Generation of Retinoid Signals During Development

描述（由申请人提供）：视黄酸（RA）是一种源自视黄醇的细胞-细胞信号分子，控制发育的几个方面。由于RA对胚胎干细胞的分化有用，因此旨在了解RA如何发挥其功能的研究对于开发基于干细胞的疾病治疗方法至关重要。为了从干细胞中产生替代器官，我们需要了解胚胎最初是如何产生器官的，包括在细胞之间发送信号的调节分子的知识。当视黄醇依次代谢为视黄醇醛，然后代谢为RA时，发生RA信号传导，RA作为核RA受体的配体发挥作用，核RA受体结合DNA并直接调节基因表达。已发现在早期胚胎发生期间控制RA合成的调节酶是视黄醇脱氢酶-2（RALDH 2），其最初仅在后中胚层中将视黄醇氧化成RA。RALDH 2在人、小鼠、鸡、青蛙和鱼中是保守的，并且是在早期小鼠胚胎中合成RA的唯一酶。Raldh 2无效突变小鼠胚胎从原始条纹晚期到尾芽期失去了通常存在于胚胎后部的所有RA信号传导活性，并且不发育超过早期尾芽期。这些突变体表现出中断体节分割和未能产生前肢芽，表明RA是这些中胚层组织的正常发育所需的。低剂量的母体膳食RA补充剂可以挽救这些缺陷。我们的实验室已经发现，一个非常有用的工具，用于解开RA功能是提供RA补充Raldh 2突变体的不同长度的时间，然后按照外源RA刺激转录使用RA报告转基因也存在于这些胚胎。这些研究使我们能够假设Raldh 2仅以细胞非自主方式发挥作用，这意味着中胚层中合成的RA被分泌并作用于邻近细胞，但在产生RA的细胞内不起作用。这一重要的信息片段，现在必须整合到我们的看法，如何RA控制中胚层发育在早期发展。在这个建议中，携带RA报告基因转基因的Raldh 2-/-小鼠将被用作揭示RA作用在中胚层发育过程中的时空机制和调控基因的模型系统。Raldh 2-/-小鼠将与各种其他无效突变小鼠杂交以解决RA作用的机制。具体目标如下：（1）我们将确定将RA作用引导至神经外胚层并阻止RA拯救的Raldh 2-l-胚胎的体前中胚层中的RA活性的机制。(2)我们将研究RA行动所需的机制，包括RA在相邻的神经外胚层和信号发送到preomitic中胚层的神经外胚层的基因调控的分析，适当的体节的形成和图案。(3)RA信号在前肢出芽中的作用将被研究，包括时间，靶组织，RA源组织和靶基因。

DESCRIPTION (provided by applicant): Retinoic acid (RA) is a cell-cell signaling molecule derived from retinol that controls several aspects of development. As RA is useful for differentiation of embryonic stem cells, studies designed to understand how RA performs its functions will be essential for development of stem cell-based treatments for disease. In order to generate replacement organs from stem cells we need to understand how embryos initially generate organs, including knowledge of the regulatory molecules that send signals between cells. RA signaling occurs when retinol is sequentially metabolized to retinaldehyde and then to RA which functions as a ligand for nuclear RA receptors that bind DNA and directly regulate gene expression. The regulatory enzyme controlling synthesis of RA during early embryogenesis has been discovered to be retinaldehyde dehydrogenase-2 (RALDH2) which oxidizes retinaldehyde to RA, initially only in posterior mesoderm. RALDH2 is conserved in human, mouse, chick, frog, and fish, and is the only enzyme synthesizing RA in early mouse embryos. Raldh2 null mutant mouse embryos lose all RA signaling activity normally present in the posterior portion of the embryo from the late primitive streak stage to the tailbud stage and do not develop beyond the early tailbud stage. These mutants exhibit disrupted somite segmentation and fail to generate forelimb buds, indicating that RA is required for proper development of these mesodermal tissues. Low-dose maternal dietary RA supplementation can rescue these defects. Our laboratory has discovered that a quite useful tool for unraveling RA function is to provide RA supplementation to Raldh2 mutants for various lengths of time and then follow where the exogenous RA stimulates transcription using an RA- reporter transgene also present in these embryos. Those studies have allowed us to hypothesize that Raldh2 functions only in a cell-nonautonomous fashion, meaning that RA synthesized in the mesoderm is secreted and acts on neighboring cells, but does not function within the cells producing RA. This important piece of information must now be integrated into our view of how RA controls mesodermal development during early development. In this proposal, Raldh2-/- mice carrying an RA-reporter transgene will be used as a model system for revealing the spatiotemporal mechanism of RA action during mesoderm development and the genes regulated. Raldh2-/- mice will be crossed with various other null mutant mice to address the mechanism of RA action. The specific aims are as follows: (1) we will determine the mechanism that directs RA action to the neuroectoderm and prevents RA activity in the presomitic mesoderm of RA-rescued Raldh2-l- embryos. (2) We will examine the mechanism of RA action required for proper somite formation and patterning including an analysis of the genes regulated by RA in the adjacent neuroectoderm and the signals sent from neuroectoderm to presomitic mesoderm. (3) The role RA signaling plays in forelimb budding will be investigated including the timing, target tissue, RA source tissue, and target genes.