Embryo structure reorganisation reduces the probability of apoptosis in preimplantation mouse embryos.

Programmed cell death plays a key role in mammalian development because the morphological events of an organism's formation are dependent on apoptosis. In the mouse development, the first apoptotic waves occur physiologically at the blastocyst stage. Cell number and the mean nucleus to cytoplasm (N/C) ratio increase exponentially throughout subsequent embryo cleavages, while cell volume concurrently decreases from the zygote to blastocyst stage. In this study we tested the hypothesis that reorganisation of the embryo structure by manipulating cell number, the N/C ratio and the cell volume of 2-cell embryos may result in the earlier and more frequent occurrence of apoptosis. The results indicate that doubling ('Aggregates' group) or halving ('Embryos 1/2' group) the initial cell number and modifying embryo volume, ploidy ('Embryos 4n' group) and the N/C ratio ('Embryos 2/1' group) reduce the probability of apoptosis in the resulting embryos. There was a higher probability of apoptosis in the inner cell mass of the blastocyst, but apoptotic cells were never observed at the morula stage in any of the experimental groups. Thus, manipulation of cell number, embryo volume, the N/C ratio and ploidy cause subtle changes in the occurrence of apoptosis, although these are mostly dependent on embryo stage and cell lineage (trophectoderm or inner cell mass), which have the greatest effect on the probability of apoptosis.

Embryonic Environmental Niche Reprograms Somatic Cells to Express Pluripotency Markers and Participate in Adult Chimaeras.

The phenomenon of the reprogramming of terminally differentiated cells can be achieved by various means, like somatic cell nuclear transfer, cell fusion with a pluripotent cell, or the introduction of pluripotency genes. Here, we present the evidence that somatic cells can attain the expression of pluripotency markers after their introduction into early embryos. Mouse embryonic fibroblasts introduced between blastomeres of cleaving embryos, within two days of in vitro culture, express transcription factors specific to blastocyst lineages, including pluripotency factors. Analysis of donor tissue marker DNA has revealed that the progeny of introduced cells are found in somatic tissues of foetuses and adult chimaeras, providing evidence for cell reprogramming. Analysis of ploidy has shown that in the chimaeras, the progeny of introduced cells are either diploid or tetraploid, the latter indicating cell fusion. The presence of donor DNA in diploid cells from chimaeric embryos proved that the non-fused progeny of introduced fibroblasts persisted in chimaeras, which is evidence of reprogramming by embryonic niche. When adult somatic (cumulus) cells were introduced into early cleavage embryos, the extent of integration was limited and only cell fusion-mediated reprogramming was observed. These results show that both cell fusion and cell interactions with the embryonic niche reprogrammed somatic cells towards pluripotency.

Differentiation of first cell lineages in mammalian embryos ­ interspecies similarities and differences / Róznicowanie pierwszych linii komórkowych w zarodkach ssaków - róznice i podobienstwa miedzygatunkowe.

The embryonic development of placental mammals takes place inside the mother's womb, which requires the formation of appropriate supportive structures by both the mother's organism and the developing embryo. The first stages of mammalian embryonic development, preceding implantation, are the period of differentiation of the first cell lineages ­ epiblast (which will give rise to the embryo proper), and extra-embryonic lineages: trophectoderm (responsible for implantation and formation of the placenta) and primitive endoderm (giving rise to the yolk sac). Their differentiation is necessary for further development, and is a common feature of the development of all placental mammals, but the timing and molecular mechanisms responsible for these processes differ between mammalian species.

Common principles of early mammalian embryo self-organisation.

Pre-implantation mammalian development unites extreme plasticity with a robust outcome: the formation of a blastocyst, an organised multi-layered structure ready for implantation. The process of blastocyst formation is one of the best-known examples of self-organisation. The first three cell lineages in mammalian development specify and arrange themselves during the morphogenic process based on cell-cell interactions. Despite decades of research, the unifying principles driving early mammalian development are still not fully defined. Here, we discuss the role of physical forces, and molecular and cellular mechanisms, in driving self-organisation and lineage formation that are shared between eutherian mammals.

Beyond the mouse: non-rodent animal models for study of early mammalian development and biomedical research.

The preimplantation development of mammals generally follows the same plan. It starts with the formation of a totipotent zygote, and through consecutive cleavage divisions and differentiation events leads to blastocyst formation. However, the intervening events may differ between species. The regulation of these processes has been extensively studied in the mouse, which displays some unique features among eutherian mammals. Farm animals such as pigs, cattle, sheep and rabbits share several similarities with one another, and with the human developmental plan. These include the timing of epigenetic reprogramming, the moment of embryonic genome activation and the developmental time-frame. Recently, efficient techniques for genetic modification have been established for large domestic animals. Genome sequences and gene manipulation tools are now available for cattle, pigs, sheep and goats, and a larger number of genetically engineered livestock is now accessible for biomedical research. Yet, these animals still make up less than 0.5% of animals in research, mainly due to our inadequate knowledge of the processes responsible for pluripotency maintenance (to date no stable naïve embryonic stem cell lines have been established) and early development. In this review, we highlight our present knowledge of the key preimplantation events in the 3 non-rodent species which present the highest potential for biomedical research related to early embryonic development: cattle, which offer an excellent model to study human in vitro embryo development, pigs which emerge as models to study the long-term effects of gene-based therapies and rabbits, which in many aspects of embryology resemble the human.

Ex Utero Culture and Imaging of Mouse Embryos.

Mouse genetic approaches when combined with live imaging tools are revolutionizing our current understanding of mammalian developmental biology. The availability and improvement of a wide variety of genetically encoded fluorescent proteins have provided indispensable tools to visualize cells and subcellular features in living organisms. It is now possible to generate genetically modified mouse lines expressing several spectrally distinct fluorescent proteins in a tissue-specific or -inducible manner. Such reporter-expressing lines make it possible to image dynamic cellular behaviors in the context of living embryos undergoing normal or aberrant development. As with all viviparous mammals, mouse embryos develop within the uterus, and so live imaging experiments require culture conditions that closely mimic the in vivo environment. Over the past decades, significant advances have been made in developing conditions for culturing both pre- and postimplantation-stage mouse embryos. In this chapter, we discuss routine methods for ex utero culture of preimplantation- and postimplantation-stage mouse embryos. In particular, we describe protocols for collecting mouse embryos of various stages, setting up culture conditions for their ex utero culture and imaging, and using laser scanning confocal microscopy to visualize live processes in mouse embryos expressing fluorescent reporters.

No evidence of involvement of E-cadherin in cell fate specification or the segregation of Epi and PrE in mouse blastocysts.

During preimplantation mouse development stages, emerging pluripotent epiblast (Epi) and extraembryonic primitive endoderm (PrE) cells are first distributed in the blastocyst in a "salt-and-pepper" manner before they segregate into separate layers. As a result of segregation, PrE cells become localised on the surface of the inner cell mass (ICM), and the Epi is enclosed by the PrE on one side and by the trophectoderm on the other. During later development, a subpopulation of PrE cells migrates away from the ICM and forms the parietal endoderm (PE), while cells remaining in contact with the Epi form the visceral endoderm (VE). Here, we asked: what are the mechanisms mediating Epi and PrE cell segregation and the subsequent VE vs PE specification? Differences in cell adhesion have been proposed; however, we demonstrate that the levels of plasma membrane-bound E-cadherin (CDH1, cadherin 1) in Epi and PrE cells only differ after the segregation of these lineages within the ICM. Moreover, manipulating E-cadherin levels did not affect lineage specification or segregation, thus failing to confirm its role during these processes. Rather, we report changes in E-cadherin localisation during later PrE-to-PE transition which are accompanied by the presence of Vimentin and Twist, supporting the hypothesis that an epithelial-to-mesenchymal transition process occurs in the mouse peri-implantation blastocyst.

Pre-implantation Development of Domestic Animals.

During the first days following fertilization, cells of mammalian embryo gradually lose totipotency, acquiring distinct identity. The first three lineages specified in the mammalian embryo are pluripotent epiblast, which later gives rise to the embryo proper, and two extraembryonic lineages, hypoblast (also known as primitive endoderm) and trophectoderm, which form tissues supporting development of the fetus in utero. Most of our knowledge regarding the mechanisms of early lineage specification in mammals comes from studies in the mouse. However, the growing body of evidence points to both similarities and species-specific differences. Understanding molecular and cellular mechanisms of early embryonic development in nonrodent mammals expands our understanding of basic mechanisms of differentiation and is essential for the development of effective protocols for assisted reproduction in agriculture, veterinary medicine, and for biomedical research. This review summarizes the current state of knowledge on key events in epiblast, hypoblast, and trophoblast differentiation in domestic mammals.

Suppression of ERK signalling abolishes primitive endoderm formation but does not promote pluripotency in rabbit embryo.

Formation of epiblast (EPI) - the founder line of all embryonic lineages - and extra-embryonic supportive tissues is one of the key events in mammalian development. The prevailing model of early mammalian development is based almost exclusively on the mouse. Here, we provide a comprehensive, stage-by-stage analysis of EPI and extra-embryonic primitive endoderm (PrE) formation during preimplantation development of the rabbit. Although we observed that rabbit embryos have several features in common with mouse embryos, including a stage-related initiation of lineage specification, our results demonstrate the existence of some key differences in lineage specification among mammals. Contrary to the current view, our data suggest that reciprocal repression of GATA6 and NANOG is not fundamental for the initial stages of PrE versus EPI specification in mammals. Furthermore, our results provide insight into the observed discrepancies relating to the role of FGF/ERK signalling in PrE versus EPI specification between mouse and other mammals.

Cell fate in animal and human blastocysts and the determination of viability.

Understanding the mechanisms underlying the first cell differentiation events in human preimplantation development is fundamental for defining the optimal conditions for IVF techniques and selecting the most viable embryos for further development. However, our comprehension of the very early events in development is still very limited. Moreover, our knowledge on early lineage specification comes primarily from studying the mouse model. It is important to recognize that although mammalian embryos share similar morphological landmarks, the timing and molecular control of developmental events may vary substantially between species. Mammalian blastocysts comprise three cell types that arise through two sequential rounds of binary cell fate decisions. During the first decision, cells located on the outside of the developing embryo form a precursor lineage for the embryonic part of the placenta: the trophectoderm and cells positioned inside the embryo become the inner cell mass (ICM). Subsequently, ICM cells differentiate into embryonic lineages that give rise to a variety of tissues in the developing foetus: either the epiblast or extraembryonic primitive endoderm. Successful formation of all three lineages is a prerequisite for implantation and development to term. A comprehensive understanding of the lineage specification processes in mammals is therefore necessary to shed light on the causes of early miscarriages and early pregnancy pathologies in humans.

The T-box transcription factor Eomesodermin is essential for AVE induction in the mouse embryo.

Reciprocal inductive interactions between the embryonic and extraembryonic tissues establish the anterior-posterior (AP) axis of the early mouse embryo. The anterior visceral endoderm (AVE) signaling center emerges at the distal tip of the embryo at embryonic day 5.5 and translocates to the prospective anterior side of the embryo. The process of AVE induction and migration are poorly understood. Here we demonstrate that the T-box gene Eomesodermin (Eomes) plays an essential role in AVE recruitment, in part by directly activating the homeobox transcription factor Lhx1. Thus, Eomes function in the visceral endoderm (VE) initiates an instructive transcriptional program controlling AP identity.

Anatomy of a blastocyst: cell behaviors driving cell fate choice and morphogenesis in the early mouse embryo.

The preimplantation period of mouse early embryonic development is devoted to the specification of two extraembryonic tissues and their spatial segregation from the pluripotent epiblast. During this period two cell fate decisions are made while cells gradually lose their totipotency. The first fate decision involves the segregation of the extraembryonic trophectoderm (TE) lineage from the inner cell mass (ICM); the second occurs within the ICM and involves the segregation of the extraembryonic primitive endoderm (PrE) lineage from the pluripotent epiblast (EPI) lineage, which eventually gives rise to the embryo proper. Multiple determinants, such as differential cellular properties, signaling cues and the activity of transcriptional regulators, influence lineage choice in the early embryo. Here, we provide an overview of our current understanding of the mechanisms governing these cell fate decisions ensuring proper lineage allocation and segregation, while at the same time providing the embryo with an inherent flexibility to adjust when perturbed.

FGF4 is required for lineage restriction and salt-and-pepper distribution of primitive endoderm factors but not their initial expression in the mouse.

The emergence of pluripotent epiblast (EPI) and primitive endoderm (PrE) lineages within the inner cell mass (ICM) of the mouse blastocyst involves initial co-expression of lineage-associated markers followed by mutual exclusion and salt-and-pepper distribution of lineage-biased cells. Precisely how EPI and PrE cell fate commitment occurs is not entirely clear; however, previous studies in mice have implicated FGF/ERK signaling in this process. Here, we investigated the phenotype resulting from zygotic and maternal/zygotic inactivation of Fgf4. Fgf4 heterozygous blastocysts exhibited increased numbers of NANOG-positive EPI cells and reduced numbers of GATA6-positive PrE cells, suggesting that FGF signaling is tightly regulated to ensure specification of the appropriate numbers of cells for each lineage. Although the size of the ICM was unaffected in Fgf4 null mutant embryos, it entirely lacked a PrE layer and exclusively comprised NANOG-expressing cells at the time of implantation. An initial period of widespread EPI and PrE marker co-expression was however established even in the absence of FGF4. Thus, Fgf4 mutant embryos initiated the PrE program but exhibited defects in its restriction phase, when lineage bias is acquired. Consistent with this, XEN cells could be derived from Fgf4 mutant embryos in which PrE had been restored and these cells appeared indistinguishable from wild-type cells. Sustained exogenous FGF failed to rescue the mutant phenotype. Instead, depending on concentration, we noted no effect or conversion of all ICM cells to GATA6-positive PrE. We propose that heterogeneities in the availability of FGF produce the salt-and-pepper distribution of lineage-biased cells.

Differential plasticity of epiblast and primitive endoderm precursors within the ICM of the early mouse embryo.

Cell differentiation during pre-implantation mammalian development involves the formation of two extra-embryonic lineages: trophoblast and primitive endoderm (PrE). A subset of cells within the inner cell mass (ICM) of the blastocyst does not respond to differentiation signals and forms the pluripotent epiblast, which gives rise to all of the tissues in the adult body. How this group of cells is set aside remains unknown. Recent studies documented distinct sequential phases of marker expression during the segregation of epiblast and PrE within the ICM. However, the connection between marker expression and lineage commitment remains unclear. Using a fluorescent reporter for PrE, we investigated the plasticity of epiblast and PrE precursors. Our observations reveal that loss of plasticity does not coincide directly with lineage restriction of epiblast and PrE markers, but rather with exclusion of the pluripotency marker Oct4 from the PrE. We note that individual ICM cells can contribute to all three lineages of the blastocyst until peri-implantation. However, epiblast precursors exhibit less plasticity than precursors of PrE, probably owing to differences in responsiveness to extracellular signalling. We therefore propose that the early embryo environment restricts the fate choice of epiblast but not PrE precursors, thus ensuring the formation and preservation of the pluripotent foetal lineage.

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.

Ex utero culture and live imaging of mouse embryos.

Mouse genetic approaches when combined with live imaging tools have the potential to revolutionize our current understanding of mammalian biology. The availability and improvement of a wide variety of fluorescent proteins have provided indispensable tools to visualize cells in living organisms. It is now possible to generate genetically modified mouse strains expressing fluorescent proteins in a tissue-specific manner. These reporter-expressing strains make it possible to image dynamic cell behaviors in the context of a living embryo. Since mouse embryos develop within the uterus, live imaging experiments require culture conditions that closely mimic those in vivo. Over the past few decades, significant advances have been made in developing conditions for culturing both pre- and postimplantation stage embryos. In this chapter, we will discuss methods for ex utero culture of preimplantation and postimplantation stage mouse embryos. In particular, we will describe protocols for collecting embryos at various stages, setting up culture conditions for imaging and using laser scanning confocal microscopy to visualize live processes in mouse embryos expressing fluorescent reporters.

The primitive endoderm lineage of the mouse blastocyst: sequential transcription factor activation and regulation of differentiation by Sox17.

Cells of the primitive endoderm (PrE) and the pluripotent epiblast (EPI), the two lineages specified within the inner cell mass (ICM) of the mouse blastocyst stage embryo, are segregated into adjacent tissue layers by the end of the preimplantation period. The PrE layer which emerges as a polarized epithelium adjacent to the blastocoel, with a basement membrane separating it from the EPI, has two derivatives, the visceral and parietal endoderm. In this study we have investigated the localization of two transcriptional regulators of the SOX family, SOX17 and SOX7, within the PrE and its derivatives. We noted that SOX17 was first detected in a salt-and-pepper distribution within the ICM, subsequently becoming restricted to the nascent PrE epithelium. This dynamic distribution of SOX17 resembled the localization of GATA6 and GATA4, two other PrE lineage-specific transcription factors. By contrast, SOX7 was only detected in PrE cells positioned in contact with the blastocoel, raising the possibility that these cells are molecularly distinct. Our observations support a model of sequential GATA6 > SOX17 > GATA4 > SOX7 transcription factor activation within the PrE lineage, perhaps correlating with the consecutive periods of cell lineage 'naïvete', commitment and sorting. Furthermore our data suggest that co-expression of SOX17 and SOX7 within sorted PrE cells could account for the absence of a detectable phenotype of Sox17 mutant blastocysts. However, analysis of implantation-delayed blastocysts, revealed a role for SOX17 in the maintenance of PrE epithelial integrity, with the absence of SOX17 leading to premature delamination and migration of parietal endoderm.

A sensitive and bright single-cell resolution live imaging reporter of Wnt/ß-catenin signaling in the mouse.

BACKGROUND: Understanding the dynamic cellular behaviors and underlying molecular mechanisms that drive morphogenesis is an ongoing challenge in biology. Live imaging provides the necessary methodology to unravel the synergistic and stereotypical cell and molecular events that shape the embryo. Genetically-encoded reporters represent an essential tool for live imaging. Reporter strains can be engineered by placing cis-regulatory elements of interest to direct the expression of a desired reporter gene. In the case of canonical Wnt signaling, also referred to as Wnt/ß-catenin signaling, since the downstream transcriptional response is well understood, reporters can be designed that reflect sites of active Wnt signaling, as opposed to sites of gene transcription, as is the case with many fluorescent reporters. However, even though several transgenic Wnt/ß-catenin reporter strains have been generated, to date, none provides the single-cell resolution favored for live imaging studies. RESULTS: We have placed six copies of a TCF/Lef responsive element and an hsp68 minimal promoter in front of a fluorescent protein fusion comprising human histone H2B to GFP and used it to generate a strain of mice that would report Wnt/ß-catenin signaling activity. Characterization of developmental and adult stages of the resulting TCF/Lef:H2B-GFP strain revealed discrete and specific expression of the transgene at previously characterized sites of Wnt/ß-catenin signaling. In support of the increased sensitivity of the TCF/Lef:H2B-GFP reporter, additional sites of Wnt/ß-catenin signaling not documented with other reporters but identified through genetic and embryological analysis were observed. Furthermore, the sub-cellular localization of the reporter minimized reporter perdurance, and allowed visualization and tracking of individual cells within a cohort, so facilitating the detailed analysis of cell behaviors and signaling activity during morphogenesis. CONCLUSION: By combining the Wnt activity read-out efficiency of multimerized TCF/Lef DNA binding sites, together with the high-resolution imaging afforded by subcellularly-localized fluorescent fusion proteins such as H2B-GFP, we have created a mouse transgenic line that faithfully recapitulates Wnt signaling activity at single-cell resolution. The TCF/Lef:H2B-GFP reporter represents a unique tool for live imaging the in vivo processes triggered by Wnt/ß-catenin signaling, and thus should help the formulation of a high-resolution understanding of the serial events that define the morphogenetic process regulated by this signaling pathway.

Distinct sequential cell behaviours direct primitive endoderm formation in the mouse blastocyst.

The first two lineages to differentiate from a pluripotent cell population during mammalian development are the extraembryonic trophectoderm (TE) and the primitive endoderm (PrE). Whereas the mechanisms of TE specification have been extensively studied, segregation of PrE and the pluripotent epiblast (EPI) has received comparatively little attention. A current model of PrE specification suggests PrE precursors exhibit an apparently random distribution within the inner cell mass of the early blastocyst and then segregate to their final position lining the cavity by the late blastocyst. We have identified platelet-derived growth factor receptor alpha (Pdgfralpha) as an early-expressed protein that is also a marker of the later PrE lineage. By combining live imaging of embryos expressing a histone H2B-GFP fusion protein reporter under the control of Pdgfra regulatory elements with the analysis of lineage-specific markers, we investigated the events leading to PrE and EPI lineage segregation in the mouse, and correlated our findings using an embryo staging system based on total cell number. Before blastocyst formation, lineage-specific factors are expressed in an overlapping manner. Subsequently, a gradual progression towards a mutually exclusive expression of PrE- and EPI-specific markers occurs. Finally, cell sorting is achieved by a variety of cell behaviours and by selective apoptosis.

Foetal fibroblasts introduced to cleaving mouse embryos contribute to full-term development.

Foetal fibroblasts (FFs) labelled with vital fluorescent dye were microsurgically introduced into eight-cell mouse embryos, three cells to each embryo. FFs were first identified in the inner cell mass (ICM) in about one-third of embryos, whereas in three quarters of embryos FFs were located among trophoblast cells. Some elimination of FFs from trophoblast occurred later on. Eventually, in blastocysts' outgrowths, an equally high contribution from FFs progeny (60%) was found in both ICM and trophoblast. Three days after manipulation, FFs resumed proliferation in vitro. More than three FFs were found in 46.2% of embryos on day 4. On the 7th day in vitro in 70% of embryos more than 12 FFs were found, proving at least three cell divisions. To study postimplantation development, the embryos with FFs were transferred to pseudopregnant recipients a day after manipulation. After implantation, FFs were identified by electrophoresis for isozymes of glucose phosphate isomerase (GPI). A single 11-day embryo delayed to day 8 proved chimeric by expressing both donor isozyme GPI-1B and recipient GPI-1A. Similar chimerism was found in the extraembryonic lineage of 11% of embryos by day 12. Starting from day 11 onwards, in 32% of normal embryos and in 57% of foetal membranes, hybrid GPI-1AB isozyme, as well as recipient isozyme, was present. Hybrid GPI-1AB can only be produced in hybrid cells derived by cell fusion, therefore, we suggest that during postimplantation development, FFs are rescued by fusion with recipient cells. In the mice born, hybrid isozyme was found in several tissues, including brain, lung, gut and kidney. We conclude that somatic cells (FFs) can proliferate in early embryonic environment until early postimplantation stages. Foetuses and the mice born are chimeras between recipient cells and hybrid cells with contributions from the donor FFs. Transdifferentiation as opposed to reprogramming by cell fusion can be considered as underlying cellular processes in these chimeras.