Gene expression is critical for animal development as cells divide and differentiate into multiple cell types. Cell-type specific gene expression is controlled by enhancers, DNA sequences that can direct expression of a target gene from hundreds of kilobases away. Gene promoters contact at least two enhancers on average, and enhancers may also contact each other. A key question is therefore how enhancers operate in this complex regulatory DNA context. It has long been assumed that enhancers act as independent modules based on their ability to drive gene expression when isolated in reporter constructs. To test assumptions of enhancer modularity, I probed interactions between two developmental enhancers from the even-skipped locus in Drosophila melanogaster blastoderm embryos. My results contradict the classic definition of enhancers; I found that the arrangement of enhancers relative to one another and the promoter influences levels of gene expression while not affecting its spatial pattern within the embryo. These results are described in Chapter 2. However, these enhancers are modular in one aspect: when fused directly together, they still direct their distinct spatial expression patterns. In Chapter 3 I describe a collaboration with Md Abul Hassan Samee in Saurabh Sinha's group at the University of Illinois Urbana-Champaign to apply computational sequence-to-expression models to my data. We found that a mechanistic model describing interactions between transcription factors was unable to fit our data well; in contrast, a phenomenological model that finds active sequences fits the data much better. These results indicate that to predict gene expression from sequence we will need to learn how enhancer boundaries are defined. In summary, I present evidence that the organization of enhancers within a locus impacts expression of the target gene. This finding overturns assumptions about enhancer modularity and emphasizes the importance of considering higher level interactions across a locus. Structural variation is common in natural populations, and our results highlight a novel way in which these sequence variants may alter gene expression. To realize the long-standing goal to predict gene expression directly from sequence we must investigate how enhancers interact within a complex locus.

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Embryonic epiboly has become an important developmental model for studying the mechanisms underlying collective movements of epithelial cells. In the last couple of decades, most studies of epiboly have utilized Xenopus or zebrafish as genetically tractable model organisms, while the avian epiboly model has received virtually no attention. Here, we re-visit epiboly in quail embryos and characterize several molecular markers of epithelial-to-mesenchymal transition (EMT) in the inner zone of the extraembryonic Area Opaca and at the blastoderm edge. Our results show that the intermediate filament vimentin, a widely-used marker of the mesenchymal phenotype, is strongly expressed in the edge cells compared to the cells in the inner zone, and that epiboly is inhibited when embryos are treated with Withaferin-A, a vimentin-targeting drug. Laminin, an extracellular matrix protein that is a major structural and adhesive component of the epiblast basement membrane, is notably absent from the blastoderm edge, and shows three distinct morphological regions approaching the leading edge. While these expression profiles are consistent with a mesenchymal phenotype, several other epithelial markers, including cytokeratin, β-catenin, and E-cadherin, were present in the blastoderm edge cells. Moreover, the results of a BrDU proliferation assay suggest that expansion of the edge cell population is primarily due to recruitment of cells from the inner zone, and not proliferation. Taken together, our data suggest that the edge cells of the avian blastoderm have characteristics of both epithelial and mesenchymal cells, and could serve as an in-vivo model for cancer and wound healing studies.

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