Fig. 5

Impaired epicardium formation following cardiomyocyte-specific inhibition of sox9b function. Control (Tg(myl7:Gal4VP16;pard3-like:EGFP)) larvae and larvae with cardiomyocyte-specific loss of sox9b function (Tg(myl7:Gal4VP16;pard3-like:EGFP;UAS:dnsox9b-2A-tRFP)) were fixed at 120 hpf and processed for fluorescent immunohistochemistry using antibodies against tRFP (red) and Alcam (blue). Larvae were mounted ventrally in low melting point agarose and imaged at 40x magnification with a confocal microscope. The myocardium is indicated by a dashed line. (a,a’) The epicardium forms normally in control larvae, as indicated by EGFP-labeled epicardial cells on the surface of the ventricular and atrial myocardium (red arrows). (b,b’) Epicardium formation is impaired when sox9b function is lost in cardiomyocytes. Very few EGFP-labeled epicardial cells can be seen on the ventricular myocardium (red arrow) and none are present on the atrial myocardium. A cluster of proepicardial progenitors is visible in the pericardial space (white arrow). (c,d) pard3-like:EGFP zebrafish embryos were injected with either a control plasmid (Tg(myl7:tRFP)) or plasmid with the dnsox9b fused to a cardiomyocyte specific promoter (Tg(myl7:dnsox9b-2A-tRFP)). Injections resulted in mosaic expression of the constructs in cardiomyocytes. Samples were fixed at 96 hpf and processed for immunohistochemistry using an antibody for tRFP (red) and DAPI to label nuclei (blue). With mosaic dnsox9b expression, epicardial cells (red arrows) are found overlying dnSox9b⁺ myocardial cells. Ventricle (V) and atrium (At) are abbreviated as indicated, and the outflow tract (bulbus arteriousus) is indicated by the yellow arrow. Images are representative phenotypes, n = 8 per group.