A Cis-regulatory Model for Neural Border Induction

描述（由申请人提供）：神经嵴是在发育中的中枢神经系统和表皮的边缘形成的多能祖细胞群。在早期发育过程中，神经嵴细胞从这个神经边界区域迁移，在整个胚胎中形成不同的衍生物，包括头部的骨骼和软骨，以及颅神经节和心脏的部分。神经嵴形成的中断导致许多人类头部、面部、心脏和神经系统的发育异常，以及神经管闭合缺陷。神经嵴发育的关键第一步是神经边缘与相邻的神经和表皮外胚层分离。该过程已显示涉及从相邻的外胚层和下面的近轴中胚层分泌的Wnt和BMP信号。这些信号通过激活“神经边界特异性”基因Zic、Pax 3/7和tfap 2的表达来诱导神经边界形成。虽然Wnt和BMP对于神经边界诱导是必不可少的，但这些途径如何整合以驱动神经边界指定符的表达尚不清楚。Wnt和BMP信号可以通过激活其典型下游效应子TCF/LEF和SMAD 1/5/8转录因子或通过几种非典型效应子来调节转录。因此，神经边界指定符的表达可能涉及TCF/LEF和SMAD 1/5/8的直接调节，非典型效应子的直接调节，或未知中间因子的间接调节。为了评估这些可能性，我们正在表征来自Pax 3，Pax 7，Zic 2/5，Zic 3/6和tfap 2a基因座的8个进化上保守的顺式调控元件（克雷斯），这些基因座概括了这些基因的早期神经边界表达。这些克雷斯的初步分析支持神经边界诱导的一般机制，其中Wnt和BMP信号通路通过TCF/LEFs和SMAD 1/5/8直接整合在进化保守的克雷斯上。拟议的工作将使用mRNA注射，诱变和染色质免疫沉淀（ChIP）来测试该模型。所提出的工作的实验目的是：1）使用这些因子的激素诱导的活化形式测试TCF/LEF和SMAD 1/5/8对神经边界指定符的调节，以及进化上保守的TCF/LEF和SMAD结合位点的诱变，以及2）使用ChIP测试TCF/LEF-ss-连环蛋白复合物和SMAD 1/5/8与克雷斯的直接结合。除了测试我们的模型外，拟议的工作还将验证用于斑马鱼胚胎ChIP的β-连环蛋白和SMAD抗体，并建立快速测试新型克雷斯的BMP和Wnt反应性的策略。未来的工作将利用这些结果来测试，如果通过直接的，典型的BMP和Wnt信号通路的组合作用的神经边界诱导是一个全基因组的机制，实现神经边界基因的表达。这些研究还将确定神经边界中BMP和Wnt信号传导的新直接靶点，从而增加我们对神经边界形成的理解的广度和深度。拟议的工作将支持一名博士后学者两年的培训。 公共卫生相关性：这项研究与公共卫生有关，因为神经边界的形成对于神经管闭合和头部，面部和心脏的正常发育至关重要。因此，了解神经边界如何形成可以帮助我们了解当神经边界和神经嵴发育受损时发生的出生缺陷和其他疾病。此外，神经嵴细胞与癌症和干细胞的遗传相似性意味着这项研究可能会揭示这些细胞类型的生物学，包括遗传学。 导致多能性和迁移能力的过程。

DESCRIPTION (provided by applicant): The neural crest is a population of multipotent progenitor cells that forms at the border of the developing central nervous system and epidermis. During early development, neural crest cells migrate from this neural border domain to form diverse derivatives throughout the embryo, including bones and cartilage in the head, and portions of the cranial ganglia and heart. Disruptions in neural crest formation cause numerous human developmental abnormalities in the head, face, heart, and nervous system, as well as neural tube closure defects. The critical first step in neural crest development is the segregation of the neural border from adjacent neural and epidermal ectoderm. This process has been shown to involve Wnt and BMP signals secreted from adjacent ectoderm and underlying paraxial mesoderm. These signals induce neural border formation by activating the expression of the 'neural border specifier' genes, Zic, Pax3/7, and tfap2. While Wnts and BMPs are essential for neural border induction, how these pathways are integrated to drive expression of the neural border specifiers is unknown. Wnt and BMP signals can regulate transcription through activation of their canonical downstream effectors, TCF/LEF and SMAD1/5/8 transcription factors, or through several non- canonical effectors. Thus, neural border specifier expression could involve direct regulation by TCF/LEF and SMAD1/5/8, direct regulation by non-canonical effectors, or indirect regulation by unknown intermediate factors. To evaluate these possibilities, we are characterizing 8 evolutionarily conserved cis-regulatory elements (CREs) from the Pax3, Pax7, Zic2/5, Zic3/6 and tfap2a loci that recapitulate early neural border expression of these genes. Preliminary analyses of these CREs support a general mechanism for neural border induction in which the Wnt and BMP signaling pathways are integrated directly on evolutionarily conserved CREs through TCF/LEFs and SMAD1/5/8. The proposed work will test this model using mRNA injections, mutagenesis, and chromatin immunoprecipitation (ChIP). The experimental aims of the proposed work are; 1) Test for regulation of the neural border specifiers by TCF/LEFs and SMAD1/5/8 using hormone- inducible, activated forms of these factors, and mutagenesis of evolutionarily conserved TCF/LEF and SMAD binding sites and, 2) Test for direct binding of the TCF/LEF-ss-catenin complex and SMAD1/5/8 to CREs using ChIP. In addition to testing our model, the proposed work will validate ss-catenin and SMAD antibodies for use in zebrafish embryo ChIP, and establish strategies for rapidly testing the BMP and Wnt responsiveness of novel CREs. Future work will leverage these results to test if neural border induction via direct, combinatorial action of canonical BMP and Wnt signaling pathways is a genome-wide mechanism for achieving neural border gene expression. These studies will also identify novel direct targets of BMP and Wnt signaling in the neural border, adding both breadth and depth to our understanding of neural border formation. The proposed work will support the training of one postdoctoral scholar for two years. PUBLIC HEALTH RELEVANCE: This research is relevant to public health because neural border formation is critical for neural tube closure and normal development of the head, face, and heart. Thus, understanding how the neural border forms can help us understand birth defects and other diseases which occur when neural border and neural crest development is impaired. In addition, the genetic similarly of neural crest cells to cancer and stem cells means that this research will likely shed light on the biology of these cell types, including the genetic processes that lead to multipotency and migratory ability.