Identification And Functional Analysis Of Genes Related To Intermuscular Bone Development Of Megalobrama Amblyphala

Intermuscular bones?IB?,which occur only in the myosepta of the lower teleosts,are segmental,serially homologous ossifications from connective tissue in the myosepta.Most of the freshwater aquaculture fishes around the world,especially Cyprinidae species,possess a certain amount of IB.These IB have brought about many adverse impacts on the deep processing and food value of a fish species,because they are difficult to remove and make the fish unpleasant to eat.Meanwhile,IB reduces the economic value of a fish species,resulting in low price and exports.Blunt snout bream?Megalobrama amblycephala,Yih,1955?,also known as Wuchang fish,belong to osteichthyes,Cypriniformes,Cyprinidae,Megalobrama,which is naturally distributed in the middle and lower reaches of the Yangtze River in Central China,is a fish species with unique cultural and high economic value,which has been farmed in China's freshwater polyculture systems since the 1960s.However,the existence of IB has also restricted the economic value and expanding farming of M.amblycephala.Almost all existing studies have only focused on the morphology of IB in fish species.Few in-depth studies have been carried out and little information has been obtained regarding the molecular mechanism for development of IB.In order to further explore the development mechanism of IB,the microstructure of IB and related organizations was firstly observed in this study and the transcriptome comparison analysis was performed based on the tissue variability.Meanwhile,mi RNA and m RNA expression profiles involved in IB development were revealed over four key developmental stages.Abundant mi RNAs/genes associated with IB development were then identified.Besides,considering the significant differences in the morphological characteristics of IB between juvenile?6-month-old?and adult M.amblyceala?3-year-old?,we performed a full-length transcriptome analysis of IB through Pac Bio single molecule real time sequencing?SMRT?to supplement the annotation of the M.megalobrama genome.Then,with this as a reference,the expression patterns of lnc RNA and mi RNA in the IB of two stages were compared and analyzed in this study,so as to explore the regulatory mechanism of non-coding RNA in the growth process of IB.Finally,the candidate gene bmpr2a/2b was functionally verified by CRISPR-Cas9 gene editing,physical phenotypic observation and expression quantification,its role in bone mineraliztition was revealed.This study lays the foundation for depth exploring the molecular mechanisms of IB development in future.Main results and conclusions are as follows:?1?The results of tissue section observation revealed the microstructural features of IB,CT and muscle tissue,indicating that IB has a potential differentiation relationship with CT.The comparative transcriptome analysis among three tissues was then performed based on the tissue variability.A total of 706,740 and 134 specific expression unigenes were further generated in three tissues,respectively.In addition,5,162?6,758 and 8,745differentially expressed unigenes were detected in pairwise comparison of IB and connective tissue,IB and muscle,connective tissue and muscle,respectively.GO and KEGG pathway analysis showed that abundant unigenes were annotated to the signal pathways related to bone development,such as TGF-?,Wnt,MAPK and so on.Meanwhile,the mi RNA comparative analysis between IB and connective tissue finally obtained a total of 218 known mi RNAs belong to 97 mi RNA families.Of them,188mi RNAs were expressed in both tissues,while 13 and 17 mi RNAs were specifically expressed in IB and connective tissue,respectively.In addition,44 known mi RNAs exhibited significant expression differences between the two libraries,with 24 and 20differentially expressed mi RNAs exhibiting higher expression in the connective tissue and IB libraries,respectively.Functional annotation results showed that metabolism is the signaling pathway for enrichment of the most mi RNAs target genes.Meanwhile,we found that abundant mi RNAs target genes were annotated to Wnt,TGF-?,MAPK and osteoclast differentiation signaling pathways,which played important roles in bone development.These results indicated that the differentially expressed mi RNAs and targets may have potential function in IB development.?2?On the basis of four key development stages of IB?S1:IB haven't emerged;S2:a few IB with small length emerged in the tail;S3:more IB of greater length gradually emerged in the tail;S4:all of the IB in the tail have a mature morphology and length.IB have also emerged throughout the body of the fish from the tail to the head?,we constructed a reference transcriptome to analyze mi RNA and m RNA expression profiles over four IB developmental stages.As results,a total of 52,918 unigenes were obtained and 24,094 unigenes were annotated to 258 KEGG signaling pathways,including MAPK,Wnt,osteoclast differentiation and TGF-?signaling pathway.Through differential expression analysis,we identified 149,492,419,723,512,808 differential expression genes and 132,120,174,194,176,241 differential expression mi RNAs from six pairwise comparisons?S1-vs-S2,S2-vs-S3,S3-vs-S4,S1-vs-S3,S2-vs-S4 and S1-vs-S4?,respectively.We also found 59,239,569 and 470 development-dependent specific expression genes in the S1,S2,S3 and S4 stages,respectively.Twenty-six TGF-?genes showed a sustained decline in expression levels,while 14 TGF-?genes showed sustained increases in expression from stages S1 to S4.By referring to the KEGG map of the TGF-?pathway,we found that the TGF-?pathway cooperated with the MAPK and extracellular signal-regulated kinase pathways to regulate and control osteoblast differentiation.Besides,analysis of mi RNA-m RNA interaction revealed lots of mi RNAs and m RNAs related to bone development,such as mi R-133/133a/133b,mi R-222,mi R-3960,CL712.Contig1,Unigene28219,Unigene2429,Unigene271,etc.The quantitative expression of mi RNA and m RNA is consistent with the RNA-Seq results,confirming the reliability of the molecular resources explored in this study.Finally,a negative regulation effect of two mi RNAs was verified through dual luciferase reporter assay.The results showed that luciferase activity for the wild-type construct?GLO-tgfbr1a,GLO-runx2a?was significantly reduced by mi R-133b-3p mimics and mi R-206-3p mimics,but not by that of the empty construct?pmir GLO?.The wild-type construct GLO-runx2b was not reduced by mi R-206-3p mimics,which showed the inaccuracy of software prediction and the necessity of experimental verification.?3?The first full-length transcriptome analysis of IB was performed and 26,302novel isoforms from known genes as well as 6,033 isoforms from novel genes were identified.A total of 10,208 alternative splicing events involved in 5,875 genes was identified and the majority of alter spliced events was skipped exon.In addition,8,541poly?A?sites in 5,639 genes,1,462 transcription factors belong to 59 families and 31,186complete open reading frames were predicted,and 4,458 fusion transcripts and 5,199lnc RNAs were identified.These new findings provided important information for improving draft genome annotation and fully characterization transcriptome of M.amblycephala,and further help advance data sharing and deepen our understanding of IB development in fish.?4?The comparative transcriptome analysis results of non-coding RNA expression in IB between between juvenile?6-month-old?and adult M.amblyceala?3-year-old?revealed 353 differentially expressed lnc RNAs?170 up-regulated and 183 down-regulated?and 126?68 up-regulated and 58 down-regulated?differentially expressed mi RNAs.Subsequently,Lnc RNA co-expression/location m RNA and potential differentially expressed mi RNAs target differentially expressed Lnc RNAs were predicted.Based on the predicted results,we conducted correlation analysis on the competitive binding regulatory relationship between lnc RNA,mi RNA and target m RNA,and explored potential regulatory networks related to IB growth,such as lncrna-017869 competitively binding mir-142a-5p and lncrna-001094 competitively binding mirna-1331a/b-3p,so as to regulate the expression of downstream genes.Quantitative expression analysis results also verified the expression patterns of non-coding RNA in different growth stages of IB.?5?Considering significant enrichment of BMP signaling pathway in previous studies,we verified the function of bmpr2a/2b receptors through CRISPR-Cas9technology and focused on its role in intermuscular bone and skeleton development.Phylogenetic and genomic structure analysis indicated that the sequence characteristics of bmpr2a and bmpr2b genes in zebrafish were differentiated in the evolutionary process.Gene knockout results showed that the loss of bmpr2a and bmpr2b function did not cause significant changes in the IB phenotype,but bmpr2b^-/-mutant showed different degrees and types of mineralized bone malformations.The significant changes in the expression levels of BMP-Smad signaling molecules and various bone development genes,and the phenotypic changes in bmpr2b^-/-individuals revealed an important role for bmpr2b in bone mineralization of zebrafish.