conditional knockout mice have been previously described (9
). A construct for the conditional allele of Pdcd10
was derived from genomic sequence obtained from a BAC clone (RP22 library; Invitrogen). The construct extended from a SacII site 5′ of exon 4 through an ApaI site 3′ of exon 8. The construct contained inserts as outlined in Supplemental Figure 1, including LoxP sequences at a KasI site just 5′ of exon 4 and at a EcoRI site just 3′ of exon 8. All mice were backcrossed into the C57BL/6J strain. Experiments performed prior to the fifth cross were performed with littermate controls. LacZ
reporter mice (R26R1), Tie2-Cre
mice, and PDGFb-iCreERT2
mice were generously provided by Phil Soriano (Fred Hutchinson Cancer Research Center, Seattle, Washington, USA, and Mount Sinai School of Medicine, New York, New York, USA), Masashi Yanagisawa (University of Texas Southwestern Medical Center at Dallas, Dallas, Texas, USA), and Marcus Fruttiger (University College London Institute of Ophthalmology, London, United Kingdom) via Holger Gerhardt (London Research Institute — Cancer Research UK, London, United Kingdom), respectively. HPRT-Cre
, and Tomato-EGFP
reporter mice were obtained from The Jackson Laboratory. Genotypes were determined by PCR analysis of genomic DNA isolated from either ear biopsies or yolk sac tissues using primers outlined in Supplemental Figure 1. Primer sequences were as follows: primer W: 5′-GCAATCCATCTTGTTCAATGGC-3′, primer X: 5′-CGTAGGTCAGGGTGGTCACG-3′, primer Y: 5′-TGAAAGAGGACATACTGGAGAGGC-3′, primer Z: 5′-AAGGACAAGAAAGCACTGTTGACC-3′. Tamoxifen (Sigma-Aldrich) was resuspended in corn oil (Sigma-Aldrich), and 40 ng was given as an i.p. injection to mouse pups on perinatal day 1.
MRI of mouse brains.
All MRI experiments were conducted on a 7T Bruker Biospec 70/30 USR scanner (Bruker Biospin MRI Inc.) equipped with a BGA12S gradients set. A combination of volume-transmit–only radio frequency coil (internal diameter: 72 mm) and a quad-surface-receive–only coil (internal diameter: 1.5 cm) were used. For live scans, mice were anesthetized in 2.5% isoflurane and then placed into the scanner on top of circulating heated water. During the scans, mice were monitored for temperature and respiration, with isoflurane concentration and water bath temperature adjusted to maintain a body temperature between 35.8°C and 37.6°C and respiration between 75 and 100 breaths/min. A gradient recalled echo sequence was used to acquire coronal slices spanning the whole brain. Sequence parameters were as follows: repetition time, 328 ms; echo time, 5.4 ms; flip-angle, 40°; 12 averages, in-plane-resolution, 125 μm × 125 μm; and slice thickness, 0.5 mm.
For postmortem specimens, mouse skulls were fixed in 4% formaldehyde for at least 3 days before the brain was imaged with the skull intact. Postmortem MRI scanning allows a high-resolution coverage of the brain at the expense of longer scan time. High-resolution 3D gradient echo was acquired using the same scanner configuration described above, with isotropic voxel size of 78 μm × 78 μm × 78 μm over 9 hours. Other sequence parameters were as follows: repetition time, 250 ms; echo time, 7.5 ms; flip angle, 30°; and 2 averages.
Embryo and mouse CCM lesions were studied with antibodies to PECAM (1:250 dilution, clone MEC13.3; BD Biosciences) and PDCD10 (1:50 dilution, Prestige Antibody; Sigma-Aldrich). Improved visualization on paraffin sections was obtained using a biotinylated tyramide signal amplification (TSA) kit (PerkinElmer) according to the manufacturer’s instructions. To demonstrate tissue specificity of PDFGb-iCreERT2
, brains from mice carrying a LacZ
reporter allele were fixed and then stained with X-gal as previously described (48
). Brains from mice carrying the Tomato-EGFP
reporter allele were fixed and then embedded into OCT (Fisher Scientific) for fluorescent frozen sections. H&E staining of embryos was performed using standard procedures.
For histology of adult mouse brains, mouse skulls were fixed in 4% formaldehyde for at least 3 days or zinc-buffered formalin overnight before the brain was removed and sliced into 4 coronal sections. These sections were embedded into paraffin by standard techniques. Prussian blue staining for iron and Masson’s trichrome staining of mouse brain sections were performed using the Artisan system (DAKO) according to manufacturer’s instructions. Staining for elastin, laminin, and collagen IV was performed by the Immunohistochemistry Research and Development Lab at ARUP Laboratories. Human CCM samples were stained in the same way as adult mouse brains except for CD34 staining, which was performed by the Central Labs at Intermountain Medical Center (Murray, Utah, USA).
Fluorescent staining of mouse retinas.
Mice were anesthetized using Avertin (2,2,2-tribromoethanol, 0.4 mg/g; Acros Organics) and perfused with fluorescently conjugated tomato lectin (Vector Labs) as previously described (49
) at a dose of 5 μg/g body weight. After 5 minutes, mice were perfused with saline and then with 4% formaldehyde. Retinas were then extracted, stained, and mounted as previously described (50
Analysis of mouse embryos.
Confocal immunofluorescence of embryos and fetal ultrasound were performed as previously described (9
Transmission electron microscopy.
Samples were fixed in 1% formaldehyde, 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer with 8 mM CaCl2 and 2.4% sucrose, pH 7.4, for at least 3 days. Samples were then rinsed in 0.1 M cacodylate buffer, postfixed in 2% osmium tetroxide in 0.1 M cacodylate buffer, rinsed in type I water, and then enbloc stained with saturated aqueous uranyl acetate. Samples were dehydrated through a graded series of ethanols, followed with final dehydration in acetone, and then infiltrated and embedded in an epoxy resin.
Thick sections and thin sections were cut on a Leica EM UC6 ultramicrotome (Leica Microsystems). The thick sections were cut with glass and the thin sections were cut with a diatome diamond knife. Thick sections were dried on glass slides and were stained with 1% toluidine blue O in 1% aqueous sodium borate. Thin sections were placed on 135 mesh copper hex grids and stained sequentially with saturated aqueous uranyl acetate and Reynolds lead citrate.
Sections were examined on an FEI Tecnai T-12 transmission electron microscope (FEI) at 80 KV. Images were taken on Kodak 4489 electron microscope film, developed with Kodak D-19 developer, fixed, washed, and dried. The negatives were scanned on an Epson Perfection Photo 4990 flatbed scanner.
HUVEC and HMVEC were obtained from Lonza and grown according to the manufacturer’s instructions in EGM-2 media (HUVEC) or EGM-2MV media (HMVEC). Human embryonic kidney (HEK 293T) cells (ATCC) were grown in DMEM (Gibco; Invitrogen) with 10% fetal bovine serum (Bio-West) supplemented with antibiotics. EAHY cells were generously provided by Andrew Weyrich (University of Utah) and were grown in DMEM with 10% fetal bovine serum and HAT supplement (Sigma-Aldrich).
Transfection of ECs with siRNAs.
siRNAs were obtained from Dharmacon. Luciferase GL2 duplex or nontargeting siRNA (Dharmacon) were used as controls. EC transfection with siRNAs was carried out in growth media with 1% serum. Details of the siRNA transfection protocol have been described previously (51
Total RNA was extracted from EC vasculogenesis assay at indicated time points or from siRNA-treated ECs using the ToTALLY RNA Isolation Kit (Ambion) according to the manufacturer’s instructions. RNA (1 μg) was reverse transcribed using AccuScript High Fidelity 1st strand cDNA synthesis kit (Stratagene; Agilent). RT-PCR amplification used the following primers: forward: 5′-AGACTTCACGGAGTCCCTTC-3′, reverse: 5′-AGAAGGTCTGAAGTATTAAGTTGG-3′. For quantitative real-time PCR, total RNA was extracted from cultured endothelial cells or from embryos using the NucleoSpin RNA II Kit (Clontech) according to the manufacturer’s instructions. Reverse transcription was performed with random primers using the RETROScript Kit (Ambion). Quantitative PCR was performed with TaqMan assays (Applied Biosystems) for human CCM2, PDCD10, GAPDH, STK24, STK25, and MST4 or mouse Pdcd10 and Gapdh. Quantification was performed by standard curve method, and CCM2 and PDCD10 transcripts were normalized to GAPDH for comparisons. RT-PCR primers for embryonic Pdcd10 transcripts were as follows: primer 4F: 5′-TTCACCGAGTCCCTCCTTCG-3′; primer 7/8R: 5′-GAACACATTTATTGCCTTGCCATC-3′; primer 1F: 5′-AAGTCCGTGCCTCAGTTGCC-3′; and primer EGFP-R: 5′-TCCTCGCCCTTGCTCACC-3′.
Immunofluorescent cell staining.
Glass chamber slides (Nalge Nunc; Thermo Fisher) were coated with human fibronectin (Biomedical Technologies Inc.), and transfected cells were seeded at 50,000 cells per well. Cells were fixed in 4% formaldehyde and actin cytoskeleton was visualized using fluorescently conjugated phalloidin (Molecular Probes; Invitrogen). Images were obtained with an Olympus FV300 confocal microscope.
Endothelial cell vasculogenesis in 3D collagen matrices.
This assay was performed as previously described (9
Immunoprecipitation and Western blotting.
An expressed sequence tag (EST) for human PDCD10 (IMAGE: 3050540) was obtained from ATCC and cloned into a pcDNA3.1 Hygro+ plasmid (Invitrogen) modified to encode an N-terminal FLAG tag. ESTs for Drosophila Pdcd10 and GCKIII were obtained from the Drosophila Genomics Resource Center and cloned into a pcDNA3.1 Hygro+ plasmid modified to encode an N-terminal FLAG tag (Pdcd10) or HA tag (GCKIII). PDCD10Δ18 constructs were made using nested PCR with primers designed to overlap and omit the 54 nucleotides encoding those 18 amino acids. Plasmids were transfected into EAHY or HEK 293T cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions, with a reduced dosage of DNA and lipofectamine for EAHY cells. Two days after transfection, cells were scraped into lysis buffer (50 mM Tris-HCl at pH 7.5, 100 mM NaCl, 0.5% Triton X-100) supplemented with protease and phosphatase inhibitors and 1 mM DTT. A portion of cell lysate was retained as whole cell lysate and the rest precleared with normal mouse IgG conjugated to agarose beads (Santa Cruz Biotechnology Inc.) at 4°C for 1 hour, then incubated with anti-FLAG resin at 4°C for 2 hours. The beads were washed 3 times with lysis buffer, and bound proteins were eluted using Laemmli sample buffer. Presence of FLAG-tagged PDCD10 proteins was detected using the anti-FLAG M2 antibody (Sigma-Aldrich). Presence of HA-tagged GCKIII was detected using an anti-HA antibody (Santa Cruz Biotechnology Inc.). Near-infrared secondary antibodies (LI-COR Biosciences) were used to probe the blots, which were visualized using the Odyssey system (LI-COR Biosciences).
Rabbit polyclonal antibody against PDCD10 was from Proteintech Group. Rabbit antibodies against phospho-myosin light chain (Thr18/Ser19), phospho-VEGFR2 (Tyr1175), total VEGFR2, phospho-PLCγ (Tyr783), total PLCγ, phospho-ERK1/2 (Thr202/Tyr204), and total ERK1/2 were from Cell Signaling Technology. α-Actinin antibody (clone H-2) was from Santa Cruz Biotechnology Inc. HRP-conjugated secondary antibodies were from Jackson ImmunoResearch. Western blots were developed using the ECL Plus Western blotting reagent (GE Healthcare) and Kodak Biomax MR film.
The GAL4/UAS system was used for tissue-specific RNAi knockdown (43
). The driver used was btl-GAL4
), and RNAi targeting constructs v109453 (against Pdcd10
), v106841 (against Pdcd10
), and v49559 (against GCKIII
) were obtained from the Vienna Drosophila RNAi Center. For rescue experiments, full-length cDNAs for Drosophila Pdcd10
were cloned into a pUAST transformation vector containing an attb site for site-specific transformation (52
) provided by Carl Thummel (University of Utah). Transgenes were injected into docking sites VK00027 (53
) and attp0016 (52
) by Genetic Services Inc., and homozygous lines were established by standard methods.
For cDNA rescue experiments, flies homozygous for the cDNA rescuing construct on the third chromosome were crossed to a homozygous btl-GAL4, UAS-GFP driver line on the second chromosome to generate transheterozygote flies (btl-GAL4, UAS-GFP/+; UAS-Rescue/+). Males of this genotype were then crossed to virgin females homozygous for the RNAi targeting construct located on the second chromosome. GFP expression indicated the presence of the btl-GAL4, UAS-GFP transgenes. All of these larvae contain 1 copy of the RNAi targeting construct, and half also have the cDNA rescue transgene. Results shown used the v109453 line, which targets the 3′ UTR of Pdcd10. The v106841 line showed similar results.
Analysis of tracheal tube formation in Drosophila larvae.
Animals were scored at the prewandering third instar larval stage for tracheal defects. Animals were graded by scoring air filling in primary sprouts emanating from the dorsal trunk. All primary sprouts were scored bilaterally (at least 12 per larva). The animals were categorized as having severe, moderate, or mild defects or wild-type if they had 3 or more of the following defects: (a) severe defects were truncations before the first bifurcation of the primary side branches or immediately after branching; (b) moderate defects represented gaps in air filling with filled tubes on both sides; (c) mild defects were loss of air filling only in the fine terminal tubes. In the event that more than 1 criterion was met, the more severe category was selected. If animals had fewer than 3 branches with a given defect, the less severe category was selected. Only animals with no defects were categorized as wild type.
Mouse and human experiments.
All mouse experiments were approved by the University of Utah Institutional Animal Care and Use Committee. Human tissue samples were obtained with written informed consent and provided by Connie Lee and Amy Akers (Angioma Alliance, Norfolk, Virginia, USA) and Randy Jensen (University of Utah). Human experiments were approved by the Institutional Review Board of the University of Utah.
For actin stress fiber content, and for in vitro lumen formation with GCKIII kinases, we performed ANOVA with Tukey’s post hoc analysis with an α value of 0.05. For in vitro lumen formation time course for PDCD10, we performed statistical comparisons between treatment groups with a 2-tailed paired-sample t test with an α value of 0.05. For the tracheal tube formation rescue experiment, we performed a χ2 test for independence with an α value of 0.05 (degrees of freedom = 9). For CCM penetrance and lesion content, we performed a 2-tailed t test with an α value of 0.05.