PMC

(a) RNA in situ hybridization for Fur1 and Fur2 in stage 10 egg chambers reveals both genes are expressed in the nurse cells (nc) and oocyte (oo). Sense controls show no staining. (b) Whole larval cuticles and head skeletons of animals with maternal knockdown (nos-Gal4) of Fur1 and Fur2 transcripts using UAS-shRNAs. Knockdown of either Fur1 or Fur2 alone has no effect. Co-knockdown of Fur1 and Fur2 results in terminal defects including a reduced posterior filzkorper (fz, arrowed) and head skeleton defects (arrowheads) similar to embryos laid by trk^Δ females. Anterior is to the left. Scale bars, 50 μm. Images are representative of at least two repeat experiments, with at least 100 individuals observed per genotype.

(a) Trk:Ch consists of the Trk coding sequence fused to three Myc epitopes and the fluorescent protein mCherry (Ch). NTrk:Ch is Trk:Ch with the cysteine-knot region removed. SP, signal peptide. (b) Immunoblot (anti-Myc) of Trk:Ch expression in embryos and S2 cells. Full-length Trk:Ch (predicted to be 55 kDa, arrowed), and three cleaved Trk:Ch species (1–3) are detectable in embryos (Emb) and both the S2 cell pellet (P) and supernatant (SN). (c) Immunoblot of NTrk:Ch expression in S2 cells. Full-length (predicted to be 44 kDa, arrowed) and cleaved (1) NTrk:Ch are detectable in the cell pellet and supernatant. (d) Immunoblot of supernatant from Trk:Ch-expressing S2 cells incubated with (+) and without (−) Furin inhibitors over 3 days. Trk:Ch cleavage is markedly reduced or absent when Furin inhibitor is present (compare species 1 level in lanes with and without inhibitor). For uncropped immunoblot see . (e) Immunoblot and quantification of NTrk:Ch in S2 cell supernatant. Cleavage is significantly reduced when Furin inhibitor is present (unpaired t-test, *P<0.05, **P<0.01). For uncropped immunoblot see . Error bars represent ±1 s.e. n=3 for each mean. Images are representative of at least three replicate immunoblots.

(a) Strong accumulation of NTrk:Ch is observed in the polar PVS in <1 h wild type but not maternal tsl null mutant embryos (tsl^Δ). Ch, mCherry. BF, brightfield. (b) sec:Ch accumulates in the polar PVS of wild-type and tsl^Δ embryos. Anterior is to the left. Scale bars, 100 μm. (c) The level of NTrk:Ch in the anterior and posterior PVS is markedly and consistently reduced in tsl^Δ embryos compared with wild type (unpaired t-test, ***P<0.0001, anterior: wild type n=197, tsl^Δ n=144; posterior: wild type n=185; tsl^Δ n=64). (d) Levels of sec:Ch are not reduced in the absence of Tsl compared with wild type (unpaired t-test, *P<0.05, anterior: wild type n=73, tsl^Δ n=57; posterior: wild type n=81; tsl^Δ n=54). ns, not significant. g.v., grey values. Error bars represent ±1 s.e. (e) Revised model for terminal patterning in Drosophila. Trk is activated by Furin cleavage within the embryo, possibly in the secretory pathway. Trk is then secreted at the embryo poles by a Tsl-dependent mechanism, and encounters the Tor receptor tyrosine kinase to initiate the signalling cascade required for terminal patterning.