Augmentation of a wound response element accompanies the origin of a Hox-regulated Drosophila abdominal pigmentation trait.

A challenge for evolutionary research is to uncover how new morphological traits evolve the coordinated spatial and temporal expression patterns of genes that govern their formation during development. Detailed studies are often limited to characterizing how one or a few genes contributed to a trait's emergence, and thus our knowledge of how entire GRNs evolve their coordinated expression of each gene remains unresolved. The melanic color patterns decorating the male abdominal tergites of Drosophila (D.) melanogaster evolved in part by novel expression patterns for genes acting at the terminus of a pigment metabolic pathway, driven by cis-regulatory elements (CREs) with distinct mechanisms of Hox regulation. Here, we examined the expression and evolutionary histories of two important enzymes in this pathway, encoded by the pale and Ddc genes. We found that while both genes exhibit dynamic patterns of expression, a robust pattern of Ddc expression specifically evolved in the lineage of fruit flies with pronounced melanic abdomens. Derived Ddc expression requires the activity of a CRE previously shown to activate expression in response to epidermal wounding. We show that a binding site for the Grainy head transcription factor that promotes the ancestral wound healing function of this CRE is also required for abdominal activity. Together with previous findings in this system, our work shows how the GRN for a novel trait emerged by assembling unique yet similarly functioning CREs from heterogeneous starting points.

A survey of the trans-regulatory landscape for Drosophila melanogaster abdominal pigmentation.

Trait development results from the collaboration of genes interconnected in hierarchical networks that control which genes are activated during the progression of development. While networks are understood to change over developmental time, the alterations that occur over evolutionary times are much less clear. A multitude of transcription factors and a far greater number of linkages between transcription factors and cis-regulatory elements (CREs) have been found to structure well-characterized networks, but the best understood networks control traits that are deeply conserved. Fruit fly abdominal pigmentation may represent an optimal setting to study network evolution, as this trait diversified over short evolutionary time spans. However, the current understanding of the underlying network includes a small set of transcription factor genes. Here, we greatly expand this network through an RNAi-screen of 558 transcription factors. We identified 28 genes, including previously implicated abd-A, Abd-B, bab1, bab2, dsx, exd, hth, and jing, as well as 20 novel factors with uncharacterized roles in pigmentation development. These include genes which promote pigmentation, suppress pigmentation, and some that have either male- or female-limited effects. We show that many of these transcription factors control the reciprocal expression of two key pigmentation enzymes, whereas a subset controls the expression of key factors in a female-specific circuit. We found the pupal Abd-A expression pattern was conserved between species with divergent pigmentation, indicating diversity resulted from changes to other loci. Collectively, these results reveal a greater complexity of the pigmentation network, presenting numerous opportunities to map transcription factor-CRE interactions that structure trait development and numerous candidate loci to investigate as potential targets of evolution.

Cis-regulatory evolution integrated the Bric-à-brac transcription factors into a novel fruit fly gene regulatory network.

Gene expression evolution through gene regulatory network (GRN) changes has gained appreciation as a driver of morphological evolution. However, understanding how GRNs evolve is hampered by finding relevant cis-regulatory element (CRE) mutations, and interpreting the protein-DNA interactions they alter. We investigated evolutionary changes in the duplicated Bric-à-brac (Bab) transcription factors and a key Bab target gene in a GRN underlying the novel dimorphic pigmentation of D. melanogaster and its relatives. It has remained uncertain how Bab was integrated within the pigmentation GRN. Here, we show that the ancestral transcription factor activity of Bab gained a role in sculpting sex-specific pigmentation through the evolution of binding sites in a CRE of the pigment-promoting yellow gene. This work demonstrates how a new trait can evolve by incorporating existing transcription factors into a GRN through CRE evolution, an evolutionary path likely to predominate newly evolved functions of transcription factors.