Whole-mount in situ Hybridization and Immunostaining of Tadpoles

Whole-mount in situ hybridization and cryosectioning of hybridized tadpoles were performed as previously described^{42 (link)–44 (link)}. Tadpoles were fixed in MEMFA solution for 2 h at room temperature and stored in 100% ethanol at − 20 °C. For anti-sense and sense probes, pDH105-inhba was linearized by BamHI and XbaI and subjected to in vitro transcription by T3 and SP6 RNA polymerases (Roche), respectively. For the whole-mount immunostaining, tadpoles were fixed in MEMFA solution for 30 min at room temperature and stored in 100% methanol at − 20 °C. Whole-mount immunostaining was carried out following our standard protocol for pH3 staining^{15 (link)} and with a minor modification for pSmad2/3 staining^{17 (link)}. The following antibodies were used at 1:500 dilution: anti-pH3 antibody (Upstate Biotechnology), anti-pSmad2/3 antibody (Cell Signaling Technology), Alexa Fluor 488 goat anti-rabbit antibody (Molecular Probes, Thermo Fisher Scientific), and Alexa Fluor 488 goat anti-mouse antibody (Molecular Probes, Thermo Fisher Scientific).