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Fig. S1 The lymphatic system in zebrafish and K. bicirrhis. Cross-sections of the fli1:EGFP zebrafish trunk region were (A) stained with H&E or (B) examined by fluorescence microscopy for EGFP expression (green). (C) Electron microscopic examination of the thoracic duct (TD). Arrow indicates a lymphatic valve of the TD. (D) A cross-section of fli1:EGFP zebrafish trunk tissue was immunohistochemically stained with anti-prox-1 (red), demonstrating overlapping positive signals with EGFP (arrows). (E) The boxed region of transparent K. bicirrhis was used to examine blood flow and the TD. (F) Whereas blood flow and red blood cells were easily detectable in the dorsal aorta (DA) and portal cardinal vein (PCV), the TD lacked blood perfusion. (G) H&E staining showed the location of the TD next to the PCV. (H) Immunostaining of K. bicirrhis trunk tissue with anti-prox-1 (red) and with DAPI to show erythrocyte nuclei (blue) showed that the TD was positive for prox-1 (arrows) and lacked erythrocytes (encircled with dashed lines). (I) Schematic presentation of the anatomy of the arterial, venous, and lymphatic systems [adapted from Steffensen JF, Lomholt JP, Vogel WOP (1986) In vivo observations on a specialized microvasculature, the primary and secondary vessels in fishes. Acta Zoologica (Stockh.) 67:193–200]. SA, segmental artery; SV, segmental vein; FR, fin ray; CLV, collecting lymphatic vessel; AFCN, anal fin capillary network. (Scale bar, 50 μm.)