Figure 1.
GSCs in spi77-20 testes cycle faster than GSCs in control testes.
(A) Cartoon depicting the organization of germ line cells and somatic cells at the tip of wildtype testes. GSCs (light green) are organized around the hub (red). CySCs (light pink) encase GSCs and are also in contact with the hub. The gonialblast (dark green) is displaced away from the hub and encased by two cyst cells (dark pink). (B) The apical tip of a w1118 testis stained with antibodies labeling the cytoplasm of the germline cells (anti-Vasa, green), the membrane of the hub cells (anti-Fasciclin III, red), and mitotic chromatin (anti-pHH3, red). Arrowheads: GSCs, arrow: GSC in mitosis, scale bar: 10 µm. The inset shows the pHH3-positive GSC next to the hub (circle). (C–H) Genotypes as indicated. >500 stem cells were scored for each genotype. (C–E) The percentage of pHH3-positive GSCs (M-phase index). (C) ***p-value<0.0001. (D) Conditions as indicated. p-value = 0.18 (E) Conditions as indicated. ***p-value<0.0001; No significant difference was noted between 18°C and 26.5°C, p-value = 0.22. (F) The percentage of pHH3-positive CySCs. No significant difference was noted. p = 0.28. (G,H) GSC S-phase indices. (G) Ex vivo labeling of testes with BrdU, ***p-value = 0.0004. (H) Flies fed a continuous diet of BrdU for 36 hours or 48 hours, ***p-value<0.0001, **p-value = 0.0074.
Figure 2.
EGF signaling from the germline to the soma decreases the frequency of GSC divisions.
(A–C) The percentage of pHH3-positive GSCs for each indicated genotype. (A) The expression of s-spi in germ cells rescues the hyper-proliferation of GSCs in spi77-20 testes. (B) GSCs in stet1/stet3 testes showed an increased M-phase index compared to w1118 testes. (C) RNAi-mediated knock-down of EGFR in the soma causes a higher GSC M-phase index, ***p-value≤0.0001.
Figure 3.
The EGF repression of GSC divisions is developmentally regulated.
(A, B) Testes from (A) w1118 or (B) w-; spi77-20/spi77-20 3rd instar larvae stained with anti-Vasa (green) and DAPI (blue). Arrows: spermatocytes, arrowheads: early stage germline cells, scale bars: 50 µm. (C) M-phase index for the GSCs of each genotype. No significant difference was detected, all p-values>0.30. (D) A model depicting the requirement for EGF signaling. EGF is required in both larvae and adults for promoting germline differentiation. In contrast, EGF is not required in larvae for the repression of GSC division frequency, demonstrating a developmental uncoupling of EGF-function.
Figure 4.
Regulation of GSC division frequency is specific to EGF signaling.
(A) Graphic demonstrating how Jak/STAT and TGFβ signaling regulate stem cell fate. Upd: unpaired, Dpp: decapentaplegic, Gbb: glass bottom boat, Sax: Saxophone. (B–D) Testes stained with anti-Vasa. (B,C) Testes with germ cell-intrinsic overexpression of (B) dpp contain excessive clusters (white circles) of small early germline cells and (C) upd iare filled with small germline cells. (D) Image of a control w1118 testis. Asterisks: apical tips of testes, arrowheads: early stage germline cells, short arrows: spermatocytes, long arrow: elongated spermatids, scale bars: 50 µm. (E) Graph showing the percentage of pHH3-positive GSCs (M-phase index), genotypes as indicated. >500 stem cells were scored for each genotype. No significant differences were observed, all p-values>0.20.