RESUMEN
Mouse embryonic stem cells have an inherent propensity to explore gene regulatory states associated with either self-renewal or differentiation. This property depends on ERK, which downregulates pluripotency genes such as Nanog. Here, we aimed at identifying repressive histone modifications that would mark Nanog for inactivation in response to ERK activity. We found that the transcription factor ZFP57, which binds methylated DNA to nucleate heterochromatin, is recruited upstream of Nanog, within a region enriched for histone H3 lysine 9 tri-methylation (H3K9me3). Whereas before differentiation H3K9me3 at Nanog depends on ERK, in somatic cells it becomes independent of ERK. Moreover, the loss of H3K9me3 at Nanog, induced by deleting the region or by knocking out DNA methyltransferases or Zfp57, is associated with reduced heterogeneity of NANOG, delayed commitment into differentiation and impaired ability to acquire a primitive endoderm fate. Hence, a network axis centred on DNA methylation, ZFP57 and H3K9me3 links Nanog regulation to ERK activity for the timely establishment of new cell identities. We suggest that establishment of irreversible H3K9me3 at specific master regulators allows the acquisition of particular cell fates during differentiation.
Asunto(s)
Células Madre Embrionarias , Endodermo , Código de Histonas , Proteína Homeótica Nanog/genética , Animales , Diferenciación Celular , Endodermo/metabolismo , Genes Homeobox , Ratones , Proteína Homeótica Nanog/metabolismoRESUMEN
Histone H3 Lysine 9 (H3K9) methylation, a characteristic mark of heterochromatin, is progressively implemented during development to contribute to cell fate restriction as differentiation proceeds. Accordingly, in undifferentiated and pluripotent mouse Embryonic Stem (ES) cells the global levels of H3K9 methylation are rather low and increase only upon differentiation. How global H3K9 methylation levels are coupled with the loss of pluripotency remains largely unknown. Here, we identify SUV39H1, a major H3K9 di- and tri-methylase, as an indirect target of the pluripotency network of Transcription Factors (TFs). We find that pluripotency TFs, principally OCT4, activate the expression of Suv39h1as, an antisense long non-coding RNA to Suv39h1. In turn, Suv39h1as downregulates Suv39h1 transcription in cis via a mechanism involving the modulation of the chromatin status of the locus. The targeted deletion of the Suv39h1as promoter region triggers increased SUV39H1 expression and H3K9me2 and H3K9me3 levels, affecting all heterochromatic regions, particularly peri-centromeric major satellites and retrotransposons. This increase in heterochromatinization efficiency leads to accelerated and more efficient commitment into differentiation. We report, therefore, a simple genetic circuitry coupling the genetic control of pluripotency with the global efficiency of H3K9 methylation associated with a major cell fate restriction, the irreversible loss of pluripotency.
Asunto(s)
Histonas , Metiltransferasas/metabolismo , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , ARN Largo no Codificante , Proteínas Represoras/metabolismo , Animales , Cromatina , Código de Histonas , Histonas/genética , Histonas/metabolismo , Metilación , Metiltransferasas/genética , Ratones , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Proteínas Represoras/genéticaRESUMEN
Mitotic bookmarking transcription factors (BFs) maintain the capacity to bind to their targets during mitosis, despite major rearrangements of the chromatin. While they were thought to propagate gene regulatory information through mitosis by statically occupying their DNA targets, it has recently become clear that BFs are highly dynamic in mitotic cells. This represents both a technical and a conceptual challenge to study and understand the function of BFs: First, formaldehyde has been suggested to be unable to efficiently capture these transient interactions, leading to profound contradictions in the literature; and second, if BFs are not permanently bound to their targets during mitosis, it becomes unclear how they convey regulatory information to daughter cells. Here, comparing formaldehyde to alternative fixatives we clarify the nature of the chromosomal association of previously proposed BFs in embryonic stem cells: While ESRRB can be considered as a canonical BF that binds at selected regulatory regions in mitosis, SOX2 and POU5F1 (also known as OCT4) establish DNA sequence-independent interactions with the mitotic chromosomes, either throughout the chromosomal arms (SOX2) or at pericentromeric regions (POU5F1). Moreover, we show that ordered nucleosomal arrays are retained during mitosis at ESRRB bookmarked sites, whereas regions losing transcription factor binding display a profound loss of order. By maintaining nucleosome positioning during mitosis, ESRRB might ensure the rapid post-mitotic re-establishment of functional regulatory complexes at selected enhancers and promoters. Our results provide a mechanistic framework that reconciles dynamic mitotic binding with the transmission of gene regulatory information across cell division.
Asunto(s)
Mitosis/genética , Nucleosomas/química , Factores de Transcripción/metabolismo , Animales , Células Cultivadas , Cromatina/metabolismo , Cromosomas de los Mamíferos , Fijadores , Formaldehído , Ratones , Receptores de Estrógenos/metabolismo , SuccinimidasRESUMEN
Cell fate decisions occur through the action of multiple factors, including signalling molecules and transcription factors. Recently, the regulation of translation has emerged as an important step for modulating cellular function and fate, as exemplified by ribosomes that play distinct roles in regulating cell behaviour. Notchless (Nle) is a conserved nuclear protein that is involved in a crucial step in ribosome biogenesis, and is required for the maintenance of adult haematopoietic and intestinal stem/progenitor cells. Here, we show that activated skeletal muscle satellite cells in conditional Nle mutant mice are arrested in proliferation; however, deletion of Nle in myofibres does not impair myogenesis. Furthermore, conditional deletion of Nle in satellite cells during homeostasis did not impact on their fate for up to 3â months. In contrast, loss of Nle function in primary myogenic cells blocked proliferation because of major defects in ribosome formation. Taken together, we show that muscle stem cells undergo a stage-specific regulation of ribosome biogenesis, thereby underscoring the importance of differential modulation of mRNA translation for controlling cell fate decisions.
Asunto(s)
Linaje de la Célula , Proteínas de la Membrana/metabolismo , Desarrollo de Músculos , Músculo Esquelético/crecimiento & desarrollo , Músculo Esquelético/metabolismo , Biogénesis de Organelos , Ribosomas/metabolismo , Animales , Ciclo Celular , Diferenciación Celular , Células Cultivadas , Ciclina E/metabolismo , Regulación del Desarrollo de la Expresión Génica , Proteínas de la Membrana/genética , Ratones Noqueados , Mutación/genética , Mioblastos/citología , Mioblastos/metabolismo , Regeneración , Células Satélite del Músculo Esquelético/citología , Células Satélite del Músculo Esquelético/metabolismoRESUMEN
The contribution of basal cellular processes to the regulation of tissue homeostasis has just started to be appreciated. However, our knowledge of the modulation of ribosome biogenesis activity in situ within specific lineages remains very limited. This is largely due to the lack of assays that enable quantitation of ribosome biogenesis in small numbers of cells in vivo. We used a technique, named Flow-FISH, combining cell surface antibody staining and flow cytometry with intracellular ribosomal RNA (rRNA) FISH, to measure the levels of pre-rRNAs of hematopoietic cells in vivo. Here, we show that Flow-FISH reports and quantifies ribosome biogenesis activity in hematopoietic cell populations, thereby providing original data on this fundamental process notably in rare populations such as hematopoietic stem and progenitor cells. We unravel variations in pre-rRNA levels between different hematopoietic progenitor compartments and during erythroid differentiation. In particular, our data indicate that, contrary to what may be anticipated from their quiescent state, hematopoietic stem cells have significant ribosome biogenesis activity. Moreover, variations in pre-rRNA levels do not correlate with proliferation rates, suggesting that cell type-specific mechanisms might regulate ribosome biogenesis in hematopoietic stem cells and progenitors. Our study contributes to a better understanding of the cellular physiology of the hematopoietic system in vivo in unperturbed situations.
Asunto(s)
Células Madre Hematopoyéticas/metabolismo , Procesamiento Postranscripcional del ARN/genética , ARN Ribosómico/biosíntesis , Ribosomas/genética , Animales , Diferenciación Celular/genética , Ratones , Precursores del ARN/genética , Proteínas Ribosómicas , Ribosomas/metabolismoRESUMEN
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.
Asunto(s)
Masa Celular Interna del Blastocisto/metabolismo , Linaje de la Célula/genética , Endodermo/metabolismo , Regulación del Desarrollo de la Expresión Génica , Fosfatidilinositol 3-Quinasas/genética , Factor de Crecimiento Derivado de Plaquetas/genética , Serina-Treonina Quinasas TOR/genética , Animales , Blastocisto , Masa Celular Interna del Blastocisto/citología , Supervivencia Celular , Endodermo/citología , Endodermo/crecimiento & desarrollo , Femenino , Factores de Crecimiento de Fibroblastos/genética , Factores de Crecimiento de Fibroblastos/metabolismo , Masculino , Ratones , Fosfatidilinositol 3-Quinasas/metabolismo , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Proteínas Proto-Oncogénicas c-kit/genética , Proteínas Proto-Oncogénicas c-kit/metabolismo , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/genética , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Transducción de Señal , Serina-Treonina Quinasas TOR/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
NOTCH signalling is an evolutionarily conserved pathway involved in intercellular communication essential for cell fate choices during development. Although dispensable for early aspects of mouse development, canonical RBPJ-dependent NOTCH signalling has been shown to influence lineage commitment during embryonic stem cell (ESC) differentiation. NOTCH activation in ESCs promotes the acquisition of a neural fate, whereas its suppression favours their differentiation into cardiomyocytes. This suggests that NOTCH signalling is implicated in the acquisition of distinct embryonic fates at early stages of mammalian development. In order to investigate in vivo such a role for NOTCH signalling in shaping cell fate specification, we use genetic approaches to constitutively activate the NOTCH pathway in the mouse embryo. Early embryonic development, including the establishment of anterior-posterior polarity, is not perturbed by forced NOTCH activation. By contrast, widespread NOTCH activity in the epiblast triggers dramatic gastrulation defects. These are fully rescued in a RBPJ-deficient background. Epiblast-specific NOTCH activation induces acquisition of neurectoderm identity and disrupts the formation of specific mesodermal precursors including the derivatives of the anterior primitive streak, the mouse organiser. In addition, we show that forced NOTCH activation results in misregulation of NODAL signalling, a major determinant of early embryonic patterning. Our study reveals a previously unidentified role for canonical NOTCH signalling during mammalian gastrulation. It also exemplifies how in vivo studies can shed light on the mechanisms underlying cell fate specification during in vitro directed differentiation.
Asunto(s)
Embrión de Mamíferos/metabolismo , Desarrollo Embrionario , Gastrulación , Receptores Notch/metabolismo , Transducción de Señal , Animales , Ectodermo/metabolismo , Implantación del Embrión , Estratos Germinativos/metabolismo , Ratones , Proteína Nodal/metabolismoRESUMEN
Over the last years, the microRNA (miRNA) pathway has emerged as a key component of the regulatory network of pluripotency. Although clearly distinct states of pluripotency have been described in vivo and ex vivo, differences in miRNA expression profiles associated with the developmental modulation of pluripotency have not been extensively studied so far. Here, we performed deep sequencing to profile miRNA expression in naive (embryonic stem cell [ESC]) and primed (epiblast stem cell [EpiSC]) pluripotent stem cells derived from mouse embryos of identical genetic background. We developed a graphical representation method allowing the rapid identification of miRNAs with an atypical profile including mirtrons, a small nucleolar RNA (snoRNA)-derived miRNA, and miRNAs whose biogenesis may differ between ESC and EpiSC. Comparison of mature miRNA profiles revealed that ESCs and EpiSCs exhibit very different miRNA signatures with one third of miRNAs being differentially expressed between the two cell types. Notably, differential expression of several clusters, including miR290-295, miR17-92, miR302/367, and a large repetitive cluster on chromosome 2, was observed. Our analysis also showed that differentiation priming of EpiSC compared to ESC is evidenced by changes in miRNA expression. These dynamic changes in miRNAs signature are likely to reflect both redundant and specific roles of miRNAs in the fine-tuning of pluripotency during development.
Asunto(s)
Células Madre Embrionarias/metabolismo , MicroARNs/biosíntesis , MicroARNs/genética , Células Madre Pluripotentes/metabolismo , Animales , Diferenciación Celular/genética , Línea Celular , Bases de Datos de Ácidos Nucleicos , Células Madre Embrionarias/citología , Perfilación de la Expresión Génica/métodos , Ratones , Células Madre Pluripotentes/citologíaRESUMEN
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.
Asunto(s)
Masa Celular Interna del Blastocisto/citología , Masa Celular Interna del Blastocisto/metabolismo , Endodermo/citología , Endodermo/metabolismo , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Transducción de Señal , Animales , Benzamidas/farmacología , Masa Celular Interna del Blastocisto/efectos de los fármacos , Inhibidores de Caspasas/farmacología , Muerte Celular/efectos de los fármacos , Linaje de la Célula/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Desarrollo Embrionario/efectos de los fármacos , Endodermo/efectos de los fármacos , Citometría de Flujo , Humanos , Imagenología Tridimensional , Mesilato de Imatinib , Ligandos , Ratones , Fenotipo , Piperazinas/farmacología , Pirimidinas/farmacología , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
H-2Z1 is an enhancer trap transgenic mouse line in which the lacZ reporter delineates the somatosensory area of the cerebral cortex where it is expressed in a subset of layer IV neurons. In the search of somatosensory specific genes or regulatory sequences, we mapped the H-2Z1 transgene insertion site to chromosome 17, 100 and 460 kb away from Tbc1d5 and Satb1 flanking genes. We show here that insertion of the H-2Z1 transgene results in three distinct outcomes. First, a genetic background-sensitive expression of lacZ in several brain and body structures. While four genes in a 1 Mb region around the insertion are expressed in the barrel cortex, H-2Z1 expression resembles more that of its two direct neighbors. Moreover, H-2Z1 closely reports most of the body and brain expression sites of the Satb1 chromatin remodeling gene including tooth buds, thymic epithelium, pontine nuclei, fastigial cerebellar nuclei, and cerebral cortex. Second, the H-2Z1 transgene causes insertional mutagenesis of Tbc1d5 and Satb1, leading to a strong decrease in their expressions. Finally, insertion of H-2Z1 affects the differentiation of a subset of cortical GABAergic interneurons, a possible consequence of downregulation of Satb1 expression. Thus, the H-2Z1 "somatosensory" transgene is inserted in the regulatory landscape of two genes highly expressed in the developing somatosensory cortex and reports for a subdomain of their expression profiles. Together, our data suggest that regulation of H-2Z1 expression results from local and remote genetic interactions.
Asunto(s)
Diferenciación Celular/genética , Corteza Cerebral/fisiología , Regulación de la Expresión Génica/genética , Interneuronas/fisiología , Operón Lac/fisiología , Proteínas de Unión a la Región de Fijación a la Matriz/biosíntesis , Corteza Somatosensorial/fisiología , Animales , Corteza Cerebral/crecimiento & desarrollo , Corteza Cerebral/metabolismo , Neuronas GABAérgicas/fisiología , Regulación de la Expresión Génica/fisiología , Interneuronas/citología , Operón Lac/genética , Ratones , Ratones Endogámicos , Ratones Transgénicos , Corteza Somatosensorial/crecimiento & desarrollo , Corteza Somatosensorial/metabolismoRESUMEN
The epiblast is the source of all mammalian embryonic tissues and of pluripotent embryonic stem cells. It differentiates alongside the primitive endoderm in a "salt and pepper" pattern from inner cell mass (ICM) progenitors during the preimplantation stages through the activity of NANOG, GATA6 and the FGF pathway. When and how epiblast lineage specification is initiated is still unclear. Here, we show that the coordinated expression of pluripotency markers defines epiblast identity. Conversely, ICM progenitor cells display random cell-to-cell variability in expression of various pluripotency markers, remarkably dissimilar from the epiblast signature and independently from NANOG, GATA6 and FGF activities. Coordination of pluripotency markers expression fails in Nanog and Gata6 double KO (DKO) embryos. Collectively, our data suggest that NANOG triggers epiblast specification by ensuring the coordinated expression of pluripotency markers in a subset of cells, implying a stochastic mechanism. These features are likely conserved, as suggested by analysis of human embryos.
Asunto(s)
Endodermo , Estratos Germinativos , Animales , Blastocisto/metabolismo , Diferenciación Celular/genética , Linaje de la Célula/genética , Endodermo/metabolismo , Regulación del Desarrollo de la Expresión Génica , Estratos Germinativos/metabolismo , Humanos , Mamíferos/genética , Proteína Homeótica Nanog/genética , Proteína Homeótica Nanog/metabolismoRESUMEN
Rat and mice are privileged tools for scientists. However, despite obvious advantages, such as a larger size, more faithful reproduction of human diseases, and utility for physiological and cognitive studies, rats have suffered from limited genetic technologies such as targeted mutagenesis. However, the gap between rat and mouse for genetic approaches will soon disappear with the recent advances of zinc finger nucleases applicable to early-stage rat embryos and the successful derivation of germ line competent rat ES cells, almost thirty years after murine ES cells. This will lead to new opportunities and to increase our capacity to model human pathologies.
Asunto(s)
Técnicas Genéticas , Ratas/genética , Animales , Animales Modificados Genéticamente , Células Cultivadas/metabolismo , ADN/genética , ADN/metabolismo , Reparación del ADN , Modelos Animales de Enfermedad , Endonucleasas/metabolismo , Predicción , Técnicas de Inactivación de Genes , Genómica , Ratones , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , Ratas/embriología , Ratas Wistar , Especificidad de la Especie , Dedos de ZincRESUMEN
The mouse preimplantation embryo is an excellent system for studying how mammalian cells organize dynamically into increasingly complex structures. Accessible to experimental and genetic manipulations, its normal or perturbed development can be scrutinized ex vivo by real-time imaging from fertilization to late blastocyst stage. High-resolution imaging of multiple embryos at the same time can be compromised by embryos displacement during imaging. We have developed an inexpensive and easy-to-produce imaging device that facilitates greatly the imaging of preimplantation embryo. In this chapter, we describe the different steps of production and storage of the imaging device as well as its use for live imaging of mouse preimplantation embryos expressing fluorescent reporters from genetically modified alleles or after in vitro transcribed mRNA transfer by microinjection or electroporation.
Asunto(s)
Blastocisto/ultraestructura , Microscopía Confocal/métodos , Animales , Electroporación/métodos , Técnicas de Cultivo de Embriones/métodos , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Microinyecciones/métodosRESUMEN
Covalent modification by SUMO regulates a wide range of cellular processes, including transcription, cell cycle, and chromatin dynamics. To address the biological function of the SUMO pathway in mammals, we generated mice deficient for the SUMO E2-conjugating enzyme Ubc9. Ubc9-deficient embryos die at the early postimplantation stage. In culture, Ubc9 mutant blastocysts are viable, but fail to expand after 2 days and show apoptosis of the inner cell mass. Loss of Ubc9 leads to major chromosome condensation and segregation defects. Ubc9-deficient cells also show severe defects in nuclear organization, including nuclear envelope dysmorphy and disruption of nucleoli and PML nuclear bodies. Moreover, RanGAP1 fails to accumulate at the nuclear pore complex in mutant cells that show a collapse in Ran distribution. Together, these findings reveal a major role for Ubc9, and, by implication, for the SUMO pathway, in nuclear architecture and function, chromosome segregation, and embryonic viability in mammals.
Asunto(s)
Núcleo Celular/metabolismo , Segregación Cromosómica , Embrión de Mamíferos/metabolismo , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Enzimas Ubiquitina-Conjugadoras/fisiología , Animales , Apoptosis , Blastocisto/citología , Blastocisto/metabolismo , Núcleo Celular/genética , Pérdida del Embrión/genética , Embrión de Mamíferos/citología , Femenino , Técnica del Anticuerpo Fluorescente , Proteínas Activadoras de GTPasa/metabolismo , Immunoblotting , Etiquetado Corte-Fin in Situ , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mitosis , Transducción de Señal , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/genética , Ubiquitinas/metabolismo , Proteína de Unión al GTP ran/metabolismoRESUMEN
Multiple cell types arise from cells in the dermomyotome of the somite that express Pax3 and Pax7, and myogenesis is regulated by Notch signaling. The asymmetric cell fate determinant Numb is thought to promote differentiation of skeletal muscle and other lineages by negatively regulating Notch signaling. We used transgenesis to overexpress Numb spatiotemporally in Pax3(+)/Pax7(+) somitic stem and progenitor cells in mouse embryos using a spatiotemporally regulated enhancer element from the Myf5 locus that can target muscle progenitor cells prior to cell commitment. Molecular analyses as well as examination of dermal and skeletal muscle cell fates in vivo show that although Numb is thought to be associated with muscle differentiation, unexpectedly the common stem/progenitor pool size for these lineages is increased in Numb-transgenic embryos. Prospective isolation of the relevant transgenic cells and analysis by quantitative reverse-transcription polymerase chain reaction demonstrated that, in this context, canonical Notch targets are not significantly downregulated. These findings were corroborated using a Notch reporter mouse during the formation of somites and prior to lineage segregation. Thus, we propose that Numb can regulate the self-renewal of dermal and muscle progenitors during a lineage progression.
Asunto(s)
Proteínas de la Membrana/fisiología , Fibras Musculares Esqueléticas/citología , Proteínas del Tejido Nervioso/fisiología , Somitos/citología , Células Madre/citología , Animales , Western Blotting , Citometría de Flujo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Inmunohistoquímica , Hibridación in Situ , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Transgénicos , Mitosis/genética , Mitosis/fisiología , Desarrollo de Músculos/genética , Desarrollo de Músculos/fisiología , Fibras Musculares Esqueléticas/enzimología , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Madre/metabolismoRESUMEN
Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is an autosomal dominant small-vessel disease of the brain caused by mutations in the NOTCH3 receptor. The highly stereotyped nature of the mutations, which alter the number of cysteine residues within the epidermal growth factor-like repeats (EGFR), predicts that all mutations share common mechanisms. Prior in vitro assays and genetic studies in the mouse support the hypothesis that common mutations do not compromise canonical Notch3 function but instead convey a non-physiological and deleterious activity to the receptor through the unpaired cysteine residue. Intriguingly, in vitro studies predict that mutations located in the Delta/Serrate/LAG-2 ligand binding domain-(EGFR10-11) may result in a loss of Notch3 receptor function. However, the in vivo relevance and functional significance of this with respect to the pathogenic mechanisms and clinical expression of the disease remain largely unexplored. To ascertain, in vivo, the functional significance of EGFR10-11 mutations, we generated transgenic mice with one representative mutation (C428S) in EGFR10 of Notch3. These mice, like those with a common R90C mutation, developed characteristic arterial accumulation of Notch3 protein and granular osmiophilic material upon aging. By introducing the mutant C428S transgene into a Notch3 null background, we found that, unlike the R90C mutant protein, the C428S mutant protein has lost wild-type Notch3 activity and exhibited mild dominant-negative activity in three different biological settings. From a large prospectively recruited cohort of 176 CADASIL patients, we identified 10 patients, from five distinct pedigrees carrying a mutation in EGFR10 or 11. These mutations were associated with significantly higher Mini-Mental State Examination and Mattis Dementia Rating Scale scores (P < 0.05), when compared with common mutations. Additionally, we found a strong effect of this genotype on the burden of white matter hyperintensities (P < 0.01). Collectively, these results highlight distinctive functional and phenotypic features of EGFR10-11 mutations relative to the common CADASIL mutations. Our findings are compatible with the hypothesis that EGFR10-11 mutations cause the disease through the same gain of novel function as the common mutations, and lead us to propose that reduced Notch3 signalling acts as a modifier of the CADASIL phenotype.
Asunto(s)
CADASIL/genética , Mutación , Receptores Notch/genética , Adulto , Anciano , Animales , Encéfalo/patología , CADASIL/metabolismo , CADASIL/patología , Arterias Cerebrales/metabolismo , Arterias Cerebrales/ultraestructura , Modelos Animales de Enfermedad , Genotipo , Humanos , Ligandos , Imagen por Resonancia Magnética/métodos , Ratones , Ratones Transgénicos , Microscopía Electrónica , Persona de Mediana Edad , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/ultraestructura , Fenotipo , Estudios Prospectivos , Receptor Notch3 , Receptores Notch/metabolismo , Receptores Notch/fisiología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodosRESUMEN
Mouse embryonic stem cells (mESCs) are a powerful model to study early mouse development. These blastocyst-derived cells can maintain pluripotency and differentiate into the three embryonic germ layers and an extraembryonic layer, the extraembryonic endoderm (ExEn), which shares similar molecular markers to the definitive endoderm. Here, we present a fast procedure to identify a differentiation defect of mESCs toward ExEn in vitro through the molecular and cellular characterization of embryoid bodies, followed by direct differentiation of mESCs into ExEn. For complete details on the use and execution of this protocol, please refer to Ngondo et al. (2018).
Asunto(s)
Diferenciación Celular/fisiología , Membranas Extraembrionarias/diagnóstico por imagen , Células Madre Embrionarias de Ratones/metabolismo , Animales , Línea Celular , Linaje de la Célula , Células Madre Embrionarias/citología , Endodermo/citología , Regulación del Desarrollo de la Expresión Génica/genética , Ratones , Células Madre Embrionarias de Ratones/fisiologíaRESUMEN
Ribosome biogenesis inhibition causes cell cycle arrest and apoptosis through the activation of tumor suppressor-dependent surveillance pathways. These responses are exacerbated in cancer cells, suggesting that targeting ribosome synthesis may be beneficial to patients. Here, we characterize the effect of the loss-of-function of Notchless (Nle), an essential actor of ribosome biogenesis, on the intestinal epithelium undergoing tumor initiation due to acute Apc loss-of-function. We show that ribosome biogenesis dysfunction strongly alleviates Wnt-driven tumor initiation by restoring cell cycle exit and differentiation in Apc-deficient progenitors. Conversely Wnt hyperactivation attenuates the cellular responses to surveillance pathways activation induced by ribosome biogenesis dysfunction, as proliferation was maintained at control-like levels in the stem cells and progenitors of double mutants. Thus, our data indicate that, while ribosome biogenesis inhibition efficiently reduces cancer cell proliferation in the intestinal epithelium, enhanced resistance of Apc-deficient stem and progenitor cells to ribosome biogenesis defects may be an important concern when using a therapeutic strategy targeting ribosome production for the treatment of Wnt-dependent tumorigenesis.
Asunto(s)
Proteína de la Poliposis Adenomatosa del Colon/fisiología , Transformación Celular Neoplásica , Mucosa Intestinal , Proteínas de la Membrana/fisiología , Ribosomas/metabolismo , Vía de Señalización Wnt , Animales , Mucosa Intestinal/citología , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Ratones , Ratones Endogámicos C57BL , Biogénesis de OrganelosRESUMEN
Stable genomic integration of exogenous transgenes is essential in neurodevelopmental and stem cell studies. Despite tools driving increasingly efficient genomic insertion with DNA vectors, transgenesis remains fundamentally hindered by the impossibility of distinguishing integrated from episomal transgenes. Here, we introduce an integration-coupled On genetic switch, iOn, which triggers gene expression upon incorporation into the host genome through transposition, thus enabling rapid and accurate identification of integration events following transfection with naked plasmids. In vitro, iOn permits rapid drug-free stable transgenesis of mouse and human pluripotent stem cells with multiple vectors. In vivo, we demonstrate faithful cell lineage tracing, assessment of regulatory elements, and mosaic analysis of gene function in somatic transgenesis experiments that reveal neural progenitor potentialities and interaction. These results establish iOn as a universally applicable strategy to accelerate and simplify genetic engineering in cultured systems and model organisms by conditioning transgene activation to genomic integration.
Asunto(s)
Expresión Génica , Técnicas de Transferencia de Gen , Células-Madre Neurales , Transgenes , Animales , Linaje de la Célula , Vectores Genéticos , Humanos , RatonesRESUMEN
The Notch signaling pathway is an evolutionarily conserved signaling system which has been shown to be essential in cell fate specification and in numerous aspects of embryonic development in all metazoans thus far studied. We recently demonstrated that several components of the Notch signaling pathway, including the four Notch receptors and their five ligands known in mammals, are expressed in mouse oocytes, in mouse preimplantation embryos, or both. This suggested a possible implication of the Notch pathway in the first cell fate specification of the dividing mouse embryo, which results in the formation of the blastocyst. To address this issue directly, we generated zygotes in which both the maternal and the zygotic expression of Rbpsuh, a key element of the core Notch signaling pathway, were abrogated. We find that such zygotes give rise to blastocysts which implant and develop normally. Nevertheless, after gastrulation, these embryos die around midgestation, similarly to Rbpsuh-null mutants. This demonstrates that the RBP-Jkappa-dependent pathway, otherwise called the canonical Notch pathway, is dispensable for blastocyst morphogenesis and the establishment of the three germ layers, ectoderm, endoderm, and mesoderm. These results are discussed in the light of recent observations which have challenged this conclusion.