Supplemental figure 1. Highly stereotyped vascular pattern in the tail.
(a) Cross section of normal tail showing the four main vascular bundles (outlined area). High power magnification of the ventral bundle (1) containing the caudal artery, the lateral bundles (2, 4) and the dorsal bundle (3), each containing one small artery (arrow) and one vein (v). In the lateral bundles, artery and vein are ventrally and dorsally located, respectively.
Scale bars: a, 890 mm; panels 1-4, 136 mm
Supplemental figure 2. Impaired postnatal maturation of the caudal artery in Notch3-/- mice.
(a-b) a-SMA staining of P0.5 caudal artery from wildtype (a) and Notch3-/- mice (b) showing that caudal arteries are surrounded by a-SMA positive mural cells. (c-h) Hematoxylin and eosin staining at P0.5 (c-d), P7 (e-f) and P28 (g-h). A maturation defect in Notch3-/- mice is apparent by P7, with ectopic aggregates of mural cells (arrows). (i,j) Electron micrographs of P28 tunica media. The wild-type artery comprises 4 cohesive smooth muscle cell layers while the Notch3-/- artery exhibits only 2 smooth cell layers adjacent to the elastica lamina. Note the irregular shape and smaller size of smooth muscle cells, particularly of those that form the ectopic cell aggregates (arrows), with fibroblasts (asterisks) intermingled between the outer and inner smooth muscle cell layers.
Supplemental figure 3: Expression of arterial SMC markers in pial vessels and aorta
Wildtype and Notch3-/- mice heterozygous for the SM22a-lacZ transgene were examined for ß galactosidase expression in pial vessels (a, c) and aorta (b,d). ß-gal staining is markedly reduced in pial arteries and retained in aorta from Notch3-/- mice. Mice shown in all panels were at P28
Supplemental figure 4. Arterial identity of endothelial cells is retained in Notch3-/- mice.
(a-b) In situ hybridization for ephrin B2 of wildtype and Notch3-/- caudal artery. EphrinB2 is detected in arterial endothelial cells at a high level and in scattered SMC at a low level; ephrinB2 expression is not affected in Notch3-/- mice. (c-d) Connexin 40 (Cx 40) immunostaining of wildtype and Notch3-/- caudal artery. Cx 40 is detected in arterial endothelial cells and Cx40 expression is not affected in Notch3-/- mice. (e-f) In situ hybridization for Notch3 of wildtype caudal artery (e) and vein (f). Notch3 RNA is detected in the presumptive arterial smooth muscle cells, but is absent in endothelial cells and venous smooth muscle cells. Mice shown in all panels were at P7.
Scale bar a-f , 34µm
Supplemental figure 5. Expression of Hes and Hey genes is unaffected in mutant tail arteries.
(a-l) In situ hybridization for Hes1 (a-c), Hey1 (d-f), Hey2 (g-i) and HeyL (j-l) of wildtype caudal artery (left column), wildtype vein (middle column) and Notch3-/- caudal artery (right column). Hes1 is expressed in scattered endothelial and smooth muscle cells from arteries and veins. Hey1 expression is essentially confined to arterial endothelial cells. Hey2 is detected in arterial endothelial cells and both arterial and venous SMC. HeyL is expressed in both arterial and venous SMC. Hes1, Hey1, Hey2 and HeyL expression are not affected in Notch3-/- mice. Hes5 is detected neither in wildtype nor in mutant tail vessels (data not shown). Mice shown in all panels were at P7. Images are representative from three wildtype and three Notch3-/- mice analyzed with each riboprobe. Scale bar a-l, 34µm. (m) Quantification of Hes1, Hey1, Hey2 and HeyL transcript levels in isolated caudal arteries from P15 wildtype and Notch3-/- mice by real-time PCR. Shown is mean mRNA copy number (± s.e.m) of the indicated genes per 1,000 copies of GAPDH mRNA. Comparison of 3 pairs of wild type and 4 pairs of mutant arteries confirmed that expression of these 4 genes is not affected in mutant arteries.
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