CrxOS maintains the self-renewal capacity of murine embryonic stem cells.

Embryonic stem (ES) cells maintain pluripotency by self-renewal. Several homeoproteins, including Oct3/4 and Nanog, are known to be key factors in maintaining the self-renewal capacity of ES cells. However, other genes required for the mechanisms underlying this process are still unclear. Here we report the identification by in silico analysis of a homeobox-containing gene, CrxOS, that is specifically expressed in murine ES cells and is essential for their self-renewal. ES cells mainly express the short isoform of endogenous CrxOS. Using a polyoma-based episomal expression system, we demonstrate that overexpression of the CrxOS short isoform is sufficient for maintaining the undifferentiated morphology of ES cells and stimulating their proliferation. Finally, using RNA interference, we show that CrxOS is essential for the self-renewal of ES cells, and provisionally identify foxD3 as a downstream target gene of CrxOS. To our knowledge, ours is the first delineation of the physiological role of CrxOS in ES cells.

p38 Mitogen-activated protein kinase controls a switch between cardiomyocyte and neuronal commitment of murine embryonic stem cells by activating myocyte enhancer factor 2C-dependent bone morphogenetic protein 2 transcription.

Many studies have shown that it is possible to use culture conditions to direct the differentiation of murine embryonic stem (ES) cells into a variety of cell types, including cardiomyocytes and neurons. However, the molecular mechanisms that control lineage commitment decisions by ES cells remain poorly understood. In this study, we investigated the role of the 3 major mitogen-activated protein kinases (MAPKs: extracellular signal-regulated kinase, c-Jun N-terminal kinase, and p38) in ES cell lineage commitment and showed that the p38 MAPK-specific inhibitor SB203580 blocks the spontaneous differentiation of ES cells into cardiomyocytes and instead induces the differentiation of these ES cells into neurons. Robust p38 MAPK activity between embryoid body culture days 3 and 4 is crucial for cardiomyogenesis of ES cells, and specific inhibition of p38 MAPK activity at this time results in ES cell differentiation into neurons rather than cardiomyocytes. At the molecular level, inhibition of p38 MAPK activity suppresses the expression of bmp-2 mRNA, whereas treatment of ES cells with bone morphogenetic protein 2 (BMP-2) inhibits the neurogenesis induced by SB203580. Further, luciferase reporter assays and chromatin immunoprecipitation experiments showed that BMP-2 expression in ES cells is regulated directly by the transcription factor myocyte enhancer factor 2C, a well-known substrate of p38 MAPK. Our findings reveal the molecular mechanism by which p38 MAPK activity in ES cells drives their commitment to differentiate preferentially into cardiomyocytes, and the conditions under which these same cells might develop into neurons.

Pax6-5a promotes neuronal differentiation of murine embryonic stem cells.

Pax6 genes are highly conserved and important for eye development. Vertebrates predominantly produce two alternatively spliced Pax6 isoforms, Pax6 and Pax6-5a. Pax6-5a differs from Pax6 by the presence of 14 additional amino acids encoded by exon 5a. These additional amino acids occur in the Pax6 paired domain and thus influence its DNA-binding properties. However, little is known about Pax6-5a's physiological functions. Here we establish murine embryonic stem (ES) cell lines in which expression of either the human Pax6 or Pax6-5a isoform is negatively controlled by tetracycline. We report that, in contrast to Pax6 expression, Pax6-5a expression strongly induces ES cells to differentiate into neurons. Moreover, using DNA microarray analysis, we have identified the transcription factor basic helix loop-helix domain containing, class b2 (bHLHb2) in Pax6-5a-expressing ES cells. Our Pax6 isoform-expressing ES cell lines may serve as useful models for identifying Pax6-regulated genes that are important for neurogenesis and/or eye development.