Growth and development symposium: Stem and progenitor cells in animal growth: The regulation of beef quality by resident progenitor cells

© The Author(s) 2019. Published by Oxford University Press on behalf of the American Society of Animal Science. All rights reserved. The intramuscular adipose tissue deposition in the skeletal muscle of beef cattle is a highly desired trait essential for high-quality beef. In contrast, the excessive accumulation of crosslinked collagen in intramuscular connective tissue contributes to beef toughness. Recent studies revealed that adipose tissue and connective tissue share an embryonic origin in mice and may be derived from a common immediate bipotent precursor in mice and humans. Having the same linkages in the development of adipose tissue and connective tissue in beef, the lineage commitment and differentiation of progenitor cells giving rise to these tissues may directly affect beef quality. It has been shown that these processes are regulated by some key transcription regulators and are subjective to epigenetic modifications such as DNA methylation, histone modifications, and microRNAs. Continued exploration of relevant regulatory pathways is very important for the identification of mechanisms influencing meat quality and the development of proper management strategies for beef quality improvement

A Single-Cell Atlas of Bovine Skeletal Muscle Reveals Mechanisms Regulating Intramuscular Adipogenesis and Fibrogenesis

Background Intramuscular 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. Methods Skeletal 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. Results A 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 \u3c 0.0001) and a perimysial fibrogenic FAP subpopulation enriched for COL1A1 and POSTN (log2FC = 1.83 and 0.87, respectively; P \u3c 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 \u3c 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 \u3c 0.05 and P \u3c 0.0001, respectively), which was positively correlated with adipogenic marker expression, including ADIPOQ (R2 = 0.303, P \u3c 0.01; and R2 = 0.348, P \u3c 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 \u3c 0.0001) and DMD patients (P \u3c 0.01) but not in aged humans. Strong interactions between vascular cells and FAPs were also identified. Conclusions Our 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

T\u3cem\u3ecf\u3c/em\u3e21 Marks Visceral Adipose Mesenchymal Progenitors and Functions as a Rate-Limiting Factor During Visceral Adipose Tissue Development

Distinct locations of different white adipose depots suggest anatomy-specific developmental regulation, a relatively understudied concept. Here, we report a population of Tcf21 lineage cells (Tcf21 LCs) present exclusively in visceral adipose tissue (VAT) that dynamically contributes to VAT development and expansion. During development, the Tcf21 lineage gives rise to adipocytes. In adult mice, Tcf21 LCs transform into a fibrotic or quiescent state. Multiomics analyses show consistent gene expression and chromatin accessibility changes in Tcf21 LC, based on which we constructed a gene-regulatory network governing Tcf21 LC activities. Furthermore, single-cell RNA sequencing (scRNA-seq) identifies the heterogeneity of Tcf21 LCs. Loss of Tcf21 promotes the adipogenesis and developmental progress of Tcf21 LCs, leading to improved metabolic health in the context of diet-induced obesity. Mechanistic studies show that the inhibitory effect of Tcf21 on adipogenesis is at least partially mediated via Dlk1 expression accentuation

Loss of \u3ci\u3eActa2\u3c/i\u3e in Cardiac Fibroblasts Does Not Prevent the Myofibroblast Differentiation or Affect the Cardiac Repair After Myocardial Infarction

In response to myocardial infarction (MI), quiescent cardiac fibroblasts differentiate into myofibroblasts mediating tissue repair. One of the most widely accepted markers of myofibroblast differentiation is the expression of Acta2 which encodes smooth muscle alpha-actin (SMαA) that is assembled into stress fibers. However, the requirement of Acta2/SMαA in the myofibroblast differentiation of cardiac fibroblasts and its role in post-MI cardiac repair remained unknown. To answer these questions, we generated a tamoxifen-inducible cardiac fibroblast-specific Acta2 knockout mouse line. Surprisingly, mice that lacked Acta2 in cardiac fibroblasts had a normal post-MI survival rate. Moreover, Acta2 deletion did not affect the function or histology of infarcted hearts. No difference was detected in the proliferation, migration, or contractility between WT and Acta2-null cardiac myofibroblasts. Acta2-null cardiac myofibroblasts had a normal total filamentous actin level and total actin level. Acta2 deletion caused a significant compensatory increase in the transcription level of non-Acta2 actin isoforms, especially Actg2 and Acta1. Moreover, in myofibroblasts, the transcription levels of cytoplasmic actin isoforms were significantly higher than those of muscle actin isoforms. In addition, we found that myocardin-related transcription factor-A is critical for myofibroblast differentiation but is not required for the compensatory effects of non-Acta2 isoforms. In conclusion, the Acta2 deletion does not prevent the myofibroblast differentiation of cardiac fibroblasts or affect the post-MI cardiac repair, and the increased expression and stress fiber formation of non-SMαA actin isoforms and the functional redundancy between actin isoforms are able to compensate for the loss of Acta2 in cardiac myofibroblasts

Progenitor Cell Isolation From Mouse Epididymal Adipose Tissue and Sequencing Library Construction

Here, we present a protocol to isolate progenitor cells from mouse epididymal visceral adipose tissue and construct bulk RNA and assay for transposase-accessible chromatin with sequencing (ATAC-seq) libraries. We describe steps for adipose tissue collection, cell isolation, and cell staining and sorting. We then detail procedures for both ATAC-seq and RNA sequencing library construction. This protocol can also be applied to other tissues and cell types directly or with minor modifications. For complete details on the use and execution of this protocol, please refer to Liu et al. (2023).1 *1 Liu, Q., Li, C., Deng, B., Gao, P., Wang, L., Li, Y., ... & Fu, X. (2023). Tcf21 marks visceral adipose mesenchymal progenitors and functions as a rate-limiting factor during visceral adipose tissue development. Cell reports, 42(3) 112166. https://doi.org/10.1016/j.celrep.2023.11216

Cloning and expression of lin-28 homolog B gene in the onset of puberty in Duolang sheep

Objective Recent studies have demonstrated that lin-28 homolog B (LIN28B)/miRNA let-7 (let-7) plays a role in the regulation of pubertal onset in mammals. However, the role of LIN28B/let-7 in the onset of ovine puberty remains unknown. We cloned the Duolang sheep Lin28B cDNA sequence, detected the expression change of LIN28B, let-7a and let-7g in hypothalamus, pituitary and ovary tissues at three different pubertal stages. Methods The reverse transcriptase polymerase chain reaction (RT-PCR) was used to clone the cDNA sequence of LIN28B gene from Duolang sheep and the bioinformatics methods were applied to analyze the amino acid sequence of LIN28B protein. The mRNA expression levels of the LIN28B gene at different pubertal stages were examined by real time RT-PCR. Results LIN28B cDNA of Duolang sheep was cloned, and two transcripts were obtained. The amino acid sequence of transcript 1 shares 99.60%, 98.78%, and 94.80% identity with those of goat, wild yak and pig, respectively. Strong LIN28B mRNA expression was detected in the hypothalamus, pituitary, ovary, oviduct and uterus, while moderate expression was found in the liver, kidney, spleen and heart, weak expression was observed in the heart. No expression was found in the lungs. Quantitative real-time PCR (QPCR) and western-blot analysis revealed that the LIN28B was highly expressed in the hypothalamus and ovary at prepuberty stages, and this expression significantly decreased from the prepuberty to puberty stages (p0.05). Conclusion These results provided a foundation for determining the functions of LIN28B/let-7 and their role in the onset of sheep puberty

Engineering Extracellular Matrix Proteins to Enhance Cardiac Regeneration After Myocardial Infarction

Engineering microenvironments for accelerated myocardial repair is a challenging goal. Cell therapy has evolved over a few decades to engraft therapeutic cells to replenish lost cardiomyocytes in the left ventricle. However, compelling evidence supports that tailoring specific signals to endogenous cells rather than the direct integration of therapeutic cells could be an attractive strategy for better clinical outcomes. Of many possible routes to instruct endogenous cells, we reviewed recent cases that extracellular matrix (ECM) proteins contribute to enhanced cardiomyocyte proliferation from neonates to adults. In addition, the presence of ECM proteins exerts biophysical regulation in tissue, leading to the control of microenvironments and adaptation for enhanced cardiomyocyte proliferation. Finally, we also summarized recent clinical trials exclusively using ECM proteins, further supporting the notion that engineering ECM proteins would be a critical strategy to enhance myocardial repair without taking any risks or complications of applying therapeutic cardiac cells

Theoretical and Experimental Study on Nonlinear Failure of an MEMS Accelerometer under Dual Frequency Acoustic Interference

In order to quantitatively study the interfered output of the accelerometer under an acoustic injection attack, a mathematical model for fitting and predicting the accelerometer output was proposed. With ADXL103 as an example, an acoustic injection attack experiment with amplitude sweeping and frequency sweeping was performed. In the mathematical model, the R-squared coefficient was R2 = 0.9990 in the acoustic injection attack experiment with amplitude sweeping, and R2 = 0.9888 with frequency sweeping. Based on the mathematical model, the dual frequency acoustic injection attack mode was proposed. The difference frequency signal caused by the nonlinear effect was not filtered by the low-pass filter. At a 115 dB sound pressure level, the maximum acceleration bias of the output was 4.4 m/s2 and the maximum amplitude of fluctuation was 4.97 m/s2. Two kinds of methods of prevention against acoustic injection attack were proposed, including changing the damping ratio of the accelerometer and adding a preposition low-pass filter

Theoretical and Experimental Study on Nonlinear Failure of an MEMS Accelerometer under Dual Frequency Acoustic Interference

In order to quantitatively study the interfered output of the accelerometer under an acoustic injection attack, a mathematical model for fitting and predicting the accelerometer output was proposed. With ADXL103 as an example, an acoustic injection attack experiment with amplitude sweeping and frequency sweeping was performed. In the mathematical model, the R-squared coefficient was R2 = 0.9990 in the acoustic injection attack experiment with amplitude sweeping, and R2 = 0.9888 with frequency sweeping. Based on the mathematical model, the dual frequency acoustic injection attack mode was proposed. The difference frequency signal caused by the nonlinear effect was not filtered by the low-pass filter. At a 115 dB sound pressure level, the maximum acceleration bias of the output was 4.4 m/s2 and the maximum amplitude of fluctuation was 4.97 m/s2. Two kinds of methods of prevention against acoustic injection attack were proposed, including changing the damping ratio of the accelerometer and adding a preposition low-pass filter