| The intermuscular bones（IBs）are ossified from tendon in myosepta and IBs are small spicule-like bones existing in the muscle fillet,which are existed only in lower teleost and have a negative effect on fish quality and aquatic product processing.Megalobrama amblycephala possessed about 130 IBs.To date,most studies have been focusing on IB number,morphology,and ossification process in blunt snout bream.However,molecular mechanism of IBs remains unclear.In order to further study the molecular formation mechanism of IBs of M.amblycephala,based on four key IBs developmental stages of M.amblycephala（S1:IBs have not emerged in the tail part;S2:several small IBs started to ossify in the tail part;S3:IBs appeared rapidly;S4:all the IBs appeared with mature morphology）,we first compared the ossification of IBs between the four stages of M.amblycephala and other three different fish with the Alizarin Red bone staining method.The results indicated that the ossification of IBs might be related with fish swimming mode.Different staining methods were used to investigate the histological structure of IBs development in M.amblycephala,and i TRAQ comparative proteomics analysis showed the differentially expressed protein and key pathway regulating IBs development were screened;Histological structures showed that one bone cavity containing osteoclasts were existed in ribs,but not in IBs of M.amblycephala.We constructed the proteomics map for fish bones including IBs and ribs,and identified the differentially expressed proteins between IBs and ribs through i TRAQ LC-MS/MS proteomic analysis.Based on our lab previous omics data,we screened out 6 genes related to tendon,mineralization,and osteoblast development（scxa,scxb,bmp2a,bmp2b,nib）,and constructed zebrafish mutant lines with CRISPR/Cas9 gene editing to study its role in the development of IBs.The scxa and nib mutants can inhibit the IBs formation.This study clarified expression pattern and function of the protein in the IBs of M.amblycephala from histology,growth and development in this study,and which laid important foundation for depth study of the further exploring molecular mechanism of IBs growth and development.The main results are as follows:（1）Alizarin red bone staining showed that in M.albus,a typical anguilliform swimmer and belonging to Synbranchidae of Synbgranchiformes,initial epicentrals were ossified at anterior part at 30 days post hatching（dph）with the body length（BL）of 40mm.Then IBs were ossified from the anterior to the posterior part.At 55dph（BL100mm）,all the IBs were basically appeared with all type“I”epicentral bones.However,in M.anguillicaudatus,carangiform swimmer and belonging to Cobitidae of Cypriniformes,initial IBs including epineurals and epipleural bones were ossified at posterior part at 27dph（BL 17mm）.Then IBs were ossified from the posterior to the anterior part.At 40 dph（BL 35mm）,all the IBs were basically appeared with the types of“I”,“Y”and“卜”.Moreover,through the use of osteoblast specific transcription factor（osterix）green fluorescent protein transgenic（osterix GFP）zebrafish（Danio rerio）,the process of development and ossification of IBs were observed in vivo and the results showed that the IBs were ossified from the posterior to the anterior parts in D.rerio.The results from this study indicated that the ossification of IBs might be related to fish swimming mode,and their morphological polymorphism may be correlated with fish swimming mode and body type.（2）Based on the information of the four key periods of the intermuscular bone development of the bream at the early stage,in this study,different staining methods and comparative proteomics were conducted to investigate the histological structure and proteome of IB development in M.amblycephala.Alcian blue and alizarin red stained results indicated that IBs were gradually formed from S2 to S4,undergoing intramembranous ossification without a cartilaginous phase.A total of 3368 proteins were identified by using the isobaric tags for relative and absolute quantitation（i TRAQ）approach.Functional annotation showed that proteins which were differentially expressed among stages were involved in calcium,MAPK,Wnt,TGF-βand osteoclast pathways which played a critical role in bone formation and differentiation.Three proteins（collagen9α1,stat1,tnc）associated with chondrocytes did not exhibit significant changes through S2 to S4;however,proteins（entpd5,casq1a,pvalb,anxa2a,anxa5）which associated with osteoblasts and bone formation and differentiation showed significantly a higher expression level from S1 to S2,as well as to S3 and S4.These further demonstrated that development of IBs did not go through a cartilaginous phase.The inhibitors of TGF-βand Wnt pathways were tested on zebrafish（sp7/e GFP）and the results indicated that both inhibitors significantly delayed IB development.This study provides a comprehensive understanding of the IB ossification pattern,which will help further elucidate the molecular mechanisms for IB development in teleosts.（2）IBs and ribs both are a part of skeletal system in teleosts,but with different developing process.In this study,Histological structures showed that ribs of M.amblycephala possessed one bone cavity containing osteoclasts,while not in IBs.We the proteomics map for IBs and ribs of M.amblycephala were constructed,and the differentially expressed proteins were also identified between IBs and ribs using i TRAQ analysis.The identified 2342 proteins among IBs and ribs at 1-to 2-year old M.amblycephala,and GO annotation results showed that 1,451 proteins were annotated in biological processes,molecular function and cellular component.In 1-IB-vs-1-Rib and2-IB-vs-2-Rib comparison groups,93 and 154 differentially expressed proteins were identified,which maybe indicate the chemical composition in ribs and IBs of M.amblycephala existed significant difference.The two proteins associated with osteoclasts differentiation（vitronectin b precursor and matrix metalloproteinase-2）exhibited higher expression in ribs compared with IBs,which was in accordance with the results from histological structure.33 and 51 differentially expressed proteins in 1-IB-vs-2-IB and 1-Rib-vs-2-Rib comparison groups were annotated with KEGG,and results indicated that some bone-related pathways played a role in bone development and differentiation,such as Calcium,MAPK,Wnt,TGF-βpathway.Moreover,in 1-IB-vs-1-Rib and 1-IB-vs-2-IB comparison groups,11 and 13 candidate bone-related proteins were confirmed with MRM method.Our present study suggested the differentially key expressed proteins had a different role in the composition and growth development of ribs and IBs in M.amblycephala.These findings provide an important reference for understanding the function of economic fish bones（ribs and IBs）.（4）IBs are ossified by tendon,and is a part of the skeletal system.IBs formation process are involved in tendon cells and osteoblasts.To study the key genes regulated IBs development in M.amblycephala,we screened out 6 genes（scxa,scxb,bmp2a,bmp2b,nib,niba）related to tendon development and osteoblast development as candidate genes based on the sequencing and experiment data of the previous our lab,and used CRISPR/Cas9 gene editing technology to construct zebrafish mutants.The alizarin red bone staining results showed that the scxa-/-mutant not only had obvious defect on rib mineralization,but also had a significant reduction in the number of IBs with close to 70%,while the scxb-/-mutant had not any obvious skeleton mutation phenotype.Although scxa-/-mutants have serious defects in rib mineralization,the mutants were viable and fertile as adult fish.To further clarify the molecular mechanism by scxa affects the IBs development,we conducted comparative transcriptome analysis of dorsal tissue samples of scxa+/+（with IBs）and scxa-/-（without IBs）,and further verified the expression of key genes with q RT-PCR.We found that some genes related with osteoblast development and mineralization had a lower expression in the scxa-/-mutant,such as entpd5a,sp7,bmp2,etc.The alizarin red bone staining results showed that the bmp2a-/-mutant had no obvious IBs mutation phenotype,but the bmp2b-/-gene mutant was lethal at 48hpf and could not survive to adult fish.Besides,we discovered another important gene that regulates the development of IBs.Nib-/-gene mutants are completely missing IBs,while other part of bones in the fish body are normally mineralized.Micro-CT analysis showed that the swimming ability,muscle volume,bone mineral density and growth of nib-/-mutant and wild type were compared and analyzed,the results showed that there was no significant difference between nib-/-mutant and wild type.While alizarin red bone staining results showed that the niba-/-mutant had no obvious IBs mutation phenotype.In a word,the study identified key genes（scxa and nib）that controlled the amount of IBs in fish,which provides a reference for the cultivation of non-IBs strains of commercial fish,and also provides a good experimental material for studying the molecular regulation mechanism of IBs. |
