Generation of Retinoid Signals during Development

描述（申请人提供）：胚胎发生过程中控制祖细胞分化的信号因子通路的研究取得了很大进展。然而，我们仍然对这些信号因子调控的发育过程和基因有初步的了解。视黄酸（RA）是一种由视黄醇衍生的分泌信号因子，视黄醇是一种必需的营养素，首先转化为视黄醛，然后通过特定的酶转化为RA。在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信息，从而产生成熟的组织和器官。RA的合成始于体轴伸展的早期阶段，通过视黄醇脱氢酶（Rdh10）和视黄醛脱氢酶（Raldh2）的连续作用，它们共同在躯干中胚层尾部前祖区产生RA。体轴延伸需要胚胎尾端祖细胞中的FGF信号和Wnt信号，对Raldh2-/-小鼠胚胎的研究表明，RA信号通过在发育主干中下调FGF和Wnt信号，设定了该祖细胞区的前侧界限。在这样做的同时，RA信号还确保以双边对称的方式生成某些点。然而，尾侧RA的作用机制尚不清楚。RA作为核RA受体与RA应答元件（RAREs）结合的配体，直接调控关键基因的转录。传统上认为RA与诱导基因表达有关，但一些研究表明RA通过RA介导的Fgf8和Wnt8a的抑制来控制体轴延伸和躯体发生，RA作用于新生成的后神经外胚层或淋巴结，而不是体前中胚层。小鼠胚胎的染色质免疫沉淀（ChIP）研究已经鉴定出Fgf8和Wnt8a启动子上游结合RA受体的RAREs，从而能够更深入地研究尾侧RA机制。在本项目中，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方法来检测Fgf8和Wnt8a启动子。本项目的目的是了解尾侧RA抑制FGF和Wnt信号以确保正常体轴延伸的机制。具体而言，我们建议：(1)利用几种遗传模型确定RA在躯体发生和体轴延伸过程中抑制FGF信号的靶组织；(2)减少RA缺陷小鼠和斑马鱼胚胎中的Wnt信号，以挽救体轴延伸缺陷，并检测RA和Wnt信号之间的串串；(3)通过体内和体外研究验证Fgf8和Wnt8a RA反应元件的抑制功能。

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.