T3 polymerase

Digoxigenin-labelled antisense riboprobes were prepared for Gfp, Fgf8 and Hoxa13. Gfp (Aequorea victoria) is the full-length open reading frame, cloned into the pGEM T easy vector (Promega) and transcribed with SP6 polymerase (Roche); Fgf8 (Gallus gallus) is the full-length open reading frame cloned into pBlueScript and transcribed with T7 polymerase (Roche); Hoxa13 (Gallus gallus) is a partial clone (80–290 of a 290 amino acid protein) in pBlueScript and transcribed with T3 polymerase (Roche). Briefly, for in situ hybridization in tissue sections, the samples were fixed overnight in 4% PFA, dehydrated, cleared, embedded in paraffin and sectioned at 7 μm. Consecutive sections were placed on separate slides to be analysed with different probes. The sections were de-paraffined, rehydrated, mildly digested with proteinase K (10 μg ml⁻¹ for 10 min) and hybridized overnight. Sections were then washed with decreasing concentrations of SSC in 50% formamide (at 65 °C), and then blocked at room temperature with 10% sheep serum before overnight incubation at 4 °C in the standard anti-digoxigenin antibody conjugated to alkaline phosphatase (Roche,1:2,000). Finally, the staining reaction was carried out with NBT/BCIP and allowed to develop for the desired time.

Fluorescence in situ hybridization experiments were performed as previously described (Bogdanović et al., 2012 (link)). Fragments of the vsx1, vsx2, ptf1a, prdm1a, gfap, prkcbb, tfec, bhlhe40 and tal1 genes were PCR amplified from zebrafish cDNA (SuperScript IV VILO Master Mix ThermoFisher Scientific, #11756050) using specific primers (Table 1). For vsx1 and vsx2 genes, the deleted region of the coding sequence in vsxKO mutants was excluded from the amplified fragment. PCR products were cloned into StrataClone PCR Cloning vector (Agilent, #240205), linearized with XbaI restriction enzyme (Takara, #1093B) and transcribed with a DIG-labeling Kit (Roche, #11277073910) using T3 polymerase (Roche, #11031163001) to obtain digoxigenin-labeled antisense probes. Probes were used at a final concentration of 2 ng/µl diluted in hybridization buffer (Thisse and Thisse, 2008 (link)). For atoh7, a colorimetric antisense digoxigenin-labeled RNA probe was prepared as reported elsewhere (Masai et al., 2000 (link)).