Hormone-independent, EcR-mediated endoreplication of SC DNA is stimulated by mating.
Males expressing GFP and other transgenes under esgtsF/O control or ecd1 mutants were cultured on EdU-containing food posteclosion and dissected at 6 days, and their AGs were probed for EdU uptake to assess DNA replication and stained with DAPI. SCs were recognised by GFP expression or by their characteristic vacuolar morphology in ecd1 mutants; selected cells are highlighted by dashed circles (red for EdU-positive, white for EdU-negative). (A) EdU is incorporated in about 30%–40% of SCs after mating (red arrows depict SCs with EdU uptake, white arrows mark those without EdU). (B, C) Almost all SCs in ecd1 males contain EdU in their nuclei after mating (B), and this is unaffected by 20-HE feeding (C). (D) SC-specific expression of shd-RNAi has no effect on the number of SCs containing EdU in mated males. (E) Histogram showing EdU incorporation into SC nuclei in different genetic backgrounds. (F) Proposed model explaining the different forms of hormone-dependent and hormone-independent, EcR-mediated SC growth in virgin and mated males. Our data reveal that EcR-dependent growth of SCs is differentially modulated by the presence of Ec according to mating status. An Ec-EcR complex, which requires a dimerisation partner other than USP (marked ?; this could be an EcR dimer, for example) is necessary for normal SC nuclear growth observed in virgin males. In mated males, SC growth is enhanced, at least in part due to new DNA synthesis driven by a hormone-independent EcR-A/USP complex. This complex must either repress or activate a subset of genes that are not EcR targets in virgin males, hence inducing cell cycle regulators like CycE either directly or indirectly. In mated males, EcR complexes that do not involve USP can also drive non-endoreplication-mediated growth, potentially via hormone-dependent and hormone-independent mechanisms, which may be coordinately regulated with endoreplication (e.g., this growth appears to increase following Usp knockdown). EcR levels and signalling are stimulated by elevated BMP signalling induced by autocrine BMP ligand Dpp through the heterodimeric Tkv/Wit receptor . Data were analysed by the Kruskal-Wallis test with Dunn’s multiple-comparisons test. *p < 0.05, **p < 0.01, ****p < 0.0001, n ≥ 8. Scale bars, 20 μm. Underlying data for this figure can be found in S1 Data. 20-HE, 20-hydroxyecdysone; AG, accessory gland; BMP, bone morphogenetic protein; CycE, Cyclin E; Dpp, Decapentaplegic; Ec, ecdysone; EcR, Ec receptor; ecd, ecdysoneless; EdU, 5-ethynyl-2′-deoxyuridine; esg, escargot; esgtsF/O, the yeast transcription factor GAL4 expressed under the control of the promoter of the gene esg in a temperature-dependent fashion; GFP, green fluorescent protein; Med, Medea; RNAi, RNA interference; SC, secondary cell; shd, shade; spo, spook; Tkv, Thick veins; USP, Ultraspiracle, Wit, Wishful thinking.