Abstract
BackgroundThe vertebrate heart consists of three cell layers: the innermost endothelium, the contractile myocardium and the outermost epicardium. The epicardium is vital for heart development and function, and forms from epicardial progenitor cells (EPCs), which migrate to the myocardium during early development. Disruptions in EPC migration and epicardium formation result in a number of cardiac malformations, many of which resemble congenital heart diseases in humans. Hence, it is important to understand the mechanisms that influence EPC migration and spreading in the developing heart. In vitro approaches heretofore have been limited to monolayer epicardial cell cultures, which may not fully capture the complex interactions that can occur between epicardial and myocardial cells in vivo.ResultsHere we describe a novel in vitro co-culture assay for assessing epicardial cell migration using embryonic zebrafish hearts. We isolated donor hearts from embryonic zebrafish carrying an epicardial-specific fluorescent reporter after epicardial cells were present on the heart. These were co-cultured with recipient hearts expressing a myocardial-specific fluorescent reporter, isolated prior to EPC migration. Using this method, we can clearly visualize the movement of epicardial cells from the donor heart onto the myocardium of the recipient heart. We demonstrate the utility of this method by showing that epicardial cell migration is significantly delayed or absent when myocardial cells lack contractility and when myocardial cells are deficient in tbx5 expression.ConclusionsWe present a method to assess the migration of epicardial cells in an in vitro assay, wherein the migration of epicardial cells from a donor heart onto the myocardium of a recipient heart in co-culture is monitored and scored. The donor and recipient hearts can be independently manipulated, using either genetic tools or pharmacological agents. This allows flexibility in experimental design for determining the role that target genes/signaling pathways in specific cell types may have on epicardial cell migration.Electronic supplementary materialThe online version of this article (doi:10.1186/s12861-015-0100-y) contains supplementary material, which is available to authorized users.
Highlights
The vertebrate heart consists of three cell layers: the innermost endothelium, the contractile myocardium and the outermost epicardium
We demonstrate key features of this assay by assessing the ability of epicardial cells from a donor heart to migrate onto the myocardial surface of a recipient heart
To confirm that recipient hearts extracted at 60 hpf lacked epicardial cells, we examined hearts from embryos with both a red epicardial marker, tcf21:DsRed2, and green myocardial marker, cmlc2:Enhanced green fluorescent protein (EGFP)
Summary
The vertebrate heart consists of three cell layers: the innermost endothelium, the contractile myocardium and the outermost epicardium. The endocardial and myocardial cells originate from populations of mesodermal cells that migrate from the midbrain-hindbrain boundary to form the linear heart tube [1, 2] These cardiogenic mesoderm cells form the ventricle, atrium, outflow tract myocardium, Yue et al BMC Developmental Biology (2015) 15:50 develops, a subset of epicardial cells undergo epithelial-tomesenchymal transition and invade the subepicardial space. These mesenchymal cells, called epicardiumderived cells (EPDCs), are important for normal heart maturation and have been shown to differentiate into interstitial cardiac fibroblasts, coronary vascular smooth muscle cells, and adventitial fibroblasts. Aberrant crosstalk between the epicardial layer and underlying myocardial and endocardial cells has been implicated in several congenital diseases, such as hypoplastic left heart syndrome and endocardial fibroelastosis [3, 7]
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