A better understanding of pluripotent state
A better understanding of pluripotent state of zebrafish embryos allows us to accurately induce differentiation to cardiomyocytes from primary embryonic cells. A central feature of our method is the use of a combination of appropriate conditions including cell density, ZF4 feeder cells, and supplements to facilitate the cardiomyocyte induction, in addition to utilizing pluripotency characteristics. This induction process is similar to in vivo cardiogenesis timing (24 hpf) in zebrafish (Kimmel et al., 1995). Among the supplements (ZEE, ZF4 CM, and INSULIN), INSULIN is an essential factor. Indeed, INSULIN and its receptor are both maternally and zygotically expressed, and knockdown of insulin receptor resulted in cardiac abnormalities in zebrafish, suggesting that INSULIN plays important roles in cardiogenesis (Papasani et al., 2006; Toyoshima et al., 2008). Treatment of mouse embryonic stem cells with INSULIN or insulin-like growth factor (IGF) 1 or 2 promoted proliferation of mesodermal cells and cardiomyocyte generation via the PI3K/AKT/TOR signaling pathway (Engels et al., 2014). IGF signaling also promotes a continued expansion of cardiac progenitor cells derived from human PSCs (Birket et al., 2015). Another feature of the induction system is that the induced cardiomyocytes have typical contractile kinetics and electrophysiological characteristics. Spontaneous beats of cardiomyocytes are driven by depolarization-repolarization cycles of the cell membrane potential. AP patterns of in vitro generated BCCs in this study resemble those of ventricles, atria, or pacemakers in adult zebrafish hearts (Nemtsas et al., 2010; Tessadori et al., 2012), and are also similar to those of human (Nemtsas et al., 2010). Functional properties of cardiomyocytes can further be manifested by rate dub inhibitor tests and drug treatments. In rate adaptation tests, BCCs adapted to the stimulations at increasing frequencies by shortening their APD, which is consistent with zebrafish ventricular myocytes (Brette et al., 2008) and human PSC-derived cardiomyocytes (He et al., 2003; Lian et al., 2012; Zhang et al., 2009). Another property is functional response to β-adrenergic stimulations, which is comparable in zebrafish hearts (Parker et al., 2014; Steele et al., 2011) and human PSC-derived cardiomyocytes (Zhang et al., 2009), suggesting the presence of functional β-adrenergic receptors in the BCCs. A more important feature is the presence of a sodium channel in the induced cardiomyocytes, as revealed by TTX response. A similar effect of TTX on the induced cardiomyocytes was also observed in adult hearts of zebrafish (Chopra et al., 2010; Nemtsas et al., 2010). Robust regenerative ability in adult cardiomyocytes makes zebrafish an ideal model for studying heart regeneration (Jopling et al., 2010; Poss et al., 2002; Raya et al., 2003). Since cardiomyocyte proliferation is a key step of zebrafish heart regeneration (Jopling et al., 2010), it is important to discover essential components that trigger or promote this process. An efficient in vitro culture of adult zebrafish cardiomyocytes has been established recently (Sander et al., 2013). In this study, we have provided a detailed method of in vitro cardiomyocyte differentiation from embryonic cells. Differentiated cardiomyocytes are functional and capable of recapitulating proliferative responses to mitogens. Generation and proliferation of cardiomyocytes were both substantially enhanced by NRG1 treatment, consistent with previous studies in mammalian and zebrafish heart regeneration (Bersell et al., 2009; Gemberling et al., 2015). Combining utilization of transgenic fish lines that specifically express fluorescent protein in cardiomyocyte nuclei, our system possesses potential in exploring novel mitogens or factors involved in heart regeneration. The advantage of our system is that it is primed to generate cardiomyocytes from lethal mutant lines that cannot survive to adulthood. As long as the mutant embryos remain viable at the oblong stage, generation of differentiated cardiomyocytes can be achieved by using the protocols provided herein, and thus gene functional studies can be carried out based on regenerative responses of the mutant cardiomyocytes. Therefore, we have determined an alternative way to gain insight into heart regeneration mechanisms in zebrafish. The primary embryonic cell-based system can be used to perform high-throughput screening of factors for heart study not merely limited to small molecules (Huang et al., 2012; Xu et al., 2013) to also include nucleic acids, proteins and lipids, and so forth, which is difficult to achieve in the zebrafish in vivo screening model. The system is also promising for the dissection of pathways of cardiac development and regeneration in lethal mutants.