DNA Methyltransferase 3b Is Dispensable for Mouse Neural Crest Development

The neural crest is a population of multipotent cells that migrates extensively throughout vertebrate embryos to form diverse structures. Mice mutant for the de novo DNA methyltransferase DNMT3b exhibit defects in two neural crest derivatives, the craniofacial skeleton and cardiac ventricular septum, suggesting that DNMT3b activity is necessary for neural crest development. Nevertheless, the requirement for DNMT3b specifically in neural crest cells, as opposed to interacting cell types, has not been determined. Using a conditional DNMT3b allele crossed to the neural crest cre drivers Wnt1-cre and Sox10-cre, neural crest DNMT3b mutants were generated. In both neural crest-specific and fully DNMT3b-mutant embryos, cranial neural crest cells exhibited only subtle migration defects, with increased numbers of dispersed cells trailing organized streams in the head. In spite of this, the resulting cranial ganglia, craniofacial skeleton, and heart developed normally when neural crest cells lacked DNMT3b. This indicates that DNTM3b is not necessary in cranial neural crest cells for their development. We conclude that defects in neural crest derivatives in DNMT3b mutant mice reflect a requirement for DNMT3b in lineages such as the branchial arch mesendoderm or the cardiac mesoderm that interact with neural crest cells during formation of these structures.     

Constitutive expression of preproendothelin in the cardiac neural crest selectively promotes expansion of the adventitia of the great vessels in vivo

Cardiac neural crest cells are essential for normal development of the great vessels and the heart, giving rise to a range of cell types, including both neuronal and non-neuronal adventitial cells and smooth muscle. Endothelin (ET) signaling plays an important role in the development of cardiac neural crest cell lineages, yet the underlying mechanisms that act to control their migration, differentiation, and proliferation remain largely unclear. We examined the expression patterns of the receptor, ET(A), and the ET-specific converting enzyme, ECE-1, in the pharyngeal arches and great vessels of the developing chick embryo. In situ hybridization analysis revealed that, while ET(A) is expressed in the pharyngeal arch mesenchyme, populated by cardiac neural crest cells, ECE-1 expression is localized to the outermost ectodermal cells of the arches and then to the innermost endothelial cells of the great vessels. This dynamic pattern of expression suggests that only a subpopulation of neural crest cells in these regions is responsive to ET signaling at particular developmental time points. To test this, retroviral gene delivery was used to constitutively express preproET-1, a precursor of mature ET-1 ligand, in the cardiac neural crest. This resulted in a selective expansion of the outermost, adventitial cell population in the great vessels. In contrast, neither differentiation nor proliferation of neural crest-derived smooth muscle cells was significantly affected. These results suggest that constitutive expression of exogenous preproET-1 in the cardiac neural crest results in expansion restricted to an adventitial cell population of the developing great vessels.     

A subpopulation of apoptosis-prone cardiac neural crest cells targets to the venous pole: multiple functions in heart development?

A well-described population of cardiac neural crest (NC) cells migrates toward the arterial pole of the embryonic heart and differentiates into various cell types, including smooth muscle cells of the pharyngeal arch arteries (but not the coronary arteries), cardiac ganglionic cells, and mesenchymal cells of the aortopulmonary septum. Using a replication-incompetent retrovirus containing the reporter gene LacZ, administered to the migratory neural crest of chicken embryos, we demonstrated another population of cardiac neural crest cells that employs the venous pole as entrance to the heart. On the basis of our present data we cannot exclude the possibility that precursors of these cells might not only originate from the dorsal part of the posterior rhombencephalon, but also from the ventral part. These NC cells migrate to locations surrounding the prospective conduction system as well as to the atrioventricular (AV) cushions. Concerning the prospective conduction system, the tagged neural crest cells can be found in regions where the atrioventricular node area, the retroaortic root bundle, the bundle of His, the left and right bundle branches, and the right atrioventricular ring bundle are positioned. The last area connects the posteriorly located AV node area with the retroaortic root bundle, which receives its neural crest cells through the arterial pole in concert with the cells giving rise to the aortopulmonary septum. The NC cells most probably do not form the conduction system proper, as they enter an apoptotic pathway as determined by concomitant TUNEL detection. It is possible that the NC cells in the heart become anoikic and, as a consequence, fail to differentiate further and merely die. However, because of the perfect timing of the arrival of crest cells, their apoptosis, and a change in electrophysiological behavior of the heart, we postulate that neural crest cells play a role in the last phase of differentiation of the cardiac conduction system. Alternatively, the separation of the central conduction system from the surrounding working myocardium is mediated by apoptotic neural crest cells. As for the presence of NC cells in both the outflow tract and the AV cushions, followed by apoptosis, a function is assigned in the muscularization of both areas, resulting in proper septation of the outflow tract and of the AV region. Failure of normal neural crest development may not only play a role in cardiac outflow tract anomalies but also in inflow tract abnormalities, such as atrioventricular septal defects.     

Gap Junction–mediated Cell–Cell Communication Modulates Mouse Neural Crest Migration

Previous studies showed that conotruncal heart malformations can arise with the increase or decrease in α₁ connexin function in neural crest cells. To elucidate the possible basis for the quantitative requirement for α₁ connexin gap junctions in cardiac development, a neural crest outgrowth culture system was used to examine migration of neural crest cells derived from CMV43 transgenic embryos overexpressing α₁ connexins, and from α₁ connexin knockout (KO) mice and FC transgenic mice expressing a dominant-negative α₁ connexin fusion protein. These studies showed that the migration rate of cardiac neural crest was increased in the CMV43 embryos, but decreased in the FC transgenic and α₁ connexin KO embryos. Migration changes occurred in step with connexin gene or transgene dosage in the homozygous vs. hemizygous α₁ connexin KO and CMV43 embryos, respectively. Dye coupling analysis in neural crest cells in the outgrowth cultures and also in the living embryos showed an elevation of gap junction communication in the CMV43 transgenic mice, while a reduction was observed in the FC transgenic and α₁ connexin KO mice. Further analysis using oleamide to downregulate gap junction communication in nontransgenic outgrowth cultures showed that this independent method of reducing gap junction communication in cardiac crest cells also resulted in a reduction in the rate of crest migration. To determine the possible relevance of these findings to neural crest migration in vivo, a lacZ transgene was used to visualize the distribution of cardiac neural crest cells in the outflow tract. These studies showed more lacZ-positive cells in the outflow septum in the CMV43 transgenic mice, while a reduction was observed in the α₁ connexin KO mice. Surprisingly, this was accompanied by cell proliferation changes, not in the cardiac neural crest cells, but in the myocardium— an elevation in the CMV43 mice vs. a reduction in the α₁ connexin KO mice. The latter observation suggests that cardiac neural crest cells may have a role in modulating growth and development of non–neural crest– derived tissues. Overall, these findings suggest that gap junction communication mediated by α₁ connexins plays an important role in cardiac neural crest migration. Furthermore, they indicate that cardiac neural crest perturbation is the likely underlying cause for heart defects in mice with the gain or loss of α₁ connexin function.     

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.     

Neural crest survival and differentiation in zebrafish depends on mont blanc/tfap2a gene function

Neural crest progenitor cells are the main contributors to craniofacial cartilage and connective tissue of the vertebrate head. These progenitor cells also give rise to the pigment, neuronal and glial cell lineages. To study the molecular basis of neural crest differentiation, we have cloned the gene disrupted in the mont blanc (mob(m610)) mutation, which affects all neural crest derivatives. Using a positional candidate cloning approach we identified an A to G transition within the 3' splice site of the sixth intron of the tfap2a gene that abolishes the last exon encoding the crucial protein dimerization and DNA-binding domains. Neural crest induction and specification are not hindered in mob(m610) mutant embryos, as revealed by normal expression of early neural crest specific genes such as snail2, foxd3 and sox10. In addition, the initial stages of cranial neural crest migration appear undisturbed, while at a later phase the craniofacial primordia in pharyngeal arches two to seven fail to express their typical set of genes (sox9a, wnt5a, dlx2, hoxa2/b2). In mob(m610) mutant embryos, the cell number of neuronal and glial derivatives of neural crest is greatly reduced, suggesting that tfap2a is required for their normal development. By tracing the fate of neural crest progenitors in live mont blanc (mob(m610)) embryos, we found that at 24 hpf neural crest cells migrate normally in the first pharyngeal arch while the preotic and postotic neural crest cells begin migration but fail to descend to the pharyngeal region of the head. TUNEL assay and Acridine Orange staining revealed that in the absence of tfap2a a subset of neural crest cells are unable to undergo terminal differentiation and die by apoptosis. Furthermore, surviving neural crest cells in tfap2a/mob(m610) mutant embryos proliferate normally and later differentiate to individual derivatives. Our results indicate that tfap2a is essential to turn on the normal developmental program in arches 2-7 and in trunk neural crest. Thus, tfap2a does not appear to be involved in early specification and cell proliferation of neural crest, but it is a key regulator of an early differentiation phase and is required for cell survival in neural crest derived cell lineages.     

Aortic arch and pharyngeal phenotype in the absence of BMP-dependent neural crest in the mouse

Neural crest cells are essential for proper development of a variety of tissues and structures, including peripheral and autonomic nervous systems, facial skeleton, aortic arches and pharyngeal glands like the thymus and parathyroids. Previous work has shown that bone morphogenic protein (BMP) signalling is required for the production of migratory neural crest cells that contribute to the neurogenic and skeletogenic lineages. We show here that BMP-dependent neural crest cells are also required for development of the embryonic aortic arches and pharynx-derived glands. Blocking formation or migration of this crest cell population from the caudal hindbrain resulted in strong phenotypes in the cardiac outflow tract and the thymus. Thymic aplasia or hypoplasia occurs despite uncompromised gene induction in the pharyngeal endoderm. In addition, when hypoplastic thymic tissue is found, it is ectopically located, but functional in thymopoiesis. Our data indicate that thymic phenotypes produced by neural crest deficits result from aberrant formation of pharyngeal pouches and impaired migration of thymic primordia because the mesenchymal content in the branchial arches is below a threshold level.     

Cytoplasmic protein methylation is essential for neural crest migration

As they initiate migration in vertebrate embryos, neural crest cells are enriched for methylation cycle enzymes, including S-adenosylhomocysteine hydrolase (SAHH), the only known enzyme to hydrolyze the feedback inhibitor of trans-methylation reactions. The importance of methylation in neural crest migration is unknown. Here, we show that SAHH is required for emigration of polarized neural crest cells, indicating that methylation is essential for neural crest migration. Although nuclear histone methylation regulates neural crest gene expression, SAHH and lysine-methylated proteins are abundant in the cytoplasm of migratory neural crest cells. Proteomic profiling of cytoplasmic, lysine-methylated proteins from migratory neural crest cells identified 182 proteins, several of which are cytoskeleton related. A methylation-resistant form of one of these proteins, the actin-binding protein elongation factor 1 alpha 1 (EF1α1), blocks neural crest migration. Altogether, these data reveal a novel and essential role for post-translational nonhistone protein methylation during neural crest migration and define a previously unknown requirement for EF1α1 methylation in migration.     

Cardiovascular malformations with normal smooth muscle differentiation in neural crest-specific type II TGFbeta receptor (Tgfbr2) mutant mice

Previous studies have demonstrated that TGFbeta induces a smooth muscle fate in primary neural crest cells in culture. By crossing a conditional allele of the type II TGFbeta receptor with the neural crest-specific Wnt1cre transgene, we have addressed the in vivo requirement for TGFbeta signaling in smooth muscle specification and differentiation. We find that elimination of the TGFbeta receptor does not alter neural crest cell specification to a smooth muscle fate in the cranial or cardiac domains, and that a smooth muscle fate is not realized by trunk neural crest cells in either control or mutant embryos. Instead, mutant embryos exhibit with complete penetrance two very specific and mechanistically distinct cardiovascular malformations--persistent truncus arteriosus (PTA) and interrupted aortic arch (IAA-B). Pharyngeal organ defects such as those seen in models of DiGeorge syndrome were not observed, arguing against an early perturbation of the cardiac neural crest cell lineage. We infer that TGFbeta is an essential morphogenic signal for the neural crest cell lineage in specific aspects of cardiovascular development, although one that is not required for smooth muscle differentiation.     

Multiple lineage-specific roles of Smad4 during neural crest development

During vertebrate development, neural crest cells are exposed to multiple extracellular cues that drive their differentiation into neural and non-neural cell lineages. Insights into the signals potentially involved in neural crest cell fate decisions in vivo have been gained by cell culture experiments that have allowed the identification of instructive growth factors promoting either proliferation of multipotent neural crest cells or acquisition of specific fates. For instance, members of the TGFβ factor family induce neurogenesis and smooth muscle cell formation at the expense of other fates in culture. In vivo, conditional ablation of various TGFβ signaling components resulted in malformations of non-neural derivatives of the neural crest, but it is unclear whether these phenotypes involved aberrant fate decisions. Moreover, it remains to be shown whether neuronal determination indeed requires TGFβ factor activity in vivo. To address these issues, we conditionally deleted Smad4 in the neural crest, thus inactivating all canonical TGFβ factor signaling. Surprisingly, neural crest cell fates were not affected in these mutants, with the exception of sensory neurogenesis in trigeminal ganglia. Rather, Smad4 regulates survival of smooth muscle and proliferation of autonomic and ENS neuronal progenitor cells. Thus, Smad signaling plays multiple, lineage-specific roles in vivo, many of which are elicited only after neural crest cell fate decision.     

Neural crest progenitors and stem cells: from early development to adulthood

In the vertebrate embryo, the neural crest forms transiently in the dorsal neural primordium to yield migratory cells that will invade nearly all tissues and later, will differentiate into bones and cartilages, neurons and glia, endocrine cells, vascular smooth muscle cells and melanocytes. Due to the amazingly diversified array of cell types it produces, the neural crest is an attractive model system in the stem cell field. We present here in vivo and in vitro studies of single cell fate, which led to the discovery and the characterization of stem cells in the neural crest of avian and mammalian embryos. Some of the key issues in neural crest cell diversification are discussed, such as the time of segregation of mesenchymal vs. neural/melanocytic lineages, and the origin and close relationships between the glial and melanocytic lineages. An overview is also provided of the diverse types of neural crest-like stem cells and progenitors, recently identified in a growing number of adult tissues in animals and humans. Current and future work, in which in vivo lineage studies and the use of injury models will complement the in vitro culture analysis, should help in unraveling the properties and function of neural crest-derived progenitors in development and disease.