Troika of the mouse blastocyst: lineage segregation and stem cells

The initial period of mammalian embryonic development is primarily devoted to cell commitment to the pluripotent lineage, as well as to the formation of extraembryonic tissues essential for embryo survival in utero. This phase of development is also characterized by extensive morphological transitions. Cells within the preimplantation embryo exhibit extraordinary cell plasticity and adaptation in response to experimental manipulation, highlighting the use of a regulative developmental strategy rather than a predetermined one resulting from the non-uniform distribution of maternal information in the cytoplasm. Consequently, early mammalian development represents a useful model to study how the three primary cell lineages; the epiblast, primitive endoderm (also referred to as the hypoblast) and trophoblast, emerge from a totipotent single cell, the zygote. In this review, we will discuss how the isolation and genetic manipulation of murine stem cells representing each of these three lineages has contributed to our understanding of the molecular basis of early developmental events.     

Suboptimal in vitro culture conditions: an epigenetic origin of long-term health effects

The foetal origins of adult diseases or Barker hypothesis suggests that there can be adverse in uterus effects on the foetus that can lead to certain diseases in adults. Extending this hypothesis to the early stages of embryo development, in particular, to preimplantation stages, it was recently demonstrated that, long-term programming of postnatal development, growth and physiology can be irreversibly affected during this period of embryo development by suboptimal in vitro culture (IVC). As an example, it was found in two recent studies that, mice derived from embryos cultured in suboptimal conditions can suffer from obesity, increased anxiety, and deficiencies on their implicit memory system. In addition, it was observed that suboptimal IVC can cause disease in mature animals by promoting alterations in their genetic imprinting during preimplantation development. Imprinting and other epigenetic mechanisms control the establishment and maintenance of gene expression patterns in the embryo, placenta and foetus. The previously described observations, suggest that the loss of epigenetic regulation during preimplantation development may lead to severe long-term effects. Although mostly tested in rodents, the hypothesis that underlies these studies can also fit assisted reproductive technology (ART) procedures in other species, including humans. The lack of information on how epigenetic controls are lost during IVC, and on the long-term consequences of ART, underscore the necessity for sustained epigenetic analysis of embryos produced in vitro and long-term tracking of the health of the human beings conceived using these procedures.     

Genome-wide analysis of epigenetic dynamics across human developmental stages and tissues

Background

Epigenome is highly dynamic during the early stages of embryonic development. Epigenetic modifications provide the necessary regulation for lineage specification and enable the maintenance of cellular identity. Given the rapid accumulation of genome-wide epigenomic modification maps across cellular differentiation process, there is an urgent need to characterize epigenetic dynamics and reveal their impacts on differential gene regulation.

Methods

We proposed DiffEM, a computational method for differential analysis of epigenetic modifications and identified highly dynamic modification sites along cellular differentiation process. We applied this approach to investigating 6 epigenetic marks of 20 kinds of human early developmental stages and tissues, including hESCs, 4 hESC-derived lineages and 15 human primary tissues.

Results

We identified highly dynamic modification sites where different cell types exhibit distinctive modification patterns, and found that these highly dynamic sites enriched in the genes related to cellular development and differentiation. Further, to evaluate the effectiveness of our method, we correlated the dynamics scores of epigenetic modifications with the variance of gene expression, and compared the results of our method with those of the existing algorithms. The comparison results demonstrate the power of our method in evaluating the epigenetic dynamics and identifying highly dynamic regions along cell differentiation process.

     

Epigenetic asymmetry in the mammalian zygote and early embryo: relationship to lineage commitment?

Epigenetic asymmetry between parental genomes and embryonic lineages exists at the earliest stages of mammalian development. The maternal genome in the zygote is highly methylated in both its DNA and its histones and most imprinted genes have maternal germline methylation imprints. The paternal genome is rapidly remodelled with protamine removal, addition of acetylated histones, and rapid demethylation of DNA before replication. A minority of imprinted genes have paternal germline methylation imprints. Methylation and chromatin reprogramming continues during cleavage divisions, but at the blastocyst stage lineage commitment to inner cell mass (ICM) or trophectoderm (TE) fate is accompanied by a dramatic increase in DNA and histone methylation, predominantly in the ICM. This may set up major epigenetic differences between embryonic and extraembryonic tissues, including in X-chromosome inactivation and perhaps imprinting. Maintaining epigenetic asymmetry appears important for development as asymmetry is lost in cloned embryos, most of which have developmental defects, and in particular an imbalance between extraembryonic and embryonic tissue development.     

Comparative aspects of early lineage specification events in mammalian embryos – insights from reverse genetics studies

Within the mammalian class, formation of the blastocyst is morphologically highly conserved among different species. The molecular and cellular events during preimplantation embryo development have been studied extensively in the mouse as model organism, because multiple genetically defined strains and a plethora of reverse genetics tools are available to dissect specific gene functions and regulatory networks. However, major differences in preimplantation developmental kinetics, implantation, and placentation exist among mammalians, and recent studies in species other than mouse showed, that even regulatory mechanisms of the first lineage differentiation events and maintenance of pluripotency are not always conserved. Here, we focus on the first and the second lineage segregation in mouse and bovine embryos, when the first differentiated cell types emerge. We outline their common features and differences in the regulation of these essential events during embryonic development with a glance at further species. In addition, we show how new reverse genetics strategies aid the study of regulatory circuits in embryos of domestic species, enhancing our overall understanding of mammalian preimplantation development.     

Effects of growth factors FGF2 and IGF2 on the development of parthenogenetic mouse embryos in utero and in vitro

We studied the effects of fibroblast growth factor 2 (FGF2) and insulin-like growth factor 2 (IGF2) on the development of parthenogenetic mouse embryos (CBA x C57BK/6)F1. The parthenogenetic embryos were treated in vitro during the preimplantation period and, at the blastocyst stage, transplanted into the uterus of pseudopregnant females. The addition of FGF2 at an optimal dose (2.5 ng/ml) to the culture medium increased twofold the number of embryos developed in utero to the somite stages as compared to the control: 18 and 43%, respectively. The parthenogenetic embryos (18-21 somites), treated and nontreated with FGF2 during the preimplantation period, were explanted for further development in vitro and treated with IGF2 at 2.5 micrograms/ml. As a result, many more parthenogenetic embryos (> 87%) of both groups developed in vitro to the stage of 30 or more somites as compared to the control (59%). The treatment of the parthenogenetic embryos with FGF2 alone at the preimplantation stages did not improve their development in vitro at the postimplantation stages. The results we obtained suggest that the treatment of parthenogenetic embryos in vitro with FGF2 during the preimplantation period increased twofold the number of somite embryos in utero, while their subsequent treatment in vitro with IGF2 leads to a significant prolongation of their development, as compared to the control.     

A genome-wide study of gene activity reveals developmental signaling pathways in the preimplantation mouse embryo

The preimplantation development of the mammalian embryo encompasses a series of critical events: the transition from oocyte to embryo, the first cell divisions, the establishment of cellular contacts, the first lineage differentiation-all the first subtle steps toward a future body plan. Here, we use microarrays to explore gene activity during preimplantation development. We reveal robust and dynamic patterns of stage-specific gene activity that fall into two major phases, one up to the 2-cell stage (oocyte-to-embryo transition) and one after the 4-cell stage (cellular differentiation). The mouse oocyte and early embryo express components of multiple signaling pathways including those downstream of Wnt, BMP, and Notch, indicating that conserved regulators of cell fate and pattern formation are likely to function at the earliest embryonic stages. Overall, these data provide a detailed temporal profile of gene expression that reveals the richness of signaling processes in early mammalian development.     

Delayed and incomplete reprogramming of chromosome methylation patterns in bovine cloned embryos

Full-term development has now been achieved in several mammalian species by transfer of somatic nuclei into enucleated oocytes [1, 2]. Although a high proportion of such reconstructed embryos can evolve until the blastocyst stage, only a few percent develop into live offspring, which often exhibit developmental abnormalities [3, 4]. Regulatory epigenetic markers such as DNA methylation are imposed on embryonic cells as normal development proceeds, creating differentiated cell states. Cloned embryos require the erasure of their somatic epigenetic markers so as to regain a totipotent state [5]. Here we report on differences in the dynamics of chromosome methylation between cloned and normal bovine embryos before implantation. We show that cloned embryos fail to reproduce distinguishable parental-chromosome methylation patterns after fusion and maintain their somatic pattern during subsequent stages, mainly by a highly reduced efficiency of the passive demethylation process. Surprisingly, chromosomes appear constantly undermethylated on euchromatin in morulae and blastocysts, while centromeric heterochromatin remains more methylated than that of normal embryos. We propose that the abnormal time-dependent methylation events spanning the preimplantation development of clones may significantly interfere with the epigenetic reprogramming, contributing to the high incidence of physiological anomalies occurring later during pregnancy or after clone birth.     

Keeping things quiet: roles of NuRD and Sin3 co-repressor complexes during mammalian development

Gene inactivation studies of mammalian histone and DNA-modifying proteins have demonstrated a role for many such proteins in embryonic development. Post-implantation embryonic lethality implies a role for epigenetic factors in differentiation and in development of specific lineages or tissues. However a handful of chromatin-modifying enzymes have been found to be required in pre- or peri-implantation embryos. This is significant as implantation is the time when inner cell mass cells of the blastocyst exit pluripotency and begin to commit to form the various lineages that will eventually form the adult animal. These observations indicate a critical role for chromatin-modifying proteins in the earliest lineage decisions of mammalian development, and/or in the formation of the first embryonic cell types. Recent work has shown that the two major class I histone deacetylase-containing co-repressor complexes, the NuRD and Sin3 complexes, are both required at peri-implantation stages of mouse development, demonstrating the importance of histone deacetylation in cell fate decisions. Over the past 10 years both genetic and biochemical studies have revealed surprisingly divergent roles for these two co-repressors in mammalian cells. In this review we will summarise the evidence that the two major class I histone deacetylase complexes in mammalian cells, the NuRD and Sin3 complexes, play important roles in distinct aspects of embryonic development.     

Lineage-specific distribution of high levels of genomic 5-hydroxymethylcytosine in mammalian development

Methylation of cytosine is a DNA modification associated with gene repression. Recently, a novel cytosine modification, 5-hydroxymethylcytosine (5-hmC) has been discovered. Here we examine 5-hmC distribution during mammalian development and in cellular systems, and show that the developmental dynamics of 5-hmC are different from those of 5-methylcytosine (5-mC); in particular 5-hmC is enriched in embryonic contexts compared to adult tissues. A detectable 5-hmC signal appears in pre-implantation development starting at the zygote stage, where the paternal genome is subjected to a genome-wide hydroxylation of 5-mC, which precisely coincides with the loss of the 5-mC signal in the paternal pronucleus. Levels of 5-hmC are high in cells of the inner cell mass in blastocysts, and the modification colocalises with nestin-expressing cell populations in mouse post-implantation embryos. Compared to other adult mammalian organs, 5-hmC is strongly enriched in bone marrow and brain, wherein high 5-hmC content is a feature of both neuronal progenitors and post-mitotic neurons. We show that high levels of 5-hmC are not only present in mouse and human embryonic stem cells (ESCs) and lost during differentiation, as has been reported previously, but also reappear during the generation of induced pluripotent stem cells; thus 5-hmC enrichment correlates with a pluripotent cell state. Our findings suggest that apart from the cells of neuronal lineages, high levels of genomic 5-hmC are an epigenetic feature of embryonic cell populations and cellular pluri- and multi-lineage potency. To our knowledge, 5-hmC represents the first epigenetic modification of DNA discovered whose enrichment is so cell-type specific.     

Centromeric DNA hypomethylation as an epigenetic signature discriminates between germ and somatic cell lineages

Germ cells have unique features strikingly distinguishable from somatic cells. The functional divergence between these two cell lineages has been postulated to result from epigenetic mechanisms. Here we show that the chromosomal centric and pericentric (C/P) regions in male and female germline cells are specifically DNA-hypomethylated, despite the hypermethylation status in somatic cells. In multipotent germline stem cells, the C/P region was initially hypomethylated and then shifted to the hypermethylation status during differentiation into somatic lineage in vitro. Moreover, the somatic-type hypermethylation pattern was maintained in the somatic cell-derived nuclear transfer embryos throughout preimplantation development. These results imply that the identity of germ cell lineage may be warranted by the hypomethylation status of the C/P region as an epigenetic signature.