Enhancer Function and Evolution in Drosophila Embryos

Abstract

As development proceeds from fertilization of a single egg cell, regulatory sequences called enhancers generate specific gene expression patterns in space and time. These patterns give rise to the many different cell types present in an adult organism. Changes in enhancer sequence underlie morphological differences between species as well as disease states in humans. It is critical, therefore, to determine how sequence variation within enhancers affect the overall expression pattern of their target gene. The enhancers that control expression of the Drosophila gene even-skipped are an excellent system for investigating regulatory logic and evolution. Decades of genetic experiments have characterized many of the transcription factors that regulate these enhancers, and quantitative gene expression measurements in Drosophila embryos have enabled computational models of enhancer function. Despite these advantages, the failure of genetic reconstitution experiments suggest that we do not currently understand these enhancers well enough to build them. This shortcoming makes it difficult to identify the sequence features that are critical for enhancer function, or to reason about how enhancer sequence may evolve. In this work, I challenged computational models of even-skipped enhancer function using genetic manipulations and quantitative measurements in Drosophila melanogaster embryos. I found that one expression pattern is generated by two different enhancers that respond differently to genetic perturbation of a particular transcription factor, hunchback. This work suggests that the same expression pattern can be built in different ways, and this feature may be important for the robustness of developmental expression patterns. I also found that the transcription factor Caudal acts as a Hunchback counter-repressor in the even-skipped stripe 2 enhancer, and that this interaction appears conserved in orthologous sequences. Finally, I present computational and experimental evidence that individual enhancers may diverge while maintaining the expression pattern of the whole locus. As a whole, my work illustrates the astonishing plasticity of regulatory information, which is consistent with the rapid pace of regulatory evolution.