| Fibroblast growth factors (FGFs) are small secreted polypeptide growth factors which have been proved to participate in a wide variety of processes, including growth, development, sex determination and differentiation, and cell differentiation, proliferation, migration and apoptosis. To date,22 FGFs (FGF1-23, FGF19 is a human ortholog of mouse FGF15) have been identified in mammals and 27 in zebrafish, but not in other species in details. Studies on the functions of FGF family members, especially in sex determination, sex differentiation and gonadal development, were focused on mammals, while a few were reported in fish. In order to study the possible roles of FGF family members in teleost sex determination, sex differentiation and gonadal development, we systematically analyzed the FGF family members in the Nile tilapia (Oreochromis niloticus) from genome-wide transcriptome analysis, and investigated the possible role of FGF9/16/20 subfamily in the development of tilapia gonad.In the present study, we isolated 33 members in Nile tilapia by homology research using transcriptome and genome sequences. Seven of them (FGF1b、4b、11b、12b、13b、14b and 23b) are newly identified. Based on the isolated Nile tilapia FGF sequences, we isolated 26-33 FGFs from stickleback, medaka, fugu, tetraodon and newly sequenced burton’s haplochromis, princess of burundi, nyererei and zebra mbuna genomes. Our data supported the non-existence of FGF9 homolog in all teleost genomes and demonstrated that there are the most members were isolated in the genomes of the five cichild fish mentioned above. Of all newly identified members, FGF23b was only isolated in the five cichlid fish genomes; FGF4b in perciform fish genomes; FGF14b in the five cichlid fish and fugu genomes, FGF1b was not isolated in fugu and zebrafish; FGF12b was not in stickleback, fugu and medaka; FGF 13b was not in stickleback, tetraodon and medaka genomes; and FGF11b could be isolated in all opened teleost genomes. The phylogenetic analysis demonstrates that vertebrate FGF family can be devided into seven subfamilies:FGF 1/2, FGF4/5/6, FGF3/7/10/22, FGF8/17/18, FGF9/16/20, FGF11/12/13/14 and FGF19/21/23, as in human and zebrafish. In this study, using both phylogenetic reconstructions and precise synteny conservation analysis, we show that all the newly isolated FGF genes are homologs of human FGFs except for FGF24 which is a fish specific FGF gene. Through analyzing the phylogeny and gene structure of known FGF8/17/18 subfamily members, we confirmed that the evolution history of FGF8/17/18 subfamily is different from other FGF subfamilies, and it seprated from other FGF subfamilies during the early evolution (possiblely in sea anemone). Through comparative transcriptome analysis, we found 20 of 33 FGFs are expressed in the Nile tilapia gonad.9 of 20 FGFs are sexual dimorphic expressed genes. Four (FGF1a,10b,16 and 20b) expressed higher in female than male, whereas five (FGF2,13a,13b,14b and 24) expressed higher in male than female.The roles of FGF9/16/20 subfamily members in the gonadal development of teleost fish have not yet been reported. Three FGFs (16,20a and 20b) of the FGF9/16/20 subfamily were cloned from the Nile tilapia by RT-PCR and RACE. Phylogenetic, bioinformatic and syntenic analyses demonstrated that these cloned FGFs are genuine FGF 16,20a and 20b. Our analyses further supported the non-existence of FGF9 ortholog and the existence of duplicated FGF20s in teleost genomes. Tissue distribution analysis by RT-PCR demonstrated that FGF16 was expressed in a wide range of tissues including the testis and ovary, FGF20b in the brain, pituitary, intestine and ovary, but not in the testis, while FGF20a in the brain, pituitary and spleen, but not in the gonad. The expression profiles of FGF 16 and FGF20b during normal and sex reversed gonadal development were investigated by real-time PCR. Both showed much higher expression in the XX ovary and 17 beta-estradiol induced XY ovary compared with the XY testis and fadrozole and tamoxifen induced XX testis, with the highest in both sexes at 120 dah. Strong signals of FGF 16 and FGF20b were detected in phaseⅡoocytes by in situ hybridization. These data suggest that FGF9/16/20 subfamily is involved in the early oocyte development of the female.In summary, we identified 33 FGFs members in the Nile tilapia genome, and seven of them are newly identified. These newly identified members are existed in the newly sequenced five cichlid fish genomes, but only few could be isolated in other opened teleost genomes. Transcriptome analysis demonstrated that 20 of 33 Nile tilapia FGFs are expressed in gonad, and their roles in the sex determination, sex differentiation and gonadal development need to be investigated. Analysis of FGF9/16/20 subfamily FGF 16 and FGF20b revealed that FGF 16 and FGF20b may be involved in regulating the development of oocyte in the ovary. |
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