SOX17 Is Not Required for the Derivation and Maintenance of Mouse Extraembryonic Endoderm Stem Cell Lines

Extraembryonic endoderm stem (XEN) cell lines can be derived and maintained in vitro and reflect the primitive endoderm cell lineage. SOX17 is thought to be required for the derivation and maintenance of mouse XEN cell lines. Here we have re-evaluated this requirement for SOX17. We derived multiple SOX17-deficient XEN cell lines from preimplantation embryos of a SOX17-Cre knockout strain and chemically converted multiple SOX17-deficient embryonic stem cell lines into XEN cell lines by transient culturing with retinoic acid and Activin A. We confirmed the XEN profile of SOX17-deficient cell lines by immunofluorescence with various markers, by NanoString gene expression analyses, and by their contribution to the extraembryonic endoderm of chimeric embryos produced by injecting these cells into blastocysts. Thus, SOX17 is not required for the derivation and maintenance of XEN cell lines.

The origin of mouse extraembryonic endoderm stem cell lines

Mouse extraembryonic endoderm stem (XEN) cell lines can be derived from preimplantation embryos (pre-XEN) and postimplantation embryos (post-XEN). XEN cells share a gene expression profile and cell lineage potential with primitive endoderm (PrE) blastocysts. However, the cellular origin of XEN cells in embryos remains unclear. Here, we report that post-XEN cell lines are derived both from the extraembryonic endoderm and epiblasts of postimplantation embryos and that pre-XEN cell lines are derived both from PrE and epiblasts of blastocysts. Our strategy consisted of deriving post-XEN cells from clumps of epiblasts, parietal endoderm (PE) and visceral endoderm (VE) and deriving pre-XEN cell lines from single PrE and single epiblasts of blastocysts. Thus, XEN cell lines in the mouse embryo originate not only from PrE and PrE-derived lineages but also from epiblast and epiblast-derived lineages of blastocysts and postimplantation embryos.

Leukemia Inhibitory Factor Stimulates Primitive Endoderm Expansion in the Bovine Inner Cell Mass

Previous work determined that bovine interleukin-6 (IL6) increases inner cell mass (ICM), primitive endoderm (PE), and total cell number in in vitro produced (IVP) bovine blastocysts. Another IL6 family member, leukemia inhibitory factor (LIF), has the potential to produce the same effects of IL6 due to the presence of its receptor in bovine blastocysts. We compared the abilities of LIF and IL6 to increase ICM cell numbers in day 7, 8, and 9 IVP bovine blastocysts. Supplementation with 100 ng/ml LIF from day 5 onward improved blastocyst formation rates on days 7 and 8 similar to what was observed when supplementing 100 ng/ml IL6. However, LIF supplementation did not cause an increase in ICM numbers like was observed after supplementing IL6. On day 9, increases in PE cell numbers were detected after LIF supplementation, but 300 ng/ml LIF was required to achieve the same effect on PE numbers that was observed by providing 100 ng/ml IL6. Collectively, these results show that LIF can mimic at least some of the effects of IL6 in bovine blastocyst.

ECM-integrin signalling instructs cellular position-sensing to pattern the early mouse embryo

Development entails patterned emergence of diverse cell types within the embryo. In mammals, cells positioned inside the embryo give rise to the inner cell mass (ICM) that eventually forms the embryo proper. Yet the molecular basis of how these cells recognise their ‘inside’ position to instruct their fate is unknown. Here we show that provision of extracellular matrix (ECM) to isolated embryonic cells induces ICM specification and alters subsequent spatial arrangement between epiblast (EPI) and primitive endoderm (PrE) cells that emerge within the ICM. Notably, this effect is dependent on integrin β1 activity and involves apical to basal conversion of cell polarity. We demonstrate that ECM-integrin activity is sufficient for ‘inside’ positional signalling and it is required for proper EPI/PrE patterning. Our findings thus highlight the significance of ECM-integrin adhesion in enabling position-sensing by cells to achieve tissue patterning.

Vacuolar-type proton ATPase is required for maintenance of apicobasal polarity of embryonic visceral endoderm

The endocytic compartments keep their interior acidic through the inward flow of protons and anions from the cytosol. Acidification is mediated by a proton pump known as vacuolar-type ATPase (V-ATPase) and transporters conferring anion conductance to the organellar membrane. In this study, we analysed the phenotype of mouse embryos lacking the V-ATPase c-subunit. The mutant embryos differentiated embryonic epithelial tissues, primitive endoderm, epiblast, and extraembryonic ectoderm; however, the organisation of these epithelia was severely affected. The apical-basal polarity in the visceral endoderm layer was not properly established in the mutant embryos, resulting in abnormal epithelial morphology. Thus, the function of V-ATPase is imperative for the establishment and/or maintenance of epithelial cell polarity, which is required for early embryogenesis.

ECM-integrin signalling instructs cellular position-sensing to pattern the early mouse embryo

Development entails patterned emergence of diverse cell types within the embryo. In mammals, cells positioned inside the embryo gives rise to the inner cell mass (ICM) that eventually forms the embryo proper. Yet the molecular basis of how these cells recognise their inside position to instruct their fate is unknown. Here we show that cells perceive their position through extracellular matrix (ECM) and integrin-mediated adhesion. Provision of ECM to isolated embryonic cells induces ICM specification and alters subsequent spatial arrangement between epiblast (EPI) and primitive endoderm (PrE) cells that emerge within the ICM. Notably, this effect is dependent on integrin β 1 activity and involves apical to basal conversion of cell polarity. We demonstrate that ECM-integrin activity is sufficient for inside positional signalling and it is required for proper sorting of EPI/PrE cells. Our findings thus highlight the significance of ECM-integrin adhesion in enabling position-sensing by cells to achieve tissue patterning.

O-169 Conventional ovarian stimulation with gonadotropins depletes the developmental proteome of mouse oocytes, reducing their size and compromising their fetal yield

Study question Does conventional ovarian stimulation with gonadotropins alter the molecular composition, size, and developmental fitness of superovulated mouse oocytes? Summary answer Ovarian stimulation perturbs 31% of developmental proteome vs. 2% of transcriptome, yielding smaller oocytes that form blastocysts with less primitive endoderm and diminished fetal yield. What is known already Prior mouse studies aimed to assess the impact of superovulation on oocyte and embryo quality provided variable results. Aberrations were observed in some studies but not in others, and were associated with a variable time spent in the oviduct until retrieval for in vitro culture. Although the natural ovarian cycle lasts 4 days in mice, the conventional stimulation protocol time spans 2 days. No genome-wide studies have been devoted yet to the global gene expression of superovulated mouse oocytes and derivative embryos, to determine if gene products accumulate to the same extent as in natural cycles. Study design, size, duration Approx. 1100 female mice were injected, half with serial equine and human chorionic gonadotropin, eCG and hCG, to induce superovulation; the other half were injected with saline as control. Both groups were mated to vasectomized or fertile males to obtain, respectively, metaphase II and fertilized oocytes. These were removed from the oviducts, and followed up in vitro to blastocyst, or in vivo to term after surgical transfer to naturally cycling females. Participants/materials, setting, methods B6C3F1 oocytes (n= ∼16000) from superovulation (10 I.U. eCG+hCG) or natural ovulation were measured for diameter, and upon fertilization, they were cultured in KSOM(aa) to collect preimplantation stages for analyses (mass spectrometry; RNA sequencing; immunofluorescence for counting of trophectoderm, epiblast, and primitive endoderm cells). Embryos at the 4-cell stage were transplanted to naturally cycling females (8/female, 45 recipients). Results were compared between natural ovulation and superovulation with 48h (conventional) or 72h interval of eCG-hCG stimulation. Main results and the role of chance Preimplantation embryos of superovulated oocytes were affected in 31% of the proteins (893 / 2855) vs. 2% of the transcripts (482 / 21784), compared to natural counterparts (adj.P<0.05, Wilcoxon test). Gene set enrichment analysis of the perturbed proteome returned the top-terms ‘thin zona pellucida’ (ZP1, ZP2, ZP3) and ‘abnormal inner cell mass apoptosis’ (DAB2, STAT3). Microscope measurements verified a thinner zona pellucida (p = 0.077, Wilcoxon test) along with a smaller diameter (p < 0.0001, Wilcoxon test) of superovulated oocytes, which gave rise to blastocysts deficient in primitive endoderm (p < 0.013, Fisher’s exact test). Since 529 of the 893 differently expressed proteins were depleted, we considered that ovarian stimulation provided insufficient time for protein accumulation. Increasing the eCG-hCG interval from 48 h (conventional) to 72 h restored oocytes’ diameters, and improved their fetal output from 25% to 59%, compared to 54% of natural ovulation (15 embryo transfers per group). Conversely, oocytes lost part of their developmental potential to the micromanipulation-assisted reduction of their volume. This study provides evidence of an additional novel effect of exposure to gonadotropins on mouse oocyte quality, whose mechanism is mediated not by the stimulated genital tract, but by the time-dependent accumulation of proteins in oocytes. Limitations, reasons for caution This is an animal model study based on one mouse strain. Ovarian stimulation protocols differ between mice and humans. There can be yet other, more subtle or long-term differences between superovulated and naturally ovulated oocytes, than those described here. Proteome and transcriptome analysis cover much, but not everything. Wider implications of the findings There is a trade-off between oocyte quantity and quality in mice subjected to superovulation. Cytological and molecular deficits define a ‘small oocyte syndrome’. Problematic is not so much the gonadotropin treatment, rather its timing. An evidence-based protocol for superovulation may be different from that used currently in mice. Trial registration number Not applicable

p38-MAPK-mediated translation regulation during early blastocyst development is required for primitive endoderm differentiation in mice

Successful specification of the two mouse blastocyst inner cell mass (ICM) lineages (the primitive endoderm (PrE) and epiblast) is a prerequisite for continued development and requires active fibroblast growth factor 4 (FGF4) signaling. Previously, we identified a role for p38 mitogen-activated protein kinases (p38-MAPKs) during PrE differentiation, but the underlying mechanisms have remained unresolved. Here, we report an early blastocyst window of p38-MAPK activity that is required to regulate ribosome-related gene expression, rRNA precursor processing, polysome formation and protein translation. We show that p38-MAPK inhibition-induced PrE phenotypes can be partially rescued by activating the translational regulator mTOR. However, similar PrE phenotypes associated with extracellular signal-regulated kinase (ERK) pathway inhibition targeting active FGF4 signaling are not affected by mTOR activation. These data indicate a specific role for p38-MAPKs in providing a permissive translational environment during mouse blastocyst PrE differentiation that is distinct from classically reported FGF4-based mechanisms.

H3K9 tri-methylation at Nanog times differentiation commitment and enables the acquisition of primitive endoderm fate

Mouse Embryonic Stem (ES) cells have an inherent propensity to explore distinct gene-regulatory states associated with either self-renewal or differentiation. This property is largely dependent on ERK activity, which promotes silencing of pluripotency genes, most notably of the transcription factor Nanog. Here, we aimed at identifying repressive histone modifications that would mark the Nanog locus for inactivation in response to ERK activity. We found histone H3 lysine 9 tri-methylation (H3K9me3) focally enriched between the Nanog promoter and its -5kb enhancer. While in undifferentiated ES cells H3K9me3 at Nanog depends on ERK activity, in somatic cells it becomes ERK-independent. Moreover, upon deletion of the region harbouring H3K9me3, ES cells display reduced heterogeneity of NANOG expression, delayed commitment into differentiation and impaired ability to acquire a primitive endoderm fate. We suggest that establishment of irreversible H3K9me3 at specific master regulators allows the acquisition of particular cell fates during differentiation.

Totipotency of mouse zygotes extends to single blastomeres of embryos at the four-cell stage

In multicellular organisms, oocytes and sperm undergo fusion during fertilization and the resulting zygote gives rise to a new individual. The ability of zygotes to produce a fully formed individual from a single cell when placed in a supportive environment is known as totipotency. Given that totipotent cells are the source of all multicellular organisms, a better understanding of totipotency may have a wide-ranging impact on biology. The precise delineation of totipotent cells in mammals has remained elusive, however, although zygotes and single blastomeres of embryos at the two-cell stage have been thought to be the only totipotent cells in mice. We now show that a single blastomere of two- or four-cell mouse embryos can give rise to a fertile adult when placed in a uterus, even though blastomere isolation disturbs the transcriptome of derived embryos. Single blastomeres isolated from embryos at the eight-cell or morula stages and cultured in vitro manifested pronounced defects in the formation of epiblast and primitive endoderm by the inner cell mass and in the development of blastocysts, respectively. Our results thus indicate that totipotency of mouse zygotes extends to single blastomeres of embryos at the four-cell stage.