Figure 4.

The 6q32 miRNA cluster. (a) Schematic representation of the imprinted rat distal 6 domain (human 14q32). The positions of maternally expressed non-coding RNA genes are indicated by pink squares and paternally expressed genes are indicated by blue squares. The positions of small nucleolar RNA (snoRNA) and miRNA genes are indicated by vertical bars and triangles, respectively. The intergenic germline-derived differentially methylated region (IG-DMR) located between Dlk1 and RGD1566401 (better known as MEG3 or GTL2) genes is methylated on the paternal chromosome (represented by circles; filled indicates hypermethylated; open indicates hypomethylated). PAT indicates paternal chromosome; MAT indicates maternal chromosome. miRNAs in dark-red font were significantly differentially expressed in the left hemisphere following treatment and miRNAs in green were also differentially expressed, although did not reach significance after correction for multiple testing. The figure is not drawn to scale. The figure is adapted from Seitz et al.⁵⁹ (b) Expression of the Ca2⁺-activated transcription factors, myocyte enhancer factor 2C and myocyte enhancer factor 2D, as validated using quantitative reverse transcription-PCR. Mef2c was significantly downregulated 9.9-fold (P=0.026) following maternal immune activation (MIA) combined with adolescent HU210 exposure, whereas Mef2d was significantly downregulated 7.6-fold (P=0.037) following HU210 exposure alone and 4.4-fold (P=0.045) following MIA combined with adolescent HU210 exposure. Bars indicate the mean fold change (treatment to control) mean±s.e.m.; (n=3–4); ^#P<0.06,*P<0.05 unpaired Student's t-test. (c) Mef2c expression plotted against the expression of 6q32 miRNA (miRs −134, −431, −433, −540, −668, −758 and −770) attained a correlation coefficient of 0.88 (P<0.0001). (d) Schematic of the KEGG mitogen-activated protein (MAP) kinase signalling pathway generated using DAVID. Putative miRNA target genes of the miRNA differentially expressed in the left hemisphere of poly I:C-treated animals, which were also exposed to HU210 during adolescence, were identified. The predicted gene lists were subjected to KEGG pathway analysis, which identified these genes as being highly enriched in the MAP kinase signalling pathway. Putative target genes are indicated with a red star.