Histological Observation And Transcriptome Analysis In Duck Bursa Of Fabricius Of Early Development Stages

The bursa of Fabricius(BF),as a central immune organ unique to birds,provides a proper microenvironment for B-cells development.The bursal B-cells undergo rapid proliferation and differentiation at embryonic stage,but 95% of them finally apoptosis after hatching.However,few studies focused on the molecular mechanism and reason for bursal B-cells apoptosis at embryonic stages of birds.We chose the BF of duck embryos and ducklings to be the experimental subjects.The three typical developmental stages of the BF in histomorphology were selected to sequence through HE staining.Through the high-throughput sequencing technology,the global genes expressions of early stages BF were obtained at each stages.By analyzing the differentially expressed genes(DEGs)among three stages,we explored mechanism of B-cells apoptosis and homeostasis regulation in duck embryonic BF.Meanwhile,we analyzed the expressions of innate immune related-genes to explore the function of innate immune related-genes in early developmental stages of duck BF.The results showed that:Histological observation explained that the bursa already formed two large fold mucous membrane at ED14.At ED22,the formed follicles of the bursa had embedded in the proper tunic.At D1,the medulla and cortex of follicles could be distinguished.Besides,the average length of BF exhibit significant differences(P<0.01)between each of the two stages.Transcriptome sequencing results showed that there were 8083,8962 and 1682 differentially expressed genes in ED14-ED22,ED14-D1 and ED22-D1 in duck BF,respectively.By KEGG enrichment analysis,we found that intestinal immune network for IgA production,glycine and cytokine-cytokine receptor interaction were always found in the three groups,which implied that they may play essential roles on the development of BF.Besides,Toll-like receptor signaling pathway,Jak-STAT signaling pathway and MAPK signaling pathways may take part in the regulation of B-cells proliferation and differentiation.Renin-angiotensin system and Ras signaling pathway may be associated with B-cells migration.The results of double immunofluorescence staining showed that the phenomenon of B-cells apoptosis were observed in the bursal follicles and follicle-associated epithelium atED22 and D1.The pathway “apoptosis” was also significantly enriched in ED14-ED22 and ED14-D1(P<0.05).Meanwhile,the expressions of Caspase-3,Caspase-7 and Bcl-2had significantly changes in ED14-ED22(P<0.01).These results demonstrated that bursal B-cells apoptosis firstly happened during ED14-ED22.Through the enrichment analysis of DEGs,intrinsic pathway for apoptosis and extrinsic pathway for apoptosis were significantly enriched(P<0.05).Besides,the results of RNA-seq and RT-PCR both found that the expressions of Bim,Bax,Bak,Cyt c,Caspase-8,Caspase-3,Caspase-7,Caspase-6,Fas,CD40,CD40 LG and Bid had significantly changes in ED14-ED22(P<0.01).TRAIL signaling pathway and its key genes(TRAIL,P53,TRAF6)were both significantly enriched(P<0.05).Meanwhile,the results of the protein to protein interaction(PPI)by using apoptosis-related genes that had significant differences(P<0.01)in ED14-ED22 showed that IGF-1,BMP4 and FOXO1 may take part in the regulation of bursal B-cells apoptosis.The significantly enriched DSBs,EBR and primary immunodeficiency implied that the false of bursal B-cells differentiation may result in genetic defects of B-cells in ED14-ED22.The expressions of TLRs,NLRs and RLRs were detected in duck bursa of Fabricius at the embryonic development stages.The results of RNA-seq,RT-PCR and Western blotting showed that the expression of RIG-? was increased with the development of BF at duck embryonic stages.At ED14,the RIG-I positive signals mostly located in the mucosal epithelium.At ED14,ED22 and D1,the RIG-I positive signals mostly distributed at FAE and the follicular middle layer between medulla and cortex.Besides,RIG-I overlapped with mature B-cells in the outer membrane since ED22.The analysis results of protein interactive relationships and cluster analysis found that TRIM25,RNF135,DHX58 and TMEM173 may induce RIG-? expression by RNA-seq.Meanwhile,the results of luciferase assays found that RXR-alpha-like and AP1 were the transcription factors of RIG-I and can positively regulate duck RIG-I expressions by combining its promoter sequence.However,expressed RIG-I cannot activate the expressions of down-stream genes in duck BF without infection.In conclusion,the bursal B-cells apoptosis firstly happened during ED14-ED22.The embryonic bursal B-cells apoptosis were mediated by mitochondrial and Fas signalingpathways,which may be induced by TRAIL.Meanwhile,BMP4,FoxO1 and IGF-1 may be the key regulators in the processes of bursal B-cells apoptosis.B-cells false differentiation may be one of the original reasons of its apoptosis at duck embryonic stages.Besides,with the development of duck,BF innate immune function was constantly improved.RIG-I was an essential preparedness of the BF immune function and a prevention to protect embryoin duck embryos and hatchlings,which suggested that the expression of PAMPs were essential members for improvement of BF innate immune function.