Molecular Mechanisms Regulating Patterning Along the Animal-Vegetal Axis

B-连环蛋白和Wnt信号通路的其他成分在海胆动物植物轴上细胞命运的决定中起着重要作用。微丝谱系的确定和微粒发出的植物诱导信号的传播都需要B-连环蛋白。由于HMG盒DNA结合蛋白的Tcf/Lef家族成员是b-catenin的潜在核伙伴，我们将探索它们是否是动物植物轴上决定细胞命运的分子途径的重要组成部分。我们将特别关注TCF是否在骨骼发育规范和微米信号转导中介导b-连环蛋白的作用，并解决以下问题：1)TCF是否中介微米血统的规范？我们将研究微体是否通过过度表达显性负性(DN TCF)而阻止它们传递b-连环蛋白信号的能力被原位分化为微体。我们将确定在表达dN-TCF的胚胎中，b-catenin在16-细胞期的微粒和分离的胚胎中的自主表达是否发生改变。我们还将测试在过度表达激活的TCF(Act-TCF)的胚胎中微米分化是否得到加强。2)为了确定TCF/b-catenin复合体是否对诱导植物区域的微米信号的繁殖和/或接收是必不可少的，我们将询问dN-TCF是否干扰微粒诱导20 o原肠的能力。将来自注入DN-TCF的胚胎的微粒移植到未注射的宿主上，并用多个原肠分化的标记来评估20个原肠的形成。为了研究b-catenin信号被阻断的宿主卵裂球是否能对植物诱导信号做出反应，将未注入的微体移植到注射了dN-TCF的宿主上，并进行了类似的评估。3)TCF功能的时间是否与植物领地的规格一致？我们将确定b-连环蛋白在决定植物细胞命运中的作用是否取决于它与TCF的相互作用。我们将通过过量表达类固醇诱导形式来检测TCF功能的时序和卵裂球对Act-TCF或dN-TCF的响应能力。4)由于口腔和流产外胚层的分化已经被证明受到不同浓度的b-catenin的影响，我们将测试外胚层规范是否也受到TCF的调节。我们将在显微注射不同浓度的DN-和ACT-TCF的胚胎中检测口腔和流产外胚层分化的程度。为了进一步了解外胚层的特性，我们还将研究不同浓度的DN-和ACT-TCF对分离的动物帽中口腔和流产外胚层分化的影响。

b-catenin and other components of the Wnt signaling pathway play an important role in the specification of cell fates along the animal vegetal axis of the sea urchin. b-catenin is required for the specification of the micromere lineage and for the propagation of the vegetal inducing signal that emanates from micromeres. Since members of the TCF/LEF family of HMG box DNA-binding proteins are potential nuclear partners of b-catenin, we will explore whether they are essential components of the molecular pathway that underlies cell fate specification along the animal vegetal axis. We will focus specifically on whether TCF mediates b-catenin's effects in skeletogenic specification and micromere signaling and address the following questions: 1) Does TCF mediate the specification of the micromere lineage? We will examine whether micromeres blocked in their ability to transduce the b-catenin signal by overexpressing dominant negative (DN TCF) differentiate as micromeres in situ. We will determine whether the autonomous expression of b-catenin in micromeres at the 16-cell stage and in dissociated embryos is altered in DN-TCF expressing embryos. We will also test whether micromere differentiation is potentiated in embryos overexpressing an activated TCF (Act-TCF).2) To determine whether a TCF/b-catenin complex is essential for the propagation and/or reception of the micromere signal that induces the vegetal territory, we will ask whether DN-TCF interferes with the ability of micromeres to induce a 2o archenteron. Micromeres from DN-TCF injected embryos will be transplanted to an uninjected host and 2o archenteron formation assessed with multiple markers of archenteron differentiation. To ask whether host blastomeres in which b-catenin signaling has been blocked can respond to the vegetal inducing signal, uninjected micromeres will be transplanted to DN-TCF injected hosts and similarly assessed. 3) Does the timing of TCF function coincide with the specification of the vegetal territory? We will determine whether the role of b-catenin in specifying vegetal cell fates is dependent on its interaction with TCF. We will examine the timing of TCF function and the competence of blastomeres to respond to Act-TCF or DN-TCF by overexpressing steroid inducible forms whose nuclear entry can be regulated.4) Since the differentiation of oral vs. aboral ectoderm has been shown to be effected by different concentrations of b-catenin we will test whether ectoderm specification is also mediated by TCF. We will examine the extent of oral and aboral ectoderm differentiation in embryos microinjected with different concentrations of DN- and Act-TCF. To further understand ectoderm specification we will also examine the effects of different concentrations of DN- and Act-TCF on the differentiation of oral vs. aboral ectoderm in isolated animal caps.