Retinoid Dehydrogenases Involved in Eye Development

维甲酸(RA)是维生素A(视黄醇)的代谢衍生物，在脊椎动物发育过程中对眼轴图案的形成是必不可少的。当视黄醇被代谢到视网膜，然后被代谢到视黄酸时，类维A酸信号就会发生，视黄醇受体是核内维甲酸受体的配体，调节基因的表达。由于妊娠期维生素A缺乏或视黄醇受体零突变导致视黄醇信号的中断，导致眼睛的背腹轴发育异常。眼球局部发生类风湿性关节炎的机制以及类风湿性关节炎如何调节眼背腹模式的机制尚不清楚。我们对视黄醇如何在生理上被激活以形成RA的了解仍然不清楚，但随着在眼睛中表达的三种维甲酸脱氢酶的发现而得到改善，即RALDH1、RALDH2和RALDH3，它们都是乙醛脱氢酶(ALDH)酶家族的成员。这个实验室和另一个实验室培育的Raldh2缺失突变小鼠的表型在怀孕中期是致命的，具有巨大的躯干和前肢发育缺陷以及异常的后脑图案，但没有观察到视泡缺陷。母系RA治疗挽救了Raldh2突变体中的许多缺陷，从而允许通过条件救援进行进一步的分析。本实验室最近培育的Raldh1缺失突变小鼠是存活的，但胚胎在背侧视网膜缺乏RA合成，这表明进一步的分析将发现视网膜缺陷。目前还没有关于Raldh3的基因研究报告。对RALDHs的进一步分析将提供了解人类发育性眼睛缺陷所需的关键信息。该项目的总体目标是利用零突变小鼠建立RALDH1、RALDH2和RALDH3在眼睛RA合成中的作用，并利用这些突变小鼠作为工具进一步研究视黄醇信号在眼睛发育中的机制。突变小鼠将接受组织学检查，并通过原位杂交检测眼睛组织中基因表达的中断。携带稀有LacZ标记基因的小鼠将被用于检测突变胚胎中的内源性RA。该项目的具体目标如下：(1)利用Raldh1突变体检测视网膜背侧缺乏RA对视网膜发育和视网膜逃逸投射轴突的影响；(2)通过比较条件拯救的Raldh2突变体和Raldh1-Raldh2双重突变体，检测RALDH1和RALDH2对眼睛发育中RA合成的贡献；(3)建立Raldh3缺失突变小鼠，以检测该基因在眼睛发育中的作用。

Retinoic acid (RA), a metabolic derivative of vitamin A (retinol), is essential for axial patterning of the eye during vertebrate development. Retinoid signaling occurs when retinol is metabolized to retinal and then to RA which serves as a ligand for nuclear retinoid receptors that regulate gene expression. Disruption of retinoid signaling either by gestational vitamin A deficiency or by creation of retinoid receptor null mutations results in abnormal dorsoventral axial development of the eye. The mechanism for generation of RA locally in the eye as well as the mechanism for how RA regulates dorsoventral patterning of the eye are not yet understood. Our understanding of how retinol is physiologically activated to form RA is still vague, but has been improved by discovery of three retinoid dehydrogenases expressed in the eye that metabolize retinal to RA, i.e. RALDH1, RALDH2, and RALDH3, all members of the aldehyde dehydrogenase (ALDH) enzyme family. Raldh2 null mutant mice generated in this laboratory and another have revealed phenotypes that are lethal at midgestation with massive trunk and forelimb developmental defects plus abnormal hindbrain patterning, but no optic vesicle defects were observed. Maternal RA administration rescues many defects in Raldh2 mutants, thus allowing further analysis by conditional rescue. Raldh1 null mutant mice recently generated in this laboratory are viable, but embryos suffer a lack of RA synthesis in the dorsal retina suggesting that retinal defects will be discovered upon further analysis. There have been no genetic studies reported on Raldh3. Further analysis of RALDHs should provide key information needed to understand human developmental eye defects. The overall goals of this project are to use null mutant mice to establish roles for RALDH1, RALDH2, and RALDH3 in eye RA synthesis, plus use these mutant mice as tools to further examine the mechanism of retinoid signaling in eye development. Mutant mice will be examined for developmental eye defects histologically and by in situ hybridization to detect disrupted gene expression in eye tissues. Mice carrying the RARE-lacZ marker gene will be used to detect endogenous RA in mutant embryos. Specific goals for this project will be as follows: (1) Use Raldh1 mutants to examine the effect of a lack of RA in the dorsal retina on retinal development and axonal pathfinding for retinofugal projections; (2) Examine the contribution of RALDH1 and RALDH2 to RA synthesis for eye development by comparison of conditionally RA-rescued Raldh2 mutants and Raldh1-Raldh2 double mutants; (3) Generate Raldh3 null mutant mice to examine the role of this gene in eye development.