The 3.0-kb genomic sequence upstream of the chick Lefty2 coding region was analyzed in silico using the software programs TRANSFAC Patch 1.0 (www.gene-regulation.com) and MacInspector Release professional 8.4.1 (www.genomatix.de) to identify potential transcription factor binding sites. The 274 bp region located between nucleotides -1709 and -1436 bp upstream the ATG (Chr3:17278480-17278753), which contains three FoxH1 binding elements, was termed the asymmetric enhancer (ASE) of chick Lefty2 gene.
Lefty2 ASE enhancer was amplified by PCR from chick genomic DNA (forward primer: 5'-CTGGAGCTCACACCCTGAATGCACCATGG-3'; reverse primer: 5'-GACACTAGTACCAGGATGAAATCTCTCCC-3') and sub-cloned into the SacI/SpeI restriction sites of the p1229-eGFP enhancer-less vector, which contains the human β-globin minimal promoter and the eGFP coding sequence. The pCAGGS-RFP vector was used as a positive control for the efficiency and extent of transfection. This vector carries the coding sequence of monomeric RFP driven by the ubiquitous CAGGS promoter.
Fertilized chicken eggs were incubated for the appropriate period in a humidified incubator at 37.5°C. Embryos were staged according to Hamburger and Hamilton (HH).
Whole-mount in situ hybridization
Chick embryos were collected at stages HH7-12, fixed overnight in 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) plus 0.1% Tween® 20 (PBT) at 4°C, dehydrated through a series of methanol solutions (25%, 50%, 75% and 100% methanol/PBT), and stored at -20°C until processed for hybridization. For this, embryos were rehydrated in PBT and bleached in 6% hydrogen peroxide in PBT for 1 h. Embryos were then rinsed 3 times in PBT, digested with proteinase K (10 μg/ml in PBT) for 5 min at room temperature, washed once in 2 mg/ml glycine/PBT and twice in PBT, and post-fixed in 4%PFA/0.2% glutaraldehyde/PBT for 20 min at room temperature. After fixation, embryos were rinsed twice in PBT and pre-hybridized for 2 h at 70°C in pre-hybridization solution (50% formamide, 5× standard saline citrate (SSC), pH 5, 0.1% Tween® 20, 50 μg/ml heparin), and incubated overnight at 70ºC in hybridization solution (pre-hybridization solution with 50 μg/ml torula RNA and 50 μg/ml salmon sperm DNA) containing 500 ng/ml of denatured riboprobe. The chick Lefty2 antisense riboprobe was generated by in vitro transcription in the presence of Digoxigenin-UTP (Roche), using the complete cDNA as template.
On the second day, embryos were washed twice in 50% formamide/4× SSC/1% SDS at 70°C and twice in 50% formamide/2× SSC at 65°C for 30 min each. Embryos were then rinsed 3 times in MABT (100 mM maleic acid, 150 mM NaCl, pH 7.5, 0.1% Tween®), blocked in 10% goat serum in MABT for 2 h at room temperature, and incubated in 1% goat serum in MABT with 1:5000 alkaline phosphatase-coupled anti-Digoxigenin antibody (Roche) overnight at 4°C. On the third day, embryos were washed 5 times in MABT for 45 min each, rinsed twice in NTMT (100 mM NaCl, 100 mM TrisHCl, pH 9.5, 50 mM MgCl2, 0.1% Tween®) for 10 min each, and incubated in BM Purple solution (Roche) in the dark for up to 12 h. Stained embryos were rinsed in PBT, fixed overnight in 4% PFA/PBT and stored in PBS at 4°C until imaging.
HH3-4 chick embryos were processed for ex ovo culture and placed inside a silicone pool containing a 2 mm square cathode (CY700-1Y electrode; Nepa Gene). After being covered with warmed Hank’s buffer (Gibco-BRL), each embryo was injected with DNA solution (3 mg/ml ASE-eGFP; 0.5 mg/ml pCAGGS-RFP; 0.1% Fast Green; Sigma) using a IM-300 microinjector (Narishige), and electroporated with 5 pulses (10 V for 50 ms at 350 ms intervals) using a 2 mm square anode (CY700-2 electrode; Nepa Gene) and a square wave electroporator (TSS20 Ovodyne; Intracel). Embryos were then transferred to 35 mm Petri dishes filled with 1.5 ml of albumen, incubated at 37.5ºC and imaged at stages HH7-11.
Whole chick embryos were observed under a Zeiss SteREO Lumar.V12 fluorescence stereomicroscope and photographed with a Zeiss MRc.Rev3 color camera and ZEN 2 Pro software (Carl Zeiss). Bright-field and fluorescence images were assembled using Adobe Photoshop (Adobe Systems).