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

描述(申请人提供)：在各种分泌的细胞间信号因子中，维甲酸(RA)是独一无二的，因为它是一种小分子，通过进入靶细胞的核并作为核受体的配体与靶基因结合来直接调节基因的表达。维生素A(视黄醇)是一种必需的营养物质，通过两步代谢途径将视黄醇转化为视黄醛，再转化为视黄醛，作为RA合成的前体。小鼠的基因敲除研究已经确定，RA信号由三个RA受体和三个视黄醛脱氢酶控制，它们执行RA合成的最后一步。只有在分析RA在胚胎发育过程中的功能时，才能成功地解开RA的生理作用，因为RA信号的消除会导致胚胎的死亡。使用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、精子生成、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，这些方法更容易产生伪影和误解，并且可能不能准确地揭示出生后或成人RA的功能。因此，本项目的目标是开发一种成功的遗传模型，用于分析出生后RA的功能。根据这一目标，我们将开发一种缺乏视黄醛合成的基因敲除小鼠。与视黄醛到视黄醛的代谢不可逆和组织特异性不同，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存在。因此，我们提出，缺乏视黄醛合成的模型生物，如果维持补充视黄醛的饮食，将能够在出生后存活。基因敲除研究支持视黄醇转化为视黄醛的三种酶的存在，视黄醇脱氢酶-10(RDH10)和两种酒精脱氢酶(ADH3和ADH4)是RA合成和生存所必需的。Rdh10基因敲除小鼠因缺乏RA合成而在妊娠期死亡，Adh3或ADH4基因敲除小鼠在缺乏维生素A的饮食中出生后死亡。为了建立成人RA功能模型，我们建议：(1)建立饮食中允许Rdh10和Rdh10基因敲除小鼠出生后存活的视黄醛处理方法；(2)从Rdh10和Rdh10基因敲除小鼠体内提取视黄醛；(3)对Rdh10和Rdh10基因敲除小鼠(视黄醛)进行基因芯片研究，以找出研究界广泛关注的各种组织中的RA靶基因。 与公共健康相关：维生素A代谢物维甲酸的基本性质已经知道很多年了，尽管我们对它在胚胎发育过程中的功能知道得很多，但我们对它在出生后和成人中的功能知之甚少。我们预测，许多已报道的基于药物研究的维甲酸成人作用将不会得到这里提出的遗传功能丧失研究的支持，但RA的其他功能将被发现，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大量研究人员产生重大影响。因此，分享在这项研究项目中产生的模型小鼠将极大地加快确定维甲酸如何在出生后和成年器官中对生理过程起作用的能力，并将导致对维生素A途径缺陷如何导致疾病的理解。

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.