A potential inhibitory function of draxin in regulating mouse trunk neural crest migration

Draxin is a repulsive axon guidance protein that plays important roles in the formation of three commissures in the central nervous system and dorsal interneuron 3 (dI3) in the chick spinal cord. In the present study, we report the expression pattern of mouse draxin in the embryonic mouse trunk spinal cord. In the presence of draxin, the longest net migration length of a migrating mouse trunk neural crest cell was significantly reduced. In addition, the relative number of apolar neural crest cells increased as the draxin treatment time increased. Draxin caused actin cytoskeleton rearrangement in the migrating trunk neural crest cells. Our data suggest that draxin may regulate mouse trunk neural crest cell migration by the rearrangement of cell actin cytoskeleton and by reducing the polarization activity of these cells subsequently.     

PlexinA1 interacts with PTK7 and is required for neural crest migration

Members of the plexin protein family are known regulators of axon guidance, but recent data indicate that they have broader functions in the regulation of embryonic morphogenesis. Here we provide further evidence of this by showing that PlexinA1 is expressed in Xenopus neural crest cells and is required for their migration. PlexinA1 expression is detected in migrating cranial neural crest cells and knockdown of PlexinA1 expression using Morpholino oligonucleotides inhibits neural crest migration. PlexinA1 likely affects neural crest migration by interaction with PTK7, a regulator of planar cell polarity that is required for neural crest migration. PlexinA1 and PTK7 interact in immunoprecipitation assays and show phenotypic interaction in co-injection experiments. Considering that plexins and PTK7 have been shown to genetically interact in Drosophila axon guidance and chick cardiac morphogenesis, our data suggest that this interaction is evolutionary conserved and may be relevant for a broad range of morphogenetic events including the migration of neural crest cells in Xenopus laevis.     

Evidence for collapsin-1 functioning in the control of neural crest migration in both trunk and hindbrain regions

Collapsin-1 belongs to the Semaphorin family of molecules, several members of which have been implicated in the co-ordination of axon growth and guidance. Collapsin-1 can function as a selective chemorepellent for sensory neurons, however, its early expression within the somites and the cranial neural tube (Shepherd, I., Luo, Y. , Raper, J. A. and Chang, S. (1996) Dev. Biol. 173, 185-199) suggest that it might contribute to the control of additional developmental processes in the chick. We now report a detailed study on the expression of collapsin-1 as well as on the distribution of collapsin-1-binding sites in regions where neural crest cell migration occurs. collapsin-1 expression is detected in regions bordering neural crest migration pathways in both the trunk and hindbrain regions and a receptor for collapsin-1, neuropilin-1, is expressed by migrating crest cells derived from both regions. When added to crest cells in vitro, a collapsin-1-Fc chimeric protein induces morphological changes similar to those seen in neuronal growth cones. In order to test the function of collapsin-1 on the migration of neural crest cells, an in vitro assay was used in which collapsin-1-Fc was immobilised in alternating stripes consisting of collapsin-Fc/fibronectin versus fibronectin alone. Explanted neural crest cells derived from both trunk and hindbrain regions avoided the collapsin-Fc-containing substratum. These results suggest that collapsin-1 signalling can contribute to the patterning of neural crest cell migration in the developing chick.     

Analysis of a repulsive axon guidance molecule, draxin, on ventrally directed axon projection in chick early embryonic midbrain

The mesencephalic V neurons and tectobulbar axons in chick embryo project over long distances that appear during the early development of the chick optic tectum. The mesencephalic V neuron and tectobulbar axonal growth begin at Hamburger and Hamilton stage 14 and stage 18, respectively. Both fibers proceed downward from the dorsal to the ventral side of the lateral wall of the optic tectum and then turn caudally and join the medial longitudinal fasciculus. Their axons appear in the most superficial layer of the tectum at early stages and do not cross the dorsal midline of the tectum. Here, we report the role of draxin, a recently identified axon guidance protein, in the formation of the ventrally directed tectum axonal tracts in chicken embryo. draxin is expressed in a high dorsal to low ventral gradient in chick optic tectum. In vitro experiments show that draxin repels neurite outgrowth from dorsal tectum explants. In vivo overexpression resulted in inhibition or misrouting of axon growth in the tectum. Therefore, draxin may be an important member of the collection of repulsive guidance molecules that regulate the formation of the ventrally directed tectum axon tracts.     

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     

Slit/Robo signaling is necessary to confine early neural crest cells to the ventral migratory pathway in the trunk

Neural crest cells migrate along two discrete pathways within the trunk of developing embryos. In the chick, early migrating crest cells are confined to a ventral pathway medial to the dermamyotome while later cells migrate on a dorsal pathway lateral to the dermamyotome. Here we show that Slits are expressed in the dermamyotome, that early migrating crest cells express the Slit receptors Robo 1 and Robo 2, that Slit2 repels migrating crest cells in an in vitro assay, and that the misexpression of a dominant-negative Robo1 receptor induces a significant fraction of early crest cells to migrate ectopically in the dorso-lateral pathway. These findings suggest that Slits, most likely those expressed in the dermamyotome, help to confine the migration of early crest cells to the ventral pathway.     

Ephrin-B ligands play a dual role in the control of neural crest cell migration

Little is known about the mechanisms that direct neural crest cells to the appropriate migratory pathways. Our aim was to determine how neural crest cells that are specified as neurons and glial cells only migrate ventrally and are prevented from migrating dorsolaterally into the skin, whereas neural crest cells specified as melanoblasts are directed into the dorsolateral pathway. Eph receptors and their ephrin ligands have been shown to be essential for migration of many cell types during embryonic development. Consequently, we asked if ephrin-B proteins participate in the guidance of melanoblasts along the dorsolateral pathway, and prevent early migratory neural crest cells from invading the dorsolateral pathway. Using Fc fusion proteins, we detected the expression of ephrin-B ligands in the dorsolateral pathway at the stage when neural crest cells are migrating ventrally. Furthermore, we show that ephrins block dorsolateral migration of early-migrating neural crest cells because when we disrupt the Eph-ephrin interactions by addition of soluble ephrin-B ligand to trunk explants, early neural crest cells migrate inappropriately into the dorsolateral pathway. Surprisingly, we discovered the ephrin-B ligands continue to be expressed along the dorsolateral pathway during melanoblast migration. RT-PCR analysis, in situ hybridisation, and cell surface-labelling of neural crest cell cultures demonstrate that melanoblasts express several EphB receptors. In adhesion assays, engagement of ephrin-B ligands to EphB receptors increases melanoblast attachment to fibronectin. Cell migration assays demonstrate that ephrin-B ligands stimulate the migration of melanoblasts. Furthermore, when Eph signalling is disrupted in vivo, melanoblasts are prevented from migrating dorsolaterally, suggesting ephrin-B ligands promote the dorsolateral migration of melanoblasts. Thus, transmembrane ephrins act as bifunctional guidance cues: they first repel early migratory neural crest cells from the dorsolateral path, and then later stimulate the migration of melanoblasts into this pathway. The mechanisms by which ephrins regulate repulsion or attraction in neural crest cells are unknown. One possibility is that the cellular response involves signalling to the actin cytoskeleton, potentially involving the activation of Cdc42/Rac family of GTPases. In support of this hypothesis, we show that adhesion of early migratory cells to an ephrin-B-derivatized substratum results in cell rounding and disruption of the actin cytoskeleton, whereas plating of melanoblasts on an ephrin-B substratum induces the formation of microspikes filled with F-actin.     

Neural crest cells and motor axons in avians: Common and distinct migratory molecules

It has long been thought that the same molecules guide both trunk neural crest cells and motor axons as these cell types grow and extend to their target regions in developing embryos. There are common territories that are navigated by these cell types: both cells grow through the rostral portion of the somitic sclerotomes and avoid the caudal half of the sclerotomes. However, these cell types seem to use different molecules to guide them to their target regions. In this Review, I will discuss the common and distinct methods of migration taken by trunk neural crest cells and motor axons as they grow and populate their target regions through chick embryos at the level of the trunk.Key words: migration, axon, motor neuron, trunk neural crest cells, chick     

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.     

Distribution and migration pathways of HNK-1-immunoreactive neural crest cells in teleost fish embryos.

Whole mounts and cross-sections of embryos from three species of teleost fish were immunostained with the HNK-1 monoclonal antibody, which recognizes an epitope on migrating neural crest cells. A similar distribution and migration was found in all three species. The crest cells in the head express the HNK-1 epitope after they have segregated from the neural keel. The truncal neural crest cells begin to express the epitope while they still reside in the dorsal region of the neural keel; this has not been observed in other vertebrates. The cephalic and anterior truncal neural crest cells migrate under the ectoderm; the cephalic cells then enter into the gill arches and the anterior truncal cells into the mesentery of the digestive tract where they cease migration. These cephalic and anterior trunk pathways are similar to those described in Xenopus and chick. The neural crest cells of the trunk, after segregation, accumulate in the dorsal wedges between the somites, however, unlike in chick and rat, they do not migrate in the anterior halves of the somites but predominantly between the neural tube and the somites, the major pathway observed in carp and amphibians; some cells migrate over the somites. The HNK-1 staining of whole-mount embryos revealed a structure resembling the Rohon-Beard and extramedullary cells, the primary sensory system in amphibians. Such a system has not been described in fish.     

Patterns of fibronectin gene expression and splicing during cell migration in chicken embryos

A variety of evidence suggests that fibronectin (FN) promotes cell migration during embryogenesis, and it has been suggested that the deposition of FN along migratory pathways may also play a role in cell guidance. In order to investigate such a role for FN, it is important to determine the relative contribution of migrating and pathway-forming cells to the FN in the migratory track, as any synthesis of FN by the migrating cells might be expected to mask guidance cues provided by the exogenous FN from pathway-forming cells. We have therefore used in situ hybridization to determine in developing chicken embryos the distribution and alternative splicing of FN mRNA during three different cell migrations known to occur through FN-rich environments; neural crest cell migration, mesenchymal cell migration in the area vasculosa and endocardial cushion cell migration in the heart. Our results show that trunk neural crest cells do not contain significant FN mRNA during their initial migration. In contrast, migrating mesenchymal cells of the area vasculosa and endocardial cushion cells both contain abundant FN mRNA. Furthermore, the FN mRNA in these migrating mesenchymal and endocardial cells appears to be spliced in a manner identical with that present in the cells adjacent to their pathways. This in vivo evidence for FN synthesis by migrating and pathway cells argues against a generalized role for exogenously produced FN as a guidance mechanism for cell migration.     

PTEN基因对早期胚胎神经嵴细胞迁移的作用研究

目的 初步探讨PTEN基因在早期神经嵴细胞迁移中的作用.方法 首先胚胎整体的原位杂交和免疫荧光方法检测鸡胚胎内源性的PTEN基因及蛋白水平的表达情况;其次,利用鸡胚胎体内半侧神经管转染的方法,使神经管一侧PTEN基因过表达,对侧神经管为正常对照侧;最后,通过Pax7的整体胚胎免疫荧光表达观察PTEN基因对其标记的部分神经嵴细胞迁移的影响.结果 内源性PTEN基因在mRNA和蛋白水平表达显示,其在早期胚胎HH4期的神经板即开始明显的表达;通过半侧过表达PTEN基因后观察到过表达PTEN基因侧的头部神经嵴细胞迁移与对照侧相比明显受到抑制,但对躯干部的影响并不明显.结论 PTEN基因可能抑制早期胚胎头部神经嵴细胞的迁移.     

Inhibitory effects of draxin on axonal outgrowth and migration of precerebellar neurons

The rhombic lip, a dorsal stripe of the neuroepithelium lining the edge of the fourth ventricle, is the site of origin of precerebellar neurons (PCN), which migrate tangentially towards the floor plate. After reaching the floor plate, they project their axons to the cerebellum. Although previous studies have shown that the guidance molecules Netrin/DCC and Slit/Robo have critical roles in PCN migration, the molecular mechanisms underlying this process remain poorly understood. Here, we report that draxin, a repulsive axon guidance protein, is involved in PCN development. We found that draxin is expressed in the rhombic lip and migratory stream of some PCN in the developing hindbrain of mice. In addition, draxin inhibited neurite outgrowth and nuclei migration from rhombic lip explants. These results suggest that draxin functions as a repulsive guidance cue for PCN migration. However, we observed no significant differences in PCN distribution between draxin^−/− and wild type embryos. Thus, draxin and other axon guidance cues may have redundant roles in PCN migration.     

Cholinergic traits in the neural crest: Acetylcholinesterase in crest cells of the chick embryo

Previous work by our group has demonstrated that mesencephalic neural crest cells at an early stage of migration are able to synthesize acetylcholine (ACh). Acetylcholinesterase (AChE), the enzyme responsible for ACh degradation, was examined in neural crest cells of the chick embryo, using cytochemical and biochemical methods. Observations at the light microscope level showed that cholinesterase activity, identified as true AChE, was present at all axial levels in presumptive crest cells of the neural folds, soon after closure of the neural tube. Subsequently, AChE activity was found in cells of the individualized neural crest and in crest cells migrating at cephalic and trunk levels. Cell counts revealed that 88–94% of the total crest population was AChE-positive. Electron microscope observations indicated that the enzyme was confined to perinuclear and endoplasmic reticulum cisternae. The AChE of migrating mesencephalic neural crest cells was identified as the dimeric form (sedimentation coefficient 6.9 S) of the catalytic subunit. These results indicate that the specific AChE is present in the majority of neural crest cells all along the neural axis. Thus the ability to synthesize and degrade ACh is expressed at least in some neural crest cells at an early stage of development.     

Novel expression patterns of Pax3/Pax7 in early trunk neural crest and its melanocyte and non‐melanocyte lineages in amniote embryos

Neural crest cells are considered a key vertebrate feature that is studied intensively because of their relevance to development and evolution. Here we report the expression of Pax7 in the dorsal non‐neural ectoderm and in the trunk neural crest of the early chick embryo. Pax7 is expressed in the trunk neural crest migrating along the ventral and dorsolateral routes. Pax7 is first downregulated in the neural crest‐derived neuronal precursors, secondly in the glial, and finally in the melanocyte precursors. Conserved developmental expression in the melanocyte lineage of both Pax3 and Pax7 was evidenced in chick and quail, but only Pax3 in mouse and rat.     

The early migration of neural crest cells in the trunk region of the avian embryo: an electron microscopic study

Four phases of neural crest migration characteristic of early avian trunk regions are described: (a) appearance, during which crest cells reside in the dorsal neural tube, but are separated from each other dorsally by large spaces; (b) condensation, during which large spaces between the crest cells become reduced, the cells elongate, flatten upon the surface of the neural tube, and become oriented tangentially (i.e., with their long axes perpendicular to the longitudinal axes of the neural tube); (c) early migration, during which the crest population expands uniformly to meet the dorsal apex of the somites; and (d) advanced migration, during which crest cells appear in the extracellular space dorsal to the somites. At the most advanced phases, the crest population at the dorsal midline decreased in number, with a concomitant loss of tangential orientation and the appearance of spaces between the cells. Extracellular components of the acellular spaces through which crest cells migrate are also described. The observations are discussed in terms of (1) those morphological changes undergone by crest cells during migration, and (2) possible factors that might delimit crest pathways. It is suggested that the operation of contact inhibition of movement within the crest population is sufficient to determine the direction of crest migration.     

Novel expression patterns of Pax3/Pax7 in early trunk neural crest and its melanocyte and non-melanocyte lineages in amniote embryos

Neural crest cells are considered a key vertebrate feature that is studied intensively because of their relevance to development and evolution. Here we report the expression of Pax7 in the dorsal non-neural ectoderm and in the trunk neural crest of the early chick embryo. Pax7 is expressed in the trunk neural crest migrating along the ventral and dorsolateral routes. Pax7 is first downregulated in the neural crest-derived neuronal precursors, secondly in the glial, and finally in the melanocyte precursors. Conserved developmental expression in the melanocyte lineage of both Pax3 and Pax7 was evidenced in chick and quail, but only Pax3 in mouse and rat.     

Slow muscle regulates the pattern of trunk neural crest migration in zebrafish

In avians and mice, trunk neural crest migration is restricted to the anterior half of each somite. Sclerotome has been shown to play an essential role in this restriction; the potential role of other somite components in specifying neural crest migration is currently unclear. By contrast, in zebrafish trunk neural crest, migration on the medial pathway is restricted to the middle of the medial surface of each somite. Sclerotome comprises only a minor part of zebrafish somites, and the pattern of neural crest migration is established before crest cells contact sclerotome cells, suggesting other somite components regulate the pattern of zebrafish neural crest migration. Here, we use mutants to investigate which components regulate the pattern of zebrafish trunk neural crest migration on the medial pathway. The pattern of trunk neural crest migration is aberrant in spadetail mutants that have very reduced somitic mesoderm, in no tail mutants injected with spadetail morpholino antisense oligonucleotides that entirely lack somitic mesoderm and in somite segmentation mutants that have normal somite components but disrupted segment borders. Fast muscle cells appear dispensable for patterning trunk neural crest migration. However, migration is abnormal in Hedgehog signaling mutants that lack slow muscle cells, providing evidence that slow muscle cells regulate the pattern of trunk neural crest migration. Consistent with this idea, surgical removal of adaxial cells, which are slow muscle precursors, results in abnormal patterning of neural crest migration; normal patterning can be restored by replacing the ablated adaxial cells with ones transplanted from wild-type embryos.