Preimplantation mouse embryo development involves temporal-spatial specification and segregation of three blastocyst cell lineages: trophectoderm, primitive endoderm and epiblast. Spatial separation of the outer-trophectoderm lineage from the two other inner-cell-mass (ICM) lineages starts with the 8- to 16-cell transition and concludes at the 32-cell stages. Accordingly, the ICM is derived from primary and secondary contributed cells; with debated relative EPI versus PrE potencies. We report generation of primary but not secondary ICM populations is highly dependent on temporal activation of mammalian target of Rapamycin (mTOR) during 8-cell stage M-phase entry, mediated via regulation of the 7-methylguanosine-cap (m7G-cap)-binding initiation complex (EIF4F) and linked to translation of mRNAs containing 5' UTR terminal oligopyrimidine (TOP-) sequence motifs, as knockdown of identified TOP-like motif transcripts impairs generation of primary ICM founders. However, mTOR inhibition-induced ICM cell number deficits in early blastocysts can be compensated by the late blastocyst stage, after inhibitor withdrawal; compensation likely initiated at the 32-cell stage when supernumerary outer cells exhibit molecular characteristics of inner cells. These data identify a novel mechanism specifically governing initial spatial segregation of mouse embryo blastomeres, that is distinct from those directing subsequent inner cell formation, contributing to germane segregation of late blastocyst lineages.

• Klíčová slova
• EIF4EBP1/4EBP1, TOP-motif, cell fate, inner cell mass/ICM and cell positioning, mTOR/mTORC1, preimplantation mouse embryo,
• MeSH
• blastocysta * MeSH
• buněčná diferenciace fyziologie   MeSH
• buněčný rodokmen MeSH
• embryo savčí * MeSH
• mTORC1 MeSH
• myši MeSH
• savci MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• mTORC1 MeSH

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.

• Klíčová slova
• DDX21, cell fate specification, p38-MAPK, preimplantation embryo development,
• MeSH
• blastocysta cytologie   metabolismus   MeSH
• buněčná diferenciace * genetika   MeSH
• buněčný rodokmen genetika   MeSH
• DEAD-box RNA-helikasy genetika   metabolismus   MeSH
• embryonální vývoj * genetika   MeSH
• fluorescenční protilátková technika MeSH
• genový knockdown MeSH
• mitogenem aktivované proteinkinasy p38 metabolismus   MeSH
• myši MeSH
• signální transdukce MeSH
• těhotenství MeSH
• transport proteinů MeSH
• vazba proteinů MeSH
• vývojová regulace genové exprese MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• těhotenství MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DDX21 protein, mouse MeSH Prohlížeč
• DEAD-box RNA-helikasy MeSH
• mitogenem aktivované proteinkinasy p38 MeSH

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.

• MeSH
• blastocysta fyziologie   MeSH
• buněčná diferenciace MeSH
• buněčný rodokmen MeSH
• DNA vazebné proteiny fyziologie   MeSH
• embryonální vývoj MeSH
• endoderm cytologie   MeSH
• mitogenem aktivované proteinkinasy p38 antagonisté a inhibitory   fyziologie   MeSH
• myši MeSH
• proteiny vázající RNA fyziologie   MeSH
• proteosyntéza * MeSH
• TOR serin-threoninkinasy fyziologie   MeSH
• transkripční faktory fyziologie   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA vazebné proteiny MeSH
• mitogenem aktivované proteinkinasy p38 MeSH
• mTOR protein, mouse MeSH Prohlížeč
• Mybbp1a protein, mouse MeSH Prohlížeč
• proteiny vázající RNA MeSH
• TOR serin-threoninkinasy MeSH
• transkripční faktory MeSH

During mouse preimplantation embryo development, the classically described second cell-fate decision involves the specification and segregation, in blastocyst inner cell mass (ICM), of primitive endoderm (PrE) from pluripotent epiblast (EPI). The active role of fibroblast growth factor (Fgf) signalling during PrE differentiation, particularly in the context of Erk1/2 pathway activation, is well described. However, we report that p38 family mitogen-activated protein kinases (namely p38α/Mapk14 and p38β/Mapk11; referred to as p38-Mapk14/11) also participate in PrE formation. Specifically, functional p38-Mapk14/11 are required, during early-blastocyst maturation, to assist uncommitted ICM cells, expressing both EPI and earlier PrE markers, to fully commit to PrE differentiation. Moreover, functional activation of p38-Mapk14/11 is, as reported for Erk1/2, under the control of Fgf-receptor signalling, plus active Tak1 kinase (involved in non-canonical bone morphogenetic protein (Bmp)-receptor-mediated PrE differentiation). However, we demonstrate that the critical window of p38-Mapk14/11 activation precedes the E3.75 timepoint (defined by the initiation of the classical 'salt and pepper' expression pattern of mutually exclusive EPI and PrE markers), whereas appropriate lineage maturation is still achievable when Erk1/2 activity (via Mek1/2 inhibition) is limited to a period after E3.75. We propose that active p38-Mapk14/11 act as enablers, and Erk1/2 as drivers, of PrE differentiation during ICM lineage specification and segregation.

• Klíčová slova
• cell signalling, cell-fate, mitogen-activated protein kinase, p38α/p38β Mapk14/Mapk11, preimplantation mouse embryo, primitive endoderm,
• MeSH
• blastocysta fyziologie   MeSH
• buněčná diferenciace MeSH
• embryonální vývoj * MeSH
• endoderm embryologie   MeSH
• fibroblastové růstové faktory metabolismus   MeSH
• messenger RNA metabolismus   MeSH
• mitogenem aktivovaná proteinkinasa 11 metabolismus   MeSH
• mitogenem aktivovaná proteinkinasa 14 metabolismus   MeSH
• myši MeSH
• signální transdukce MeSH
• zárodečné listy fyziologie   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• fibroblastové růstové faktory MeSH
• messenger RNA MeSH
• mitogenem aktivovaná proteinkinasa 11 MeSH
• mitogenem aktivovaná proteinkinasa 14 MeSH

During mouse preimplantation embryo development, three distinct cell lineages are formed, represented by the differentiating trophectoderm (TE), primitive endoderm (PrE) and the pluripotent epiblast (EPI). Classically, lineage derivation has been presented as a two-step process whereby outer TE cells are first segregated from inner-cell mass (ICM), followed by ICM refinement into either the PrE or EPI. As ICM founders can be produced following the fourth or fifth cleavage divisions, their potential to equally contribute to EPI and PrE is contested. Thus, modelling the early sequestration of ICM founders from TE-differentiation after the fourth cleavage division, we examined ICM lineage contribution of varying sized cell clones unable to initiate TE-differentiation. Such TE-inhibited ICM cells do not equally contribute to EPI and PrE and are significantly biased to form EPI. This bias is not caused by enhanced expression of the EPI marker Nanog, nor correlated with reduced apical polarity but associated with reduced expression of PrE-related gene transcripts (Dab2 and Lrp2) and down-regulation of plasma membrane associated Fgfr2. Our results favour a unifying model were the three cell lineages are guided in an integrated, yet flexible, fate decision centred on relative exposure of founder cells to TE-differentiative cues.

• MeSH
• blastocysta cytologie   fyziologie   MeSH
• buněčná diferenciace fyziologie   MeSH
• embryo savčí cytologie   embryologie   MeSH
• embryonální vývoj fyziologie   MeSH
• kultivované buňky MeSH
• myši inbrední C57BL MeSH
• myši knockoutované MeSH
• myši MeSH
• vývojová regulace genové exprese fyziologie   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Heterogeneous expression of the transcription factor NANOG has been linked to the existence of various functional states in pluripotent stem cells. This heterogeneity seems to arise from fluctuations of Nanog expression in individual cells, but a thorough characterization of these fluctuations and their impact on the pluripotent state is still lacking. Here, we have used a novel fluorescent reporter to investigate the temporal dynamics of NANOG expression in mouse embryonic stem cells (mESCs), and to dissect the lineage potential of mESCs at different NANOG states. Our results show that stochastic NANOG fluctuations are widespread in mESCs, with essentially all expressing cells showing fluctuations in NANOG levels, even when cultured in ground-state conditions (2i media). We further show that fluctuations have similar kinetics when mESCs are cultured in standard conditions (serum plus leukemia inhibitory factor) or ground-state conditions, implying that NANOG fluctuations are inherent to the pluripotent state. We have then compared the developmental potential of low-NANOG and high-NANOG mESCs, grown in different conditions, and confirm that mESCs are more susceptible to enter differentiation at the low-NANOG state. Further analysis by gene expression profiling reveals that low-NANOG cells have marked expression of lineage-affiliated genes, with variable profiles according to the signalling environment. By contrast, high-NANOG cells show a more stable expression profile in different environments, with minimal expression of lineage markers. Altogether, our data support a model in which stochastic NANOG fluctuations provide opportunities for mESCs to explore multiple lineage options, modulating their probability to change functional state.

• Klíčová slova
• Gene expression heterogeneity, Lineage priming, Nanog, Pluripotency, Stem cells,
• MeSH
• analýza hlavních komponent MeSH
• buněčné klony MeSH
• buněčné linie MeSH
• buněčný rodokmen genetika   MeSH
• časosběrné zobrazování MeSH
• embryonální kmenové buňky cytologie   metabolismus   MeSH
• genetická transkripce MeSH
• homeodoménové proteiny genetika   metabolismus   MeSH
• kinetika MeSH
• messenger RNA genetika   metabolismus   MeSH
• myši MeSH
• nanog MeSH
• pluripotentní kmenové buňky cytologie   metabolismus   MeSH
• proliferace buněk MeSH
• průtoková cytometrie MeSH
• stanovení celkové genové exprese MeSH
• stochastické procesy MeSH
• vývojová regulace genové exprese MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• homeodoménové proteiny MeSH
• messenger RNA MeSH
• Nanog protein, mouse MeSH Prohlížeč
• nanog MeSH