Targeting of endothelin receptor-B to the neural crest

Endothelin receptor B (Ednrb) plays a critical role in the development of melanocytes and neurons and glia of the enteric nervous system. These distinct neural crest-derived cell types express Ednrb and share the property of intercalating into tissues, such as the intestine whose muscle precursor cells also express Ednrb. Such widespread Ednrb expression has been a significant obstacle in establishing precise roles for Ednrb in development. We describe here the production of an Ednrb allele floxed at exon 3 and its use in excising the receptor from mouse neural crest cells by use of Cre-recombinase driven by the Wnt1 promoter. Mice born with neural crest-specific excision of Ednrb possess aganglionic colon, lack trunk pigmentation, and die within 5 weeks due to megacolon. Ednrb receptor expression in these animals is absent only in the neural crest but present in surrounding smooth muscle cells. The absence of Ednrb from crest cells also results in a compensatory upregulation of Ednrb expression in other cells within the gut. We conclude that Ednrb loss only in neural crest cells is sufficient to produce the Hirschsprungs disease phenotype observed with genomic Ednrb mutations.     

Involvement of the neuregulins and their receptors in cardiac and neural development

The neuregulin gene encodes a series of polypeptide growth factors that can influence the growth state of target vertebrate cells in culture. Recently, three studies have explored the in vivo function of the neuregulin signaling system in mice by disrupting the genes encoding the neuregulin ligand⁽¹⁾ and two of its receptors, ErbB2⁽²⁾ and ErbB4⁽³⁾. Each of the genes is essential for development, and aberrations in cardiac and neural development are particularly prominent in mutant embryos. The observed defects, together with the localization of expression of the neuregulin signaling components within these tissues, highlight a paracrine mechanism for neuregulin-mediated intercellular communication.     

Dgcr8 controls neural crest cells survival in cardiovascular development

DiGeorge syndrome (DGS), characterized genetically by a deletion within chromosome 22q11.2, is associated with a constellation of congenital heart defects. DiGeorge critical region 8 (Dgcr8), a gene that maps to the common deletion region of DGS, encodes a double stranded RNA-binding protein that is essential for miRNA biogenesis. To address the potential contribution of Dgcr8 insufficiency to cardiovascular development, we have inactivated Dgcr8 in cardiac neural crest cells (cNCCs). Dgcr8 mutants displayed a wide spectrum of malformations, including persistent truncus arteriosus (PTA) and ventricular septal defect (VSD). Interestingly, Dgcr8-null cNCCs that properly migrated into the cardiac outflow tract (OFT), proliferate normally and differentiate into vascular smooth muscle cells. However, loss of Dgcr8 causes a significant portion of the cNCCs to undergo apoptosis, causing a decrease in the pool of progenitors required for OFT remodeling. Our data uncover a new role of Dgcr8 in cardiovascular morphogenesis, plausibly as part of transmission mechanism for FGF-dependent survival cue for migrating cNCCs.     

Appearance of nerve growth factor receptors on cultured neural crest cells

Light microscopic radioautography of differentiating quail neural crest cultures (1 to 2 weeks after explanation) incubated with Iodine-125-labeled nerve growth factor (125I-NGF) revealed that approximately 35% of the cells bound NGF. The binding was specific and saturable; it was blocked by an excess of nonradioactive NGF, and was not detected following incubation with biologically inactive 125I-NGF. In addition, the binding did not appear to be blocked or diminished by insulin. Cell cultures prepared from somites or notochord showed no specific binding of 125I-NGF. Melanocytes comprised approximately 10% of the cell population in these cultures and appeared to be unlabeled. The subpopulation of cells with NGF receptors that were morphologically similar to other non-melanocyte unlabeled cells present in the neural crest cultures are probably the targets of the factor during differentiation and development. In contrast, there was no evidence of 125I-NGF binding by premigratory neural crest (adherent to the isolated neural tube) or by early migratory neural crest cells (24 hr after explantation). Both of these types of neural crest cells are relatively undifferentiated. The cells of the neural tube were also unlabeled. The binding of 125I-NGF to differentiating neural crest cells was not noticeably diminished by a brief pretreatment with trypsin or Dispase, enzymes used in the isolation of neural tubes. Hence, the absence of NGF receptors on premigratory neural crest and early migratory neural crest cultures was not due to enzymatic alterations of the receptor. It seems, therefore, that receptors for NGF appear on neural crest cells during the time when these cells are acquiring their phenotypic characteristics.     

Involvement of central endothelin receptors in neonatal morphine withdrawal

The involvement of central endothelin (ET) receptors in neonatal morphine tolerance has been demonstrated. The present study investigates the role of central ET receptors in morphine withdrawal in neonatal rats. The aim was to determine whether activation of G-proteins coupled to opioid and ET receptors by morphine and various ET receptor modulators is affected during morphine withdrawal in neonatal rats. Pregnant female rats were rendered tolerant to morphine by chronic exposure to morphine pellets during 7 days. On Day 8, pellets were removed and rats were allowed to undergo withdrawal for 24 hrs. Rat pups were delivered by cesarean section. G-protein stimulation induced by morphine; ET-1; the ET(A) receptor antagonist, BMS182874; and the ET(B) receptor agonist, IRL1620, were determined in the brain of neonatal rats undergoing morphine withdrawal by [35S]GTPgammaS binding assay. Morphine produced higher (P < 0.05) maximal stimulation of G-protein in the morphine-withdrawal group (83.60%) compared with the placebo group (66.81%). ET-1-induced G-protein stimulation was also altered, and the median effective concentration (EC50) during morphine withdrawal (170.60 nM) was significantly higher than placebo (62.5 nM; P< 0.05). The maximal stimulation induced by the ET(A) receptor antagonist, BMS182874, in the morphine-withdrawal group (86.07%; EC50 = 31.25 nM) was significantly higher than in the placebo group (EC50 > 1000 nM). The ET(B) agonist, IRL1620, induced G-protein stimulation was similar in placebo (73.43%, EC50 = 13.26 nM) and morphine-withdrawal groups (75.08%, EC(50) = 11.70 nM), respectively. To our knowledge, this is the first report indicating involvement of central ET(A) receptors in neonatal morphine withdrawal.     

Epigenetic regulation in neural crest development

The neural crest (NC) is a multipotent, migratory cell population that arises from the developing dorsal neural fold of vertebrate embryos. Once their fates are specified, neural crest cells (NCCs) migrate along defined routes and differentiate into a variety of tissues, including bone and cartilage of the craniofacial skeleton, peripheral neurons, glia, pigment cells, endocrine cells, and mesenchymal precursor cells (Santagati and Rijli,2003; Dupin et al.,2006; Hall,2009). Abnormal development of NCCs causes a number of human diseases, including ear abnormalities (including deafness), heart anomalies, neuroblastomas, and mandibulofacial dysostosis (Hall,2009). For more than a century, NCCs have attracted the attention of geneticists and developmental biologists for their stem cell-like properties, including self-renewal and multipotent differentiation potential. However, we have only begun to understand the underlying mechanisms responsible for their formation and behavior. Recent studies have demonstrated that epigenetic regulation plays important roles in NC development. In this review, we focused on some of the most recent findings on chromatin-mediated mechanisms for vertebrate NCC development.     

Cochleovestibular nerve development is integrated with migratory neural crest cells

The cochleovestibular (CV) nerve, which connects the inner ear to the brain, is the nerve that enables the senses of hearing and balance. The aim of this study was to document the morphological development of the mouse CV nerve with respect to the two embryonic cells types that produce it, specifically, the otic vesicle-derived progenitors that give rise to neurons, and the neural crest cell (NCC) progenitors that give rise to glia. Otic tissues of mouse embryos carrying NCC lineage reporter transgenes were whole mount immunostained to identify neurons and NCC. Serial optical sections were collected by confocal microscopy and were compiled to render the three dimensional (3D) structure of the developing CV nerve. Spatial organization of the NCC and developing neurons suggest that neuronal and glial populations of the CV nerve develop in tandem from early stages of nerve formation. NCC form a sheath surrounding the CV ganglia and central axons. NCC are also closely associated with neurites projecting peripherally during formation of the vestibular and cochlear nerves. Physical ablation of NCC in chick embryos demonstrates that survival or regeneration of even a few individual NCC from ectopic positions in the hindbrain results in central projection of axons precisely following ectopic pathways made by regenerating NCC.     

Molecular regulation of neural crest development

The neural crest is a transient embryonic structure that gives rise to a multitude of different cell types in the vertebrate. As such, it is an iideal model to study the processes of vertebrate differentiation and development. This review focuses on two major questionsrelated to neural crest development. The first question concerns the degree and time of commitment of the neural crest cellsto differntt cell lineages and the emerging role of the homebox containing genes in regulating this process. Evidence from the cephalic crest suggests that the commitment process does start before the neural crest cells migrate away from the neural tube and gene ablation experiments suggest that different homeobox genes are required for the development of neural and mesenchymal tissue derivatives. However, clonal analysis of neural crest cell before migration suggests that many of the cells remain multi-potential indicating that the final determinative steps occur progressively during migration and in association with environmental influences. The second question concerns the nature of the environmental factors that determine the differentiation of neural crest cells into discrete lineages. Evidence is provided, mainly from in vitro experiments, that purified growth factors selectively promote the differentiation of neural crest cells down either sympathetic, adrenal, sensory, or melanocytic cell lineages.     

Roles of Eph receptors and ephrins in neural crest pathfinding

Ichthyophis kohtaoensis, a member of the limbless Gymnophiona, has a specialized subterranean burrowing mode of life and a predominantly olfactory-guided orientation. The only visually guided behavior seems to be negative phototaxis. As these animals possess extremely small eyes (only 540 μm in diameter in adults), functional investigations of single retinal cells by electrophysiological methods have so far failed. Therefore, the content and distribution of retinal transmitters have been investigated as indications for a functioning sense organ in an animal that is supposed to be blind. In this study, the organization and development of the dopaminergic system have been examined in the retinae of embryonic, larval, and adult I. kohtaoensis, by using an antiserum against tyrosine hydroxylase, the rate-limiting enzyme in the catecholamine synthetic pathway, and an antiserum against dopamine itself. Labeled somata are situated in the inner nuclear layer and in the ganglion cell layer. Dopamine-positive fibers form a dense diffuse plexus, that covers the whole inner plexiform layer, whereas tyrosine hydroxylase-immunoreactive processes show a tendency to arborize in a stratified manner. Tyrosine-hydroxylase-immunolabeled fibers can occasionally be observed in the optic nerve head of larval stages. During ontogenesis and larval development, the distribution of transmitter-expressing cells changes and their number decreases, but no general degeneration of the visual system is detectable. Adult Ichthyophis still have retinal transmitters, indicating that the eyes, although obviously playing a minor role in a subterranean ecological niche, retain all the elements of functioning sense organs. Received: 20 November 1996 / Accepted: 23 February 1997     

Migration and proliferation of cultured neural crest cells in W mutant neural crest chimeras.

Chimeric mice, generated by aggregating preimplantation embryos, have been instrumental in the study of the development of coat color patterns in mammals. This approach, however, does not allow for direct experimental manipulation of the neural crest cells, which are the precursors of melanoblasts. We have devised a system that allows assessment of the developmental potential and migration of neural crest cells in vivo following their experimental manipulation in vitro. Cultured C57Bl/6 neural crest cells were microinjected in utero into neurulating Balb/c or W embryos and shown to contribute efficiently to pigmentation in the host animal. The resulting neural crest chimeras showed, however, different coat pigmentation patterns depending on the genotype of the host embryo. Whereas Balb/c neural crest chimeras showed very limited donor cell pigment contribution, restricted largely to the head, W mutant chimeras displayed extensive pigmentation throughout, often exceeding 50% of the coat. In contrast to Balb/c chimeras, where the donor melanoblasts appeared to have migrated primarily in the characteristic dorsoventral direction, in W mutants the injected cells appeared to migrate in the longitudinal as well as the dorsoventral direction, as if the cells were spreading through an empty space. This is consistent with the absence of a functional endogenous melanoblast population in W mutants, in contrast to Balb/c mice, which contain a full complement of melanocytes. Our results suggest that the W mutation disturbs migration and/or proliferation of endogenous melanoblasts. In order to obtain information on clonal size and extent of intermingling of donor cells, two genetically marked neural crest cell populations were mixed and coinjected into W embryos. In half of the tricolored chimeras, no co-localization of donor crest cells was observed, while, in the other half, a fine intermingling of donor-derived colors had occurred. These results are consistent with the hypothesis that pigmented areas in the chimeras can be derived from extensive proliferation of a few donor clones, which were able to colonize large territories in the host embryo. We have also analyzed the development of pigmentation in neural crest cultures in vitro, and found that neural tubes explanted from embryos carrying wt or weak W alleles produced pigmented melanocytes while more severe W genotypes were associated with deficient pigment formation in vitro.     

Temporally distinct requirements for endothelin receptor B in the generation and migration of gut neural crest stem cells

Loss of Endothelin-3/Endothelin receptor B (EDNRB) signaling leads to aganglionosis of the distal gut (Hirschsprung's disease), but it is unclear whether it is required primarily for neural crest progenitor maintenance or migration. Ednrb-deficient gut neural crest stem cells (NCSCs) were reduced to 40% of wild-type levels by embryonic day 12.5 (E12.5), but no further depletion of NCSCs was subsequently observed. Undifferentiated NCSCs persisted in the proximal guts of Ednrb-deficient rats throughout fetal and postnatal development but exhibited migration defects after E12.5 that prevented distal gut colonization. EDNRB signaling may be required to modulate the response of neural crest progenitors to migratory cues, such as glial cell line-derived neurotrophic factor (GDNF). This migratory defect could be bypassed by transplanting wild-type NCSCs directly into the aganglionic region of the Ednrb(sl/sl) gut, where they engrafted and formed neurons as efficiently as in the wild-type gut.     

Cadherins in neural crest cell development and transformation

Cadherins constitute a superfamily of cell adhesion molecules involved in cell-cell interaction, histogenesis and cellular transformation. They have been implicated in the development of various lineages, including derivatives of the neural crest. Neural crest cells (NCC) emerge from the dorsal part of the neural tube after an epithelio-mesenchymal transition (EMT) and migrate through the embryo. After homing and differentiation, NCC give rise to many cell types, such as neurons, Schwann cells and melanocytes. During these steps, the pattern of expression of the various cadherins studied is very dynamic. Cadherins also display plasticity of expression during the transformation of neural crest cell derivatives. Here, we review the pattern of expression and the role of the main cadherins involved in the development and transformation of neural crest cell derivatives.     

Gene-regulatory interactions in neural crest evolution and development

In this review, we outline the gene-regulatory interactions driving neural crest development and compare these to a hypothetical network operating in the embryonic ectoderm of the cephalochordate amphioxus. While the early stages of ectodermal patterning appear conserved between amphioxus and vertebrates, later activation of neural crest-specific factors at the neural plate border appears to be a vertebrate novelty. This difference may reflect co-option of genetic pathways which conferred novel properties upon the evolving vertebrate neural plate border, potentiating the evolution of definitive neural crest.     

Differential development of cholinergic neurons from cranial and trunk neural crest cells in vitro

Several studies have suggested that the development of cholinergic properties in cranial parasympathetic neurons is determined by these cells' axial level of origin in the neural crest. All cranial parasympathetic neurons normally derive from cranial neural crest. Trunk neural crest cells give rise to sympathetic neurons, most of which are noradrenergic. To determine if there is an intrinsic difference in the ability of cranial and trunk neural crest cells to form cholinergic neurons, we have compared the development of choline acetyltransferase (ChAT)-immunoreactive cells in explants of quail cranial and trunk neural crest in vitro. Both cranial and trunk neural crest explants gave rise to ChAT-immunoreactive cells in vitro. In both types of cultures, some of the ChAT-positive cells also expressed immunoreactivity for the catecholamine synthetic enzyme tyrosine hydroxylase. However, several differences were seen between cranial and trunk cultures. First, ChAT-immunoreactive cells appeared two days earlier in cranial than in trunk cultures. Second, cranial cultures contained a higher proportion of ChAT-immunoreactive cells. Finally, a subpopulation of the ChAT-immunoreactive cells in cranial cultures exhibited neuronal traits, including neurofilament immunoreactivity. In contrast, neurofilament-immunoreactive cells were not seen in trunk cultures. These results suggest that premigratory cranial and trunk neural crest cells differ in their ability to form cholinergic neurons.