Maternal care boosted by paternal imprinting in mammals.

In mammals, mothers are the primary caregiver, programmed, in part, by hormones produced during pregnancy. High-quality maternal care is essential for the survival and lifelong health of offspring. We previously showed that the paternally silenced imprinted gene pleckstrin homology-like domain family A member 2 (Phlda2) functions to negatively regulate a single lineage in the mouse placenta called the spongiotrophoblast, a major source of hormones in pregnancy. Consequently, the offspring's Phlda2 gene dosage may influence the quality of care provided by the mother. Here, we show that wild-type (WT) female mice exposed to offspring with three different doses of the maternally expressed Phlda2 gene-two active alleles, one active allele (the extant state), and loss of function-show changes in the maternal hypothalamus and hippocampus during pregnancy, regions important for maternal-care behaviour. After birth, WT dams exposed in utero to offspring with the highest Phlda2 dose exhibit decreased nursing and grooming of pups and increased focus on nest building. Conversely, 'paternalised' dams, exposed to the lowest Phlda2 dose, showed increased nurturing of their pups, increased self-directed behaviour, and a decreased focus on nest building, behaviour that was robustly maintained in the absence of genetically modified pups. This work raises the intriguing possibility that imprinting of Phlda2 contributed to increased maternal care during the evolution of mammals

Unified behavioral scoring for preclinical models

Preclinical mental health research relies upon animal models, and whilst many encouraging advances are being made, reproducibility and translational relevance may be limited by sub-optimal testing or model choices. Animal behaviors are complex and test batteries should be designed to include their multifaceted nature. However, multiple behavioral testing is often avoided due to cost, availability or statistical rigor. Additionally, despite the disparity in the incidence of mental health problems between the sexes, a move toward reducing animal numbers could be a deterrent to including both male and female animals. The current study introduces a unified scoring system for specific behavioral traits with the aim of maximizing the use of all data generated whilst reducing the incidence of statistical errors. Female and male mice from two common background strains were tested on behavior batteries designed to probe multiple aspects of anxiety-related and social behavioral traits. Results for every outcome measure were normalized to generate scores for each test and combined to give each mouse a single unified score for each behavioral trait. The unified behavioral scores revealed clear differences in the anxiety and stress-related, and sociability traits of mice. Principle component analysis of data demonstrated significant clustering of animals into their experimental groups. In contrast, individual tests returned an ambiguous mixture of non-significant trends and significant effects for various outcome measures. Utilizing a range of behavioral measures and combining all outcome measure data to produce unified scores provides a useful tool for detecting subtle behavioral traits in preclinical models

Placental endocrine insufficiency programs anxiety, deficits in cognition and atypical social behaviour in offspring

Abnormally elevated expression of the imprinted PHLDA2 gene has been reported in the placenta of human babies that are growth restricted in utero in several studies. We previously modelled this gene alteration in mice and found that just two-fold increased expression of Phlda2 resulted in placental endocrine insufficiency. In addition, elevated Phlda2 was found to drive fetal growth restriction (FGR) of transgenic offspring and impaired maternal care by their wild type mothers. Being born small and being exposed to suboptimal maternal care have both been associated with the increased risk of mental health disorders in human populations. In the current study we probed behavioural consequences of elevated Phlda2 for the offspring. We discovered increased anxiety-like behaviours, deficits in cognition and atypical social behaviours, with the greatest impact on male offspring. Subsequent analysis revealed alterations in the transcriptome of the adult offspring hippocampus, hypothalamus, and amygdala, regions consistent with these behavioural observations. The inclusion of a group of fully wild type controls raised in a normal maternal environment allowed us to attribute behavioural and molecular alterations to the adverse maternal environment induced by placental endocrine insufficiency rather than the specific gene change of elevated Phlda2. Our work demonstrates that a highly common alteration reported in human fetal growth restriction is associated with negative behavioural outcomes later in life. Importantly, we also establish the experimental paradigm that placental endocrine insufficiency can program atypical behaviour in offspring highlighting the under-appreciated role of placental endocrine insufficiency in driving disorders of later life behaviour

Regulatory feedback from nascent RNA to chromatin and transcription

Transcription and chromatin function are regulated by proteins that bind to DNA, nucleosomes or RNA polymerase II, with specific non-coding RNAs (ncRNAs) functioning to modulate their recruitment or activity. Unlike ncRNAs, nascent pre-mRNA was considered to be primarily a passive player in these processes. In this Opinion article, we describe recently identified interactions between nascent pre-mRNAs and regulatory proteins, highlight commonalities between the functions of nascent pre-mRNA and nascent ncRNA, and propose that both types of RNA have an active role in transcription and chromatin regulation

Maternal care boosted by paternal imprinting in mammals.

In mammals, mothers are the primary caregiver, programmed, in part, by hormones produced during pregnancy. High-quality maternal care is essential for the survival and lifelong health of offspring. We previously showed that the paternally silenced imprinted gene pleckstrin homology-like domain family A member 2 (Phlda2) functions to negatively regulate a single lineage in the mouse placenta called the spongiotrophoblast, a major source of hormones in pregnancy. Consequently, the offspring's Phlda2 gene dosage may influence the quality of care provided by the mother. Here, we show that wild-type (WT) female mice exposed to offspring with three different doses of the maternally expressed Phlda2 gene-two active alleles, one active allele (the extant state), and loss of function-show changes in the maternal hypothalamus and hippocampus during pregnancy, regions important for maternal-care behaviour. After birth, WT dams exposed in utero to offspring with the highest Phlda2 dose exhibit decreased nursing and grooming of pups and increased focus on nest building. Conversely, 'paternalised' dams, exposed to the lowest Phlda2 dose, showed increased nurturing of their pups, increased self-directed behaviour, and a decreased focus on nest building, behaviour that was robustly maintained in the absence of genetically modified pups. This work raises the intriguing possibility that imprinting of Phlda2 contributed to increased maternal care during the evolution of mammals

Alterations in priorities of ‘paternalised (0x)’ and ‘maternalised (2x)’ dams during the nest building task.

<p>(A) Nursing behaviours during the 1-hour nest building task. Duration of nursing (left panels) presented as total nursing duration and duration of crouched, arched, or passive nursing. Number of pup nursing events (right panels) presented as total events and by each category. (B) Duration spent grooming pups (left panel) and number of pup grooming events (right panel). (C) Duration spent self-grooming (left panel) and number of self-grooming events (right panel). (D) Ethovision tracking of distance travelled by dams during the nest building task. (E) Number of visits to the food zone during the nest building task. Additional data in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2006599#pbio.2006599.s002" target="_blank">S2 Table</a>. Numerical data can be found at <a href="https://osf.io/543jg/" target="_blank">https://osf.io/543jg/</a> “RAW NUMERICAL DATA.xlsx”, Sheets labelled Nest_Building_Behaviour. Data are shown as mean ± SEM. Significance determined using one-way ANOVA Dunnet post hoc test. Statistical significance: *<i>p</i> < 0.05, **<i>p</i> < 0.01, and ***<i>p</i> < 0.005. WT(0x), WT dams carrying and caring for all <i>Phlda2</i>^−/+ embryos (maternal inheritance of targeted allele, <i>Phlda2</i> loss of function); WT(1x), WT dams carrying and caring for all WT embryos; WT(2x), WT dams carrying >60% <i>Phlda2</i>^+/+BACx1 embryos generated by crossing <i>Phlda2</i>^+/+BACx1 dams and <i>Phlda2</i>^+/+BACx1 studs.</p

Gene alteration of prenatal maternal hypothalamus and hippocampus exposed to 0, 1, or 2 doses of offspring <i>Phlda2</i>.

<p>(A) Schematic for tissue generation. WT maternal brain tissues from <i>n</i> = 3–4 dams exposed to decreasing doses of offspring <i>Phlda2</i> were collected on E16.5 of pregnancy. (B) Gene expression data generated using the Affymetrix GeneChip platform. Three-way heat map showing differential gene expression levels between the cohorts for the hypothalamus (left panel) and hippocampus (right panel; >0.05 significance). Venn diagram from Limma analysis of significantly altered probes below. (C) Top left panel shows pathways’ perturbation versus overrepresentation for WT(2x) versus WT(0x) hypothalamus. Top right panel shows top-5 pathways plotted by evidence computed by iPathwayGuide: overrepresentation on x-axis (labelled ‘pORA’) and total pathway accumulation on y-axis (labelled ‘pAcc’). Each pathway is represented by coloured dot, with the size proportional to the size of the pathway represented. <i>P</i> values are shown as negative log (base 10) values. Biological processes and molecular functions: bar plots of top-20 genes out of the total number of differentially expressed genes. Up-regulated in red, down-regulated in blue. Box and whisker plots summarise distribution of differentially expressed genes annotated to the GO term. Box represents the first quartile, the median, and the third quartile; outliers are represented by circles. (D) Pathway analysis for WT(2x) versus WT(0x) hippocampus as described above. Additional data in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2006599#pbio.2006599.s005" target="_blank">S2</a> and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2006599#pbio.2006599.s006" target="_blank">S3</a> Figs. Raw data accession number: GSE115276. E, embryonic day; GO, gene ontology; <i>Phlda2</i>, <i>pleckstrin homology-like domain family A member 2</i>; WT, wild-type; WT(0x), WT dams carrying and caring for all <i>Phlda2</i>^−/+ embryos (maternal inheritance of targeted allele, <i>Phlda2</i> loss of function); WT(1x), WT dams carrying and caring for all WT embryos; WT(2x), WT dams carrying >60% <i>Phlda2</i>^+/+BACx1 embryos generated by crossing <i>Phlda2</i>^+/+BACx1 dams and <i>Phlda2</i>^+/+BACx1 studs.</p

Classic tests of maternal behaviour reveal altered behaviour in WT dams exposed to different doses of offspring <i>Phlda2</i>.

<p>(A) Schematic of behavioural paradigm. WT(0x) <i>n</i> = 13, WT(1x) <i>n</i> = 14, and WT(2x) <i>n</i> = 11 experimental dams carrying and caring for pups with 0, 1, or 2 active copies of <i>Phlda2</i>. (B) EPM performed on P2. Percentage of time spent in open and closed arms, number of entries in the open and closed arms, distance moved, and mean velocity measured over 5 minutes. (C) Pup retrieval task on P3. Time taken to sniff and retrieve the first pup measured over 20 minutes. (D) Activity tracking during a 23-hour period after pup-retrieval task. Distance travelled per hour during each phase and total distance travelled during the light phase and dark phases. (E) Time spent on the nest over 23 hours and total time spent in nest during the light phase and dark phase. (F) Nest building task on P4. Dams scored on their ability to build a nest during a 1-hour test (no nest built; nest built/no pups moved into nest; nest built/pups moved into nest). Of the dams, 7/11 WT(2x), 3/14 WT(1x), and 1/13 WT(0x) built a nest and placed at least 1 pup inside within the time limit. (G) Time spent building nest (left panel) and number of nest building events (right panel). Numerical data can be found at <a href="https://osf.io/543jg/" target="_blank">https://osf.io/543jg/</a> “RAW NUMERICAL DATA.xlsx”, Sheets labelled EPM, Pup_Retrieval_Nest_Building, Ethovision_23hr. Data shown as mean ± SEM. Significance determined using one-way ANOVA Dunnet post hoc test. Statistical significance: *<i>p</i> < 0.05, **<i>p</i> < 0.01, and ***<i>p</i> < 0.005. EPM, elevated plus maze; P, postnatal day; <i>Phlda2</i>, <i>pleckstrin homology-like domain family A member 2</i>; WT, wild-type; WT(0x), WT dams carrying and caring for all <i>Phlda2</i>^−/+ embryos (maternal inheritance of targeted allele, <i>Phlda2</i> loss of function); WT(1x), WT dams carrying and caring for all WT embryos; WT(2x), WT dams carrying >60% <i>Phlda2</i>^+/+BACx1 embryos generated by crossing <i>Phlda2</i>^+/+BACx1 dams and <i>Phlda2</i>^+/+BACx1 studs.</p

<i>Phlda2</i> imprinting may have increased maternal care provision during the evolution of mammals.

<p><i>Phlda2</i> regulates the production of placental hormones by controlling lineage allocation in the placenta. Decreasing <i>Phlda2</i> expression through imprinting increases placental hormone production, programming the mother to increase her focus on nurturing her young. Maternal silencing of <i>Phlda2</i> occurred after marsupials diverged from eutherian mammals more than 160 million years ago. Postpartum development in marsupials predominantly takes place in a pouch, with maternal care being most prominent after the young leave the pouch. In contrast, the eutherian mother is primed during pregnancy to display sophisticated maternal nurturing from birth, raising the intriguing possibility that imprinting of <i>Phlda2</i> contributed to the evolution of maternal care in mammals. <i>Phlda2</i>, <i>pleckstrin homology-like domain family A member 2</i>.</p