QTL Hotspot on Mouse Distal Chromosome 1 Figure 7. Expression of FMN2 protein in hippocampal neurons. (A) Neurons exhibited pronounced fine granular immunoreactivity for FMN2. The cell body had the strongest signal. The fine granular staining extended into apical and distal dendrites (arrows). Thin axon-like processes were also labeled (arrow head). (B) The fine granular staining is not detected in controls of sister cultures processed in parallel without the first antibody. doi:10.1371/journal.pgen.1000260.g007 Discussion Qrr1 is a complex regulatory region that modulates expression of many genes and classical phenotypes. By exploiting a variety of microarray datasets and by applying a combination of high- resolution mapping, sequence analysis, and multiple cross analysis, we have dissected Qrr1 into segments that are primarily responsible for variation in the expression of functionally coherent sets of transcripts. The distal portion of Qrr1 (Qrr1d) has a strong trans- Figure 8. Expression patterns of seizure related genes with cis- and trans-QTLs in Qrr1p. Candidate gene Kcnj9 (A) has heavy expression in neurons. Kcnj9 shows a regionally restricted expression in the hippocampus with intense labeling in dentate gyrus, strong labeling in CA1, and relatively weak labeling in CA2 and CA3. Candidate gene Kcnj10 (B) has a more diffused pattern and expressed primarily in glial cells. There is almost no labeling for Kcnj10 in the hippocampus. Transcripts of seizure-related genes, Cacna1g (C) and Socs2 (D), have trans-QTLs in Qrr1p. Both genes show high expression in neurons. Cacna1g matches the expression of Kcnj9 with strong labeling in dentate gyrus and CA1, and weak labeling in CA2 and CA3. Socs2 complements the expression of Kcnj9 and Cacna1g with intense labeling in CA2 and CA3. In Situ expression data are from the Allen Brain Atlas. doi:10.1371/journal.pgen.1000260.g008 effect on RNA metabolism, translation, tRNA aminoacylation, and transcripts that are transported into neuronal dendrites. Fmn2, Rgs7, and a cluster of tRNAs are strong candidates in Qrr1d. We analyzed gene expression changes in the CNS of Fmn2-null mice and detected a profound effect on the expression of a small number of transcripts that map to Qrr1d, particularly on the expression of the transcription factor Pou6f1. We have shown that the FMN2 protein is highly expressed in the cell body and processes of neurons, and is a high priority candidate in Qrr1d. Kcnj9 vs. Kcnj10 and Seizure Susceptibility The two inwardly rectifying potassium channel genes—Kcnj9 and Kcnj10—are strong candidates for the seizure susceptibility QTL in Qrr1p that has been unambiguously narrowed to the short interval from Atp1a2 to Kcnj10 [74]. In BXD CNS datasets, Qrr1 also modulates the expression of a set of genes implicated in the etiology of seizure and epilepsy, including Pnpo, Scn1b, Kcnma1, Socs2, and Cacna1g. Polymorphisms in the Kcnj9/Kcnj10 interval that influence expression of these genes are excellent candidates for the Szs1 locus. The in situ expression data in the ABA shows a striking spatial correlation between expression of Kcnj9 and other seizure-related transcripts that have trans-QTLs in Qrr1p. The complementary expression of Kcnj9 and the seizure-related transcripts (figure 8) make Kcnj9 a stronger candidate than Kcnj10. Kcnj9 has over a 2- fold higher expression in D2 [our data, and cf. 26,86], a seizure prone strain, compared to B6, a relatively seizure resistant strain, suggesting that the proximal cause of Szs1 may be high expression of this gene, perhaps due to the promoter polymorphism discovered by Hitzemann and colleagues [26]. Multiple Loci in a Major QTL Interval Fine mapping of complex traits have often yielded multiple constituent loci within a QTL interval [87,88]. Our mapping analyses of expression traits also show that multiple gene variants, rather than one master regulatory gene, cause the aggregation of expression QTLs in Qrr1. Subgroups of genes with tight coexpression can be dissected from the dense cluster of QTLs. Most notable is the strong trans-regulatory effect of Qrr1d on genes involved in amino acid metabolism and translation, including a host of ARS transcripts. However, there are limits to our ability to dissect Qrr1, and genes associated with protein degradation and RNA metabolism map throughout the region. In part this may be due to inadequate mapping resolution, but it may also reflect PLoS Genetics | www.plosgenetics.org 12 November 2008 | Volume 4 | Issue 11 | e1000260