Fig. 4

Cxcr4a-Cxcl12b Expression during Coronary Vessel Formation

Confocal images of transgenic fish Tg(myl7:DsRed2-NLS; fli1:EGFP; cxcl12b:YFP) (A?C, E), Tg(kdrl:mCherry; fli1:EGFP; cxcl12b:YFP) (F?H), Tg(myl7:DsRed2-NLS; fli1:EGFP; cxcr4a:YFP) (I?M), Tg(dll4:EGFP; cxcr4a:YFP) (N), Tg(dll4:EGFP; cxcr4a:YFP; kdrl:mCherry) (O), and Tg(kdrl:mCherry; fli1:EGFP; cxcr4a:YFP) (P). Ventricular, but not atrial cardiomyocytes express cxcl12b:YFP prior to and during vessel formation (A, 28 dpf; B, 56 dpf; C, 77 dpf). cxcl12b:YFP labeling is found within the cytoplasm of cells containing a myl7-positive nuclei (red) and flanked by fli1a-positive (green) endothelial cells (A, inset, from confocal section, side image). Levels of cxcl12b:YFP expression vary over the ventricle and between hearts, but are generally higher toward the base (A?D). Quantification of cxcl12b:YFP intensity (blue, normalized mean 14 dpf to 84 dpf, ± SEM) and vessel density (green, mean values of individual time points 7 dpf to 420 dpf) between base and apex of ventricular myocardium (D), both p < 0.0001 (Wilcoxon signed rank test on paired values). In addition, expression is observed on the surface of the bulbous arteriosus (ba, A?C, F). cxcl12b:YFP expression is downregulated following establishment of the vessel network in cardiomyocytes, but is later expressed in the mural cells of mature vessels (E, inset arrowhead). cxcl12b:YFP-expressing mural cells surround large arterial vessels (F?H, blue), which express high levels of kdrl:mCherry (F?H, red). cxcr4a:YFP is not expressed prior to the emergence of endothelial cells on the surface of the ventricle (I, 28 dpf). Endothelial cells of angiogenic sprouts express cxcr4a:YFP as they migrate over the surface of the myocardium (J, 42 dpf, image showing tip cell; K, 70 dpf; brackets label cells displaying active migration morphology; L, intensity of cxcr4a:YFP in endothelial cells). The majority of vessels do not express cxcr4a:YFP after their formation (K?P). Larger arterial vessels do maintain cxcr4a:YFP expression, which overlaps with high levels of kdrl:mCherry and dll4:EGFP expression in these vessels (N?P). Scale bars represent 50 µm.