when the sik1flp/flp EBs reached the same percentage of beating EBs as wild-type cells (Fig. 2A). The EBs derived from sik1+/flp cells showed an intermediate phenotype compared to sik1flp/flp EBs, which can be attributed to a gene-dosage effect. We then tested whether functional rescue of cardiomyocyte differentiation can be achieved by gene re-introduction. To this purpose, we generated stable clones overexpressing the HA-tagged sik1 transgene. Several drug-resistant clones were amplified and RNA extracted to measure the expression of HA-sik1 by qPCR. Sik1 transgene expression was detected in all clones analyzed; however, none of them reached the basal level of endogenous sik1 expression (Fig. 2B). The clone B6, which showed the highest level of transgene expression, was induced to differentiate into cardiomy- ocytes. The HA-sik1(B6) overexpressing clone only partially restored the appearance of the beating cardiomyocytes (Fig. 2A). Since over-expression experiments do not allow the modulation of kinase activity both in terms of timing and signal strength and, moreover, since the levels of transgene expression in selected clones were lower than in the heterozygous cells, the combination of these drawbacks might explain the incomplete rescue (Fig. 2A). Taken together, these data indicate that the sik1flp/flp population can differentiate into contracting cardiomyocytes, but showing a consistent delay in cardiomyogenesis. Having shown that lack of sik1 led to a delayed differentiation, we assessed the proliferative potential of sik1flp/flp ES cells by BrdU incorporation, postulating that the lack of sik1 might increase the proliferation rate of these cells. In line with this hypothesis, the rate of sik1flp/flp cell proliferation significantly increased (15%) compared to the wild-type cells (Fig. 2C). These data were also confirmed by cell counting (data not shown). The increased proliferation rate of sik1flp/flp ES cells resulted from a shortened G1 phase (wt 37% vs. sik1flp/flp 23%, Fig. 2D) and accumulation in S- phase, (wt 55% vs. sik1flp/flp 72%, Fig. 2D). sik1 Deficiency Specifically Affects Cardiac Differentiation of ES Cells We evaluated the expression of pluripotency genes as well as of mesoderm induction and specification markers. We measured the expression of Oct-3/4 transcript, a pluripotency marker, and Brachyury transcript, which is required for correct mesoderm formation and patterning [17]. As shown in figure 3A, sik1 deficiency did not affect Oct-3/4 expression, which was progres- sively downregulated in both cell populations, nor Brachyury, which was nearly absent in the undifferentiated ES cells and transiently expressed during embryoid body formation peaking at day 4. At the same time, committed mesodermal cells started to express similar levels of pre-cardiac mesodermal genes (Mesp1, Mesp2 and Islet-1 [18] in both the wild-type and in the sik1flp/flp differentiating populations. Given the delay in cardiomyogenesis (Fig. 2A), we studied cardiac differentiation in sik1flp/flp differentiating EBs measuring the levels of cardiogenic transcription factors (Nkx2.5, Tbx5 and Mef2C), which are first expressed in cardiomyoblast cells [19]. These factors then act in combination to activate the expression of several cardiac structural genes, i.e. myosin heavy chain (a-MyHC), myosin light chain (Mlc2V), calcium channel (aCach) and cardiac troponin (cTnI) [20]. We measured the expression of these genes both in the wild-type and in the sik1flp/flp differentiating EBs. Nkx2.5, Tbx5 and Mef2C expression started at day 5 in the wild- type EBs and progressively increased. In the sik1flp/flp differenti- ating EBs, the expression of these genes was impaired in this time window; in line with the morphological analysis, the expression of the cardiac structural genes (a-MyHC, Mlc2V, aCach and cTnI) was Sik1 Controls Cardiomyogenesis impaired from day 6 to day 8, whereas it increased from day 9 onward to reach levels comparable to wt cells. (Fig. 3A), Next, we also examined the influence of sik1 on the induction of the hematopoietic/endothelial lineage, measuring the expression of Runx1, VegfC and Flk1. As shown in figure 3B, the levels of expression of these genes were not significantly impaired in the sik1flp/flp differentiating population. Altogether, these data suggested that mesoderm formation and patterning were not altered ab inicio, as demonstrated by the correct expression of Mesp1, Mesp2 and Isl1, but the cardiogenic programme of these cells was delayed, as showed by the downregulation of Nkx2.5, Tbx5 and Mef2C genes as well as of markers of terminal cardiac differentiation (aMyHC, Mlc2v, aCach, cTnI). In order to ascertain whether lack of sik1 affected the number of cardiac cells, we quantified the percentage of cardiomyocytes derived from wild-type and sik1flp/flp EBs throughout the differentiation by FACS analysis. To this end, we used the sarcomeric myosin (MF-20) staining (Fig. 4A, 4B). In line with our hypothesis, from day 5 to day 7, the sik1flp/flp -derived EBs showed a reduced number of MF-20 positive cells, compared to the wt EBs. However, at day 9, the number of cardiomyocytes was fully rescued in the sik1flp/flp EBs, according to the morphological and qPCR analysis reported above (Fig. 2A–3A). Furthermore, we analyzed the FACS data by discriminating high fluorescence (hf) vs. low fluorescence (lf) cells expressing sarcomeric myosin (MF-20). Remarkably, while a significant number of hf cells already appeared at d5 in wt EBs and the hf vs. lf ratio was comparable throughout the differentiation, in sik1flp/flp EBs, consistent number of hf cells can be detected only starting from day 9 onward (Fig. 4C). All together, these data supported the idea that the appearance of cardiomyocytes in sik1flp/flp EBs occurred as a slower wave of differentiation. Worth noting, in many cell types the differentiation is strictly dependent on the cell cycle withdrawal. We thus reasoned that the difference observed in the two cell populations may be due to a defect of the mechanism that regulates cell cycle exit toward differentiation of cardiomyocytes. To directly address this issue, we carried out FACS analysis to study the concurrent cell cycle withdrawal and differentiation in the cardiomyocyte population in both groups and in the most critical days of differentiation (d5–d7). To this end, we performed double immunostaining studies using an antibody against a cardiac sarcomeric protein (cTnI) coupled with the analysis of cell cycle activity (BrdU). On day 5, double positive cells dramatically increased in sik1flp/flp vs. wt population, indicating that cell cycle exit of cardiomyocytes was impaired in absence of sik1 (Fig. 4C). Furthermore, at day 6, few double- positive cells were detected in the wt population, which almost disappeared at day 7. On the contrary, in the sik1flp/flp population, the MF20/BrdU-positive cells decreased in number at day 6 but were still present at day 7, supporting the idea of a delayed cell cycle withdrawal in absence of sik1. Lack of sik1 Does Not Affect Neuronal Differentiation In order to exclude a general effect of sik1 deficiency on ESC differentiation, we evaluated whether sik1 deficiency impairs neuronal differentiation. As shown in figure 5A, both the wild- type and the sik1flp/flp EBs were able to generate a dense network of neurite growth [21]. The neural nature of these cells was confirmed by staining with an antibody against the neuron- specific form of class bIII-tubulin (Fig. 5A). We also measured the expression of the neuron- and glia-specific markers NFM and GFAP (Fig. 5B). The expression of NFM and GFAP was similar in PLoS ONE | www.plosone.org 4 February 2010 | Volume 5 | Issue 2 | e9029