The impairment of cardiac differentiation resulting from Cit

The impairment of cardiac differentiation resulting from Cited2 depletion at the onset of ESC differentiation may be due to a consequent downregulation of Brachyury and Mesp1 expression. The decrease of Isl1, Nkx2.5, Gata4, and Tbx5 expression that we observed in Cited2-depleted cells at D5 of differentiation, which corresponds to the time when CPC emerge, might be a consequence of Mesp1 downregulation, since these genes are activated by MESP1 to promote cardiogenesis and CPC specification during cardiac development and ESC differentiation (Bondue et al., 2008; David et al., 2011). On the other hand, we show that Cited2 expression is enriched in CPC, and CITED2 protein is recruited to Isl1 promoter at D5 of differentiation during CPC specification. This suggests that CITED2 may play a role in CPC specification, proliferation, and/or differentiation. In agreement with this hypothesis, flag-CITED2 overexpression promoted both the emergence of CPC and terminal cardiomyocyte differentiation from ESC, and increased Isl1, Nkx2.5, Gata4, and Tbx5 expression in undifferentiated ESC. Fluctuations in the expression of genes encoding transcription factors with capacities to instruct lineage specification have been evidenced in undifferentiated pluripotent ESC and, in accordance with the transcripts expressed, subsets of ESC might be prone to undergo particular differentiation programs (Lanner and Rossant, 2010). The overexpression of CITED2 in undifferentiated ESC might specify and promote CPC and cardiac differentiation by raising the expression of the pro-cardiogenic factors Isl1, Nkx2.5, Gata4, and Tbx5. Interestingly, the transdifferentiation of human dermal fibroblasts into cardiac progenitors by expression of MESP1 and ETS2 has been shown to stimulate the expression of CITED2 (Islas et al., 2012), suggesting that CITED2 may play a role in cardiac progenitor specification and/or functions. Therefore, CITED2 overexpression might be instrumental for specification of CPC and cardiomyocyte differentiation from pluripotent stem cells. We also provided evidence for a privileged regulatory interaction between Cited2 and Isl1. Indeed, our ChIP assays show that CITED2 is specifically recruited at the Isl1 promoter. Furthermore, supplementation of Cited2-null ESC with CITED2 recombinant protein restores the expression of Isl1 to normal levels at D5 of ESC differentiation. In addition, ESC overexpressing flag-CITED2 are enriched for H3triMeK4 at the Isl1 regulatory elements, which marks transcriptionally active chromatin. The mechanisms by which flag-CITED2 overexpression contribute to the enrichment of H3triMeK4 at the Isl1 loci and increase Gata4 and Nkx2.5 expression in undifferentiated ESC remain to be elucidated. Interestingly, at the protein level we demonstrated that ISL1 and CITED2 interact, delineated the Dinaciclib 66–214 of CITED2 as part of the ISL1 interacting domain, and by transient transfection assays established that CITED2 increased ISL1-mediated Mef2c-enhancer activity and that ISL1 and CITED2 have a synergistic effect on cardiac cells derived from ESC. The exact molecular mechanism by which CITED2 and ISL1 cooperate in cardiac specification remains to be clarified, but it has been recently demonstrated that in mouse embryonic hearts and cardiac progenitors ISL1 interacts with p300 to selectively promote H3 acetylation at the Mef2c promoter (Yu et al., 2013). Since CITED2 interacts strongly with p300, it would be of interest to determine whether CITED2 cooperates with p300 to promote its functional interaction with ISL1. Altogether, our results indicated that Cited2 contributes to the expression of a subset of pivotal cardiopoietic genes involved in mesoderm and cardiac progenitor specification (Figure 5F). During mouse embryonic development, Cited2 expression was detected in the early mesoderm- and cardiac-derived structures (Dunwoodie et al., 1998), but rather surprisingly a Brachyury/T-Cre or Mesp1-Cre conditional Cited2 knockout only resulted in infrequent and minor heart developmental defects of mouse embryos, while Cited2 knockout in the epiblast consistently caused heart malformations (Bamforth et al., 2001; MacDonald et al., 2008). In the present report, we show that Cited2 depletion at the onset of differentiation causes the most severe impact on cardiac differentiation, and results also in the impairment of Brachyury and Mesp1 expression at D3. Interestingly, the cardiogenic defects due to the loss of Cited2 expression at D0 were reversed by supplementation of the 8R-CITED2 at D2 of differentiation. On the other hand, Cited2 depletion at later time points (D2, D4, and D6) had little or no effect on cardiogenesis. Together, these observations indicate that Cited2 function is important for early commitment of ESC to mesoderm and/or cardiac specification, or at least contribute to the correct expression of Brachyury and Mesp1, or other genes crucial for these processes. Therefore, the lack of a strong phenotype in Brachyury/T-Cre and Mesp1-CreCited2 conditional knockout might be because the depletion of Cited2 in these embryos was triggered after Brachyury and Mesp1 were activated and after the requirement for Cited2. Of particular interest, we show that CITED2 stimulates the expression of Isl1, a marker of SHF cardiac progenitors, and binds to its promoter at the time of CPC specification. We also show that Cited2 expression is enriched in SHF cardiac progenitors derived either from ESC or mouse E9.5 embryonic hearts. Moreover, Cited2-null embryos display a variety of cardiac developmental defects such as ventricular septal defects with overriding aorta or double-outlet right ventricle, outflow tract defects, and transposition of the great arteries, which may result from anomalies of SHF and cardiac neural crest cell progenitors known to express ISL1 (Bamforth et al., 2001; Bruneau, 2013). Therefore, CITED2 and ISL1 may also interact during heart development and play a critical role for the fulfillment of CPC functions in this process. It would be of interest to investigate the contribution of the CITED2-ISL1 interaction in CPC functions both in vitro and in vivo.