- Skeletal muscle is the largest organ in the body by mass, comprising roughly 40% of total bodyweight in adults. It plays diverse and unique roles that include movement, locomotion, and support for posture and internal organs, among others. The structural foundation for all skeletal muscle in adults is formed early in development, emphasizing the importance of understanding the mechanisms of skeletal muscle development. This is especially important since adult skeletal muscle is limited in its ability to regenerate, but the regeneration mechanism reactivates certain developmental pathways.
Skeletal muscle formation in the vertebrate forelimb occurs in distinct phases during embryogenesis. Beginning around embryonic day (E) 10.5 in mice, embryonic myogenic progenitor cells (EMPCs) express the gene Pax3, which triggers migration into the limb bud. Once settled, between E10.5 and E12.5, embryonic myoblasts fuse with each other to form embryonic myotubes. Between E12.5 and E17.5 fetal myoblasts fuse with both embryonic myotubes, and each other, to form fetal myofibers, which serve as the structural foundation of all skeletal muscle in the forelimb. Not much is known regarding the molecular mechanisms behind this process, except that they significantly overlap with the mechanisms responsible for skeletal muscle regeneration in adults. Knowledge gained about myogenesis can also be applied to muscle regeneration in adults, to both accelerate wound healing, or reverse muscle-wasting diseases, called myopathies. Two sequence specific transcription factors, Pitx2 and Pax3, are fundamental to skeletal muscle development. Mice carrying locus specific alterations for both genes are used to molecularly dissect the skeletal muscle formation in time and space.
Pitx2 is required for the embryonic to fetal transition of skeletal muscle formation. ChIP-Seq (Chromatin-Immunoprecipitation followed by sequencing) approach was used to compare the chromatin state of E12.5 embryonic myoblasts from mice in which Pitx2 was present or had been deleted. ChIP-seq data were integrated with previous gene expression profiling data of the forelimb transcriptome to identify changes in the chromatin state of embryonic myoblasts at Pitx2-target genes. We observed significant disruption in the chromatin state of genes related to neurogenesis and cytoskeletal organization, implying Pitx2 regulates the cytoskeletal rearrangements during myogenesis.
Pax3 marks all skeletal muscle myoblasts as they migrate into the forelimb, beginning at E9.5 in the mouse. Whole-transcriptome profiling of pure forelimb isolated myoblasts was performed, via fluorescence activated cell sorting (FACS), from Pax3Cre|RosaEGFP mice. Myoblasts were isolated at 4 embryonic states (E11.5, E12.5, E13.5, E14.5), bracketing the embryonic to fetal transition during myogenesis. The increased expression of genes involved in cell-adhesion, angiogenesis, and immune system during fetal myogenesis, implying there is communication between different organ systems even when limited to what was thought to be a myogenic lineage. Additionally, coexpression network analysis revealed two distinct subnetworks present during all stages of myogenesis, but both expressed highest during fetal myogenesis. One network was enriched in genes that are involved in cell-adhesion, and the second was enriched in genes involving the immune-response, suggesting consistent interplay between the immune system and skeletal muscle. Our studies emphasize the complexity of myogenesis, with multiple different systems developing and communicating in parallel, and will serve as a base for future studies to explore the effect of specific perturbations during forelimb myogenesis. These perturbations will result in knowledge and techniques that can be used to enhance or reactivate skeletal muscle regeneration in mature muscle.