Connexin 43 expression reflects neural crest patterns during cardiovascular development

We used transgenic mice in which the promoter sequence for connexin 43 linked to a lacZ reporter was expressed in neural crest but not myocardial cells to document the pattern of cardiac neural crest cells in the caudal pharyngeal arches and cardiac outflow tract. Expression of lacZ was strikingly similar to that of cardiac neural crest cells in quail-chick chimeras. By using this transgenic mouse line to compare cardiac neural crest involvement in cardiac outflow septation and aortic arch artery development in mouse and chick, we were able to note differences and similarities in their cardiovascular development. Similar to neural crest cells in the chick, lacZ-positive cells formed a sheath around the persisting aortic arch arteries, comprised the aorticopulmonary septation complex, were located at the site of final fusion of the conal cushions, and populated the cardiac ganglia. In quail-chick chimeras generated for this study, neural crest cells entered the outflow tract by two pathways, submyocardially and subendocardially. In the mouse only the subendocardial population of lacZ-positive cells could be seen as the cells entered the outflow tract. In addition lacZ-positive cells completely surrounded the aortic sac prior to septation, while in the chick, neural crest cells were scattered around the aortic sac with the bulk of cells distributed in the bridging portion of the aorticopulmonary septation complex. In the chick, submyocardial populations of neural crest cells assembled on opposite sides of the aortic sac and entered the conotruncal ridges. Even though the aortic sac in the mouse was initially surrounded by lacZ-positive cells, the two outflow vessels that resulted from its septation showed differential lacZ expression. The ascending aorta was invested by lacZ-positive cells while the pulmonary trunk was devoid of lacZ staining. In the chick, both of these vessels were invested by neural crest cells, but the cells arrived secondarily by displacement from the aortic arch arteries during vessel elongation. This may indicate a difference in derivation of the pulmonary trunk in the mouse or a difference in distribution of cardiac neural crest cells. An independent mouse neural crest marker is needed to confirm whether the differences are indeed due to species differences in cardiovascular and/or neural crest development. Nevertheless, with the differences noted, we believe that this mouse model faithfully represents the location of cardiac neural crest cells. The similarities in location of lacZ-expressing cells in the mouse to that of cardiac neural crest cells in the chick suggest that this mouse is a good model for studying mammalian cardiac neural crest and that the mammalian cardiac neural crest performs functions similar to those shown for chick.     

Xenopus brain factor-2 controls mesoderm, forebrain and neural crest development

The forkhead type Brain Factor 2 from mouse and chicken help pattern the forebrain, optic vesicle and kidney. We have isolated a Xenopus homolog (Xbf2) and found that during gastrulation it is expressed in the dorsolateral mesoderm, where it helps specify this territory by downregulating BMP-4 and its downstream genes. Indeed, Xbf2 overexpression caused partial axis duplication. Interference with BMP-4 signaling also occurs in isolated animal caps, since Xbf2 induces neural tissue. Within the neurula forebrain, Xbf2 and the related Xbf1 gene are expressed in the contiguous diencephalic and telencephalic territories, respectively, and each gene represses the other. Finally, Xbf2 seems to participate in the control of neural crest migration. Our data suggest that XBF2 interferes with BMP-4 signaling, both in mesoderm and ectoderm.     

Dicer is required for survival of differentiating neural crest cells

The neural crest (NC) lineage gives rise to a wide array of cell types ranging from neurons and glia of the peripheral nervous system to skeletal elements of the head. The mechanisms regulating NC differentiation into such a large number of cell types remain largely unknown. MicroRNAs (miRNAs) play key roles in regulating developmental events suggesting they may also play a role during NC differentiation. To determine what roles miRNAs play in differentiation of NC-derived tissues, we deleted the miRNA processing gene Dicer in NC cells using the Wnt1-Cre deleter line. We show that deletion of Dicer soon after NC cells have formed does not affect their migration and colonization of their targets in the embryo. However, the post-migratory NC is dependent on Dicer for survival. In the head, loss of Dicer leads to a loss of NC-derived craniofacial bones while in the trunk, cells of the enteric, sensory and sympathetic nervous systems are lost during development. We found that loss of Dicer does not prevent the initial differentiation of NC but as development progresses, NC derivatives are lost due to apoptotic cell death. When Dicer is deleted, both Caspase-dependent and -independent apoptotic pathways are activated in the sensory ganglia but only the Caspase-dependent apoptotic program was activated in the sympathetic nervous system showing that the specific endogenous apoptotic programs are turned on by loss of Dicer. Our results show that Dicer and miRNAs, are required for survival of NC-derived tissues by preventing apoptosis during differentiation.     

FGF8 signaling is chemotactic for cardiac neural crest cells

Cardiac neural crest cells migrate into the pharyngeal arches where they support development of the pharyngeal arch arteries. The pharyngeal endoderm and ectoderm both express high levels of FGF8. We hypothesized that FGF8 is chemotactic for cardiac crest cells. To begin testing this hypothesis, cardiac crest was explanted for migration assays under various conditions. Cardiac neural crest cells migrated more in response to FGF8. Single cell tracing indicated that this was not due to proliferation and subsequent transwell assays showed that the cells migrate toward an FGF8 source. The migratory response was mediated by FGF receptors (FGFR) 1 and 3 and MAPK/ERK intracellular signaling. To test whether FGF8 is chemokinetic and/or chemotactic in vivo, dominant negative FGFR1 was electroporated into the premigratory cardiac neural crest. Cells expressing the dominant negative receptor migrated slower than normal cardiac neural crest cells and were prone to remain in the vicinity of the neural tube and die. Treating with the FGFR1 inhibitor, SU5402 or an FGFR3 function-blocking antibody also slowed neural crest migration. FGF8 over-signaling enhanced neural crest migration. Neural crest cells migrated to an FGF8-soaked bead placed dorsal to the pharynx. Finally, an FGF8 producing plasmid was electroporated into an ectopic site in the ventral pharyngeal endoderm. The FGF8 producing cells attracted a thick layer of mesenchymal cells. DiI labeling of the neural crest as well as quail-to-chick neural crest chimeras showed that neural crest cells migrated to and around the ectopic site of FGF8 expression. These results showing that FGF8 is chemotactic and chemokinetic for cardiac neural crest adds another dimension to understanding the relationship of FGF8 and cardiac neural crest in cardiovascular defects.     

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.     

The mych gene is required for neural crest survival during zebrafish development

Background

Among Myc family genes, c-Myc is known to have a role in neural crest specification in Xenopus and in craniofacial development in the mouse. There is no information on the function of other Myc genes in neural crest development, or about any developmental role of zebrafish Myc genes.

Principal Findings

We isolated the zebrafish mych (myc homologue) gene. Knockdown of mych leads to severe defects in craniofacial development and in certain other tissues including the eye. These phenotypes appear to be caused by cell death in the neural crest and in the eye field in the anterior brain.

Significance

Mych is a novel factor required for neural crest cell survival in zebrafish.     

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.     

Inactivation of Cdc42 in neural crest cells causes craniofacial and cardiovascular morphogenesis defects

Neural crest cells (NCCs) are physically responsible for craniofacial skeleton formation, pharyngeal arch artery remodeling and cardiac outflow tract septation during vertebrate development. Cdc42 (cell division cycle 42) is a Rho family small GTP-binding protein that works as a molecular switch to regulate cytoskeleton remodeling and the establishment of cell polarity. To investigate the role of Cdc42 in NCCs during embryonic development, we deleted Cdc42 in NCCs by crossing Cdc42 flox mice with Wnt1-cre mice. We found that the inactivation of Cdc42 in NCCs caused embryonic lethality with craniofacial deformities and cardiovascular developmental defects. Specifically, Cdc42 NCC knockout embryos showed fully penetrant cleft lips and short snouts. Alcian Blue and Alizarin Red staining of the cranium exhibited an unfused nasal capsule and palatine in the mutant embryos. India ink intracardiac injection analysis displayed a spectrum of cardiovascular developmental defects, including persistent truncus arteriosus, hypomorphic pulmonary arteries, interrupted aortic arches, and right-sided aortic arches. To explore the underlying mechanisms of Cdc42 in the formation of the great blood vessels, we generated Wnt1Cre-Cdc42-Rosa26 reporter mice. By beta-galactosidase staining, a subpopulation of Cdc42-null NCCs was observed halting in their migration midway from the pharyngeal arches to the conotruncal cushions. Phalloidin staining revealed dispersed, shorter and disoriented stress fibers in Cdc42-null NCCs. Finally, we demonstrated that the inactivation of Cdc42 in NCCs impaired bone morphogenetic protein 2 (BMP2)-induced NCC cytoskeleton remodeling and migration. In summary, our results demonstrate that Cdc42 plays an essential role in NCC migration, and inactivation of Cdc42 in NCCs impairs craniofacial and cardiovascular development in mice.     

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.     

Fate of cranial neural crest cells during craniofacial development in endothelin-A receptor-deficient mice

Most of the bone, cartilage and connective tissue of the lower jaw is derived from cranial neural crest cells (NCCs) arising from the posterior midbrain and hindbrain. Multiple factors direct the patterning of these NCCs, including endothelin-1-mediated endothelin A receptor (Edn1/Ednra) signaling. Loss of Ednra signaling results in multiple defects in lower jaw and neck structures, including homeotic transformation of lower jaw structures into upper jaw-like structures. However, since the Ednra gene is expressed by both migrating and post-migrating NCCs, the actual function of Ednra in cranial NCC development is not clear. Ednra signaling could be required for normal migration or guidance of NCCs to the pharyngeal arches or in subsequent events in post-migratory NCCs, including proliferation and survival. To address this question, we performed a fate analysis of cranial NCCs in Ednra-/- embryos using the R26R;Wnt1-Cre reporter system, in which Cre expression within NCCs results in permanent beta-galactosidase activity in NCCs and their derivatives. We find that loss of Ednra does not detectably alter either migration of most cranial NCCs into the mandibular first arch and second arch or their subsequent proliferation. However, mesenchymal cell apoptosis is increased two fold in both E9.5 and E10.5 Ednra-/- embryos, with apoptotic cells being present in and just proximal to the pharyngeal arches. Based on these studies, Ednra signaling appears to be required by most cranial NCCs after they reach the pharyngeal arches. However, a subset of NCCs appear to require Ednra signaling earlier, with loss of Ednra signaling likely leading to premature cessation of migration into or within the arches and subsequent cell death.     

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.     

Involvement of endothelin receptors in normal and pathological development of neural crest cells

Endothelin receptors (Ednr) are G-protein-coupled receptors with seven membrane-spanning domains and are involved in various physiological processes in adults. We review here the function of these receptors during the development and transformation of the neural crest cell-specific lineage. Neural crest cells (NCC) may be classified according to their location in the body. In particular, there are clear differences between the neural crest cells arising from the cephalic part of the embryo and those arising from the vagal and truncal part. The development of cranial and cardiac NCC requires the endothelin-1/Ednra system to be fully functional whereas the development of more posterior NCC requires full functionality of the endothelin-3/Ednrb system. Mutations have been found in the genes corresponding to these systems in mammals. These mutations principally impair pigmentation and enteric ganglia development. The precise patterns of expression of these receptors and their ligands have been determined in avian and mammalian models. Data obtained in vitro and in vivo have provided insight into the roles of these proteins in cell proliferation, migration, differentiation and transformation.     

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.