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Developmental Cell, Volume 24

Supplemental Information

Peripheral Nerve-Derived CXCL12 and VEGF-A

Regulate the Patterning of Arterial

Vessel Branching in Developing Limb Skin

Wenling Li, Hiroshi Kohara, Yutaka Uchida, Jennifer M. James, Kosha Soneji,

Darran G. Cronshaw, Yong-Rui Zou, Takashi Nagasawa, and Yoh-suke

Mukouyama

INVENTORY OF SUPPLEMENTAL INFORMATION

Supplemental Figure S1, related to Figure 1:

Figure S1. Expression of Cxcl12 and its receptors Cxcr4 and Cxcr7 in the

developing DRG and limb skins

Supplemental Figure S2, related to Figures 2, 4:

Figure S2. Requirement of Cxcl12-Cxcr4 signaling for nerve-vessel alignment but

not for sensory nerve development

Supplemental Figure S3, related to Figure 2:

Figure S3. No significant morphological change in forelimb vessels, developing

heart and trunk vasculature in Cxcl12 and Cxcr4 homozygous mutants

Supplemental Figure S4, related to Figure 4:

Figure S4. Vascular branching pattern and arteriogenesis in Cxcr7 homozygous

mutants

Supplemental Figure S5, related to Figure 5:

Figure S5. VEGF-A is independent of Cxcl12-Cxcr4 signaling in promoting

arterial d ifferentiation

Supplemental Figure S6, related to Figure 6:

Figure S6. VEGF-A enhances arterial marker expression in MSS31 endothelial

cells in vitro.

Supplemental Experimental Procedures

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SUPPLEMENTAL FIGURES AND TEXT

Figure S1 (related to Fig. 1). Expression of Cxcl12 and its receptors Cxcr4 and

Cxcr7 in the developing DRG and limb skins

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(A and B) Expression of Cxcl12 and its receptors Cxcr4 and Cxcr7 mRNA was

detected by semi-quantitative RT-PCR assay. FACS-isolated limb skin

endothelial cells expressed Cxcr4 and Cxcr7, whereas DRG sensory neurons and

Schwann cells express their ligand Cxcl12 at E13.5 and E15.5. Note that increased

expression of Cxcr4 mRNA was detected from E13.5 to E15.5. (C and D)

Expression of arterial and venous markers in freshly isolated PECAM-1+/ Cxcr4

+

and PECAM-1+/ Cxcr4

- endothelial cells from E13.5 limb skins. RT-PCR

experiments indicate the arterial transcription factors Hey1 and Hey2 mRNAs

and the venous restricted genes EphB4, CoupTFII and BMX mRNAs. The data

presented are representative of three independent experiments. (E and F) Whole-

mount triple immunofluorescence analysis of limb skin from Cxcl12GFP

embryos

with antibodies to Cxcl12 (Cxcl12GFP

, green), III tubulin (Tuj1, red) and PECAM-

1 (blue) is shown. In a skin area where no association between nerves and vessels

was yet evident, nerves (specifically, Schwann cells in nerves) have already

expressed Cxcl12 (open arrows). Scale bars are 50 m. (G and H) Whole-mount

immunohistochemical analysis of limb skin from ephrinB2taulacZ/+

embryos with

antibodies to Cxcr4 (red , arrowheads), the arterial marker ephrinB2 (ß -gal, green)

and III tubulin (Tuj1, blue), is shown. Cxcr4 expression was detected in nerve-

associated ephrinB2+ arteries (arrowheads). (I-L) Cxcr4 is expressed by vessels

associating with the d isorganized nerves in Sema3A homozygous mutants.

Whole-mount immunohistochemical analysis of limb skin with antibodies to

Cxcr4 (red , arrowheads), PECAM-1 (blue) and III tubulin (Tuj1, green, open

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arrows) is shown. Cxcr4 expression was detected in nerve-associated vessels

(arrowheads) in both Sema3A-/-

mutants (J and L) and control littermates (I and K).

In Sema3A-/-

mutants the pattern of nerves is clearly d ifferent from that in control

littermates. Note that the anti-Cxcr4 antibody also stained hair follicles in the

skin (open arrowheads). Scale bars are 100 m.

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Figure S2 (related to Figs. 2 and 4). Requirement of Cxcl12-Cxcr4 signaling for

nerve-vessel alignment but not for sensory nerve development

(A-I) Whole-mount triple immunofluorescence labeling of limb skin with

antibodies to the Schwann cell marker BFABP (red , arrows), III tubulin (Tuj1,

green) and PECAM-1 (blue) in Cxcl12-/- mutants (B, E and H), Cxcr4

-/- mutants (C,

F and I), or control littermates (A, D and G) at E15.5 is shown. Normal

innervation accompanied by BFABP+ migrating Schwann cells was observed in

Cxcl12-/- and Cxcr4

-/- mutants, compared to control littermates. The numbers of

BFABP+ Schwann cells per 100µm of branched nerves (J) is shown (n=3 embryos

per genotype; bars represent mean ± SEM). (K-N) Whole-mount double

immunofluorescence labeling of limb skin with antibodies to PECAM-1 (K-L,

blue; M-N, white, arrowheads) and III tubulin (Tuj1, K-L, green, open arrows)

in Tie2-Cre; Cxcr4flox/-

and control littermates is shown. Compared to control

littermates, d isrupted nerve-vessel alignment was observed in Tie2-Cre; Cxcr4flox/-

mutants (K versus L, M versus N, arrowheads and open arrows). (O)

Quantification of the nerve-vessel alignment as the percentage of nerve-length

aligned with vessels was performed. Tie2-Cre; Cxcr4flox/-

mutants exhibit defective

nerve-vessel alignment. Asterisk indicates statistically significant d ifference

(P<0.05) in the mutants compared with control littermates according to Student’s

t-test (n=5 per genotype; bars represent mean ± SEM). (P-S) Whole-mount triple

immunofluorescence labeling of limb skin with antibodies to BFABP (red ,

arrows), III tubulin (Tuj1, green) and PECAM-1 (blue) in Tie2-Cre; Cxcr4flox/-

and

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control littermates is shown. Normal innervation accompanied by BFABP+

migrating Schwann cells was observed in Tie2-Cre; Cxcr4flox/-

, compared to control

littermates. (T) The average number of BFABP+ Schwann cells per the length of

branched nerves is shown (n=3 per genotype; bars represent mean ± SEM). Scale

bars are 100 m.

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Figure S3 (related to Fig. 2). No significant morphological change in forelimb

vessels, developing heart and trunk vasculature in Cxcl12 and Cxcr4

homozygous mutants

Triple immunofluorescence labeling of forelimb with antibodies to PECAM-1

(blue), neurofilament (2H3, green) and the smooth muscle cell marker αSMA (red ,

arrowheads) in Cxcl12-/- mutants (B), Cxcr4

-/- mutants (C) or control littermates (A)

is shown. No significant d ifference in gross morphology and d istribution of

major arteries is detected in the forelimb (A versus B and C, arrowheads). (D-F)

H&E staining of trunk sections of Cxcl12-/- mutants (E), Cxcr4

-/- mutants (F) or

control littermates (D) is shown. (G-O) Triple immunofluorescence labeling of

trunk sections with antibodies to the lymphatic endothelial cell marker LYVE-1

(blue) in addition to PECAM-1 (green) and αSMA (red) in Cxcl12-/- mutants (H, K,

N), Cxcr4-/- mutants (I, L, O) or control littermates (G, J, M) is shown. Close-up

images (J-L) show the boxed regions in G, H, I. Compared to control littermates,

gross morphology of the thoracic aorta (G versus H and I, J versus K and L,

arrowheads) and LYVE-1+ lymphatic vasculature in trunk region appeared

normal in Cxcl12-/-

and Cxcr4-/- mutants (G versus H and I, J versus K and L, blue).

In the heart, endocard ium lining and myocardial trabeculation appeared

unaffected in these mutants at E15.5 (M versus N and O, open arrowheads). Note

that based on our transverse sections through E15.5 heart, no detectable

ventricular septal defect was observed . Scale bars are 100 m.

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Figure S4 (related to Fig. 4). Vascular branching pattern and arteriogenesis in

Cxcr7 homozygous mutants

(A-D) Whole-mount double immunofluresence labeling of limb skins with

antibodies to PECAM-1 (A and B, blue; C and D, white, arrowheads) and III

tubulin (Tuj1, A and B, green, open arrows) in Cxcr7-/- mutants (B and D) or

control littermates (A and C) at E15.5 is shown. Vascular remodeling occurred

normally and many remodelled vessels aligned with nerves (A versus B, C

versus D, arrowheads and open arrows). Some smaller -d iameter vessel branches

failed to associate with nerves (B and D, arrows and open arrowheads). (E)

Quantification of the nerve-vessel alignment as the percentage of nerve-length

aligned with vessels. Asterisk indicates statistically significant d ifference (P<0.05)

in the mutants compared with control littermates according to Student’s t-test

(n=3 per genotype; bars represent mean ± SEM). (F-I) Whole-mount triple

immunofluorescence labeling of limb skins with antibodies to BFABP (red ,

arrows), III tubulin (Tuj1, green) and PECAM-1 (blue) in Cxcr7-/- mutants (G and

I) or control littermates (F and H) at E15.5 is shown. Normal innervation

accompanied by BFABP+ migrating Schwann cells was observed in the mutants,

compared to control littermates. (J) The average number of BFABP+ Schwann

cells per the length of branched nerves is shown (n=3 embryos per genotype;

bars represent mean ± SEM). (K-N and P-S) Whole-mount triple

immunofluorescence labeling of limb skins with antibodies to arterial markers

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Nrp1 (K-N, red , arrowheads) and αSMA (P-S, red , arrowheads), in addition to

PECAM-1 (K, L, P, Q, blue) and neurofilament (2H3, K, L, P, Q, green, open

arrows) in Cxcr7-/- mutants and control littermates is shown. The expression of

Nrp1 (K versus L, M versus N, arrowheads) and αSMA+ smooth muscle cell

coverage appeared unaffected in Cxcr7-/- mutants (P versus Q, R versus S,

arrowheads). Quantification analysis of Nrp1 expression (O) or αSMA+ smooth

muscle cell coverage (T) in small-d iameter branched vessels was performed (n=3

per genotype; bars represent mean ± SEM). Scale bars are 100 m.

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Figure S5 (related to Fig. 5). VEGF-A is independent of Cxcl12-Cxcr4 signaling

in promoting arterial differentiation

(A) Schematic illustrating experimental procedure of endothelial cell isolation

from E10.5-E12.5 Cxcr4-/-

embryos and arterial d ifferentiation assay in culture. (B-

D) PECAM-1+/ ephrinB2

- endothelial cells isolated by flow cytometry from Cxcr4

-

/- mutant embryos were cultured in 10ng/ ml bFGF (B) or with 10ng/ ml VEGF-A

plus 10ng/ ml bFGF (C and D) for 2 days, followed by double-staining with anti-

ephrinB2 (green) and anti-PECAM-1 (red) antibodies. VEGF-A can promote

arterial d ifferentiation in Cxcr4 deficient endothelial cells (C and D, green,

arrows). Note that PECAM-1 appears to be expressed at d ifferent levels in

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endothelia cells in cu lture (B), and PECAM-1 staining is relatively weak in

ephrinB2+ endothelial cells (C and D). Scale bars are 100 m.

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Figure S6 (related to Fig. 6). VEGF-A enhances arterial marker expression in

MSS31 endothelial cells in v it ro.

Schematic illustrating experimental procedure for arterial d ifferentiation assay

for MSS31 endothelial cells (A). MSS31 cells were cultured with 10ng/ ml or

30ng/ ml VEGF-A for 1 day, followed by FACS analysis with Alexa647-

conjugated anti-ephrinB2 antibody. VEGF-A stimulates ephrinB2 expression in

MSS31 endothelial cells. Comparison of mean fluorescence intensity of ephrinB2

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expression is shown (B). The data presented are representative of three

independent experiments.

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SUPPLEMENTAL EXPERIMENTAL PROCEDURES

RT-PCR

Total RNA was extracted from freshly-isolated endothelial cells from E13.5 and

E15.5 limb skin as well as E13.5 DRG using Trizol Reagent (Invitrogen) and then

reverse-transcribed into first-strand cDNA with SuperScript III first-strand

synthesis supermix kit (Invitrogen) according to manufacture’s instruction. The

results of mRNA expression of Cxcl12, Cxcr4 and Cxcr7 were confirmed by semi-

quantitative RT-PCR. mRNA expression of Hey1, Hey2, EphB4, COUTFII and

BMX was also detected . mRNA expression of GAPDH was used as an internal

control. Primer sequences used for PCR are: 5′-CCA TGG AGA AGG CTG GGG-

3′ (sense) and 5′-CAA AGT TGT CAT GGA TGA CC -3′ (antisense) for GAPDH;

5’-GTT CTG GAG ACT ATG ACT CC -3’ (sense) and 5’-CAC AGA TGT ACC

TGT CAT CC-3’ (antisense) for Cxcr4; 5’-GGT CAG TCT CGT GCA GCA TA-3’

(sense) and 5’-GTG CCG GTG AAG TAG GTG AT-3’ (antisense) for Cxcr7; 5’-

CAC TCC AAA CTG TGC CCT TCA-3’ (sense) and 5’-CAC TTT AAT TTC GGG

TCA ATG C-3’ (antisense) for Cxcl12; 5’-GAA GCG CCG ACG AGA CCG AAT

CAA-3’ (sense) and 5’-CAG GGC GTG CGC GTC AAA ATA ACC-3’ (antisense)

for Hey1; GTG GGG AGC GAG AAC AAT TAC CCT GG (sense) and TGC TGA

GAT GAG AGA CAA GGC GCA CG (antisense) for Hey2; 5’-CAG GTG GTC

AGC GCT CTG GAC-3’ (sense) and 5’-ATC TGC CAC GGT GGT GAG TCC-3’

(antisense) for EphB4; 5’-AAG CTG TAC AGA GAG GCA GGA-3’ (sense) and 5’-

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AGA GCT TTC CGA ACC GTG TT-3’ (antisense) for COUPTFII; 5’-GCA ACA

TAC GCT ATA TTC CA-3’ (sense) and 5’-AAG CAT GCA GAT TTT CCT CT-3’

(antisense) for BMX .

Whole-mount immunohistochemistry of limb skin

Forelimb skin tissue was d issected from embryos (E13.5~E15.5), fixed in 4%

paraformaldehyde/ PBS at 4°C overnight, and dehydrated in 100% methanol at -

20°C. Staining was performed using anti-GFP antibody (rat monoclonal antibody,

Nacalai Tesque, 1:1000 overnight at 4°C) to detect Cxcl12, anti-PECAM-1

antibody (rat monoclonal antibody, clone MEC13.3, BD Pharmingen, 1:300

overnight at 4°C) to detect endothelial cells; anti-Nrp1 antibody (rabbit

polyclonal antibody, A.L. Kolodkin, 1:3000, overnight at 4°C) and anti-Cx40

antibody (rabbit polyclonal antibody, Alpha Diagnostic Intl. Inc., 1:300, overnight

at 4°C) as arterial endothelial cell markers; Cy3-conjugated anti-αSMA antibody

(mouse monoclonal antibody, clone 1A4, Sigma, 1:500, 1 hr at room temperature)

to detect smooth muscle cells; anti-Neurofilament antibody (mouse monoclonal

antibody, clone 2H3, Developmental Studies Hybridoma Bank, 1:200, 1 hr at

room temperature) and anti-β-tubulin(βIII) antibody (mouse monoclonal

antibody, clone TuJ1, Covance, 1:500, 1 hr at room temperature) to detect nerve

fibers; anti-BFABP antibody (rabbit polyclonal antibody, T. Müller, 1:1000,

overnight at 4°C) to detect Schwann cells; anti-Cxcr4 antibody (rabbit polyclonal

antibody, Biotrend , 1:10 overnight at 4°C) and anti-Cxcr7 antibody (rabbit

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polyclonal antibody, Abcam, 1:300, overnight at 4°C) to detect Cxcr4 and Cxcr7

expressions separately. For immunofluorescent detection, eith er Alexa-488-,

Alexa-568-, Cy3- or Dylight 649-conjugated secondary antibodies (Invitrogen

1:250 or Jackson, 1:300, 1 hr at room temperature) were used . All confocal

microscopy was carried out on a Leica TCS SP5 confocal (Leica).

The quantification of degree of alignment/ misalignment shows the

percentage of total nerve length aligned with blood vessels (the proximity gap

between nerve and vessel should be less than 10µm). Correctly recognized nerve

length was measured as a total manually traced lengths of visible nerves using

NIH imageJ software.

The numbers of BFABP+ Schwann cells were normalized to the total cells

per 100 µm of branched nerves.

NIH imageJ software was used to quantify fluorescence signal intensity.

The vessel area is selected using the freehand selection tool and the mean value

as an indicator of fluorescence intensity is obtained . The d isplayed arbitrary

fluorescence units (average mean fluorescence) are representative of four

embryos.

Number of embryos is ind icated as “n” in the FIGURE LEGENDS. In

statistics, comparisons between the means of two independent group s were

made by 2-tailed Student’s t test.

Section immunohistochemistry

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Embryos were fixed with 4% paraformaldehyde/ PBS at 4°C overnight, sunk in

30% sucrose/ PBS at 4°C and then embedded in OCT compound. Embryos were

cryosectioned at 10-12µm thickness and collected on pre-cleaned slides

(Matsunami, Japan). Staining was performed using anti-PECAM-1 antibody to

detect endothelial cells; Cy3-conjugated anti-αSMA antibody to detect smooth

muscle cells; anti-LYVE-1 antibody (rabbit polyclonal antibody, Abcam, 1:200,

overnight at 4°C) to detect lymphatic endothelial cells; and anti-Neurofilament

antibody to detect nerve fibers. For immunofluorescent detection, either Alexa -

488-, Alexa-568-, Cy3- or Dylight 649-conjugated secondary antibodies

(Invitrogen 1:250 or Jackson, 1:300, 1 hr at room temperature) were used . All

confocal microscopy was carried out on a Leica TCS SP5 confocal (Leica).

Flow cytometry and culture methods

The isolation and culture of ephrinB2-negative, eprhinB2-negative Cxcr4 positive,

ephrinB2 negative Cxcr4 negative endothelial cells from E11.5 ephrinB2lacZ/+

embryos and ephrinB2-negative endothelial cells from E10-11.5 Cxcr4 mutant

embryos were performed as described previously (Mukouyama et al., 2005;

Mukouyama et al., 2002). All cell sorts and analyses were performed on a MoFlo

(Beckman Coulter, Fort Collins, CO.). The freshly isolated endothelial cells were

plated on type 4 collagen coated 35 mm dishes (BD Bioscience) with cloning

cylinders (6x8 mm, Fisher). The culture medium contained EBM-2 (Clonetics)

with 15% FBS (Hyclone Laboratories), Penicillin/ Streptomycin (Gibco), and 10

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ng/ ml bFGF (NCI BRB Preclinical Repository). The endothelial cells were

challenged with d ifferent concentrations of VEGF164

(NCI BRB Preclinical

Repository, 1-10 ng/ ml) and Cxcl12 (R&D systems, 100-500 ng/ ml). Cultures

were incubated for 2 days in a reduced oxygen environment to more closely

approximate physiological oxygen levels using a gas-tight modular incubator

chamber (Billups-Rothenberg), which was flushed daily with a custom gas

mixture of 5%CO2/ 1%O

2. After 2 days, cultures were fixed with 0.25%

glutalaldehyde/ PBS for X-gal staining and/ or anti-ephrinB2 antibody (rat

monoclonal antibody, N. Takakura) immunohistochemistry as previously

described (Mukouyama et al., 2005; Mukouyama et al., 2002). The ephrinB2-lacZ-

positive or negative cells were counted in PECAM-1 positive cells and statistical

significance was assessed using the Student’s t-test.

The expression of arterial and venous surface markers in PECAM-

1+/ Cxcr4

+ and PECAM-1

+/ Cxcr4

- endothelial cells from E13.5 limb skins was

analyzed on the BD LSRII workstation (BD Bioscience). The d issociated skin cells

were stained with d irectly conjugated antibodies to ephrinB2 (Alexa 647; the

antibody was conjugated to Alexa647 by Alexa Fluor 647 Monoclonal Antibody

Labeling Kit; Molecular Probe) in addition to PE-conjugated anti-PECAM-1 and

FITC- or APC- conjugated anti-Cxcr4 antibodies.

Chemotactic migration assay

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Chemotactic migration assay was performed with a 48-well modified Boyden

chamber (NeuroProbe) using a polycarbonate membrane with a 8 µm pore size.

The membrane was coated with 10 ng/ ml fibronectin (Biomedical Technologies)

overnight at 4°C. In the upper chamber, starved , non-treated or treated MSS31

endothelial cells were treated with anti-IgG2b subtype control (20 ng/ µl, R&D

systems), neutralizing antibody against CXCR4 (20 ng/ µl, R&D systems), CXCR4

antagonist AMD3100 (1µg/ µl, Sigma) or G-protein inhibitor Pertussis toxin (PTX,

2 ng/ ml, Sigma). In the bottom chamber, the DRG supernatant, CXCL12 knock-

down DRG supernatant, CXCL12 (300ng/ ml, R&D systems), VEGF-A (10ng/ ml,

R&D system), Flt1-Fc (VEGFR1-Fc, 100ng/ ml, R&D system) or IgG1 subtype Fc

control (100ng/ ml, R&D system) was added. After 6 hr incubation at 37°C, the

chamber was d isassembled , and cells were removed mechanically by Q -tip from

the upper side of the filter membrane. The membrane was fixed in 100%

methanol, and stained with hematoxylin solution. The number of cells that

migrate to the bottom side was counted , and statistical significance was assessed

using the Student’s t-test.