Under the supervision of Pascal Maire team Neuromuscular Development, Genetics and Physiopathology
Four SIX homeoproteins display a combinatorial expression throughout embryonic developmental myogenesis and they modulate the expression of the myogenic regulatory factors. However, the specific requirement of each of the SIX homeoproteins on each of the craniofacial, hypaxial or epaxial embryonic territories is not defined. Here we define the redundancies between those paralogs and show that Six1 and Six2 are required for craniofacial myogenesis where their KO leads to a complete absence of craniofacial musculature, not observed in their respective single KO, and we demonstrate that they regulate the myogenic commitment of progenitor cells in the branchial arches and on the extraocular muscles level. However, on the hypaxial level, it is Six1 and Six4 that are essential to activate the myogenic fate of PAX3+ progenitor cells and their migration to the growing limb buds. In fact, we demonstrate that their double KO results in the loss of the myogenic identity of PAX3+ cells in favor an endothelial and smooth muscle fate, whereas their migration is redirected under the dorsal aorta instead of the growing limb buds. Finally, we investigated the requirement of all the myogenic SIX homeoproteins by generating SIX quadruple KO fetuses. Interestingly, those mutant fetuses still form highly reduced and disorganized muscle masses at the epaxial and neck levels, and we show that the progenitor cells fail to self-renew and eventually differentiate prematurely, which leads to their exhaustion by the end of fetal development. We thus refined the involvement of SIX homeoproteins in the genetic cascades operating at the head level and in the genesis of myogenic stem cells and uncovered distinct cross-talks required between these transcription factors the different myogenic territories. In our search for new potential SIX1 cofactors, we identified LRRFIP2, that was already known to be highly expressed in the skeletal muscle, as one of them. We characterized three different LRRFIP2 isoforms in the skeletal muscle, generated by alternative splicing events as well as their differential expression and subcellular localization in standard and hypoxic conditions. We then generated a conditional Lrrfip2 KO in muscle stem cells (SC) to investigate its role during muscle regeneration and found that LRRFIP2 refrains the exit of SC from quiescence at the basal state, where the number of PAX7+ cells and the expression level of Pax7 are both reduced in the mutant mice. This defect led to a shift in the timeframe of regeneration and to an earlier differentiation and formation of new myofibers accompanied by an earlier return of SC to quiescence; effect we report to the dialog between the Lrrfip2 mutant regenerated myofiber and the mutant SC. Finally, we highlight the anti-inflammatory role of Lrrfip2 in SC through TLR4 signaling and MyD88 activation and show that the nuclear LRRFIP2 isoforms switch we observe in hypoxia is conserved in nuclear human muscle extracts, opening insights into studying the precise role of each of those isoforms in the skeletal muscle and the SC.