Genome scans for SWD traits in <i>Gria4</i> deficient backcross mice.

<p>Shown are genome-wide interval mapping LOD score plots from 89 backcross <i>Gria4</i> mutant mice for two observed spike-wave discharge (SWD) traits - SWD incidence (panel A), SWD length (panel B) - and their first two principal components (panels C and D). The insets of panels A and B show the frequency distribution of the respective raw traits, although for interval mapping all four traits were first rank- and quartile-normalized prior to analysis. The chromosomes are shown at the bottom, with tick marks indicating positions of individual markers within each chromosome. Dotted lines show positions of genome-wide significance thresholds determined from 1000 permutations.</p

Genotypes and features of inbred and congenic strains used in this study.

<p>Genotypes and features of inbred and congenic strains used in this study.</p

C3H/HeJ IAP-1Δ1 insertions absent from C3HeB/FeJ.

a<p>To detect both IAP insertion allele and the pre-insertion allele, the IAP5′LTR-F oligonucleotide is used in a 3′-primer assay with both the locus-specific forward and reverse primers. In cases where the product sizes of both alleles were identical, the IAP5′LTR-F+gene-F (or gene -R, depending upon IAP orientation) for the insertion allele and gene -F+gene -R primers are run separately for the pre-insertion allele.</p

TALEN-induced frameshift alleles of <i>Pcnxl2</i> and suppression of <i>Gria4</i> SWD.

<p>A. Exon-intron structure of mouse <i>Pcnxl2</i> (from UCSC genome browser, version m38 mouse genome assembly), highlighting the position of the HeJ-specific IAP-1Δ1 insertion in intron 19, the two exons (exon 16 and exon 29, respectively) targeted for TALEN mutagenesis and the wildtype FeJ sequence of each together with the sequence of the respective frameshift alleles in TALEN mutations A+1 (FS2), A−11 (FS1), and B−2 (FS3). The asterisk indicates a premature translational stop codon in each mutant allele. B. Spike-wave discharge (SWD) incidence (top) and length (bottom) for parent FeJ-<i>Gria4</i>^IAP congenic and HeJ-<i>Gria4</i>^IAP inbred strain colonies, compared with littermate wildtype (wt), heterozygous or homozygous respective TALEN mutations created and maintained on the isogenic FeJ-<i>Gria4</i>^IAP congenic strain background. Asterisks indicate where homozygous mutant genotypes were significantly different (<i>p</i><0.01) from wildtype control. Sample sizes: FeJ-<i>Gria4</i>^IAP (9), HeJ- <i>Gria4</i>^IAP (18), wt (16), FS1 het (6), FS1 hom (6), FS2 het (9), FS2 hom (9), FS3 het (6), FS3 hom (6). Error bars are SEM. C. Spike-wave discharge (SWD) incidence (left) and length (right) for littermate TalA-11 genotypes (hom, het, wt) isogenic on the FeJ strain, in double mutant combination with <i>Gabrg2</i>^tm1Spet or <i>Scn8a</i>^8J/+ from a congenic FeJ strain background. None of the <i>Pcnxl2</i> mutant allele homozygotes were significantly different from the other <i>Pcnxl2</i> genotypes for either mutation or SWD measure. Sample sizes: <i>Gabrg2</i>^tm1Spet (3 hom, 12 het, 2 wt); <i>Scn8a</i>^8J/+ (2 hom, 4 het, 2 wt).</p

IAP-1Δ1 element diversity in C3H substrains.

<p>A. Strategy for detection of IAP-1Δ1 elements. For detection of host-IAP-1Δ1 5′ <i>Bgl</i>II-restricted junction fragments by dried gel hybridization, a 31-nt oligonucleotide hybridization probe (IAPd1oligo1-R) was designed to straddle the previously described 1.2 kb IAP-1Δ1 element common deletion <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004454#pgen.1004454-Ishihara1" target="_blank">[22]</a>. For cloning of IAP-1Δ1 elements by inverse PCR, a complementary oligonucleotide (IAPd1oligo2F) was designed to pair with an oligonucleotide specific for the IAP LTR (IAPLTR5′) for amplification of circularized genomic <i>Bgl</i>II fragments. B. Dried gel detection of IAP-1Δ1 5′ junction fragments from C3H substrains (HeJ, HeOuJ, HeSnJ, FeJ) plus the outlier B6J. Bands estimated as unique to HeJ are indicated with asterisks on the left. C. Example of an inverse PCR experiment from HeJ and FeJ substrains showing <i>Bgl</i>II restricted genomic DNA (two lanes each), unligated controls (one lane each), and location of apparent HeJ-specific bands prepared. for cloning and Sanger sequencing. In additional experiments not shown, higher molecular weight <i>Bgl</i>II restricted fragments were similarly processed. Also shown with connecting lines are locus identities for specific bands after sequencing, the sizes of which correspond to the junction fragment lengths determined from the sequence, and dashed lines indicate the corresponding region from the dried gel, for the purpose of highlighting the similar banding pattern (<i>N.b.</i> the absolute sizes of fragments in panel C are smaller because the portion of the IAP genome between primers is expectedly absent from the inverse PCR product). Sizes of <i>Hind</i>III-digested bacteriophage lambda DNA are shown on the right, of panel B, and <i>Hae</i>III-digested bacteriophage phi-X DNA markers are shown at the left of panel C.</p

Quantitative RT-PCR in <i>Pcnxl2</i> mutants.

a<p>Fold-difference for each pair of rows was calculated as 2^{(top row ΔCt – bottom row ΔCt)}, and is signed relative to the bottom row (e.g. <i>Pcnxl2</i> transcript is 2.0 fold higher in FeJ-<i>Gria4</i>^IAP compared to HeJ.</p

SWD phenotypes conferred by <i>G4swdm1</i>, a Chr 15 strain modifier of <i>Gria4</i> absence seizures.

<p>A. LOD score plot, similar to those in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004454#pgen-1004454-g003" target="_blank">Figure 3</a>, except only for Chr 15 in 84 N₉F₁ or N₁₀F₁ intercross congenic mice. At the bottom are Mb (mouse genome assembly m38) positions in of Chr 15 markers used for this analysis and the genome-wide significance threshold is also shown, as determined by 5000 permutations. B. For the marker <i>D15Mit93</i> corresponding to the peak maximum likelihood location from panel A, quartile plots with data points for average SWD incidence or SWD length from individual mice, showing additivity for both traits. C. EEG from a <i>Gria4</i>^KO/KO, <i>G4swdm1^Fe/FeJ</i> double homozygote on the B6J congenic strain, showing a particularly long (15 second) SWD. D. EEG from a <i>Gria4</i>^KO/IAP compound heterozygous, <i>G4swdm1^Fe/FeJ</i> homozygous double mutant genotype on a (B6J.FeJ×FeJ)F₁ hybrid background, showing a particularly long (46 second) SWD. For panels C and D, two of the six recorded channels are shown (LF-RB – left front, right back; LB-RB – left back, right back, corresponding to the electrode placement relative to Bregma and midline). 1 s: 1 second.</p

Significance and direction of effect for synaptic CELF targets enriched between cell body and neuropil.

<p>This figure, together with <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003067#pgen-1003067-t003" target="_blank">Table 3</a>, considers the subset of 142 CELF4 targets selectively enriched for differential expression between hippocampal CA1 cell body vs. neuropil, as derived from the GO categories “Regulation of synaptic plasticity”, “Synapse part” and “Cell adhesion”, from <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003067#pgen-1003067-g008" target="_blank">Figure 8</a>. The relative significance is shown for the three proxy categories (non-solid columns) and various subcategories (solid columns) of molecular function (panel A) and subcellular location (panel B), using the difference in the log <i>p</i> value derived from Fisher's Exact test for each (Δ log <i>p</i>), as an indicator (Y-axis). Categories were selected based on having at least 12 CELF4 targets in each, and also only one category is shown if closely-related GO categories had the same members in them (e.g. “transporter activity” was shown but not “ion transporter activity”, “transmembrane transporter activity” which had the same members). For molecular function, the largest effect is seen for proteins that associate with “lipid binding”, transporter activity” and “channel activity”, with the next category being almost an order of magnitude less significant. For subcellular localization, the subcategories that had the largest effect were “synaptic membrane”, and “presynaptic membrane”, although various other structures were almost as significant. The full list of genes, categories, sample sizes and expression data can be found in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003067#pgen.1003067.s010" target="_blank">File S8</a>.</p

CELF4 cosediments with polysomes and large RNA granules.

<p>A. Cortical brain tissue lysates from 4-week old mice were fractionated on 15–55% linear sucrose gradients. Fractions were collected and analyzed by immunoblot with antibodies against CELF4 and ribosomal S6 (S6) protein. Lysates were treated with either EDTA (B) or RNase A (C) in parallel. Sedimentation is show from left to right, with the positions of monosomes (80S), polysomes, and RNA granules indicated.</p

<i>Celf4</i> genotype-dependent shifts (“Interaction”) of CELF4 target mRNAs.

<p><i>Celf4</i> genotype-dependent shifts (“Interaction”) of CELF4 target mRNAs.</p