Rosalind M. John

Background: Imprinted genes are expressed from one parental allele as a result of epigenetic events initiated in the male and female germlines. We previously identified a role for imprinted genes as master regulators of placental endocrine lineage development in mice. We further reported that loss-of-function of the paternally expressed imprinted gene Peg3 resulted in sexually dimorphic placental and behavioural phenotypes, with the male being relatively more impacted in both cases. Here, we asked whether loss-of-imprinting of the maternally expressed imprinted gene Phlda2 similarly had a sexually dimorphic impact on placental endocrine lineages, potentially underlying male-biased behavioural outcomes that arise when this gene is abnormally expressed. Methods: C57BL/6 wildtype (WT) female mice were mated with Phlda2 ^BACx1 transgenic studs to generate WT control and loss-of-imprinting model samples overexpressing Phlda2 . Placental and fetal weight, placental endocrine lineages and hormone gene expression were characterised at embryonic day (E) 14.5, together with fetal and placenta weight data at E18.5. Results: RNAscope-aided identification of the placental endocrine lineages revealed a significant loss of spongiotrophoblast cells from the junctional zone, with a substantially greater impact on female mutant placenta compared to male. Glycogen cell and parietal trophoblast giant cell number was also diminished but without detectable sexual dimorphism. Reverse-transcription quantitative PCR (RT-qPCR) and RNA sequence analysis supported a female-biased reduction in the expression of placental hormones expressed by the spongiotrophoblast in response to Phlda2 “loss-of-imprinting”. Placental changes were associated with reduced fetal growth in females but not in males late in gestation. Conclusions: Phlda2 expression is known to increase in response to maternal adversity, including nutritional deficiencies, thereby reducing placental “demand” signalling by limiting hormone production. Our new findings identify a striking biological asymmetry whereby female fetuses reduce their demands on the mother in response to Phlda2 loss-of-imprinting, whereas male fetuses do not. Such asymmetries may be linked to sex-specific reproductive strategies in mammals in response to prenatal adversity. Our findings also have implications for human pregnancy research. Elevated placental PHLDA2 has repeatedly been linked to fetal growth restriction and low birthweight but without consideration of the sex of the infant. Our findings that this imprinted gene can exert sex-specific effects on fetal growth may therefore be of translational importance.

The neurodevelopmental disorder Prader–Willi syndrome (PWS) is caused by paternally derived loss of gene expression from the imprinted interval on chromosome 15q11-q13. Recently, it has been suggested that the abnormal feeding-related behaviours characteristic of PWS can, in part, be developmentally programmed in utero via abnormal placental function. Here, we report that several PWS-associated genes were expressed in mouse placenta with three PWS-associated RNA transcripts, i.e. genes Magel2 and Necdin, and the lncRNA Sngh14, colocalised to the Kdr-positive (Kdr+) foetal endothelial cells of the labyrinth zone central to nutrient transport. In a novel PWS mouse model (Large+/−) we found markedly reduced expression of PWS-associated genes in the placenta and an associated ∼25% reduction in Kdr+ foetal endothelial cells. Although this did not directly translate into a significant reduction in foetal growth late in gestation, these data suggest that placental function and nutrient transfer from mother to foetus could be compromised in PWS contributing to later post-natal phenotypes.

The neurodevelopmental disorder Prader-Willi syndrome (PWS) is caused by loss of paternally-derived gene expression from the imprinted interval on chromosome 15q11-q13. Recently, it has been suggested that the abnormal feeding-related behaviours characteristic of PWS may, in part, be developmentally programmed in utero via abnormal placental function. Here we report that several PWS-genes are expressed in mouse placenta with three PWS-transcripts Magel2 , Necdin and the lncRNA Sngh14 , co-localising to the Kdr -positive fetal endothelial cells of the labyrinth zone central to nutrient transport. In a novel PWS deletion mouse model (Large ^+/− ) we find markedly reduced expression of PWS genes in the placenta and an associated ∼25% reduction in Kdr -positive fetal endothelial cells. Although this did not directly translate into a significant reduction in fetal growth late in gestation, these data suggest that placental function and nutrient transfer from mother to fetus could be compromised in PWS contributing to later post-natal phenotypes. Summary statement: Reduced expression of PWS-associated genes in the mouse placenta results in a 25% loss of the fetal endothelial cells that play a central role in nutrient and gas exchange.

Introduction: Imprinted genes are expressed from one parental allele as a consequence of epigenetic processes initiated in the germline. Consequently, their ability to influence phenotype depends on their parent-of-origin. Recent research suggests that the sex of the individual expressing the imprinted gene is also important. We have previously reported that genetically wildtype (WT) dams carrying and caring for pups mutant for PEG3 exhibit anxiety-like behaviours and their mutant pups show a reduction in ultrasonic vocalisation when separated from their mothers. Sex-specificity was not examined.Methods: WT female mice were mated with WT, heterozygous Peg3^−/+ or homozygous Peg3^−/− studs to generate all WT (control), 50:50 mixed or 100% mutant litters, respectively, followed by behavioural assessment of both dams and their pups.Results: We reproduced our original finding that WT dams carrying and caring for 100% mutant litters exhibit postpartum anxiety-like symptoms and delayed pup retrieval. Additionally, these WT dams were found to allocate less time to pup-directed care behaviours relative to controls. Male Peg3-deficient pups demonstrated significantly reduced vocalisation with a more subtle communication deficit in females. Postweaning, male mutants exhibited deficits across a number of key social behaviours as did WT males sharing their environment with mutants. Only modest variations in social behaviour were detected in experimental females.Discussion: We have experimentally demonstrated that Peg3 deficiency confined to the offspring causes anxiety in mouse mothers and atypical behaviour including deficits in communication in their male offspring. A male-specific reduction in expression PEG3 in the fetally-derived placenta has previously been associated with maternal depression in human pregnancy. Maternal mood disorders such as depression and anxiety are associated with delays in language development and neuroatypical behaviour more common in sons. Peg3 deficiency could drive the association of maternal and offspring behavioural disorders reported in humans.

Imprinted genes are subject to germline epigenetic modification resulting in parental-specific allelic silencing. Although genomic imprinting is thought to be important for maternal behaviour, this idea is based on serendipitous findings from a small number of imprinted genes. Here, we undertook an unbiased systems biology approach, taking advantage of the recent delineation of specific neuronal populations responsible for controlling parental care, to test whether imprinted genes significantly converge to regulate parenting behaviour. Using single-cell RNA sequencing datasets, we identified a specific enrichment of imprinted gene expression in a recognised “parenting hub”, the galanin-expressing neurons of the preoptic area. We tested the validity of linking enriched expression in these neurons to function by focusing on MAGE family member L2 (Magel2), an imprinted gene not previously linked to parenting behaviour. We confirmed expression of Magel2 in the preoptic area galanin expressing neurons. We then examined the parenting behaviour of Magel2-null^(+/p) mice. Magel2-null mothers, fathers and virgin females demonstrated deficits in pup retrieval, nest building and pup-directed motivation, identifying a central role for this gene in parenting. Finally, we show that Magel2-null mothers and fathers have a significant reduction in POA galanin expressing cells, which in turn contributes to a reduced c-Fos response in the POA upon exposure to pups. Our findings identify a novel imprinted gene that impacts parenting behaviour and, moreover, demonstrates the utility of using single-cell RNA sequencing data to predict gene function from expression and in doing so here, have identified a purposeful role for genomic imprinting in mediating parental behaviour.

Genomic imprinting refers to the parent-of-origin expression of genes, which originates from epigenetic events in the mammalian germ line. The evolution of imprinting may reflect a conflict over resource allocation early in life, with silencing of paternal genes in offspring soliciting increased maternal provision and silencing of maternal genes limiting demands on the mother. Parental caregiving has been identified as an area of potential conflict, with several imprinted genes serendipitously found to directly influence the quality of maternal care. Recent systems biology approaches, based on single-cell RNA sequencing data, support a more deliberate relationship, which is reinforced by the finding that imprinted genes expressed in the offspring influence the quality of maternal caregiving. These bidirectional, reiterative relationships between parents and their offspring are critical both for short-term survival and for lifelong wellbeing, with clear implications for human health.

Imprinted genes are subject to germline epigenetic modification resulting in parental-specific allelic silencing. Although genomic imprinting is thought to be important for maternal behaviour, this idea is based on serendipitous findings from a small number of imprinted genes. Here, we undertook an unbiased systems biology approach, taking advantage of the recent delineation of specific neuronal populations responsible for controlling parental care, to test whether imprinted genes significantly converge to regulate parenting behaviour. Using single-cell RNA sequencing datasets, we identified a specific enrichment of imprinted gene expression in a recognised “parenting hub”, the galanin-expressing neurons of the preoptic area. We tested the validity of linking enriched expression in these neurons to function by focusing on MAGE family member L2 (Magel2), an imprinted gene not previously linked to parenting behaviour. We confirmed expression of Magel2 in the preoptic area galanin expressing neurons. We then examined the parenting behaviour of Magel2 ^+/- null mice. Magel 2-null mothers, fathers and virgin females demonstrated deficits in pup retrieval, nest building and pup-directed motivation, identifying a central role for this gene in parenting. Finally, we show that Magel2- null mothers and fathers have a significant reduction in POA galanin expressing cells, which in turn contributes to a reduced c-Fos response in the POA upon exposure to pups. Our findings identify a novel imprinted gene that impacts parenting behaviour and, moreover, demonstrates the utility of using single-cell RNA sequencing data to predict gene function from expression and in doing so here, have identified a purposeful role for genomic imprinting in mediating parental behaviour.

Placental lactogen (hPL) is a key hormone of pregnancy responsible for inducing maternal adaptations critical for a successful pregnancy. Low levels of placental lactogen have been associated with lower birth weight as well as symptoms of maternal depression and anxiety. Lower placental lactogen has been reported in women with higher body mass index (BMI) but it is unclear whether prenatal health behaviours predict hPL levels or if hPL is associated with infant weight outcomes. This study utilised data from the longitudinal Grown in Wales cohort, based in South Wales. Participants were recruited at the pre-surgical appointment for an elective caesarean section. This study incorporates data from recruitment, post-delivery and a 12 month follow-up. Measures of maternal serum hPL were available for 248 participants. Analysis included unadjusted and adjusted linear and binary regression. Unadjusted, prenatal smoking and a Health Conscious dietary pattern were associated with hPL levels, however this was lost on adjustment for BMI at booking, Welsh Index of Multiple Deprivation (WIMD) score and placental weight. When stratified by maternal BMI at booking, a Health Conscious dietary pattern remained associated with increased hPL levels in women with a healthy BMI (p=.024, B=.59. 95% CI=.08,1.11) following adjustment for WIMD score and placental weight. When adjusted for a wide range of confounders, maternal hPL was also associated with increased custom birthweight centiles (CBWC) (p=.014, B=1.64. 95% CI=.33,2.94) and increased odds of large for gestational age deliveries (p=<.001, Exp(B)=1.42. 95% CI=1.17,1.72). This study identified that consuming a Health Conscious dietary pattern in pregnancy was associated with increased hPL, within women of a healthy BMI. Moreover, higher hPL levels were associated with increased CBWC and increased odds of delivering a large for gestational age infant. This improves the current limited evidence surrounding the nature of hPL in these areas.