Opposing gene regulatory programs governing myofiber development and maturation revealed at single nucleus resolution

Abstract

Skeletal muscle fibers express distinct gene programs during development and maturation, but the underlying gene regulatory networks that confer stage-specific myofiber properties remain unknown. To decipher these distinctive gene programs and how they respond to neural activity, we generated a combined multi-omic single-nucleus RNA-seq and ATAC-seq atlas of mouse skeletal muscle development at multiple stages of embryonic, fetal, and postnatal life. We found that Myogenin, Klf5, and Tead4 form a transcriptional complex that synergistically activates the expression of muscle genes in developing myofibers. During myofiber maturation, the transcription factor Maf acts as a transcriptional switch to activate the mature fast muscle gene program. In skeletal muscles of mutant mice lacking voltage-gated L-type Ca2+ channels (Cav1.1), Maf expression and myofiber maturation are impaired. These findings provide a transcriptional atlas of muscle development and reveal genetic links between myofiber formation, maturation, and contraction.

Deregulation of multiple mechanisms shapes the onset ofLAMA2-congenital muscular dystrophy

“…This is consistent with our findings showing that Lama2 -deficiency leads to a simultaneous decrease in Myog ( Figure 3A ), MYF5 ( Figure 3B,C ) and Cacna1s ( Figure 6J ). Our in vivo findings from the RNAseq analysis ( Figure 4A ), and subsequent validation ( Figure 6H ), support the idea that Cacna1s expression is not only required for muscle differentiation, but also for its maturation 45 . Moreover, downregulation of Cacna1s has also been shown to occur in C2C12 cells treated with fibroblast growth factor 9 (FGF9) 58 , and correlated with defective differentiation.…”

“…To validate the important defect in muscle cell differentiation identified in our RNAseq analysis (Figure 4), we analyzed four top hits: the fast embryonic MyHC gene Myh2, previously shown to be implicated in skeletal muscle differentiation, and the embryonic slow myosin and myocyte marker (Myh7) [39][40][41][42] , the MYOD target gene Tmem182, a negative regulator of muscle growth 43 , but required for muscle cell fusion 44 , and the α1 s subunit of voltage-gated L-type Ca 2+ channel (Cav1.1) (Cacna1s), recently shown to be required for muscle fiber maturation 45 (Figure 6). Comparison of RNAseq normalized counts (Figures 6A-D) with qPCR of isolated muscle fibers from E17.5 wildtype and dy W fetuses ( Figures 6E-H) confirmed the significant downregulation of these genes, providing further evidence of a defective muscle differentiation and fusion signature in dy W fetuses.…”

Fibroblast diversification is an embryonic process dependent on muscle contraction

“…Additionally, analysis of transcriptional data from other models of paralyzed muscles (RyR and Cacna1s knockout) at E18.5 found a decrease in expression of genes associated with Ly6a+ fibroblasts, but not with Smoc2+ or Cldn1+ fibroblasts (Dos Santos et al, 2023;Filipova et al, 2018). These findings indicate that the failure to diversify is not inherent to the mdg model, supporting our hypothesis that Ly6a+ fibroblasts' diversification is dependent on muscle contraction during development.…”

“…Concomitant with the emergence of Ly6a + fibroblasts is the transition from embryonic to fetal myogenesis at around E14.5. Remarkably, this transition is accompanied by maturation of the muscle contractile units, MTJ formation, and initiation of myofiber contractions, which are known to be crucial for limb patterning and muscle transcriptional changes during development (Dos Santos et al, 2023; Felsenthal & Zelzer, 2017; Huang et al, 2015; Lipp et al, 2023). Therefore, to test whether muscle contraction could impact fibroblasts diversification we analyzed limbs of muscle dysgenic ( mdg ) embryos, harboring a lethal point mutation in the skeletal muscle specific Cacna1s gene which encodes the alpha subunit of Cav1.1 calcium channel, a lethal mutation resulting in paralyzed non-contractile skeletal muscles.…”

“…Our results indicate fibroblasts to have started diversifying by E15.5, accompanying the initiation of muscle contraction following the transition from primary to secondary myogenesis. Muscle contraction has been reported to be crucial for the development of several components of the musculoskeletal system in the embryo, including bone morphogenesis (Felsenthal & Zelzer, 2017), tendon patterning (Huang et al, 2015), MTJ and myofiber maturation (Dos Santos et al, 2023; Lipp et al, 2023). Results from the paralyzed mdg mutant showed loss of expression of Ly6a+ fibroblasts markers in the muscle periphery, suggesting that muscle contraction initiates these fibroblasts’ diversification.…”