Assessing changes induced by in utero low protein dietary exposure in offspring development and behaviour

Embryonic development involves temporal and spatial coordination of epigenetic processes, which are exquisitely sensitive to environmental influences and can have life-long implications for disease if disrupted. In this thesis, I examined the impact of gestational diet, specifically protein restriction, on the expression of cyclin-dependent kinase inhibitor C1 (Cdkn1c), a gene that is epigenetically regulated through genomic imprinting, during development and post-natally, with an allele-specific bioluminescent reporter mouse model. I showed that in utero low protein diet (LPD) exposure affects behaviour in adolescence and adulthood, through changes in the dopamine system at a transcriptional, cellular and metabolic level. Sensitisation experiments suggest that adult LPD mice are hypersensitive to cocaine, as they show increased locomotor activity compared to controls, and increased stereotypy at higher doses. Adult LPD mice also show hyperactivity and reduced anxiety, which are phenotypes frequently observed in hyperdopaminergic mouse models. Adolescent LPD mice demonstrated impaired motor function, in contrast to age-matched controls, based on open field and rotarod performance. However, I have shown this deficit to be transient and resolved by adulthood. Juvenile and adult LPD-exposed mice displayed a 25% increase in midbrain dopaminergic neuron numbers. Moreover, RNA analysis from adult midbrains showed a downregulation of the gene that encodes dopamine transporter (DAT) and upregulation of dopamine receptor 5 (D5) in LPD mice, with many other dopamine-related genes unaffected. A pilot RNA-seq experiment on midbrain neuronal nuclei identified 123 differentially expressed genes in LPD mice, affecting cholesterol synthesis and markers linked to neurodegeneration and early-life stress. Lastly, μPET imaging revealed elevated striatal dopamine synthesis capacity in adult LPD mice, recapitulating one of the hallmarks of schizophrenia, and in accordance with reports of gestational exposure to famine inferring increased susceptibility to schizophrenia. This work sheds light on consequences of early-life adversity and epigenetic disruption on embryonic brain development, behaviour and disease risk.Open Acces

Protein restriction during pregnancy alters Cdkn1c silencing, dopamine circuitry and offspring behaviour without changing expression of key neuronal marker genes

We tracked the consequences of in utero protein restriction in mice throughout their development and life course using a luciferase-based allelic reporter of imprinted Cdkn1c. Exposure to gestational low-protein diet (LPD) results in the inappropriate expression of paternally inherited Cdkn1c in the brains of embryonic and juvenile mice. These animals were characterised by a developmental delay in motor skills, and by behavioural alterations indicative of reduced anxiety. Exposure to LPD in utero resulted in significantly more tyrosine hydroxylase positive (dopaminergic) neurons in the midbrain of adult offspring as compared to age-matched, control-diet equivalents. Positron emission tomography (PET) imaging revealed an increase in striatal dopamine synthesis capacity in LPD-exposed offspring, where elevated levels of dopamine correlated with an enhanced sensitivity to cocaine. These data highlight a profound sensitivity of the developing epigenome to gestational protein restriction. Our data also suggest that loss of Cdkn1c imprinting and p57KIP2 upregulation alters the cellular composition of the developing midbrain, compromises dopamine circuitry, and thereby provokes behavioural abnormalities in early postnatal life. Molecular analyses showed that despite this phenotype, exposure to LPD solely during pregnancy did not significantly change the expression of key neuronal- or dopamine-associated marker genes in adult offspring

Protein restriction during pregnancy alters Cdkn1c silencing, dopamine circuitry and offspring behaviour without changing expression of key neuronal marker genes.

Acknowledgements: We would like to thank Chad Whilding for assistance with immunofluorescence image analysis, Justyna Glegola for advice on mouse behavioural testing and George Young for bioinformatics support. We thank David Bonsall, Sac-Pham Tang and the rest of the radiochemistry and biology teams at Invicro for assistance with F-DOPA imaging. This work was supported by funding from the Medical Research Council (MC_U120027516 (AGF and MM), MC_UP_1605/12 (AGF), MC_UP_1605/11 (MM) and MC-A654-5QB40 (DJW). For the purpose of open access, the author has applied a CC BY public copyright licence to any Author Accepted Manuscript version arising from this submission.We tracked the consequences of in utero protein restriction in mice throughout their development and life course using a luciferase-based allelic reporter of imprinted Cdkn1c. Exposure to gestational low-protein diet (LPD) results in the inappropriate expression of paternally inherited Cdkn1c in the brains of embryonic and juvenile mice. These animals were characterised by a developmental delay in motor skills, and by behavioural alterations indicative of reduced anxiety. Exposure to LPD in utero resulted in significantly more tyrosine hydroxylase positive (dopaminergic) neurons in the midbrain of adult offspring as compared to age-matched, control-diet equivalents. Positron emission tomography (PET) imaging revealed an increase in striatal dopamine synthesis capacity in LPD-exposed offspring, where elevated levels of dopamine correlated with an enhanced sensitivity to cocaine. These data highlight a profound sensitivity of the developing epigenome to gestational protein restriction. Our data also suggest that loss of Cdkn1c imprinting and p57KIP2 upregulation alters the cellular composition of the developing midbrain, compromises dopamine circuitry, and thereby provokes behavioural abnormalities in early postnatal life. Molecular analyses showed that despite this phenotype, exposure to LPD solely during pregnancy did not significantly change the expression of key neuronal- or dopamine-associated marker genes in adult offspring

Epigenetic changes induced by in utero dietary challenge result in phenotypic variability in successive generations of mice.

Transmission of epigenetic information between generations occurs in nematodes, flies and plants, mediated by specialised small RNA pathways, modified histones and DNA methylation. Similar processes in mammals can also affect phenotype through intergenerational or trans-generational mechanisms. Here we generate a luciferase knock-in reporter mouse for the imprinted Dlk1 locus to visualise and track epigenetic fidelity across generations. Exposure to high-fat diet in pregnancy provokes sustained re-expression of the normally silent maternal Dlk1 in offspring (loss of imprinting) and increased DNA methylation at the somatic differentially methylated region (sDMR). In the next generation heterogeneous Dlk1 mis-expression is seen exclusively among animals born to F1-exposed females. Oocytes from these females show altered gene and microRNA expression without changes in DNA methylation, and correct imprinting is restored in subsequent generations. Our results illustrate how diet impacts the foetal epigenome, disturbing canonical and non-canonical imprinting mechanisms to modulate the properties of successive generations of offspring