Zebrafish models in cardiac development and congenital heart birth defects

Abstract

The zebrafish has become an ideal vertebrate animal system for investigating cardiac development due to its genetic tractability, external fertilization, early optical clarity and ability to survive without a functional cardiovascular system during development. In particular, recent advances in imaging techniques and the creation of zebrafish transgenics now permit the in vivo analysis of the dynamic cellular events that transpire during cardiac morphogenesis. As a result, the combination of these salient features provides detailed insight as to how specific genes may influence cardiac development at the cellular level. In this review, we will highlight how the zebrafish has been utilized to elucidate not only the underlying mechanisms of cardiac development and human congenital heart diseases (CHDs), but also potential pathways that may modulate cardiac regeneration. Thus, we have organized this review based on the major categories of CHDs—structural heart, functional heart, and vascular/great vessel defects, and will conclude with how the zebrafish may be further used to contribute to our understanding of specific human CHDs in the future.

Introduction

The development of the heart is a dynamic and complex process starting with the specification of myocardial, endocardial, and vascular/endothelial precursors and culminating in the morphogenesis of these differentiated cells/tissues into a functional cardiac pumping organ. Because of the exquisite coordination of these events, the slightest cardiac developmental perturbation (genetic or environmental) can easily lead to catastrophic heart defects and subsequent embryonic/fetal demise. Thus, congenital heart diseases (CHDs), including both structural and functional cardiovascular defects, are amongst the most common and most devastating birth defects in humans, occurring in about 5% of live births, and resulting in significant mortality and morbidity (Pierpont et al., 2007).

Identifying suitable vertebrate animal model systems that permit the detailed investigations of cardiac development and the mechanisms underlying CHDs can be challenging. However, the zebrafish is particularly well suited to studies of cardiovascular development because, unlike mouse and chick embryos, they do not completely depend on a functional cardiovascular system. The zebrafish's small embryonic/larval size allows them to receive sufficient oxygen by passive diffusion even when manifesting the most severe cardiovascular defects (Stainier, 2001). Furthermore, zebrafish embryos develop rapidly and are relatively easy to analyze because of their optical transparency and external fertilization (Stainier, 2001). In particular, these specific advantages offer the possibility of live in vivo imaging of the cellular and physiologic processes during cardiac morphogenesis, and recent imaging advances have been developed and utilized to study these dynamic events (Huisken et al., 2004, Huisken and Stainier, 2009). Additionally, because of their large offspring numbers and rapid development, the zebrafish is genetically amenable to forward genetic screens, which have led to the identification of a wide range of cardiovascular mutant phenotypes including many that recapitulate known CHDs (Stainier et al., 1996, Sehnert and Stainier, 2002, Chen et al., 1996, Beis et al., 2005, Weinstein et al., 1996, Jin et al., 2007, Chi et al., 2008). Conversely, a number of tools, including morpholinos (Bill et al., 2009), TILLING (Wienholds et al., 2003), TALEN (Huang et al., 2011), and zinc finger nucleases (Meng et al., 2008), have been created to perturb specific genes of interest (reverse genetics) and subsequently used to model candidate CHD genes. Moreover, the zebrafish is particularly sensitive to small molecule treatment and thus suitable to chemical genetic studies and screens to identify additional cellular and molecular pathways, which may regulate cardiovascular development (Peterson and Fishman, 2004). Finally, zebrafish and mammalian hearts exhibit fairly well conserved structures including atria, ventricles, cardiac valves and a cardiac conduction system (Beis et al., 2005, Chi et al., 2008, Sedmera et al., 2003, Walsh and Stainier, 2001, Stainier et al., 1993, Yelon and Stainier, 1999). These features are remarkably useful in discovering zebrafish cardiovascular mutants that provide insight into human cardiovascular diseases. Thus, the combination of these advantages makes the zebrafish an attractive vertebrate model organism to complement human and other mammalian studies of CHDs and cardiac development.