A co-culture assay of embryonic zebrafish hearts to assess migration of epicardial cells in vitro

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.

Proepicardium: Current Understanding of its Structure, Induction, and Fate

“…Determining the induction, differentiation, and fate of proepicardial cells is not easy due to the fact that the cells forming the PE are heterogeneous and express various markers (Maya‐Ramos et al, ). There are a number of articles describing the isolation of mouse, chick, and zebrafish proepicardia for in vitro studies and addressing PE‐derived cell differentiation and epicardium formation (Guadix et al, ; Buermans et al, ; Garriock et al, ; Yue et al, ). Also mouse embryonic stem cell (ESC) systems were established to obtain proepicardial progenitor cells.…”

sox9b is required in cardiomyocytes for cardiac morphogenesis and function

“…Embryonic hearts were extracted as previously described 44 , 73 , 74 . Hearts expressing tRFP were collected from the resulting filtrate using a micropipettor with the aid of an Olympus SZX16 epifluorescence stereomicroscope.…”

The Last Half Century of Fish Explant and Organ Culture

“…McCormick (1989) 143 Salmon Stadtländer (1990) 1 Salmon Shrimpton (1999) 144 Trout Bury (1998) 145 Tilapia Holland (1998) 146 Trout Nematollahi (2003) 147 Trout Mazon (2004) 148 Trout Kiilerich (2007) 149 Salmon Lazado (2020) 35 Salmon Gonads Ovary Tyler (1990) 4 Trout Pankhurst (2000) 150 Flounder Todo (2008) 151 Wrasse Ogiwara (2010) 152 Medaka Tsai (2010) 153 Zebrafish Wang (2020) 154 Sterlet Testis Bonnin (1975) 155 Goby Miura (1991) 156 Eel Amer (2001) 157 Huchen Bouma (2003) 158 Trout Bouma (2005) 159 Trout Iwasaki (2009) 160 Medaka Leal (2009) 33 Zebrafish Skaar (2011) 161 Zebrafish Kang (2019) 162 Medaka Ovary and testis de Clercq (1977) 163 Goldfish Sakai (2008) 164 Tilapia Beitel (2014) 165 Pike, sucker, walleye (continued) 167 Salmon Jirillo (2007) 168 Trout Barato (2016) 169 Tilapia Vasquez-Machado (2019) 170 Tilapia Coccia (2019) 171 Trout Heart Kitambi (2012) 172 Zebrafish Pieperhoff (2014) 121 Zebrafish Yue (2015) 119 Zebrafish Cao (2016) 120 Zebrafish Rastgar (2019) 173 Grass carp Yip (2020) 96 Zebrafish Kidney Trump (1977) 174 Flounder de Ruiter (1981) 175 Stickleback Jones (1982)…”