The Function Of T-box Transcription Factor Eomesb In The Development Of Dorsal Fin Of Zebrafish

Life originated in the water.In the evolution from aquatic to terrestrial vertebrates,their appendages underwent a series of processes,including the change from paired fins to limbs,the evolution from limbs to wings,and even the loss of limbs,such as snakes and whales.The fins of fish can be divided into median fins and paired fins.The median fins are the pioneers of the evolution of paired fins.Paired fins are homologous to the limbs of tetrapods.At present,the pattern of formation and development regulation of limbs and paired fins tends to be clear,but little is known about the development mechanism of odd fins.The development of vertebrate appendage morphology has been a typical example of the morphological evolution of species.Many t-box genes,including T,Eomesodermin?Eomes?,all members of the Tbx2 subfamily and three members of the Tbx1 subfamily,play important roles in appendage development.Eomes,a member of the Tbr1 family,are involved in pattern formation and morphogenesis in frogs,fishes,and mice.The two paralogous genes of zebrafish eomes,eomesa and eomesb,showed49.3%amino acid similarity in the coding region and 88.2%amino acid similarity in the T-box DNA binding region.There are more studies on the eomesa gene.A point mutation in the eomesa gene causes zebrafish homozygous mutants to develop normally to sexual maturity,but females are unable to mate and lay eggs naturally,and both lacked dorsal fins.This study studied the role of eomesb in the development of zebrafish.In this study,the mutant of zebrafish eomesb gene was constructed by using CRISPR/Cas9 technology,with two different target sites?T4,target site 4 and T3,target site 3?.Among them,T4 screened two homozygous mutants?-14 bp and+13bp?;T3 screened two homozygous mutants?-10 bp and+10 bp?.The zebrafish eomesb T4 homozygous mutant constructed in this study not only could develop normally to sexual maturity,but also had normal fin development.Through the q-PCR and whole-embryo in situ hybridization technologies,we found that the eomesb mutation did not affect the expression of eomesa at all,and the eomesa^-/-;eomesb^-/-double mutant did not show any additional phenotypes.So,we speculated that there may not be functional redundancy between eomesa and eomesb during the developmental process of dorsal fin and eomesb may not be involved in dorsal fin development.In addition,the result from whole embryo in situ hybridization experiments showed that the eomesb mutation might not affect the early expression of its homologous genes tbr1a and tbr1b.The reason why the homozygous mutant of eomesb does not present any phenotype may be because either the function affected by the mutation does not result in obvious phenotype at all or other T-box genes have the effect of genetic compensation response to the mutation.During the screening of zebrafish eomesb T3?-10 bp?homozygous mutants,the phenotype of incomplete or complete absence of dorsal fin was found,but the fish with phenotype were irregularly distributed in the heterozygotes of F₂population and even WT?wild tipe?.The dorsal fin mutant fishes were incrossed and found that their offsprings also had the phenotype of dorsal fin mutation,which indicated that the mutant phenotype of dorsal fin might not be caused by the environment,but was caused by the gene mutation.Interestingly,like eomesa^-/-in F₂population,females without dorsal fin can develop normally to sexual maturity,but cannot mate and lay eggs naturally.Therefore,it is speculated that mutant gene may be similar to eomesa and also affect ovarian development.In order to study the effect of eomesb gene in the development of zebrafish,we used CRISPR/Cas9 technology to build the eomesb mutants.The homozygous mutant of eomesb did not show obvious phenotype,but a dorsal finless zebrafish was obtained by accident,which provided new experimental materials for studying of the development of median fin.