Dissecting Notch signaling in quiescent and activated muscle stem cells. – MyoNotch22

Stem cells maintain tissues and repair tissue injuries. They remain quiescent when they are not needed, but they can be activated for regeneration upon demand. Thus, by responding to environmental cues, stem cells shuttle between quiescent and activated states. Dissecting the mechanisms that regulate the transition between quiescence and activation is critical for understanding tissue maintenance and repair, but also for the manipulation of stem cells in the context of regenerative medicine. The adult skeletal muscle tissue has an extraordinary capacity to regenerate after injury. Muscle stem cells (MuSCs) are the cellular source for repair which reside in a specialized microenvironment called the niche. Partners 1 and 3 showed previously that Notch signalling is critical for two distinct and seemingly contradictory processes that depend on the state of MuSCs, first the maintenance of the quiescent state of MuSCs which requires the construction of their niche, and second the suppression of differentiation of activated MuSCs that ensures that they can proliferate and self-renew. Our planned experiments combine mouse genetic models with high-throughput screenings to identify novel factors participating in quiescence and activation of MuSCs. To date, the majority of our analyses of MuSC quiescence and self-renewal is based on transcriptomic analyses as a convenient proxy of the proteome, and we used transcriptomics to infer lineage identity, active biochemical pathways and cellular functions. However, protein interactions and reversible posttranslational modifications are sensors that modulate entire cellular pathways and adopt them to changing conditions. Therefore, in this Consortium, we have teamed up with Partner 2, a specialist on cutting edge proteomic methodologies. Combining expertise in mouse genetics, Notch and ERK1/2 signalling with modern proteomic approaches, we propose to conduct together a comprehensive study on signal integration and Notch signalling in quiescent and activated muscle stem cells. We hypothesize that the diverse roles of Notch reflect the outcome of crosstalk with other signalling pathways, which adjusts MuSCs responses to Notch in quiescent and activated states. Thus, we will study how crosstalk affects interacting partners and the dynamics of Notch signalling propagation. We focus on Notch and ERK, two fundamental signalling pathways in MuSC activation: ERK proliferation signals initiate MuSC activation and proceed the initiation of the Notch-regulated myogenic program. We aim to identify binding protein partners of Notch signalling components in myogenic cells and to study how these are impacted by manipulations of ERK signals. Moreover, we will determine specific roles of RBPJ and HES1 and define targets that differ in quiescent and activated MuSCs.