The Effect Of Eomesa Functional Deficiency On Reproduction Disorder In Female Zebrafish

The absence of the dorsal fin is a crucial feature of Ranchu,an important ornamental fish in China.Studies have shown that eomesa plays an essential role in fin development.The mutation of eomesa causes dorsal fin deficiency in zebrafish,Cyprinus carpio var.color,and Carassius auratus,but also causes reproductive disorders in female zebrafish,which hinders the breeding of new goldfish varieties with dorsal finless by utilizing eomesa.eomesa is a critical transcription factor in the T-box gene family which plays an essential role in the development of embryos.However,the mechanism of eomesa that affects reproductive disorders in female zebrafish is unknown.In this study,we constructed eomesa mutant lines using CRISPR/Cas9 technology and analyzed the effects of eomesa loss-of-function on female zebrafish reproduction through reproductive capacity analysis,reproductive behavior observation,comparative ovarian histological analysis,RNA-FISH and q RT-PCR of genes related to gonadal development;moreover,the transcriptome of ovarian in wild-type and mutants were carried out to explore molecular mechanisms of eomesa to the zebrafish reproduction.1.The eomesa homozygous mutant lines(eomesa^-/-)were constructed using CRISPR/Cas9 gene editing technology.Observing body shape and skeletal staining showed two distinct fin patterns in eomesa-/-zebrafish（absence of dorsal fin and lack of dorsal and anal fins）.Reproductive experiments showed that the number of males in the mutated line was significantly higher than that in the wildtype line,and the male could reproduce naturally;eomesa^-/-females could engage in courtship pairing behavior and were not able to ovulate naturally but could expel mature eggs by artificial pressure on the abdomen which could be produced offspring by artificial insemination.Histological analysis of the ovaries revealed that the morphology of ovarian and oviductal was normal in eomesa^-/-zebrafish;however,the development of oocytes was delayed by histological section analysis.The observation of embryo development also showed that the epiboly of the gastrula was postponed,and the malformation rate of embryos was significantly increased.（P<0.001）.In addition,the body length of mutants at 90dpf（days post fertilization）was significantly increased compared to that of the wild-type zebrafish（P<0.0001）.In summary,the deficiency of eomesa resulted in the absence of the dorsal and anal fins,a significant increment in body length,as well as an imbalance of sex ratio,and delayed development of oocytes;the mutation of eomesa also prevented natural ovulation and affected embryo’s development with a high rate of embryo malformation.2.The spatial analysis of eomesa and vasa,nanos3,the primordial germ cell development-related genes,and dmrt1,a critical gene for spermatogonia development,in the gonad of 90dpf wild-type zebrafish by RNA-FISH showed that eomesa was expressed in all periods of ovarian development and co-expressed with all genes in the primary growth phase of the oocytes;vasa was co-expressed with the eomesa gene in the spermatogonia of the spermathecae.The expression levels of six gonadal development-related genes were analyzed by q RT-PCR in mutant 12hpf embryos and three post-embryonic developmental stages,45dpf,60dpf,and 90dpf.The results showed that the primordial germ cell marker genes dnd1 and nanos3 were expressed in the somatic stage of embryonic development（12hpf）.The expression of dnd1,vasa,and nanos3 in mutant ovarian tissues at 45dpf was significantly higher than that of the wild type（P<0.01）;the expression of cyp17a1 and amh genes in mutant ovarian tissues at 90dpf was significantly lower than that of the wild type（P<0.01）.The expression of cyp17a1 and amh genes in the mutant ovarian tissues at 90dpf was significantly lower than that of the wild type（P<0.01）;In comparison,the expression of dmrt1 in the mutant was significantly higher than in the wild type（P<0.0001）.These results suggested that eomesa was involved in the gonadal development and affected egg maturation.The knockout of eomesa interfered with the development and differentiation of germ cells,as well as ovulation mechanisms.It might cause abnormal male-female differentiation by affecting the expression of cyp17a1 and dmrt1.3.Transcriptome analysis of ovarian tissues of 110dpf wild type and two homozygous mutated lines（dorsal fin absent mutated line,named mut1,and both dorsal and anal fin absent mutated line,named mut2）showed that 121 and 60 significantly upregulated genes were obtained in mut1 and mut2,respectively,and 109 and 75significantly down-regulated genes were obtained in mut1 and mut2,respectively,a total of 33 DEGs in mut1 vs mut2.The enrichment analysis showed that 67 GO terms were significantly enriched by DEGs,mainly involved in cellular processes and the immune system.Forty-two KEGG pathways（P<0.05）were significantly enriched by DEGs,including the Intestinal immune network for lg A production,cell adhesion molecules,One carbon pool by folate,Phagosome,and Herpes simplex virus 1 infection.The gene concept network map was constructed with GO enrichment results,and 10 genes were characterized as hub genes,including b2ml,b2m,mhc2a,mhc2dhb,and rnf168,involved in the immune system,slc25a32a,pou5f3,and bmp7a,involved in cell fate commit engaged in the formation of the primary germ layer,endodermal cell differentiation,specification,and cell fate commit.In addition,the deletion of eomesa function also resulted in the downregulation of atp2b1b,affecting Ca²⁺homeostasis in ovarian tissue.It is suggested that eomesa may regulates the gonadal development through the immune system,Ca²⁺homeostasis,and primordial germ layer cell fate differentiation.The loss of function of eomesa affects the oocyte’s development and maturation by inhibiting the expression of bmp7a and pou5f3,consequence in delayed oocyte development.In addition,it affects the quality of oocytes by upregulating the expression of slc25a32a.In summary,this study revealed the spatial expression pattern of eomesa in zebrafish gonadal tissues and constructed eomesa knockout mutant lines,this study also preliminarily explored the effects and molecular mechanisms of the eomesa on the reproduction of zebrafish females,these results provide a theoretical basis for the molecular breeding of Ranchu and sterile varieties of Cyprinus carpio by utilizing eomesa.