Generation of Retinoid Signals During Development

描述（由申请人提供）：视黄酸（RA）是一种细胞-细胞信号分子，来源于视黄醇，控制发育的几个方面。由于类风湿关节炎对胚胎干细胞的分化是有用的，因此研究类风湿关节炎如何发挥其功能将对基于干细胞的疾病治疗的发展至关重要。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，包括了解在细胞之间发送信号的调节分子。当视黄醇依次代谢为视黄醛，然后再代谢为RA时，RA信号就会发生。RA作为核RA受体的配体，结合DNA并直接调节基因表达。在胚胎发生早期控制RA合成的调节酶是视黄醛脱氢酶-2 (RALDH2)，它将视黄醛氧化为RA，最初只在后中胚层发生。RALDH2在人、小鼠、鸡、蛙和鱼中均有保守表达，是唯一能在小鼠早期胚胎中合成RA的酶。Raldh2缺失突变小鼠胚胎从原始条纹期晚期到尾芽期失去了正常存在于胚胎后部的所有RA信号活动，并且在尾芽期之后不再发育。这些突变体表现出体分裂中断，不能产生前肢芽，表明RA是这些中胚层组织正常发育所必需的。低剂量的RA膳食补充可以挽救这些缺陷。我们的实验室已经发现，揭示RA功能的一个非常有用的工具是为Raldh2突变体提供不同长度的RA补充，然后使用这些胚胎中也存在的RA报告基因跟踪外源RA刺激转录的地方。这些研究允许我们假设Raldh2仅以细胞非自主方式起作用，这意味着在中胚层合成的RA分泌并作用于邻近细胞，但在产生RA的细胞内不起作用。这一重要信息现在必须整合到我们对RA如何在早期发育期间控制中胚层发育的看法中。本研究将以携带RA报告基因的Raldh2-/-小鼠作为模型系统，揭示RA在中胚层发育过程中的时空作用机制和受调控的基因。Raldh2-/-小鼠将与各种其他零突变小鼠杂交，以研究RA作用的机制。具体目的如下：(1)我们将确定RA作用于神经外胚层的机制，并阻止RA在RA拯救的raldh2 - 1 -胚胎的体前中胚层中的活性。(2)我们将研究RA的作用机制，包括分析邻近神经外胚层中RA调控的基因，以及神经外胚层向体前中胚层发送的信号。(3)研究RA信号在前肢出芽中的作用，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.