Functional Analysis of WNT-Signaling in Embryonic Stem Cells

包括环节动物在内的所有现存动物门中，有近三分之一的动物通过一种称为螺旋卵裂的定型程序产生胚胎细胞谱系。 这种发育程序产生了许多不同的胚胎干细胞。 这些细胞的大小和位置允许跨门明确鉴定同源胚胎干细胞。 尽管事实上，这种广泛使用的模式的细胞命运规范有许多祖先的功能，潜在的分子机制还没有阐明。本项目的目的是了解的作用wnt信号在早期细胞命运规范和分割的环节动物Helobdella triseralis的胚胎是适合拟议的实验。 在脊椎动物、昆虫、线虫和棘皮动物中，wnt信号通路被证明控制着早期细胞的命运决定，这表明它是一种古老的模式化机制。 为了研究wnt信号在螺旋分裂胚胎中的作用，将分析wnt信号的位点和功能。 该途径的两个下游组分和关键介质，β-连环蛋白和LEF-1直系同源物，已经从Helobdella中回收。 针对两者的特异性抗血清将用于定位早期胚胎中的wnt反应细胞。 wnt途径的功能将通过两种分子在不同胚胎干细胞中的显性负性和显性活性形式的过表达来确定，以抑制或引发靶细胞谱系中的wnt应答。 拟议的研究将提供信息，以确定在胚胎发生的祖先类型的wnt信号的作用，从而将提供一个基本的发育程序，在轴的形成和分割在现存的双侧动物胚胎的进化历史的见解。

Nearly one third of all extant animal phyla including the annelids produce their embryonic cell lineages via a stereotypic program, called spiral cleavage. This developmental program generates a number of distinct embryonic stem cells. The size and location of those cells allows the unequivocal identification of homologous embryonic stem cells across phyla. Despite the fact that this widely used mode of cell fate specification has many ancestral features, the underlying molecular mechanisms have not been elucidated.The purpose of this project is to understand the role of wnt-signaling in early cell fate specification and segmentation of the annelid Helobdella triseralis whose embryos are suitable for the proposed experiments. The wnt-pathway has been shown to control early cell fate decisions in vertebrates, insects, nematodes and echinoderms which suggests that it is an ancient patterning mechanisms. In order to investigate the role of wnt-signaling in a spiral cleaving embryo, the sites and function of wnt-signaling will be analyzed. Two downstream components and key mediators of the pathway, a beta-catenin and a LEF-1 ortholog, have been already recovered from Helobdella. Specific antisera against both will be used to localize wnt-responsive cells in early embryos. The function of the wnt-pathway will be determined by the overexpression of dominant negative and dominant active forms of both molecules in distinct embryonic stem cells to inhibit or elicit wnt-responses in a targeted cell lineage. The proposed research will provide information to determine the role of wnt-signaling in an ancestral type of embryogenesis and thereby will give insights into the evolutionary history of a fundamental developmental program that operates during axis formation and segmentation in extant embryos of bilaterian animals.