A Cis-regulatory Model for Neural Border Induction

神经边界诱导的顺式调节模型

• 批准号: 8448593
• 负责人: DANIEL MEULEMANS MEDEIROS
• 金额: $ 10.98万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-04-01 至 2014-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8448593
• 关键词: Antibodies; Binding; Binding Sites; Biochemistry; Biology; Cartilage; Cell Separation; Cephalic; Chromatin; Collaborations; Colorado; Complex; Congenital Abnormality; Connective Tissue; DNA; Data; Defect; Development; DiGeorge Syndrome; Disease; Dysplasia; Ectoderm; Embryo; Epidermis; Face; Family member; Funding; Future; Ganglia; Gene Expression; Genes; Genetic; Genetic Processes; Genetic Transcription; Head; Heart; Hormones; Human; Human Pathology; In Situ Hybridization; Injection of therapeutic agent; Lateral; Lead; Letters; Ligands; Light; Mediating; Messenger RNA; Modeling; Multipotent Stem Cells; Mutagenesis; Nervous system structure; Neural Crest; Neural Crest Cell; Neural Tube Closure; Neuraxis; Paraxial Mesoderm; Pathology; Pathway interactions; Population; Precipitation; Process; Public Health; Regulation; Regulatory Element; Reporter; Research; Role; Signal Pathway; Signal Transduction; Specific qualifier value; Stem cells; Syndrome; Techniques; Testing; Tooth structure; Training Support; Universities; Work; Writing; Zebrafish; base; blastomere structure; bone; cancer stem cell; cell type; chromatin immunoprecipitation; combinatorial; craniofacial; genome-wide; in vivo; migration; neural model; neuromechanism; neuroregulation; novel; prevent; relating to nervous system; segregation; transcription factor

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.

描述（由申请人提供）：神经rest是在发展中心神经系统和表皮边界形成的多能细胞的群体。在早期发育过程中，神经rest细胞从这个神经边界结构域迁移，在整个胚胎中形成各种衍生物，包括头部的骨骼和软骨，以及颅神经节和心脏的一部分。神经rest形成的破坏会导致头部，面部，心脏和神经系统的许多人类发育异常以及神经管闭合缺陷。神经rest发育的关键第一步是神经边界与相邻神经和表皮外胚层的分离。该过程已显示出涉及从相邻外胚层和基础近期中胚层分泌的Wnt和BMP信号。这些信号通过激活“神经边框”基因的表达ZIC，PAX3/7和TFAP2来诱导神经边界形成。虽然WNT和BMP对于神经边界诱导至关重要，但这些途径如何集成以驱动神经边界规范的表达。 Wnt和BMP信号可以通过激活其规范下游效应子，TCF/LEF和SMAD1/5/8转录因子或通过几个非规范效应子来调节转录。因此，神经边界规范仪的表达可能涉及由TCF/LEF和SMAD1/5/8，非传统效应子进行直接调控，或者通过未知中间因素进行间接调节。为了评估这些可能性，我们正在表征来自PAX3，PAX7，ZIC2/5，ZIC3/6和TFAP2A基因座的8个进化保守的顺式调节元件（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信号通路直接通过TCF/LEFS和SMAD1/5/8直接集成在进化保守的CRE上。提出的工作将使用mRNA注射，诱变和染色质免疫沉淀（CHIP）测试该模型。拟议工作的实验目的是： 1）测试TCF/LEFS和SMAD1/5/8对这些因素的激素，激活形式的调节，以及进化保守的TCF/LEF和SMAD结合位点的诱变，以及2）直接测试使用芯片的TCF/LEF-SS-catenin复合物和Smad1/5/8的结合。除了测试我们的模型外，所提出的工作还将验证用于斑马鱼胚胎芯片的SS-catenin和Smad抗体，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是否通过直接的，典型BMP和Wnt信号通路的直接，组合作用是全基因组来实现神经边界基因表达的一种机制。这些研究还将确定神经边界中BMP和Wnt信号传导的新型直接靶标，从而为我们对神经边界形成的理解增加了广度和深度。拟议的工作将支持一名博士后学者两年的培训。

项目成果

Gene regulatory evolution and the origin of macroevolutionary novelties: insights from the neural crest.

• DOI: 10.1002/dvg.22403
• 发表时间: 2013-07
• 期刊: GENESIS
• 影响因子: 1.5
• 作者: Van Otterloo, Eric;Cornell, Robert A.;Medeiros, Daniel Meulemans;Garnett, Aaron T.
• 通讯作者: Garnett, Aaron T.