Regulation of opposite Wnt target genes in C. elegans embryonic development

项目摘要 胚胎模式需要细胞间信号通路来触发分子开关，以精确地 控制细胞的命运。开发的一个基本步骤是将身体轴线图案化。动物前后部 构图需要Wnt途径，该途径通过转录因子(Tf)Tcf及其共激活因子β- 连锁素。关于这一途径的经典靶点是如何转录激活的 WNT。WNT诱导β-连环蛋白的核积聚，它与TcF络合并将其从 转录抑制因子转变为激活剂。然而，许多目标反而被WNT压制，而 人们对这种压制的机制知之甚少。WNT可诱导转录因子的表达 抑制子，导致间接靶抑制，但从小鼠，果蝇和C. Elgans已经确定了直接的“相反”靶标，由Tcf单独激活并被Tcf抑制：β-catenin in 对信号的响应。线虫胚胎是定义这些调节机制的理想系统。 与WNT目标相反。几乎所有的细胞分裂都是由Wnt构图的；在每个细胞分裂后，后姐妹 细胞具有较高的Wnt途径活性，而前姊妹细胞Wnt途径处于不活跃状态。我们的实验室有 鉴定了数十个在前或后姊妹细胞来源的谱系中特异表达的基因，并 表明后姐妹特异性基因是经典的Wnt靶标，当Wnt激活时激活，并且 当WNT处于非活动状态时被抑制。我的初步数据显示，许多基因在前姐妹细胞中表达 是由Wnt途径调节的，我建议检验这些基因是直接相反的假设 WNT目标。我将首先测试POP-1/Tcf、Sys-1/β-catenin和Wnt配体和受体是否是必需的 正确激活或抑制五个前特异性基因中的每一个，并确定控制 他们的表情。我将在整个基因组中识别与POP-1结合的位点，并测试是否有 对于正确表达相同的五个相反的Wnt靶标以及整个基因组来说都是必要的。对于目标 在没有POP-1结合的情况下，暗示由Wnt调节的抑制子进行间接调控，我将结合详细的顺-1- 利用我们的谱系特异性转铁蛋白和经典Wnt靶向表达目录对其增强子的调控分析 寻找Wnt通过转录抑制因子间接调控的前体基因。这项工作将考验 在线虫中，TCF的直接调控是否是调节相反Wnt靶标的常见机制， 将开始确定这一规定的机制，并将为这一制度和其他制度的未来工作提供信息。

Project Summary Embryonic patterning requires intercellular signaling pathways that trigger molecular switches to precisely control cell fates. One fundamental step in development is patterning the body axes. Animal anterior-posterior patterning requires the Wnt pathway, which acts through the transcription factor (TF) TCF and its coactivator β- catenin. Much is known about how classic targets of this pathway are transcriptionally activated in response to Wnt. Wnt induces nuclear accumulation of β-catenin, which complexes with TCF and converts it from a transcriptional repressor to an activator. Many targets, however, are instead repressed by Wnt, and the mechanisms for this repression are less well understood. Wnt can induce expression of transcriptional repressors, leading to indirect target repression, but emerging evidence from mouse, Drosophila, and C. elegans has identified direct “opposite” targets, activated by TCF alone and repressed by TCF:β-catenin in response to signaling. The C. elegans embryo is an ideal system to define regulatory mechanisms of these opposite Wnt targets. Almost all cell divisions are patterned by Wnt; after each cell division the posterior sister cell has high Wnt pathway activity, and the anterior sister cell has the Wnt pathway inactive. Our laboratory has identified dozens of genes expressed specifically in either anterior or posterior sister cell-derived lineages and showed that the posterior sister-specific genes are classic Wnt targets, activated when Wnt is active and repressed when Wnt is inactive. My preliminary data indicate that many genes expressed in anterior sister cells are regulated by the Wnt pathway and I propose to test the hypothesis that these genes are direct opposite Wnt targets. I will first test if POP-1/TCF, SYS-1/ β-catenin, and Wnt ligands and receptors are necessary for proper activation or repression of each of five anterior-specific genes and identify the enhancers controlling their expression. I will identify sites bound by POP-1 throughout the genome, and test whether any are necessary for proper expression of the same five opposite Wnt targets as well as genome-wide. For targets without POP-1 binding, suggesting indirect regulation by Wnt-regulated repressors, I will combine detailed cis- regulatory analysis of their enhancers with our catalog of lineage-specific TF and classic Wnt target expression to identify anterior genes indirectly regulated by Wnt through transcriptional repressors. This work will test whether direct regulation by TCF is a common mechanism for regulation of opposite Wnt targets in C. elegans, will begin to identify mechanisms for this regulation, and will inform future work in this and other systems.