Fig 2. The Rbf/E2F1 axis maintains adult SC binucleation state.

(A-G) Nuclei are stained with DAPI (blue). Dashed red ellipses mark the outline of mononucleate SCs; dashed yellow ellipses mark the outline of binucleate SCs; red arrows mark the nuclei of mononucleate SCs; yellow arrows mark the nuclei of binucleate SCs. (A-D) Images show distal tip of AGs from 6-day-old adult virgin males of controls or of flies expressing Rbf^CA either under the control of the esg^tsF/O driver (A, C; SC driver) or the Acp26Aa-GAL4 driver (B, D; MC driver), which activates nuclear GFP production (also stains cytoplasm in A, C). Orange boxes outline the area zoomed in the insets C’, D’. (E) Image shows distal tip of an AG from a 3-day-old adult virgin male expressing Rbf^CA in SCs under esg^tsF/O control. (F) Image shows distal tip of an AG from a 9-day-old virgin male, which expressed Rbf^CA in SCs for 3 days following eclosion, and was then incubated for 6 days at 25°C to block further Rbf^CA expression. No GFP was expressed during the 6-day follow-up period, so SCs were identified using their well-characterised vacuolar morphology ². (G) Image shows the distal tip of an AG of a 6-day-old multiply-mated male expressing Rbf^CA under the control of the Acp26Aa-GAL4 driver. The AG was stained for EdU presence, revealing that mononucleate SCs are still able to endoreplicate. (H-J) Bar charts depicting the mean % of mononucleate SCs per gland of virgin males in controls or males expressing transgenes either in SCs (H, J) or MCs (I). Expression of Rbf^CA either in SCs or MCs induces SC mononucleation in a reversible manner, but this transgene has no effect in MCs. Knocking down E2F1 in SCs promotes SC mononucleation but no effect is observed when knocked down in MCs. Kruskal Wallis test; Dunn’s post hoc test; n≥6 glands (H-J). (K) Schematic detailing nucleation state regulation in adult SCs. Expression of a constitutively active form of Rbf, Rbf^CA, which has mutations in three of its four CDK phosphorylation sites, in SCs is able to induce mononucleation. This phenotype is negatively regulated by E2F1. Additionally, expression of Rbf^CA in MCs is able to activate mononucleation in SCs. In one model, it might induce secretion of ‘X’, which could activate an unknown receptor (?) that drives SC mononucleation; alternatively, Rbf^CA may suppress secretion of a molecule, which promotes binucleation. It is possible that Rbf^CA when expressed in SCs also at least partly promotes mononucleation via the same mechanism. Scale bars correspond to 50 μm (A-G) and 10 μm (C’, D’). 0.0001≤***p≤0.001; ****p≤0.0001.