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. 2022 May 5;13:2464. doi: 10.1038/s41467-022-30022-2

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© The Author(s) 2022

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 5 — a Schematic for generational studies following HFD exposure. Gestationally exposed animals (Dlk1-FLucLacZ F1^mat-HFD) were bred with wt (CD-fed) mates, maintained on CD, and F2 and F3 offspring examined. b BL signal (blue) in F2 offspring (F2^mat-HFD) derived from F1 HFD-exposed females. Signal was variable and ectopic. c Abdominal BL signal in P56 F2^mat-HFD males (open-circles) and females (filled-circles), from six F1^mat-HFD females and wt^CD males (litters 1–5, no litter from female 6) or two F1^mat-HFD males and wt^CD females (litters 7–8). KI^mat-CD and KI^pat-CD signal shown for comparison. Litter 4 is represented in (b). Source data are provided as a Source Data file. d Dlk1 expression (QRT-PCR, left) in tissues from P56 males (uterus from females) whose mothers were exposed in utero to CD (F2^mat-CD, black) or HFD (F2^mat-HFD, red). Expression normalised to β-Actin, 18S and Hprt (bars show geometric mean with geometric SD; N = 4 + 4 individual mice; Two-way ANOVA on delta-Ct values (Tissue p < 0.0001, Diet p = 0.002, Interaction p < 0.0001); Holm-Šídák’s multiple comparisons follow-up test for diet in each tissue: ****padj < 0.0001, ns=not significant). Allelic Dlk1 analysis in F2^mat-HFD mice (right) showed reduced paternal (dark grey) versus maternal (light grey) expression bias, compared to control conditions (bars indicate mean allelic contribution ±SD; N = 4 + 4 individual mice). Source data are provided as a Source Data file. e Bisulphite analysis in male P56 F2^mat-HFD liver showed Dlk1 sDMR hyper-methylation, increased IG-DMR methylation (padj = 0.078) and slightly reduced Gtl2 sDMR methylation, compared to F1^mat-CD (Fig. 4a). Closed circles: methylated CpG, open circles: un-methylated CpG. Rows show individual clones from a representative individual, percentages indicate total methylation from two animals. (Kolmogorov-Smirnov test comparing clonal methylation levels, using Holm-Šídák’s correction for multiple comparisons: *padj = 0.025, ***padj = 0.0001, ns=not significant). Source data are provided as a Source Data file. f Summary of altered Dlk1 expression following gestational HFD. Dlk1 is silent (white) when transmitted maternally and expressed (blue) when transmitted paternally. Gestational HFD exposure provokes LOI in F1 offspring (blue, box). F1 females transmit altered Dlk1 expression to F2 offspring (blue, box), whereas F1 males and F2 females transmit Dlk1 appropriately.