DIFFERENTIAL DISPLAY OF mRNAs FROM THE ATRIOVENTRICULAR REGION OF DEVELOPING CHICKEN HEARTS AT STAGES 15 AND 21

Department of Biological Sciences, University of Iowa, Iowa City, Iowa 52242-1324

Received 11/15/95; Accepted 11/28/95; On-line 1/1/96

Tissue Collection and Total RNA Isolation
Fertilized White Leghorn chicken eggs (Hy-Vac, Adel, Iowa) were incubated at 38°C in a humidified incubator with automatic rotation. All embryos were staged according to Hamburger and Hamilton (16). Hearts of stage 15 and stage 21 embryos were quickly dissected and tissues from atrioventricular (AV) canal region were collected and frozen in liquid nitrogen, then stored in -70°C freezer, or immediately used for isolating total RNA. Total cellular RNAs were isolated either by using Tri Reagent RNA isolation kit ( Molecular Research Center, Inc., Cincinnati, OH ) according to manufacturer's manual, or by using guanidinium thiocyanate method (17,18). Total RNAs were also prepared from both stage embryos without hearts and used for the comparison of differentially expressed genes.

Differential mRNA Display
Before use, total RNA samples were treated with RNase-free DNase I ( Promega, Madison, WI ) to remove potential chromosomal DNA contamination. About 2µg each of total RNAs from stage 15 and stage 21 AV canal tissues or embryos without hearts were used for reverse transcription. Reverse transcription and subsequent PCRs were performed as previously described (11,19) with some modifications. The PCR cycling parameters were as follows: 94°C for 45 sec, 42°C for 1 min and 30 sec, 72°C for 45 sec for 30 cycles followed by 72°C for 5 min. The PCR products together with size markers of known sequence were then separated on a 6% denaturing polyacrylamide gel. Differentially expressed gene fragments detected in either stage 15 or stage 21 AV canal tissues were identified and further compared to the displayed patterns obtained from stage 15 and stage 21 embryo tissues without hearts. Only those fragments with clear differential expression and not detected in embryos lacking hearts were selected for further cloning and characterization. The sequencing gel was run to resolve cDNA fragments sized from 200bp to 500bp. Bands of interest were cut out from the gel and cDNA fragments eluted by boiling the gel pieces in 100µl H²O for 15 min were used as templates for PCR re-amplification using the same condition as described above. A portion of the re-amplified PCR product was analyzed on a 2% agarose gel to check the efficiency of re-amplification and to confirm the size of cDNA fragments. All primers used for reverse transcription and PCR were synthesized by Oligo Etc. Inc. (Guilford, CT). Primers used included: AP1 (T₁₁CA), AP2 (T₁₁GC), AP3 (T₁₁AG), AP6 (T₁₁AA), AP8 (T₁₁AT), AR1 (CTGATCCATG), AR2 (CTTGATTGCC), AR3 (CTGCTCTCAA). AmpliTaq DNA polymerase was purchased from Perkin-Elmer Corp. (Norwalk, CT.). alpha-[³⁵S]dATP (1200Ci/mmole) was obtained from New England Nuclear (Boston, MA.).

cDNA Cloning and Sequencing
Re-amplified cDNA fragments were cloned into either pBluescript II SK (pBK) vector (Stratagene, San Diego, CA) or the pCRII vector using the TA cloning system from Invitrogen (San Diego, CA). For cloning into pBluescript II SK vector, the vector DNAs were linearized at the EcoRV site and a T residue was tailed to the blunt ends according to Marchuk et al.(20) to facilitate the direct cloning of PCR fragments. DNA sequencing was performed using Sequenase Kit Version 2.0 (United States Biochemical, Cleveland, OH). The nucleotide sequences obtained were compared with GenBank databases using the GCG (Genetics Computer Group) FASTA program software (Madison, WI).

Northern Blot Analysis
For Northern blot analysis, total RNAs were isolated from stage 15 and stage 21 chicken hearts. 10 µg of total RNA was loaded on a 0.8% formaldehyde/agarose gel. After electrophoresis, the RNA was eletrophoretically transferred to Zetaprobe nylon membrane (Bio-Rad, Richmond, CA). DNA probes were labeled either by nick translation (Promega, Madison, WI) for longer cDNA fragments or by radioactive PCR-labeling protocol (21) for shorter fragments. Hybridization and washes were performed as described previously (21).

PhosphorImager Analysis After hybridization and washing, the Northern blot membranes were exposed to a PhosphorImager plate (Molecular Dynamics, Sunnyvale, CA.). The radioactivity on the plate was scanned and analyzed by a PhosphorImager 450 (Molecular Dynamics, Sunnyvale, CA.).

Whole-Mount in situ Hybridization
For the in situ hybridization, plasmids containing cDNA fragments in either pBK or pCRII vector were used as templates for the preparation of digoxigenin(DIG)-labeled riboprobes. DIG-labeling kit was purchased from Boehringer Mannheim (Indianapolis, IN) and riboprobes were synthesized according to the manufacturer's protocols. Both sense and antisense DIG-labeled RNA probes were synthesized by the in vitro transcription of linearized DNA templates, in the presence of either SP6, T3, or T7 RNA polymerase using DIG-labeled uridine-triphosphate (DIG-UTP) as one of nucleotide substrates.

Whole-mount in situ hybridization was performed generally according to the methods of Hemmati-Brivanlou et al.(22) and Riddle et al. (23) with some modifications. Briefly, staged chicken embryos were dissected in PBS buffer and fixed in M buffer (100mM MOPS pH 7.4, 2mM EGTA, 1mM MgSO₄, and 3.7% formaldehyde) at 4°C overnight. After bleaching in M buffer with 6% hydrogen peroxide for 48 hr at room temperature, embryos were dehydrated through an ascending methanol series in PBS ( 25, 50, 75 and 100% methanol) and stored in 100% methanol at -20°C until needed. Embryos were rehydrated through a descending methanol series in PBT (PBS plus 1% Tween 20) and PBT, permeabilized in PBT containing proteinase K (µg/ml in PBT) for 30 minutes at room temperature. After post-fixing in 4% paraformaldehyde in PBT, embryos were washed three times in PBT, rinsed once each in 0.1M triethanolamine (pH 8) and in 0.1M triethanolamine plus acetic anhydride (2.5µl/ml). After incubation with prehybridization solution (0.3M NaCl, 20mM Tris pH 8.0, 1mM EDTA, 0.1M DTT, 1X Denharts, 10% dextran sulfate, 50% formamide, 250µg/ml yeast tRNA, and 100µg/ml salmon sperm DNA) for 2 hr at 50°C or 60°C, embryos were hybridized with DIG-labeled riboprobes in hybridization solution ( prehybridization solution plus 0.5-1µg/ml riboprobe ) at 50°C or 60°C overnight. After hybridization, embryos were washed two times (30 min each) with prehybridization solution at the hybridization temperature, followed by washing two times (30 min each) with 50% formamide, 2X SSC at the same temperature. Embryos were then treated with RNase A (1µg/ml) and RNase T1(1 unit/ml) for 30 min at 37°C. After a final wash with 0.2X SSC, 1% Tween 20 at 50°C or 60°C, embryos were incubated with PBT plus 10% heat inactivated sheep serum (Sigma, St.Louis, MO) for 2 hr at room temperature and then with alkaline phosphatase conjugated Fab anti-DIG solution (1 to 2000 dilution in PBT plus 10% sheep serum) overnight at 4°C. Embryos were washed at least 8 times with PBT (1 hour each) and left in PBT overnight at 4°C to completely wash out excess amounts of alkaline phosphatase conjugated Fab. Color reaction was performed by washing embryos three times for 10 min each with buffer A containing 100 mM Tris pH 9.5, 50mM MgCl₂, 100mM NaCl, 1% Tween 20, and 2mM levamisol, followed by incubating with detection solution (buffer A plus 4.5µl/ml nitroblue tetrazolium and 3.5µl/ml 5-bromo-4-chloro-3-indolyl-phosphate toluidinium ) for various times at room temperature. When the color reaction was complete, embryos were washed twice with buffer A, once with PBT, and then post-fixed in 4% paraformaldehyde in PBT. After several washes with PBT, embryos were observed and photographed with a Wild M420(Swiss) stereo microscope using either bright-field or dark-field optics with either Kodak Ektar 64 or Royal Golden 100 film. Selected embryos were embedded in Paraffin Plus (Oxford, St. Louis, MO) and sectioned at 10-15µm. After deparaffinizing, rehydrating and mounting on Super frost/Plus slides (Fisher), sections were photographed with bright-field or phase-contrast optics using a Leitz Laborlux 12 microscope ( Leitz Wetzlar, Germany ) and Kodak Royal Golden 100 or 25 films.