Intermuscular bones(IBs)are mineralized spicules,present in the myosepta of many,but not all,teleost species.IBs are often small and sharp,and they consequently limit how the fish can be processed;the IBs may cause injury or trauma if lodged in consumers'throats or mouths,and therefore affect the appeal of the fish to many consumers.The development of IBs in teleosts is still not fully understood and the molecular basis of IB development remains to be established.Here,the characteristics of IB tissue are evaluated based on single-cell transcriptomics in wild-type zebrafish.The analysis defined 18 distinct cell types.Differentiation trajectories showed that IBs are derived from tendons and that a core tendon-osteoblast cell lineage is related to IB formation.In particular,the functions of 10 candidate genes were evaluated via CRISPR-Cas9 mutants.Among those,runx2b-/-mutants completely lost IBs,while swimming performance,growth and bone mineral density were not significantly different from runx2b+/+zebrafish.Comparative single-cell RNA sequencing(scRNA-seq)analysis in runx2b-/-and runx2b+/+zebrafish revealed the role of osteoblasts in IB formation.In addition,differentially expressed genes were enriched in the transforming growth factor β/bone morphogenetic protein(TGF-β/BMP)pathway after runx2b deletion.This study provides evidence for the crucial role of runx2b regulation in IB formation.Genetic breeding can target runx2b regulation and generate strains of commercial fish species without IBs,which can improve the safe consumption and economic value of many farmed fish species.

Chun-Hong Nie;Shi-Ming Wan;Yu-Long Chen;Ann Huysseune;Ya-Ming Wu;Jia-Jia Zhou;Alexandre Wagner Silva Hilsdorf;Wei-Min Wang;Paul Eckhard Witten;Qiang Lin;Ze-Xia Gao

College of Fisheries Key Lab of Freshwater Animal Breeding,Ministry of Agriculture and Rural Affairs/Key Lab of Agricultural Animal Genetics Breeding and Reproduction of Ministry of Education/Engineering Research Center of Green Development for Conventional Aquatic Biological Industry in the Yangtze River Economic Beit Ministry of Education,Huazhong Agricultural University,Wuhan 430070,China;Hubei Hongshan Laboratory,Wuhan 430070,China;CAS Key Laboratory of Tropical Marine Bio-Resources and Ecology,South China Sea Institute of Oceanology Innovation Academy of South China Sea Ecology and Environmental Engineering,Chinese Academy of Sciences,Guangzhou,510301,China;Department of Biology,Ghent University,GhentB-9000 Belgium;Unit of Biotechnology,University of Mogidas Cruzes Mogidas Cruzes San Paulo 08780-911,Brazil;Guangdong Laboratory for Lingnan Modern Agriculture,Guangzhou 510000,China

国家科学评论（英文版）

[1]Chun-Hong Nie;Shi-Ming Wan;Yu-Long Chen;Ann Huysseune;Ya-Ming Wu;Jia-Jia Zhou;Alexandre Wagner Silva Hilsdorf;Wei-Min Wang;Paul Eckhard Witten;Qiang Lin;Ze-Xia Gao-.Single-cell transcriptomes and runx2b-/-mutants reveal the genetic signatures of intermuscular bone formation in zebrafish)[J].国家科学评论（英文版）,2022(11):92-102

runx2b,intermuscular,Intermuscular,IBs,myosepta,lodged,mouths,runx2b+,+zebrafish

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