The molecular evolution of the Drosophila neurogenic ectodermal enhancers

The control of gene expression is central to developmental biology, pattern formation, and phenotypic evolution. Enhancers are a class of regulatory DNAs that control when and where genes are turned ON or OFF. This highly regulated process is controlled by transcription factors (TFs), a class of proteins that bind to specific DNA sequences in a concentration-dependent manner. Despite the centrality of enhancers to development, little is currently understood concerning enhancer structure and function. The Neurogenic Ectodermal Enhancers (NEEs) are a collection of enhancers found in D. melanogaster, that are co-regulated in response to the morphogenic TF Dorsal. The NEEs share similar organizations of specialized binding sites for several TFs including, Dorsal, Twist, Snail, and Su(H). This thesis explores the molecular evolution of the NEEs in order to understand how regulatory information is encoded in DNA. Using the D. melanogaster transgenic system, we found that the NEEs from D. melanogaster, D. pseudoobscura, and D. virilis, have evolved in parallel, in response to lineage-specific selective pressures. We found that these adaptations are encoded in the length of DNA separating the specialized Dorsal and Twist binding sites. Altering the length between these elements fine-tunes the Dorsal-threshold transcriptional response. Extending these studies to the D. willistoni and D. ananassae genomes, we mapped the full functional range of the Dorsal-to-Twist threshold-encoding spacer variable. Additionally, we found that the majority of TF-motifs found in the NEEs appear to be non-functional, divergent elements. We propose that the process of continuously evolving new lineage-specific threshold encodings, results in the production of non-functional sites, which we have termed necro-elements. Finally, we found that the Drosophila vnd NEE sequences are repressed by the Decapentaplegic pathway via a conserved Schnurri/Mad/Medea silencer element, demonstrating how two complementary morphogen gradients can be integrated by an enhancer. In conclusion, this thesis demonstrates that evolution acts efficiently on the precise arrangement of specialized TF-binding sites to modify patterns of gene expression, this has important implications for genome-wide enhancer detection and for understanding the genetic basis of morphological evolution.