Animal Model to Study Retinoic Acid Function in Postnatal and Adult Tissues

描述（申请人提供）：在各种分泌型细胞间信号因子中，视黄酸（RA）的独特之处在于它是一种小分子，通过进入靶细胞的细胞核并通过作为核受体的配体与靶基因结合来直接调节基因表达。维生素 A（视黄醇）是一种必需营养素，可作为 RA 合成的前体，通过两步代谢途径合成 RA，其中视黄醇转化为视黄醛，然后再转化为 RA。小鼠基因敲除研究已确定 RA 信号传导由三种 RA 受体和三种视黄醛脱氢酶控制，它们执行 RA 合成的最后一步。阐明 RA 生理作用的重大成功仅在于分析胚胎发育过程中的 RA 功能，因为消除 RA 信号传导会导致胚胎致死。服用 RA 或抑制 RA 合成或受体活性的药物表明 RA 还可能控制许多成人功能，包括海马神经发生、免疫功能、精子发生、干细胞功能、皮肤和毛囊再生以及预防癌症。然而，与基因敲除方法相比，这些方法更容易出现伪影和误解，并且可能无法准确揭示出生后或成人 RA 功能。因此，该项目的目标是开发一个成功的遗传模型来分析出生后 RA 功能。根据这个目标，我们将开发一种缺乏视黄醛合成的基因敲除小鼠。视黄醛向 RA 的代谢是不可逆的且具有组织特异性，与之相反，视黄醇和视黄醛的相互转化是可逆的，并且在整个生物体中广泛发生。因此，我们提出，如果维持补充视黄醛的饮食，缺乏视黄醛合成的模型生物将能够在出生后存活。基因敲除研究支持 RA 合成和生存所需的三种将视黄醇转化为视黄醛的酶的存在，即视黄醇脱氢酶 10 (RDH10) 和两种乙醇脱氢酶 (ADH3 和 ADH4)。 Rdh10 敲除小鼠在妊娠期间因缺乏 RA 合成而死亡，而 Adh3 或 Adh4 敲除小鼠在缺乏维生素 A 的饮食中会在出生后死亡。为了生成成人 RA 功能的模型，我们建议：（1）建立饮食视黄醛治疗，使 Rdh10 敲除和 Rdh10；缺乏 RDH10 和所有形式 ADH 的 Adh-del 复合敲除的产后存活； (2) 出生后从Rdh10和Rdh10;Adh-del基因敲除小鼠中取出视黄醛，检查存活情况并确定哪些组织RA活性降低或消除； (3) 对 Rdh10 和 Rdh10;Adh-del 敲除小鼠（视黄醛）进行微阵列研究，以鉴定研究界广泛感兴趣的各种组织中的 RA 靶基因。 公共健康相关性：维生素 A 代谢物视黄酸的基本性质早已为人所知，尽管我们对它在胚胎发生过程中的功能了解很多，但我们对其在出生后和成人中的功能知之甚少。我们预测，许多基于药物研究报道的视黄酸对成人的作用将不会得到本文提出的遗传功能丧失研究的支持，但 RA 的其他功能将会被发现，从而对包括神经科学家、免疫学家、生殖生物学家、干细胞生物学家和癌症研究人员在内的广大研究人员产生重大影响。因此，共享本研究项目中生成的模型小鼠将大大加快确定视黄酸如何促进各种产后和成人器官的生理过程的能力，并将导致了解维生素 A 途径的缺陷如何导致疾病。

DESCRIPTION (provided by applicant): Among the various secreted intercellular signaling factors, retinoic acid (RA) is unique in that it is a small molecule that directly regulates gene expression by entering the nucleus of target cells and binding to target genes by acting as a ligand for nuclear receptors. Vitamin A (retinol) is an essential nutrient that serves as a precursor for RA synthesis through a two step metabolic pathway in which retinol is converted to retinaldehyde which is then converted to RA. Gene knockout studies in mice have determined that RA signaling is controlled by three RA receptors and three retinaldehyde dehydrogenases that perform the last step of RA synthesis. Major success in unraveling the physiological roles of RA has come only for analysis of RA function during embryonic development since elimination of RA signaling leads to embryonic lethality. Administration of RA or drugs that inhibit RA synthesis or receptor activity suggests that RA may also control many adult functions including hippocampal neurogenesis, immune function, spermatogenesis, stem cell function, skin and hair follicle regeneration, and prevention of cancer. However, these approaches are more prone to artifacts and misinterpretation compared with gene knockout approaches, and may not reveal an accurate view of postnatal or adult RA function. Thus, the goal of this project is to develop a successful genetic model for analysis of RA function postnatally. Pursuant to this goal, we will develop a knockout mouse lacking retinaldehyde synthesis. In contrast to metabolism of retinaldehyde to RA, which is irreversible and tissue-specific, interconversion of retinol and retinaldehyde is reversible and occurs widely throughout the organism. Therefore, we propose that a model organism lacking retinaldehyde synthesis will be able to survive postnatally if maintained on a retinaldehyde-supplemented diet. Gene knockout studies support the existence of three enzymes that convert retinol to retinaldehyde needed for RA synthesis and survival, i.e. retinol dehydrogenase-10 (RDH10) and two alcohol dehydrogenases (ADH3 and ADH4). Rdh10 knockout mice die during gestation due to lack of RA synthesis, and Adh3 or Adh4 knockout mice die postnatally when placed on a vitamin A deficient diet. In order to generate a model for adult RA function, we propose to: (1) Establish a dietary retinaldehyde treatment that will allow postnatal survival of Rdh10 knockouts and Rdh10;Adh-del compound knockouts lacking RDH10 and all forms of ADH; (2) Withdraw retinaldehyde from Rdh10 and Rdh10;Adh-del knockout mice postnatally to examine survival and determine what tissues have a reduction or elimination of RA activity; (3) Perform microarray studies on Rdh10 and Rdh10;Adh-del knockout mice ( retinaldehyde) to identify RA target genes in a variety of tissues that are of broad interest to the research community. PUBLIC HEALTH RELEVANCE: The essential nature of the vitamin A metabolite retinoic acid has been known for many years, and although we know quite a lot about its function during embryogenesis, we know little about how it functions postnatally and in the adult. We predict that many reported adult actions of retinoic acid based on drug studies will not be supported by genetic loss-of-function studies proposed here, but other functions of RA will be discovered, thus having a major impact on a large community of researchers including neuroscientists, immunologists, reproductive biologists, stem cell biologists, and cancer researchers. Sharing of the model mice generated in this research project will thus greatly accelerate the ability to determine how retinoic acid contributes to physiological processes in a wide variety of postnatal and adult organs, and will lead to an understanding of how defects in the vitamin A pathway contribute to disease.