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First published online 8 August 2006
doi: 10.1242/jcs.03081

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Redundant roles of Sox17 and Sox18 in postnatal angiogenesis in mice

Toshiyasu Matsui^1,*, Masami Kanai-Azuma^2,*, Kenshiro Hara¹, Shogo Matoba¹, Ryuji Hiramatsu¹, Hayato Kawakami², Masamichi Kurohmaru¹, Peter Koopman³ and Yoshiakira Kanai^1,

¹ Department of Veterinary Anatomy, The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-8657, Japan
² Department of Anatomy, Kyorin University School of Medicine, Mitaka, Tokyo 181-8611, Japan
³ Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland 4072, Australia

View larger version (136K): [in a new window]   Fig. 1. Whole-mount in situ hybridization analysis showing Sox17 (left panels) and Sox18 (right panels) 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). Arrowheads indicate small blood vessels around hindgut. (C-H) Embryos at early organogenic stage (E9.5); whole embryo (C,D) and histological sections (E-H). (I-K) Embryos at E11.5; whole embryo (I) and histological sections (J,K). Sox18-positive endothelial cells appear to be more widely distributed in the smaller microvasculatures (arrowheads in D,E,K). The red broken arrows (A,C,I) and boxed area (C,E,G,J) indicate the planes of sections and the magnified area in the designated panel, respectively. da, dorsal aorta; fg, foregut; gb, presumptive gallbladder; hg, hindgut; isv, intersomitic vessels; nt, neural tube. Bars, 50 µm.

View larger version (142K): [in a new window]   Fig. 2. Gross anatomical and histopathological analyses showing vascular anomalies in the liver (A-E), kidney (F-J), scrotum (K,L) and ovary (M,N) of the Sox17+/--Sox18-/- mice that survived to adulthood. (A-E) Gross-anatomy and HE-staining of the Sox17+/--Sox18-/- liver showing severe atrophy (right in A,C,D). The blood vessels running at the edge of the liver lobules (arrows in B) are missing in the Sox17+/--Sox18-/- livers. Their hepatocytes were smaller in size (C,D). The corrosion-cast SEM analysis visualized sparse sinusoidal microvasculatures with narrow lumina in the Sox17+/--Sox18-/- liver (arrowheads in E). bd, interlobular bile duct; cv, central vein; ia, interlobular artery; iv, interlobular vein. (F-J) The Sox17+/--Sox18-/- kidneys showing the hypoplasia of the outer medulla ('om' in G,H) attended with variable degrees of hydronephrosis (asterisk in F). The vascular bundles (arrows in G; arrowheads in H,I) are missing in the Sox17+/--Sox18-/- kidney (right in G-I). The corrosion-cast SEM analyses reveal the smaller capillaries of presumptive interbundle plexus in the affected outer medulla in the Sox17+/--Sox18-/- kidneys (right in I,J). (J) Higher magnification images indicated by the boxed area in I. aa, arcuate artery; ct, cortex; gl, glomerulus; im, inner medulla; om, outer medulla; rp, renal papilla. (K,L) Protruded and enlarged scrotums of the Sox17+/--Sox18-/- male (K). In the Sox17+/--Sox18-/- spermatic cords, the pampiniform plexus is less-tortuous (arrows) and enlarged in its lumen (L). (M,N) The Sox17+/--Sox18-/- ovaries exhibit less blood contents (M) and are smaller (N) than the Sox18-/- ovaries. +/+ and +/-indicate Sox17+/+-Sox18-/- and Sox17+/--Sox18-/- organs of the same littermates, respectively. Bar, 50 µm.

View larger version (128K): [in a new window]   Fig. 3. Liver atrophy and kidney outer medulla hypoplasia in the Sox17+/--Sox18-/- neonates at P7 and P14. (A-D) HE-staining of the affected Sox17+/--Sox18-/- livers at P7 (A,B) and P14 (C,D) showing atrophy of the hepatocytes (asterisks in A,B and whole area in C,D). cv, central vein; hc, hematopoietic cells. (E-H) HE-staining of the affected Sox17+/--Sox18-/- kidneys at P7 (E,F) and P14 (G,H), showing the hypoplasia of the outer medulla that might result in variable degrees of hydronephrosis. In the affected Sox17+/--Sox18-/- kidneys, their outer medulla showed dysmorphogenesis (E,G), in which the running patterns of vasa recta appeared to be irregular (arrowheads and arrows in F,H). ct, cortex; im, inner medulla; om, outer medulla; rp, renal papilla. The boxed area encompasses the area magnified in the designated panel. Bars, 50 µm (for A-D,F,H) and 100 µm (for E,G).

View larger version (89K): [in a new window]   Fig. 4. Immunohistochemical and immunoblot analyses showing increased expression of HIF1 in the affected Sox17+/--Sox18-/- livers at P7. (A,B) Anti-HIF1 staining of Sox18-/- (+/+) and affected Sox17+/--Sox18-/- (+/-) livers at P7. White and black arrowheads indicates HIF1-positive and HIF1-negative hepatocytes, respectively. (B) Magnified images of respective panels in A. (C) Immunoblot analysis of Sox18-/- (left), mild Sox17+/--Sox18-/- (middle) and severe Sox17+/--Sox18-/- (right) livers at P7 (two independent samples in each group), showing increased expression of HIF1 in the Sox17+/--Sox18-/- livers in a phenotype-dependent manner. Numbers indicate relative HIF1 expression levels normalized to ACTIN level ± standard error (amount of HIF1 in the Sox18-/- liver is set as 1.0; n=3). Bar, 50 µm.

View larger version (115K): [in a new window]   Fig. 5. Transmission electron micrographs showing ischemic necrosis of hepatocyte and renal tubular epithelia in the affected livers (A-C) and kidneys (D-F) of the P7 Sox17+/--Sox18-/-neonates. (A-C) Enlarged sinusoidal lumen and Disse's space in the affected Sox17+/--Sox18-/- livers (right in A-C). Their surrounding hepatocytes (hc) are smaller in size, and some of them displayed necrosis and vacuolation with swollen mitochondria and dilated rough endoplasmic reticulum (asterisks in A,B). Ds, Disse's space; ec, endothelial cell; hc, hepatocyte. (D-F) Normal vascular structures of vasa recta around renal tubular epithelia in the affected Sox17+/--Sox18-/- kidneys at P7 (D,E). However, some tubular epithelia display necrotic morphology (asterisk in F). ec, endothelial cell; te, renal tubular epithelial cells. Bars, 5 µm.

View larger version (88K): [in a new window]   Fig. 6. Section in situ hybridization (A,B) and semi-quantitative RT-PCR (C) analyses showing the expression profiles of Sox17, Sox18 and Sox7 in the livers, kidneys and other organs of the wildtype mice at P7 and adult stages. (A) The sections of the wildtype livers at P7, showing that Sox17 and Sox18, but not Sox7, are expressed in the endothelial cells of central veins (cv) and their closely associated sinusoidal vessels (arrowheads). Lower panels show the magnified view of central veins. cv, central veins; hc, hematopoietic cell. (B) The sagittal sections of the wildtype kidneys at P7, showing the expressions of Sox17 and Sox18, but not Sox7, in some populations of the outer medulla vasa recta (arrowheads and inset in bottom panles). Bottom panels show the magnified view of the outer medulla region and the outer medulla vasa recta (inset). ct, cortex; gl; glomerulus; im, inner medulla; om, outer medulla. (C) Semi-quantitative RT-PCR analyses showing relative expression levels of Sox17, Sox18 and Sox7 in various tissues of the wild-type mice at P7 and adult stages. Arrows indicate position of Sox products. Bars indicate position of G3pdh products as an internal control. Bars, 50 µm.

View larger version (14K): [in a new window]   Fig. 7. (A-E) RT-PCR analysis showing the differences of Vcam1 (A), Tie1 (B), Tie2 (C), Ang1 (D) and Ang2 (E) transcript levels in the livers (left) and kidneys (right) of severe Sox17+/--Sox18-/- (), mild Sox17+/--Sox18-/- () and Sox18-/- () groups at P7. Vertical axis represents relative expression levels of each vascular endothelial cell marker gene per G3pdh amplicon ratio; horizontal axis represents the severe or mild Sox17+/--Sox18-/- group or the Sox18-/- group. Each value indicates the expression level in each sample (one neonate); average of the values indicate the bar. Each asterisk on the average bar indicates a significant difference (*P<0.05; **P<0.01) compared with the data of the Sox18-/- group.

View larger version (160K): [in a new window]   Fig. 8. Immunohistochemical analyses showing a drastic reduction of positive signals for VCAM1 (B,G,H), but not PECAM1 (A,E,F) and TIE2 (C,D,I,J), in vascular endothelial cells of the affected livers (A-D) and kidneys (E-J) in the P7 Sox17+/--Sox18-/- neonates. Anti-PECAM1 staining shows sparse and dilated liver sinusoids (right in A) and irregular kidney vasa recta (right in E) in the severe Sox17+/--Sox18-/- group. Anti-VCAM1 staining reveals that its signal intensities are clearly reduced in the sinusoids and central veins of the livers (B) and in the vasa recta of the kidney outer medulla (arrows in G,H) of the affected Sox17+/--Sox18-/- pups. Its positive signals were found in the glomerular afferent and efferent arterioles (arrowheads) and some microvasculatures located in their cortex interstitium (G). Anti-TIE-2 staining shows no difference in their immunoreactivity in the liver sinusoids (C, arrows in D) and the kidney outer medulla vasa recta (arrows in I,J) between the severe Sox17+/--Sox18-/- and Sox18-/- groups. (D) Higher magnification of TIE2-positive sinusoids (broken line). (F,H,J) Higher magnification of the outer medulla vasa recta shown in E,G and I, respectively. Asterisks indicate non-specific staining in apical surface of the hepatic capsule (A-C) and in lumen of the renal tubular epithelia (G). ct, cortex; gl, glomerulus; im, inner medulla; om, outer medulla. Bar, 50 µm.

View larger version (81K): [in a new window]   Fig. 9. Quantitative analyses showing a drastic reduction of capillary densities of the affected Sox17+/--Sox18-/- livers and kidneys at P7. (A,B) Anti-PECAM-1 staining visualizes sparse sinusoids in the livers (A) and poorly-developed vascular bundles in kidney (arrowheads in B) of the severe Sox17+/--Sox18-/- group at P7. (C,D) Quantitative analyses showing the densities (C) and the numbers (D) of PECAM-positive microvasculatures per unit area in the surface (S) and proximal (P) zones of the livers, and in the cortex (C), outer medulla (OM) and inner medulla (IM) of the kidneys in the severe phenotype Sox17+/--Sox18-/- (solid bar) and Sox18-/- (open bar) groups at P7. Significance levels are indicated by **P<0.01 and *P<0.05. asterisk, non-specific staining in the apical surface of the hepatic capsule; ct, cortex; cv, central vein; gl, glomerulus; ia, interlobular artery; im, inner medulla; om, outer medulla. Bar, 50 µm.

View larger version (77K): [in a new window]   Fig. 10. In vitro angiogenesis assay using primary vascular endothelial cells isolated from the Sox17+/--Sox18-/- livers at P7, showing defective formation of the vascular-like structures on Matrigel. (A,B) Diff-Quik staining of the vascular endothelail cells cultured for 6 hours on Matrigel. PECAM1-positive endothelial cells were prepared from Sox18-/- (left) livers and mild (middle) and severe (right) Sox17+/--Sox18-/- livers at P7. (B) Higher magnified image of the panel above in A. (C) Quantitative analyses showing the number of aggregated nodes per area (left), the branch number per node (middle), and vessel length per area (right) in the 6-hour cultures of the liver vascular endothelial cells isolated from Sox18-/- (open bar), mild Sox17+/--Sox18-/- (gray bar) and severe Sox17+/--Sox18-/- (solid bar) groups (**P<0.01; n=8 in Sox18-/- and mild Sox17+/--Sox18-/- groups; n=4 in severe Sox17+/--Sox18-/- group). Bars, 100 µm.