MECHANISMS OF RETINOID EFFECTS ON SKELETOGENESIS

类维生素A影响骨骼形成的机制

• 批准号: 6240356
• 负责人: DOUGLAS Frank PAULSEN
• 金额: $ 6.84万
• 依托单位: MOREHOUSE SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-08-01 至 1998-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6240356
• 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.

尽管肢芽作为一种发育模型被仔细研究了一半以上， 一个世纪，肢体畸形仍然困扰着超过一个每一个 2,000个活产婴儿。这些现象背后的机制往往 毁灭性的缺陷尚不清楚，部分原因是我们仍然有太多的 了解正常的肢体发育 也许最根本的 肢体发育的一个方面是向外生长。 项目的长期性 目的了解脊椎动物肢芽发育的调控 结果 当前的目标是检验这个假设，即井- 肢芽顶端外胚层的能力是已知的，但了解甚少 脊（AER），以促进肢体生长涉及的能力， 细胞视黄酸结合蛋白（CRABP）在细胞中的表达 从而限制游离视黄酸的量 核RA受体（如RA受体β）遇到的酸（RA） （RAR-beta），在这些细胞中。 有待检验的推论假设是 CRABP的作用涉及促进游离RA的代谢失活。 由于RA在生理浓度下的作用可以在很大程度上 一般认为通过与 限制RA的可用性可以保护干细胞 在AER的早熟分化的影响范围内。 提出了四个具体目标来检验这一假设。 目的1是 确定AER对下肢芽中CRABP表达的影响 体内间充质。 将采取两种方法CRABP表达式将 在其AER已被切除的鸡肢中进行检查， 肢体长出的阶段，以及在已经有额外AER的肢体中 嫁接的 未手术的对侧肢体将作为对照。 目的2 目的是确定AER对CRABP表达的影响， 来自不同翅芽区域的间充质培养物。 CRABP 将在翅间充质的微团培养物中检测表达 被孤立的AER覆盖。 对照将包括缺乏 AER和其他类型外胚层覆盖的培养物。的能力 还将测试外源性RA以阻碍外胚层效应。 目标3 在无血清培养中确定AER对RA代谢的影响 翅芽间充质的不同肢体区域。 放射性标记RA 将添加到显示不同CRABP的选定培养物类型中 Aim 2的表达水平和/或不同水平的RAR-β 目标4中的表达。 将分析处理培养物的浸提液， 揭示了RA代谢、CRABP表达和RA代谢之间的任何相关性。 激活RA驱动的基因。 目的4是确定AER的效果 在体内和体外RA激活基因的表达。 Aer 对RAR-β表达的影响将采用相同的方法进行检查。 用于分析对目的1中CRABP表达的影响的方法（体内） 和2（体外）。 所述方法将包括原位杂交， 免疫组织化学检查CRABP和RAR-β的模式 表达以及北方和西方印迹以提供信息 关于CRABP和RAR-β亚型的信息 维甲酸代谢将是 使用反相HPLC法对用以下物质处理的培养物的提取物进行评估： 放射性标记的类维生素A，HPLC馏分的闪烁计数，和 峰保留时间与标准品的比较。 目标5是引入 少数民族生物医学科学学生结合经典和分子 研究发育生物学中的重要问题。