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

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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.