Gene regulatory network underlying neural crest formation

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Early- and late-migrating cranial neural crest cell populations have equivalent developmental potential in vivo

We present the first in vivo study of the long-term fate and potential of early-migrating and late-migrating mesencephalic neural crest cell populations, by performing isochronic and heterochronic quail-to-chick grafts. Both early- and late-migrating populations form melanocytes, neurons, glia, cartilage and bone in isochronic, isotopic chimeras, showing that neither population is lineage-restricted. The early-migrating population distributes both dorsally and ventrally during normal development, while the late-migrating population is confined dorsally and forms much less cartilage and bone. When the late-migrating population is substituted heterochronically for the early-migrating population, it contributes extensively to ventral derivatives such as jaw cartilage and bone. Conversely, when the early-migrating population is substituted heterochronically for the late-migrating population, it no longer contributes to the jaw skeleton and only forms dorsal derivatives. When the late-migrating population is grafted into a late-stage host whose neural crest had previously been ablated, it migrates ventrally into the jaws. Thus, the dorsal fate restriction of the late-migrating mesencephalic neural crest cell population in normal development is due to the presence of earlier-migrating neural crest cells, rather than to any change in the environment or to any intrinsic difference in migratory ability or potential between early- and late-migrating cell populations. These results highlight the plasticity of the neural crest and show that its fate is determined primarily by the environment

A critical role for Cadherin6B in regulating avian neural crest emigration

Neural crest cells originate in the dorsal neural tube but subsequently undergo an epithelial-to-mesenchymal transition (EMT), delaminate, and migrate to diverse locations in the embryo where they contribute to a variety of derivatives. Cadherins are a family of cell–cell adhesion molecules expressed in a broad range of embryonic tissues, including the neural tube. In particular, cadherin6B (Cad6B) is expressed in the dorsal neural tube prior to neural crest emigration but is then repressed by the transcription factor Snail2, expressed by premigratory and early migrating cranial neural crest cells. To examine the role of Cad6B during neural crest EMT, we have perturbed Cad6B protein levels in the cranial neural crest-forming region and have examined subsequent effects on emigration and migration. The results show that knock-down of Cad6B leads to premature neural crest cell emigration, whereas Cad6B overexpression disrupts migration. Our data reveal a novel role for Cad6B in controlling the proper timing of neural crest emigration and delamination from the neural tube of the avian embryo

Corneal Plasticity: Characterization of the Multipotentiality of Human Keratocytes

Purpose: To determine the cell properties of adult human corneal keratocytes when challenged in the chick embryonic environment. Methods: Cultured human keratocytes were injected along cranial neural crest migratory pathways in chick embryos. Human keratocytes were also cultured under various conditions and differentiated into either fibroblasts or myofibroblasts, then transplanted into the chick embryo. Migration of the injected cells was determined by immunohistochemistry using human cell-specific markers and markers of crest derivatives. Results: Injected human keratocytes proliferated and migrated ventrally adjacent to host neural crest cells. They contributed to numerous neural crest-derived tissues including cranial blood vessels, ocular tissues, musculature of the mandibular process, and cardiac cushion tissue. Conclusions: Adult human corneal keratocytes that have undergone terminal differentiation can be induced to form cranial neural crest derivatives when grafted into an embryonic environment

Stem-Cell Properties of Human Corneal Keratocytes

Purpose: To determine the stem cell properties of human corneal stromal keratocytes when challenged in the chick embryonic environment. Methods: Stromal keratocytes isolated from human corneas were injected along cranial neural crest migratory pathways and in the periocular mesenchyme in chick embryos. Localization Migration of the injected cells stromal keratocytes was determined at various stages of development by immunohistochemistry using human cell-specific markers. Differentiation of the human keratocytes into other neural crest-derived tissues was determined by immunohistochemistry with tissue cell-specific markers. Results: Human keratocytes injected along cranial neural crest pathways proliferated and migrated ventrally adjacent to host neural crest cells. They contributed to numerous neural crest-derived tissues including cranial blood vessels, ocular tissues, and cardiac cushion tissue mesenchyme. Keratocytes injected into the periocular mesenchyme region contributed to the corneal stroma and endothelial layers. Conclusions: Adult human corneal stromal keratocytes exhibit stem cell characteristics. They can be induced to form cranial neural crest derivatives, including other anterior ocular structures, when grafted into an embryonic environment

Relationship between spatially restricted Krox-20 gene expression in branchial neural crest and segmentation in the chick embryo hindbrain

Previous studies have suggested that the rostrocaudal patterning of branchial arches in the vertebrate embryo derives from a coordinate segmental specification of gene expression in rhombomeres (r) and neural crest. However, expression of the Krox-20 gene is restricted to neural crest cells migrating to the third branchial arch, apparently from r5, whereas this rhombomere contributes cells to both the second and third arches. We examined in the chick embryo how this spatially restricted expression is established. Expression occurs in precursors in both r5 and r6, and we show by cell labelling that both rhombomeres contribute to Krox-20-expressing neural crest, emigration occurring first from r6 and later caudally from r5. Krox-20 transcripts are not detected in some precursors in rostral r5, presaging the lack of expression in cells migrating rostrally from this rhombomere. After transposition of r6 to the position of r4 or r5, many Krox-20-expressing cells migrate rostral to the otic vesicle, whereas when r5 is transplanted to the position of r4, only a small number of migrating cells express Krox-20. These results indicate that, in the chick, Krox-20 expression in branchial neural crest does not correlate with rhombomeric segmentation, and that there may be intrinsic differences in regulation between the r5 and r6 Krox-20-expressing populations

Early regulative ability of the neuroepithelium to form cardiac neural crest

The cardiac neural crest (arising from the level of hindbrain rhombomeres 6–8) contributes to the septation of the cardiac outflow tract and the formation of aortic arches. Removal of this population after neural tube closure results in severe septation defects in the chick, reminiscent of human birth defects. Because neural crest cells from other axial levels have regenerative capacity, we asked whether the cardiac neural crest might also regenerate at early stages in a manner that declines with time. Accordingly, we find that ablation of presumptive cardiac crest at stage 7, as the neural folds elevate, results in reformation of migrating cardiac neural crest by stage 13. Fate mapping reveals that the new population derives largely from the neuroepithelium ventral and rostral to the ablation. The stage of ablation dictates the competence of residual tissue to regulate and regenerate, as this capacity is lost by stage 9, consistent with previous reports. These findings suggest that there is a temporal window during which the presumptive cardiac neural crest has the capacity to regulate and regenerate, but this regenerative ability is lost earlier than in other neural crest populations

Distribution of a putative cell surface receptor for fibronectin and laminin in the avian embryo

The cell substratum attachment (CSAT) antibody recognizes a 140-kD cell surface receptor complex involved in adhesion to fibronectin (FN) and laminin (LM) (Horwitz, A., K. Duggan, R. Greggs, C. Decker, and C. Buck, 1985, J. Cell Biol., 101:2134-2144). Here, we describe the distribution of the CSAT antigen along with FN and LM in the early avian embryo. At the light microscopic level, the staining patterns for the CSAT receptor and the extracellular matrix molecules to which it binds were largely codistributed. The CSAT antigen was observed on numerous tissues during gastrulation, neurulation, and neural crest migration: for example, the surface of neural crest cells and the basal surface of epithelial tissues such as the ectoderm, neural tube, notochord, and dermomyotome. FN and LM immunoreactivity was observed in the basement membranes surrounding many of these epithelial tissues, as well as around the otic and optic vesicles. In addition, the pathways followed by cranial neural crest cells were lined with FN and LM. In the trunk region, FN and LM were observed surrounding a subpopulation of neural crest cells. However, neither molecule exhibited the selective distribution pattern necessary for a guiding role in trunk neural crest migration. The levels of CSAT, FN, and LM are dynamic in the embryo, perhaps reflecting that the balance of surface-substratum adhesions contributes to initiation, migration, and localization of some neural crest cell populations