Figure 5

Egr and Grnd are essential for ectopic Zfh-1 expression by protein refeeding. (A–D’) Testes immunostained for Zfh-1 (green in A–D’), co-stained for Vasa (red). (A–B’) Zfh-1-positive cells were restricted to the apical region in testes of OR and w¹¹¹⁸ flies fed for 24 days. (C–D’) Ectopic Zfh-1 expression (arrows) outside the apical region was found in testes from OR and w¹¹¹⁸ males starved for 18 days and re-fed for 6 days. (E) Percentages of testes exhibiting ectopic Zfh-1 expression. Refeeding induced ectopic Zfh-1 expression in testes from OR and w¹¹¹⁸. Ectopic Zfh-1 expression was rarely observed in testes from refed males of egr¹ homozygous or heterozygous. Ectopic Zfh-1 expression was almost not observed in males fed for 18–24 days. (F) Percentages of testes showing ectopic Zfh-1 expression. Testes were form males starved for 12–18 days, followed by refeeding for 6 days. (G–H’) Testes immunostained for DNA (blue in G and H, white in G’ and H’), co-stained for Vasa (red). Early germ cells characterized by intense DNA staining were overproduced (arrows) in the testes from males starved for 18 days followed by refeeding for 6 days (H). Overproduction of early germ cells was not found in testes from males fed for 24 days (G). (I–J’) Testes immunostained for Grnd (green in I and J, white in I’ and J’), con-stained for Arm (cyst cell cytoplasm, red) and DNA (blue). Anti-Grnd staining was observed specifically on the cyst cells. (K) Box-and-whisker plots showing relative anti-Grnd/anti-Arm signal intensity in cyst cells, normalized to the median value of anti-Grnd/anti-Arm intensity in the testes from fed males. Significantly higher levels of anti-Grnd staining were observed in the re-fed males (S18R6) compared to the age-matched fed males (fed 24). P value was calculated by Mann–Whitney nonparametric test. (L) Percentages of testes exhibiting ectopic Zfh-1 expression. One copy of grnd null allele (grnd^minos) dominantly suppressed the ectopic Zfh-1 expression in the testes of the refed males (S18R6). (M) Percentages of the testes with overproduction of early germ cells. Early germ cell overproduction by refeeding was specifically observed in wild-type (OR and w¹¹¹⁸) males, but not in egr¹ mutants. (N) Knockdown of egr in testis muscles via tara-Gal4 suppressed ectopic Zfh-1 expression induced by refeeding. Testes are dissected from refed (S18R6) males. P value was calculated by Chi-squared test.