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Real-Time RT-PCR. GenBank mouse and human cDNA sequences were used in PRIMER EXPRESS 1.0 (Applied Biosystems) and PRIMER 3 (www-genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi) for primer design. The following oligonucleotides were used (Quantum RNA classical 18S internal standard kit, Ambion, Austin, TX):

mWnt-1 forward, 5'-CTTCGGCAAGATCGTCAACC-3';

mWnt-1 reverse, 5'-GCGAAGATGAACGCTGTTTCT-3';

mWnt-3a forward, 5'-GAACGCGACCTGGTCTACTACG-3';

mWnt-3a reverse, 5'-GTTAGGTTCGCAGAAGTTGGGT-3';

mWnt-5a forward, 5'-aataaccctgttcagatgtca-3';

mWnt-5a reverse, 5'-tactgcatgtggtcctgata-3';

Tyrosine hydroxylase forward, 5'-AGTACTTTGTGCGCTTCGAGGTG-3';

Tyrosine hydroxylase reverse, 5'-CTTGGGAACCAGGGAACCTTG-3';

Fz8 forward, 5'-TTGGAAGTGACCTCGCTCCTAG-3';

Fz8 reverse, 5'-GGTTGGGCATGTAAGTGTAGTTGT-3';

Cyclin D1 forward, 5'-ACCCTGACACCAATCTCCTCAAC-3';

Cyclin D1 reverse, 5'-GTAAGATACGGAGGGCGCACAG-3';

Cyclin D2 forward, 5'-ACTGATGTGGATTGTCTCAAAGCCT-3';

Cyclin D2 reverse, 5'-CGTTATGCTGCTCTTGACGGAA-3';

Cyclin D3 forward: 5'-GGCTATGAACTACCTGGATCGCTA-3';

Cyclin D3 reverse: 5'-ACGGTACCTAGAAGCTGCAATTG-3';

p21 forward: 5'-AGCAAAGTATGCCGTCGTCTGT-3';

p21 reverse: 5'-TCTCAGTGGCGAAGTCAAAGTTC-3';

p27 forward: 5'-TTAATTGGGTCTCAGGCAAACTCT-3';

p27 reverse: 5'-CTAACCCAGCCTGATTGTCTGAC-3';

p57 forward: 5'-GAGGACCAGAACCGCTGGGACTT-3';

p57 reverse: 5'-ACTCGCTGTCCACCTCCATCCA-3';

Ptx3 forward, 5'-AGGGTGGACTCCTACAGATTGG-3';

Ptx3 reverse, 5'-CCGATCCCAGATATTGAAGCC-3';

c-ret forward, 5'-ATGCACAATTACAGGCTGGTTCT-3';

c-ret reverse, 5'-GTCATTGACCAGGACTACTAGCTGC-3';

NCAM forward, 5'-CACTTCGTGTTCAGGACTTCAGC-3';

NCAM reverse, 5'-GGACGAAAATGACAATGAGGATG-3';

GFRa 1 forward, 5'-GTCTGAGAATGAGATCCCCACAC-3';

GFRa 1 reverse, 5'-ACACATTGGATTTCAGCTTCTGAG-3'.

Apart from 18S, all the remaining primers were purchased from Eurogentec (Seraing, Belgium) and DNA Technology (Aarhus, Denmark).

Total RNA was isolated with the RNeasy Mini extraction kit (Qiagen, Hilden, Germany) from ventral and dorsal mesencephalic tissue obtained from embryonic day (E)10.5, E11.5, E13.5, E15.5, and postnatal day 1 (P1) rats; tissue dissected from E10.5, E11.5, E13.5, E15.5, and P1 ventral and dorsal mesencephalon; and E14, E16, E18, and P1 cortex. For reverse transcription, 1 m g of total RNA was initially treated with 1 unit of RQ1 RNase-free DNase (Promega) for 40 min. The DNase was inactivated by the addition of 1 m l of 0.02 M EDTA and incubation at 65ºC for 10 min. Random primers (0.5 m g; Life Technologies, Grand Island, NY) were then added, and the mixture was incubated at 70ºC for 10 min. Each sample was then equally divided in two tubes: a cDNA-reaction tube and a negative-control tube. A master mixture [1× first-strand buffer (Life Technologies)/0.01 M DTT (Life Technologies)/0.5 mM deoxynucleoside triphosphates (Promega)] was then added to both cDNA and reverse transcriptase (RT)– tubes and incubated at 25ºC for 10 min and then at 42ºC for 2 min. Two-hundred units of SuperScript II RT (Life Technologies) was then added only to the cDNA tubes, and all samples were incubated at 42ºC for 50 min. SuperScript II was inactivated by incubation for 10 min at 70ºC. Both cDNA and RT– were then diluted 10 times for further analysis.

Real-time RT-PCR was performed in triplicate with 1 m l of cDNA (1:10 dilution) and RT in a total volume of 25 μl. Each PCR consisted of 1Χ PCR buffer (Life Technologies) containing 3 mM MgCl₂ (Life Technologies), 0.2 mM deoxynucleoside triphosphates (Promega), 0.3 m M each of the forward and reverse primers, 1 unit of Platinum Taq DNA polymerase (Life Technologies), and 1× SYBR green (Molecular Probes). The PCR was performed with the ABI Prism 5700 detection system (Applied Biosystems) and started with 94°C for 2 min, followed by 35–40 cycles, which each consisted of 94°C for 30 s, 60ºC for 30 s , 72°C for 45 s, and 80ºC for 15 s. Other annealing temperatures included: 54ºC for Wnt-5a, 62ºC for cyclin D1, 61ºC for cyclin D2, 65ºC for p57, and 57ºC for p27. A melting curve was obtained for each PCR product after each run to confirm that the SYBR green signal corresponded to a unique and specific amplicon. The specificity of the PCR product was verified by sequencing.

Standard curves were generated in every 96-well plate for every real-time PCR run by using serial 3-fold dilutions of a reverse transcribed RNA or plasmid containing the sequence of interest for every probe. The resulting standard-curve plots were then used to convert the Cts (number of PCR cycles needed for a given template to be amplified to an established fluorescence threshold) into arbitrary quantities of initial template of a given sample.

The expression levels were obtained by subtracting the RT– value for each sample from the corresponding RT+ value and then dividing that number by the value of the housekeeping gene encoding 18S rRNA, obtained for every sample in parallel assays.

Each assay for a particular gene was repeated twice or three times in triplicate. Assays of 18S rRNA were run for each sample at the beginning and once or twice in the middle of assays to verify the integrity of the samples. The specificity of PCR primers was determined by BLAST run of the primer sequences. All PCR products were run in a 2% agarose gel to verify the size of the amplicon. The specificity of the PCR product was determined by sequencing the amplicon in random samples.

Statistical analysis of the results was performed by one-way ANOVA. A Fisher's protected least significant difference test was used post hoc to identify specific points at which the different developmental stages differed from the earliest stage only when significantly different interactions occurred. Significant difference for all tests was assumed at the level of P < 0.05 (*, P < 0.05; **, P < 0.001; ***, P < 0.0001).

Wnt Conditioned Medium (CM) Preparation, Characterization, and Purification. Rat B1A fibroblast lines stably overexpressing hemagglutanin-tagged Wnt-1a, -3a, or -5a (1) were grown in standard complete medium (DMEM/10% FBS) supplemented with 100 m g/ml geneticin. For collection of CM, cells were replated at low density in complete medium and allowed to reach 50–75% confluency, at which point cells were washed in 1× PBS, and medium was replaced with serum-free N2 (GIBCO; with 10 m M sodium butyrate) for 24 h. CM from sister flasks was then harvested, pooled as lots, and stored at –80°C for up to 2 months. Compared with fresh CM, no loss of activity was observed when using this collection and storage routine.

For concentration, individual lots of CM were thawed at room temperature, divided into 80-ml aliquots, loaded onto Centricon Plus-80 columns (Millipore), and concentrated by centrifugation according to the manufacturer’s instructions. After concentration, aliquots were repooled and frozen at –80° C after a sample was taken for determination of protein content and Western blot analysis. In brief, 20 m g of protein was loaded onto a 10% polyacrylamide minigel and run under denaturing conditions at 150 V for ≈30 min. After dry electroblot transfer onto poly(vinylidene difluoride) membranes (Hybond P, Amersham Biosciences) and preincubation for 30 min in a blocking buffer consisting of 3% BSA in Tris-buffered saline with 0.1% Triton X-100, blots were incubated with mouse anti-hemagglutinin (Babco, Richmond, CA), diluted to 1:1,000 in Tris-buffered saline with Tween 20 overnight at 4° C. After washing, blots were incubated with alkaline phosphatase-coupled goat anti-mouse IgG (Santa Cruz Biotechnology), diluted to 1:1,000 in Tris-buffered saline with Tween 20 for 1 h at room temperature. Blots were visualized by using an ECF (enhanced chemifluorescence) reagent (Amersham Biosciences), and blue fluorescence was quantitated by using the Storm 840 phosphorimager (Molecular Devices; see Fig. 6, published as supporting information on the PNAS web site).

Fz8 Cysteine-Rich Domain (Fz8-CRD) CM Preparation, Characterization, and Purification. B1A fibroblast lines were cotransfected with the mouse Fz8-CRD–IgG/pRK5 (a generous gift of J.-C. Hsieh, The Johns Hopkins University, Baltimore) and the pIRESpuro 2 (Clontech) plasmids by using Lipofectamine Plus reagent (Invitrogen) according to the manufacturer’s instructions. On selection for puromycin resistance (1 m g/ml; Sigma), clones were isolated and expanded. A clone overexpressing the Fz8-CRD 700× (real-time RT-PCR analysis) was identified. Harvesting and partial purification of Fz8-CRD CM were performed as described previously. In all blocking experiments performed, the Fz8-CRD and B1A control partial purified CM were used at a final concentration of 125 m g/ml.

1. Shimizu, H., Julius, M. A., Giarre, M., Zheng, Z., Brown, A. M. & Kitajewski, J. (1997) Cell Growth Differ. 8, 1349–1358.