scholarly journals rRNA biogenesis regulates mouse 2C-like state by 3D structure reorganization of peri-nucleolar heterochromatin

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Hua Yu ◽  
Zhen Sun ◽  
Tianyu Tan ◽  
Hongru Pan ◽  
Jing Zhao ◽  
...  
Keyword(s):  
Cell Fate ◽  
Ribosome Biogenesis ◽  
3D Structure ◽  
Embryonic Stem ◽  
Natural State ◽  
Cell Stage ◽  
Nucleolar Stress ◽  
Stage Embryo ◽  
Mes Cells ◽  
Liquid Liquid Phase Separation

AbstractThe nucleolus is the organelle for ribosome biogenesis and sensing various types of stress. However, its role in regulating stem cell fate remains unclear. Here, we present evidence that nucleolar stress induced by interfering rRNA biogenesis can drive the 2-cell stage embryo-like (2C-like) program and induce an expanded 2C-like cell population in mouse embryonic stem (mES) cells. Mechanistically, nucleolar integrity maintains normal liquid-liquid phase separation (LLPS) of the nucleolus and the formation of peri-nucleolar heterochromatin (PNH). Upon defects in rRNA biogenesis, the natural state of nucleolus LLPS is disrupted, causing dissociation of the NCL/TRIM28 complex from PNH and changes in epigenetic state and reorganization of the 3D structure of PNH, which leads to release of Dux, a 2C program transcription factor, from PNH to activate a 2C-like program. Correspondingly, embryos with rRNA biogenesis defect are unable to develop from 2-cell (2C) to 4-cell embryos, with delayed repression of 2C/ERV genes and a transcriptome skewed toward earlier cleavage embryo signatures. Our results highlight that rRNA-mediated nucleolar integrity and 3D structure reshaping of the PNH compartment regulates the fate transition of mES cells to 2C-like cells, and that rRNA biogenesis is a critical regulator during the 2-cell to 4-cell transition of murine pre-implantation embryo development.

scholarly journals rRNA Biogenesis Regulates Mouse 2C-like State by 3D Structure Reorganization of Peri-Nucleolar Heterochromatin

2021 ◽  
Author(s):  
Hua Yu ◽  
Zhen Sun ◽  
Tianyu Tan ◽  
Hongru Pan ◽  
Jing Zhao ◽  
...  
Keyword(s):  
Cell Fate ◽  
Ribosome Biogenesis ◽  
3D Structure ◽  
Embryonic Stem ◽  
Cell Stage ◽  
Nucleolar Stress ◽  
Stage Embryo ◽  
Mes Cells ◽  
Cell Transition ◽  
Liquid Liquid Phase Separation

Nucleolus is the organelle for ribosome biogenesis and for sensing various types of stress. Its role in regulating stem cell fate is unclear. Here, we present multiple lines of evidence that nucleolar stress induced by interfering rRNA biogenesis can drive 2-cell stage embryo-like (2C-like) transcriptional program and induce an expanded 2C-like cell population in mouse embryonic stem (mES) cells. Mechanistically, the nucleolar integrity mediated by rRNA biogenesis maintains the normal liquid-liquid phase separation (LLPS) of nucleolus and the formation of peri-nucleolar heterochromatin (PNH). Upon rRNA biogenesis defect, the natural LLPS of nucleolus is disrupted, causing dissociation of NCL/TRIM28 complex from PNH and changes of epigenetic states and reorganization of the 3D structure of PNH, which leads to Dux, a 2C program transcription factor gene, to be released from the PNH region and activation of 2C-like program. Correspondingly, embryos with rRNA biogenesis defect are incompatible to develop from 2-cell (2C) to 4-cell embryos, with delayed repression of 2C/ERV genes and a transcriptome skewed toward earlier cleavage embryo signatures. Our results highlight that rRNA-mediated nucleolar integrity and 3D structure reshaping of PNH compartment regulates the fate transition of mES cells to 2C-like cells, and that rRNA biogenesis is a critical regulator during the 2-cell-to-4-cell transition of murine pre-implantation embryo development.

scholarly journals The molecular and cellular features of 2-cell-like cells: a reference guide

Development ◽  
2020 ◽  
Vol 147 (16) ◽  
pp. dev189688 ◽  
Author(s):  
Marion Genet ◽  
Maria-Elena Torres-Padilla
Keyword(s):  
Stem Cells ◽  
Cell Fate ◽  
Embryonic Stem ◽  
The Body ◽  
Cell Stage ◽  
Developmental Potential ◽  
Culture Models ◽  
Stage Embryo ◽  
Induced Pluripotent

ABSTRACTCurrently, two main cell culture models predominate pluripotent stem cell research: embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). Thanks to their ability to contribute to and form all tissues within the body, ESCs and iPSCs have proven invaluable in understanding pluripotent states, early embryonic development and cell differentiation, as well as in devising strategies for regenerative medicine. Comparatively little is known about totipotency – a cellular state with greater developmental potential. In mice, only the zygote and the blastomeres of the 2-cell-stage embryo are truly totipotent, as they alone can develop to form the embryo and all of its supportive extra-embryonic tissues. However, the discovery of a rare subpopulation of cells in murine ESC cultures, possessing features of 2-cell embryo blastomeres and expanded cell fate potential, has provided a biochemically tractable model to enable the in vitro study of totipotency. Here, we summarize current known features of these 2-cell-like cells (2CLCs) in an effort to provide a reference for the community, and to clarify what we know about their identity so far.

XIPOU 2, a noggin-inducible gene, has direct neuralizing activity

Development ◽  
1995 ◽  
Vol 121 (3) ◽  
pp. 721-730 ◽  
Author(s):  
S.E. Witta ◽  
V.R. Agarwal ◽  
S.M. Sato
Keyword(s):  
Cell Fate ◽  
Neural Induction ◽  
Mesoderm Induction ◽  
Class Iii ◽  
Cell Stage ◽  
Neuronal Phenotype ◽  
Pou Domain ◽  
Spemann's Organizer ◽  
Stage Embryo ◽  
Synthetic Mrna

XIPOU 2, a member of the class III POU domain family, is expressed initially in Spemann's organizer, and later, in discrete regions of the developing nervous system in Xenopus laevis. XIPOU 2 may act downstream from initial neural induction events, since it is activated by the neural inducer, noggin. To determine if XIPOU 2 participates in the early events of neurogenesis, synthetic mRNA was microinjected into specific blastomeres of the 32-cell stage embryo. Misexpression of XIPOU 2 in the epidermis causes a direct switch in cell fate from an epidermal to a neuronal phenotype. In the absence of mesoderm induction, XIPOU 2 has the ability to induce a neuronal phenotype in uncommitted ectoderm. These data demonstrate the potential of XIPOU 2 to act as a master regulator of neurogenesis.

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

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.

scholarly journals Consistent apparent Young’s modulus of human embryonic stem cells and derived cell types stabilized by substrate stiffness regulation promotes lineage specificity maintenance

2020 ◽  
Vol 9 (1) ◽  
Author(s):  
Anqi Guo ◽  
Bingjie Wang ◽  
Cheng Lyu ◽  
Wenjing Li ◽  
Yaozu Wu ◽  
...  
Keyword(s):  
Stem Cell ◽  
Cell Fate ◽  
Cell Expansion ◽  
Embryonic Stem ◽  
Cell Types ◽  
Substrate Stiffness ◽  
Cell Stage ◽  
Cytoskeletal Organization ◽  
Lineage Specificity

Abstract Background Apparent Young’s modulus (AYM), which reflects the fundamental mechanical property of live cells measured by atomic force microscopy and is determined by substrate stiffness regulated cytoskeletal organization, has been investigated as potential indicators of cell fate in specific cell types. However, applying biophysical cues, such as modulating the substrate stiffness, to regulate AYM and thereby reflect and/or control stem cell lineage specificity for downstream applications, remains a primary challenge during in vitro stem cell expansion. Moreover, substrate stiffness could modulate cell heterogeneity in the single-cell stage and contribute to cell fate regulation, yet the indicative link between AYM and cell fate determination during in vitro dynamic cell expansion (from single-cell stage to multi-cell stage) has not been established. Results Here, we show that the AYM of cells changed dynamically during passaging and proliferation on substrates with different stiffness. Moreover, the same change in substrate stiffness caused different patterns of AYM change in epithelial and mesenchymal cell types. Embryonic stem cells and their derived progenitor cells exhibited distinguishing AYM changes in response to different substrate stiffness that had significant effects on their maintenance of pluripotency and/or lineage-specific characteristics. On substrates that were too rigid or too soft, fluctuations in AYM occurred during cell passaging and proliferation that led to a loss in lineage specificity. On a substrate with ‘optimal’ stiffness (i.e., 3.5 kPa), the AYM was maintained at a constant level that was consistent with the parental cells during passaging and proliferation and led to preservation of lineage specificity. The effects of substrate stiffness on AYM and downstream cell fate were correlated with intracellular cytoskeletal organization and nuclear/cytoplasmic localization of YAP. Conclusions In summary, this study suggests that optimal substrate stiffness regulated consistent AYM during passaging and proliferation reflects and contributes to hESCs and their derived progenitor cells lineage specificity maintenance, through the underlying mechanistic pathways of stiffness-induced cytoskeletal organization and the downstream YAP signaling. These findings highlighted the potential of AYM as an indicator to select suitable substrate stiffness for stem cell specificity maintenance during in vitro expansion for regenerative applications.

scholarly journals Piezo1 forms a slowly-inactivating mechanosensory channel in mouse embryonic stem cells

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Josefina Inés del Mármol ◽  
Kouki K Touhara ◽  
Gist Croft ◽  
Roderick MacKinnon
Keyword(s):  
Motor Neurons ◽  
Es Cells ◽  
Embryonic Stem ◽  
Inactivation Kinetics ◽  
Fast Inactivation ◽  
Cell Stage ◽  
Slow Kinetics ◽  
Aminoacid Sequence ◽  
Reduced Rate ◽  
Mes Cells

Piezo1 is a mechanosensitive (MS) ion channel with characteristic fast-inactivation kinetics. We found a slowly-inactivating MS current in mouse embryonic stem (mES) cells and characterized it throughout their differentiation into motor-neurons to investigate its components. MS currents were large and slowly-inactivating in the stem-cell stage, and became smaller and faster-inactivating throughout the differentiation. We found that Piezo1 is expressed in mES cells, and its knockout abolishes MS currents, indicating that the slowly-inactivating current in mES cells is carried by Piezo1. To further investigate its slow inactivation in these cells, we cloned Piezo1 cDNA from mES cells and found that it displays fast-inactivation kinetics in heterologous expression, indicating that sources of modulation other than the aminoacid sequence determine its slow kinetics in mES cells. Finally, we report that Piezo1 knockout ES cells showed a reduced rate of proliferation but no significant differences in other markers of pluripotency and differentiation.

scholarly journals Ribosome and Translational Control in Stem Cells

Cells ◽  
2020 ◽  
Vol 9 (2) ◽  
pp. 497 ◽  
Author(s):  
Mathieu Gabut ◽  
Fleur Bourdelais ◽  
Sébastien Durand
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Cell Fate ◽  
Chromatin Remodeling ◽  
Translational Control ◽  
Ribosome Biogenesis ◽  
Adult Stem Cells ◽  
Embryonic Stem ◽  
Cell Homeostasis ◽  
Cell Functions

Embryonic stem cells (ESCs) and adult stem cells (ASCs) possess the remarkable capacity to self-renew while remaining poised to differentiate into multiple progenies in the context of a rapidly developing embryo or in steady-state tissues, respectively. This ability is controlled by complex genetic programs, which are dynamically orchestrated at different steps of gene expression, including chromatin remodeling, mRNA transcription, processing, and stability. In addition to maintaining stem cell homeostasis, these molecular processes need to be rapidly rewired to coordinate complex physiological modifications required to redirect cell fate in response to environmental clues, such as differentiation signals or tissue injuries. Although chromatin remodeling and mRNA expression have been extensively studied in stem cells, accumulating evidence suggests that stem cell transcriptomes and proteomes are poorly correlated and that stem cell properties require finely tuned protein synthesis. In addition, many studies have shown that the biogenesis of the translation machinery, the ribosome, is decisive for sustaining ESC and ASC properties. Therefore, these observations emphasize the importance of translational control in stem cell homeostasis and fate decisions. In this review, we will provide the most recent literature describing how ribosome biogenesis and translational control regulate stem cell functions and are crucial for accommodating proteome remodeling in response to changes in stem cell fate.

scholarly journals 246 EXPRESSION PATTERN OF NANOG AND Par3 GENES IN IN VITRO-DERIVED BOVINE EMBRYOS

2006 ◽  
Vol 18 (2) ◽  
pp. 231 ◽  
Author(s):  
F. Gandolfi ◽  
F. Cillo ◽  
S. Antonini ◽  
S. Colleoni ◽  
I. Lagutina ◽  
...  
Keyword(s):  
Cell Fate ◽  
Cdna Sequence ◽  
Inner Cell Mass ◽  
Expression Patterns ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Bovine Embryos ◽  
Cell Stage ◽  
Genome Activation

Homeobox genes have been demonstrated to be important in patterning and lineage specification during early embryogenesis. Nanog belongs to the family of DNA-binding transcription factors and has been shown to maintain pluripotency of embryonic stem cells, both in murine and human. Par3 plays an essential role in determining cell fate of the early mouse embryo, leading to the generation of the inner cell mass and the trophectoderm. No information is available on these genes in the bovine; therefore, the aim of the present study was to identify and characterize Nanog and Par3 expression in bovine embryos. Oocytes recovered from slaughterhouse ovaries were matured for 22 h, fertilized in vitro and then cultured in mSOFaa medium. RNA was extracted from pools of five oocytes and embryos at different stages of development (2-, 4-, 8-, 16-cell, morula and blastocyst). It was then reverse transcribed, and PCR runs were carried out with primers specifically designed for Nanog and Par3, based on the sequence data bank available. The amplified products were separated on a 2% TAE agarose gel, purified, sequenced and aligned using Clustal W. Comparison of the bovine Nanog cDNA sequence (EMBL AM039957) with databases revealed a 84% degree of homology with the human, 97% with the mouse, and 82% with the goat genes. IVF bovine embryos express Nanog only upon genome activation, becoming detectable from the 8-cell stage onward indicating that Nanog is zygotically expressed in the bovine similar to what happens in mouse, pig and goat. Bovine Par3 cDNA sequence (EMBL AM039956) shows a high degree of homology with human (83%), mouse (81%), and rat (79%). Also Par3 is expressed only upon the maternal to embryonic transition (MET) at the 8-cell stage. As opposed to the expression patterns of other early embryo genes, like Oct-4 and Zar-1, Nanog and Par3 expression patterns in bovine embryos closely resemble those described in the mouse. Since both are absent in the ooplasm and before MET, they represent useful markers for genome activation. This work was supported by FIRB RBNE01HPMX, FIRST 2004 and ESF-EuroStells.

scholarly journals Origin of cellular asymmetries in the pre-implantation mouse embryo: a hypothesis

2014 ◽  
Vol 369 (1657) ◽  
pp. 20130536 ◽  
Author(s):  
Katsuyoshi Takaoka ◽  
Hiroshi Hamada
Keyword(s):  
Dna Methylation ◽  
Cell Fate ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Cell Fate Decision ◽  
Mouse Development ◽  
Cell Stage ◽  
Stage Embryo ◽  
A Cell ◽  
Fate Decision

The first cell fate decision during mouse development concerns whether a blastomere will contribute to the inner cell mass (ICM; which gives rise to the embryo proper) or to trophectoderm (TE; which gives rise to the placenta). The position of a cell within an 8- to 16-cell-stage embryo correlates with its future fate, with outer cells contributing to TE and inner cells to the ICM. It remains unknown, however, whether an earlier pre-pattern exists. Here, we propose a hypothesis that could account for generation of such a pre-pattern and which is based on epigenetic asymmetry (such as in histone or DNA methylation) between maternal and paternal genomes in the zygote.

scholarly journals Single-Cell Transcriptome Analysis as a Promising Tool to Study Pluripotent Stem Cell Reprogramming

2021 ◽  
Vol 22 (11) ◽  
pp. 5988
Author(s):  
Hyun Kyu Kim ◽  
Tae Won Ha ◽  
Man Ryul Lee
Keyword(s):  
Stem Cells ◽  
Single Cell ◽  
Cell Fate ◽  
Human Cell ◽  
Transcriptome Analysis ◽  
Single Cell Analysis ◽  
Embryonic Stem ◽  
Single Cell Level ◽  
Cell Analysis ◽  
Cell Level

Cells are the basic units of all organisms and are involved in all vital activities, such as proliferation, differentiation, senescence, and apoptosis. A human body consists of more than 30 trillion cells generated through repeated division and differentiation from a single-cell fertilized egg in a highly organized programmatic fashion. Since the recent formation of the Human Cell Atlas consortium, establishing the Human Cell Atlas at the single-cell level has been an ongoing activity with the goal of understanding the mechanisms underlying diseases and vital cellular activities at the level of the single cell. In particular, transcriptome analysis of embryonic stem cells at the single-cell level is of great importance, as these cells are responsible for determining cell fate. Here, we review single-cell analysis techniques that have been actively used in recent years, introduce the single-cell analysis studies currently in progress in pluripotent stem cells and reprogramming, and forecast future studies.

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