Abstract
The specification of the neural crest progenitor cell (NCPC) population in the early vertebrate embryo requires an elaborate network of signaling pathways, one of which is the Bone Morphogenetic Protein (BMP) pathway. Based on alterations in neural crest gene expression in zebrafish BMP pathway component mutants, we previously proposed a model in which the gastrula BMP morphogen gradient establishes an intermediate level of BMP activity establishing the future NCPC domain. Here, we tested this model and show that an intermediate level of BMP signaling acts directly to specify the NCPC. We quantified the effects of reducing BMP signaling on the number of neural crest cells and show that neural crest cells are significantly increased when BMP signaling is reduced and that this increase is not due to an increase in cell proliferation. In contrast, when BMP signaling is eliminated, NCPC fail to be specified. We modulated BMP signaling levels in BMP pathway mutants with expanded or no NCPCs to demonstrate that an intermediate level of BMP signaling specifies the NCPC. We further investigated the ability of Smad5 to act in a graded fashion by injecting smad5 antisense morpholinos and show that increasing doses first expand the NCPCs and then cause a loss of NCPCs, consistent with Smad5 acting directly in neural crest progenitor specification. Using Western blot analysis, we show that P-Smad5 levels are dose-dependently reduced in smad5 morphants, consistent with an intermediate level of BMP signaling acting through Smad5 to specify the neural crest progenitors. Finally, we performed chimeric analysis to demonstrate for the first time that BMP signal reception is required directly by NCPCs for their specification. Together these results add substantial evidence to a model in which graded BMP signaling acts as a morphogen to pattern the ectoderm, with an intermediate level acting in neural crest specification.
Highlights
Neural crest cells are a multipotent population derived from embryonic ectoderm
To investigate if these defects are due to defects in neural crest progenitor cells (NCPC) specification, we examined foxd3 expression at the end of gastrulation in these Bone Morphogenetic Protein (BMP) pathway mutants
We found that NCPCs were greatly reduced to absent in swr/bmp2b mutants (Fig. 1B); greatly expanded in sbn/smad5 (Fig. 1C); and moderately expanded in snhty68a/bmp7a mutant embryos (Fig. 1D) compared to wild-type (Fig. 1A)
Summary
During neurulation neural crest cells undergo an epithelial-to-mesenchymal transition, delaminate from the dorsal neural tube, and migrate throughout the embryo, contributing to a variety of tissues including craniofacial skeleton, pigment cells, and the peripheral nervous system In both frog and chick, juxtaposition of explanted neural and non-neural ectoderm gives rise to neural crest cells [1,2,3]. In Xenopus, fate map analysis reveals that the prospective neural crest population lies adjacent to the dorsolateral marginal zone at an early gastrula stage [5] Consistent with these studies, the earliest genes expressed within the neural crest progenitor cells (NCPC), e.g. snail, AP2, and foxd, are localized to lateral regions of the neural plate adjacent to the non-neural ectoderm [6,7,8,9,10,11,12]
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