Regional differences in neural crest morphogenesis

Neural crest cells are pluripotent cells that emerge from the neural epithelium, migrate extensively, and differentiate into numerous derivatives, including neurons, glial cells, pigment cells and connective tissue. Major questions concerning their morphogenesis include: 1) what establishes the pathways of migration and 2) what controls the final destination and differentiation of various neural crest subpopulations. These questions will be addressed in this review. Neural crest cells from the trunk level have been explored most extensively. Studies show that melanoblasts are specified shortly after they depart from the neural tube, and this specification directs their migration into the dorsolateral pathway. We also consider other reports that present strong evidence for ventrally migrating neural crest cells being similarly fate restricted. Cranial neural crest cells have been less analyzed in this regard but the preponderance of evidence indicates that either the cranial neural crest cells are not fate-restricted, or are extremely plastic in their developmental capability and that specification does not control pathfinding. Thus, the guidance mechanisms that control cranial neural crest migration and their behavior vary significantly from the trunk. The vagal neural crest arises at the axial level between the cranial and trunk neural crest and represents a transitional cell population between the head and trunk neural crest. We summarize new data to support this claim. In particular, we show that: 1) the vagal-level neural crest cells exhibit modest developmental bias; 2) there are differences in the migratory behavior between the anterior and the posterior vagal neural crest cells reminiscent of the cranial and the trunk neural crest, respectively; 3) the vagal neural crest cells take the dorsolateral pathway to the pharyngeal arches and the heart, but the ventral pathway to the peripheral nervous system and the gut. However, these pathways are not rigidly specified because of prior fate restriction. Understanding the molecular, cellular and behavioral differences between these three populations of neural crest cells will be of enormous assistance when trying to understand the evolution of the neck.     

Regional differences in neural crest morphogenesis

Neural crest cells are pluripotent cells that emerge from the neural epithelium, migrate extensively and differentiate into numerous derivatives, including neurons, glial cells, pigment cells and connective tissue. Major questions concerning their morphogenesis include: (1) what establishes the pathways of migration? And (2), what controls the final destination and differentiation of various neural crest subpopulations? These questions will be addressed in this Review. Neural crest cells from the trunk level have been explored most extensively. Studies show that melanoblasts are specified shortly after they depart from the neural tube and this specification directs their migration into the dorsolateral pathway. We also consider other reports that present strong evidence for ventrally migrating neural crest cells being similarly fate restricted. Cranial neural crest cells have been less analyzed in this regard but the preponderance of evidence indicates that either the cranial neural crest cells are not fate-restricted or are extremely plastic in their developmental capability and that specification does not control pathfinding. Thus, the guidance mechanisms that control cranial neural crest migration and their behavior vary significantly from the trunk.The vagal neural crest arises at the axial level between the cranial and trunk neural crest and represents a transitional cell population between the head and trunk neural crest. We summarize new data to support this claim. In particular, we show that: (1) the vagal-level neural crest cells exhibit modest developmental bias; (2) there are differences in the migratory behavior between the anterior and the posterior vagal neural crest cells reminiscent of the cranial and the trunk neural crest, respectively and (3) the vagal neural crest cells take the dorsolateral pathway to the pharyngeal arches and the heart, but take the ventral pathway to the peripheral nervous system and the gut. However, these pathways are not rigidly specified because of prior fate restriction. Understanding the molecular, cellular and behavioral differences between these three populations of neural crest cells will be of enormous assistance when trying to understand the evolution of the neck.Key words: neural crest, morphogenesis, cell migration, chicken embryo, fate restriction, vagal neural crest, pathways     

Do vertebrate neural crest and cranial placodes have a common evolutionary origin?

Two embryonic tissues-the neural crest and the cranial placodes-give rise to most evolutionary novelties of the vertebrate head. These two tissues develop similarly in several respects: they originate from ectoderm at the neural plate border, give rise to migratory cells and develop into multiple cell fates including sensory neurons. These similarities, and the joint appearance of both tissues in the vertebrate lineage, may point to a common evolutionary origin of neural crest and placodes from a specialized population of neural plate border cells. However, a review of the developmental mechanisms underlying the induction, specification, migration and cytodifferentiation of neural crest and placodes reveals fundamental differences between the tissues. Taken together with insights from recent studies in tunicates and amphioxus, this suggests that neural crest and placodes have an independent evolutionary origin and that they evolved from the neural and non-neural side of the neural plate border, respectively.     

Wnt and BMP signaling govern lineage segregation of melanocytes in the avian embryo

Recent studies show that specification of some neural crest lineages occurs prior to or at the time of migration from the neural tube. We investigated what signaling events establish the melanocyte lineage, which has been shown to migrate from the trunk neural tube after the neuronal and glial lineages. Using in situ hybridization, we find that, although Wnts are expressed in the dorsal neural tube throughout the time when neural crest cells are migrating, the Wnt inhibitor cfrzb-1 is expressed in the neuronal and glial precursors and not in melanoblasts. This expression pattern suggests that Wnt signaling may be involved in specifying the melanocyte lineage. We further report that Wnt-3a-conditioned medium dramatically increases the number of pigment cells in quail neural crest cultures while decreasing the number of neurons and glial cells, without affecting proliferation. Conversely, BMP-4 is expressed in the dorsal neural tube throughout the time when neural crest cells are migrating, but is decreased coincident with the timing of melanoblast migration. This expression pattern suggests that BMP signaling may be involved in neural and glial cell differentiation or repression of melanogenesis. Purified BMP-4 reduces the number of pigment cells in culture while increasing the number of neurons and glial cells, also without affecting proliferation. Our data suggest that Wnt signaling specifies melanocytes at the expense of the neuronal and glial lineages, and further, that Wnt and BMP signaling have antagonistic functions in the specification of the trunk neural crest.     

Sequential actions of Pax3 and Pax7 drive xanthophore development in zebrafish neural crest

The Pax3/7 gene family has a fundamental and conserved role during neural crest formation. In people, PAX3 mutation causes Waardenburg syndrome, and murine Pax3 is essential for pigment formation. However, it is unclear exactly how Pax3 functions within the neural crest. Here we show that pax3 is expressed before other pax3/7 members, including duplicated pax3b, pax7 and pax7b genes, early in zebrafish neural crest development. Knockdown of Pax3 protein by antisense morpholino oligonucleotides results in defective fate specification of xanthophores, with complete ablation in the trunk. Other pigment lineages are specified and differentiate. As a consequence of xanthophore loss, expression of pax7, a marker of the xanthophore lineage, is reduced in neural crest. Morpholino knockdown of Pax7 protein shows that Pax7 itself is dispensable for xanthophore fate specification, although yellow pigmentation is reduced. Loss of xanthophores after reduction of Pax3 correlates with a delay in melanoblast differentiation followed by significant increase in melanophores, suggestive of a Pax3-driven fate switch within a chromatophore precursor or stem cell. Analysis of other neural crest derivatives reveals that, in the absence of Pax3, the enteric nervous system is ablated from its inception. Therefore, Pax3 in zebrafish is required for specification of two specific lineages of neural crest, xanthophores and enteric neurons.     

Single cell transplantation reveals interspecific cell communication in Drosophila chimeras

Cell-cell communication is not only a common strategy for cell fate specification in vertebrates, but plays important roles in invertebrate development as well. We report here on experiments testing the compatibility of mechanisms specifying cell fate among six different Drosophila species. Following interspecific transplantation, the development of single ectodermal cells was traced in order to test their abilities to proliferate and differentiate in a heterologous environment. Despite considerable differences in cell size and length of cell cycle among some of the species, the transplants gave rise to fully differentiated clones that were integrated into the host tissue. Clones comprised cells of epidermal and/or neural histotypes, indicating that mechanisms mediating the epidermal/neural dichotomy in the ectoderm are conserved between the species. Cells of the neural lineages differentiated into neurones, glia, or both. Moreover, heterologous neurones sent out axons that followed major pathways along nerves and within the neuropile, demonstrating their ability to recognize positional cues in the heterologous CNS of the host.     

Anterior/Posterior Influences on Neural Crest-Derived Pigment Cell Differentiation

The neural crest of vertebrate embryos has been used to elucidate steps involved in early embryonic cellular processes such as differentiation and migration. Neural crest cells form a ridge along the dorsal midline and subsequently they migrate throughout the embryo and differentiate into a wide variety of cell types. Intrinsic factors and environmental cues distributed along the neural tube, along the migratory pathways, and/or at the location of arrest influence the fate of neural crest cells. Although premigratory cells of the cranial and trunk neural crest exhibit differences in their differentiation potentials, premigratory trunk neural crest cells are generally assumed to have equivalent developmental potentials. Axolotl neural crest cells from different regions of origin, different stages of development, and challenged with different culture media have been analyzed for differentiation preferences pertaining to the pigment cell lineages. We report region-dependent differentiation of chromatophores from trunk neural crest at two developmental stages. Also, dosage with guanosine produces region-specific influences on the production of xanthophores from wild-type embryos. Our results support the hypothesis that spatial and temporal differences among premigratory trunk neural crest cells found along the anteroposterior axis influence developmental potentials and diminish the equivalency of axolotl neural crest cells.     

Cephalic neural crest cells and the evolution of craniofacial structures in vertebrates: morphological and embryological significance of the premandibular-mandibular boundary

The vertebrate head characteristically has two types of mesenchyme: the neural crest-derived ectomesenchyme and the mesoderm derived mesenchyme. Conserved patterns of development in various animal taxa imply the presence of shared inductive events for cephalic mesenchyme. These developmental programs can serve as developmental constraints that emerge as morphological homology of embryonic patterns. To understand the evolutionary changes in the developmental programs that shape the skull, we need to separate ancestral and derived patterns of vertebrate craniogenesis. This review deals with the terminology for neural crest cell subpopulations at each developmental stage, based on the topographical relationships and possible mechanisms for specification. The aim is to identify the changes that could have occurred in the evolutionary history of vertebrates. From comparisons of a lamprey species, Lethenteron japonicum, with gnathostomes it is clear that the initial distribution of cephalic crest cells is identical in the two animal lineages. In all vertebrate embryos, the trigeminal crest (TC) cells of an early pharyngula are subdivided into three subpopulations. At this stage, only the posterior subpopulation of the TC cells is specified as the mandibular arch, as compared to the more rostral components, the 'premandibular crest cells'. Later in development, the local specification patterns of the lamprey and the gnathostomes differ, so that homology cannot be established in the craniofacial primordia, including the oral apparatus. Therefore, embryological terminology should reflect these hierarchical patterns in developmental stages and phylogeny.     

Indian hedgehog signaling is required for proper formation, maintenance and migration of Xenopus neural crest

Neural crest induction is the result of the combined action at the neural plate border of FGF, BMP, and Wnt signals from the neural plate, mesoderm and nonneural ectoderm. In this work we show that the expression of Indian hedgehog (Ihh, formerly named Banded hedgehog) and members of the Hedgehog pathway occurs at the prospective neural fold, in the premigratory and migratory neural crest. We performed a functional analysis that revealed the requirement of Ihh signaling in neural crest development. During the early steps of neural crest induction loss of function experiments with antisense morpholino or locally grafted cyclopamine-loaded beads suppressed the expression of early neural crest markers concomitant with the increase in neural and epidermal markers. We showed that changes in Ihh activity produced no alterations in either cell proliferation or apoptosis, suggesting that this signal involves cell fate decisions. A temporal analysis showed that Hedgehog is continuously required not only in the early and late specification but also during the migration of the neural crest. We also established that the mesodermal source of Ihh is important to maintain specification and also to support the migratory process. By a combination of embryological and molecular approaches our results demonstrated that Ihh signaling drives in the migration of neural crest cells by autocrine or paracrine mechanisms. Finally, the abrogation of Ihh signaling strongly affected only the formation of cartilages derived from the neural crest, while no effects were observed on melanocytes. Taken together, our results provide insights into the role of the Ihh cell signaling pathway during the early steps of neural crest development.     

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.     

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.