Active cell movements coupled to positional induction are involved in lineage segregation in the mouse blastocyst

In the mouse blastocyst, some cells of the inner cell mass (ICM) develop into primitive endoderm (PE) at the surface, while deeper cells form the epiblast. It remained unclear whether the position of cells determines their fate, such that gene expression is adjusted to cell position, or if cells are pre-specified at random positions and then sort. We have tracked and characterised dynamics of all ICM cells from the early to late blastocyst stage. Time-lapse microscopy in H2B-EGFP embryos shows that a large proportion of ICM cells change position between the surface and deeper compartments. Most of this cell movement depends on actin and is associated with cell protrusions. We also find that while most cells are precursors for only one lineage, some give rise to both, indicating that lineage segregation is not complete in the early ICM. Finally, changing the expression levels of the PE marker Gata6 reveals that it is required in surface cells but not sufficient for the re-positioning of deeper cells. We provide evidence that Wnt9A, known to be expressed in the surface ICM, facilitates re-positioning of Gata6-expressing cells. Combining these experimental results with computer modelling suggests that PE formation involves both cell sorting movements and position-dependent induction.     

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.     

Roles of ERα during mouse trophectoderm lineage differentiation: revealed by antagonist and agonist of ERα

During mouse early embryogenesis, blastomeres increase in number by the morula stage. Among them, the outer cells are polarized and differentiated into trophectoderm (TE), while the inner cells remain unpolarized and give rise to inner cell mass (ICM). TE provides an important liquid environment for ICM development. In spite of extensive research, the molecular mechanisms underlying TE formation are still obscure. In order to investigate the roles of estrogen receptor α (ERα) in this course, mouse 8‐cell embryos were collected and cultured in media containing ERα specific antagonist MPP and/or agonist PPT. The results indicated that MPP treatment inhibits blastocyst formation in a dose‐dependent manner, while PPT, at proper concentration, promotes the cavitation ratio of mouse embryos. Immunofluorescence staining results showed that MPP significantly decreased the nuclear expression of CDX2 in morula, but no significant changes of OCT4 were observed. Moreover, after MPP treatment, the expression levels of the genes related to TE specification, Tead4, Gata3 and Cdx2, were significantly reduced. Overall, these results indicated that ERα might affect mouse embryo cavitation by regulating TE lineage differentiation.     

CDX2 in the formation of the trophectoderm lineage in primate embryos

The first lineage decision during mammalian development is the establishment of the trophectoderm (TE) and the inner cell mass (ICM). The caudal-type homeodomain protein Cdx2 is implicated in the formation and maintenance of the TE in the mouse. However, the role of CDX2 during early embryonic development in primates is unknown. Here, we demonstrated that CDX2 mRNA levels were detectable in rhesus monkey oocytes, significantly upregulated in pronuclear stage zygotes, diminished in early cleaving embryos but restored again in compact morula and blastocyst stages. CDX2 protein was localized to the nucleus of TE cells but absent altogether in the ICM. Knockdown of CDX2 in monkey oocytes resulted in formation of early blastocyst-like embryos that failed to expand and ceased development. However, the ICM lineage of CDX2-deficient embryos supported the isolation of functional embryonic stem cells. These results provide evidence that CDX2 plays an essential role in functional TE formation during primate embryonic development.     

BRG1 Governs Nanog Transcription in Early Mouse Embryos and Embryonic Stem Cells via Antagonism of Histone H3 Lysine 9/14 Acetylation

During mouse preimplantation development, the generation of the inner cell mass (ICM) and trophoblast lineages comprises upregulation of Nanog expression in the ICM and its silencing in the trophoblast. However, the underlying epigenetic mechanisms that differentially regulate Nanog in the first cell lineages are poorly understood. Here, we report that BRG1 (Brahma-related gene 1) cooperates with histone deacetylase 1 (HDAC1) to regulate Nanog expression. BRG1 depletion in preimplantation embryos and Cdx2-inducible embryonic stem cells (ESCs) revealed that BRG1 is necessary for Nanog silencing in the trophoblast lineage. Conversely, in undifferentiated ESCs, loss of BRG1 augmented Nanog expression. Analysis of histone H3 within the Nanog proximal enhancer revealed that H3 lysine 9/14 (H3K9/14) acetylation increased in BRG1-depleted embryos and ESCs. Biochemical studies demonstrated that HDAC1 was present in BRG1-BAF155 complexes and BRG1-HDAC1 interactions were enriched in the trophoblast lineage. HDAC1 inhibition triggered an increase in H3K9/14 acetylation and a corresponding rise in Nanog mRNA and protein, phenocopying BRG1 knockdown embryos and ESCs. Lastly, nucleosome-mapping experiments revealed that BRG1 is indispensable for nucleosome remodeling at the Nanog enhancer during trophoblast development. In summary, our data suggest that BRG1 governs Nanog expression via a dual mechanism involving histone deacetylation and nucleosome remodeling.     

Allocation of inner cells to epiblast vs primitive endoderm in the mouse embryo is biased but not determined by the round of asymmetric divisions (8→16- and 16→32-cells)

The epiblast (EPI) and the primitive endoderm (PE), which constitute foundations for the future embryo body and yolk sac, build respectively deep and surface layers of the inner cell mass (ICM) of the blastocyst. Before reaching their target localization within the ICM, the PE and EPI precursor cells, which display distinct lineage-specific markers, are intermingled randomly. Since the ICM cells are produced in two successive rounds of asymmetric divisions at the 8→16 (primary inner cells) and 16→32 cell stage (secondary inner cells) it has been suggested that the fate of inner cells (decision to become EPI or PE) may depend on the time of their origin. Our method of dual labeling of embryos allowed us to distinguish between primary and secondary inner cells contributing ultimately to ICM. Our results show that the presence of two generations of inner cells in the 32-cell stage embryo is the source of heterogeneity within the ICM. We found some bias concerning the level of Fgf4 and Fgfr2 expression between primary and secondary inner cells, resulting from the distinct number of cells expressing these genes. Analysis of experimental aggregates constructed using different ratios of inner cells surrounded by outer cells revealed that the fate of cells does not depend exclusively on the timing of their generation, but also on the number of cells generated in each wave of asymmetric division. Taking together, the observed regulatory mechanism adjusting the proportion of outer to inner cells within the embryo may be mediated by FGF signaling.     

BMP4 plays a role in apoptosis during human preimplantation development

Comprehensive understanding of lineage differentiation and apoptosis processes is important to increase our knowledge of human preimplantation development in vitro. We know that BMP signaling is important for different processes during mammalian development. In mouse preimplantation embryos, BMP signaling has been shown to play a role in the differentiation into extra‐embryonic trophectoderm (TE) and primitive endoderm (PE). In this study, we aimed to investigate the effect of bone morphogenetic protein 4 (BMP4) supplementation on human preimplantation embryos cultured in vitro. The BMP4 treatment impaired human blastocyst formation. No differences in the expression of the early lineage markers NANOG, CDX2, GATA3, and GATA6 were found between BMP4‐treated embryos and controls. Instead, BMP4 supplementation triggered apoptosis in the human blastocyst. We focused on P53, which is known to play a major role in the apoptosis. In BMP4‐treated embryos, the P53 responsive gene expression was not altered; however, the P53 deacetylase SIRT1 was downregulated and acetylated P53 was increased in mitochondria. Altogether, our findings suggest that BMP4 plays a role in the apoptosis during human preimplantation development.     

Individual blastomeres of 16- and 32-cell mouse embryos are able to develop into foetuses and mice

Cell and developmental studies have clarified how, by the time of implantation, the mouse embryo forms three primary cell lineages: epiblast (EPI), primitive endoderm (PE), and trophectoderm (TE). However, it still remains unknown when cells allocated to these three lineages become determined in their developmental fate. To address this question, we studied the developmental potential of single blastomeres derived from 16- and 32-cell stage embryos and supported by carrier, tetraploid blastomeres. We were able to generate singletons, identical twins, triplets, and quadruplets from individual inner and outer cells of 16-cell embryos and, sporadically, foetuses from single cells of 32-cell embryos. The use of embryos constitutively expressing GFP as the donors of single diploid blastomeres enabled us to identify their cell progeny in the constructed 2n↔4n blastocysts. We showed that the descendants of donor blastomeres were able to locate themselves in all three first cell lineages, i.e., epiblast, primitive endoderm, and trophectoderm. In addition, the application of Cdx2 and Gata4 markers for trophectoderm and primitive endoderm, respectively, showed that the expression of these two genes in the descendants of donor blastomeres was either down- or up-regulated, depending on the cell lineage they happened to occupy. Thus, our results demonstrate that up to the early blastocysts stage, the destiny of at least some blastomeres, although they have begun to express markers of different lineage, is still labile.     

Expression of fibroblast growth factor receptors in peri-implantation mouse embryos

FGF receptor (FGFR) function is essential during peri-implantation mouse development. To understand which receptors are functioning, we tested for the expression of all four FGF receptors in peri-implantation blastocysts. By RT-PCR, FGFR-3 and FGFR-4 were detected at high levels, FGFR-2 at lower levels, and FGFR-1 was detected at background levels compared to control tissues. Because FGFR-3 and FGFR-4 were detected at the highest levels, we studied these in detail. Between 3.5 days after fertilization (E3.5) and E6.0, FGFR-4 mRNA was detected ubiquitously in the peri-implantation embryo, restricted to the inner cell mass (ICM) and its derivatives and primitive endoderm by E6.0, and was not detected at E6.5. FGFR-3 mRNA was detected ubiquitously in the peri-implantation embryo with a tendency towards extraembryonic cells. We tested blastocyst outgrowths, a model for implantation, for FGFR-3 and FGFR-4 protein. FGFR-3 protein was detected in all cells early during the outgrowth. Later, FGFR-3 was detected in the extraembryonic endoderm and trophoblast giant cells (TGC), but not in the ICM. FGFR-4 protein was detected in all cells of the implanting embryo, but was restricted to the ICM/primitive endoderm in later stage outgrowths. The distribution of the receptor proteins in the blastocyst outgrowths is similar to the distribution of the mRNA detected by in situ hybridization of sections of embryos. The data suggest roles for FGFR-3 and FGFR-4 in peri-implantation development. Mol. Reprod. Dev. 51:254–264, 1998. © 1998 Wiley-Liss, Inc.     

Trophoblast-specific DNA methylation occurs after the segregation of the trophectoderm and inner cell mass in the mouse periimplantation embryo

The first cell differentiation in the mammalian development separates the trophoblast and embryonic cell lineages, resulting in the formation of the trophectoderm (TE) and inner cell mass (ICM) in blastocysts. Although a lower level of global DNA methylation in the genome of the TE compared with ICM has been suggested, the dynamics of the DNA methylation profile during TE/ICM differentiation has not been elucidated. To address this issue, first we identified tissue-dependent and differentially methylated regions (T-DMRs) between trophoblast stem (TS) and embryonic stem (ES) cells. Most of these TS–ES T-DMRs were also methylated differentially between trophoblast and embryonic tissues of embryonic day (E) 6.5 mouse embryos. Furthermore, we found that the human genomic regions homologous to mouse TS–ES T-DMRs were methylated differentially between human placental tissues and ES cells. Collectively, we defined them as cell-lineage-based T-DMRs between trophoblast and embryonic cell lineages (T–E T-DMRs). Then, we examined TE and ICM cells isolated from mouse E3.5 blastocysts. Interestingly, all T-DMRs examined, including the Elf5, Pou5f1 and Nanog loci, were in the nearly unmethylated status in both TE and ICM and exhibited no differences. The present results suggest that the establishment of DNA methylation profiles specific to each cell lineage follows the first morphological specification. Together with previous reports on asymmetry of histone modifications between TE and ICM, the results of the current study imply that histone modifications function as landmarks for setting up cell-lineage-specific differential DNA methylation profiles.     

Trichostatin A-treated eight-cell bovine embryos had increased histone acetylation and gene expression, with increased cell numbers at the blastocyst stage

The objective was to determine the effects of trichostatin A (TSA), a potent histone deacetylase inhibitor, on eight-cell bovine embryos. That treatment increased histone acetylation was confirmed by immunostaining with anti-AcH4K5 and AcH4K8 antibodies. Embryos treated with TSA (100 nM) for various intervals (4, 8, and 12 h) developed to the blastocyst stage as frequently as untreated embryos (average development rate, 49.5%). Treatment with TSA for 12 h increased (P < 0.05) the numbers of inner cell mass (ICM) cells and total cells (TC), as well as the ICM/TC ratio in the blastocyst, but the number of cells in the trophectoderm decreased (P < 0.05). Treated embryos had increased relative abundance (RA) of OCT3/4 and E-CADHERIN mRNA relative to controls at the morula stage (P < 0.05), however, the RA of CDX2 mRNA was unchanged. In conclusion, TSA-treated eight-cell stage embryos had increased histone acetylation and gene expression, which increased ICM and TC numbers and the ICM/TC ratio, but significantly decreased the number of cells in the trophectoderm of resulting blastocysts.     

DDX21 is a p38-MAPK-sensitive nucleolar protein necessary for mouse preimplantation embryo development and cell-fate specification

Successful navigation of the mouse preimplantation stages of development, during which three distinct blastocyst lineages are derived, represents a prerequisite for continued development. We previously identified a role for p38-mitogen-activated kinases (p38-MAPK) regulating blastocyst inner cell mass (ICM) cell fate, specifically primitive endoderm (PrE) differentiation, that is intimately linked to rRNA precursor processing, polysome formation and protein translation regulation. Here, we develop this work by assaying the role of DEAD-box RNA helicase 21 (DDX21), a known regulator of rRNA processing, in the context of p38-MAPK regulation of preimplantation mouse embryo development. We show nuclear DDX21 protein is robustly expressed from the 16-cell stage, becoming exclusively nucleolar during blastocyst maturation, a localization dependent on active p38-MAPK. siRNA-mediated clonal Ddx21 knockdown within developing embryos is associated with profound cell-autonomous and non-autonomous proliferation defects and reduced blastocyst volume, by the equivalent peri-implantation blastocyst stage. Moreover, ICM residing Ddx21 knockdown clones express the EPI marker NANOG but rarely express the PrE differentiation marker GATA4. These data contribute further significance to the emerging importance of lineage-specific translation regulation, as identified for p38-MAPK, during mouse preimplantation development.