Figure legends supplemental figures Supplemental figure 1. (a) Staining pattern of cre-protein is similar to GFAP-staining of astrocytes but does not resemble NeuN-staining of neurons in mouse hippocampus. (b) Cre protein does not colocalize with NeuN-positve neurons (scalebar 100µm) (c) Upper: GFAPcre+/VHL+f/+f are smaller than widtype littermates. Lower: A dome-shaped head in GFAPcre+/VHL+f/+f mice (arrowhead) indicates the presence of hydrocephalus in those animals. (d) Analysis of Evans blue (EB) leakage into the CNS indicates a significantly impaired blood-brain barrier function in GFAPcre+/VHL+f/+f (1.73 ± 0.2fold increase (n=7) of OD620/g brain) and GFAPcre+/VHL+f/+f/HIF-1α+f/+f (1.46 ± 0.2fold increase (n=4)) compared to GFAPcre-negative mice. GFAPcre+/VHL+f/+f/HIF2α+f/+f mice have no significantly altered blood-brain barrier permeability (1.22 ± 0.1fold change (n=5; Mann-Whitney-test)) (e) Upper graph: hearts of GFAPcre+/VHL+f/+f (167.1 ± 10.8 mg (n=7)) and of GFAPcre+/VHL+f/+f/HIF-1α+f/+f (158.0 ± 7.9 (n=5)) are significantly larger than hearts of control (GFAPcre-; 130.0 ± 2.5 mg (n=9)) and of GFAPcre+/VHL+f/+f/HIF-2α+f/+f mice (135.0 ± 8.9 mg (n=4; Mann-Whitney-test)). Lower graph: spleens are significantly enlarged in GFAPcre+/VHL+f/+f (244.2 ± 31.9 mg (n=6)) and in GFAPcre+/VHL+f/+f/HIF-1α+f/+f animals (173.0 ± 14.5 mg (n=5)) compared to control mice (GFAPcre-: 77.7 ± 8.4 mg (n=9)). Spleens are not significantly different in GFAPcre+/VHL+f/+f/HIF-2α+f/+f animals (88 ± 0.01 mg (n=4); data represents means ± S.E.M.). (f) Hydrocephaly is detectable in three week old GFAPcre+/VHL+f/+f and GFAPcre+/VHL+f/+f/HIF-1α+f/+f animals. In GFAPcre+/VHL+f/+f/HIF-2α+f/+f mice ventricles are not enlarged (representative Nissl staining, 50x magnification). Supplemental figure 2. Expression of VEGF mRNA (a) and GLUT-1 mRNA (b) in livers is not significantly different between control (GFAPcre-; n=11), GFAPcre+/VHL+f/+f (n=6), GFAPcre+/VHL+f/+f/HIF-1α+f/+f (n=6) and GFAPcre+/VHL+f/+f/HIF-2α+f/+f (n=5) animals (data represents means ± S.E.M.). (c) VEGF mRNA is significantly upregulated in brains of GFAPcre+/VHL+f/+f (n=10) and GFAPcre+/VHL+f/+f/HIF-1α+f/+f mice (n=6) compared to control animals (GFAPcre-; n=8). Double knockout of VHL and HIF-1α does not change VEGF expression significantly. In contrast, double knockout of VHL and HIF-2α (n=10) significantly reduces VEGF upregulation, and double knockout of VHL and VEGF (n=5) reduces it further to wildtype levels (data represent means ± S.E.M.). (d) Immunofluorescence staining in the hippocampus shows that VEGF upregulation in GFAPcre+/ GFAPcre+/VHL+f/+f mice increases vessel numbers as visualized by the endothelial marker CD34 (red). Double knockout of VHL and HIF-2α reduces vessel numbers whereas double knockout of VHL and HIF-1α has essentially no effect. In GFAPcre+/VHL+f/+f/VEGF+f/+f mice vessel number is further reduced (representative picture of 3 animals each genotype; astrocytes are stained with anti-GFAP (green); nuclei are stained with DAPI; scalebars 100µm). (e) 2Photon microscopy shows the increased cerebral vessel number in GFAPcre+/VHL+f/+f and GFAPcre+/VHL+f/+f/HIF-1α+f/+f mutant animals. No difference is observed between wildtype and GFAPcre+/VHL+f/+f/HIF-2α+f/+f animals (representative images of cranial windows; larger image 400x, scalebar 50µm; inset 200x, scalebar 200µm). (f) In vivo measurements of brain capillary diameter (right) and the blood velocity in the brain capillaries (left) by 2-photon microscopy demonstrate that both GFAPcre+/VHL+f/+f and GFAPcre+/VHL+f/+f/HIF-1α+f/+f animals have significantly thicker capillaries and that bloodflow is significantly slower compared to wildtype (GFAPcre-) and GFAPcre+/VHL+f/+f/HIF-2α+f/+f mice (n=4 each genotype; Kruskal-Wallis-test). Supplemental figure 3. (a) Survival of GFAPcre+/VHL+f/+f/VEGF+f/+f mice (n=19) is significantly reduced compared to wildtype mice (GFAPcre-; n=62), but extended compared to GFAPcre+/VHL+f/+f animals (data represents median survival (weeks)). (b) The hydrocephaly of the GFAPcre+/VHL+f/+f mice is completely rescued in GFAPcre+/VHL+f/+f/VEGF+f/+f double knockout animals (representative T1 weighed fMRI picture). Supplementary Methods: Primer Sequences for RNA analyses: EPO: (F): 5′-TGCGACAGTCGAGTTCTGGA-3′ (R): 5′-TCTGCACAACCCATCGTGAC-3′ Probe: 5′- (6~FAM)AGGTACATCTTAGAGGCCAAGGAGGCAGAAA-(BHQ)-3′ VEGF (F): 5′-ATCCGCATGATCTGCATGG-3′ (R): 5′-AGTCCCATGAAGTGATCAAGTTCA-3′ Probe: 5′-(6~FAM)TGCCCACGTCAGAGAGCAACATCAC-(BHQ)-3′ Primer Sequences for DNA recombination analyses: Location of the primers see (42) VHL: VHL F1: 5′-CTGGTACCCACGAAACTGTC-3′ VHL F2: 5′-CTAGGCACCGAGCTTAGAGGTTTGCG-3′ VHL R: 5′-CTGACTTCCACTGATGCTTGTCACAG-3′ Product size: VHL 2-lox allele: 460bp, 1-lox allele: 260bp; wt allele 290bp HIF-1α: HIF-1α F1: 5′-TTGGGGATGAAAACATCTGC-3′ HIF-1α F2: 5′-GCAGTTAAGAGCACTAGTTG-3′ HIF-1α R: 5′-GGAGCTATCTCTCTAGACC-3′ Product size: HIF-1α 1-lox allele: 270bp; 2-lox allele 260bp; wt allele 240bp HIF-2α: HIF-2α F1: 5′-CAGGCAGTATGCCTGGCTAAT TCCAGTT-3′ HIF-2α F2: 5′-CTTCTTCCATCATCTGGGATCTGGGACT-3′ HIF-2α R: 5′-GCTAACACTGTACTGTCTGAAAGAGTAGC-3′ Product size: HIF-2α 1-lox allele: 360bp; 2-lox allele 460bp; wt allele 430bp Supplemental Figure 1 GFAP b NeuN GFAPcre- c NeuN / Cre GFAPcre- OD620nm/g brain (fold increase) GFAPcre+/VHL n.s. Heart weight (g) n.s. 3.0 p=0.029 p=0.0011 2.5 2.0 1.5 1.0 0.5 0 - + GFAPcre- + + p=0.0079 0.18 0.16 0.14 0.12 n.s. GFAPcre- GFAPcre+/VHL+f/+f GFAPcre+/ VHL+f/+f/HIF-2α+f/+f GFAPcre+/ VHL+f/+f/HIF-1α+f/+f Spleen weight (g) p=0.0010 0.4 p=0.0004 0.3 0.2 0.1 0.0 GFAPcre- GFAPcre+/ GFAPcre+/ VHL+f/+f/ VHL+f/+f HIF-1α+f/+f GFAPcre+/ VHL+f/+f/ HIF-2α+f/+f + GFAPcre+/ GFAPcre+/ GFAPcre+/ VHL+f/+f VHL+f/+f/ VHL+f/+f/ HIF-1α+f/+f HIF-2α+f/+f e p=0.0010 0.20 Evans blue permeability into CNS EB +f/+f Cortex NeuN / Cre d GFAPcre+/VHL+f/+f d Hippocampus GFAP-cre+ Cre GFAP-cre+ a Supplemental Figure 2 b Liver 1.50 1.25 n.s. n.s. n.s. 1.00 0.75 0.50 0.25 0.00 GFAP-cre+/ GFAP-creVHL+f/+f rel. VEGF expression c GFAP-cre+/ GFAP-cre+/ VHL+f/+f/ VHL+f/+f/ HIF-1α+f/+f HIF-2α+f/+f Brain 17.5 15.0 Liver rel. Glut-1 expression rel. VEGF expression a 2.00 1.75 1.50 n.s. n.s. n.s. 1.25 1.00 0.75 0.50 0.25 0.00 GFAP-cre- GFAP-cre+/ VHL+f/+f GFAP-cre+/ GFAP-cre+/ VHL+f/+f/ VHL+f/+f/ HIF-1α+f/+f HIF-2α+f/+f d n.s. p<0.0001 p<0.0001 n.s. 12.5 10.0 GFAP-cre- GFAP-cre+/VHL+f/+f/HIF-2α+f/+f GFAP-cre+/VHL+f/+f/VEGF+f/+f 7.5 GFAP/ CD34/ DAPI 5.0 2.5 0.0 GFAPcre- GFAPcre+/ GFAPcre+/ GFAPcre+/ GFAPcre+/ VHL+f/+f/ VHL+f/+f/ VHL+f/+f/ VHL+f/+f +f/+f +f/+f HIF-1α HIF-2α VEGF+f/+f GFAP-cre+/VHL+f/+f GFAP-cre+/VHL+f/+f/HIF-1α+f/+f Supplemental Figure 2 GFAPcre+/VHL e GFAPcre- +f/+f GFAPcre+/VHL+f/+f/HIF-2α +f/+f f GFAPcre+/VHL+f/+f/HIF-1α +f/+f 5 p<0.0001 n.s. 4 3 2 1 0 GFAPcre- GFAPcre+/ GFAPcre+/ GFAPcre+/ VHL+f/+f VHL+f/+f/ VHL+f/+f/ HIF-1α+f/+f HIF-2α+f/+f Velocity (mm/sec) Diameter (µm) n.s. p<0.0001 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0 n.s. p<0.0001 p<0.0001 n.s. GFAPcre- GFAPcre+/ GFAPcre+/ GFAPcre+/ VHL+f/+f VHL+f/+f/ VHL+f/+f/ HIF-1α+f/+f HIF-2α+f/+f Supplemental figure 3 a b GFAPcreGFAPcre+/VHL+f/+f/VEGF+f/+f GFAPcre+/VHL+f/+f Percent Survival 100 p<0.0001 80 60 40 20 0 GFAPcre+/VHL+f/+f/VEGF+f/+f 0 5 10 15 Age (weeks) 20