Tcf Function in Spinal Cord Patterning

描述（由申请人提供）：脊椎动物中枢神经系统（CNS）是由控制细胞命运规范的环境信号形成的。了解神经系统中细胞命运规范的机制对我们诊断疾病和设计再生治疗方法的能力至关重要。发育中的中枢神经系统中的一个重要信号是由wnt产生的，wnt通过下游效应物β -连环蛋白和Tcf调节转录。Wnt/B-catenin信号在脊髓模式中发挥重要作用，但Wnt信号在脊髓组织中的细胞和分子靶点尚不清楚，也不清楚Wnt信号是否仅促进细胞分裂或直接决定细胞命运。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，我们将验证Tcf介导的转录直接调节脊髓祖细胞命运规范的假设。首先，我们将测试Tcf7和Tcf3是否为脊髓祖结构域特异性基因的表达所必需。我们的初步数据表明，当Tcf3活性丢失时，至少有1个中间结构域被错误指定。我们现在将研究其他祖细胞结构域标记是否也需要Tcf3，背侧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而起作用。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，或两者兼而有之。我们将研究Tcf3是否与内源性β -连环蛋白活性重叠，以及是否需要Tcf3，确定Tcf3突变形式是否可以表型或挽救Tcf3功能丧失表型，并询问Tcf3是否对典型Wnt信号传导具有协同或拮抗作用。这些实验将支持一种模型，即Tcf3仅作为抑制因子发挥作用，或者它也激活靶基因。第三，通过染色质免疫沉淀（ChIP）分析测试候选靶基因作为Tcf3信号的直接转录靶点。我们将结合已知基因、计算基因组分析和无偏见筛选来确定候选基因。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祖细胞规范中的作用。总的来说，这些研究将使我们了解在脊髓发育过程中基因是如何被调节的，最终导致脊髓神经元的正确位置和连接。这一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.