Common and Distinctive Functions of the Hippo Effectors Taz and Yap in Skeletal Muscle Stem Cell Function.

Hippo pathway downstream effectors Yap and Taz play key roles in cell proliferation and regeneration, regulating gene expression especially via Tead transcription factors. To investigate their role in skeletal muscle stem cells, we analyzed Taz in vivo and ex vivo in comparison with Yap. Small interfering RNA knockdown or retroviral-mediated expression of wild-type human or constitutively active TAZ mutants in satellite cells showed that TAZ promoted proliferation, a function shared with YAP. However, at later stages of myogenesis, TAZ also enhanced myogenic differentiation of myoblasts, whereas YAP inhibits such differentiation. Functionally, while muscle growth was mildly affected in Taz (gene Wwtr1-/- ) knockout mice, there were no overt effects on regeneration. Conversely, conditional knockout of Yap in satellite cells of Pax7Cre-ERT2/+ : Yapfl °x/fl °x :Rosa26Lacz mice produced a regeneration deficit. To identify potential mechanisms, microarray analysis showed many common TAZ/YAP target genes, but TAZ also regulates some genes independently of YAP, including myogenic genes such as Pax7, Myf5, and Myod1 (ArrayExpress-E-MTAB-5395). Proteomic analysis revealed many novel binding partners of TAZ/YAP in myogenic cells, but TAZ also interacts with proteins distinct from YAP that are often involved in myogenesis and aspects of cytoskeleton organization (ProteomeXchange-PXD005751). Neither TAZ nor YAP bind members of the Wnt destruction complex but both regulated expression of Wnt and Wnt-cross talking genes with known roles in myogenesis. Finally, TAZ operates through Tead4 to enhance myogenic differentiation. In summary, Taz and Yap have overlapping functions in promoting myoblast proliferation but Taz then switches to enhance myogenic differentiation. Stem Cells 2017;35:1958-1972.

Isolation, Cryosection and Immunostaining of Skeletal Muscle.

Adult skeletal muscle is maintained and repaired by resident stem cells called satellite cells, located between the plasmalemma of a muscle fiber, and the surrounding basal lamina. When needed, satellite cells are activated to form proliferative myoblasts, that then differentiate and fuse to existing muscle fibers, or fuse together to form replacement myofibers. In parallel, a proportion of satellite cells self-renew, to maintain the stem cell pool. To date, Pax7 is the marker of choice for identifying quiescent satellite cells. Co-immunostaining of skeletal muscle with Pax7 and laminin allows both identification of satellite cells, and the myofiber that they are associated with. Furthermore, satellite cells can be followed through the early stages of the myogenic program by co-immunostaining with myogenic regulatory factors such as MyoD. To test genetically modified mice for satellite cell expression, co-immunostaining can be performed for Pax7 and reporter genes such as eGFP. Here, we describe a method for identification of satellite cells in skeletal muscle sections, including muscle isolation, cryosectioning and co-immunostaining for Pax7 and laminin.