Abstract

Novel morphological traits originate largely from the novel expression patterns of genes controlled by enhancers during development. Enhancers bind and integrate the spatial-temporal activity of transcription factors, and their combinatorial interplay determines the time, location and levels of transcriptional output. New enhancers can arise through enhancer co-option by reusing some of the regulatory information from a preexisting enhancer. While enhancer co-option is thought to be a fast and likely way to evolve new enhancers, its genetic and molecular mechanisms still remain elusive. In this context, this thesis investigates the genetic origin of a novel enhancer and the regulatory logic underlying its function. I used the spot enhancer of the gene yellow as a model, which underlies the evolution of a morphological trait, the wing spot in Drosophila biarmipes. I sought to understand how the novel spot enhancer has evolved and what regulatory logic governs its function. Specifically: In the first chapter, I examined the evolutionary mechanism of spot enhancer in the context of the preexisting wing blade enhancer. By revisiting the entire D. biarmipes yellow 5´ region with a comprehensive and quantitative method, I mapped the full activities of the novel spot and preexisting wing blade enhancers to a much larger region (3.5 kb) than previously described (1.1 kb together). Within the region, the regulatory information necessary and sufficient for the spot activity was inseparable from, and extensively overlapping with the wing blade activity. Further dissection of the shared core region revealed a pleiotropic binding site that contributed to both activities by regulating the local chromatin accessibility. I therefore confirmed that the novel spot activity originates from the co-option of the preexisting wing blade activity. The pleiotropic site for chromatin accessibility suggests a possible model where a new enhancer could evolve by co-option of chromatin accessibility input from the ancestral element, and that might facilitate the emergence and diversification of morphological traits. In the second chapter, I investigated how the various aspects of regulatory information encoded in the spot enhancer sequences influenced its activity. Through introducing systematic mutations along the enhancer sequences and implementing a quantitative framework, the spatial activities on the wing of all the mutant enhancers were measured. The analysis showed an unexpected density of regulatory information within the spot enhancer. Moreover, it reveals an unanticipated regulatory logic underlying the activity of this enhancer and how it reads the wing trans-regulatory landscape to encode a spatial pattern. The gene yellow is required for black pigment production and its expression in late pupal stage prefigures the adult wing spot pigmentation pattern. Therefore, understanding the dynamics of yellow expression is essential to elucidate the process of yellow enhancer regulation as well as pigment formation during development. Chapter three investigates the process of pigment formation in space and time using the pigment gene yellow in D. melanogaster. Firstly, a fluorescent protein-tagged yellow allele was generated, then the dynamics of yellow expression and cellular localization in relationship to the process of pigment formation was examined during development. It was found that yellow is expressed in a few neurons in the brain and the ventral nerve chord from the second larval instar to adult stage, indicating a neuro-developmental function of yellow. In addition, the results mainly showed how yellow expression in the adult cuticle is determined by regulated developmental processes affecting the body color, and suggested a structural role of Yellow in the establishment of pigmentation patterns.