Myelinating Schwann cells and Netrin-1 control intra-nervous vascularization of the developing mouse sciatic nerve
- Center for Interdisciplinary Research in Biology (CIRB), College de France, CNRS, INSERM, Université PSL, France
- Institut Mondor de Recherche Biomédicale. UMR U955 INSERM UPEC, France
Figures
Figure 1
Intra-nervous vascular system (INV) develops rapidly starting from embryonic day E16 and angiogenesis is still ongoing at P0.
(A) Whole-mount immunofluorescence staining of sciatic nerve showing axons (TUJ-1, green) and blood vessels (endothelial cells expressing CD31, magenta) at embryonic day E15.5. (B) Snapshot of a 3D …
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Figure 1—source data 1
Quantification of the number of angiogenic sprouts.
- https://cdn.elifesciences.org/articles/64773/elife-64773-fig1-data1-v2.xlsx
Figure 2
Intra-nervous vascular system (INV) matures during post-natal stages with arterial differentiation and pericytes recruitment.
(A) Whole-mount immunofluorescence staining of a sciatic nerve from a connexin-40 (Cx-40)-GFP reporter mouse at P0 showing an artery composed of endothelial cells expressing the protein Cx-40 (in …
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Figure 2—source data 1
Quantification of Cx-40-GFP+ and NG2-Dsred+ coverage.
- https://cdn.elifesciences.org/articles/64773/elife-64773-fig2-data1-v2.xlsx
Figure 3
Intra-nervous vascular system (INV) density decreases during post-natal development.
(A) Immunofluorescence staining of sciatic nerve’s transversal sections from a P0 mouse and from a young adult mouse (B) showing blood vessels (CD31, magenta) and axons (TUJ-1, green) inside the …
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Figure 3—source data 1
Quantification of intra-nervous vascular system (INV) density.
- https://cdn.elifesciences.org/articles/64773/elife-64773-fig3-data1-v2.xlsx
Figure 4 with 2 supplements
Schwann cells ablation disrupt sciatic nerve vasculature.
(A) Whole-mount immunofluorescence staining of sciatic nerve from control Wnt1-Cre mouse at P5, the blood vessels are stained with CD31 and shown in red. (B) Vasculature of a sciatic nerve from a Wnt…
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Figure 4—source data 1
Quantification of nerve area and intra-nervous vascular system (INV) length and branch points in Wnt1-Cre and Wnt1-Cre;Krox20GFP(DT)/+ mice.
- https://cdn.elifesciences.org/articles/64773/elife-64773-fig4-data1-v2.xlsx
Figure 4—figure supplement 1
Schwann cells (SC) genetic ablation in Wnt1-Cre;Krox20GFP(DT)/+ mutant mice.
(A, A’) Whole-mount immunofluorescence staining of sciatic nerves from control Wnt1-Cre mouse and Wnt1-Cre;Krox20GFP(DT)/+ mutant at E19, SC expressing SOX10 are visible in white. (B) Quantification …
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Figure 4—figure supplement 1—source data 1
Quantification of nerve area and sox10+ area in Wnt1-Cre and Wnt1-Cre;Krox20GFP(DT)/+ mice.
- https://cdn.elifesciences.org/articles/64773/elife-64773-fig4-figsupp1-data1-v2.xlsx
Figure 4—figure supplement 2
Intra-nervous vascular system (INV) disruption after Schwann cells genetic ablation in Wnt1-Cre;Krox20GFP(DT)/+ mutant mice.
(A, A’) Whole-mount immunofluorescence staining of a sciatic nerves from a control Wnt1-Cre mouse and from a Wnt1-Cre;Krox20GFP(DT)/+ mouse at P0, the blood vessels are stained with CD31 and shown …
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Figure 4—figure supplement 2—source data 1
Quantification of nerve area and CD31+ area in Wnt1-Cre and Wnt1-Cre;Krox20GFP(DT)/+ mice.
- https://cdn.elifesciences.org/articles/64773/elife-64773-fig4-figsupp2-data1-v2.xlsx
Figure 5 with 1 supplement
Sciatic nerve is hypervascularized upon myelination inhibition.
(A) Schematic illustrating Krox20 knock-out leading to myelination inhibition. (B, B’) Whole-mount immunofluorescence staining of sciatic nerves from a Krox20Cre/+ control mouse and from a Krox20Cre/…
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Figure 5—source data 1
Quantification of nerve area, intra-nervous vascular system (INV) length, and branch points, Ki67+ area in Krox20Cre/Fl mice.
- https://cdn.elifesciences.org/articles/64773/elife-64773-fig5-data1-v2.xlsx
Figure 5—figure supplement 1
Krox20 protein is detectable after birth in the sciatic nerve.
(A) Krox20 Western blotting analysis in protein extracts prepared from sciatic nerves from wild-type (WT) mice at E18, P1, P2, and P8. The red box shows the band corresponding to Krox20 expression …
Figure 6 with 2 supplements
Netrin-1 and UNC5B control intra-nervous vascularization.
(A) Whole-mount X-Gal staining (showed in green) of sciatic nerve from Ntn1Lacz/+ mouse at P2 and vasculature is showed in magenta (CD31). (B) Immunofluorescence staining of β-galactosidase …
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Figure 6—source data 1
Quantification of nerve area and CD31+ area in Ntn1+/+ and Ntn1LacZ/LacZ embryos; quantification of level of expression of unc5b, neo1, and dcc by isolated endothelial cells from sciatic nerves from P2 mice; quantification of CD31+ area in sciatic nerves from pups injected with anti-UNC5B antibodies at P0.
- https://cdn.elifesciences.org/articles/64773/elife-64773-fig6-data1-v2.xlsx
Figure 6—figure supplement 1
Netrin-1 expression and sciatic nerve size from Ntn1LacZ/LacZ mice.
(A) Slice view of a cleared limb from an Ntn1LacZ/+ embryo at E16. The sciatic nerve is labelled in green (Tuj-1) and the β-galactosidase in red. (B, B’) Slice view of a cleared limb from an Ntn1LacZ…
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Figure 6—figure supplement 1—source data 1
Quantification of the average diameter of sciatic nerve from Ntn1LacZ/LacZ and Ntn1+/+ embryos.
- https://cdn.elifesciences.org/articles/64773/elife-64773-fig6-figsupp1-data1-v2.xlsx
Figure 6—figure supplement 2
Sural, phrenic, intercostal, and cutaneous nerves are devoid of intra-nervous vascular system (INV).
(A) Schematic representation of the sciatic nerve organization with its origin and different branches. In red are represented the axonal projections forming the sciatic nerve and more distally the …
Figure 7
Methodology used to quantify nerve area.
(A) Whole-mount immunofluorescent staining of a sciatic nerve showing axons in white (neurofilament [NF]) at P0. Red dotted lines represent nerve area. (B) Snapshot of a 3D visualization of a …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Mus musculus; male/female) | Krox20cre | Jackson laboratory | Egr2tm2(cre)Pch/J | B6D2 background |
| Strain, strain background (Mus musculus; male/female) | Krox20fl/+ | Decker et al., 2006;DOI: 10.1523/JNEUROSCI.0716–06.2006 | B6D2 background | |
| Strain, strain background (Mus musculus; male/female) | Krox20GFP(DT) | Vermeren et al., 2003 DOI: 10.1016/S0896-6273(02)01188–1 | B6D2 background | |
| Strain, strain background (Mus musculus; male/female) | Wnt1-Cre | Jackson laboratory | B6.Cg-E2f1tg(Wnt1-cre)2Sor/JRRID:IMSR_JAX:022501 | B6D2 background |
| Strain, strain background (Mus musculus; male/female) | Cx-40-GFP | Miquerol et al., 2004;DOI:10.1016/j.cardiores.2004.03.007 | CD1 background | |
| Strain, strain background (Mus musculus; male/female) | Ng2-DsRed | Jackson laboratory | (TgCspg4-DsRed.T1)1Akik/J | C57/Bl6 background |
| Strain, strain background (Mus musculus; male/female) | Ntn1LacZ/+ | Serafini et al., 1996;DOI: 10.1016/S0092-8674(00)81,795X | CD1 background | |
| Antibody | CD31(rat monoclonal) | BD Pharmigen | Cat# 553370, RRID:AB_394816 | (1:400) |
| Antibody | CD31(goat polyclonal) | R&D | Cat# AF3628, RRID:AB_2161028 | (1:400) |
| Antibody | Tuj-1(mouse monoclonal IgG2a) | R&D | Cat# BAM1195, RRID:AB_356859 | (1:500) |
| Antibody | Tuj-1(mouse monoclonal IgG2a) | Biolegend | Cat# 801213, RRID:AB_2728521 | (1:400) |
| Antibody | SMA-cy3(mouse monoclonal) | Sigma | Cat# C6198, RRID:AB_476856 | (1:500) |
| Antibody | Neurofilament Heavy chain (chicken polyclonal) | Abcam | Cat# ab4680, RRID:AB_304560 | (1:1000) |
| Antibody | Ki67(rabbit polyclonal) | Abcam | Cat# ab15580, RRID:AB_443209 | (1:500) |
| Antibody | Sox10(Mouse monoclonal IgG2a) | Proteintech | Cat# 66786–1-Ig, RRID:AB_2882131 | (1:200) |
| Antibody | S100β(rabbit polyclonal) | Proteintech | Cat# 15146–1-AP, RRID:AB_2254244 | (1:400) |
| Antibody | β-Galactosidase (chicken polyclonal) | Abcam | Cat# ab9361, RRID:AB_307210 | (1:500) |
| Antibody | Goat anti-Rat IgG (H + L) Alexa Fluor 555(goat polyclonal) | Thermo Fisher | Cat# A-21434, RRID:AB_2535855 | (1:400) |
| Antibody | Goat anti-Chicken IgG (H + L) Alexa Fluor 647(goat polyclonal) | Thermo Fisher | Cat# A32933, RRID:AB_2762845 | (1:400) |
| Antibody | Donkey anti-Rabbit IgG (H + L) Alexa Fluor 488(donkey polyclonal) | Thermo Fisher | Cat# A-21206, RRID:AB_2535792 | (1:400) |
| Antibody | Donkey anti-Goat IgG (H + L) Alexa Fluor 555(donkey polyclonal) | Thermo Fisher | Cat# A-21432, RRID:AB_2535853 | (1:400) |
| Antibody | Rat UNC5H2/UNC5B Antibody | Bio-Techne | AF1006 | (dose = 4 μg/g of body weight)IF (1:200) |
| Commercial assay or kit | RNAscope Multiplex Fluorescent V2 Assay | ACD | 323100 | |
| Sequence-based reagent | RNAscope Probe - Mm-Ntn1 | ACD | 407621 | |
| Sequence-based reagent | RNAscope Probe - Mm-Fap-C3 | ACD | 423881-C3 | |
| Sequence-based reagent | RNAscope Probe - Mm-Pecam1-C3 | ACD | 316721-C3 | |
| Sequence-based reagent | RNAscope Probe - Mm-Unc5b-No-XHs | ACD | 482481 | |
| Sequence-based reagent | PrimePCR Template for SYBR Green Assay: Pecam1, Mouse | Bio-Rad | qMmuCID0005317 | |
| Sequence-based reagent | PrimePCR Template for SYBR Green Assay: Dcc, Mouse | Bio-Rad | qMmuCED0051027 | |
| Sequence-based reagent | PrimePCR Template for SYBR Green Assay: neo1, Mouse | Bio-Rad | qMmuCID0011752 | |
| Sequence-based reagent | PrimePCR Template for SYBR Green Assay: unc5b, Mouse | Bio-Rad | qMmuCID0016421 | |
| Commercial assay or kit | NucleoSpin RNA Plus XS, Micro kit for RNA purification | Macherey-Nagel | REF 740990.50 |
