Satellite cells (SCs) are tissue-specific stem cells responsible for adult skeletal muscle regeneration and maintenance. SCs function is critically dependent on two families of transcription factors: the paired box (Pax) involved in specification and maintenance and the Muscle Regulatory Factors (MRFs), which orchestrate myogenic commitment and differentiation. In turn, signaling events triggered by extrinsic and intrinsic stimuli control their function via post-translational modifications, including ubiquitination and phosphorylation. In this context, the Abelson non-receptor tyrosine kinase (c-Abl) mediates the activation of the p38 α/β MAPK pathway, promoting myogenesis. c-Abl also regulates the activity of the transcription factor MyoD during DNA-damage stress response, pausing differentiation. However, it is not clear if c-Abl modulates other key transcription factors controlling SC function. This work aims to determine the role of c-Abl in SCs myogenic capacity via loss of function approaches in vitro and in vivo. Here we show that c-Abl inhibition or deletion results in a down-regulation of Pax7 mRNA and protein levels, accompanied by decreased Pax7 transcriptional activity, without a significant effect on MRF expression. Additionally, we provide data indicating that Pax7 is directly phosphorylated by c-Abl. Finally, SC-specific c-Abl ablation impairs muscle regeneration upon acute injury. Our results indicate that c-Abl regulates myogenic progression in activated SCs by controlling Pax7 function and expression.

Glucose is a major energy source consumed by proliferating mammalian cells. Therefore, in general, proliferating cells have the preference of high glucose contents in extracellular environment. Here, we showed that high glucose concentrations impede the proliferation of satellite cells, which are muscle-specific stem cells, under adherent culture conditions. We found that the proliferation activity of satellite cells was higher in glucose-free DMEM growth medium (low-glucose medium with a glucose concentration of 2 mM) than in standard glucose DMEM (high-glucose medium with a glucose concentration of 19 mM). Satellite cells cultured in the high-glucose medium showed a decreased population of reserve cells, identified by staining for Pax7 expression, suggesting that glucose concentration affects cell fate determination. In conclusion, glucose is a factor that decides the cell fate of skeletal muscle-specific stem cells. Due to this unique feature of satellite cells, hyperglycemia may negatively affect the regenerative capability of skeletal muscle myofibers and thus facilitate sarcopenia.

Myogenic differentiation, muscle stem cell functionality, and regeneration of skeletal muscle are cellular processes under tight control of various signaling pathways. Here, we investigated the role of non-canonical NF-κB signaling in myogenic differentiation, muscle stem cell functionality, and regeneration of skeletal muscle. We stimulated non-canonical NF-κB signaling with an agonistically acting antibody of the lymphotoxin beta receptor (LTβR). Interestingly, we found that stimulation of non-canonical NF-κB signaling through the LTβR agonist impairs myogenic differentiation, muscle stem cell function, and regeneration of skeletal muscle. Furthermore, we show that stimulation of non-canonical NF-κB signaling by the LTβR agonist coincides with activation of canonical NF-κB signaling. We suggest a direct crosstalk between canonical and non-canonical NF-κB signaling during myogenic differentiation which is required for proper myogenic differentiation and thereby regeneration of skeletal muscle.

Producing an adequate number of muscle stem cells (MuSCs) with robust regenerative potential is essential for the successful cell therapy of muscle-wasting disorders. We have recently developed a method to produce skeletal myogenic cells with exceptional engraftability and expandability through an in vivo pluripotent stem cell (PSC) differentiation approach. We have subsequently mapped engraftment and gene expression and found that leukemia inhibitory factor receptor (Lifr) expression is positively correlated with engraftability. We therefore investigated the effect of LIF, the endogenous ligand of LIFR, on cultured MuSCs and examined their engraftment potential. We found that LIF-treated MuSCs exhibited elevated expression of PAX7, formed larger colonies from single cells, and favored the retention of PAX7+ "reserve cells" upon myogenic differentiation. This suggested that LIF promoted the maintenance of cultured MuSCs at a stem cell stage. Moreover, LIF enhanced the engraftment capability of MuSCs that had been expanded in vitro for 12 days by 5-fold and increased the number of MuSCs that repopulated the stem cell pool post-transplantation. These results thereby demonstrated the effectiveness of our in vivo PSC differentiation platform to identify positive regulators of the engraftability of cultured MuSCs.

The knowledge of the molecular mechanisms that regulate embryonic myogenesis from early myogenic progenitors to myoblasts, as well as the emergence of adult satellite stem cells (SCs) during development, are key concepts to understanding the genesis and regenerative abilities of the skeletal muscle. Several previous pieces of evidence have revealed that the transcription factor Pitx2 might be a player within the molecular pathways controlling somite-derived muscle progenitors' fate and SC behavior. However, the role exerted by Pitx2 in the progression from myogenic progenitors to myoblasts including SC precursors remains unsolved. Here, we show that Pitx2 inactivation in uncommitted early myogenic precursors diminished cell proliferation and migration leading to muscle hypotrophy and a low number of SCs with decreased myogenic differentiation potential. However, the loss of Pitx2 in committed myogenic precursors gave rise to normal muscles with standard amounts of SCs exhibiting high levels of Pax7 expression. This SC population includes few MYF5+ SC-primed but increased amount of less proliferative miR-106b+cells, and display myogenic differentiation defects failing to undergo proper muscle regeneration. Overall our results demonstrate that Pitx2 is required in uncommitted myogenic progenitors but it is dispensable in committed precursors for proper myogenesis and reveal a role for this transcription factor in the generation of diverse SC subpopulations.

Loss of ALX1 function causes the frontonasal dysplasia syndrome FND3, characterized by severe facial clefting and microphthalmia. Whereas the laboratory mouse has been the preeminent animal model for studying developmental mechanisms of human craniofacial birth defects, the roles of ALX1 in mouse frontonasal development have not been well characterized because the only previously reported Alx1 mutant mouse line exhibited acrania due to a genetic background-dependent failure of cranial neural tube closure. Using CRISPR/Cas9-mediated genome editing, we have generated an Alx1-deletion mouse model that recapitulates the FND craniofacial malformations, including median orofacial clefting and disruption of development of the eyes and alae nasi. In situ hybridization analysis showed that Alx1 is strongly expressed in frontonasal neural crest cells that give rise to periocular and frontonasal mesenchyme. Alx1 del/del embryos exhibited increased apoptosis of periocular mesenchyme and decreased expression of ocular developmental regulators Pitx2 and Lmxb1 in the periocular mesenchyme, followed by defective optic stalk morphogenesis. Moreover, Alx1 del/del embryos exhibited disruption of frontonasal mesenchyme identity, with loss of expression of Pax7 and concomitant ectopic expression of the jaw mesenchyme regulators Lhx6 and Lhx8 in the developing lateral nasal processes. The function of ALX1 in patterning the frontonasal mesenchyme is partly complemented by ALX4, a paralogous ALX family transcription factor whose loss-of-function causes a milder and distinctive FND. Together, these data uncover previously unknown roles of ALX1 in periocular mesenchyme development and frontonasal mesenchyme patterning, providing novel insights into the pathogenic mechanisms of ALX1-related FND.