Erk2 signaling and early embryo stem cell self-renewal

Differentiation of the mammalian blastocyst generates two distinct cell lineages: the trophectoderm, which contributes to the trophoblast layers of the placenta, and the inner cell mass, which forms the embryo. We and others recently demonstrated that the MAP kinase ERK2 is essential for trophoblast development. Erk2 mutant embryos fail to form extra-embryonic ectoderm and the ectoplacental cone, suggesting a role for ERK2 activation in the proliferation of trophoblast stem (TS) cells. Previous studies have documented that ERK1/2 activity is dispensable for proliferation of embryonic stem (ES) cells and rather interferes with self-renewal. Thus, signaling by the ERK1/2 MAP kinase pathway appears to be critical for the regulation of self-renewal and propagation of early embryo stem cell populations.     

An essential function of the mitogen-activated protein kinase Erk2 in mouse trophoblast development

The closely related mitogen-activated protein kinase isoforms extracellular signal-regulated kinase 1 (ERK1) and ERK2 have been implicated in the control of cell proliferation, differentiation and survival. However, the specific in vivo functions of the two ERK isoforms remain to be analysed. Here, we show that disruption of the Erk2 locus leads to embryonic lethality early in mouse development after the implantation stage. Erk2 mutant embryos fail to form the ectoplacental cone and extra-embryonic ectoderm, which give rise to mature trophoblast derivatives in the fetus. Analysis of chimeric embryos showed that Erk2 functions in a cell-autonomous manner during the development of extra-embryonic cell lineages. We also found that both Erk2 and Erk1 are widely expressed throughout early-stage embryos. The inability of Erk1 to compensate for Erk2 function suggests a specific function for Erk2 in normal trophoblast development in the mouse, probably in regulating the proliferation of polar trophectoderm cells.     

The possible dual origin of the ectoderm of the chorion in the mouse embryo.

The origin of the extraembryonic ectoderm of the chorion in the mouse embryo has long been the source of some controversy. Various manipulative studies suggested that it arose from the trophectoderm and not the inner cell mass (ICM) of the blastocyst. However, recent studies on the development of isolated ICMs in vitro have reported the formation of tissues morphologically resembling extraembryonic ectoderm. One explanation not excluded by previous studies is that the chorionic ectoderm is of dual origin, from both ICM and trophectoderm. The present study provides a more detailed analysis than previously possible of the in vivo fate of ICMs in chimeras, using a sensitive assay for glucose phosphate isomerase (GPI) isozymes which permits study of the chorionic ectoderm alone. In a large series of blastocyst injection chimeras, no donor ICM contribution to the mature chorionic ectoderm could be detected, donor activity appearing only in the embryonic fraction. Thus, the in vitro results cannot be readily explained by dual origin of the chorionic ectoderm and remain in conflict with existing in vivo data. Analysis of most ICM/morula chimeras revealed the same pattern, but a few showed ICM contributions to the trophoblast fractions, suggesting that some ICM cells retain the potential to form trophectoderm derivatives in vivo.     

Phagocytosis reveals a reversible differentiated state early in the development of the mouse embryo

Mural trophectoderm cells of the mouse embryo possess a phagocytic potential as early as 3.5 days post coitum (d.p.c.). This first differentiated function shows a graded variation along the embryonic-abembryonic axis, from a maximal activity in the non-dividing cells of the abembryonic pole to a complete lack of activity in the replicating polar trophectoderm overlying the inner cell mass (ICM). This pattern can be explained by a negative control exerted by the ICM. Addition of FGF4, a factor secreted by ICM cells, strongly inhibited phagocytosis while inducing resumption of DNA synthesis in mural trophectoderm cells, revealing a reversible, FGF4-dependent differentiation state. Under conditions in which a small cluster of mural trophectoderm cells (<10) had internalized large particles, these otherwise morphologically normal embryos could not implant in the uterus, indicating that cells at the abembryonic pole have a critical role in initiating the implantation process. At post-implantation stages (6.5-8.5 d.p.c.), the ectoplacental cone and secondary giant cells derived from the polar trophectoderm also contained active phagocytes, but at that stage, differentiation was not reversed by FGF4.     

Expression of Cre recombinase in early diploid trophoblast cells of the mouse placenta

An increasing number of genes known to be critical for cell cycle control, differentiation, and tumor suppression have been found to impact development of the placenta. To elucidate how these genes contribute to development of embryonic and extra-embryonic lineages, we generated a transgenic mouse in which the Cre transgene is driven by placenta-specific regulatory sequences from the human CYP19 gene. Using ROSA26 conditional reporter mice, we could detect expression of the CYP19-Cre transgene throughout the extra-embryonic ectoderm and in the ectoplacental cone at embryonic day 6.5 (E6.5). By E11.5, recombination of LoxP reporter sites was detected in all derivatives of trophoblast stem cells, including spongiotrophoblast, giant cells, and labyrinth trophoblasts. We conclude that the CYP19-Cre transgenic mouse developed here can be used in combination with conditional alleles to distinguish between embryonic and extra-embryonic gene function, and to begin to map the period of time when gene function is critical during development.     

Control of trophectoderm differentiation by inner cell mass-derived fibroblast growth factor-4 in mouse blastocysts and corrective effect of FGF-4 on high glucose-induced trophoblast disruption

Previous studies have suggested that fibroblast growth factor-4 (FGF-4) may be a paracrine signal used by inner cell mass (ICM) cells to maintain adjacent trophectoderm (TE) cells in an undifferentiated state. In the present work, immunocytochemical analysis of mouse blastocysts confirmed that FGF-4 was predominantly detected in the ICM before and after spreading over a fibronectin-coated culture substrate. Addition of human recombinant FGF-4 did not influence morphological progression, cell allocation and proliferation in ICM and TE lineages or mitosis and karyorhexis frequencies during blastocyst expansion. Addition of FGF-4 to outgrowing blastocysts, in contrast, induced a significant decrease in the surface of the trophoblast outgrowths formed by the TE cells and in the proportion of giant trophoblasts per outgrowth. The fact that blastocysts display excessive trophoblast expansion and spreading over their culture substrate upon pre-exposure to high concentrations of glucose in vitro was used to further assess the regulatory effect of FGF-4. Addition of FGF-4 was indeed found to fully neutralize the disruptive impact of high glucose on trophoblast outgrowths. Altogether, our data indicate that ICM-derived FGF-4 participates actively in the regulation of trophoblast development.     

The effects of cell size and ploidy on cell allocation in mouse chimaeric blastocysts

In a previous study of mouse tetraploid<-->diploid chimaeric blastocysts, tetraploid cells were found to be more abundant in the trophectoderm than the inner cell mass (ICM) and more abundant in the mural trophectoderm than the polar trophectoderm. This non-random allocation of tetraploid cells to different regions of the chimaeric blastocyst may contribute to the restricted tissue distribution seen in post-implantation stage tetraploid<-->diploid chimaeras. However, the tetraploid and diploid embryos that were aggregated together differed in several respects: the tetraploid embryos had fewer cells and these cells were bigger and differed in ploidy. Each of these factors might underlie a non-random allocation of tetraploid cells to the chimaeric blastocyst. A combination of micromanipulation and electrofusion was used to produce two series of chimaeras that distinguished between the effects of cell size and ploidy on the allocation of cells to different tissues in chimaeric blastocysts. When aggregated cells differed in cell size but not ploidy, the derivatives of the larger cell contributed significantly more to the mural trophectoderm and polar trophectoderm than the ICM. When aggregated cells differed in ploidy but not cell size, the tetraploid cells contributed significantly more to the mural trophectoderm than the ICM. In both experiments the contributions to the polar trophectoderm tended to be intermediate between those of the mural trophectoderm and ICM. These experiments show that both the larger size and increased ploidy of tetraploid cells could have contributed to the non-random cell distribution that was observed in a previous study of tetraploid<-->diploid chimaeric blastocysts.     

Developmental changes in the ploidy of mouse implanting trophoblast cells in vitro

Shortly after the onset of implantation, polar mouse trophoblast cells proliferate and give rise to the ectoplacental cone, constituted by two distinct cell populations: undifferentiated, diploid cells and giant cells. Giant cells characteristically exhibit exaggerated dimensions and polyploid nuclei. In this study, we employ ectoplacental cones as a dynamic source of trophoblast giant cells to analyze cell proliferation, cell death, and ploidy under in vitro conditions. Our results show that DNA synthesis and the increase in the cell number are relevant only during the first 24 h of culture. Subsequently, DNA synthesis still occurs, mainly in the giant cell compartment, while the number of cells gradually decreases. Cell death by injury and apoptosis was also observed in the non-giant cell compartment of the ectoplacental cone. These findings suggest that the first 24 h of culture are crucial to the mitotic activity of the ectoplacental cone cells that gradually ceases, favoring the endoreduplication process. The DNA synthesis index during the subsequent experimental intervals emphasizes accumulation of DNA for the polyploidization. There was clear correlation between DNA content and nuclear dimension. The ploidy values for the trophoblast giant cells varied from 2C up to 368C in the giant cells, but were not as expressive as those known from in vivo conditions, probably due to the absence of regulatory factors specific to the embryonic-maternal interface. In situ hybridization and histochemistry for the nucleolus-organizing region showed that trophoblast nuclei have only two marker signals, indicative of a typical polytenic process. This present study elucidates important aspects of trophoblast behavior and provides new information on trophoblast physiology in vivo and in vitro.     

Fate of the inner cell mass in mouse embryos as studied by microinjection of lineage tracers

We microinjected horseradish peroxidase and rhodamine-conjugated dextran into single inner cell mass (ICM) cells of preimplantation mouse embryos to study their fate in culture. Simultaneous iontophoresis of both lineage markers allowed immediate localization of the injected cell by epifluorescence, followed by microdrop culture of individual embryos. After 24 hr in culture, labeled descendants were found in the polar trophectoderm, ICM, and parietal endoderm, providing direct evidence that the ICM contributes descendants to the trophectoderm and the endoderm in the intact mouse embryo. Our results substantiate the totipotency of the ICM during the expanding blastocyst stage and further demonstrate that the ICM is a stem cell population from which cells are recruited into these tissue lineages during growth of the blastocyst.     

Amino acids and preimplantation development of the mouse in protein-free potassium simplex optimized medium

Development of outbred CF1 mouse zygotes in vitro was studied in a chemically defined, protein-free medium both with and without amino acids. The addition of amino acids to protein-free potassium simplex optimized medium (KSOM) had little effect on the proportion of embryos that developed at least to the zona-enclosed blastocyst stage. In contrast, amino acids stimulated very significantly, in a dilution-dependent way, the proportion of blastocysts that at least partially or completely hatched. Amino acids also stimulated cell proliferation in both the trophectoderm and inner cell mass (ICM) cells, at rates that favored proliferation of cells in the ICM; had no effect on the incidence of cell death (oncosis or apoptosis); and improved development of the basement membranes, which form on the blastocoelic surface of the trophectoderm and between the primitive endoderm and the primitive ectoderm. Thus, KSOM, supplemented with amino acids but containing no protein supplements, supports development of a newly fertilized ovum to the late blastocyst stage, in which its normal, three-dimensional structure is preserved and in which the ICM has been partitioned into the primitive ectoderm and primitive endoderm.     

Potential of isolated mouse inner cell masses to form trophectoderm derivatives in vivo

Recent in vitro experiments on immunosurgically isolated mouse inner cell masses (ICMs) have suggested that some ICM cells may retain the potential to form trophectoderm after initial blastocyst formation. These experiments relied almost solely on in vitro morphology for identification of trophectoderm derivatives and provided no proof that the putative trophectoderm cells were capable of functioning in utero. We present clear in vivo evidence that at least some cells in ICMs isolated from early blastocysts do retain the potential to form postimplantation trophectoderm derivatives. Early ICMs occasionally contributed to trophoblast fractions in ICM/morula aggregation chimeras. More strikingly, when aggregated with each other, these ICMs were capable of implanting in the uterus, a property of trophectoderm cells alone. Indeed, some aggregates reconstituted complete egg cylinders. However, ICMs isolated from later blastocysts rarely produced in vivo trophoblast, and it appears that the ability to form trophectoderm is lost around the 16–19 cell ICM stage. These results are discussed in relation to changing patterns of gene activity in early development.     

Integrity of the preimplantation pig blastocyst during expansion and loss of polar trophectoderm (Rauber cells) and the morphology of the embryoblast as an indicator for developmental stage

The embryonic ectoderm of the pig differentiated and became part of the outer barrier of the blastocyst (earlier formed by the trophectoderm alone) before shedding of the overlying polar trophectoderm around Day 10, thus securing the integrity of the rapidly expanding blastocyst. Ferritin, added to the medium of the blastocyst, was taken up rapidly by trophectoderm cells, but did not reach the blastocoele, and consequently no tracer was found within hypoblast cells. Embryonic ectoderm cells did not absorb the macromolecule, before or after loss of the polar trophectoderm. When ferritin was injected into the blastocoele, trophectoderm, hypoblast and embryoblast cells all absorbed the tracer. At Day 11, blastocyst diameter and embryoblast cell number varied widely and were hardly correlated. We suggest that embryoblast development may be a more reliable indicator for the developmental stage of a blastocyst than its diameter, which may merely be an indication of the viability of the trophoblast.     

Multiple developmental roles of Ahnak are suggested by localization to sites of placentation and neural plate fusion in the mouse conceptus

Ahnak is a gigantic (700 kD) phosphoprotein with a unique structure whose expression and cellular localization are dynamically regulated during cell cycle progression. Here, we report that Ahnak is localized to sites of major morphogenesis during mouse placentation and neurulation. Ahnak was found in: (i) derivatives of trophectoderm, including chorionic ectoderm prior to and during union with the ectoplacental cone, presumptive syncytiotrophoblast cells in the chorionic labyrinth, and giant cells at the trophoblast-uterine interface; (ii) the allantois prior to, during, and after union with the chorion; and (iii) the tips of the neural plate during formation of the neural tube. On the basis of these observations, we suggest that Ahnak may play heretofore unrecognized roles in tissue union during normal mouse development.     

Multiple developmental roles of Ahnak are suggested by localization to sites of placentation and neural plate fusion in the mouse conceptus

Ahnak is a gigantic (700 kD) phosphoprotein with a unique structure whose expression and cellular localization are dynamically regulated during cell cycle progression. Here, we report that Ahnak is localized to sites of major morphogenesis during mouse placentation and neurulation. Ahnak was found in: (i) derivatives of trophectoderm, including chorionic ectoderm prior to and during union with the ectoplacental cone, presumptive syncytiotrophoblast cells in the chorionic labyrinth, and giant cells at the trophoblast-uterine interface; (ii) the allantois prior to, during, and after union with the chorion; and (iii) the tips of the neural plate during formation of the neural tube. On the basis of these observations, we suggest that Ahnak may play heretofore unrecognized roles in tissue union during normal mouse development.     

Expression of mouse PDGF-A and PDGF alpha-receptor genes during pre- and post-implantation development: evidence for a developmental shift from an autocrine to a paracrine mode of action.

We examined the expression of platelet-derived growth factor (PDGF)-A and the PDGF alpha-receptor in pre-implantation and early post-implantation mouse embryos. At two-cell and blastocyst stages, all cells express mRNA and protein for both ligand and receptor. In contrast, early post-implantation embryos express PDGF-A chain mRNA in both embryonic ectoderm and in the ectoderm lining the ectoplacental cavity, while mRNA for PDGF alpha-receptor is localized to the mesoderm layers of both embryonic and extra-embryonic membranes. At days 3.5 and 7.5, receptors are demonstrably functional in response to exogenous PDGF-AA. We propose that chronic autostimulation of PDGF alpha-receptors occurs in pre-implantation embryos, whereas, following implantation, early mesoderm development is dependent on stimulation by ectodermally produced PDGF-A.