Qrr1d—Fmn2 and Rgs7—we implemented a partial correlation analysis [68] in which the effect of genotype at Qrr1d was controlled. For this analysis, we computed the partial correlation coefficient between cis-regulated transcripts and each trans- regulated transcript after regression against the Qrr1d genotype. This partial correlation reveals residual variance that links cis candidates with trans targets, independent of genetic variance at Qrr1d. We computed the partial correlation between Rgs7 and Fmn2, and 14 transcripts representative of the different GOs that map to Qrr1d (dataset S4). The highest partial correlations are between Fmn2 and Rnf6 (r = 0.68, p-value,10213), Atf4 (r = 0.6, p- value,1029), Asns (r = 0.55, p-value,1027), Ube2d3 (r = 0.5, p- value,1026), Hnrpk (r = 0.5, p-value = 1025), Rab2 (r = 20.5, p- value = 1025), and Gars (r = 0.5, p-value = 1025). The strongest correlate of Fmn2 is Rnf6, a gene involved in regulating actin dynamics in axonal growth cones [69]. Although not unequivocal, this analysis provides stronger support for Fmn2 than for Rgs7. Effect of Fmn2 Deletion on Gene Expression Fmn2 is almost exclusively expressed in the nervous system [70] and is a strong candidate for a trans-effect specific to neural tissues. However, its precise function in the brain has not been established. Fmn2-null mice do not have notable CNS abnormalities [71], but to evaluate a possible role of Fmn2 on expression of genes that map to Qrr1d, we generated array data from brains of Fmn2-null (Fmn22/2) and coisogenic (Fmn2+/+) 129/SvEv controls. At a stringent statistical threshold (Bonferroni corrected p,0.05), only eight genes have significant expression differences between Fmn22/2 and Fmn2+/+ genotypes (table 6). Five out of the eight genes, including Pou6f1, Usp53, and Slc11a, have trans-QTLs in Qrr1d. Deletion of Fmn2 had the most drastic effect on the expression of the transcription factor gene Pou6f1, a gene implicated in CNS development and regulation of brain-specific gene expression [72,73]. Expression of Pou6f1 maps as a trans-QTL (at LOD score of 3) to Qrr1d in the hippocampus dataset, and its expression was down-regulated more than 44-fold in the Fmn22/2 line. While the expression analysis of Fmn2-null mice does not definitively link all the trans-QTLs to Fmn2, variation in this gene is likely to underlie some of the trans-QTLs in Qrr1d. The possible compensatory mechanism in the Fmn2-null CNS, and the different genetic background of the mice (129/SvEv) are factors that may have Gene Pou6f1 Zfp420 Txnl1 Usp53 LOC331139 Slc11a2 Pgbd5 6330569M22Rik ProbeIDa ILM6200168 ILM2570632 ILM2850148 ILM103190068 ILM103170273 ILM104050242 ILM103940435 ILM104570300 QTL Hotspot on Mouse Distal Chromosome 1 contributed to the weak detection of trans-effects in the knockout line. Sub-Cellular Localization of FMN2 Protein in Hippocampal Neurons We examined the intracellular distribution of FMN2 protein in neurons using immunocytochemical techniques. All hippocampal pyramidal neurons on a culture dish exhibited distinct and fine granular immunoreactivity for FMN2. The cell body itself had the strongest signal (figure 7A). This fine punctate labeling extended into proximal dendrites and could be followed into distal dendrites. In some instances very thin processes, possibly the axons, were also labeled. Linking Expression and Classical QTLs: Szs1 The strong trans-effect that Qrr1 has on gene expression is a likely basis for the classical QTLs that map to this region. For example, the major seizure susceptibility QTL (Szs1) has been precisely narrowed to Qrr1p [74]. We found that 10 genes already known to be associated with seizure or epilepsy have trans-QTLs with peak LOD scores near Szs1 and in Qrr1p. These include Scn1b, Cacna1g, Pnpo, and Dapk1 (Table S2) [75–84]. In every case, the D allele has the positive additive effect on the expression of these seizure related transcripts, increasing expression 5% to 20%. The two potassium channel genes, Kcnj9 and Kcnj10, are the primary candidates [74]. Both are strongly cis- regulated. The tight linkage between these genes (within 100 kb) limits further genetic dissection, but in situ expression data from the Allen Brain Atlas (ABA, www.brain-map.org) provides us with a powerful complementary approach to evaluate these candidates [85]. Kcnj9 (figure 8A) is expressed most heavily in neurons within the dentate gyrus, whereas Kcnj10 (figure 8B) is expressed diffusely in glial cells in all parts of the CNS. The seizure-related transcripts with trans- QTLs near Szs1 are most highly expressed in neurons, and all have comparatively high expression in the hippocampus. Furthermore, expression patterns of six of the seizure transcripts that map to Qrr1p show spatial correlations with Kcnj9. Dapk1 and Cacna1g (figure 8C) have expression pattern that match Kcnj9 with strong labeling in the dentate gyrus and CA1, and weaker labeling in CA2 and CA3. In contrast, Socs2 (figure 8D), Adora1, Pnpo, and Kcnma1 complement the expression of Kcnj9 with comparatively strong expression in CA2 and CA3, and weak expression in CA1 and dentate gyrus. Table 6. Genes that have significant expression difference between Fmn2+/+ and Fmn22/2. aIllumina probe ID. bPhysical location of gene. cAverage expression signal in Fmn2-null and wild-type lines. dFold difference in expression between Fmn2-null and wild-type lines eBonferroni adjusted p-values; corrected for 46,620 tests. fHighest LOD in Qrr1 and dataset in which transcript has highest LOD in Qrr1. doi:10.1371/journal.pgen.1000260.t006 PLoS Genetics | www.plosgenetics.org 11 November 2008 | Volume 4 | Issue 11 | e1000260 Chrb Fmn2+/+c Fmn22/2c Foldd LODf Datasetf 15 7 18 15 3 4 8 3 11.96 10.12 10.72 7.17 14.45 9.92 13.40 6.42 6.48 7.70 6.70 9.32 10.59 9.17 12.12 10.63 45 5 16 4 15 2 2 18 pe 361026 0.002 0.002 0.009 0.01 0.02 0.02 0.03 3.0 3.0 3.3 3.9 3.3 BXD Hippocampus B6D2F2 striatum BXD Hippocampus BXD Hippocampus BXD HBP Striatum