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The placenta performs a range of crucial functions that support fetal growth during pregnancy, including facilitating the supply of oxygen and nutrients to the fetus, removal of waste products from the fetus and the endocrine modulation of maternal physiology. The placenta also stores glucose in the form of glycogen, the function of which remains unknown. Aberrant placental glycogen storage in humans is associated with maternal diabetes during pregnancy and pre-eclampsia, thus linking placental glycogen storage and metabolism to pathological pregnancies. To understand the role of placental glycogen in normal and complicated pregnancies, we must turn to animal models. Over 40 targeted mutations in mice demonstrate the defects in placental cells that store glycogen and suggest that placental glycogen represents a source of readily mobilized glucose required during periods of high fetal demand. However, direct functional evidence is currently lacking. Here, we evaluate these genetic mouse models with placental phenotypes that implicate glycogen trophoblast cell differentiation and function to illuminate the common molecular pathways that emerge and to better understand the relationship between placental glycogen and fetal growth. We highlight the current limitations in exploring the key questions regarding placental glycogen storage and metabolism and define how to experimentally overcome these constraints.

Abnormal uptake or metabolism of folate increases risk of human pregnancy complications, though the mechanism is unclear. Here, we explore how defective folate metabolism influences early development by analysing mice with the hypomorphic Mtrr gt mutation. MTRR is necessary for methyl group utilisation from folate metabolism, and the Mtrr gt allele disrupts this process. We show that the spectrum of phenotypes previously observed in Mtrr gt/gt conceptuses at embryonic day (E) 10.5 is apparent from E8.5 including developmental delay, congenital malformations, and placental phenotypes. Notably, we report misalignment of some Mtrr gt conceptuses within their implantation sites from E6.5. The degree of misorientation occurs across a continuum, with the most severe form visible upon gross dissection. Additionally, some Mtrr gt/gt conceptuses display twinning. Therefore, we implicate folate metabolism in blastocyst orientation and spacing at implantation. Skewed growth likely influences embryo development since developmental delay and heart malformations (but not defects in neural tube closure or trophoblast differentiation) associate with severe misalignment of Mtrr gt/gt conceptuses. Typically, the uterus is thought to guide conceptus orientation. To investigate a uterine effect of the Mtrr gt allele, we manipulate the maternal Mtrr genotype. Misaligned conceptuses were observed in litters of Mtrr +/+ , Mtrr +/gt , and Mtrr gt/gt mothers. While progesterone and/or BMP2 signalling might be disrupted, normal decidual morphology, patterning, and blood perfusion are evident at E6.5 regardless of conceptus orientation. These observations argue against a post-implantation uterine defect as a cause of conceptus misalignment. Since litters of Mtrr +/+ mothers display conceptus misalignment, a grandparental effect is explored. Multigenerational phenotype inheritance is characteristic of the Mtrr gt model, though the mechanism remains unclear. Genetic pedigree analysis reveals that severe conceptus skewing associates with the Mtrr genotype of either maternal grandparent. Moreover, the presence of conceptus skewing after embryo transfer into a control uterus indicates that misalignment is independent of the peri- and/or post-implantation uterus and instead is likely attributed to an embryonic mechanism that is epigenetically inherited. Overall, our data indicates that abnormal folate metabolism influences conceptus orientation over multiple generations with implications for subsequent development. This study casts light on the complex role of folate metabolism during development beyond a direct maternal effect., Lister Institute of Preventative Medicine Newton International Fellowship, Royal Society AG Leventis Fund

Endoplasmic reticulum (ER) stress promotes placental dysmorphogenesis and is associated with poor pregnancy outcomes. We show that unfolded protein response signalling pathways located in the ER drive differentiation of mouse trophoblast stem cells into trophoblast subtypes involved in development of the placental labyrinth zone and trophoblast invasion. In a mouse model of chronic ER stress (Eif2s1tm1RjK ), higher ER stress in homozygous blastocysts is accompanied by reduced trophectoderm cell number, developmental delay, and is associated with an increased incidence of early pregnancy loss. Administration of the chemical chaperone, tauroursodeoxycholic acid, to Eif2s1tm1RjK heterozygous females during pregnancy alleviated ER stress in the mutant placenta, restored normal trophoblast populations and reduced the frequency of early pregnancy loss. Our results suggest that alleviation of intrauterine ER stress could provide a potential therapeutic target to improve pregnancy outcome in women with pre-gestational metabolic or gynaecologic conditions. ABSTRACT: Women with pre-gestational health conditions (e.g., obesity, diabetes) or gynaecological problems (e.g., endometriosis) are at increased risk of adverse pregnancy outcomes including miscarriage, preeclampsia and fetal growth restriction. Increasing evidence suggests that unfavourable intrauterine conditions leading to poor implantation and/or defective placentation are a possible causative factor. The endoplasmic reticulum (ER) unfolded protein response (UPRER ) signalling pathways are a convergence point of various physiological stress stimuli that can be triggered by an unfavourable intrauterine environment. Therefore, we explored the impact of ER stress on mouse trophoblast differentiation in vitro, mouse blastocyst formation and early placenta development in the Eif2s1tm1RjK mutant mouse model of chronic ER stress. Chemically-manipulated ER stress or activation of UPRER pathways in a mouse trophoblast stem cell line promoted lineage-specific differentiation. Co-treatment with specific UPRER pathway inhibitors rescued this effect. While the inner cell mass was unaffected, the trophectoderm of homozygous Eif2s1tm1RjK blastocysts exhibited ER stress associated with a reduced cell number. Furthermore, one-third of Eif2s1tmRjK homozygous blastocysts exhibited severe developmental defects. We have previously reported a reduced trophoblast population and premature trophoblast differentiation in Eif2s1tm1RjK homozygous placentas at mid-gestation. Here, we demonstrate that treatment of Eif2s1+/tm1RjK heterozygous pregnant females with the chemical chaperone tauroursodeoxycholic acid alleviated ER stress, restored the trophoblast population, and reduced the frequency of embryonic lethality. Our data suggest that therapeutic targeting of ER stress may improve pregnancy outcome in women with pre-gestational metabolic or gynaecologic conditions. This article is protected by copyright. All rights reserved.

Introduction Throughout pregnancy, the placenta dynamically changes as trophoblast progenitors differentiate into mature trophoblast cell subtypes. This process is in part controlled by epigenetic regulation of DNA methylation leading to the inactivation of ‘progenitor cell’ genes and the activation of ‘differentiation’ genes. TET methylcytosine dioxygenases convert 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC) during DNA demethylation events. Here, we determine the spatiotemporal expression of TET1, TET2, and TET3 in specific trophoblast cell populations of mouse and human placentas throughout gestation, and consider their role in trophoblast cell differentiation and function. Methods In situ hybridization analysis was conducted to localize Tet1, Tet2, and Tet3 mRNA at key stages of mouse placental development. The distribution of 5-mC and 5-hmC in these samples was also evaluated. In comparison, expression patterns of TET1, TET2, and TET3 protein in human placentas were determined in first trimester and term pregnancies. Results In mouse, Tet1-3 mRNA was widely expressed in trophoblast cell populations from embryonic (E) day 8.5 to E12.5 including in progenitor and differentiated cells. However, expression became restricted to specific trophoblast giant cell subtypes by late gestation (E14.5 to E18.5). This coincided with cellular changes in 5-mC and 5-hmC levels. In human, cell columns, extravillous trophoblast and syncytiotrophoblast expressed TET1-3 whereas only TET3 was expressed in villus cytotrophoblast cells in first trimester and term placentas. Discussion Altogether, our data suggest that TET enzymes may play a dynamic role in the regulation of transcriptional activity of trophoblast progenitors and differentiated cell subtypes in mouse and human placentas.

Assisted reproduction technologies (ARTs) are becoming increasingly common. Therefore, how these procedures influence gene regulation and foeto-placental development are important to explore. Here, we assess the effects of blastocyst transfer on mouse placental growth and transcriptome. C57Bl/6 blastocysts were transferred into uteri of B6D2F1 pseudopregnant females and dissected at embryonic day 10.5 for analysis. Compared to non-transferred controls, placentas from transferred conceptuses weighed less even though the embryos were larger on average. This suggested a compensatory increase in placental efficiency. RNA sequencing of whole male placentas revealed 543 differentially expressed genes (DEGs) after blastocyst transfer: 188 and 355 genes were downregulated and upregulated, respectively. DEGs were independently validated in male and female placentas. Bioinformatic analyses revealed that DEGs represented expression in all major placental cell types and included genes that are critical for placenta development and/or function. Furthermore, the direction of transcriptional change in response to blastocyst transfer implied an adaptive response to improve placental function to maintain foetal growth. Our analysis revealed that CpG methylation at regulatory regions of two DEGs was unchanged in female transferred placentas and that DEGs had fewer gene-associated CpG islands (within ~20 kb region) compared to the larger genome. These data suggested that altered methylation at proximal promoter regions might not lead to transcriptional disruption in transferred placentas. Genomic clustering of some DEGs warrants further investigation of long-range, cis-acting epigenetic mechanisms including histone modifications together with DNA methylation. We conclude that embryo transfer, a protocol required for ART, significantly impacts the placental transcriptome and growth.

The agouti viable yellow (A

Recent research has focussed on the significance of folate metabolism in male fertility. Knocking down the mouse gene Mtrr impedes the progression of folate and methionine metabolism and results in hyperhomocysteinaemia, dysregulation of DNA methylation and developmental phenotypes (e.g. neural tube, heart and placenta defects). The Mtrrgt mouse line is a model of transgenerational epigenetic inheritance (TEI), the hypothesised cause of which is the inheritance of a yet-to-be determined epigenetic factor via the germline. We investigated Mtrrgt/gt testes and sperm function compared with control C57Bl/6J testes to explore potential defects that might confound our understanding of TEI in the Mtrrgt model. Histological analysis revealed that adult Mtrrgt/gt testes are more spherical in shape than C57Bl/6J testes, though serum testosterone levels were normal and spermatogenesis progressed in a typical manner. Spermatozoa collected from the cauda epididymis showed normal morphology, counts, and viability in Mtrrgt/gt males. Correspondingly, Mtrrgt spermatozoa contributed to normal pregnancy rates. Similar parameters were assessed in Mtrr+/+ and Mtrr+/gt males, which were normal compared with controls. Overall, our data showed that the Mtrrgt allele is unlikely to alter spermatogenesis or male fertility. Therefore, it is improbable that these factors confound the mechanistic study of TEI in Mtrrgt mice.

Key points Folate (folic acid) deficiency and mutations in folate‐related genes in humans result in megaloblastic anaemia.Folate metabolism, which requires the enzyme methionine synthase reductase (MTRR), is necessary for DNA synthesis and the transmission of one‐carbon methyl groups for cellular methylation.In this study, we show that the hypomorphic Mtrrgt/gt mutation in mice results in late‐onset and sex‐specific blood defects, including macrocytic anaemia, extramedullary haematopoiesis and lymphopenia.Notably, when either parent carries an Mtrrgt allele, blood phenotypes result in their genetically wildtype adult daughters, the effects of which are parent specific.Our data establish a new model for studying the mechanism of folate metabolism in macrocytic anaemia aetiology and suggest that assessing parental folate status might be important when diagnosing adult patients with unexplained anaemia. Abstract The importance of the vitamin folate (also known as folic acid) in erythrocyte formation, maturation and/or longevity is apparent since folate deficiency in humans causes megaloblastic anaemia. Megaloblastic anaemia is a type of macrocytic anaemia whereby erythrocytes are enlarged and fewer in number. Folate metabolism is required for thymidine synthesis and one‐carbon metabolism, though its specific role in erythropoiesis is not well understood. Methionine synthase reductase (MTRR) is a key enzyme necessary for the progression of folate metabolism since knocking down the Mtrr gene in mice results in hyperhomocysteinaemia and global DNA hypomethylation. We demonstrate here that abnormal folate metabolism in mice caused by Mtrrgt/gt homozygosity leads to haematopoietic phenotypes that are sex and age dependent. Specifically, Mtrrgt/gt female mice displayed macrocytic anaemia, which might be due to defective erythroid differentiation at the exclusion of haemolysis. This was associated with increased renal Epo mRNA expression, hypercellular bone marrow, and splenic extramedullary haematopoiesis. In contrast, the male response differed since Mtrrgt/gt male mice were not anaemic but did display erythrocytic macrocytosis and lymphopenia. Regardless of sex, these phenotypes were late onset. Remarkably, we also show that when either parent carries an Mtrrgt allele, a haematological defect results in their adult wildtype daughters. However, the specific phenotype was dependent upon the sex of the parent. For instance, wildtype daughters of Mtrr+/gt females displayed normocytic anaemia. In contrast, wildtype daughters of Mtrr+/gt males exhibited erythrocytic microcytosis not associated with anaemia. Therefore, abnormal folate metabolism affects adult haematopoiesis in an age‐, sex‐ and parent‐specific manner., Key points Folate (folic acid) deficiency and mutations in folate‐related genes in humans result in megaloblastic anaemia.Folate metabolism, which requires the enzyme methionine synthase reductase (MTRR), is necessary for DNA synthesis and the transmission of one‐carbon methyl groups for cellular methylation.In this study, we show that the hypomorphic Mtrrgt/gt mutation in mice results in late‐onset and sex‐specific blood defects, including macrocytic anaemia, extramedullary haematopoiesis and lymphopenia.Notably, when either parent carries an Mtrrgt allele, blood phenotypes result in their genetically wildtype adult daughters, the effects of which are parent specific.Our data establish a new model for studying the mechanism of folate metabolism in macrocytic anaemia aetiology and suggest that assessing parental folate status might be important when diagnosing adult patients with unexplained anaemia.

Elucidating how the environment affects human health will further our understanding of disease risk and prevention, and adaptation. Recent evidence reveals that exposure to environmental stressors in our lifetime might affect disease risk in our descendants. This phenomenon known as transgenerational epigenetic inheritance involves the transmission of epigenetic information via the germline over multiple generations, independent of the DNA base sequence. Epigenetic factors (e.g., DNA methylation, histone modifications, non-coding RNA) are responsive to environmental stressors and can be transmitted between generations. However, very little is currently understood about the mechanism involved in epigenetic inheritance. Here, we explore key plant and animal models that will ultimately help us understand the epigenetic intricacies of this phenomenon and relate it to disease inheritance observed in human populations exposed to famine, toxicants, and psychological stress.

The exposure to adverse environmental conditions (e.g. poor nutrition) may lead to increased disease risk in an individual and their descendants. In some cases, the results may be sexually dimorphic. A range of phenotypes has been associated with deficiency in or defective metabolism of the vitamin folate. However, the molecular mechanism linking folate metabolism to development is still not well defined nor is it clear whether phenotypes are sex-specific. The enzyme methionine synthase reductase (MTRR) is required for the progression of folate metabolism and the utilization of methyl groups from the folate cycle. Previously, we showed that the hypomorphic Mtrrgt mutation in mice results in metabolic disruption, epigenetic instability, and a wide spectrum of developmental phenotypes (e.g. growth defects, congenital malformations) at midgestation that appear in subsequent wild-type generations. This transgenerational effect only occurs through the maternal lineage. Here, we explore whether the phenotypes that result from either intrinsic or ancestral Mtrr deficiency are sexually dimorphic. We found that no sexual dimorphism is apparent in either situation when the phenotypes were broadly or specifically defined. However, when we focused on the group of phenotypically normal conceptuses derived from maternal grandparental Mtrr deficiency, we observed an apparent increase in placental efficiency in each subsequent generation leading to F4 generation female embryos that weigh more than controls. These data suggest that ancestral abnormal folate metabolism may lead to male grandprogeny that are less able to adapt or female grandprogeny that are programmed to become more sensitive to folate availability in subsequent generations.

SummaryThe importance of maternal folate consumption for normal development is well established, yet the molecular mechanism linking folate metabolism to development remains poorly understood. The enzyme methionine synthase reductase (Mtrr) is necessary for utilization of methyl groups from the folate cycle. We found that a hypomorphic mutation of the mouse Mtrr gene results in intrauterine growth restriction, developmental delay, and congenital malformations, including neural tube, heart, and placental defects. Importantly, these defects were dependent upon the Mtrr genotypes of the maternal grandparents. Furthermore, we observed widespread epigenetic instability associated with altered gene expression in the placentas of wild-type grandprogeny of Mtrr-deficient maternal grandparents. Embryo transfer experiments revealed that Mtrr deficiency in mice lead to two distinct, separable phenotypes: adverse effects on their wild-type daughters’ uterine environment, leading to growth defects in wild-type grandprogeny, and the appearance of congenital malformations independent of maternal environment that persist for five generations, likely through transgenerational epigenetic inheritance.PaperFlick

How maternal diet influences offspring metabolism is unclear, as it is difficult to distinguish between the effects of the in utero environment and epigenetic factors contributed by the oocyte. In a mouse model of high-fat diet, a new study teases apart these mechanisms by using in vitro fertilization and shows that susceptibility of offspring to metabolic disorder can likely be attributed to epigenetic inheritance via the oocyte.

Suboptimal intrauterine conditions, including poor nutrition, during critical periods of growth may lead to lifelong changes in the body's organs and tissues, thus providing a physiological basis for adult-onset disease. Remarkably, recent evidence suggests that the long-term consequences of adverse conditions during early development may not be limited to one generation, but may lead to poor health in the generations to follow, even if these individuals develop in normal conditions themselves. For example, the diet of a pregnant mother may affect the development and disease risk of her children and even her grandchildren. There is limited evidence for this in humans since studies of multiple generations are difficult to maintain. However, recent animal models have been generated to investigate this phenomenon and will be instrumental in the future for assessing the underlying mechanisms of intergenerational and transgenerational transmission of disease. These mechanisms remain unclear, though environmental, metabolic and epigenetic factors are likely involved. Researchers have begun to address how changes in metabolism and epigenetic regulation of gene expression caused by poor nutrition can be passed from one generation to the next. Ultimately, these findings will shed light on the transmission of diabetes, obesity and cardiovascular disease that are rapidly expanding in Western countries. Public health strategies that focus on improved maternal nutrition may provide a means of promoting cardiovascular and metabolic health. However, the full impact of these strategies may not be apparent for decades. (C) 2012 Elsevier Ltd. All rights reserved

There are numerous benefits to elucidating how our environment affects our health: from a greater understanding of adaptation to disease prevention. Evidence shows that stressors we are exposed to during our lifetime might cause disease in our descendants. Transgenerational epigenetic inheritance involves the transmission of 'information' over multiple generations via the gametes independent of the DNA base sequence. Despite extensive research, the epigenetic mechanisms remain unclear. Analysis of model organisms exposed to environmental insults (e.g., diet manipulation, stress, toxin exposure) or carrying mutations in the epigenetic regulatory machinery indicates that inheritance of altered DNA methylation, histone modifications, or non-coding RNAs are key mechanisms. Tracking inherited epigenetic information and its effects for multiple generations is a significant challenge to overcome.

Defects in protein-folding and -degradation machinery have been identified as a major cause of intracellular protein aggregation and of aggregation-associated diseases. In general, it remains unclear how these aggregates are harmful to normal cellular function. We demonstrate here that,in the developing placenta of the mouse, the absence of the Mrj (Dnajb6)co-chaperone prevents proteasome degradation of keratin 18 (K18; Krt18)intermediate filaments, resulting in the formation of keratin inclusion bodies. These inclusions in chorionic trophoblast cells prevent chorioallantoic attachment during placental development. We show further that keratin-deficient embryos undergo chorioallantoic attachment and that, by genetically reducing keratin expression in Mrj-/-conceptuses, chorioallantoic attachment was rescued. Therefore, the chorioallantoic attachment phenotype in Mrj mutants is not due to a deficiency of the normal keratin cytoskeleton, but rather is cytotoxicity caused by keratin aggregates that disrupt chorion trophoblast cell organization and function.

The keratin cytoskeleton is present in all trophoblast cell subtypes of the mouse and human placenta and is required to maintain the structural integrity of these cells. Recently, various genetic mouse models have shown that a normal keratin network is necessary for placental development. Keratin-deficiency leads to trophoblast giant cell fragility, breaking the barrier between the conceptus and the maternal blood circulation. Alternatively, keratin aggregation prevents chorioallantoic attachment, a key developmental milestone required for the formation of the labyrinth within the mouse placenta. These models give us insight into cytokeratin function in human trophoblast cell subtypes and suggest that defects in the keratin cytoskeleton may result in intrauterine growth restriction or miscarriage.

The placenta is essential for sustaining the growth of the fetus during gestation, and defects in its function result in fetal growth restriction or, if more severe, fetal death. Several molecular pathways have been identified that are essential for development of the placenta, and mouse mutants offer new insights into the cell biology of placental development and physiology of nutrient transport.

The placenta is a highly specialized organ whose primary function is to promote the exchange of nutrients and oxygen between maternal and fetal blood, essential for survival and growth of the baby. The surface area for nutrient transport is a highly convoluted villous structure that forms by branching morphogenesis. In mice, this process begins after embryonic day 8.5, following attachment of allantoic mesoderm to the chorion, and continues through the end of gestation. Gene targeting studies in mice have identified a large number of genes that are essential for chorioallantoic development to give rise to the layer of the placenta called the labyrinth. Collectively, these studies reveal that a number of signaling pathways regulate four distinct phases of labyrinth development: chorioallantoic attachment (involving VCAM1 and its receptor alpha4 integrin, Bmp5/7, and Wnt7b, as well as the cochaperone Mrj), initiation of branching (involving the Gcm1 transcription factor to select sites of branch initiation), extension of villous branching (involving FGF, EGF, and HGF/Met signaling, through the Grb2/Sos1/Mek1/p38alpha MAPK pathway), followed by vascularization of the villous tree. The restricted expression and/or action of the signaling components indicate that a series of intercellular interactions regulate chorioallantoic development.

During development, a fetus and its placenta must respond to a changing maternal environment to ensure normal growth is achieved and survival is maintained. The mechanisms behind developmental programming involve complex interactions between epigenetic and physiological processes, which are not well understood. Importantly, when programming goes awry, it puts the fetus at risk for disease later in life and may, in some instances, affect subsequent generations via epigenetic processes including DNA methylation. The one-carbon metabolism, which includes the folate, methionine and choline pathways, provides methyl groups nec- essary for DNA methylation and a normal epigenetic landscape. Accordingly, disruptions in this pathway affect placental develop- ment and function leading to altered fetal programming. Remarkably, recent studies have revealed that abnormal folate metabolism causes transgenerational effects probably through epigenetic inheritance. The epigenetic mechanisms behind this phe- nomenon are not well understood but they have important implications for the influence of the metabolic environment on epige- netic stability and non-genetic inheritance of disease. Importantly, there are increasing concerns that assisted reproductive technologies cause aberrant epigenetic profiles in embryos leading to abnormal fetal programming. How the negative epigenetic consequences of assisted reproduction treatment affect subsequent generations requires further investigation. RBMOnline

It is well understood that maternal folate deficiency can cause abnormal fetal development. However, the extent to which placental development and function are also dependent upon folate uptake and metabolism remains unclear. To understand which trophoblast cell types may be affected by folate deficiency or abnormal folate metabolism, we completed a comprehensive spatial and temporal protein expression analysis of folate receptor (Folr), folate transporters (proton-coupled folate receptor [Slc46a1 or PCFT] and reduced folate carrier-1 [Rfc1]) and folate metabolic enzymes (5,10-methylenetetrahydrofolate reductase [Mthfr] and methionine synthase [Mtr]) in histological sections of mouse placentas from early development (E8.5) until term (E18.5). We observed that the highest level of protein expression was during early development (E8.5-E10.5), prior to the formation of the three main layers of the mature placenta suggesting that folate uptake and metabolism may be required for placental development, itself. As expected, the labyrinth trophoblast cells, which are responsible for nutrient transport, expressed these proteins throughout pregnancy, including robust expression in the sinusoidal trophoblast giant cells that line the maternal blood spaces. Other trophoblast giant cell (TGC) subtypes (parietal-TGCs and canal-TGCs), whose function does not include nutrient transport, expressed folate transporters and enzymes from E8.5 onwards. Remarkably, these proteins were also detected in glycogen trophoblast cells from E12.5–E18.5 suggesting a new role in folate uptake and metabolism for these cells. Together, these data provide evidence that folate may be necessary for normal placental development and function, and perturbations in its availability or metabolism may lead to secondary effects on fetal development.

We recently reported the first evidence of placental endoplasmic reticulum (ER) stress in the pathophysiology of human intrauterine growth restriction. Here, we used a mouse model to investigate potential underlying mechanisms. Eif2s1tm1RjK mice, in which Ser51 of eukaryotic initiation factor 2 subunit alpha (eIF2α) is mutated, display a 30% increase in basal translation. In Eif2s1tm1RjK placentas, we observed increased ER stress and anomalous accumulation of glycoproteins in the endocrine junctional zone (Jz), but not in the labyrinthine zone where physiological exchange occurs. Placental and fetal weights were reduced by 15% (97 mg to 82 mg, p < 0.001) and 20% (1009 mg to 798 mg, p < 0.001), respectively. To investigate whether ER stress affects bioactivity of secreted proteins, mouse embryonic fibroblasts (MEFs) were derived from Eif2s1tm1RjK mutants. These MEFs exhibited ER stress, grew 50% slower, and showed reduced Akt–mTOR signalling compared to wild-type cells. Conditioned medium (CM) derived from Eif2s1tm1RjK MEFs failed to maintain trophoblast stem cells in a progenitor state, but the effect could be rescued by exogenous application of FGF4 and heparin. In addition, ER stress promoted accumulation of pro-Igf2 with altered glycosylation in the CM without affecting cellular levels, indicating that the protein failed to be processed after release. Igf2 is the major growth factor for placental development; indeed, activity in the Pdk1–Akt–mTOR pathways was decreased in Eif2s1tm1RjK placentas, indicating loss of Igf2 signalling. Furthermore, we observed premature differentiation of trophoblast progenitors at E9.5 in mutant placentas, consistent with the in vitro results and with the disproportionate development of the labyrinth and Jz seen in placentas at E18.5. Similar disproportion has been reported in the Igf2-null mouse. These results demonstrate that ER stress adversely affects placental development, and that modulation of post-translational processing, and hence bioactivity, of secreted growth factors contributes to this effect. Placental dysmorphogenesis potentially affects fetal growth through reduced exchange capacity. Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Early placental development in mice involves patterning of the chorion into distinct layers, though little is understood regarding the interactions that regulate its organization. Here we demonstrate that keratin aggregates found in Mrj(-/-) chorionic trophoblast cells are associated with abnormal cell morphology, collapse of the actin cytoskeleton, E-cadherin and β-catenin misexpression and extracellular matrix (ECM) disorganization. Accordingly, Mrj(-/-) trophoblast cells in vitro are nonadherent and display erratic migratory behavior. These cells also fail to differentiate into syncytiotrophoblast cells since Rhox4b expression, a marker of syncytiotrophoblast progenitors, was maintained and Gcm1, Synb, and Syna expression failed to increase. This differentiation defect was not solely attributable to E-cadherin misexpression or ECM disorganization. However, plating Mrj-deficient cells on exogenous laminin-511 normalized their cell behavior. Lastly, we show that Mrj(-/-) chorions at embryonic day 8.5 have expanded Rhox4b expression domains and do not form normal layers of gene expression suggesting that chorion patterning requires Mrj.

The Mrj co-chaperone is expressed throughout the mouse conceptus, yet its requirement for placental development has prohibited a full understanding of its embryonic function. Here, we show that Mrj−/− embryos exhibit neural tube defects independent of the placenta phenotype, including exencephaly and thin-walled neural tubes. Molecular analyses revealed fewer proliferating cells and a down-regulation of early neural progenitor (Pax6, Olig2, Hes5) and neuronal (Nscl2, SCG10) cell markers in Mrj−/− neuroepithelial cells. Furthermore, Mrj−/− neurospheres are significantly smaller and form fewer secondary neurospheres indicating that Mrj is necessary for self-renewal of neural stem cells. However, the molecular function of Mrj in this context remains elusive because Mrj does not colocalize with Bmi-1, a self-renewal protein. Furthermore, unlike in Mrj−/− placentas, intermediate filament-containing aggregates do not accumulate in Mrj−/− neuroepithelium, ruling out nestin as a substrate for Mrj. Regardless, Mrj plays an important role in neural stem cell self-renewal. Developmental Dynamics 238:2564–2574, 2009. © 2009 Wiley-Liss, Inc.

Hyperhomocyst(e)inemia is a metabolic derangement that is linked to the distribution of folate pools, which provide one-carbon units for biosynthesis of purines and thymidylate and for remethylation of homocysteine to form methionine. In humans, methionine synthase deficiency results in the accumulation of methyltetrahydrofolate at the expense of folate derivatives required for purine and thymidylate biosynthesis. Complete ablation of methionine synthase activity in mice results in embryonic lethality. Other mouse models for hyperhomocyst(e)inemia have normal or reduced levels of methyltetrahydrofolate and are not embryonic lethal, although they have decreased ratios of AdoMet/AdoHcy and impaired methylation. We have constructed a mouse model with a gene trap insertion in the Mtrr gene specifying methionine synthase reductase, an enzyme essential for the activity of methionine synthase. This model is a hypomorph, with reduced methionine synthase reductase activity, thus avoiding the lethality associated with the absence of methionine synthase activity. Mtrr(gt/gt) mice have increased plasma homocyst(e)ine, decreased plasma methionine, and increased tissue methyltetrahydrofolate. Unexpectedly, Mtrr(gt/gt) mice do not show decreases in the AdoMet/AdoHcy ratio in most tissues. The different metabolite profiles in the various genetic mouse models for hyperhomocyst(e)inemia may be useful in understanding biological effects of elevated homocyst(e)ine.

We have previously shown that the targeted deletions of both type I keratins (K) 18 and 19 cause lethality by embryonic day (e) 9.5 due to fragility and cytolysis of trophoblast giant cells. The development of the embryo proper appeared to be unaffected and its death was caused by nutrient deficiency. In order to address the function of keratins within the embryo proper, lethality due to extraembryonic tissue failure must be overcome. One approach to rescue doubly deficient embryos is by aggregating knockout embryos with tetraploid wild-type embryos. As a general tool, tetraploid aggregation can be used to rescue embryonic lethality caused by defects in extraembryonic tissues like the placenta, trophoblast or yolk sac. We rescued K18-/- K19-/- embryos until e11.5, using this approach, proving that the loss of the keratin cytoskeleton causes defects in the trophoblast giant cell layer, but has no effect on early development of the embryo proper.

The agouti viable yellow (Avy) allele is an insertional mutation in the mouse genome caused by a variably methylated intracisternal A particle (VM-IAP) retrotransposon. Avy expressivity is sensitive to a range of early-life chemical exposures and nutritional interventions, suggesting that environmental perturbations can have long-lasting effects on the methylome. However, the extent to which VM-IAP elements are environmentally labile with phenotypic implications is unknown. Using a recently identified repertoire of VM-IAPs, we assessed the epigenetic effects of different environmental contexts. A longitudinal aging analysis indicated that VM-IAPs are stable across the murine lifespan, with only small increases in DNA methylation detected for a subset of loci. No significant effects were observed after maternal exposure to the endocrine disruptor bisphenol A, an obesogenic diet or methyl donor supplementation. A genetic mouse model of abnormal folate metabolism exhibited shifted VM-IAP methylation levels and altered VM-IAP-associated gene expression, yet these effects are likely largely driven by differential targeting by polymorphic KRAB zinc finger proteins. We conclude that epigenetic variability at retrotransposons is not predictive of environmental susceptibility.