CRANIOFACIAL CHONDROGENESIS AND RETINOID RECEPTORS

这项建议的主要主题是提高我们对如何 维甲酸及其受体有助于头面部发育。 研究的第一阶段将定义时间和空间 维甲酸受体和细胞维甲酸的表达 胚胎小鼠颅面间充质中的酸结合蛋白。 这将通过原位杂交和Northern杂交来完成。受体 不同面部区域的基因表达将进行定量比较 使用图像分析系统。受体的表达也将被研究 在暴露于致畸剂量的外源性维甲酸的胚胎中。这个 第二阶段的调查将分析维甲酸的表达 高密度微团细胞培养中的酸受体和CRABP 取自不同头面部的间充质组织，有无 外源性维甲酸。本研究将提供有关以下方面的信息 维甲酸受体在调控中胚层发育中的作用 软骨生成。在这两个阶段的研究中，特别强调 将在观察第一臂和第二臂之间的任何差异时做出 弓间充质，因为它们对 在小鼠、非人类灵长类动物和人类中发现了外源性维甲酸。 第三阶段通过研究维甲酸在头面部发育中的作用 阻断维甲酸受体基因的表达。反义 含有受体反义片段的寡核苷酸或逆转录病毒 CDNA将被用来阻断受体的表达。反义词 将向微团中胚层细胞添加寡核苷酸 文化。逆转录病毒将被注射到小鼠的神经折叠中 胚胎，为了将一些注定要成为脑神经的细胞 克雷斯特。然后，这些胚胎将被允许在整个胚胎中生长， 微团细胞培养。这一系列的实验应该会提供一些见解 视黄酸受体和CRABP的发育调控 正常颅面软骨形成期间及外源性软骨形成后 维甲酸在颅面神经损伤中的应用 畸形。

The major theme of this proposal is to improve our understanding of how retinoic acid and its receptors contribute to craniofacial development. The first phase of the study will define the temporal and spatial expressions of the specific retinoic acid receptors and cellular retinoic acid binding protein (CRABP) in embryonic mouse craniofacial mesenchyme. This will be done by in situ and Northern blot hybridization. Receptor gene expression from different facial areas will be quantitatively compared using an image analyzer system. Receptor expression will also be studied in embryos exposed to teratogenic doses of exogenous retinoic acid. The second phase of the investigation will analyze the expression of retinoic acid receptors and CRABP in high density micromass cell cultures of mesenchyme taken from different craniofacial areas, with and without exogenous retinoic acid. This study will provide information concerning the role of specific retinoic acid receptors in controlling mesectodermal chondrogenesis. In the studies of both of these phases, special emphasis will be made on observing any differences between first and second brachial arch mesenchyme, because consistent differences in their responses to exogenous retinoids have been found in mice, nonhuman primates, and humans. The third phase probes retinoic acid's role in craniofacial development by blocking expression of retinoic acid receptor genes. Antisense oligonucleotides or retroviruses containing antisense fragments of receptor cDNAs will be used to block receptor expression. The antisense oligonucleotides will be added to the micromass mesectodermal cell cultures. The retroviruses will be injected into the neural fold of mouse embryos, in order to transfect some cells destined to become cranial neural crest. These embryos will then be allowed to grow in whole embryo and micromass cell culture. This series of experiments should provide insight into the developmental regulation of retinoic acid receptors and CRABP during normal craniofacial chondrogenesis and following exogenous application of retinoic acid that is known to induce craniofacial malformations.