Synthesis and functionalization of coiled carbon filaments
For closely-related species, development begins at a very similar state yet the adult organisms display an array of distinguishing morphological traits. A major focus in evolutionary developmental biology is to understand what genetic steps were taken on evolutionary paths towards this array of traits. Historically, early studies into morphological diversity emphasized differences through new genes and changes to their protein-coding sequence. In the genome-era of genetics research, it has become clear that many species protein-coding sequence identities are very similar and changes in gene numbers have been somewhat modest. Thus, another type of genetic change must have contributed largely to diversity. In recent years, a plethora of case studies have been reported in which genetic alterations responsible for morphological evolution were found that modify how genes are expressed. These alterations occur in non-protein coding sequences called cis-regulatory elements (CREs), which control gene expression through their interactions with transcription factor proteins. Moreover, the transcription factor to CRE interactions connects genes into a regulatory network. At the onset of my dissertation research, little was known about the paths of CRE evolution, and how gene regulatory networks evolve to alter morphology. Moreover, tools were inadequate to study both CREs and gene regulatory networks. My dissertation research focused on gaining insights on the mechanistic underpinnings of the evolution of a CRE known as the dimorphic element (Chapter 3), which functions in an evolved gene regulatory network for patterning Drosophila (D.) melanogaster fruit flies abdomen pigmentation (Chapter 4). These studies required the establishment of a quantitative method for comparing the gene regulatory capabilities of CREs (Chapter 2). In Chapter 2, a protocol, utilized throughout my dissertation, was developed that enables the quantification of CRE activity by measuring the level of Green Fluorescent Protein (GFP) production within D. melanogaster. In Chapter 3, this method showed that evolved differences in abdomen pigmentation recurrently involved function-altering mutations in the dimorphic element for D. melanogaster populations and closely-related species. Many of these key mutations did not overlap known transcription factor binding sites. This outcome may be due to pleiotropic constraints on these conserved binding sites while other transcription factors binding sites were perhaps gained or loss. In order to find potential transcription factors for these evolved binding sites, I led a genetic screen to find pigmentation network transcription factors by RNA interference. We found twenty-four novel transcription factors controlling abdomen pigmentation (Chapter 4). These results show that the abdominal pigmentation network is quite complex and future studies are needed to connect these factors to the CREs they regulate. A remaining obstacle to understand CRE function and evolution is to understand the in vivo effects of mutations. In Chapter 5, a protocol CRISPR CREam is presented which I have been developing to remove a CRE from its endogenous gene context and replace it with a variant CRE. Collectively, my dissertation has furthered the understanding of CREs and a model gene regulatory network. With the development of new genetic tools, CRE and network biology should be poised for drastic progress.