TROPHOBLAST CELL MOTILITY

胚胎植入子宫和由此产生的形成 是哺乳动物胎盘的独特和关键特征， 发展 植入是由细胞的侵入行为介导的。 滋养层细胞，从滋养外胚层细胞分化而来， 囊胚 滋养细胞的侵袭行为是发育性的 前体滋养外胚层细胞是静止的上皮细胞 随着分化获得运动性侵入行为的细胞。 的 本提案的目标是检查和定义单元格中的更改 伴随滋养层分化的行为，并且， 最近定义的用于控制培养细胞运动的范例 细胞，以测试小GTP结合蛋白的Rho家族的作用 在滋养层细胞行为的发育调节中的作用。 胚胎在正常培养基中培养和在 抑制剂的存在将用于确定何时运动 行为通常是在什么时候开始的， 转录和翻译是其发生所必需的。 结果 将与滋养层粘附性和分子生物学分析进行比较。 区分，以确定这些组件之间的关系 入侵行为。这些结果将更好地定义 滋养层分化，并提供解释的基础 并扩大转基因实验。 GTP结合蛋白的Rho家族的成员已经在文献中显示。 调节丝状伪足突起形成的培养细胞 （Cdc42Hs）、缺肢（Rac）和应力纤维以及局灶接触 （Rho）。 为了确定这三种蛋白质在调节 滋养层细胞运动，基因转移，通过重组 腺病毒感染将用于过表达显性阴性或 植入前期各蛋白质的组成型活性形式 胚胎 显性负性过表达实验将 证明每个Rho家族成员是否参与了 控制滋养层运动，而组成性活跃 过表达实验将解决这些蛋白质是否作为 控制点的时间调节滋养层运动。 的 这些实验的结果将阐明调节机制 滋养层行为和分化，并将提供一个模型， 对于后来的，不太容易获得的发育事件， 在其他系统中的转换。

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.