Novel role of Hox-expressing interstitial cells in myogenesis during muscle growth, homeostasis and regeneration - PROJECT SUMMARY Adult skeletal muscle possesses remarkable regenerative capacity. This ability is attributed to tissue- specific muscle stem cells (MuSCs), also called satellite cells. Upon activation, MuSCs are capable of exiting quiescence, proliferating, differentiating into muscle, and self-renewing. In addition to MuSCs, other cell types are known to support muscle development, growth, and regeneration. For instance, interstitial stromal cells that lie outside the basal lamina of muscle fibers are present from the earliest stages of muscle development and play critical roles in muscle patterning. However, muscle fibers and MuSCs derive from somites whereas interstitial/stromal cells derive from the lateral plate mesoderm. These lineages have been assumed to remain distinct throughout life. A handful of recent publications have challenged this paradigm and support the existence of a subset of interstitial stromal cells that have myogenic potential. We have previously reported that Hox11 genes are expressed in interstitial stromal cells that surround the muscle fibers of the zeugopod-attached muscles (radius/ulna, tibia/fibula) at embryonic stages and that loss of Hox11 function leads to disrupted muscle patterning. Recently published work from the Wellik laboratory reveals that Hox11 expression in the muscle interstitial stroma continues throughout life. Using a Hoxa11- CreERT2 allele generated in my laboratory to induce lineage labeling, we show that Hoxa11-expressing muscle interstitial cells contribute to muscle fibers beginning at postnatal stages. This contribution is MuSC- independent; MuSCs are not labeled using the Hoxa11-CreERT2 lineage reporter, and the lineage does not appear at any time post-induction in MuSCs. Use of Hoxa11-CreERT2 to induce nuclear lineage labeling (ROSA-LSL-H2BmCherry) supports interstitial cell nuclei contribution into muscle fibers (i.e. not just cytoplasmic contents). When cultured alone in myogenic media in vitro, Hoxa11 lineage-positive cells are unable to differentiate into myofibers, but when cultured with C2C12 or Pax7-lineage cells, Hoxa11-lineage cells are capable of contributing to existing myofibers, including in the presence of extracellular vesicle inhibitors. Our recent publication and new preliminary data supports the existence of a highly understudied set of Hox-expressing myoprogenitor cells. Further study of this novel subset of cells will fill gaps in our knowledge of muscle biology and could lead to new regenerative avenues for ameliorating the loss of muscle that occurs in dystrophinapathies. In this proposal, we will interrogate Hoxa11-lineage potential using multiple approaches in vivo, including in response to injury and induced hypertrophy (Aim 1). Using our collection of genetic tools, we will perform Hoxa11-CreERT2-mediated cell ablation studies, loss of Hox11 function studies and interrogate functional subsets and cellular mechanisms using various -omic technologies (Aim 2). We will define the in vitro cellular behavior and the ex vivo transplantation potential of Hoxa11 lineage-positive cells and compare them to other cells with myogenic capacity (Aim 3).
