Preparation of primary mouse brain ECs
Mouse brain microvascular fragments were processed as described previously (Liebner et al., 2000b
; Calabria et al., 2006
). Capillary fragments were seeded in culture medium (DME, 20% FCS, 100 μg/ml heparin, 5 μg/ml EC growth supplement [homemade from calf brain], 100 U/L penicillin/streptomycin, 2 mM glutamin, 1× MEM, and 1 mM Na-pyruvate) at a density of 1.5 × 104
cells/ml on collagen I–coated wells. ECs were selected for puromycin (4 μg/ml) resistance for 2 d, grown to confluency and passaged 1:2, and grown for an additional 6 d in the presence of control, Wnt3a, or Wnt5aCM. Cells were fixed with methanol for immunofluorescent staining or lysed for RNA and protein, respectively.
The lentiviral construct LefΔN-βCTA lacking the armadillo repeats with constitutive TCF–β-cat transcriptional activity was used. The day after splitting, MBEs were infected with lenti-GFP or lenti–LefΔN-βCTA (provided by K. Vleminckx, University of Gent, Gent, Belgium; Vleminckx et al., 1999
) and fixed or extracted after 1 wk. Lentiviral and packaging plasmids were provided by L. Naldini (San Raffaele Telethon Institute for Gene Therapy, Milan, Italy) and produced as described previously (Dull et al., 1998
). Adenoviral dnTCF4 was provided by S.J. George (Bristol Heart Institute, Bristol, England, UK; Quasnichka et al., 2006
). The day after splitting, one set of cells was treated with Wnt3aCM, and when they reached confluency, they were infected with Ad-GFP or Ad-dnTCF4. 72 h later, the infection cells were fixed or extracted for quantitative RT-PCR.
Conditional deletion/activation of β-cat in vivo and in vitro
ECs were treated with 50 μg/ml TAT-Cre for 60 min in DME without serum followed by 100 μM chloroquine for 30 min (Wadia et al., 2004
). The cells were washed with DME and cultured with complete medium with or without WntCM.
For β-cat deletion, in vivo Pdgfb-iCreERT2 (Claxton et al., 2008
) lines were crossed to mice harboring floxed alleles of β-cat (β-catlox/lox
; Brault et al., 2001
) to obtain homozygous floxed β-catlox/lox
pups, of which 50% also carried one Pdgfb-iCreERT2 allele. 4OHT (Sigma-Aldrich) was dissolved in absolute ethanol at 20 mg/ml, diluted to 4 mg/ml in sterile peanut oil, and injected i.p. at 20 μl/g into pups 3 d before tissue harvesting. When harvesting embryonic tissues at E13.5, the mothers were injected with 1 mg 4OHT at E11.5 and 12.5. For control experiments on promoter activity and recombination frequency, see Online supplemental material.
Evans blue permeability
A 1% sterile solution of Evans blue (Sigma-Aldrich) in PBSa was injected i.p. 18–24 h before the animals were harvested. To remove the Evans blue from the vasculature, the mice were perfused with PBSa while under terminal anesthesia. Frozen 12-μm sections of brain and 15-μm sections of skin were dried onto slides for approximately 10 min at 37°C (provided by G. Elia, Cancer Research Institute, London, England, UK). They were fixed in 4°C acetone for 2 min, air dried, dipped in xylene, and mounted in dibutyl polystyrene xylene (Hamer et al., 2002
The following mouse mAbs were used: anti–α-tubulin and anti-Cldn5 (Invitrogen), anti–β-cat (BD Biosciences), and antiactive β-cat (8E7; Millipore). Rat anti-PECAM/CD31 clone MEC13.3 mAb has been described previously (Vecchi et al., 1994
). Rat anti–VE-cad clone BV13 mAb has been described previously (Lampugnani et al., 2002
). Rat monoclonal anti–MECA-32/Plvap mAb was provided by R. Hallmann (University of Münster, Münster, Germany; Hallmann et al., 1995
The following polyclonal antibodies were used: goat anti–β-galactosidase (Abcam), rabbit anti-Cldn3 and rabbit anti–ZO-1 (Invitrogen), rabbit anti–collagen IV (AbD Serotec), biotinylated isolectin B4 (Sigma-Aldrich), and Alexa Fluor direct-conjugated isolectin 568 and 488 (Invitrogen).
The following secondary antibodies were used: appropriated antibodies and streptavidin conjugates Alexa Fluor 350, 488, 555, 568, and 633 were purchased from Invitrogen (the anti–mouse antibodies were highly cross-absorbed against nonspecific mouse proteins). For nuclear stain on brain sections, Hoechst 333258 was diluted to 1 μg/ml in PBS/0.2% Tween 20 and incubated at RT for 5 min. Alternatively, TOTO-3 (1:1,000; Invitrogen) was used.
β-Galactosidase staining of BAT-Gal embryos was performed as previously described (Liebner et al., 2004
). Hindbrains were dissected out from PFA-fixed embryos, washed in PBS, and subjected to isolectin. LacZ was either detected as reflection of the 488-nm laser or by antibody staining as indicated. Eyes were fixed in PFA for 5 min on ice, and retinas were subsequently dissected in cold PBSa. Retinas were fixed in 4°C methanol and stored at −20°C. Before staining, the retinas were placed in PBSa to rehydrate. Retinas were incubated in blocking buffer (1% BSA + 0.5% Triton X-100 in PBSa) for 1 h. Immunofluorescent staining with anti–collagen IV, Cldn5, MECA-32/Plvap, VE-cad, ZO-1, and β-cat was performed as previously described (Gerhardt et al., 2003
). Appropriate Alexa Fluor–conjugated secondary antibodies were used for detection.
Brains were isolated, placed in Tissue Tek (Sakura Finetek), and slowly frozen in liquid nitrogen vapor. 10–12-μm cryosections were fixed and stained as described previously (Liebner et al., 2000a
Flat mounted hindbrains were analyzed by fluorescence microscopy using a microscope (80i; Nikon;) with a 20× NA 0.75 or 40× NA 1.4 oil/RT objective equipped with a digital camera (DS-5Mc; Nikon). Measurements were performed by using NIS Elements software (version 3.0; Nikon). Alternatively, images were captured by confocal laser-scanning microscopy using a C1si microscope (Nikon) with a 40× NA 1.4 oil/RT objective or an LCS NT microscope (Leica). Retinas and brain sections were analyzed at RT by confocal laser-scanning microscopy using an upright microscope (LSM 510; Carl Zeiss, Inc.) with 405-, 488-, 543-, and 633-nm laser lines. The lenses used were C Apochromat 10× NA 0.45w, Plan-Neofluar 25× NA 0.80w, and C Apochromat 40× NA 1.2w. Digital zoom factor two or three was used for detail panels. Images and stacks were processed using ImageJ (National Institutes of Health) and Photoshop (CS; Adobe).
RNA preparation and quantitative RT-PCR
Total RNA from cultured cells was prepared with the RNeasy Mini kit (QIAGEN) according to the manufacturer's protocol. RNA was reverse transcribed with the Transcriptor First Strand cDNA Sythesis kit (Roche) using 1 μg of total RNA per 20-μl reaction. For real-time PCR reactions, Absolute Quantitative RT-PCR SYBR Green Fluorescein Mix (Thermo Fisher Scientific) was used as described in the protocol on a DNA Engine Opticon 2 (Bio-Rad Laboratories).
The following primers were used for identification: Cldn1 (sense 5′-GATGTGGATGGCTGTCATTGG-3′, antisense 5′-ACACCTCCCAGAAGGCAGAGG-3′), Cldn3 (sense 5′-CGTACAAGACGAGACGGCCAAG-3′, antisense 5′-CACGTACAACCCAGCTCCCATC-3′), Cldn5 (sense 5′-ATGTCGTGCGTGGTGCAGAGT-3′, antisense 5′-GCGCCGGTCAAGGTAACAAAG-3′), Cldn12 (sense 5′-CAGACAGGCTGCTTGGAGAAAC-3′, antisense 5′-AGGCAATACCACACAGGAAGGA-3′), Ocln (sense 5′-GTGAATGGCAAGCGATCATACC-3′, antisense 5′-TGCCTGAAGTCATCCACACTCA-3′), Plvap (sense 5′-GACTACGCGACGTGAGATGGA-3′, antisense 5′-AGGATGATAGCGGCGATGAAG-3′), and Axin2 (sense 5′-GCCGACCTCAAGTGCAAACTC-3′, antisense 5′-GGCTGGTGCAAAGACATAGCC-3′) and, as reference for the house keeping genes, RNA polymerase II (sense 5′-ATGAGCTGGAACGGGAATTTGA-3′, antisense 5′-ACCACTTTGATGGGATGCAGGT-3′) and glucose-6-phosphate dehydrogenase (sense 5′-GGACGACATCCGAAAGCAGAGT-3′, antisense 5′-GAATAGACGGTTGGCCTGCATC-3′). Quantitative RT-PCR conditions were 15 min at 95°C, 40 cycles of 15 s at 94°C, 30 s at 60°C, and 30 s at 72°C. For intron less genes, negative RT controls were performed to exclude the possibility of genomic background.
Western blot analysis was performed according to standard protocols. In brief, confluent cells were washed with ice-cold PBS and scraped in lysis buffer (50 mM Tris and 150 mM NaCl, pH 7.4, containing 1% Triton X-100, 1% Nonidet P-40, 0.5% sodium-deoxycholate, 0.1% sodium-dodecyl-sulfate, 1 mM phenylmethylsulfonyl fluoride, 15 μg/ml leupeptin, 71 μg/ml phenanthrolyne, and 20 U/ml aprotine [Sigma-Aldrich]). The insoluble material was removed by centrifugation at 12,000 rpm for 10 min. Alternatively, cultured cells were lysated by boiling in a modified laemli sample buffer (2% SDS, 20% glycerol, and 125 mM Tris-HCl, pH 6.8). The protein content was measured according to the bicinchoninic acid method (Thermo Fisher Scientific). Total cell lysates were separated by SDS-PAGE under reducing conditions, transfered to a nitrocellulose membrane, and analyzed by immunoblotting with specific antibodies.
Freeze fracturing on glutaraldehyde-fixed ECs was performed as previously described (Liebner et al., 2000b
). Negatives were digitized, and images were arranged in Photoshop.
Results are presented as mean ± SEM. A two-tailed Student's t test was used to analyze the difference between two groups. Values were regarded as significant at P < 0.05.
Online supplemental material
Fig. S1 shows that conditional inactivation of β-cat in ECs of postnatal mice led to down-regulation of Cldn3 and Cldn5 and increased permeability in brain capillaries. Fig. S2 shows that conditional activation of β-cat transcriptional activity induced Cldn3 only in brain vessels of early postnatal mice. Fig. S3 shows that activated β-cat can induce Cldn3 and TJ formation in a nonbrain-derived endothelioma cell line. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200806024/DC1