Figure S1. Notch signaling does not regulate NG2 pericyte coverage

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Figure S1. Notch signaling does not regulate NG2 + pericyte coverage in vivo Balb/c pups were treated with the γ-secretase inhibitor DAPT from P2–P5 to block Notch signaling. Retinas were harvested at P6 and immunostained with antibodies against CD31 (red) and NG2 (green). DAPT treatment does not affect NG2 + pericyte coverage of vessels in developing retina. Scale bar: 300 μm. Figure S2. Jag1 is arterially restricted in the retina Representative images of P12 retina immunostained with CD31 to label the endothelium (white), Jag1 (red), and αSMA to label vascular smooth muscle cells (green). Jag1 is localized to arteries and arterioles. Artery “A”, vein “V”. Scale bar, 150 μm Figure S3. Endothelial Jag1 is required for SM22α + perivascular cell coverage during retinal angiogenesis An alternative VSMC marker was used to examine VSMC loss in retinas lacking endothelial Jag1. Shown are representative images of the vasculature in P8 retinas from mice (A) with (Jag1 lox/lox VECAD-Cre negative, “wild type”) and (B) without (Jag1 lox/lox VECAD-Cre positive, “Jag1 ECKO”) Jag1 in the endothelium. Retinas were immunostained with antibodies to CD31 (green) to label the endothelium and SM22α (red) to label VSMCs. The arteries, “A” are marked with arrows to highlight the lack of arterial SM22α in Jag1 ECKO mice compared to wild type. Scale bars, 70 μm. Figure S4. Loss of Notch ligand Jag1 in the endothelium does not affect pericyte coverage Representative images of vessels in P7 retinas from mice with (Jag1 lox/lox VECAD-Cre negative, “wild type”) and without (Jag1 lox/lox VECAD-Cre positive, “JAG1 ECKO”) Jag1 in the endothelium. Retinas were immunostained with antibodies to NG2 (green) and αSMA (blue) to label perivascular cells, and GS-IB4 from Griffonia simplciifolia (“Lectin,” red). While the absence of Jag1 in the endothelium decreases perivascular αSMA + cell coverage, it does not affect general pericyte coverage as marked by NG2. Arteries are labeled “A”, veins “V”. Scale bars, 300 μm. Figure S5. NG2 + pericyte coverage is not regulated by either vWF or integrin β3 expression in the retina Representative images of retinas from P5 mice, immunostained with antibodies to NG2 (green) and αSMA (blue) to label perivascular cells, and GS-IB4 from Griffonia simplciifolia (lectin, red) to label endothelial cells. The NG2 + perivascular cell coverage is consistent between wild type (c57Bl/6, A, B), vWF knockout (C, D), and DiYF (E, F) mice. Scale bars, 300 μm. Figure S6. Intravitreal injection of integrin β3 blocking antibody reduces arterial VSMC coverage (A) Pharmacological blockade of integrin β3 reduces αSMA + arterial VSMC coverage compared to (B) injection with isotype control. As expected, functional blockade of β3 resulted in reduced angiogenesis in the deep plexus (D) compared to control (C). Images A and C, B and D were taken at different depths in the same fields of view. Scale bars, A–D: 275 um.

Transcript of Figure S1. Notch signaling does not regulate NG2 pericyte coverage

Figure S1. Notch signaling does not regulate NG2+ pericyte coverage in vivo Balb/c pups were treated with the γ-secretase inhibitor DAPT from P2–P5 to block Notch signaling. Retinas were harvested at P6 and immunostained with antibodies against CD31 (red) and NG2 (green). DAPT treatment does not affect NG2+ pericyte coverage of vessels in developing retina. Scale bar: 300 µm. Figure S2. Jag1 is arterially restricted in the retina Representative images of P12 retina immunostained with CD31 to label the endothelium (white), Jag1 (red), and αSMA to label vascular smooth muscle cells (green). Jag1 is localized to arteries and arterioles. Artery “A”, vein “V”. Scale bar, 150 μm Figure S3. Endothelial Jag1 is required for SM22α+ perivascular cell coverage during retinal angiogenesis An alternative VSMC marker was used to examine VSMC loss in retinas lacking endothelial Jag1. Shown are representative images of the vasculature in P8 retinas from mice (A) with (Jag1 lox/lox VECAD-Cre negative, “wild type”) and (B) without (Jag1 lox/lox VECAD-Cre positive, “Jag1 ECKO”) Jag1 in the endothelium. Retinas were immunostained with antibodies to CD31 (green) to label the endothelium and SM22α (red) to label VSMCs. The arteries, “A” are marked with arrows to highlight the lack of arterial SM22α in Jag1 ECKO mice compared to wild type. Scale bars, 70 μm. Figure S4. Loss of Notch ligand Jag1 in the endothelium does not affect pericyte coverage Representative images of vessels in P7 retinas from mice with (Jag1 lox/lox VECAD-Cre negative, “wild type”) and without (Jag1 lox/lox VECAD-Cre positive, “JAG1 ECKO”) Jag1 in the endothelium. Retinas were immunostained with antibodies to NG2 (green) and αSMA (blue) to label perivascular cells, and GS-IB4 from Griffonia simplciifolia (“Lectin,” red). While the absence of Jag1 in the endothelium decreases perivascular αSMA+ cell coverage, it does not affect general pericyte coverage as marked by NG2. Arteries are labeled “A”, veins “V”. Scale bars, 300 μm. Figure S5. NG2+ pericyte coverage is not regulated by either vWF or integrin β3 expression in the retina Representative images of retinas from P5 mice, immunostained with antibodies to NG2 (green) and αSMA (blue) to label perivascular cells, and GS-IB4 from Griffonia simplciifolia (lectin, red) to label endothelial cells. The NG2+ perivascular cell coverage is consistent between wild type (c57Bl/6, A, B), vWF knockout (C, D), and DiYF (E, F) mice. Scale bars, 300 μm. Figure S6. Intravitreal injection of integrin β3 blocking antibody reduces arterial VSMC coverage (A) Pharmacological blockade of integrin β3 reduces αSMA+ arterial VSMC coverage compared to (B) injection with isotype control. As expected, functional blockade of β3 resulted in reduced angiogenesis in the deep plexus (D) compared to control (C). Images A and C, B and D were taken at different depths in the same fields of view. Scale bars, A–D: 275 um.

Figure S7. Arterial VSMC outgrowth co-patterns with vWF deposition in the endothelial basement membrane Representative images of retinas from postnatal Balb/c pups harvested over a time course during the process of blood vessel outgrowth and maturation, from postnatal days (P)4–P16. Retinas were immunostained with CD31 to label blood vessels, αSMA to label VSMC (green), and vWF (red). VSMC coverage of arteries is limited to areas of significant vWF accumulation in the arterial basement membrane. Scale bars, 200 μm. Figure S8. Notch signaling in VSMCs enhances the ability of VSMCs to adhere to vWF and to the endothelial basement membrane (A) The ability of VSMCs to adhere to a vWF-coated substrate was quantified following VSMC culture on control (IgG) or Jag1 coated plates (to stimulate Notch signaling). Addition of the γ-secretase inhibitor DAPT to block Notch signaling prevented adhesion to vWF in vitro. (B–E) Representative images of in vitro fibrin bead angiogenesis assays in which HUVECs (green) were cultured on microcarrier beads and suspended in a fibrin gel with VSMCs (red). Addition of the γ-secretase inhibitor DAPT to the culture media (D, E) prevented association of VSMC with endothelial tubes compared to vehicle (B, C). Arrows point to regions magnified, shown in C, E. (F) Blocking Notch signaling with DAPT resulted in a 49% reduction in VSMC-endothelial co-patterning. Values represent means ± s.e.m. DMSO control (n=6); DAPT (n=12); where n is the number of beads analyzed; p=0.002. Scale bars, B, D, 325 μm; C, E, 150 μm. Figure S9. Notch 3 knockdown in VSMCs reduces integrin β3 expression Knockdown of Notch signaling in VSMCs by transfection of siRNA specific to Notch 3 leads to a ~40% reduction of integrin ß3 expression, as analyzed by western blot (A) and real-time qPCR (B). Figure S10. Schematic representation of vessel maturation as a result of Notch signaling Nascent arteries are stabilized by immature perivascular cells, which, when stimulated by the Notch ligand Jag1 on the endothelium, upregulate their expression of integrin αvβ3. Separately, endothelial cells accumulate vWF, which is deposited into the endothelial basement membrane. As they mature, more vWF accumulates in the basement membrane. The VSMCs adhere to this vWF via αvβ3, thereby providing the appropriate mural cell coverage to the mature artery.

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