ANALYSIS OF PRIMARY PALATE FORMATION

这项研究计划的长期目标是确定 形成、扩大和发展的机制 面部原基的结合。目前具体目标代表 上皮-间充质关系的继续分析 细胞通讯和细胞增殖的相互作用和模式。 先前的研究表明，上皮细胞的存在对 对间充质细胞活性和增殖的影响。 荧光示踪剂显微注射到红豆杉外植体细胞中 重组组织显示缝隙连接通讯的模式 下层间充质。其他研究，使用免疫荧光 缝隙连接蛋白(27 kDa和43 kDa)抗体的定位 展示了在特定地点的独特分布模式，如 作为鼻部胎盘(27 KDa)，以及相关的分布模式 细胞增殖(43 KDa)。根据这些调查结果， 将完成以下工作。具体目标#1：通信能力不强的细胞 人口将在器官培养系统中进行准备和使用 检验假设：a)上皮细胞和 间充质通过缝隙连接传递b)缝隙连接的阻塞 上皮-间充质交界处的沟通将影响 邻近具有沟通能力的间充质细胞增殖。 特定目标#2：免疫细胞化学，使用43 kDa抗体 缝隙连接蛋白，将被用来检验假设a) 43 kDa蛋白存在于面部原基中，分布于 区域特定方式b)其分布与先前的 面部原基细胞增殖率的测定 分布是一致的(或不一致的) 27 kDa缝隙连接蛋白。具体目标3：假设 27 kDa基因的表达受发育调控 通过a)确定该基因的表达是否 在鼻部胎盘中的区域特异性b)决定是否表达 该基因在鼻部胎盘形成过程中受时间调控。 确定该基因的表达是否与 鼻胎盘发育过程中的特殊形态发生事件。 上皮-间充质分离和重组，器官培养， 显微注射、电穿孔、流式细胞术、免疫细胞化学、In 将利用原位杂交和放射自显影技术。

The long range goal of this research program is to identify the developmental mechanisms responsible for the formation, enlargement and union of the facial primordia. The present specific aims represent continuing analysis of the relationship between epithelial-mesenchymal interaction and patterns of cell communication and cell proliferation. Prior studies indicated that the presence of epithelium had a profound effect on the viability and proliferation of subjacent mesenchyme. Microinjection of fluorescent tracers into cells of explants of recombined tissues revealed patterns of gap junctional communication in subjacent mesenchyme. Other studies, employing immunofluorescent localization with antibodies to gap junction proteins (27kDa and 43kDa) demonstrated unique patterns of distribution in specific locations, such as the nasal placode (27kDa), and patterns of distribution associated with cell proliferation (43kDa). In light of these findings, the following will be done. Specific Aim #1: Communication-incompetent cell populations will be prepared and employed in organ culture systems to test the hypotheses: a) developmental signals between epithelium and mesenchyme are transmitted by gap junctions b) blockage of gap junctional communication at the epithelial-mesenchymal interface will affect proliferation of the adjacent communication-competent mesenchyme. Specific Aim #2: Immunocytochemistry, employing antibodies to the 43kDa gap junction protein, will be used to test the hypotheses a) that the 43kDa protein is present in the facial primordia and is distributed in a region-specific manner b) its distribution is correlated with previously determined rates of cell proliferation in the facial primordia c) the distribution is concordant (or discordant) with the distribution of the 27kDa gap junction protein. Specific Aim #3: The hypothesis that expression of the gene for the 27kDa is developmentally regulated will be tested by a) determining whether expression of the gene is region-specific in the nasal placode b) determining whether expression of the gene is temporally regulated during nasal placode formation c) determining whether expression of the gene is associated with a particular morphogenetic event during nasal placode development. Epithelial-mesenchymal separation and recombination, organ culture, microinjection, electroporation, flow cytometry, immunocytochemistry, in situ hybridization, and autoradiography will be utilized.