Identification And Comprehensive Analysis Of Genes,lncRNAs And MiRNAs During Chicken Embryonic Gonadal Development

In chicken,sex differentiation are activated at embryonic day 5.5-6.5,then two important events occur.One is that a specific "trigger" trigger the glands toward to testes or ovaries.The other is the high speed of proliferation in primordial germ cells(PGCs).Sexual dimorphism in chicken which has a Z/W sex chromosome system is thought to arise largely as a consequence of sex-biased gene expression,including thousands of protein-coding genes and several micro RNAs(mi RNAs),and most of them are in the gonads.The genes belong to the chicken gonad differentiation pathway are activated at embryonic day 4.5-6,but the sexbiased genes are expressed as early as 12 hours of incubation.Therefore,the genes related to sex differentiation may be independent of the sex-biased genes,and some sex-biased genes may be in the gonad prior to differentiation.The development of chicken embryonic gonads(ovaries,testes)includes the migration and proliferation of PGCs,the proliferation of oocytes or spermatogonia,and the meiosis of oocytes.However,few researches on the growth environment of PGCs in vivo leads to cumbersome and high cost of in vitro culture conditions,which greatly limits the use of PGCs.Therefore,further research on the growth environment of PGCs in vivo is urgently needed.Studies have shown that mi RNA and long non coding RNA(lnc RNA)are involved in organogenesis and cell differentiation.Therefore,this study(1)collected E5(embryo 5)-E9 gonads(ovaries or testes)for morphological observation to understand the differentiation of chicken ovary and testis,and the proliferation of PGCs in the ovary and testis by immunofluorescence situation;(2)Collected gonadal tissues during sex differentiation(E5,E6 and E9),then constructed lnc RNA library and small RNA library for sequencing to identify the expression profiles of genes,lnc RNA and mi RNA;Compared the differentially expressed genes,lnc RNAs and mi RNAs between females and males in the three periods to identify sex-biased genes,lnc RNAs and mi RNAs;(4)Identified the genes,lnc RNAs and mi RNAs involved in the ovarian and testicular differentiation;(5)Combined with the developmental phenomenon of PGCs during gonad differentiation,analyzed and selected genes,lnc RNA and mi RNA that may be related to the self-renewal of PGCs,and constructed an lnc RNA-mi RNA-m RNA interaction network;(6)Prepared pluripotent PGCs,RT-q PCR can identify genes and mi RNA target cells that may be related to the development of PGCs.The main results are as follows.1.Morphological observation results showed that before sex differentiation(E5),the gonads were neither male nor female.After gonadal differentiation(E8 and E9),males develop bilateral testes,whereas female develop only a left ovary.2.We collected more than 2,000 gonads,then constructed 36 lnc RNA libraries and 36 small RNA libraries for sequencing.The results showed that 17348,16909 and 17081 genes,4730,4283 and 4473 known lnc RNAs,and 1095,1112,and 1078 known mi RNAs were identified at E5,E6 and E9,respectively.3.A total of 253,966,5058 differentially expressed genes(DEGs),23,32,336 differentially expressed genes Known lnc RNA(DELs),20,7,41 differentially expressed known mi RNAs(DEMs)were identified by comparing the female and male(F5 vs M5,F6 vs M6,F9 vs M9),respectively.4.By comparing the left ovary(F5L vs F9L)and bilateral testes(M5 vs M9),3685 genes,208 lnc RNAs and 87 mi RNAs were identified and may be related to ovarian differentiation,1678 genes,275 lnc RNAs and 112 mi RNAs may be related to testicular differentiation.During ovarian differentiation,genes related to testicular differentiation or development are down-regulated,and genes related to ovarian development are upregulated(CYP19A1,FSHR,EMB,etc.).During testicular differentiation,genes related to ovarian differentiation are not activated,and genes related to testicular differentiation are up-regulated(AMH,SOX9,DMRT1,etc.).Functional enrichment analysis found that a large number of gene-enriched functions and pathways are related to organ development and cell development,such as gonad development,cell adhesion,and ECM-receptor interaction.Six genes(TGFB2 and Fox family)potentially causing ovarian or testicular differentiation were screened,two mi RNAs and one lnc RNA that could potentially initiate CYP19A1 expression.5.We analyzed the DEGs,DELs,and DEMs in the comparison between the dual(F5L vs F5 R,F6L vs F6R)and(F9L vs F9 R,M9L vs M9R),and constructed lnc RNA-m RNA,mi RNA-m RNA interaction networks.Finally,there were several significant enrichments,31 genes,3 and 13 candidate lnc RNAs and mi RNAs related to PGCs development were selected as well as five lnc RNA-mi RNA-m RNA interaction networks that may be related to the development of PGCs.6.We successfully isolated and cultivated PGCs.We performed genomic manipulation on the obtained PGCs,and injected them into the recipient chicken,which can continue to develop in the recipient chicken.RT-q PCR results identified the target cell of candidate genes and mi RNA related to PGCs development.Among them,CYP19A1 gene was only expressed in E9 left ovarian fibroblasts,STK31,DDX4 and TDRD9 genes were expressed both in PGCs and E9 left ovarian fibroblasts.The target cells of mi R-302b-3p,mi R-302b-5p and mi R-302 d were PGCs.In summary,this study obtained the expression profiles of genes,lnc RNAs and mi RNAs in the gonads during the sex differentiation,and obtained the response to different events in the sex differentiation process(gender undifferentiated state,testicular differentiation,ovarian differentiation and PGCs development).The results provided a theoretical basis for the developmental fate of testes,ovaries and PGCs during the process of chicken sex differentiation.