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Fig. S1. Analysis of integrin-dependent endothelial cell proliferation. (A,B) Retinas from P4 wild-type (A) or β8−/− (B) littermates were labeled with anti-CD31 and anti-pH3 antibodies to analyze endothelial cell proliferation. Comparable numbers of proliferating endothelial cells (arrows) were present in both wild-type and β8−/− retinas. Proliferating cells that are CD31-negative are likely to be retinal macrophages and/or astrocytes. (C,D) Retinas from P14 wild-type (C) or β8−/− (D) littermates were labeled with anti-CD31 and anti-pH3 antibodies. Comparable numbers of proliferating endothelial cells (arrows) were present in both wild-type and β8−/− retinas. Arrowheads in D indicate β8−/− vascular tufts. Images are shown at 200× magnification.
Fig. S2. Ablation of αv integrin gene expression in astrocytes leads to endothelial cell sprouting defects. (A,B) Retinas from P5 Nestin-Cre;αvflox/+ control (A) or Nestin-Cre;αvflox/flox mutant (B) mice were labeled with anti-CD31 or anti-NG2 to label endothelial cells and pericytes, respectively, within the primary vascular plexus. Note that blood vessels in Nestin-Cre;αvflox/flox mutant samples contain pericytes but have endothelial tip cells with blunted morphologies (arrows in B). (C,D) Retinas from P5 Nestin-Cre;αvflox/+ control (C) or Nestin-Cre;αvflox/flox mutant (D) mice were labeled with anti-CD31 or anti-GFAP to label endothelial cells and astrocytes, respectively. Note the apparently normal astrocyte cytoarchitecture. (E) Quantification of CD31-expressing filopodia per endothelial tip cell in retinas of P5 Nestin-Cre;αvflox/+ control and Nestin-Cre;αvflox/flox mutant mice, *P<0.001. Error bars represent s.d. Images are shown at 400× magnification.
Fig. S3. Ablation of β8 integrin gene expression in astrocytes leads to retinal endothelial cell sprouting defects. (A,B) Retinas from P5 Nestin-Cre;β8flox/+ control (A) or Nestin-Cre;β8flox/flox mutant (B) mice were labeled with anti-CD31 or anti-NG2 antibodies to visualize endothelial cells and pericytes, respectively, within the primary vascular plexus. Note the blunted endothelial tip cells in Nestin-Cre;β8flox/flox mutant samples (arrows in B) that are associated with pericytes. (C,D) Retinas from P5 Nestin-Cre;β8flox/+ control (C) or Nestin-Cre;β8flox/flox mutant (D) mice were labeled with anti-CD31 or anti-GFAP to detect endothelial cells and astrocytes, respectively. Although there are obvious endothelial cell defects in Nestin-Cre;β8flox/flox mutant samples (arrows in D), note the apparently normal astrocyte cytoarchitecture. (E) Quantification of CD31-expressing filopodia per endothelial tip cell in retinas of P5 Nestin-Cre;β8flox/+ control and Nestin-Cre;β8flox/flox mutant mice. *P<0.001. Error bars represent s.d. Images are shown at 400× magnification.
Fig. S4. Tamoxifen-inducible deletion of β8 integrin in astrocytes leads to retinal vascular pathologies. (A,B) GFAP-CreERT2;Rosa26-lox-STOP-lox-YFP mice were injected with 50 µg tamoxifen from P1-P3 and 10 days after the last injection animals were sacrificed and YFP expression patterns were analyzed. Note the mosaic patterns of Cre activation in GFAP-expressing astrocytes (A) but not CD31-expressing endothelial cells (B). (C,D) GFAP-CreERT2;β8flox/+ (C) or GFAP-CreERT2;β8flox/flox (D) pups were injected with tamoxifen from P1-P4 and 10 days after the last injection animals were sacrificed. Retinal endothelial cells and astrocytes were visualized by double immunofluorescence staining with anti-CD31 and anti-GFAP antibodies, respectively. Note that inducible deletion of β8 integrin expression in astrocytes leads to blood vessel morphogenesis defects (arrows in D). Images are shown at 200× magnification.
Fig. S5. Tamoxifen-inducible deletion of β8 integrin in astrocytes leads to cerebral vascular pathologies. (A-D) Paraffin-embedded sagittal brain sections from GFAP-CreERT2;β8flox/+ (A,B) or GFAP-CreERT2;β8flox/flox (C,D) mice were analyzed by Hematoxylin & Eosin staining (A,C) or double immunofluorescence labeling with anti-laminin and anti-GFAP antibodies (B,D) to visualize vascular basement membranes and astrocytes, respectively. Note that inducible deletion of β8 integrin gene expression leads to defective blood vessel morphologies and perivascular reactive gliosis. Images are shown at 200× magnification.
Fig. S6. Lack of retinal endothelial cell sprouting defects after ablation of TGFβ signaling in astrocytes. (A,B) Retinas from P5 Nestin-Cre/+;Tgfbr2flox/+ control (A) and Nestin-Cre/+;Tgfbr2flox/flox mutant (B) littermates were stained with anti-CD31 antibodies. Note the apparently normal endothelial tip cell morphologies in control and mutant mice. (C) Quantification of CD31-expressing filopodia per endothelial tip cell in retinas of P5 Nestin-Cre;Tgfbr2flox/+ control and Nestin-Cre;Tgfbr2flox/flox mutant mice reveals no statistically significant differences. Images are shown at 400× magnification. Error bars represent s.d.
