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. Author manuscript; available in PMC: 2015 May 8.
Published in final edited form as: Cell Rep. 2014 Apr 18;7(3):715–721. doi: 10.1016/j.celrep.2014.03.058

Figure 3. Ectopic CycD-Cdk4 expression in the hub promotes hub cell proliferation and conversion to CySCs.

Figure 3

(A–D) Single confocal sections through the apex of testes expressing CycD and Cdk4 in hub cells, immunostained as indicated. Nuclei are counterstained with DAPI (blue). (A –B) E132-Gal4>CycD, Cdk4 testes immunostained with anti-Vasa (red; germ cells), anti-Arm (green at cell cortex; hub cells), and anti-PH3 (green; mitotic nuclei). (A) Before CycD-Cdk4 induction (18°C), PH3-positive cells (arrow) are found outside but never inside the hub (n = 127 testes). (B) After CycD-Cdk4 expression (7 d, 31°C), 42% of testes (n = 35/83) contain PH3-positive hub cells (two shown, arrowheads). (C–D) E132-Gal4>CycD, Cdk4 testes co-expressing Fas3-GFP (marking hub cells), immunostained with anti-Tj (red; hub cells, CySCs and early cyst cells) and anti-GFP (green). (C) Before CycD-Cdk4 expression (18°C), GFP expression is restricted to the hub. (D-D’) After CycD-Cdk4 expression (6 d, 31°C) and recovery (3 d, 18°C), GFP is observed in Tj-positive cells outside the hub (arrows) in 23% of testes (n = 7/30). (E, F) X-gal staining revealing lineage traced hub cells (outlined) in the absence or presence of ectopic CycD-Cdk4. Testes contain either (E) marked hubs (example is from a control male, genotype: E132-gal4;;UAS-Flp/Act5c>Stop>lacZ), or (F) marked hubs and marked CySCs and their descendants (F, arrows), referred to herein as “CySC clones” (example is from an experimental male, genotype: E132-gal4; UAS-CycD, UAS-Cdk4; UAS-Flp/Act5c>Stop>lacZ). (G) Bar graph showing that flies expressing CycD-Cdk4 in hub cells have significantly more CySC clones than age-matched uninduced flies of the same genotype (bars 4 vs. 6), and than flies lacking ectopic CycD-Cdk4 that are processed in parallel (bars 5 vs. 6). The low percentage of CySC clones in testes from newly eclosed males represents background levels of labeling; this does not change significantly over time (bars 1,3,5). ns, not significant. *** P value < 0.0001 (two-tailed Fisher’s exact test). Scale bars, 20 µm (A–D) or 100 µm (E–F). See also Table S4.