1. Cell Biology
  2. Developmental Biology
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YAP and TAZ regulate adherens junction dynamics and endothelial cell distribution during vascular development

  1. Filipa Neto
  2. Alexandra Klaus-Bergmann
  3. Yu Ting Ong
  4. Silvanus Alt
  5. Anne-Clémence Vion
  6. Anna Szymborska
  7. Joana R Carvalho
  8. Irene Hollfinger
  9. Eireen Bartels-Klein
  10. Claudio A Franco
  11. Michael Potente  Is a corresponding author
  12. Holger Gerhardt  Is a corresponding author
  1. Max-Delbrück-Center for Molecular Medicine, Germany
  2. London Research Institute – Cancer Research UK, United Kingdom
  3. DZHK (German Center for Cardiovascular Research), Germany
  4. Max Planck Institute for Heart and Lung Research, Germany
  5. Faculdade de Medicina da Universidade de Lisboa, Portugal
  6. International Institute of Molecular and Cell Biology, Poland
  7. Vesalius Research Center, Belgium
  8. KU Leuven, Belgium
  9. Berlin Institute of Health, Germany
Research Article
Cite this article as: eLife 2018;7:e31037 doi: 10.7554/eLife.31037
12 figures, 1 table and 9 additional files

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 endothelial membrane marker Isolectin-B4 (IB4; blue) and with antibodies against the endothelial nuclei marker ERG (red). White dotted lines, outline of endothelial nuclei. Yellow dotted lines, outline of perivascular cells’ nuclei. Green arrowheads, nuclear localisation of YAP and TAZ. Red arrowheads, cytoplasmic localisation of YAP and TAZ. Yellow arrowheads, junctional localisation of YAP and TAZ. Images correspond to single confocal planes. n > 3 animals for each staining. Scale bar: 10 μm.

https://doi.org/10.7554/eLife.31037.002
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 TAZ with VE-Cadherin. Images correspond to single confocal planes. n > 3 pups per staining. Scale bar A-C and D-F 10 μm, A’-C’ and D’-F’ 3 μm.

https://doi.org/10.7554/eLife.31037.003
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 radial expansion (G), vessel density (H), branching frequency (I) and area of vessel loops (J) in Yap iEC-KO, Taz iEC-KO and YapTaz iEC-KO. Results are shown as percentage of the respective controls. Data are mean ±SD. n ≥ 5 pups. p values were calculated using unpaired t-test. *p<0.05; **p<0.01; ****p<0.0001. (K, L), Quantification of the standard deviation of the area (K) and circularity (L) of the vessels loops in Yap iEC-KO, Taz iEC-KO and YapTaz iEC-KO retinas. Results are shown as percentage of the respective controls. Data are mean ±SD. n ≥ 5 pups. p values were calculated using unpaired t-test. *p<0.05; **p<0.01; ***p<0.001****p<0.0001.

https://doi.org/10.7554/eLife.31037.004
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 at P1 and P3. At P6, mouse retinas were stained for YAP (grey, B,D), TAZ (grey, F,H), and with Isolectin B4 (IB4; red, A,C,E,G). n > 3 pups. Scale bar: 20 μm.

https://doi.org/10.7554/eLife.31037.005
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 stack. C,D,C‘,D’ correspond to single confocal planes. n > 3 pups. Scale bar: A-D 50 μm, A’-D’ 20 μm.

https://doi.org/10.7554/eLife.31037.006
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 mice (A). A’,B’, mask of Erg +cells indicating endothelial nuclei. (A’’, B’’) mask of Erg + and EdU + cells indicating proliferating endothelial cells. (C) Quantification of endothelial proliferation in Yap iEC-KO (n = 3 control/4 KO pups), Taz iEC-KO (n = 5 control/5 KO pups) and YapTaz iEC-KO (n = 8 control/7 KO pups). Number of EdU-positive and ERG-positive cells per IB4 labelled vascular area was calculated for each genotype and results are shown in percentage of the respective controls. Data are mean ±SD. p values were calculated using unpaired t-test. ns, p>0.05; *p<0.05; **p<0.01. Scale bar: 50 μm. (D, E) P6 retinal vessels labelled with IB4 (grey) and stained for cleaved caspase 3 (red) in YapTaz iEC-KO (E) and littermate control mice (D).D’, E’, magnification of boxed area in D,E. Red arrowheads, cleaved caspase 3 positive endothelial cell. Black arrowheads, cleaved caspase 3 outside vessels. D’’,E’’, mask of cleaved caspase 3 positive endothelial cells. (F) quantification of endothelial apoptosis in Yap iEC-KO (n = 7 control/7 KO pups), Taz iEC-KO (n = 4 control/4KO pups) and YapTaz iEC-KO (n = 5 control/4 KO pups). Data are mean ±SD. p values were calculated using unpaired t-test. ns, p>0.05. Scale bar: D-E 100 μm, D’-E’ 50 μm. (G) Quantification of endothelial proliferation with increasing concentrations of VEGF treatment in YAP, TAZ and YAP/TAZ knockdown cells and control. HUVECs were treated with 0, 40, 200 or 1000 ng/mL VEGF for 24 hr and the percentage of cells in S phase was determined by flow cytometry. Graph shows the mean +SD fold change in percentage of S phase positive cells relative to 0 ng/mL of VEGF treatment. n = 3 independent experiments;>50.000 cells analysed per experiment per condition. (H) Quantification of endothelial proliferation after stretch in in YAP, TAZ, YAP/TAZ and VE-Cadherin knockdown cells and control. HUVECs were subjected to cyclic stretch for 24 hr and percentage of cells in S phase was determined by EdU pulsing and immunofluorescence staining. Graph shows the mean +SD fold change in percentage of S phase positive cells of stretched to non stretched cells for each knockdown condition. n = 5 independent experiments, >100 cells counted per experiment per condition. p values were calculated using unpaired t-test. ns, p>0.05; *p<0.05.

https://doi.org/10.7554/eLife.31037.008
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.

Each value corresponds to the average of several measurements for one animal (see Material and methods for details). Values for quantification of endothelial proliferation in vitro in response to VEGF (Figure 3G) and stretch (Figure 3H) in YAP, TAZ and YAPTAZ knockdown cells.

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 nuclei with DAPI (magenta) 72 hr after siRNA transfection. Scale bar: 10 μm. I, Western blot for YAP/TAZ and GAPDH 72 hr after siRNA transfection.

https://doi.org/10.7554/eLife.31037.009
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) Quantification of nuclear to cytoplasmic ratio of YAP (I) or TAZ (J) with VEGF treatment of one representative experiment. Nuclear/cytoplasmic ratio >1, YAP/TAZ nuclear; nuclear/cytoplasmic ratio <1, YAP/TAZ cytoplasmic. At least 200 cells quantified per condition per experiment. n = 2 independent experiments.

https://doi.org/10.7554/eLife.31037.010
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. A’,B’, magnification of boxed areas in A and B. n = 9 control/9 KO pups. Scale bar: A,B 100 μm, A’, B’ 25 μm. (C, D) P6 retinal vessels labelled with IB4 in YapTaz iEC-KO (D) and littermate control mice (E). Red arrowheads, vessel crosses. (C’, D’) magnification of boxed areas in C,D. C’’,D’’, depiction of vessels in C’ and D‘; different colours represent vessels in different 3D planes. n = 4 control/4 KO pups. Scale bar: C,D 100 μm, C’-D’ 20 μm. (E, F) P6 retinal vessels labelled with IB4 (green) and stained for TER119 (magenta, marking red blood cells) in YapTaz iEC-KO (F) and littermate control mice (E). Red arrowheads, haemorrhages. E’,F’, magnification of boxed areas in E and F. n = 4 control/5 KO pups. Scale bar: E,F 1000 μm, E’, F’ 100 μm. (G, H), P6 retinal vessels labelled with IB4 (green) and stained for VE-Cadherin (magenta) in YapTaz iEC-KO (H) and littermate control mice (G). Red arrowheads, no longitudinal VE-Cadherin labelled junction along vessel axis denoting unicellular vessel segments. (G’,H’, G’’,H’’) magnification of boxed areas in G and H. n = 4 control/4 KO pups. Scale bar: G,H 25 μm, G’,H’ 5 μm, G”,H” 10 μm.

https://doi.org/10.7554/eLife.31037.012
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 using unpaired t-test. *p<0.05.

https://doi.org/10.7554/eLife.31037.013
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 for manual morphological classification of adherens junctions in five categories: straight junctions, thick junctions, thick to reticular junctions, reticular junctions and fingers. (F) Morphological analysis of VE-Cadherin labelled cell junctions in HUVECs knocked down for YAP, TAZ and YAP/TAZ. Data are mean percentage ±SD of 3 independent experiments (two for siTAZ). n > 140 patches of VE-Cadherin stained HUVECs per knockdown condition per experiment. p values were calculated using unpaired t-test between knocked down cells for YAP, TAZ and YAP/TAZ and control. *p<0.05; **p<0.01. (G) Permeability of YAP, TAZ and YAP/TAZ knockdown monolayers of HUVECs to 250 kDa fluorescent dextran molecules. Data are mean +SD of 3 independent experiments. p values were calculated using unpaired t-test between knocked down cells for YAP, TAZ and YAP/TAZ and control. RFU, relative fluorescence units. *p<0.05. (H, I) HUVECs knocked down for YAP/TAZ (I) and control (H) triple labelled with DAPI (blue), pulsed VE-Cadherin 55-7HI (red, VE-Cadherin pulse), and surface VE-Cadherin (green, VE-Cadherin staining). VE-Cadherin 55-7HI pulse was done for 30 min and cells were fixed 2 hr after end of pulse. Scale bar: 20 μm. (J) Representative patches used for manual classification of junctions into high, intermediate and low turnover. (K) Quantification of junctional turnover in YAP/TAZ knockdown cells and control. (L) Quantification of the percentage of high turnover junctions in each morphological category in YAP/TAZ knockdown cells and control. (K, L) Data are mean ±SD of 3 independent experiments. n > 70 patches per knockdown condition per experiment. Fewer then five patches were reticular in YAP/TAZ knockdown, not allowing for reliable assessment of percentages between high, intermediate and low turnover. p values were calculated using unpaired t-test. *p<0.05. (M, N) Fluorescence loss after photoconversion of VE-Cadherin mEos in straight junctions of YAP/TAZ knockdown HUVECs and control HUVECs. M, VE-Cadherin mEos immobile fraction. N, VE-Cadherin mEos half-life of fluorescence loss. Data are mean ±SD of 3 independent experiments. n = 15 control cells and 16 cells YAP/TAZ knockdown cells. p values were calculated using unpaired t-test between knocked down cells for YAP/TAZ and control. **p<0.01. (O, P) HUVECs knocked down for YAP/TAZ (P) and control (O) double stained for VE-Cadherin (red) and f-actin (green, phalloidin). (O’, P’) f-actin (black, phalloidin). Scale bar: 50 μm.

https://doi.org/10.7554/eLife.31037.014
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.

Values for quantification of permeability of YAP, TAZ and YAP/TAZ knockdown monolayers of HUVECs to 250 kDa fluorescent dextran molecules (Figure 5G). Values for quantification of VE-Cadherin mEos immobile fraction (Figure 5M) and half-life of fluorescence loss (Figure 5N).

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 correspond to small junction associated intermediate lamellipodia. Scale bar 20 μm.

https://doi.org/10.7554/eLife.31037.016
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,F,H). (I), Quantification of wound closure at 16 hr. Data are mean ±SD of 3 independent experiments (8–9 biological replicates). p values were calculated using unpaired t-test between knocked down cells for YAP, TAZ or YAP/TAZ and control. ****p<0.0001. (J, K), Phase contrast images of YAP/TAZ knockdown monolayer of HUVECs (K) and control (J). (L, M), Fluorescence labelling of nuclei with DAPI of YAP/TAZ knockdown monolayer of HUVECs (M) and control (L). Scale bar: 100 mm. (L’,M’) Longest axis of nuclei. (N) Alignment score between nuclei pairs used for quantification of cell coordination in O. Angles made by the nuclei longest axis of a pair of nuclei were calculated; angles of 0, 45 and 90 degrees scored 1,0 and −1 in alignment. (O) Coordination plot of monolayers of HUVECs knocked down for YAP, TAZ and YAP and TAZ and control. Graph shows mean alignment score of all pairs of cells in the monolayer plotted against distance between them. Randomly aligned cells score 0 in mean alignment. n = 3 independent experiments,>10.000 pairs of nuclei analysed per knockdown condition per experiment.

https://doi.org/10.7554/eLife.31037.017
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 nuclear EGFP (nEGFP, green) marking cells expressing the TAZ gain of function mutation TAZS89A. Yellow asterisks mark sprouts. Images correspond to maximum projection of z stack. Scale bar: 50 μm. (C) Quantification of number of sprouts per 100 μm of sprouting front extension at P6 in Taz iEC-GOF mice (n = 6 pups) and littermate control mice (n = 6 pups). Data are mean ±SD. p values were calculated using unpaired t-test. **p<0.01. (D) Quantification of branching frequency (i.e. number of branching points per field of view) in Taz iEC-GOF mice (n = 6 pups) and littermate control mice (n = 6 pups). Data are mean ±SD. p values were calculated using unpaired t-test. **p<0.01. (E), Reverse transcriptase PCR of HUVECs transduced with adenoviruses carrying YAP (AdYAP) and TAZ (AdTAZ) constitutively active forms and control (AdGFP). Data are mean ±SD of 3 independent experiments. p values were calculated using unpaired t-test between AdGFP and AdYAP or AdTAZ. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. (F) Reverse transcriptase PCR of YAP/TAZ knockdown HUVECs and control. Data are mean ±SD of 3 independent experiments. p values were calculated using unpaired t-test. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. (G,H), P6 retinal vessels labelled with IB4 (green) and stained for DLL4 (magenta) in YapTaz iEC-KO mice (H) and littermate control mice (G). Images correspond to maximum projection of z stack. Scale bar: 50 μm. (I,J), P6 retinal vessels labelled with IB4 (blue) and stained for ERG (red, marking endothelial nuclei) and pSMAD1/5/8 (green) in YapTaz iEC-KO (J) and littermate control mice (I). Images correspond to single confocal planes. (I’,J’) magnification of boxed areas in I and J. Red arrowheads, endothelial nuclei negative for pSMAD1/5/8. Green arrowheads, endothelial nuclei positive for pSMAD1/5/8. Scale bar: I,J 50 μm, I’, J’ 10 μm. (K) Quantification of endothelial cells positive for pSMAD1/5/8 at the sprouting front of the P6 retina in YapTaz iEC-KO (n = 3 pups) and littermate control mice (n = 3 pups). Data are mean percentage ±SD. p values were calculated using unpaired t-test. ****p<0.0001.

https://doi.org/10.7554/eLife.31037.019
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.

RT-PCR values of YAP and TAZ gain of function (Figure 7E) and loss of function (Figure 7F) HUVECs for Notch and BMP genes. Values for quantification of pSMAD1/5/8 staining in P6 retinas of YapTaz iEC-KO (Figure 7K).

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 (IRES) and a nuclear-localized enhanced green fluorescence protein (nEGFP) for monitoring transgene expression. To allow Cre-dependent expression of 3xFLAG-TAZS89A and of the EGFP reporter, a floxed transcriptional STOP cassette was incorporated between the 3xFLAG-TAZS89A-IRES-nEGFP sequence and the CAG promoter.

https://doi.org/10.7554/eLife.31037.020
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.

https://doi.org/10.7554/eLife.31037.021
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 using unpaired t-test. n ≥ 3 experiments for Notch reporter, three experiments for BMP reporter,≥6 experiments for TEAD reporter. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

https://doi.org/10.7554/eLife.31037.022
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 YapTaz iEC-KO.

https://doi.org/10.7554/eLife.31037.023
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 biological replicates. (B) Quantification of wound closure at 16 hr for HUVECs knocked down for YAP/TAZ and treated with 0.1 μM DBZ or DMSO. Data are mean ±SD. p values were calculated using unpaired t-test. n = 6 biological replicates. (C) Schematic of the BMP inhibitors used depicting preferential sites of inhibition. Alk1fc, ENGecd and Gremlin preferentially bind extracellular BMPs. K02288 and Ldn193189 are kinase inhibitors. (D) Luciferase reporter assay for BMP activity in YAP/TAZ knockdown HUVECs and controls treated with 25 ng/mL Alk1fc, 0.25 μg/mL ENGecd, 0.1 μg/mL Gremlin, 1 μM K02288, 1 μM Ldn193189 and DMSO. Data are mean ±SEM. p values were calculated using unpaired t-test. n ≥ 3 biological replicates. (E) Quantification of wound closure at 16 hr for HUVECs knocked down for YAP/TAZ and treated with 1 μM Ldn193189 or DMSO. Data are mean ±SD. p values were calculated using unpaired t-test. n = 6–7 biological replicates. (F) Permeability of HUVECs knocked down for YAP/TAZ and treated with 1 μM Ldn193189 or DMSO to 250 kDa fluorescent dextran molecules. Data are mean ±SD. RFU, relative fluorescence units. p values were calculated using unpaired t-test. n = 6 biological replicates. (G, H) HUVECs knocked down for YAP/TAZ and treated with 1 μM Ldn193189 (H) or DMSO (G) stained for VE-Cadherin. (G’, H’) different colours mark different cells. Red arrowheads, fingers. Red arrow, reticular junction. Scale bar G, H, 50 μm. Scale bar G’, H’, 10 μm. (I) Morphological analysis of VE-Cadherin labelled cell junctions in HUVECs knocked down for YAP/TAZ and treated with 1 μM Ldn193189 or DMSO control. Data are mean ±SD. p values were calculated using unpaired t-test. n = 3 biological replicates; n ≥ 45 patches of VE-Cadherin stained HUVECs per condition per replicate.

https://doi.org/10.7554/eLife.31037.025
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.

https://doi.org/10.7554/eLife.31037.026
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) and DAPI (not shown). I,J, Quantification of nuclear to cytoplasmic ratio of YAP (I) or TAZ (J) with VEGF treatment. Nuclear/cytoplasmic ratio > 1, YAP/TAZ nuclear; nuclear/cytoplasmic < 1, YAP/TAZ cytoplasmic. At least 200 cells quantified.

https://doi.org/10.7554/eLife.31037.038
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 (B, D, F, H) or vehicle (A, C, E, G). Acid wash treatment (C, D, G, H) removes cell surface antibody revealing internalised VE-cadherin 55/7H1. Scale bar 50μm.

https://doi.org/10.7554/eLife.31037.039
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 5mM EGTA (C, D) or vehicle (A, B). Acid wash treatment was performed prior to fixing cells. Cells were co-stained with the early endosome marker EEA1 (green) and nuclei marker DAPI (blue). Yellow arrowheads, co-localisation of VE-cadherin 55/7H1-A647 with EEA1. Scale bar A-D, 10μm, A’-D’, 5μm.

https://doi.org/10.7554/eLife.31037.040

Tables

Key resources table
Reagent type (species)
or resource
DesignationSource or referenceIdentifiersAdditional information
strain, strain background
(Mus musculus, C57BL/6J)
WTThe 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 paperCloning information in
Material and methods
and Figure 7—figure supplement 1
cell line (human)HUVECPromoCell and Lonza
transfected construct (human)VE-Cadherin EGFPPMID: 24658686
transfected construct (human)VE-Cadherin mEos3.2This paperCloning information in
Material and methods
transfected construct (human)pCMV-flag S127A YAPAddgene, 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 reporterPMID: 7566092
transfected construct (murine)BRE Luciferase reporterPMID: 11729207
transfected construct (human)FOPflash Luciferase
reporter
PMID: 9065401
transfected construct (Renilla)Renilla Luciferase
control reporter
Promega, E2241
antibodyYap (rabbit polyclonal)ThermoFisher Scientific,
PA1-461894
Dilution 1:100
antibodyTaz (rabbit polyclonal)Sigma, HPA007415Dilution 1:100
antibodyErg (goat polyclonal)Santa Cruz Biotechnology,
sc-18136
Dilution 1:100
antibodyErg (rabbit monoclonal)Abcam, Ab92513Dilution 1:1000
antibodyVE-Cadherin
(rat monoclonal)
BD Biosciences, 555289Dilution 1:100
antibodyVE-Cadherin
(goat polyclonal)
Santa Cruz Biotechnology,
sc-6458
Dilution 1:100
antibodyVE-Cadherin 55–7 H1 -
Alexa-Fluor 647 Conjugate
BD Biosciences, 561567Dilution 1:200
antibodyTER-119 (rat monoclonal)R and D Systems, MAB1125Dilution 1:100
antibodyPECAM-1 (goat polyclonal)R and D Systems, AF3628Dilution 1:200
antibodyCleaved caspase 3
(rabbit polyclonal)
R and D Systems, AF835Dilution 1:200
antibodyDll4 (goat polyclonal)R and D Systems, AF1389Dilution 1:100
antibodypSMAD1/5/8
(rabbit monoclonal)
Cell Signalling, 13820SDilution 1:1000
antibodyPhalloidin-
Alexa-Fluor 488
ThermoFisher
Scientific, A12379
Dilution 1:100
antibodyIb4-Alexa-Fluor 647
Conjugate
ThermoFisher
Scientific, I32450
Dilution 1:1000
antibodyIb4-Alexa-Fluor 488
Conjugate
ThermoFisher
Scientific, I21411
Dilution 1:1000
antibodyIb4-Alexa-Fluor 568
Conjugate
ThermoFisher
Scientific, I21412
Dilution 1:1000
antibodyYAP 63.7
(mouse monoclonal)
Santa Cruz Biotechnology,
sc-101199
Dilution 1:1000
antibodyGAPDH
(mouse monoclonal)
Millipore, MAB374Dilution 1:4000
sequence-based reagentSMART pool: siGENOME
siRNA YAP
Dharmacon, M-012200-00-0005
sequence-based reagentSMART pool: siGENOME
siRNA TAZ
Dharmacon, M-016083-00-0005
sequence-based reagentSMART pool: siGENOME
siRNA VE-Cadherin
Dharmacon, M-003641-01-0005
sequence-based reagentSMART pool: siGENOME
siRNA Non targeting 1
Dharmacon, D001206-13-05
sequence-based reagentTaqman probes
for RT-qPCR
TaqmanSupplementary file 3
commercial assay or kitPermeability assay -
Transwell membranes
Costar, 3460
commercial assay or kitScratch wound assay -
Culture-Insert 2
Well in µ-Dish 35 mm
Ibidi, 81176
commercial assay or kitClick-iT EdU Alexa
Fluor 647 Imaging Kit
ThermoFisher Scientific,
C10340
commercial assay or kitPropidium Iodide (PI)/
RNase Staining Solution
Cell Signalling, 4087
commercial assay or kitRneasy Mini KitQuiagen, 74104
commercial assay or kitM-MLV reverse transcriptaseThermoFisher Scientific,
28025013
commercial assay or kitRevertAid First Strand
cDNA Synthesis Kit
ThermoFisher Scientific,
K1621
commercial assay or kitAgilent RNA 6000 Nano KitAgilent, 5067–1511
commercial assay or kitGeneChip Human
Gene 2.0 ST Array
ThermoFisher Scientific,
902113
chemical compound, drug250 kDa FITC DextranSigma, FD250
chemical compound, drugLipofectamine 2000ThermoFisher Scientific,
11668019
chemical compound, drugDharmafect 1
transfection reagent
Dharmacon, T-2001
chemical compound, drugPolybreneSanta Cruz, sc-134220
chemical compound, drugHydroxytamoxifenSigma, 7904
chemical compound, drugDBZCayman chemicals 14627
chemical compound, drugRecombinant-hGremlinR and D Systems, 5190-GR
chemical compound, drugRecombinant-hEndoglinR and D Systems, 1097-EN
chemical compound, drugLDN-193189Cayman chemicals, 19396
chemical compound, drugK02288Cayman chemicals, 16678
chemical compound, drugRecombinant hAlk1fcR and D Systems,
370-AL-100
chemical compound, drugVEGF-165 (murine)Prepotech, 450–32
software, algorithmFIJIFIJI
software, algorithmCytoplasm to nucleus
translocation assay
Cell Profiler, adapted
from PMID: 17076895
software, algorithmMouse retina
regularity script
This paperSource code 1
software, algorithmVE-Cadherin turnover
analysis script
This paperSource code 2
software, algorithmPatching scriptThis paperSource code 3
software, algorithmCell coordination
analysis script
This paperSource code 4
software, algorithmDll4 gradient
analysis script
This paperSource code 5

Additional files

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
Source code 2

VE-Cadherin turnover analysis script.

Used in Figure 5K,L. Written in Python.

https://doi.org/10.7554/eLife.31037.029
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
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
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
Supplementary file 1

List of reagents used to manipulate Notch and BMP signaling in cell culture.

https://doi.org/10.7554/eLife.31037.033
Supplementary file 2

List of primary antibodies and dyes used.

https://doi.org/10.7554/eLife.31037.034
Supplementary file 3

List of the TaqMan primers (Applied Biosystems) used.

https://doi.org/10.7554/eLife.31037.035
Transparent reporting form
https://doi.org/10.7554/eLife.31037.036

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