YAP and TAZ regulate adherens junction dynamics and endothelial cell distribution during vascular development
- Max-Delbrück-Center for Molecular Medicine, Germany
- London Research Institute – Cancer Research UK, United Kingdom
- DZHK (German Center for Cardiovascular Research), Germany
- Max Planck Institute for Heart and Lung Research, Germany
- Faculdade de Medicina da Universidade de Lisboa, Portugal
- International Institute of Molecular and Cell Biology, Poland
- Vesalius Research Center, Belgium
- KU Leuven, Belgium
- Berlin Institute of Health, Germany
Figures
Figure 1 with 1 supplement
YAP and TAZ are expressed throughout the vasculature of developing mouse retinas, and localise to the nucleus of sprouting endothelial cells.
Immunofluorescence staining of YAP (green, A–D and A’–D’) and TAZ (green, E–H and E’–H’) was performed in wild-type mouse retinas at post-natal day 6 (P6). Retinas were co-stained with the …
Figure 1—figure supplement 1
YAP and TAZ localise at endothelial adherens junctions in the mouse retina.
Immunofluorescence stainings of YAP (green, A–C,A’–C’), TAZ (green, D–F, D’–F’) and VE-Cadherin (red, B,E,B’,E’) were performed in wild-type mouse retinas at P6. Arrows, co-localisation of YAP or …
Figure 2 with 2 supplements
Endothelial YAP and TAZ are required for vessel growth, branching and homogeneity of the plexus.
(A–F,) Retinas from P6 Yap iEC-KO (B), Taz iEC-KO (D) and YapTaz iEC-KO (F), and respective control pups (A,C,E) were stained with Isolectin B4 (IB4). Scale bar: 200 μm. (G–J), Quantification of …
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Figure 2—source data 1
Values for quantification of radial expansion (Figure 2G), vessel density (Figure 2H), branching frequency (Figure 2I), area of gaps (Figure 2J) and standard deviation of area (Figure 2K) and circularity (Figure 2L) of gaps in P6 Yap iEC-KO, Taz iEC-KO and YapTaz iEC-KO and respective control pups.
Each value corresponds to the average of several measurements for one animal (see Material and methods for details).
- https://doi.org/10.7554/eLife.31037.007
Figure 2—figure supplement 1
YAP and TAZ proteins are lost upon Cre-mediated genetic deletion in P6 mouse retinas.
Yap iEC-KO (Yapfl/fl Pdgfb-iCreERT2+/wt), Taz iEC-KO (Tazfl/fl Pdgfb-iCreERT2+/wt) and respective littermate control mice (YapControl, Yapfl/fl and TazControl, Tazfl/fl) were injected with tamoxifen …
Figure 2—figure supplement 2
TAZ compensates for the loss of YAP in endothelial cells in vivo.
Retinas from P6 Yap iEC-KO (B,D and B’,D’) and littermate controls (A,C and A’, C’) were immunostained for TAZ. Green arrowheads, nuclear Taz. A,B, A’,B’ images correspond to maximum projection of z …
Figure 3 with 2 supplements
YAP and TAZ are required for endothelial cell proliferation in vivo and endothelial cell proliferation in response to mechanical stretch in vitro.
(A, B) P6 retinal vessels labelled with IB4 (grey) and stained for EdU (red, marking S phase positive cells) and Erg (green, marking endothelial nuclei) in YapTaz iEC-KO (B) and littermate control …
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Figure 3—source data 1
Values for quantification of endothelial proliferation (Figure 3C) and apoptosis (Figure 3F) in P6 Yap iEC-KO, Taz iEC-KO and YapTaz iEC-KO and respective control pups.
- https://doi.org/10.7554/eLife.31037.011
Figure 3—figure supplement 1
YAP and TAZ proteins are lost after gene knockdown by siRNA in HUVECs.
HUVECs were treated with non targeting siRNA (siCTR) or siRNA targeting YAP, TAZ and YAP +TAZ for 24 hr. A-H, Immunofluorescence staining for YAP (green, A–D) or TAZ (green, E–H) and labelling of …
Figure 3—figure supplement 2
VEGF treatment does not affect YAP and TAZ subcellular localisation.
(A–H), Confluent HUVECs were treated with 40 ng/mL of VEGF for 30 min (B, F), 1 hr (C,G) and 3 hr (D,H) or control (A,E) and stained for YAP (A–D) or TAZ (E–H) and DAPI (not shown). (I,J) …
Figure 4 with 1 supplement
Combined loss of YAP and TAZ leads to decreased sprouting numbers and shape defects, vessel crosses, haemorrhages at the sprouting front and adherens junctions’ defects in vivo.
(A, B) P6 retinal vessels labelled with IB4 (green) and stained for ERG (magenta, marking endothelial nuclei) in YapTaz iEC-KO (B) and littermate control mice (A). Yellow asterisks mark sprouts. …
Figure 4—figure supplement 1
Combined loss of YAP and TAZ leads to decreased number of sprouts in the developing mouse retina.
Quantification of number of sprouts per 100 μm of sprouting front extension at P6 in YapTaz iEC-KO (n = 9 pups) and littermate control mice (n = 9 pups). Data are mean ±SD. p values were calculated …
Figure 5 with 1 supplement
YAP and TAZ regulate adherens junctions’ morphology, monolayer permeability and VE-Cadherin turnover in vitro.
(A–D) HUVECs knocked down for YAP (B), TAZ (C) and YAP/TAZ (D) and control (A) stained for VE-Cadherin. Red arrowheads, discontinuous VE-Cadherin. Scale bar: 50 μm. (E) Representative patches used …
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Figure 5—source data 1
Values for quantification of morphological (Figure 5F) and junctional turnover (Figure 5K) analysis of VE-Cadherin in HUVECs knocked down for YAP, TAZ and YAP/TAZ.
- https://doi.org/10.7554/eLife.31037.015
Figure 5—video 1
Reticular junctions correspond to junction associated intermediate lamellipodia.
Live imaging of VE-Cadherin-EGFP expressing HUVECs. Reticular junctions were identified as junction associated intermediate lamellipodia from their common outline shape. Thick to reticular junctions …
Figure 6
YAP and TAZ are required for uncoupled, individual cell migration.
(A–H), Phase contrast images of YAP (C,D), TAZ (E,F) and YAP/TAZ (G,H) knockdown HUVECs and control (A,B) immediately after removing barrier to create a cell free space (A,C,E,G) and 16 hr later (B,D…
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Figure 6—source data 1
Values for quantification of wound closure at 16 hr in YAP, TAZ and YAP/TAZ knockdown HUVECs and control (Figure 6I).
- https://doi.org/10.7554/eLife.31037.018
Figure 7 with 4 supplements
Nuclear YAP and TAZ inhibit Notch and BMP signalling in endothelial cells.
(A–B) Retinas from P6 Taz iEC-GOF (B) and control pups (A) were stained for the endothelial marker PECAM (blue) and the endothelial nuclei marker ERG (red). Taz iEC-GOF mice express mosaically …
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Figure 7—source data 1
Values for quantification of number of sprouts (Figure 7C) and branching frequency (Figure 7D) in Taz iEC-GOF mice and controls.
- https://doi.org/10.7554/eLife.31037.024
Figure 7—figure supplement 1
Targeting strategy used for the generation of the conditional TAZ gain-of-function mouse model.
cDNA coding for a 3xFLAG-tagged human TAZ S89A was inserted into a Rosa26 targeting vector downstream of the ubiquitous CAG promoter. The cDNA also included an internal ribosome entry sequence …
Figure 7—figure supplement 2
Microarray of YAP and TAZ gain of function mutant cells.
Heatmaps of Notch, BMP and Hippo pathway genes in control (AdGFP), AdYAP and AdTAZ HUVECs.
Figure 7—figure supplement 3
YAP and TAZ knockdown increases Notch and BMP reporter activities in vitro.
(A–C) Luciferase reporter assays in YAP, TAZ and YAP/TAZ knockdown HUVECs and controls for Notch reporter (A), BMP reporter (B) and TEAD reporter (C). Data are mean ±SD. p values were calculated …
Figure 7—figure supplement 4
DLL4 intensity in YapTaz iEC-KO.
Graph shows mean DLL4 staining intensity in the vascular retina of control (green) and YapTaz iEC-KO (red) P6 pups normalised to the background intensity. Data are mean ±SEM. n = 3 control and 3 YapT…
Figure 8 with 1 supplement
BMP inhibition partially rescues the cellular defects of YAPTAZ knockdown HUVECs.
(A) Luciferase reporter assay for Notch activity in YAP/TAZ knockdown HUVECs and controls treated with 0.1 μM DBZ or DMSO. Data are mean ±SEM. p values were calculated using unpaired t-test. n ≥ 3 …
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Figure 8—source data 1
Values of luciferase reporter assays for Notch (Figure 8A) and BMP (Figure 8D) activity in YAP/TAZ knockdown HUVECs and controls treated with Notch or BMP inhibitors.
Values for quantification of wound closure at 16 hr in YAP/TAZ knockdown HUVECs treated with Notch (Figure 8B) and BMP (Figure 8E) inhibitors. Values for quantification of permeability of YAP/TAZ knockdown HUVECs treated with 1 μM Ldn193189 (Figure 8F). Values for quantification of morphological analysis of VE-Cadherin in YAP/TAZ knockdown HUVECs treated with 1 μM Ldn193189 (Figure 8I).
- https://doi.org/10.7554/eLife.31037.027
Figure 8—figure supplement 1
Nuclear YAP and TAZ increase the expression of BMP inhibitors.
Heatmap of BMPs, BMP receptors and co-receptors and BMP inhibitors genes in control (AdGFP), AdYAP and AdTAZ gain of function mutant cells.
Author response image 1
VEGF treatment does not affect YAP and TAZ subcellular localisation.
Confluent HUVECs were starved in FBS free media (EBM2 0.1% BSA) and treated with 40ng/mL of VEGF for 30 min (B, F), 1h (C,G) and 3h (D, H) or control (A, E) and stained for YAP (A-D) or TAZ (E-H) …
Author response image 2
Acid wash treatment removes cell surface bound antibody.
HUVECs knocked down for YAP/TAZ (E-H) and control (A-D) were pulse labelled with VE-cadherin 55/7H1 for 30 mins at 4C and internalisation was allowed for 30 min at 37C in the presence of 5mM EGTA …
Author response image 3
Internalised VE-cadherin 55/7H1 co-localises with EEA1 in normal conditions, but not after disruption of cell junctions by calcium depletion.
HUVECs knocked down for YAP/TAZ (B, D) and control (A, C) were pulse labelled with VE-cadherin 55/7H1-A647 (red) for 30 min at 4C and internalisation was allowed for 30 min at 37C in the presence of …
Author response image 4
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| strain, strain background (Mus musculus, C57BL/6J) | WT | The Jackson laboratories | ||
| genetic reagent (Mus musculus) | Yap iEC-KO, Yapfl/fl Pdgfb-iCreERT2 | PMID: 27215660, PMID: 18257043 | ||
| genetic reagent (Mus musculus) | Taz iEC-KO, Tazfl/fl Pdgfb-iCreERT2 | PMID: 27215660, PMID: 18257043 | ||
| genetic reagent (Mus musculus) | YapTaz iEC-KO, Yapfl/fl Tazfl/fl Pdgfb-iCreERT2 | PMID: 27215660, PMID: 18257043 | ||
| genetic reagent (Mus musculus) | Taz iEC-GOF, TAZ S89A EGFP Pdgfb-iCreERT2 | This paper | Cloning information in Material and methods and Figure 7—figure supplement 1 | |
| cell line (human) | HUVEC | PromoCell and Lonza | ||
| transfected construct (human) | VE-Cadherin EGFP | PMID: 24658686 | ||
| transfected construct (human) | VE-Cadherin mEos3.2 | This paper | Cloning information in Material and methods | |
| transfected construct (human) | pCMV-flag S127A YAP | Addgene, plasmid 27370 | ||
| transfected construct (human) | 3xFLAG-pCMV5-TOPO TAZ(S89A) | Addgene, plasmid 24815 | ||
| transfected construct (human) | TEF-1 Luciferase reporter (GTIIC) | PMID: 15628970 | ||
| transfected construct (murine) | RBPj Luciferase reporter | PMID: 7566092 | ||
| transfected construct (murine) | BRE Luciferase reporter | PMID: 11729207 | ||
| transfected construct (human) | FOPflash Luciferase reporter | PMID: 9065401 | ||
| transfected construct (Renilla) | Renilla Luciferase control reporter | Promega, E2241 | ||
| antibody | Yap (rabbit polyclonal) | ThermoFisher Scientific, PA1-461894 | Dilution 1:100 | |
| antibody | Taz (rabbit polyclonal) | Sigma, HPA007415 | Dilution 1:100 | |
| antibody | Erg (goat polyclonal) | Santa Cruz Biotechnology, sc-18136 | Dilution 1:100 | |
| antibody | Erg (rabbit monoclonal) | Abcam, Ab92513 | Dilution 1:1000 | |
| antibody | VE-Cadherin (rat monoclonal) | BD Biosciences, 555289 | Dilution 1:100 | |
| antibody | VE-Cadherin (goat polyclonal) | Santa Cruz Biotechnology, sc-6458 | Dilution 1:100 | |
| antibody | VE-Cadherin 55–7 H1 - Alexa-Fluor 647 Conjugate | BD Biosciences, 561567 | Dilution 1:200 | |
| antibody | TER-119 (rat monoclonal) | R and D Systems, MAB1125 | Dilution 1:100 | |
| antibody | PECAM-1 (goat polyclonal) | R and D Systems, AF3628 | Dilution 1:200 | |
| antibody | Cleaved caspase 3 (rabbit polyclonal) | R and D Systems, AF835 | Dilution 1:200 | |
| antibody | Dll4 (goat polyclonal) | R and D Systems, AF1389 | Dilution 1:100 | |
| antibody | pSMAD1/5/8 (rabbit monoclonal) | Cell Signalling, 13820S | Dilution 1:1000 | |
| antibody | Phalloidin- Alexa-Fluor 488 | ThermoFisher Scientific, A12379 | Dilution 1:100 | |
| antibody | Ib4-Alexa-Fluor 647 Conjugate | ThermoFisher Scientific, I32450 | Dilution 1:1000 | |
| antibody | Ib4-Alexa-Fluor 488 Conjugate | ThermoFisher Scientific, I21411 | Dilution 1:1000 | |
| antibody | Ib4-Alexa-Fluor 568 Conjugate | ThermoFisher Scientific, I21412 | Dilution 1:1000 | |
| antibody | YAP 63.7 (mouse monoclonal) | Santa Cruz Biotechnology, sc-101199 | Dilution 1:1000 | |
| antibody | GAPDH (mouse monoclonal) | Millipore, MAB374 | Dilution 1:4000 | |
| sequence-based reagent | SMART pool: siGENOME siRNA YAP | Dharmacon, M-012200-00-0005 | ||
| sequence-based reagent | SMART pool: siGENOME siRNA TAZ | Dharmacon, M-016083-00-0005 | ||
| sequence-based reagent | SMART pool: siGENOME siRNA VE-Cadherin | Dharmacon, M-003641-01-0005 | ||
| sequence-based reagent | SMART pool: siGENOME siRNA Non targeting 1 | Dharmacon, D001206-13-05 | ||
| sequence-based reagent | Taqman probes for RT-qPCR | Taqman | Supplementary file 3 | |
| commercial assay or kit | Permeability assay - Transwell membranes | Costar, 3460 | ||
| commercial assay or kit | Scratch wound assay - Culture-Insert 2 Well in µ-Dish 35 mm | Ibidi, 81176 | ||
| commercial assay or kit | Click-iT EdU Alexa Fluor 647 Imaging Kit | ThermoFisher Scientific, C10340 | ||
| commercial assay or kit | Propidium Iodide (PI)/ RNase Staining Solution | Cell Signalling, 4087 | ||
| commercial assay or kit | Rneasy Mini Kit | Quiagen, 74104 | ||
| commercial assay or kit | M-MLV reverse transcriptase | ThermoFisher Scientific, 28025013 | ||
| commercial assay or kit | RevertAid First Strand cDNA Synthesis Kit | ThermoFisher Scientific, K1621 | ||
| commercial assay or kit | Agilent RNA 6000 Nano Kit | Agilent, 5067–1511 | ||
| commercial assay or kit | GeneChip Human Gene 2.0 ST Array | ThermoFisher Scientific, 902113 | ||
| chemical compound, drug | 250 kDa FITC Dextran | Sigma, FD250 | ||
| chemical compound, drug | Lipofectamine 2000 | ThermoFisher Scientific, 11668019 | ||
| chemical compound, drug | Dharmafect 1 transfection reagent | Dharmacon, T-2001 | ||
| chemical compound, drug | Polybrene | Santa Cruz, sc-134220 | ||
| chemical compound, drug | Hydroxytamoxifen | Sigma, 7904 | ||
| chemical compound, drug | DBZ | Cayman chemicals 14627 | ||
| chemical compound, drug | Recombinant-hGremlin | R and D Systems, 5190-GR | ||
| chemical compound, drug | Recombinant-hEndoglin | R and D Systems, 1097-EN | ||
| chemical compound, drug | LDN-193189 | Cayman chemicals, 19396 | ||
| chemical compound, drug | K02288 | Cayman chemicals, 16678 | ||
| chemical compound, drug | Recombinant hAlk1fc | R and D Systems, 370-AL-100 | ||
| chemical compound, drug | VEGF-165 (murine) | Prepotech, 450–32 | ||
| software, algorithm | FIJI | FIJI | ||
| software, algorithm | Cytoplasm to nucleus translocation assay | Cell Profiler, adapted from PMID: 17076895 | ||
| software, algorithm | Mouse retina regularity script | This paper | Source code 1 | |
| software, algorithm | VE-Cadherin turnover analysis script | This paper | Source code 2 | |
| software, algorithm | Patching script | This paper | Source code 3 | |
| software, algorithm | Cell coordination analysis script | This paper | Source code 4 | |
| software, algorithm | Dll4 gradient analysis script | This paper | Source code 5 |
Additional files
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Source code 1
Mouse retina regularity script.
Determines the regularity of the gaps in the mouse retina vasculature Used in Figure 2r,K,L. Written in Python.
- https://doi.org/10.7554/eLife.31037.028
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Source code 2
VE-Cadherin turnover analysis script.
Used in Figure 5K,L. Written in Python.
- https://doi.org/10.7554/eLife.31037.029
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Source code 3
Patching script.
Used in Figure 5F,K,L and Figure 8I. Written in Python.
- https://doi.org/10.7554/eLife.31037.030
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Source code 4
Cell coordination analysis script.
Segments images of DAPI stained cell nuclei in a confluent monolayer and assesses the alignment between cells as a function of their distance. Used in Figure 6N,O. Written in Python.
- https://doi.org/10.7554/eLife.31037.031
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Source code 5
Dll4 gradient analysis script.
Analyses Dll4 intensity in the mouse retina as a function of the distance to the sprouting front. Used in Figure 7—figure supplement 4. Written in Python.
- https://doi.org/10.7554/eLife.31037.032
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Supplementary file 1
List of reagents used to manipulate Notch and BMP signaling in cell culture.
- https://doi.org/10.7554/eLife.31037.033
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Supplementary file 2
List of primary antibodies and dyes used.
- https://doi.org/10.7554/eLife.31037.034
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Supplementary file 3
List of the TaqMan primers (Applied Biosystems) used.
- https://doi.org/10.7554/eLife.31037.035
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Transparent reporting form
- https://doi.org/10.7554/eLife.31037.036
