Mechanisms governing context-dependent Wnt activity in C. elegans embryogenesis

描述（由申请人提供）：少数细胞外信号通路通过改变靶细胞的转录来控制发育和稳态过程中的多种过程。通过信号通路调控基因的一个重要问题是相同的信号如何激活不同细胞中的不同靶标。Wnt信号通路及其在发育中的作用在所有后生动物中都是保守的，该通路的破坏会导致人类的发育缺陷和疾病，包括癌症。我建议通过分析Wnt靶基因的增强子和研究合成增强子的表达，揭示在不同胚胎环境下决定哪些基因受Wnt通路调节的基本规则。我将利用在酵母中开创的合成增强子策略，系统地研究秀丽隐杆线虫胚胎发育过程中Wnt通路靶点的增强子功能。为了做到这一点，我将使用一个强大的自动化谱系追踪系统，在一个活的发育胚胎中定量测量动态表达模式；因为秀丽隐杆线虫有一个不变的谱系，我可以在细胞水平上直接比较报告者之间的表达，并确定细胞命运，而不需要单独的标记。在前期工作中，我确定了16个作为Wnt通路靶点的转录因子，发现与之前的想法相反，一些靶点只需要Wnt效应转录因子TCF来抑制非信号细胞中的表达，而另一些靶点只需要TCF来激活信号细胞中的表达。此外，我发现对Wnt信号的反应在整个胚胎中并不均匀；相反，接收到Wnt信号的亲本细胞对Wnt信号的反应强于未接收到Wnt信号的亲本细胞。这表明细胞具有Wnt信号的传递记忆，这是一个新的发现。在本研究的指导阶段，我将描述作为Wnt信号直接靶点的增强子，并确定TCF和上下文转录因子如何促进Wnt靶基因的调节。在第一个目标中，我

DESCRIPTION (provided by applicant): A small number of extracellular signaling pathways control a diverse array of processes during development and homeostasis by altering transcription in target cells. An important question in the regulation of genes by signaling pathways is how the same signal can activate different targets in different cells. The Wnt signaling pathway and its role in development are conserved among all metazoans and disruption of the pathway causes developmental defects and disease in humans, including cancer. I propose to uncover the fundamental rules that determine which genes are regulated by the Wnt pathway in different embryonic contexts by analyzing the enhancers of Wnt target genes and investigating the expression of synthetic enhancers. I will leverage synthetic enhancer strategies pioneered in yeast to systematically study enhancer function for the targets of the Wnt pathway over the course of developmental time in C. elegans embryos. To do this, I will use a powerful automated lineage tracing system that measures dynamic expression patterns quantitatively in a live developing embryo; because C. elegans has an invariant lineage, I can directly compare expression between reporters on a cellular level and identify cell fates without the need for separate markers. In preliminary work, I identified sixteen transcription factors that are targets of the Wnt pathway and found that contrary to previous thinking, some targets required the Wnt effector transcription factor, TCF, only to repress expression in unsignaled cells, while others required TCF only to activate expression in signaled cells. Furthermore, I found that the response to Wnt signaling is not uniform throughout the embryo; instead, cells with parents who received a Wnt signal exhibit a stronger response to a Wnt signal than cells with unsignaled parents. This indicates the cells possess transmitotic memory of Wnt signaling, a novel finding. In the mentored phase of this study, I will characterize enhancers that are direct targets of Wnt signaling and determine how TCF and context transcription factors contribute to the regulation of Wnt target genes. In the first aim, I will identify sites bound by TCF during embryogenesis and evaluate the ability of TCF binding sites to drive expression in synthetic enhancers. In the second aim, I will characterize the enhancers of Wnt target genes, identify the context factors that co-regulate them, and use synthetic enhancers to determine how context factors sites combine with TCF binding sites to regulate expression. The data I gather and techniques I learn in the mentored phase will help me be successful in undertaking the third aim in the independent phase. For this third aim, I will use synthetic enhancers to identify the properties that are essential for Wnt targets, including the numbers, strength, orientation, and spacing of TCF and context factor binding sites. I will then predict and test the effect of targeted mutations on endogenous target enhancers and generate synthetic enhancers with novel expression patterns to demonstrate understanding of the underlying rules. This research will significantly advance our ability to identify Wnt target genes and predict their expression by sequence alone. Due to evolutionary conservation, some of the rules that govern Wnt regulation in C. elegans will likely be universal principles that also govern regulation of this and other important pathways in vertebrates. During the initial phase of this award, I will receive mentoring from my primary mentor, Dr. John Murray, my co-mentor, Dr. Klaus Kaestner, and two additional faculty members with relevant expertise. This mentorship committee will assist me with learning new techniques, including genome engineering, ChIP-seq, and bioinformatic enhancer identification that will be essential in my future career as an independent investigator in developmental genetics. My training will also include coursework, grant writing, and a mentored faculty position search to allow me to transition to an independent career. The proposed training will take place at the Perelman School of Medicine at the University of Pennsylvania, which possesses outstanding facilities, an excellent collaborative environment with many opportunities for postdoctoral fellows to present their research, and an office of Biomedical Postdoctoral Programs that organizes training in career development and responsible conduct in research.