Many motor control systems generate multiple movements using a common set of muscles. How are premotor circuits able to flexibly generate diverse movement patterns? Here, we characterize the neuronal circuits that drive the distinct courtship songs of Drosophila melanogaster. Male flies vibrate their wings towards females to produce two different song modes – pulse and sine song – which signal species identity and male quality. Using cell-type specific genetic reagents and the connectome, we provide a cellular and synaptic map of the circuits in the male ventral nerve cord that generate these songs and examine how activating or inhibiting each cell type within these circuits affects the song. Our data reveal that the song circuit is organized into two nested feed-forward pathways, with extensive reciprocal and feed-back connections. The larger network produces pulse song, the more complex and ancestral song form. A subset of this network produces sine song, the simpler and more recent form. Such nested organization may be a common feature of motor control circuits in which evolution has layered increasing flexibility on to a basic movement pattern.

The density and distribution of regulatory information in non-coding DNA of eukaryotic genomes is largely unknown. Evolutionary analyses have estimated that ∼60% of nucleotides in intergenic regions of the D. melanogaster genome is functionally relevant. This estimate is difficult to reconcile with the commonly accepted idea that enhancers are compact regulatory elements that generally encompass less than 1 kilobase of DNA. Here, we approached this issue through a functional dissection of the regulatory region of the gene shavenbaby (svb). Most of the ∼90 kilobases of this large regulatory region is highly conserved in the genus Drosophila, though characterized enhancers occupy a small fraction of this region. By analyzing the regulation of svb in different contexts of Drosophila development, we found that the regulatory architecture that drives svb expression in the abdominal pupal epidermis is organized in a dramatically different way than the information that drives svb expression in the embryonic epidermis. While in the embryonic epidermis svb is activated by compact and dispersed enhancers, svb expression in the pupal epidermis is driven by large regions with enhancer activity, which occupy a great portion of the svb cis-regulatory DNA. We observed that other developmental genes also display a dense distribution of putative regulatory elements in their regulatory regions. Furthermore, we found that a large percentage of conserved non-coding DNA of the Drosophila genome is contained within putative regulatory DNA. These results suggest that part of the evolutionary constraint on non-coding DNA of Drosophila is explained by the density of regulatory information.