METHOD DETAILS

Genomic DNA extracted from ear or tail biopsies was used to determine the genotype of each mouse. DreamTaq Green Master Mix was used to perform the amplification (35 cycles with an annealing temperature of 59°C). The primers used for the genotyping PCR are listed in Table S1 and their position is indicated in Figure S1B.

DRGs were collected from mutant and wild-type animals at E12.5 from both Prdm12 lines (Prdm12^+/+, Prdm12^+/−, Prdm12^−/−, Prdm12^+/LZ and Prdm12^LZ/LZ). mRNA was isolated using the RNeasy® Lipid Tissue Mini Kit (QIAGEN, Hilden, Germany), following the manufacturer’s instructions. Tissue was homogenized in QIAzol® Lysis Reagent using a TissueLyser II and optional DNase digestion was performed according to the RNeasy® Lipid Tissue Handbook (QIAGEN). Reverse transcription (RT)-PCR was performed using PrimeScript RT reagent kit manual (Takara Bio Inc., Kusatsu, Japan) employing equal amounts of RNA among samples. qPCR was performed based on the SYBR® Green System (F. Hoffmann-La Roche AG, Basel, Switzerland) using the Applied Biosystems 7500 Real-Time PCR System (Thermo Fisher Scientific, Inc.). Relative mRNA expression of TrkA/Ntrk1, Ngn1, Neurod1, Pou4f1, Rbfox3, and Prdm12 were analyzed. The obtained expression levels were normalized using the expression of Actb as reference gene (the Prdm12^+/+ values for normalized mRNA expression of each gene were arbitrarily set to 1).

Embryos were collected, decapitated and fixed in 4% paraformaldehyde (PFA) in PBS for 1–6hrs (depending on the stage) at 4°C, washed in PBS, cryopreserved in 30% sucrose in PBS and subsequently embedded in NEG-50 (Thermo Fisher Scientific, Inc.). Cryosections were collected onto Superfrost Plus slides at 14 μm thickness. Sections were washed in PBS prior to incubation in blocking solution (5% BSA, 1% normal donkey serum, 0.02% NaAz in PBS or 2% donkey serum, 0.5% Triton X-100 in PBS) for 1h at room temperature (RT). Nuclei were counterstained with DAPI (1:10.000 in PBS) and mounted in VECTASHIELD® mounting medium for fluorescence (Vector Laboratories, Inc., Burlingame, CA, US) or DAKO fluorescent mounting medium. In some cases, antigen retrieval procedure (S1699, DAKO) was applied before the blocking step. Single plane images were acquired at 20x magnification and identical settings between mutant samples and controls were used. Stainings were documented by confocal microscopy (Zeiss LSM700 and LSM800) Optical sections were 2 μm in 20X overview pictures unless specified.

In situ hybridization assay (ISH, RNAscope®, Advanced Cell Diagnostics, Inc., ACDBio, Newark, CA, US) was performed on embryo cryosections (fixed overnight in 4% PFA in PBS) at E10.5 (brachial (Br) and Lumbar (L)), E11.5, E12.5 and E18.5 on Prdm12^+/+ and Prdm12^LZ/LZ, Ngn1^+/+, Ngn1^−/−, Ngn2^+/+ and Ngn2^−/−. RNAscope® was performed using the manufacturer’s instructions (ACDBio). The following probes have been used in this study: Mm-Prdm12 # 524371, Mm-Neurog1 #414211 and Mm-Neurog2 #417291. For fluorophore labeling, the AMP 4 Alt C-FL option was employed resulting in probe labeling with Atto 550 (Figures 1B and S1G) and the subsequent immunohistochemistry (IHC) protocol described above was performed with some minor alterations (all primary antibody concentrations were 1:100). Alternatively, we used for fluorophore labeling the TSA Cy3, Cy5 and Fluorescein evaluation kit (Perkin Elmer; Figures 3E and ​and3F).3F). Images were acquired as described above.

Whole-mount immunofluorescent staining was previously described¹⁴ and performed as followed. Following samples fixation (4% PFA for 4h at 4°C with rotation 72 rpm) and PBS washes (with rotation 72 rpm, 3 times 10min), autofluorescence from the blood was beached with incubation in Dent’s bleach (one part 30% H₂O₂, two parts Dent’s fix) (Dent’s Fix: 20% DMSO, 80% MetOH) at 4°C for 24h. Next the samples were washed in MetOH at room temperature 5 times, 10min each and then fixed in Dent’s Fix solution for 24h at 4°C.

On the day of the staining, the samples were rinsed in PBS three times and then washed in PBS at room temperature with rotation. Samples were then incubated with the primary antibodies (anti-Peripherin, anti-TrkA and anti-Neurofilament 160, see Key Resources Table for details) into blocking solution (5% normal donkey serum, 20% DMSO in PBS) for a week, at room temperature with rotation (72 rpm). The samples were rinsed in PBS and washed 6 times in washing buffer (0.1% Triton X-100, PBS) for 30min each and incubated in secondary antibodies in blocking solution for 2–3 days at room temperature with rotation. Finally, the samples were rinsed then washed six times in washing buffer at room temperature, 30min each and dehydrated in 50% MetOH/PBS and then, 3 times 20min in 100% MetOH. Prior to imaging, the samples were cleared in BABB solution (one part benzyl alcohol, two parts benzyl benzoate) and imaged in BABB. Z stacks images were collected and 2D projections were created using the Zeiss LSM image processing software.

KEY RESOURCES TABLE

A control plasmid pCAGeGFP or our construct of interest pCAG-FLAG-mPRDM12-IRES-puro¹⁵, were injected in ovo into the neural tube of HHst13/14 chick embryos (plasmids concentration 1 μg/μl. Electroporation as described previously¹⁶ and performed as followed. Five pulses of 50V/cm was performed using a square wave electroporator (BTX). Embryos were collected at 96h post electroporation and processed for immunostaining. The transfection efficiency was high in both condition, eGFP and FLAG-PRDM12, as seen with the numerous eGFP⁺ and FLAG-labeled PRDM12⁺ cells in the electroporated side of the neural tube.

Sciatic nerves from E18.5 old Prdm12^−/− and control mice were used. Nerves were fixed with 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.3; over-night at 4°C) and then treated with 1% OsO4 in sodium cacodylate buffer at 4°C for 4h. Samples were embedded in epoxy resin according to the manufacturer’s (Sigma) protocol, cut and contrasted by lead citrate and uranyl acetate for ultra-thin analysis. Imaging was performed with the FEI Tecnai Spirit BioTWIN electron microscope (Thermo Fischer Scientific).