Hedgehog signaling via its ligand DHH acts as cell fate determinant during skeletal muscle regeneration.

Successful muscle regeneration relies on the interplay of multiple cell populations. However, the signals required for this coordinated intercellular crosstalk remain largely unknown. Here, we describe how the Hedgehog (Hh) signaling pathway controls the fate of fibro/adipogenic progenitors (FAPs), the cellular origin of intramuscular fat (IMAT) and fibrotic scar tissue. Using conditional mutagenesis and pharmacological Hh modulators in vivo and in vitro, we identify DHH as the key ligand that acts as a potent adipogenic brake by preventing the adipogenic differentiation of FAPs. Hh signaling also impacts muscle regeneration, albeit indirectly through induction of myogenic factors in FAPs. Our results also indicate that ectopic and sustained Hh activation forces FAPs to adopt a fibrogenic fate resulting in widespread fibrosis. In this work, we reveal crucial post-developmental functions of Hh signaling in balancing tissue regeneration and fatty fibrosis. Moreover, they provide the exciting possibility that mis-regulation of the Hh pathway with age and disease could be a major driver of pathological IMAT formation.

A Guide to Examining Intramuscular Fat Formation and its Cellular Origin in Skeletal Muscle.

Fibro-adipogenic progenitors (FAPs) are mesenchymal stromal cells that play a crucial role during skeletal muscle homeostasis and regeneration. FAPs build and maintain the extracellular matrix that acts as a molecular myofiber scaffold. In addition, FAPs are indispensable for myofiber regeneration as they secrete a multitude of beneficial factors sensed by the muscle stem cells (MuSCs). In diseased states, however, FAPs are the cellular origin of intramuscular fat and fibrotic scar tissue. This fatty fibrosis is a hallmark of sarcopenia and neuromuscular diseases, such as Duchenne Muscular Dystrophy. One significant barrier in determining why and how FAPs differentiate into intramuscular fat is effective preservation and subsequent visualization of adipocytes, especially in frozen tissue sections. Conventional methods of skeletal muscle tissue processing, such as snap-freezing, do not properly preserve the morphology of individual adipocytes, thereby preventing accurate visualization and quantification. To overcome this hurdle, a rigorous protocol was developed that preserves adipocyte morphology in skeletal muscle sections allowing visualization, imaging, and quantification of intramuscular fat. The protocol also outlines how to process a portion of muscle tissue for RT-qPCR, enabling users to confirm observed changes in fat formation by viewing differences in the expression of adipogenic genes. Additionally, it can be adapted to visualize adipocytes by whole-mount immunofluorescence of muscle samples. Finally, this protocol outlines how to perform genetic lineage tracing of Pdgfrα-expressing FAPs to study the adipogenic conversion of FAPs. This protocol consistently yields high-resolution and morphologically accurate immunofluorescent images of adipocytes, along with confirmation by RT-qPCR, allowing for robust, rigorous, and reproducible visualization and quantification of intramuscular fat. Together, the analysis pipeline described here is the first step to improving our understanding of how FAPs differentiate into intramuscular fat, and provides a framework to validate novel interventions to prevent fat formation.