CRANIOFACIAL CHONDROGENESIS AND RETINOID RECEPTORS

本提案的主题是提高我们对以下问题的认识： 视黄酸及其受体有助于颅面发育。 研究的第一阶段将确定时间和空间 特异性视黄酸受体和细胞视黄酸的表达 酸结合蛋白（CRABP）在胚胎小鼠颅面间质。 这将通过原位和北方印迹杂交进行。 受体 将定量比较来自不同面部区域的基因表达 使用图像分析系统。 还将研究受体表达 胚胎暴露于致畸剂量的外源性视黄酸。 的 研究的第二阶段将分析维甲酸的表达， 酸受体和CRABP在高密度微团细胞培养物中的作用 间充质取自不同的颅面区域，有和没有 外源性视黄酸 这项研究将提供有关 特异性视黄酸受体在中外胚层调控中的作用 软骨形成 在这两个阶段的研究中，特别强调 将在观察第一和第二肱动脉之间的任何差异时进行 弓间充质，因为他们的反应一致的差异， 在小鼠、非人灵长类动物和人中发现了外源性类视色素。 第三阶段探讨视黄酸在颅面发育中的作用 阻断视黄酸受体基因的表达。 反义 含有受体反义片段的寡核苷酸或逆转录病毒 cDNA将用于阻断受体表达。 反义 将寡核苷酸加入微团中外胚层细胞 cultures. 将逆转录病毒注射到小鼠的神经褶中， 胚胎，以去除一些注定要成为颅神经细胞的细胞。 冠。 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能提供 视黄酸受体和CRABP的发育调节 在正常颅面软骨形成过程中， 应用视黄酸，已知其可诱导颅面 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.