Dissecting of the Tbx20 Regulatory Network

对协调心脏发育的遗传通路层级的认识仍然不完整，部分原因是很难识别心脏形成过程中基因区域化表达所需的关键顺式作用序列。在这里，我们提出了一个整合的系统来识别TBX20调控网络的关键组件，TBX20基因在心脏发育中发挥重要作用。为了实现这一点，我们建议利用体内斑马鱼和小鼠报告试验相结合的方法来鉴定TBX20基因座上的心脏增强剂。我们将对TBX20基因座中的增强子进行饱和扫描，系统地测试序列，而不考虑它们的进化保守模式，目的是识别在心脏发生过程中协调TBX20表达的关键增强子。我们将对这些心脏增强剂在胚胎发育过程中的空间和时间域进行详细的表征，从而使人们对TBX20调控网络的组成部分有一个广泛的了解。我们将测试每个增强子对TBX20正确表达的必要性。使用工程细菌人工染色体(BAC)，我们将删除在心脏多个区域驱动表达的每个增强子。这一实验设计将使我们能够推断调控网络的单个组件对于适当的时间、空间和数量表达的必要性。 综上所述，我们将使用斑马鱼和小鼠体内试验来表征在心脏发生过程中协调TBX20表达的顺式调控网络，并评估该网络的各个组件如何协调动态而复杂的TBX20表达模式。

The recognition of the hierarchies of genetic pathways coordinating heart development remains incomplete, in part because of the difficulties to identify the critical cis-acting sequences that are required for the regionalized expression of genes during cardiogenesis. Here we propose an integrative system to identify the critical components of the regulatory network of TBX20, a gene that plays important roles in heart development. To achieve this, we propose to utilize a combination of an in vivo zebrafish and mouse reporter assay to identify heart enhancers in the TBX20 locus. We will carry out a saturation scan for enhancers in the TBX20 locus, systematically testing sequences regardless of their pattern of evolutionary conservation, with the goal of identifying the critical enhancers that coordinate TBX20 expression during cardiogenesis. We will carry out a detailed characterization of the spatial and temporal domains of these heart enhancers during embryogenesis, leading to a broad understanding of the components of the TBX20 regulatory network. We will test the necessity of each of these enhancers for the proper expression of TBX20. Using engineered Bacterial Artificial Chromosomes (BACs), we will delete each enhancer that drives expression in multiple domains of the heart. This experimental design will allow us to infer the necessity of individual components of the regulatory network for the proper temporal, spatial and quantitative expression of TBX20. In summary, we will use zebrafish and mouse in vivo assays to characterize the cis-regulatory network that coordinates TBX20 expression during cardiogenesis, and evaluate how the individual components of the network orchestrate the dynamic and complex pattern of TBX20 expression.