Department of Cell and Developmental Biology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, United States of America.
PLoS One. 2011;6(11):e27403. doi: 10.1371/journal.pone.0027403. Epub 2011 Nov 15.
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.
神经嵴祖细胞（NCPC）群体在早期脊椎动物胚胎中的特化需要一个精细的信号通路网络，其中之一是骨形态发生蛋白（BMP）通路。基于斑马鱼 BMP 通路成分突变体中神经嵴基因表达的改变，我们之前提出了一个模型，即原肠胚期的 BMP 形态发生梯度建立了未来 NCPC 区域的中间 BMP 活性水平。在这里，我们测试了这个模型，并表明中间水平的 BMP 信号直接作用于指定 NCPC。我们量化了降低 BMP 信号对神经嵴细胞数量的影响，并表明当 BMP 信号降低时，神经嵴细胞显著增加，并且这种增加不是由于细胞增殖增加所致。相比之下，当 BMP 信号被消除时，NCPC 则无法被指定。我们用扩展或没有 NCPC 的 BMP 通路突变体来调节 BMP 信号水平，以证明中间水平的 BMP 信号指定 NCPC。我们进一步研究了 Smad5 以梯度方式作用的能力，通过注射 Smad5 反义 morpholino，并表明增加剂量首先扩展 NCPC，然后导致 NCPC 的丧失，与 Smad5 直接作用于神经嵴祖细胞特化一致。使用 Western blot 分析，我们表明 smad5 形态发生素中的 P-Smad5 水平呈剂量依赖性降低，与中间水平的 BMP 信号通过 Smad5 作用于神经嵴祖细胞特化一致。最后，我们进行了嵌合体分析，首次证明 BMP 信号接收直接由 NCPC 用于其特化。这些结果共同为一个模型提供了大量证据，即分级 BMP 信号作为形态发生素作用于外胚层，中间水平作用于神经嵴特化。
