Study On The Gonad Function Of Foxl2 Gene In Medaka

Objective:foxl2,a member of the forkhead transcription factor family,was originally reported and cloned in patients with blepharophimosis/ptosis/epicanthus inversus syndrome(BPES),a rare developmental disorder affecting the ovary.Researchers have investigated the biological function of the Foxl2 gene during ovarian development in mice and other mammals.Medaka(Oryzias latipes)is an important model organism in biological research,especially in reproductive research,who is the same as human in sex determination.In order to comprehensively explain the function of Foxl2 gene in gonadal development,this study intends to use gene editing technology to establish a new mutant material for the study of foxl2 gene function.According to phenotypic analysis,histological analysis and gene expression analysis between wildtype and foxl2 mutants,the function of foxl2 gene on the gonads would be firstly explored in Medaka.It provided a new animal model for further interpretation of the function of Foxl2 gene in gonadal development.Method:The CRISPR target site of foxl2 gene was designed,and the disruption of foxl2 gene was established in Medaka using CRISPR/Cas9 technology.The frameshift mutation of foxl2 gene were identified in FO generation by PCR detection and sequencing,who were selected as male parent crossing with WT female parent.After foxl2 parents crossing,the foxl2 gene heterozygous mutants and homozygous mutants were detected and obtained in F2 generation.The phenotypic characteristics and gonads were analyzed among wild-type males,wild-type females,foxl2 heterozygous mutants(foxl2 +/-)and foxl2 homozygous mutants(foxl2-/1-)The cell types,cell distribution and quantity in gonad were analyzed using tissue section-staining.The expression of gonadal development-related genes is analyzed by quantitative real-time PCR.Result:The foxl2 gene was successfully knocked out by CRISPR/Cas9 technology.4 base-pairs deletion and 11 base-pairs insertion mutation type of the foxl2 gene were selected as parents,to generate foxl2 mutants.Predicting both types of foxl2 gene mutations based on amino acids,it resulted in the loss function of FOXL2 protein that normally has a Forkhead domain.According to the phenotype,foxl2-/-mutant is male in physiology,in contrast,foxl2-/-mutant is female in genetics,who is XX type in the type of sex chromosome.The tissue analysis showed that the gonad offoxl2-/-mutant were different in gonad both the wild type ovary and wild type testis.There were degenerate nucleolar peripheral oocytes and sperm in gonad of mutants.Quantitative real time PCR analysis of the expression of gonadal differentiation and developmental related genes revealed that the loss of foxl2 gene resulted in upregulating the expression of sox9b,gsdf,and amh,in contrast,downregulating the expression of cyp19a1a.The disruption of foxl2 gene cause gonadal developmental disorder in Medaka.Conclusion:In this study,the foxl2 gene was successfully knocked out by CRISPR/Cas9 in Medaka.A new foxl2 gene mutant organism was generated.Phenotypic characteristics,genetic phenotype,gonadal histology and gene expression were performed among wild-type female,wild-type male,foxl2+/-mutants and foxl2-/-mutants,these results preliminarily revealed the characteristics of foxl2-/-mutants is male in physiology,who is female in hereditary.The results showed that the loss of foxl2 caused the up-regulating expression of sox9b,gsdf,amh and the downregulating expression of cyp19a1a by quantitative real time PCR.In summary,the disruption of foxl2 gene may cause the degradation or even disappearance of ovary function,which proves that the function of foxl2 gene is conservative in ovarian development from lower vertebrates to mammals.