Warren et al. (2010) revisited singing-driven gene regulation in area X and found 474 known genes (represented by 807 probes) that were regulated over the course of 0.5–7 hr of singing. Three hundred of these genes were in our network, with subsets enriched in the three song modules (blue: 71 genes, with, e.g., SHC3, SMEK2, and NTRK2 having the highest GS.motifs.X, p < 4e-28; orange: 17 genes, e.g., CSRNP3, SCN3B,

http://www.selleckchem.com/erk.html and PLCB1, p < 3e-6; dark green: 38 genes, e.g., BSDC1, VLDLR, and RORA, p < 5e-5; Fisher's exact test; Table S2) and in one other module (yellow: 104 genes, p < 5e-7; Table S2). Compared to the rest of the network, probes for all 300 genes had greater expression increases (p = 1.9e-12, Kruskal-Wallis test; 882 probes total), higher GS.motifs.X (p = 7.8e-11), and higher GS.singing.X (p = 2.7e-11; Table S2). These genes were also more interconnected in their respective modules throughout the network (kIN.X, p = 4.2e-4), especially in the blue song GW-572016 nmr module (p = 3.8e-14). A separate aspect of the study revealed enrichment for the functional annotation term “ion channel activity” in 49 genes posited to have undergone positive selection in zebra finches, which are also suppressed in the auditory forebrain during song perception. Of these, 42/49 were in our network (114 probes; Table S2),

with six in the orange song module (p < 3.3e-4, Fisher's exact test). One of the ion channel genes, TRPV1 (dark green/salmon modules), was highly connected and strongly suppressed by singing in our data, and thus selected for validation in area X in vivo (see below and Table S2). We previously showed that FoxP2 mRNA and protein are lower in area X following 2 hr of undirected singing compared to nonsinging, with the magnitude of downregulation correlated to singing (Miller et al., 2008, Teramitsu and White, 2006 and Teramitsu et al., 2010). This finding was reproduced here; expression levels for all 12 FOXP2 probes in the network were negatively correlated with the number of motifs sung ( Figure S5). Although our study

used an indirect approach, i.e., a behavioral paradigm in which the birds’ natural singing behavior significantly alters FoxP2 levels within area X ( Miller until et al., 2008, Teramitsu and White, 2006 and Teramitsu et al., 2010), we predicted that this paradigm coupled with WGCNA would reveal FoxP2 transcriptional targets in area X singing-related modules. To test this, we screened the network for direct FOXP2 targets previously identified by three studies. Of 175 targets found in human fetal basal ganglia ( Spiteri et al., 2007), 56 were in our network (149 probes total; Table S2). These had relatively high MM in the orange song module (p = 0.05, Kruskal-Wallis; Table S2), which contained genes that were downregulated with continued singing, including 9/12 probes for FOXP2. Of 302 targets found by a second study in SY5Y cells ( Vernes et al.