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NRP1 Regulates CDC42 Activation to Promote Filopodia Formation in Endothelial Tip Cells.

Fantin A, Lampropoulou A, Gestri G, Raimondi C, Senatore V, Zachary I, Ruhrberg C - Cell Rep (2015)

Bottom Line: Instead, we find that NRP1 is essential to form the filopodial bursts that distinguish tip cells morphologically from neighboring stalk cells, because it enables the extracellular matrix (ECM)-induced activation of CDC42, a key regulator of filopodia formation.Accordingly, NRP1 knockdown and pharmacological CDC42 inhibition similarly impaired filopodia formation in vitro and in developing zebrafish in vivo.We conclude that NRP1 enables ECM-induced filopodia formation for tip cell function during sprouting angiogenesis.

View Article: PubMed Central - PubMed

Affiliation: UCL Institute of Ophthalmology, University College London, 11-43 Bath Street, London EC1V 9EL, UK.

No MeSH data available.


Related in: MedlinePlus

NRP1 Promotes Vessel Ingression Into and Growth Within the Neural Parenchyma(A–C) PECAM immunohistochemistry of E10.5 mouse hindbrains of the indicated genotypes, flat-mounted with the ventricular side facing up; the position of individual rhombomeres (r) is indicated. Scale bar, 500 μm. (A′–C″) Higher magnification of the boxed areas in (AC); arrowheads indicate vessels in rhombomere boundaries.(D–I) PECAM immunohistochemistry of E12.5 hindbrains of the indicated genotypes, flat-mounted to visualize radial (D–F) or SVP (G–I) vessels. Clear arrowheads indicate vascular tufts and arrows vascular bridges between radial vessels in deep brain layers (below the focal plane). Scale bar, 100 μm.(J and K) Quantitation of radial vessel (J) or SVP branchpoint (K) number at E12.5, shown as mean ± SD, n ≥ 8 hindbrains each; asterisks indicate ∗p < 0.05 and ∗∗∗p < 0.001.
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fig1: NRP1 Promotes Vessel Ingression Into and Growth Within the Neural Parenchyma(A–C) PECAM immunohistochemistry of E10.5 mouse hindbrains of the indicated genotypes, flat-mounted with the ventricular side facing up; the position of individual rhombomeres (r) is indicated. Scale bar, 500 μm. (A′–C″) Higher magnification of the boxed areas in (AC); arrowheads indicate vessels in rhombomere boundaries.(D–I) PECAM immunohistochemistry of E12.5 hindbrains of the indicated genotypes, flat-mounted to visualize radial (D–F) or SVP (G–I) vessels. Clear arrowheads indicate vascular tufts and arrows vascular bridges between radial vessels in deep brain layers (below the focal plane). Scale bar, 100 μm.(J and K) Quantitation of radial vessel (J) or SVP branchpoint (K) number at E12.5, shown as mean ± SD, n ≥ 8 hindbrains each; asterisks indicate ∗p < 0.05 and ∗∗∗p < 0.001.

Mentions: Using the mouse embryo hindbrain as a model for angiogenesis (Fantin et al., 2013b), we have recently shown that NRP1 confers a selective advantage to ECs competing for the tip cell position in growing vessel sprouts (Fantin et al., 2013a). To define specific roles for NRP1 in endothelial tip cells during angiogenesis, we first determined the emergence and nature of vascular defects in Nrp1-deficient mouse embryo hindbrains in greater detail than previously done. Immunohistochemistry for the vascular endothelial marker platelet endothelial cell adhesion molecule (PECAM) showed that vessel ingression was mildly delayed in hindbrains carrying one, and severely delayed in hindbrains carrying two, Nrp1 alleles (Figures 1A–1C). Thus, the wild-type subventricular vascular plexus (SVP) appeared well developed at embryonic day 10.5 (E10.5) (Figures 1A′ and 1A″) but was less dense in heterozygous mutants (Figures 1B′ and 1B″) and had not formed in homozygous mutants at this stage (Figures 1C′ and 1C″). Instead, vessel growth in homozygous mutants was largely restricted to the boundary regions between neighboring hindbrain segments. The preferential vascularization of regions corresponding to rhombomere boundaries may reflect their high content of growth-factor-binding proteoglycans (Heyman et al., 1993, 1995) and agrees with findings in zebrafish (Ulrich et al., 2011). Importantly, heterozygous and homozygous mutants, similar to wild-type littermates, had an average of 35 somites at E10.5, suggesting that neither were affected by a general developmental delay at this stage. The comparison of wild-type, heterozygous, and homozygous mutant hindbrains therefore demonstrates a dose-dependent role for NRP1 in promoting vessel ingression into neural parenchyma.


NRP1 Regulates CDC42 Activation to Promote Filopodia Formation in Endothelial Tip Cells.

Fantin A, Lampropoulou A, Gestri G, Raimondi C, Senatore V, Zachary I, Ruhrberg C - Cell Rep (2015)

NRP1 Promotes Vessel Ingression Into and Growth Within the Neural Parenchyma(A–C) PECAM immunohistochemistry of E10.5 mouse hindbrains of the indicated genotypes, flat-mounted with the ventricular side facing up; the position of individual rhombomeres (r) is indicated. Scale bar, 500 μm. (A′–C″) Higher magnification of the boxed areas in (AC); arrowheads indicate vessels in rhombomere boundaries.(D–I) PECAM immunohistochemistry of E12.5 hindbrains of the indicated genotypes, flat-mounted to visualize radial (D–F) or SVP (G–I) vessels. Clear arrowheads indicate vascular tufts and arrows vascular bridges between radial vessels in deep brain layers (below the focal plane). Scale bar, 100 μm.(J and K) Quantitation of radial vessel (J) or SVP branchpoint (K) number at E12.5, shown as mean ± SD, n ≥ 8 hindbrains each; asterisks indicate ∗p < 0.05 and ∗∗∗p < 0.001.
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Related In: Results  -  Collection

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fig1: NRP1 Promotes Vessel Ingression Into and Growth Within the Neural Parenchyma(A–C) PECAM immunohistochemistry of E10.5 mouse hindbrains of the indicated genotypes, flat-mounted with the ventricular side facing up; the position of individual rhombomeres (r) is indicated. Scale bar, 500 μm. (A′–C″) Higher magnification of the boxed areas in (AC); arrowheads indicate vessels in rhombomere boundaries.(D–I) PECAM immunohistochemistry of E12.5 hindbrains of the indicated genotypes, flat-mounted to visualize radial (D–F) or SVP (G–I) vessels. Clear arrowheads indicate vascular tufts and arrows vascular bridges between radial vessels in deep brain layers (below the focal plane). Scale bar, 100 μm.(J and K) Quantitation of radial vessel (J) or SVP branchpoint (K) number at E12.5, shown as mean ± SD, n ≥ 8 hindbrains each; asterisks indicate ∗p < 0.05 and ∗∗∗p < 0.001.
Mentions: Using the mouse embryo hindbrain as a model for angiogenesis (Fantin et al., 2013b), we have recently shown that NRP1 confers a selective advantage to ECs competing for the tip cell position in growing vessel sprouts (Fantin et al., 2013a). To define specific roles for NRP1 in endothelial tip cells during angiogenesis, we first determined the emergence and nature of vascular defects in Nrp1-deficient mouse embryo hindbrains in greater detail than previously done. Immunohistochemistry for the vascular endothelial marker platelet endothelial cell adhesion molecule (PECAM) showed that vessel ingression was mildly delayed in hindbrains carrying one, and severely delayed in hindbrains carrying two, Nrp1 alleles (Figures 1A–1C). Thus, the wild-type subventricular vascular plexus (SVP) appeared well developed at embryonic day 10.5 (E10.5) (Figures 1A′ and 1A″) but was less dense in heterozygous mutants (Figures 1B′ and 1B″) and had not formed in homozygous mutants at this stage (Figures 1C′ and 1C″). Instead, vessel growth in homozygous mutants was largely restricted to the boundary regions between neighboring hindbrain segments. The preferential vascularization of regions corresponding to rhombomere boundaries may reflect their high content of growth-factor-binding proteoglycans (Heyman et al., 1993, 1995) and agrees with findings in zebrafish (Ulrich et al., 2011). Importantly, heterozygous and homozygous mutants, similar to wild-type littermates, had an average of 35 somites at E10.5, suggesting that neither were affected by a general developmental delay at this stage. The comparison of wild-type, heterozygous, and homozygous mutant hindbrains therefore demonstrates a dose-dependent role for NRP1 in promoting vessel ingression into neural parenchyma.

Bottom Line: Instead, we find that NRP1 is essential to form the filopodial bursts that distinguish tip cells morphologically from neighboring stalk cells, because it enables the extracellular matrix (ECM)-induced activation of CDC42, a key regulator of filopodia formation.Accordingly, NRP1 knockdown and pharmacological CDC42 inhibition similarly impaired filopodia formation in vitro and in developing zebrafish in vivo.We conclude that NRP1 enables ECM-induced filopodia formation for tip cell function during sprouting angiogenesis.

View Article: PubMed Central - PubMed

Affiliation: UCL Institute of Ophthalmology, University College London, 11-43 Bath Street, London EC1V 9EL, UK.

No MeSH data available.


Related in: MedlinePlus