TROPHOBLAST CELL MOTILITY

胚胎植入子宫及由此形成的胚胎 胎盘是哺乳动物独特而重要的特征 发展。植入是由人的侵袭行为介导的 滋养层细胞，由滋养层细胞分化而来 胚泡。滋养层细胞的侵袭行为是发育中的 调节；前体滋养外胚层细胞是静止的上皮细胞 通过分化获得运动性侵袭行为的细胞。这个 本提案的目标是检查和定义单元格中的更改 伴随着滋养层细胞分化的行为，并且，遵循 新近确定的控制培养细胞运动的范例 细胞，以测试Rho家族小GTP结合蛋白的作用 在滋养层细胞行为的发育调控中的作用。 正常培养液中培养的胚胎的延时视频分析 抑制剂的存在将被用来确定何时运动 行为通常是启动的，在发展的哪个时间点 它的出现需要转录和翻译。结果是 将与滋养层细胞粘附性和分子分析进行比较 区分，以确定这些组件之间的关系 具有侵犯性的行为。这些结果将更好地定义 并为解释滋养层细胞分化提供了基础 以及延长转基因实验。 Rho家族的GTP结合蛋白的成员已经在 培养细胞调节丝状突起的形成 (Cdc42Hs)、IamelLipodia(RAC)、应力纤维和焦点接触 (Rho)。以确定这三种蛋白在调节中的作用 通过重组基因转移滋养层细胞的运动性 腺病毒感染将被用来过度表达显性阴性或 植入前各蛋白的组成活性形式 胚胎。显性的负向过度表达实验将 证明Rho家族的每个成员是否都参与了 控制滋养层细胞的运动，而结构性活跃 过度表达实验将解决这些蛋白质是否作为 滋养层细胞运动时间调节的控制点。这个 这些实验的结果将阐明调控机制。 滋养层细胞的行为和分化，并将提供一个模型 对于较晚的、较难获得的发育事件，以及对于侵袭性的 其他系统的转型。

Implantation of the embryo into the uterus and the resultant formation of the placenta are unique and critical features of mammalian development. Implantation is mediated by the invasive behavior of the trophoblast cells, which differentiate from the trophectoderm cells of the blastocyst. Trophoblast invasive behavior is developmentally regulated; the precursor trophectoderm cells are quiescent epithelial cells which acquire motile invasive behavior with differentiation. The goals of this proposal are to examine and define the changes in cell behavior that accompany trophoblast differentiation, and, following the paradigms recently defined for the control of cell motility in cultured cells, to test the role of the Rho family of small GTP-binding proteins in the developmental regulation of trophoblast cell behavior. Time-lapse video analysis of embryos cultured in normal medium and in the presence of inhibitors will be used to determine when motile behavior is normally initiated, and at what point in development transcription and translation are required for its onset. The results will be compared to analyses of trophoblast adhesivity and molecular differentiation, to determine the relationship between these components of invasive behavior. These results will better define the process of trophoblast differentiation, and provide the foundation for interpreting and extending the transgenic experiments. Members of the Rho family of GTP-binding proteins have been shown in cultured cells to regulate formation of filopodial protrusions (Cdc42Hs), Iamellipodia (Rac) and stress fibers and focal contacts (Rho). To determine the role of these three proteins in the regulation of trophoblast cell motility, gene transfer through recombinant adenovirus infection will be used to overexpress dominant negative or constitutively active forms of each protein in preimplantation stage embryos. The dominant negative overexpression experiments will demonstrate whether each of the Rho family members is involved in the control of trophoblast motility, while the constitutively active overexpression experiments will address whether these proteins serve as control points for temporal regulation of trophoblast motility. The results of these experiments will clarify the mechanisms regulating trophoblast behavior and differentiation, and will provide a model both for later, less accessible developmental events, and for invasive transformation in other systems.