Postimplantation Mouse Embryos Research Articles

Transient Accumulation of 5-Carboxylcytosine Indicates Involvement of Active Demethylation in Lineage Specification of Neural Stem Cells

5-Methylcytosine (5mC) is an epigenetic modificationinvolved in regulation of gene activity during differentiation. Tet dioxygenases oxidize 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). Both 5fC and 5caC can be excised from DNA by thymine-DNA glycosylase (TDG) followed by regeneration of unmodified cytosine via the base excision repair pathway. Despite evidence that this mechanism is operative in embryonic stem cells, the role of TDG-dependent demethylation in differentiation and development is currently unclear. Here, we demonstrate that widespread oxidation of 5hmC to 5caC occurs in postimplantation mouse embryos. We show that 5fC and 5caC are transiently accumulated during lineage specification of neural stem cells (NSCs) in culture and invivo. Moreover, 5caC is enriched at the cell-type-specific promoters during differentiation of NSCs, and TDG knockdown leads to increased 5fC/5caC levels in differentiating NSCs. Our data suggest that active demethylation contributes to epigenetic reprogramming determining lineage specification in embryonic brain.

A microdevice to locally electroporate embryos with high efficiency and reduced cell damage.

The ability to follow and modify cell behaviour with accurate spatiotemporal resolution is a prerequisite to study morphogenesis in developing organisms. Electroporation, the delivery of exogenous molecules into targeted cell populations through electric permeation of the plasma membrane, has been used with this aim in different model systems. However, current localised electroporation strategies suffer from insufficient reproducibility and mediocre survival when applied to small and delicate organisms such as early post-implantation mouse embryos. We introduce here a microdevice to achieve localised electroporation with high efficiency and reduced cell damage. In silico simulations using a simple electrical model of mouse embryos indicated that a dielectric guide-based design would improve on existing alternatives. Such a device was microfabricated and its capacities tested by targeting the distal visceral endoderm (DVE), a migrating cell population essential for anterior-posterior axis establishment. Transfection was efficiently and reproducibly restricted to fewer than four visceral endoderm cells without compromising cell behaviour and embryo survival. Combining targeted mosaic expression of fluorescent markers with live imaging in transgenic embryos revealed that, like leading DVE cells, non-leading ones send long basal projections and intercalate during their migration. Finally, we show that the use of our microsystem can be extended to a variety of embryological contexts, from preimplantation stages to organ explants. Hence, we have experimentally validated an approach delivering a tailor-made tool for the study of morphogenesis in the mouse embryo. Furthermore, we have delineated a comprehensive strategy for the development of ad hoc electroporation devices.

Senescence‐associated β‐galactosidase activity marks the visceral endoderm of mouse embryos but is not indicative of senescence

Senescence-associated β-galactosidase (SA-β-gal) activity is widely used as a marker of cellular senescence and as an indicator of organismal aging. Here, we report that SA-β-gal activity is present in the visceral endoderm layer of early postimplantation mouse embryos in predictable patterns that vary as the embryo progresses in development. However, determination of the mitotic index and analysis of the expression of Cdkn1a (p21), a marker of senescent cells, do not indicate cellular senescence. Instead, analysis of embryos in culture revealed the presence of SA-β-gal activity in apical vacuoles of visceral endoderm cells likely a reflection of acidic β-galactosidase function in these organelles. SA-β-gal serves as a practical marker of the dynamics of the visceral endoderm that can be applied to developmental as well as functional studies of early mammalian embryos.

Neural stem cells derived from epiblast stem cells display distinctive properties

Pluripotent stem cells can be derived from preimplantation and postimplantation mouse embryos. Embryonic stem cells (ESCs) derived from blastocysts are in a “naive” pluripotent state and meet all of the criteria for pluripotency, including the ability to generate live pups through tetraploid complementation. Epiblast stem cells (EpiSCs) derived from postimplantation epiblasts are in a “primed” pluripotent state. ESCs and EpiSCs show different phenotypes and gene expression patterns, and EpiSCs are thought to be less pluripotent than ESCs. In this study, we addressed whether EpiSCs can be differentiated into specialized cell types in vitro. To do this, we first derived EpiSCs from E5.5–6.5 mouse embryos containing the Oct4-GFP transgene. We found that EpiSCs expressed pluripotency markers and differentiated into all three germ layers in intro and in vivo. Interestingly, EpiSCs also efficiently differentiated into a homogenous population of neural stem cells (NSCs) in vitro. The EpiSC-derived NSCs (EpiSC-NSCs) expressed NSC markers (Nestin, Sox2, and Musashi), self-renewed for more than 20 passages, and differentiated into neuronal and glial neural cell subtypes in vitro. We then transplanted the EpiSC-NSCs into the neonatal mouse brains, and found that they were able to survive and differentiate into robust neurons and glial cells in the mouse brains, demonstrating that primed pluripotent EpiSCs efficiently form functional NSCs. We compared the global gene expression patterns of NSCs differentiated from EpiSC-NSCs, ESCs, and brain tissue and found that the expression patterns of most genes, including pluripotency and NSC specificity, were similarly clustered, but that the developmental process-related genes were distantly clustered. Moreover, the global gene expression pattern of brain-derived NSCs was more similar to that of ESC-derived NSCs than that of EpiSC-derived NSCs. Taken together, these results indicate that although NSCs, regardless of their origins, display very similar in vitro and in vivo differentiation properties, their global gene expression profiles may differ, depending on the pluripotency state, i.e., naive or primed.

Somatic Donor Cell Type Correlates with Embryonic, but Not Extra-Embryonic, Gene Expression in Postimplantation Cloned Embryos

The great majority of embryos generated by somatic cell nuclear transfer (SCNT) display defined abnormal phenotypes after implantation, such as an increased likelihood of death and abnormal placentation. To gain better insight into the underlying mechanisms, we analyzed genome-wide gene expression profiles of day 6.5 postimplantation mouse embryos cloned from three different cell types (cumulus cells, neonatal Sertoli cells and fibroblasts). The embryos retrieved from the uteri were separated into embryonic (epiblast) and extraembryonic (extraembryonic ectoderm and ectoplacental cone) tissues and were subjected to gene microarray analysis. Genotype- and sex-matched embryos produced by in vitro fertilization were used as controls. Principal component analysis revealed that whereas the gene expression patterns in the embryonic tissues varied according to the donor cell type, those in extraembryonic tissues were relatively consistent across all groups. Within each group, the embryonic tissues had more differentially expressed genes (DEGs) (>2-fold vs. controls) than did the extraembryonic tissues (P<1.0×10–26). In the embryonic tissues, one of the common abnormalities was upregulation of Dlk1, a paternally imprinted gene. This might be a potential cause of the occasional placenta-only conceptuses seen in SCNT-generated mouse embryos (1–5% per embryos transferred in our laboratory), because dysregulation of the same gene is known to cause developmental failure of embryos derived from induced pluripotent stem cells. There were also some DEGs in the extraembryonic tissues, which might explain the poor development of SCNT-derived placentas at early stages. These findings suggest that SCNT affects the embryonic and extraembryonic development differentially and might cause further deterioration in the embryonic lineage in a donor cell-specific manner. This could explain donor cell-dependent variations in cloning efficiency using SCNT.

Epiblast Ground State Is Controlled by Canonical Wnt/β-Catenin Signaling in the Postimplantation Mouse Embryo and Epiblast Stem Cells

Epiblast stem cells (EpiSCs) are primed pluripotent stem cells and can be derived from postimplantation mouse embryos. We now show that the absence of canonical Wnt/β-catenin signaling is essential for maintenance of the undifferentiated state in mouse EpiSCs and in the epiblast of mouse embryos. Attenuation of Wnt signaling with the small-molecule inhibitor XAV939 or deletion of the β-catenin gene blocked spontaneous differentiation of EpiSCs toward mesoderm and enhanced the expression of pluripotency factor genes, allowing propagation of EpiSCs as a homogeneous population. EpiSCs were efficiently established and propagated from single epiblast cells in the presence of both XAV939 and the Rho kinase (ROCK) inhibitor Y27632. Cell transplantation revealed that EpiSCs were able to contribute to primordial germ cells and descendants of all three germ layers in a host embryo, suggesting that they maintained pluripotency, even after prolonged culture with XAV939. Such an improvement in the homogeneity of pluripotency achieved with the use of a Wnt inhibitor should prove advantageous for manipulation of primed pluripotent stem cells.

Unusual chromatin status and organization of the inactive X chromosome in murine trophoblast giant cells

Mammalian X-chromosome inactivation (XCI) enables dosage compensation between XX females and XY males. It is an essential process and its absence in XX individuals results in early lethality due primarily to extra-embryonic defects. This sensitivity to X-linked gene dosage in extra-embryonic tissues is difficult to reconcile with the reported tendency of escape from XCI in these tissues. The precise transcriptional status of the inactive X chromosome in different lineages has mainly been examined using transgenes or in in vitro differentiated stem cells and the degree to which endogenous X-linked genes are silenced in embryonic and extra-embryonic lineages during early postimplantation stages is unclear. Here we investigate the precise temporal and lineage-specific X-inactivation status of several genes in postimplantation mouse embryos. We find stable gene silencing in most lineages, with significant levels of escape from XCI mainly in one extra-embryonic cell type: trophoblast giant cells (TGCs). To investigate the basis of this epigenetic instability, we examined the chromatin structure and organization of the inactive X chromosome in TGCs obtained from ectoplacental cone explants. We find that the Xist RNA-coated X chromosome has a highly unusual chromatin content in TGCs, presenting both heterochromatic marks such as H3K27me3 and euchromatic marks such as histone H4 acetylation and H3K4 methylation. Strikingly, Xist RNA does not form an overt silent nuclear compartment or Cot1 hole in these cells. This unusual combination of silent and active features is likely to reflect, and might underlie, the partial activity of the X chromosome in TGCs.

Regulation of embryonic size in early mouse development in vitro culture system

Mammals self-regulate their body size throughout development. In the uterus, embryos are properly regulated to be a specific size at birth. Previously, size and cell number in aggregated embryos, which were made from two or more morulae, and half embryos, which were halved at the 2-cell stage, have been analysed in vivo in preimplantation and post-implantation development in mice. Here, we examined whether or not the mouse embryo has the capacity to self-regulate growth using an in vitro culture system. To elucidate embryonic histology, cells were counted in aggregated or half embryos in comparison with control embryos. Both double- and triple-aggregated embryos contained more cells than did control embryos during all culture periods, and the relative growth ratios showed no growth inhibition in an in vitro culture system. Meanwhile, half embryos contained fewer cells than control embryos, but the number grew throughout the culture period. Our data suggest that the growth of aggregated embryos is not affected and continues in an in vitro culture system. On the other hand, the growth of half embryos accelerates and continues in an in vitro culture system. This situation, in turn, implied that post-implantation mouse embryos might have some potential to regulate their own growth and size as seen by using an in vitro culture system without uterus factors. In conclusion, our results indicated that embryos have some ways in which to regulate their own size in mouse early development.

Grand challenges in stem cell treatments.

Grand challenges in stem cell treatments.

E-Cadherin Promotes Incorporation of Mouse Epiblast Stem Cells into Normal Development

Mouse epiblast stem cells (mEpiSCs) are pluripotent stem cells derived from epiblasts of postimplantation mouse embryos. Their pluripotency is distinct from that of mouse embryonic stem cells (mESCs) in several cell biological criteria. One of the distinctions is that mEpiSCs contribute either not at all or at much lower efficiency to chimeric embryos after blastocyst injection compared to mESCs. However, here we showed that mEpiSCs can be incorporated into normal development after blastocyst injection by forced expression of the E-cadherin transgene for 2 days in culture. Using this strategy, mEpiSCs gave rise to live-born chimeras from 5% of the manipulated blastocysts. There were no obvious signs of reprogramming of mEpiSCs toward the mESC-like state during the 2 days after induction of the E-cadherin transgene, suggesting that mEpiSCs possess latent ability to integrate into the normal developmental process as its origin, epiblasts.

Protection against De Novo Methylation Is Instrumental in Maintaining Parent-of-Origin Methylation Inherited from the Gametes

SummaryIdentifying loci with parental differences in DNA methylation is key to unraveling parent-of-origin phenotypes. By conducting a MeDIP-Seq screen in maternal-methylation free postimplantation mouse embryos (Dnmt3L-/+), we demonstrate that maternal-specific methylation exists very scarcely at midgestation. We reveal two forms of oocyte-specific methylation inheritance: limited to preimplantation, or with longer duration, i.e. maternally imprinted loci. Transient and imprinted maternal germline DMRs (gDMRs) are indistinguishable in gametes and preimplantation embryos, however, de novo methylation of paternal alleles at implantation delineates their fates and acts as a major leveling factor of parent-inherited differences. We characterize two new imprinted gDMRs, at the Cdh15 and AK008011 loci, with tissue-specific imprinting loss, again by paternal methylation gain. Protection against demethylation after fertilization has been emphasized as instrumental in maintaining parent-of-origin methylation inherited from the gametes. Here we provide evidence that protection against de novo methylation acts as an equal major pivot, at implantation and throughout life.

Initiating head development in mouse embryos: integrating signalling and transcriptional activity

The generation of an embryonic body plan is the outcome of inductive interactions between the progenitor tissues that underpin their specification, regionalization and morphogenesis. The intercellular signalling activity driving these processes is deployed in a time- and site-specific manner, and the signal strength must be precisely controlled. Receptor and ligand functions are modulated by secreted antagonists to impose a dynamic pattern of globally controlled and locally graded signals onto the tissues of early post-implantation mouse embryo. In response to the WNT, Nodal and Bone Morphogenetic Protein (BMP) signalling cascades, the embryo acquires its body plan, which manifests as differences in the developmental fate of cells located at different positions in the anterior–posterior body axis. The initial formation of the anterior (head) structures in the mouse embryo is critically dependent on the morphogenetic activity emanating from two signalling centres that are juxtaposed with the progenitor tissues of the head. A common property of these centres is that they are the source of antagonistic factors and the hub of transcriptional activities that negatively modulate the function of WNT, Nodal and BMP signalling cascades. These events generate the scaffold of the embryonic head by the early-somite stage of development. Beyond this, additional tissue interactions continue to support the growth, regionalization, differentiation and morphogenesis required for the elaboration of the structure recognizable as the embryonic head.

Generation and Characterization of LIF-dependent Canine Induced Pluripotent Stem Cells from Adult Dermal Fibroblasts

Dogs provide a more clinically relevant model of human disease than rodents, particularly with respect to hereditary diseases. Thus, the availability of canine stem cells will greatly facilitate the use of the dog in the development of stem cell-based gene therapies and regenerative medicine. In this study we describe the production of canine induced pluripotent stem cells (ciPSCs) from adult dermal fibroblasts. These cells have a morphology resembling previously described canine embryonic stem cells, a normal karyotype, and express pluripotency markers including alkaline phosphatase, Nanog, Oct4, Telomerase, SSEA1, SSEA4, TRA1-60, TRA1-81, and Rex1. Furthermore, the inactive X chromosome is reactivated indicating a ground-state pluripotency. In culture they readily form embryoid bodies, which in turn give rise to cell types from all 3 embryonic germ layers, as indicated by expression of the definitive endoderm markers Cxcr4 and α-fetoprotein, mesoderm markers Collagen IIA and Gata2, and ectoderm markers βIII-tubulin, Enolase, and Nestin. Of particular significance is the observation that these ciPSCs are dependent only on leukemia inhibitory factor (LIF), making them similar to mouse and canine embryonic stem cells, but strikingly unlike the ciPSCs recently described in two other studies, which were dependent on both basic fibroblast growth factor and LIF in order to maintain their pluripotency. Thus, our ciPSCs closely resemble mouse ESCs derived from the inner cell mass of preimplantation embryos, while the previously described ciPSCs appear to be more representative of cells from the epiblast of mouse postimplantation embryos.

BMP4 signaling directs primitive endoderm-derived XEN cells to an extraembryonic visceral endoderm identity

The visceral endoderm (VE) is an epithelial tissue in the early postimplantation mouse embryo that encapsulates the pluripotent epiblast distally and the extraembryonic ectoderm proximally. In addition to facilitating nutrient exchange before the establishment of a circulation, the VE is critical for patterning the epiblast. Since VE is derived from the primitive endoderm (PrE) of the blastocyst, and PrE-derived eXtraembryonic ENdoderm (XEN) cells can be propagated in vitro, XEN cells should provide an important tool for identifying factors that direct VE differentiation. In this study, we demonstrated that BMP4 signaling induces the formation of a polarized epithelium in XEN cells. This morphological transition was reversible, and was associated with the acquisition of a molecular signature comparable to extraembryonic (ex) VE. Resembling exVE which will form the endoderm of the visceral yolk sac, BMP4-treated XEN cells regulated hematopoiesis by stimulating the expansion of primitive erythroid progenitors. We also observed that LIF exerted an antagonistic effect on BMP4-induced XEN cell differentiation, thereby impacting the extrinsic conditions used for the isolation and maintenance of XEN cells in an undifferentiated state. Taken together, our data suggest that XEN cells can be differentiated towards an exVE identity upon BMP4 stimulation and therefore represent a valuable tool for investigating PrE lineage differentiation.

Distinct Developmental Ground States of Epiblast Stem Cell Lines Determine Different Pluripotency Features

Epiblast stem cells (EpiSCs) are pluripotent stem cells derived from mouse postimplantation embryos at embryonic day (E) 5.5-E7.5 at the onset of gastrulation, which makes them a valuable tool for studying mammalian postimplantation development in vitro. EpiSCs can also be reprogrammed into a mouse embryonic stem cell (mESC)-like state. Some reports have shown that the reversion of EpiSCs requires transcription factor overexpression, whereas others have suggested that use of stringent mESC culture conditions alone is sufficient for the reversion of EpiSCs. To clarify these discrepancies, we systematically compared a panel of independent EpiSC lines. We found that--regardless of the embryonic day of derivation--the different EpiSC lines shared a number of defining characteristics such as the ability to form teratomas. However, despite use of standard EpiSC culture conditions, some lines exhibited elevated expression of genes associated with mesendodermal differentiation. Pluripotency (Oct4) and mesodermal (Brachyury) marker genes were coexpressed in this subset of lines. Interestingly, the expression of mesendodermal marker genes was negatively correlated with the cells' ability to efficiently undergo neural induction. Moreover, these mesodermal marker gene-expressing cell lines could not be efficiently reverted to an mESC-like state by using stringent mESC culture conditions. Conversely, Brachyury overexpression diminished the reversion efficiency in otherwise Brachyury-negative lines. Overall, our data suggest that different EpiSC lines may undergo self-renewal into distinct developmental states, a finding with important implications for functional readouts such as reversion of EpiSCs to an mESC-like state as well as directed differentiation.

Developmental toxicity of retrorsine on mouse embryos in vitro

To investigate the fetotoxicity of retrorsine. Mouse whole embryo culture (WEC) was applied. Post-implantation (8.5 d) mouse embryos were isolated from their mothers and put into the medium of immediately centrifuged serum (ICS) prepared from rats. Different concentrations of retrorsine (12.5, 25, 50, 100 mg x L(-1)) were added into the WEC culture. Development (yolk sac diameter, crown-rump length, head length, somite number) and organic morphodifferentiation (yolk sac circulation, allantois, embryonic flexion, heart, brain, optic-otic-olfactory organ, branchial arch, maxillary, mandible, bud) of embryos were observed at 48 h after treatment. Obvious fetotoxicity could be observed in various retrorsine treatment groups in a dose-dependent manner. Development of embryos was delayed significantly at dose 12.5-100 mg x L(-1). Malformations were shown in all organic morphodifferentiation indexes, especially in otic-olfactory organ, branchial arch, maxillary, mandible, bud. Retrorsine had obvious fetotoxicity in vitro WEC culture, indicating that exposure of pregnant mice to retrorsine may have potential risk on fetals.

Lineage-specific distribution of high levels of genomic

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.

Time-Lapse Imaging of Postimplantation Mouse Embryos

INTRODUCTIONGenetic manipulation methods allow fluorescent labeling of virtually any cell type or protein of interest in developing embryos, providing powerful insights into morphogenetic events at cellular and subcellular resolutions. The development of ex vivo embryo culture methods combined with high-resolution imaging provides a strong platform for observing morphogenetic events as they occur within the developing embryo. In this protocol, postimplantation mouse embryos are cultured directly on the microscope stage under conditions that carefully control temperature, humidity, and gas exchange. This permits time-lapse imaging of developing mouse embryos.

Preparation of Postimplantation Mouse Embryos for Imaging: Figure 1.

INTRODUCTIONGenetic manipulation methods allow fluorescent labeling of virtually any cell type or protein of interest in developing embryos, providing powerful insights into morphogenetic events at cellular and subcellular resolutions. The development of ex vivo embryo culture methods combined with high-resolution imaging provides a strong platform for observing morphogenetic events as they occur within the developing embryo. This protocol details the dissection and immobilization of postimplantation mouse embryos in preparation for live imaging.

Primed for Pluripotency

To date, pluripotent epiblast stem cells (EpiSCs) had only been derived from postimplantation mouse embryos. In this issue of Cell Stem Cell, Najm et al. (2011) demonstrate that EpiSCs can be routinely derived from preimplantation embryos, showing that both human and mouse blastocysts can produce the same class of primed pluripotent cells.