Digoxigenin

Unless otherwise noted, asexual planarians 1–5 mm in length were processed for WISH essentially as described [21 (link)] with the following significant modifications: the reduction step prior to dehydration was omitted. Bleaching was performed for 2 hours in formamide bleaching solution (1.2% H₂O₂, 5% formamide, and 0.5xSSC [32 ]). For regenerating planarians, the Proteinase K/post fixation steps were replaced with a 10 minute boiling step in 10 mM sodium citrate pH 6.0 with 0.05% Tween20, followed by a 20 minute room temperature incubation in PBSTx (Phosphate Buffered Saline [32 ], 0.3% Triton X-100) with 1% SDS. Blocking and antibody incubation for peroxidase-conjugated anti-digoxigenin (1:2,000 [Roche]), anti-fluorescein (1:2,000 [Roche]), and anti-dinitrophenol (1:300 [PerkinElmer]) were performed with 5% horse serum and 0.5% RWBR in TNTx (100 mM Tris pH 7.5, 150 mM NaCl, 0.3% Triton X-100). For chromogenic detection using alkaline phosphatase-conjugated anti-digoxigenin antibody (1:2,000 [Roche]), antibody incubation and blocking were performed with 5% horse serum in TNTx, and post-antibody washes were with TNTx prior to development as described in [21 (link)].

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).

DNA was obtained using the QiaAmp DNA Mini kit (Qiagen #51304) according to the manufacturer’s instructions and concentration was determined by Nanodrop (Thermo Scientific) measurement. Array comparative genomic hybridisation (arrayCGH) was performed using 105 K (amadid#019015) or 180 K (amadid#023363) Human Genome CGH Microarray slides from Agilent Technologies (Santa Clara, CA, USA) following the manufacturer’s protocols. For shallow whole genome sequencing, DNA extraction was performed on 10 µm-thick FFPE tissue sections, using the QIAamp DNA FFPE tissue kit (Qiagen, Hilden, Germany) and protocol and deparaffinization solution. Covaris’ adaptive focused acoustics technology and M220 focused ultrasonicator (Covaris, Woburn, MA) were used to prepare fragmented DNA with fragment sizes of 200 bp. DNA libraries were constructed with the NEXTflex rapid DNA-Seq kit and protocol and NEXTflex DNA barcodes (Bioo Scientific, Austin, TX) using 200 ng of fragmented DNA as starting material, size selection, and eight PCR cycles. Cluster generation and sequencing were accomplished by, respectively, a cBot 2 and HiSeq 3000 system (Illumina, Essex, UK). The minimal number of reads (single-end; 50-cycle mode) per sample was intended to be at least 10 million (mean coverage of ~ 0.15 ×).
Copy number data were processed, analysed and visualised using VIVAR^{31 (link)}. For fluorescent in situ hybridization (FISH), four-micron-thick tissue sections were cut onto positively charged slides. The unstained slides were deparaffinized in xylene and dehydrated in graded alcohols. Cell conditioning was performed in a 1 M sodium thiocyanate water bath at 80 °C for 30 min, followed by a washing step in 2 × saline-sodium citrate (SSC) buffer and an incubation step using proteinase K (Roche, Indianapolis, IN, USA) for 20 min at 37 °C. Probes for for the SOX11 locus (CTD-2037E22) was applied, following heat block denaturation at 80 °C for 5 min, and hybridization at 37 °C for 14 to 18 h. The coverslip was removed by washing in 2 × SSC buffer. Excess probe was eliminated with 0.5 × SSC buffer stringency washes, followed by similar graded stringency washes. The digoxigenin-labeled probes were visualized using fluorescein isothiocyanate-antidigoxigenin (Roche). Using 4′,6-diamidino-2-phenylindole counterstain, nucleated cells were highlighted. A microscope, equipped with a dual-pass filter (Green/Orange; Vysis) and two single-pass filters (Green; Vysis, and Orange, Vysis), was employed to ultimately observe FISH signals. SOX11 amplifications and high-level focal gains were identified as copy number segments overlapping with the SOX11 locus with log2 ratio > = 2 and > = 0.3 respectively and a maximal size of 5 Mb.