Neuropeptides PDF and DH31 hierarchically regulate free-running rhythmicity in Drosophila circadian locomotor activity

Neuropeptides play pivotal roles in modulating circadian rhythms. Pigment-dispersing factor (PDF) is critical to the circadian rhythms in Drosophila locomotor activity. Here, we demonstrate that diuretic hormone 31 (DH31) complements PDF function in regulating free-running rhythmicity using male flies. We determined that Dh31 loss-of-function mutants (Dh31(#51)) showed normal rhythmicity, whereas Dh31(#51);Pdf(01) double mutants exhibited a severe arrhythmic phenotype compared to Pdf-null mutants (Pdf(01)). The expression of tethered-PDF or tethered-DH31 in clock cells, posterior dorsal neurons 1 (DN1ps), overcomes the severe arrhythmicity of Dh31(#51);Pdf(01) double mutants, suggesting that DH31 and PDF may act on DN1ps to regulate free-running rhythmicity in a hierarchical manner. Unexpectedly, the molecular oscillations in Dh31(#51);Pdf(01) mutants were similar to those in Pdf(01) mutants in DN1ps, indicating that DH31 does not contribute to molecular oscillations. Furthermore, a reduction in Dh31 receptor (Dh31r) expression resulted in normal locomotor activity and did not enhance the arrhythmic phenotype caused by the Pdf receptor (Pdfr) mutation, suggesting that PDFR, but not DH31R, in DN1ps mainly regulates free-running rhythmicity. Taken together, we identify a novel role of DH31, in which DH31 and PDF hierarchically regulate free-running rhythmicity through DN1ps.