Fig. 1
Rbfox1 levels depend on miR-980 and stress. a Schematics of ovaries and a single ovariole (lower panel), where the germline (blue) is surrounded by the somatic follicular epithelium cells, in which at st.7, Notch signaling-dependent mitotic-to-endocycle and at st.10b, endocycle-to-amplification transitions occur. These phases are distinguished by Cut (red), expression of which is repressed by activated Notch signaling. Upon stress, egg production is sharply terminated at the nutritional stress checkpoint, resulting in small ovaries. Images of sated and 10-day protein-starved Control and miR-980 ovaries. Starvation response is not as robust in miR-980 mutants, their ovaries are larger and contain later stage egg chambers. b miR-980 overexpression significantly reduces expression of luciferase reporters (P1 and P2), containing parts of the extended Rbfox1 3′UTR with different miR-980 binding sites. c miR-980 ovaries have significantly increased Rbfox1 mRNA levels. d Nutritional stress (2-day) influences miR-980 and Rbfox1 expression in ovaries: miR-980 is significantly downregulated, while Rbfox1 is elevated. e Rbfox1 levels in wild type (WT), miR-980, and Rbfox1-overexpressing (hsFlp; act > CD2 > Gal4 UAS-GFP/UAS-Rbfox1-RE) ovaries from sated and protein-deprived animals detected by western blotting. Colored arrows point to Rbfox1 isoforms that were used for Rbfox1 expression quantification shown in the bar graph. f Rbfox1 expression pattern is temporally and spatially dynamic. At mitotic stages (st.3–6), Rbfox1 is nuclear; at early endocycling (st.6–8), its levels are strongly decreased; at late endocycling (st.9–10a), nuclear Rbfox1 reappears. At amplification stages (st.11–14), Rbfox1 nuclear expression is strongly reduced, but the moderate cytosolic remains. g In miR-980Ex1-2, Rbfox1 is elevated in comparison to Control egg chambers of the corresponding stages. h Assembly of Control and miR-980 ovarioles under normal and starvation stress conditions. Delayed termination of egg production in miR-980 mutants is rescued by Rbfox1 downregulation. i Quantification of ovarioles with no response to starvation. Introduction of one copy of Rbfox1 mutation to the miR-980 loss-of-function background rescues the phenotype. DAPI (green), Cut (blue), Rbfox1 (red, rabbit anti-Rbfox1). f, g maximum intensity projections of multiple Z-sections. Scale bars 5 µm. AVE ± SD are reported from triplicates, two-tailed Student’s t-test was applied for statistics in b–d, and Chi-square test in i. See Supplementary Tables 1–3. ** p ≤ 0.01, *** p ≤ 0.001
