Fig. 4. Conditional deletion of v integrin in both CNS neurons and glia leads to cerebral hemorrhage. (A) Generation of the conditional v mutant allele in CNS glia and neurons. (B) PCR performed using DNA from mutant tail (T) or brain (Br) tissue. In a sample from the brain, the intensity of the 350 bp band is significantly reduced, owing to Cre-mediated recombination of the v-flox allele. We confirmed deletion of exon four using a primer pair that detects the deleted v-flox cassette (data not shown). (C) Lysates from the cerebellum and cortex were immunoblotted with anti-v antibody. There is marked reduction in v protein levels in the mutant brains, indicating Cre-mediated deletion of v gene expression. (D-G,I) Control or mutant brains dissected from E17.5 embryos (D,E) and P7 neonates (F,G,I). Unlike the control brains (D,F), there are obvious regions of cerebral hemorrhage present throughout the forebrain and midbrain regions of the mutant brains (arrows in E,G,I). Significantly more severe cerebral hemorrhage is seen in the complete v-nulls (H). (J,K) Horizontal semi-thin sections from the ganglionic eminence region of E14.5 control (J) and mutant (K) brains. Vessels in the control brain are closely juxtaposed to the surrounding parenchyma (arrows in J). There are distended and tortuous vessels that separate from the surrounding neural tissue in the v conditional mutant brain (arrows in K). vz, ventricular zone. (L,M) Horizontal sections from the ganglionic eminence of E14.5 control (L) and mutant (M) embryonic brains stained with anti-PECAM (green) and anti-NG2 (red) antibodies to reveal endothelial cells and pericytes/vSMCs, respectively. The mutant vessels lined with vascular endothelium have a distended appearance and there is surrounding coverage by pericytes (arrows in M). (N-Q) Coronal sections through the cerebral cortex of control (N) or v mutant (O) P7 brains stained with H&E; normal neuronal patterning is observed in both. However, focal regions of hemorrhage are associated with many blood vessels in the mutant cerebral cortex (arrows in O). Coronal sections were stained with an anti-GFAP antibody to visualize astrocytes in control (P) and mutant (Q) cerebral cortex. Elevated numbers of astrocytes are present throughout the cortex of v mutant brain (arrows in Q).