scholarly journals CNOT3 interacts with the Aurora B and MAPK/ERK kinases to promote survival of differentiating mesendodermal progenitor cells

2021 ◽  
Author(s):  
Moumita Sarkar ◽  
Matteo Martufi ◽  
Monica Roman-Trufero ◽  
Yi-Fang Wang ◽  
Chad Whilding ◽  
...  
Keyword(s):  
Es Cells ◽  
Primitive Streak ◽  
Embryoid Bodies ◽  
Aurora B ◽  
Double Mutation ◽  
Mouse Es Cells ◽  
Intermediate Progenitors ◽  
Metastatic Lung ◽  
Localization Signal ◽  
Cell Cycle Kinase

Mesendoderm cells are key intermediate progenitors that form at the early primitive streak (PrS) and give rise to mesoderm and endoderm in the gastrulating embryo. We have identified an interaction between CNOT3 and the cell cycle kinase Aurora B, which requires sequences in the NOT box domain of CNOT3, and regulates MAPK/ERK signalling during mesendoderm differentiation. Aurora B phosphorylates CNOT3 at two sites located close to a nuclear localization signal and promotes localization of CNOT3 to the nuclei of mouse ES cells (ESCs) and metastatic lung cancer cells. ESCs that have both sites mutated give rise to embryoid bodies that are largely devoid of mesoderm and endoderm and are composed mainly of cells with ectodermal characteristics. The mutant ESCs are also compromised in their ability to differentiate into mesendoderm in response to FGF2, BMP4 and Wnt3 due to reduced survival and proliferation of differentiating mesendoderm cells. We also show that the double mutation alters the balance of interaction of CNOT3 with Aurora B and with ERK and reduces phosphorylation of ERK in response to FGF2. Our results identify a potential adaptor function for CNOT3 that regulates the Ras/MEK/ERK pathway during embryogenesis. [Media: see text]

scholarly journals A signaling axis involving CNOT3, Aurora B and ERK promotes mesendodermal differentiation of ES cells in response to FGF2 and BMP4

2019 ◽  
Author(s):  
Moumita Sarkar ◽  
Matteo Martufi ◽  
Monica Roman-Trufero ◽  
Yi-Fang Wang ◽  
Chad Whilding ◽  
...  
Keyword(s):  
Es Cells ◽  
Primitive Streak ◽  
Embryoid Bodies ◽  
Aurora B ◽  
Double Mutation ◽  
Mouse Es Cells ◽  
Intermediate Progenitors ◽  
Metastatic Lung ◽  
Signaling Axis ◽  
Localization Signal

ABSTRACTMesendodermal cells are key intermediate progenitors that form at the early primitive streak (PrS) and give rise to mesoderm and endoderm in the gastrulating embryo. We have identified an interaction between CNOT3 and the cell cycle kinase Aurora B that regulates MAPK/ERK signalling during mesendodermal differentiation. Aurora B phosphorylates CNOT3 at two sites that are located close to a nuclear localization signal and promotes nuclear localisation of CNOT3 in mouse ES cells (ESCs) and metastatic lung cancer cells. Mutation of these sites in ESCs gives reduced numbers of embryoid bodies that are largely composed of ectoderm and interferes with differentiation of ESCs into mesendoderm in response to FGF2, BMP4, Wnt3 and Activin. The double mutation affects interaction of CNOT3 with Aurora B and ERK and reduces phosphorylation of ERK in response to FGF2. Our results identify a signalling axis involving CNOT3 that regulates a key pathway in embryogenesis and cancer.

scholarly journals Functional role of Tet-mediated RNA hydroxymethylcytosine in mouse ES cells and during differentiation

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Jie Lan ◽  
Nicholas Rajan ◽  
Martin Bizet ◽  
Audrey Penning ◽  
Nitesh K. Singh ◽  
...  
Keyword(s):  
Es Cells ◽  
Consensus Sequence ◽  
Embryonic Stem ◽  
Embryoid Bodies ◽  
Messenger Rnas ◽  
Related Factors ◽  
Mouse Es Cells ◽  
Mrna Targets ◽  
Tet Enzymes ◽  
And Function

Abstract Tet-enzyme-mediated 5-hydroxymethylation of cytosines in DNA plays a crucial role in mouse embryonic stem cells (ESCs). In RNA also, 5-hydroxymethylcytosine (5hmC) has recently been evidenced, but its physiological roles are still largely unknown. Here we show the contribution and function of this mark in mouse ESCs and differentiating embryoid bodies. Transcriptome-wide mapping in ESCs reveals hundreds of messenger RNAs marked by 5hmC at sites characterized by a defined unique consensus sequence and particular features. During differentiation a large number of transcripts, including many encoding key pluripotency-related factors (such as Eed and Jarid2), show decreased cytosine hydroxymethylation. Using Tet-knockout ESCs, we find Tet enzymes to be partly responsible for deposition of 5hmC in mRNA. A transcriptome-wide search further reveals mRNA targets to which Tet1 and Tet2 bind, at sites showing a topology similar to that of 5hmC sites. Tet-mediated RNA hydroxymethylation is found to reduce the stability of crucial pluripotency-promoting transcripts. We propose that RNA cytosine 5-hydroxymethylation by Tets is a mark of transcriptome flexibility, inextricably linked to the balance between pluripotency and lineage commitment.

Human embryonic stem cells: prototypical pluripotential progenitors

2002 ◽  
Vol 10 (3) ◽  
pp. 187-199 ◽  
Author(s):  
R Mollard ◽  
BJ Conley ◽  
AO Trounson
Keyword(s):  
Inner Cell Mass ◽  
Es Cells ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Embryoid Bodies ◽  
Es Cell ◽  
Germ Layers ◽  
Mouse Es Cells ◽  
Morphological Criteria ◽  
Human Es Cells

Embryonic stem (ES) cells are a primitive cell type derived from the inner cell mass (ICM) of the developing embryo. When cultured for extended periods, ES cells maintain a high telomerase activity, normal karyotype and the pluripotential developmental capacity of their ICM derivatives. Such capacity is best demonstrated by mouse ES cells which can contribute to all tissues of the developing embryo following either their injection into host blastocysts or tetraploid embryo complimentation (for a review see Robertson). For both practical and ethical reasons it is not possible to inject human ES cells into blastocysts for the development of a term fetus. However, when injected beneath the testicular capsule of severe combined immunodeficient (SCID) mice, human ES cells form teratomas comprising tissue representatives of all three embryonic germ layers (ectoderm, mesoderm and endoderm) thus attesting to their pluripotency. Based upon morphological criteria, neuronal, cardiac, bone, squamous epithelium, skeletal muscle, gut and respiratory epithelia are readily identifiable within the human ES-cell-derived teratomas. With the demonstrated capability to isolate and maintain pluripotent human ES cells in vitro, their ability to give rise to tissue representatives of all three embryonic germ layers and the technical advances made possible by research on mouse ES cells, a rapid increase in human ES cell research aimed at drug discovery and human cell and gene therapies has occurred. Indeed in the mouse, dissociated embryoid bodies (EBs) have already been demonstrated capable of repopulating the haematopoietic system of recipient animals (for a review see Keller) and mouse ES cells are currently being used in attempts to repair mouse neural degenerative lesions.

scholarly journals An interplay between extracellular signalling and the dynamics of the exit from pluripotency drives cell fate decisions in mouse ES cells

2013 ◽  
Author(s):  
David A Turner ◽  
Jamie Trott ◽  
Penelope Hayward ◽  
Pau Rué ◽  
Alfonso Martinez Arias
Keyword(s):  
Cell Fate ◽  
Neural Development ◽  
Es Cells ◽  
Initial Period ◽  
Embryonic Stem ◽  
Primitive Streak ◽  
Wnt Signalling ◽  
Dependent Manner ◽  
Cell Fate Decisions ◽  
Mouse Es Cells

Embryonic Stem cells derived from the epiblast tissue of the mammalian blastocyst retain the capability to differentiate into any adult cell type and are able to self-renew indefinitely under appropriate culture conditions. Despite the large amount of knowledge that we have accumulated to date about the regulation and control of self-renewal, efficient directed differentiation into specific tissues remains elusive. In this work, we have analysed in a systematic manner the interaction between the dynamics of loss of pluripotency and Activin/Nodal, BMP4 and Wnt signalling in fate assignment during the early stages of differentiation of mouse ES cells in culture. During the initial period of differentiation, cells exit from pluripotency and enter an Epi-like state. Following this transient stage, and under the influence of Activin/Nodal and BMP signalling, cells face a fate choice between differentiating into neuroectoderm and contributing to Primitive Streak fates. We find that Wnt signalling does not suppress neural development as previously thought and that it aids both fates in a context dependent manner. Our results suggest that as cells exit pluripotency they are endowed with a primary neuroectodermal fate and that the potency to become endomesodermal rises with time. We suggest that this situation translates into a ?race for fates? in which the neuroectodermal fate has an advantage.

scholarly journals Localization of Liv2 as an Immature Hepatocyte Marker in EB Outgrowth

2009 ◽  
Vol 9 ◽  
pp. 190-199 ◽  
Author(s):  
Ikkei Takashimizu ◽  
Yoshiki Tanaka ◽  
Susumu Yoshie ◽  
Yoshiya Kano ◽  
Hinako Ichikawa ◽  
...  
Keyword(s):  
Cell Membrane ◽  
Es Cells ◽  
Embryonic Stem ◽  
Embryoid Bodies ◽  
Surface Marker ◽  
Neuronal Marker ◽  
Mouse Es Cells ◽  
Undifferentiated State ◽  
Hanging Drop Method ◽  
Selection Of

The objective of this study was to establish Liv2, a surface marker of mouse immature hepatocytes (hepatoblasts), as a selection tool for embryonic stem (ES) cell–derived immature hepatocytes by acquiring basic data on Liv2 in normal mouse embryos and by confirming Liv2 expression in mouse ES-derived cells. The estimated molecular weight of Liv2 was 40–45 kDa, and immunoreactivity was definitively detected in the cell membrane of fetal hepatocytes on embryonic day (E) 9.5, declined gradually until E12.5, and subsequently became undetectable. Liv2 was localized on and close to the cell membrane. Embryoid bodies (EB) were formed from mouse ES cells whose undifferentiated state was confirmed with immunostaining of Nanog by the hanging drop method. A few Liv2-positive cells occurred as a cluster in EB outgrowth on day 7, but only some of these were albumin (ALB)-positive on day 13. These cells had the same pattern of immunoreactivity, i.e., localization on the cell membrane, as immature hepatocytes in the developing liver, although there were other types of cells with a different pattern of immunoreactivity that were seen only as a granular pattern in the cytoplasm and without ALB or the neuronal marker nestin. These results suggest that Liv2 may be useful as a surface marker for immature hepatocytes derived from ES cells. This application would allow for the sole selection of immature hepatocytes and provide a useful tool for regenerative medicine.

Developmental Potentials of Human Embryonic Stem Cells Lacking PIG-A and GPI-Anchored Proteins.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1314-1314
Author(s):  
Guibin Chen ◽  
Zhaohui Ye ◽  
Xiaobing Yu ◽  
Robert A. Brodsky ◽  
Linzhao Cheng
Keyword(s):  
Stem Cells ◽  
Cell Surface ◽  
Es Cells ◽  
Surface Expression ◽  
Embryoid Bodies ◽  
Cell Surface Expression ◽  
Hematopoietic Stem ◽  
Body Formation ◽  
Mouse Es Cells ◽  
Hes Cells

Abstract Paroxysmal Nocturnal Hemoglobinuria (PNH) is a clonal disorder of hematopoietic stem cells (HSCs) acquiring mutations in the PIG-A gene. Its product (PIG-A protein) is required for biosynthesis of dozens of cell surface proteins to be linked to glycosyl-phosphatidyl-inositol (GPI) molecule and anchored on plasma membrane. The underlying mechanisms of PIG-A mutant clonal dominance in PNH patients and the close relationship of PNH to other bone marrow failure diseases (aplastic anemia and myelodysplasia syndrome) and leukemia remain unknown. Establishing a mutation of PIG-A gene in human HSCs from healthy donors remains unfeasible due to the current inability to expand and select clonal HSCs in culture. Although mouse models have been generated by disrupting conditionally the mouse Pig-a gene, the existing Pig-a null mice indeed lacking GPI-APs in blood cells did not replicate faithfully PNH pathological symptoms. To create a human cell-based, prospective experimental system to investigate the effects of PIG-A/GPI-AP deficiency and pathophysiology of PNH, we made mutated hES cells lacking GPI-APs. FACS analysis revealed that two independent hES clones lack cell-surface expression of CD55 and CD59, as well as CD90/Thy and Cripto that are preferentially expressed in undifferentiated hES cells. However, the cell-surface expression of these GPI-APs can be restored by a lentiviral vector inducibly expressing the PIG-A cDNA. Like mouse ES cells, lack of PIG-A/GPI-APs did not affect the growth of undifferentiated hES cells in culture. Unlike mouse ES cells, however, PIG-A/GPI-AP deficient hES cells formed embryoid bodies normally in culture. RT-PCR analysis of marker gene expression indicated that commitment to the 3 (somatic) germ layers appeared normal within embryonic bodies from either the mutated or wildtype hES cells. However, formation of extra-embryonic cells such as trophoblasts from the PIG-A/GPI-AP deficient hES cells is defective in both embryoid body formation and BMP4-induced assays. Expression of trophoectoderm-specific genes such as hCGalpha could not be induced in PIG-A/GPI-AP deficient hES cells upon BMP4 induced trophoectodermal differentiation. The induction of other trophoectoderm markers such as hCGbeta and CDX2 was also significantly reduced after the BMP4 treatment. The lack of tropho-ectoderm was further confirmed by lacking of hCG hormone production. The defect in trophoectoderm differentiation from the PIG-A/GPI-AP deficient hES cells was restored by the expression of the PIG-A cDNA in the mutated hES cells. For somatic cell differentiation, we are currently examining the effects of PIG-A/GPI-AP deficiency beyond the initial differentiation commitment during embryonic body formation stage. Along mesodermal and hematopoietic differentiation, we found that the PIG-A/GPI-AP deficiency in hES cells had little effect on the formation of CD34+ cells, a precursor cell population for human hematopoietic and endothelial lineages. We are currently examining the effects of PIG-A/GPI-AP deficiency on properties of hematopoietic cells derived from the mutated and normal hES cells. This study represents one of first cases that hES cells may provide a prospective research model to investigate genetic and developmental basis of human diseases.

scholarly journals Brachyury cooperates with Wnt/β-Catenin signalling to elicit Primitive Streak like behaviour in differentiating mouse ES cells.

2014 ◽  
Author(s):  
David Andrew Turner ◽  
Pau Rué ◽  
Jonathan P Mackenzie ◽  
Eleanor Davies ◽  
Alfonso Martinez Arias
Keyword(s):  
Epithelial To Mesenchymal Transition ◽  
Es Cells ◽  
Single Cells ◽  
Embryonic Stem ◽  
Primitive Streak ◽  
Mouse Development ◽  
Mesenchymal Transition ◽  
Mouse Es Cells ◽  
Cellular Events ◽  
Early Mouse

The formation of the Primitive Streak is the first visible sign of gastrulation, the process by which the three germ layers are formed from a single epithelium during early development. Embryonic Stem Cells (ESCs) provide a good system to understand the molecular and cellular events associated with these processes. Previous work, both in embryos and in culture, has shown how converging signals from both Nodal/TGFβR and Wnt/β-Catenin signalling pathways specify cells to adopt a Primitive Streak like fate and direct them to undertake an epithelial to mesenchymal transition (EMT). However, many of these approaches have relied on genetic analyses without taking into account the temporal progression of events within single cells. In addition, it is still unclear as to what extent events in the embryo are able to be reproduced in culture. Here, we combine flow-cytometry and a quantitative live single-cell imaging approach to demonstrate how the controlled differentiation of mouse ESCs (mESCs) towards a Primitive Streak fate in culture results in cells displaying many of the characteristics observed during early mouse development including transient Brachyury expression, EMT and increased motility. We also find that the EMT initiates the process, and this is both fuelled and terminated by the action of Bra, whose expression is dependent on the EMT and β-Catenin activity. As a consequence of our analysis, we propose that a major output of Brachyury expression is in controlling the velocity of the cells that are transiting out of the Primitive Streak.

scholarly journals Specification of the Retinal Fate of Mouse Embryonic Stem Cells by Ectopic Expression of Rx/rax, a Homeobox Gene

2004 ◽  
Vol 24 (10) ◽  
pp. 4513-4521 ◽  
Author(s):  
Yoko Tabata ◽  
Yasuo Ouchi ◽  
Haruyuki Kamiya ◽  
Toshiya Manabe ◽  
Ken-ichi Arai ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Fluorescent Protein ◽  
Es Cells ◽  
Horizontal Cell ◽  
Ectopic Expression ◽  
Embryonic Stem ◽  
Embryoid Bodies ◽  
Retinal Cells ◽  
Mouse Es Cells ◽  
Explant Cultures

ABSTRACT With the goal of generating retinal cells from mouse embryonic stem (ES) cells by exogenous gene transfer, we introduced the Rx/rax transcription factor, which is expressed in immature retinal cells, into feeder-free mouse ES cells (CCE). CCE cells expressing Rx/rax as well as enhanced green fluorescent protein (CCE-RX/E cells) proliferated and remained in the undifferentiated state in the presence of leukemia inhibitory factor, as did parental ES cells. We made use of mouse embryo retinal explant cultures to address the differentiation ability of grafted ES cells. Dissociated embryoid bodies were treated with retinoic acid for use as donor cells and cocultured with retina explants for 2 weeks. In contrast to the parental CCE cells, which could not migrate into host retinal cultures, CCE-RX/E cells migrated into the host retina and extended their process-like structures between the host retinal cells. Most of the grafted CCE-RX/E cells became located in the ganglion cell and inner plexiform layers and expressed ganglion and horizontal cell markers. Furthermore, these grafted cells had the electrophysiological properties expected of ganglion cells. Our data thus suggest that subpopulations of retinal neurons can be generated in retinal explant cultures from grafted mouse ES cells ectopically expressing the transcription factor Rx/rax.

Gene Targeting

1999 ◽  
Keyword(s):  
Homologous Recombination ◽  
Gene Targeting ◽  
Mammalian Cells ◽  
Es Cells ◽  
Practical Approach ◽  
Simple Method ◽  
Mouse Es Cells ◽  
Cell Clones ◽  
Previous Edition ◽  
Pros And Cons

Since the publication of the first edition of Gene Targeting: A Practical Approach in 1993 there have been many advances in gene targeting and this new edition has been thoroughly updated and rewritten to include all the major new techniques. It provides not only tried-and-tested practical protocols but detailed guidance on their use and applications. As with the previous edition Gene Targeting: A Practical Approach 2e concentrates on gene targeting in mouse ES cells, but the techniques described can be easily adapted to applications in tissue culture including those for human cells. The first chapter covers the design of gene targeting vectors for mammalian cells and describes how to distinguish random integrations from homologous recombination. It is followed by a chapter on extending conventional gene targeting manipulations by using site-specific recombination using the Cre-loxP and Flp-FRT systems to produce 'clean' germline mutations and conditionally (in)activating genes. Chapter 3 describes methods for introducing DNA into ES cells for homologous recombination, selection and screening procedures for identifying and recovering targeted cell clones, and a simple method for establishing new ES cell lines. Chapter 4 discusses the pros and cons or aggregation versus blastocyst injection to create chimeras, focusing on the technical aspects of generating aggregation chimeras and then describes some of the uses of chimeras. The next topic covered is gene trap strategies; the structure, components, design, and modification of GT vectors, the various types of GT screens, and the molecular analysis of GT integrations. The final chapter explains the use of classical genetics in gene targeting and phenotype interpretation to create mutations and elucidate gene functions. Gene Targeting: A Practical Approach 2e will therefore be of great value to all researchers studying gene function.

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