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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.


Related in: MedlinePlus

Nrp2 deficient LECs fail to respond to Sema3G in culture. (A) Immunoblot analysis probed for NRP2 and β-actin of control or Nrp2 knockdown (KD) human LECs (HMVEC-dLy-Neo). (B-G) Cell contraction assay for control or Nrp2 knockdown human LECs with 1 nM of AP control protein (B-D) or AP-SEMA3G (E-G). Phalloidin-AF568 staining shows cell shape and nuclei are stained by TO-PRO-3. (H) Quantification of total cell area in the cell contraction assay. Control KD LECs treated with AP control, N=263 cells; control KD LECs treated with AP-SEMA3G, N=296; Nrp2 KD #1 LECs treated with AP control, N=187 cells; Nrp2 KD #1 LECs treated with AP-SEMA3G; N=221; Nrp2 KD #2 LECs treated with AP control, N=227; Nrp2 KD #2 LECs treated with AP-SEMA3G, N=223; two independent experiments). (I-L) Sprouting assay for control or Nrp2 knockdown human LECs in response to VEGFC with 0.5 nM of AP control protein (I,J) or AP-SEMA3G (K,L). Phalloidin-AF568 staining shows cell shape and nuclei are stained by TO-PRO-3. (M) Quantification of total number of LEC sprouts per one aggregate. (N=8 from two independent experiments, bars represent mean±s.d.). *P<0.01; NS, not significant by one-way ANOVA with Tukey-HSD multiple comparison test.
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BIO012302F6: Nrp2 deficient LECs fail to respond to Sema3G in culture. (A) Immunoblot analysis probed for NRP2 and β-actin of control or Nrp2 knockdown (KD) human LECs (HMVEC-dLy-Neo). (B-G) Cell contraction assay for control or Nrp2 knockdown human LECs with 1 nM of AP control protein (B-D) or AP-SEMA3G (E-G). Phalloidin-AF568 staining shows cell shape and nuclei are stained by TO-PRO-3. (H) Quantification of total cell area in the cell contraction assay. Control KD LECs treated with AP control, N=263 cells; control KD LECs treated with AP-SEMA3G, N=296; Nrp2 KD #1 LECs treated with AP control, N=187 cells; Nrp2 KD #1 LECs treated with AP-SEMA3G; N=221; Nrp2 KD #2 LECs treated with AP control, N=227; Nrp2 KD #2 LECs treated with AP-SEMA3G, N=223; two independent experiments). (I-L) Sprouting assay for control or Nrp2 knockdown human LECs in response to VEGFC with 0.5 nM of AP control protein (I,J) or AP-SEMA3G (K,L). Phalloidin-AF568 staining shows cell shape and nuclei are stained by TO-PRO-3. (M) Quantification of total number of LEC sprouts per one aggregate. (N=8 from two independent experiments, bars represent mean±s.d.). *P<0.01; NS, not significant by one-way ANOVA with Tukey-HSD multiple comparison test.

Mentions: Knockdown of NRP2 expression in LECs was successfully carried out in culture (Fig. 6A). Nrp2-deficient LECs failed to respond to SEMA3G such that the cells did not show SEMA3G-induced cell contraction (Fig. 6B-H). Furthermore, the Nrp2 deficiency diminished SEMA3G-mediated inhibition of VEGFC-induced LEC sprouting, although Nrp2-deficient LECs sprout more than control LECs (Fig. 6I-M). These results suggest that NRP2 is essential for SEMA3G-mediated inhibition of lymphatic sprouting.Fig. 6.


Class 3 semaphorins negatively regulate dermal lymphatic network formation.

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

Nrp2 deficient LECs fail to respond to Sema3G in culture. (A) Immunoblot analysis probed for NRP2 and β-actin of control or Nrp2 knockdown (KD) human LECs (HMVEC-dLy-Neo). (B-G) Cell contraction assay for control or Nrp2 knockdown human LECs with 1 nM of AP control protein (B-D) or AP-SEMA3G (E-G). Phalloidin-AF568 staining shows cell shape and nuclei are stained by TO-PRO-3. (H) Quantification of total cell area in the cell contraction assay. Control KD LECs treated with AP control, N=263 cells; control KD LECs treated with AP-SEMA3G, N=296; Nrp2 KD #1 LECs treated with AP control, N=187 cells; Nrp2 KD #1 LECs treated with AP-SEMA3G; N=221; Nrp2 KD #2 LECs treated with AP control, N=227; Nrp2 KD #2 LECs treated with AP-SEMA3G, N=223; two independent experiments). (I-L) Sprouting assay for control or Nrp2 knockdown human LECs in response to VEGFC with 0.5 nM of AP control protein (I,J) or AP-SEMA3G (K,L). Phalloidin-AF568 staining shows cell shape and nuclei are stained by TO-PRO-3. (M) Quantification of total number of LEC sprouts per one aggregate. (N=8 from two independent experiments, bars represent mean±s.d.). *P<0.01; NS, not significant by one-way ANOVA with Tukey-HSD multiple comparison test.
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BIO012302F6: Nrp2 deficient LECs fail to respond to Sema3G in culture. (A) Immunoblot analysis probed for NRP2 and β-actin of control or Nrp2 knockdown (KD) human LECs (HMVEC-dLy-Neo). (B-G) Cell contraction assay for control or Nrp2 knockdown human LECs with 1 nM of AP control protein (B-D) or AP-SEMA3G (E-G). Phalloidin-AF568 staining shows cell shape and nuclei are stained by TO-PRO-3. (H) Quantification of total cell area in the cell contraction assay. Control KD LECs treated with AP control, N=263 cells; control KD LECs treated with AP-SEMA3G, N=296; Nrp2 KD #1 LECs treated with AP control, N=187 cells; Nrp2 KD #1 LECs treated with AP-SEMA3G; N=221; Nrp2 KD #2 LECs treated with AP control, N=227; Nrp2 KD #2 LECs treated with AP-SEMA3G, N=223; two independent experiments). (I-L) Sprouting assay for control or Nrp2 knockdown human LECs in response to VEGFC with 0.5 nM of AP control protein (I,J) or AP-SEMA3G (K,L). Phalloidin-AF568 staining shows cell shape and nuclei are stained by TO-PRO-3. (M) Quantification of total number of LEC sprouts per one aggregate. (N=8 from two independent experiments, bars represent mean±s.d.). *P<0.01; NS, not significant by one-way ANOVA with Tukey-HSD multiple comparison test.
Mentions: Knockdown of NRP2 expression in LECs was successfully carried out in culture (Fig. 6A). Nrp2-deficient LECs failed to respond to SEMA3G such that the cells did not show SEMA3G-induced cell contraction (Fig. 6B-H). Furthermore, the Nrp2 deficiency diminished SEMA3G-mediated inhibition of VEGFC-induced LEC sprouting, although Nrp2-deficient LECs sprout more than control LECs (Fig. 6I-M). These results suggest that NRP2 is essential for SEMA3G-mediated inhibition of lymphatic sprouting.Fig. 6.

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