The Function And Application Of T-box Transcription Factor Eomesa In The Development Of Median Fin

Appendages are an important organ of motion in vertebrates for survive.Various appendages make vertebrates adapt well to the three niches of water,land and air.The evolution and development of appendages has been a major scientific issue and a long-standing controversy topic in the fields of paleontology,comparative anatomy,developmental biology and evolutionary biology.The appendages of fish consist of median fins（including dorsal,caudal and anal fin）and paired fins（including pectoral and pelvic fin）.The pectoral fin and pelvic fin of fish are homologous to the tetrapod limbs.At present,the formation and development regulation patterns of limbs and paired fins are becoming more and more clear,but little is known about the developmental mechanism of median fins.In evolution,the median fin is the pioneer of the paired fin,and the dorsal fin in the median fins is an important organ for fish swimming.The dorsal fin is related to the movement behavior of fish such as maintaining stability,turning and braking during swimming.The swimming ability and predation ability of the non-dorsal fin fish such as“egg”goldfish are greatly reduced,but this kind of fish is popular among the ornamental fish enthusiasts due to its sleek body shape and slow swimming speed.The results of biological research in recent years have revealed that the T-box transcription factor family plays a key role in the formation of vertebrate appendages.And the eomesa point mutation homozygote of zebrafish appeared phenotype of adult dorsal fin missing.Goldfish and common carp are valuable aquatic fishes.In this study,we identify44 members of goldfish and 47 members of carp using the goldfish and carp genome database,respectively,and divided them into five subfamilies.Finally,we compared the number variation of T-box gene family members between amphioxus,mammals（human and mouse）and teleost fish（zebrafish,goldfish and common carp）.The results showed that the number of T-box genes in amphioxus was 11,which was obvious less than that in mammals（17）and about half of the number in zebrafish（26）.The number of T-box genes in goldfish and common carp was nearly twice that of zebrafish.We speculated that the effect of teleost-specific whole genome duplication（3R-WGD）and lineage-specific whole genome duplication（4R-WGD）on the number of T-box family genes in vertebrate.We also preliminarily studied the effect of T-box transcription factor Eomes on the development of median fins in common carp.The Eomes gene in mammals has two homologous genes eomesa and eomesb in zebrafish.And four homologous genes of eomesa1,eomesa2,eomesb1 and eomesb2 were identified in common carp.We identified these sequences by bioinformatics analysis,and designed four knock-out target sites.Then simultaneously knock out the eomesa1 and eomesa2 genes of Oujiang color common carp by CRISPR/Cas9 system.The 24 hpf efficiency evaluation showed that the population knockout efficiencies of eomesa1 gene four targets were between30-50%,30-40%,20-50%and 2-13%,respectively;and that of eomesa2 gene were between 40-50%,35-50%,15-70%and 7-15%,respectively.The 7 dpf efficiency evaluation showed that the population knockout efficiencies of eomesa1 gene four targets were 40%,80%,70%and 20%,respectively;the individual knockout efficiencies were 83.3%,76.7%,85.3%and 13.3%,respectively.The population knockout efficiencies of eomesa2 gene four targets were 30%,80%,70%and 20%,respectively;the individual knockout efficiencies were 76.7%,77.7%,50.0%and31.3%,respectively.The dorsal fin of color common carp was examined on 4 months after the injection.We examined all the fins of F₀ juveniles（n=145）in detail,and finally found that there were partial defects in the dorsal fins of twelve mutants,the anal fin of the one mutants（knockout efficiency of both eomesa genes are 80%）were completely absence,and the dorsal fin and anal fin dysplasia was observed in two mosaic mutant（knockout efficiency of both eomesa genes are great than or equal to90%）.It takes two years for the Oujiang color common carp to mature and the period of obtaining homozygous mutant is long.Therefore,we observed the development of dorsal fins of zebrafish,which is also belong to the Cyprinidae.We found that the wild-type zebrafish had formed a dorsal fin bud at a standard body length of about 6.3 mm,and the dorsal fin had formed substantially at a standard body length of about 7.5 mm.In the zebrafish homozygous mutant with point mutation in eomesa,the dorsal fin buds could not develop normally,and finally the dorsal fin exoskeleton fin-rays of adult zebrafish completely disappeared.The results of Alizarin red and Alcian blue double staining showed that 3 phenotypes were found in 30 mutants:the first one retains part of the endoskeletal fin rays and exoskeleton fin rays,accounting for 6.7%;the second is to leave only a part of the endoskeletal fin rays,accounting for 56.7%;the third is that the endoskeletal fins and exoskeleton fins disappeared,accounting for 36.7%.These results lay the foundation for studying the formation and development of median fins and understanding the similarities and differences between median fins and paired fins formation mechanisms.We also provide a theoretical basis for the possible mechanism of evolutionary amphibious median fins disappearing.Finally,it has provided a new technical method to transform the median fins of ornamental fish and improve its ornamental value.