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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. [ABSTRACT FROM AUTHOR]