Figures and data
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Pericyte-deficient larval brain vasculature displays abnormal patterning but an intact blood-brain barrier
a, Confocal projections of pericytes (TgBAC(pdgfrb:egfp)ncv22) and brain vasculature (Tg(kdrl:Hsa.HRAS-mCherry)s843) in sibling and pdgfrbuq30bh mutants at 7 dpf.
a′, Zoomed section from a (dashed rectangle) showing the midbrain central arteries.
b, Midbrain central arteries in sibling and pdgfrbuq30bh mutants at 14 dpf.
c, Representative images of vascular tracing at 7 dpf using Imaris 10.1 software. Lumenized blood vessels branching from the middle mesencephalic central arteries in the midbrain were traced.
d,e, Quantification of midbrain vessel length (d) and branching points (e). N=8 per group, unpaired t-test, *P<0.05, **P<0.01.
f, Fluorescent tracer assays in the midbrain of zebrafish larvae. 10 kDa Dextran–Cascade Blue and 70 kDa Dextran–Fluorescein were used to detect tracer extravasation to the brain parenchyma (white squares) in separate experiments and were coinjected with 2000 kDa Dextran–Tetramethylrhodamine to normalize vascular tracer intensity. Representative image of 2000 kDa Dextran was taken from 70 kDa Dextran coinjection, and 10 kDa Dextran coinjection is shown in Extended Data Fig. 1.
g, Quantification of brain parenchymal 70 kDa and 10 kDa Dextran intensity normalized to the vascular 2000 kDa Dextran intensity. N=15 in sibling and n=7 in pdgfrbuq30bh for 70 kDa Dextran intensity, n=8 in sibling, n=9 in pdgfrbuq30bh for 10 kDa Dextran intensity. Unpaired t-test, ns=not significant.
a,a′, b, c, f, Scale bars: 100 μm.
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pdgfrbuq30bh mutants develop aneurysms but display an intact blood-brain barrier at 2 months of age.
a, Confocal projections of whole brain imaging showing mural cells (TgBAC(pdgfrb:egfp)ncv22) and brain vasculature (Tg(kdrl:Hsa.HRAS-mCherry)s843) in sibling and pdgfrbuq30bh mutants at 5-months of age.
b, Restricted regions from the whole brain imaging in a (approximate location of dotted line) indicating pericyte loss and abnormal capillary network patterning. 100-µm thick maximum intensity projections (MIP) were generated using the continuation of the left middle mesencephalic central artery (arrow) as an anatomical anchor point.
c, d, Quantification of capillary branching points, large vessel and capillary diameter using the area shown in b. Large vessel diameter was quantified by averaging 5 points per vessel and the capillary diameter was quantified by averaging 10 capillary diameter per sample. N=3 per group, unpaired t-test, **P<0.01, ns=not significant.
e, Fluorescent tracer assays in the midbrain of 2-month-old zebrafish. 70 kDa Dextran– Fluorescein and 2000 kDa Dextran–Tetramethylrhodamine were co-injected to detect the tracer extravasation to the brain parenchyma and the tracer within the vasculature, respectively. Arrowheads indicate examples of aneurysms.
e′, Confocal projections zoomed in on the approximate area shown in e (dashed rectangle). 30-µm thick MIPs were generated to examine the capillary leakage, starting 30 µm below the most superficial point of the left hemisphere of the brain to prevent the potential leakage from the superficial vessels.
f, Quantification of the severity of aneurysms in sibling (n=11) or pdgfrbuq30bh(n=11). For examples of "severe" and "mild" see Extended Data Fig. 2.
g, Quantification of the brain parenchymal 70 kDa Dextran intensity normalized to the vascular 2000 kDa Dextran intensity, n=5 in sibling, n=4 in pdgfrbuq30bh mutants, unpaired t-test, ns=not significant.
a, b, e, e′, Scale bars: 250 μm.
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Adult pdgfrbuq30bh mutants display aneurysms and vascular integrity defects.
a, Representative images of coronal midbrain sections obtained from 3 month-old sibling and pdgfrbuq30bh animals.
b, Fluorescent tracer assays in 3-month-old sibling and pdgfrbuq30bhmutant animals. 10 kDa Dextran–Cascade Blue (cyan) was retro-orbitally injected, and Tg(kdrl:Hsa.HRAS-mCherry)s843 was used to label the blood vessels (magenta). Scale bar: 250 μm.
c, Zoomed regions from a and b (dotted squares) displaying blood and tracer accumulation (arrows).
d, Zoomed regions from b (dashed rectangle) displaying vasculature and tracer dye leakage from medial (large vessel) to lateral (capillary region) locations. The area was divided into 10 regions every 100 µm for tracer leakage quantification.
e, Quantification of brain parenchymal dextran intensity at medial (large vessel) to lateral (capillary region) locations shown in d. N=5 in sibling, n=4 in pdgfrbuq30bh, Two-sample Kolmogorov-Smirnov test, error bars represent SEM.
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Adult pdgfrb mutants display structural endothelial cell defects, vessel rupture and tracer accumulation at aneurysm hotspots.
a, Sections from whole brain imaging showing brain vasculature (Tg(kdrl:Hsa.HRAS-mCherry)s843) and 70 kDa Dextran Fluorescein at 5-months of age. Inset images within dashed rectangle show areas of hot spot leakage sites (arrows).
b, Quantification of hotspot leakage sites per animal in the midbrain region. N=3 for each group, unpaired t-test, ***P<0.01.
c, d, High resolution imaging of large calibre vessels and capillary zones in brain regions of pdgfrb crispants and uninjected siblings at 10-week-old stage. 10 kDa Dextran–Cascade Blue (cyan) injected, blood vessels (Tg(kdrl:EGFP)s843) and red blood cells (Tg(gata1:DsRed)sd2) are shown in MIPs and single Z-sections (c). Hotspot leakage sites are indicated by arrows. Capillary zones are shown in MIPs for all three channels and the single 10 kDa Dextran channel showing a lack of hotspot leakage site (d).
e, Transmission electron microscopy images of sectioned adult zebrafish brain vessels. pdgfrbuq30bh mutants showed basement membrane thickening and breakdown (cyan, black arrow), serum accumulation outside the vessels (white arrow), increased abluminal endothelial caveolae (magenta). Pseudo colours shown are ECs (purple), basement membrane (cyan), caveolae (magenta) and vesicles larger than 100 nm (green). Scale bars: 2 µm.
f, Zoomed regions from (e) showing intact tight junctions (arrowheads) in both siblings and pdgfrbuq30bh mutants. Scale bar: 1 µm.
g, Quantification of endothelial caveolae and basement membrane thickness. Caveolae were defined as uncoated spherical profiles <100 nm in diameter and scored as luminal or abluminal (see methods). Measurements were made for n=3 vessels for each group and a total of 8 different cellular regions measured for siblings and 19 for mutants. Basement membrane thickness was scored in 6 different regions per vessel with n=8 vessels in siblings and n=4 vessels in pdgfrbuq30bh mutants. Unpaired t-test, ***P<0.001, ****P<0.0001, ns= not significant.
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pdgfrbuq30bh larvae exhibit decreased cerebrovascular complexity but an intact BBB.
a, Confocal projections of pericytes (TgBAC(pdgfrb:egfp)ncv22) and brain vasculature (Tg(kdrl:Hsa.HRAS-mCherry)s843) in sibling and pdgfrbuq30bh mutants at 3-5 dpf.
b, Quantification of branching points of midbrain central arteries demonstrating the first detectable vessel patterning phenotype is seen at 5 dpf. N=10 per group at 3 dpf, n=15 in sibling and n=9 in pdgfrbuq30bhat 4 dpf, n=15 in sibling and n=11 in pdgfrbuq30bh at 5 dpf. Unpaired t-test, ns=not significant, *P<0.05.
c, d, Fluorescent tracer leakage assays in the midbrain of zebrafish larvae at 7 (c) and 14 dpf (d). 70 kDa Dextran–Fluorescein, 10 kDa Dextran–Cascade Blue and 1 kDa NHS Ester–Alexa Fluor 405 were used to detect tracer extravasation to the brain parenchyma in separate experiments and were coinjected with 2000 kDa Dextran–Tetramethylrhodamine to normalize vascular tracer intensity.
e, Quantification of brain parenchymal 70- and 10- and 1 kDa tracer intensity normalized to vascular 2000 kDa Dextran intensity. N=12 for sibling and n=7 in pdgfrbuq30bh 1 kDa NHS Ester intensity at 7 dpf, n=10 in sibling and n=9 in pdgfrbuq30bh for 70 kDa intensity at 14dpf, n=6 for sibling and n=4 for pdgfrbuq30bh for 10 kDa intensity at 14 dpf. Unpaired t-test, ns=not significant.
a, c, d, Scale bars: 100 μm.
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Examples of aneurysms in pdgfrbuq30bh mutants at 2-month-old stage.
a, Fluorescent tracer assays in the midbrain of 2-month-old zebrafish displaying examples of aneurysm severity in major vessels. 70 kDa Dextran–Fluorescein and 2000 kDa Dextran– Tetramethylrhodamine were co-injected to detect tracer extravasation to the brain parenchyma and tracer within the vasculature, respectively.
a′, Confocal projections zoomed in on the area shown in a (dashed square). Aneurysm (arrowheads) phenotypes were qualitatively categorised based on severity; normal, mild and severe and these categories used in Fig. 2.
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Adult pdgfrbuq30bhmutant animals display leakage and tracer accumulation localised at major vessel aneurysms.
a, Fluorescent tracer leakage assays in 5-month-old sibling and pdgfrbuq30bh animals. 70 kDa Dextran–Fluorescein (green) was retro-orbitally injected, and Tg(kdrl:Hsa.HRAS-mCherry)s843 was used to label the blood vessels (magenta). Scale bar: 250 μm.
a′, Zoomed regions (dashed rectangles) from a highlighting the tracer accumulation outside the large calibre vessels with aneurysm. Scale bar: 250 μm.
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Adult pdgfrb mutant rupture hotspots at aneurysms and unchanged endothelial ultrastructure in capillaries.
a, Whole brain imaging showing midbrain vasculature (Tg(kdrl:Hsa.HRAS-mCherry)s843) and 70 kDa Dextran–Fluorescein (green) in siblings and pdgfrbuq30bh mutants at 5-months of age. Hotspot leakage sites are indicated by white arrowheads.
b, Confocal projections of pericytes (TgBAC(pdgfrb:egfp)ncv22) and brain vasculature (Tg(kdrl:Hsa.HRAS-mCherry)s843) in sibling and pdgfrb crispants at 5 dpf, showing loss of pericytes in the pdgfrb crispants (phenocopying null mutants).
c, Quantification of endothelial caveolae in capillaries of siblings and pdgfrbuq30bh mutants. n=5 vessels per group, unpaired t-test ns= not significant.