Life Sciences Research for Lifelong Health

Publications myriam-hemberger

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The H19 induction triggers trophoblast lineage commitment in mouse ES cells.
H Fujimori, H Mukai, Y Murakami, M Hemberger, Y Hippo, M Masutani

Trophoblast lineage differentiation is properly regulated to support embryogenesis. Besides normal developmental process, during germ cell tumor formation or development of other reproductive system diseases, unregulated trophoblast differentiation is also observed and affects the pathogenesis of the diseases. During normal embryogenesis, cell fate of late-stage blastcyst is regulated by a reciprocal repression of the key transcriptional factors; Oct3/4 dominancy inhibits Cdx2 expression in inner cell mass (ICM) and leads them to epiblast/primitive ectoderm but Cdx2 dominancy in trophectoderm (TE) leads them to trophoblast lineage. In contrast during early blastcyst stage, the Cdx2 expression is restricted in TE and not present in ICM, although Oct3/4 signaling does not inhibit the Cdx2 expression in ICM, implying that some factors could be inactivated leading to the suppressed Cdx2 expression in ICM of early blastcyst. ES cells (ESCs), which are derived from ICM, could be a unique model to study trophoblast differentiation in an ectopic context. We previously showed that poly(ADP-ribose) polymerase-1 (Parp-1) deficient ESCs highly expressed non-coding RNA H19 and could differentiate into trophoblast lineage. The expression of H19 is known to start at pre-blastcyst stage during mouse development, and the gene shows high expression only in trophoectoderm (TE) at blastcyst stage. However, its role in trophoblast differentiation has not been clarified yet. Thus, we hypothesized that the H19 activation may act as a trigger for induction of trophoblast differentiation cascade in mouse ESCs. To investigate this issue, we asked whether a forced H19 expression drives ESCs into trophoblast lineage or not. We demonstrated that the H19 induction leads to trophoblast lineage commitment through induction of the Cdx2 expression. We also showed that the expression of Cdx2 is induced in ESCs by forced H19 expression even under a high level of Oct3/4, which could act as a suppressor for Cdx2 expression. It is thus suggested that the H19 induction promotes trophoblast lineage commitment against the repression pressure by Oct3/4 in differentiating ESCs. Taken together, this study suggests that the H19 expression is able to function as a cascade activator of trophoblast lineage commitment possibly by overriding the Oct3/4 action in ESCs.

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Biochemical and biophysical research communications, 436, 2, 313-8, 2013

PMID: 23743205
DOI: 10.1016/j.bbrc.2013.05.100

Deciphering the Mechanisms of Developmental Disorders (DMDD): a new programme for phenotyping embryonic lethal mice.
T Mohun, DJ Adams, R Baldock, S Bhattacharya, AJ Copp, M Hemberger, C Houart, ME Hurles, E Robertson, JC Smith, T Weaver, W Weninger

International efforts to test gene function in the mouse by the systematic knockout of each gene are creating many lines in which embryonic development is compromised. These homozygous lethal mutants represent a potential treasure trove for the biomedical community. Developmental biologists could exploit them in their studies of tissue differentiation and organogenesis; for clinical researchers they offer a powerful resource for investigating the origins of developmental diseases that affect newborns. Here, we outline a new programme of research in the UK aiming to kick-start research with embryonic lethal mouse lines. The 'Deciphering the Mechanisms of Developmental Disorders' (DMDD) programme has the ambitious goal of identifying all embryonic lethal knockout lines made in the UK over the next 5 years, and will use a combination of comprehensive imaging and transcriptomics to identify abnormalities in embryo structure and development. All data will be made freely available, enabling individual researchers to identify lines relevant to their research. The DMDD programme will coordinate its work with similar international efforts through the umbrella of the International Mouse Phenotyping Consortium [see accompanying Special Article (Adams et al., 2013)] and, together, these programmes will provide a novel database for embryonic development, linking gene identity with molecular profiles and morphology phenotypes.

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Disease models & mechanisms, 6, 3, 562-6, 2013

PMID: 23519034
DOI: 10.1242/dmm.011957

Open Access

Immune balance at the foeto-maternal interface as the fulcrum of reproductive success.
M Hemberger

Viviparity has many evolutionary advantages but brings with it the problem of the semi-allogeneic foetus having to coexist with the mother for the duration of pregnancy. In species with haemochorial placentation this problem is particularly evident as foetal trophoblast cells are extensively intermingled with maternal tissue and are directly exposed to maternal blood. Fascinating adaptations on both the foetal and maternal side have allowed for this interaction to be re-directed away from an immune rejection response not only towards immunotolerance, but in fact towards actively supporting reproductive success. Recent data have shown that some of these remarkable adaptations are conserved between mice and humans. Thus, a subset of trophoblast cells that is directly exposed to the maternal uterine environment shares the feature of expressing an unusual antigen repertoire on their surface. Paternal antigens can be recognized by maternal immune cells, in particular uterine natural killer cells that express cognate receptors, to regulate the extensive remodelling events that take place at the implantation site. Detailed genetic dissection experiments in the mouse have further demonstrated the direct impact of antigenic dissimilarity on foetal growth. With the availability of inbred strains, in vitro culture systems of trophoblast stem cells, and in-depth genetic, genomic and epigenomic data the mouse will be a valuable model system to study the intricate immune crosstalk at the foeto-maternal boundary. These insights will pave the way towards unravelling the mutual and synergistic interactions between trophoblast and its surrounding maternal environment, and in doing so help understand pregnancy pathologies.

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Journal of reproductive immunology, 97, 1, 36-42, 2013

PMID: 23432870
DOI: 10.1016/j.jri.2012.10.006

DNA methylation profiles define stem cell identity and reveal a tight embryonic-extraembryonic lineage boundary.
CE Senner, F Krueger, D Oxley, S Andrews, M Hemberger

Embryonic (ES) and epiblast (EpiSC) stem cells are pluripotent but committed to an embryonic lineage fate. Conversely, trophoblast (TS) and extraembryonic endoderm (XEN) stem cells contribute predominantly to tissues of the placenta and yolk sac, respectively. Here we show that each of these four stem cell types is defined by a unique DNA methylation profile. Despite their distinct developmental origin, TS and XEN cells share key epigenomic hallmarks, chiefly characterized by robust DNA methylation of embryo-specific developmental regulators, as well as a subordinate role of 5-hydroxymethylation. We also observe a substantial methylation reinforcement of pre-existing epigenetic repressive marks that specifically occurs in extraembryonic stem cells compared to in vivo tissue, presumably due to continued high Dnmt3b expression levels. These differences establish a major epigenetic barrier between the embryonic and extraembryonic stem cell types. In addition, epigenetic lineage boundaries also separate the two extraembryonic stem cell types by mutual repression of key lineage-specific transcription factors. Thus, global DNA methylation patterns are a defining feature of each stem cell type that underpin lineage commitment and differentiative potency of early embryo-derived stem cells. Our detailed methylation profiles identify a cohort of developmentally regulated sequence elements, such as orphan CpG islands, that will be most valuable to uncover novel transcriptional regulators and pivotal "gatekeeper" genes in pluripotency and lineage differentiation.

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Stem cells (Dayton, Ohio), 30, 12, 2732-45, 2012

PMID: 23034951
DOI: 10.1002/stem.1249

Open Access

Endoplasmic reticulum stress disrupts placental morphogenesis: implications for human intrauterine growth restriction.
HW Yung, M Hemberger, ED Watson, CE Senner, CP Jones, RJ Kaufman, DS Charnock-Jones, GJ Burton

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. Eif2s1(tm1RjK) mice, in which Ser51 of eukaryotic initiation factor 2 subunit alpha (eIF2α) is mutated, display a 30% increase in basal translation. In Eif2s1(tm1RjK) 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 Eif2s1(tm1RjK) 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 Eif2s1(tm1RjK) 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 Eif2s1(tm1RjK) 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.

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The Journal of pathology, , , , 2012

PMID: 22733590
DOI: 10.1002/path.4068

Open Access

A placenta for life.
R John, M Hemberger

The chorioallantoic placenta is the defining organ of eutherians that has enabled prolonged intrauterine gestation. As such, normal placental development and function are essential for mammalian reproductive success. Reflecting the key role of this organ in providing nutrients to the embryo, the characteristic cell type that forms substantial parts of the placenta is called 'trophoblast' (from Greek trephein 'to feed' and blastos 'germinator'). However, in addition to regulating nutrient supply, the placenta also exerts a number of other pivotal functions that highlight the importance of normal trophoblast differentiation for a successful pregnancy. In this guest symposium, 'Trophoblast Development', several contributors summarize insights gained from recent studies in the mouse that have advanced our understanding of trophoblast biology. This includes how the earliest trophoblast cells are set aside to expand in a stem- or progenitor-cell compartment under tight genetic and epigenetic control and how subsequent differentiation into the various placental cell types is controlled to ensure normal placentation. The relevance of these contributions range from early developmental cell fate decisions, stem cell biology and placental development for healthy pregnancy to the impact of placental failures on long-term health, with important clinical implications for assisted reproductive technology procedures and pregnancy-associated complications.

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Reproductive biomedicine online, 25, 1, 5-11, 2012

PMID: 22578825
DOI: 10.1016/j.rbmo.2012.03.018

Health during pregnancy and beyond: Fetal trophoblast cells as chief co-ordinators of intrauterine growth and reproductive success.
M Hemberger

Abstract Differentiation of extra-embryonic tissues and organs, notably the placenta, is vital for embryonic development and growth throughout gestation, starting from a few days after fertilization when the trophoblast cell lineage arises until parturition. In utero metabolic programming events may even extend the impact of placental function well into adulthood as they may predispose the offspring to common pathologies such as diabetes and cardiovascular disease. This review summarizes key steps that lead up to formation of a functional placenta. It highlights recent insights that have advanced our view of how early trophoblast expansion is achieved and how sufficient maternal blood supply to the developing fetus is secured. Exciting cumulative data have revealed the importance of a close cross-talk between the embryo proper and extra-embryonic trophoblast cells that involves extracellular matrix components in the establishment of a stem cell-like niche and proliferation compartment. Remarkably, placental function also relies on beneficial interactions between trophoblast cells and maternal immune cells at the implantation site. Our growing knowledge of the molecular mechanisms involved in trophoblast differentiation and function will help to devise informed approaches aimed at deciphering how placentation is controlled in humans as an essential process for reproductive success and long-term health.

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Annals of medicine, 44, 4, 325-37, 2012

PMID: 22409432
DOI: 10.3109/07853890.2012.663930

Pluripotency factor binding and Tsix expression act synergistically to repress Xist in undifferentiated embryonic stem cells.
TB Nesterova, CE Senner, J Schneider, T Alcayna-Stevens, A Tattermusch, M Hemberger, N Brockdorff

Expression of Xist, the master regulator of X chromosome inactivation, is extinguished in pluripotent cells, a process that has been linked to programmed X chromosome reactivation. The key pluripotency transcription factors Nanog, Oct4 and Sox2 are implicated in Xist gene extinction, at least in part through binding to an element located in Xist intron 1. Other pathways, notably repression by the antisense RNA Tsix, may also be involved.

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Epigenetics & chromatin, 4, 1, 17, 2011

PMID: 21982142
DOI: 10.1186/1756-8935-4-17

Open Access

Lineage-specific function of the noncoding Tsix RNA for Xist repression and Xi reactivation in mice.
T Ohhata, CE Senner, M Hemberger, A Wutz

The noncoding Tsix RNA is an antisense repressor of Xist and regulates X inactivation in mice. Tsix is essential for preventing the inactivation of the maternally inherited X chromosome in extraembryonic lineages where imprinted X-chromosome inactivation (XCI) occurs. Here we establish an inducible Tsix expression system for investigating Tsix function in development. We show that Tsix has a clear functional window in extraembryonic development. Within this window, Tsix can repress Xist, which is accompanied by DNA methylation of the Xist promoter. As a consequence of Xist repression, reactivation of the inactive X chromosome (Xi) is widely observed. In the parietal endoderm, Tsix represses Xist and causes reactivation of an Xi-linked GFP transgene throughout development, whereas Tsix progressively loses its Xist-repressing function from embryonic day 9.5 (E9.5) onward in trophoblast giant cells and spongiotrophoblast, suggesting that Tsix function depends on a lineage-specific environment. Our data also demonstrate that the maintenance of imprinted XCI requires Xist expression in specific extraembryonic tissues throughout development. This finding shows that reversible XCI is not exclusive to pluripotent cells, and that in some lineages cell differentiation is not accompanied by a stabilization of the Xi.

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Genes & development, 25, 16, 1702-15, 2011

PMID: 21852535
DOI: 10.1101/gad.16997911

Open Access

BRACHYURY and CDX2 mediate BMP-induced differentiation of human and mouse pluripotent stem cells into embryonic and extraembryonic lineages.
AS Bernardo, T Faial, L Gardner, KK Niakan, D Ortmann, CE Senner, EM Callery, MW Trotter, M Hemberger, JC Smith, L Bardwell, A Moffett, RA Pedersen

BMP is thought to induce hESC differentiation toward multiple lineages including mesoderm and trophoblast. The BMP-induced trophoblast phenotype is a long-standing paradox in stem cell biology. Here we readdressed BMP function in hESCs and mouse epiblast-derived cells. We found that BMP4 cooperates with FGF2 (via ERK) to induce mesoderm and to inhibit endoderm differentiation. These conditions induced cells with high levels of BRACHYURY (BRA) that coexpressed CDX2. BRA was necessary for and preceded CDX2 expression; both genes were essential for expression not only of mesodermal genes but also of trophoblast-associated genes. Maximal expression of the latter was seen in the absence of FGF but these cells coexpressed mesodermal genes and moreover they differed in cell surface and epigenetic properties from placental trophoblast. We conclude that BMP induces human and mouse pluripotent stem cells primarily to form mesoderm, rather than trophoblast, acting through BRA and CDX2.

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Cell stem cell, 9, 2, 144-55, 2011

PMID: 21816365
DOI: 10.1016/j.stem.2011.06.015

Open Access

Paternal MHC expression on mouse trophoblast affects uterine vascularization and fetal growth.
Z Madeja, H Yadi, R Apps, S Boulenouar, SJ Roper, L Gardner, A Moffett, F Colucci, M Hemberger

The mammalian fetus represents a semiallograft within the maternal uterus yet is not rejected. This situation is particularly pronounced in species with a hemochorial type of placentation, such as humans and rodents, where maternal tissues and blood are in direct contact with fetal trophoblast and thus potentially with paternal antigens. The main polymorphic antigens responsible for graft rejection are MHC antigens. In humans the trophoblast cells invading into the decidua have a unique pattern of MHC class I expression characterized by both classical (HLA-C) and nonclassical (HLA-G and HLA-E) molecules. Whether such an unusual MHC repertoire on the surface of trophoblast is a conserved feature between species with hemochorial placentation has not been resolved. Here we demonstrate, using a range of methods, that C57BL/6 mouse trophoblast predominantly expresses only one MHC class I antigen, H2-K, at the cell surface of giant cells but lacks expression of nonclassical MHC molecules. Antigenic disparity between parental MHCs affects trophoblast-induced transformation of the uterine vasculature and, consequently, placental and fetal gowth. Maternal uterine blood vessels were more dilated, allowing for increased blood supply, in certain combinations of maternal and paternal MHC haplotypes, and these allogeneic fetuses and placentas were heavier at term compared with syngeneic controls. Thus, maternal-fetal immune interactions are instrumental to optimize reproductive success. This cross-talk has important implications for human disorders of pregnancy, such as preeclampsia and fetal growth restriction.

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Proceedings of the National Academy of Sciences of the United States of America, 108, 10, 4012-7, 2011

PMID: 21300875
DOI: 10.1073/pnas.1005342108

Open Access

Utility of dried blood spot sampling and storage for increased stability of photosensitive compounds.
Bowen CL, Hemberger MD, Kehler JR, Evans CA

Compound stability remains a major point of concern within pharmaceutical development. In attempts to minimize degradation, scientists may utilize acidification of samples prior to storage, dark chambers, decreased freezer temperatures and a variety of other stabilization techniques. All of these steps require additional procedures, increased costs and increased validation steps. Dried blood spots (DBS) are becoming a popular alternative to plasma sampling in many small- and even large-molecule applications. An investigation was performed in order to establish if DBS would provide storage advantages over liquid-based matrices for two light-sensitive compounds, nifedipine and omeprazole, to prevent or minimize photodegradation.

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Bioanalysis, 2, 1757-6199, 1823-8, 2010

PMID: 21083490

Stem cells. Epigenome disruptors.
M Hemberger, R Pedersen

Science (New York, N.Y.), 330, 6004, 598-9, 2010

PMID: 21030637
DOI: 10.1126/science.1199006

PI3K signaling through the dual GTPase-activating protein ARAP3 is essential for developmental angiogenesis.
L Gambardella, M Hemberger, B Hughes, E Zudaire, S Andrews, S Vermeren

One function of phosphoinositide 3-kinase α (PI3Kα), which generates the lipid second messenger phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P(3)], is its regulation of angiogenesis in the developing embryo and in pathological situations. ARAP3 is a PtdIns(3,4,5)P(3)- and Rap-activated guanosine triphosphatase (GTPase)-activating protein (GAP) for the small GTPases RhoA and Arf6. Here, we show that deleting Arap3 in the mouse caused embryonic death in mid-gestation due to an endothelial cell-autonomous defect in sprouting angiogenesis. Explants taken at a developmental stage at which no defect was yet present reproduced this phenotype ex vivo, demonstrating that the defect was not secondary to hypoxia, placental defects, or organ failure. In addition, knock-in mice expressing an ARAP3 point mutant that cannot be activated by PtdIns(3,4,5)P(3) had angiogenesis defects similar to those of Arap3(-/-) embryos. Our work delineates a previously unknown signaling pathway that controls angiogenesis immediately downstream of PI3Kα through ARAP3 to the Rho and Arf family of small GTPases.

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Science signaling, 3, 145, ra76, 2010

PMID: 20978237
DOI: 10.1126/scisignal.2001026

Open Access

Regulation of early trophoblast differentiation - lessons from the mouse.
CE Senner, M Hemberger

The earliest stages of trophoblast differentiation are of tremendous importance to mediate implantation and to lay the anatomical foundations for normal placental development and function throughout gestation. Yet our molecular insights into these early developmental processes in humans have been limited by the inaccessibility of material and the unavailability of trophoblast cell lines that fully recapitulate the behaviour of early placental trophoblast. In this review we highlight recent advances that have come from the study of distinct stem cell types representative of the embryonic and extraembryonic lineages in the mouse, and from the study of mouse mutants. These models have revealed the presence of intricate transcriptional networks that are set up by signalling pathways, translating extracellular growth factor and cell positional information into distinct lineage-specific transcriptional programmes. The trophoblast specificity of these networks is ensured by epigenetic mechanisms including DNA methylation and histone modifications that complement each other to define trophoblast cell fate and differentiation. Despite the anatomical differences between mouse and human placentas, it seems that important aspects of early trophoblast specification are conserved between both species. Thus we may be able to build on our insights from the mouse to better understand early trophoblast differentiation in the human conceptus which is important for improving assisted reproductive technologies and may enable us in the future to derive human trophoblast stem cell lines. These advances will facilitate the investigation of genetic, epigenetic and environmental influences on early trophoblast differentiation in normal as well as in pathological conditions.

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Placenta, 31, 11, 944-50, 2010

PMID: 20797785
DOI: 10.1016/j.placenta.2010.07.013

Cell type-specific thalamic innervation in a column of rat vibrissal cortex.
Meyer HS, Wimmer VC, Hemberger M, Bruno RM, de Kock CP, Frick A, Sakmann B, Helmstaedter M

This is the concluding article in a series of 3 studies that investigate the anatomical determinants of thalamocortical (TC) input to excitatory neurons in a cortical column of rat primary somatosensory cortex (S1). We used viral synaptophysin-enhanced green fluorescent protein expression in thalamic neurons and reconstructions of biocytin-labeled cortical neurons in TC slices to quantify the number and distribution of boutons from the ventral posterior medial (VPM) and posteromedial (POm) nuclei potentially innervating dendritic arbors of excitatory neurons located in layers (L)2-6 of a cortical column in rat somatosensory cortex. We found that 1) all types of excitatory neurons potentially receive substantial TC input (90-580 boutons per neuron); 2) pyramidal neurons in L3-L6 receive dual TC input from both VPM and POm that is potentially of equal magnitude for thick-tufted L5 pyramidal neurons (ca. 300 boutons each from VPM and POm); 3) L3, L4, and L5 pyramidal neurons have multiple (2-4) subcellular TC innervation domains that match the dendritic compartments of pyramidal cells; and 4) a subtype of thick-tufted L5 pyramidal neurons has an additional VPM innervation domain in L4. The multiple subcellular TC innervation domains of L5 pyramidal neurons may partly explain their specific action potential patterns observed in vivo. We conclude that the substantial potential TC innervation of all excitatory neuron types in a cortical column constitutes an anatomical basis for the initial near-simultaneous representation of a sensory stimulus in different neuron types.

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Cerebral cortex (New York, N.Y. : 1991), 20, 1460-2199, 2287-303, 2010

PMID: 20534783

Open Access

ELF5-enforced transcriptional networks define an epigenetically regulated trophoblast stem cell compartment in the human placenta.
M Hemberger, R Udayashankar, P Tesar, H Moore, GJ Burton

The first definitive cell fate decision in development occurs at the blastocyst stage with establishment of the trophoblast and embryonic cell lineages. In the mouse, lineage commitment is achieved by epigenetic regulation of a critical gatekeeper gene, the transcription factor Elf5, that reinforces placental cell fate and is necessary for trophoblast stem (TS) cell self-renewal. In humans, however, the epigenetic lineage boundary seems to be less stringent since human embryonic stem (ES) cells, unlike their murine counterparts, harbour some potential to differentiate into trophoblast derivatives. Here, we show that ELF5 is expressed in the human placenta in villous cytotrophoblast cells but not in post-mitotic syncytiotrophoblast and invasive extravillous cytotrophoblast cells. ELF5 establishes a circuit of mutually interacting transcription factors with CDX2 and EOMES, and the highly proliferative ELF5(+)/CDX2(+) double-positive subset of cytotrophoblast cells demarcates a putative TS cell compartment in the early human placenta. In contrast to placental trophoblast, however, ELF5 is hypermethylated and largely repressed in human ES cells and derived trophoblast cell lines, as well as in induced pluripotent stem cells and murine epiblast stem cells. Thus, these cells exhibit an embryonic lineage-specific epigenetic signature and do not undergo an epigenetic reprogramming to reflect the trophoblast lineage at key loci such as ELF5. Our identification of the trophoblast-specific transcriptional circuit established by ELF5 will be instrumental to derive human TS cell lines that truly reflect early placental trophoblast and that will be most beneficial to gain insights into the aetiology of common pregnancy complications, including intra-uterine growth restriction and pre-eclampsia.

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Human molecular genetics, 19, 12, 2456-67, 2010

PMID: 20354077
DOI: 10.1093/hmg/ddq128

Open Access

Genetic-epigenetic intersection in trophoblast differentiation: implications for extraembryonic tissue function.
M Hemberger

Recent years have seen considerable advances in our understanding of early mammalian development leading up to the establishment of the first cell lineages, with important implications for the behavior of stem cells derived from the early embryo. Dramatic new insights have also propelled the field of epigenetics with the identification of 5-hydroxymethylcytosine as an additional base modification and the pervasiveness of asymmetrical non-CG DNA methylation specifically in ES cells. Prompted by our findings on the role of DNA methylation in cell lineage commitment, this review highlights recent insights into the genetic-epigenetic intersection in the establishment of the placental trophoblast lineage that is essential for embryo implantation, nutrition and survival. The unique trophoblast epigenotype is instrumental for normal trophoblast differentiation and placental function, and consequently trophoblast is particularly susceptible to regrogramming failures.

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Epigenetics : official journal of the DNA Methylation Society, 5, 1, 24-9, 2010

PMID: 20083894

Open Access

Activin promotes differentiation of cultured mouse trophoblast stem cells towards a labyrinth cell fate.
DR Natale, M Hemberger, M Hughes, JC Cross

Prolonged maintenance of trophoblast stem (TS) cells requires fibroblast growth factor (FGF) 4 and embryonic fibroblast feeder cells or feeder cell-conditioned medium. Previous studies have shown that TGF-beta and Activin are sufficient to replace embryonic fibroblast-conditioned medium. Nodal, a member of the TGF-beta superfamily, is also known to be important in vivo for the maintenance of TS cells in the developing placenta. Our current studies indicate that TS cells do not express the Nodal co-receptor, Cripto, and do not respond directly to active Nodal in culture. Conversely, Activin subunits and their receptors are expressed in the placenta and TS cell cultures, with Activin predominantly expressed by trophoblast giant cells (TGCs). Differentiation of TS cells in the presence of TGC-conditioned medium or exogenous Activin results in a reduction in the expression of TGC markers. In line with TGC-produced Activin representing the active component in TGC-conditioned medium, this differentiation-inhibiting effect can be reversed by the addition of follistatin. Additional experiments in which TS cells were differentiated in the presence or absence of exogenous Activin or TGF-beta show that Activin but not TGF-beta results in the maintenance of expression of TS cell markers, prolongs the expression of syncytiotrophoblast markers, and significantly delays the expression of spongiotrophoblast and TGC markers. These results suggest that Activin rather than TGF-beta (or Nodal) acts directly on TS cells influencing both TS cell maintenance and cell fate, depending on whether the cells are also exposed to FGF4.

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Developmental biology, 335, 1, 120-31, 2009

PMID: 19716815
DOI: 10.1016/j.ydbio.2009.08.022

Open Access

Down-regulation of Cdx2 in colorectal carcinoma cells by the Raf-MEK-ERK 1/2 pathway.
F Krueger, Z Madeja, M Hemberger, M McMahon, SJ Cook, SJ Gaunt

Cdx2 is a homeodomain transcription factor that regulates normal intestinal cell differentiation. Cdx2 is frequently lost during progression of colorectal cancer (CRC) and is widely viewed as a colorectal tumour suppressor. A previous study suggested that activation of protein kinase C (PKC) may be responsible for Cdx2 down-regulation in CRC cells. Here we show that activation of PKC does indeed promote down-regulation of Cdx2 at both the mRNA and protein levels. However, PKC-dependent loss of Cdx2 is dependent upon activation of the Raf-MEK-ERK1/2 pathway. Indeed, specific activation of the ERK1/2 pathway using the conditional kinase DeltaRaf-1:ER is sufficient to inhibit Cdx2 transcription. The Raf-MEK-ERK1/2 pathway is hyper-activated in a large fraction of colorectal cancers due to mutations in K-Ras and we show that treatment of CRC cell lines with MEK inhibitors causes an increase in Cdx2 expression. Furthermore, activation of the ERK1/2 pathway promotes the phosphorylation and proteasome-dependent degradation of the Cdx2 protein. The inhibitory effect of ERK1/2 upon Cdx2 in CRC cells is in sharp contrast to its stimulatory effect upon Cdx2 expression in trophectoderm and trophoblast stem cells. These results provide important new insights into the regulation of the Cdx2 tumour suppressor by linking it to ERK1/2, a pathway which is frequently activated in CRC.

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Cellular signalling, 21, 12, 1846-56, 2009

PMID: 19686845
DOI: 10.1016/j.cellsig.2009.07.020

Epigenetic dynamics of stem cells and cell lineage commitment: digging Waddington's canal.
M Hemberger, W Dean, W Reik

Cells of the early mammalian embryo, including pluripotent embryonic stem (ES) cells and primordial germ cells (PGCs), are epigenetically dynamic and heterogeneous. During early development, this heterogeneity of epigenetic states is associated with stochastic expression of lineage-determining transcription factors that establish an intimate crosstalk with epigenetic modifiers. Lineage-specific epigenetic modification of crucial transcription factor loci (for example, methylation of the Elf5 promoter) leads to the restriction of transcriptional circuits and the fixation of lineage fate. The intersection of major epigenetic reprogramming and programming events in the early embryo creates plasticity followed by commitment to the principal cell lineages of the early conceptus.

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Nature reviews. Molecular cell biology, 10, 8, 526-37, 2009

PMID: 19603040
DOI: 10.1038/nrm2727

Defining pathways that enforce cell lineage specification in early development and stem cells.
S Roper, M Hemberger

The molecular processes that govern the first cell lineage decisions after fertilization also dictate the developmental potency of stem cells derived from the early mouse embryo. Our understanding of these mechanisms is therefore instrumental for stem cell biology and regenerative medicine. A number of transcription factors are known that determine a cell's fate towards either the embryonic or extraembryonic trophoblast lineages. Recent insights have shown that the definitive fixation of cell lineage fate is achieved by an epigenetic restriction through DNA methylation of the transcription factor Elf5. Lineage crossover can be induced, however, by manipulation of lineage determinants and gatekeepers, or their epigenetic regulation. Here we summarize the accumulating number of experimental conditions where such 'transdifferentiation' is observed that shed light onto the genetic and epigenetic pathways involved in lineage separation and the developmental potential of stem cells.

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Cell cycle (Georgetown, Tex.), 8, 10, 1515-25, 2009

PMID: 19377304

Open Access

The RNA-binding protein Elavl1/HuR is essential for placental branching morphogenesis and embryonic development.
V Katsanou, S Milatos, A Yiakouvaki, N Sgantzis, A Kotsoni, M Alexiou, V Harokopos, V Aidinis, M Hemberger, DL Kontoyiannis

HuR is an RNA-binding protein implicated in a diverse array of pathophysiological processes due to its effects on the posttranscriptional regulation of AU- and U-rich mRNAs. Here we reveal HuR's requirement in embryonic development through its genetic ablation. Obligatory HuR-null embryos exhibited a stage retardation phenotype and failed to survive beyond midgestation. By means of conditional transgenesis, we restricted HuR's mutation in either embryonic or endothelial compartments to demonstrate that embryonic lethality is consequent to defects in extraembryonic placenta. HuR's absence impaired the invagination of allantoic capillaries into the chorionic trophoblast layer and the differentiation of syncytiotrophoblast cells that control the morphogenesis and vascularization of the placental labyrinth and fetal support. HuR-null embryos rescued from these placental defects proceeded to subsequent developmental stages but displayed defects in skeletal ossification, fusions in limb elements, and asplenia. By coupling gene expression measurements, data meta-analysis, and HuR-RNA association assays, we identified transcription and growth factor mRNAs controlled by HuR, primarily at the posttranscriptional level, to guide morphogenesis, specification, and patterning. Collectively, our data demonstrate the dominant role of HuR in organizing gene expression programs guiding placental labyrinth morphogenesis, skeletal specification patterns, and splenic ontogeny.

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Molecular and cellular biology, 29, 10, 2762-76, 2009

PMID: 19307312
DOI: 10.1128/MCB.01393-08

Open Access

Unique receptor repertoire in mouse uterine NK cells.
H Yadi, S Burke, Z Madeja, M Hemberger, A Moffett, F Colucci

Uterine NK (uNK) cells are a prominent feature of the uterine mucosa and regulate placentation. NK cell activity is regulated by a balance of activating and inhibitory receptors, however the receptor repertoire of mouse uNK cells is unknown. We describe herein two distinct subsets of CD3(-)CD122(+) NK cells in the mouse uterus (comprising decidua and mesometrial lymphoid aggregate of pregnancy) at mid-gestation: a small subset indistinguishable from peripheral NK cells, and a larger subset that expresses NKp46 and Ly49 receptors, but not NK1.1 or DX5. This larger subset reacts with Dolichus biflores agglutinin, a marker of uNK cells in the mouse, and is adjacent to the invading trophoblast. By multiparametric analysis we show that the phenotype of uNK cells is unique and unprecedented in terms of adhesion, activation, and MHC binding potential. Thus, the Ly49 repertoire and the expression of other differentiation markers strikingly distinguish uNK cells from peripheral NK cells, suggesting that a selection process shapes the receptor repertoire of mouse uNK cells.

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Journal of immunology (Baltimore, Md. : 1950), 181, 9, 6140-7, 2008

PMID: 18941204

Open Access

Epigenetic restriction of embryonic cell lineage fate by methylation of Elf5.
RK Ng, W Dean, C Dawson, D Lucifero, Z Madeja, W Reik, M Hemberger

Mouse ES cells can differentiate into all three germ layers of the embryo but are generally excluded from the trophoblast lineage. Here we show that ES cells deficient in DNA methylation can differentiate efficiently into trophoblast derivatives. In a genome-wide screen we identified the transcription factor Elf5 as methylated and repressed in ES cells, and hypomethylated and expressed in TS and methylation-deficient ES cells. Elf5 creates a positive-feedback loop with the TS cell determinants Cdx2 and Eomes that is restricted to the trophoblast lineage by epigenetic regulation of Elf5. Importantly, the late-acting function of Elf5 allows initial plasticity and regulation in the early blastocyst. Thus, Elf5 functions as a gatekeeper, downstream of initial lineage determination, to reinforce commitment to the trophoblast lineage or to abort this pathway in epiblast cells. This epigenetic restriction of cell lineage fate provides a molecular mechanism for Waddington's concept of canalization of developmental pathways.

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Nature cell biology, 10, 11, 1280-90, 2008

PMID: 18836439
DOI: 10.1038/ncb1786

Open Access