Changes in gene regulation underlie much of phenotypic evolution. However, our understanding of the potential for regulatory evolution is biased, because most evidence comes from either natural variation or limited experimental perturbations. Using an automated robotics pipeline, we surveyed an unbiased mutation library for a developmental enhancer in Drosophila melanogaster. We found that almost all mutations altered gene expression and that parameters of gene expression-levels, location, and state-were convolved. The widespread pleiotropic effects of most mutations may constrain the evolvability of developmental enhancers. Consistent with these observations, comparisons of diverse Drosophila larvae revealed apparent biases in the phenotypes influenced by the enhancer. Developmental enhancers may encode a higher density of regulatory information than has been appreciated previously, imposing constraints on regulatory evolution.

Pigmentation divergence between Drosophila species has emerged as a model trait for studying the genetic basis of phenotypic evolution, with genetic changes contributing to pigmentation differences often mapping to genes in the pigment synthesis pathway and their regulators. These studies of Drosophila pigmentation have tended to focus on pigmentation changes in one body part for a particular pair of species, but changes in pigmentation are often observed in multiple body parts between the same pair of species. The similarities and differences of genetic changes responsible for divergent pigmentation in different body parts of the same species thus remain largely unknown. Here we compare the genetic basis of pigmentation divergence between Drosophila elegans and D. gunungcola in the wing, legs, and thorax. Prior work has shown that regions of the genome containing the pigmentation genes yellow and ebony influence the size of divergent male-specific wing spots between these two species. We find that these same two regions of the genome underlie differences in leg and thorax pigmentation; however, divergent alleles in these regions show differences in allelic dominance and epistasis among the three body parts. These complex patterns of inheritance can be explained by a model of evolution involving tissue-specific changes in the expression of Yellow and Ebony between D. elegans and D. gunungcola.

It is unclear how regulatory genes establish neural circuits that compose sex-specific behaviors. The Drosophila melanogaster male courtship song provides a powerful model to study this problem. Courting males vibrate a wing to sing bouts of pulses and hums, called pulse and sine song, respectively. We report the discovery of male-specific thoracic interneurons—the TN1A neurons—that are required specifically for sine song. The TN1A neurons can drive the activity of a sex-non-specific wing motoneuron, hg1, which is also required for sine song. The male-specific connection between the TN1A neurons and the hg1 motoneuron is regulated by the sexual differentiation gene doublesex. We find that doublesex is required in the TN1A neurons during development to increase the density of the TN1A arbors that interact with dendrites of the hg1motoneuron. Our findings demonstrate how a sexual differentiation gene can build a sex-specific circuit motif by modulating neuronal arborization.

•Doublesex-expressing TN1 neurons are necessary and sufficient for the male sine song•A subclass of TN1 neurons, TN1A, contributes to the sine song•TN1A neurons are functionally coupled to a sine song motoneuron, hg1•Doublesex regulates the connectivity between the TN1A and hg1 neurons

It is unclear how developmental regulatory genes specify sex-specific behaviors. Shirangi et al. demonstrate that the Drosophila sexual differentiation gene doublesex encodes a sex-specific behavior—male song—by promoting the connectivity between the male-specific TN1A neurons and the sex-non-specific hg1 neurons, which are required for production of the song.

Courtship rituals serve to reinforce reproductive barriers between closely related species. Drosophila melanogaster and Drosophila simulans exhibit reproductive isolation, owing in part to the fact that D. melanogaster females produce 7,11-heptacosadiene, a pheromone that promotes courtship in D. melanogaster males but suppresses courtship in D. simulans males. Here we compare pheromone-processing pathways in D. melanogaster and D. simulans males to define how these sister species endow 7,11-heptacosadiene with the opposite behavioural valence to underlie species discrimination. We show that males of both species detect 7,11-heptacosadiene using homologous peripheral sensory neurons, but this signal is differentially propagated to P1 neurons, which control courtship behaviour. A change in the balance of excitation and inhibition onto courtship-promoting neurons transforms an excitatory pheromonal cue in D. melanogaster into an inhibitory cue in D. simulans. Our results reveal how species-specific pheromone responses can emerge from conservation of peripheral detection mechanisms and diversification of central circuitry, and demonstrate how flexible nodes in neural circuits can contribute to behavioural evolution.

Genetically wired neural mechanisms inhibit mating between species because even naive animals rarely mate with other species. These mechanisms can evolve through changes in expression or function of key genes in sensory pathways or central circuits. Gr32a is a gustatory chemoreceptor that, in D. melanogaster, is essential to inhibit interspecies courtship and sense quinine. Similar to D. melanogaster, we find that D. simulans Gr32a is expressed in foreleg tarsi, sensorimotor appendages that inhibit interspecies courtship, and it is required to sense quinine. Nevertheless, Gr32a is not required to inhibit interspecies mating by D. simulans males. However, and similar to its function in D. melanogaster, Ppk25, a member of the Pickpocket family, promotes conspecific courtship in D. simulans. Together, we have identified distinct evolutionary mechanisms underlying chemosensory control of taste and courtship in closely related Drosophila species.

Pheromones, chemical signals that convey social information, mediate many insect social behaviors, including navigation and aggregation. Several studies have suggested that behavior during the immature larval stages of Drosophila development is influenced by pheromones, but none of these compounds or the pheromone-receptor neurons that sense them have been identified. Here we report a larval pheromone-signaling pathway. We found that larvae produce two novel long-chain fatty acids that are attractive to other larvae. We identified a single larval chemosensory neuron that detects these molecules. Two members of the pickpocket family of DEG/ENaC channel subunits (ppk23 and ppk29) are required to respond to these pheromones. This pheromone system is evolving quickly, since the larval exudates of D. simulans, the sister species of D. melanogaster, are not attractive to other larvae. Our results define a new pheromone signaling system in Drosophila that shares characteristics with pheromone systems in a wide diversity of insects.

Biological systems display extraordinary robustness. Robustness of transcriptional enhancers results mainly from clusters of binding sites for the same transcription factor, and it is not clear how robust enhancers can evolve loss of expression through point mutations. Here, we report the high-resolution functional dissection of a robust enhancer of the shavenbaby gene that has contributed to morphological evolution. We found that robustness is encoded by many binding sites for the transcriptional activator Arrowhead and that, during evolution, some of these activator sites were lost, weakening enhancer activity. Complete silencing of enhancer function, however, required evolution of a binding site for the spatially restricted potent repressor Abrupt. These findings illustrate that recruitment of repressor binding sites can overcome enhancer robustness and may minimize pleiotropic consequences of enhancer evolution. Recruitment of repression may be a general mode of evolution to break robust regulatory linkages.

From 1980 to 1992, a series of influential papers reported on the discovery, genetics, and evolution of a periodic cycling of the interval between Drosophila male courtship song pulses. The molecular mechanisms underlying this periodicity were never described. To reinitiate investigation of this phenomenon, we previously performed automated segmentation of songs but failed to detect the proposed rhythm [Arthur BJ, et al. (2013) BMC Biol 11:11; Stern DL (2014) BMC Biol 12:38]. Kyriacou et al. [Kyriacou CP, et al. (2017) Proc Natl Acad Sci USA 114:1970-1975] report that we failed to detect song rhythms because (i) our flies did not sing enough and (ii) our segmenter did not identify many of the song pulses. Kyriacou et al. manually annotated a subset of our recordings and reported that two strains displayed rhythms with genotype-specific periodicity, in agreement with their original reports. We cannot replicate this finding and show that the manually annotated data, the original automatically segmented data, and a new dataset provide no evidence for either the existence of song rhythms or song periodicity differences between genotypes. Furthermore, we have reexamined our methods and analysis and find that our automated segmentation method was not biased to prevent detection of putative song periodicity. We conclude that there is no evidence for the existence of Drosophila courtship song rhythms.

Transcriptional enhancers are regions of DNA that drive precise patterns of gene expression. While many studies have elucidated how individual enhancers can evolve, most of this work has focused on what are called "minimal" enhancers, the smallest DNA regions that drive expression that approximates an aspect of native gene expression. Here we explore how the Drosophila erecta even-skipped (eve) locus has evolved by testing its activity in the divergent D. melanogaster genome. We found, as has been reported previously, that the D. erecta eve stripe 2 enhancer (eveS2) fails to drive appreciable expression in D. melanogaster (1). However, we found that a large transgene carrying the entire D. erecta eve locus drives normal eve expression, including in stripe 2. We performed a functional dissection of the region upstream of the D. erecta eveS2 region and found multiple Zelda motifs that are required for normal expression. Our results illustrate how sequences outside of minimal enhancer regions can evolve functionally through mechanisms other than changes in transcription factor binding sites that drive patterning.