Figure 2.

Follistatin acts downstream of Dp/E2f1 in CySCs to maintain hub cells

(A) Model of the Activin pathway. Fs (purple) inhibits Activin ligands (red) binding to Activin receptors, Baboon (Babo, type I receptor) and Punt (Put, type II receptor). Receptor activation causes phosphorylation (orange P star) of the SMAD3 homolog Smox (inactive Smox is gray; active Smox is brown). Active Smox associates with the Co-SMAD Medea (Med, yellow) at regulatory sites of target genes to alter transcription.

(B and C) A control tj-GAL4 (labeled tj>LacZ, LacZ) adult testis has a normal number of hub cells (B), whereas a tj>Fs-RNAi, LacZ (C) adult testis has only 2 hub cells. Both testes were isolated after 14 days at 29°C to induce maximal GAL4 activity. Fas3 (green) labels hub cells, Vasa (red) marks germ cells, and DNA marked by DAPI is white.

(D) Graph showing the average number of hub cells at 0 and 14 days at 29°C in tj>LacZ, LacZ (gray bars, n = 49 and n = 22, respectively), tj>Fs-RNAi, LacZ (purple bars, n = 32 and n = 28, respectively), tj>Fs-RNAi, UAS-Fs (pink bars, n = 31 and n = 34, respectively), or tj>LacZ, UAS-Fs (blue bars, n = 37 and n = 31, respectively). See Table S1 for n values.

(E) Graph showing the average number of hub cells at 0 days and 4 weeks in Fs^null mutant (n = 18 and n = 31, respectively).

(F) Graph showing the fertility (brood size) at 4 weeks in a control and Fs^null mutant (n = 50 in both cases).

(G and H) Expression at 0 days of adulthood of Fs-GAL4 in a control testis (Fs>GFP, LacZ) or a testis in which Dp was depleted throughout development (Fs>GFP, Dp-RNAi). Fs-GAL4 is expressed strongly in CySCs and early cyst cells and weakly in hub cells (G) but its expression is substantially reduced when Dp is depleted (H). GFP (green) labels Fs-GAL4-expressing cells, Zfh1 (red) marks CySCs, Fas3 (blue) marks hub cells, and DNA marked by DAPI is white. Blue line surrounds hub cells and green line marks 25 μM away from the hub.

(I) Graph of relative GFP intensity in Fs>GFP, LacZ testes (labeled LacZ, gray bars, n = 13) or Fs>GFP, Dp-RNAi testes (labeled Dp-RNAi, brown bars, n = 13) in CySCs and early cyst cells (i.e., area between blue and green lines in (G and H) (labeled “25 μM ring around the hub”) and in hub cells (i.e., the area within the blue line, labeled “Hub”).

(J) Graph of the number of hub cells in testes from control tj-GAL4, hh-GAL80 males (gray bar, labeled “tj>+,” n = 27) or these males expressing Dp-RNAi and LacZ (brown bar, n = 8) or expressing Dp-RNAi and UAS-Fs (yellow bar, n = 17). UAS-Fs significantly rescues hub cell number compared with Dp-RNAi alone.

Error bars represent the data range. ^∗∗∗∗ p < 0.0001; ^∗∗∗ p < 0.001. ns = not significant as assessed by Student’s t test.

See also Tables S1 and S2; Figures S3 and S4. Scale bar, 10 μM.