RESUMEN
Sperm production and function require the correct establishment of DNA methylation patterns in the germline. Here, we examined the genome-wide DNA methylation changes during human spermatogenesis and its alterations in disturbed spermatogenesis. We found that spermatogenesis is associated with remodeling of the methylome, comprising a global decline in DNA methylation in primary spermatocytes followed by selective remethylation, resulting in a spermatids/sperm-specific methylome. Hypomethylated regions in spermatids/sperm were enriched in specific transcription factor binding sites for DMRT and SOX family members and spermatid-specific genes. Intriguingly, while SINEs displayed differential methylation throughout spermatogenesis, LINEs appeared to be protected from changes in DNA methylation. In disturbed spermatogenesis, germ cells exhibited considerable DNA methylation changes, which were significantly enriched at transposable elements and genes involved in spermatogenesis. We detected hypomethylation in SVA and L1HS in disturbed spermatogenesis, suggesting an association between the abnormal programming of these regions and failure of germ cells progressing beyond meiosis.
Asunto(s)
Metilación de ADN , Genoma Humano , Espermatogénesis , Humanos , Espermatogénesis/genética , Masculino , Espermátides/metabolismo , Espermatocitos/metabolismo , Elementos Transponibles de ADN/genética , Espermatozoides/metabolismo , Meiosis/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Embryonic stem cells (ESCs) comprise at least two populations of cells with divergent states of pluripotency. Here, we show that epiblast stem cells (EpiSCs) also comprise two distinct cell populations that can be distinguished by the expression of a specific Oct4-GFP marker. These two subpopulations, Oct4-GFP positive and negative EpiSCs, are capable of converting into each other in vitro. Oct4-GFP positive and negative EpiSCs are distinct from ESCs with respect to global gene expression pattern, epigenetic profile, and Oct4 enhancer utilization. Oct4-GFP negative cells share features with cells of the late mouse epiblast and cannot form chimeras. However, Oct4-GFP positive EpiSCs, which only represent a minor EpiSC fraction, resemble cells of the early epiblast and can readily contribute to chimeras. Our findings suggest that the rare ability of EpiSCs to contribute to chimeras is due to the presence of the minor EpiSC fraction representing the early epiblast.
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Estratos Germinativos/citología , Ratones/embriología , Células Madre/citología , Animales , Femenino , Perfilación de la Expresión Génica , Masculino , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Factor 3 de Transcripción de Unión a Octámeros/análisis , Factor 3 de Transcripción de Unión a Octámeros/genética , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismoRESUMEN
Hematopoietic stem and progenitor cell (HSPC) function in bone marrow (BM) is controlled by stroma-derived signals, but the identity and interplay of these signals remain incompletely understood. Here, we show that sympathetic nerve-derived dopamine directly controls HSPC behavior through D2 subfamily dopamine receptors. Blockade of dopamine synthesis, as well as pharmacological or genetic inactivation of D2 subfamily dopamine receptors, leads to reduced HSPC frequency, inhibition of proliferation, and low BM transplantation efficiency. Conversely, treatment with a D2-type receptor agonist increases BM regeneration and transplantation efficiency. Mechanistically, dopamine controls expression of the lymphocyte-specific protein tyrosine kinase (Lck), which, in turn, regulates MAPK-mediated signaling triggered by stem cell factor in HSPCs. Our work reveals critical functional roles of dopamine in HSPCs, which may open up new therapeutic options for improved BM transplantation and other conditions requiring the rapid expansion of HSPCs.
Asunto(s)
Dopamina/metabolismo , Células Madre Hematopoyéticas/citología , Receptores de Dopamina D2/metabolismo , Transducción de Señal , Animales , Trasplante de Médula Ósea , Proliferación Celular , Células Cultivadas , Células Madre Hematopoyéticas/metabolismo , RatonesRESUMEN
STUDY QUESTION: Is the vertebrate protein Dead end (DND1) a causative factor for human infertility and can novel in vivo assays in zebrafish help in evaluating this? SUMMARY ANSWER: Combining patient genetic data with functional in vivo assays in zebrafish reveals a possible role for DND1 in human male fertility. WHAT IS KNOWN ALREADY: About 7% of the male population is affected by infertility but linking specific gene variants to the disease is challenging. The function of the DND1 protein was shown to be critical for germ cell development in several model organisms but a reliable and cost-effective method for evaluating the activity of the protein in the context of human male infertility is still missing. STUDY DESIGN, SIZE, DURATION: Exome data from 1305 men included in the Male Reproductive Genomics cohort were examined in this study. A total of 1114 of the patients showed severely impaired spermatogenesis but were otherwise healthy. Eighty-five men with intact spermatogenesis were included in the study as controls. PARTICIPANTS/MATERIALS, SETTING, METHODS: We screened the human exome data for rare, stop-gain, frameshift, splice site, as well as missense variants in DND1. The results were validated by Sanger sequencing. Immunohistochemical techniques and, when possible, segregation analyses were performed for patients with identified DND1 variants. The amino acid exchange in the human variant was mimicked at the corresponding site of the zebrafish protein. Using different aspects of germline development in live zebrafish embryos as biological assays, we examined the activity level of these DND1 protein variants. MAIN RESULTS AND THE ROLE OF CHANCE: In human exome sequencing data, we identified four heterozygous variants in DND1 (three missense and one frameshift variant) in five unrelated patients. The function of all of the variants was examined in the zebrafish and one of those was studied in more depth in this model. We demonstrate the use of zebrafish assays as a rapid and effective biological readout for evaluating the possible impact of multiple gene variants on male fertility. This in vivo approach allowed us to assess the direct impact of the variants on germ cell function in the context of the native germline. Focusing on the DND1 gene, we find that zebrafish germ cells, expressing orthologs of DND1 variants identified in infertile men, failed to arrive correctly at the position where the gonad develops and exhibited defects in cell fate maintenance. Importantly, our analysis facilitated the evaluation of single nucleotide variants, whose impact on protein function is difficult to predict, and allowed us to distinguish variants that do not affect the protein's activity from those that strongly reduce it and could thus potentially be the primary cause for the pathological condition. These aberrations in germline development resemble the testicular phenotype of azoospermic patients. LIMITATIONS, REASONS FOR CAUTION: The pipeline we present requires access to zebrafish embryos and to basic imaging equipment. The notion that the activity of the protein in the zebrafish-based assays is relevant for the human homolog is well supported by previous knowledge. Nevertheless, the human protein may differ in some respects from its homologue in zebrafish. Thus, the assay should be considered only one of the parameters used in defining DND1 variants as causative or non-causative for infertility. WIDER IMPLICATIONS OF THE FINDINGS: Using DND1 as an example, we have shown that the approach described in this study, relying on bridging between clinical findings and fundamental cell biology, can help to establish links between novel human disease candidate genes and fertility. In particular, the power of the approach we developed is manifested by the fact that it allows the identification of DND1 variants that arose de novo. The strategy presented here can be applied to different genes in other disease contexts. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by the German Research Foundation, Clinical Research Unit, CRU326 'Male Germ Cells'. There are no competing interests. TRIAL REGISTRATION NUMBER: N/A.
Asunto(s)
Infertilidad Masculina , Pez Cebra , Animales , Humanos , Masculino , Pez Cebra/genética , Infertilidad Masculina/genética , Infertilidad Masculina/patología , Testículo/patología , Fertilidad , Fenotipo , Proteínas de Neoplasias/genéticaRESUMEN
During implantation, the murine embryo transitions from a "quiet" into an active metabolic/proliferative state, which kick-starts the growth and morphogenesis of the post-implantation conceptus. Such transition is also required for embryonic stem cells to be established from mouse blastocysts, but the factors regulating this process are poorly understood. Here, we show that Ronin plays a critical role in the process by enabling active energy production, and the loss of Ronin results in the establishment of a reversible quiescent state in which naïve pluripotency is promoted. In addition, Ronin fine-tunes the expression of genes that encode ribosomal proteins and is required for proper tissue-scale organisation of the pluripotent lineage during the transition from blastocyst to egg cylinder stage. Thus, Ronin function is essential for governing the metabolic capacity so that it can support the pluripotent lineage's high-energy demands for cell proliferation and morphogenesis.
Asunto(s)
Desarrollo Embrionario , Células Madre Embrionarias , Animales , Blastocisto/metabolismo , Implantación del Embrión/fisiología , Embrión de Mamíferos/metabolismo , Desarrollo Embrionario/genética , Células Madre Embrionarias/metabolismo , RatonesRESUMEN
Endothelial cells line blood and lymphatic vessels and form intercellular junctions, which preserve vessel structure and integrity. The vascular endothelial cadherin, VE-cadherin, mediates endothelial adhesion and is indispensible for blood vessel development and permeability regulation. However, its requirement for lymphatic vessels has not been addressed. During development, VE-cadherin deletion in lymphatic endothelial cells resulted in abortive lymphangiogenesis, edema, and prenatal death. Unexpectedly, inducible postnatal or adult deletion elicited vessel bed-specific responses. Mature dermal lymph vessels resisted VE-cadherin loss and maintained button junctions, which was associated with an upregulation of junctional molecules. Very different, mesenteric lymphatic collectors deteriorated and formed a strongly hyperplastic layer of lymphatic endothelial cells on the mesothelium. This massive hyperproliferation may have been favored by high mesenteric VEGF-C expression and was associated with VEGFR-3 phosphorylation and upregulation of the transcriptional activator TAZ Finally, intestinal lacteals fragmented into cysts or became highly distended possibly as a consequence of the mesenteric defects. Taken together, we demonstrate here the importance of VE-cadherin for lymphatic vessel development and maintenance, which is however remarkably vessel bed-specific.
Asunto(s)
Antígenos CD/metabolismo , Cadherinas/metabolismo , Dermis/embriología , Regulación del Desarrollo de la Expresión Génica , Linfangiogénesis , Vasos Linfáticos/metabolismo , Mesenterio/embriología , Animales , Antígenos CD/genética , Cadherinas/genética , Células Endoteliales/metabolismo , Eliminación de Gen , Ratones , Factor C de Crecimiento Endotelial Vascular/genética , Factor C de Crecimiento Endotelial Vascular/metabolismo , Receptor 3 de Factores de Crecimiento Endotelial Vascular/genética , Receptor 3 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
OBJECTIVE: Vascular endothelial (VE)-cadherin is of dominant importance for the formation and stability of endothelial junctions, yet induced gene inactivation enhances vascular permeability in the lung but does not cause junction rupture. This study aims at identifying the junctional adhesion molecule, which is responsible for preventing endothelial junction rupture in the pulmonary vasculature in the absence of VE-cadherin. Approach and Results: We have compared the relevance of ESAM (endothelial cell-selective adhesion molecule), JAM (junctional adhesion molecule)-A, PECAM (platelet endothelial cell adhesion molecule)-1, and VE-cadherin for vascular barrier integrity in various mouse tissues. Gene inactivation of ESAM enhanced vascular permeability in the lung but not in the heart, skin, and brain. In contrast, deletion of JAM-A or PECAM-1 did not affect barrier integrity in any of these organs. Blocking VE-cadherin with antibodies caused lethality in ESAM-/- mice within 30 minutes but had no such effect in JAM-A-/-, PECAM-1-/- or wild-type mice. Likewise, induced gene inactivation of VE-cadherin caused rapid lethality only in the absence of ESAM. Ultrastructural analysis revealed that only combined interference with VE-cadherin and ESAM disrupted endothelial junctions and caused massive blood coagulation in the lung. Mechanistically, we could exclude a role of platelet ESAM in coagulation, changes in the expression of other junctional proteins or a contribution of cytoplasmic signaling domains of ESAM. CONCLUSIONS: Despite well-documented roles of JAM-A and PECAM-1 for the regulation of endothelial junctions, only for ESAM, we detected an essential role for endothelial barrier integrity in a tissue-specific way. In addition, we found that it is ESAM which prevents endothelial junction rupture in the lung when VE-cadherin is absent.
Asunto(s)
Antígenos CD/metabolismo , Cadherinas/metabolismo , Permeabilidad Capilar/fisiología , Moléculas de Adhesión Celular/metabolismo , Muerte Celular/fisiología , Endotelio Vascular/metabolismo , Pulmón/metabolismo , Animales , Coagulación Sanguínea/fisiología , Adhesión Celular , Células Cultivadas , Cricetinae , Endotelio Vascular/ultraestructura , Femenino , Immunoblotting , Pulmón/irrigación sanguínea , Ratones , Ratones Endogámicos C57BL , Microscopía Electrónica , Modelos Animales , Transducción de SeñalRESUMEN
Tissue regeneration depends on proliferative cells and on cues that regulate cell division, differentiation, patterning and the restriction of these processes once regeneration is complete. In planarians, flatworms with high regenerative potential, muscle cells express some of these instructive cues. Here, we show that members of the integrin family of adhesion molecules are required for the integrity of regenerating tissues, including the musculature. Remarkably, in regenerating ß1-integrin RNAi planarians, we detected increased numbers of mitotic cells and progenitor cell types, as well as a reduced ability of stem cells and lineage-restricted progenitor cells to accumulate at wound sites. These animals also formed ectopic spheroid structures of neural identity in regenerating heads. Interestingly, those polarized assemblies comprised a variety of neural cells and underwent continuous growth. Our study indicates that integrin-mediated cell adhesion is required for the regenerative formation of organized tissues and for restricting neurogenesis during planarian regeneration.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Integrina beta1/fisiología , Neurogénesis/fisiología , Planarias/fisiología , Regeneración/fisiología , Animales , Tipificación del Cuerpo , Adhesión Celular , Diferenciación Celular , Proliferación Celular , Hibridación in Situ , Neuronas/citología , Filogenia , Interferencia de ARN , Transducción de Señal , Células Madre/citologíaRESUMEN
Multiple sclerosis (MS) is the most frequent demyelinating disease in young adults and despite significant advances in immunotherapy, disease progression still cannot be prevented. Promotion of remyelination, an endogenous repair mechanism resulting in the formation of new myelin sheaths around demyelinated axons, represents a promising new treatment approach. However, remyelination frequently fails in MS lesions, which can in part be attributed to impaired differentiation of oligodendroglial progenitor cells into mature, myelinating oligodendrocytes. The reasons for impaired oligodendroglial differentiation and defective remyelination in MS are currently unknown. To determine whether intrinsic oligodendroglial factors contribute to impaired remyelination in relapsing-remitting MS (RRMS), we compared induced pluripotent stem cell-derived oligodendrocytes (hiOL) from RRMS patients and controls, among them two monozygous twin pairs discordant for MS. We found that hiOL from RRMS patients and controls were virtually indistinguishable with respect to remyelination-associated functions and proteomic composition. However, while analyzing the effect of extrinsic factors we discovered that supernatants of activated peripheral blood mononuclear cells (PBMCs) significantly inhibit oligodendroglial differentiation. In particular, we identified CD4+ T cells as mediators of impaired oligodendroglial differentiation; at least partly due to interferon-gamma secretion. Additionally, we observed that blocked oligodendroglial differentiation induced by PBMC supernatants could not be restored by application of oligodendroglial differentiation promoting drugs, whereas treatment of PBMCs with the immunomodulatory drug teriflunomide prior to supernatant collection partly rescued oligodendroglial differentiation. In summary, these data indicate that the oligodendroglial differentiation block is not due to intrinsic oligodendroglial factors but rather caused by the inflammatory environment in RRMS lesions which underlines the need for drug screening approaches taking the inflammatory environment into account. Combined, these findings may contribute to the development of new remyelination promoting strategies.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Esclerosis Múltiple Recurrente-Remitente/inmunología , Oligodendroglía/patología , Remielinización/inmunología , Diferenciación Celular/fisiología , Humanos , Células Madre Pluripotentes Inducidas , Interferón gamma/inmunología , Células Precursoras de Oligodendrocitos/patologíaRESUMEN
Primordial germ cells (PGCs) develop only into sperm and oocytes in vivo. The molecular mechanisms underlying human PGC specification are poorly understood due to inaccessibility of cell materials and lack of in vitro models for tracking the earliest stages of germ cell development. Here, we describe a defined and stepwise differentiation system for inducing pre-migratory PGC-like cells (PGCLCs) from human pluripotent stem cells (PSCs). In response to cytokines, PSCs differentiate first into a heterogeneous mesoderm-like cell population and then into PGCLCs, which exhibit minimal PRDM14 expression. PGC specification in humans is similar to the murine process, with the sequential activation of mesodermal and PGC genes, and the suppression of neural induction and of de novo DNA methylation, suggesting that human PGC formation is induced via epigenesis, the process of germ cell specification via inductive signals from surrounding somatic cells. This study demonstrates that PGC commitment in humans shares key features with that of the mouse, but also highlights key differences, including transcriptional regulation during the early stage of human PGC development (3-6 weeks). A more comprehensive understanding of human germ cell development may lead to methodology for successfully generating PSC-derived gametes for reproductive medicine.
Asunto(s)
Diferenciación Celular/genética , Células Germinativas/fisiología , Células Madre Pluripotentes/fisiología , Proteínas Represoras/genética , Activinas/farmacología , Animales , Proteína Morfogenética Ósea 4/farmacología , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Proteínas de Unión al ADN , Epigénesis Genética , Factor 2 de Crecimiento de Fibroblastos/farmacología , Células Germinativas/citología , Humanos , Ratones , Análisis por Micromatrices , Células Madre Pluripotentes/efectos de los fármacos , Proteínas de Unión al ARN , Factores de Transcripción , Transcriptoma/efectos de los fármacosRESUMEN
In mammals, postnatal haematopoiesis occurs in the bone marrow (BM) and involves specialized microenvironments controlling haematopoietic stem cell (HSC) behaviour and, in particular, stem cell dormancy and self-renewal. While these processes have been linked to a number of different stromal cell types and signalling pathways, it is currently unclear whether BM has a homogenous architecture devoid of structural and functional partitions. Here, we show with genetic labelling techniques, high-resolution imaging and functional experiments in mice that the periphery of the adult BM cavity harbours previously unrecognized compartments with distinct properties. These units, which we have termed hemospheres, were composed of endothelial, haematopoietic and mesenchymal cells, were enriched in CD150+ CD48- putative HSCs, and enabled rapid haematopoietic cell proliferation and clonal expansion. Inducible gene targeting of the receptor tyrosine kinase VEGFR2 in endothelial cells disrupted hemospheres and, concomitantly, reduced the number of CD150+ CD48- cells. Our results identify a previously unrecognized, vessel-associated BM compartment with a specific localization and properties distinct from the marrow cavity.
Asunto(s)
Células de la Médula Ósea/citología , Células de la Médula Ósea/fisiología , Proliferación Celular , Hematopoyesis/fisiología , Células Madre Adultas/citología , Células Madre Adultas/fisiología , Animales , Médula Ósea/metabolismo , Diferenciación Celular/fisiología , Separación Celular , Células Cultivadas , Células Clonales/fisiología , Femenino , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/fisiología , Células Madre Mesenquimatosas/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Modelos BiológicosRESUMEN
The control over the acquisition of cell motility is central for a variety of biological processes in development, homeostasis, and disease. An attractive in vivo model for investigating the regulation of migration initiation is that of primordial germ cells (PGCs) in zebrafish embryos. In this study, we show that, following PGC specification, the cells can polarize but do not migrate before the time chemokine-encoded directional cues are established. We found that the regulator of G-protein signaling 14a protein, whose RNA is a newly identified germ plasm component, regulates the temporal relations between the appearance of the guidance molecules and the acquisition of cellular motility by regulating E-cadherin levels.
Asunto(s)
Movimiento Celular , Proteínas RGS/metabolismo , Transducción de Señal , Proteínas de Pez Cebra/metabolismo , Pez Cebra/metabolismo , Animales , Cadherinas/metabolismo , Movimiento Celular/genética , Polaridad Celular/genética , Regulación del Desarrollo de la Expresión Génica , Células Germinativas/citología , Células Germinativas/metabolismo , Proteínas RGS/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transducción de Señal/genética , Factores de Tiempo , Pez Cebra/embriología , Pez Cebra/genética , Proteínas de Pez Cebra/genéticaRESUMEN
The spleen tyrosine kinase family members Syk and Zap-70 are pivotal signal transducers downstream of antigen receptors and exhibit overlapping expression patterns at early lymphocytic developmental stages. To assess their differential kinase fitness in vivo, we generated mice, which carry a Zap-70 cDNA knock-in controlled by intrinsic Syk promoter elements that disrupts wild-type Syk expression. Kinase replacement severely compromised Erk1/2-mediated survival and proper selection of developing B cells at central and peripheral checkpoints, demonstrating critical dependence on BCR signalling quality. Furthermore, ITAM- and hemITAM-mediated activation of platelets and neutrophils was completely blunted, while surprisingly FcγR-mediated phagocytosis in macrophages was retained. The alteration in BCR signalling quality resulted in preferential development and survival of marginal zone B cells and prominent autoreactivity, causing the generation of anti-insulin antibodies and age-related glomerulonephritis. Development of concomitant fasting glucose intolerance in knock-in mice highlights aberrant B cell selection as a potential risk factor for type 1 diabetes, and suggests altered BCR signalling as a mechanism to cause biased cellular and Ig repertoire selection, ultimately contributing to B cell-mediated autoimmune predisposition.
Asunto(s)
Enfermedades Autoinmunes/genética , Estado Prediabético/genética , Proteínas Proto-Oncogénicas c-bcr/fisiología , Animales , Linfocitos B/inmunología , Linfocitos B/metabolismo , Linfocitos B/fisiología , Células Cultivadas , Técnicas de Sustitución del Gen , Reordenamiento Génico de Linfocito B/genética , Predisposición Genética a la Enfermedad , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas Tirosina Quinasas/genética , Proteínas Proto-Oncogénicas c-bcr/genética , Proteínas Proto-Oncogénicas c-bcr/metabolismo , Transducción de Señal/genética , Quinasa Syk , Proteína Tirosina Quinasa ZAP-70/genéticaRESUMEN
Syk and Zap-70 constitute a closely related nonreceptor protein tyrosine kinase family, of which both members are functionally indispensable for conferring their respective antigen receptors with enzymatic activity. In this study, we analyze the impact of altering BCR signaling output on B-cell germinal center (GC) fate selection by constitutive, as well as inducible, monoallelic Syk kinase loss in the presence of a Zap-70 knock-in rescue allele. Cre-mediated Syk deletion in Syk(flox/Zap-70) B cells lowers pErk, but not pAkt-mediated signaling. Surprisingly, the use of a B-cell-specific constitutive mb1-cre deleter mouse model showed that a small cohort of peripheral Syk(flox/Zap-70);mb1-cre B cells efficiently circumvents deletion, which ultimately favors these Syk-sufficient cells to contribute to the GC reaction. Using a developmentally unbiased Syk(flox/Zap-70);mb1-creER(T2) approach in combination with an inducible tdRFP allele, we further demonstrate that this monoallelic deletion escape is not fully explained by leakiness of Cre expression, but is possibly the result of differential Syk locus accessibility in maturing B cells. Altogether, this underscores the importance of proper Syk kinase function not only during central and peripheral selection processes, but also during GC formation and maintenance.
Asunto(s)
Linfocitos B/metabolismo , Centro Germinal/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Receptores de Antígenos de Linfocitos B/metabolismo , Proteína Tirosina Quinasa ZAP-70/metabolismo , Alelos , Animales , Linfocitos B/citología , Linfocitos B/inmunología , Quinasas MAP Reguladas por Señal Extracelular/genética , Quinasas MAP Reguladas por Señal Extracelular/inmunología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Regulación de la Expresión Génica , Prueba de Complementación Genética , Centro Germinal/citología , Centro Germinal/inmunología , Integrasas/genética , Integrasas/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/inmunología , Ratones , Ratones Transgénicos , Fosforilación , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/inmunología , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/inmunología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptores de Antígenos de Linfocitos B/genética , Receptores de Antígenos de Linfocitos B/inmunología , Transducción de Señal , Quinasa Syk , Proteína Tirosina Quinasa ZAP-70/genética , Proteína Tirosina Quinasa ZAP-70/inmunologíaRESUMEN
Functional endothelial cells and their progenitors are required for vascular development, adequate vascular function, vascular repair and for cell-based therapies of ischemic diseases. Currently, cell therapy is limited by the low abundance of patient-derived cells and by the functional impairment of autologous endothelial progenitor cells (EPCs). In the present study, murine germline-derived pluripotent stem (gPS) cells were evaluated as a potential source for functional endothelial-like cells. Cells displaying an endothelial cell-like morphology were obtained from gPS cell-derived embryoid bodies using a combination of fluorescence-activated cell sorting (FACS)-based selection of CD31-positive cells and their subsequent cultivation on OP9 stromal cells in the presence of VEGF-A. Real-time reverse transcriptase polymerase chain reaction, FACS analysis and immunofluorescence staining showed that the gPS cell-derived endothelial-like cells (gPS-ECs) expressed endothelial cell-specific markers including von Willebrand Factor, Tie2, VEGFR2/Flk1, intercellular adhesion molecule 2 and vascular endothelial-cadherin. The high expression of ephrin B2, as compared to Eph B4 and VEGFR3, suggests an arterial rather than a venous or lymphatic differentiation. Their capability to take up Dil-conjugated acetylated low-density lipoprotein and to form capillary-like networks on matrigel confirmed their functionality. We conclude that gPS cells could be a novel source of endothelial cells potentially suitable for regenerative cell-based therapies for ischemic diseases.
Asunto(s)
Células Madre Adultas/citología , Células Madre Adultas/fisiología , Células Endoteliales/citología , Células Endoteliales/fisiología , Ingeniería de Tejidos/métodos , Animales , Técnicas de Cultivo de Célula/métodos , Diferenciación Celular/fisiología , Línea Celular , Proliferación Celular , Supervivencia Celular/fisiología , RatonesRESUMEN
The conversion of the nuclear program of a somatic cell from a differentiated to an undifferentiated state can be accomplished by transplanting its nucleus to an enucleated oocyte (somatic cell nuclear transfer [SCNT]) in a process termed "reprogramming." This process achieves pluripotency and occasionally also totipotency. Exploiting the obstacle of tetraploidy to full development in mammals, we show that mouse ooplasts transplanted with two somatic nuclei simultaneously (double SCNT) support preimplantation development and derivation of novel tetraploid SCNT embryonic stem cells (tNT-ESCs). Although the double SCNT embryos do not recapitulate the expression pattern of the pluripotency-associated gene Oct4 in fertilized embryos, derivative tNT-ESCs have characteristics of genuine pluripotency: in vitro they differentiate into neurons, cardiomyocytes, and endodermal cells; in vivo, tNT-ESCs form teratomas, albeit at reduced rates compared to diploid counterparts. Global transcriptome analysis revealed only few specific alterations, for example, in the quantitative expression of gastrulation-associated genes. In conclusion, we have shown that the oocyte's reprogramming capacity is in excess of a single nucleus and that double nucleus-transplanted embryos and derivative ESCs are very similar to their diploid counterparts. These results have key implications for reprogramming studies based on pluripotency: while reprogramming in the tetraploid state was known from fusion-mediated reprogramming and from fetal and adult hepatocyte-derived induced pluripotent stem cells, we have now accomplished it with enucleated oocytes.
Asunto(s)
Reprogramación Celular/fisiología , Células Madre Embrionarias/fisiología , Oocitos/fisiología , Células Madre Pluripotentes/fisiología , Animales , Núcleo Celular/genética , Núcleo Celular/metabolismo , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Femenino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Técnicas de Transferencia Nuclear , Oocitos/citología , Oocitos/metabolismo , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , TetraploidíaRESUMEN
Myelofibrosis and osteosclerosis are fibrotic diseases disrupting bone marrow function that occur in various leukemias but also in response to non-malignant alterations in hematopoietic cells. Here we show that endothelial cell-specific inactivation of the Lats2 gene, encoding Hippo kinase large tumor suppressor kinase 2, or overexpression of the downstream effector YAP1 induce myofibroblast formation and lead to extensive fibrosis and osteosclerosis, which impair bone marrow function and cause extramedullary hematopoiesis in the spleen. Mechanistically, loss of LATS2 induces endothelial-to-mesenchymal transition, resulting in increased expression of extracellular matrix and secreted signaling molecules. Changes in endothelial cells involve increased expression of serum response factor target genes, and, strikingly, major aspects of the LATS2 mutant phenotype are rescued by inactivation of the Srf gene. These findings identify the endothelium as a driver of bone marrow fibrosis, which improves understanding of myelofibrotic and osteosclerotic diseases, for which drug therapies are currently lacking.
RESUMEN
Embryonic diapause is a reproductive adaptation that enables some mammalian species to halt the otherwise continuous pace of embryonic development. In this dormant state, the embryo exploits poorly understood regulatory mechanisms to preserve its developmental potential for prolonged periods of time. Here, using mouse embryos and single-cell RNA sequencing, we molecularly defined embryonic diapause at single-cell resolution, revealing transcriptional dynamics while the embryo seemingly resides in a state of suspended animation. Additionally, we found that the dormant pluripotent cells rely on integrin receptors to sense their microenvironment and preserve their viability via Yap/Taz-mediated prosurvival signaling.
RESUMEN
Cell segregation allows the compartmentalization of cells with similar fates during morphogenesis, which can be enhanced by cell fate plasticity in response to local molecular and biomechanical cues. Endothelial tip cells in the growing retina, which lead vessel sprouts, give rise to arterial endothelial cells and thereby mediate arterial growth. Here, we have combined cell type-specific and inducible mouse genetics, flow experiments in vitro, single-cell RNA sequencing and biochemistry to show that the balance between ephrin-B2 and its receptor EphB4 is critical for arterial specification, cell sorting and arteriovenous patterning. At the molecular level, elevated ephrin-B2 function after loss of EphB4 enhances signaling responses by the Notch pathway, VEGF and the transcription factor Dach1, which is influenced by endothelial shear stress. Our findings reveal how Eph-ephrin interactions integrate cell segregation and arteriovenous specification in the vasculature, which has potential relevance for human vascular malformations caused by EPHB4 mutations.
Asunto(s)
Células Endoteliales , Efrinas , Ratones , Humanos , Animales , Células Endoteliales/metabolismo , Efrina-B2/genética , Efrina-B2/metabolismo , Arterias/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Separación Celular , Receptor EphB4/genética , Receptor EphB4/metabolismoRESUMEN
Molecular mechanisms underlying the commitment of cells to the germ cell lineage during mammalian embryogenesis remain poorly understood due to the limited availability of cellular materials to conduct in vitro analyses. Although primordial germ cells (PGCs)--precursors to germ cells--have been generated from embryonic stem cells (ESCs)--pluripotent stem cells derived from the inner cell mass of the blastocyst of the early embryo in vitro-the simultaneous expression of cell surface receptors and transcription factors complicates the detection of PGCs. To date, only a few genes that mark the onset of germ cell commitment in the epiblast--the outer layer of cells of the embryo--including tissue non-specific alkaline phosphatase (TNAP), Blimp1, Stella and Fragilis--have been used with some success to detect PGC formation in in vitro model systems. Here, we identified 11 genes (three of which are novel) that are specifically expressed in male and female fetal germ cells, both in vivo and in vitro, but are not expressed in ESCs. Expression of these genes allows us to distinguish committed germ cells from undifferentiated pluripotent cell populations, a prerequisite for the successful derivation of germ cells and gametes in vitro.