UNRAVELLING THE MOLECULAR SIGNALLING REGULATING EMBRYONIC AND FETAL MYOGENESIS: THE ROLE OF NFIX AS ¿MASTER¿ GENE IN REGULATING FETAL GENETIC PROGRAM

Skeletal muscle development occurs through successive developmental phases, named embryonic and fetal myogenesis, involving the differentiation of distinct myogenic populations: the embryonic and fetal myoblasts. An important work developed in 2007 identified specific features of embryonic and fetal myoblasts, demonstrating that these two populations of muscle progenitors represent intrinsically different myogenic lineages. The identification of the transcriptional factor Nfix was the major step in understanding how muscle progenitor fate decisions are mediated. Nfix, expressed in fetal but not in embryonic muscles, is necessary for the acquisition of fetal myogenic identity, activating fetal-specific genes and repressing the expression of embryonic one, such as slow myosin heavy chain (MyHC-I). Another repressor of MyHC-I, during fetal myogenesis, is the transcription factor Sox6, which inhibits MyHC-I by direct binding to the proximal promoter of MyHC-I gene. Interestingly, Sox6 at variance with Nfix is also expressed during embryonic myogenesis, when embryonic myoblasts form fibers that express high level of MyHC-I. We demonstrated that Sox6 has opposite roles in regulating MyHC-I expression between embryonic and fetal myogenesis. Specifically, during embryonic myogenesis, Sox6 indirectly promotes MyHC-I expression via transcriptional activation of Mef2C. On the contrary, during fetal myogenesis, Nfix allows the proper binding of Sox6 to the MyHC-I promoter with the consequent repression of MyHC-I expression. In addition, we provide evidence that the functional interplay of Nfix and Sox6 is conserved also in zebrafish. Although Nfix functions were partially characterized, nowadays the mechanisms that regulate its expression during fetal myogenesis are still unknown. Another transcription factor more expressed during fetal myogenesis is JunB. We assessed that JunB is required and sufficient to induce the expression of Nfix, acting as direct activator of it. To better elucidate this pathway, we started to study the possible involvement of RhoA and its major kinase ROCK, since growing investigations have shown that RhoA/ROCK regulate skeletal muscle differentiation. We demonstrated that RhoA/ROCK are active only during embryonic myogenesis and their inhibition in embryonic myoblasts increased the expression of both Nfix and JunB. The interference with the RhoA/ROCK signalling led also to the increased activation of the ERK kinases, which we show are necessary for Nfix and JunB up-regulation. In summary, we have identified the RhoA/ROCK axis as an important negative regulator of JunB and Nfix expression during embryonic myogenesis, through the inhibition of ERK activity. Conversely, during fetal myogenesis, the ERK kinases are active and allow JunB and Nfix expression. Finally, Nfix is sufficient to activate the fetal genetic program and to permit the complete maturation of prenatal skeletal muscles.