A Cis-regulatory Model for Neural Border Induction

神经边界诱导的顺式调节模型

• 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
• 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

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(申请人提供)：神经脊是在发育中的中枢神经系统和表皮交界处形成的一群多能祖细胞。在早期发育期间，神经脊细胞从这个神经边界域迁移，在整个胚胎中形成各种各样的衍生品，包括头部的骨骼和软骨，以及部分脑神经节和心脏。神经脊形成的中断会导致人类头部、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。神经脊发育的关键第一步是将神经边缘与相邻的神经和表皮外胚层分离。这一过程已经被证明涉及从相邻的外胚层和潜在的旁轴中胚层分泌的Wnt和BMP信号。这些信号通过激活神经边界说明符基因Zic、Pax3/7和tfap2的表达来诱导神经边界的形成。虽然WNTs和BMPs对于神经边界诱导是必不可少的，但这些通路如何整合以驱动神经边界说明符的表达尚不清楚。WNT和BMP信号可以通过激活其典型的下游效应因子TCF/Lef和Smad1/5/8或通过几个非典型的效应因子来调节转录。因此，神经边界说明子的表达可能涉及TCF/Lef和Smad1/5/8的直接调节，非规范效应器的直接调节，或未知中间因子的间接调节。为了评估这些可能性，我们描述了来自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)，它们概括了这些基因的早期神经边界表达。对这些CRE的初步分析支持一种通用的神经边界诱导机制，即WNT和BMP信号通路通过TCF/LEF和Smad1/5/8直接整合到进化保守的CRE上。拟议的工作将使用mRNA注射、突变和染色质免疫沉淀(ChIP)来测试这一模型。这项工作的实验目标是：1)使用激素诱导的这些因子的激活形式来测试TCF/LEF和Smad1/5/8对神经边界说明物的调节，以及进化上保守的Tcf/Lef和SMAD结合位点的突变，以及2)使用芯片测试Tcf/Lef-ss-catenin复合体和Smad1/5/8与Cres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芯片的ss-catenin和SMAD抗体，并建立快速测试新型Cres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规范的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BMP和Wnt信号在神经边界的新的直接靶点，增加我们对神经边界形成的理解的广度和深度。拟议的工作将支持一名博士后学者为期两年的培训。 公共卫生相关性：这项研究与公共健康相关，因为神经边界的形成对神经管闭合和头部、面部和心脏的正常发育至关重要。因此，了解神经边界是如何形成的，可以帮助我们了解当神经边界和神经脊发育受损时发生的出生缺陷和其他疾病。此外，神经脊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，包括基因 导致多能性和迁徙能力的过程。