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    • br Introduction Cardiac morphogenesis results from the speci

    2018-10-24


    Introduction Cardiac morphogenesis results from the specification, differentiation, and migration of spatially and temporally distinct sets of cardiac precursor 740 Y-P that give rise to the mature cardiac tissue. The first multipotent cardiogenic cells originate from the mesoderm formed at early stages of gastrulation when cells of the epiblast ingress to the primitive streak (Costello et al., 2011; David et al., 2011; Garry and Olson, 2006; Kitajima et al., 2000; Tam et al., 1997). The mesoderm first expresses the markers BRACHYURY and FLK1 and subsequently the cardiogenic marker MESP1 (Bondue et al., 2008; Chan et al., 2013; Ishitobi et al., 2011; Saga et al., 1999). The first (FHF) and second (SHF) heart fields then arise from the cardiogenic mesoderm to ultimately generate the atria, ventricles, and outflow tract of the nascent heart (Cai et al., 2003; Domian et al., 2009; Moretti et al., 2006). Both FHF and SHF progenitors express the transcription factors NKX2.5, TBX5, GATA4, MEF2C, and ISL1, although TBX5 is predominantly present in cells of the FHF, and Isl1 expression is a hallmark of SHF progenitors (Laugwitz et al., 2005; Moretti et al., 2006; Vincent et al., 2010). In mouse, the transcriptional modulator CITED2 is required for normal embryogenesis. Deletion of Cited2 in the epiblast results in embryonic lethality associated with multiple cardiovascular defects (Bamforth et al., 2001, 2004; MacDonald et al., 2008, 2013; Weninger et al., 2005; Yin et al., 2002). Of important note, however, although Cited2 is expressed in the early mesoderm (Dunwoodie et al., 1998), conditional deletion of Cited2 in BRACHYURY-expressing mesoderm cells or MESP1-expressing cardiogenic mesoderm progenitors did not significantly affect cardiac development (MacDonald et al., 2008). In humans, mutations in the gene encoding CITED2 are associated with congenital heart disease (Chen et al., 2012; Sperling et al., 2005). The specification and differentiation of cardiac progenitor cells (CPC) and mature cardiovascular cells during the in vitro differentiation of pluripotent stem cells recapitulate the cellular and molecular processes of embryonic development (Blin et al., 2010; Bondue et al., 2008, 2011; Christoforou et al., 2008; Gai et al., 2009; Kattman et al., 2011; Kouskoff et al., 2005; Laugwitz et al., 2005; Moretti et al., 2006; Van Vliet et al., 2012; Yang et al., 2008). In mouse, an acute Cited2 depletion reduces the self-renewal capacity of most embryonic stem cells (ESC), but a small population of Cited2-null ESC with apparent characteristics of undifferentiated cells adapt to the loss of Cited2 (Kranc et al., 2015; Li et al., 2012). Interestingly, Cited2-null ESC showed an impairment of differentiation, including cardiac commitment (Li et al., 2012). To better understand the role of Cited2 at early stages of mouse ESC differentiation, we here employ Cited2 loss- and gain-of-function approaches to examine the role of Cited2 during cardiac differentiation. Cited2 depletion at the onset of differentiation significantly impairs the expression of Brachyury, Mesp1, Isl1, Gata4, and Tbx5. Conversely, CITED2 overexpression stimulates the expression of these genes in undifferentiated ESC and promotes cardiac lineage commitment and differentiation. We further show that Cited2 expression is highly associated with CPC populations, particularly cardiac progenitors of the SHF. Finally, we show that CITED2 is recruited to the promoter of the Isl1 gene, and provide evidence that the human CITED2 and ISL1 proteins physically interact and synergize to promote cardiogenesis from ESC. Collectively our results show that Cited2 is a key regulator of early cardiac lineage commitment and differentiation of ESC.
    Results
    Discussion In this study, we show that Cited2 is required for early cardiac commitment of mouse ESC, in agreement with a previous study (Li et al., 2012). We and others have demonstrated that Cited2 expression promotes mouse ESC self-renewal (Chen et al., 2012; Kranc et al., 2015; Pritsker et al., 2006). Interestingly, we show that the deletion of Cited2 in ESC or depletion of Cited2 at the onset of differentiation impairs cardiac lineage commitment. Remarkably, we determine that endogenous expression of Cited2 transcripts is biphasic during ESC differentiation, starting with a decrease from D0 to D2. This decline of Cited2 expression upon differentiation might be necessary for ESC to switch from a non-permissive to a permissive differentiation state. In a second phase, Cited2 expression increases from D3 of differentiation onward, implying that Cited2 might be required for subsequent differentiation processes. This is corroborated by the rescue of the cardiac differentiation defects in Cited2-depleted ESC with the supplementation of the recombinant CITED2 protein to the cells at D2 of differentiation, a day before the levels of endogenous Cited2 start to increase during differentiation.