Functional Identification Of Foxl2 Gene In Temperature-dependent Sex Determination In Trachemys Scripta

Temperature-dependent sex determination?TSD? is a notable model of phenotypic plasticity,which mainly exists in some reptiles,such as Trachemys scripta,Mississippi crocodiles,Eublepharis macularius,etc.These reptiles can determine the sex of the organism according to the range of incubation temperature,which have the characteristics of losing heteromorphic sex chromosomes and adapting to the temperature so quickly.Among them,red-eared slider turtles can produce offspring with different sex ratios at a smaller incubation temperature range?26??32??.Compared with genetic sex determination?GSD?,which has been studied extensively in mammals and birds,the underlying mechanism of TSD remain unclear.In this study,the gonads of red-eared turtles were not formed before stage 15.As the gonads develop to the temperature sensitive period from stage 15 to stage 19?also known as the sex determination period?,it will show the temperature-dependent differential expression of genes?such as Dmrt1,Sox9,AMH,etc.?.Foxl2 is a highly conserved female gene throughout the phylum of vertebrates.Many investigations just analyzed the expression pattern of Foxl2,without providing the functional evidence.In this study,transcriptome sequencing was performed on the early embryonic gonads of the T.scripta.Lots of related genes involving sexual differentiation were screened,among these genes,Foxl2was selected to be further cloned and its expression profile was analyzed.Most importantly,the functional characterization of Foxl2 was performed via the loss of and gain of gene approach.The detailed results are described as below:?1?Transcriptome analysis of the embryonic gonad of T.scriptaIn this study,transcriptome sequencing was performed on the early embryonic gonadal tissues of T.scripta,and a total of 12 cDNA libraries?F16 and M16,F17 and M17 respectively?were constructed.After comparative analysis,the filter condition is set to padj<0.05.Compared with the FPT gonads,474 and 810 differentially expressed genes were obtained in the stage 16 and 17 bisexual gonads,respectively.Among stage16,193 genes were up-regulated in FPT?female-producing temperature?and 283 genes were down-regulated in MPT?male-producing temperature?.At stage 17,412 genes were significantly up-regulated and 298 genes were significantly down-regulated.According to GO enrichment and KEGG pathway enrichment,the function of differential genes was analyzed,and four genes related to sex determination and gender differentiation?Foxl2,Dmrt1,Kdm6B,AMH?were selected based on the enrichment situation and related reports in other vertebrates.Among them,the FPKM value of Foxl2 in FPT gonadal glands at stage 16 and 17 was significantly higher than that of MPT gonadal glands.?2?cDNA sequence cloning and expression analysis of Foxl2The Foxl2 gene cDNA sequence with a length of 2324 bp was obtained by RACE cloning technology,in which 1?258 bp was 5'non-coding region?UTR?,1164?2324 bp was 3'non-coding region?UTR?,and 259?1164 bp was open reading frame?ORF??906bp in total?,and a total of 301 amino acids were encoded.Semi-quantitative PCR data showed that Foxl2 was highly expressed in adult ovarian tissues,and was also slightly expressed in liver and spleen,but not in testis,kidney and muscle.q RT-PCR further demonstrated that Foxl2 expression in adult ovaries was significantly higher than that in testis,while only weak or no Foxl2 expression was detected in some tissues?heart,liver,spleen,kidney and muscle?.q RT-PCR showed that Foxl2 began to show FPT-specific expression in gonads of stage 16 and 17 while the expression level in MPT gonads remained low,exhibitinga typical temperature-dependent expression pattern.In addition,the temperature shift experiment showed that the expression of Foxl2 was rapidly up-regulated on the 3rd day after MPT shift to FPT.Foxl2 gene expression also increased significantly in the gonads of stage 16 and 17 MPT embryos after E₂induction of estrogen,indicating that Foxl2 can rapidly respond to changes in temperature and sex hormones.?3?The functional loss-of and gain-of Foxl2By constructing lentivirus Foxl2 knockdown and overexpression vector systems and subsequently injecting into FPT and MPT embryos of T.scripta respectively before sex differentiation,the knockdown and knockin effects of the gene were examined.Morphological tissue observation and molecular examination show that,the structure of FPT gonads after Foxl2 knockdown were obviously masculinized,accompanied by the upregulation of testicular regulators Dmrt1 and Sox9.Immunofluorescence showed that AMH and SOX9 protein were detected in the masculinized medulla area,with lots of germ cells locating in the cortex,showing a female-to-male sex reversal.On the contrary,after Foxl2 overexpression,the MPT gonads differentiated toward females,cortical areas were highly developed and medullary areas were degraded.The expression level of Dmrt1 and Sox9 was significantly down-regulated,the expression level of Rspo1 was increased,and AMH and SOX9 protein were almost disappeared.The germ cells are completely distributed in the developed cortical region,showing a female typic distribution pattern.This study provides a solid evidence that Foxl2 is both necessary and sufficient for female sex determination in T.scripta.Herein a female reptilian sex determing gene was functionally identified for the first time,which laid a foundation for the further illustration of molecular mechanism of TSD.