Geula Hanin

Early-life factors, including postnatal nutrition, shape long-term health outcomes with epigenetic effects being implicated but poorly understood in the process. Zinc-finger protein 57 (ZFP57), is an epigenetic regulator of genomic imprinting, a process controlling gene expression based on parental origin with a vital role in prenatal growth. Here, we report an imprinting-independent function of ZFP57 in postnatal resource control via the mammary gland. ZFP57 influences multiple mammary gland phenotypes, including ductal branching and cellular homeostasis with its absence leading to significant differential gene expression related to alveologenesis and lactogenesis and altered milk composition. Zfp 57 ^-/- dams attenuate offspring growth, with impacts on offspring metabolic health; effects exacerbated when pups are raised by a dam of a different genotype than their birth mother. The study identifies ZFP57 as a major regulator of both pre and postnatal resource control in mammals, offering new insights into early factors influencing lifelong health.

Lactation is a cornerstone of mammalian life, providing nutrient-rich milk that supports offspring growth and development. Breastfeeding offers immediate and long-term benefits for infant and maternal health, shaping life-course health outcomes and reducing disease risks. Milk is a complex and dynamic fluid containing nutrients and bioactive molecules, and its composition is affected by environmental and genetic factors. Research on human milk falls into two primary areas: its biochemistry, which explores milk composition, and epidemiology, which examines the impact of lactation and breastfeeding on health. Together, these fields provide insights into the function of milk components. However, the genetic mechanisms that determine and influence milk composition remain poorly understood. Most findings linking genetics and milk composition have emerged from agricultural studies, where the mechanistic understanding is limited. In-vivo studies focus on mammary gland development, lactation, and cancer mechanisms, yet these interconnected topics are often divided across different research fields. Emerging evidence also suggests a role for genomic imprinting in the regulation of postnatal resource allocation via lactation and milk composition. This review aims to provide a summary of current knowledge on genetic influences affecting milk composition, offering a holistic perspective that unifies mammary gland physiology, milk biochemistry, lactation epidemiology, and genetics.

The mammary gland, which primarily develops postnatally, undergoes significant changes during pregnancy and lactation to facilitate milk production. Through the generation and analysis of 480 transcriptomes, we provide the most detailed allelic expression map of the mammary gland, cataloguing cell-type-specific expression from ex-vivo purified cell populations over 10 developmental stages, enabling comparative analysis. The work identifies genes involved in the mammary gland cycle, parental-origin-specific and genetic background-specific expression at cellular and temporal resolution, genes associated with human lactation disorders and breast cancer. Genomic imprinting, a mechanism regulating gene expression based on parental origin, is crucial for controlling gene dosage and stem cell potential throughout development. The analysis identified 25 imprinted genes monoallelically expressed in the mammary gland, with several showing allele-specific expression in distinct cell types. No novel imprinted genes were identified and the absence of biallelically expressed imprinted genes suggests that, unlike in brain, selective absence of imprinting does not regulate gene dosage in the mammary gland. This research highlights transcriptional dynamics within mammary gland cells and identifies novel candidate genes potentially significant in the tissue during pregnancy and lactation. Overall, this comprehensive atlas represents a valuable resource for future studies on expression and transcriptional dynamics in mammary cells.

The mammary gland, which primarily develops postnatally, undergoes significant changes during pregnancy and lactation to facilitate milk production. Through the generation and analysis of 480 transcriptomes, we provide the most detailed allelic expression map of the mammary gland, cataloguing cell-type-specific expression from ex-vivo purified cell populations over 10 developmental stages, enabling comparative analysis. The work identifies genes involved in the mammary gland cycle, parental-origin-specific and genetic background-specific expression at cellular and temporal resolution, genes associated with human lactation disorders and breast cancer. Genomic imprinting, a mechanism regulating gene expression based on parental origin, is crucial for controlling gene dosage and stem cell potential throughout development. The analysis identified 25 imprinted genes monoallelically expressed in the mammary gland, with several showing allele-specific expression in distinct cell types. No novel imprinted genes were identified and the absence of biallelically expressed imprinted genes suggests that, unlike in brain, selective absence of imprinting does not regulate gene dosage in the mammary gland. This research highlights transcriptional dynamics within mammary gland cells and identifies novel candidate genes potentially significant in the tissue during gestation and lactation. Overall, this comprehensive atlas represents a valuable resource for future studies on expression and transcriptional dynamics in mammary cells.

Early-life factors, including nutrition, shape long-term health outcomes. Despite the essential role of lactation in maternal nutritional support, the influence of epigenetic factors on lactation and postnatal growth remains poorly understood. Zinc-finger protein 57 (ZFP57), is an epigenetic regulator of genomic imprinting, a process that directs gene expression based on parental origin, playing a vital role in mammalian prenatal growth. Here, we identify a novel function of ZFP57 in regulating the mammary gland, where it serves as a key modulator of postnatal resource control, independently of imprinted genes. ZFP57 influences multiple aspects of mammary gland function, including ductal branching and cellular homeostasis. Its absence leads to significant differential gene expression, related to alveologenesis, lactogenesis and milk synthesis, associated with delayed lactation and altered milk composition. This results in life-long impacts on offspring including the development of metabolic syndrome. Cross-fostering reveals intricate dynamics between mother and offspring during lactation. Pups raised by a dam of a different genotype than their birth mother exhibit exacerbated metabolic features in adulthood, providing additional insight into the programming of offspring long-term health by maternal context. This study deepens our understanding of the interplay between epigenetic factors, lactation, and postnatal resource control and identifies ZFP57 as a major regulator of both pre and postnatal resource control in mammals.

The mammary gland is a vital exocrine organ that has evolved in mammals to secrete milk and provide nutrition to ensure the growth and survival of the neonate The mouse mammary gland displays extraordinary plasticity each time the female undergoes pregnancy and lactation, including a sophisticated process of tertiary branching and alveologenesis to form a branched epithelial tree and subsequently milk-producing alveoli. Upon the cessation of lactation, the gland remodels back to a simple ductal architecture via highly regulated involution processes. At the cellular level, the plasticity is characterised by proliferation of mammary cell populations, differentiation and apoptosis, accompanied by major changes in cell function and morphology. The mammary epithelium requires a specific stromal environment to grow, known as the mammary fat pad. Mammary adipocytes are one of the most prominent cell types in the fat pad, but despite their vast proportion in the tissue and their crucial interaction with epithelial cells, their physiology remains largely unknown. Over the past decade, the need to understand the properties and contribution of mammary adipocytes has become more recognised. However, the development of adequate methods and protocols to study this cellular niche is still lagging, partially due to their fragile nature, the difficulty of isolating them, the lack of reliable cell surface markers and the heterogenous environment in this tissue, which differs from other adipocyte depots. Here, we describe a new rapid and simple flow cytometry protocol specifically designed for the analysis and isolation of mouse mammary adipocytes across mammary gland developmental stages.

Intracisternal A-particles (IAPs) are endogenous retroviruses (ERVs) responsible for most insertional mutations in the mouse. Full-length IAPs harbour genes flanked by long terminal repeats (LTRs). Here, we identify a solo LTR IAP variant ( C57iap1 ^solo ) recently formed in the inbred C57BL/6J mouse strain. In contrast to the C57BL/6J full-length IAP at this locus ( C57iap1 ^full ), C57iap1 ^solo lacks DNA methylation and H3K9 trimethylation. The distinct DNA methylation levels between the two alleles are established during preimplantation development, likely due to loss of KRAB zinc finger protein binding at the C57iap1 ^solo variant. C57iap1 ^solo methylation increases and becomes more variable in a hybrid genetic background yet is unresponsive to maternal dietary methyl supplementation. Differential epigenetic modification of the two variants is associated with metabolic differences and tissue-specific changes in adjacent gene expression. Our characterisation of C57iap1 as a genetically-induced epiallele with functional consequences establishes a new model to study transposable element repression and host-element co-evolution.

Genomic imprinting is an epigenetically regulated process leading to gene expression according to its parental origin. Imprinting is essential for prenatal growth and development, regulating nutritional resources to offspring, and contributing to a favored theory about the evolution of imprinting being due to a conflict between maternal and paternal genomes for the control of prenatal resources—the so‐called kinship hypothesis. Genomic imprinting has been mainly studied during embryonic and placental development; however, maternal nutrient provisioning is not restricted to the prenatal period. In this context, the mammary gland acts at the maternal‐offspring interface providing milk to the newborn. Maternal care including lactation supports the offspring, delivering nutrients and bioactive molecules protecting against infections and contributing to healthy organ development and immune maturation. The normal developmental cycle of the mammary gland—pregnancy, lactation, involution—is vital for this process, raising the question of whether genomic imprinting might also play a role in postnatal nutrient transfer by controlling mammary gland development. Characterizing the function and epigenetic regulation of imprinted genes in the mammary gland cycle may therefore provide novel insights into the evolution of imprinting since the offspring's paternal genome is absent from the mammary gland, in addition to increasing our knowledge of postnatal nutrition and its relation to life‐long health.This article is categorized under: Developmental Biology > Developmental Processes in Health and Disease

Epilepsy is a common neurological disease, manifested in unprovoked recurrent seizures. Epileptogenesis may develop due to genetic or pharmacological origins or following injury, but it remains unclear how the unaffected brain escapes this susceptibility to seizures. Here, we report that dynamic changes in forebrain microRNA (miR)-211 in the mouse brain shift the threshold for spontaneous and pharmacologically induced seizures alongside changes in the cholinergic pathway genes, implicating this miR in the avoidance of seizures. We identified miR-211 as a putative attenuator of cholinergic-mediated seizures by intersecting forebrain miR profiles that were Argonaute precipitated, synaptic vesicle target enriched, or differentially expressed under pilocarpine-induced seizures, and validated TGFBR2 and the nicotinic antiinflammatory acetylcholine receptor nAChRa7 as murine and human miR-211 targets, respectively. To explore the link between miR-211 and epilepsy, we engineered dTg-211 mice with doxycycline-suppressible forebrain overexpression of miR-211. These mice reacted to doxycycline exposure by spontaneous electrocorticography-documented nonconvulsive seizures, accompanied by forebrain accumulation of the convulsive seizures mediating miR-134. RNA sequencing demonstrated in doxycycline-treated dTg-211 cortices overrepresentation of synaptic activity, Ca²⁺ transmembrane transport, TGFBR2 signaling, and cholinergic synapse pathways. Additionally, a cholinergic dysregulated mouse model overexpressing a miR refractory acetylcholinesterase-R splice variant showed a parallel propensity for convulsions, miR-211 decreases, and miR-134 elevation. Our findings demonstrate that in mice, dynamic miR-211 decreases induce hypersynchronization and nonconvulsive and convulsive seizures, accompanied by expression changes in cholinergic and TGFBR2 pathways as well as in miR-134. Realizing the importance of miR-211 dynamics opens new venues for translational diagnosis of and interference with epilepsy. Significance Acute traumatic stress increases the sensitivity to develop epileptic seizures in certain people. It is therefore important to discover physiological mechanisms that avoid epilepsy. To test if rapidly inducible microRNAs (miRs) could mediate such protection, we combined mouse engineering, RNA sequencing, electric recording of brain activity, and learning tests. We discovered that miR-211, originating from an epilepsy-related genomic locus, may be involved, and therefore engineered mice produce a drug-suppressible excess of brain miR-211. In these mice, suppressing miR-211 excess to the original expression levels in normal brains led to electrically recorded epilepsy and hypersensitivity to epilepsy-inducing compounds; it also modified acetylcholine receptor composition. The functional impact of miR-211 dynamics on seizure threshold may enable future development of miR-211–directed therapeutics.