Integrated computational and experimental study of embryonic patterning by sequential and oscillatory gene regulatory mechanisms

从信号中心发出的基因表达的顺序波和循环波是胚胎发生(如脊椎动物的体细胞发育、神经管发育和肢体发育)中常见的现象。最近，我描述了在甲虫Tribolium Castaneum的后组织者区域中，GAP基因表达的成对规则的循环波和非周期波。在拟议的项目中，我使用Tribolium前后轴模式中的GAP和配对规则基因调控作为一个简单易处理的模型系统来研究发育过程中基因表达波的分子基础。我采取一种假设驱动的方法，测试我最近设计的一个分子/计算模型的预测，该模型是我最近为调节这种波而设计的：“渐进式增强子开关模型”。在这个模型中，两组增强子参与基因调控：动态增强子和静态增强子。动态增强子负责振荡/顺序的基因表达，静态增强子负责将这些波冻结成稳定的空间模式。计算机模型显示，在这两组增强剂之间的这种渐进切换(由形态因子梯度介导)可以产生基因表达波。该模型得到了遗传数据的支持，但分子证据仍然缺乏。在拟议的项目中，我设计了活胚胎中的报告分析方法，以及测试和修改我的模型的计算方法。将使用WT中的MS2-MCP系统和各种RNAi敲除背景，在活胚胎中进行增强子活性的详细分析。实验数据将被分析并集成到使用电子进化技术的计算模型中。拟议的项目扩展了目前理解基因调控网络在发育过程中如何工作的方法。基因之间的相互作用通常被视为简单的激活/抑制关系，并且通常是通过计算来模拟的。然而，最近的研究表明，单个基因往往受到多个增强子的调控，形成复杂而动态的3D染色质结构，并驱动不同的表达，这些表达通常在空间和时间上重叠。这种复杂的转录机制可能被用来增加基因调控网络的计算能力。我的长期目标是解开发展中的基因调控网络中顺式调控机制的计算能力。在提议的项目中，我从第一层复杂性开始，即基因的多增强子调控。在未来，我的目标是探索顺式调控区的3D染色质结构在调节发育过程中模式形成的重要性。

Sequential and cyclic waves of gene expression emanating from signaling centers are commonly observed phenomena in embryogenesis (e.g. somitogenesis, neural tube development, and limb development in vertebrates). Recently, I have described cyclic waves of pair-rule, and aperiodic waves of gap gene expression in the posterior organizer region of the beetle Tribolium castaneum. In the proposed project, I use gap and pair-rule gene regulation during the anterior-posterior axis patterning in Tribolium as a simple and tractable model system to study the molecular underpinnings of gene expression waves in development. I take a hypothesis-driven approach, where I test the predictions of a molecular/computational model I recently devised for the regulation of such waves: the "gradual enhancer switching model". In this model, two sets of enhancers are involved in gene regulation: dynamic enhancers and static enhancers. The dynamic enhancers are responsible for the oscillatory/sequential gene expression, and the static enhancer for freezing these waves into stable spatial patterns. Computer models show that such gradual switching (mediated by a morphogen gradient) between these two sets of enhancers can generate gene expression waves. The model is supported by genetic data, but a molecular evidence is still lacking.In the proposed project, I devise reporter assay approaches in live embryos as well as computational approaches to test and modify my models. Detailed analyses of enhancer activities will be carried out in live embryos using the MS2-MCP system in WT and various RNAi knockdown backgrounds. Experimental data will be analyzed and integrated into computational models using in silico evolution techniques.The proposed project expands the current approach of understanding how gene regulatory networks work during development. Interactions between genes are usually viewed as simple activation/repression relationships, and are usually computationally modeled as such. However, recent studies show that a single gene is often regulated by multiple enhancers, forming a complex and dynamic 3D chromatin structures, and driving different expressions that usually overlap in space and time. This complex transcriptional machinery is probably utilized to increase the computational power of gene regulatory networks. My long-term goal is to unravel the computational power of the cis-regulatory machinery within gene regulatory networks in development. In the proposed project, I start with the first tier of complexity, namely the multi-enhancer regulation of genes. In the future, I aim to probe the importance of the 3D chromatin structure of cis-regulatory regions to mediate pattern formation in development.