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，在整个胚胎中形成各种各样的衍生物，包括头部的骨骼和软骨，以及部分颅神经节和心脏。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。这一过程涉及邻近外胚层和旁轴中胚层分泌的Wnt和BMP信号。这些信号通过激活“神经边界指示基因”Zic、Pax3/7和tfap2的表达诱导神经边界的形成。虽然wnt和bmp对于神经边界诱导至关重要，但这些通路如何整合以驱动神经边界指示因子的表达尚不清楚。Wnt和BMP信号可以通过激活其典型下游效应因子TCF/LEF和SMAD1/5/8转录因子，或通过几种非典型效应因子来调节转录。因此，神经边界指示物的表达可能涉及TCF/LEF和SMAD1/5/8的直接调控，非规范效应物的直接调控，或未知中间因子的间接调控。为了评估这些可能性，我们从Pax3、Pax7、Zic2/5、Zic3/6和tfap2a基因座中鉴定了8个进化上保守的顺式调控元件（cre），这些元件概括了这些基因的早期神经边界表达。这些cre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SMAD1/5/8直接整合在进化保守的cre上。这项工作将使用mRNA注射、诱变和染色质免疫沉淀（ChIP）来测试该模型。提出的工作的实验目的是；1)利用激素诱导、激活形式检测TCF/LEF和SMAD1/5/8对神经边界指示物的调控作用，以及对进化保守的TCF/LEF和SMAD结合位点的诱变；2)利用ChIP检测TCF/LEF-ss-catenin复合物和SMAD1/5/8与cre的直接结合。除了测试我们的模型，我们还将验证ss-catenin和SMAD抗体在斑马鱼胚胎芯片中的应用，并建立快速测试新型cre的BMP和Wnt响应性的策略。未来的工作将利用这些结果来测试通过典型BMP和Wnt信号通路的直接组合作用来诱导神经边界是否是实现神经边界基因表达的全基因组机制。这些研究还将确定神经边界中BMP和Wnt信号的新的直接靶点，增加我们对神经边界形成的理解的广度和深度。拟开展的工作将支持培养一名博士后，为期两年。

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.