Control of Neural Crest Development in Xenopus

描述（由申请人提供）：神经嵴是一种多能细胞群，具有在整个胚胎中迁移的能力，并产生广泛的衍生物。由于其对多谱系的贡献，NC的异常发育可导致影响多器官系统的一系列看似无关的临床表现，如巨结肠病（色素沉着和神经节巨结肠）和DiGeorge综合征（颅面和心脏缺陷）。因此，专注于调控NC出现的分子机制的研究对于进一步加深我们对广泛的人类先天性畸形的理解至关重要，并且是开发可能有助于逆转这些缺陷的新疗法的起点。在响应由Bmp、Wnt和Fgf家族分子介导的信号事件时，许多转录因子在神经板边界被依次诱导。首先，一组基因被激活，被称为“神经板边界指示物”，其中包括转录调节因子Zic， Pax， Dlx和Msx家族的成员。这些在神经板边缘广泛表达的因子反过来又负责激活具有更有限表达域的基因子集，称为“NC指示物”，其中包括SoxE蛋白（Sox8、9和10）。在这里，我们建议在监管级联的三个不同层次上分析NC规范。在非洲爪蟾中，NC的诱导依赖于Bmp信号，该信号必须被Bmp拮抗剂部分减弱，以及一个由典型Wnt或Fgf介导的单独信号。虽然外胚层的Bmp衰减似乎是NC诱导的先决条件，但尚不清楚Wnt和Fgf如何在神经板边界相互作用以产生NC。我们建议解决Fgf和Wnt信号对NC诱导的相对贡献这一悬而未决的问题。2- morpholino介导的神经板边界指示因子Pax3或Zic1的敲低表明这些因子都是NC形成所独立需要的。此外，它们协同激活NC命运。我们的初步结果表明，通过控制动物外植体中Pax3和Zic1的水平，我们可以独立于诱导其他神经板边界细胞类型而产生NC祖细胞。我们建议使用Pax3/Zic1注射动物外植体制剂来鉴定发育中的NC中Pax3/Zic1协同激活的基因。由于单个SoxE家族成员的异位表达独立诱导NC祖细胞，因此有人提出这些因素在功能上是等同的。然而，也有证据表明SoxE蛋白对NC谱系有差异调节。例如，Sox9和sox10缺陷小鼠表现出严重但明显的NC缺陷，这表明单个SoxE蛋白在NC发育中起着独特的作用。我们将通过系统分析单个SoxE蛋白挽救Sox8-、Sox9-或sox10 -耗尽胚胎的NC表型的潜力，来定义单个SoxE蛋白在NC多样化过程中的共同和独特功能。

DESCRIPTION (provided by applicant): The neural crest is a multipotent population of cells that has the ability to migrate throughout the embryo and give rise to a broad range of derivatives. Because of its contribution to multiple lineages, abnormal development of the NC can result in a wide array of seemingly unrelated clinical manifestations affecting multiple organ systems, as observed in Hirschsprung disease (hypopigmentation and aganglionic megacolon) and DiGeorge syndrome (craniofacial and heart defects). Therefore, studies focusing on the molecular mechanisms regulating the emergence of the NC are critical for furthering our understanding of a broad range of human congenital malformations and are the starting point for the development of new therapeutics that might serve to reverse these defects. In response to signaling events mediated by molecules of the Bmp, Wnt and Fgf families a number of transcription factors are sequentially induced at the neural plate border. First, a group of genes is activated, referred as "neural plate border specifiers", which include members of the Zic, Pax, Dlx and Msx families of transcriptional regulators. These factors, which are broadly expressed at the neural plate border, are in turn responsible for the activation of a subset of genes with more restricted expression domains, known as "NC specifiers" among which are the SoxE proteins (Sox8, 9 and 10). Here we propose to analyze NC specification at three different levels in the regulatory cascade. 1- In Xenopus NC induction depends on a Bmp signal, which must be partially attenuated by Bmp antagonists, and a separate signal mediated by either a canonical Wnt or Fgf. While Bmp attenuation in the ectoderm appears to be a pre-requisite for NC induction, it is still unclear how Wnt and Fgf interact at the neural plate border to generate the NC. We propose to address the outstanding question of the relative contribution of Fgf and Wnt signaling to NC induction. 2- Morpholino-mediated knockdown of the neural plate border specifiers Pax3 or Zic1 indicates that these factors are both independently required for NC formation. Moreover, they synergistically activate NC fate. Our preliminary results indicate that by manipulating the levels of Pax3 and Zic1 in animal explants, we can generate NC progenitors independently of the induction of other neural plate border cell types. We propose to use the Pax3/Zic1 injected animal explants preparation to identify genes synergistically activated by Pax3/Zic1 in the developing NC. 3- Because ectopic expression of individual SoxE family members independently induces NC progenitors, it has been proposed that these factors are functionally equivalent. However, there is also evidence that SoxE proteins differentially regulate NC lineages. For example Sox9- and Sox10-deficient mice show severe but distinct NC defects suggesting that individual SoxE proteins play unique roles in NC development... We will define the common and unique functions of individual SoxE proteins during NC diversification by systematically analyzing the potential for individual SoxE proteins to rescue the NC phenotype of Sox8-, Sox9- or Sox10-depleted embryos. PUBLIC HEALTH RELEVANCE: Neural crest cells have the remarkable ability to contribute to a broad range of tissues in the embryo. Defects in the specification or differentiation of these cells may have very dramatic consequences on the development and function of many organ systems. Defining the factors that regulate the fate of these cells is critical to understand the molecular basis underlying these pathologies.