Dioxin Inhibits Zebrafish Epicardium and Proepicardium Development

Abstract

Embryonic exposure to the environmental contaminant and aryl hydrocarbon receptor agonist, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin), disrupts cardiac development and function in fish, birds, and mammals. In zebrafish, the temporal window of sensitivity to the cardiotoxic effects of TCDD coincides with epicardium formation. We hypothesized that this TCDD-induced heart failure results from disruption of epicardial development. To determine whether embryonic TCDD exposure inhibits epicardium and proepicardium (PE) development in zebrafish, we used histology and fluorescence immunocytochemistry to examine the epicardium formation in fish exposed to TCDD. TCDD exposure prevented epicardium formation. Using live imaging and in situ hybridization, we found that TCDD exposure blocked the formation of the PE cluster. In situ hybridization experiments showed that TCDD exposure also prevented the expression of the PE marker tcf21 at the site where the PE normally forms. TCDD also inhibited expansion of the epicardial layer across the developing heart: Exposure after PE formation was completed prevented further expansion of the epicardium. However, TCDD exposure did not affect epicardial cells already present. Because TCDD blocks epicardium formation, but is not directly toxic to the epicardium once complete, we propose that inhibition of epicardium formation can account for the window of sensitivity to TCDD cardiotoxicity in developing zebrafish. Epicardium development is crucial to heart development. Loss of this layer during development may account for most if not all of the TCDD-induced cardiotoxicity in zebrafish.

Sox9bIs Required for Epicardium Formation and Plays a Role in TCDD-Induced Heart Malformation in Zebrafish

“…Whole mount in situ hybridization was conducted as previously described (Plavicki et al, 2013). A 560-base pair fragment of sox9b was amplified from embryonic zebrafish cDNA and subcloned into a pCRII-TOPO vector (Invitrogen).…”

“…In zebrafish, TCDD-induced heart malformation is associated with the loss of epicardium and the proepicardium (PE) (Plavicki et al, 2013). During the period before 48 hours postfertilization (hpf), TCDD exposure has no discernible effect on development of the zebrafish heart.…”

“…However, after 48 hpf, TCDD-exposed hearts begin to deteriorate and unloop. The manifestation of cardiotoxicity corresponds to the timing of epicardium formation (Plavicki et al, 2013). The epicardium and epicardium-derived progenitor cells are thought to play a critical role in cardiomyocyte proliferation, valve development, heart looping, generation of fibroblasts, cardiac morphogenesis, development of the coronary vasculature, and adult cardiac regeneration (Lepilina et al, 2006;Lie-Venema et al, 2007;Olivey and Svensson, 2010;Svensson, 2010).…”

Retene causes multifunctional transcriptomic changes in the heart of rainbow trout ( Oncorhynchus mykiss ) embryos

“…It is possible that the main factor behind the developmental cardiovascular toxicity and other adverse effects of TCDD is the down-regulated sox9 in epicardium (Mathew et al, 2008,Xiong et al, 2008,Mehta et al, 2008,Hofsteen et al, 2013.…”

“…The AhR agonists disturb the development of the heart and the vasculature in fishes, and cardiovascular pathology is the first sign of dioxin-like toxicity in zebrafish (Danio rerio) and medaka (Oryzias latipes) (Henry et al, 1997,Hornung et al, 1999,Antkiewicz et al, 2005,Carney et al, 2006,Mehta et al, 2008,Plavicki et al, 2013,Scott, 2009). The most studied AhR agonist, TCDD, prevents cardiac valve formation, inhibits epicardial and proepicardial development, causes altered looping of the heart, and reduces the volume and number of cardiomyocytes in fishes (Hornung et al, 1999,Antkiewicz et al, 2005,Carney et al, 2006,Mehta et al, 2008,Plavicki et al, 2013. Similarly, other AhR agonists (e.g., PCB126, retene, benz [a]anthracene) disturb the development of the heart in zebrafish and medaka (Incardona et al, 2006,Grimes et al, 2008,Scott et al, 2011,Scott, 2009).…”