Figure 5.
Increased Activin signaling is responsible for the decline of hub cell numbers during normal aging
(A–C) Expression at 0 days (A) and 4 weeks (B) of adulthood of Fs-GAL4, UAS-GFP in a control testis (Fs>GFP). Fs-GAL4 is expressed strongly in CySCs and early cyst cells and weakly in hub cells (A), and its expression is substantially reduced at 4 weeks of age (B). GFP (green) labels Fs-GAL4-expressing cells, Zfh1 (red) marks CySCs, Fas3 (blue) marks hub cells, and DNA labeled with DAPI is white. Blue line surrounds hub cells and green line marks a 25 μM ring from the hub.
(C) Graph of relative GFP intensity in Fs>GFP testes at 0 days (light gray bars, n = 15) or 4 weeks (dark gray bars, n = 17) in CySCs and early cyst cells (i.e., area between blue and green lines in (A) and (B) (labeled “25 μM ring around the hub”) and in hub cells (i.e., the area within the blue line, labeled “Hub”).
(D) Graph showing the reduction in hub cell number after 4 weeks relative to newly eclosed flies in control tj>LacZ (gray bar, n = 49 at 0 days and n = 66 at 4 weeks) and in tj>Fs (purple bar, n = 37 at 0 days and n = 42 at 4 weeks).
(E) Graph showing the reduction in hub cell number after 4 weeks relative to newly eclosed flies in control upd>lacZ (gray bar, n = 45 at 0 days and n = 84 at 4 weeks); upd>Smox-RNAi (pink bar, n = 19 at 0 days and n = 47 at 4 weeks); upd>babo-RNAi (blue bar, n = 38 at 0 days and n = 26 at 4 weeks); upd>daw RNAi-HMS01110 (first green bar, n = 33 at 0 days and n = 45 at 4 weeks); upd>daw RNAi-HMJ03135 (second green bar, n = 29 at 0 days and n = 46 at 4 weeks).
(F and G) Expression of daw in the testis tip, as detected with a protein trap (F) or enhancer trap (G). Both reporters show weak expression in the hub (marked by Fas3, red in F, blue in G, and indicated with a dotted line). Daw-GFP protein is also detected outside the hub, and the enhancer trap reveals expression in Zfh1-positive CySCs (red, G).
(H) Expression of daw mRNA increases with age in wild-type testes, as measured by qPCR (n = 8).
Error bars represent the data range. ∗∗∗∗ p < 0.0001; ∗∗∗ p < 0.001; ∗∗ p < 0.01; ∗ p < 0.05; ns = not significant, as assessed by Student’s t test.