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. 2012 Aug 3;111(4):437-45.
doi: 10.1161/CIRCRESAHA.112.269316. Epub 2012 Jun 21.

Semaphorin3A, Neuropilin-1, and PlexinA1 are required for lymphatic valve formation

Karine Bouvrée  1 , Isabelle Brunet, Raquel Del Toro, Emma Gordon, Claudia Prahst, Brunella Cristofaro, Thomas Mathivet, Yunling Xu, Jihane Soueid, Vitor Fortuna, Nayoki Miura, Marie-Stéphane Aigrot, Charlotte H Maden, Christiana Ruhrberg, Jean Léon Thomas, Anne Eichmann
Affiliations

Semaphorin3A, Neuropilin-1, and PlexinA1 are required for lymphatic valve formation

Karine Bouvrée et al. Circ Res. .

Abstract

Rationale: The lymphatic vasculature plays a major role in fluid homeostasis, absorption of dietary lipids, and immune surveillance. Fluid transport depends on the presence of intraluminal valves within lymphatic collectors. Defective formation of lymphatic valves leads to lymphedema, a progressive and debilitating condition for which curative treatments are currently unavailable. How lymphatic valve formation is regulated remains largely unknown.

Objective: We investigated if the repulsive axon guidance molecule Semaphorin3A (Sema3A) plays a role in lymphatic valve formation.

Methods and results: We show that Sema3A mRNA is expressed in lymphatic vessels and that Sema3A protein binds to lymphatic valves expressing the Neuropilin-1 (Nrp1) and PlexinA1 receptors. Using mouse knockout models, we show that Sema3A is selectively required for lymphatic valve formation, via interaction with Nrp1 and PlexinA1. Sema3a(-/-) mice exhibit defects in lymphatic valve formation, which are not due to abnormal lymphatic patterning or sprouting, and mice carrying a mutation in the Sema3A binding site of Nrp1, or deficient for Plxna1, develop lymphatic valve defects similar to those seen in Sema3a(-/-) mice.

Conclusions: Our data demonstrate an essential direct function of Sema3A-Nrp1-PlexinA1 signaling in lymphatic valve formation.

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Figures

Figure 1
Figure 1. Sema3A expression and binding to lymphatic valves expressing Nrp1 and PlexinA1
A, In situ hybridization with Sema3a antisense riboprobe on P0 mesenteric vessels. Note specific labeling of lymphatic vessels (L) including valve-forming areas (arrow) but not arteries (A) or veins (V). B and C, Sema3A-AP (B) and VEGFA-AP (C) fusion protein binding to whole-mount mesenteries from wild-type mice at P0. Sema3A-AP binds to lymphatic valves (B, arrows). VEGFA-AP binds to arteries and veins but not to lymphatics (C). D through F, In situ hybridization of whole-mount mesenteries at P0 with the indicated antisense riboprobes. Nrp1 and Plxna1 label valve-forming areas of lymphatic vessels (D and E, arrows). Note strong Nrp2 staining in lymphatic vessels but absence of signal in valve-forming areas (arrows, F). G, HDLEC protein extracts subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blots were incubated with the indicated antibodies. HDLEC express Nrp1, PlexinA1, and Sema3A. H, HDLEC were serum-starved and stimulated with Sema3A for 15 minutes. Nrp1 was immunoprecipitated from cultures, and Western blots were probed with indicated antibodies. Quantification of 3 independent experiments shows that Sema3A treatment enhances Nrp1/PlexinA1 complex formation. Bars represent SEM. *P<0.05 (t test). Scale bars: 50 μm.
Figure 2
Figure 2. Abnormal lymphatic valves in Sema3a−/−, Nrp1sema−/− and Plxna1−/− mice
Confocal images of whole-mount mesenteries stained with the indicated antibodies. A and B, Anti-Foxc2 (A, green) and anti-Itgα9 (A and B, red) staining in wild-type mesentery lymphatics show normal valve leaflets. C, D, and F, Staining of mutant mouse mesenteries with the indicated markers. Note abnormal valves in Sema3a−/− (C), Nrp1sema−/− (D), and Plxna1−/− (F) compared with wild-type (A and B). E, Itgα9-positive valve area was measured using 8 to 10 images from 3 mice of each genotype at P0. Note reduction of valve area in all 3 mutant mouse lines. Quantification was performed using ImageJ (Mann-Whitney U test, P<0.05). Scale bars: 90 μm.
Figure 3
Figure 3. Normal lymphatic sprouting in Sema3a−/− mice
A through B‴, Confocal images of lymphatic sprouts in E14.5 skin from Vegfr3::YFP, WT (A) or Vegfr3::YFP, Sema3a−/− (B) embryos, labeled with Nrp2 (red), and anti-CD31 (blue). Note normal appearance of lymphatic sprouts in WT and in Sema3a−/− mice. Hair follicles: orange arrows. C and D, Live imaging of lymphatic vessel sprouting in Vegfr3::YFP, WT (C through C‴) and Vegfr3::YFP, Sema3a−/− (D through D‴) mice. Single images were captured every day in the same position in eyelid skin, between P0 and P3 in Vegfr3::YFP, WT mice (C through C‴) and in Vegfr3::YFP, Sema3a−/− mice (D through D‴). Orange and blue markers indicate the same position among the lymphatic capillaries. E and F, Quantification of total length (E) and number of branch points (F) of growing lymphatic capillaries from P0 to P2 in Vegfr3::YFP, WT and Vegfr3::YFP, Sema3a−/− mice. Note similar increase in length and branch point number in animals of both genotypes. Quantification was performed using ImageJ (P>0.05); n=3 mice/genotype. Scale bars: 57 μm (A through B‴); 426 μm (C through D‴).
Figure 4
Figure 4. Development of sensory nerves, arteries, and lymphatics in Sema3a−/− mice
Confocal images of whole-mount skin from embryos at E15.5 (E through L) or E14.5 (A through D) stained with the indicated antibodies. A through D, In wild-type, Nrp2-positive collecting lymphatic vessels (labeled as l* in A) develop along the main branches of a Cx40-positive artery (labeled as a in B). Note Nrp2 expression in hair follicles (orange arrows, A). Only the small-diameter arteries are aligned with TuJ1-positive sensory axons (arrows in B through D). E through L, Normal development of collecting lymphatics (labeled as l*) alongside main arterial Nrp1-positive branches in wild-type (E through H) and Sema3a−/− (I through L) mice. Lymphatics are labeled with a Vegfr3::YFP reporter. Scale bars: 208 μm.
Figure 5
Figure 5. Abnormal smooth muscle coating in Sema3a−/− mice and mice treated with anti-Nrp1A
Confocal images of mouse mesenteries stained with antibodies against SMA (red) and Itgα9 (green). A and B, Note absence of anti-SMA staining in anti–Itgα9-positive lymphatic valves (arrows) in P4 wild-type mice. C and D, Sema3a−/− littermates show SMA-positive valve areas (arrows). E and F, Wild-type mice injected intraperitoneally with anti-Nrp1A, which blocks Sema3A binding to Nrp1, exhibit SMA-positive lymphatic valves (arrows). G and H, SMA-positive valves are not seen after injection of anti-Nrp1B (arrows), which blocks VEGF binding to Nrp1. I, Quantification of valve smooth muscle cell coverage. Itgα9-positive valve area covered by SMA-positive staining was measured. Note increase in SMC coverage of Sema3a−/− mice and Nrp1A-injected mice compared with wild-type or PBS-injected counterparts. Quantification was performed using ImageJ (*P<0.05); n>4 mice per genotype. Scale bars: 20 μm.
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