Limits...
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.


Related in: MedlinePlus

SEMA3-NRP2 signaling is required for lymphatic network formation in limb skin. (A) Schematic diagram illustrating forelimb skin dissection and whole-mount limb skin staining for analysis of lymphatic network formation. The forelimb is dissected at the base of shoulder (dashed line) from E15.5 embryos, and then limb skin is peeled off along the dashed line. Whole-mount immunolabeling of limb skin is performed with antibodies to the LEC markers PROX1 and LYVE1, and the pan-endothelial cell markers PECAM1. For multiple quantification measurements, we defined an image area (white box, 1.45 mm×1.45 mm) in 20× confocal tiled z-stack images using the position of large-diameter blood vessels (white dashed line) as a frame of reference. The PROX1 staining visualizes nuclei of LECs that allows us to measure LEC number in the lymphatic vasculature. The LYVE1 staining allows us to measure lymphatic branching points. Note that the LYVE1 staining also detects tissue macrophages in the skin. (B-F′) Whole-mount staining of limb skin from E15.5 mutants and wild-type (WT) controls with PROX1 (red) and LYVE1 (green). The boxed regions in (B-F) are magnified in (B′-F′), respectively: The magnified images show PROX1 only. Scale bars: 100 µm. (G-J) Quantification of lymphatic branching points (G) and total LEC number (H) per area (mm2). Lymphatic vessel width at the middle of lymphatic branching points represented as box and whisker plot (I) (N=1404, WT controls; N=980, Nrp2 mutants; N=1856, Sema3f mutants; N=2388, Sema3g mutants; N=3318, Sema3f;Sema3g double mutants). Quantification of lymphatic tip cells in total LECs (J). Bars represent mean±s.e.m. and sample numbers (the number of limb skins we analyzed) are shown in the bars. *P<0.05; **P<0.01; NS, not significant by one-way ANOVA with Tukey-HSD multiple comparison test.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4582121&req=5

BIO012302F3: SEMA3-NRP2 signaling is required for lymphatic network formation in limb skin. (A) Schematic diagram illustrating forelimb skin dissection and whole-mount limb skin staining for analysis of lymphatic network formation. The forelimb is dissected at the base of shoulder (dashed line) from E15.5 embryos, and then limb skin is peeled off along the dashed line. Whole-mount immunolabeling of limb skin is performed with antibodies to the LEC markers PROX1 and LYVE1, and the pan-endothelial cell markers PECAM1. For multiple quantification measurements, we defined an image area (white box, 1.45 mm×1.45 mm) in 20× confocal tiled z-stack images using the position of large-diameter blood vessels (white dashed line) as a frame of reference. The PROX1 staining visualizes nuclei of LECs that allows us to measure LEC number in the lymphatic vasculature. The LYVE1 staining allows us to measure lymphatic branching points. Note that the LYVE1 staining also detects tissue macrophages in the skin. (B-F′) Whole-mount staining of limb skin from E15.5 mutants and wild-type (WT) controls with PROX1 (red) and LYVE1 (green). The boxed regions in (B-F) are magnified in (B′-F′), respectively: The magnified images show PROX1 only. Scale bars: 100 µm. (G-J) Quantification of lymphatic branching points (G) and total LEC number (H) per area (mm2). Lymphatic vessel width at the middle of lymphatic branching points represented as box and whisker plot (I) (N=1404, WT controls; N=980, Nrp2 mutants; N=1856, Sema3f mutants; N=2388, Sema3g mutants; N=3318, Sema3f;Sema3g double mutants). Quantification of lymphatic tip cells in total LECs (J). Bars represent mean±s.e.m. and sample numbers (the number of limb skins we analyzed) are shown in the bars. *P<0.05; **P<0.01; NS, not significant by one-way ANOVA with Tukey-HSD multiple comparison test.

Mentions: To examine whether mutants lacking SEMA3s-mediated signaling exhibit defective lymphatic network formation in the embryonic skin, we first established a whole-mount imaging of embryonic limb skin lymphatic vasculature with quantification measurements (Fig. 3A). Using this method, we examined what happens to dermal lymphatic vessel development in mutants lacking Nrp2, Sema3f or Sema3g. Interestingly, Nrp2taugfp/taugfp, Sema3f−/−, Sema3glacZ/lacZ or Sema3f−/−;Sema3glacZ/lacZ double mutants showed different phenotypes in lymphatic branching morphogenesis and LEC growth (Fig. 3B-F). Analysis of branching point phenotypes revealed that Nrp2taugfp/taugfp mutants exhibited decreased lymphatic branching complexity (Fig. 3B vs C; G). In contrast, both Sema3f−/− and Sema3glacZ/lacZ mutants exhibited increased lymphatic branching complexity, albeit more branching in Sema3glacZ/lacZ mutants than Sema3f−/− mutants (Fig. 3B vs D and E; G). Furthermore, Sema3f−/−;Sema3glacZ/lacZ double mutants displayed a synergistic increase in lymphatic branching points (Fig. 3B vs D vs E vs F; G), which indicates both SEMA3F and SEMA3G cooperatively inhibit lymphatic sprouting in in vivo. Quantification analysis of PROX1+ LEC number revealed that Nrp2taugfp/taugfp mutants exhibited increased LEC number resulting in lymphatic hyperplasia (Fig. 3B′ vs C′; H). Like Nrp2taugfp/taugfp mutants, Sema3f−/− mutants, but not Sema3glacZ/lacZ mutants, exhibited a significant increase in LEC number (Fig. 3B′ vs D′ vs E′; H). Interestingly, Sema3f−/−;Sema3glacZ/lacZ double mutants exhibited increased PROX1+ LEC number, but not a synergistic increase (Fig. 3B′ vs D′ vs E′ vs F′; H), almost similar number with Sema3f−/− mutants. This result suggests that SEMA3F but not SEMA3G is responsible to control PROX1+ LEC number in vivo. Furthermore, Nrp2taugfp/taugfp mutants exhibited an increase lymphatic width whereas Sema3glacZ/lacZ had a decrease width (Fig. 3B vs C vs E; I). The increased branching complexity of Sema3f−/−;Sema3glacZ/lacZ double mutants may be due to an increased tip cell formation (Fig. 3J).Fig. 3.


Class 3 semaphorins negatively regulate dermal lymphatic network formation.

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

SEMA3-NRP2 signaling is required for lymphatic network formation in limb skin. (A) Schematic diagram illustrating forelimb skin dissection and whole-mount limb skin staining for analysis of lymphatic network formation. The forelimb is dissected at the base of shoulder (dashed line) from E15.5 embryos, and then limb skin is peeled off along the dashed line. Whole-mount immunolabeling of limb skin is performed with antibodies to the LEC markers PROX1 and LYVE1, and the pan-endothelial cell markers PECAM1. For multiple quantification measurements, we defined an image area (white box, 1.45 mm×1.45 mm) in 20× confocal tiled z-stack images using the position of large-diameter blood vessels (white dashed line) as a frame of reference. The PROX1 staining visualizes nuclei of LECs that allows us to measure LEC number in the lymphatic vasculature. The LYVE1 staining allows us to measure lymphatic branching points. Note that the LYVE1 staining also detects tissue macrophages in the skin. (B-F′) Whole-mount staining of limb skin from E15.5 mutants and wild-type (WT) controls with PROX1 (red) and LYVE1 (green). The boxed regions in (B-F) are magnified in (B′-F′), respectively: The magnified images show PROX1 only. Scale bars: 100 µm. (G-J) Quantification of lymphatic branching points (G) and total LEC number (H) per area (mm2). Lymphatic vessel width at the middle of lymphatic branching points represented as box and whisker plot (I) (N=1404, WT controls; N=980, Nrp2 mutants; N=1856, Sema3f mutants; N=2388, Sema3g mutants; N=3318, Sema3f;Sema3g double mutants). Quantification of lymphatic tip cells in total LECs (J). Bars represent mean±s.e.m. and sample numbers (the number of limb skins we analyzed) are shown in the bars. *P<0.05; **P<0.01; NS, not significant by one-way ANOVA with Tukey-HSD multiple comparison test.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4582121&req=5

BIO012302F3: SEMA3-NRP2 signaling is required for lymphatic network formation in limb skin. (A) Schematic diagram illustrating forelimb skin dissection and whole-mount limb skin staining for analysis of lymphatic network formation. The forelimb is dissected at the base of shoulder (dashed line) from E15.5 embryos, and then limb skin is peeled off along the dashed line. Whole-mount immunolabeling of limb skin is performed with antibodies to the LEC markers PROX1 and LYVE1, and the pan-endothelial cell markers PECAM1. For multiple quantification measurements, we defined an image area (white box, 1.45 mm×1.45 mm) in 20× confocal tiled z-stack images using the position of large-diameter blood vessels (white dashed line) as a frame of reference. The PROX1 staining visualizes nuclei of LECs that allows us to measure LEC number in the lymphatic vasculature. The LYVE1 staining allows us to measure lymphatic branching points. Note that the LYVE1 staining also detects tissue macrophages in the skin. (B-F′) Whole-mount staining of limb skin from E15.5 mutants and wild-type (WT) controls with PROX1 (red) and LYVE1 (green). The boxed regions in (B-F) are magnified in (B′-F′), respectively: The magnified images show PROX1 only. Scale bars: 100 µm. (G-J) Quantification of lymphatic branching points (G) and total LEC number (H) per area (mm2). Lymphatic vessel width at the middle of lymphatic branching points represented as box and whisker plot (I) (N=1404, WT controls; N=980, Nrp2 mutants; N=1856, Sema3f mutants; N=2388, Sema3g mutants; N=3318, Sema3f;Sema3g double mutants). Quantification of lymphatic tip cells in total LECs (J). Bars represent mean±s.e.m. and sample numbers (the number of limb skins we analyzed) are shown in the bars. *P<0.05; **P<0.01; NS, not significant by one-way ANOVA with Tukey-HSD multiple comparison test.
Mentions: To examine whether mutants lacking SEMA3s-mediated signaling exhibit defective lymphatic network formation in the embryonic skin, we first established a whole-mount imaging of embryonic limb skin lymphatic vasculature with quantification measurements (Fig. 3A). Using this method, we examined what happens to dermal lymphatic vessel development in mutants lacking Nrp2, Sema3f or Sema3g. Interestingly, Nrp2taugfp/taugfp, Sema3f−/−, Sema3glacZ/lacZ or Sema3f−/−;Sema3glacZ/lacZ double mutants showed different phenotypes in lymphatic branching morphogenesis and LEC growth (Fig. 3B-F). Analysis of branching point phenotypes revealed that Nrp2taugfp/taugfp mutants exhibited decreased lymphatic branching complexity (Fig. 3B vs C; G). In contrast, both Sema3f−/− and Sema3glacZ/lacZ mutants exhibited increased lymphatic branching complexity, albeit more branching in Sema3glacZ/lacZ mutants than Sema3f−/− mutants (Fig. 3B vs D and E; G). Furthermore, Sema3f−/−;Sema3glacZ/lacZ double mutants displayed a synergistic increase in lymphatic branching points (Fig. 3B vs D vs E vs F; G), which indicates both SEMA3F and SEMA3G cooperatively inhibit lymphatic sprouting in in vivo. Quantification analysis of PROX1+ LEC number revealed that Nrp2taugfp/taugfp mutants exhibited increased LEC number resulting in lymphatic hyperplasia (Fig. 3B′ vs C′; H). Like Nrp2taugfp/taugfp mutants, Sema3f−/− mutants, but not Sema3glacZ/lacZ mutants, exhibited a significant increase in LEC number (Fig. 3B′ vs D′ vs E′; H). Interestingly, Sema3f−/−;Sema3glacZ/lacZ double mutants exhibited increased PROX1+ LEC number, but not a synergistic increase (Fig. 3B′ vs D′ vs E′ vs F′; H), almost similar number with Sema3f−/− mutants. This result suggests that SEMA3F but not SEMA3G is responsible to control PROX1+ LEC number in vivo. Furthermore, Nrp2taugfp/taugfp mutants exhibited an increase lymphatic width whereas Sema3glacZ/lacZ had a decrease width (Fig. 3B vs C vs E; I). The increased branching complexity of Sema3f−/−;Sema3glacZ/lacZ double mutants may be due to an increased tip cell formation (Fig. 3J).Fig. 3.

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