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Fig. 4 Effects of hey2 mRNA injections on the interrenal tissue and its neighboring artery and vein. A: Effects of hey2 mRNA on the expressions of flt4 and eph-B2a in the peri-interrenal region. Uninjected control (a, g) and injected embryos (b-f, h-l) were fixed at 35 hpf and subject to two-color ISH for detecting the expression of ff1b together with that of either flt4 (a-f) or eph-B2a (g-l). All panels in A are ventral views with anterior oriented to the top. The flt4 expression at the peri-interrenal area is absent in all phenotypic classes of injected embryos (classes I-V). B: Sets of confocal images display the interrenal tissues as detected by 3 β-Hsd activity staining (left panels of each set, a-l), and the neighboring endothelium as labeled by green fluorescence (middle panels of each set, a′-l′), of 34-hpf Tg(kdrl:EGFP)s843 embryos uninjected (a, b) or injected with hey2 mRNA (c-l, classes I-V). The merged images of 3 β-Hsd activity staining and GFP are shown in the right panels of each set (a″-l″). Each fluorescent image depicting the vascular morphology represents a projection of a consecutive z-stack encompassing the depth of the interrenal tissue. a, c, e, g, i, k are dorsal views with anterior oriented to the top, while b, d, f, h, j, l are dorsolateral views with anterior to the right. The venous endothelium at the peri-interrenal area is severely reduced in all classes of hey2 mRNA-injected embryos, which is accompanied by various expressivities of the interrenal morphogenetic defect. White and orange arrows, ff1b-expressing and steroidogenic interrenal tissues; red and blue arrowheads, artery and vein, respectively; blue and yellow dotted lines, boundaries of venous and arterial structures, respectively; white dotted lines, position of the midline; DA, dorsal aorta; PCV, posterior cardinal vein; G, glomerulus. Scale bar = 50 μM.