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Fig. 4 (a) Analysis of zCOX-1 and zCOX-2 transcript expression in adult zebrafish. We assessed constitutive zCOX isoform expression in adult zebrafish by reverse transcriptase-PCR using total RNA from freshly dissected adult organs. PCR products (500 bp and 162 bp) and the corresponding negative controls were visualized by agarose gel-electrophoresis. Expression of zCOX-1 (b, e, h, k, n, and q), zCOX-2 (c, f, i, l, o, and r), and flk-1 (d, g, j, m, p, and s) by whole-mount in situ hybridization in zebrafish larvae at 96 hpf. zCOX-1 (b), zCOX-2 (c), and the endothelial marker flk-1 (d) are expressed in the carotid artery (ca) and the pharyngeal arches (white arrow) (lateral view). zCOX-1 (b) and flk-1 (d) are also present in cranial arteries (arrowheads). (e-g) Ventral views showing expression in the pharyngeal arches (arrowheads). While flk-1 staining (g) highlights the vasculature in the center of the arches [arch arteries 1 and 2 (opercular artery); 3-6,ha, hypobranchial artery (35)], zCOX-1 (e) and zCOX-2 (f) appear to be expressed in more peripheral structures of the arches. (h-j) Oblique ventral views. zCOX-1 (h), zCOX-2 (i), and flk-1 (j) colocalize and are expressed within sprouting gill arteries (*) that are apparent as protrusions at the caudal surface of arch arteries 3-6. (k-m) Histological cross sections through these vessels (*, section rotated by 90°) show flk-1 (m) expression in endothelial cells. zCOX-2 is highly expressed in the vessel wall of the sprouting gill vasculature (l). zCOX-1 expression is less intense and appears to be present in both endothelial and wall structures (k). (n-p) Endothelial expression of zCOX-1 was seen the ciliary arteries of the eye (n, arrowheads), which were also visible in flk-1 (p), but not in zCOX-2-stained embryos (o). zCOX-1 and zCOX-2 were both differentially expressed in other structures of the developing eye (n and o). (q-s) All three genes were expressed in the vasculature of the intestine. Endothelial cells are highlighted by arrowheads. (Magnifications: (b-j) x200, (k-m and q-s) x630, n-p) x400.)