Selective ablation of v integrins in the central nervous system leads to cerebral hemorrhage, seizures, axonal degeneration and premature death

DEV01551 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. The av integrin subunit is not expressed on newly differentiated neurons in the embryonic brain. Horizontal sections through diencephalon of E13.5 av^+/– embryo labeled with pre-absorbed specific anti-av (A) and anti-b-tubulin III antibodies (B). av integrin is expressed on glial cell processes in av^+/– embryonic brain sections (A). However, av expression is not detected on newly differentiated, migrating neurons (C).

• Supplemental Figure 2 -

  Fig. S2. Adult GFAP-Cre conditional av mutants do not display obvious blood-brain barrier defects. (A,B) Three-month-old wild-type (A) or GFAP conditional av mutant (B) mice were anesthetized and cardiac perfused with 5 ml PBS, 15 ml of primary amine-reactive biotin (1 mg/ml), followed by perfusion with 4% paraformaldehyde. Frozen sections from cerebral cortex were labeled with HRP-conjugated streptavidin and a peroxidase chromogen. In both control (A) and mutant (B) brains, reactive biotin labeled cerebral endothelial cells (arrows), but did not cross the blood-brain barrier and label brain parenchymal tissue.

• Supplemental Figure 3 -

  Fig. S3. Adult Nestin-Cre conditional av mutants do not display persistent cerebral hemorrhage, and do not have grossly obvious disruption of neuronal layers within the cerebral cortex. (A,B) Hematoxylin and Eosin-stained coronal sections from ganglionic eminences of 7-month-old control (A) and mutant (B) mice. There is normal cortical layering and cytoarchitecture in both control (A) and mutant (B) (arrows mark intact cerebral blood vessels). (C,D) Coronal sections from control (C) or mutant (D) adult mice were immunostained with an antibody recognizing the neuronal nuclear marker NeuN, showing no obvious cortical layering defects in the Nestin-Cre conditional av mutants. (E,F) Silver-stained coronal sections from the cerebral cortex of 7-month-old control (E) and av mutant (F) mice. Normal axonal arborizations exist in both control and mutant sections (arrows in E,F). (G,H) Coronal sections from control (G) or mutant (H) P7 neonatal mice were immunostained with an antibody recognizing the neuronal nuclear marker NeuN. Despite the obvious region of hemorrhage and vascular distension (arrows in H), the developing cortical layers show no indication of dysplasia or ectopia. (I-L) Analysis of glial endfeet attachment to the pial basement membrane in Nestin-Cre conditional av mutant neonates. Sagittal sections through the cerebral cortex of PO control (I) or mutant (J) neontates immunolabeled with antibody directed against the glial marker GLAST. Note the appearance of normal glial end-feet attaching to the meningeal boundary in both control and mutant cortex (arrows in I,J). Anti-GLAST was also used to visualize Bergmann glial processes in the developing cerebellum from either control (K) or mutant (L) P0 neonates. No obvious defects in glial endfeet attachment were present in the Nestin-Cre conditional av mutants (arrows in L).

• Supplemental Figure 4 -

  Fig. S4. Adult Nestin-Cre conditional av mutants do not develop hindlimb muscular atrophy and do not display peripheral nerve abnormalities. (A,B) Hematoxylin and Eosin-stained hindlimb cross-sections from 7-month-old control and mutant mice. A normal hind-limb musculature is present in both control (A) and mutant (B) quadriceps, with no indication of atrophy, inflammation or fibrosis, which are indicative of myopathy. (C,D) Cross-sections from the lumbar spinal region of control (D) and mutant (E) adult mice stained with Luxol fast Blue to visualize myelin. No obvious peripheral axonal dystrophy or demyelination is present in the mutant (E).

• Movie 1 -

  Movie 1. Adult Nestin-Cre conditional av mutants are ataxic and display defects in hindlimb coordination. A 6-month-old control mouse displays a normal gait and fluid movement.

• Movie 2 -

  Movie 2. Adult Nestin-Cre conditional av mutants are ataxic and display defects in hindlimb coordination. A 6-month-old conditional av mutant littermate, however, has an abnormal gait, is ataxic, and displays hind limb rigidity.