The bHLH protein MyoR inhibits the differentiation of early embryonic endoderm

MyoR is a bHLH protein whose expression was reported to be almost exclusively restricted to the precursors of the skeletal muscle lineage where it was postulated to function as a transcriptional repressor of myogenesis. However, previous studies in our laboratory suggested a much broader role for MyoR in embryonic cell differentiation. We demonstrated that, besides being expressed in several adult tissues of non-muscle lineage, MyoR was expressed at a much earlier stage in mammalian development than had previously been reported, that is, as early as the blastocyst stage, well before skeletal muscle specification. We also found that, as in skeletal muscle precursor cells, MyoR expression is inversely correlated with the cellular differentiative state of ectodermal, non-muscle embryonal carcinoma (EC) cells. Retinoic acid (RA) treatment of ectodermal EC or embryonal stem (ES) cells promotes their differentiation into primitive endoderm. However, in the present study, we show that the RA-induced expression of endodermal markers such as EndoA, collagen IV, and t-PA are inhibited by exogenous MyoR expression and that the level of inhibition of these markers correlates with the level of MyoR expressed. Conversely, knock-down of MyoR expression via RNA interference enhances RA-induced differentiation of EC cells, promoting earlier and much higher expression of the above-mentioned endodermal markers following RA treatment. Finally, we have narrowed the period of exogenous MyoR-induced embryonic lethality to between 3.5 and 5.5 days post-coitum (dpc), the period during which embryonic endoderm differentiates from the embryonic ectoderm. Our results suggest, therefore, that inhibition of endodermal differentiation between 3.5 and 5.5 dpc contributes to the embryonic death of mouse embryos overexpressing exogenous MyoR and consequently that MyoR may serve as a repressor of embryonal endoderm differentiation.