RETINOIC ACID AND ITS RECEPTORS IN EARLY EMBRYOGENESIS

早期胚胎发生中的视黄酸及其受体

• 批准号: 3331040
• 负责人: TSUNG-CHIEH J WU
• 金额: $ 16.56万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-07-01 至 1995-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3331040
• 关键词: DNA replication; autoradiography; cell differentiation; cell growth regulation; densitometry; developmental genetics; early embryonic stage; gel electrophoresis; gene expression; histochemistry /cytochemistry; immunochemistry; laboratory mouse; northern blottings; polymerase chain reaction; protein structure function; receptor expression; retinoate

Retinoic acid (RA) exhibits diverse functions in cell differentiation, proliferation, and pattern formation. The actions of RA are mediated through retinoic acid receptors (RARs) which are composed of two gene families (RAR and RXR). The presence of multiple receptor types for one ligand indicates that each kind of receptor has its own biological function. The broad, long-term objective of this project is to understand the roles of RA and RARs in early pregnancy. The specific aims of this proposal are: 1) to examine the cullular distribution of RARalpha, beta, and gamma in preimplantation mouse embryos, 2) to study the effect of RA on preimplantation embryos, 3) to study the effect of RA on RAR gene expression in preimplantation embryos, 4) to analyze the function of RARalpha, beta, and gamma in F9 mouse teratocarcinoma cells and embryos. To achieve these, the stage specificity and cellular specificity of RAR gene expression will be examined in preimplantation embryos using immunohistochemistry. Different amounts of RA will be added to growing two-cell embryos, in culture media, and their beneficial and detrimental effects will be assessed. The RA-regulated RAR gene expression, a presumed mechanism. by which RA controls its own effects, will be examined in embryos using reverse transcriptase-competitive polymerase chain reaction. Therefore, the effects of RA on its own receptor gene expression during embryonic development can be determined. Furthermore, the function of each RAR type in both F9 mouse teratocarcinoma cells and preimplantation embryos will be examined. Antisense DNA specific for RARalpha, beta, or gamma will be introduced into F9 cells to block the expression of RAR. The successful blockage of each receptor type will be confirmed by degradation of RNA using Northern blot hybridization, reverse transcriptase-polymerase chain reaction, or by absence of the receptor protein using immunohistochemistry. After a specific RAR expression is blocked, its impact on F9 cell differentiation will be examined. Similarly, after an antisense oligonucleotide specific to each RAR type is introduced, into early embryos, its effect on embryogenesis will be determined. These approaches will allow us to reveal which RAR type is essential for specific embryonic development and cell differentiation. Since RA exerts diverse effects in embryos, the outcomes from our proposed studies should be able to reveal the biochemical/molecular mechanisms by which RA and RARs regulate embryogenesis, cell differentiation and gene expression.

视黄酸（RA）在细胞分化中表现出多种功能， 增殖和模式形成。 RA 的作用是介导的 通过视黄酸受体（RAR），它由两个基因组成 系列（RAR 和 RXR）。 一种受体存在多种类型 配体表明每种受体都有其自身的生物学特性 功能。 该项目的广泛、长期目标是 了解 RA 和 RAR 在妊娠早期的作用。 具体的 该提案的目的是：1）检查 植入前小鼠胚胎中的 RARα、β 和 γ，2) 研究 RA对植入前胚胎的影响，3）研究RA的影响 着床前胚胎中 RAR 基因表达的影响，4) 分析 RARalpha、beta 和 gamma 在 F9 小鼠畸胎癌细胞中的功能 和胚胎。 为了实现这些，阶段特异性和细胞 RAR 基因表达的特异性将在植入前进行检查 使用免疫组织化学检测胚胎。 不同数量的 RA 将 添加到培养基中生长的双细胞胚胎中，及其有益的 并将评估有害影响。 RA调控的RAR基因 表达，一种推测的机制。 RA 通过它来控制其自身的影响， 将使用逆转录酶竞争性在胚胎中进行检查 聚合酶链式反应。 因此，RA对其自身的影响 可以确定胚胎发育过程中受体基因的表达。 此外，每种 RAR 类型在 F9 小鼠中的功能 将检查畸胎癌细胞和植入前胚胎。 将引入针对 RARalpha、beta 或 gamma 的反义 DNA 进入F9细胞以阻断RAR的表达。 成功封堵 每种受体类型将通过使用 Northern 降解 RNA 来确认 印迹杂交、逆转录酶-聚合酶链式反应，或 使用免疫组织化学检测受体蛋白的缺失。 经过一个 特异性RAR表达被阻断，其对F9细胞分化的影响 将接受检查。 类似地，在特定的反义寡核苷酸之后 将每种 RAR 类型引入早期胚胎中，其对 胚胎发生将被确定。 这些方法将使我们能够 揭示哪种 RAR 类型对于特定胚胎发育至关重要 细胞分化。 由于 RA 对胚胎产生不同的影响， 我们提出的研究结果应该能够揭示 RA 和 RAR 调节的生化/分子机制 胚胎发生、细胞分化和基因表达。