Scatter factor/hepatocyte growth factor as a regulator of skeletal muscle and neural crest development.

Factors that regulate cellular migration during embryonic development are essential for tissue and organ morphogenesis. Scatter factor/hepatocyte growth factor (SF/HGF) can stimulate motogenic and morphogenetic activities in cultured epithelial cells expressing the Met tyrosine kinase receptor and is essential for development; however, the precise physiological role of SF/HGF is incompletely understood. Here we provide functional evidence that inappropriate expression of SF/HGF in transgenic mice influences the development of two distinct migratory cell lineages, resulting in ectopic skeletal muscle formation and melanosis in the central nervous system, and patterned hyperpigmentation of the skin. Committed TRP-2 positive melanoblasts were found to be situated aberrantly within defined regions of the transgenic embryo, including the neural tube, which overproduced SF/RGF. Our data strongly suggest that SF/HGF possesses physiologically relevant scatter activity, and functions as a true morphogenetic factor by regulating migration and/or differentiation of select populations of premyogenic and neural crest cells during normal mammalian embryogenesis.

Endothelin 3 promotes neural crest cell proliferation and mediates a vast increase in melanocyte number in culture.

Mutations in the endothelin 3 (EDN3) gene severely affect the development of neural crest-derived melanocytes. In this paper, we report the action of EDN3 on neural crest cells in vitro. The presence of EDN3 leads to a large increase in the number of cells, the majority of which eventually differentiate into melanocytes that aggregate to form a reproducible pigmentation pattern. Quantitative analysis of the effect of different culture conditions revealed that EDN3 initially promotes neural crest cell proliferation. This phase of expansion, which can be prolonged for a few weeks if the cells are replaced regularly, is followed by both a decrease in cell proliferation and the onset of melanocytic differentiation. Therefore, EDN3 is a potent mitogen for early neural crest cell precursors that can give rise to melanocytes.

Serotonin regulates mouse cranial neural crest migration.

Serotonergic agents (uptake inhibitors, receptor ligands) cause significant craniofacial malformations in cultured mouse embryos suggesting that 5-hydroxytryptamine (serotonin) (5-HT) may be an important regulator of craniofacial development. To determine whether serotonergic regulation of cell migration might underly some of these effects, cranial neural crest (NC) explants from embryonic day 9 (E9) (plug day = E1) mouse embryos or dissociated mandibular mesenchyme cells (derived from NC) from E12 embryos were placed in a modified Boyden chamber to measure effects of serotonergic agents on cell migration. A dose-dependent effect of 5-HT on the migration of highly motile cranial NC cells was demonstrated, such that low concentrations of 5-HT stimulated migration, whereas this effect was progressively lost as the dose of 5-HT was increased. In contrast, most concentrations of 5-HT inhibited migration of less motile, mandibular mesenchyme cells. To investigate the possible involvement of specific 5-HT receptors in the stimulation of NC migration, several 5-HT subtype-selective antagonists were used to block the effects of the most stimulatory dose of 5-HT (0.01 microM). Only NAN-190 (a 5-HT1A antagonist) inhibited the effect of 5-HT, suggesting involvement of this receptor. Further evidence was obtained by using immunohistochemistry with 5-HT receptor antibodies, which revealed expression of the 5-HT1A receptor but not other subtypes by migrating NC cells in both embryos and cranial NC explants. These results suggest that by activating appropriate receptors 5-HT may regulate migration of cranial NC cells and their mesenchymal derivatives in the mouse embryo.

The development and growth of tissues derived from cranial neural crest and primitive mesoderm is dependent on the ligation status of retinoic acid receptor γ:evidence that retinoic acid receptor γ functions to maintain stem/progenitor cells in the absence of retinoic acid

Retinoic acid (RA) signaling is important to normal development. However, the function of the different RA receptors (RARs)-RARα, RARβ, and RARγ-is as yet unclear. We have used wild-type and transgenic zebrafish to examine the role of RARγ. Treatment of zebrafish embryos with an RARγ-specific agonist reduced somite formation and axial length, which was associated with a loss of hoxb13a expression and less-clear alterations in hoxc11a or myoD expression. Treatment with the RARγ agonist also disrupted formation of tissues arising from cranial neural crest, including cranial bones and anterior neural ganglia. There was a loss of Sox 9-immunopositive neural crest stem/progenitor cells in the same anterior regions. Pectoral fin outgrowth was blocked by RARγ agonist treatment. However, there was no loss of Tbx-5-immunopositive lateral plate mesodermal stem/progenitor cells and the block was reversed by agonist washout or by cotreatment with an RARγ antagonist. Regeneration of the caudal fin was also blocked by RARγ agonist treatment, which was associated with a loss of canonical Wnt signaling. This regenerative response was restored by agonist washout or cotreatment with the RARγ antagonist. These findings suggest that RARγ plays an essential role in maintaining stem/progenitor cells during embryonic development and tissue regeneration when the receptor is in its nonligated state.

Endothelin receptor B and neural crest derived melanocyte development

Neural Crest cells (NCC) constitute a unique embryonic cell population that arises between the prospective epidermis and the dorsal aspect of the neural tube of vertebrates. NCC migrate ventromedially and dorsolaterally throughout the developing embryo giving rise to the peripheral nervous system constituents and melanocytes that ultimately reside in the skin and hair follicles respectively. Mice and humans with mutations in the Endothelin receptor b (Ednrb) gene manifest strikingly similar phenotypes characterized by hypopigmentation, hearing loss and megacolon these are due to absence of melanocytes in the skin and inner ear and lack of enteric ganglia in the distal part of the gut, respectively. Piebald lethal mice and humans with Hirschsprung's disease or Waardenburg syndrome carry different mutations in the Ednrb gene. The major goals of this project were to determine whether the action of Ednrb in NCC is required prior to commitment of these cells to the melanocytic lineage and to investigate its potential participation in the actual process of commitment. In order to achieve these goals transgenic mice that express Ednrb under two different regulatory elements were created. The first, Dct-Ednrb, expresses Ednrb under the control of the DOPAchrome tautomerase (Dct) promoter to direct expression to already committed melanocyte precursors. The second, Nes-Ednrb, expresses Ednrb under the regulation of the human nestin gene second enhancer to direct expression to pre-migratory NCC. Crosses of the Dct-Ednrb mouse with piebald lethal showed that the transgene was capable of rescuing the hypopigmentation phenotype of the later. This result indicates that the action of Ednrb after NCC commit to the melanocytic lineage is sufficient for normal melanocyte development. The Dct-Ednrb was further crossed with two other hypopigmentation mutants that carry mutations in the transcription factors Sox10 and Pax3. The transgene rescued the phenotype of the Sox10 mutant only. This suggests that Ednrb interacts with Sox10 but not with Pax3 during melanocyte development. The Nes-Ednrb mice developed a hypopigmentation phenotype that was augmented when crossed with piebald lethal or lethal spotting (mutation in Edn3, the ligand for Ednrb) mice but was rescued by over expression of Edn3. These results suggest that alterations in Ednrb expression early in development affect melanocyte development. This study provides novel information necessary to better understand the early embryonic development of NCC, clarifies specific interactions between different melanogenic genes and, could eventually help in the implementation of therapies for human pigmentary genetic disorders. ^

Mus Musculus Neural Crest Development: Is Pax3 Regulating the Expression of Endothelin Receptor B?

The vertebrate Neural Crest (NC) is formed during early embryonic development at the neurulation stage. This group of multi potent cells gives rise to a variety of derivatives such as the skin's pigmented cells (Melanocytes), the peripheral nervous system with its associated components, and the endocrine cells of the adrenal medulla amongst others. There are several molecular mechanisms that underlie the development and migration of NC derived cells. For example, during melanocyte differentiation and migration the Endothelin Receptor B and its ligand Endothelin 3 (EdnrB/Edn3), the kit/ Steel factor and the FGF receptor I FGF pathways amongst others play important roles. Additionally, several transcription factors such as Pax3, SoxlO and Mitfalso intervene during the NC cells differentiation processes. In this work, the possible regulatory interaction of Pax3 and EdnrB was assessed by in situ hybridization methods with EdnrB, SoxlO and Dct riboprobes in Pax3 homozygous embryos. To further characterize this interaction, genetic crosses between Pax3 heterozygous mutants and EdnrB heterozygous animals were established. Coat pigmentation was used as an indicator of genetic interaction on the progeny. Experimental results indicated that Pax3 does not directly regulate the expression of EdnrB during neural crest development but interact to produce normal coat color. I propose two possible models to explain the epistatic relationship of Pax3 and EdnrB during normal melanocyte development.

Cardiac Neural Crest Cells Affect the Development of the Myocardium and Conduction System in the Mouse

Neural crest cells originate from the dorsal most region of the embryonic neural tube. These cells migrate into several embryonic locations and differentiate into a variety of cell types. Cardiac neural crest (CNC) cells are a set of neural crest progenitors that aid in the proper formation of the cardiac septum, which separates the pulmonary from the systemic circulation. We have used Splotch mice to investigate whether the murine CNC cells play a role during the development oft he myocardium and the conduction system. Splotch mice carry a mutation in the P AX3 transcription factor, and display a problem in CNC cell migration. A scanning-electron-microscopy analysis of Splotch mutant-embryonic-hearts reveals abnormalities in the interventricular septum. In addition, the right and left ventricular cavities appear dilated relative to a wild type heart. Hoechst nuclei staining of Splotch heart cryosections demonstrates a decreased number of cardiomyocytes and a corresponding thinner ventricular wall. The absence of Connexin 40 in the ventricles of Splotch mutants, suggests conduction system defects. These results support the evidence that CNC cell signaling plays a role in modulating the growth and development of murine cardiomyocytes and their differentiation into conductile cells.

Cardiac Neural Crest Cells and Their Role in Murine Purkinje Fiber Development

The heart beat is regulated by the cardiac conduction system (CCS), a specialized group of cells that transmit electrical impulses around the heart chambers. During development, ventricular CCS cells originate from embryonic cardiomyocytes and not from the neural crest. Nonetheless, discoveries in chick implied that the cardiac neural crest (CNC) cells contribute to proper development of the ventricular CCS. In this report, the Splotch mouse mutant (Pax3sp), in which the CNC cells do not migrate to the heart, was used to investigate whether these cells also affect proper CCS development in mammals. Homozygote mutants (Pax3Sp!Sp) are lethal on 111 Embryonic Day 13 (E13), and can be phenotyped by spina bifida and exencephaly. Pax3Spi+ mice were crossed to obtain wild type, Pax3 Spi+ and Pax3 Sp!Sp embryos. Comparison of hematoxylin and eosin stained histological sections showed less trabeculation in El2.5 cardiac ventricles of Pax3Sp!Sp. Furthermore, immunofluorescence analysis with the Purkinje fiber marker Cx40 showed a qualitative difference between wild type and mutant hearts. Quantitative analysis indicated that Pax3 Sp!Sp ventricles had fewer Cx40 expressing cells, as well as less Cx40 being expressed per cell when compared to wild type ventricles. Immunofluorescence with the H3 histome mitosis antibody showed fewer proliferating cells in the ventricles of mutant embryos when compared to controls. These results suggest that CNCC affect the morphogenesis of cardiac ventricles and the development of the ventricular CCS by contributing cellular proliferation.

Epigenetic regulation in neural crest development : role of DNA methyltransferases 3A and 3B

The neural crest is a group of migratory, multipotent stem cells that play a crucial role in many aspects of embryonic development. This uniquely vertebrate cell population forms within the dorsal neural tube but then emigrates out and migrates long distances to different regions of the body. These cells contribute to formation of many structures such as the peripheral nervous system, craniofacial skeleton, and pigmentation of the skin. Why some neural tube cells undergo a change from neural to neural crest cell fate is unknown as is the timing of both onset and cessation of their emigration from the neural tube. In recent years, growing evidence supports an important role for epigenetic regulation as a new mechanism for controlling aspects of neural crest development. In this thesis, I dissect the roles of the de novo DNA methyltransferases (DNMTs) 3A and 3B in neural crest specification, migration and differentiation. First, I show that DNMT3A limits the spatial boundary between neural crest versus neural tube progenitors within the neuroepithelium. DNMT3A promotes neural crest specification by directly mediating repression of neural genes, like Sox2 and Sox3. Its knockdown causes ectopic Sox2 and Sox3 expression at the expense of neural crest territory. Thus, DNMT3A functions as a molecular switch, repressing neural to favor neural crest cell fate. Second, I find that DNMT3B restricts the temporal window during which the neural crest cells emigrate from the dorsal neural tube. Knockdown of DNMT3B causes an excess of neural crest emigration, by extending the time that the neural tube is competent to generate emigrating neural crest cells. In older embryos, this resulted in premature neuronal differentiation. Thus, DNMT3B regulates the duration of neural crest production by the neural tube and the timing of their differentiation. My results in avian embryos suggest that de novo DNA methylation, exerted by both DNMT3A and DNMT3B, plays a dual role in neural crest development, with each individual paralogue apparently functioning during a distinct temporal window. The results suggest that de novo DNA methylation is a critical epigenetic mark used for cell fate restriction of progenitor cells during neural crest cell fate specification. Our discovery provides important insights into the mechanisms that determine whether a cell becomes part of the central nervous system or peripheral cell lineages.

Insights into Neural Crest Evolution

Neural crest cells are unique to vertebrates and essential to the development and evolution of the craniofacial skeleton. Using a combination of DiI cell lineage tracing, transcriptomics, and analysis of key transcription factors of the Sox Family, I examined neural crest development in the sea lamprey, Petromyzon marinus, as the most basal extant vertebrate from which it is possible to get embryos. The results have uncovered distinct cranial and trunk neural crest subpopulations along the anterior-posterior axis of the lamprey embryo, with a clear separation between the two. However, no evidence of the presence of an intermediate vagal neural crest population was uncovered. Comparing cranial neural crest genes between lamprey and chick, either by examining individual candidate genes or whole genome transcriptome analysis, reveals significant changes in the cranial neural crest gene regulatory network of lamprey compared with chick. In particular, the lamprey cranial neural crest is "missing" several gnathostome cranial crest genes. We speculate that these may underlie the evolutionary divergence of craniofacial development between jawed and jawless vertebrates. Despite the absence of vagal neural crest, DiI-labeling shows that trunk neural crest-derived cells, likely homologous to mammalian Schwann cell precursors, contribute to the lamprey enteric nervous system, potentially representing the most primitive form of neural crest cells contribution to the ENS. Finally, I characterized key members of the Sox Family (Sox B-F) due to their importance in neural crest specification in other species. In comparative studies of the SoxC genes (Sox4, Sox11, and Sox12) in both lamprey and Xenopus, I found similar expression patterns and a novel key role in early neural crest specification, suggesting a conserved role of the SoxC genes amongst vertebrates. Taken together, this work represents important progress in characterizing the early evolution of the neural crest in vertebrates and its role in the transition from jawless to jawed vertebrates.

Expression of PROKR1 and PROKR2 in Human Enteric Neural Precursor Cells and Identification of Sequence Variants Suggest a Role in HSCR

Background. The enteric nervous system (ENS) is entirely derived from neural crest and its normal development is regulated by specific molecular pathways. Failure in complete ENS formation results in aganglionic gut conditions such as Hirschsprung's disease (HSCR). Recently, PROKR1 expression has been demonstrated in mouse enteric neural crest derived cells and Prok-1 was shown to work coordinately with GDNF in the development of the ENS. Principal Findings. In the present report, ENS progenitors were isolated and characterized from the ganglionic gut from children diagnosed with and without HSCR, and the expression of prokineticin receptors was examined. Immunocytochemical analysis of neurosphere-forming cells demonstrated that both PROKR1 and PROKR2 were present in human enteric neural crest cells. In addition, we also performed a mutational analysis of PROKR1, PROKR2, PROK1 and PROK2 genes in a cohort of HSCR patients, evaluating them for the first time as susceptibility genes for the disease. Several missense variants were detected, most of them affecting highly conserved amino acid residues of the protein and located in functional domains of both receptors, which suggests a possible deleterious effect in their biological function. Conclusions. Our results suggest that not only PROKR1, but also PROKR2 might mediate a complementary signalling to the RET/GFRα1/GDNF pathway supporting proliferation/survival and differentiation of precursor cells during ENS development. These findings, together with the detection of sequence variants in PROKR1, PROK1 and PROKR2 genes associated to HSCR and, in some cases in combination with RET or GDNF mutations, provide the first evidence to consider them as susceptibility genes for HSCR.

An epigenetic signature of developmental potential in neural stem cells and early neurons.

A cardinal property of neural stem cells (NSCs) is their ability to adopt multiple fates upon differentiation. The epigenome is widely seen as a read-out of cellular potential and a manifestation of this can be seen in embryonic stem cells (ESCs), where promoters of many lineage-specific regulators are marked by a bivalent epigenetic signature comprising trimethylation of both lysine 4 and lysine 27 of histone H3 (H3K4me3 and H3K27me3, respectively). Bivalency has subsequently emerged as a powerful epigenetic indicator of stem cell potential. Here, we have interrogated the epigenome during differentiation of ESC-derived NSCs to immature GABAergic interneurons. We show that developmental transitions are accompanied by loss of bivalency at many promoters in line with their increasing developmental restriction from pluripotent ESC through multipotent NSC to committed GABAergic interneuron. At the NSC stage, the promoters of genes encoding many transcriptional regulators required for differentiation of multiple neuronal subtypes and neural crest appear to be bivalent, consistent with the broad developmental potential of NSCs. Upon differentiation to GABAergic neurons, all non-GABAergic promoters resolve to H3K27me3 monovalency, whereas GABAergic promoters resolve to H3K4me3 monovalency or retain bivalency. Importantly, many of these epigenetic changes occur before any corresponding changes in gene expression. Intriguingly, another group of gene promoters gain bivalency as NSCs differentiate toward neurons, the majority of which are associated with functions connected with maturation and establishment and maintenance of connectivity. These data show that bivalency provides a dynamic epigenetic signature of developmental potential in both NSCs and in early neurons. Stem Cells 2013;31:1868-1880.

Highly efficient neural differentiation of human somatic stem cells, isolated by minimally invasive periodontal surgery

Neural stem cells (NSCs) are potential sources for cell therapy of neurodegenerative diseases and for drug screening. Despite their potential benefits, ethical and practical considerations limit the application of NSCs derived from human embryonic stem cells (ES) or adult brain tissue. Thus, alternative sources are required to satisfy the criteria of ready accessibility, rapid expansion in chemically defined media and reliable induction to a neuronal fate. We isolated somatic stem cells from the human periodontium that were collected during minimally invasive periodontal access flap surgery as part of guided tissue regeneration therapy. These cells could be propagated as neurospheres in serum-free medium, which underscores their cranial neural crest cell origin. Culture in the presence of epidermal growth factor (EGF) and fibroblast growth factor-2 (FGF-2) under serum-free conditions resulted in large numbers of nestin-positive/Sox-2-positive NSCs. These periodontium-derived (pd) NSCs are highly proliferative and migrate in response to chemokines that have been described as inducing NSC migration. We used immunocytochemical techniques and RT-PCR analysis to assess neural differentiation after treatment of the expanded cells with a novel induction medium. Adherence to substrate, growth factor deprivation, and retinoic acid treatment led to the acquisition of neuronal morphology and stable expression of markers of neuronal differentiation by more than 90% of the cells. Thus, our novel method might provide nearly limitless numbers of neuronal precursors from a readily accessible autologous adult human source, which could be used as a platform for further experimental studies and has potential therapeutic implications.

BCL2 regulates neural differentiation.

A main function attributed to the BCL2 protein is its ability to confer resistance against apoptosis. In addition to the constitutively high expression of BCL2, caused by gene rearrangement in follicular lymphomas, elevated expression of the BCL2 gene has been found in differentiating hematopoietic, neural, and epithelial tissues. To address the question of whether the expression of BCL2 is a cause or consequence of cell differentiation, we used a human neural-crest-derived tumor cell line, Paju, that undergoes spontaneous neural differentiation in vitro. The Paju cell line displays moderate expression of BCL2, the level of which increases in parallel with further neural differentiation induced by treatment with phorbol 12-myristate 13-acetate. Transfection of normal human BCL2 cDNA in sense and antisense orientations had a dramatic impact on the differentiation of the Paju cells. Overexpression of BCL2 cDNA induced extensive neurite outgrowth, even in low serum concentrations, together with an increased expression of neuron-specific enolase. Paju cells expressing the anti-sense BCL2 cDNA construct, which reduced the endogenous levels of BCL2, did not undergo spontaneous neural differentiation. These cells acquired an epithelioid morphology and up-regulated the intermediate filament protein nestin, typically present in primitive neuroectodermal cells. The manipulated levels of BCL2 did not have appreciable impact on cell survival in normal culture. Our findings demonstrate that the BCL2 gene product participates in the regulation of neural differentiation.

Retinoic acid disrupts anterior ectodermal and endodermal development in ascidian larvae and postlarvae

In vertebrates, excess all-trans retinoic acid (RA) applied during axis formation leads to the apparent truncation of anterior structures. In this study we sought to determine the type of defects caused by ectopic RA on the development of the ascidian Herdmania curvata. We demonstrate that H. curvata embryos cultured in the presence of RA develop into larvae whose trunks are shortened and superficially resemble those of early metamorphosing postlarvae. Despite RA-treated larvae lacking papillar structures they respond normally to natural cues that induce metamorphosis, indicating that chemosensory functionality previously mapped to the most anterior region of normal larvae is unaffected by RA. Excess RA applied during postlarval development leads to a graded loss of the juvenile pharynx, apparently by respecifying anterior endoderm to a more posterior fate. This structure is considered homologous to the gill slits of amphioxus. which are also lost upon RA treatment. This suggests that RA may have had a role in the development of the pharynx of the ancestral chordate and that this function has been maintained in ascidians and cephalochordates and lost in vertebrates.