Wnt ligand required for regulating vertebrate heart development via the canonical Wnt pathway, but surprisingly discovered a function at later stages of development, during organogenesis preceding cardiomyocyte differentiation

INTRODUCTION During vertebrate embryonic development, heart progenitors acquire the potential to subsequently differentiate as cardiomyocytes in the myocardium and form the functional heart muscle (Mohun et al., 2000; Mohun et al., 2003; Nakajima et al., 2009). Identifying how these cardiac progenitors are controlled and the source of signals regulating differentiation is of fundamental importance for our understanding of embryonic heart development. Moreover, injury to the adult heart, for instance after myocardial infarction, can cause serious damage to mature cardiomyocytes. Even though cardiomyocytes have some potential to proliferate and there is encouraging evidence for resident cardiac progenitors in the adult heart, this is normally insufficient to repair the damage. Understanding how to promote cardiomyocyte differentiation from endogenous progenitor cells in order to replace damaged cardiomyocytes and regenerate damaged heart muscle would be an exciting prospect and impact greatly on clinical treatments of heart patients (reviewed by Mummery et al., 2010). The Wnt signalling pathway is a key regulator of heart development and particularly of cardiomyocyte differentiation (reviewed by Gessert and Kühl, 2010). Experimental manipulation of Wnt ligands and extracellular Wnt inhibitors was shown to influence heart development (e.g. Marvin, 2001; Schneider and Mercola, 2001). Furthermore, the endogenous expression of some of these factors in, or close to, embryonic heart tissue further supported their possible role in this process (e.g. Monaghan et al., 1999). However, evidence of the requirement for specific endogenous Wnt signalling component genes in early embryonic heart development is slow to emerge. We set out to identify the important endogenous Wnt signalling components required for vertebrate heart development and to uncover the Wnt-regulated molecular gene regulatory mechanisms promoting myocardium development and cardiomyocyte differentiation. This will prove paramount for understanding heart development in the embryo but also for developing future regenerative therapies for heart patients. However, Wnt signalling in heart development appears complicated. So-called canonical β-catenin-dependent Wnt signalling has apparently opposing effects at different developmental stages on subsequent cardiomyocyte differentiation (reviewed by Tzahor, 2007), which may be linked to a positive effect on progenitor expansion and a brake on differentiation (reviewed by Bergmann, 2010). Additionally, non-canonical (i.e. βcatenin-independent) Wnt signalling mechanisms also regulate heart development (e.g. Eisenberg and Eisenberg, 1999; Pandur et al., 2002; Afouda et al., 2008; Cohen et al., 2012; Onizuka et al., 2012). The Xenopus model system has been instrumental in uncovering fundamental molecular mechanisms and conserved functions in vertebrate heart development (reviewed by Warkman and Krieg, 2007), particularly the role of Wnt signalling during the early specification of heart development (e.g. Marvin, 2001; Schneider and Mercola, 2001). We identified in Wnt6 the first endogenous Wnt ligand required for regulating vertebrate heart development via the canonical Wnt pathway, but surprisingly discovered a function at later stages of development, during organogenesis preceding cardiomyocyte differentiation (Lavery et al., 2008b). The identification of a specific Wnt ligand that is expressed close to, but outside of, the cardiac mesoderm (Lavery et al., 2008a) allowed us to consider a spatial model of how heart muscle differentiation is regulated in the embryo. The cardiogenic mesoderm is further patterned into muscular (i.e. myocardium) and non-muscular (e.g. pericardium) tissues (e.g. Raffin et al., 2000). We wondered whether Institute of Medical Sciences, University of Aberdeen, Aberdeen AB25 2ZD, UK.