31 Harnessing developmental cues for cardiomyocyte production 2 muscle development is induced and erythropoiesis is suppressed (Marvin et al., 2001, Naito et al., 2006). Meanwhile, ectopic expression of WNT8 or WNT3a in precardiac mesoderm inhibits heart muscle formation and promotes erythropoiesis (Marvin et al., 2001). In mouse embryonic stem cells, Wnt/β-catenin signaling acts biphasically; early treatment with Wnt3a stimulates mesoderm induction and cardiac differentiation, whereas late activation of β-catenin signaling impairs cardiac differentiation (Ueno et al., 2007). In combination with BMP and FGF, Wnt inhibition results in the activation of key upstream cardiac transcriptional regulatory genes NKX2-5, GATA4 and TBX5, which are required for the initiation of cardiac-like gene expression (Kelly et al., 2014). The formation of the linear heart tube is mostly initiated by the contribution of the FHF, which eventually gives rise to the inflow tract and the majority of the left ventricle (Brade et al., 2013). FHF progenitors at this stage specifically express TBX5 and HCN4 (Später et al., 2013; Bruneau et al., 1999). The SHF develops slightly later and is less differentiated, providing cardiac progenitors that proliferate to promote the expansion of the heart tube (Kelly, 2012). The right ventricle and outflow tract are exclusively generated by the SHF (Buckingham et al., 2005). These SHF cardiomyocytes are marked with TBX1, ISL1 and HAND2 (Moretti et al., 2006; Stanley et al., 2002). Together, these studies demonstrate that Wnt signals in different parts of the mesoderm are repressed as required for cardiac specification of these regions. Most in vitro protocols for directed cardiac differentiation of pluripotent stem cells incorporate this inhibition of Wnt signaling through the application of porcupine small-molecule inhibitors including IWP-2, IWR1 and Wnt-C59 (Lian et al., 2012; Burridge et al., 2014). Developmental heart growth Organ size regulation is an important aspect of cardiac development. The heart must grow large enough to generate sufficient cardiac output, and regional under- or overgrowth may result in septal wall defects, hypoplastic ventricle(s) or obstruction of the outflow tract(s) (Heallen et al., 2011). Heart growth during development is regulated by a combination of cardiomyocyte differentiation, proliferation and hypertrophy. The human heart undergoes a dramatic proliferation period from CS9-CS16, resulting in a 600fold increase in heart volume in just 3 weeks (de Bakker et al., 2016). First, the cardiac crescent fuses at the midline and gives rise to the FHF-derived linear heart tube, which subsequently commences beating and undergoes looping. Hereafter, the linear heart tube expands by drastic proliferation and recruitment of SHF cardiac progenitor cells that migrate from the pericardial cavity to the dorsal and caudal heart tube regions while undergoing differentiation (Kelly et al., 2014). Their rapid proliferation is regulated by canonical Wnt signaling (Günthel et al., 2018; Kwon et al., 2007). Upon the presence of the receptor-bound ligands WNT5A and WNT11, active β-catenin enters the nucleus, where it acts as a transcriptional co-activator of
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