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Class 3 semaphorins negatively regulate dermal lymphatic network formation.

Uchida Y, James JM, Suto F, Mukouyama YS - Biol Open (2015)

Bottom Line: In contrast, Sema3f;Sema3g double mutants display increased lymphatic branching, while Nrp2 mutants exhibit reduced lymphatic branching.Our results provide the first evidence that SEMA3F and SEMA3G function as a negative regulator for dermal lymphangiogenesis in vivo.The reciprocal phenotype in lymphatic branching between Sema3f;Sema3g double mutants and Nrp2 mutants suggest a complex NRP2 function that regulates LEC behavior both positively and negatively, through a binding with VEGFC or SEMA3s.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Stem Cell and Neuro-Vascular Biology, Genetics and Developmental Biology Center, National Heart, Lung, and Blood Institute, National Institutes of Health, Building 10/6C103, 10 Center Drive, Bethesda, MD 20892, USA.

No MeSH data available.


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SEMA3-PlexinA signaling is required for lymphatic network formation in limb skin. (A) RT-PCR analysis of mRNA from FACS-isolated PECAM1+/LYVE1+ LECs from E16.5 embryos. (B) Whole-mount staining of E16.5 limb skin with antibodies to PlexinA1 (red) together with LYVE1 (green). (C-E′) Whole-mount staining of limb skins from E15.5 PlexinA1, PlexinA2 mutants and WT controls with PROX1 (red) and LYVE1 (green). The boxed regions in (C-E) are magnified in (C′-E′), respectively: The magnified images show PROX1 only (C′-E′). Scale bars: 100 µm. (F,G) Quantification of lymphatic branching points (F) and total LEC number (G) per area (mm2). Bars represent mean±s.e.m. and sample numbers (the number of limb skins we analyzed) are shown in the bars. *P<0.01; NS, not significant by one-way ANOVA with Tukey-HSD multiple comparison test.
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BIO012302F5: SEMA3-PlexinA signaling is required for lymphatic network formation in limb skin. (A) RT-PCR analysis of mRNA from FACS-isolated PECAM1+/LYVE1+ LECs from E16.5 embryos. (B) Whole-mount staining of E16.5 limb skin with antibodies to PlexinA1 (red) together with LYVE1 (green). (C-E′) Whole-mount staining of limb skins from E15.5 PlexinA1, PlexinA2 mutants and WT controls with PROX1 (red) and LYVE1 (green). The boxed regions in (C-E) are magnified in (C′-E′), respectively: The magnified images show PROX1 only (C′-E′). Scale bars: 100 µm. (F,G) Quantification of lymphatic branching points (F) and total LEC number (G) per area (mm2). Bars represent mean±s.e.m. and sample numbers (the number of limb skins we analyzed) are shown in the bars. *P<0.01; NS, not significant by one-way ANOVA with Tukey-HSD multiple comparison test.

Mentions: The increased branching in Sema3f and Sema3g mutants are opposite to what we expected based on our finding of decreased branching in Nrp2 mutants (Fig. 3G). The phenotypic differences between Sema3f/g double mutants and Nrp2 mutants might reflect a dual function of NRP2 that regulates lymphatic sprouting and LEC proliferation both positively and negatively, through a binding with VEGFC or SEMA3s. To further address the role of SEMA3s-mediated signaling, we decided to examine whether loss of PlexinA, a signal transducer and receptor complex with NRP2 for SEMA3s, influences lymphatic branching and LEC number. Among PlexinA family members, PlexinA1 and PlexinA2 are mainly expressed by FACS-isolated LECs (Fig. 5A). We further confirmed PlexinA1 expression in dermal lymphatic vasculature by anti-PlexinA1 specific antibody (Fig. 5B). PlexinA1−/− mutants exhibited an increase in lymphatic branching points and LEC number (Fig. 5C vs D; C′ vs D′; F,G), whereas PlexinA2−/− mutants exhibited only an increase in lymphatic branching points (Fig. 4C vs E; C′ vs E′; F,G). Interestingly, PlexinA1−/− mutants exhibit almost identical, albeit milder, phenotypes with Sema3f−/−;Sema3glacZ/lacZ double mutants. These observations support that PlexinA functions as a physiological receptor for SEMA3F and SEMA3G in vivo.Fig. 5.


Class 3 semaphorins negatively regulate dermal lymphatic network formation.

Uchida Y, James JM, Suto F, Mukouyama YS - Biol Open (2015)

SEMA3-PlexinA signaling is required for lymphatic network formation in limb skin. (A) RT-PCR analysis of mRNA from FACS-isolated PECAM1+/LYVE1+ LECs from E16.5 embryos. (B) Whole-mount staining of E16.5 limb skin with antibodies to PlexinA1 (red) together with LYVE1 (green). (C-E′) Whole-mount staining of limb skins from E15.5 PlexinA1, PlexinA2 mutants and WT controls with PROX1 (red) and LYVE1 (green). The boxed regions in (C-E) are magnified in (C′-E′), respectively: The magnified images show PROX1 only (C′-E′). Scale bars: 100 µm. (F,G) Quantification of lymphatic branching points (F) and total LEC number (G) per area (mm2). Bars represent mean±s.e.m. and sample numbers (the number of limb skins we analyzed) are shown in the bars. *P<0.01; NS, not significant by one-way ANOVA with Tukey-HSD multiple comparison test.
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BIO012302F5: SEMA3-PlexinA signaling is required for lymphatic network formation in limb skin. (A) RT-PCR analysis of mRNA from FACS-isolated PECAM1+/LYVE1+ LECs from E16.5 embryos. (B) Whole-mount staining of E16.5 limb skin with antibodies to PlexinA1 (red) together with LYVE1 (green). (C-E′) Whole-mount staining of limb skins from E15.5 PlexinA1, PlexinA2 mutants and WT controls with PROX1 (red) and LYVE1 (green). The boxed regions in (C-E) are magnified in (C′-E′), respectively: The magnified images show PROX1 only (C′-E′). Scale bars: 100 µm. (F,G) Quantification of lymphatic branching points (F) and total LEC number (G) per area (mm2). Bars represent mean±s.e.m. and sample numbers (the number of limb skins we analyzed) are shown in the bars. *P<0.01; NS, not significant by one-way ANOVA with Tukey-HSD multiple comparison test.
Mentions: The increased branching in Sema3f and Sema3g mutants are opposite to what we expected based on our finding of decreased branching in Nrp2 mutants (Fig. 3G). The phenotypic differences between Sema3f/g double mutants and Nrp2 mutants might reflect a dual function of NRP2 that regulates lymphatic sprouting and LEC proliferation both positively and negatively, through a binding with VEGFC or SEMA3s. To further address the role of SEMA3s-mediated signaling, we decided to examine whether loss of PlexinA, a signal transducer and receptor complex with NRP2 for SEMA3s, influences lymphatic branching and LEC number. Among PlexinA family members, PlexinA1 and PlexinA2 are mainly expressed by FACS-isolated LECs (Fig. 5A). We further confirmed PlexinA1 expression in dermal lymphatic vasculature by anti-PlexinA1 specific antibody (Fig. 5B). PlexinA1−/− mutants exhibited an increase in lymphatic branching points and LEC number (Fig. 5C vs D; C′ vs D′; F,G), whereas PlexinA2−/− mutants exhibited only an increase in lymphatic branching points (Fig. 4C vs E; C′ vs E′; F,G). Interestingly, PlexinA1−/− mutants exhibit almost identical, albeit milder, phenotypes with Sema3f−/−;Sema3glacZ/lacZ double mutants. These observations support that PlexinA functions as a physiological receptor for SEMA3F and SEMA3G in vivo.Fig. 5.

Bottom Line: In contrast, Sema3f;Sema3g double mutants display increased lymphatic branching, while Nrp2 mutants exhibit reduced lymphatic branching.Our results provide the first evidence that SEMA3F and SEMA3G function as a negative regulator for dermal lymphangiogenesis in vivo.The reciprocal phenotype in lymphatic branching between Sema3f;Sema3g double mutants and Nrp2 mutants suggest a complex NRP2 function that regulates LEC behavior both positively and negatively, through a binding with VEGFC or SEMA3s.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Stem Cell and Neuro-Vascular Biology, Genetics and Developmental Biology Center, National Heart, Lung, and Blood Institute, National Institutes of Health, Building 10/6C103, 10 Center Drive, Bethesda, MD 20892, USA.

No MeSH data available.


Related in: MedlinePlus