br Results br Discussion Here we

Results
Discussion Here we established a role for HES5 in instructing a cardiac fate while repressing the hemogenic program in early mesodermal progenitors. In Hes5-KD the first hematopoietic wave of primitive erythropoiesis overrides the cardiac potential, whereas a stage-specific Hes5 overexpression results in enhanced cardiac differentiation. Importantly, we show Hes5 expression in early mesoderm at the onset of gastrulation, which closely resembles the transient pattern of Mesp1 (E6.5–E7.0) and correlates with the time cardiac progenitors are specified (Saga et al., 1999). Moreover, the unaltered frequency of cell proliferation and apoptosis after Hes5 induction further imply an instructive rather than a selective role in primitive mesoderm. To date, a role for HES5 in cardiac development has not been demonstrated. Hes5-null embryos have no apparent cardiovascular phenotype, but to our knowledge studies specifically addressing heart malformations in the mutants have not been performed. Nonetheless, HES5 function may be compensated by another HES/HEY protein, as functional redundancy between members of these families has been shown at later stages of heart morphogenesis (Fischer et al., 2007; Kokubo et al., 2005). For example, Hey1, Hey2, and Heyl play important roles in heart formation but, with exception of Hey2-null mice, only the double mutants show cardiac abnormalities (reviewed in Nemir and Pedrazzini, 2008). Repression of the hematopoietic/erythroid program to ensure cardiac fate determination in mesodermal (±)-Nutlin-3 Supplier has been previously demonstrated (Caprioli et al., 2011). Our data show that HES5 binds regulators of vascular/hematopoietic and heart development, including Scl and Isl1, which likely mediate in part preferential cardiac cell fate decision downstream of HES5. Homozygous Isl1-null mice have several heart abnormalities, dying by E10.5 (Cai et al., 2003), and Isl1 overexpression in mESCs enhances specification of cardiac progenitors (Dorn et al., 2015). ISL1 has been referred as an SHF marker; however, the expression in pre-cardiac mesoderm and in the cardiac crescent, as well as the contribution to derivatives of both heart fields, suggest ISL1 to be a pan-cardiac progenitor marker (Brade et al., 2007; Ma et al., 2008; Prall et al., 2007; Yuan and Schoenwolf, 2000). Scl-deficient embryos show ectopic cardiogenesis in prospective hemogenic endothelium (Van Handel et al., 2012) and die by E9.5, lacking hematopoiesis in the yolk sac (Robb et al., 1995). In the embryo brain, HES5 maintains undifferentiated neural stem cells, which are then capable to differentiate after downregulation (Ohtsuka et al., 2001). We demonstrate a similar role in cardiogenesis, since sustained Hes5 impairs the emergence of contracting colonies, whereas downregulation after cardiac induction allows cardiomyocytic differentiation. These findings further imply a transient role for HES5 during early mesodermal specification to its derivatives, which is corroborated by the pattern of expression we observed in gastrulating embryos. Thus HES5 plays a dual role in cardiogenesis, promoting cardiac fate specification as a pulse, while impairing differentiation to cardiomyocytes when activated in a sustained manner. Our results are in line with previous evidence for an inhibitory effect of Notch in cardiogenesis, which appears to correlate with activation at the level of development and maturation of the cardiac lineage but not upon its induction (Chen et al., 2008; Liu et al., 2014; Rones et al., 2000; Watanabe et al., 2006). Interestingly, and suggesting a conserved mechanism throughout evolution, esr9, a member of the Hes5-like subfamily expressed in Xenopus (Li et al., 2003), regulates the timing of heart field specification, repressing cardiac differentiation when induced after gastrulation (Miazga and McLaughlin, 2009). Herein, we identified an important player downstream of NICD1 that sets in motion cardiogenesis and represses hematopoietic commitment in early mesodermal precursors. Notably, the effect of Hes5 on primitive erythropoiesis is consistent with the required inhibition of Notch signaling for development of this lineage (Cheng et al., 2008), while NOTCH1 is essential for definitive hematopoiesis (Kumano et al., 2003). Furthermore, our data reinforce the context-specific and time-dependent nature of HES proteins, i.e., not only the timing of induction but also the duration of the signal affects cell fate determination and differentiation.