Patient clinical evaluation and sample collection
Patients with EvC syndrome were evaluated by echocardiography for structural heart malformations, physical exam for stature and post-axial polydactyly and by medical record review. Family history, gender and ethnicity were also noted on a multi-generation pedigree. A 10 mL intravenous blood sample was collected from each participant in acid-citrate dextrose (ACD) tubes. Informed consent was obtained for all participants. This study was approved by the Institutional Review Board of Cincinnati Children’s Hospital Medical Center.
Genomic extraction and polymerase chain reaction
The Puregene DNA Isolation Kit (Gentra Systems, Mineapolis, MN, USA) was used to extract genomic DNA from whole blood. PCR was carried out using EVC
(Supplementary Material, Table S2
) and LBN
) designed to amplify the coding sequence and flanking intronic sequence. PCR was carried out on the PTC-225 Peltier Thermal Cycler (Bio-Rad, Hercules, CA, USA) for 35 cycles of denaturation at 95°C for 30 s, primer annealing at 55–65°C for 30 s depending on the primer pair and extension at 72°C for 60 s. PCR products were electrophoresed on a 1–2% Agarose gel, and then purified with the QIAquick Gel Extraction Kit (Qiagen Inc., Valencia, CA, USA). The BigDye Terminator Cycle Sequencing Kit (v3.0) was used to generate fluorescently labeled fragments for analysis on the Applied Biosystem’s 3100 Capillary Gel Electrophoresis DNA Analyzer (Applied Biosystems, Foster City, CA, USA). Sequences were compared with consensus EVC
(AF216184) and LBN
(NM 147127) transcripts from the National Center for Biotechnology Information (NCBI). One mutation resulted in a 23 bp deletion and was verified by PCR followed by gel electrophoresis. All other mutations were verified by restriction enzyme digest. Mutation origin was traced in families when parental DNA was available. Identification of putative protein domains was based on previously reported domains (4
), or Ensembl (24
) or Motif Scan (25
Genotyping with polymorphic short-tandem repeat markers
Five microsatellite markers, D4S412, D4S3023, D4S431, D4S2366 and D4S2983, encompassing ~4.5 Mb of the EvC locus (4p16.2) were used to genotype participants in three families where the proband had a homozygous mutation. Genomic DNA was amplified with a 32P-labeled primer in a 10 µL reaction containing 4 pmols of each primer, 2 pmols dNTPs and 0.28 U of AmpliTaq Gold DNA polymerase (Applied Biosystems). Samples were denatured at 94°C for 15 s, annealed at 57°C for 30 s and extended for 45 s at 72°C for 10 cycles, then denatured at 94°C for 15 s, annealed at 57°C for 30 s and extended at 72°C for 45 s for another 25 cycles. Final extension was for 10 min at 72°C. PCR products were loaded to a 6% polyacrylimide gel and electrophoresed for 2 h and 15 min, then exposed for 3 h on a phosphoimager cassette (Molecular Dynamics, Sunnyvale, CA, USA). Manually constructed haplotypes were then examined for evidence of homozygosity.
Quantitative RT–PCR analysis of gene expression in mice
Total RNA was extracted from NIH3T3 cells and embryonic hearts using Trizol reagent (Invitrogen, Carlsbad, CA, USA) and chloroform. RNA was precipitated overnight in isopropanol and 5 µg of glycogen (Ambion, Austin, TX, USA) then quantified and diluted to 3 ng/µL. cDNA generation and PCR amplification was performed with the iScript One-Step RT–PCR Kit With SYBR Green (Bio-Rad, Hercules, CA, USA). Products were detected and quantified with the MJ Research Opticon 2 (Bio-Rad). qRT–PCR primers designed across exons to prevent amplification of genomic DNA were as follows: Evc 5′-AAGACGAAGGAGCTGCAGAAC-3′and 5′-AAGACGAAGGAGCTGCAGAAC-3′, Lbn 5′-AAGACGAAGGAGCTGCAGAAC-3′and 5′-AAGACGAAGGAGCTGCAGAAC-3′, and mL7 5′-GAAGCTCATCTATGAGAAGGC-3′ and 5′-AAGACGAAGGAGCTGCAGAAC-3′. Evc and Lbn gene expression levels were quantified with respect to control treatment using the Stealth RNAi Negative Control LO GC (Invitrogen) for gene knockdown experiments or an empty pcDNA3.1 (−) vector (Invitrogen) for overexpression studies. A standard curve was generated for all primer pairs using RNA extracted from untreated NIH3T3 cells. Standardized RNA was further normalized to amplification with the murine L7 primer pair to account for spectrophotometer error. Quantitative RT–PCR experiments were carried out three times, each time in triplicate.
Preparation of mouse tissues for in situ hybridization or immunohistofluorescence
Timed matings of C57Bl/6 mice were set up, with the morning of a vaginal mucous plug considered to be 0.5 dpc. Females were sacrificed by CO2 asphyxiation and the embryos were extracted between 9.5 and 17.5 dpc. Mouse embryos were dissected and fixed overnight in 4% paraformaldehyde/PBS at 4°C. In preparation for in situ hybridization, mouse embryos were snap frozen in Optimal Cutting Temperature (OCT) medium (Tissue-Tek, Torrance, CA, USA) and sectioned at 14 µM. Tissues to be used for immunofluorescence were dehydrated through a methanol/PBS gradient and stored in 100% MeOH at −20°C. Prior to staining, tissues were hydrated in an ethanol series, cleared in xylene and washed three times for 1 h in paraffin at 65°C, then paraffin embedded and sectioned at 8 µM. All animal procedures were carried out according to Institutional Animal Care and Use Committee (IACUC)-approved methods.
In situ hybridization
To generate antisense RNA probes, mouse Evc
(AK148262) and Lbn
(AK165051) products were first amplified from mouse whole embryo cDNA with the following primers: Evc 5′°-AGCTGTACTGTAGCACCATG-3′ and 5′-GACACTAGTGCTGGTCACAT-3′, and Lbn 5′-TACTCAGGATGGAATCCAGACT-3′ and 5′-CTCATCTCTAGGTCACACTCTG-3′. All primers were designed to produce amplicons crossing exons so only mRNA would be detected after hybridization. PCR products were cloned into the pCR 2.1-Topo Vector with the TOPO TA Cloning Kit (Invitrogen) then moved to pBluecript SK+ (Stratagene, La Jolla, CA, USA) where sequence accuracy was confirmed. Digoxigenin labeled antisense and sense probes were generated using the T7 and the T3 promoter, respectively (26
). Both Evc
sense probes were used as controls. Figure L–N and U–W are Evc
sense slides which are representative of all sense control slides.
In situ hybridization was carried out on formaldehyde-fixed, OCT-embedded tissues cut at 14 µM and mounted on Superfrost Plus microscope slides (Fisher Scientific, Pittsburg, PA, USA). Slides were brought to room temperature prior to running on the Ventana Discovery XT (Ventana Medical Systems Inc., Tuscon, AZ, USA). The colorimetric reaction was carried out using the ChromoMap Blue Kit (Ventana Medical Systems Inc). Slides were counterstained with Nuclear Fast Red. Signal was visualized with an Olympus BX60 microscope (Olympus Imaging American Inc., Center Valley, PA, USA).
Polyclonal antibody production
Mouse peptide-specific antibodies were generated to keyhole limpet hemocyanin (KLH)-conjugated oligopeptides representing either EVC or LBN (Zymed/Invitrogen Custom Antibody Production). Peptide sequences were identified based on the likelihood of the region to be on the protein’s exterior and avoidance of regions with high homology to other mouse and chicken proteins. The EVC peptide (mEvc: KLH-CKARRRQRETTRDED) was injected into goat, while the LBN peptide (mLbn: KLH-CENESSGEQAQAEQSKRRKH) was introduced to rabbits. The use of two different host species allowed for simultaneous staining of protein products.
NIH3T3 cells were grown on cover slips, rinsed twice with PBS then fixed in 3.7% formaldehyde/PBS. Following fixation, cells were again rinsed with PBS then blocked for 1 h with 2% horse serum, 2%BSA and 1% NP-40 in PBS. Cells were incubated with anti-EVC (1:100 dilution), anti-LBN (1:100 dilution), anti-acetylated Tubulin (Sigma, St Louis, MO, USA) and/or anti-gamma-tubulin (Sigma) antibodies overnight at 4°C. Cells were washed five times for 5 min with 1% NP-40/PBS before secondary antibodies, Alexafluor 568 rabbit anti-goat and Alexafluor 488 donkey anti-rabbit (Invitrogen) were used to detect EVC and LBN, respectively. TO-PRO-3 iodide (Molecular Probes/Invitrogen) was used to counter stain the nucleus. Fluorescence was visualized with a Nikon inverted Eclipse TE 300 (Nikon Instruments Inc., Mellville, NY, USA).
Paraffin-embedded heart and whole embryo sections were hydrated, rinsed with PBST, and boiled in antigen unmasking solution (Vector Labs, Burlingame, CA, USA) prior to blocking for 1 h in immunoblock (1% BSA, 0.1% cold water fish skin gelatin, 0.1% TWEEN-20 in PBS and 0.05% NaN3). After blocking, the primary antibody was added overnight at 4°C. Slides were rinsed in PBST, then incubated in the dark with the appropriate secondary AlexaFluor (Invitrogen) antibody for 1 h. After rinse, TO-PRO-3 Iodide was applied at a 1:1000 dilution for 20 min before final rinses and mounting with Vectashield Mounting Media for Fluorescence (Vector Labs). Confocal images were acquired with either the Nikon inverted Eclipse TE 300 (Nikon Instruments) or the Axio plane LSM 510 (Carl Zeiss MicroImaging, Thornwood, NY, USA). The Isl-1 antibody made by Thomas M. Jessell was obtained from the Developmental Studies Hybridoma Bank created under the auspices of the NICHD and maintained by The University of Iowa, Department of Biological Sciences. Reactions were carried out with secondary only negative controls for Evc (Anti-Goat-488 Only) and Lbn (Anti-Rb-488 Only). Figure Q–S, which is Anti-Goat-488 only, displays representative negative control slides.
Obtaining cDNA of murine Evc and Lbn
pCMV-sport6-mLbn (MGC-47318) was purchased from ATCC (Manassas, VA). The Expand Long Template PCR system (Roche Scientific) was used to amplify Evc from mouse whole embryo cDNA with primers mEvcF_LR: TGAGCCCTCCATGACCTGCACTAAAGATGC and mEvcR_LR: GTCCCAAGAGGAAGTGAGGTGCGTGCAG. The PCR product was gel purified and cloned into the pCR 2.1-Topo Vector. Cloned Evc sequence was compared with accession number AK148262. The Evc open reading frame was then cut out of Topo-mEvc with EcoRV and XbaI and ligated to pCMV-sport6 with T4 DNA Ligase (Invitrogen, Carlsbad, CA, USA) to ensure similar degrees of overexpression in cell culture.
Transfections for overexpression
NIH3T3 cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) modified with 4 mm l-glutamine and 4.5 g/L glucose (Gibco/Invitrogen), 1.5 g/L sodium bicarbonate and 10% fetal bovine serum. Media was changed to OptiMEM I Reduced Serum Medium (Invitrogen) prior to transfection with the Lipofectamine reagent (Invitrogen). Transfection was carried out according to the manufacturer’s guidelines. Briefly, plasmid purified with Qiafilter Plasmid Maxi Kit (Qiagen) was incubated with Optimem and Lipofectamine for 20 min prior to dropwise addition to cells. After 48 h, transfection efficiency was measured by counting the number of cells expressing pcDNA3.1-eGFP and dividing by the total number of cells in the field. Transcript overexpression was verified by quantitative RT–PCR.
The Stealth™ Select RNAi Card for Evc and Lbn were tested for efficacy and the following sequences were chosen for experimentation: Evc 5′-ACAAGCUGUGGAAGAAGCAAGAAUU-3′ and Lbn 5′-CCUCACAGAACAGAACUCAGCUAAA-3′. All siRNA transfections were carried out at a 100 nM concentration using Lipofectamine 2000 reagent (Invitrogen). Transfected cells were incubated for 48 h before collection in Trizol reagent. The transfection efficiency was determined by counting the number of cells containing the green labeled BLOCK-iT Fluorescent Oligo (Invitrogen) and dividing by the total number of cells in the same field. The percent transcript knockdown was determined by quantitative RT–PCR.
Significance values were Bonferroni adjusted to account for the multiple testing strategy. To achieve statistical significance the P-value had to be <0.0125. Due to non-normal distribution, overexpression data were analyzed with the non-parametric Wilcoxon sign-rank. The remaining comparisons were made with a Student’s t-test.
Cells grown in a 100 mm dish were washed twice with 1× PBS and lysed in M-PER Mammalian Protein Extraction Reagent (Pierce, Rockford, IL, USA) plus Complete protease inhibitor (Roche Diagnostics, Indianapolis, IN). After three cycles of freeze–thaw, debris was removed by centrifugation at 18 000g for 10 min and protein was stored temporarily at −20°C. For western Blot, Laemmli sample buffer (Bio-Rad) plus ß-Mercaptoethanol was added to 50 µg of protein and loaded on a 4–12% Tris–Glycine SDS gel (Invitrogen, Carlsbad, CA, USA). Gels were run at room temperature for 90–120 min at 100 V. Membranes were blocked for 1 h in 5% powdered milk in PBST and antibody was added overnight at a 1:100 dilution. Membranes were rinsed repeatedly before addition of ECL Donkey Anti-Rabbit IgG Horseradish Peroxidase linked whole antibody (Amersham, Piscataway, NJ, USA). Amersham ECL Plus Western Blotting Detection System was used to detect HRP activity (Amersham, Piscataway, NJ, USA).