Regulation of the onset of neural crest migration by coordinated activity of BMP4 and Noggin in the dorsal neural tube.

For neural crest cells to engage in migration, it is necessary that epithelial premigratory crest cells convert into mesenchyme. The mechanisms that trigger cell delamination from the dorsal neural tube remain poorly understood. We find that, in 15- to 40-somite-stage avian embryos, BMP4 mRNA is homogeneously distributed along the longitudinal extent of the dorsal neural tube, whereas its specific inhibitor noggin exists in a gradient of expression that decreases caudorostrally. This rostralward reduction in signal intensity coincides with the onset of emigration of neural crest cells. Hence, we hypothesized that an interplay between Noggin and BMP4 in the dorsal tube generates graded concentrations of the latter that in turn triggers the delamination of neural crest progenitors. Consistent with this suggestion, disruption of the gradient by grafting Noggin-producing cells dorsal to the neural tube at levels opposite the segmental plate or newly formed somites, inhibited emigration of HNK-1-positive crest cells, which instead accumulated within the dorsal tube. Similar results were obtained with explanted neural tubes from the same somitic levels exposed to Noggin. Exposure to Follistatin, however, had no effect. The Noggin-dependent inhibition was overcome by concomitant treatment with BMP4, which when added alone, also accelerated cell emigration compared to untreated controls. Furthermore, the observed inhibition of neural crest emigration in vivo was preceded by a partial or total reduction in the expression of cadherin-6B and rhoB but not in the expression of slug mRNA or protein. Altogether, these results suggest that a coordinated activity of Noggin and BMP4 in the dorsal neural tube triggers delamination of specified, slug-expressing neural crest cells. Thus, BMPs play multiple and discernible roles at sequential stages of neural crest ontogeny, from specification through delamination and later differentiation of specific neural crest derivatives.     

Analysis of neural crest cell lineage and migration.

In this review, we describe the results of recent experiments designed to investigate various aspects of neural crest cell lineage and migration. We have analyzed the lineage of individual premigratory neural crest cells by injecting a fluorescent lineage tracer dye, lysinated fluorescein dextran, into cells within the dorsal neural tube. Individual clones contained cells that were located in very diverse sites consistent with their being sensory neurons, prepigment cells, Schwann cells, adrenergic cells, and neural tube cells. These results suggest that some neural crest cells in the trunk and cranial regions are multipotent prior to their emigration from the neural tube. The environment through which neural crest cells move influences both the pattern and direction of their migration. We have shown that the sclerotomal portion of the somites are responsible for the rostrocaudal pattern of trunk neural crest cell movement, whereas the neural tube appears to govern the dorsoventral position of neural crest-derived ganglia. In addition, the notochord inhibits the movement of neural crest cells. In order to understand necessary cell-matrix interactions in neural crest migration, we have performed perturbation experiments, in which antibodies directed against cell surface or extracellular matrix molecules were introduced along neural crest pathways. We find that integrins, fibronectin, laminin, and tenascin all play some role in cranial neural crest emigration. Thus, multiple factors may be involved in controlling neural crest cell migration, and different factors may be important for migration in different regions of the embryo.     

机械阻断神经管的闭合影响爪蟾神经管辐射状切入及神经嵴细胞定向迁移(英文)

神经管闭合缺陷 (NTDs)是一种严重的先天畸形疾病,在新生儿中有千分之一的发病率。神经管融合前后,多种组织参与形态发生运动。神经管一经融合,神经嵴细胞就会向背侧中线方向产生单极突出并向此方向迁移形成神经管的顶部。与此同时,神经管从腹侧开始发生辐射状切入以实现单层化。在此,我们在非洲爪蟾的移植体中机械阻断神经管的闭合以检测其细胞运动及随后的图式形成。结果显示神经管闭合缺陷的移植体不能形成单层化的神经管,并且神经嵴细胞滞留在侧面区域不能向背侧中线迁移,而对神经前体标记基因的检测显示神经管的背腹图式形成并未受到影响。以上结果表明神经管的融合对于辐射状切入和神经嵴细胞向背侧中线方向的迁移过程是必需的,而对于神经管的沿背腹轴方向的图式形成是非必需的。     

A vital dye analysis of the timing and pathways of avian trunk neural crest cell migration

To permit a more detailed analysis of neural crest cell migratory pathways in the chick embryo, neural crest cells were labelled with a nondeleterious membrane intercalating vital dye, DiI. All neural tube cells with endfeet in contact with the lumen, including premigratory neural crest cells, were labelled by pressure injecting a solution of DiI into the lumen of the neural tube. When assayed one to three days later, migrating neural crest cells, motor axons, and ventral root cells were the only cells types external to the neural tube labelled with DiI. During the neural crest cell migratory phase, distinctly labelled cells were found along: (1) a dorsolateral pathway, under the epidermis, as well adjacent to and intercalating through the dermamyotome; and (2) a ventral pathway, through the rostral portion of each sclerotome and around the dorsal aorta as described previously. In contrast to those cells migrating through the sclerotome, labelled cells on the dorsolateral pathway were not segmentally arranged along the rostrocaudal axis. DiI-labelled cells were observed in all truncal neural crest derivatives, including subepidermal presumptive pigment cells, dorsal root ganglia, and sympathetic ganglia. By varying the stage at which the injection was performed, neural crest cell emigration at the level of the wing bud was shown to occur from stage 13 through stage 22. In addition, neural crest cells were found to populate their derivatives in a ventral-to-dorsal order, with the latest emigrating cells migrating exclusively along the dorsolateral pathway.     

Notochord grafts do not suppress formation of neural crest cells or commissural neurons.

Grafting experiments previously have established that the notochord affects dorsoventral polarity of the neural tube by inducing the formation of ventral structures such as motor neurons and the floor plate. Here, we examine if the notochord inhibits formation of dorsal structures by grafting a notochord within or adjacent to the dorsal neural tube prior to or shortly after tube closure. In all cases, neural crest cells emigrated from the neural tube adjacent to the ectopic notochord. When analyzed at stages after ganglion formation, the dorsal root ganglia appeared reduced in size and shifted in position in embryos receiving grafts. Another dorsal cell type, commissural neurons, identified by CRABP and neurofilament immunoreactivity, differentiated in the vicinity of the ectopic notochord. Numerous neuronal cell bodies and axonal processes were observed within the induced, but not endogenous, floor plate 1 to 2 days after implantation but appeared to be cleared with time. These results suggest that dorsally implanted notochords cannot prevent the formation of neural crest cells or commissural neurons, but can alter the size and position of neural crest-derived dorsal root ganglia.     

How to become neural crest: from segregation to delamination

The development of the neural crest up to the stage where they leave the neural tube can be observed as a series of concatenated but independent events that involve dorsalization of the neural plate/neural tube, neural crest induction, segregation and stabilization, epithelial to mesenchymal transition and delamination. During all these processes, the nascent neural crest cells are subjected to the influence of different signals and have to overcome competition for cell fate and apoptotic signals. In addition, striking rostrocaudal differences unveil how the regulatory cascades are somehow different but still can lead to the production of bona fide neural crest cells.     

Cellular retinoic acid-binding protein and the role of retinoic acid in the development of the chick embryo

The distribution of cellular retinoic acid-binding protein (CRABP) in four stages of chick development is described using an affinity-purified antibody against rat CRABP. CRABP is the protein to which retinoic acid (RA) binds when it enters cells and may reflect the requirement of those cells for RA. We found several discrete cell populations which showed high levels of immunoreactivity. Some were in the neural tube such as the commissural neurons and the dorsal roof plate. Some were of neural crest origin such as the dorsal root ganglia, sensory axons, sympathetic ganglia, and enteric ganglia. The remaining populations were certain connective tissue cells, limb bud cells, and the myotome. These results suggest that certain organ systems, particularly the nervous system, have a requirement for RA during development and they may further our understanding of the teratogenic effects of retinoids on the embryo.     

Neural Crest Cell Fate

The neural crest, the intriguing cell population that gives rise to a panoply of derivatives in the vertebrate embryo, including the mesenchymal structures in the head, melanocytes and most of the peripheral nervous system, still proves to be an important yet enigmatic developmental cell population to study with applications in stem cell biology, cancer biology and clinical medicine. Albeit our knowledge base is rich due to a strong history of experimentation, the fact that we have yet to decipher so many key aspects of neural crest cell (NCC) behavior speaks to the challenging complexity of this transient yet vital cell population. With the advent of new fluorescent tracing techniques, we have reexamined the migratory behaviors and ultimate fate of ventrally migrating avian NCCs within a late wave of emigration and identified a subpopulation of lineally restricted NCCs who migrate to the contralateral dorsal root ganglia (DRG) and therein give rise to mitotically active progenitor cells that ultimately produce the majority of the nociceptive sensory neurons in the DRG. These data provide evidence for the fate prespecification of subsets of NCCs while still resident in the neural tube.     

Netrin‐1 is required for efficient neural tube closure

During neural tube formation, neural plate cells migrate from the lateral aspects of the dorsal surface towards the midline. Elevation of the lateral regions of the neural plate produces the neural folds which then migrate to the midline where they fuse at their dorsal tips, generating a closed neural tube comprising an apicobasally polarized neuroepithelium. Our previous study identified a novel role for the axon guidance receptor neogenin in Xenopus neural tube formation. We demonstrated that loss of neogenin impeded neural fold apposition and neural tube closure. This study also revealed that neogenin, via its interaction with its ligand, RGMa, promoted cell–cell adhesion between neural plate cells as the neural folds elevated and between neuroepithelial cells within the neural tube. The second neogenin ligand, netrin‐1, has been implicated in cell migration and epithelial morphogenesis. Therefore, we hypothesized that netrin‐1 may also act as a ligand for neogenin during neurulation. Here we demonstrate that morpholino knockdown of Xenopus netrin‐1 results in delayed neural fold apposition and neural tube closure. We further show that netrin‐1 functions in the same pathway as neogenin and RGMa during neurulation. However, contrary to the role of neogenin‐RGMa interactions, neogenin‐netrin‐1 interactions are not required for neural fold elevation or adhesion between neuroepithelial cells. Instead, our data suggest that netrin‐1 contributes to the migration of the neural folds towards the midline. We conclude that both neogenin ligands work synergistically to ensure neural tube closure. © 2012 Wiley Periodicals, Inc., 2013     

Chicken protocadherin-1 functions to localize neural crest cells to the dorsal root ganglia during PNS formation

In vertebrate embryos, neural crest cells emerge from the dorsal neural tube and migrate along well defined pathways to form a wide diversity of tissues, including the majority of the peripheral nervous system (PNS). Members of the cadherin family of cell adhesion molecules play key roles during the initiation of migration, mediating the delamination of cells from the neural tube. However, a role for cadherins in the sorting and re-aggregation of the neural crest to form the PNS has not been established. We report the requirement for a protocadherin, chicken protocadherin-1 (Pcdh1), in neural crest cell sorting during the formation of the dorsal root ganglia (DRG). In embryos, cPcdh1 is highly expressed in the developing DRG, where it co-localizes with the undifferentiated and mitotically active cells along the perimeter. Pcdh1 can promote cell adhesion in vivo and disrupting Pcdh1 function in embryos results in fewer neural crest cells localizing to the DRG, with a concomitant increase in cells that migrate to the sympathetic ganglia. Furthermore, those cells that still localize to the DRG, when Pcdh1 is inhibited, are no longer found at the perimeter, but are instead dispersed throughout the DRG and are now more likely to differentiate along the sensory neuron pathway. These results demonstrate that Pcdh1-mediated cell adhesion plays an important role as neural crest cells coalesce to form the DRG, where it serves to sort cells to the mitotically active perimeter.     

Pax3-expressing trigeminal placode cells can localize to trunk neural crest sites but are committed to a cutaneous sensory neuron fate

The cutaneous sensory neurons of the ophthalmic lobe of the trigeminal ganglion are derived from two embryonic cell populations, the neural crest and the paired ophthalmic trigeminal (opV) placodes. Pax3 is the earliest known marker of opV placode ectoderm in the chick. Pax3 is also expressed transiently by neural crest cells as they emigrate from the neural tube, and it is reexpressed in neural crest cells as they condense to form dorsal root ganglia and certain cranial ganglia, including the trigeminal ganglion. Here, we examined whether Pax3+ opV placode-derived cells behave like Pax3+ neural crest cells when they are grafted into the trunk. Pax3+ quail opV ectoderm cells associate with host neural crest migratory streams and form Pax3+ neurons that populate the dorsal root and sympathetic ganglia and several ectopic sites, including the ventral root. Pax3 expression is subsequently downregulated, and at E8, all opV ectoderm-derived neurons in all locations are large in diameter, and virtually all express TrkB. At least some of these neurons project to the lateral region of the dorsal horn, and peripheral quail neurites are seen in the dermis, suggesting that they are cutaneous sensory neurons. Hence, although they are able to incorporate into neural crest-derived ganglia in the trunk, Pax3+ opV ectoderm cells are committed to forming cutaneous sensory neurons, their normal fate in the trigeminal ganglion. In contrast, Pax3 is not expressed in neural crest-derived neurons in the dorsal root and trigeminal ganglia at any stage, suggesting either that Pax3 is expressed in glial cells or that it is completely downregulated before neuronal differentiation. Since Pax3 is maintained in opV placode-derived neurons for some considerable time after neuronal differentiation, these data suggest that Pax3 may play different roles in opV placode cells and neural crest cells.     

Neural crest development is regulated by the transcription factor Sox9

The neural crest is a transient migratory population of stem cells derived from the dorsal neural folds at the border between neural and non-neural ectoderm. Following induction, prospective neural crest cells are segregated within the neuroepithelium and then delaminate from the neural tube and migrate into the periphery, where they generate multiple differentiated cell types. The intrinsic determinants that direct this process are not well defined. Group E Sox genes (Sox8, Sox9 and Sox10) are expressed in the prospective neural crest and Sox9 expression precedes expression of premigratory neural crest markers. Here, we show that group E Sox genes act at two distinct steps in neural crest differentiation. Forced expression of Sox9 promotes neural-crest-like properties in neural tube progenitors at the expense of central nervous system neuronal differentiation. Subsequently, in migratory neural crest cells, SoxE gene expression biases cells towards glial cell and melanocyte fate, and away from neuronal lineages. Although SoxE genes are sufficient to initiate neural crest development they do not efficiently induce the delamination of ectopic neural crest cells from the neural tube consistent with the idea that this event is independently controlled. Together, these data identify a role for group E Sox genes in the initiation of neural crest development and later SoxE genes influence the differentiation pathway adopted by migrating neural crest cells.     

Dorsal patterning defects in the hindbrain, roof plate and skeleton in the dreher (dr(J)) mouse mutant

dreher is a spontaneous mouse mutation in which adult animals display a complex phenotype associated with hearing loss, neurological, pigmentation and skeletal abnormalities. During early embryogenesis, the neural tube of dreher mutants is abnormally shaped in the region of the rhomboencephalon, due to problems in the formation of a proper roof plate over the otic hindbrain. We have studied the expression of Hox/lacZ transgenic mouse strains in the dreher background and shown that primary segmentation of the neural tube is not altered in these mutants, although correct morphogenesis is affected resulting in misshapen rhombomeres. Neural crest derivatives from rhombomere 6, such as the glossopharyngeal ganglion, are defective, and the dorsal neural tube marker Wnt1 is absent from this segment. Selected trunk neural crest populations are also altered, as there is a lack of pigmentation in the thoracic region of mutant mice. Skeletal defects include abnormal cranial bones of neural crest origin, and improper fusion of the dorsal aspects of cervical and thoracic vertebrae. Taken together, the gene affected in the dreher mutant is responsible for correct patterning of the dorsal-most cell types of the neural tube, that is, the neural crest and the roof plate, in the hindbrain region. Axial skeletal defects could reflect inductive influence of the dorsal neural tube on proper fusion of the neural arches. It is possible that a common precursor population for both neural crest and roof plate is the cellular target of the dreher mutation.