Fig. 1: Characterization and conditional ablation of mural cells covering skin vasculature in adult zebrafish.

A Confocal z-projection images of skin vasculature in Tg(kdrl:eGFP);NTRmC^MC adult zebrafish. Upper left, NTRmC^MC image (red); upper middle, merged image of NTRmC^MC (red) and eGFP (green). The boxed areas, labeled “AV” and “C”, represent arterioles/venules, and capillaries, respectively, and are shown enlarged at the bottom. The boxed areas in the enlarged images are further enlarged as z-projection images extracted at approximately the vascular level on the right. Arrowheads indicate pericytes covering arteriole, venule and capillaries. The arrow indicates a vessel structure which does not contain circulating erythrocytes. Note that mesh pericytes cover the pre-capillary arterioles and post-capillary venules, while thin strand pericytes surround the capillaries. B Confocal z-projection images of the skin vasculature in the Tg(kdrl:mScarlet-I);TgBAC (abcc9:gal4FF);Tg(UAS:eGFP) adult zebrafish. Left, abcc9:gal4FF,UAS:eGFP (abcc9:eGFP) image (eGFP, green); right, merged image of abcc9:eGFP (eGFP, green) and kdrl:mScarlet-I (mScarlet, red). The boxed areas, labeled “A”, “V,” and “C,” represent arterioles, venules, and capillaries, respectively, and are shown enlarged on the right. Arrowheads indicate abcc9^high pericytes covering the capillaries. Arrows indicate abcc9-positive cells covering the vessel structures which do not contain circulating erythrocytes. C Confocal z-projection images of skin vasculature in Tg(kdrl:eGFP);Tg(acta2:mCherry) adult zebrafish. Left, acta2:mCherry (mCherry, red); right, merged image of acta2:mCherry (mCherry, red) and kdrl:eGFP (eGFP, green). The boxed areas, labeled “A”, “V,” and “C,” are shown as in B. Arrowheads indicate acta2^high pericytes covering the pre-capillary arterioles. The arrow indicates a vessel structure which does not contain circulating erythrocytes. D Confocal z-projection images of the skin vasculature in Tg(kdrl:mScarlet-I);TgBAC (tagln:eGFP) adult zebrafish. Left, tagln:eGFP (eGFP, green); right, merged image of tagln:eGFP (eGFP, green) and kdrl:mScarlet-I (mScarlet, red). The boxed areas, labeled “AV” and “C,” represent arterioles/venules and capillaries, respectively, and are shown enlarged at the bottom. Arrowheads indicate tagln^high pericytes surrounding the pre-capillary arterioles and post-capillary venules, while double-arrowheads indicate tagln^low pericytes covering the capillary. The arrow indicates a vessel structure which does not contain circulating erythrocytes. E Schematic diagram showing the classification of pericytes covering the skin vasculatures in the adult zebrafish. F Experimental scheme of MTZ-induced ablation of mural cells in NTRmC^MC adult zebrafish. G Confocal z-projection images of skin vasculatures in Tg(kdrl:eGFP);NTRmC^MC adult zebrafish in the absence (left) or presence (right) of MTZ. Images taken before (Day 0), and at 1 (Day 1) and 2 (Day 2) days after the initiation of MTZ treatment are shown. Upper row, NTRmC^MC images shown in inverted grayscale; middle row, merged images of NTRmC^MC (red) and eGFP (green). The boxed areas in the middle row are enlarged at the bottom. H Quantification of MTZ-induced pericyte ablation in NTRmC^MC adult zebrafish, as shown in G. Blue and red lines indicate the number of pericytes in NTRmC^MC fish treated without and with MTZ, respectively. The number of NTRmC^MC-positive pericytes covering the skin vasculature is expressed as a ratio relative to the number observed before MTZ treatment. Data are presented as mean ± standard deviation (n = 6 in each group). *P < 0.05, by Wilcoxson test at each time point with Bonferroni adjustment. Scale bars: 100 μm and 20 μm (enlarged images in right column of A).