Adenine

The target RNA probe sequences of Cdh2^{42 (link)} and Vangl2^{43 (link)} were amplified by PCR using KOD plus (Toyobo, Osaka, Japan), adenine tailed, and inserted into the pGEM-T easy vector (Promega, Madison, WI). DNA templates were amplified from pGEM-T-Cdh2 or Vangl2 by PCR, using M13 primers and ExTaq (TaKaRa Bio, Kusatsu, Japan). Amplified samples were purified using the QIAquick PCR Purification Kit (QIAGEN, Hilden, Germany). RNA transcription was carried out using the DIG RNA labelling mix (Roche) and T7 or SP6 RNA polymerase (Roche) at 37 °C for 2 h. The products were purified using NucleoSEQ columns (Macherey–Nagel, Düren, Germany) and an equal volume of formamide was added before storage at − 20 °C.
Mouse E8.5 Embryos were fixed in 4% PFA in phosphate buffered saline (PBS) at 4 °C overnight. The embryos were dehydrated using a methanol gradient (25, 50, 75 and 100% methanol) and 1% Tween-20 in PBS (PBT) for a period of 5 min in each solution. After rehydrating samples in a 75, 50 and 25% methanol/PBT gradient, samples were washed with PBT twice. Embryos were bleached with 6% hydrogen peroxide in PBT for 1 h at room temperature followed by washing with PBT thrice. They were subsequently incubated with 20 μg/ml proteinase K in PBT for 6 min at room temperature, followed by post-fixation treatment with PBT containing 4% PFA and 0.2% glutaraldehyde for 20 min and washing with PBT twice. Embryos were next washed with a 1:1 mixture of hybridization solution (50% formamide, 1% SDS, 50 μg/ml yeast tRNA, 50 μg/ml heparin, 5 × SSC, pH 4.5)/PBT and hybridization buffer for 10 min each at room temperature. The embryos were further incubated at 70 °C in hybridization solution for 1 h, followed by replacement of the solution with fresh hybridization solution containing the RNA probe and incubated overnight at 70 °C. The next day embryos were washed with 5 × SSC, pH 4.5 containing 50% formamide and 1% SDS thrice for 30 min each at 70 °C followed by three washes with 2 × SSC, pH 4.5, containing 50% formamide for 30 min each at 65 °C and two washes with RNase buffer containing 0.5 M NaCl, 1% Tween-20 and 0.1 M Tris–Cl, pH 7.5 for 5 min. Subsequently, embryos were incubated with 20 μg/ml RNase A for 30 min at 37 °C and washed thrice with Tris buffered saline containing 1% Tween-20 (TBST) at room temperature. The buffer was then replaced with TBST containing 10% sheep serum and 1% blocking reagent (Roche) for 1 h at room temperature, followed by incubation with anti-digoxigenin-AP Fab fragments (Roche) diluted in a blocking solution overnight at 4 °C. Embryos were then washed thrice with TBST for 5 min at room temperature, five times for 1 h at room temperature, and once overnight at 4 °C. The next day, embryos were washed with 100 mM Tris–Cl, pH 9.5 containing 100 mM NaCl, 1% Tween-20 and 2 mM Levamisole thrice for 5 min at room temperature, followed by replacement of the buffer containing 250 μg/ml NBT and 125 μg/ml BCIP. Whole-mount samples were visualized using a stereomicroscope (SZ9, Olympus, Tokyo, Japan) and images were recorded using a digital camera (DP-50, Olympus). Frozen sections were prepared by embedding stained samples in OCT compound (Sakura Finetek) and immediately freezing on frosted dry ice, followed by perpendicular sectioning against the anterior–posterior axis using a cryostat (CM1850, Leica) set at 10 μm thickness at − 20 °C. The sectioned samples were visualized under an all-in-one microscope BZ-9000 (Keyence, Osaka, Japan).