Fig 7. Egr induces ectopic Zfh-1 expression from testis sheath.

(A-C) Percentages of testes with ectopic Zfh-1 expression (A and B) and accumulation of early germ cells (C) from mated 5w-old (A) and 4w-old (B and C) males. (A) One copy of egr¹ allele significantly reduced the percentage of mated testes exhibiting ectopic Zfh-1 expression at 5w-old. N = number of the testes scored. (B-C) Suppression of reproduction-induced ectopic Zfh-1 expression (B) and accumulation of early germ cells (C) via ubiquitous knockdown of egr. (D-G) Testes from 1-3-day-old egr-Gal4>UAS-lacZ (egr>lacZ) males. (D) Testis immunostained for β-galactosidase (red), co-stained for F-actin (green) and DNA (blue). LacZ was expressed in testis muscle characterized by a chevron-like F-actin pattern. (E and F) An egr>lacZ testis immunostained for β-Galactosidase (red), co-stained for FasIII and Eya (green) and DNA (blue). (E) Thin optical section of the testis surface. LacZ was detected in the nuclei of muscles (arrows) and pigment cells (arrowhead). (F) Thin optical section of the testis interior. LacZ was not expressed in the cyst cells (Eya-positive cells, green). (G) LacZ was not expressed in the germ cells (Vasa-positive, green). (H) A testis from a 1-3-day old egr>nGFP male immunostained for DNA (red). GFP fluorescence was detected in the nuclei of testis muscles (arrows). (I) A testis from 1-3-day old w¹¹¹⁸ male immunostained for F-actin (red), co-stained for Arm (green) and Vasa (blue). Cyst cell cytoplasm located right beneath the muscles (arrows). (J-M) Expression patterns of tara-Gal4 (J and K) and fz2-Gal4 (L and M). UAS-LacZ expression driven by the two Gal4 lines were activated in testicular muscles characterized with the Chevron-like F-actin patter (J and L). LacZ expression was absent in the interior of testes (K and M). (N and O) Suppression of ectopic Zfh-1 expression (N) and accumulation of early germ cells (O) by depletion of egr via tara-Gal4 and fz2-Gal4. (P-O’) Testes from 14-day-old unmated males (30 males vs. 0 female) immunostained for Zfh-1 (green in P and Q; white in P’ and Q’), co-stained for FasIII and Eya (red in P and Q), and Vasa (blue in P and Q). Ectopic Zfh-1 expression in Eya-positive cyst cells was induced by egr overexpression via tara^ts driver (P-P’). Overexpression of egr in early cyst cells failed to trigger ectopic Zfh-1 expression (Q and Q’). Asterisks mark the hubs. P-values from Chi-squared test are shown in A-C, N, and O. *p<0.05, **p<0.01, and ***p<0.001. Mass-mating was conducted for results shown in A-C, N, and O.