Litters of wildtype C57Bl/6J and Nfib-deficient mice, bred at The University of Queensland with approval from the institutional Animal Ethics Committee, were used in this study. The Nfib-/- allele  was backcrossed for more than ten generations onto the C57Bl/6J background. Nfib+/- mice were bred to obtain wildtype, Nfib+/- and Nfib-/-progeny. No midline defects were detected in wildtype or heterozygote animals. Timed-pregnant females were obtained by placing male and female mice together overnight. The following day was designated as E0 if the female had a vaginal plug. Embryos were genotyped by PCR as previously described .
On the required gestational day, embryos were drop-fixed in 4% paraformaldehyde (PFA; E14 and below) or transcardially perfused with 0.9% phosphate buffered saline, followed by 4% PFA (E15 to E18). They were then postfixed in 4% PFA at 4°C until sectioning.
Brains of E18 wildtype C57Bl/6J or Nfib-/- embryos were dissected from the skull, blocked in 3% noble agar (Difco, Sparks, MD, USA), and then sectioned coronally at 45 μm on a vibratome (Leica, Nussloch, Germany). Sections were then mounted and stained with Mayer's haematoxylin using standard protocols.
Immunohistochemistry on floating sections
Brains were sectioned as described above, then processed free-floating for immunohistochemistry using the chromogen 3,3' diaminobenzidine as described previously . Primary antibodies used for immunohistochemistry were anti-GAP43 (mouse monoclonal, 1/100,000; Chemicon, Bedford, MA, USA), anti-GFAP (rabbit polyclonal, 1/50,000; DAKO, Glostrup, Denmark), anti-cleaved caspase 3 (rabbit polyclonal, 1/1,000; Cell Signaling Technology, Danvers, MA, USA), anti-GLAST (rabbit polyclonal, 1/50,000; a gift from Niels Danbolt, University of Oslo), anti-nestin (mouse monoclonal, 1/1,500; Developmental Studies Hybridoma Bank), anti-tenascin C (rabbit polyclonal, 1/2,000; Chemicon), anti-Tbr1 (rabbit polyclonal, 1/100,000; a gift from Robert Hevner, University of Washington), anti-NFIA (rabbit polyclonal, 1/30,000; Active Motif, Carlsbad, CA, USA), anti-Emx1 (rabbit polyclonal, 1/30,000; a gift from Giorgio Corte, The University of Genova Medical School), anti-Npn1 (rabbit polyclonal, 1/75,000; a gift from David Ginty, Johns Hopkins University) and anti-DCC (rabbit polyclonal, 1/30,000; a gift from Helen Cooper, Queensland Brain Institute). Secondary antibodies used were biotinylated goat-anti-rabbit IgG (1/1,000; Vector Laboratories, Burlingame, CA, USA) and biotinylated donkey-anti-mouse IgG (1/1,000; Jackson ImmunoResearch, West Grove, PA, USA). To perform immunofluorescent labelling, sections were incubated overnight with the primary antibody at 4°C. They were then washed and incubated in secondary antibody, before being washed again and mounted. The primary antibodies used for immunofluorescent labelling were anti-phosphohistone H3 (rabbit polyclonal, 1/1,000; Millipore, Billerica, MA, USA), anti β-galactosidase (1/1,000; Promega, Madison, WI, USA), anti-GAP43 (1/5,000), anti-DCC (1/1,000), anti-GFAP (1/2,000), anti-Satb2 (1/1,000; Abcam, Cambridge, UK) and anti-NFIB (1/1,000). The secondary antibodies used were goat-anti-rabbit IgG AlexaFluor488 and goat-anti-mouse IgG AlexaFluor594 (both 1/1,000; Invitrogen, Carlsbad, CA).
Immunohistochemistry on paraffin sections
E18 wildtype brains were perfused as above and embedded in paraffin wax. Brains were sectioned at a thickness of 6 μm. Antigen retrieval was performed using a 10 mM, pH 6 sodium citrate solution, and immunohistochemistry was performed as described above using 3,3' diaminobenzidine as the chromogen. The primary antibody used for immunohistochemistry was anti-NFIB (1/1,000, Active Motif), and a biotinylated goat-anti-rabbit IgG secondary antibody (Vector Laboratories) was used at 1/1,000.
Image acquisition and analysis
Following immunohistochemistry, sections were imaged using an upright microscope (Zeiss Z1, Zeiss, Goettingen, Germany) attached to a digital camera (Zeiss AxioCam HRc). AxioVision software (Zeiss) was used to capture images. When comparing wildtype to knockout tissue, sections from matching positions along the rostro-caudal axis were selected.
Quantification of proliferation
To quantify proliferation at the developing cortical midline, sections from E13, E14 and E15 wildtype C57Bl/6J or Nfib-/- embryos were labelled with an anti-phosphohistone H3 antibody as described above. Sections were imaged using an upright fluorescence microscope (Zeiss Z1) attached to a digital camera (Zeiss AxioCam HRc). Eight to ten optical sections encompassing the entire 45-μm section were captured with an ApoTome (Zeiss). To calculate the total number of phosphohistone H3-positive cells per unit area at the cortical midline, a 300 μm2-boxed region, encompassing the presumptive glial wedge area, was generated using AxioVision software (Zeiss). The number of immunolabelled cells in focus in each optical section of this region was counted and pooled (n = 3 for both wildtype and knockout at all ages).
For all experiments described in this study, sections from three different brains of each genotype were analysed. Statistical analyses were performed using a two-tailed unpaired t- test. Error bars represent standard error of the mean.
Carbocyanine tract tracing
E18 wildtype and Nfib-/- brains were fixed in 4% PFA as described above. A small injection of DiI (in a 10% solution of dimethylformamide; Invitrogen) was then made into the neocortex using a pulled glass pipette attached to a picospritzer. Brains were stored in the dark at 37°C in 4% PFA for at least 4 weeks to allow dye transport. They were then sectioned coronally at 45 μm using a vibratome, and imaged using an upright fluorescence microscope (Zeiss Z1). Nuclei were counterstained with 4',6-diamidino-2-phenylindole (DAPI; blue). Three brains were analysed for each genotype.
Retrograde labelling under ultrasound guidance
Pregnant mice were anaesthetised with isofluorane (2%) for the duration of the microinjection procedures. The uterine horn was exposed through an incision in the abdominal midline for the purpose of ultrasound- imaging and guided microinjections (Vevo770, VisualSonics, Toronto, Canada). Retrograde labelling of callosal axons with True Blue chloride (Invitrogen) was performed as described previously, with modifications appropriate for ultrasound-guided microinjection in utero. Embryos were visualised under a 40 MHz transducer probe (RMV711) and a small volume of the tracer (approximately 250 nL of a 1 μg/μL solution) was injected into the cortex of wildtype E17 embryos in utero through the uterine wall with the aid of a nanojector (Nanoject II, Drummond Scientific, Broomall, PA, USA). Once embryos were injected, the uterine horn was returned to the abdominal cavity, and the incision was sutured. Then, 24 hours later (E18) the embryos were perfused transcardially as described above and processed for NFIB immunohistochemistry. Fluorescence images were obtained with an upright microscope (Zeiss Z1) as described above.
In situ hybridisation
In situ hybridisation was performed as described previously , with minor modifications. An antisense riboprobe specific to Slit2 was hybridised to coronal brain sections at 65°C overnight. The colour reaction solution was BM Purple (Roche, Mannheim, Germany).
Reverse transcription and quantitative real-time PCR
The reverse transcription was performed using Superscript III (Invitrogen). Briefly, 0.5 μg total RNA was reverse transcribed with random hexamers. qPCR reactions were carried out in a Rotor-Gene 3000 (Corbett Life Science, Sydney, Australia) using the SYBR Green PCR Master Mix (Invitrogen). All the samples were diluted 1/100 with water and 5 μL of these dilutions were used for each SYBR Green PCR reaction containing 10 μL SYBR Green PCR Master Mix, 10 μM of each primer, and deionised water. The reactions were incubated for 10 minutes at 95°C followed by 40 cycles with 15 seconds denaturation at 95°C, 20 seconds annealing at 60°C, and 30 seconds extension at 72°C. Primer sequences are available on request.
Quantitative real-time PCR data expression and analysis
After completion of the PCR amplification, the data were analysed with the Rotor-Gene software (Corbett Life Science) and Microsoft Excel. In order to quantify the mRNA expression levels, the housekeeping gene HPRT was used as a relative standard. All the samples were tested in triplicate. By means of this strategy, we achieved a relative PCR kinetic of the standard and the sample. For all qPCR analyses, RNA from three independent replicates for both wildtype and Nfib mutants were interrogated. Statistical analyses were performed using a two-tailed unpaired t- test. Error bars represent the standard error of the mean.
Diffusion-weighted magnetic resonance imaging and tractography
Following perfusion fixation and phosphate-buffered saline washing, diffusion-weighted images were acquired with the samples immersed in Fomblin Y-LVAC fluid (Solvay Solexis, Italy), using a 16.4 Tesla Bruker scanner and a 10 mm quadrature birdcage coil. A three-dimensional diffusion-weighted spin-echo sequence was acquired using a repetition time of 400 ms, an echo time of 22.8 ms and an imaging resolution of 0.08 × 0.08 × 0.08 mm with a signal average of 1. Each dataset was composed of two Bo and thirty direction diffusion-weighted images (b value of 5,000 s/mm2, δ/Δ = 2.5/14 ms). Reconstruction and tractography were performed with Diffusion Toolkit  according to high angular resolution diffusion (HARDI) and Q-ball models . Tractography limits were set at fractional anisotropy values greater than 0.1 and a turning angle ≤ 45°. Hippocampal commissure tractography was performed using hand-drawn regions-of-interest on colour-coded fractional anisotropy maps in TrackVis .