A Cis-regulatory Model for Neural Border Induction

神经边界诱导的顺式调节模型

基本信息

批准号：
8301929
负责人：
DANIEL MEULEMANS MEDEIROS
金额：
$ 11.44万
依托单位：
UNIVERSITY OF COLORADO
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-04-01 至 2014-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8301929
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

项目摘要

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.

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界处形成的多能祖细胞群。在早期发育过程中，神经嵴细胞从该神经边界域迁移，在整个胚胎中形成多种衍生物，包括头部的骨骼和软骨，以及颅神经节和心脏的部分。神经嵴形成的破坏会导致人类头部、面部、心脏和神经系统的许多发育异常，以及神经管闭合缺陷。神经嵴发育的关键第一步是神经边界与邻近的神经和表皮外胚层的分离。该过程已被证明涉及从邻近的外胚层和下面的轴旁中胚层分泌的 Wnt 和 BMP 信号。这些信号通过激活“神经边界指定”基因 Zic、Pax3/7 和 tfap2 的表达来诱导神经边界形成。虽然 Wnt 和 BMP 对于神经边界诱导至关重要，但如何整合这些途径来驱动神经边界指定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子、TCF/LEF 和 SMAD1/5/8 转录因子或通过几个非典型效应子来调节转录。因此，神经边界特异性表达可能涉及 TCF/LEF 和 SMAD1/5/8 的直接调节、非典型效应器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，我们对来自 Pax3、Pax7、Zic2/5、Zic3/6 和 tfap2a 位点的 8 个进化保守的顺式调控元件 (CRE) 进行了表征，这些位点概括了这些基因的早期神经边界表达。对这些 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/LEF 和 SMAD1/5/8 直接整合到进化上保守的 CRE 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。拟议工作的实验目标是： 1) 使用这些因子的激素诱导型、活化形式，以及进化上保守的 TCF/LEF 和 SMAD 结合位点的诱变，测试 TCF/LEF 和 SMAD1/5/8 对神经边界指定因子的调节，2) 直接测试使用 ChIP 将 TCF/LEF-ss-catenin 复合物和 SMAD1/5/8 与 CRE 结合。除了测试我们的模型之外，拟议的工作还将验证 ss-catenin 和 SMAD 抗体在斑马鱼胚胎 ChIP 中的使用，并建立快速测试新型 CRE 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通过经典 BMP 和 Wnt 信号通路的直接组合作用进行的神经边界诱导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 信号传导的新直接靶标，从而增加我们对神经边界形成的理解的广度和深度。拟议的工作将支持培养一名博士后学者两年。公共健康相关性：这项研究与公共健康相关，因为神经边界的形成对于神经管闭合以及头部、面部和心脏的正常发育至关重要。因此，了解神经边界的形成方式可以帮助我们了解出生缺陷以及神经边界和神经嵴发育受损时发生的其他疾病。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，包括遗传导致多能性和迁移能力的过程。