Generation of Retinoid Signals during Development

描述(由申请人提供)：在识别胚胎发育过程中控制祖细胞分化的信号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一个初步的了解。视黄酸(RA)是一种由视黄醇产生的分泌型信号因子，视黄醇是一种必需的营养物质，它首先被转化为视黄醛，然后被特定的酶转化为视黄酸。脊椎动物胚胎发育过程中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。视黄醇脱氢酶(Rdh10)和视黄醛脱氢酶(Raldh2)共同作用于躯干中胚层尾侧祖细胞前部的RA合成，在体轴伸展的早期阶段开始合成RA。体轴的延伸需要胚胎尾端的前体细胞中的成纤维细胞生长因子信号和Wnt信号，对Raldh2-/-小鼠胚胎的研究表明，RA信号通过作用于发育中的躯干下调成纤维细胞生长因子和Wnt信号，设定了这一前体区域的前限。在这样做的同时，RA信号也确保了体节以双边对称的方式产生。然而，尾侧RA的作用机制仍不清楚。RA作为核内RA受体与RA反应元件(RARES)结合的配基，直接调节关键基因的转录。RA传统上与基因表达的诱导有关，但一些研究表明，RA通过RA介导的对Fgf8和WNT8a的抑制来控制体轴的伸展和躯体的发生，并且RA作用于新生成的后神经外胚层或结节，而不是卵裂期前的中胚层。对小鼠胚胎的染色质免疫沉淀(CHIP)研究发现，与RA受体结合的Fgf8和Wnt8a启动子上游的稀有元素，使我们能够更深入地研究RA的尾部机制。在这个项目中，我们计划使用几个小鼠和斑马鱼的遗传模型来消除或减少RA、成纤维细胞生长因子和Wnt信号，以及转基因和芯片方法来检测Fgf8和Wnt8a启动子。本项目的目的是了解尾侧RA抑制成纤维细胞生长因子和Wnt信号以确保正常的体轴伸展的机制。具体地说，我们建议：(1)使用几种遗传模型来确定在体细胞发生和体轴延伸过程中RA抑制成纤维细胞生长因子信号的靶组织；(2)减少RA缺陷小鼠和斑马鱼胚胎中的Wnt信号，以挽救体轴延伸中的缺陷，并检测RA和Wnt信号之间的串扰；(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。 公共卫生相关性：研究重点是了解胚胎内组织的正常发育情况，为合理设计各种人类疾病的再生治疗方法提供了重要信息。理解这一点的关键是利用小鼠和斑马鱼的遗传学研究来了解胚胎祖细胞在组织和器官的生成过程中如何通过分泌的信号分子相互沟通。通过确定信号分子维甲酸如何调节祖细胞中的另外两个重要的信号分子(成纤维细胞生长因子和Wnt)，该项目将帮助我们更好地了解胚胎发育过程，并将提供关于这些信号因子在寻找有效的再生治疗方法以对抗人类疾病或衰老方面的潜在用处的重要线索。

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. PUBLIC HEALTH RELEVANCE: Studies focused on understanding how tissues normally develop within the embryo provide important information useful in the rational design of regenerative treatments for various human diseases. Key to this understanding is the use of genetic studies in mouse and zebrafish to learn how embryonic progenitor cells communicate with one another via secreted signaling molecules during generation of tissues and organs. By determining how the signaling molecule retinoic acid functions to regulate two other important signaling agents in progenitor cells (Fgf and Wnt), this project will help us better understand the process of embryogenesis and will provide important clues about the potential usefulness of these signaling factors in the search for effective regenerative treatments that can be used to combat human disease or aging.