Bioscience Methods 2025, Vol.16, No.2, 83-99 http://bioscipublisher.com/index.php/bm 88 proteomics to reveal the important role of calcium ion signaling pathways in determining skeletal muscle growth potential. They found that there were significant differences in the expression of genes related to intracellular calcium homeostasis regulation in lean and obese pig muscle cells, suggesting that Ca2+ signals may be the key determinant of muscle tissue's choice of myogenesis or adipogenesis fate. This finding is also instructive for species such as goats, because the balance between intramuscular fat deposition and muscle fiber growth directly affects meat quality and meat production. Single-cell data helps us determine which signaling molecules mediate the interaction between muscle cells and fat precursors. Secondly, the identification of key transcription factors is one of the important results of single-cell transcriptomics. Through comprehensive single-cell gene regulatory network analysis and functional verification, Zhu et al. (2024) identified the transcription factor TCF7L2 as a central regulator in early adipogenesis in goat skeletal muscle. They not only inferred from the gene regulatory network of single-cell data that TCF7L2 may control the differentiation of fat precursors, but also confirmed through in vitro intervention experiments that inhibiting TCF7L2 would affect the differentiation process of adipocytes. This is a typical case of single-cell analysis guiding functional experiments, which shows that we can lock potential key factors from massive single-cell transcription information and then verify them at the cellular or animal level. In addition, there are similar experiences in bovine muscle research: for example, Zhang et al. (2020) found that a new lncRNA affects myogenesis by mediating the KRAS/Myf6 pathway. This discovery was also based on the analysis of transcriptome data and subsequent functional experiments. For goat muscle, due to the late start of genomic and transcriptional regulation research, the discovery of such key factors is particularly valuable, which provides specific targets for further genetic improvement. Third, the combination of single-cell and bulk transcriptomes also revealed new regulatory molecules related to muscle development. In addition to the transcription factors such as TCF7L2 mentioned above, the functions of many non-coding RNAs are also gradually being clarified. For example, Han et al. (2022) identified goat muscle lncRNAs that were differentially expressed at multiple developmental stages, some of which affected the proliferation and differentiation of muscle cells by competing with mRNA for the "sponge" effect of miRNA. Another study on goat fetal skeletal muscle found the involvement of natural antisense long-chain RNA (nat lncRNA) through transcriptome analysis. These natural antisense RNAs were negatively correlated with the expression of corresponding positive chain genes (such as muscle structural genes), and may regulate muscle development through chromatin modification or transcriptional interference mechanisms. In addition, in terms of small RNAs, Shen et al. (2022) compared skeletal muscle miRNAs of different goat breeds and found that some miRNAs (such as miR-432 and miR-133 families) were highly expressed in high intramuscular fat breeds, and it was speculated that they promoted adipogenesis and inhibited muscle fiber proliferation, thereby leading to higher intramuscular fat deposition. 4 Epigenetic Mechanisms of Muscle Development 4.1 Chromatin accessibility and histone modification Epigenetic regulation plays a key role in muscle development by affecting gene expression without changing the DNA sequence. One important aspect is chromatin accessibility. The degree of chromatin openness determines whether transcription factors and transcription machinery can bind to DNA and initiate gene transcription. During the transition of muscle progenitor cells to differentiated muscle cells, the chromatin accessibility of related muscle gene promoters and enhancers will change significantly. For example, in a study of Hu sheep (Hu sheep), ATAC-seq (analysis of chromatin open regions) was compared on skeletal muscles of different ages, and 3742 differentially accessible regions were found to change during development (Cao et al., 2023). These open regions are significantly associated with genes related to muscle development pathways. For example, the open regulatory elements that appear at 3-6 months of age are enriched with transcription factor binding sites related to muscle hypertrophy and myofiber conversion, including ARID5B, MYOG, etc., which promote myofiber growth; while other open regions are enriched with binding sites of factors such as NR1D1 and ZFP36L2, which are related to myofiber type conversion. It can be seen that the dynamic changes in chromatin accessibility directly affect the fate determination of muscle cells. Through technologies such as single-cell ATAC-seq or 10x Multiome, the
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