Unraveling Transcriptional Dynamics in Early Drosophila Embryos

Abstract

Signaling pathways are crucial to development in living organisms. They control the expression of genes that affect downstream processes including cell differentiation and proliferation. Disruption of these pathways can lead to abnormal development, disease, and sometimes death. Understanding how signals lead to changes in gene expression will inform us about the underlying mechanisms involved in proper development. The Drosophila melanogaster embryo is an ideal system for studying how signaling pathways establish gene regulatory networks due to their very distinct spatiotemporal patterns of gene expression. In this thesis, we studied the interactions of 5 genes responsive to the ERK signaling pathway that are involved in differentiation of the Drosophila gut. Through fluorescence in situ hybridization, we established a genetic map of these genes, i.e. where the patterns formed and the overlap or lack thereof between the genes. To obtain a deeper understanding of the dynamics of how the genes interact with each other and how the patterns develop, we utilized the MS2:MCP system to track gene transcription throughout development. A pipeline was created for processing these MS2 movies so that quantitative results could be deduced from this data in the future. Once the patterning in wild type embryos was established, we looked at how it changes when a perturbation to ERK signaling is introduced. The experiments above were repeated in embryos with reduced functional Capicua, a transcriptional repressor downstream of ERK that represses most of the genes of interest. This disruption resulted in expanded expression of all 5 genes and showed different areas of overlap than were seen in the wild type embryos. With the understanding we gained through this process, we plan to continue deciphering the signaling interactions that determine cell fate.