With the advent of whole genome sequences, biologists are confronted with the problem of understanding the non protein-coding portion of the genome. This remains a difficult task due to the many enigmatic features of cis- controlling sequences. In this thesis, I will present the genomic tools, methodologies, and observations I have made in the course of studying the peripheral nervous system of Drosophila melanogaster, a model system well suited for the study of transcriptional regulatory networks. In the first chapter, I will describe a software tool that I created to facilitate the use of genome sequences in wet- lab experiments. This tool, GenePalette, is particularly well suited for inspecting genomic regions that have been implicated by whole-genome analysis (searches for transcription factor binding sites, core promoter sequences, microRNA target sites, etc), and it was used extensively in the analyses presented in each subsequent chapter. In the second chapter, I describe a technique I developed for searching a genome for biologically relevant transcription factor binding site clusters. Using this methodology, we came to the surprising realization that many known Notch targets have statistically significant clusters of binding sites for Suppressor of Hairless [Su(H)], the transcription factor responsible for transducing the Notch signal. In chapter three, I validate a novel cluster of Su(H) binding sites, identified by my in silico study, residing within the numb gene. The investigation of this enhancer not only validates my bioinformatic approach, but also serves to illuminate several as of yet unappreciated aspects of bristle development. In chapter four, I take a different approach to finding new cis-regulatory sequences. By composing a hypothetical dual input code for neural precursors, we identify a cluster of binding sites that implements this code through a candidate gene approach. Finally, in chapter five, I present the discovery of an ancient transcription factor binding site, conserved for >700 million years. This finding establishes that although cis- regulatory change is a major engine for evolution, some transcriptional linkages can withstand the constant erosion of sequence turnover for extremely long periods

Comparative sequence analysis methods, such as phylogenetic footprinting, represent one of the most effective ways to decode regulatory sequence functions based upon DNA sequence information alone. The laborious task of assembling orthologous sequences to perform these comparisons is a hurdle to these analyses, which is further aggravated by the relative paucity of tools for visualization of sequence comparisons in large genic regions. Here, we describe a second-generation implementation of the GenePalette DNA sequence analysis software to facilitate comparative studies of gene function and regulation. We have developed an automated module called OrthologGrabber (OG) that performs BLAT searches against the UC Santa Cruz genome database to identify and retrieve segments homologous to a region of interest. Upon acquisition, sequences are compared to identify high-confidence anchor-points, which are graphically displayed. The visualization of anchor-points alongside other DNA features, such as transcription factor binding sites, allows users to precisely examine whether a binding site of interest is conserved, even if the surrounding region exhibits poor sequence identity. This approach also aids in identifying orthologous segments of regulatory DNA, facilitating studies of regulatory sequence evolution. As with previous versions of the software, GenePalette 2.1 takes the form of a platform-independent, single-windowed interface that is simple to use.

The molecular and genetic basis for the evolution of anatomical diversity is a major question that has inspired evolutionary and developmental biologists for decades. Because morphology takes form during development, a true comprehension of how anatomical structures evolve requires an understanding of the evolutionary events that alter developmental genetic programs. Vast gene regulatory networks (GRNs) that connect transcription factors to their target regulatory sequences control gene expression in time and space and therefore determine the tissue-specific genetic programs that shape morphological structures. In recent years, many new examples have greatly advanced our understanding of the genetic alterations that modify GRNs to generate newly evolved morphologies. Here, we review several aspects of GRN evolution, including their deep preservation, their mechanisms of alteration, and how they originate to generate novel developmental programs.

The vinegar fly Drosophila melanogaster is a pivotal model for invertebrate development, genetics, physiology, neuroscience, and disease. The whole family Drosophilidae, which contains over 4,400 species, offers a plethora of cases for comparative and evolutionary studies. Despite a long history of phylogenetic inference, many relationships remain unresolved among the genera, subgenera, and species groups in the Drosophilidae. To clarify these relationships, we first developed a set of new genomic markers and assembled a multilocus data set of 17 genes from 704 species of Drosophilidae. We then inferred a species tree with highly supported groups for this family. Additionally, we were able to determine the phylogenetic position of some previously unplaced species. These results establish a new framework for investigating the evolution of traits in fruit flies, as well as valuable resources for systematics.