The cDNA clones of Sp-Tbx2/3, Sp-IrxA, Sp-Dlx, and Sp-Chordin were obtained by PCR or cDNA library screening as previously described (Su et al.,2009). These clones were used directly for probe synthesis. For Sp-Hmx and Sp-Nk2.2, 3′RACE was performed using the FirstChoice RLM-RACE Kit (Ambion, Austin, TX). A 3′ Sp-Nk2.2 clone was successfully isolated, and a fragment containing both the partial coding sequence (CDS) and the 3′UTR was PCR amplified for probe synthesis. Because of a cluster of multiple adenosine monophosphates found in the middle of the Sp-Hmx CDS, the 3′RACE resulted in a fragment containing a partial 5′UTR and CDS that was used for probe synthesis. For Sp-Msx and Sp-Hox7, the 5′UTR sequences were extended by 5′RACE. The full-length sequence of the Sp-Klf7 transcript was obtained by 5′ and 3′ RACE, and the sequence was deposited in the GenBank with the accession number HM347349. Primers used to construct the clones for probe synthesis and the positions of the probes are listed in Supp. Table S1, which is available online. Antisense riboprobes were synthesized using digoxigenin (DIG) or fluorescein RNA Labeling Mix (Roche) with T7 or SP6 RNA polymerases (Promega, Madison, WI). For Sp-Klf7, because of its low expression level, a dinitrophenol (DNP)-labeled probe was made using the LabelIT DNP Labeling Kit (Mirus, Madison, WI).
In situ hybridization was performed as previously described (Walton et al.,2006; Yu and Holland,2009) with the following modifications. Embryos were fixed in 4% paraformaldehyde in FSW with 10 mM 4-(2-Hydroxyethyl)-1-piperazinepropanesulfonic acid (EPPS; Sigma E1894, St. Louis, MO), pH 8, at 4°C overnight. Next, they were washed with FSW and dehydrated with cold methanol. Stored embryos were rehydrated with phosphate buffer saline containing 0.1% Tween-20 (PBST) and pre-hybridized at 60°C with hybridization buffer (50% formamide, 5× SSC pH 7, 1 mg/ml of yeast RNA, 100 μg/ml of heparin, 1× Denhardt's, 0.1% Tween-20, and 5 mM EDTA). Embryos were then hybridized with DIG, fluorescein, or DNP-labeled probe overnight at 60°C. After hybridization, embryos were washed with the hybridization buffer once at 60°C for 10 min, then four times with wash solution (50% formamide, 5× SSC, 0.1% Tween-20, and 5 mM EDTA) at 60°C, followed by two MABT (100 mM maleic acid, pH 7.5, 150 mM NaCl, and 0.1% Tween-20) washes at room temperature. Washed embryos were blocked in MAB blocking buffer (1× MABT, 2% blocking reagent [Roche], and 10% sheep serum) for 1 hr at room temperature and then incubated in alkaline phosphatase (AP) conjugated anti-DIG (Roche), anti-fluorescein (Roche), or anti-DNP antibody (Abcam, Cambridge, MA) overnight at 4°C. Embryos were then washed with MABT and AP buffer (100 mM Tris, pH 9.5, 50 mM MgCl2, 100 mM NaCl, 0.1% Tween-20, and 2 mM levamisole). The AP-conjugated antibodies were detected using 190 μg/ml of 4-Nitro blue tetrazolium chloride (NBT) and 175 μg/ml of BCIP 4-toluidine salt as substrates. For double fluorescent in situ hybridization (FISH), post-hybridization washes with MABT were replaced with wash solution/2× SSCT (1:1), 2× SSCT, 0.2× SSCT, and TBST (100 mM Tris, 150 mM NaCl, pH 7.5, and 0.1% Tween-20) washes at 60°C. TBST blocking buffer (1×X TBST, 0.5% blocking reagent) was used before adding horse-radish peroxidase (POD) conjugated anti-DIG (Roche), anti-Fluorescein (Roche), or anti-DNP antibody (PerkinElmer). The fluorescent signal was amplified by the TSA Plus Cyanine 3 & Fluorescein system (PerkinElmer, Waltham, MA). Two-channel fluorescence and DIC images of the embryo were visualized and photographed using a Zeiss (Thornwood, NY) Axio Imager.A1 microscope or a Leica (Bannockburn, IL) TCS-SP5-AOBS confocal microscope.