Tcf Function in Spinal Cord Patterning

脊椎动物中枢神经系统（CNS）由控制中枢神经系统的环境信号模式化。 细胞命运的规范。了解神经系统中细胞命运的机制 系统对于我们诊断疾病和设计再生治疗方法的能力至关重要。 发育中的CNS中的一个重要信号是由Wnt产生的，Wnt通过转录调控因子调节转录。 下游效应物（i-catenin和Tcf. Wnt/B-catenin信号在脊髓中起重要作用 图案，但在这个组织中Wnt信号传导的细胞和分子靶点是未知的， Wnt是否仅起促进细胞分裂的作用或也直接分配细胞命运。斑马鱼现在给了我们 一个理想的模型系统来解决这个问题。在这项研究中，我们将测试的假设，Tcf- 介导的转录直接调节脊髓中的祖细胞命运特化。 首先，我们将测试Tcf 7和Tcf 3是否是表达脊髓祖细胞特异性结构域所必需的。 基因.我们的初步数据表明，至少有一个中间域是错误的指定时，Tcf 3 活动丢失。我们现在将检查其他祖域标记是否也需要Tcf 3， 背侧祖细胞特别需要Tcf 7活性，以及这些分子是否独立于 细胞周期调控 第二，我们将测试Tcf 3是否通常作为一个转录激活剂，抑制剂，或两者兼而有之。我们将 检查Tcf 3是否与内源性B-连环蛋白活性重叠并为内源性B-连环蛋白活性所需， Tcf的突变形式可以复制或挽救Tcf 3功能丧失的表型，并询问Tcf 3是否 与经典Wnt信号传导协同或拮抗地起作用。这些实验将支持 一个模型，其中Tcf 3功能完全作为一个阻遏物，或其中它也激活靶基因。 第三，将测试候选靶基因作为染色质Tcf 3信号传导的直接转录靶标 免疫沉淀（ChIP）分析。我们将使用已知基因的组合，计算基因组， 分析，以及无偏见的筛选，以确定候选人。这些实验将产生Tcf 3的图像， 体内靶点，以及B-连环蛋白信号通路在脊髓祖细胞特化中的作用。 总的来说，这些研究将使我们了解基因是如何在脊髓发育过程中受到调控的。 发育，最终导致脊髓神经元的正确定位和布线。这 了解这些将有助于治疗和修复脊髓损伤和疾病。

The vertebrate central nervous system (CNS) is patterned by environmental signals that control the specification of cell fate. Understanding the mechanism of cell fate specification in the nervous system is vital for our ability to diagnose disease and design regenerative therapeutic treatments. One important signal in the developing CNS is produced by Wnts, which regulate transcription through the downstream effectors (i-catenin and Tcf. Wnt/B-catenin signaling plays an important role in spinal cord patterning, but the cellular and molecular targets of Wnt signaling in this tissue are unknown, and it is unclear whether Wnts act only to promote cell division or also directly assign cell fate. The zebrafish now gives us an ideal model system to address this problem. In this study, we will test the hypothesis that Tcf- mediated transcription directly regulates progenitor cell fate specification in the spinal cord. First, we will test whether Tcf7 and Tcf3 are required for the expression of spinal progenitor domain-specific genes. Our preliminary data suggest that at least one intermediate domain is mis-specified when Tcf3 activity is lost. We will now examine whether other progenitor domain markers also require Tcf3, whether dorsal progenitors specifically require Tcf7 activity, and whether these molecules function independently of cell-cycle control. Second, we will test whether Tcf3 normally acts as a transcriptional activator, represser, or both. We will examine whether Tcf3 overlaps with and is required for endogenous B-catenin activity, determine whether mutant forms of Tcf can phenocopy or rescue Tcf3 loss-of-function phenotypes, and ask whether Tcf3 functions synergistically or antagonistically to canonical Wnt signaling. These experiments will support either a model in which Tcf3 functions exclusively as a represser, or one in which it also activates target genes. Third, candidate target genes will be tested as direct transcriptional targets of Tcf3 signaling by chromatin immunoprecipitation (ChIP) analysis. We will use a combination of known genes, computational genomic analysis, and an unbiased screen to identify candidates. These experiments will yield a picture of Tcf3 targets in vivo, and the roles of the B-catenin signaling pathway in spinal cord progenitor specification. Overall, these studies will allow us to understand how genes are regulated during spinal cord development, ultimately resulting in the correct placement and wiring of spinal neurons. This understanding will help in the treatment and repair of spinal cord injuries and disease.