Genetic Changes to a Transcriptional Silencer Element Confers Phenotypic Diversity within and between Drosophila Species

The modification of transcriptional regulation has become increasingly appreciated as a major contributor to morphological evolution. However, the role of negative-acting control elements (e.g. silencers) in generating morphological diversity has been generally overlooked relative to positive-acting “enhancer” elements. The highly variable body coloration patterns among Drosophilid insects represents a powerful model system in which the molecular alterations that underlie phenotypic diversity can be defined. In a survey of pigment phenotypes among geographically disparate Japanese populations of Drosophila auraria, we discovered a remarkable degree of variation in male-specific abdominal coloration. In testing the expression patterns of the major pigment-producing enzymes, we found that phenotypes uniquely correlated with differences in the expression of ebony, a gene required for yellow-colored cuticle. Assays of ebony’s transcriptional control region indicated that a lightly pigmented strain harbored cis-regulatory mutations that caused correlated changes in its expression. Through a series of chimeric reporter constructs between light and dark strain alleles, we localized function-altering mutations to a conserved silencer that mediates a male-specific pattern of ebony repression. This suggests that the light allele was derived through the loss of this silencer’s activity. Furthermore, examination of the ebony gene of D. serrata, a close relative of D. auraria which secondarily lost male-specific pigmentation revealed the parallel loss of this silencer element. These results demonstrate how loss-of-function mutations in a silencer element resulted in increased gene expression. We propose that the mutational inactivation of silencer elements may represent a favored path to evolve gene expression, impacting morphological traits

Recurrent Modification of a Conserved Cis-Regulatory Element Underlies Fruit Fly Pigmentation Diversity

The development of morphological traits occurs through the collective action of networks of genes connected at the level of gene expression. As any node in a network may be a target of evolutionary change, the recurrent targeting of the same node would indicate that the path of evolution is biased for the relevant trait and network. Although examples of parallel evolution have implicated recurrent modification of the same gene and cis-regulatory element (CRE), little is known about the mutational and molecular paths of parallel CRE evolution. In Drosophila melanogaster fruit flies, the Bric-à-brac (Bab) transcription factors control the development of a suite of sexually dimorphic traits on the posterior abdomen. Female-specific Bab expression is regulated by the dimorphic element, a CRE that possesses direct inputs from body plan (ABD-B) and sex-determination (DSX) transcription factors. Here, we find that the recurrent evolutionary modification of this CRE underlies both intraspecific and interspecific variation in female pigmentation in the melanogaster species group. By reconstructing the sequence and regulatory activity of the ancestral Drosophila melanogaster dimorphic element, we demonstrate that a handful of mutations were sufficient to create independent CRE alleles with differing activities. Moreover, intraspecific and interspecific dimorphic element evolution proceeded with little to no alterations to the known body plan and sex-determination regulatory linkages. Collectively, our findings represent an example where the paths of evolution appear biased to a specific CRE, and drastic changes in function were accompanied by deep conservation of key regulatory linkages. © 2013 Rogers et al

DrosoPhyla: Resources for Drosophilid Phylogeny and Systematics.

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

Logic, genomics, and evolution of the peripheral nervous system transcriptional network of Drosophila

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 period

Experimental Approaches to Evaluate the Contributions of Candidate Cis-regulatory Mutations to Phenotypic Evolution

Elucidating the molecular bases by which phenotypic traits have evolved provides a glimpse into the past, allowing the characterization of genetic changes that cumulatively contribute to evolutionary innovations. Historically, much of the experimental attention has been focused on changes in protein-coding regions that can readily be identified by the genetic code for translating gene coding sequences into proteins. Resultantly, the role of noncoding sequences in trait evolution has remained more mysterious. In recent years, several studies have reached an unprecedented level of detail in describing how noncoding mutations in gene cis-regulatory elements contribute to morphological evolution. Based on these and other studies, we describe an experimental framework and some of the genetic and molecular methods to connect a particular cis-regulatory mutation to the evolution of any phenotypic trait

Raw Data from Manuscript "A genetic screen of transcription factors in the <i>Drosophila melanogaster</i> abdomen performed in an undergraduate laboratory course"

The raw image files and supplemental material for the manuscript "A genetic screen of transcription factors in the Drosophila melanogaster abdomen identifies novel pigmentation genes", are included here. This manuscript details a genetic screen in which we targeted 71 transcription factor genes in the Drosophila abdomen, either overexpressing or knocking out the target gene. In our screen, we used a transgenic CRISPR/Cas9 approach to overexpress or knockout a transcription factor gene in the pannier expression domain, which drives expression in the middle of the abdomen and thorax.Supplemental material includes Figures S1, S2, and S3; Tables S1 and S2; and Supplementary File 1.Image files are named to include 1) the gene being targeted, indicated by its abbreviation; 2) whether the treatment is overexpression (e) or knockout (ko); 3) the Bloomington ID numbers of the parents; and 4) whether the fly is male or female.For example, if a file is named "ab_e_67040_83608_f0001", the "ab" indicates the gene targeted, in this case, abrupt; the "e" indicates that this was an overexpression cross; 67040 is the Bloomington ID for the female parent of the cross; 83608 is the Bloomington ID for the male parent of the cross; and the "f0001" indicates that the fly in the image is female.The numbers 67040 and 67077 are the IDs for the overexpression and knockout lines, respectively. In all cases, the female parent was the one that contained the pannier-Gal4 driver. The male parent contained the guide RNAs for the target genes.For the controls, overexpression controls are labeled "gof_control_f/m", and knockout controls are labeled "lof_control_f/m".Raw numerical measurements of these images is included in file Supplementary File 1. Measurements from overexpression experiments and from knockout experiments are on separate, labeled tabs. In this file, the darkness measurement has been converted to percent darkness using the following formula:(255 - x) / 255where x is the "mean A4 darkness" value.</p