Sik1 Controls Cardiomyogenesis Figure 5. Neuronal differentiation of wild-type and sik1flp/flp ES cells. (A) Immunofluorescence analysis of wild-type and sik1flp/flp EBs at day 16 of differentiation using an anti-bIII-tubulin antibody reveals the presence of neurons. (B) Quantitative real-time PCR analysis to measure the expression of the neuron- and glial-specific markers NFM and GFAP, respectively, in wild-type and sik1flp/flp EBs at different time points during differentiation, n = 3. doi:10.1371/journal.pone.0009029.g005 could be involved in the sik1 deficiency -dependent delayed cardiomyocyte differentiation. In order to visualize the temporal regulation of p57Kip2 throughout the differentiation process, we first evaluated the transcript levels of Cdkn1c gene in undifferentiated ES cells, in differentiating EBs and in fully differentiated cardiomyocytes, in both the wild-type and sik1flp/flp cells. We also investigated the expression levels of the two other members of the KIP family, Cdkn1a (p21) and Cdkn1b (p27), during cardiomyocyte differenti- ation. As shown in figure 6A, p57Kip2 was expressed at very low levels in undifferentiated wild-type ES cells and in day 1–5 differentiating EBs. From day 5 onward, p57Kip2 expression increased, and its expression correlated to the appearance of beating EBs. It is thus tempting to hypothesize that p57Kip2 is upregulated to progressively withdraw cells from the cell cycle and direct them toward differentiation. In sik1flp/flp cells, the expression levels of p57Kip2 were strongly downregulated throughout differentiation, being barely detectable (Fig. 6A). The levels of the two other members of the KIP family, p21 and p27, in the sik1flp/flp cells were comparable to those seen in wild-type cells, although a reduction in p21 was observed (Fig. 6A). We confirmed these results using Western immunoblotting (Fig. 6B). As shown by the relative quantitative analysis (Fig. 6C), the protein levels were in accordance with the mRNA levels. To further prove the specific effect of sik1 deficiency in the cardiogenic program, we measured the temporal expression of p57Kip2 throughout neuronal differentiation of both wild-type and sik1flp/flp ES cells. As opposed to what we observed in cardiac differentiation, the temporal regulation of cdkn1c gene was not affected throughout the neuronal differentiation in sik1flp/flp population (Fig. 6D). These results are in line with our previous data showing that sik1 deficiency does not affect neuronal differentiation (Fig. 5). Sik1 Directly Regulates p57Kip2 p57Kip2 and other CDKIs have pivotal roles in the regulation of the transition between cell cycle and terminal differentiation in many cell types [24]. To prove that downregulation of p57Kip2 directly affect the cardiogenic program, we evaluated whether re- expression of p57Kip2 in sik1flp/flp cells might rescue the cardiogenic defect of these cells. To this end, we generated transgenic sik1flp/flp cells over- expressing p57Kip2. After selection of positive clones both by PCR (data not shown) and by western blot (Fig. 7A), we evaluated the effects of the p57Kip2 over-expression on cardiac differentiation. Remarkably, the over-expression of p57Kip2 in sik1flp/flp cells was able to fully rescue the cardiac phenotype, as shown by FACS and qPCR analysis. Upon forced expression of p57 the number of cardiomyocytes (MF-20 positive cells) significantly increased in the crucial time window (d5–d7) in sik1flp/flp-derived EBs (Fig. 7B). Expression data supported the FACS analysis (Fig. 7C), demon- strating that p57Kip2 over-expression has the ability to fully rescue the delayed phenotype observed in the sik1flp/flp cells. Altogether, these findings demonstrated that sik1 regulated cardiomyocytes development via the p57Kip2 signaling. PLoS ONE | www.plosone.org 7 February 2010 | Volume 5 | Issue 2 | e9029