Roles Of The Sox9 Transcription Factor In The Development Of Gonad And Cartilage In Nile Tilapia

Gonad is a reproductive organ in the vertebrates.Gonadal development is a very complicated biological process,and the characterization and roles of the related genes have always been attractive.Craniofacial malformation is a chondrogenesis-related disease,and deciphering the regulatory mechanism underlying chondrogenesis will be helpful for elucidating the genetic basis of human diseases.The Sry-related HMG-box(Sox)gene family are closely related to mammalian sex determination gene of Y chromosome(Sry).As a member of the Sox transcription factor family,Sox9 plays key roles in sex determination and differentiation,gonadal development,chondrocyte differentiation and formation processes in the vertebrates.In teleosts,such as Nile tilapia(Oreochromis niloticus,hereinafter referred to as tilapia),due to whole-genome duplication,there are two copies for the Sox9 gene,namely Sox9 a and Sox9 b.The tilapia gonadal transcriptome data revealed that the Sox9 a expression was low in the early stages of gonadal development in both male and female,and could not be detected in the later stages.The Sox9 b expression was low at the early stages but increased at 90 days after hatching(dah)and 180 dah.However,the localization and the function of Sox9 a and Sox9 b in the tilapia gonad or other tissues like cartilage remain elusive.In this study,we used the in situ hybridization approach to analyze the expression of Sox9 a and Sox9 b in the gonad and cartilage of the tilapia.Furthermore,based on CRISPR/Cas9 system,morphological observation,histological analysis,EdU test and immunofluorescence,we elucidated the roles and potential regulatory mechanism of Sox9 a and Sox9 b in the development of the gonad and cartilage.The main results are as follows: 1)Spatio-temporal expression of the Sox9 gene in tilapiaWe detected the expression of Sox9 in normal gonad at the different stages of development by FISH.The results showed that Sox9 a expression was weak in the somatic cells surrounding the germ cells at 5 dah;and was increased in male and female somatic cells at 20 dah and 30 dah,but could not be detected at 60 dah and later.In addition,the Sox9 b expression was low in the somatic cells surrounding germ cells and showed no significant difference between male and female tilapia at 5 dah and 30 dah.Nevertheless,Sox9 b was highly expressed in the somatic cells around the germ cells in male fishes at 90 dah and 180 dah.We also analyzed the expression pattern of Sox9 via WISH at 3 dah,a critical period for chondrogenesis at larval period.The results revealed that Sox9 a was highly expressed in the craniofacial cartilage,pectoral fin,vertebral cartilage and caudal bud cartilage.Sox9 b expressed highly in the brain but lowly in the cartilage during larval period.2)Roles of Sox9 a and Sox9 b in tilapia gonadal developmentGiven that Sox9 a and Sox9 b exhibit a differential expression profile in tilapia gonads,to define the roles of Sox9 a and Sox9 b in gonadal development,we performed knockout of Sox9 a and Sox9 b gene in tilapia through CRISPR/Cas9 technique,respectively.Based on Sox9 a homozygous mutant,we carried out immunofluorescence to detect the expression of marker gene during critical period of sex determination(5 dah)in tilapia.The results showed that the expressions of both Cyp19a1a(a highly expressed ovarian factor)and Gsdf(a highly expressed testis factor)could be detected in the female and male gonads of Sox9 a homozygous mutants,respectively.This implies that Sox9 a should not be involved in the primary sex determination and differentiation in gonads of tilapia.To explore whether the Sox9 a homozygous mutation could affect the proliferation of germ cells,we used Vasa antibody to mark the germ cells in the gonad and performed statistical analysis of the wild-type fish,Sox9 a heterozygous mutants and Sox9 a homozygous mutants at 8 dah,12 dah(the beginning of germ cell proliferation in female gonads),16 dah(two days after the beginning of germ cell proliferation in male gonads)and 20 dah.These results showed that there was no significant difference at 8 dah and 12 dah among three types.But at 16 dah and 20 dah,the number of germ cells in the Sox9 a homozygous mutants was significantly reduced,compared with the wild-type fishes.Furthermore,we detected cell proliferation case in gonads with EdU experiment at 15 dah and found that some somatic cells surrounding germ cells in the gonads of wild-type fishes had much positive signals,indicating that these cells were proliferating.But few positive signals in the gonads of Sox9 a homozygous mutants were detected.These results suggest that Sox9 a maybe plays a crucial role in the proliferation of somatic cells around germ cells in the gonads of juvenile tilapia,and then affects the proliferation of the germ cells.Due to Sox9 a homozygous mutants died at about 20 dah,we therefore analyzed the gonads of Sox9 a heterozygous mutants at the later stage of gonadal development.Our result showed that the size of testis in the Sox9 a heterozygous male mutants was significantly smaller than that of the wild-type male fishes at 180 dah,as demonstrated by gonadal somatic index(GSI,the ratio between gonad weight and body weight).Moreover,histological analysis revealed that the number of spermatogonia was significantly but the number of spermatocytes,spermatids and sperms was significantly decreased in Sox9 a heterozygous male mutants,compared with the wild-type fishes.These results suggest that Sox9 a maybe plays a vital role in the process of maintaining proliferation of germ cells.Meanwhile,we successfully induced Sox9 b mutation by CRISPR/Cas9 and obtained heterozygous mutants.Considering the high expression of Sox9 b in the testis,we focused on analyzing the effect of the Sox9 b heterozygous mutation on the development of the tilapia testis.Histological analysis revealed that spermatocytes(after onset of meiosis)were present in the testis of wild-type male fishes at 90 dah,while only spermatogonia cells were present in the testis of Sox9 b heterozygous male mutants.However,Spermatogenic cells at all levels has appeared in the testis of Sox9 b heterozygous male mutants at 150 dah.These results suggest that the Sox9 b heterozygous mutation may delay rather than block the initiation of meiosis in the germ cells of the testis.3)Roles of Sox9 a transcription factor in tilapia chondrogenesisGiven that Sox9 a expression was high in various cartilage tissues,we further analyzed the effects of Sox9 a mutation on the development of cartilage.Our results demonstrated that Sox9 a homozygous mutants could not eat food normally due to craniofacial bone deformity and died at about 20 dah.At 10 dah,morphological observation and Alcian Blue Staining showed that compared with the control,the craniofacial bone was severely deformed in the Sox9 a homozygous mutants,including absence of the Meckel's cartilage,the first branchial arch,the second branchial arch and the third branchial arch,resulting in obvious changes in craniofacial bone morphology.At 5 dah(during osteoid deposition and calcification),Calcein staining was performed with a fluorescent dye complexed calcium ions in vivo showed that the spinal of some homozygous mutant fish was curved and tail bud bones were smaller and curved and the craniofacial bone was severely deformed.These results indicate that Sox9 a may play an important role in the development of tilapia cartilage.By whole-embryo in situ hybridization experiment,we found that the chondrocyte differentiation marker gene Col2a1 a was highly expressed in the jaw cartilage,skull cartilage and pectoral fin of wild-type tilapia at 3 dah,which was similar to the expression pattern of Sox9 a in tilapia at 3 dah.However,the expression of Col2a1 a was significantly down-regulated in craniofacial cartilage of Sox9 a homozygous mutants,indicating that Sox9 a affects craniofacial cartilage development by up-regulating the expression of the Col2a1 a gene.In summary,we demonstrated the cellular localization of Sox9 expression in the development of tilapia gonad and cartilage,and revealed that Sox9 a played a significant role in the proliferation and maintenance of germ cells and somatic cells,but was not involved in primary sex determination and differentiation.Moreover,we confirmed that Sox9 a upregulated the expression of Col2a1 a to affect correct formation of craniofacial cartilage in tilapia.We also found Sox9 b might play vital role in the process of initiating meiosis in testis germ cells.These data will be helpful for better understanding the role of Sox9 transcription factor in the development of gonad and cartilage in teleosts,even in vertebrates.