Generation of Retinoid Signals during Development

描述（由申请人提供）：在鉴定胚胎发生过程中控制祖细胞分化的信号传导因子途径方面取得了很大进展。然而，我们仍然对这些信号因子调节的发育过程和基因有初步的了解。视黄酸（RA）是一种分泌性信号因子，来源于视黄醇，视黄醇是一种必需的营养素，首先转化为视黄醇，然后通过特定的酶转化为RA。脊椎动物胚胎发生过程中RA合成的组织特异性位置和时间提供了刺激祖细胞分化所需的细胞间信号传导信息，从而产生成熟的组织和器官。RA合成启动在体轴延伸的早期阶段，通过视黄醇脱氢酶（Rdh 10）和视黄醇脱氢酶（Raldh 2）的顺序行动，一起产生RA的躯干中胚层前尾祖区。体轴延伸需要胚胎尾尖处祖细胞中的FGF信号传导和Wnt信号传导，并且对Raldh 2-/-小鼠胚胎的研究表明RA信号传导通过在发育中的躯干中起作用以下调FGF和Wnt信号传导来设置该祖细胞区的前部限制。在这样做的同时，RA信号也确保体节以双侧对称的方式产生。然而，尾部RA的作用机制仍不清楚。RA通过作为与RA反应元件（RARE）结合的核RA受体的配体直接调节关键基因的转录。RA传统上与基因表达的诱导相关，但一些研究表明，RA通过RA介导的Fgf 8和Wnt 8a的抑制来控制体轴延伸和体节发生，并且RA作用于新产生的后神经外胚层或节点而不是前体节中胚层。对小鼠胚胎的染色质免疫沉淀（ChIP）研究已经确定了结合RA受体的Fgf 8和Wnt 8a启动子上游的RARE，从而能够更深入地研究尾部RA机制。在这个项目中，我们计划使用几种小鼠和斑马鱼遗传模型来消除或减少RA，FGF和Wnt信号，以及转基因和ChIP方法来检查Fgf 8和Wnt 8a启动子。本项目的目的是了解尾部RA通过抑制FGF和Wnt信号传导来确保正常体轴延伸的机制。具体而言，我们建议：（1）使用几种遗传模型确定在体节发生和体轴延伸期间FGF信号传导的RA抑制的靶组织;（2）减少RA缺陷的小鼠和斑马鱼胚胎中的Wnt信号传导以挽救体轴延伸中的缺陷并检查RA和Wnt信号传导之间的串扰;（3）通过体内和体外研究确定Fgf 8和Wnt 8a RA响应元件的抑制功能。

DESCRIPTION (provided by applicant): Great progress has been made identifying signaling factor pathways controlling differentiation of progenitor cells during embryogenesis. However, we still have a rudimentary understanding of what developmental processes and genes these signaling factors regulate. Retinoic acid (RA) is a secreted signaling factor derived from retinol, an essential nutrient that is converted first to retinaldehyde and then to RA by specific enzymes. The tissue-specific location and timing of RA synthesis during vertebrate embryogenesis provides intercellular signaling information needed to stimulate differentiation of progenitor cells, thus generating mature tissues and organs. RA synthesis initiates during the early stages of body axis extension through the sequential actions of retinol dehydrogenase (Rdh10) and retinaldehyde dehydrogenase (Raldh2) that together generate RA in trunk mesoderm just anterior to the caudal progenitor zone. Body axis extension requires FGF signaling and Wnt signaling in progenitor cells at the caudal tip of the embryo, and studies on Raldh2-/- mouse embryos suggest that RA signaling sets the anterior limit of this progenitor zone by acting in the developing trunk to down-regulate FGF and Wnt signaling. While doing this, RA signaling also ensures that somites are generated in a bilaterally symmetric fashion. However, the mechanism of caudal RA action is still unclear. RA directly regulates transcription of key genes by serving as a ligand for nuclear RA receptors bound to RA response elements (RAREs). RA has traditionally been associated with induction of gene expression, but some studies suggest that RA controls body axis extension and somitogenesis through RA-mediated repression of Fgf8 and Wnt8a, and that RA acts in newly generated posterior neuroectoderm or the node rather than presomitic mesoderm. Chromatin immunoprecipitation (ChIP) studies on mouse embryos have identified RAREs upstream of the Fgf8 and Wnt8a promoters that bind RA receptors, enabling a deeper examination of the caudal RA mechanism. In this project we plan to use several mouse and zebrafish genetic models to eliminate or reduce RA, FGF, and Wnt signaling, as well as transgenic and ChIP approaches to examine Fgf8 and Wnt8a promoters. The goal of this project is to understand the mechanism through which caudal RA represses FGF and Wnt signaling to ensure normal body axis extension. Specifically, we propose to: (1) Determine the target tissue for RA repression of FGF signaling during somitogenesis and body axis extension using several genetic models; (2) Reduce Wnt signaling in RA-deficient mouse and zebrafish embryos to rescue defects in body axis extension and to examine crosstalk between RA and Wnt signaling; (3) Validate repressive functions of Fgf8 and Wnt8a RA response elements through in vivo and in vitro studies.