Gata6, Nanog and Erk signaling control cell fate in the inner cell mass through a tristable regulatory network

AbstractDuring the mammalian preimplantation phase, cells undergo two subsequent cell fate decisions. During the first decision, the trophectoderm and the inner cell mass are formed. Subsequently, the inner cell mass segregates into the epiblast and the primitive endoderm. Inner cell mass organoids represent an experimental model system, mimicking the second cell fate decision. It has been shown that cells of the same fate tend to cluster stronger than expected for random cell fate decisions. Three major processes are hypothesised to contribute to the cell fate arrangements: (1) chemical signalling; (2) cell sorting; and (3) cell proliferation. In order to quantify the influence of cell proliferation on the observed cell lineage type clustering, we developed an agent-based model accounting for mechanical cell–cell interaction, i.e. adhesion and repulsion, cell division, stochastic cell fate decision and cell fate heredity. The model supports the hypothesis that initial cell fate acquisition is a stochastically driven process, taking place in the early development of inner cell mass organoids. Further, we show that the observed neighbourhood structures can emerge solely due to cell fate heredity during cell division.

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p38-MAPK mediated rRNA processing and translation regulation enables PrE differentiation during mouse blastocyst maturation

10.1101/2020.11.30.403931 ◽

2020 ◽

Author(s):

Pablo Bora ◽

Lenka Gahurova ◽

Tomáš Mašek ◽

Andrea Hauserova ◽

David Potěšil ◽

...

Keyword(s):

Cell Fate ◽

P38 Mapk ◽

Ribosomal Proteins ◽

De Novo ◽

Inner Cell Mass ◽

Cell Mass ◽

Translation Regulation ◽

Placental Development ◽

Mapk Activity ◽

Live Embryo

AbstractBackgroundp38-MAPKs are stress-activated kinases necessary for placental development and nutrient and oxygen transfer during murine post-implantation development. In preimplantation development, p38-MAPK activity is required for blastocyst formation. Additionally, we have previously reported its role in regulating specification of inner cell mass (ICM) towards primitive endoderm (PrE), although a comprehensive mechanistic understanding is currently limited. Adopting live embryo imaging, proteomic and transcriptomic approaches, we report experimental data that directly address this deficit.ResultsChemical inhibition of p38-MAPK activity during blastocyst maturation causes impaired blastocyst cavity expansion, most evident between the third and tenth hours post inhibition onset. We identify an overlapping minimal early blastocyst maturation window of p38-MAPKi inhibition (p38-MAPKi) sensitivity, that is sufficient to impair PrE cell fate by the late blastocyst (E4.5) stage. Comparative proteomic analyses reveal substantial downregulation of ribosomal proteins, the mRNA transcripts of which are also significantly upregulated. Ontological analysis of the differentially expressed transcriptome during this developmental period reveals “translation” related gene transcripts as being most significantly, yet transiently, affected by p38-MAPKi. Moreover, combined assays consistently report concomitant reductions in de novo translation that are associated with accumulation of unprocessed rRNA precursors. Using a phosphoproteomic approach, ± p38-MAPKi, we identified Mybpp1a, an rRNA transcription and processing regulator gene, as a potential p38-MAPK effector. We report that siRNA mediated clonal knockdown of Mybpp1a is associated with significantly diminished PrE contribution. Lastly, we show that defective PrE specification caused by p38-MAPKi (but not MEK/ERK signalling inhibition) can be partially rescued by activating the archetypal mTOR mediated translation regulatory pathway.ConclusionsActivated p38-MAPK controls blastocyst maturation in an early and distinctly transient developmental window by regulating gene functionalities related to translation, that creates a permissive environment for appropriate specification of ICM cell fate.

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Specific expression of a retinoic acid-regulated, zinc-finger gene, Rex-1, in preimplantation embryos, trophoblast and spermatocytes

Development ◽

10.1242/dev.113.3.815 ◽

1991 ◽

Vol 113 (3) ◽

pp. 815-824 ◽

Cited By ~ 6

Author(s):

M.B. Rogers ◽

B.A. Hosler ◽

L.J. Gudas

Keyword(s):

Retinoic Acid ◽

Germ Cell ◽

Cell Fate ◽

Zinc Finger ◽

Zinc Finger Protein ◽

Inner Cell Mass ◽

Cell Mass ◽

Embryonic Stem ◽

Preimplantation Embryos ◽

Specific Expression

We have previously isolated a cDNA clone for a gene whose expression is reduced by retinoic acid (RA) treatment of F9 embryonal carcinoma cells. The nucleotide sequence indicated that this gene, Rex-1, encodes a zinc-finger protein and thus may be a transcriptional regulator. The Rex-1 message level is high in two lines of embryonic stem cells (CCE and D3) and is reduced when D3 cells are induced to differentiate using four different growth conditions. As expected for a stem-cell-specific message, Rex-1 mRNA is present in the inner cell mass (ICM) of the day 4.5 mouse blastocyst. It is also present in the polar trophoblast of the blastocyst. One and two days later, Rex-1 message is found in the ectoplacental cone and extraembryonic ectoderm of the egg cylinder (trophoblast-derived tissues), but its abundance is much reduced in the embryonic ectoderm which is directly descended from the ICM. Rex-1 is expressed in the day 18 placenta (murine gestation is 18 days), a tissue which is largely derived from trophoblast. The only tested adult tissue that contains detectable amounts of Rex-1 mRNA is the testis. In situ hybridization and northern analyses of RNA from germ-cell-deficient mouse testis and stage-specific germ cell preparations suggest that Rex-1 expression is limited to spermatocytes (germ cells undergoing meiosis). These results suggest that Rex-1 is involved in trophoblast development and spermatogenesis, and is a useful marker for studies of early cell fate determination in the ICM.

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102. THE EFFECT OF INSULIN ON EMBRYONIC STEM CELL PROGENITOR CELLS IN THE MOUSE BLASTOCYST

Reproduction Fertility and Development ◽

10.1071/srb09abs102 ◽

2009 ◽

Vol 21 (9) ◽

pp. 21

Author(s):

J. M. Campbell ◽

I. Vassiliev ◽

M. B. Nottle ◽

M. Lane

Keyword(s):

Progenitor Cells ◽

Cell Fate ◽

Culture Medium ◽

Inner Cell Mass ◽

Cell Mass ◽

Embryonic Stem ◽

Cell Number ◽

Human Embryos ◽

Cell Stage ◽

Stage Of Development

Human ESCs are produced from embryos donated at the mid-stage of pre-implantation development. This cryostorage reduced viability. However, it has been shown that this can be improved by the addition of growth factors to culture medium. The aim of the present study was to examine whether the addition of insulin to embryo culture medium from the 8-cell stage of development increases the number of ES cell progenitor cells in the epiblast in a mouse model. In vivo produced mouse zygotes (C57Bl6 strain) were cultured in G1 medium for 48h to the 8-cell stage, followed by culture in G2 supplemented with insulin (0, 0.17, 1.7 and 1700pM) for 68h, at 37 o C , in 5% O2, 6%CO2, 89% N2 . The number of cells in the inner cell mass (ICM) and epiblast was determined by immunohistochemical staining for Oct4 and Nanog. ICM cells express Oct4, epiblast cells express both Oct4 and Nanog. The addition of insulin at the concentrations examined did not increase the ICM. However, at 1.7pM insulin increased the number of epiblast cells (6.6±0.5 cells vs 4.1±0.5, P=0.001) in the ICM, which increased the proportion of the ICM that was epiblast (38.9±3.7% compared to 25.8±3.4% in the control P=0.01). This indicates that the increase in the epiblast is brought about by a shift in cell fate as opposed to an increase in cell division. The effect of insulin on the proportion of cells in the epiblast was investigated using inhibitors of phosphoinositide3-kinase (PI3K) (LY294002, 50µM); one of insulin's main second messengers, and p53 (pifithrin-α, 30µg/ml); a pro-apoptotic protein inactivated by PI3K. Inhibition of PI3K eliminated the increase caused by insulin (4.5±0.3 cells versus 2.2±0.3 cells, P<0.001), while inhibition of p53 increased the epiblast cell number compared to the control (7.1±0.8 and 4.1±0.7 respectively P=0.001). This study shows that insulin increases epiblast cell number through the activation of PI3K and the inhibition of p53, and may be a strategy for improving ESC isolation from human embryos.

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G9a regulates temporal preimplantation developmental program and lineage segregation in blastocyst

eLife ◽

10.7554/elife.33361 ◽

2018 ◽

Vol 7 ◽

Cited By ~ 14

Author(s):

Jan J Zylicz ◽

Maud Borensztein ◽

Frederick CK Wong ◽

Yun Huang ◽

Caroline Lee ◽

...

Keyword(s):

Cell Fate ◽

Inner Cell Mass ◽

Cell Mass ◽

Blastocyst Stage ◽

Vital Role ◽

Mouse Development ◽

Cell Stage ◽

Developmental Progression ◽

Early Mouse ◽

The Impact

Early mouse development is regulated and accompanied by dynamic changes in chromatin modifications, including G9a-mediated histone H3 lysine 9 dimethylation (H3K9me2). Previously, we provided insights into its role in post-implantation development (Zylicz et al., 2015). Here we explore the impact of depleting the maternally inherited G9a in oocytes on development shortly after fertilisation. We show that G9a accumulates typically at 4 to 8 cell stage to promote timely repression of a subset of 4 cell stage-specific genes. Loss of maternal inheritance of G9a disrupts the gene regulatory network resulting in developmental delay and destabilisation of inner cell mass lineages by the late blastocyst stage. Our results indicate a vital role of this maternally inherited epigenetic regulator in creating conducive conditions for developmental progression and on cell fate choices.

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Epigenetic Modification Affecting Expression of Cell Polarity and Cell Fate Genes to Regulate Lineage Specification in the Early Mouse Embryo

Molecular Biology of the Cell ◽

10.1091/mbc.e10-01-0053 ◽

2010 ◽

Vol 21 (15) ◽

pp. 2649-2660 ◽

Cited By ~ 42

Author(s):

David-Emlyn Parfitt ◽

Magdalena Zernicka-Goetz

Keyword(s):

Cell Fate ◽

Cell Polarity ◽

Mouse Embryo ◽

Inner Cell Mass ◽

Epigenetic Modification ◽

Cell Mass ◽

Time Lapse ◽

Cell Polarization ◽

Asymmetric Divisions

Formation of inner and outer cells of the mouse embryo distinguishes pluripotent inner cell mass (ICM) from differentiating trophectoderm (TE). Carm1, which methylates histone H3R17 and R26, directs cells to ICM rather that TE. To understand the mechanism by which this epigenetic modification directs cell fate, we generated embryos with in vivo–labeled cells of different Carm1 levels, using time-lapse imaging to reveal dynamics of their behavior, and related this to cell polarization. This shows that Carm1 affects cell fate by promoting asymmetric divisions, that direct one daughter cell inside, and cell engulfment, where neighboring cells with lower Carm1 levels compete for outside positions. This is associated with changes to the expression pattern and spatial distribution of cell polarity proteins: Cells with higher Carm1 levels show reduced expression and apical localization of Par3 and a dramatic increase in expression of PKCII, antagonist of the apical protein aPKC. Expression and basolateral localization of the mouse Par1 homologue, EMK1, increases concomitantly. Increased Carm1 also reduces Cdx2 expression, a transcription factor key for TE differentiation. These results demonstrate how the extent of a specific epigenetic modification could affect expression of cell polarity and fate-determining genes to ensure lineage allocation in the mouse embryo.

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Controlling embryonic stem cell proliferation and pluripotency: the role of PI3K- and GSK-3-dependent signalling

Biochemical Society Transactions ◽

10.1042/bst0390674 ◽

2011 ◽

Vol 39 (2) ◽

pp. 674-678 ◽

Cited By ~ 24

Author(s):

Melanie J. Welham ◽

Emmajayne Kingham ◽

Yolanda Sanchez-Ripoll ◽

Benjamin Kumpfmueller ◽

Michael Storm ◽

...

Keyword(s):

Cell Fate ◽

Molecular Mechanisms ◽

Inner Cell Mass ◽

Glycogen Synthase ◽

Cell Mass ◽

Embryonic Stem ◽

Cell Types ◽

Specific Cell ◽

Human Escs ◽

Phosphoinositide 3 Kinase

ESCs (embryonic stem cells) are derived from the inner cell mass of pre-implantation embryos and are pluripotent, meaning they can differentiate into all of the cells that make up the adult organism. This property of pluripotency makes ESCs attractive as a model system for studying early development and for the generation of specific cell types for use in regenerative medicine and drug screening. In order to harness their potential, the molecular mechanisms regulating ESC pluripotency, proliferation and differentiation (i.e. cell fate) need to be understood so that pluripotency can be maintained during expansion, while differentiation to specific lineages can be induced accurately when required. The present review focuses on the potential roles that PI3K (phosphoinositide 3-kinase) and GSK-3 (glycogen synthase kinase 3)-dependent signalling play in the co-ordination and integration of mouse ESC pluripotency and proliferation and contrast this with our understanding of their functions in human ESCs.

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Cell Fate Specification Based on Tristability in the Inner Cell Mass of Mouse Blastocysts

Biophysical Journal ◽

10.1016/j.bpj.2015.12.020 ◽

2016 ◽

Vol 110 (3) ◽

pp. 710-722 ◽

Cited By ~ 28

Author(s):

Laurane De Mot ◽

Didier Gonze ◽

Sylvain Bessonnard ◽

Claire Chazaud ◽

Albert Goldbeter ◽

...

Keyword(s):

Cell Fate ◽

Inner Cell Mass ◽

Cell Mass ◽

Cell Fate Specification ◽

Fate Specification

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ICM conversion to epiblast by FGF/ERK inhibition is limited in time and requires transcription and protein degradation

10.1101/157537 ◽

2017 ◽

Author(s):

Sylvain Bessonnard ◽

Sabrina Coqueran ◽

Sandrine Vandormael-Pournin ◽

Alexandre Dufour ◽

Jerome Artus ◽

...

Keyword(s):

Time Window ◽

Inner Cell Mass ◽

Late Phase ◽

Cell Mass ◽

Erk Signaling ◽

Primitive Endoderm ◽

Short Time Window ◽

Erk Inhibition ◽

Short Time ◽

Kinetics Of

Inner cell Mass (ICM) specification into epiblast (Epi) and primitive endoderm (PrE) is an asynchronous and progressive process taking place between E3.0 to E3.75 under the control of the FGF/ERK signaling pathway. Here, we have analyzed in details the kinetics of specification and found that ICM cell responsiveness to the up and down regulation of FGF signaling activity are temporally distinct. We also showed that PrE progenitors are generated later than Epi progenitors. We further demonstrated that, during this late phase of specification, a 4 hours period of FGF/ERK inhibition prior E3.75 is sufficient to convert ICM cells into Epi. Finally, we showed that ICM conversion into Epi in response to inhibition during this short time window requires both transcription and proteasome degradation. Collectively, our data give new insights into the timing and mechanisms involved in the process of ICM specification.

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Dynamic changes in Sox2 spatio-temporal expression promote the second cell fate decision through Fgf4/Fgfr2 signaling in preimplantation mouse embryos

Biochemical Journal ◽

10.1042/bcj20170418 ◽

2018 ◽

Vol 475 (6) ◽

pp. 1075-1089 ◽

Cited By ~ 12

Author(s):

Tapan Kumar Mistri ◽

Wibowo Arindrarto ◽

Wei Ping Ng ◽

Choayang Wang ◽

Leng Hiong Lim ◽

...

Keyword(s):

Cell Fate ◽

Target Genes ◽

Inner Cell Mass ◽

Cell Mass ◽

Correlation Spectroscopy ◽

Binding Interaction ◽

Regulatory Motifs ◽

Sox2 Expression ◽

Spatio Temporal ◽

Fate Decision

Oct4 and Sox2 regulate the expression of target genes such as Nanog, Fgf4, and Utf1, by binding to their respective regulatory motifs. Their functional cooperation is reflected in their ability to heterodimerize on adjacent cis regulatory motifs, the composite Sox/Oct motif. Given that Oct4 and Sox2 regulate many developmental genes, a quantitative analysis of their synergistic action on different Sox/Oct motifs would yield valuable insights into the mechanisms of early embryonic development. In the present study, we measured binding affinities of Oct4 and Sox2 to different Sox/Oct motifs using fluorescence correlation spectroscopy. We found that the synergistic binding interaction is driven mainly by the level of Sox2 in the case of the Fgf4 Sox/Oct motif. Taking into account Sox2 expression levels fluctuate more than Oct4, our finding provides an explanation on how Sox2 controls the segregation of the epiblast and primitive endoderm populations within the inner cell mass of the developing rodent blastocyst.

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