The importance of the finding that the prostaglandin pathway

The importance of the finding that the prostaglandin pathway is selectively downregulated by MSX2, but not MSX1, is 2-fold; i) this functional disparity between MSX1 and MSX2 provides a mechanistic insight into a cellularization event during muscle dedifferentiation and ii) it also demonstrates that these two homeodomain-containing homolog proteins are not completely redundant and have different functionalities. Previous studies and sequence comparisons have shown differential binding affinities of MSX1 and MSX2 homeodomains to DNA and differences in primary protein sequence at the N-terminus (Catron et al., 1996). Our results indicate that these differences lead to functional and qualitative distinction of MSX1 and MSX2. This idea is also strengthened by our observation that MHC transcript levels are decreased only by Msx2 expression in myotubes, but not MSX1. Our finding about the role of the cAMP pathway in myotube cellularization and phenotypic dedifferentiation connects MSX2-induced cytosolic signaling with the nuclear changes and opens up several questions about the downstream and parallel signaling events. Which GPCRs are activated upon MSX2-induction and are responsible for signaling to the nucleus for gene expression changes? What downstream effectors, secondary transcription factors and epigenetic modifiers mediate this signaling? How does the role of cAMP signaling during dedifferentiation compare with its role during differentiation? Previous studies have shown that intracellular cAMP levels decrease upon myoblast fusion and sustained cAMP signaling inhibits myoblast fusion and differentiation (Hu and Olson, 1988; Li et al., 1992; Tsai et al., 1997). Our results indicate that cAMP signaling is a major regulator of the downregulation of myotube-specific gene expression profile, as well as the consecutive morphological changes induced by ectopic Msx2 expression. Previous studies on newt myotubes undergoing dedifferentiation suggested that etizolam vendor re-entry was uncoupled from phenotypic dedifferentiation and cellularization. (Velloso et al., 2000). Furthermore, several reports aimed at inducing cell cycle re-entry in myotubes identified 1) CyclinD1 and CDK4 activation; 2) Rb and ARF downregulation, or 3) sustained ERK activity as necessary events (Pajcini et al., 2010; Latella et al., 2001; Yun et al., 2014). Our findings showing extensive phenotypic dedifferentiation and cellularization along with partial cell cycle re-entry suggest a similar uncoupling between the first two and the latter events for mammalian myotubes. On the other hand, the pathways leading to the events related to cell cycle re-entry are not entirely activated during Msx2 expression, with the exception of CyclinD1 upregulation. A recent study showed that a combination of BMPs and FGFs was sufficient to form the blastema in a newt limb regeneration model (Makanae et al., 2014). Although this study analyzed a complex heterogeneous tissue such as blastema, the results from our pharmacological treatments that recapitulate phenotypic dedifferentiation and cellularization events in isolated myotubes suggest that BMP and FGF signaling are likely to contribute to muscle dedifferentiation within blastema, as well. Both the genetic and the pharmacological inducers that we used to engineer the amphibian dedifferentiation response into mammalian myotubes have been previously implicated in the inhibition of differentiation. Expression of Msx genes have been associated with multipotent progenitor cells and inhibition of differentiation of various mesenchymal progenitor cells including the muscle progenitor cells (Hu et al., 2001; Kodaka K et al., 2015; Takahashi et al., 2001). Similarly, BMP signaling stimulates muscle progenitor cell expansion and inhibits myogenic differentiation (Ono et al., 2011; Shi et al., 2011). Interestingly, Msx2 is a downstream target gene for BMP signaling (Brugger et al., 2004). FGF signaling is also required to maintain the proliferative state of the muscle progenitor cells and blocks differentiation (Olwin and Rapraeger, 1992; Rando and Blau, 1994). These inhibitory functions of MSX2, BMPs and FGFs on differentiation and their association with the maintenance of the proliferative progenitor cells further strengthens their potential to reverse the differentiated state of myotubes when ectopically expressed or activated in those cells. Our single and combinatorial treatments also suggest that when combined with molecules that allow for a more accessible chromatin, as in the case of TSA, mammalian myotubes exhibit an enhanced response to these signaling molecules for cell fate reversal.