单细胞测序分析牛骨骼肌发育的转录和开放染色质。

Transcriptional and open chromatin analysis of bovine skeletal muscle development by single-cell sequencing.

机构信息

Key Laboratory of Animal Genetics, Breeding and Reproduction of Shaanxi Province, College of Animal Science and Technology, Northwest A&F University, Yangling, China.

Guyuan Branch, Ningxia Academy of Agriculture and Forestry Sciences, Guyuan, China.

出版信息

Cell Prolif. 2023 Sep;56(9):e13430. doi: 10.1111/cpr.13430. Epub 2023 Mar 1.

Abstract

Skeletal muscle is a complex heterogeneous tissue and characterizing its cellular heterogeneity and transcriptional and epigenetic signatures are important for understanding the details of its ontogeny. In our study, we applied scRNA-seq and scATAC-seq to investigate the cell types, molecular features, transcriptional and epigenetic regulation, and patterns of developing bovine skeletal muscle from gestational, lactational and adult stages. Detailed molecular analyses were used to dissect cellular heterogeneity, and we deduced the differentiation trajectory of myogenic cells and uncovered their dynamic gene expression profiles. SCENIC analysis was performed to demonstrate key regulons during cell fate decisions. We explored the future expression states of these heterogeneous cells by RNA velocity analysis and found extensive networks of intercellular communication using the toolkit CellChat. Moreover, the transcriptomic and chromatin accessibility modalities were confirmed to be highly concordant, and integrative analysis of chromatin accessibility and gene expression revealed key transcriptional regulators acting during myogenesis. In bovine skeletal muscle, by scRNA-seq and scATAC-seq analysis, different cell types such as adipocytes, endothelial cells, fibroblasts, lymphocytes, monocytes, pericyte cells and eight skeletal myogenic subpopulations were identified at the three developmental stages. The pseudotime trajectory exhibited a distinct sequential ordering for these myogenic subpopulations and eight distinct gene clusters were observed according to their expression pattern. Moreover, specifically expressed TFs (such as MSC, MYF5, MYOD1, FOXP3, ESRRA, BACH1, SIX2 and ATF4) associated with muscle development were predicted, and likely future transcriptional states of individual cells and the developmental dynamics of differentiation among neighbouring cells were predicted. CellChat analysis on the scRNA-seq data set then classified many ligand-receptor pairs among these cell clusters, which were further categorized into significant signalling pathways, including BMP, IGF, WNT, MSTN, ANGPTL, TGFB, TNF, VEGF and FGF. Finally, scRNA-seq and scATAC-seq results were successfully integrated to reveal a series of specifically expressed TFs that are likely to be candidates for the promotion of cell fate transition during bovine skeletal muscle development. Overall, our results outline a single-cell dynamic chromatin/transcriptional landscape for normal bovine skeletal muscle development; these provide an important resource for understanding the structure and function of mammalian skeletal muscle, which will promote research into its biology.

摘要

骨骼肌是一种复杂的异质组织,对其细胞异质性以及转录组和表观遗传特征进行鉴定对于了解其发生过程的细节非常重要。在我们的研究中,我们应用 scRNA-seq 和 scATAC-seq 来研究从妊娠、哺乳和成年阶段的牛骨骼肌中的细胞类型、分子特征、转录和表观遗传调控以及发育模式。详细的分子分析用于剖析细胞异质性,我们推断出肌细胞的分化轨迹,并揭示了它们的动态基因表达谱。SCENIC 分析用于展示细胞命运决定过程中的关键调控因子。我们通过 RNA 速度分析探索这些异质细胞的未来表达状态,并使用 CellChat 工具包发现了广泛的细胞间通讯网络。此外,转录组和染色质可及性模式高度一致,对染色质可及性和基因表达的综合分析揭示了在肌发生过程中起作用的关键转录调节因子。在牛骨骼肌中,通过 scRNA-seq 和 scATAC-seq 分析,在三个发育阶段鉴定出不同的细胞类型,如脂肪细胞、内皮细胞、成纤维细胞、淋巴细胞、单核细胞、周细胞和 8 个骨骼肌肌源性亚群。伪时间轨迹显示这些肌源性亚群具有明显的顺序排列,根据其表达模式观察到 8 个不同的基因簇。此外,预测了与肌肉发育相关的特定表达 TFs(如 MSC、MYF5、MYOD1、FOXP3、ESRRA、BACH1、SIX2 和 ATF4),并预测了单个细胞的可能未来转录状态和相邻细胞之间分化的发育动态。然后对 scRNA-seq 数据集进行 CellChat 分析,对这些细胞簇中的许多配体-受体对进行分类,这些配体-受体对进一步分为有意义的信号通路,包括 BMP、IGF、WNT、MSTN、ANGPTL、TGFB、TNF、VEGF 和 FGF。最后,成功整合 scRNA-seq 和 scATAC-seq 结果以揭示一系列可能成为牛骨骼肌发育过程中促进细胞命运转变的候选特定表达 TFs。总的来说,我们的研究结果概述了正常牛骨骼肌发育的单细胞动态染色质/转录景观;这些为理解哺乳动物骨骼肌的结构和功能提供了重要资源,将促进对其生物学的研究。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/c2e8/10472525/f289c2d48675/CPR-56-e13430-g001.jpg

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