Control of Neural Crest Development in Xenopus

描述(申请人提供)：神经脊是一个多能的细胞群体，具有在整个胚胎中迁移的能力，并产生一系列的衍生品。由于其对多个谱系的贡献，NC的异常发展可导致一系列看似无关的临床表现，影响到多个器官系统，如先天性巨结肠(色素减退和无神经节细胞巨结肠)和DiGeorge综合征(颅面和心脏缺陷)。因此，对NC发生的分子调控机制的研究对于加深我们对广泛的人类先天性畸形的理解是至关重要的，也是开发新的治疗方法以逆转这些缺陷的起点。作为对BMP、WNT和FGF3个家族分子介导的信号事件的响应，一系列转录因子在神经板边界被顺序地诱导。首先，一组基因被激活，被称为“神经板边界说明子”，其中包括转录调控基因Zic、Pax、DLX和MSX家族的成员。这些在神经板边界广泛表达的因子，反过来又负责激活具有更多受限表达结构域的基因子集，称为“NC说明子”，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面上分析NC规范。1-在非洲爪哇中，NC的诱导依赖于BMP信号，该信号必须被BMP拮抗剂部分减弱，以及由规范的Wnt或Fgf介导的单独信号。虽然外胚层中BMP的衰减似乎是NC诱导的先决条件，但Wnt和FGF如何在神经板边界相互作用产生NC仍不清楚。我们建议解决一个悬而未决的问题，即成纤维细胞生长因子和Wnt信号在NC诱导中的相对贡献。2-Morpholino介导的神经板边界说明子Pax3或Zic1的敲除表明这两个因子都是NC形成所独立需要的。此外，它们还协同激活了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，我们可以独立于其他神经板边缘细胞类型的诱导而产生NC祖细胞。我们建议使用Pax3/Zic1注射的动物外植体制备来鉴定在发育中的NC中Pax3/Zic1协同激活的基因。3.由于SoxE家族单个成员的异位表达独立地诱导NC祖细胞，因此有人提出这些因素在功能上是等价的。然而，也有证据表明SoxE蛋白对NC谱系有不同的调节作用。例如，Sox9和Sox10基因缺陷的小鼠表现出严重但明显的NC缺陷，这表明单独的SoxE蛋白在NC的发育中发挥着独特的作用。我们将通过系统分析单个SoxE蛋白拯救Sox8、Sox9或Sox10缺失胚胎的NC表型的潜力，确定单个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：神经脊细胞具有为胚胎中的广泛组织做出贡献的非凡能力。这些细胞的规格或分化缺陷可能会对许多器官系统的发育和功能产生非常严重的影响。确定控制这些细胞命运的因素对于理解这些病理背后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.