BackgroundIntramuscular fat (IMF) and intramuscular connective tissue (IMC) are often seen in human myopathies and are central to beef quality. The mechanisms regulating their accumulation remain poorly understood. Here, we explored the possibility of using beef cattle as a novel model for mechanistic studies of intramuscular adipogenesis and fibrogenesis.MethodsSkeletal muscle single‐cell RNAseq was performed on three cattle breeds, including Wagyu (high IMF), Brahman (abundant IMC but scarce IMF), and Wagyu/Brahman cross. Sophisticated bioinformatics analyses, including clustering analysis, gene set enrichment analyses, gene regulatory network construction, RNA velocity, pseudotime analysis, and cell–cell communication analysis, were performed to elucidate heterogeneities and differentiation processes of individual cell types and differences between cattle breeds. Experiments were conducted to validate the function and specificity of identified key regulatory and marker genes. Integrated analysis with multiple published human and non‐human primate datasets was performed to identify common mechanisms.ResultsA total of 32 708 cells and 21 clusters were identified, including fibro/adipogenic progenitor (FAP) and other resident and infiltrating cell types. We identified an endomysial adipogenic FAP subpopulation enriched for COL4A1 and CFD (log2FC = 3.19 and 1.92, respectively; P < 0.0001) and a perimysial fibrogenic FAP subpopulation enriched for COL1A1 and POSTN (log2FC = 1.83 and 0.87, respectively; P < 0.0001), both of which were likely derived from an unspecified subpopulation. Further analysis revealed more progressed adipogenic programming of Wagyu FAPs and more advanced fibrogenic programming of Brahman FAPs. Mechanistically, NAB2 drives CFD expression, which in turn promotes adipogenesis. CFD expression in FAPs of young cattle before the onset of intramuscular adipogenesis was predictive of IMF contents in adulthood (R2 = 0.885, P < 0.01). Similar adipogenic and fibrogenic FAPs were identified in humans and monkeys. In aged humans with metabolic syndrome and progressed Duchenne muscular dystrophy (DMD) patients, increased CFD expression was observed (P < 0.05 and P < 0.0001, respectively), which was positively correlated with adipogenic marker expression, including ADIPOQ (R2 = 0.303, P < 0.01; and R2 = 0.348, P < 0.01, respectively). The specificity of Postn/POSTN as a fibrogenic FAP marker was validated using a lineage‐tracing mouse line. POSTN expression was elevated in Brahman FAPs (P < 0.0001) and DMD patients (P < 0.01) but not in aged humans. Strong interactions between vascular cells and FAPs were also identified.ConclusionsOur study demonstrates the feasibility of beef cattle as a model for studying IMF and IMC. We illustrate the FAP programming during intramuscular adipogenesis and fibrogenesis and reveal the reliability of CFD as a predictor and biomarker of IMF accumulation in cattle and humans.
White adipose tissue plays an important role in energy storage. Excessive adiposity especially in the visceral adipose depot however has a stronger correlation with metabolic diseases such as insulin resistance. The specific anatomical locations of the visceral adipose tissue (VAT) suggest that it is subjective to depot‐specific regulation during development. Here, using a specific inducible lineage‐tracing mouse line, we identified that Tcf21 is specifically expressed in VAT but not in subcutaneous tissue. In VAT, Tcf21 is expressed in mesenchymal progenitor cells but not in differentiated adipocytes. Tcf21 lineage cells actively proliferate followed by differentiation into adipocytes during neonatal development but have a limited adipogenic capacity in adult mice even after high‐fat diet treatment. Bulk RNAseq and ATACseq analyses of Tcf21 lineage cells isolated from mice of different ages revealed the dynamic gene expression and chromatin accessibility in Tcf21 lineage cells. In particular, elevated expression of inflammatory genes and fibrotic genes were observed in Tcf21 lineage cells as the adiposity of mice increased. Using the transcriptomic and motif enrichment data, we predicted a gene regulatory network mediating the gene expression changes in Tcf21 lineage cells. Single‐cell RNAseq (scRNAseq) and immunostaining identified multiple subpopulations of Tcf21 lineage cells including 2 major subpopulations consisting of a mesothelial subpopulation and an interstitial subpopulation, as well as a small population that expressed select inflammatory genes exclusively in obese mice. Using an inducible cell‐type‐specific Tcf21 knockout mouse line, we identified that neonatal deletion of Tcf21 in mice led to increased adipogenesis of Tcf21 lineage cells during postnatal development and improved metabolism after high‐fat diet treatment. In vitro loss‐of‐function and gain‐of‐function studies showed that Tcf21 inhibits the adipogenic differentiation of VAT progenitor cells. Bulk RNAseq and scRNAseq showed that Tcf21 lineage cells from Tcf21 knockout mice were developmentally in advance of those from their WT littermates. Mechanistic studies identified that Tcf21 inhibits adipogenesis through promoting the expression of Dlk1, a known negative regulator of adipogenesis, in the interstitial subpopulation of Tcf21 lineage cells.
Human and mouse studies have shown that fibro/adipogenic progenitors (FAPs) are a major source of intramuscular fat (IMF) and extracellular matrix (ECM) proteins. IMF and ECM proteins directly influence the palatability of beef, suggesting an essential role of FAPs in beef quality determination, which is still largely unexplored. We performed single-cell RNAseq (scRNAseq) using cells isolated from full blood Wagyu and Brahman cattle and Wagyu/Brahman cross cattle, which identified 21 cell clusters representing FAPs, several endothelial cell types, vascular smooths muscle cells, satellite cells, muscle fibers, and multiple immune cell types. More abundant FAPs were identified in the muscle of Brahman cattle, while a larger number of endothelial cells were identified in the muscle of Wagyu cattle. Further analysis of FAPs identified multiple FAP subpopulations with distinct gene expression profiles and anatomic locations. GSEA analysis revealed adipogenic and fibrogenic FAP subpopulations. A comparison of FAP subpopulations among different breeds showed higher complement system activity in the adipogenic FAP subpopulation of Wagyu cattle. Forced activation of the complement system in FAPs enhanced their adipogenic efficiency in vitro. In addition, cell-cell communication analysis identified active interactions between FAPs and other cell types through direct contact and secreted factors, many of which may affect FAP activities. In conclusion, our study revealed the single-cell atlas of bovine skeletal muscle and identified mechanisms regulating bovine intramuscular adipogenesis and fibrogenesis.
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