CRISPR/Cas9-induced disruption of wt1a and wt1b reveals their different roles in kidney and gonad development in Nile tilapia.

Wilms tumor 1 (Wt1) is an essential factor for urogenital system development. Teleosts have two wt1s, named as wt1a and wt1b. In this study, the expression pattern of wt1a and wt1b and their functions on the urogenital system were analyzed by in situ hybridization and CRISPR/Cas9. wt1a was found to be expressed in the glomerulus at 3 dah (days after hatching), earlier than wt1b. wt1a and wt1b were simultaneously expressed in the somatic cells of gonads at 3 dah, while their cell locations were similar, but not identical in adult fish gonads. The wt1a-/- fish displayed pericardial edema and yolk sac edema at 3 dah and subsequently expanded as general body edema at 6 dah, failed to develop glomerulus and died during 6-10 dah, whereas the wt1b-/- fish were phenotypically normal. Immunohistochemical analyses revealed that the germ cell marker Vasa was expressed, while somatic cell genes Cyp19a1a, Amh, Gsdf and Dmrt1 were not expressed in the wt1a-/- gonads at 6 dah. The sex phenotypes of XX and XY in the wt1b-/- fish were not affected. Real-time PCR revealed that the ovarian cyp19a1a expression was up-regulated in XX wt1b-/- fish, compared with XX control at 90 dah. Serum estradiol-17β level was also up-regulated in XX wt1b-/- fish at 90 and 180 dah. The XY wt1b-/- fish had normal serum estradiol-17β and 11-ketotestosterone levels and remained fertile. These results suggest that Wt1a and Wt1b have different functions in the kidneys and gonads of tilapia.