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

Abstract

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.

Editing the duplicated insulin-like growth factor binding protein-2b gene in rainbow trout (Oncorhynchus mykiss)

“…Advancements in gene editing technology, particularly using the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system, has expanded the capacity for targeted gene mutagenesis in many animals, including fish 35 , 36 . This technology has been successfully performed in several aquacultured species, including Atlantic salmon 37 , 38 , catfish 39 , 40 , tilapia 41 , 42 , and carp 43 , 44 to induce a range of phenotypes related to fertility, muscle growth, and disease resistance. In Atlantic salmon the CRISPR/Cas9 system is efficient at inducing bi-allelic mutations in the F0 generation; although both homozygous and heterozygous mutants are produced that result in a proportion of individuals displaying a mosaic phenotype 37 , 38 .…”

Histological and sex-determining genes expression effects of 17α-methyltestosterone on mandarin fish Siniperca chuatsi gonad development

“…The expression of sox9 in the testis, ovary, brain, liver and heart of rainbow trout were detected and the results showed that the gene was only significantly expressed in the testis and brain [60]. Similar to rainbow trout, sox9b were found to be expressed at higher levels in the testes than ovaries in Nile tilapia [61,62].In orange-spotted grouper, the expression level of sox9 upregulated significantly and the results of tissue section showed that the gonad of it changed from ovary to testis during MT induction, indicating that sox9 may be an important factor in triggering and maintaining masculinity of orange-spotted grouper [63]. Our results suggest that sox9 may play an important role in the differentiation and development of testis.…”

Genomics to accelerate genetic improvement in tilapia

“…Gene editing methods have already been developed for Nile tilapia (Feng et al, 2015a;Li et al, 2015aLi et al, , 2015bXie et al, 2016;Wu et al, 2016aWu et al, , 2016bJiang et al, 2017;Li et al, , 2019. Targeted mutagenesis using TALENs and CRISPR/Cas9 technology have been successfully applied to understand the genetic basis of sex determination and sex differentiation in Nile tilapias.…”