MECHANISMS OF RETINOID EFFECTS ON SKELETOGENESIS

每2000名活产儿中就有一名以上患有肢体缺陷。 这些往往具有破坏性的缺陷背后的机制尚不清楚。一个 肢体发育的基本方面是生长。该项目耗时很长 术语的目标是了解这一过程的规则。这个 眼下的目标是检验这样一种假设，即肢体的能力 用于促进肢体生长的巴德顶端外胚层脊(AER)涉及 维甲酸结合蛋白(CRABP)的维持能力 在脊下肢间充质中的表达，从而限制视黄酸 这些细胞中的核RA受体所遇到的酸(RA)。推论 要检验的假设是1)AER信号维持CRABP 表达，2)AER信号是成纤维细胞生长因子-8，3)CRABP促进RA 降解，4)RA降解阻止干细胞分化，5) 阻止干细胞分化可阻止成纤维细胞生长因子受体(FGFR) 异构体转换，以及6)阻止成纤维细胞生长因子受体异构体转换 维持肢体间充质能力以应对AER。六个具体的 为了检验这一假说，我们提出了目标。目标1是记录AER CRABP在体内对下肢芽间充质细胞表达的影响 我们将采取两种方法。将检测CRABP在雏鸡中的表达 在肢体生长的不同阶段切除了AER的肢体， 以及移植了额外AER的四肢。未操作 对侧肢体将作为对照。目标2是确定 Aer对维甲酸代谢的影响。完好无损的远端肢体碎片 或之前手术切除的AER将被孵化在 2/H-RA体外培养。培养的组织和培养基萃取物将通过 高效液相色谱比较不同来源的 准备工作。目的3确定RA对CRABP表达的影响。血清- 肢体间充质细胞的游离培养将用全反式ERA处理， 9-顺式-RA或3，4-二氢-RA。CRABP蛋白水平将通过IN进行评估 培养物抽提物的原位酶联免疫吸附试验和免疫印迹试验。目标4是确定 成纤维细胞生长因子对CRABP表达的影响。不同来源间充质细胞的微培养 在含有成纤维细胞生长因子-2、成纤维细胞生长因子-4、成纤维细胞生长因子-8或 成纤维细胞生长因子-10将接受整装原位杂交 地高辛标记的CRABP核糖核酸探针。目标5是为了 确定CRABP对维甲酸反应的影响。微文化 瞬时转染含有AFP基因的真核表达载体 700个BP。CRABP cDNA构建，将用全反式、9-顺式或 3，4-二氢-维甲酸及其对生长、软骨形成和FGFR的影响 表情将被测量。目的6是确定CRABP对RA的影响 新陈代谢。雏鸡肢芽间充质细胞将一过性 用含700bpCRABP的真核表达载体转染 CDNA.转基因细胞将在无血清的情况下收获、洗涤、悬浮 培养基，并与放射性标记的RA孵育至高比活性。

Limb defects afflict more than one out of every 2,000 live human births. The mechanisms underlying these often devastating defects are unclear. A fundamental aspect of limb development is outgrowth. The project's long term objective is to understand the regulation of this process. The immediate goal is to test the hypothesis that the 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 sub-ridge limb mesenchyme and thereby to limit the retinoic acid (RA) encountered by nuclear RA receptors in these cells. Corollary hypothesis to be tested are 1) than an AER signal maintains CRABP expression, 2) that the AER signal is FGF-8, 3) that CRABP promotes RA degradation, 4) that RA degradation prevents stem-cell differentiation, 5) that preventing stem-cell differentiation prevents FGF-receptor (FGFR) isoform switching, and 6) that preventing FGF-R isoform switching maintains limb mesenchymal competence to respond to the AER. Six specific aims are proposed to test this hypothesis. Aim 1 is to document AER effects 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 limbs into which extra AERs have been grafted. Unoperated contralateral limbs will serve as controls. Aim 2 is to determine the AER's effects on retinoic acid metabolism. Distal limb fragments, intact or with their AER's previously surgically removed, will be incubated in vitro with 2/H-RA. Incubated tissue and media extracts will be analyzed by HPLC to compare metabolite profiles produced by the different preparations. Aim 3 is to determine RA effects on CRABP expression. Serum- free micro-cultures of limb mesenchyme will be treated with all-trans-ERA, 9-cis-RA, or 3,4-didehydro-RA. CRABP protein levels will be assessed by in situ ELISA and Western blots of culture extracts. Aim 4 is to determine FGF effects on CRABP expression. Micro-cultures of mesenchyme from various limb regions grown in serum-free medium containing FGF-2, FGF-4, FGF-8, or FGF-10 will be subjected to whole-mount in situ hybridization using a digoxigenin-labeled CRABP riboprobe in the wells of the plate. Aim 5 is to determine CRABP's effect on responses to retinoic acid. Micro-cultures transiently transfected with a eukaryotic expression vector containing a 700 bp. CRABP cDNA construct, will be treated with all-trans, 9-cis-, or 3,4-didehydro-RA, and effects on growth, chondrogenesis, and FGFR expression will be measured. Aim 6 is to determine CRABP's effect on RA metabolism. Chick limb-bud mesenchymal cells will be transiently transfected with a eukaryotic expression vector containing a 700 bp CRABP cDNA. Transfected cells will be harvested, washed, suspended in serum-free medium, and incubated with RA radio-labeled to high specific activity.