Primitive Endoderm Lineages Research Articles

PI3K/AKT signaling controls ICM maturation and proper epiblast and primitive endoderm specification in mice.

The inner cell mass (ICM) of early mouse embryos is specified into epiblast (Epi) and primitive endoderm (PrE) lineages during blastocyst formation. The antagonistic transcription factors (TFs) NANOG and GATA-binding protein 6 (GATA6) in combination with fibroblast growth factor (FGF)/extracellular-signal-regulated kinase (ERK) signaling are central actors in ICM fate choice. However, what initiates the specification of ICM progenitors into Epi or PrE and whether other factors are involved in this process has not been fully understood yet. Here, we show that phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) is constitutively active during preimplantation development. Using pharmacological inhibition, we demonstrate that PI3K/AKT enables the formation of a functional ICM capable of giving rise to both the Epi and the PrE: it maintains the expression of the TF NANOG, which specifies the Epi, and confers responsiveness to FGF4, which is essential for PrE specification. Our work thus identifies PI3K/AKT signaling as an upstream regulator controlling the molecular events required for both Epi and PrE specification.

Loss of KANSL3 leads to defective inner cell mass and early embryonic lethality.

e-Lysine acetylation is a prominent histone mark found at transcriptionally active loci. Among many lysine acetyl transferases, nonspecific lethal complex (NSL) members are known to mediate the modification of histone H4. In addition to histone modifications, the KAT8 regulatory complex subunit 3 gene (Kansl3), a core member of NSL complex, has been shown to be involved in several other cellular processes such as mitosis and mitochondrial activity. Although functional studies have been performed on NSL complex members, none of the four core proteins, including Kansl3, have been studied during early mouse development. Here we show that homozygous knockout Kansl3 embryos are lethal at peri-implantation stages, failing to hatch out of the zona pellucida. When the zona pellucida is removed in vitro, Kansl3 null embryos form an abnormal outgrowth with significantly disrupted inner cell mass (ICM) morphology. We document lineage-specific defects at the blastocyst stage with significantly reduced ICM cell number but no difference in trophectoderm cell numbers. Both epiblast and primitive endoderm lineages are altered with reduced cell numbers in null mutants. These results show that Kansl3 is indispensable during early mouse embryonic development and with defects in both ICM and trophectoderm lineages.

Evidence for Functional Roles of MicroRNAs in Lineage Specification During Mouse and Human Preimplantation Development.

Proper formation of the blastocyst, including the specification of the first embryonic cellular lineages, is required to ensure healthy embryo development and can significantly impact the success of assisted reproductive technologies (ARTs). However, the regulatory role of microRNAs in early development, particularly in the context of preimplantation lineage specification, remains largely unknown. Taking a cross-species approach, this review aims to summarize the expression dynamics and functional significance of microRNAs in the differentiation and maintenance of lineage identity in both the mouse and the human. Findings are consolidated from studies conducted using in vitro embryonic stem cell models representing the epiblast, trophectoderm, and primitive endoderm lineages (modeled by naïve embryonic stem cells, trophoblast stem cells, and extraembryonic endoderm stem cells, respectively) to provide insight on what may be occurring in the embryo. Additionally, studies directly conducted in both mouse and human embryos are discussed, emphasizing similarities to the stem cell models and the gaps in our understanding, which will hopefully lead to further investigation of these areas. By unraveling the intricate mechanisms by which microRNAs regulate the specification and maintenance of cellular lineages in the blastocyst, we can leverage this knowledge to further optimize stem cell-based models such as the blastoids, enhance embryo competence, and develop methods of non-invasive embryo selection, which can potentially increase the success rates of assisted reproductive technologies and improve the experiences of those receiving fertility treatments.

TimeTalk uses single-cell RNA-seq datasets to decipher cell-cell communication during early embryo development

Early embryonic development is a dynamic process that relies on proper cell-cell communication to form a correctly patterned embryo. Early embryo development-related ligand-receptor pairs (eLRs) have been shown to guide cell fate decisions and morphogenesis. However, the scope of eLRs and their influence on early embryo development remain elusive. Here, we developed a computational framework named TimeTalk from integrated public time-course mouse scRNA-seq datasets to decipher the secret of eLRs. Extensive validations and analyses were performed to ensure the involvement of identified eLRs in early embryo development. Process analysis identified that eLRs could be divided into six temporal windows corresponding to sequential events in the early embryo development process. With the interpolation strategy, TimeTalk is powerful in revealing paracrine settings and studying cell-cell communication during early embryo development. Furthermore, by using TimeTalk in the blastocyst and blastoid models, we found that the blastoid models share the core communication pathways with the epiblast and primitive endoderm lineages in the blastocysts. This result suggests that TimeTalk has transferability to other bio-dynamic processes. We also curated eLRs recognized by TimeTalk, which may provide valuable clues for understanding early embryo development and relevant disorders.

Localization of embryonic stem cells lacking E-cadherin in a mouse blastocyst

Abstract Here we present our results regarding the role of E-cadherin (CDH1, cadherin 1) in the specification of embryonic stem cells (ESCs) in the embryo environment. It has been previously shown that, when forming embryoid bodies with wild-type ESCs, ESCs lacking a functional copy of the gene encoding the adhesive protein E-cadherin (Ecad−/−) preferably sort out to the outside compartment, thus forming the primitive endoderm (PrE) lineage. However, little or no information is available regarding the dynamics of Ecad−/− cells in the actual blastocyst, and so the aim of this work was to determine in which of the three blastocyst cell lines - trophoblast (TE), epiblast (Epi), or PrE - Ecad−/− cells would be located in the embryo environment. For this purpose we injected ESCs into embryos at various stages of pre-implantation development. We used a H2B-GFP cell line expressing histone H2B conjugated with green fluorescent protein (H2BEGFP), as well as a Ecad−/− cell line, in which cells exhibit weaker adhesive properties than wild-type Esc because of their allelic deficiency in the locus encoding cadherin CDH1. We have demonstrated that these cells exhibit a trend to locate in the TE and much less frequently in the Epi, but never in the PrE. We propose that this may be due to differences in the expression of genes characteristic of these cell lines within the Ecad−/− cell colony.

Influence of FGF4 and BMP4 on FGFR2 dynamics during the segregation of epiblast and primitive endoderm cells in the pre-implantation mouse embryo.

Specification of the epiblast (EPI) and primitive endoderm (PE) in the mouse embryo involves fibroblast growth factor (FGF) signaling through the RAS/MAP kinase pathway. FGFR1 and FGFR2 are thought to mediate this signaling in the inner cell mass (ICM) of the mouse blastocyst and BMP signaling can also influence PE specification. In this study, we further explored the dynamics of FGFR2 expression through an enhanced green fluorescent protein (eGFP) reporter mouse line (FGFR2-eGFP). We observed that FGFR2-eGFP is present in the late 8-cell stage; however, it is absent or reduced in the ICM of early blastocysts. We then statistically correlated eGFP expression with PE and EPI markers GATA6 and NANOG, respectively. We detected that eGFP is weakly correlated with GATA6 in early blastocysts, but this correlation quickly increases as the blastocyst develops. The correlation between eGFP and NANOG decreases throughout blastocyst development. Treatment with FGF from the morula stage onwards did not affect FGFR2-eGFP presence in the ICM of early blastocysts; however, late blastocysts presented FGFR2-eGFP in all cells of the ICM. BMP treatment positively influenced FGFR2-eGFP expression and reduced the number of NANOG-positive cells in late blastocysts. In conclusion, FGFR2 is not strongly associated with PE precursors in the early blastocyst, but it is highly correlated with PE cells as blastocyst development progresses, consistent with the proposed role for FGFR2 in maintenance rather than initiating the PE lineage.

Transcriptional network governing extraembryonic endoderm cell fate choice

The mechanism by which transcription factor (TF) network instructs cell-type-specific transcriptional programs to drive primitive endoderm (PrE) progenitors to commit to parietal endoderm (PE) versus visceral endoderm (VE) cell fates remains poorly understood. To address the question, we analyzed the single-cell transcriptional signatures defining PrE, PE, and VE cell states during the onset of the PE-VE lineage bifurcation. By coupling with the epigenomic comparison of active enhancers unique to PE and VE cells, we identified GATA6, SOX17, and FOXA2 as central regulators for the lineage divergence. Transcriptomic analysis of cXEN cells, an in vitro model for PE cells, after the acute depletion of GATA6 or SOX17 demonstrated that these factors induce Mycn, imparting the self-renewal properties of PE cells. Concurrently, they suppress the VE gene program, including key genes like Hnf4a and Ttr, among others. We proceeded with RNA-seq analysis on cXEN cells with FOXA2 knockout, in conjunction with GATA6 or SOX17 depletion. We found FOXA2 acts as a potent suppressor of Mycn while simultaneously activating the VE gene program. The antagonistic gene regulatory activities of GATA6/SOX17 and FOXA2 in promoting alternative cell fates, and their physical co-bindings at the enhancers provide molecular insights to the plasticity of the PrE lineage. Finally, we show that the external cue, BMP signaling, promotes the VE cell fate by activation of VE TFs and repression of PE TFs including GATA6 and SOX17. These data reveal a putative core gene regulatory module that underpins PE and VE cell fate choice.

The Wnt/TCF7L1 transcriptional repressor axis drives primitive endoderm formation by antagonizing naive and formative pluripotency

Early during preimplantation development and in heterogeneous mouse embryonic stem cells (mESC) culture, pluripotent cells are specified towards either the primed epiblast or the primitive endoderm (PE) lineage. Canonical Wnt signaling is crucial for safeguarding naive pluripotency and embryo implantation, yet the role and relevance of canonical Wnt inhibition during early mammalian development remains unknown. Here, we demonstrate that transcriptional repression exerted by Wnt/TCF7L1 promotes PE differentiation of mESCs and in preimplantation inner cell mass. Time-series RNA sequencing and promoter occupancy data reveal that TCF7L1 binds and represses genes encoding essential naive pluripotency factors and indispensable regulators of the formative pluripotency program, including Otx2 and Lef1. Consequently, TCF7L1 promotes pluripotency exit and suppresses epiblast lineage formation, thereby driving cells into PE specification. Conversely, TCF7L1 is required for PE specification as deletion of Tcf7l1 abrogates PE differentiation without restraining epiblast priming. Taken together, our study underscores the importance of transcriptional Wnt inhibition in regulating lineage specification in ESCs and preimplantation embryo development as well as identifies TCF7L1 as key regulator of this process.

Chemical conversion of human conventional PSCs to TSCs following transient naive gene activation

In human embryos, naive pluripotent cells of the inner cell mass (ICM) generate epiblast, primitive endoderm and trophectoderm (TE) lineages, whence trophoblast cells derive. In vitro, naive pluripotent stem cells (PSCs) retain this potential and efficiently generate trophoblast stem cells (TSCs), while conventional PSCs form TSCs at low efficiency. Transient histone deacetylase and MEK inhibition combined with LIF stimulation is used to chemically reset conventional to naive PSCs. Here, we report that chemical resetting induces the expression of both naive and TSC markers and of placental imprinted genes. A modified chemical resetting protocol allows for the fast and efficient conversion of conventional PSCs into TSCs, entailing shutdown of pluripotency genes and full activation of the trophoblast master regulators, without induction of amnion markers. Chemical resetting generates a plastic intermediate state, characterised by co‐expression of naive and TSC markers, after which cells steer towards one of the two fates in response to the signalling environment. The efficiency and rapidity of our system will be useful to study cell fate transitions and to generate models of placental disorders.

Generation of Mouse Primitive Endoderm Stem Cells.

The blastocysts consist of dozens of cells of three distinct lineages: epiblast (Epi), trophoblast (TB), and primitive endoderm (PrE). All embryonic and extraembryonic tissues are derived from Epi, TB, and PrE. Stem cell lines representing preimplantation Epi and TB have been established and are known as embryonic stem cells (ESCs) and trophoblast stem cells (TSCs). Extraembryonic endoderm cells (XENCs) constitute a cell line that has been established from PrE. Although in vivo, PrE gives rise to visceral endoderm (VE), parietal endoderm (PE), and marginal zone endoderm (MZE); XENCs, on blastocyst injection into chimeras, primarily contribute to the distal region of PE. Here, we provide a comprehensive protocol for the establishment of fully potent primitive endoderm stem cell (PrESC) lines. PrESCs are established and maintained on mouse embryonic fibroblast (MEF) feeder cells in a serum-free medium supplemented with fibroblast growth factor 4 (FGF4), heparin, CHIR99021, and platelet-derived growth factor-AA (PDGF-AA). PrESCs co-express markers indicative of pluripotency and endoderm lineage commitment, exhibiting characteristics akin to those of PrE. On transplantation of PrESCs into blastocysts, they demonstrate a high efficiency in contributing to VE, PE, and MZE. PrESCs serve as a valuable model for studying PrE, sharing similarities in gene expression profiles and differentiation potential. PrESCs constitute a pivotal cornerstone for in vitro analysis of early developmental mechanisms and for studies of embryo reconstitution in vitro, particularly in conjunction with ESCs and TSCs. Key features • Establishment and maintenance of primitive endoderm stem cell (PrESCs) capable of recapitulating the developmental prowess inherent to PrE. • Offering a source of PrE lineage for embryo-like organoid reconstitution studies.

GATA6 regulates expression of annexin A10 (ANXA10) associated with epithelial–mesenchymal transition of oral squamous cell carcinoma

Oral squamous cell carcinoma (OSCC) can disturb oral function and quality of life and is associated with poor survival, likely due to the development of cervical lymph node metastases. Epithelial-mesenchymal transition (EMT) is a process in which cells acquire molecular alterations that facilitate cell motility and invasion, and has been associated with tumor metastasis. EMT changes also play important roles in the induction of lymph node metastasis in OSCC. GATA6 is known as the earliest marker of the primitive endoderm lineages. GATA6 inhibits de-differentiation and EMT in human pancreatic ductal adenocarcinoma cells and promotes EMT. However, in OSCC, the expression and function of GATA6 in EMT and lymph node metastasis remains unclear. Therefore, this study aimed to clarify the targets of GATA6 in OSCC cells and whether the change in GATA6 expression affects EMT in OSCC cells, as well as the association between GATA6 and lymph node metastasis. The results showed that GATA6 knockdown OSCC cells promoted EMT and increased lymph node metastasis compared with control cells, whereas the overexpression of GATA6 inhibited the induction of EMT and reduced lymph node metastasis. In addition, annexin A10 (ANXA10) which is the largest type of Ca2+-regulated phospholipid-binding protein in eukaryotic cells was detected as a target gene for GATA6 and ANXA10 suppressed Vimentin expression in EMT in OSCC. Therefore, the GATA6/ANXA10 cascade may be a potential therapeutic approach for the treatment of lymph node metastases in OSCC patients.

Transcriptional heterogeneity and cell cycle regulation as central determinants of Primitive Endoderm priming.

During embryonic development cells acquire identity as they proliferate, implying that an intrinsic facet of cell fate choice requires coupling lineage decisions to cell division. How is the cell cycle regulated to promote or suppress heterogeneity and differentiation? We explore this question combining time lapse imaging with single-cell RNA-seq in the contexts of self-renewal, priming, and differentiation of mouse embryonic stem cells (ESCs) towards the Primitive Endoderm (PrE) lineage. Since ESCs are derived from the inner cell mass (ICM) of the mammalian blastocyst, ESCs in standard culture conditions are transcriptionally heterogeneous containing dynamically interconverting subfractions primed for either of the two ICM lineages, Epiblast and PrE. Here, we find that differential regulation of cell cycle can tip the balance between these primed populations, such that naïve ESC culture promotes Epiblast-like expansion and PrE differentiation stimulates the selective survival and proliferation of PrE-primed cells. In endoderm differentiation, this change is accompanied by a counter-intuitive increase in G1 length, also observed in vivo. While fibroblast growth factor/extracellular signal-regulated kinase (FGF/ERK) signalling is a key regulator of ESC differentiation and PrE specification, we find it is not just responsible for ESCs heterogeneity, but also the inheritance of similar cell cycles between sisters and cousins. Taken together, our results indicate a tight relationship between transcriptional heterogeneity and cell cycle regulation in lineage specification, with primed cell populations providing a pool of flexible cell types that can be expanded in a lineage-specific fashion while allowing plasticity during early determination.

Post-gastrulation synthetic embryos generated ex utero from mouse naive ESCs

SummaryIn vitro cultured stem cells with distinct developmental capacities can contribute to embryonic or extraembryonic tissues after microinjection into pre-implantation mammalian embryos. However, whether cultured stem cells can independently give rise to entire gastrulating embryo-like structures with embryonic and extraembryonic compartments remains unknown. Here, we adapt a recently established platform for prolonged ex utero growth of natural embryos to generate mouse post-gastrulation synthetic whole embryo models (sEmbryos), with both embryonic and extraembryonic compartments, starting solely from naive ESCs. This was achieved by co-aggregating non-transduced ESCs, with naive ESCs transiently expressing Cdx2 or Gata4 to promote their priming toward trophectoderm and primitive endoderm lineages, respectively. sEmbryos adequately accomplish gastrulation, advance through key developmental milestones, and develop organ progenitors within complex extraembryonic compartments similar to E8.5 stage mouse embryos. Our findings highlight the plastic potential of naive pluripotent cells to self-organize and functionally reconstitute and model the entire mammalian embryo beyond gastrulation.

Extensive co-binding and rapid redistribution of NANOG and GATA6 during emergence of divergent lineages

Fate-determining transcription factors (TFs) can promote lineage-restricted transcriptional programs from common progenitor states. The inner cell mass (ICM) of mouse blastocysts co-expresses the TFs NANOG and GATA6, which drive the bifurcation of the ICM into either the epiblast (Epi) or the primitive endoderm (PrE), respectively. Here, we induce GATA6 in embryonic stem cells–that also express NANOG–to characterize how a state of co-expression of opposing TFs resolves into divergent lineages. Surprisingly, we find that GATA6 and NANOG co-bind at the vast majority of Epi and PrE enhancers, a phenomenon we also observe in blastocysts. The co-bound state is followed by eviction and repression of Epi TFs, and quick remodeling of chromatin and enhancer-promoter contacts thus establishing the PrE lineage while repressing the Epi fate. We propose that co-binding of GATA6 and NANOG at shared enhancers maintains ICM plasticity and promotes the rapid establishment of Epi- and PrE-specific transcriptional programs.

BET proteins are essential for the specification and maintenance of the epiblast lineage in mouse preimplantation embryos

BackgroundDuring mammalian preimplantation development, as the fertilized egg develops and differentiates, three cell lineages become specified: trophectoderm (TE), epiblast, and primitive endoderm (PrE). Through two steps of cell fate decisions, 16-cell blastomeres develop into TE and an inner cell mass (ICM), and thereafter, the latter differentiates into pluripotent epiblast and PrE. Although bromodomain and extra-terminal domain (BET) proteins, such as BRD4, are necessary for the transcriptional activation of genes involved in the maintenance of mouse embryonic stem cells by occupying their enhancers, their roles in the development of mouse preimplantation are unknown.ResultsTo evaluate the effect of BET protein deficiency on cell lineage formation, we cultured preimplantation embryos in the presence of JQ1, which blocks the binding of BET bromodomains to acetylated-histones. We found BET inhibition blocked the transcriptional activation of genes, such as Nanog, Otx2, and Sox2, important for the formation of the epiblast lineage in blastocysts. Expression studies with lineage-specific markers in morulae and blastocysts revealed BET proteins were essential for the specification and maintenance of the epiblast lineage but were dispensable for the formation of primarily extraembryonic TE and PrE lineages. Additional Ingenuity Pathway Analysis and expression studies with a transcriptionally active form of signal transducer and activator of the transcription 3 (STAT3) suggested BET-dependent activation was partly associated with the STAT3-dependent pathway to maintain the epiblast lineage. To identify BET proteins involved in the formation of the epiblast lineage, we analyzed mutant embryos deficient in Brd4, Brd2, and double mutants. Abolishment of NANOG-positive epiblast cells was only evident in Brd4/Brd2 double-deficient morulae. Thus, the phenotype of JQ1-treated embryos is reproduced not by a Brd4- or Brd2-single deficiency, but only Brd4/Brd2-double deficiency, demonstrating the redundant roles of BRD2 and BRD4 in the specification of the epiblast lineage.ConclusionsBET proteins are essential to the specification and maintenance of the epiblast lineage by activating lineage-specific core transcription factors during mouse preimplantation development. Among BET proteins, BRD4 plays a central role and BRD2 a complementary role in the specification and maintenance of epiblast lineages. Additionally, BET-dependent maintenance of the epiblast lineage may be partly associated with the STAT3-dependent pathway.

Klf4 methylated by Prmt1 restrains the commitment of primitive endoderm.

The second cell fate decision in the early stage of mammalian embryonic development is pivotal; however, the underlying molecular mechanism is largely unexplored. Here, we report that Prmt1 acts as an important regulator in primitive endoderm (PrE) formation. First, Prmt1 depletion promotes PrE gene expression in mouse embryonic stem cells (ESCs). Single-cell RNA sequencing and flow cytometry assays demonstrated that Prmt1 depletion in mESCs contributes to an emerging cluster, where PrE genes are upregulated significantly. Furthermore, the efficiency of extraembryonic endoderm stem cell induction increased in Prmt1-depleted ESCs. Second, the pluripotency factor Klf4 methylated at Arg396 by Prmt1 is required for recruitment of the repressive mSin3a/HDAC complex to silence PrE genes. Most importantly, an embryonic chimeric assay showed that Prmt1 inhibition and mutated Klf4 at Arg 396 induce the integration of mouse ESCs into the PrE lineage. Therefore, we reveal a regulatory mechanism for cell fate decisions centered on Prmt1-mediated Klf4 methylation.

Development of Pre-implantation Mammalian Blastocyst.

The preimplantation mammalian embryo is a simplistic, self-contained, and a superior model for investigating the inherent complexities of cell fate decision mechanisms. All mammals begin their humble journey from a single-cell fertilized zygote contained within a proteinaceous coat called the zona pellucida. The zygote embarks on a series of well-orchestrated events, beginning with the activation of embryonic genome, transition from meiotic to mitotic divisions, spatial organization of the cells, timely differentiation into committed trophectoderm (TE) and primitive endoderm (PrE), and ultimately escape from zona pellucida for implantation into the uterus. The entire development of preimplantation embryo can be studied in vitro using a minimalistic and defined culture system. The ease of culture along with the ability to manipulate gene expression and image the embryos makes them an ideal model system for investigation into the first two of several cell fate decisions made by the embryo that result in a pluripotent epiblast (EPI) and differentiated TE and PrE lineages. This chapter reviews our latest knowledge of preimplantation embryo development, setting the stage for understanding placental development in subsequent chapters in this Book.

ZC3H4-a novel Cys-Cys-Cys-His-type zinc finger protein-is essential for early embryogenesis in mice†.

Zinc finger domains of the Cys-Cys-Cys-His (CCCH) class are evolutionarily conserved proteins that bind nucleic acids and are involved in various biological processes. Nearly 60 CCCH-type zinc finger proteins have been identified in humans and mice, most have not been functionally characterized. Here, we provide the first in vivo functional characterization of ZC3H4-a novel CCCH-type zinc finger protein. Our results show that although Zc3h4 mutant embryos exhibit normal morphology at E3.5 blastocyst stage, they cannot be recovered at E7.5 early post-gastrulation stage, suggesting implantation failure. Outgrowth assays reveal that mutant blastocysts either fail to hatch from the zona pellucida, or can hatch but do not form a typical inner cell mass colony, the source of embryonic stem cells (ESCs). Although there is no change in levels of reactive oxygen species, Zc3h4 mutants display severe DNA breaks and reduced cell proliferation. Analysis of lineage specification reveals that both epiblast and primitive endoderm lineages are compromised with severe reductions in cell number and/or specification in the mutant blastocysts. In summary, these findings demonstrate the essential role of ZC3H4 during early mammalian embryogenesis.

Loss of RBBP4 results in defective inner cell mass, severe apoptosis, hyperacetylated histones and preimplantation lethality in mice†.

Retinoblastoma-binding protein 4 (RBBP4) (also known as chromatin-remodeling factor RBAP48) is an evolutionarily conserved protein that has been involved in various biological processes. Although a variety of functions have been attributed to RBBP4 in vitro, mammalian RBBP4 has not been studied in vivo. Here we report that RBBP4 is essential during early mouse embryo development. Although Rbbp4 mutant embryos exhibit normal morphology at E3.5 blastocyst stage, they cannot be recovered at E7.5 early post-gastrulation stage, suggesting an implantation failure. Outgrowth (OG) assays reveal that mutant blastocysts cannot hatch from the zona or can hatch but then arrest without further development. We find that while there is no change in proliferation or levels of reactive oxygen species, both apoptosis and histone acetylation are significantly increased in mutant blastocysts. Analysis of lineage specification reveals that while the trophoblast is properly specified, both epiblast and primitive endoderm lineages are compromised with severe reductions in cell number and/or specification. In summary, these findings demonstrate the essential role of RBBP4 during early mammalian embryogenesis.

P38-Mitogen Activated Kinases Mediate a Developmental Regulatory Response to Amino Acid Depletion and Associated Oxidative Stress in Mouse Blastocyst Embryos.

Maternal starvation coincident with preimplantation development has profound consequences for placental-fetal development, with various identified pathologies persisting/manifest in adulthood; the ‘Developmental Origin of Health and Disease’ (DOHaD) hypothesis/model. Despite evidence describing DOHaD-related incidence, supporting mechanistic and molecular data relating to preimplantation embryos themselves are comparatively meager. We recently identified the classically recognized stress-related p38-mitogen activated kinases (p38-MAPK) as regulating formation of the extraembryonic primitive endoderm (PrE) lineage within mouse blastocyst inner cell mass (ICM). Thus, we wanted to assay if PrE differentiation is sensitive to amino acid availability, in a manner regulated by p38-MAPK. Although blastocysts appropriately mature, without developmental/morphological or cell fate defects, irrespective of amino acid supplementation status, we found the extent of p38-MAPK inhibition induced phenotypes was more severe in the absence of amino acid supplementation. Specifically, both PrE and epiblast (EPI) ICM progenitor populations remained unspecified and there were fewer cells and smaller blastocyst cavities. Such phenotypes could be ameliorated, to resemble those observed in groups supplemented with amino acids, by addition of the anti-oxidant NAC (N-acetyl-cysteine), although PrE differentiation deficits remained. Therefore, p38-MAPK performs a hitherto unrecognized homeostatic early developmental regulatory role (in addition to direct specification of PrE), by buffering blastocyst cell number and ICM cell lineage specification (relating to EPI) in response to amino acid availability, partly by counteracting induced oxidative stress; with clear implications for the DOHaD model.