Characterisation of the F2 CU hepatic and placental transcriptome.

<p>(A) Schematic of the F2 generation, the transcriptome of the CU and CC F2 crosses was assessed at E16.5. (B) Distribution of the ranked difference in gene expression in CU E16.5 liver according to FDR q-value. A list of genes comprising a network involved in lipid metabolism, identified as enriched in CU liver using IPA software was used as a positive control. Imprinted genes most closely resemble randomly selected genes. (C) Distribution of genes differentially expressed in CU E16.5 placenta according to FDR q-value. A list of genes comprising a network involved in lipid metabolism, identified as enriched in CU placenta using IPA software was used as a positive control. Imprinted genes most closely resemble randomly selected genes.</p

Expression of candidate imprinted genes at E16.5 in the F2 generation and assessment of F1 germline imprints.

<p>(A) Schematic of the F2 generation, expression was assessed at E16.5. (B) There are no significant differences in the F2 hepatic expression of imprinted genes at E16.5. Error bars denote SEM. (C) At E16.5 CU individuals demonstrate a significant increased in placental expression of <i>Igf2P0</i> (One-way ANOVA, Bonferroni's multiple comparison post-test. P<0.05), while UU placentas significant up-regulate <i>Snrpn</i> (One-way ANOVA, Bonferroni's multiple comparison post-test. P<0.01). Per condition n≥24, 6 litters. Error bars denote SEM. (D, E) Methylation was assessed by pyrosequencing at the paternally methylated <i>H19</i> (A) and <i>Dlk1/Dio3</i> (B) germline ICRs. Sperm from both control and <i>in utero</i> undernourished males showed the expected hypermethylation in comparison to somatic tissues (liver). Controls: n = 12, 5 litters. Undernourished n = 11, 4 litters. Error bars denote SEM. (F, G) Pyrosequencing assessment of methylation at the maternally methylated <i>Peg3</i> (C) and <i>Snrpn</i> (D) germline DMRs shows that these regions are unmethylated in the sperm of both control and <i>in utero</i> undernourished males. Controls: n = 12, 5 litters Undernourished n = 11, 4 litters. Error bars denote SEM.</p

Analysis of F2 placental candidate imprinted gene expression by two-way ANOVA.

<p>Analysis of F2 placental candidate imprinted gene expression by two-way ANOVA.</p

Characterisation of the E16.5 hepatic and placental transcriptome response to undernourishment.

<p>(A) Schematic of the experimental design: F1 generation: On pregnancy day 12.5, dams were randomly assigned to either control or undernutrition groups and food intake of undernutrition mothers was restricted to 50% that of controls. After delivery litter size was equalized to eight pups and dams received 9F chow <i>ad libitum</i>. Pups nursed freely and were weaned at 3 weeks onto 9F chow <i>ad libitum</i>. F2 generation: control and undernourished females from the F1 generation were mated at age 2 months with nonsibling control or undernourished males. After confirmation of pregnancy, females were caged individually and fed <i>ad libitum</i> throughout pregnancy to produce the four experimental F2 generation groups: CC – both parents are controls. CU – control dam, <i>in utero</i> undernourished sire; UC - <i>in utero</i> undernourished dam, control sire; UU - <i>in utero</i> undernourished dam, <i>in utero</i> undernourished sire. (B, C) Gene ontology (GO) analysis of the F1 undernourished hepatic transcriptome at E16.5: Functional enrichment analysis using (B) DAVID <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002605#pgen.1002605-Dennis1" target="_blank">[27]</a>–<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002605#pgen.1002605-Huangda1" target="_blank">[28]</a> and (C) Ingenuity Pathway Analysis functional analysis tools. Among genes upregulated in the liver, both tools identify significant enrichment (after Benjamini-Hochberg correction for multiple testing) of gene groups associated with metabolism, particularly of lipids. Among genes downregulated in the liver, both tools identify significant enrichment of categories related to cell-cycle and the control of proliferation. (D) Transcriptome analysis of E16.5 undernourished versus control liver. Distribution of the ranked difference in gene expression according to FDR q-value. Lists of housekeeping genes and adult hepatic fasting-response genes curated from the literature were used as negative and positive controls respectively and their rankings shown. Imprinted genes most closely resemble randomly selected genes. (E) Transcriptome analysis of E16.5 undernourished versus control placenta; distribution of genes according to FDR q-value. The placental response to maternal undernourishment is undetermined, and is different to that of the liver, as fasting response genes are largely unperturbed. Therefore a network of genes involved in RNA post-transcriptional modification, identified as enriched in undernourished placenta by IPA analysis; was used as a positive control. Imprinted genes most closely resemble randomly selected genes.</p

Gene set enrichment analysis of E16.5 F1 hepatic transcriptome response to undernutrition.

<p>Gene set enrichment analysis of E16.5 F1 hepatic transcriptome response to undernutrition.</p