Decoding lineage and fate specification in the C. elegans embryo

摘要 发育调控转录因子的组合调控在细胞内起着重要作用， 确定在发育和疾病过程中每个细胞中哪些基因受到调控，并允许相同的因子 在不同的细胞中扮演不同的角色。梭线虫胚胎是一个理想的系统， 谱系历史在细胞命运的背景依赖性调节中的作用，因为它的谱系是不变的， 强大的实验工具。我们最近开发了自动化谱系追踪和表达映射 方法C. elegans胚胎发生，并建立了“小杆线虫表达模式”（EPIC） 包含超过250种用于转录因子（TF）表达的荧光报告基因表达的数据库 在发育中的胚胎的每个细胞中。在我们过去的工作中，我们已经展示了这种资源作为一种 确定控制发展的机制的起点。虽然我们将继续映射表达式 新的监管机构，这项建议的主要重点是使用这个数据库和我们的方法，以解决不良 理解发育生物学中的问题。1)细胞如何知道它们的谱系历史并翻译 转化成正确的终末细胞命运我们已经确定了一组约15个谱系特异性TF， 表达区分了一组来自“ABpxp”的不同组织的祖细胞。 卵裂球我们计划测试这些TF单独和组合是否是必要和足够的， 通过成像和基因组学，确定这些祖细胞的谱系身份，并确定其靶点 功能丧失和获得突变体的分析。我们将通过对顺式的详细分析来补充这一点- 控制终末分化基因的调控序列，以鉴定其上游调控子。这些 “自上而下”和“自下而上”的办法最终应汇聚成一个共同的监管网络。2)什么 这种机制允许在不同的开发环境中重复使用相同的调节剂用于不同的目的。 不同的背景，比如不同的血统？我们已经确定了两组基因，表达在后或 细胞分裂后的前子代细胞，两者都受Wnt通路调节。我们将联合收割机 增强子精细图谱、合成增强子的表达图谱以及全基因组结合测定 Wnt调节的TF POP-1/TCF，以确定每个基因对此的反应（激活或抑制） 在不同的细胞中，这条通路的调节方式不同。3)冗余的基因和增强子 影响发展的稳健性？红细胞在早期胚胎中非常常见，我们将 检验这种冗余存在的假设，以确保在面临环境问题时的稳健发展。 通过在不同条件下对突变体进行详细的表型分析，总之，我们建议的工作将 开始完成早期胚胎的调控网络，并回答有关的原则， 可能在动物中保存的发展。

ABSTRACT Combinatorial regulation by developmentally regulated transcription factors play a central role in defining which genes are regulated in each cell during development and disease, and allows the same factors to play different roles in different cells. The C. elegans embryo is an ideal system for a comprehensive study of the role of lineage history in the context-dependent regulation of cell fate because of its invariant lineage and powerful experimental tools. We recently developed automated lineage tracing and expression mapping methods for C. elegans embryogenesis and have built the “Expression Patterns in Caenorhabditis” (EPIC) database that contains the expression of over 250 fluorescent reporters for transcription factor (TF) expression in every cell of developing embryos. In our past work, we have shown the potential for this resource as a starting point for defining mechanisms controlling development. While we will continue to map the expression of novel regulators, the main focus of this proposal is to use this database and our methods to address poorly understood questions in developmental biology. 1) How do cells know their lineage history and translate this information into correct terminal cell fates? We have identified a set of ~15 lineage-specific TFs whose expression distinguishes a group of progenitor cells for diverse tissues descended from the “ABpxp” blastomeres. We plan to test whether these TFs are necessary and sufficient individually and in combination to specify the lineage identity of these progenitor cells, and to identify their targets, by imaging and genomics analysis of loss and gain-of-function mutants. We will complement this by a detailed analysis of the cis- regulatory sequences controlling terminal differentiation genes to identify their upstream regulators. These “top-down” and “bottom-up” approaches should eventually converge to a common regulatory network. 2) What mechanisms allow reuse of the same regulator(s) for different purposes in different developmental contexts, such as different lineages? We have identified two sets of genes, expressed in either posterior or anterior daughter cells after cell division, both of which are regulated by the Wnt pathway. We will combine enhancer fine-mapping, expression mapping of synthetic enhancers, and genome-wide binding assays for the Wnt-regulated TF POP-1/TCF to determine how each gene's response (activation or repression) to this pathway is regulated differently in different cells. 3) How does redundancy of genes and enhancers influence developmental robustness? Redundancy is extremely common in the early embryo, and we will test the hypothesis that this redundancy exists to ensure robust development in the face of environmental variability by detailed phenotyping of mutants in different conditions. In summary, the work we propose will begin to complete the early embryonic regulatory network and answer important questions about principles of development that are likely to be conserved across animals.