All animals were used in accordance with an animal protocol approved by the Animal Care and Use Committee of the Institute for Developmental Research, Aichi Developmental Disability Center. Timed-pregnant ICR mice were purchased from Japan SLC (Hamamatsu, Japan).
We raised an antibody against mouse SON by immunizing rabbits with a keyhole limpet hemocyanin-fused mouse SON peptide (NM_178880, residues 4-23) (Biomatik, Wilmington, DE). The other antibodies used in this study were as follows: anti-HA (6E2, 2367) (Cell Signaling Technology, Beverly, MA), anti-β-actin (AC-15, A5441), anti-SRSF2/SC35 (SC-35, SAB4200725) (Sigma-Aldrich, St. Louis, MO), anti-green fluorescent protein (GFP) (A10262), Alexa-conjugated secondary antibodies (Invitrogen, Waltham, MA), and horseradish peroxidase (HRP)-conjugated secondary antibodies (Jackson ImmunoResearch, West Grove, PA).
Short hairpin RNA (shRNA) vectors were constructed by inserting cDNAs of the following shRNAs into the pLLC vector : shRNA#1 (5’-AGGCTCAATTACTTGAAATA-3’)  and shRNA#2 (5’-GCTGAGCGTTCTATGATGT-3’) . The pCAGGS vector, kindly provided by Dr. Miyazaki in Osaka University, was engineered to express HA-tagged human SON (hSON), shRNA-resistant human SON (hSONr), or a disease-associated mutant SON (hSONm1 or hSONm2). hSONr was derived from SON cDNA containing three nucleotide substitutions within the target sequence of shRNA#1.
Neuro-2a and HEK293 cells were obtained from ATCC (Manassas, VA) and RIKEN BRC (Tsukuba, Japan), respectively. Each cell line was maintained with DMEM supplemented with 10% FBS, penicillin, and streptomycin under standard conditions. The expression plasmids were transfected with polyethyleneimine (Polysciences, Inc., Warrington, PA) or Lipofectamine 2000 (Invitrogen), according to manufacturer’s directions.
In Utero Electroporation (IUE)
Various combinations of plasmids were transfected into neural progenitors on the lateral ventricular surface of E14.5 embryos by IUE as previously described . Electroporation was performed by administering five consequent electronic pulses at an intensity of 35 V for a duration of 50 ms with 450-ms intervals using a NEPA21 SuperElectroporator (NEPA Gene, Chiba, Japan). For neuronal migration analysis, 1 mg of shRNA vector with 1.5 μg of the pCAGGS vectors harboring the various forms of human SON cDNA described above were applied. For dendritic spine formation analysis, a plasmid mixture containing 1 mg of shRNA vector with or without 0.5 μg of the hSON expression vectors was applied.
Immunocytochemistry and Immunohistochemistry
Neuro-2a cells were grown on poly-L-lysine coated glass coverslips. The cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. Immunocytochemical staining was performed with the anti-SON antibody and Alexa-conjugated secondary antibodies (Invitrogen).
Paraffin-embedded brain tissues were sectioned at a thickness of 4 mm and subjected to immunohistochemical staining and hematoxylin and eosin (HE) staining. Anti-SON immunoreactivity was visualized using EnVision (Dako, Glostrup, Denmark). All images were acquired with BX60 microscope (Olympus, Tokyo, Japan).
Neuronal Migration Analysis and Spine Density Analysis
Mice subjected to IUE were sacrificed at E18.5 or P60, and perfused with 4% paraformaldehyde in PBS. Then the mice were dissected and obtained brains were postfixed in paraformaldehyde solution for 2-24 hours. The brains were embedded in 3% agarose and sectioned at a thickness of 50-100 mm using a VT1200S vibrating microtome (Leica Microsystems Wetzlar, Germany). The coronal sections were stained with an anti-GFP antibody to visualize Son knockdown cells. The distribution of GFP-positive cells at E18.5 was examined to assess neuronal migration. For spine density analysis, dendrites of pyramidal neurons in cortical layer II/III at P60 were examined. The number of spines on each dendrite at between 30 mm and 80 mm from the soma was counted, and the spine density was represented as the number of spines per dendrite length of 10 mm. All images were acquired using a FV1000 (Olympus) or LSM880 (Carl Zeiss, Göttingen, Germany) confocal laser scanning microscope. Image processing was performed with Fiji (http://fiji.sc) and Photoshop (Adobe Systems, San Jose, CA).
Statistical significance was determined using one-way ANOVA followed by a Dunnett’s post hoc test for multiple comparisons using Prism 8 (GraphPad Software, San Diego, CA).