Animal species display enormous variation for innate behaviours, but little is known about how this diversity arose. Here, using an unbiased genetic approach, we map a courtship song difference between wild isolates of Drosophila simulans and Drosophila mauritiana to a 966 base pair region within the slowpoke (slo) locus, which encodes a calcium-activated potassium channel. Using the reciprocal hemizygosity test, we confirm that slo is the causal locus and resolve the causal mutation to the evolutionarily recent insertion of a retroelement in a slo intron within D. simulans. Targeted deletion of this retroelement reverts the song phenotype and alters slo splicing. Like many ion channel genes, slo is expressed widely in the nervous system and influences a variety of behaviours; slo-null males sing little song with severely disrupted features. By contrast, the natural variant of slo alters a specific component of courtship song, illustrating that regulatory evolution of a highly pleiotropic ion channel gene can cause modular changes in behaviour.

We used live imaging to visualize the transcriptional dynamics of the Drosophila melanogaster even-skipped gene at single-cell and high-temporal resolution as its seven stripe expression pattern forms, and developed tools to characterize and visualize how transcriptional bursting varies over time and space. We find that despite being created by the independent activity of five enhancers, even-skipped stripes are sculpted by the same kinetic phenomena: a coupled increase of burst frequency and amplitude. By tracking the position and activity of individual nuclei, we show that stripe movement is driven by the exchange of bursting nuclei from the posterior to anterior stripe flanks. Our work provides a conceptual, theoretical and computational framework for dissecting pattern formation in space and time, and reveals how the coordinated transcriptional activity of individual nuclei shapes complex developmental patterns.

We used live imaging to visualize the transcriptional dynamics of the Drosophila melanogaster even-skipped gene at single-cell and high temporal resolution as its iconic seven stripe expression pattern forms. We developed a rigorous quantitative framework for analyzing and visualizing these data, that allows us to infer the temporal dynamics of the transcriptional state of roughly 3,000 nuclei, and characterize how the process varies over time and space. We show that despite being created by the largely independent activity of five discrete enhancers, the seven eve stripes are sculpted by the same basic kinetic phenomena: the suppression of bursting outside of stripes, a coupled increase of burst frequency and burst duration within stripes over time and across stripes from their margins to their centers, and the exchange of active nuclei from the posterior to anterior flanks to reposition stripes over time.