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）由控制细胞命运规范的环境信号形成模式。了解神经系统中细胞命运特化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。在发育中的CNS中的1个重要信号由Wnt产生，Wnt通过下游效应子β-连环蛋白和Tcf调节转录。Wnt/B-连环蛋白信号在脊髓模式中起着重要作用，但Wnt信号在该组织中的细胞和分子靶点尚不清楚，并且不清楚Wnt是否仅起促进细胞分裂的作用或也直接分配细胞命运。斑马鱼现在为我们提供了解决这个问题的理想模型系统。在这项研究中，我们将测试的假设，Tcf介导的转录直接调节祖细胞的命运规格在脊髓。首先，我们将测试Tcf 7和Tcf 3是否需要脊髓祖细胞结构域特异性基因的表达。我们的初步数据表明，至少有一个中间域是错误指定时，Tcf 3活性丢失。我们现在将检查是否其他祖域标记也需要Tcf 3，背祖细胞是否特别需要Tcf 7活性，以及这些分子是否独立于细胞周期控制发挥作用。第二，我们将测试Tcf 3是否通常作为转录激活因子，抑制因子，或两者兼而有之。我们将研究Tcf 3是否与内源性β-连环蛋白活性重叠，并确定Tcf的突变形式是否可以表型复制或拯救Tcf 3功能丧失表型，并询问Tcf 3是否协同或拮抗经典Wnt信号传导。这些实验将支持Tcf 3仅作为阻遏物发挥作用的模型，或Tcf 3也激活靶基因的模型。第三，通过染色质免疫沉淀（ChIP）分析，将候选靶基因作为Tcf 3信号传导的直接转录靶点进行测试。我们将使用已知基因，计算基因组分析和无偏筛选的组合来识别候选人。这些实验将产生Tcf 3在体内的靶点，和β-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在脊髓发育过程中是如何调节的，最终导致脊髓神经元的正确定位和布线。这种理解将有助于治疗和修复脊髓损伤和疾病。

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.