Expression patterns of ambra1 mRNAs and pathological findings in the zebrafish ambra1a and ambra1b KO lines. (A) RT-qPCR analysis of ambra1a and ambra1b mRNA expression in different adult zebrafish tissues. Data were generated from different biological replicates, each consisting of tissue samples from single individual (intestine, liver, ovary n = 4; brain, muscle n = 3; testis n = 2; intestine, liver, brain and muscle were a mix of female and male tissues). qPCR data were analyzed by 2−∆CT method and actb2 was selected as reference gene for normalization. Values represent the mean ± SEM. Statistical analysis was performed using Student’s t-test. * P < 0.05; *** P < 0.001. (B, C) Whole mount in situ hybridization with ambra1b (B) and ambra1a probes (C) in ovaries of 6-month-old WT zebrafish. Arrowheads point at somatic cells surrounding oocytes. Poc1, primary oocytes stage 1; Poc2, primary oocytes stage 2; Poc3, previtellogenic oocytes; Poc4, vitellogenic oocytes. (D) Representative ovary of a 18-mpf ambra1a−/− female showing follicular degeneration and presence of yolk free in the coelomic cavity as a result of degenerated follicles (H&E; scale bar, 200 μm). (E) Higher magnification of panel D, showing a moderate granulomatous reaction towards proteinaceous material (H&E; scale bar, 400 μm). (F) Testis of a 20-mpf ambra1b−/− male, showing diffuse cystic degeneration of the coelomic cavity (H&E; scale bar, 400 μm). (G) Testis of a 15-mpf ambra1b−/− male, showing diffuse ectasia of seminiferous tubules and hyperplasia of spermatogonia lining the tubules (H&E; scale bar, 200 μm). (H) Testis of 18-mpf ambra1a−/− male, displaying seminoma (H&E; scale bar, 200 μm). (I) Testis of a 17-mpf ambra1b−/− male, containing an undifferentiated germ cell tumor (H&E; scale bar, 200 μm). AF, atretic follicle; CST, cystic seminiferous tubule; GR, granulomatous reaction; I, intestine; L, liver; Y, yolk.
