EphB4 forward signalling regulates lymphatic valve development
Identifieur interne : 000926 ( Pmc/Curation ); précédent : 000925; suivant : 000927EphB4 forward signalling regulates lymphatic valve development
Auteurs : Gu Zhang [États-Unis] ; John Brady [États-Unis] ; Wei-Ching Liang [États-Unis] ; Yan Wu [États-Unis] ; Mark Henkemeyer [États-Unis] ; Minhong Yan [États-Unis]Source :
- Nature Communications [ 2041-1723 ] ; 2015.
Abstract
Bidirectional signalling is regarded as a notable hallmark of the Eph-ephrin signalling system: Eph-dependent forward signalling in Eph-expressing cells and ephrin-dependent reverse signalling in Ephrin-expressing cells. The notion of ephrin-dependent reverse signalling derives from genetic experiments utilizing mice carrying mutations in the intracellular region of ephrinBs. Here we show that EphB4-dependent forward signalling regulates lymphatic valve development, a process previously thought to be regulated by ephrinB2-dependent reverse signalling. We develop antibodies that selectively target EphB4 and ephrinB2. We find that mice bearing genetically altered cytoplasmic region of ephrinB2 have significantly altered EphB4-dependent forward signalling. Selective inhibition of EphB4 using a functional blocking antibody results in defective lymphatic valve development. Furthermore, a chemical genetic approach is used to unequivocally show that the kinase activity of EphB4 is essential for lymphatic valve development.
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DOI: 10.1038/ncomms7625
PubMed: 25865237
PubMed Central: 4403310
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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">EphB4 forward signalling regulates lymphatic valve development</title><author><name sortKey="Zhang, Gu" sort="Zhang, Gu" uniqKey="Zhang G" first="Gu" last="Zhang">Gu Zhang</name><affiliation wicri:level="1"><nlm:aff id="a1"><institution>Department of Molecular Oncology, Division of Research, Genentech Inc.</institution>, 1 DNA Way, South San Francisco, CA 94080,<country>USA</country></nlm:aff><country xml:lang="fr">États-Unis</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Brady, John" sort="Brady, John" uniqKey="Brady J" first="John" last="Brady">John Brady</name><affiliation wicri:level="1"><nlm:aff id="a1"><institution>Department of Molecular Oncology, Division of Research, Genentech Inc.</institution>, 1 DNA Way, South San Francisco, CA 94080,<country>USA</country></nlm:aff><country xml:lang="fr">États-Unis</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Liang, Wei Ching" sort="Liang, Wei Ching" uniqKey="Liang W" first="Wei-Ching" last="Liang">Wei-Ching Liang</name><affiliation wicri:level="1"><nlm:aff id="a2"><institution>Department of Antibody Engineering, Division of Research, Genentech Inc.</institution>, 1 DNA Way, South San Francisco, California 94080,<country>USA</country></nlm:aff><country xml:lang="fr">États-Unis</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Wu, Yan" sort="Wu, Yan" uniqKey="Wu Y" first="Yan" last="Wu">Yan Wu</name><affiliation wicri:level="1"><nlm:aff id="a2"><institution>Department of Antibody Engineering, Division of Research, Genentech Inc.</institution>, 1 DNA Way, South San Francisco, California 94080,<country>USA</country></nlm:aff><country xml:lang="fr">États-Unis</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Henkemeyer, Mark" sort="Henkemeyer, Mark" uniqKey="Henkemeyer M" first="Mark" last="Henkemeyer">Mark Henkemeyer</name><affiliation wicri:level="1"><nlm:aff id="a3"><institution>Department of Developmental Biology, University of Texas Southwestern Medical Center</institution>, Dallas, Texas 75390,<country>USA</country></nlm:aff><country xml:lang="fr">États-Unis</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Yan, Minhong" sort="Yan, Minhong" uniqKey="Yan M" first="Minhong" last="Yan">Minhong Yan</name><affiliation wicri:level="1"><nlm:aff id="a1"><institution>Department of Molecular Oncology, Division of Research, Genentech Inc.</institution>, 1 DNA Way, South San Francisco, CA 94080,<country>USA</country></nlm:aff><country xml:lang="fr">États-Unis</country><wicri:regionArea># see nlm:aff country 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Oncology, Division of Research, Genentech Inc.</institution>, 1 DNA Way, South San Francisco, CA 94080,<country>USA</country></nlm:aff><country xml:lang="fr">États-Unis</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Brady, John" sort="Brady, John" uniqKey="Brady J" first="John" last="Brady">John Brady</name><affiliation wicri:level="1"><nlm:aff id="a1"><institution>Department of Molecular Oncology, Division of Research, Genentech Inc.</institution>, 1 DNA Way, South San Francisco, CA 94080,<country>USA</country></nlm:aff><country xml:lang="fr">États-Unis</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Liang, Wei Ching" sort="Liang, Wei Ching" uniqKey="Liang W" first="Wei-Ching" last="Liang">Wei-Ching Liang</name><affiliation wicri:level="1"><nlm:aff id="a2"><institution>Department of Antibody Engineering, Division of Research, Genentech Inc.</institution>, 1 DNA Way, South San Francisco, California 94080,<country>USA</country></nlm:aff><country xml:lang="fr">États-Unis</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Wu, Yan" sort="Wu, Yan" uniqKey="Wu Y" first="Yan" last="Wu">Yan Wu</name><affiliation wicri:level="1"><nlm:aff id="a2"><institution>Department of Antibody Engineering, Division of Research, Genentech Inc.</institution>, 1 DNA Way, South San Francisco, California 94080,<country>USA</country></nlm:aff><country xml:lang="fr">États-Unis</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Henkemeyer, Mark" sort="Henkemeyer, Mark" uniqKey="Henkemeyer M" first="Mark" last="Henkemeyer">Mark Henkemeyer</name><affiliation wicri:level="1"><nlm:aff id="a3"><institution>Department of Developmental Biology, University of Texas Southwestern Medical Center</institution>, Dallas, Texas 75390,<country>USA</country></nlm:aff><country xml:lang="fr">États-Unis</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Yan, Minhong" sort="Yan, Minhong" uniqKey="Yan M" first="Minhong" last="Yan">Minhong Yan</name><affiliation wicri:level="1"><nlm:aff id="a1"><institution>Department of Molecular Oncology, Division of Research, Genentech Inc.</institution>, 1 DNA Way, South San Francisco, CA 94080,<country>USA</country></nlm:aff><country xml:lang="fr">États-Unis</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author></analytic><series><title level="j">Nature Communications</title><idno type="eISSN">2041-1723</idno><imprint><date when="2015">2015</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p>Bidirectional signalling is regarded as a notable hallmark of the Eph-ephrin signalling system: Eph-dependent forward signalling in Eph-expressing cells and ephrin-dependent reverse signalling in Ephrin-expressing cells. The notion of ephrin-dependent reverse signalling derives from genetic experiments utilizing mice carrying mutations in the intracellular region of ephrinBs. Here we show that EphB4-dependent forward signalling regulates lymphatic valve development, a process previously thought to be regulated by ephrinB2-dependent reverse signalling. We develop antibodies that selectively target EphB4 and ephrinB2. We find that mice bearing genetically altered cytoplasmic region of ephrinB2 have significantly altered EphB4-dependent forward signalling. Selective inhibition of EphB4 using a functional blocking antibody results in defective lymphatic valve development. Furthermore, a chemical genetic approach is used to unequivocally show that the kinase activity of EphB4 is essential for lymphatic valve development.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Alitalo, K" uniqKey="Alitalo K">K. 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<front><journal-meta><journal-id journal-id-type="nlm-ta">Nat Commun</journal-id><journal-id journal-id-type="iso-abbrev">Nat Commun</journal-id><journal-title-group><journal-title>Nature Communications</journal-title></journal-title-group><issn pub-type="epub">2041-1723</issn><publisher><publisher-name>Nature Pub. Group</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">25865237</article-id><article-id pub-id-type="pmc">4403310</article-id><article-id pub-id-type="pii">ncomms7625</article-id><article-id pub-id-type="doi">10.1038/ncomms7625</article-id><article-categories><subj-group subj-group-type="heading"><subject>Article</subject></subj-group></article-categories><title-group><article-title>EphB4 forward signalling regulates lymphatic valve development</article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Zhang</surname><given-names>Gu</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Brady</surname><given-names>John</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Liang</surname><given-names>Wei-Ching</given-names></name><xref ref-type="aff" rid="a2">2</xref></contrib><contrib contrib-type="author"><name><surname>Wu</surname><given-names>Yan</given-names></name><xref ref-type="aff" rid="a2">2</xref></contrib><contrib contrib-type="author"><name><surname>Henkemeyer</surname><given-names>Mark</given-names></name><xref ref-type="aff" rid="a3">3</xref></contrib><contrib contrib-type="author"><name><surname>Yan</surname><given-names>Minhong</given-names></name><xref ref-type="corresp" rid="c1">a</xref><xref ref-type="aff" rid="a1">1</xref></contrib><aff id="a1"><label>1</label><institution>Department of Molecular Oncology, Division of Research, Genentech Inc.</institution>, 1 DNA Way, South San Francisco, CA 94080,<country>USA</country></aff><aff id="a2"><label>2</label><institution>Department of Antibody Engineering, Division of Research, Genentech Inc.</institution>, 1 DNA Way, South San Francisco, California 94080,<country>USA</country></aff><aff id="a3"><label>3</label><institution>Department of Developmental Biology, University of Texas Southwestern Medical Center</institution>, Dallas, Texas 75390,<country>USA</country></aff></contrib-group><author-notes><corresp id="c1"><label>a</label><email>minhong@gene.com</email></corresp></author-notes><pub-date pub-type="epub"><day>13</day><month>04</month><year>2015</year></pub-date><volume>6</volume><elocation-id>6625</elocation-id><history><date date-type="received"><day>05</day><month>08</month><year>2014</year></date><date date-type="accepted"><day>12</day><month>02</month><year>2015</year></date></history><permissions><copyright-statement>Copyright © 2015, Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved.</copyright-statement><copyright-year>2015</copyright-year><copyright-holder>Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved.</copyright-holder><license license-type="open-access" xlink:href="http://creativecommons.org/licenses/by/4.0/"><pmc-comment>author-paid</pmc-comment>
<license-p>This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit <ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by/4.0/">http://creativecommons.org/licenses/by/4.0/</ext-link></license-p></license></permissions><abstract><p>Bidirectional signalling is regarded as a notable hallmark of the Eph-ephrin signalling system: Eph-dependent forward signalling in Eph-expressing cells and ephrin-dependent reverse signalling in Ephrin-expressing cells. The notion of ephrin-dependent reverse signalling derives from genetic experiments utilizing mice carrying mutations in the intracellular region of ephrinBs. Here we show that EphB4-dependent forward signalling regulates lymphatic valve development, a process previously thought to be regulated by ephrinB2-dependent reverse signalling. We develop antibodies that selectively target EphB4 and ephrinB2. We find that mice bearing genetically altered cytoplasmic region of ephrinB2 have significantly altered EphB4-dependent forward signalling. Selective inhibition of EphB4 using a functional blocking antibody results in defective lymphatic valve development. Furthermore, a chemical genetic approach is used to unequivocally show that the kinase activity of EphB4 is essential for lymphatic valve development.</p></abstract><abstract abstract-type="web-summary"><p><inline-graphic id="i1" xlink:href="ncomms7625-i1.jpg"></inline-graphic>The bidirectional Eph-ephrin signalling regulates a myriad of developmental programmes. Zhang <italic>et al</italic>. show that EphB4 forward signalling is crucial for lymphatic valve development, providing new insight into this important developmental process previously thought to be regulated by ephrinB2-dependent reverse signalling.</p></abstract></article-meta></front><floats-group><fig id="f1"><label>Figure 1</label><caption><title>Characterization of anti-ephrinB2 and anti-EphB4 antibodies.</title><p>(<bold>a</bold>) Biotinylated α-EphB4 selectively binds to EphB4-Fc, but not other EphB proteins (left); and biotinylated α-ephrinB2 selectively binds to ephrinB2-Fc but not to other ephrinB proteins (right). (<bold>b</bold>) Blocking activity of α-ephrinB2 measured by western blot (WB) analysis of EphB4 phosphorylation in HUVECs stimulated by overlaid ephrinB2-expressing 3T3 cells. (<bold>c</bold>) Agonistic activity of α-EphB4 measured by WB (left) and ELISA (right) of EphB4 phosphorylation in EphB4-expressing 3T3 cells treated with ephrinB2-Fc or α-EphB4. Dotted line indicates a cropped lane (full WB data in <xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 11</xref>). (<bold>d</bold>) Antagonistic activity of α-EphB4 Fab measured by ELISA of EphB4 phosphorylation in EphB4-expressing 3T3 cells. (<bold>a</bold>,<bold>c</bold>,<bold>d</bold>) Error bars, s.d. of technical triplicates. Ctrl, control; IP, immunoprecipitation.</p></caption><graphic xlink:href="ncomms7625-f1"></graphic></fig><fig id="f2"><label>Figure 2</label><caption><title>Anti-ephrinB2 causes dramatic lymphatic defects in neonatal mice.</title><p>Treatment was started at P1 and mice were examined on P7. Scale bar, 1 mm. (<bold>a</bold>) Chylothorax in α-ephrinB2-treated mice. Exterior (<bold>b</bold>) and interior (<bold>c</bold>) views of the leg skins following injection of FITC-dextran into the hindlimb footpad. The main lymphatic collecting vessels are marked (arrowheads). Anti-ephrinB2 (α-ephrinB2)-treated animals exhibited abnormal outflow of FITC-dextran from collecting vessels to the pre-collector vessel branches. (<bold>d</bold>) Collecting vessels running between the lumbar (LLN) lymph nodes and renal lymph nodes (RLN). In control (Ctrl) mice, only a pair of collecting lymphatic vessel trunks was highlighted by FITC-dextran (left). In anti -ephrinB2-treated animals (right), FITC-dextran visibly diffused into the surrounding lymphatic network.</p></caption><graphic xlink:href="ncomms7625-f2"></graphic></fig><fig id="f3"><label>Figure 3</label><caption><title>Effect of anti-ephrinB2 and anti-EphB4 antibodies on lymphatic valves.</title><p>Visualization of lymphatic vessels and valves (arrows, not all valves are marked) by footpad injection of FITC-lectin following antibody administration from P1. (<bold>a</bold>) Defective lymphatic valves with ring-like appearance (arrow heads) in P5 leg skins of mice treated with function-blocking anti-ephrinB2 (α-ephrinB2) antibody. Right panel, quantification of lymphatic valves. (<bold>b</bold>) Higher magnification view of defective lymphatic valves in P5 leg skins of mice treated with function-blocking α-ephrinB2 antibody. (<bold>c</bold>) Agonistic anti-EphB4 (α-EphB4) reverses lymphatic valve defects caused by function-blocking α-ephrinB2. P6 leg skins of neonatal mice are shown. Lower panel, quantification of lymphatic valves. (<bold>d</bold>) Abnormal lymphatic valves with ring-like appearance (arrow heads) in P7 leg skins of mice treated with antagonistic anti-EphB4 Fab (α-EphB4 Fab). Lower panel, quantification of lymphatic valves. Scale bars: (<bold>a</bold>), (<bold>c</bold>) and (<bold>d</bold>), 500 μm; (<bold>b</bold>), 100 μm. ****<italic>P</italic>< 0.0001, ***<italic>P</italic>< 0.001, **<italic>P</italic>< 0.01 (two-tailed, unpaired Student's <italic>t</italic>-test), <italic>n</italic>=3 per treatment group (error bars, s.d.). NS, not significant.</p></caption><graphic xlink:href="ncomms7625-f3"></graphic></fig><fig id="f4"><label>Figure 4</label><caption><title>EphB4 activation rescues lymphatic valve defects in <italic>ephrinB2</italic> mutants.</title><p>(<bold>a</bold>) Schematic representation of ephrinB2 mutants. (<bold>b</bold>,<bold>d</bold>,<bold>e</bold>) Visualization of mesenteric lymphatic vessels and valves (arrows) by immuno-staining for Prox-1. Strong α-smooth muscle actin (αSMA) staining highlights blood vessels. Scale bar, 500 μm. (<bold>b</bold>) Defective mesenteric lymphatic valve development in E18 <italic>ephrinB2</italic><sup>6YFΔV/6YFΔV</sup> embryos. (<bold>c</bold>) Compromised EphB4 activation in <italic>ephrinB2</italic><sup>6YFΔV/6YFΔV</sup> embryos. Total tissue lysates from E12.5 embryos were subjected to phospho-EphB4 (pEphB4) and total-EphB4 immunoblotting analysis. Ratios of pEphB4:EphB4 are graphed (right panel). (<bold>d</bold>) <italic>In utero</italic> treatment with agonist anti-EphB4 (α-EphB4) restores lymphatic valves in the mesentery of P0 <italic>ephrinB2</italic><sup>6YFΔV/6YFΔV</sup> mice. Right panel, quantification of mesenteric lymphatic valves, <italic>n</italic>=3 per genotype. (<bold>e</bold>) Normal mesenteric lymphatic valve development in <italic>ephrinB2</italic><sup><italic>lac</italic>Z/6YFΔV</sup> neonatal mice (P3). Right panel, quantification of mesenteric lymphatic valves, <italic>n</italic>=3 per genotype. Statistical analysis in (<bold>c</bold>–<bold>e</bold>), two-tailed, unpaired Student's <italic>t</italic>-test, error bars, s.d. NS, not significant.</p></caption><graphic xlink:href="ncomms7625-f4"></graphic></fig><fig id="f5"><label>Figure 5</label><caption><title>Lymphatic valves are present in <italic>ephrinB2</italic><sup><italic>lac</italic>Z/<italic>lacZ</italic></sup> mice.</title><p>(<bold>a</bold>) Visualization of mesenteric lymphatic vessels and valves by immunostaining for Prox-1 in E18 embryos. Scale bar, 500 μm. Right panel, quantification of lymphatic valves, two-tailed, unpaired student's <italic>t</italic>-test, <italic>n</italic>=3 per genotype (error bars, s.d.). (<bold>b</bold>) Elevated EphB4 phosphorylaiton in <italic>ephrinB2</italic><sup><italic>lac</italic>Z/<italic>lacZ</italic></sup> embryos. Total tissue lysates from E12.5 embryos were subjected to phospho-EphB4 and total EphB4 immunoblotting analysis. Ratios of pEphB4:total EphB4 are graphed. **<italic>P</italic><0.01 (two-tailed, unpaired student's <italic>t</italic>-test), <italic>n</italic>=6 for <italic>ephrinB2</italic><sup>+/<italic>+</italic></sup>, <italic>n</italic>=4 for <italic>ephrinB2</italic><sup><italic>lac</italic>Z/<italic>lacZ</italic></sup>, error bars, s.d. NS, not significant.</p></caption><graphic xlink:href="ncomms7625-f5"></graphic></fig><fig id="f6"><label>Figure 6</label><caption><title>Kinase activity of EphB4 is required for lymphatic valve development.</title><p>(<bold>a</bold>) Visualization of mesenteric lymphatic vessels and valves (arrows) by staining for Prox-1 and VEGFR3 2 days following treatment of P3 neonatal mice with NVP-BHG712, a selective EphB4 inhibitor. Blood vessels are highlighted by strong α-smooth muscle actin (αSMA) staining. Scale bar, 200 μm. (<bold>b</bold>) Quantification of mesenteric lymphatic valves, ***<italic>P</italic>< 0.001 (two-tailed, unpaired student's <italic>t</italic>-test), <italic>n</italic>=3 per treatment group (error bars, s.d.). (<bold>c</bold>) NVP-BHG712 inhibits EphB4 phosphorylation in P2 neonatal mice. Lung tissue lysates were subjected to anti-EphB4 immunoprecipitation followed by anti-pY or anti-EphB4 immunoblotting. Ratios of pEphB4 to total EphB4 (pEphB4: EphB4) are graphed. *<italic>P</italic><0.05 (two-tailed, unpaired student's <italic>t</italic>-test), <italic>n</italic>=4 per treatment group (error bars, s.d.).</p></caption><graphic xlink:href="ncomms7625-f6"></graphic></fig><fig id="f7"><label>Figure 7</label><caption><title>Chemical genetic inhibition of EphB4 in EphB4 ASKA mice.</title><p>(<bold>a</bold>–<bold>c</bold>) Visualization of mesenteric lymphatic vessels (L) and valves (arrows) by immunostaining for Prox-1 following treatments starting from P2. Blood vessels are highlighted by strong α-smooth muscle actin (αSMA) staining. Scale bar, 200 μm. (<bold>a</bold>) Agonistic anti-EphB4 (α-EphB4) reverses lymphatic valve defects caused by function-blocking anti-ephrinB2 (α-ephrinB2) in EphB4 ASKA (<italic>EphB4</italic><sup><italic>T693A/T693A</italic></sup>) neonatal mice. P7 mesenteric vessels are shown. Right panel, quantification of lymphatic valves. ****<italic>P</italic><0.0001 (two-tailed, unpaired Student's <italic>t</italic>-test), <italic>n</italic>=3 per treatment group (error bars, s.d.). (<bold>b</bold>) NaPP1 treatment results in lymphatic valve defect in <italic>EphB4</italic><sup><italic>T693A/T693A</italic></sup> neonatal mice. P4 mesenteric vessels are shown. Right panel, quantification of lymphatic valves. ****<italic>P</italic><0.0001(two-tailed, unpaired Student's <italic>t</italic>-test), <italic>n</italic>=3 per treatment group (error bars, s.d.). (<bold>c</bold>) NaPP1 treatment has no effect on lymphatic valves in wild-type neonatal mice. P4 mesenteric vessels are shown. Right panel, quantification of lymphatic valves, <italic>n</italic>=3 per treatment group, two-tailed, unpaired Student's <italic>t</italic>-test, error bars, s.d. (<bold>d</bold>) Schematic representation of EphB4 ASKA mutant (T693A). EphB4 ASKA is susceptible to inhibition by NaPP1. 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