Tcf Function in Spinal Cord Patterning

描述(申请人提供)：脊椎动物的中枢神经系统(CNS)是由控制细胞命运的环境信号形成的。了解神经系统中细胞命运指定的机制对于我们诊断疾病和设计再生治疗方法的能力至关重要。1中枢神经系统发育中的重要信号是由WNTS产生的，它通过下游的效应物β-连环素和Tcf来调节转录。WNT/B-catenin信号在脊髓构型中起重要作用，但Wnt信号在该组织中的细胞和分子靶点尚不清楚，目前尚不清楚Wnts是仅起促进细胞分裂的作用，还是直接分配细胞命运。斑马鱼现在为我们提供了一个理想的模型系统来解决这个问题。在这项研究中，我们将检验Tcf介导的转录直接调控脊髓祖细胞命运的假设。首先，我们将测试Tcf7和Tcf3是否是表达脊髓祖细胞特定结构域基因所必需的。我们的初步数据表明，当Tcf3活性丧失时，至少有一个中间结构域被错误指定。我们现在将检查是否其他祖细胞结构域标记也需要Tcf3，背部祖细胞是否特别需要Tcf7活性，以及这些分子是否独立于细胞周期控制发挥作用。其次，我们将测试TCF3是否正常地作为转录激活因子、抑制因子，或者两者兼而有之。我们将研究Tcf3是否与内源性β-连环素活性重叠，是否需要内源性β-连环素活性，确定突变形式的Tcf是否可以复制或拯救Tcf3功能丧失的表型，并询问Tcf3对经典的Wnt信号是协同还是拮抗。这些实验将支持一种模型，在该模型中，Tcf3只起到抑制因子的作用，或者1，在该模型中，Tcf3还激活目标基因。第三，通过染色质免疫沉淀(ChIP)分析，将候选靶基因作为Tcf3信号的直接转录靶基因进行测试。我们将使用已知基因、计算基因组分析和无偏见筛选的组合来确定候选基因。这些实验将产生体内Tcf3靶点的图像，以及β-连环蛋白信号通路在脊髓前体细胞规格中的作用。总体而言，这些研究将使我们能够了解基因在脊髓发育过程中是如何调控的，最终导致脊髓神经元的正确位置和连接。这种认识将有助于脊髓损伤和疾病的治疗和修复。

DESCRIPTION (provided by applicant): 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. 1 important signal in the developing CNS is produced by Wnts, which regulate transcription through the downstream effectors beta-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 1 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, repressor, or both. We will examine whether Tcf3 overlaps with and is required for endogenous beta-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 repressor, or 1 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 beta-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.