G·A·B·B·A

One fundamental question in biology lies in the understanding of the genetic and molecular bases of the extant morphological diversity in animals. Changes in the expression patterns of developmental genes are known to correlate with morphological changes between animal species. Deciphering the molecular mechanisms that govern gene expression change is therefore a crucial challenge in evolutionary developmental biology.
The regulatory changes in gene expression pattern may occur through the modification of regulatory DNA and/or the deployment or activity of the upstream trans-acting factors. In this context, the evolution of cis-regulatory DNA encoding spatiotemporal regulatory information contributes to morphological diversity in animals. It is unclear how the base pair composition of enhancer sequences, particularly binding site organization, can influence the resulting gene expression pattern. Consequently, it is unknown how regulatory DNA can evolve to generate new regulatory functions and thus novel gene expression patterns. In my thesis I tried to unravel what are the sufficient evolutionary steps to generate a novel gene expression pattern, both at the cis-regulatory DNA and transcription factor levels. Additionally, I was also interested in exploring the structure-function relation of enhancers.
To answer these basic biological questions, I studied the regulation of the yellow (y) gene, the expression pattern of which foreshadows a pigmentation spot present on the wings of some Drosophila species (the wing spot). In D. biarmipes, a species in which the wing spot is present, y expression pattern has changed through the evolution of an enhancer: the y spot element. My initial objective was to map the binding sites present in the y spot element, as well as to identify the transcription factors that activate and repress it.
Through the scanning the y spot element with point mutations and the analysis of the resulting expression pattern in transgenic flies, I identified some of the functionally relevant sites that compose it. Some levels of the organization of these sites in the y spot element is also shown to be important for its function.
In parallel, I performed a genetic screen that enabled the identification of several candidates for the activation of the spot expression pattern. Combining these two sets of data pointed to a genetic candidate for spot activator: Distal-less (Dll). The combination of in vitro gelshift assays with in vivo reporter gene experiments further showed that Dll is a direct activator of the y spot element. These experiments also enabled the identification of Dll binding sites in the y spot element. The comparative analysis of Dll expression patterns in spotted and non-spotted species showed that Dll determines the pattern of the y spot element.
Taken together, the results I obtained suggest two necessary steps for the evolution of a novel gene expression pattern and the consequent novel pigmentation morphology: i) the establishment of a regulatory link between Dll and y through the evolution of Dll binding sites in a functional enhancer; ii) alterations in Dll expression pattern with the consequent shaping of y expression pattern and of the resulting pigmentation pattern. These results have implications in the general understanding of how gene expression patterns and the consequent new morphologies evolve.