RÉSUMÉ
The embryonic development of the pig comprises a long in utero pre- and peri-implantation development, which dramatically differs from mice and humans. During this peri-implantation period, a complex series of paracrine signals establishes an intimate dialogue between the embryo and the uterus. To better understand the biology of the pig blastocyst during this period, we generated a large dataset of single-cell RNAseq from early and hatched blastocysts, spheroid and ovoid conceptus and proteomic datasets from corresponding uterine fluids. Our results confirm the molecular specificity and functionality of the three main cell populations. We also discovered two previously unknown subpopulations of the trophectoderm, one characterised by the expression of LRP2, which could represent progenitor cells, and the other, expressing pro-apoptotic markers, which could correspond to the Rauber's layer. Our work provides new insights into the biology of these populations, their reciprocal functional interactions, and the molecular dialogue with the maternal uterine environment.
Sujet(s)
Blastocyste , Protéomique , Grossesse , Humains , Femelle , Suidae , Souris , Animaux , Blastocyste/métabolisme , Implantation embryonnaire/physiologie , Développement embryonnaire/génétique , Analyse de profil d'expression de gènesRÉSUMÉ
Generating hematopoietic stem cells (HSCs) from pluripotent stem cells (PSCs) has been a long-lasting quest in the field of hematopoiesis. Previous studies suggested that enforced expression of BCR-ABL, the unique oncogenic driver of chronic myelogeneous leukemia (CML), in embryonic stem cells (ESCs)-derived hematopoietic cells is sufficient to confer long-term in vivo repopulating potential. To precisely uncover the molecular events regulated by the tyrosine kinase activity of BCR-ABL1 (p210) during the course of hematopoietic differentiation, we engineered a Tet-ON inducible system to modulate its expression in murine ESCs (mESCs). We showed in unique site-directed knock-in ESC model that BCR-ABL expression tightly regulated by doxycycline (dox) controls the formation and the maintenance of immature hematopoietic progenitors. Interestingly, these progenitors can be expanded in vitro for several passages in the presence of dox. Our analysis of cell surface markers and transcriptome compared with wild-type fetal and adult HSCs unraveled a similar molecular signature. Long-term culture initiating cell (LTC-IC) assay confirmed their self-renewal capacities albeit with a differentiation bias toward erythroid and myeloid cells. Collectively, our novel Tet-ON system represents a unique in vitro model to shed lights on ESC-derived hematopoiesis, CML initiation, and maintenance.
Sujet(s)
Leucémie myéloïde chronique BCR-ABL positive , Souris , Animaux , Leucémie myéloïde chronique BCR-ABL positive/génétique , Leucémie myéloïde chronique BCR-ABL positive/métabolisme , Protéines de fusion bcr-abl/génétique , Protéines de fusion bcr-abl/métabolisme , Cellules souches hématopoïétiques/métabolisme , Différenciation cellulaire , Cellules souches embryonnaires/métabolisme , Doxycycline/pharmacologie , Doxycycline/métabolismeRÉSUMÉ
Chronic myeloid leukemia (CML) is a clonal hematopoietic malignancy driven by the BCR-ABL1 fusion oncoprotein. The development of tyrosine kinase inhibitors (TKIs) has deeply increased long-term survival of CML patients. Nonetheless, one patient out of four will switch TKI off owing either to drug intolerance or resistance partly due to amplification or mutations of BCR-ABL1 oncogene and alteration in ATP-binding cassette (ABC) transporters. Increasing evidence suggests the involvement of the microRNA miR-495-3p in cancer-associated chemoresistance through multidrug resistance 1 (MDR1) gene, which encodes an ATP-dependent efflux pump. Our study aimed at investigating the potential role of miR-495-3p in CML TKI chemo-sensitivity and determining the underlying molecular circuitry involved. We first observed that miR-495-3p expression was lower in BCR-ABL1-expressing cellular models in vitro. Notably, loss-of-function experiments showed increased proliferation associated with a decreased number of nondividing cells (G0/G1) and resistance to Imatinib. Conversely, our data showed that miR-495-3p overexpression hindered leukemic cell growth and TKI resistance in Imatinib-resistant T315I-mutant cells, as well as drug efflux activity through MDR1 regulation. Further investigating the role of miR-495-3p in CML patients, we found that predicted miR-495-3p targets were upregulated in patients in blast crisis that were involved in protein phosphorylation and associated with the worst prognosis. Taken together, our results demonstrate that downregulation of miR-495-3p expression is important in the malignant phenotype of CML and TKI resistance mechanisms and could be a useful biomarker and a potential therapeutic target to eradicate CML.
Sujet(s)
Leucémie myéloïde chronique BCR-ABL positive , microARN , Humains , Mésilate d'imatinib/pharmacologie , Protéines de fusion bcr-abl/génétique , Protéines de fusion bcr-abl/métabolisme , Inhibiteurs de protéines kinases/pharmacologie , Inhibiteurs de protéines kinases/usage thérapeutique , Lignée cellulaire tumorale , Résistance aux médicaments antinéoplasiques/génétique , Leucémie myéloïde chronique BCR-ABL positive/traitement médicamenteux , Leucémie myéloïde chronique BCR-ABL positive/génétique , Leucémie myéloïde chronique BCR-ABL positive/anatomopathologie , microARN/génétique , microARN/métabolisme , Multirésistance aux médicaments , Adénosine triphosphateRÉSUMÉ
Cancer is maintained by the activity of a rare population of self-renewing "cancer stem cells" (CSCs), which are resistant to conventional therapies. CSCs over-express several proteins shared with induced pluripotent stem cells (iPSCs). We show here that allogenic or autologous murine iPSCs, combined with a histone deacetylase inhibitor (HDACi), are able to elicit major anti-tumor responses in a highly aggressive triple-negative breast cancer, as a relevant cancer stemness model. This immunotherapy strategy was effective in preventing tumor establishment and efficiently targeted CSCs by inducing extensive modifications of the tumor microenvironment. The anti-tumoral effect was correlated with the generation of CD4+, CD8+ T cells, and CD44+ CD62L- CCR7low CD127low T-effector memory cells, and the reduction of CD4+ CD25+FoxP3+ Tregs, Arg1+ CD11b+ Gr1+, and Arg1+ and CD11b+ Ly6+ myeloid-derived suppressor cell populations within the tumor. The anti-tumoral effect was associated with a reduction in metastatic dissemination and an improvement in the survival rate. These results demonstrate for the first time the clinical relevance of using an off-the-shelf allogeneic iPSC-based vaccine combined with an HDACi as a novel pan-cancer anti-cancer immunotherapy strategy against aggressive tumors harboring stemness features with high metastatic potential.
RÉSUMÉ
In ungulates, early embryonic development differs dramatically from that of mice and humans and is characterized by an extended period of pre- and peri-implantation development in utero. After hatching from the zona pellucida, the ungulate blastocyst will stay free in the uterus for many days before implanting within the uterine wall. During this protracted peri-implantation period, an intimate dialog between the embryo and the uterus is established through a complex series of paracrine signals. The blastocyst elongates, leading to extreme growth of extra-embryonic tissues, and at the same time, the inner cell mass moves up into the trophoblast and evolves into the embryonic disc, which is directly exposed to molecules present in the uterine fluids. In the peri-implantation period, uterine glands secrete a wide range of molecules, including enzymes, growth factors, adhesion proteins, cytokines, hormones, and nutrients like amino and fatty acids, which are collectively referred to as histotroph. The identification, role, and effects of these secretions on the biology of the conceptus are still being described; however, the studies that have been conducted to date have demonstrated that histotroph is essential for embryonic development and serves a critical function during the pre- and peri implantation periods. Here, we present an overview of current knowledge on the molecular dialogue among embryonic, extraembryonic, and maternal tissues prior to implantation. Taken together, the body of work described here demonstrates the extent to which this dialog enables the coordination of the development of the conceptus with respect to the establishment of embryonic and extra-embryonic tissues as well as in preparation for implantation.
Sujet(s)
Artiodactyla/embryologie , Blastocyste/physiologie , Développement embryonnaire , Perissodactyla/embryologie , Utérus/physiologie , Animaux , FemelleRÉSUMÉ
Receptor tyrosine kinase signaling pathways are key regulators for the formation of the primitive endoderm (PrE) and the epiblast (Epi) from the inner cell mass (ICM) of the mouse preimplantation embryo. Among them, FGF signaling is critical for PrE cell specification, whereas PDGF signaling is critical for the survival of committed PrE cells. Here, we investigated possible functional redundancies among FGF, PDGF, and KIT signaling and showed that only PDGF signaling is involved in PrE cell survival. In addition, we analyzed the effectors downstream of PDGFRα. Our results suggest that the role of PDGF signaling in PrE cell survival is mediated through PI3K-mTOR and independently from p53. Lastly, we uncovered a role for PI3K-mTOR signaling in the survival of Epi cells. Taken together, we propose that survival of ICM cell lineages relies on the regulation of PI3K-mTOR signaling through the regulation of multiple signaling pathways. Stem Cells 2019;37:888-898.
Sujet(s)
Cellules de la masse interne du blastocyste/métabolisme , Lignage cellulaire/génétique , Endoderme/métabolisme , Régulation de l'expression des gènes au cours du développement , Phosphatidylinositol 3-kinases/génétique , Facteur de croissance dérivé des plaquettes/génétique , Sérine-thréonine kinases TOR/génétique , Animaux , Blastocyste , Cellules de la masse interne du blastocyste/cytologie , Survie cellulaire , Endoderme/cytologie , Endoderme/croissance et développement , Femelle , Facteurs de croissance fibroblastique/génétique , Facteurs de croissance fibroblastique/métabolisme , Mâle , Souris , Phosphatidylinositol 3-kinases/métabolisme , Facteur de croissance dérivé des plaquettes/métabolisme , Protéines proto-oncogènes c-kit/génétique , Protéines proto-oncogènes c-kit/métabolisme , Récepteur au PDGF alpha/génétique , Récepteur au PDGF alpha/métabolisme , Transduction du signal , Sérine-thréonine kinases TOR/métabolisme , Protéine p53 suppresseur de tumeur/génétique , Protéine p53 suppresseur de tumeur/métabolismeRÉSUMÉ
Aryl Hydrocarbon Receptor (AHR) is an ubiquitous basic helix-loop-helix transcription factor, which is ligand-activated and involved in numerous biological processes including cell division, cell quiescence and inflammation. It has been shown that AHR is involved in normal hematopoietic progenitor proliferation in human cells. In addition, loss of AHR in knockout mice is accompanied by a myeloproliferative syndrome-like disease, suggesting a role of AHR in hematopoietic stem cell (HSC) maintenance. To study the potential role of AHR pathway in CML progenitors and stem cells, we have first evaluated the expression of AHR in UT-7 cell line expressing BCR-ABL. AHR expression was highly reduced in UT-7 cell expressing BCR-ABL as compared to controls. AHR transcript levels, quantified in primary peripheral blood CML cells at diagnosis (n = 31 patients) were found to be significantly reduced compared to healthy controls (n = 15). The use of StemRegenin (SR1), an AHR antagonist, induced a marked expansion of total leukemic cells and leukemic CD34+ cells by about 4- and 10-fold respectively. SR1-treated CML CD34+ cells generated more colony-forming cells and long-term culture initiating cell (LTC-IC)-derived progenitors as compared to non-SR1-treated counterparts. Conversely, treatment of CML CD34+ cells with FICZ, a natural agonist of AHR, induced a 3-fold decrease in the number of CD34+ cells in culture after 7 days. Moreover, a 4-day FICZ treatment was sufficient to significantly reduce the clonogenic potential of CML CD34+ cells and this effect was synergized by Imatinib and Dasatinib treatments. Similarly, a 3-day FICZ treatment contributed to hinder significantly the number of LTC-IC-derived progenitors without synergistic effect with Imatinib. The analysis of molecular circuitry of AHR signaling in CML showed a transcriptional signature in CML derived CD34+ CD38- primitive cells with either low or high levels of AHR, with an upregulation of myeloid genes involved in differentiation in the "AHR low" fraction and an upregulation of genes involved in stem cell maintenance in the "AHR high" fraction. In conclusion, these findings demonstrate for the first time that down-regulation of AHR expression, a major cell cycle regulator, is involved in the myeloproliferative phenotype associated with CML. AHR agonists inhibit clonogenic and LTC-IC-derived progenitor growth and they could be used in leukemic stem cell targeting in CML.
Sujet(s)
Facteurs de transcription à motif basique hélice-boucle-hélice/métabolisme , Leucémie myéloïde chronique BCR-ABL positive/métabolisme , Cellules souches tumorales/métabolisme , Récepteurs à hydrocarbure aromatique/métabolisme , Facteurs de transcription à motif basique hélice-boucle-hélice/agonistes , Facteurs de transcription à motif basique hélice-boucle-hélice/génétique , Carbazoles/pharmacologie , Études cas-témoins , Lignée cellulaire tumorale , Prolifération cellulaire/effets des médicaments et des substances chimiques , Protéines de fusion bcr-abl/génétique , Protéines de fusion bcr-abl/métabolisme , Régulation de l'expression des gènes tumoraux/effets des médicaments et des substances chimiques , Humains , Leucémie myéloïde chronique BCR-ABL positive/traitement médicamenteux , Leucémie myéloïde chronique BCR-ABL positive/génétique , Cellules souches tumorales/effets des médicaments et des substances chimiques , Cellules souches tumorales/anatomopathologie , Purines/pharmacologie , ARN messager/génétique , ARN messager/métabolisme , ARN tumoral/génétique , ARN tumoral/métabolisme , Récepteurs à hydrocarbure aromatique/agonistes , Récepteurs à hydrocarbure aromatique/génétique , Transduction du signal/effets des médicaments et des substances chimiques , Test clonogénique de cellules souches tumoralesRÉSUMÉ
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 Fibroblast Growth Factor (FGF)/Extracellular signal-Regulated Kinase (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.
Sujet(s)
Cellules de la masse interne du blastocyste/physiologie , Différenciation cellulaire/physiologie , Facteurs de croissance fibroblastique/métabolisme , Feuillets embryonnaires/physiologie , Système de signalisation des MAP kinases/physiologie , Animaux , Plasticité cellulaire/physiologie , Souris , Protéolyse , Facteurs temps , Transcription génétique/physiologieRÉSUMÉ
During early development, the mammalian embryo undergoes a series of profound changes that lead to the formation of two extraembryonic tissues--the trophectoderm and the primitive endoderm. These tissues encapsulate the pluripotent epiblast at the time of implantation. The current model proposes that the formation of these lineages results from two consecutive binary cell fate decisions. The first controls the formation of the trophectoderm and the inner cell mass, and the second controls the formation of the primitive endoderm and the epiblast within the inner cell mass. While early mammalian embryos develop with extensive plasticity, the embryonic pattern prior to implantation is remarkably reproducible. Here, we review the molecular mechanisms driving the cell fate decision between primitive endoderm and epiblast in the mouse embryo and integrate data from recent studies into the current model of the molecular network regulating the segregation between these lineages and their subsequent differentiation.
Sujet(s)
Blastocyste/physiologie , Souris/embryologie , Animaux , Blastocyste/cytologie , Cellules de la masse interne du blastocyste/cytologie , Cellules de la masse interne du blastocyste/métabolisme , Blastomères/physiologie , Différenciation cellulaire , Lignage cellulaire , Mouvement cellulaire , Cellules cultivées , Cellules souches embryonnaires/cytologie , Endoderme/cytologie , Protéines foetales/physiologie , Facteurs de transcription GATA/physiologie , Régulation de l'expression des gènes au cours du développement , Protéines à homéodomaine/physiologie , Mammifères/génétique , Morphogenèse , Morula/cytologie , Morula/physiologie , Protéine homéotique Nanog , Spécificité d'espèceRÉSUMÉ
At the end of the preimplantation period, the inner cell mass (ICM) of the mouse blastocyst is composed of two distinct cell lineages, the pluripotent epiblast (EPI) and the primitive endoderm (PrE). The current model for their formation involves initial co-expression of lineage-specific markers followed by mutual-exclusive expression resulting in a salt-and-pepper distribution of lineage precursors within the ICM. Subsequent to lineage commitment, cell rearrangements and selective apoptosis are thought to be key processes driving and refining the emergence of two spatially distinct compartments. Here, we have addressed a role for Platelet Derived Growth Factor (PDGF) signaling in the regulation of programmed cell death during early mouse embryonic development. By combining genetic and pharmacological approaches, we demonstrate that embryos lacking PDGF activity exhibited caspase-dependent selective apoptosis of PrE cells. Modulating PDGF activity did not affect lineage commitment or cell sorting, suggesting that PDGF is involved in the fine-tuning of patterning information. Our results also indicate that PDGF and fibroblast growth factor (FGF) tyrosine kinase receptors exert distinct and non-overlapping functions in PrE formation. Taken together, these data uncover an early role of PDGF signaling in PrE cell survival at the time when PrE and EPI cells are segregated.
Sujet(s)
Cellules de la masse interne du blastocyste/cytologie , Cellules de la masse interne du blastocyste/métabolisme , Endoderme/cytologie , Endoderme/métabolisme , Facteur de croissance dérivé des plaquettes/métabolisme , Transduction du signal , Animaux , Benzamides/pharmacologie , Cellules de la masse interne du blastocyste/effets des médicaments et des substances chimiques , Inhibiteurs des caspases/pharmacologie , Mort cellulaire/effets des médicaments et des substances chimiques , Lignage cellulaire/effets des médicaments et des substances chimiques , Survie cellulaire/effets des médicaments et des substances chimiques , Développement embryonnaire/effets des médicaments et des substances chimiques , Endoderme/effets des médicaments et des substances chimiques , Cytométrie en flux , Humains , Imagerie tridimensionnelle , Mésilate d'imatinib , Ligands , Souris , Phénotype , Pipérazines/pharmacologie , Pyrimidines/pharmacologie , Récepteur au PDGF alpha/métabolisme , Transduction du signal/effets des médicaments et des substances chimiquesRÉSUMÉ
The emergence of pluripotent epiblast (EPI) and primitive endoderm (PrE) lineages within the inner cell mass (ICM) of the mouse blastocyst involves initial co-expression of lineage-associated markers followed by mutual exclusion and salt-and-pepper distribution of lineage-biased cells. Precisely how EPI and PrE cell fate commitment occurs is not entirely clear; however, previous studies in mice have implicated FGF/ERK signaling in this process. Here, we investigated the phenotype resulting from zygotic and maternal/zygotic inactivation of Fgf4. Fgf4 heterozygous blastocysts exhibited increased numbers of NANOG-positive EPI cells and reduced numbers of GATA6-positive PrE cells, suggesting that FGF signaling is tightly regulated to ensure specification of the appropriate numbers of cells for each lineage. Although the size of the ICM was unaffected in Fgf4 null mutant embryos, it entirely lacked a PrE layer and exclusively comprised NANOG-expressing cells at the time of implantation. An initial period of widespread EPI and PrE marker co-expression was however established even in the absence of FGF4. Thus, Fgf4 mutant embryos initiated the PrE program but exhibited defects in its restriction phase, when lineage bias is acquired. Consistent with this, XEN cells could be derived from Fgf4 mutant embryos in which PrE had been restored and these cells appeared indistinguishable from wild-type cells. Sustained exogenous FGF failed to rescue the mutant phenotype. Instead, depending on concentration, we noted no effect or conversion of all ICM cells to GATA6-positive PrE. We propose that heterogeneities in the availability of FGF produce the salt-and-pepper distribution of lineage-biased cells.
Sujet(s)
Endoderme/physiologie , Facteur de croissance fibroblastique de type 4/métabolisme , Régulation de l'expression des gènes au cours du développement , Animaux , Blastocyste/cytologie , Techniques de culture cellulaire/méthodes , Différenciation cellulaire , Lignage cellulaire , Facteur de transcription GATA-6/métabolisme , Marqueurs génétiques , Feuillets embryonnaires/cytologie , Hétérozygote , Protéines à homéodomaine/métabolisme , Traitement d'image par ordinateur , Souris , Mutation , Protéine homéotique Nanog , Transduction du signalRÉSUMÉ
Recent studies have shown that factors involved in transcription-coupled mRNA processing are important for the maintenance of genome integrity. How these processes are linked and regulated in vivo remains largely unknown. In this study, we addressed in the mouse model the function of Omcg1, which has been shown to participate in co-transcriptional processes, including splicing and transcription-coupled repair. Using inducible mouse models, we found that Omcg1 is most critically required in intestinal progenitors. In absence of OMCG1, proliferating intestinal epithelial cells underwent abnormal mitosis followed by apoptotic cell death. As a consequence, the crypt proliferative compartment of the small intestine was quickly and totally abrogated leading to the rapid death of the mice. Lack of OMCG1 in embryonic stem cells led to a similar cellular phenotype, with multiple mitotic defects and rapid cell death. We showed that mutant intestinal progenitors and embryonic stem cells exhibited a reduced cell cycle arrest following irradiation, suggesting that mitotic defects may be consecutive to M phase entry with unrepaired DNA damages. These findings unravel a crucial role for pre-mRNA processing in the homeostasis of the small intestine and point to a major role of OMCG1 in the maintenance of genome integrity.
RÉSUMÉ
The inner cell mass of the mouse pre-implantation blastocyst comprises epiblast progenitor and primitive endoderm cells of which cognate embryonic (mESCs) or extra-embryonic (XEN) stem cell lines can be derived. Importantly, each stem cell type retains the defining properties and lineage restriction of their in vivo tissue of origin. Recently, we demonstrated that XEN-like cells arise within mESC cultures. This raises the possibility that mESCs can generate self-renewing XEN cells without the requirement for gene manipulation. We have developed a novel approach to convert mESCs to XEN cells (cXEN) using growth factors. We confirm that the downregulation of the pluripotency transcription factor Nanog and the expression of primitive endoderm-associated genes Gata6, Gata4, Sox17 and Pdgfra are necessary for cXEN cell derivation. This approach highlights an important function for Fgf4 in cXEN cell derivation. Paracrine FGF signalling compensates for the loss of endogenous Fgf4, which is necessary to exit mESC self-renewal, but not for XEN cell maintenance. Our cXEN protocol also reveals that distinct pluripotent stem cells respond uniquely to differentiation promoting signals. cXEN cells can be derived from mESCs cultured with Erk and Gsk3 inhibitors (2i), and LIF, similar to conventional mESCs. However, we find that epiblast stem cells (EpiSCs) derived from the post-implantation embryo are refractory to cXEN cell establishment, consistent with the hypothesis that EpiSCs represent a pluripotent state distinct from mESCs. In all, these findings suggest that the potential of mESCs includes the capacity to give rise to both extra-embryonic and embryonic lineages.
Sujet(s)
Cellules souches embryonnaires/cytologie , Endoderme/cytologie , Endoderme/embryologie , Cellules souches pluripotentes/cytologie , Activines/administration et posologie , Animaux , Protéine morphogénétique osseuse de type 4/pharmacologie , Différenciation cellulaire/effets des médicaments et des substances chimiques , Lignage cellulaire , Cellules cultivées , Cellules souches embryonnaires/effets des médicaments et des substances chimiques , Cellules souches embryonnaires/métabolisme , Endoderme/métabolisme , Facteur de croissance fibroblastique de type 4/déficit , Facteur de croissance fibroblastique de type 4/génétique , Facteur de croissance fibroblastique de type 4/métabolisme , Facteur de transcription GATA-4/génétique , Facteur de transcription GATA-6/génétique , Régulation de l'expression des gènes au cours du développement , Protéines HMGB/génétique , Souris , Souris knockout , Souris transgéniques , Modèles biologiques , Communication paracrine , Cellules souches pluripotentes/effets des médicaments et des substances chimiques , Cellules souches pluripotentes/métabolisme , Récepteur au PDGF alpha/génétique , Facteurs de transcription SOX-F/génétique , Trétinoïne/administration et posologieRÉSUMÉ
The initial period of mammalian embryonic development is primarily devoted to cell commitment to the pluripotent lineage, as well as to the formation of extraembryonic tissues essential for embryo survival in utero. This phase of development is also characterized by extensive morphological transitions. Cells within the preimplantation embryo exhibit extraordinary cell plasticity and adaptation in response to experimental manipulation, highlighting the use of a regulative developmental strategy rather than a predetermined one resulting from the non-uniform distribution of maternal information in the cytoplasm. Consequently, early mammalian development represents a useful model to study how the three primary cell lineages; the epiblast, primitive endoderm (also referred to as the hypoblast) and trophoblast, emerge from a totipotent single cell, the zygote. In this review, we will discuss how the isolation and genetic manipulation of murine stem cells representing each of these three lineages has contributed to our understanding of the molecular basis of early developmental events.
Sujet(s)
Blastocyste/physiologie , Développement embryonnaire , Endoderme/physiologie , Feuillets embryonnaires/physiologie , Cellules souches pluripotentes/physiologie , Trophoblastes/physiologie , Animaux , Différenciation cellulaire , Lignage cellulaire , SourisRÉSUMÉ
The visceral endoderm (VE) is an epithelial tissue in the early postimplantation mouse embryo that encapsulates the pluripotent epiblast distally and the extraembryonic ectoderm proximally. In addition to facilitating nutrient exchange before the establishment of a circulation, the VE is critical for patterning the epiblast. Since VE is derived from the primitive endoderm (PrE) of the blastocyst, and PrE-derived eXtraembryonic ENdoderm (XEN) cells can be propagated in vitro, XEN cells should provide an important tool for identifying factors that direct VE differentiation. In this study, we demonstrated that BMP4 signaling induces the formation of a polarized epithelium in XEN cells. This morphological transition was reversible, and was associated with the acquisition of a molecular signature comparable to extraembryonic (ex) VE. Resembling exVE which will form the endoderm of the visceral yolk sac, BMP4-treated XEN cells regulated hematopoiesis by stimulating the expansion of primitive erythroid progenitors. We also observed that LIF exerted an antagonistic effect on BMP4-induced XEN cell differentiation, thereby impacting the extrinsic conditions used for the isolation and maintenance of XEN cells in an undifferentiated state. Taken together, our data suggest that XEN cells can be differentiated towards an exVE identity upon BMP4 stimulation and therefore represent a valuable tool for investigating PrE lineage differentiation.
Sujet(s)
Plan d'organisation du corps/effets des médicaments et des substances chimiques , Protéine morphogénétique osseuse de type 4/pharmacologie , Endoderme/effets des médicaments et des substances chimiques , Endoderme/embryologie , Membranes extraembryonnaires/cytologie , Transduction du signal/effets des médicaments et des substances chimiques , Viscères/embryologie , Animaux , Plan d'organisation du corps/génétique , Polarité de la cellule/effets des médicaments et des substances chimiques , Forme de la cellule/effets des médicaments et des substances chimiques , Régulation négative/effets des médicaments et des substances chimiques , Endoderme/cytologie , Épithélium/effets des médicaments et des substances chimiques , Épithélium/embryologie , Épithélium/métabolisme , Précurseurs érythroïdes/cytologie , Précurseurs érythroïdes/effets des médicaments et des substances chimiques , Précurseurs érythroïdes/métabolisme , Membranes extraembryonnaires/effets des médicaments et des substances chimiques , Membranes extraembryonnaires/embryologie , Régulation de l'expression des gènes au cours du développement/effets des médicaments et des substances chimiques , Protéines à fluorescence verte/métabolisme , Facteur inhibiteur de la leucémie/pharmacologie , Souris , Séquençage par oligonucléotides en batterie , Transduction du signal/génétique , Régulation positive/effets des médicaments et des substances chimiques , Viscères/cytologie , Viscères/effets des médicaments et des substances chimiquesRÉSUMÉ
Mammalian cells have three ATP-dependent DNA ligases, which are required for DNA replication and repair. Homologues of ligase I (Lig1) and ligase IV (Lig4) are ubiquitous in Eukarya, whereas ligase III (Lig3), which has nuclear and mitochondrial forms, appears to be restricted to vertebrates. Lig3 is implicated in various DNA repair pathways with its partner protein Xrcc1 (ref. 1). Deletion of Lig3 results in early embryonic lethality in mice, as well as apparent cellular lethality, which has precluded definitive characterization of Lig3 function. Here we used pre-emptive complementation to determine the viability requirement for Lig3 in mammalian cells and its requirement in DNA repair. Various forms of Lig3 were introduced stably into mouse embryonic stem (mES) cells containing a conditional allele of Lig3 that could be deleted with Cre recombinase. With this approach, we find that the mitochondrial, but not nuclear, Lig3 is required for cellular viability. Although the catalytic function of Lig3 is required, the zinc finger (ZnF) and BRCA1 carboxy (C)-terminal-related (BRCT) domains of Lig3 are not. Remarkably, the viability requirement for Lig3 can be circumvented by targeting Lig1 to the mitochondria or expressing Chlorella virus DNA ligase, the minimal eukaryal nick-sealing enzyme, or Escherichia coli LigA, an NAD(+)-dependent ligase. Lig3-null cells are not sensitive to several DNA-damaging agents that sensitize Xrcc1-deficient cells. Our results establish a role for Lig3 in mitochondria, but distinguish it from its interacting protein Xrcc1.
Sujet(s)
DNA ligases/métabolisme , Réparation de l'ADN , ADN mitochondrial/métabolisme , Protéines de liaison à l'ADN/métabolisme , Mitochondries/enzymologie , Mitochondries/génétique , Animaux , Biocatalyse , Survie cellulaire , Altération de l'ADN , DNA ligase ATP , DNA ligases/composition chimique , DNA ligases/déficit , DNA ligases/génétique , Cellules souches embryonnaires/métabolisme , Gènes essentiels , Test de complémentation , Humains , Souris , Mitochondries/anatomopathologie , Protéines liant le poly-adp-ribose , Structure tertiaire des protéines , Échange de chromatides soeurs/effets des médicaments et des substances chimiques , Protéine-1 de complémentation croisée de la réparation des lésions induites par les rayons X , Protéines de XénopeRÉSUMÉ
Cells of the primitive endoderm (PrE) and the pluripotent epiblast (EPI), the two lineages specified within the inner cell mass (ICM) of the mouse blastocyst stage embryo, are segregated into adjacent tissue layers by the end of the preimplantation period. The PrE layer which emerges as a polarized epithelium adjacent to the blastocoel, with a basement membrane separating it from the EPI, has two derivatives, the visceral and parietal endoderm. In this study we have investigated the localization of two transcriptional regulators of the SOX family, SOX17 and SOX7, within the PrE and its derivatives. We noted that SOX17 was first detected in a salt-and-pepper distribution within the ICM, subsequently becoming restricted to the nascent PrE epithelium. This dynamic distribution of SOX17 resembled the localization of GATA6 and GATA4, two other PrE lineage-specific transcription factors. By contrast, SOX7 was only detected in PrE cells positioned in contact with the blastocoel, raising the possibility that these cells are molecularly distinct. Our observations support a model of sequential GATA6 > SOX17 > GATA4 > SOX7 transcription factor activation within the PrE lineage, perhaps correlating with the consecutive periods of cell lineage 'naïvete', commitment and sorting. Furthermore our data suggest that co-expression of SOX17 and SOX7 within sorted PrE cells could account for the absence of a detectable phenotype of Sox17 mutant blastocysts. However, analysis of implantation-delayed blastocysts, revealed a role for SOX17 in the maintenance of PrE epithelial integrity, with the absence of SOX17 leading to premature delamination and migration of parietal endoderm.
Sujet(s)
Blastocyste/physiologie , Différenciation cellulaire , Lignage cellulaire , Endoderme/physiologie , Protéines HMGB/physiologie , Facteurs de transcription SOX-F/physiologie , Facteurs de transcription/physiologie , Animaux , Blastocyste/cytologie , Mouvement cellulaire , Polarité de la cellule , Endoderme/cytologie , Femelle , Protéines HMGB/analyse , Mâle , Souris , Souris de lignée ICR , Facteurs de transcription SOX-F/analyse , Facteurs de transcription SOX-F/génétiqueRÉSUMÉ
From the hybrid creatures of the Greek and Egyptian mythologies, the concept of the chimera has evolved and, in modern day biology, refers to an organism comprises of at least two populations of genetically distinct cells. Mouse chimeras have proven an invaluable tool for the generation of genetically modified strains. In addition, chimeras have been extensively used in developmental biology as a powerful tool to analyze the phenotype of specific mutations, to attribute function to gene products and to address the question of cell autonomy versus noncell autonomy of gene function. This chapter describes a simple and economical technique used to generate mouse chimeras by embryo aggregation. Multiple aggregation combinations are described each of which can be tailored to answer particular biological questions.
Sujet(s)
Diploïdie , Embryon de mammifère/cytologie , Cellules souches embryonnaires/cytologie , Cellules souches embryonnaires/métabolisme , Tétraploïdie , Animaux , Chimère , Femelle , Mâle , SourisRÉSUMÉ
During S phase, the replisome has to overcome many physical obstacles that can cause replication fork stalling and compromise genome integrity. Transcription is an important source of replicative stress and consequently, maintenance of genome integrity requires the protection of chromosomes from the deleterious effects arising from the interaction between nascent RNAs and template DNA, leading to stable DNA-RNA hybrids (R-loop) formation. We previously reported the essential role of Omcg1 (Ovum Mutant Candidate Gene) for cell cycle progression during early embryonic development. Here, we show that OMCG1 is a target of the cell cycle checkpoint kinases ATR/ATM and is essential for S phase progression in mouse embryonic fibroblasts. Using a conditional gene inactivation strategy, we demonstrate that OMCG1 depletion impairs cell viability as a consequence of DSB formation, checkpoint activation and replication fork collapse. We also show that no chromosome breaks were generated in non-cycling Omcg1-deficient cells. Furthermore, increased RNaseH expression significantly alleviated genomic instability in deficient fibroblasts suggesting that cotranscriptional R-loops formation contributes to the genesis of replication-dependent DSBs in these cells. Together with recent reports describing its participation to complexes involved in cotanscriptional processes, our results suggest that OMCG1 plays a role in the tight coupling between mRNA processing pathways and maintenance of genome integrity during cell cycle progression.
Sujet(s)
Chimère/génétique , Réplication de l'ADN/génétique , Instabilité du génome/génétique , Protéines nucléaires/déficit , ARN messager/génétique , Animaux , Protéines du cycle cellulaire/génétique , Protéines du cycle cellulaire/physiologie , Survie cellulaire/génétique , Cellules cultivées , Femelle , Souris , Cellules NIH 3T3 , Protéines nucléaires/génétique , Protéines nucléaires/physiologie , Grossesse , ARN messager/physiologieRÉSUMÉ
BACKGROUND: Initial specification of cardiomyocytes in the mouse results from interactions between the extraembryonic anterior visceral endoderm (AVE) and the nascent mesoderm. However the mechanism by which AVE activates cardiogenesis is not well understood, and the identity of specific cardiogenic factors in the endoderm remains elusive. Most mammalian studies of the cardiogenic potential of the endoderm have relied on the use of cell lines that are similar to the heart-inducing AVE. These include the embryonal-carcinoma-derived cell lines, END2 and PYS2. The recent development of protocols to isolate eXtraembryonic ENdoderm (XEN) stem cells, representing the extraembryonic endoderm lineage, from blastocyst stage mouse embryos offers new tools for the genetic dissection of cardiogenesis. METHODOLOGY/PRINCIPAL FINDINGS: Here, we demonstrate that XEN cell-conditioned media (CM) enhances cardiogenesis during Embryoid Body (EB) differentiation of mouse embryonic stem (ES) cells in a manner comparable to PYS2-CM and END2-CM. Addition of CM from each of these three cell lines enhanced the percentage of EBs that formed beating areas, but ultimately, only XEN-CM and PYS2-CM increased the total number of cardiomyocytes that formed. Furthermore, our observations revealed that both contact-independent and contact-dependent factors are required to mediate the full cardiogenic potential of the endoderm. Finally, we used gene array comparison to identify factors in these cell lines that could mediate their cardiogenic potential. CONCLUSIONS/SIGNIFICANCE: These studies represent the first step in the use of XEN cells as a molecular genetic tool to study cardiomyocyte differentiation. Not only are XEN cells functionally similar to the heart-inducing AVE, but also can be used for the genetic dissection of the cardiogenic potential of AVE, since they can be isolated from both wild type and mutant blastocysts. These studies further demonstrate the importance of both contact-dependent and contact-independent factors in cardiogenesis and identify potential heart-inducing proteins in the endoderm.