Primary myogenesis in the sand lizard (Lacerta agilis) limb bud

Our studies conducted on reptilian limb muscle development revealed, for the first time, early forelimb muscle differentiation at the morphological and molecular level. Sand lizard skeletal muscle differentiation in the early forelimb bud was investigated by light, confocal, and transmission electron microscopy as well as western blot. The early forelimb bud, filled with mesenchymal cells, is surrounded by monolayer epithelium cells. The immunocytochemical analysis revealed the presence of Pax3- and Lbx-positive cells in the vicinity of the ventro-lateral lip (VLL) of the dermomyotome, suggesting that VLL is the source of limb muscle progenitor cells. Furthermore, Pax3- and Lbx-positive cells were observed in the dorsal and ventral myogenic pools of the forelimb bud. Skeletal muscle development in the early limb bud is asynchronous, which is manifested by the presence of myogenic cells in different stages of differentiation: multinucleated myotubes with well-developed contractile apparatus, myoblasts, and mitotically active premyoblasts. The western blot analysis revealed the presence of MyoD and Myf5 proteins in all investigated developmental stages. The MyoD western blot analysis showed two bands corresponding to monomeric (mMyoD) and dimeric (dMyoD) fractions. Two separate bands were also detected in the case of Myf5. The observed bands were related to non-phosphorylated (Myf5) and phosphorylated (pMyf5) fractions of Myf5. Our investigations on sand lizard forelimb myogenesis showed that the pattern of muscle differentiation in the early forelimb bud shares many features with rodents and chicks.