Methods

Animals
Animal experiments were approved by the Animal Experiment Committee of the RIKEN Brain Science Institute (approval no. H22-EP068), and the mice were maintained by the institute's Research Resource Center. Mutant mice heterozygous for the Zic2kd allele (Zic2kd/+) were described previously8912. Zic2kd was generated by the insertion of the neomycin resistant cassette into an intron of mouse Zic2, resulting in the 20% of the wild-type allele8, and were maintained in C57BL/6J background.

Behavioral tests
Home cage activity measurement, open field test, classical fear conditioning, Y-maze test were done as described5051.

Resident-intruder test
Group-reared mice were kept in isolation for 5 days before the test. The test was carried out in a dark phase (0:30 to 2:30) in a chamber that keeps the cage under dim (infrared) light at 25°C. Video recording from two opposite directions was initiated once the intruder mice had been gently placed in a vacant spot in the cage of the resident mice. The behaviors of the resident mouse were recorded for 10 min. The duration and number of times the resident mice spent sniffing, in active contact with, and in pursuit and attack with the intruder mice were measured by observers who were blinded to the genotypes of the mice.

Social dominance tube test
A wild type and a Zic2kd/+ mice were placed in a head-to-head position first at the opposite ends of a clear plexiglass tube (3.4 cm inner diameter, 30 cm in length) in which two shutter plates were inserted at a distance of 13 cm from each end. The tests were begun by removing the shutters and ended when one mouse completely retreated from the tube. The mouse that retreated first was designated as the loser, and the remaining mouse was judged as the winner. The maximal test time was set to 2 min.

Magnetic resonance imaging (MRI) based volumetric analysis
MRI images of the adult male mice were acquired by subjecting anesthetized mice to an MRI scan using a vertical bore 9.4-T Bruker AVANCE 400WB imaging spectrometer (Bruker BioSpin, Rheinstetten, Germany). Animals were anesthetized with 3% and 1.5% isoflurane in air (2 L/min flow rate) for induction and maintenance, respectively. MRI images were obtained by using the FISP-3D protocol of Paravision software 5.0, by setting the following parameter values: Effective TE = 4.0 ms, TR = 8.0 ms, Flip angle = 15 degree, Average number = 5, Acquisition Matrix = 256 × 256 × 256, FOV = 25.6 × 25.6 × 25.6 mm. Manual measurements were made on the 3-dimensional (3D) MRI data to calculate total brain volume, hippocampus volume and lateral ventricle volume using the InsightITK-Snap software52. Regional volumetric changes were measured by voxel-based morphometry (VBM) using the Statistical Parametric Mapping (SPM) software package (http://www.fil.ion.ucl.ac.uk/spm/software/) for MATLAB (Mathworks, Natick, MA, USA) for pilot survey (data not shown).

Histology and immunostaining
Histological examination was done as described9. For the morphometric analysis, serial coronal sections were prepared, and analyzed at the following positions: +0.74 to +1.10 for the septum, diagonal band, striatum and the motor cortex; −0.34 to −0.82 for the substantia innominata, the basal nucleus of the Meynert, and the somatosensory cortex [anterior(+) to posterior(−) distance (mm) from bregma according to Paxinos et al.53]. The sections were stained with cresyl violet or by utilizing endogenous acetylcholine esterase activity54 for histological examination.
Immunostaining was performed as previously described55. The primary antibodies were rabbit mouse anti-choline acetyltransferase (ChAT) polyclonal antibody (Chemicon, Temecula, CA, USA), mouse monoclonal anti-parvalbumin (PV) (Sigma, St. Louis, MO, USA), and rabbit anti-pan-Zic antibodies10.

Resequencing analysis of ZIC2 in human subjects
We performed resequencing analysis of ZIC2 in 278 patients with schizophrenia who were of Japanese descent. The diagnosis of schizophrenia including the samples below was made on the basis of Diagnostic and Statistical Manual of Mental Disorders criteria (DSM-IV), by at least two expert psychiatrists. We then determined the allele frequencies of detected mutations using an expanded sample panel of schizophrenia patients (967 subjects: 457 men, 510 women; mean age 47.3 ± 13.8 [SD] years) and 1060 controls (502 men, 558 women; mean age 47.7 ± 13.6 years) who were documented as being free of mental disorders following brief interviews by expert psychiatrists. Our recruitment of schizophrenia and control subjects did not involve structured or semi-structured instruments. This study was approved by the ethics committees of RIKEN.
Protein-coding regions and exon/intron boundaries within the ZIC2 gene were screened for polymorphisms by direct sequencing of PCR products using the BigDye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems, Foster City, CA, USA) and the ABI PRISM 3730xl Genetic Analyzer (Applied Biosystems).

Molecular and functional analysis
Mouse Zic2 variants that have the same missense mutations as the human ZIC2 nonsynonymous mutations (Zic2A95T for ZIC2A95T, Zic2R408P for ZIC2R409P, and Zic2S443R for ZIC2S444R) were generated by PCR56 using pEFBOS-Zic231 or pcDNA3-HA-Zic2 as templates. Hereinafter, we refer to them as Zic2-A95T (Zic2A95T), Zic2-R409P (Zic2R408P) and Zic2-S444R (Zic2S443R), respectively. Expression plasmids for these wild-type Zic2 and Zic2 variants were constructed pcDNA3.1 (Invitrogen, Carlsband, CA, USA) and pCMVtag2 (Stratagene, La Jolla, CA, USA). pGL4-ZBS was constructed by inserting a mouse genomic DNA clone containing Zic2-binding sequences (Ishiguro et al., unpublished) into pGL4 (Promega, Madison, WI, USA). Cell culture, transfection, immunoblot analysis. luciferase reporter assay, gel shift assay, and immunoprecipitation were performed as previously described3133.

Statistical analysis
Parametric data were analyzed by using the two-sided Student's t-test (t-test) and non-parametric data were analyzed by using the Mann–Whitney's U-test (U-test). The P values refer to the t-test, unless otherwise specified. We also used the repeated measure two-way analysis of variance (RMANOVA) or the chi-square test for homogeneity. Differences were defined as statistically significant when P < 0.05.