Single-cell transcriptomes and runx2b−/− mutants reveal the genetic signatures of intermuscular bone formation in teleosts

ABSTRACT: Purpose: Intermuscular bones (IBs) are hard spicules, mainly existing in the myosepta of recent vertebrates. This evolutionary trait has puzzled biologists and consumers, and the molecular basis of IB development remains unclear. The goals of this study are to acquire characteristic single-cell maps of gene expression landscapes and lineages and to elucidate the differentiation trajectory of cell clusters related to IB formation and obtain the key regulated gene of IB formation. Methods: The scRNA-seq profiles of 60 dpf wild-type zebrafish and 60 dpf runx2b-/- zebrafish were generated, using the 10X Genomics Chromium system. The bulk RNA-seq profiles of 60 dpf wild-type zebrafish and 60 dpf runx2b-/- zebrafish were generated, using mirVana™ miRNA ISOlation Kit. The scRNA-seq data were filted and clsutered by the Seurat package. The cell differentiation trajectorie was constructed, using Monocle analysis. Runx2b knockdown was preformed by RNA interference and runx2b was inducted by neuropeptide substance P. qRT–PCR validation was performed using SYBR Green assays. The mutant zebrafish lines were constructed, using cripsr cas9 technology.The Phenotypic observation was perfomed by Hematoxylin and Eosin (HE), Alizarin red, masson trichrome staining and mico-CT. The nutritent content was also obtained including fatty acid content and anio acid content. Results: we reveal that teleost fish IBs originate from tendons in the skeletal muscle and that the differentiation trajectory from tendon progenitors and defined runx2b as the key rugulated gene of IB formaiton, using cripsr cas9 technology. Conclusions: This study is the first to reveal that teleost fish IBs originate from tendons in the skeletal muscle and that the differentiation trajectory from tendon progenitors to the osteoblast lineage is key for IB formation. Large-scale gene function analysis using CRISPR-Cas9 identified the decisive role of runx2b in IB formation. The loss of runx2b significantly restricted osteoblast differentiation and inhibited IB formation, which potentially provides a basis for breeding strains of fish without IBs to improve the safe consumption and economic value of many aquaculture species and in this way, boost the proportion of fish for the supply of animal protein worldwide (Fig. 6). For this reason, we needed to verify that the content of fatty acid in the runx2b−/− mutant was higher than that in runx2b+/+ fish, as well as the expression of key genes related to IBs in the runx2b−/− and runx2b+/+ strains obtained by scRNA-seq data. This study also provides materials and directions for further study to clarify the specific regulatory mechanism of IB formation and will help to discover more specific genetic resources that can be used for IB trait improvement in fish.

ORGANISM(S): Danio rerio

PROVIDER: GSE186957 | GEO | 2021/11/04

REPOSITORIES: GEO