MECHANISMS OF RETINOID EFFECTS ON SKELETOGENESIS

类维生素A影响骨骼形成的机制

• 批准号: 5212001
• 负责人: DOUGLAS Frank PAULSEN
• 金额: --
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/5212001
• 关键词: chick embryo; ectoderm; embryo /fetus; growth /development; high performance liquid chromatography; immunocytochemistry; in situ hybridization; limbs; mesenchyme; northern blottings; nucleoproteins; nutrition related tag; retinoate; retinoid binding proteins; scintillation counter; transforming growth factors; vitamin metabolism; vitamin receptor; western blottings

Despite the limb bud's scrutiny as a model of development for over half a century, limb malformations still afflict more than one out of every 2,000 live human births. The mechanisms underlying these often devastating defects are unclear, partly because we still have so much to learn about normal limb development. Perhaps the most fundamental aspect of limb development is outgrowth. The project's long term objective is to understand the regulation of vertebrate limb-bud outgrowth. The immediate goal is to test the hypothesis that the well- known, but poorly understood ability of the limb bud's apical ectodermal ridge (AER) to promote limb outgrowth involves an ability to maintain cellular retinoic acid-binding protein (CRABP) expression in the subjacent mesenchyme and thereby to limit the amount of free retinoic acid (RA) encountered by nuclear RA receptors, such as RA receptor-beta (RAR-beta), in these cells. A corollary hypothesis to be tested is that CRABP's effect involves promoting metabolic inactivation of free RA. Since RA's effects at physiologic concentrations can be largely generalized as promoting differentiation through interactions with nuclear receptors, limiting RA's availability could protect stem cells within the AER's zone of influence from precocious differentiation. Four specific aims are proposed to test this hypothesis. Aim 1 is to determine the AER's effect on CRABP expression in the subjacent limb-bud mesenchyme in vivo. Two approaches will be taken CRABP expression will be examined in chick limbs whose AERs have been excised at different stages of limb outgrowth, and in limbs to which extra AERs have been grafted. Unoperated contralateral limbs will serve as controls. Aim 2 is to determine the AER's effect on CRABP expression in serum-free cultures of mesenchyme from different wing-bud regions. CRABP expression will be examined in micromass cultures of wing mesenchyme overlain by isolated AERs. Controls will include cultures lacking an AER and cultures covered with other types of ectoderm. The ability of exogenous RA to thwart the ectodermal effect will also be tested. Aim 3 is to determine the AER's effect on RA metabolism in serum-free cultures of wing-bud mesenchyme from different limb regions. Radiolabeled RA will be added to selected culture types that exhibited different CRABP expression levels in Aim 2 and/or different levels of RAR-beta expression in Aim 4. Extracts of treated cultures will be analyzed to reveal any correlation between RA metabolism, CRABP expression and the activation of RA-driven genes. Aim 4 is to determine the AER's effect on the expression of RA-activated genes in vivo and in vitro. AER effects on the expression of RAR-beta will be examined taking the same approach used to analyze effects on CRABP expression in Aims 1 (in vivo) and 2 (in vitro). The methods will include in situ hybridization and immunohistochemistry to examine patterns of CRABP and RAR-beta expression and Northern and Western blotting to provide information about CRABP and RAR-beta isoforms present. Retinoid metabolism will be assessed using reverse phase HPLC of extracts from cultures treated with radiolabeled retinoids, scintillation counting of HPLC fractions, and comparison of peak retention times to standards. Aim 5 is to introduce minority biomedical science students to combined classic and molecular approaches to important problems in development biology.

尽管肢芽被认为是超过一半的发育模式 一个世纪以来，肢体畸形仍然困扰着每一个人中的一个以上 2,000个活产人类。这些问题背后的机制往往 毁灭性的缺陷尚不清楚，部分原因是我们还有很多东西要做 了解肢体正常发育情况。也许最基本的 肢体发育的一个方面是生长。该项目的长期 目的：了解脊椎动物肢芽的调控。 外延生长。直接的目标是测试油井的假设- 已知但知之甚少的肢芽顶端外胚层的能力 促进肢体生长的脊椎(AER)包括一种保持能力 细胞维甲酸结合蛋白(CRABP)在血管内皮细胞的表达 从而限制游离维甲酸的量 核内RA受体如RA受体-β所遇到的酸(RA) (RAR-β)，在这些细胞中。一个需要检验的推论是 CRABP的作用包括促进游离RA的代谢失活。 由于RA在生理浓度下的影响可以在很大程度上 概括为通过与 核受体，限制RA的可用性可以保护干细胞 在AER的早熟分化影响范围内。 为了检验这一假说，本文提出了四个具体目标。目标1是 测定AER对下肢芽CRABP表达的影响 体内的间充质细胞。将采取两种方法CRABP表达 在不同部位切除AER的雏鸡四肢上进行检查 肢体生长的阶段，以及额外的AER已经到达的肢体 嫁接的。未手术的对侧肢体将作为对照。目标2 目的是确定AER对无血清条件下CRABP表达的影响 不同翅芽地区间充质细胞的培养。CRABP 将在微团培养的翼间充质细胞中检测其表达 被孤立的AER覆盖。控制将包括缺少 以及被其他类型的外胚层覆盖的培养物。的能力 外源性RA抑制外胚层的作用也将得到检验。目标3 是为了确定AER对无血清培养中RA代谢的影响 来自不同肢体区域的翼芽间充质细胞。放射性标记RA 将添加到显示不同CRABP的选定区域性类型 AIM 2和/或不同水平的RAR-β的表达水平 在AIM 4中的表达。处理后的培养物的提取物将被分析以 揭示RA代谢、CRABP表达与 RA驱动基因的激活。目标4是确定AER的效果 关于RA激活基因在体内和体外的表达。艾尔 用同样的方法检测对RAR-β表达的影响 分析AIMS-1对CRABP表达影响的方法(体内) 和2(体外)。这些方法将包括原位杂交和 免疫组织化学方法检测CRABP和RAR-β的表达 表达和Northern和Western印迹提供信息 关于CRABP和RAR-β亚型的存在。类维A酸代谢将会是 反相高效液相色谱检测细菌培养物中的提取物 放射性标记的维甲酸，高效液相色谱组分的闪烁计数，以及 峰保留时间与标准品的比较。目标5是介绍 少数民族生物医学专业学生将经典与分子相结合 研究发育生物学中重要问题的方法。