Early lineage segregation of primary myotubes from secondary myotubes and adult muscle stem cells

Myogenesis in amniotes unfolds through two consecutive waves. The primary myotube lineage is characterized by the expression of slow myosin, sometimes in combination with fast myosin, and may serve as a scaffold for the secondary lineage, which expresses exclusively fast myosin. The embryonic origin of these two lineages, their relationship, and their connection to adult muscle stem cells are unknown. Here, we employed innovative strategies, combining novel TCF-LEF/β-catenin signaling reporters with the precise spatiotemporal control of in vivo electroporation in avian embryos, to track limb muscle progenitors from early migration to late fetal stages. Strikingly, we uncovered two distinct progenitor populations co-existing from the earliest stages of limb myogenesis, with specific developmental fates: reporter-positive progenitors exclusively form primary myotubes, while reporter-negative progenitors generate secondary myotubes and adult muscle stem cells. Furthermore, we uncovered a novel function of TCF-LEF/β-catenin signaling in regulating the spatial organization of the primary myotube lineage via CXCR4-mediated control of myoblast migration, likely contributing to its proposed organizing function. By redefining the embryonic origins of these myogenic populations, our findings not only resolve a longstanding question in muscle biology but also provide a crucial molecular entry point for understanding the cellular and molecular underpinnings of muscle fiber type diversity and function. Dissection of electroporated limb bud of chicken embryos with a reporter plasmid (dTomato+) and a transcriptional reporter for TCF/LEF transcription (mVenus+), shorting of the dTomato+ cells and analysis with 10X single-cell RNA-sequencing