FGF4 is a limiting factor controlling the proportions of primitive endoderm and epiblast in the ICM of the mouse blastocyst

The primitive endoderm (PE) and epiblast (EPI) are two lineages derived from the inner cell mass (ICM) of the E3.5 blastocyst. Although it has been shown that FGF signaling is necessary and sufficient for PE specification in the ICM, it is unknown what mechanisms control the PE/EPI proportion in the embryo. Because modulation of FGF signaling alone is sufficient to convert all ICM cells to either PE or EPI, a model has been proposed in which the amount of FGF in the embryo controls the PE/EPI proportion. To test this model, we reduced the amount of FGF4, the major FGF in the preimplantation embryo, using various genotypes of Fgf4 mutants. We observed a maternal contribution of Fgf4 in PE specification, but it was dispensable for development. In addition, upon treatment of Fgf4 mutant embryos with exogenous FGF4, we observed a progressive increase of PE proportions in an FGF4 dose dependent manner, regardless of embryo genotype. We conclude that the amount of FGF4 is limited and regulates PE/EPI proportions in the mouse embryo.     

The dual roles of geminin during trophoblast proliferation and differentiation

Geminin is a protein involved in both DNA replication and cell fate acquisition. Although it is essential for mammalian preimplantation development, its role remains unclear. In one study, ablation of the geminin gene (Gmnn) in mouse preimplantation embryos resulted in apoptosis, suggesting that geminin prevents DNA re-replication, whereas in another study it resulted in differentiation of blastomeres into trophoblast giant cells (TGCs), suggesting that geminin regulates trophoblast specification and differentiation. Other studies concluded that trophoblast differentiation into TGCs is regulated by fibroblast growth factor-4 (FGF4), and that geminin is required to maintain endocycles. Here we show that ablation of Gmnn in trophoblast stem cells (TSCs) proliferating in the presence of FGF4 closely mimics the events triggered by FGF4 deprivation: arrest of cell proliferation, formation of giant cells, excessive DNA replication in the absence of DNA damage and apoptosis, and changes in gene expression that include loss of Chk1 with up-regulation of p57 and p21. Moreover, FGF4 deprivation of TSCs reduces geminin to a basal level that is required for maintaining endocycles in TGCs. Thus, geminin acts both like a component of the FGF4 signal transduction pathway that governs trophoblast proliferation and differentiation, and geminin is required to maintain endocycles.     

Signaling pathways and preimplantation development of mammalian embryos

The mammalian preimplantation embryo is a critical and unique stage in embryonic development. This stage includes a series of crucial events: the transition from oocyte to embryo, the first cell divisions, and the establishment of cellular contacts. These events are regulated by multiple signal-transduction pathways. In this article we describe patterns of stage-specific expression in several signal-transduction pathways and try to give a profile of the signaling transduction network in preimplantation development of mammalian embryo.     

Changes in requirements and utilization of nutrients during mammalian preimplantation embryo development and their significance in embryo culture

Along with the transition from maternal to embryonic genome control the mammalian preimplantation embryo undergoes significant changes in its physiology during development. Concomitant with these changes are altering patterns of nutrient uptake and differences in the subsequent fate of such nutrients. The most significant nutrients to the developing mammalian preimplantation embryo are carbohydrates and amino acids, which serve not only to provide energy but also to maintain embryo function by preventing cellular stress induced by suboptimal culture conditions in vitro. It is subsequently proposed that optimal development of the mammalian embryo in culture requires the use of two or more media, each designed to cater for the changing requirements of the embryo. Importantly, culture conditions that maintain the early embryo are not ideal for the embryo post-compaction, and conditions that support excellent development and differentiation of the blastocyst can actually be inhibitory to the zygote. A marker of in vitro-induced cellular stress to the embryo is the relative activity of the metabolic pathways used to generate energy for development. Quantification of embryo energy metabolism may therefore serve as a valuable marker of embryo development and viability.     

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.     

Automated microinjection of recombinant BCL-X into mouse zygotes enhances embryo development

Progression of fertilized mammalian oocytes through cleavage, blastocyst formation and implantation depends on successful implementation of the developmental program, which becomes established during oogenesis. The identification of ooplasmic factors, which are responsible for successful embryo development, is thus crucial in designing possible molecular therapies for infertility intervention. However, systematic evaluation of molecular targets has been hampered by the lack of techniques for efficient delivery of molecules into embryos. We have developed an automated robotic microinjection system for delivering cell impermeable compounds into preimplantation embryos with a high post-injection survival rate. In this paper, we report the performance of the system on microinjection of mouse embryos. Furthermore, using this system we provide the first evidence that recombinant BCL-XL (recBCL-XL) protein is effective in preventing early embryo arrest imposed by suboptimal culture environment. We demonstrate that microinjection of recBCL-XL protein into early-stage embryos repairs mitochondrial bioenergetics, prevents reactive oxygen species (ROS) accumulation, and enhances preimplantation embryo development. This approach may lead to a possible treatment option for patients with repeated in vitro fertilization (IVF) failure due to poor embryo quality.     

Cytoskeleton in preimplantation mouse development

This paper reviews the constituents of the cytoskeleton in the cells of the preimplantation mouse embryo and how they change as the development proceeds. The cytoskeleton can be divided into two distinct groups, that in the cytosplasm and that associated with the membrane. The first and better-known group contains microfilaments, microtubules and intermediate filaments, the second such components of the cell and nuclear membrane as spectrin-like protein and nuclear lamin. The filamentous components of the cytoplasmic cytoskeleton adhere to the nuclear and cell membrane at attachment points where specific proteins such as vinculin may mediate the interaction. Each cell of the early embryo has all of these components, but their morphological organization and molecular constitution alter as the embryo develops. These modifications are especially pronounced when the cleavage-stage embryo compacts and when the blastocysts forms and differentiates. These events represent the most critical stages of morphogenesis and cytodifferentiation in the preimplantation embryo. The cytoskeleton may thus have an important role in the control of the early mammalian development.     

Regulation of proliferation and apoptosis during development of the preimplantation embryo and the placenta

The preimplantation embryo starts as a single cell, the zygote. The first cell divisions do not lead to volume expansion, but rather to an increasing number of small cells. At the morula stage the first two cell lineages differentiate into the trophoblast and the inner cells mass/embryoblast. During development of the preimplantation embryo, apoptosis occurs only after the onset of the embryonic genome. It has become clear that the development of a healthy child requires not only very high rates of proliferation and differentiation, but also apoptosis, which is a crucial mechanism for morphogenesis and the development of the inner organs. Furthermore, the generation of specific cell types, such as lens cells, erythrocytes, and thrombocytes, depends on the apoptosis pathways. This is also true later in gestation, when the trophoblasts form the placenta and provide the epithelial cover of the villous trees of the placenta. This layer is in direct contact with maternal blood and, as do all epithelia, displays a continuous turnover of cells. Thus, apoptosis is a normal constituent of survival in this layer as well, and changes in the regulation and rate of apoptosis have deleterious effects on the trophoblast and consequently the developing embryo or fetus. Here we present a very brief overview of the importance of apoptosis for the development of the preimplantation embryo and the maintenance of placental trophoblasts. Furthermore, we highlight what happens when regulation of proliferation or apoptosis fails in these systems, and attempt to show that apoptosis is only the consequence of poor embryo or trophoblast development -- not its cause.     

Implication of nucleolar protein SURF6 in ribosome biogenesis and preimplantation mouse development

The step-wise assembly of a functional nucleolus, which occurs over the first few cell cycles during preimplantation development, is poorly understood. In this study, we examined the function of the evolutionary conserved nucleolar protein SURF6 in preimplantation mouse embryo development. Immunocytochemical analyses revealed that the localization of SURF6 was similar but not identical to those of fibrillarin and B23/nucleophosmin 1, which are involved in rRNA processing and ribosome biogenesis in mammalian somatic cells. Surf6 mRNA, which is expressed in oocytes and maternally inherited in the zygote, reached a peak level of expression during the 8-cell stage of embryo development, at which time rDNA is highly transcribed. Knock-down of Surf6 mRNA by RNAi led to a decrease in both the mRNA and protein levels, and resulted in developmental arrest at the 8-cell/morula stage, as well as a decrease in the level of 18S rRNA. These results suggest that Surf6 is essential for mouse preimplantation development, presumably by regulating ribosome biogenesis.     

Characterization of Trophoblast and Extraembryonic Endoderm Cell Lineages Derived from Rat Preimplantation Embryos

Background

Previous attempts to isolate pluripotent cell lines from rat preimplantation embryo in mouse embryonic stem (ES) cell culture conditions (serum and LIF) were unsuccessful, however the resulting cells exhibited the expression of such traditional pluripotency markers as SSEA-1 and alkaline phosphatase. We addressed the question, which kind of cell lineages are produced from rat preimplantation embryo under “classical” mouse ES conditions.

Results

We characterized two cell lines (C5 and B10) which were obtained from rat blastocysts in medium with serum and LIF. In the B10 cell line we found the expression of genes known to be expressed in trophoblast, Cdx-2, cytokeratin-7, and Hand-1. Also, B10 cells invaded the trophectodermal layer upon injection into rat blastocysts. In contrast to mouse Trophoblast Stem (TS) cells proliferation of B10 cells occurred independently of FGF4. Cells of the C5 line expressed traditional markers of extraembryonic-endoderm (XEN) cells, in particular, GATA-4, but also the pluripotency markers SSEA-1 and Oct-4. C5 cell proliferation exhibited dependence on LIF, which is not known to be required by mouse XEN cells.

Conclusions

Our results confirm and extend previous findings about differences between blastocyst-derived cell lines of rat and mice. Our data show, that the B10 cell line represents a population of FGF4-independent rat TS-like cells. C5 cells show features that have recently become known as characteristic of rat XEN cells. Early passages of C5 and B10 cells contained both, TS and XEN cells. We speculate, that mechanisms maintaining self-renewal of cell lineages in rat preimplantation embryo and their in vitro counterparts, including ES, TS and XEN cells are different than in respective mouse lineages.     

Beyond the centrosome: The mystery of microtubule organising centres across mammalian preimplantation embryos

Mammalian preimplantation embryogenesis depends on the spatio-temporal dynamics of the microtubule cytoskeleton to enable exceptionally fast changes in cell number, function, architecture, and fate. Microtubule organising centres (MTOCs), which coordinate the remodelling of microtubules, are therefore of fundamental significance during the first days of a new life. Despite its indispensable role during early mammalian embryogenesis, the origin of microtubule growth remains poorly understood.In this review, we summarise the most recent discoveries on microtubule organisation and function during early human embryogenesis and compare these to innovative studies conducted in alternative mammalian models. We emphasise the differences and analogies of centriole inheritance and their role during the first cleavage. Furthermore, we highlight the significance of non-centrosomal MTOCs for embryo viability and discuss the potential of novel in vitro models and light-inducible approaches towards unravelling microtubule formation in research and assisted reproductive technologies.     

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.     

Simultaneous analysis of chromosomes and chromosome-associated proteins in mammalian oocytes and embryos

Cytogenetic analyses of mammalian eggs and preimplantation embryos have been limited by the difficult and tedious task of preparing chromosomes from single cells or small numbers of cells. In this report we describe a new technique that is both reliable and comparatively simple. Further, since the technique does not use the conventional 3:1 methanol:acetic acid fixative, it has the advantage of producing high-resolution chromosome preparations without destroying chromosome-associated proteins. Thus, this method provides a sensitive means of conducting studies of a heretofore inaccessible period of mammalian development, and of studying proteins thought to mediate both meiotic chromosome segregation and chromatin modifications in the preimplantation embryo.     

Laser Fusion of Mouse Embryonic Cells and Intra-Embryonic Fusion of Blastomeres without Affecting the Embryo Integrity

Manipulation with early mammalian embryos is the one of the most important approach to study preimplantation development. Artificial cell fusion is a research tool for various biotechnological experiments. However, the existing methods have various disadvantages, first of them impossibility to fuse selected cells within multicellular structures like mammalian preimplantation embryos. In our experiments we have successfully used high repetition rate picosecond near infrared laser beam for fusion of pairs of oocytes and oocytes with blastomeres. Fused cells looked morphologically normal and keep their ability for further divisions in vitro. We also fused two or three blastomeres inside four-cell mouse embryos. The presence of one, two or three nuclei in different blastomeres of the same early preimplantation mouse embryo was confirmed under UV-light after staining of DNA with the vital dye Hoechst-33342. The most of established embryos demonstrated high viability and developed in vitro to the blastocyst stage. We demonstrated for the first time the use of laser beam for the fusion of various embryonic cells of different size and of two or three blastomeres inside of four-cell mouse embryos without affecting the embryo’s integrity and viability. These embryos with blastomeres of various ploidy maybe unique model for numerous purposes. Thus, we propose laser optical manipulation as a new tool for investigation of fundamental mechanisms of mammalian development.     

FGF ligand family mRNA expression profile for mouse preimplantation embryos, early gestation human placenta, and mouse trophoblast stem cells

Signaling by fibroblast growth factor (FGF) is essential is for trophoblast stem (TS) cells and preimplantation embryos. FGF4 provides essential signaling, but the expression of the complete set of 23 FGF family members has not been analyzed. Here, semi-quantitative RT-PCR and microarray analyses were used to define expression of all FGF ligand mRNA. RT-PCR was done for developmentally important FGF subfamilies, FGF10/FGF22 and FGF8/FGF17/FGF18 as well as FGF11. FGF4 and FGF18 are detected at highest levels by RT-PCR and microarrays. FGF10 was detected at low levels in both assays. FGF11 was detected at moderate levels by microarray, but not by RT-PCR. FGF17 was detected at low levels by array and moderate levels by RT-PCR. FGF8 and FGF22 were detected by RT-PCR, but not by microarrays during late cleavage divisions. FGF8, FGF5, and FGF9 were detected in the oocyte by microarray. FGF2, FGF3, and FGF7 were not detected by RT-PCR or microarrays and FGF13, FGF14, and FGF23 were not detected by microarray. Since a major role of FGF is to maintain TS cells, we tested human and mouse placental cell lines and early gestation human placenta for expression of FGF ligands. Expression in mouse TS cells was compared with preimplantation embryos, and human placental cell line expression was compared with human placenta, to infer which ligands are expressed in placental lineage vs. other cell lineages. The data suggest that human and mouse placenta share FGF18 and its high expression suggests preimplantation and early placental function.     

The role of FGF signaling in guiding coordinate movement of cell groups

Cell migration influences cell-cell interactions to drive cell differentiation and organogenesis. To support proper development, cell migration must be regulated both temporally and spatially. Mesoderm cell migration in the Drosophila embryo serves as an excellent model system to study how cell migration is controlled and influences organogenesis. First, mesoderm spreading transforms the embryo into a multilayered form during gastrulation and, subsequently, cells originating from the caudal visceral mesoderm (CVM) migrate along the entire length of the gut. Here we review our studies, which have focused on the role of fibroblast growth factor (FGF) signaling, and compare and contrast these two different cell migration processes: mesoderm spreading and CVM migration. In both cases, FGF acts as a chemoattractant to guide cells’ directional movement but is likely not the only signal that serves this role. Furthermore, FGF likely modulates cell adhesion properties since FGF mutant phenotypes share similarities with those of cell adhesion molecules. Our working hypothesis is that levels of FGF signaling differentially influence cells’ response to result in either directional movement or changes in adhesive properties.