function of Brg1 Chromatin Remodeling Factor in Sonic Hedgehog-Dependent Medulloblastoma Initiation and Maintenance

DTIC Science & Technology

2013-10-01

Remodeling Factor in Sonic Hedgehog -Dependent Medulloblastoma Initiation and Maintenance PRINCIPAL INVESTIGATOR: Xuanming Shi CONTRACTING...5a. CONTRACT NUMBER W81XWH-12-1-0527 Function of Brg1 Chromatin Remodeling Factor in Sonic Hedgehog -Dependent 5b. GRANT NUMBER W81XWH-12-1...drug development and therapy of pediatric brain tumor and other Shh- dependent tumors. 15. SUBJECT TERMS Medulloblastoma, Sonic Hedgehog , Chromatin

Targeting of Cancer Stem Cells and Their Microenvironment in Early-Stage Mutant K-ras Lung Cancer

DTIC Science & Technology

2016-12-01

Aldefluor reagent. (B) A549 control lung cancer cells were incubated with Alde- fluor regent and DEAB, an inhibitor of aldehyde dehydro- genase. (C...increase in liquid colony formation or in cell proliferation compared to SHH- cells. Therefore, we turned to identify aldehyde dehydrogenase (ALDH...in which a green fluorescent BODIPY moiety is linked to aminoacetaldehyde, an aldehyde dehydrogenase substrate, and thus, cells expressing ALDH

Sequential and direct ionic excitation in the strong-field ionization of 1-butene molecules.

PubMed

Schell, Felix; Boguslavskiy, Andrey E; Schulz, Claus Peter; Patchkovskii, Serguei; Vrakking, Marc J J; Stolow, Albert; Mikosch, Jochen

2018-05-18

We study the Strong-Field Ionization (SFI) of the hydrocarbon 1-butene as a function of wavelength using photoion-photoelectron covariance and coincidence spectroscopy. We observe a striking transition in the fragment-associated photoelectron spectra: from a single Above Threshold Ionization (ATI) progression for photon energies less than the cation D0-D1 gap to two ATI progressions for a photon energy greater than this gap. For the first case, electronically excited cations are created by SFI populating the ground cationic state D0, followed by sequential post-ionization excitation. For the second case, direct sub-cycle SFI to the D1 excited cation state contributes significantly. Our experiments access ionization dynamics in a regime where strong-field and resonance-enhanced processes can interplay.

Pax1 and Pax9 activate Bapx1 to induce chondrogenic differentiation in the sclerotome.

PubMed

Rodrigo, Isabel; Hill, Robert E; Balling, Rudi; Münsterberg, Andrea; Imai, Kenji

2003-02-01

We have previously shown that the paired-box transcription factors Pax1 and Pax9 synergistically act in the proper formation of the vertebral column. Nevertheless, downstream events of the Pax1/Pax9 action and their target genes remain to be elucidated. We show, by analyzing Pax1;Pax9 double mutant mice, that expression of Bapx1 in the sclerotome requires the presence of Pax1 and Pax9, in a gene dose-dependent manner. By using a retroviral system to overexpress Pax1 in chick presomitic mesoderm explants, we show that Pax1 can substitute for Shh in inducing Bapx1 expression and in initiating chondrogenic differentiation. Furthermore, we demonstrate that Pax1 and Pax9 can transactivate regulatory sequences in the Bapx1 promoter and that they physically interact with the Bapx1 promoter region. These results strongly suggest that Bapx1 is a direct target of Pax1 and Pax9. Together, we conclude that Pax1 and Pax9 are required and sufficient for the chondrogenic differentiation of sclerotomal cells.

Sequential chemical-biological processes for the treatment of industrial wastewaters: review of recent progresses and critical assessment.

PubMed

Guieysse, Benoit; Norvill, Zane N

2014-02-28

When direct wastewater biological treatment is unfeasible, a cost- and resource-efficient alternative to direct chemical treatment consists of combining biological treatment with a chemical pre-treatment aiming to convert the hazardous pollutants into more biodegradable compounds. Whereas the principles and advantages of sequential treatment have been demonstrated for a broad range of pollutants and process configurations, recent progresses (2011-present) in the field provide the basis for refining assessment of feasibility, costs, and environmental impacts. This paper thus reviews recent real wastewater demonstrations at pilot and full scale as well as new process configurations. It also discusses new insights on the potential impacts of microbial community dynamics on process feasibility, design and operation. Finally, it sheds light on a critical issue that has not yet been properly addressed in the field: integration requires complex and tailored optimization and, of paramount importance to full-scale application, is sensitive to uncertainty and variability in the inputs used for process design and operation. Future research is therefore critically needed to improve process control and better assess the real potential of sequential chemical-biological processes for industrial wastewater treatment. Copyright © 2013 Elsevier B.V. All rights reserved.

A real-time comparison between direct control, sequential pattern recognition control and simultaneous pattern recognition control using a Fitts’ law style assessment procedure

PubMed Central

2014-01-01

Background Pattern recognition (PR) based strategies for the control of myoelectric upper limb prostheses are generally evaluated through offline classification accuracy, which is an admittedly useful metric, but insufficient to discuss functional performance in real time. Existing functional tests are extensive to set up and most fail to provide a challenging, objective framework to assess the strategy performance in real time. Methods Nine able-bodied and two amputee subjects gave informed consent and participated in the local Institutional Review Board approved study. We designed a two-dimensional target acquisition task, based on the principles of Fitts’ law for human motor control. Subjects were prompted to steer a cursor from the screen center of into a series of subsequently appearing targets of different difficulties. Three cursor control systems were tested, corresponding to three electromyography-based prosthetic control strategies: 1) amplitude-based direct control (the clinical standard of care), 2) sequential PR control, and 3) simultaneous PR control, allowing for a concurrent activation of two degrees of freedom (DOF). We computed throughput (bits/second), path efficiency (%), reaction time (second), and overshoot (%)) and used general linear models to assess significant differences between the strategies for each metric. Results We validated the proposed methodology by achieving very high coefficients of determination for Fitts’ law. Both PR strategies significantly outperformed direct control in two-DOF targets and were more intuitive to operate. In one-DOF targets, the simultaneous approach was the least precise. The direct control was efficient in one-DOF targets but cumbersome to operate in two-DOF targets through a switch-depended sequential cursor control. Conclusions We designed a test, capable of comprehensively describing prosthetic control strategies in real time. When implemented on control subjects, the test was able to capture statistically significant differences (p < 0.05) in control strategies when considering throughputs, path efficiencies and reaction times. Of particular note, we found statistically significant (p < 0.01) improvements in throughputs and path efficiencies with simultaneous PR when compared to direct control or sequential PR. Amputees could readily achieve the task; however a limited number of subjects was tested and a statistical analysis was not performed with that population. PMID:24886664

A real-time comparison between direct control, sequential pattern recognition control and simultaneous pattern recognition control using a Fitts' law style assessment procedure.

PubMed

Wurth, Sophie M; Hargrove, Levi J

2014-05-30

Pattern recognition (PR) based strategies for the control of myoelectric upper limb prostheses are generally evaluated through offline classification accuracy, which is an admittedly useful metric, but insufficient to discuss functional performance in real time. Existing functional tests are extensive to set up and most fail to provide a challenging, objective framework to assess the strategy performance in real time. Nine able-bodied and two amputee subjects gave informed consent and participated in the local Institutional Review Board approved study. We designed a two-dimensional target acquisition task, based on the principles of Fitts' law for human motor control. Subjects were prompted to steer a cursor from the screen center of into a series of subsequently appearing targets of different difficulties. Three cursor control systems were tested, corresponding to three electromyography-based prosthetic control strategies: 1) amplitude-based direct control (the clinical standard of care), 2) sequential PR control, and 3) simultaneous PR control, allowing for a concurrent activation of two degrees of freedom (DOF). We computed throughput (bits/second), path efficiency (%), reaction time (second), and overshoot (%)) and used general linear models to assess significant differences between the strategies for each metric. We validated the proposed methodology by achieving very high coefficients of determination for Fitts' law. Both PR strategies significantly outperformed direct control in two-DOF targets and were more intuitive to operate. In one-DOF targets, the simultaneous approach was the least precise. The direct control was efficient in one-DOF targets but cumbersome to operate in two-DOF targets through a switch-depended sequential cursor control. We designed a test, capable of comprehensively describing prosthetic control strategies in real time. When implemented on control subjects, the test was able to capture statistically significant differences (p < 0.05) in control strategies when considering throughputs, path efficiencies and reaction times. Of particular note, we found statistically significant (p < 0.01) improvements in throughputs and path efficiencies with simultaneous PR when compared to direct control or sequential PR. Amputees could readily achieve the task; however a limited number of subjects was tested and a statistical analysis was not performed with that population.

Biomarkers of Renal Tumor Burden and Progression in TSC

DTIC Science & Technology

2013-09-01

found no significant increase in CKD in those TSC patients with cysts versus those with normal renal anatomy , excluding patients with PKD, 8 and...similarly no significant increase in CKD in patients with “undisturbed” AMLs versus those with cysts or normal renal anatomy . Biopsy of AMLs had no...SMDF (NRG1 Isoform), Soggy-1, Sonic Hedgehog (ShhN-terminal), SPARC, Spinesin, TACI (TNFRSF13B), Tarc, TCCR (WSX-1), TECK (CCL25), TFPI, TGF alpha, TGF

Integrated Proteomic and Transcriptomic-Based Approaches to Identifying Signature Biomarkers and Pathways for Elucidation of Daoy and UW228 Subtypes.

PubMed

Higdon, Roger; Kala, Jessie; Wilkins, Devan; Yan, Julia Fangfei; Sethi, Manveen K; Lin, Liang; Liu, Siqi; Montague, Elizabeth; Janko, Imre; Choiniere, John; Kolker, Natali; Hancock, William S; Kolker, Eugene; Fanayan, Susan

2017-02-03

Medulloblastoma (MB) is the most common malignant pediatric brain tumor. Patient survival has remained largely the same for the past 20 years, with therapies causing significant health, cognitive, behavioral and developmental complications for those who survive the tumor. In this study, we profiled the total transcriptome and proteome of two established MB cell lines, Daoy and UW228, using high-throughput RNA sequencing (RNA-Seq) and label-free nano-LC-MS/MS-based quantitative proteomics, coupled with advanced pathway analysis. While Daoy has been suggested to belong to the sonic hedgehog (SHH) subtype, the exact UW228 subtype is not yet clearly established. Thus, a goal of this study was to identify protein markers and pathways that would help elucidate their subtype classification. A number of differentially expressed genes and proteins, including a number of adhesion, cytoskeletal and signaling molecules, were observed between the two cell lines. While several cancer-associated genes/proteins exhibited similar expression across the two cell lines, upregulation of a number of signature proteins and enrichment of key components of SHH and WNT signaling pathways were uniquely observed in Daoy and UW228, respectively. The novel information on differentially expressed genes/proteins and enriched pathways provide insights into the biology of MB, which could help elucidate their subtype classification.

Extracellular Vesicles as Therapeutic Tools in Cardiovascular Diseases

PubMed Central

Fleury, Audrey; Martinez, Maria Carmen; Le Lay, Soazig

2014-01-01

Extracellular vesicles (EVs), including microvesicles (MVs) and exosomes, are small vesicles secreted from a wide variety of cells. Whereas MVs are particles released by the outward budding of the plasma membrane, exosomes are derived from endocytic compartments. Secretion of EVs can be enhanced by specific stimuli, and increased plasma circulating levels of EVs have been correlated with pathophysiological situations. MVs, already present in the blood of healthy individuals, are considerably elevated in several cardiovascular diseases associated with inflammation, suggesting that they can mediate deleterious effects such as endothelial dysfunction or thrombosis. Nonetheless, very recent studies also demonstrate that MVs may act as biological information vectors transferring proteins or genetic material to maintain cell homeostasis, favor cell repair, or even promote angiogenesis. Additionally, exosomes have also been shown to have pro-angiogenic and cardio-protective properties. These beneficial effects, therefore, reveal the potential therapeutical use of EVs in the field of cardiovascular medicine and regenerative therapy. In this review, we will provide an update of cellular processes modulated by EVs of specific interest in the treatment of cardiovascular pathologies. A special focus will be made on the morphogen sonic hedgehog (Shh) associated with EVs (EVsShh+), which have been shown to mediate many pro-angiogenic effects. In addition to offer a potential source of cardiovascular markers, therapeutical potential of EVs reveal exciting opportunities to deliver specific agents by non-immunogenic means to cardiovascular system. PMID:25136343

Vertebrate limb development: moving from classical morphogen gradients to an integrated 4-dimensional patterning system.

PubMed

Bénazet, Jean-Denis; Zeller, Rolf

2009-10-01

A wealth of classical embryological manipulation experiments taking mainly advantage of the chicken limb buds identified the apical ectodermal ridge (AER) and the zone of polarizing activity (ZPA) as the respective ectodermal and mesenchymal key signaling centers coordinating proximodistal (PD) and anteroposterior (AP) limb axis development. These experiments inspired Wolpert's French flag model, which is a classic among morphogen gradient models. Subsequent molecular and genetic analysis in the mouse identified retinoic acid as proximal signal, and fibroblast growth factors (FGFs) and sonic hedgehog (SHH) as the essential instructive signals produced by AER and ZPA, respectively. Recent studies provide good evidence that progenitors are specified early with respect to their PD and AP fates and that morpho-regulatory signaling is also required for subsequent proliferative expansion of the specified progenitor pools. The determination of particular fates seems to occur rather late and depends on additional signals such as bone morphogenetic proteins (BMPs), which indicates that cells integrate signaling inputs over time and space. The coordinate regulation of PD and AP axis patterning is controlled by an epithelial-mesenchymal feedback signaling system, in which transcriptional regulation of the BMP antagonist Gremlin1 integrates inputs from the BMP, SHH, and FGF pathways. Vertebrate limb-bud development is controlled by a 4-dimensional (4D) patterning system integrating positive and negative regulatory feedback loops, rather than thresholds set by morphogen gradients.

[Medulloblastoma. Pathology].

PubMed

Siegfried, A; Delisle, M-B

2018-04-24

Medulloblastomas, embryonal neuroepithelial tumors developed in the cerebellum or brain stem, are mainly observed in childhood. The treatment of WHO-Grade IV tumors depends on stratifications that are usually based on postoperative data, histopathological subtype, tumor extension and presence of MYC or NMYC amplifications. Recently, molecular biology studies, based on new technologies (i.e. sequencing, transcriptomic, methylomic) have introduced genetic subtypes integrated into the latest WHO-2016 neuropathological classification. According to this classification, the three genetic groups WNT, SHH, with or without mutated TP53 gene, and non-WNT/non-SHH, comprising subgroups 3 and 4, are recalled in this review. The contribution of immunohistochemistry to define these groups is specified. The four histopathological groups are detailed in comparison to the WHO-2007 classification and the molecular data: classic medulloblastoma, desmoplastic/nodular medulloblastoma, medulloblastoma with extensive nodularity, and large cell/anaplastic medulloblastoma. The groups defined on genetic and histopathological grounds are not strictly concordant. Depending on the age of the patients, their correlations are different, as well as their role in the management and prognosis of these tumors. Other embryonal tumors, for which new classifications are in progress and gliomas may be confused with a medulloblastoma and the elements of the differential diagnosis of these entities are discussed. This evolution in classification fully justifies ongoing structuring procedures such as histopathological review (RENOCLIP) and the organization of molecular biology platforms. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Shh signaling from the nucleus pulposus is required for the postnatal growth and differentiation of the mouse intervertebral disc.

PubMed

Dahia, Chitra Lekha; Mahoney, Eric; Wylie, Christopher

2012-01-01

Intervertebral discs (IVD) are essential components of the vertebral column. They maintain separation, and provide shock absorbing buffers, between adjacent vertebrae, while also allowing movements between them. Each IVD consists of a central semi-liquid nucleus pulposus (NP) surrounded by a multi-layered fibrocartilagenous annulus fibrosus (AF). Although the IVDs grow and differentiate after birth along with the vertebral column, little is known about the mechanism of this. Understanding the signals that control normal IVD growth and differentiation would also provide potential therapies for degenerative disc disease, which is the major cause of lower back pain and affects a large proportion of the population. In this work, we show that during postnatal growth of the mouse, Sonic hedgehog (Shh) signaling from the NP cells controls many aspects of growth and differentiation of both the NP cells themselves and of the surrounding AF, and that it acts, at least partly, by regulating other signaling pathways in the NP and AF. Recent studies have shown that the NP cells arise from the embryonic notochord, which acts as a major signaling center in the embryo. This work shows that this notochord-derived tissue continues to carry out a major signaling function in the postnatal body and that the IVDs are signaling centers, in addition to their already known functions in the mechanics of vertebral column function.

Purmorphamine as a Shh Signaling Activator Small Molecule Promotes Motor Neuron Differentiation of Mesenchymal Stem Cells Cultured on Nanofibrous PCL Scaffold.

PubMed

Bahrami, Naghmeh; Bayat, Mohammad; Mohamadnia, Abdolreza; Khakbiz, Mehrdad; Yazdankhah, Meysam; Ai, Jafar; Ebrahimi-Barough, Somayeh

2017-09-01

There is variety of stem cell sources but problems in ethical issues, contamination, and normal karyotype cause many limitations in obtaining and using these cells. The cells in Wharton's jelly region of umbilical cord are abundant and available stem cells with low immunological incompatibility, which could be considered for cell replacement therapy. Small molecules have been presented as less expensive biologically active compounds that can regulate different developmental process. Purmorphamine (PMA) is a small molecule that, according to some studies, possesses certain differentiation effects. In this study, we investigated the effect of the PMA on Wharton's jelly mesenchymal stem cell (WJ-MSC) differentiation into motor neuronal lineages instead of sonic hedgehog (Shh) on PCL scaffold. After exposing to induction media for 15 days, the cells were characterized for expression of motor neuron markers including PAX6, NF-H, Islet1, HB9, and choline acetyl transferase (ChAT) by quantitative reverse transcription (PCR) and immunocytochemistry. Our results demonstrated that induced WJ-MSCs with PMA could significantly express motor neuron markers in RNA and protein levels 15 days post induction. These results suggested that WJ-MSCs can differentiate to motor neuron-like cells with PMA on PCL scaffold and might provide a potential source in cell therapy for nervous system.

A sequential analysis of classroom discourse in Italian primary schools: the many faces of the IRF pattern.

PubMed

Molinari, Luisa; Mameli, Consuelo; Gnisci, Augusto

2013-09-01

A sequential analysis of classroom discourse is needed to investigate the conditions under which the triadic initiation-response-feedback (IRF) pattern may host different teaching orientations. The purpose of the study is twofold: first, to describe the characteristics of classroom discourse and, second, to identify and explore the different interactive sequences that can be captured with a sequential statistical analysis. Twelve whole-class activities were video recorded in three Italian primary schools. We observed classroom interaction as it occurs naturally on an everyday basis. In total, we collected 587 min of video recordings. Subsequently, 828 triadic IRF patterns were extracted from this material and analysed with the programme Generalized Sequential Query (GSEQ). The results indicate that classroom discourse may unfold in different ways. In particular, we identified and described four types of sequences. Dialogic sequences were triggered by authentic questions, and continued through further relaunches. Monologic sequences were directed to fulfil the teachers' pre-determined didactic purposes. Co-constructive sequences fostered deduction, reasoning, and thinking. Scaffolding sequences helped and sustained children with difficulties. The application of sequential analyses allowed us to show that interactive sequences may account for a variety of meanings, thus making a significant contribution to the literature and research practice in classroom discourse. © 2012 The British Psychological Society.

Sequential strand displacement beacon for detection of DNA coverage on functionalized gold nanoparticles.

PubMed

Paliwoda, Rebecca E; Li, Feng; Reid, Michael S; Lin, Yanwen; Le, X Chris

2014-06-17

Functionalizing nanomaterials for diverse analytical, biomedical, and therapeutic applications requires determination of surface coverage (or density) of DNA on nanomaterials. We describe a sequential strand displacement beacon assay that is able to quantify specific DNA sequences conjugated or coconjugated onto gold nanoparticles (AuNPs). Unlike the conventional fluorescence assay that requires the target DNA to be fluorescently labeled, the sequential strand displacement beacon method is able to quantify multiple unlabeled DNA oligonucleotides using a single (universal) strand displacement beacon. This unique feature is achieved by introducing two short unlabeled DNA probes for each specific DNA sequence and by performing sequential DNA strand displacement reactions. Varying the relative amounts of the specific DNA sequences and spacing DNA sequences during their coconjugation onto AuNPs results in different densities of the specific DNA on AuNP, ranging from 90 to 230 DNA molecules per AuNP. Results obtained from our sequential strand displacement beacon assay are consistent with those obtained from the conventional fluorescence assays. However, labeling of DNA with some fluorescent dyes, e.g., tetramethylrhodamine, alters DNA density on AuNP. The strand displacement strategy overcomes this problem by obviating direct labeling of the target DNA. This method has broad potential to facilitate more efficient design and characterization of novel multifunctional materials for diverse applications.

Sequential growth for lifetime extension in biomimetic polypyrrole actuator systems

NASA Astrophysics Data System (ADS)

Sarrazin, J. C.; Mascaro, Stephen A.

2015-04-01

Electroactive polymers (EAPs) present prospective use in actuation and manipulation devices due to their low electrical activation requirements, biocompatibility, and mechanical performance. One of the main drawbacks with EAP actuators is a decrease in performance over extended periods of operation caused by over-oxidation of the polymer and general polymer degradation. Synthesis of the EAP material, polypyrrole with an embedded metal helix allows for sequential growth of the polymer during operation. The helical metal electrode acts as a scaffolding to support the polymer, and direct the 3-dimensional change in volume of the polymer along the axis of the helix during oxidative and reductive cycling. The metal helix also provides a working metal electrode through the entire length of the polymer actuator to distribute charge for actuation, as well as for sequential growth steps during the lifetime of operation of the polymer. This work demonstrates the method of sequential growth can be utilized after extended periods of use to partially restore electrical and mechanical performance of polypyrrole actuators. Since the actuation must be temporarily stopped to allow for a sequential growth cycle to be performed and reverse some of the polymer degradation, these actuator systems more closely mimic natural muscle in their analogous maintenance and repair.

Parallel solution of closely coupled systems

NASA Technical Reports Server (NTRS)

Utku, S.; Salama, M.

1986-01-01

The odd-even permutation and associated unitary transformations for reordering the matrix coefficient A are employed as means of breaking the strong seriality which is characteristic of closely coupled systems. The nested dissection technique is also reviewed, and the equivalence between reordering A and dissecting its network is established. The effect of transforming A with odd-even permutation on its topology and the topology of its Cholesky factors is discussed. This leads to the construction of directed graphs showing the computational steps required for factoring A, their precedence relationships and their sequential and concurrent assignment to the available processors. Expressions for the speed-up and efficiency of using N processors in parallel relative to the sequential use of a single processor are derived from the directed graph. Similar expressions are also derived when the number of available processors is fewer than required.

Structural insights into photocatalytic performance of carbon nitrides for degradation of organic pollutants

NASA Astrophysics Data System (ADS)

Oh, Junghoon; Shim, Yeonjun; Lee, Soomin; Park, Sunghee; Jang, Dawoon; Shin, Yunseok; Ohn, Saerom; Kim, Jeongho; Park, Sungjin

2018-02-01

Degradation of organic pollutants has a large environmental impact, with graphitic carbon nitride (g-C3N4) being a promising metal-free, low cost, and environment-friendly photocatalyst well suited for this purpose. Herein, we investigate the photocatalytic performance of g-C3N4-based materials and correlate it with their structural properties, using three different precursors (dicyandiamide, melamine, and urea) and two heating processes (direct heating at 550 °C and sequential heating at 300 and 550 °C) to produce the above photocatalysts. We further demonstrate that sequential heating produces photocatalysts with grain sizes and activities larger than those of the catalysts produced by direct heating and that the use of urea as a precursor affords photocatalysts with larger surface areas, allowing efficient rhodamine B degradation under visible light.

Imaging sequential dehydrogenation of methanol on Cu(110) with a scanning tunneling microscope.

PubMed

Kitaguchi, Y; Shiotari, A; Okuyama, H; Hatta, S; Aruga, T

2011-05-07

Adsorption of methanol and its dehydrogenation on Cu(110) were studied by using a scanning tunneling microscope (STM). Upon adsorption at 12 K, methanol preferentially forms clusters on the surface. The STM could induce dehydrogenation of methanol sequentially to methoxy and formaldehyde. This enabled us to study the binding structures of these products in a single-molecule limit. Methoxy was imaged as a pair of protrusion and depression along the [001] direction. This feature is fully consistent with the previous result that it adsorbs on the short-bridge site with the C-O axis tilted along the [001] direction. The axis was induced to flip back and forth by vibrational excitations with the STM. Two configurations were observed for formaldehyde, whose structures were proposed based on their characteristic images and motions.

Development of Cranial Bone Surrogate Structures Using Stereolithographic Additive Manufacturing

DTIC Science & Technology

2017-09-29

shown in Fig. 5. With each cycle, a blade is passed across the platform to create a uniform layer of resin. The resin layer is exposed to a UV laser...due to the direction in which the layers are deposited. In both cases, the sequential layers run parallel to the loading direction of the tensile

Comparing Parent-Child Interactions in the Clinic and at Home: An Exploration of the Validity of Clinical Behavior Observations Using Sequential Analysis

ERIC Educational Resources Information Center

Shriver, Mark D.; Frerichs, Lynae J.; Williams, Melissa; Lancaster, Blake M.

2013-01-01

Direct observation is often considered the "gold standard" for assessing the function, frequency, and intensity of problem behavior. Currently, the literature investigating the construct validity of direct observation conducted in the clinic setting reveals conflicting results. Previous studies on the construct validity of clinic-based…

Effect of Sequential Treatment with Bisphosphonates After Teriparatide in Ovariectomized Rats: A Direct Comparison Between Risedronate and Alendronate.

PubMed

Yano, Tetsuo; Yamada, Mei; Inoue, Daisuke

2017-07-01

Teriparatide (TPTD), a recombinant human parathyroid hormone N-terminal fragment (1-34), is a widely used bone anabolic drug for osteoporosis. Sequential treatment with antiresorptives such as bisphosphonates after TPTD discontinuation is generally recommended. However, relative effects of bisphosphonates have not been determined. In the present study, we directly compared effects of risedronate (RIS) and alendronate (ALN) on bone mineral density (BMD), bone turnover, structural property and strength in ovariectomized (OVX) rats, when administered after TPTD. Female Sprague Dawley rats were divided into one sham-operated and eight ovariectomized groups. TPTD, RIS, and ALN were given subcutaneously twice per week for 4 or 8 weeks after 4 week treatment with TPTD. TPTD significantly increased BMD (+9.6%) in OVX rats after 4 weeks of treatment. 8 weeks after TPTD withdrawal, vehicle-treated group showed a blunted BMD increase of +8.4% from the baseline. In contrast, 8 weeks of treatment with RIS and ALN significantly increased BMD to 17.4 and 21.8%, respectively. While ALN caused a consistently larger increase in BMD, sequential treatment with RIS resulted in lower Tb.Sp compared to ALN in the fourth lumbar vertebra as well as in greater stiffness in compression test. In conclusion, the present study demonstrated that sequential therapy with ALN and RIS after TPTD both improved bone mass and structure. Our results further suggest that RIS may have a greater effect on improving bone quality and stiffness than ALN despite less prominent effect on BMD. Further studies are necessary to determine clinical relevance of these findings to fracture rate.

Direct Synthesis of Medium-Bridged Twisted Amides via a Transannular Cyclization Strategy

PubMed Central

Szostak, Michal; Aubé, Jeffrey

2009-01-01

The sequential RCM to construct a challenging medium-sized ring followed by a transannular cyclization across a medium-sized ring delivers previously unattainable twisted amides from simple acyclic precursors. PMID:19708701

Understanding Selective Downregulation of c-Myc Expression through Inhibition of General Transcription Regulators in Multiple Myeloma

DTIC Science & Technology

2015-06-01

Love, and S. Gupta at the Whitehead Genome Core for assistance with genome sequencing . This research was supported by NIH K08 HL105678, The Wat...efficient alignment of short DNA sequences to the human genome . Genome Bioi. 10, R25. LeRoy, G., Rickards, B., and Flint, S.J. (2008). The double...of the beginning. Nature reviews. Cancer 12, 818-834, doi:10.1038/nrc3410 (2012). 12 Kool, M. et al. Genome sequencing of SHH medulloblastoma

The Role of the Sonic Hedgehog Pathway for Prostate Cancer Progression

DTIC Science & Technology

2007-02-01

hepatocellular carcinomas : through transcriptional activation of the ligand Shh (Carcinogenesis 27: 1334-40, 2006). Second, our studies of Su(Fu...in hepatocellular carcinomas . Fig. 3 shows that the sonic hedgehog promoter activity is high in Huh7 cells but low in HepG2 cells. In the presence of... hepatocellular carcinomas and prostate cancer. LNCaP cells 0 50 100 150 200 250 300 Vector (-1800 to -200) (-680 to -200) R el at iv e lu ci fe ra

Goal-Directed Decision Making with Spiking Neurons.

PubMed

Friedrich, Johannes; Lengyel, Máté

2016-02-03

Behavioral and neuroscientific data on reward-based decision making point to a fundamental distinction between habitual and goal-directed action selection. The formation of habits, which requires simple updating of cached values, has been studied in great detail, and the reward prediction error theory of dopamine function has enjoyed prominent success in accounting for its neural bases. In contrast, the neural circuit mechanisms of goal-directed decision making, requiring extended iterative computations to estimate values online, are still unknown. Here we present a spiking neural network that provably solves the difficult online value estimation problem underlying goal-directed decision making in a near-optimal way and reproduces behavioral as well as neurophysiological experimental data on tasks ranging from simple binary choice to sequential decision making. Our model uses local plasticity rules to learn the synaptic weights of a simple neural network to achieve optimal performance and solves one-step decision-making tasks, commonly considered in neuroeconomics, as well as more challenging sequential decision-making tasks within 1 s. These decision times, and their parametric dependence on task parameters, as well as the final choice probabilities match behavioral data, whereas the evolution of neural activities in the network closely mimics neural responses recorded in frontal cortices during the execution of such tasks. Our theory provides a principled framework to understand the neural underpinning of goal-directed decision making and makes novel predictions for sequential decision-making tasks with multiple rewards. Goal-directed actions requiring prospective planning pervade decision making, but their circuit-level mechanisms remain elusive. We show how a model circuit of biologically realistic spiking neurons can solve this computationally challenging problem in a novel way. The synaptic weights of our network can be learned using local plasticity rules such that its dynamics devise a near-optimal plan of action. By systematically comparing our model results to experimental data, we show that it reproduces behavioral decision times and choice probabilities as well as neural responses in a rich set of tasks. Our results thus offer the first biologically realistic account for complex goal-directed decision making at a computational, algorithmic, and implementational level. Copyright © 2016 the authors 0270-6474/16/361529-18$15.00/0.

Goal-Directed Decision Making with Spiking Neurons

PubMed Central

Lengyel, Máté

2016-01-01

Behavioral and neuroscientific data on reward-based decision making point to a fundamental distinction between habitual and goal-directed action selection. The formation of habits, which requires simple updating of cached values, has been studied in great detail, and the reward prediction error theory of dopamine function has enjoyed prominent success in accounting for its neural bases. In contrast, the neural circuit mechanisms of goal-directed decision making, requiring extended iterative computations to estimate values online, are still unknown. Here we present a spiking neural network that provably solves the difficult online value estimation problem underlying goal-directed decision making in a near-optimal way and reproduces behavioral as well as neurophysiological experimental data on tasks ranging from simple binary choice to sequential decision making. Our model uses local plasticity rules to learn the synaptic weights of a simple neural network to achieve optimal performance and solves one-step decision-making tasks, commonly considered in neuroeconomics, as well as more challenging sequential decision-making tasks within 1 s. These decision times, and their parametric dependence on task parameters, as well as the final choice probabilities match behavioral data, whereas the evolution of neural activities in the network closely mimics neural responses recorded in frontal cortices during the execution of such tasks. Our theory provides a principled framework to understand the neural underpinning of goal-directed decision making and makes novel predictions for sequential decision-making tasks with multiple rewards. SIGNIFICANCE STATEMENT Goal-directed actions requiring prospective planning pervade decision making, but their circuit-level mechanisms remain elusive. We show how a model circuit of biologically realistic spiking neurons can solve this computationally challenging problem in a novel way. The synaptic weights of our network can be learned using local plasticity rules such that its dynamics devise a near-optimal plan of action. By systematically comparing our model results to experimental data, we show that it reproduces behavioral decision times and choice probabilities as well as neural responses in a rich set of tasks. Our results thus offer the first biologically realistic account for complex goal-directed decision making at a computational, algorithmic, and implementational level. PMID:26843636

Temporal texture of associative encoding modulates recall processes.

PubMed

Tibon, Roni; Levy, Daniel A

2014-02-01

Binding aspects of an experience that are distributed over time is an important element of episodic memory. In the current study, we examined how the temporal complexity of an experience may govern the processes required for its retrieval. We recorded event-related potentials during episodic cued recall following pair associate learning of concurrently and sequentially presented object-picture pairs. Cued recall success effects over anterior and posterior areas were apparent in several time windows. In anterior locations, these recall success effects were similar for concurrently and sequentially encoded pairs. However, in posterior sites clustered over parietal scalp the effect was larger for the retrieval of sequentially encoded pairs. We suggest that anterior aspects of the mid-latency recall success effects may reflect working-with-memory operations or direct access recall processes, while more posterior aspects reflect recollective processes which are required for retrieval of episodes of greater temporal complexity. Copyright © 2013 Elsevier Inc. All rights reserved.

Using Priced Options to Solve the Exposure Problem in Sequential Auctions

NASA Astrophysics Data System (ADS)

Mous, Lonneke; Robu, Valentin; La Poutré, Han

This paper studies the benefits of using priced options for solving the exposure problem that bidders with valuation synergies face when participating in multiple, sequential auctions. We consider a model in which complementary-valued items are auctioned sequentially by different sellers, who have the choice of either selling their good directly or through a priced option, after fixing its exercise price. We analyze this model from a decision-theoretic perspective and we show, for a setting where the competition is formed by local bidders, that using options can increase the expected profit for both buyers and sellers. Furthermore, we derive the equations that provide minimum and maximum bounds between which a synergy buyer's bids should fall in order for both sides to have an incentive to use the options mechanism. Next, we perform an experimental analysis of a market in which multiple synergy bidders are active simultaneously.

A Developmental Perspective on Peer Rejection, Deviant Peer Affiliation, and Conduct Problems Among Youth.

PubMed

Chen, Diane; Drabick, Deborah A G; Burgers, Darcy E

2015-12-01

Peer rejection and deviant peer affiliation are linked consistently to the development and maintenance of conduct problems. Two proposed models may account for longitudinal relations among these peer processes and conduct problems: the (a) sequential mediation model, in which peer rejection in childhood and deviant peer affiliation in adolescence mediate the link between early externalizing behaviors and more serious adolescent conduct problems; and (b) parallel process model, in which peer rejection and deviant peer affiliation are considered independent processes that operate simultaneously to increment risk for conduct problems. In this review, we evaluate theoretical models and evidence for associations among conduct problems and (a) peer rejection and (b) deviant peer affiliation. We then consider support for the sequential mediation and parallel process models. Next, we propose an integrated model incorporating both the sequential mediation and parallel process models. Future research directions and implications for prevention and intervention efforts are discussed.

A Developmental Perspective on Peer Rejection, Deviant Peer Affiliation, and Conduct Problems among Youth

PubMed Central

Chen, Diane; Drabick, Deborah A. G.; Burgers, Darcy E.

2015-01-01

Peer rejection and deviant peer affiliation are linked consistently to the development and maintenance of conduct problems. Two proposed models may account for longitudinal relations among these peer processes and conduct problems: the (a) sequential mediation model, in which peer rejection in childhood and deviant peer affiliation in adolescence mediate the link between early externalizing behaviors and more serious adolescent conduct problems; and (b) parallel process model, in which peer rejection and deviant peer affiliation are considered independent processes that operate simultaneously to increment risk for conduct problems. In this review, we evaluate theoretical models and evidence for associations among conduct problems and (a) peer rejection and (b) deviant peer affiliation. We then consider support for the sequential mediation and parallel process models. Next, we propose an integrated model incorporating both the sequential mediation and parallel process models. Future research directions and implications for prevention and intervention efforts are discussed. PMID:25410430

Implementation of Temperature Sequential Controller on Variable Speed Drive

NASA Astrophysics Data System (ADS)

Cheong, Z. X.; Barsoum, N. N.

2008-10-01

There are many pump and motor installations with quite extensive speed variation, such as Sago conveyor, heating, ventilation and air conditioning (HVAC) and water pumping system. A common solution for these applications is to run several fixed speed motors in parallel, with flow control accomplish by turning the motors on and off. This type of control method causes high in-rush current, and adds a risk of damage caused by pressure transients. This paper explains the design and implementation of a temperature speed control system for use in industrial and commercial sectors. Advanced temperature speed control can be achieved by using ABB ACS800 variable speed drive-direct torque sequential control macro, programmable logic controller and temperature transmitter. The principle of direct torque sequential control macro (DTC-SC) is based on the control of torque and flux utilizing the stator flux field orientation over seven preset constant speed. As a result of continuous comparison of ambient temperature to the references temperatures; electromagnetic torque response is particularly fast to the motor state and it is able maintain constant speeds. Experimental tests have been carried out by using ABB ACS800-U1-0003-2, to validate the effectiveness and dynamic respond of ABB ACS800 against temperature variation, loads, and mechanical shocks.

Research on parallel algorithm for sequential pattern mining

NASA Astrophysics Data System (ADS)

Zhou, Lijuan; Qin, Bai; Wang, Yu; Hao, Zhongxiao

2008-03-01

Sequential pattern mining is the mining of frequent sequences related to time or other orders from the sequence database. Its initial motivation is to discover the laws of customer purchasing in a time section by finding the frequent sequences. In recent years, sequential pattern mining has become an important direction of data mining, and its application field has not been confined to the business database and has extended to new data sources such as Web and advanced science fields such as DNA analysis. The data of sequential pattern mining has characteristics as follows: mass data amount and distributed storage. Most existing sequential pattern mining algorithms haven't considered the above-mentioned characteristics synthetically. According to the traits mentioned above and combining the parallel theory, this paper puts forward a new distributed parallel algorithm SPP(Sequential Pattern Parallel). The algorithm abides by the principal of pattern reduction and utilizes the divide-and-conquer strategy for parallelization. The first parallel task is to construct frequent item sets applying frequent concept and search space partition theory and the second task is to structure frequent sequences using the depth-first search method at each processor. The algorithm only needs to access the database twice and doesn't generate the candidated sequences, which abates the access time and improves the mining efficiency. Based on the random data generation procedure and different information structure designed, this paper simulated the SPP algorithm in a concrete parallel environment and implemented the AprioriAll algorithm. The experiments demonstrate that compared with AprioriAll, the SPP algorithm had excellent speedup factor and efficiency.

Bursts and heavy tails in temporal and sequential dynamics of foraging decisions.

PubMed

Jung, Kanghoon; Jang, Hyeran; Kralik, Jerald D; Jeong, Jaeseung

2014-08-01

A fundamental understanding of behavior requires predicting when and what an individual will choose. However, the actual temporal and sequential dynamics of successive choices made among multiple alternatives remain unclear. In the current study, we tested the hypothesis that there is a general bursting property in both the timing and sequential patterns of foraging decisions. We conducted a foraging experiment in which rats chose among four different foods over a continuous two-week time period. Regarding when choices were made, we found bursts of rapidly occurring actions, separated by time-varying inactive periods, partially based on a circadian rhythm. Regarding what was chosen, we found sequential dynamics in affective choices characterized by two key features: (a) a highly biased choice distribution; and (b) preferential attachment, in which the animals were more likely to choose what they had previously chosen. To capture the temporal dynamics, we propose a dual-state model consisting of active and inactive states. We also introduce a satiation-attainment process for bursty activity, and a non-homogeneous Poisson process for longer inactivity between bursts. For the sequential dynamics, we propose a dual-control model consisting of goal-directed and habit systems, based on outcome valuation and choice history, respectively. This study provides insights into how the bursty nature of behavior emerges from the interaction of different underlying systems, leading to heavy tails in the distribution of behavior over time and choices.

Increased efficacy of photodynamic therapy via sequential targeting

NASA Astrophysics Data System (ADS)

Kessel, David; Aggarwal, Neha; Sloane, Bonnie F.

2014-03-01

Photokilling depends on the generation of death signals after photosensitized cells are irradiated. A variety of intracellular organelles can be targeted for photodamage, often with a high degree of specificity. We have discovered that a low level of photodamage directed against lysosomes can sensitize both a murine hepatoma cell line (in 2D culture) and an inflammatory breast cancer line of human origin (in a 3D model) to subsequent photodamage directed at mitochondria. Additional studies were carried out with hepatoma cells to explore possible mechanisms. The phototoxic effect of the `sequential targeting' approach was associated with an increased apoptotic response. The low level of lysosomal photodamage did not lead to any detectable migration of Fe++ from lysosomes to mitochondria or increased reactive oxygen species (ROS) formation after subsequent mitochondrial photodamage. Instead, there appears to be a signal generated that can amplify the pro-apoptotic effect of subsequent mitochondrial photodamage.

Comprehension of Navigation Directions

NASA Technical Reports Server (NTRS)

Healy, Alice F.; Schneider, Vivian I.

2002-01-01

Subjects were shown navigation instructions varying in length directing them to move in a space represented by grids on a computer screen. They followed the instructions by clicking on the grids in the locations specified. Some subjects repeated back the instructions before following them, some did not, and others repeated back the instructions in reduced form, including only the critical words. The commands in each message were presented simultaneously for half of the subjects and sequentially for the others. For the longest messages, performance was better on the initial commands and worse on the final commands with simultaneous than with sequential presentation. Instruction repetition depressed performance, but reduced repetition removed this disadvantage. Effects of presentation format were attributed to visual scanning strategies. The advantage for reduced repetition was attributable either to enhanced visual scanning or to reduced output interference. A follow-up study with auditory presentation supported the visual scanning explanation.

Development of an efficient genetic manipulation strategy for sequential gene disruption and expression of different heterologous GFP genes in Candida tropicalis.

PubMed

Zhang, Lihua; Chen, Xianzhong; Chen, Zhen; Wang, Zezheng; Jiang, Shan; Li, Li; Pötter, Markus; Shen, Wei; Fan, You

2016-11-01

The diploid yeast Candida tropicalis, which can utilize n-alkane as a carbon and energy source, is an attractive strain for both physiological studies and practical applications. However, it presents some characteristics, such as rare codon usage, difficulty in sequential gene disruption, and inefficiency in foreign gene expression, that hamper strain improvement through genetic engineering. In this work, we present a simple and effective method for sequential gene disruption in C. tropicalis based on the use of an auxotrophic mutant host defective in orotidine monophosphate decarboxylase (URA3). The disruption cassette, which consists of a functional yeast URA3 gene flanked by a 0.3 kb gene disruption auxiliary sequence (gda) direct repeat derived from downstream or upstream of the URA3 gene and of homologous arms of the target gene, was constructed and introduced into the yeast genome by integrative transformation. Stable integrants were isolated by selection for Ura + and identified by PCR and sequencing. The important feature of this construct, which makes it very attractive, is that recombination between the flanking direct gda repeats occurs at a high frequency (10 -8 ) during mitosis. After excision of the URA3 marker, only one copy of the gda sequence remains at the recombinant locus. Thus, the resulting ura3 strain can be used again to disrupt a second allelic gene in a similar manner. In addition to this effective sequential gene disruption method, a codon-optimized green fluorescent protein-encoding gene (GFP) was functionally expressed in C. tropicalis. Thus, we propose a simple and reliable method to improve C. tropicalis by genetic manipulation.

Ochratoxin A at nanomolar concentration perturbs the homeostasis of neural stem cells in highly differentiated but not in immature three-dimensional brain cell cultures.

PubMed

Zurich, Marie-Gabrielle; Honegger, Paul

2011-08-28

Ochratoxin A (OTA), a fungal contaminant of basic food commodities, is known to be highly cytotoxic, but the pathways underlying adverse effects at subcytotoxic concentrations remain to be elucidated. Recent reports indicate that OTA affects cell cycle regulation. Therefore, 3D brain cell cultures were used to study OTA effects on mitotically active neural stem/progenitor cells, comparing highly differentiated cultures with their immature counterparts. Changes in the rate of DNA synthesis were related to early changes in the mRNA expression of neural stem/progenitor cell markers. OTA at 10nM, a concentration below the cytotoxic level, was ineffective in immature cultures, whereas in mature cultures it significantly decreased the rate of DNA synthesis together with the mRNA expression of key transcriptional regulators such as Sox2, Mash1, Hes5, and Gli1; the cell cycle activator cyclin D2; the phenotypic markers nestin, doublecortin, and PDGFRα. These effects were largely prevented by Sonic hedgehog (Shh) peptide (500ngml(-1)) administration, indicating that OTA impaired the Shh pathway and the Sox2 regulatory transcription factor critical for stem cell self-renewal. Similar adverse effects of OTA in vivo might perturb the regulation of stem cell proliferation in the adult brain and in other organs exhibiting homeostatic and/or regenerative cell proliferation. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Selection of the in vitro culture media influences mRNA expression of Hedgehog genes, Il-6, and important genes regarding reactive oxygen species in single murine preimplantation embryos.

PubMed

Pfeifer, N; Baston-Büst, D M; Hirchenhain, J; Friebe-Hoffmann, U; Rein, D T; Krüssel, J S; Hess, A P

2012-01-01

The aim of this paper was to determine the influence of different in vitro culture media on mRNA expression of Hedgehog genes, il-6, and important genes regarding reactive oxygen species in single mouse embryos. Reverse transcription of single embryos either cultured in vitro from day 0.5 until 3.5 (COOK's Cleavage medium or Vitrolife's G-1 PLUS medium) or in vivo until day 3.5 post coitum. PCR was carried out for β-actin followed by nested-PCR for shh, ihh, il-6, nox, gpx4, gpx1, and prdx2. The number of murine blastocysts cultured in COOK medium which expressed il-6, gpx4, gpx1, and prdx2 mRNA differed significantly compared to the in vivo group. Except for nox, the mRNA profile of the Vitrolife media group embryos varied significantly from the in vivo ones regarding the number of blastocysts expressing the mRNA of shh, ihh, il-6, gpx4, gpx1 and prdx2. The present study shows that different in vitro culture media lead to different mRNA expression profiles during early development. Even the newly developed in vitro culture media are not able to mimic the female reproductive tract. The question of long-term consequences for children due to assisted reproduction techniques needs to be addressed in larger studies.

Sirenomelia Phenotype in Bmp7;Shh Compound Mutants: A Novel Experimental Model for Studies of Caudal Body Malformations

PubMed Central

Garrido-Allepuz, Carlos; González-Lamuño, Domingo; Ros, Maria A.

2012-01-01

Sirenomelia is a severe congenital malformation of the lower body characterized by the fusion of the legs into a single lower limb. This striking external phenotype consistently associates severe visceral abnormalities, most commonly of the kidneys, intestine, and genitalia that generally make the condition lethal. Although the causes of sirenomelia remain unknown, clinical studies have yielded two major hypotheses: i) a primary defect in the generation of caudal mesoderm, ii) a primary vascular defect that leaves the caudal part of the embryo hypoperfused. Interestingly, Sirenomelia has been shown to have a genetic basis in mice, and although it has been considered a sporadic condition in humans, recently some possible familial cases have been reported. Here, we report that the removal of one or both functional alleles of Shh from the Bmp7-null background leads to a sirenomelia phenotype that faithfully replicates the constellation of external and internal malformations, typical of the human condition. These mutants represent an invaluable model in which we have analyzed the pathogenesis of sirenomelia. We show that the signaling defect predominantly impacts the morphogenesis of the hindgut and the development of the caudal end of the dorsal aortas. The deficient formation of ventral midline structures, including the interlimb mesoderm caudal to the umbilicus, leads to the approximation and merging of the hindlimb fields. Our study provides new insights for the understanding of the mechanisms resulting in caudal body malformations, including sirenomelia. PMID:23028704

Hair growth-promoting activity of hot water extract of Thuja orientalis.

PubMed

Zhang, Nan-nan; Park, Dong Ki; Park, Hye-Jin

2013-01-10

Thuja orientalis has been traditionally used to treat patients who suffer from baldness and hair loss in East Asia. The present study sought to investigate the hair growth-promoting activity of T. orientalis hot water extract and the underlying mechanism of action. After T. orientalis extract was topically applied to the shaved dorsal skin of telogenic C57BL/6 N mice, the histomorphometric analysis was employed to study induction of the hair follicle cycle. To determine the effect of T. orientalis extract on the telogen to anagen transition, the protein expression levels of β-catenin and Sonic hedgehog (Shh) in hair follicles were determined by immunohistochemistry. We observed that T. orientalis extract promoted hair growth by inducing the anagen phase in telogenic C57BL/6 N mice. Specifically, the histomorphometric analysis data indicates that topical application of T. orientalis extract induced an earlier anagen phase and prolonged the mature anagen phase, in contrast to either the control or 1% minoxidil-treated group. We also observed increases in both the number and size of hair follicles of the T. orientalis extract-treated group. Moreover, the immunohistochemical analysis reveals earlier induction of β-catenin and Shh proteins in hair follicles of the T. orientalis extract-treated group, compared to the control or 1% minoxidil-treated group. These results suggest that T. orientalis extract promotes hair growth by inducing the anagen phase in resting hair follicles and might therefore be a potential hair growth-promoting agent.

Kruppel-like factor 5 is Required for Formation and Differentiation of the Bladder Urothelium

PubMed Central

Bell, Sheila. M.; Zhang, Liqian; Mendell, Angela; Xu, Yan; Haitchi, Hans Michael; Lessard, James L.; Whitsett, Jeffrey A.

2011-01-01

SUMMARY Kruppel-like transcription factor 5 (Klf5) was detected in the developing and mature murine bladder urothelium. Herein we report a critical role of KLF5 in the formation and terminal differentiation of the urothelium. The ShhGfpCre transgene was used to delete the Klf5floxed alleles from bladder epithelial cells causing prenatal hydronephrosis, hydroureter, and vesicoureteric reflux. The bladder urothelium failed to stratify and did not express terminal differentiation markers characteristic of basal, intermediate, and umbrella cells including keratins 20, 14, and 5, and the uroplakins. The effects of Klf5 deletion were unique to the developing bladder epithelium since maturation of the epithelium comprising the bladder neck and urethra were unaffected by the lack of KLF5. mRNA analysis identified reductions in Pparγ, Grhl3, Elf3, and Ovol1expression in Klf5 deficient fetal bladders supporting their participation in a transcriptional network regulating bladder urothelial differentiation. KLF5 regulated expression of the mGrhl3 promoter in transient transfection assays. The absence of urothelial Klf5 altered epithelial-mesenchymal signaling leading to the formation of an ectopic alpha smooth muscle actin positive layer of cells subjacent to the epithelium and a thinner detrusor muscle that was not attributable to disruption of SHH signaling, a known mediator of detrusor morphogenesis. Deletion of Klf5 from the developing bladder urothelium blocked epithelial cell differentiation, impaired bladder morphogenesis and function causing hydroureter and hydronephrosis at birth. PMID:21803035

Gene Expression Analyses of the Spatio-Temporal Relationships of Human Medulloblastoma Subgroups during Early Human Neurogenesis

PubMed Central

Hooper, Cornelia M.; Hawes, Susan M.; Kees, Ursula R.; Gottardo, Nicholas G.; Dallas, Peter B.

2014-01-01

Medulloblastoma is the most common form of malignant paediatric brain tumour and is the leading cause of childhood cancer related mortality. The four molecular subgroups of medulloblastoma that have been identified – WNT, SHH, Group 3 and Group 4 - have molecular and topographical characteristics suggestive of different cells of origin. Definitive identification of the cell(s) of origin of the medulloblastoma subgroups, particularly the poorer prognosis Group 3 and Group 4 medulloblastoma, is critical to understand the pathogenesis of the disease, and ultimately for the development of more effective treatment options. To address this issue, the gene expression profiles of normal human neural tissues and cell types representing a broad neuro-developmental continuum, were compared to those of two independent cohorts of primary human medulloblastoma specimens. Clustering, co-expression network, and gene expression analyses revealed that WNT and SHH medulloblastoma may be derived from distinct neural stem cell populations during early embryonic development, while the transcriptional profiles of Group 3 and Group 4 medulloblastoma resemble cerebellar granule neuron precursors at weeks 10–15 and 20–30 of embryogenesis, respectively. Our data indicate that Group 3 medulloblastoma may arise through abnormal neuronal differentiation, whereas deregulation of synaptic pruning-associated apoptosis may be driving Group 4 tumorigenesis. Overall, these data provide significant new insight into the spatio-temporal relationships and molecular pathogenesis of the human medulloblastoma subgroups, and provide an important framework for the development of more refined model systems, and ultimately improved therapeutic strategies. PMID:25412507

Use of Drawing Lithography-Fabricated Polyglycolic Acid Microneedles for Transdermal Delivery of Itraconazole to a Human Basal Cell Carcinoma Model Regenerated on Mice

NASA Astrophysics Data System (ADS)

Zhang, Jennifer; Wang, Yan; Jin, Jane Y.; Degan, Simone; Hall, Russell P.; Boehm, Ryan D.; Jaipan, Panupong; Narayan, Roger J.

2016-04-01

Itraconazole is a triazole agent that is routinely used for treatment of nail infections and other fungal infections. Recent studies indicate that itraconazole can also inhibit the growth of basal cell carcinoma (BCC) through suppression of the Sonic Hedgehog (SHH) signaling pathway. In this study, polyglycolic acid microneedle arrays and stainless steel microneedle arrays were used for transdermal delivery of itraconazole to a human BCC model which was regenerated on mice. One-by-four arrays of 642- μm-long polyglycolic acid microneedles with sharp tips were prepared using injection molding and drawing lithography. Arrays of 85 stainless steel 800- μm-tall microneedles attached to syringes were obtained for comparison purposes. Skin grafts containing devitalized split-thickness human dermis that had been seeded with human keratinocytes transduced to express human SHH protein were sutured to the skin of immunodeficient mice. Mice with this human BCC model were treated daily for 2 weeks with itraconazole dissolved in 60% dimethylsulfoxane and 40% polyethylene glycol-400 solution; transdermal administration of the itraconazole solution was facilitated by either four 1 × 4 polyglycolic acid microneedle arrays or stainless steel microneedle arrays. The epidermal tissues treated with polyglycolic acid microneedles or stainless steel microneedles were markedly thinner than that of the control (untreated) graft tissue. These preliminary results indicate that microneedles may be used to facilitate transdermal delivery of itraconazole for localized treatment of BCC.

Indian and sonic hedgehogs regulate synchondrosis growth plate and cranial base development and function.

PubMed

Young, Blanche; Minugh-Purvis, Nancy; Shimo, Tsuyoshi; St-Jacques, Benoit; Iwamoto, Masahiro; Enomoto-Iwamoto, Motomi; Koyama, Eiki; Pacifici, Maurizio

2006-11-01

The synchondroses consist of mirror-image growth plates and are critical for cranial base elongation, but relatively little is known about their formation and regulation. Here we show that synchondrosis development is abnormal in Indian hedgehog-null mice. The Ihh(-/-) cranial bases displayed reduced growth and chondrocyte proliferation, but chondrocyte hypertrophy was widespread. Rather than forming a typical narrow zone, Ihh(-/-) hypertrophic chondrocytes occupied an elongated central portion of each growth plate and were flanked by immature collagen II-expressing chondrocytes facing perichondrial tissues. Endochondral ossification was delayed in much of the Ihh(-/-) cranial bases but, surprisingly, was unaffected most posteriorly. Searching for an explanation, we found that notochord remnants near incipient spheno-occipital synchondroses at E13.5 expressed Sonic hedgehog and local chondrocytes expressed Patched, suggesting that Shh had sustained chondrocyte maturation and occipital ossification. Equally unexpected, Ihh(-/-) growth plates stained poorly with Alcian blue and contained low aggrecan transcript levels. A comparable difference was seen in cultured wild-type versus Ihh(-/-) synchondrosis chondrocytes. Treatment with exogenous Ihh did not fully restore normal proteoglycan levels in mutant cultures, but a combination of Ihh and BMP-2 did. In summary, Ihh is required for multiple processes during synchondrosis and cranial base development, including growth plate zone organization, chondrocyte orientation, and proteoglycan production. The cranial base appears to be a skeletal structure in which growth and ossification patterns along its antero-posterior axis are orchestrated by both Ihh and Shh.

Selection of the In Vitro Culture Media Influences mRNA Expression of Hedgehog Genes, Il-6, and Important Genes regarding Reactive Oxygen Species in Single Murine Preimplantation Embryos

PubMed Central

Pfeifer, N.; Baston-Büst, D. M.; Hirchenhain, J.; Friebe-Hoffmann, U.; Rein, D. T.; Krüssel, J. S.; Hess, A. P.

2012-01-01

Background. The aim of this paper was to determine the influence of different in vitro culture media on mRNA expression of Hedgehog genes, il-6, and important genes regarding reactive oxygen species in single mouse embryos. Methods. Reverse transcription of single embryos either cultured in vitro from day 0.5 until 3.5 (COOK's Cleavage medium or Vitrolife's G-1 PLUS medium) or in vivo until day 3.5 post coitum. PCR was carried out for β-actin followed by nested-PCR for shh, ihh, il-6, nox, gpx4, gpx1, and prdx2. Results. The number of murine blastocysts cultured in COOK medium which expressed il-6, gpx4, gpx1, and prdx2 mRNA differed significantly compared to the in vivo group. Except for nox, the mRNA profile of the Vitrolife media group embryos varied significantly from the in vivo ones regarding the number of blastocysts expressing the mRNA of shh, ihh, il-6, gpx4, gpx1 and prdx2. Conclusions. The present study shows that different in vitro culture media lead to different mRNA expression profiles during early development. Even the newly developed in vitro culture media are not able to mimic the female reproductive tract. The question of long-term consequences for children due to assisted reproduction techniques needs to be addressed in larger studies. PMID:22919324

Gene expression analyses of the spatio-temporal relationships of human medulloblastoma subgroups during early human neurogenesis.

PubMed

Hooper, Cornelia M; Hawes, Susan M; Kees, Ursula R; Gottardo, Nicholas G; Dallas, Peter B

2014-01-01

Medulloblastoma is the most common form of malignant paediatric brain tumour and is the leading cause of childhood cancer related mortality. The four molecular subgroups of medulloblastoma that have been identified - WNT, SHH, Group 3 and Group 4 - have molecular and topographical characteristics suggestive of different cells of origin. Definitive identification of the cell(s) of origin of the medulloblastoma subgroups, particularly the poorer prognosis Group 3 and Group 4 medulloblastoma, is critical to understand the pathogenesis of the disease, and ultimately for the development of more effective treatment options. To address this issue, the gene expression profiles of normal human neural tissues and cell types representing a broad neuro-developmental continuum, were compared to those of two independent cohorts of primary human medulloblastoma specimens. Clustering, co-expression network, and gene expression analyses revealed that WNT and SHH medulloblastoma may be derived from distinct neural stem cell populations during early embryonic development, while the transcriptional profiles of Group 3 and Group 4 medulloblastoma resemble cerebellar granule neuron precursors at weeks 10-15 and 20-30 of embryogenesis, respectively. Our data indicate that Group 3 medulloblastoma may arise through abnormal neuronal differentiation, whereas deregulation of synaptic pruning-associated apoptosis may be driving Group 4 tumorigenesis. Overall, these data provide significant new insight into the spatio-temporal relationships and molecular pathogenesis of the human medulloblastoma subgroups, and provide an important framework for the development of more refined model systems, and ultimately improved therapeutic strategies.

Sma- and Mad-related protein 7 (Smad7) is required for embryonic eye development in the mouse.

PubMed

Zhang, Rui; Huang, Heng; Cao, Peijuan; Wang, Zhenzhen; Chen, Yan; Pan, Yi

2013-04-12

Smad7 is an intracellular inhibitory protein that antagonizes the signaling of TGF-β family members. Deletion of Smad7 in the mouse leads to an abnormality in heart development. However, whether Smad7 has a functional role in the development of other organs has been elusive. Here we present evidence that Smad7 imparts a role to eye development in the mouse. Smad7 is expressed in both the lens and retina in the developing embryonic eye. Depletion of Smad7 caused various degrees of coloboma and microphthalmia with alterations in cell apoptosis and proliferation in eyes. Smad7 was implicated in lens differentiation but was not required for the induction of the lens placode. The development of the periocular mesenchyme was retarded with the down-regulation of Bmp7 and Pitx2 in mutant mice. Retinal spatial patterning was affected by Smad7 deletion and was accompanied by altered bone morphogenetic protein (BMP) signaling. At late gestation stages, TGF-β signaling was up-regulated in the differentiating retina. Smad7 mutant mice displayed an expanded optic disc with increasing of sonic hedgehog (SHH) signaling. Furthermore, loss of Smad7 led to a temporal change in retinal neurogenesis. In conclusion, our study suggests that Smad7 is essential for eye development. In addition, our data indicate that alterations in the signaling of BMP, TGF-β, and SHH likely underlie the defects in eye development caused by Smad7 deletion.

Evaluation of motor neuron differentiation potential of human umbilical cord blood- derived mesenchymal stem cells, in vitro.

PubMed

Yousefi, Behnam; Sanooghi, Davood; Faghihi, Faezeh; Joghataei, Mohammad Taghi; Latifi, Nourahmad

2017-04-01

Many people suffer from spinal cord injuries annually. These deficits usually threaten the quality of life of patients. As a postpartum medically waste product, human Umbilical Cord Blood (UCB) is a rich source of stem cells with self- renewal properties and neural differentiation capacity which made it useful in regenerative medicine. Since there is no report on potential of human umbilical cord blood-derived mesenchymal stem cells into motor neurons, we set out to evaluate the differentiation properties of these cells into motor neuron-like cells through administration of Retinoic Acid(RA), Sonic Hedgehog(Shh) and BDNF using a three- step in vitro procedure. The results were evaluated using Real-time PCR, Flowcytometry and Immunocytochemistry for two weeks. Our data showed that the cells changed into bipolar morphology and could express markers related to motor neuron; including Hb-9, Pax-6, Islet-1, NF-H, ChAT at the level of mRNA and protein. We could also quantitatively evaluate the expression of Islet-1, ChAT and NF-H at 7 and 14days post- induction using flowcytometry. It is concluded that human UCB-MSCs is potent to express motor neuron- related markers in the presence of RA, Shh and BDNF through a three- step protocol; thus it could be a suitable cell candidate for regeneration of motor neurons in spinal cord injuries. Copyright © 2017. Published by Elsevier B.V.

Activation of retinal stem cells in the proliferating marginal region of RCS rats during development of retinitis pigmentosa.

PubMed

Jian, Qian; Xu, Haiwei; Xie, Hanping; Tian, Chunyu; Zhao, Tongtao; Yin, ZhengQin

2009-11-06

Retinal stem cells (RSCs) have been demonstrated at the proliferating marginal regions from the pars plana of ciliary body to the ciliary marginal zone (CMZ) in adult lower vertebrates and mammals. Investigations in the lower vertebrates have provided some evidence that RSCs can proliferate following retinal damage; however, the evidence that this occurs in mammals is not clear. In this study, we explored RSCs proliferation potential of adult mammalian in proliferating marginal regions of Royal College of Surgeons (RCS) rats, an animal model for retinitis pigmentosa (RP). The proliferation was evaluated using BrdU labeling, and Chx-10 as markers to discern progenitor cell of CMZ in Long-Evan's and RCS rats at different postnatal day (PND) after eye opening. We found that few Chx-10 and BrdU labeled cells in the proliferating marginal regions of Long-Evan's rats, which significantly increased in RCS rats at PND30 and PND60. Consistent with this, Chx-10/Vimentin double staining cells in the center retina of RCS rats increased significantly at PND30 after eye opening. In addition, mRNA expression of Shh, Ptch1 and Smo was up-regulated in RCS rats at PND60 compared to age-matched Long-Evan's rats, which revealed Shh/ptc pathway involving in the activation of RSCs. These results suggest that RSCs in the mammalian retinal proliferating marginal regions has the potential to regenerate following degeneration.

BMP inhibition by DAN in Hensen's node is a critical step for the establishment of left-right asymmetry in the chick embryo.

PubMed

Katsu, Kenjiro; Tokumori, Daisuke; Tatsumi, Norifumi; Suzuki, Atsushi; Yokouchi, Yuji

2012-03-01

During left-right (L-R) axis formation, Nodal is expressed in the node and has a central role in the transfer of L-R information in the vertebrate embryo. Bone morphogenetic protein (BMP) signaling also has an important role for maintenance of gene expression around the node. Several members of the Cerberus/Dan family act on L-R patterning by regulating activity of the transforming growth factor-β (TGF-β) family. We demonstrate here that chicken Dan plays a critical role in L-R axis formation. Chicken Dan is expressed in the left side of the node shortly after left-handed Shh expression and before the appearance of asymmetrically expressed genes in the lateral plate mesoderm (LPM). In vitro experiments revealed that DAN inhibited BMP signaling but not NODAL signaling. SHH had a positive regulatory effect on Dan expression while BMP4 had a negative effect. Using overexpression and RNA interference-mediated knockdown strategies, we demonstrate that Dan is indispensable for Nodal expression in the LPM and for Lefty-1 expression in the notochord. In the perinodal region, expression of Dan and Nodal was independent of each other. Nodal up-regulation by DAN required NODAL signaling, suggesting that DAN might act synergistically with NODAL. Our data indicate that Dan plays an essential role in the establishment of the L-R axis by inhibiting BMP signaling around the node. Copyright © 2012. Published by Elsevier Inc.

Making teeth to order: conserved genes reveal an ancient molecular pattern in paddlefish (Actinopterygii)

PubMed Central

Smith, Moya M.; Johanson, Zerina; Butts, Thomas; Ericsson, Rolf; Modrell, Melinda; Tulenko, Frank J.; Davis, Marcus C.; Fraser, Gareth J.

2015-01-01

Ray-finned fishes (Actinopterygii) are the dominant vertebrate group today (+30 000 species, predominantly teleosts), with great morphological diversity, including their dentitions. How dental morphological variation evolved is best addressed by considering a range of taxa across actinopterygian phylogeny; here we examine the dentition of Polyodon spathula (American paddlefish), assigned to the basal group Acipenseriformes. Although teeth are present and functional in young individuals of Polyodon, they are completely absent in adults. Our current understanding of developmental genes operating in the dentition is primarily restricted to teleosts; we show that shh and bmp4, as highly conserved epithelial and mesenchymal genes for gnathostome tooth development, are similarly expressed at Polyodon tooth loci, thus extending this conserved developmental pattern within the Actinopterygii. These genes map spatio-temporal tooth initiation in Polyodon larvae and provide new data in both oral and pharyngeal tooth sites. Variation in cellular intensity of shh maps timing of tooth morphogenesis, revealing a second odontogenic wave as alternate sites within tooth rows, a dental pattern also present in more derived actinopterygians. Developmental timing for each tooth field in Polyodon follows a gradient, from rostral to caudal and ventral to dorsal, repeated during subsequent loss of teeth. The transitory Polyodon dentition is modified by cessation of tooth addition and loss. As such, Polyodon represents a basal actinopterygian model for the evolution of developmental novelty: initial conservation, followed by tooth loss, accommodating the adult trophic modification to filter-feeding. PMID:25788604

Shale Frac Sequential Flowback Analyses and Reuse Implications, March 30, 2011

EPA Pesticide Factsheets

Water re-use challenges and solutions have direct and indirect influences in the design of hydraulic fracturing fluid systems and products used in High Volume, High Rate (HVHR) hydraulic fracturing of shale wells (1,2).

Polymeric assay film for direct colorimetric detection

DOEpatents

Charych, Deborah; Nagy, Jon; Spevak, Wayne

2002-01-01

A lipid bilayer with affinity to an analyte, which directly signals binding by a changes in the light absorption spectra. This novel assay means and method has special applications in the drug development and medical testing fields. Using a spectrometer, the system is easily automated, and a multiple well embodiment allows inexpensive screening and sequential testing. This invention also has applications in industry for feedstock and effluent monitoring.

Polymeric assay film for direct colorimetric detection

DOEpatents

Charych, Deborah; Nagy, Jon; Spevak, Wayne

1999-01-01

A lipid bilayer with affinity to an analyte, which directly signals binding by a changes in the light absorption spectra. This novel assay means and method has special applications in the drug development and medical testing fields. Using a spectrometer, the system is easily automated, and a multiple well embodiment allows inexpensive screening and sequential testing. This invention also has applications in industry for feedstock and effluent monitoring.

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

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

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

2013-05-01

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

Variability of haemocyte and haemolymph parameters in European flat oyster Ostrea edulis families obtained from brood stocks of different geographical origins and relation with infection by the protozoan Bonamia ostreae.

PubMed

Mirella da Silva, Patricia; Comesaña, Pilar; Fuentes, José; Villalba, Antonio

2008-05-01

A research project to compare productive traits (growth and mortality), disease susceptibility and immune capability between Ostrea edulis stocks was performed. This article reports the results on the immune capability and its relation with infection by the intrahaemocytic protozoan Bonamia ostreae. Four to five oyster spat families were produced from each of four European flat oyster populations (one from Ireland, one from Greece and two from Galicia, Spain) in a hatchery. The spat were transferred to a raft in the Ría de Arousa (Galicia) for on growing for 2 years. Total haemocyte count (THC) and differential haemocyte count (DHC) were estimated monthly through the second year of growing-out. Three types of haemocytes were distinguished: granulocytes (GH), large hyalinocytes (LHH) and small hyalinocytes (SHH). Significant correlations between the mean relative abundance of GH and SHH of the families and the mean prevalence of B. ostreae, the overall incidence of pathological conditions and the cumulative mortality of the families were found; these correlations supported the hypothesis that high %GH and low %SHH would enhance oyster immune ability and, consequently, would contribute to lower susceptibility to disease and longer lifespan. Infection by B. ostreae involved a significant increase of circulating haemocytes, which affected more markedly the LHH type. The higher the infection intensity the higher the %LHH. This illustrates the ability of B. ostreae to modulate the immune responses of the O. edulis to favour its own multiplication. A significant reduction of the phenoloxidase activity in the haemolymph of oysters O. edulis infected by B. ostreae was observed. Nineteen enzymatic activities in the haemolymph of O. edulis and Crassostrea gigas (used as a B. ostreae resistant reference) were measured using the kit api ZYM, Biomerieux. Qualitative and quantitative differences in enzyme activities in both haemocyte and plasma fractions between B. ostreae noninfected O. edulis from different origins were recorded. However, no clear positive association between enzyme activity and susceptibility to bonamiosis was found. The only enzyme detected in the resistant species C. gigas that was not found in the susceptible one O. edulis was beta-glucosidase (in plasma). B. ostreae infected O. edulis showed significant increase of some enzyme activities and the occurrence of enzymes that were not detected in noninfected oysters. These changes could be due to infection-induced enzyme synthesis by the host or to enzyme synthesis by the parasite.