Ovarian NPFR controls mating-induced GSC proliferation.

(A, B, D, E) Frequency of germaria containing 1, 2, and 3 GSCs (left axis) and the average number of GSCs per germarium (right axis) in virgin (v) and mated (m) female flies. The addition of synthetic NPF peptide (panel A), but not of sNPF (panel B), to the ex vivo ovary culture medium was sufficient to increase GSC number. (C) NPFR transcripts were detected in the ovary, although expression levels were much lower than in the head and midgut. (D) NPFR RNAi driven by tj-GAL4, e22c-GAL4, or c587-GAL4 (targeting germarium somatic cells) inhibited the mating-induced increase in GSC number. The increase in GSC number after mating was not altered by NPFR RNAi driven by nos-GAL4, bab1-GAL4, or c306-GAL4 (germ, cap, and posterior follicle cells, respectively). (E) Overexpressing NPF or NPFR in the somatic cells of the germaria was sufficient to increase GSC number in virgin female flies. The GSC proliferation defect in NPFR genetic null mutants was rescued by overexpressing the NPFR transgene under the control of tj-GAL4. Rescue, tj-GAL4/UAS-NPFR; NPFRsk8/Df(3R)BSC464. Control, tj-GAL4/+; NPFRsk8/Df(3R)BSC464 or UAS-NPFR/+; NPFRsk8/Df(3R)BSC464. (F) Mating increased the frequency of GSCs in M (measured by pH3 labeling) and S (measured by BrdU incorporation) phases, but not G1/S or G2/M fusome morphologies. Cell cycle promotion in GSCs after mating was suppressed in tj-GAL4>NPFRRNAi animals. (G) The total number of eggs produced by females within 48 hours was reduced in tj-GAL4>NPFRRNAi animals. (H) Receptivity of virgin females was scored as the percentage of females that copulated within 24 hours. The number of germaria analyzed is shown inside the bars in panel A, B, D–F, and H. For statistical analysis, a Wilcoxon rank sum test with Holm’s correction was used for A, B, D, and E. Fisher’s exact test with Holm’s correction was used for panel F and H. Student t test was used for panel G. ***P ≤ 0.001, **P ≤ 0.01, and *P ≤ 0.05; NS, nonsignificant (P > 0.05). Underlying data can be found in S1 Data. See also S6 Fig. BrdU, bromodeoxyuridine; CpC, cap cell; GSC, germline stem cell; NPF, neuropeptide F; NPFR, neuropeptide F receptor; OSC, ovarian somatic cell; pH3, phospho-histone H3; RNAi, RNA interference; sNPF, short neuropeptide F precursor.