Skeletal muscle possesses remarkable regenerative potential due to satellite cells, an injury-responsive stem cell population located beneath the muscle basal lamina that expresses Pax7. cell involved in post-natal muscle growth and regeneration. Introduction Skeletal muscle is among the most regenerative buy 123447-62-1 adult tissues. Its remarkable regenerative capacity originates from a population of resident stem cells, termed satellite cells (SCs), located beneath the muscle basal lamina 1. SCs are marked by expression of Pax7, a transcription factor critical for muscle regeneration 1. In response to injury and disease, SCs become activated and undergo self-renewal and differentiation to form new myofibers 1-3. While SCs are essential for muscle regeneration, their their genetic ablation in adult mice does not accelerate sarcopenia 4-6. Thus, additional mechanisms or cell types might contribute to maintenance of muscle mass during aging. Skeletal muscle is composed of heterogeneous myofiber types that differ in contractile and metabolic properties and expression of distinctive myosin isoforms. Four major fiber types are present in rodent muscles: one type of slow-twitch fiber (type I) and 3 types of fast-twitch fibers (type buy 123447-62-1 IIa, IIx/d, and IIb). While type I and type IIa fibers exhibit oxidative metabolism and high endurance; type IIx and buy 123447-62-1 IIb fibers are glycolytic and display low endurance 7. Slow and fast twitch fibers also differ in their responses to hypertrophic or atrophic stimuli. For example, type IIb and IIx myofibers are more susceptible than slow twitch fibers to a variety of atrophic signals such as denervation, nutrient deprivation, buy 123447-62-1 cancer cachexia, and chronic heart failure 8-10. While SCs can fuse into all myofiber types in injured muscle 11, it remains unknown whether fiber-type specific myogenic progenitors might also exist. The Drosophila basic helix-loop-helix transcription factor Twist is expressed in muscle progenitors during embryogenesis and is essential for the formation of mesoderm and muscle 12-14. Within the adult musculature of Drosophila, Twist expression is restricted to muscle precursors that are normally quiescent but are activated by extracellular cues to regenerate the adult musculature during metamorphosis 15-17. Two mammalian Twist genes, Twist1 (Tw1) and Twist2 (Tw2), are expressed in various mesenchymal cell types, but not in differentiated myofibers18, 19. Tw1 and Tw2 have been shown to block myogenesis in vitro 19-22,23, but their potential roles in muscle formation or regeneration in mammals have not been explored. Here, we traced the fate of Tw2-dependent cell lineages in mice and discovered that Tw2 expression marks a previously unrecognized interstitial myogenic progenitor cell that forms type IIb/x myofibers in adult muscle. Tw2-expressing progenitors represent a population of myogenic progenitor cells that contributes to specific fiber types during muscle homeostasis and regeneration, highlighting the ancestral functions of Twist as a regulator of muscle formation. Results Twist Expression in Interstitial Cells Within Adult Skeletal Muscle In adult muscle, Tw2 transcript is barely detectable in whole G/P muscle at 1, 2 and 4 months of age by RNA-seq analysis, in contrast to MyoD and Myh4 that are readily detected (Supplementary Fig. 1a). Real-time RT-PCR revealed that Tw2 was highly enriched in mononuclear non-myofiber cells compared to whole quadriceps muscle (Supplementary Fig. 1b). Immunostaining of transverse sections of gastrocnemius muscle from 3 months old wild-type (WT) mice revealed Tw2 protein in interstitial buy 123447-62-1 cells outside of the myofibers, but not within myofibers (Fig. 1a). Furthermore, Tw2 protein was not co-localized with Pax7, which was restricted to SCs beneath the basal lamina (Fig. 1a and b). Similar mutual exclusivity of expression of Tw2 and Pax7 was observed in muscles of 12 month-old mice (Supplementary Fig. 1c). We conclude that Tw2 is expressed in the myofiber interstitium and not in mature myofibers or SCs in adult muscle. Figure 1 Tw2-expressing and Pax7-expressing cells are distinct Flt3 cell types in skeletal muscle To analyze Tw2 expression during muscle regeneration, we performed cardiotoxin (CTX) injury on tibialis anterior (TA) muscle of WT mice, and harvested muscles on.