Molecular Mechanisms Regulating Patterning Along the Animal-Vegetal Axis

沿动植物轴调节图案的分子机制

• 批准号: 9985769
• 负责人: Judith Venuti
• 金额: $ 36万
• 依托单位: Columbia University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-01-01 至 2001-01-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9985769&HistoricalAwards=false
• 关键词: Molecular; Mechanisms; Regulating; Patterning; Along

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.

β-连环蛋白和Wnt信号传导途径的其它组分在沿着海胆的动物植物轴沿着的细胞命运的特化中起重要作用。 β-连环蛋白对于微粒谱系的特化和对于从微粒发出的植物诱导信号的传播是必需的。由于HMG盒DNA结合蛋白的TCF/LEF家族成员是b-连环蛋白的潜在核伴侣，因此我们将探索它们是否是沿着动物植物轴的细胞命运规范的分子途径的重要组成部分。我们将特别关注TCF是否介导b-连环蛋白在骨骼发育特化和微粒信号传导中的作用，并解决以下问题：1）TCF是否介导微粒谱系的特化？我们将研究是否通过过度表达显性阴性（DN TCF）阻断了微小颗粒抑制b-连环蛋白信号的能力，使其在原位分化为微小颗粒。 我们将确定在DN-TCF表达胚胎中，在16细胞期的微粒体和解离胚胎中b-连环蛋白的自主表达是否改变。 我们还将测试在过表达活化的TCF（Act-TCF）的胚胎中是否增强了微节分化。2）为了确定TCF/b-连环蛋白复合物是否对于诱导植物领域的微节信号的传播和/或接收是必需的，我们将询问DN-TCF是否干扰微节诱导2 o原肠的能力。 将来自DN-TCF注射的胚胎的微粒移植到未注射的宿主中，并用多个原肠分化标志物评估2 o原肠形成。 为了询问b-连环蛋白信号传导已被阻断的宿主卵裂球是否可以响应植物诱导信号，将未注射的微粒移植到DN-TCF注射的宿主中并进行类似的评估。3)TCF功能的时间是否与植物区的规格一致？ 我们将确定β-连环蛋白在指定植物细胞命运中的作用是否依赖于其与TCF的相互作用。 我们将检查TCF功能的时间和卵裂球通过过表达类固醇诱导形式（其核进入可以被调节）对Act-TCF或DN-TCF作出反应的能力。4）由于口腔与离口外胚层的分化已被证明受到不同浓度的b-连环蛋白的影响，我们将测试外胚层特化是否也由TCF介导。 我们将研究不同浓度的DN-和Act-TCF显微注射胚胎的口腔和反口腔外胚层分化的程度。为了进一步了解外胚层特化，我们还将检查不同浓度的DN-和Act-TCF对离体动物帽中的口外胚层与离口外胚层的分化的影响。