Genetic and cellular analyses of zebrafish atrioventricular cushion and valve development

Abstract

Defects in cardiac valve morphogenesis and septation of the heart chambers constitute some of the most common human congenital abnormalities. Some of these defects originate from errors in atrioventricular (AV) endocardial cushion development. Although this process is being extensively studied in mouse and chick, the zebrafish system presents several advantages over these models, including the ability to carry out forward genetic screens and study vertebrate gene function at the single cell level. In this paper, we analyze the cellular and subcellular architecture of the zebrafish heart during stages of AV cushion and valve development and gain an unprecedented level of resolution into this process. We find that endocardial cells in the AV canal differentiate morphologically before the onset of epithelial to mesenchymal transformation, thereby defining a previously unappreciated step during AV valve formation. We use a combination of novel transgenic lines and fluorescent immunohistochemistry to analyze further the role of various genetic (Notch and Calcineurin signaling) and epigenetic (heart function)pathways in this process. In addition, from a large-scale forward genetic screen we identified 55 mutants, defining 48 different genes, that exhibit defects in discrete stages of AV cushion development. This collection of mutants provides a unique set of tools to further our understanding of the genetic basis of cell behavior and differentiation during AV valve development.

Rapid three‐dimensional imaging and analysis of the beating embryonic heart reveals functional changes during development

“…Previous studies have suggested that heart cushions are functional by 48 hpf because obvious regurgitation between the atrium and the ventricle was not noted in wildtype embryos, but at the same stage, regurgitation is evident in jekyll mutants (Walsh and Stainier, 2001;Beis et al, 2005). Our studies show that there is significant regurgitation across the AV boundary at this stage in normal embryos, indicating that the contractile ring at the AV boundary does not create a one-way valve to limit flow.…”

“…Time-lapse imaging with the high spatial and temporal resolution provided by the methods described here show that unidirectional flow between chambers is not observed until after considerable thickening of the endocardial cushions is evident. Careful studies of the developing valves indicate that cells undergo significant changes in cell morphology, extracellular matrix composition and cell adhesion molecule expression during the first 5-7 days and begin transformation into valve leaflets by 105 hpf (Beis et al, 2005). Taken together, the results presented here suggest that one-way valve function emerges only as the cushions transform into leaflets.…”

“…Many factors have been identified that play a role in patterning and morphogenesis in the early heart (Olson, 2002;Armstrong and Bischoff, 2004;Olson, 2004) and normal heart function is also necessary for proper cardiac development (Beis et al, 2005). For instance, blocking flow through the zebrafish heart leads to severe dysmorphology (Hove et al, 2003), and mutants that lack contractile function in the early heart also have defects in valve formation and show an absence of endocardial thickening at the AV boundary (Sehnert et al, 2002;Bartman et al, 2004).…”

“…In the zebrafish, cardiac progenitors from both sides of the midline fuse and give rise to a luminized heart tube; the heart begins to beat at the linear tube stage, moving blood cells through this conduit, before the emergence of clearly defined heart chambers, valves, or septae (Stainier and Fishman, 1994;Trinh and Stainier, 2004). The vertebrate heart develops in the presence of mechanical and hemodynamic stimuli; thus, the question arises whether the structure of the heart influences its function, or, conversely, whether the flows and mechanical forces in the heart influence its form (Hove et al, 2003;Bartman et al, 2004;Beis et al, 2005). Although a great number of methods have been developed to understand the genetic contributions to heart formation, methods to study epigenetic contributions have been difficult to establish, mainly because of rapid motions that are involved (typically millimeters per second for distances of a few hundred microns).…”

Illuminating cardiac development: Advances in imaging add new dimensions to the utility of zebrafish genetics

“…Next, localized bulges emerge from the walls of the heart tube: this process, called ballooning, creates the characteristic curvatures of the ventricle and the atrium ( Figure 1E,J). Chambers are further demarcated by the distinctive constriction of the atrioventricular (AV) canal; morphogenetic changes also occur inside this portion of the heart, where endocardial cushions (ECs) form and are then remodeled into AV valve leaflets [24]. Altogether, cardiac morphogenesis is a dynamic and multifaceted process that must be carefully regulated to insure proper cardiac structure.…”