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. 2018 Sep 24;16(9):e2005004. doi: 10.1371/journal.pbio.2005004

Fig 3. Mating induces NPF accumulation in EECs, and exogenous NPF increases GSC.

Fig 3

(A–D) Levels of NPF protein and NPF transcript in the middle midgut. (A, C) Representative images of anti-NPF immunostaining in the middle midgut are shown on the left. Quantification of anti-NPF signal intensity in the middle midgut is shown on the right graph. (A) Anti-NPF signal accumulated in virgin female flies and females mated with SP-null mutant males (SP0/SPΔ130). (B) Transcript abundance of NPF in the middle midgut did not change after mating with wild-type or SP-null male flies. (C) NPF release was induced without mating by overexpressing mSP in ppk-positive neurons (ppk-GAL4>mSP). (D) Transcript abundance of NPF in the middle midgut was not changed by this manipulation. (E, F) Fraction 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. (E) Microinjection of synthetic NPF peptide, but not sNPF (Bm-sNPF), into virgin female flies induced an increase in GSC number. NPF-dependent increase in GSC number occurred after injecting 3–3,000 pM (but not 0–0.3 pM) NPF. (F) The GSC-number phenotype of NPF RNAi animals was rescued by injecting synthetic NPF peptide. Dots represent the relative signal intensity of anti-NPF in single middle midguts (panel A and C) or the relative expression levels of NPF in the middle midgut (panel B and D); lines represent the median, and whiskers represent the interquartile range. The number of germaria analyzed is shown inside the bars in panel E and F. For statistical analysis, a Wilcoxon rank sum test with Holm’s correction was used for panel A, C, E, and F. Student t test with Holm’s correction was used for panel B and D. ***P ≤ 0.001, **P ≤ 0.01, and *P ≤ 0.05; NS, nonsignificant (P > 0.05). Scale bar = 50 μm in panel A and C. Underlying data can be found in S1 Data. See also S5 Fig. GSC, germline stem cell; mSP, membrane-tethered SP; NPF, neuropeptide F; ppk, pickpocket; RNAi, RNA interfererence; sNPF, short neuropeptide F precursor; SP, sex peptide; Tkg-GAL4, Tk-gut-GAL4.