Our bioinformatics study using the results from the global gene expression analysis of human cells (GSM412342-41344 and GSM201137-201145 at http://www.ncbi.nlm.nih.gov/geo
) nominated Grem1 as a candidate gene that may participate in cardiomyogenesis. By using CL6 embryonic cells as a model of cardiomyogenesis, we obtained two major findings: the first is that Grem1 enhanced cardiomyogenic differentiation of DMSO-induced CL6 cells at the early stage; the second is that Wnt/β-catenin and BMP signaling activity had developmental stage-specific effects on cardiomyogenesis (). Wnt/β-catenin activity at the early stage enhanced embryonic cell differentiation into cardiomyocytes, while suppressing this activity by BMP2 or BMP4 proteins as reported in the avian embryo 
. In contrast, BMP signaling activity in the late stage enhanced cardiomyocytic differentiation. Grem1 regulated the stage-specific Wnt/β-catenin and BMP signaling activity on cardiomyogenesis.
Grem1-accelerated CL6 cardiomyogenesis through regulation of BMP- and Wnt/β-catenin-signaling pathways.
Many studies have indicated that Grem1 is involved in cell differentiation and development, such as osteogenesis 
, lung morphogenesis 
, myogenesis 
, and limb formation 
, through inhibition of BMP2 and BMP4. Grem1-null mice show intact heart development, despite impairment of lung and kidney 
, and therefore Grem1 is considered not to be involved in cardiogenesis, or supplementary factors such as Noggin 
, with a similar function, may compensate Grem1 during development. Grem1 had an enhancing or promoting activity in in vitro
cardiomyogenesis, as is the case with platelet-derived growth factor as a promoter of cell growth 
. In this study, Grem1 was involved in cardiomyocyte differentiation. However Grem1 alone could not induce cardiomyocytic differentiation of CL6 cells in the absence of DMSO (), suggesting that Grem1 is solely a promoter of cardiomyogenic differentiation. One of the possible mechanisms for Grem1-enhanced cardiomyogenesis at the early stage is inhibition of the BMP signaling pathway 
. Alternatively, Grem1-enhanced cardiomyogenesis may be mediated through proliferation of cardiac progenitor cells, as is the case of myogenic progenitor proliferation by Grem1 
, and this possibility is supported by an increased number of sarcomeric myosin-positive CL6 cardiomyocytes ().
The stage specificity of the Grem1 effect is possibly correlated with the biphasic and antagonistic effect of Wnt/β-catenin signaling on cardiomyogenesis, depending on the stage of development in vitro 
and in vivo 
. CL6 cells differentiated into cardiomyocytes via mesodermal induction by the Wnt/β-catenin signaling pathway at the early stage, and CL6 mesodermal cells differentiated into cardiomyocytes induced by BMP2 at the late stage. It is conceivable that embryonic cells, such as CL6 cells and ES cells, differentiate into cardiomyocytes by inhibiting BMP signaling via putative “mesodermal cells” or “cardiomyogenic progenitors”, or differentiation stages corresponding to these cells (, Figure S2
). The early stage process from embryonic cells to mesodermal cells was mediated via Wnt/β-catenin signaling (), and was assessed by expression of BrachyuryT
genes (), which are target genes for Wnt/β-catenin signaling 
. BMP signaling antagonizes the cell fate-inducing activity of Wnt/β-catenin 
. When embryonic cells or cardiomyogenic progenitors are induced to become mature cardiomyocytes by cytokines and growth factors, we must be careful with respect to the stage of cell differentiation because of the biphasic differential action of the factors which are dependent upon the differentiation stage.
In conclusion, we have demonstrated that Grem1 enhances the commitment or determined path to cardiogenic differentiation of CL6 teratocarcinoma cells. Apart from a role in development, Grem1 may serve a clinical use in cardiology, like granulocyte colony-stimulating factor that accelerates production of granulocytes in both peripheral blood and bone marrow. Nomination of Grem1 as a cardiomyogenic factor is based on hierarchical clustering analysis using global gene expression data of human cells. This bioinformatics approach may be useful for identifying morphogens/factors that can induce differentiation of other cell types/tissues/organs.