Local angiogenic interplay of Vegfc/d and Vegfa controls brain region-specific emergence of fenestrated capillaries
- Department of Neurosciences, Lerner Research Institute, Cleveland Clinic, United States
- Department of Molecular Medicine, Cleveland Clinic Lerner College of Medicine, Case Western Reserve University, United States
- Departments of Molecular Cell Biology and Molecular Neuroscience, The Weizmann Institute of Science, Israel
- Laboratory of Neurovascular Signaling, Department of Molecular Biology, ULB Neuroscience Institute, Université libre de Bruxelles, Belgium
Figures
Figure 1 with 2 supplements
Genetic loss of Wnt/β-catenin signaling leads to severely impaired angiogenesis in the brain parenchyma without any apparent defect in fenestrated choroid plexus (CP) vasculature.
(A, A’) Dorsal (A) and lateral (A’) views of a 72 hours post fertilization (hpf) Et(cp:EGFP);Tg(kdrl:ras-mCherry) larval head point to the locations of the diencephalic and myelencephalic CP (dCP …
Figure 1—figure supplement 1
Characterization of endothelial marker and β-catenin reporter expression in zebrafish larval brain vasculature.
(A–A”) Dorsal views of a 10 days post fertilization (dpf) Tg(plvap:EGFP) zebrafish head immunostained for Glut1, an endothelial marker for the blood-brain barrier (BBB) state. Tg(plvap:EGFP)+ blood …
Figure 1—figure supplement 2
Characterization of the reckulb3 allele.
(A) Schematic representation of the domain architecture of Reck, with from N- to C-terminus, five cysteine knot (CK) motifs, a Frizzled-like cysteine-rich domain (CRD), two epidermal growth …
Figure 2
Wnt/β-catenin signaling deficiency does not cause any apparent defect in fenestrated capillary formation in the neurohypophysis (NH) and organum vasculosum of the lamina terminalis (OVLT).
(A) Schematic diagram of vasculature in the ventral brain at around 5–10 days post fertilization (dpf), illustrating the locations of the OVLT, the NH, and distinct blood vessels used for …
Figure 3
Developmental angiogenesis at the interface of the diencephalic choroid plexus (dCP) and pineal gland (PG), and vegf expression patterns during the vascularization of this interface.
(A) Schematic representation of the dorsal view of the zebrafish larval head at 10 days post fertilization (dpf), indicating the location of the dCP and PG interface (red boxed area). (B–C”’) 3D (B),…
Figure 4
Heterogeneous endothelial requirements for Vegfs-dependent angiogenesis at the diencephalic choroid plexus (dCP)/pineal gland (PG) interface.
(A–H) Dorsal views of 10 days post fertilization (dpf) wild-type (WT) (A), vegfab-/- (B), vegfd-/- (C), vegfc-/- (D), vegfab-/-;vegfd-/- (E), vegfc-/-;vegfd-/- (F), vegfab-/-;vegfc-/- (G), and vegfab…
Figure 5
Endothelial cell-autonomous and cell non-autonomous requirements of Vegfc for vascularization of the diencephalic choroid plexus (dCP)/pineal gland (PG) interface.
(A–E) Dorsal views of 72 hours post fertilization (hpf) wild-type (WT) (A), vegfcum18/um18 (B), vegfab-/- (C), and vegfab-/-;vegfcum18/um18 (D, E) cranial vasculature visualized by Tg(kdrl:EGFP) …
Figure 6 with 1 supplement
BAC transgenic analysis of vegf expression in the ventral brain around the neurohypophysis (NH)/organum vasculosum of the lamina terminalis (OVLT).
(A–D) Dorsal views of 72 hours post fertilization (hpf) TgBAC(vegfaa:EGFP) (A), TgBAC(vegfab:EGFP) (B), TgBAC(vegfc:EGFP) (C), and TgBAC(vegfd:EGFP) (D) ventral brain of the larvae carrying the Tg(kd…
Figure 6—figure supplement 1
Co-localization of the pituicyte marker cyp26b1 and vegfaa or vegfab BAC transgenic reporter expression at early larval stages.
(A–A”) Single confocal z-plane images of 75 hours post fertilization (hpf) TgBAC(vegfaa:EGFP) ventral brain following in situ hybridization of cyp26b1, showing overlapping signals between the EGFP+ …
Figure 7
Heterogeneous endothelial requirements for Vegfs-dependent angiogenesis in the ventral brain around the neurohypophysis (NH)/organum vasculosum of the lamina terminalis (OVLT).
(A–H) Dorsal views of 120 hours post fertilization (hpf) wild-type (WT) (A), vegfab-/- (B), vegfd-/- (C), vegfc-/- (D), vegfab-/-;vegfd-/- (E), vegfc-/-;vegfd-/- (F), vegfab-/-;vegfc-/- (G), and vegf…
Figure 8
Temporal inhibition of Vegfa signaling by sFlt1 overexpression is sufficient to cause impaired formation of the hypophyseal artery (HyA) and Hy loop.
(A–C) Dorsal views of 72 hours post fertilization (hpf) wild-type (WT) (A), vegfcum18/um18 (B), and vegfcum18/um18;vegfab-/- (C) ventral brain vasculature visualized by Tg(kdrl:EGFP) expression. vegf…
Figure 9
Normal choriocapillaris formation in zebrafish deficient for Wnt/β-catenin signaling, and conserved expression of Vegfa paralogs in retinal pigment epithelium (RPE).
(A) Dissected eye from 10 days post fertilization (dpf) Tg(plvap:EGFP);Tg(glut1b:mCherry) zebrafish immunostained for GFP and DsRed shows strong Tg(plvap:EGFP) and absent Tg(glut1b:mCherry) …
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Figure 9—source data 1
Quantification of the number of endothelial cells that comprised the choroidal vascular plexus (CVP) in wild-type (WT), gpr124-/-, reck-/-, and wnt7aa-/- at 6 days post fertilization (dpf).
- https://cdn.elifesciences.org/articles/86066/elife-86066-fig9-data1-v2.xlsx
Figure 10
Zebrafish Vegfa paralogs are redundantly required for fenestrated choroidal vascular plexus (CVP) formation.
(A–D) Wild-type (WT) (A), vegfab-/- (B), vegfaa-/- (C), and vegfab-/-;vegfaa-/- (D) CVP visualized by Tg(kdrl:EGFP) and Tg(kdrl:NLS-mCherry) expression at 72 hours post fertilization (hpf). (E) …
-
Figure 10—source data 1
Quantification of the number of endothelial cells that comprised the choroidal vascular plexus (CVP) in wild-type (WT) and various vegf mutants at 6 days post fertilization (dpf).
- https://cdn.elifesciences.org/articles/86066/elife-86066-fig10-data1-v2.xlsx
Figure 11
Comparative analysis of vascularization across fenestrated central nervous system (CNS) vascular beds reveals crucial angiogenic interplay of Vegfc/d and Vegfa in choroid plexus (CP) and circumventricular organ (CVO) vascularization.
(A) Schematic diagram of a mid-sagittal section of the larval zebrafish brain indicates the location of the CPs and CVOs examined in our current and previous studies. MO: medulla oblongata, OB: …
Videos
Video 1
Fenestrated vasculature at the pineal gland (PG)/diencephalic choroid plexus (dCP) interface.
Ten days post fertilization (dpf) Et(cp:EGFP);Tg(kdrl:ras-mCherry) head immunostained for rhodopsin shows the 3D spatial relationship between dCP epithelial cells (green), pineal photoreceptor cells …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Genetic reagent (Danio rerio) | Tg(kdrl:EGFP)s843 | Jin et al., 2005 | ZFIN: s843 | |
| Genetic reagent (Danio rerio) | Tg(kdrl:Has.HRAS-mcherry)s896 | Chi et al., 2008 | ZFIN: s896 | |
| Genetic reagent (Danio rerio) | Tg(kdrl:NLS-mCherry)is4 | Wang et al., 2010 | ZFIN: is4 | |
| Genetic reagent (Danio rerio) | Tg(UAS:EGFP)nkuasgfp1a | Asakawa et al., 2008 | ZFIN: nkuasgfp1a | |
| Genetic reagent (Danio rerio) | Tg(UAS:EGFP-CAAX)m1230 | Fernandes et al., 2012 | ZFIN: m1230 | |
| Genetic reagent (Danio rerio) | Tg(hsp70l:sflt1, cryaa-cerulean)bns80 | Matsuoka et al., 2016 | ZFIN: bns80 | |
| Genetic reagent (Danio rerio) | Tg(hsp70l:sflt4, cryaa-cerulean)bns82 | Matsuoka et al., 2016 | ZFIN: bns82 | |
| Genetic reagent (Danio rerio) | TgBAC(vegfab:gal4ff)bns273 | Mullapudi et al., 2019 | ZFIN: bns273 | |
| Genetic reagent (Danio rerio) | TgBAC(vegfaa:gal4ff)lri96 | Parab et al., 2021 | ZFIN: lri96 | |
| Genetic reagent (Danio rerio) | TgBAC(vegfc:gal4ff)bns270 | Parab et al., 2021 | ZFIN: bns270 | |
| Genetic reagent (Danio rerio) | TgBAC(vegfd:gal4ff)lri95 | Parab et al., 2021 | ZFIN: lri95 | |
| Genetic reagent (Danio rerio) | Tg(glut1b:mCherry)sj1 | Umans et al., 2017 | ZFIN: sj1 | |
| Genetic reagent (Danio rerio) | Tg(plvapb:EGFP)sj3 | Umans et al., 2017 | ZFIN: sj3 | |
| Genetic reagent (Danio rerio) | Et(cp:EGFP)sj2 | Henson et al., 2014 | ZFIN: sj2 | |
| Genetic reagent (Danio rerio) | Tg(fli1:GAL4-Hsa.TCF7L2-2A-mCherry)ncv15 | Kashiwada et al., 2015 | ZFIN: ncv15 | |
| Genetic reagent (Danio rerio) | Tg(fli1:MYR-mCherry)ncv1 | Kwon et al., 2013 | ZFIN: ncv1 | |
| Genetic reagent (Danio rerio) | vegfaabns1 | Rossi et al., 2016 | ZFIN: bns1 | |
| Genetic reagent (Danio rerio) | vegfabbns92 | Rossi et al., 2016 | ZFIN: bns92 | |
| Genetic reagent (Danio rerio) | vegfchu6410 | Helker et al., 2013 | ZFIN: hu6410 | |
| Genetic reagent (Danio rerio) | vegfcum18 | Villefranc et al., 2013 | ZFIN: um18 | |
| Genetic reagent (Danio rerio) | vegfdbns257 | Gancz et al., 2019 | ZFIN: bns257 | |
| Genetic reagent (Danio rerio) | gpr124s984 | Vanhollebeke et al., 2015 | ZFIN: s984 | |
| Genetic reagent (Danio rerio) | wnt7aaulb2 | Martin et al., 2022 | ZFIN: ulb2 | |
| Genetic reagent (Danio rerio) | reckulb3 | This manuscript | ZFIN: ulb3 | |
| Antibody | Anti-GFP (chicken polyclonal) | Aves Labs | Cat#: GFP-1010 | IF (1:1000) |
| Antibody | Anti-GFP (rabbit polyclonal) | Thermo Fisher Scientific | Cat#: A-11122 | IF (1:200) |
| Antibody | Anti-GFP (chicken polyclonal) | Thermo Fisher Scientific | Cat#: A10262 | IF (1:200) |
| Antibody | Anti-DsRed (rabbit polyclonal) | Clontech Labs | Cat#: 632496 | IF (1:300) |
| Antibody | Anti-Claudin-5 (mouse monoclonal) | Thermo Fisher Scientific | Cat#: 35–2500 | IF (1:500) |
| Antibody | Anti-ZO-1 (mouse monoclonal) | Thermo Fisher Scientific | Cat#: 33–9100 | IF (1:500) |
| Antibody | Anti-P-glycoprotein (mouse monoclonal) | Thermo Fisher Scientific | Cat#: MA1-26528 | IF (1:100) |
| Antibody | Anti-Glut1 (rabbit polyclonal) | Novus Biologicals | Cat#: NB300-666 | IF (1:200) |
| Sequence-based reagent | reck sgRNA | This paper | Single-guide RNA | CCTGACAGTACTCACGAC |
| Commercial assay or kit | MEGAshortscript T7 Transcription | Thermo Fisher Scientific | Cat#: AM1354 | |
| Commercial assay or kit | mMessage mMachine T3 Transcription Kit | Thermo Fisher Scientific | Cat#: AM1348 | |
| Commercial assay or kit | DIG RNA Labeling Kit | MilliporeSigma | Cat#: 11277073910 | |
| Chemical compound, drug | Dextran, Tetramethylrhodamine, 3000 MW | Thermo Fisher Scientific | Cat#: D3308 | |
| Chemical compound, drug | Dextran, Alexa Fluor 647; 10,000 MW | Thermo Fisher Scientific | Cat#: D22914 | |
| Software, algorithm | LAS X Version 3.7.0.20979 | Leica Microsystems | ||
| Software, algorithm | ZEN 2.3 Blue Edition | Carl Zeiss Microscopy | ||
| Software, algorithm | GraphPad Prism 8.1.1 | GraphPad Software | ||
| Software, algorithm | Adobe Photoshop CS6 | Adobe | ||
| Software, algorithm | Adobe Illustrator CS6 | Adobe |
