Study On The Function And Regulation Mechanism Of SMAD2 Alternative Splicing Form(SMAD2W_?11)in Chicken Sex Determination

Sex determination is a complex and plastic developmental process,which has been a long-term research hotspot in the fields of biology,medicine,and animal husbandry.Studying the mechanism of sex determination not only is important for understanding the mechanism of development and differentiation process,but also could effectively improve the production efficiency of economic animals.Specifically,the sex of chickens has a prodigious relationship with their productivity.In poultry production,the production efficiency of females in commercial laying chickens is much higher than that of male individuals;while in the Broiler chicken production,males grow faster,produce more meat than female individuals,and have more significant feed rewards.Therefore,if the sex of chickens can be effectively controlled,the production efficiency would be greatly improved,and the waste of resources and related ethical issues caused by the elimination of chicks at birth can be avoided.Its importance and significance are self-evident.Hence,the establishment of efficient and accurate sex control methods has always been a hotspot in poultry production research.However,the bona-fide mechanism of chicken sex determination is still unclear,which heavily restricts the research and development of sex control technology in poultry production.In recent years,studies have shown that alternative splicing,as an important regulatory mechanism,not only grandly involved in the life activities of organisms,but also played an important role in the sex determination process.For instance,the alternative splicing event of Sxl in Drosophila and Sry gene in Mus musculus have been proved to regulate the process of sex determination.Although current studies have pointed out that there are a wide range of alternative splicing events in the development of chicken embryos,indicating that alternative splicing events are involved in the embryo's sex determination process,the role and function of alternative splicing in chicken sex determination still need further exploration.Based on this,this study employed the latest single-molecule real-time sequencing technology PacBio Sequel ? and the traditional next-generation sequencing technology Illumina HiSeq system to study the gene expression and alternative splicing differences at key time points during the sex determination process of chicken embryos.Then the chicken sex chromosome homologous gene SMAD2 on the W chromosome was identified with specific alternative splicing expression SMAD2W?11.Furthermore,we investigated its biological function in sex determination in ovo and in vitro.Finally,we explored the underlying mechanism of SMAD2W?11's role in sex determination.The Results not only provide a reference for studying chicken(bird)sex determination mechanisms,but also suggest the theoretical support for the subsequent in-depth study of the function and mechanism of other gene alternative splicing events in the process of chicken sex determination.The results of the study are as follows:(1)The sequencing results showed that the W chromosome gene SMAD2 has specific expression transcripts during the sex determination process in chicken embryos.Both male and female tissues samples at different stages during the sex determination,such as embryos(E0,HH Stage 1),gonads(E3.5-E6.5,HH Stage 21-HH Stage 30)and gonads(E18.5,HH Stage 44),were sequenced.The experimental results found that the overall transcription level difference between all male and female samples was small,and further analysis showed that there are 84 differentially expressed genes in the embryonic stage(E0,HH Stage 1),37 highly expressed genes in males and 47 highly expressed genes in females,62 of which are on the sex chromosomes(Z:30,W:32);the numbers of genes involved in the process of tissue determination are differentially expressed in male and female gonads at different stages are 122(E3.5,male 40,female 82),59(E4.5,male 14,female 45),59(E5.5,male 22,female 37)and 124(E6.5,male 46,female 78).Among them,the numbers of differentially expressed genes on the sex chromosomes are 65(E3.5,Z:27,W:38),44(E4.5,Z:8,W:36),22(E5.5,Z:1,W:21)and 53(E6.5,Z:16,W:37).A total of 3000 differentially expressed genes were observed between male and female gonadal tissues(E18.5,HH Stage 44),1952 highly expressed in males and 1048 highly expressed in females,243 out of which are on sex chromosomes(Z:206,W:37).We further collected tissues of male and female embryos(E0,HH Stage 1),gonads(E3.5-E6.5,HH Stage 21-HH Stage 30)and gonads(E18.5,HH Stage 44).The samples were mixed and divided into two groups for database construction and sequencing according to sex,male and female.A total of 56,844,862 subreads were detected in the male SMRT library while 53,056,064 in the female SMRT library.After further data processing,26,089 male non-redundant transcripts and female 23,889 non-redundant transcripts were obtained.The analysis results showed that full-length transcripts between two sexes exhibited small differences in the sex determination process,but they varied a lot between two sex chromosomes.In males,417 specific expression transcripts were observed on the Z chromosome while 0 on the W chromosome;in females,there were 208 transcripts specifically expressed on the Z chromosome and 60 on the W chromosome.The analysis results of differentially expressed genes showed that there are 12 genes with female-specific expression transcripts on the W chromosome,namely UBP2,UBE2R2,ZSWIM6,SPIN and SMAD2 and other genes.In next-generation sequencing with a combination of transcript levels in different periods and genders,it was found that SMAD2 gene on W chromosome has a specific variable splicing and differentially expressed during the sex determination process.At day 0 of the embryonic development,SMAD2 transcript levels on the Z chromosome in both sexes didn't exhibit a difference,while higher level of SMAD2Z transcripts in males than females was observed in sex differentiation stages(E3.5,E4.5,E5.5 and E6.5)with a significant increase at E18.5.On the contrary,the expression of SMAD2W was not detected in males during the entire developmental stage.In females,SMAD2W was expressed from E3.5 to E6.5 with a relative high expression at 18.5 days.The results of sequencing and co-analysis indicated that SMAD2Z and SMAD2W may play different roles in the development of female chicken embryos.(2)Study the function of SMAD2Z and SMAD2W in the sex determination process of chicken embryos in ovo and in vitro.The full-length and alternatively sliced SMAD2Z and SMAD2W were identified and cloned.qRT-PCR results presented splice variants ofSMAD2Z in both male and females and that of SMADW in females during early bisexual embryo development.Most splice variants of SMAD2Z are SMAD2ZFL and SMAD2Z?3,and the majority of SMAD2W splice variants are SMAD2WFL,SMAD2W?3 and SMAD2W?11.Further results proved that the specific expression in female ovaries of SMAD2W variable splicing body,SMAD2W?11,was further confirmed by the Northern Blotting.The lentiviral overexpression vector constructed with the sequences of SMAD2Z or SMAD2W and alternative splicing forms was successfully constructed for and verified.At the same time,three SMAD2 interfering vectors were constructed according to their consensus sequence part.The vector with the best interference efficiency was selected for further lentivirus packaging.The male and female CEF cell line was successfully isolated and identified in vitro.The function of full-length SMAD2Z and SMAD2W?11 was verified in DF-1 and male and female CEF cells,respectively.SMAD2 inference in DF-1 or amphiphilic CEF cells downregulated male-related genes and upregulated female-related genes.Conversely,SMAD2Z overexpression promoted the expression of male-related genes but inhibited that of female-related genes.At the same time,the expression of female-related genes was upregulated while that of male-related genes was downregulated upon SMAD2W?11 overexpression.An efficient in-vivo injection model of chicken embryos was established and used to verify SMAD2Z and SMAD2W in the sex determination process of chicken embryos.Histological results showed that interfering SMAD2 induced the sex reversal of gonads from male to female(7/34,20.59%),and overexpressing SMAD2W?11 also promoted the reversal of gonads from male to female(10/46,21.73%).;The collective effect of SMAD2 inference and SMAD2W?11 overexpression further contributed this process(23/41,56.10%).On the contrary,SMAD2Z overexpression alone induced gonad reversal from female to male(21/44,47.73%),which was confirmed by qRT-PCR and western blotting analyses of sex-related genes.These results together indicate that SMAD2Z contributes to the sex differentiation of embryos into males,while SMAD2W?11 triggers the development of embryos into females.(3)Investigate the molecular regulation mechanism of SMAD2W?11 in the female sex determination of chicken embryos.SMAD2Z and SMAD2W?11 His tag prokaryotic fusion expression vectors were constructed.The fusion protein was induced and purified.His-Pulldown and Mass Spectrometry(MS)technologies were utilized to verify the protein interaction of SMAD2Z and SMAD2Z in ovaries and testis,respectively.It is found that the differentially binding proteins are enrichment in pathways related to sex differentiation and development,hormone synthesis and post-protein modification processes.A crucial gene of the sex hormone synthesis pathway,CYP19A1,was found to specifically bind to SMAD2W?11 in ovaries,which was further confirmed by Co-IP.The function of CYP19A1 in the sex determination process of chicken embryos was verified in ovo and results showed that CYP19A1 overexpression effectively promoted the sex differentiation of chicken embryos towards females whileCYP19A1 interference triggered this process towards males.In conclusion,the specific splice variant SMAD2?11 on the W chromosome promotes the sex determination process of chicken embryos towards females by binding to CYP19A1.The underlying mechanism still needs further study.