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. 2019 Jul 11;15(7):e1008062. doi: 10.1371/journal.pgen.1008062

Fig 10. JNK signaling is required to maintain CySCs and early cyst cells.

Fig 10

(A) Averages of Zfh-1-postive cell numbers per testis from unmated males at 0-, 5-, and 10-day-old. Numbers of testes scored are shown at the bottom of each column. Error bars represente SD. (B-E) puc knockdown testes. Animals were maintained at 25°C during development. (B and C) Testes immunostained for Zfh-1 (green), co-stained for Vasa (red) and Eya (blue). Testes were from males shifted from 25°C to 29°C for 4 days (B) or 10 days (C). 10-day incubation at 29°C led to excessive accumulation of Zfh-1-positive cells and small germ cells (C). (D) Testis labeled for EdU (red) incorporation and co-stained for Zfh-1 (green). Arrows mark Zfh-1 and EdU double-positive cells away from the hub, suggesting S phase progression in ectopic Zfh-1-positive cells. (E) Testis immunostained for Adducin to label spectrosomes (arrowheads) and thin fusomes (arrows). Numerous spectrosome-positive GSC/Gb-like cells and thin fusome-positive spermatogonia were observed in puc knockdown testes. (F-G’) Testes from 1w-old puc-lacZB48 (F and F’) and TRE-EGFP (G and G’) males. (F and F’) Testis immunostained for β-Galactosidase (green in F and white in F’), co-stained for DE-cad (blue) and Vasa (red). LacZ was detected in nucleus of CySCs (arrows). (G and G’) Testis immunostained for GFP (green in G and white in G’), co-stained for Br-C (red) to mark CySCs and early cyst cells. GFP was detected in nucleus and cytoplasm of CySCs (arrows). (H-K) Testes with control or bsk1 clones marked by MARCM system. Asterisks indicate the hubs. Arrows indicate the GFP-positive CySC clones. Arrowheads mark the cyst cells with elongated cytoplasmic GFP. Scale bars = 50μm. (H and I) When dissected 2 days after clone induction (ACI), CySC clones (arrows) were observed in both FRT40A control (H) and FRT40A bsk1/+ (I) testes. (J and K) When dissected 5 days ACI, cyst cell clones with elongated cytoplasm (arrowheads) were observed in both FRT40A control (J) and FRT40A bsk1/+ (K) testes. CySC clones were lost in some FRT40A bsk1/+ testes (K). P-values were calculated with student t test in A. ns: p>0.05, *p<0.05, and ***p<0.001.