Gpc1- heterozygous mutants were bred extensively onto CD1 and C57BL/6 backgrounds prior to breeding inter se to produce homozygous mutant animals. PCR primers specific for the Gpc1- allele were 5'-AGCCGGCTTTTGTTGTCTC-3' and 5'-CACGAGTGTGCTAGGATAGGG-3'. Primers specific for the Gpc1 wild-type allele were 5'-CAGCGAAGTCCGCCAGAT-3' and 5'-CAGACCTCCCGAGTGCTAGG-3'.
The following additional mutant alleles were used in this study: gene-trap alleles of Gpc1 (GPC1lacZ; Baygenomics ID GST062 San Francisco, CA, USA) and Gpc4 (GPC4lacZ; Baygenomics ID Ex194) [100, 101]; a targeted null mutation in Gpc2 (S Saunders and ADL, unpublished); Fgf2 (Jackson Laboratory, Bar Harbor, Maine, USA), a hypomorphic allele of Fgf8 (Fgf8
), Fgf17 () and a LacZ-reporter of canonical Wnt signaling (Bat-gal ). Wild type CD1 and C57BL/6 mice were from Charles River (San Diego, CA, USA) Genotypes were determined by PCR of tail DNA.
For production of staged embryos, timed matings were used and noon of the day of vaginal plug was considered as E0.5. At early embryonic stages, more precise staging was obtained from somite number. To obtain BrdU-labeled embryos, pregnant mice were injected intraperitoneally with 50 μg of BrdU (B5002; Sigma-Aldrich St. Louis, MO, USA)per gram body weight and embryos were collected 24 hours later.
Mouse colonies were maintained, and all animal experimentation conducted, in accordance with the policies and guidelines of the Institutional Animal Care and Use Committee (IACUC) of the University of California, Irvine. (IACUC protocol number 1998-1656).
Histology and histochemistry
Adult brains were fast-frozen in 2-methyl-butane prior to cryomicrotome sectioning at 20 μm. Embryos were dissected in cold phosphate-buffered saline (PBS), fixed in 4% paraformaldehyde in PBS at 4°C overnight, cryoprotected in 30% sucrose in PBS at 4°C, and cryomicrotome sectioned at 10 to 20 μm. Sections were stored at -20°C prior to immunohistochemistry or Cresyl-Violet staining. For BrdU staining, cryosections were treated with 2 M HCl for 1 hour at 37°C. Sections were then blocked with 5% goat serum +10% bovine serum albumin/PBS +0.2% Tween20 and incubated with primary antibody diluted in blocking solution at 4°C overnight (anti-Gpc1 , 1:500; rabbit anti-phosphohistone H3 (anti-PHH3; Millipore, Billerica, MA, USA, 5 μg/ml, 1:500; Tuj1 (R&D systems, 1:1,000, Minneapolis, MN, USA); anti-BrdU (Abcam, 1:100, Cambridge, MA, USA). Secondary antibodies (alexaFluor goat anti-rabbit IgG, 2 μg/ml alexaFluor goat anti-mouse IgG, 10 μg/ml; Cy3-goat anti mouse, 7 μg/ml (Jackson Immunoresearch, West Grove, PA, USA); or Cy2-goat anti rat, 14 μg/ml (Jackson Immunoresearch)) were applied for 1 hour at room temperature. For quantification of apoptosis, fluorescent TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) assays (Apotag Kit, Serologicals, Norcross, GA, USA) were performed on cyrosections. Hoechst33258 was used at 2 μg/ml for nuclear counterstaining. Fluorescence images were analyzed with a Ziss Axiovert S100 microscope and Hamamatsu C4742-95 digital camera. Wholemount staining for beta-galactosidase activity was performed as described in .
In situ hybridization
E8.5-E9.5 embryos, fixed by immersion in 4% paraformaldehyde, were gradually dehydrated in methanol and stored in 100% methanol at -20°C. Wholemount RNA in situ hybridization was performed as described  with probes synthesized using digoxigenin-labeled NTP mix (Roche, Indianapolis, IN, USA). Probes for glypicans (Glypicans 1 to 6) were obtained by RT-PCR from E13.5 brain total RNA, using the following primer pairs, and subcloned into the PCRII-TOPO vector (Invitrogen, Carlsbad, CA, USA): Gpc1, 5'-GCTACATCTCCATCTTCCTTGAC-3' and 5'-AACACACATTATCCACTGACACC-3'; Gpc2, 5'-AGTCTGGCGAGGGGTTAGAT-3' and 5'-GGCTACATTGAGGCAGAAGC-3'; Gpc3, 5'-GGATGGTGAAAGTGAAGAATCAAC-3' and 5'-GAGAGAAAGAGAAAAGAGGGAAAC-3'; Gpc4, 5'-CATGGCACGCTTAGGCTTGCTCGC-3' and 5'-TGGTTGCACTGTTCGCTGACCACG-3'; Gpc5, 5'-CGCCAGGATGTTAGTCCATT-3' and 5'-AATTTCTGCCCATTGAGGTG-3'; Gpc6, 5'-GCTGTGTATTCTTGCTCTCTCCGGG-3' and 5'-GTACAGCATCCCGTAGGTCCGGAC-3'.
The following additional RNA probes were used: Pax6 (335 to 595 bp MN_013627), Spry2 (probe used in ), Pyst1 (probe used in ), Ptc1 (probe used in ), Msx1 (Eco RI fragment from IMAGE clone 903377). Controls for in situ hybridization consisted of sense probes derived from the same DNA fragments.
Measurement of brain size and DNA content
Postnatal and adult brains were freshly dissected. After removal of olfactory bulbs and remaining spinal cord (at the level of the posterior margin of the cerebellum), brains were immediately weighed on a laboratory scale.
Images of fresh embryos were collected using a Leica MZFLIII stereomicroscope and a SPOT camera (Diagnostic Instruments, Inc. Sterling Heights, MI, USA). For embryos at E11.5 or older, brain height, depth and width were separately measured from lateral and frontal images (Additional file 2), and multiplied to produce a volume estimate. For E8.5 and E9.5 embryos, measurements of area were obtained from perimeter tracings of lateral views using Image J analysis software . At these stages the central nervous system comprises the majority of head tissue, so such tracings included the entire head, stopping ventrally at the rostral border of the first branchial arch, and dorsally at the top of mesencephalon. Volume was then estimated as area3/2. In some cases, volume was also estimated by the procedures outlined above for older embryos, and qualitatively similar results were obtained.
DNA content in brain homogenates was measured by enhancement of bisbenzimid fluorescence at 458 nm, as described by Labarca and Paigen . A linear standard curve (1 to 10 μg/ml) was obtained using salmon sperm DNA (Invitrogen).
Forebrain vesicles of E9.5 and E8.5 wild-type and mutant mice were dissected in ice-cold PBS, and RNA was isolated and column purified (Aurum Total RNA Mini Kit, Bio-Rad, Herculeus, CA, USA) according to the manufacturer's instructions. cDNA was generated by reverse transcription with a mixture of oligo dT and random hexamers (Superscript First-Strand Synthesis kit, Invitrogen). PCR quality controls, experimental runs and statistical methods were performed as described [110, 111]. Quantification of total mRNA expression was performed with an Opticon System (MJ Systems CFD-3200, Calgary, Denver, USA) and SYBR-Green (Bio-Rad).
All measurements were normalized to values for 18S RNA in the same samples. All cDNA samples were validated for reverse transcription reaction efficiency and minimal genomic DNA contamination (cDNA/genomic target ratio >105) for 40 cycles in duplicates. Average of duplicated cycle threshold (Ct) values were normalized as ΔCt (Ctgene of interest - Ctreference(18S)). Relative levels were converted using the 2-ΔΔCt method: ΔΔCt = ΔCtmutant - ΔCtwild-type  Averages of duplicate Ct, normalized ΔCt, ΔΔCt and relative level 2-ΔΔCt and standard errors were calculated using Microsoft Excel.
Measurement of Erk activity in embryonic explant cultures
E9.5 dorsal telencephalon explants were isolated and cultured as previously described . After 1 hour of incubation at 37°C, FGF2 (R&D Systems) was added at the concentrations indicated for 15 minutes. Explants were briefly washed with 1× PBS and individually homogenized in lysis buffer (1 mM EGTA, 1% Triton X-100, 150 mM NaCl, 50 mM Tris-Cl pH7.4, 1% NP40, 1 μg/ml phenylmethylsulphonyl fluoride (PMSF), 1 μg/ml leupeptin, 1 μg/ml pepstatin, 1 μg/ml aprotinin, 25 μg/ml N-ethylmaleimide (NEM), and phosphatase inhibitors (1 mM NaF, 1 mM Na3VO4)) with a disposable pestle (Knotes Scientific, Vineland, NJ, USA) Lysed samples were stored at -80°C until use. Erk activity was quantified using an in vitro phosphorylation assay (MAP Kinase/Erk Assay kit; Upstate Biotechnology) following the manufacturer's instructions, with or without the 20 μM Erk inhibitor FR180204 (Calbiochem, Gibbstownm, NJ, USA) treatment for 10 minutes prior to the assay. Values in the presence of FR180204 were taken to represent non-ERK phosphorylation activity, and subtracted from each data point. Data were normalized to protein concentration determined by a bicinchoninic acid (BCA) assay .
Subcellular fractionation and analysis of proteoglycan content
Adult brains were dissected in ice-cold PBS and immediately homogenized. to obtain membrane and soluble fractions as described . For SDS-PAGE analysis, samples prepared in this way were digested for 30 minutes at 37°C with Heparinase III or with Heparinase III plus Chondroitinase ABC (all used at 1.5 U/mg of protein; both enzymes were purchased from Seikagaku Corp., Tokyo, Japan) along with a proteinase inhibitor mixture (10 μg/ml pepstatin A, 20 μg/ml leupeptin, 2.5 mg/ml NEM, and PMSF in 50 mM Tris-hydroxyaminomethane, 15 mM phosphoric acid, pH7.3). Digested samples were boiled for 10 minutes in SDS-PAGE sample buffer and loaded at 50 μg protein per lane onto 7.5% SDS-polyacrylamide gels, and subjected to electrophoresis. Gels were transferred to PVDF membrane (Millipore, Billerica, MA, USA) and probed with rabbit anti-glypican-1 (1:3,000) antibody or mouse 3G10 monoclonal antibody (1:2,000; USBiological, Swampscott, MA, USA). Samples without enzyme treatment, or subjected to single enzyme treatment, were used where indicated. Blots were incubated with horseradish peroxidase-conjugated goat anti-rabbit or donkey anti-mouse antibody, as appropriate, and visualized using enhanced chemiluminescence.