Glial cell line-derived neurotrophic factor promotes the development of adrenergic neurons in mouse neural crest cultures

Growth of mouse neural crest cultures in the presence of glial cell line-derived neurotrophic factor (GDNF) resulted in a dramatic dose-dependent increase in the number of tyrosine hydroxylase (TH)-positive cells that developed when 5% chicken embryo extract was present in the medium. In contrast, growth in the presence of bone morphogenetic protein (BMP)-2, BMP-4, BMP-6, transforming growth factor (TGF) β1, TGF-β2, and TGF-β3 elicited no increase in the number of TH-positive cells. The TH-positive cells that developed in the presence of GDNF had neuronal morphology and contained the middle and low molecular weight neurofilament proteins. Numerous TH-negative cells with the morphology of neurons also were observed in GDNF-treated cultures. Analysis revealed that the period from 6 to 12 days in vitro was the critical time for exposure to GDNF to generate the increase in TH-positive cell number. The growth factors neurotrophin-3 and fibroblast growth factor-2 elicited increases in the number of TH-positive cells similar to that seen in response to GDNF. In contrast, nerve growth factor was unable to substitute for GDNF. These findings extend the previously reported biological activities of GDNF by showing that it can act on mouse neural crest cultures to promote the development of neurons.

Neural crest-directed gene transfer demonstrates Wnt1 role in melanocyte expansion and differentiation during mouse development

Wnt1 signaling has been implicated as one factor involved in neural crest-derived melanocyte (NC-M) development. Mice deficient for both Wnt1 and Wnt3a have a marked deficiency in trunk neural crest derivatives including NC-Ms. We have used cell lineage-directed gene targeting of Wnt signaling genes to examine the effects of Wnt signaling in mouse neural crest development. Gene expression was directed to cell lineages by infection with subgroup A avian leukosis virus vectors in lines of transgenic mice that express the retrovirus receptor tv-a. Transgenic mice with tva in either nestin-expressing neural precursor cells (line Ntva) or dopachrome tautomerase (DCT)-expressing melanoblasts (line DCTtva) were analyzed. We overstimulated Wnt signaling in two ways: directed gene transfer of Wnt1 to Ntva+ cells and transfer of β-catenin to DCTtva+ NC-M precursor cells. In both methods, NC-M expansion and differentiation were effected. Significant increases were observed in the number of NC-Ms [melanin+ and tyrosinase-related protein 1 (TYRP1)+ cells], the differentiation of melanin− TYRP1+ cells to melanin+ TYRP1+ NC-Ms, and the intensity of pigmentation per NC-M. These data are consistent with Wnt1 signaling being involved in both expansion and differentiation of migrating NC-Ms in the developing mouse embryo. The use of lineage-directed gene targeting will allow the dissection of signaling molecules involved in NC development and is adaptable to other mammalian developmental systems.

Neural crest cell survival is dependent on Rho kinase and is required for development of the mid face in mouse embryos.

Neural crest cells (NCC) give rise to much of the tissue that forms the vertebrate head and face, including cartilage and bone, cranial ganglia and teeth. In this study we show that conditional expression of a dominant-negative (DN) form of Rho kinase (Rock) in mouse NCC results in severe hypoplasia of the frontonasal processes and first pharyngeal arch, ultimately resulting in reduction of the maxilla and nasal bones and severe craniofacial clefting affecting the nose, palate and lip. These defects resemble frontonasal dysplasia in humans. Disruption of the actin cytoskeleton, which leads to abnormalities in cell-matrix attachment, is seen in the RockDN;Wnt1-cre mutant embryos. This leads to elevated cell death, resulting in NCC deficiency and hypoplastic NCC-derived craniofacial structures. Rock is thus essential for survival of NCC that form the craniofacial region. We propose that reduced NCC numbers in the frontonasal processes and first pharyngeal arch, resulting from exacerbated cell death, may be the common mechanism underlying frontonasal dysplasia.

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.

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.

Wild-type ALK and activating ALK-R1275Q and ALK-F1174L mutations upregulate Myc and initiate tumor formation in murine neural crest progenitor cells.

The anaplastic lymphoma kinase (ALK) gene is overexpressed, mutated or amplified in most neuroblastoma (NB), a pediatric neural crest-derived embryonal tumor. The two most frequent mutations, ALK-F1174L and ALK-R1275Q, contribute to NB tumorigenesis in mouse models, and cooperate with MYCN in the oncogenic process. However, the precise role of activating ALK mutations or ALK-wt overexpression in NB tumor initiation needs further clarification. Human ALK-wt, ALK-F1174L, or ALK-R1275Q were stably expressed in murine neural crest progenitor cells (NCPC), MONC-1 or JoMa1, immortalized with v-Myc or Tamoxifen-inducible Myc-ERT, respectively. While orthotopic implantations of MONC- 1 parental cells in nude mice generated various tumor types, such as NB, osteo/ chondrosarcoma, and undifferentiated tumors, due to v-Myc oncogenic activity, MONC-1-ALK-F1174L cells only produced undifferentiated tumors. Furthermore, our data represent the first demonstration of ALK-wt transforming capacity, as ALK-wt expression in JoMa1 cells, likewise ALK-F1174L, or ALK-R1275Q, in absence of exogenous Myc-ERT activity, was sufficient to induce the formation of aggressive and undifferentiated neural crest cell-derived tumors, but not to drive NB development. Interestingly, JoMa1-ALK tumors and their derived cell lines upregulated Myc endogenous expression, resulting from ALK activation, and both ALK and Myc activities were necessary to confer tumorigenic properties on tumor-derived JoMa1 cells in vitro.

Alternative generation of CNS neural stem cells and PNS derivatives from neural crest-derived peripheral stem cells

Neural crest-derived stem cells (NCSCs) from the embryonic peripheral nervous system (PNS) can be reprogrammed in neurosphere (NS) culture to rNCSCs that produce central nervous system (CNS) progeny, including myelinating oligodendrocytes. Using global gene expression analysis we now demonstrate that rNCSCs completely lose their previous PNS characteristics and acquire the identity of neural stem cells derived from embryonic spinal cord. Reprogramming proceeds rapidly and results in a homogenous population of Olig2-, Sox3-, and Lex-positive CNS stem cells. Low-level expression of pluripotency inducing genes Oct4, Nanog, and Klf4 argues against a transient pluripotent state during reprogramming. The acquisition of CNS properties is prevented in the presence of BMP4 (BMP NCSCs) as shown by marker gene expression and the potential to produce PNS neurons and glia. In addition, genes characteristic for mesenchymal and perivascular progenitors are expressed, which suggests that BMP NCSCs are directed toward a pericyte progenitor/mesenchymal stem cell (MSC) fate. Adult NCSCs from mouse palate, an easily accessible source of adult NCSCs, display strikingly similar properties. They do not generate cells with CNS characteristics but lose the neural crest markers Sox10 and p75 and produce MSC-like cells. These findings show that embryonic NCSCs acquire a full CNS identity in NS culture. In contrast, MSC-like cells are generated from BMP NCSCs and pNCSCs, which reveals that postmigratory NCSCs are a source for MSC-like cells up to the adult stage.

A novel role for neural crest cells in thymus organogenesis

Classical ablation studies have shown that neural crest cells (NCC) are critical for thymus organogenesis, though their role in this process has never been determined. We have used a mouse model deficient in NCC near the thymus rudiment to investigate the role of NCC in thymus organogenesis. Splotch mice exhibit a lack of NCC migration due to mutation in the gene encoding the transcription factor Pax 3. Homozygous mutants, designated Pax3Sp/Sp, display a range of phenotypes including spina bifida, cardiac outflow tract deformities, and craniofacial deformities. Pax3Sp/Sp, mice have also been reported to have hypoplastic and abnormal thymi, which is consistent with the expected result based on the classical ablation studies. However, in contrast to the dogma, we find that the thymus lobes in Pax3Sp/Sp, mice are even larger in size than those of littermate controls, although they fail to migrate and are therefore ectopic. Differentiation of the thymic epithelial compartments occurs normally, including the ability to import hematopoietic precursors, until the embryos die at embryonic day E13.0. We also investigated the patterning of the third pharyngeal pouch which gives rise to both the thymus and the parathyroid. Using RNA probes to detect expression of transcription factors exclusively expressed in the ventral, thymus- or dorsal, parathyroidfated domains of the E11.5 third pouch, we show that the parathyroid domain is restricted and the thymus-fated domain is expanded in Pax3Sp/Sp, embryos. Furthermore, mixing of the boundary between these domains occurs at E12.0. These results necessitate reconsideration of the previously accepted role for NCC in thymus organogenesis. NCC are not required for outgrowth of the thymus up to E13.0, and most strikingly, we have discovered a novel role for NCC in establishing parathyroid versus thymus fate boundaries in the third pharyngeal pouch. ^

Xenopus Zic3, a primary regulator both in neural and neural crest development

Xenopus Zic3 is a Xenopus homologue of mouse Zic and Drosophila pair-rule gene, odd-paired. We show here that Zic3 has significant roles both in neural and neural crest development in Xenopus embryo. Expression of Zic3 is first detected in prospective neural plate region at gastrulation. Onset of the expression was earlier than most proneural genes and followed chordin expression. The expression was induced by blockade of BMP4 signal. Overexpression of Zic3 resulted in hyperplastic neural and neural crest derived tissue. In animal cap explant, the overexpression of Zic3 induced expression of all the proneural genes and neural crest marker genes. These findings suggest that Zic3 can determine the ectodermal cell fate and promote the earliest step of neural and neural crest development.

Endothelin 3 selectively promotes survival and proliferation of neural crest-derived glial and melanocytic precursors in vitro

Genetic data in the mouse have shown that endothelin 3 (ET3) and its receptor B (ETRB) are essential for the development of two neural crest (NC) derivatives, the melanocytes and the enteric nervous system. We report here the effects of ET3 in vitro on the differentiation of quail trunk NC cells (NCC) in mass and clonal cultures. Treatment with ET3 is highly mitogenic to the undifferentiated NCC population, which leads to expansion of the population of cells in the melanocytic, and to a lesser extent, the glial lineages. The effect of ET3 on these two NC derivatives was confirmed by the quantitative analysis of clones derived from individual NCC subjected to ET3: we found a large increase in the survival and proliferation of unipotent and bipotent precursors for glial cells and melanocytes, with no significant effect on multipotent cells generating neurons. ET3 first stimulates expression of both ETRB and ETRB2 by cultured NCC. Then, under prolonged exposure to ET3, ETRB expression decreases and switches toward an ETRB2-positive melanogenic cell population. We therefore propose that the present in vitro experiments (long-lasting exposure to a high concentration of ET3) mimic the environment encountered by NCC in vivo when they migrate to the skin under the ectoderm that expresses ET3.

Endothelin-B receptor is expressed by neural crest cells in the avian embryo.

Disruptions of the genes encoding endothelin 3 (EDN3) and its receptor endothelin-B receptor (EDNRB) in the mouse result in defects of two neural crest (NC)-derived lineages, the melanocytes, and the enteric nervous system. To assess the mechanisms through which the EDN3/EDNRB signaling pathway can selectively act on these NC derivatives, we have studied the spatiotemporal expression pattern of the EDNRB gene in the avian embryo, a model in which NC development has been extensively studied. For this purpose, we have cloned the quail homologue of the mammalian EDNRB cDNA. EDNRB transcripts are present in NC cells before and during their emigration from the neural tube at all levels of the neuraxis. At later developmental stages, the receptor remains abundantly expressed in the peripheral nervous system including the enteric nervous system. In a previous study, we have shown that EDN3 enhances dramatically the proliferation of NC cells when they are at the pluripotent stage. We propose that the selective effect of EDN3 or EDNRB gene inactivation is due to the fact that both melanocytes and enteric nervous system precursors have to colonize large embryonic areas (skin and bowel) from a relatively small population of precursors that have to expand considerably in number. It is therefore understandable that a deficit in one of the growth-promoting pathways of NC cells has more deleterious effects on long-range migrating cells than on the NC derivatives which develop close to the neural primordium like the sensory and sympathetic ganglia.

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. ^

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.

Tyrosinase-Cre mice for tissue-specific gene ablation in neural crest and neuroepithelial-derived tissues

This study describes the derivation of two new lines of transgenic mice that express Cre recombinase under the control of tyrosinase transcriptional elements. To determine the suitability of the Tyrosinase-Cre transgene for tissue-specific gene ablation studies, a fate map of Cre expression domains was determined using the Z/AP reporter strain. It was shown that Cre-expressing cells contribute to a wide array of neural crest and neuroepithelial-derived lineages. The melanocytes of the harderian gland and eye choroid, sympathetic cephalic ganglia, leptomeninges of the telencephalon, as well as cranial nerves (V), (VII), and (IX) are derived either fully or partly from Cre-expressing cephalic crest. The cells contributing to the cranial nerves were the first to exhibit Cre expression at E10.5 as they were migrating into the branchial arches. The melanocytes, chromaffin cells of the adrenal medulla, and dorsal root ganglia are derived from trunk neural crest that either express Cre or were derived from Cre-expressing precursors. An array of brain tissue including the basal forebrain, hippocampus, olfactory bulb, and the granule cell layer of the lateral cerebellum, as well as the retinal pigmented epithelium and glia of the optic nerve originate from Cre-expressing neuroepithelial cells. (C) 2003 Wiley-Liss, Inc.

Tbx1 and Bmp2 in the development of the ear, neural crest and pharyngeal system in mice

Dissertation presented to obtain the Ph.D degree in Biology