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并产生广泛衍生物的能力。由于其对多个谱系的贡献，NC的异常发育可导致影响多个器官系统的各种看似无关的临床表现，如在先天性巨结肠（色素减退和无神经节巨结肠）和DiGeorge综合征（颅面和心脏缺陷）中观察到的。因此，研究重点是调节NC出现的分子机制，对于进一步了解广泛的人类先天性畸形至关重要，并且是开发可能有助于逆转这些缺陷的新疗法的起点。响应于由Bmp、Wnt和Fgf家族的分子介导的信号传导事件，在神经板边界处顺序地诱导许多转录因子。首先，一组基因被激活，称为“神经板边界指定符”，其包括转录调节因子的Zic、Pax、Dlx和Msx家族的成员。这些因子在神经板边缘广泛表达，反过来又负责激活具有更受限制的表达结构域的基因子集，称为“NC指定符”，其中包括SoxE蛋白（Sox 8、9和10）。在这里，我们建议分析NC规格在监管级联的三个不同的水平。1-在非洲爪蟾NC诱导依赖于BMP信号，这必须部分减弱BMP拮抗剂，和一个单独的信号介导的经典Wnt或FGF。虽然外胚层中的BMP衰减似乎是NC诱导的先决条件，但仍不清楚Wnt和Fgf如何在神经板边缘相互作用以产生NC。我们建议解决悬而未决的问题的相对贡献的成纤维细胞生长因子和Wnt信号NC诱导。2-吗啉介导的敲除的神经板边界指定Pax 3或Zic 1表明，这些因素都是独立需要的NC形成。此外，它们协同激活NC命运。我们的初步结果表明，通过操纵动物外植体中Pax 3和Zic 1的水平，我们可以产生独立于其他神经板边缘细胞类型的诱导的NC祖细胞。我们建议使用Pax 3/Zic 1注射的动物外植体制备，以确定基因协同激活的Pax 3/Zic 1在发展中的NC。3-由于单个SoxE家族成员的异位表达独立地诱导NC祖细胞，因此已经提出这些因子在功能上是等同的。然而，也有证据表明，SoxE蛋白差异调节NC谱系。例如，Sox 9和Sox 10缺陷小鼠表现出严重但明显的NC缺陷，表明单个SoxE蛋白在NC发育中发挥独特作用。我们将通过系统分析单个SoxE蛋白拯救Sox 8-，Sox 9-或Sox 10-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。 公共卫生相关性：神经嵴细胞具有显著的能力，有助于胚胎中广泛的组织。这些细胞的特化或分化缺陷可能对许多器官系统的发育和功能产生非常显著的影响。确定调节这些细胞命运的因素对于理解这些病理的分子基础至关重要。

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.