JCS03081 Supplementary Material

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• Supplemental Figure 1 - Fig. S1. Whole-mount in situ hybridization analysis showing Sox7 expression in developing blood vessels of mouse organogenic embryos. (A,B) Embryos at early somite stage (embryonic day 8.5 (E8.5); whole embryos; (A) and histological sections (B). Sox7 is highly expressed in the endothelial cells of both dorsal aorta (da) and small blood vessels around hindgut (arrowheads in B). (C-H) Embryos at early organogenesis stage (E9.5); whole embryo (C, D) and histological sections (E-H). Sox7 expression is found in the developing vascular endothelial cells of the surface smaller microvasculatures (arrowheads in D, E), the intersomite blood vessels (‘isv’ in G, H) and the dorsal aorta (‘da’ in D, E; F). The red broken arrows in A and C, or broken rectangles in E and G indicate the planes of sections or the magnified area in the designated panel, respectively. nt, neural tube; da, dorsal aorta; isv, intersomitic vessels; fg, foregut; hg, hindgut. Bars, 50 μm.

• Supplemental Figure 2 -

  Fig. S2. Histopathological analyses showing the abnormal vasculatures in the pampiniform plexus (A) and ovary (B-D) of the Sox17^+/−-Sox18^−/− adult mice. (A) HE staining of transverse sections of the Sox17^+/−-Sox18^−/− spermatic cord, showing dilated lumina of both arteries (a) and veins (v) and the thinner vein walls of the pampiniform venous plexus than those of the Sox1^−/− ones. (B-D) HE staining of the sagittal sections of the Sox17^+/−-Sox18^−/− ovary showing the reduced number of both corpus lutea and growing follicles (B). Histological (HE staining; B, C) and immunohistochemical analyses using a vascular endothelial cell marker, vWF (von Willebrand factor) (D), demonstrating a drastic reduction of small blood vessels located near the developing follicles (arrows in C, D). cl, corpus luteum; Gf, Graafian follicle; sf, secondary follicle. +/+ and +/− indicate the Sox17^+/+-Sox18^−/− and Sox17^+/−-Sox18^−/− organs of the same littermate, respectively. Bar, 50 μm.

• Supplemental Figure 3 -

  Fig. S3. Immunohistochemical analysis showing no increase of HIF1α expression in the outer medulla region of the affected Sox17^+/−-Sox18^−/− kidneys at P7. Anti-HIF1α signals are observed in the nucleus of some renal tubular epithelia and glomeruli located in the kidney cortex, but no appreciable difference is detectable between the normal Sox18^−/− (+/+) and affected Sox17^+/−-Sox18^−/− (+/−) kidneys (A; upper panel in B). Moreover, no positive signals for anti-HIF1α staining are detectable in the atrophic outer medulla region of the affected Sox17^+/−-Sox18^−/− kidneys (lower right panel in B). Each panel in B shows a higher magnified image of the cortex (upper) and outer medulla (lower) shown in A. ct, cortex; gl, glomerulus; om, outer medulla. Bar, 50 μm.

• Supplemental Figure 4 -

  Fig. S4. Immunostaining for vascular smooth muscle marker (α-SMA) and lymphatic endothelial marker (LYVE-1), showing no defects in the arteries and lymphatic vessels of the affected Sox17^+/−-Sox18^−/− livers and kidneys at P7. Bouins-fixed, deparaffinized sections were used for immunostaining for anti-αSMA (α smooth muscle actin) and anti-LYVE-1 antibodies. (A, B) Anti-α-SMA staining, visualizing the developing arteries of the livers (A) and kidneys (B) in the severe Sox17^+/−-Sox18^−/− (right) and Sox18^−/− (left) groups at P7. There is no appreciable defects in interlobular arteries in the liver (‘ia’ in A) and the inter- and/or intralobular arteries (‘ia’ in B) and glomerular arterioles (arrowheads in B) in the kidneys. (C, D) Immunostaining for the lymphatic endothelial marker LYVE-1, visualizing the developing lymphatic vessels (arrows) running along the large portal veins in the livers (‘pv’ in C) and the interlobular veins of the kidney (‘iv’ in D) at P7. No appreciable difference is detectable in the lymphatic vessels between the severe Sox17^+/−-Sox18^−/− (right) and Sox18^−/− (left) groups. +/+ and +/− indicate the Sox17^+/+-Sox18^−/− and Sox17^+/−-Sox18^−/− genotypes, respectively. gl, glomerulus; ia, interlobular artery; iv, interlobular vein; om, outer medulla; pv, portal vein. Bar, 50 μm.

• Supplemental Figure 5 -

  Fig. S5. BrdU labeling (A, B) and TUNEL staining (C, D) analyses showing no defects in the proliferation and maintenance of the liver sinusoids in the severely affected Sox17^+/−-Sox18^−/− neonates at P7. (A,B) Double staining using anti-BrdU (black) and anti-vWF (brown) Abs with hematoxylin couterstaining (blue) in the severely affected Sox17^+/−-Sox18^−/− and Sox18^−/− livers (A). The mutant mice were injected intraperitoneally with BrdU (100μg/g body weight). A quantitative analysis of BrdU-labeling index of vWF-positive cells (arrows in A) in the severely affected Sox17^+/−-Sox18^−/− livers revealed no defective proliferation of vWF-positive sinusoidal endothelial cells: a slight increase of BrdU-labeling index in the Sox17^+/−-Sox18^−/− vascular endothelial cells (n=4) (B). Asterisks in A indicate BrdU-positive hepatocytes. ec, sinusoidal endothelial cells. (C, D) TUNEL staining showing no appreciable increase of the apoptotic cells in the liver sinusoids in the severely affected Sox17^+/−-Sox18^−/− neonates. Only a few TUNEL-positive cells were detectable even in the severely affected Sox17^+/−-Sox18^−/− liver (C). A quantitative analysis supports no significant difference of the TUNEL labeling index in the sinusoidal cells between the severely affected Sox17^+/−-Sox18^−/− and Sox18^−/− livers (n=4) (D). +/+ and +/− indicate the Sox17^+/+-Sox18^−/− and Sox17^+/−-Sox18^−/− tissues of the same littermate, respectively. Bar, 50 μm.

• Supplemental Figure 6 -

  Fig. S6. In vivo angiogenesis assay by using the subcutaneous implantation of Matrigel including VEGF, showing no defects in neovascularization of the adult Sox17^+/−-Sox18^−/− mice. (A) HE staining of the Matrigel pellets which were subcutaneously implanted under dorsal skins for 4 days. The microvasculatures are frequently found to invade into the Matrigel pellets in both Sox17^+/−-Sox1^−-/− and Sox18^−/− mice. (B) Quantitative analyses showing no significant changes in the relative number of endothelial cells between the Sox17^+/−Sox18^−/− (solid bar) and normal Sox18^−/− (open bar) mice (n=4). ec, endothelial cells. Bar, 50 μm.

• Supplemental Figure 7 -

  Fig. S7. Immunostaining showing increased reactivity for vascular endothelial growth factor (VEGF; A) and no changes in its receptor FLK1 expression (B) in the affected Sox17^+/−-Sox18^−/− livers at P7. PFA-fixed, deparaffinized sections were used for anti-VEGF staining, while acetone-fixed cryosections were used for anti-FLK1 staining. In these Sox17^+/−-Sox18^−/− and Sox18^−/− samples, all procedures for fixation and immunostaining were performed under the same conditions in order to evaluate the relative signal intensities. Anti-VEGF staining displays slightly increased immunoreactivity in hepatocytes of the affected Sox17^+/−-Sox18^−/− livers as compared with those of the normal Sox18^−/− livers. Anti-FLK1 staining visualizes a sparse and enlarged sinusoidal endothelial cells of the Sox17^+/−-Sox18^−/− livers, but no appreciable changes are detected in immunoreactivity between the affected Sox17^+/−-Sox18^−/− and normal Sox18^−/− livers. asterisk, non-specific staining in apical surface of the hepatic capsule. +/+ and +/− indicate the Sox17^+/+-Sox18^−/− and Sox17^+/−-Sox18^−/− genotypes, respectively. Bar, 50 μm.