Fertilized chicken eggs were purchased from Case Western Reserve University Squire Valleevue and Valley Ridge Farms and the Department of Animal Sciences at Ohio State University, so no animal husbandry was required on our part. Only early stage, pre-hatch chicken embryos were used for these studies, which are not subject to federal regulation and do not require approval from the Cleveland Clinic Institutional Animal Care and Use Committee. All embryos used in this study were taken from stages prior to the development of a differentiated nervous system, so no special measures to minimize pain and distress were needed.
Explants, siRNA transfection, and treatments
Fertilized Babcock B-300 (Case Western Reserve University Squire Valleevue and Valley Ridge Farms) or Hy-line W-36 (Ohio State University) White Leghorn chicken eggs were incubated in 50% humidity at 100°F until desired stages were reached. Embryos were staged according to Hamburger and Hamilton . Collagen gels were prepared as previously described .
For siRNA experiments, AVC or OFT regions were excised, transfected, and boosted with anti-MBNL1 or siGLO control siRNAs as previously described . Knockdown of MBNL1 transcripts with anti-MBNL1 siRNA is typically ~60% relative to mock-treated explants . For anti-TGFβ antibody experiments, M199+ alone [1X Medium 199 (Mediatech) supplemented with 1% each ITS (Invitrogen), pen/strep antibiotic solution, and chick serum] or 10 μg/ml anti-TGFβ antibody diluted in M199+ [anti-TGF-β1, -β2, -β3 pan antibody (R&D Systems, catalog no. MAB1835), anti-TGFβ2 antibody (R&D Systems, catalog no. AB-112-NA), or anti-TGFβ3 antibody (R&D Systems, catalog no. AF-243-NA)] was added to explants. For exogenous growth factor experiments, 0, 5, 25, 50, 100, or 200 ng/ml active human recombinant TGFβ3 (R&D Systems, catalog no. 243-B3) was diluted in serum-free M199.
To measure invasion, AVC explants were fixed with 4% paraformaldehyde for 45 min at room temperature and invaded cells (i.e., cells wholly within the gel matrix) were counted using a Leica DMIRB microscope fitted with Hoffman Modulation Contrast optics. To measure EMT in OFT explants, both activated cells (i.e., isolated cells on the surface of the gel) and invaded cells were counted. When determining the percentage of explants with significant invasion, a threshold of three invaded cells was used, consistent with earlier studies [7, 10, 23, 24]. OFT explants were imaged using QCapture Pro 6.0 software.
Enzyme-linked immunosorbent assay (ELISA)
M199+ was added to explants and medium was collected after conditioning for 12 or 20 hours as indicated. Secreted TGFβ levels in the conditioned media were measured by sandwich ELISA. 96-well plates were coated with 2 μg/ml of a pan-TGFβ capture antibody (R&D Systems, catalog no. MAB1835) diluted in phosphate buffered saline (PBS), overnight at room temperature. Plates were washed five times with PBS + 0.05% tween-20 and blocked in PBS + 1% BSA for one hour at room temperature. After five more washes, 100 μl of sample (conditioned supernatant diluted 1:1 in PBS + 1% BSA) was added per well. A serial dilution of rTGFβ3 (R&D Systems, catalog no. 243-B3) or rTGFβ2 (R&D Systems, catalog no. 302-B2) was used to generate a standard curve. M199+ alone was diluted 1:1 in PBS + 1% BSA to establish the baseline level in unconditioned medium. Samples were incubated for two hours at room temperature. Plates were washed five times, and 0.4 μg/ml biotinylated anti-TGFβ3 (R&D Systems, catalog no. BAF243) or anti-TGFβ2 (R&D Systems, catalog no. BAF302) detection antibody diluted in PBS + 1% BSA was added. Plates were incubated two hours at room temperature, then washed five times. Streptavidin-HRP (R&D Systems, catalog no. DY-998) was diluted 1:200 in PBS + 1% BSA, added to wells, and incubated for 20 min at room temperature in the dark. Plates were washed five times, and 1-Step Slow TMB-ELISA substrate (Pierce) was added. Plates were incubated at room temperature until a strong color reaction was observed in the standards, usually about 20 min, then the reaction was stopped by addition of 1 M H2SO4. Absorbances were read at 450 nm and concentrations were calculated from the standard curve. Baseline measurements from M199+ alone were subtracted from conditioned supernatant measurements prior to comparison between experimental samples, except for values shown in Figure 4 where absolute measurements were compared directly to baseline levels in M199+ alone.
After 38 hrs in culture, AVC explants were fixed in 4% paraformaldehyde for 45 min at room temperature. Explants were washed twice with PBS and then incubated in permeabilization/blocking solution (3% BSA and 0.2% Triton X-100 in PBS) for 30 min at room temperature. Explants were incubated in 10 μg/mL monoclonal mouse anti-TGFβ3 (R&D Systems, catalog no. MAB463) in 3% BSA-PBS overnight at 4°C. The explants were washed once with PBS and incubated in FITC-conjugated goat anti-mouse secondary antibody (Jackson ImmunoResearch Laboratories, catalog no. 515-095-003) diluted 1:200 in 3% BSA-PBS for 2 hrs at room temperature. Explants were washed twice with PBS and counterstained (5 μg/mL Hoechst 33342 and 0.4 μg/mL DAPI in PBS) for 30 min at room temperature (Hoechst/DAPI staining not shown). Following a final wash with PBS, explants were mounted using VECTASHIELD Hard-Set Mounting Medium (Vector Laboratories) and imaged on a Leica DMIRB microscope using QCapture Pro 6.0 software.
To evaluate TGFβ3, ACTA2, and FBN2 expression in explants, the myocardium was removed and total RNA was harvested from the remaining endocardial monolayer and endocardially-derived mesenchyme. To evaluate MBNL1 levels during development, total RNA was harvested from AVC segments excised from stage 14, 15, 16, 18, and 23 hearts. RNA was extracted using Trizol Reagent (Invitrogen) and quantified using a NanoDrop 1000 (Thermo Scientific). At least five explants or AVC segments were pooled per sample, and each pooled sample was treated as a single biological replicate in real-time RT-PCR analyses. For each pooled sample, 1 μg RNA was converted to cDNA using the SuperScript VILO cDNA Synthesis Kit (Invitrogen) and cDNA was quantified using the Quant-iT OliGreen ssDNA Assay Kit (Invitrogen). Real-time PCR reactions were carried out using TaqMan Gene Expression Assays on the StepOnePlus platform (Life Technologies), with the following reagents: 1X TaqMan Gene Expression Master Mix, 0.25X TGFβ3 (Gg03371524_m1), 0.25X ACTA2 (Gg03352404_m1), or 0.25X FBN2 (Gg03323683_m1) FAM-labeled probe duplexed with 1X GUSB VIC-labeled probe (Gg03358465_m1; Primer Limited), and 5 ng cDNA. Each sample was run in triplicate and normalized to beta glucuronidase (GUSB), which did not vary in response to treatments or between developmental stages. Relative expression values for biological replicates were standardized using the method described by Willems et al. prior to statistical analysis .
Embryos were fixed in 4% paraformaldehyde in PBS at 4°C overnight and dehydrated the next day by sequentially washing with 25, 50, 75, and 100% methanol in PBSw (PBS with 0.1% Tween-20). To embed, embryos were incubated in the following sequence of solutions: isopropanol at room temperature for 15 min, fresh isopropanol at 68°C for 15 min, isopropanol/paraplast (1:1) at 68°C for 30 min, liquid paraplast at 68°C twice for one hour each and once for 30 min, and solidified at room temperature. Ten micron thick sagittal sections mounted on charged slides were de-waxed and rehydrated in xylene and a 25, 50, 75, and 100% ethanol series then rinsed in 2X SSPE (300 mM NaCl, 20 mM NaH2PO4, 2 mM EDTA, pH 7.4). Slides were re-fixed in 4% paraformaldehyde in PBSw at room temperature for 15 min, rinsed in 2X SSPE, incubated in Proteinase K (3 μg/ml in PBSw) at 37°C for 30 min, and rinsed in 2X SSPE. Slides were incubated in 0.2 M HCl in PBSw at room temp for 15 min and rinsed in 2X SSPE. Slides were then incubated in a humid chamber with hybridization buffer [1% boehringer block (Roche), 50% formamide, 5X SSC (0.75 M NaCl, 0.075 M sodium citrate, pH 7.0), 12% DEPC water, 1 mg/ml Torula RNA (Roche) filtered, 0.1 mg/ml heparin in 1X SSC, 5 mM EDTA, 0.1% Tween-20, 0.1% CHAPS] at 65°C for 2 hrs after which they were incubated with RNA probe in hybridization buffer diluted 3:1000 at 65°C overnight. Sense and antisense MBNL1 probes were previously described . The next day, slides were rinsed in 2X SSPE before being incubated in hybridization buffer at room temp for 5 min. Sections were then incubated in post-hybridization solution [5 ml hybridization buffer, 4.7 ml 2X SSPE, 0.3 ml 10% CHAPS] at room temp for 10 min, and then 0.3% CHAPS in 2X SSPE at room temp for 20 min. Slides were soaked in 2X SSPE at room temp for 20 min, and then rinsed with PBSw at room temp three times for 10 min each. Sections were incubated in Antibody Buffer (PBS with 10% heat inactivated goat serum, 1% boehringer block, 0.1% Tween-20) at room temp for 2 hrs. During this time, Antibody Buffer was pre-blocked with anti-dig AP fab fragments (Roche) diluted 1:1000 rocking at 4°C. Slides were then incubated in the pre-blocked Antibody Buffer at room temp for 1 hr. Sections were rinsed with 0.1% BSA in PBSw at room temp three times for 10 min each, then in AP1 Buffer (0.1 M NaCl, 0.1 M Tris pH 9.5, 50 mM MgCl2) at room temperature for 10 min. Slides were stained with BM Purple (Roche) at 4°C in the dark until desired strength of staining was reached. Slides were then placed in Stop Solution (100 mM Tris pH 7.4, 1 mM EDTA) at 4°C in the dark for 15 minutes before being dehydrated in a 25, 50, 75, and 100% ethanol series and xylene and finally mounted with Permount (Fisher Scientific). Sections were imaged on a Leica DM2500 microscope using QCapture Pro 6.0 software. Brightness, contrast, and color balance were adjusted using Adobe Photoshop software to match the appearance of the sections when viewed through the microscope by eye.
Data are reported as the mean plus the standard error of the mean unless otherwise noted. Real-time RT-PCR data are reported as the mean flanked by the upper and lower 95% confidence intervals. Comparisons between means were performed via one-tailed t-tests assuming unequal variances. Comparisons between proportions of explants with significant invasion (Figure 5) were performed via z-test. Differences were considered statistically significant when P ≤ 0.05.