EphrinB2-EphB4 signalling provides Rho-mediated homeostatic control of lymphatic endothelial cell junction integrity
- Uppsala University, Department of Immunology, Genetics and Pathology, Sweden
- University Medical Center Hamburg-Eppendorf, Institute of Clinical Chemistry and Laboratory Medicine, Germany
- Biocenter Oulu, University of Oulu, Finland
- Lymphatic Development Laboratory, Cancer Research UK London Research Institute, United Kingdom
- Discovery Biology, Discovery Sciences, Biopharmaceuticals R&D, AstraZeneca, Sweden
- Oulu Centre for Cell-Matrix Research, Faculty of Biochemistry and Molecular Medicine, University of Oulu, Finland
- Crystallography and Protein Engineering Unit, Spanish National Cancer Research Centre (CNIO), Spain
- Max Planck Institute for Molecular Biomedicine, Germany
Figures
Figure 1 with 1 supplement
Endothelial deletion of Ephb4 or Efnb2 selectively disrupts dermal collecting lymphatic vessels.
(A) Experimental scheme for Ephb4 deletion in the mature vasculature by three consecutive intraperitoneal (i.p.) 4-OHT injections (arrowheads) into 3-week-old Ephb4flox;R26-mTmG;Pdgfb-CreERT2 mice. …
-
Figure 1—source data 1
Flow cytometric analysis of endothelial cell proliferation in postnatal mouse ear skin.
- https://cdn.elifesciences.org/articles/57732/elife-57732-fig1-data1-v1.xlsx
Figure 1—figure supplement 1
Pdgfb-CreERT2-mediated deletion of Efnb2 in mature collecting vessels leads to defective cell-cell junctions in lymphatic capillaries.
(A) Flow cytometric analysis of dermal BEC (PECAM1+PDPN-) and LEC (PECAM1+PDPN+) proliferation in the ear skin of 3 day (n = 5), 10 day (n = 3), 3-week-old (n = 6) and 5-week-old (n = 4) mice. Data …
-
Figure 1—figure supplement 1—source data 1
Measurement of blood vessel permeability in Efnb2 mutants and control littermates.
- https://cdn.elifesciences.org/articles/57732/elife-57732-fig1-figsupp1-data1-v1.xlsx
Figure 2 with 3 supplements
Lymphatic endothelial specific deletion of Efnb2 or Ephb4 disrupts endothelial cell-cell junctions in several organs.
(A) Experimental scheme for LEC-specific deletion of Efnb2 and Ephb4 in the remodeling lymphatic vasculature (LV) using the Prox1-CreERT2 line and a single 4-OHT treatment at P12 (arrowhead). Ear …
-
Figure 2—source data 1
Quantification of LEC junction parameters in the skin.
- https://cdn.elifesciences.org/articles/57732/elife-57732-fig2-data1-v1.xlsx
Figure 2—figure supplement 1
Efnb2 deletion in 4-week-old and 18-week-old mice.
(A) Relative mRNA expression levels of Efnb2 in freshly isolated LECs from 4-week-old mutant (n = 3) and heterozygote/wildtype control (n = 4) ear skin. (B) Experimental scheme for LEC-specific …
-
Figure 2—figure supplement 1—source data 1
qRT-PCR analysis of Efnb2 expression in FACS sorted dermal LECs.
- https://cdn.elifesciences.org/articles/57732/elife-57732-fig2-figsupp1-data1-v1.xlsx
Figure 2—figure supplement 2
Dysfunctional lymphatic valves and chylothorax in Efnb2 and Ephb4 mutant mice.
Whole-mount immunofluorescence of P11 mesenteric collecting vessels (on the left) and incidence of chylotorax (on the right) in Ephb4flox;Prox1-CreERT2 (A) and Efnb2flox;Prox1-CreERT2 (B) mice …
-
Figure 2—figure supplement 2—source data 1
Incidence of chylothorax in Ephb4 and Efnb2 mutant mice.
- https://cdn.elifesciences.org/articles/57732/elife-57732-fig2-figsupp2-data1-v1.xlsx
Figure 2—figure supplement 3
Abnormal morphology of LEC junctions in the inguinal lymph node capsule of Efnb2 mutant mice.
Quantification of lymphatic junctional morphology in CLDN5-stained lymph nodes upon Efnb2 deletion. Junctional categories are defined as 1. linear/thick/reticular, 2. jagged and 3. discontinuous …
-
Figure 2—figure supplement 3—source data 1
Quantification of LEC junction morphology in the inguinal lymph node capsule.
- https://cdn.elifesciences.org/articles/57732/elife-57732-fig2-figsupp3-data1-v1.xlsx
Figure 3 with 1 supplement
Lymphatic valve-specific deletion of Efnb2 leads to valve defects but unaltered LEC junctions on the collecting vessel wall.
(A) Whole-mount immunofluorescence of mesenteric lymphatic vessels of P11 Efnb2GFP mice using antibodies against GFP (green) and FOXC2 (red). Enlarged view of the boxed area with single channel …
-
Figure 3—source data 1
Quantification of LEC junction morphology in the mesentery.
- https://cdn.elifesciences.org/articles/57732/elife-57732-fig3-data1-v1.xlsx
Figure 3—figure supplement 1
Valve-specific deletion of Efnb2 using the Cldn11-CreERT2 line.
(A) Whole mount immunofluorescence of mesentery of a R26-mTmG;Cldn11-CreERT2 mesentery showing GFP expression (Cre-mediated recombination) in lymphatic valves. Boxed area of the PECAM1-stained …
-
Figure 3—figure supplement 1—source data 1
Quantification of valve phenotype upon valve-specific deletion of Efnb2.
- https://cdn.elifesciences.org/articles/57732/elife-57732-fig3-figsupp1-data1-v1.xlsx
Figure 4 with 4 supplements
Loss of junctional CLDN5 occurs after EphrinB2 blockade in vitro but does not lead to breakdown of LEC junctions in vivo.
(A) Top: Western blot of EphrinB2-Fc-precipitated EphB4 HDLECs for phosphotyrosine (4G10) and EphB4. Cells were treated with control (siCTRL) or EphrinB2 siRNA (siEFNB2), and stimulated with …
-
Figure 4—source data 1
Quantification of the effects of EphrinB2 inhibition on EphB4 phosphorylation, endothelial monolayer permeability and junctional CLDN5.
- https://cdn.elifesciences.org/articles/57732/elife-57732-fig4-data1-v1.xlsx
Figure 4—figure supplement 1
Loss of junctional CLDN5 after long-term EphrinB2 inhibition in primary LECs.
Immunofluorescence of HDLECs treated with EphrinB2 blocking antibody (B11) for 16 hr using antibodies against VE-cadherin (red) and CLDN5 (green). Single channel images are depicted in grey. Note …
Figure 4—figure supplement 2
The effect EFNB2 silencing on CLDN5 and VE-cadherin.
(A) Western blot analysis of total cell lysates (TCL) of HDLECs treated for 48 hr with control (siCTRL) or EFNB2 siRNA (siEFNB2). Note reduced CLDN5 but unchanged VE-cadherin levels in EFNB2-silenced…
Figure 4—figure supplement 3
Silencing of CDH5 and CLDN5 in HDLECs.
Western blot analysis of total cell lysates (TCL) of HDLECs treated for 72 hr with control (siCTRL), CDH5 siRNA (siCDH5) and CLDN5 siRNA (siCLDN5). Western blot is shown as representative of 2 …
Figure 4—figure supplement 4
Generation of conditional Cldn5 knock-out mice.
(A) Schematic of the Cldn5 wild type allele, targeted ‘Knockout-First’ allele, conditional allele (floxed) and deletion allele. The ‘Knockout-First’ allele Cldn5tm1a(EUCOMM)Wtsi contains an …
Figure 5
Basal EphrinB2/EphB4 signalling controls the stability of LEC junctions through regulation of Rho-mediated cytoskeletal contractility.
Immunofluorescence of HDLECs for VE-cadherin (red) and phalloidin (green), showing increase in actin stress fibres after 3 h-treatment with the EphrinB2 blocking antibody (B11) compared to untreated …
-
Figure 5—source data 1
Quantification of the effect of EphrinB2 blockade on the actin cytoskeleton and Rac1 activity.
- https://cdn.elifesciences.org/articles/57732/elife-57732-fig5-data1-v1.xlsx
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Genetic reagent (Mus musculus) | Efnb2flox | Grunwald et al., 2004 | Efnb2tm4Kln; RRDI: MGI:2182626 | |
| Genetic reagent (Mus musculus) | Ephb4flox | Martin-Almedina et al., 2016 | ||
| Genetic reagent (Mus musculus) | Pdgfb-iCreERT2iresGFP | Claxton et al., 2008 | Tg(Pdgfb-icre/ERT2)1Frut; RRDI: MGI:3793852 | |
| Genetic reagent (Mus musculus) | R26-mTmG | Muzumdar et al., 2007 | Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP)Luo; RRDI: MGI:3716464 | |
| Genetic reagent (Mus musculus) | Prox1-CreERT2 | Bazigou et al., 2011 | Tg(Prox1-cre/ERT2)1Tmak; RRDI: MGI:5617984 | |
| Genetic reagent (Mus musculus) | Efnb2GFP | Davy and Soriano, 2007 | Efnb2tm2Sor; RRDI: MGI:3526818 | |
| Genetic reagent (Mus musculus) | Cldn11-CreERT2 | This paper | H Ortsäter and T Mäkinen, manuscript in preparation. | |
| Genetic reagent (Mus musculus) | Cldn5flox | This paper | Cldn5tm1a(EUCOMM)Wtsi; MGI:5473167 | Obtained from The European Conditional Mouse Mutagenesis Program (EUCOMM). |
| Cell line (Homo sapiens) | Dermal lymphatic endothelial cell (normal, juvenile, male) | PromoCell | Cat# C12216 | Primary cell line isolated from foreskin, tested negative for mycoplasma contamination |
| Antibody | anti-EphrinB2 (Human single chain variable fragment) | Abéngozar et al., 2012 | B11 | (80 μg/ml) |
| Antibody | anti-human IgG (Goat polyclonal) | Jackson ImmunoResearch | Cat# 109-005-098 | (5 μg/ml) |
| Antibody | anti-human/mouse/rat EphrinB2 (Goat polyclonal) | R and D Systems | Cat# AF496 | (0.5 μg/ml) |
| Antibody | anti-human EphB4 (Goat polyclonal) | R and D Systems | Cat# AF3038 | (0.5 μg/ml) |
| Antibody | anti-human VE-cadherin (Goat polyclonal) | Santa Cruz Biotechnology | C19, Cat# sc-6458 | (IF: 0.2 μg/ml; WB 2 μg/ml) |
| Antibody | anti-human/mouse CLDN5 (Rabbit polyclonal) | Invitrogen | Cat# 34–1600 | (0.5 μg/ml) |
| Antibody | anti-phosphotyrosine 4G10 (Mouse monoclonal) | Merck | Cat# 05–321 | (0.5 μg/ml) |
| Antibody | anti-β-actin (Rabbit polyclonal) | Cell Signalling Technologies | Cat# 4967 | (0.1 μg/ml) |
| Antibody | anti-mouse Podoplanin (Syrian hamster monoclonal) | eBioscience | eBio8.1.1, PE; Cat# 12-5381-82; RRDI:AB_1907439 | (2 µg/ml) |
| Antibody | anti-mouse CD31 (Rat monoclonal) | eBioscience | 390, PE-Cy7, Cat# A14715; RRDI:AB_2534231 | (0.7 µg/ml) |
| Antibody | anti-mouse CD45 (Rat monoclonal) | eBioscience | 30-F11, PerCP-Cyanine5.5; Cat# 5-0451-82, RRDI:AB_1107002 or eFluor450; Cat# 48-0451-82; RRDI:AB_1518806 | (4 µg/ml) |
| Antibody | anti-mouse CD11b (Rat monoclonal) | eBioscience | M1/70, PerCP-Cyanine5.5; Cat# 45-0112-82; RRDI:AB_953558 or eFluor450; Cat# 48-0112-82; RRDI:AB_1582236 | (4 µg/ml) |
| Antibody | anti-TER-119 (Rat monoclonal) | eBioscience | TER-119, eFluor450; Cat# 48-5921-82; RRDI:AB_1518808 | (4 µg/ml) |
| Antibody | anti-Ki-67 (Rat monoclonal) | eBioscience | SolA15, eFluor 660; Cat# 50-5698-82; RRDI:AB_2574235 | (1:100) |
| Antibody | anti-human VE-cadherin (Mouse monoclonal) | Santa Cruz Biotechnology | F-8, Cat# sc-9989 | (2 μg/ml) |
| Antibody | anti-mouse LYVE1 (Rat monoclonal) | R and D Systems | Cat# MAB2125 | (1 μg/ml) |
| Antibody | anti-GFP (Rabbit polyclonal) | Abcam | Cat# ab290 | (1 μg/ml) |
| Antibody | anti-mouse FoxC2 (Sheep polyclonal) | R and D Systems | Cat# AF6989 | (2 μg/ml) |
| Sequence-based reagent | TaqMan probe: Gapdh | ThermoFisher Scientific | Cat# Mm_03302249_g1 | |
| Sequence-based reagent | TaqMan probe: Efnb2 | ThermoFisher Scientific | Cat# Mm00438670_m1 | |
| Sequence-based reagent | siRNA: human CLDN5 | Dharmacon | Cat# D-011409-03-0010 | |
| Sequence-based reagent | siRNA: human CDH5 | Dharmacon | Cat# D-003641-03-0010 | |
| Sequence-based reagent | siRNA: human EFNB2 | Dharmacon | Cat# L-003659-00-0005 | |
| Sequence-based reagent | siRNA: negative control, human | Qiagen | Cat# 1027281 | |
| Peptide, recombinant protein | Human EphrinB2-Fc | R and D Systems | Cat# 7397-EB | (0.5 μg/ml: activation assay, 3 μg: IP) |
| Chemical compound, drug | ROCK inhibitor (Y-27632) | Sigma | Cat# SCM075 | (10 µM) |
| Commercial assay or kit | Rac1-GTP pulldown assay | Cytoskeleton Inc | Cat# BK030 |
