Figure 1.
Vascular defects in perlecan-null embryos.
(A) Schematic view of the region of the forebrain shown in (B–E). (B–C) Nissl stained coronal sections of E12.5 forebrain: the cerebral microvasculature is tightly embedded into the neuronal tissue of E12.5 wild type embryos (B); the perlecan-deficient vessels are dilated and loosely incorporated in the tissue (C). (D–E) Laminin immunostained BMs of neuroepithelium and blood vessels in ventral forebrain sections. A region with a blood leakage in a mutant embryo is shown (arrow in E). (F–G) Whole-mount picture of wild type (F) and perlecan mutant (G) at E17.5. Hemorrhages (arrow in G) and edema in the skin is evident in the mutant embryo. (H–I) Hematoxilin and eosin staining of the skin sections of E16.5 embryos. The skin of the perlecan-null embryo shows increased interstitial spaces (arrowheads) and dilated vessels (arrow). Bars: (B and C) 125 µm; (D and E) 40 µm; (H and I) 50 µm.
Figure 2.
Immunostaining and ultra-structural analysis of microvessels.
(A–B) Perlecan is expressed in the sub-endothelial BMs of normal brain capillaries and is absent in the mutant tissue. (C–D) Laminin-1 is present in both wild type and perlecan-null BMs. (E–F) SMA is expressed in the microvessels sprouting into the brain parenchyma in wild type E17.5 (E), as well as mutant brains (F). (G–I) Ultrastructural analysis of endothelial cells from E14.5 brain capillaries. Note the tight association between the endothelial cell (ec) and the directly adjacent cells (ac) in the wild type brain capillary (G). The BM is visible on the upper surface of the adjacent cells (arrows in G). In the mutant brains, gaps are evident between the endothelial cells and the adjacent cells and pericytes (p) (arrows in H and I). (J–L) Electron micrographs of E16.5 skin microvessels. Electron dense material (arrows) is deposited at the abluminar side and appears as a BM-like structure in the wild type (J) as well as in the perlecan-null vessels (K,L).Vessel lumen (VL). Bars: (A–F) 250 µm; (G–L) 400 nm.
Figure 3.
Histological and immunohistochemical analysis of teratomas.
Hematoxylin/eosin staining shows teratomas derived from wild type (A) and mutant (B) ES cells that are composed of a variety of differentiated cells. (C–D) Perlecan immunofluorescent labeling in wild type (C) and mutant (D) teratomas. In wild type perlecan is expressed in the stroma (s), and in BMs surrounding vessels (arrow in C) and other structures including glands. Perlecan-null teratomas also have perlecan expression in the tumor stroma (s) and vessels. (E–F) Double fluorescent labeling with PECAM-1 in red (E) and perlecan in green (F) reveal that the vasculature of the perlecan-null tumors is composed of a mixture of perlecan-positive (host-derived) and perlecan-negative (ES cell-derived; arrowheads) endothelial cells. (G) Quantification of perlecan-positive and perlecan-negative vessels per area in wild type (n = 10) and mutant teratomas (n = 10). Bars: (A–D) 250 µm; (E–F) 125 µm.
Figure 4.
In vitro analysis of vessel formation in embryoid bodies.
EBs were generated from control (+/+) and perlecan mutant (−/−) ES cells. (A–F) The data are shown from PECAM-1 immunostainings of 5+12 days EBs cultured in 5% FCS-containing DMEM. A prominent vascular network is formed in wild type EBs (A), while mutants form endothelial networks that are less dense and smaller (B). (C–D) Treatment with 20 ng/ml VEGF165 stimulates the formation of large PECAM-positive vascularized areas both in control and mutant EBs. (E–F) Treatment with 20 ng/ml FGF-2 strongly induces the formation of extensive vascular networks in both control and perlecan-deficient EBs. (G) Binding of FGFR1-AP to control (+/+) and perlecan mutant (−/−) 5+9 days EBs. The EBs were grown until confluency in 96-well tissue culture plates and fixed. The fixed EBs were incubated with increasing concentration of FGF-2 and exposed to FGFR1-AP at a concentration of 300 µg/ml. After washing to remove unbound receptor bound FGFR1-AP was measured with AP chromogenic substrate. The data are means of 8 EBs per data-point. Statistical differences were tested by Students t-test (***, p<0.001). The experiment was repeated 3 times. Bars: (A–F) 1 mm.
Table 1.
Number of PECAM-positive vessel branches and size of vascularized areas in wild type and perlecan-null EBs.