RESUMEN
Thymic epithelial cells (TECs) are crucial to the ability of the thymus to generate T cells for the adaptive immune system in vertebrates. However, no in vitro system for studying TEC function exists. Overexpressing the transcription factor FOXN1 initiates transdifferentiation of fibroblasts into TEC-like cells (iTECs) that support T-cell differentiation in culture or after transplant. In this study, we have characterized iTEC programming at the cellular and molecular level in mouse to determine how it proceeds, and have identified mechanisms that can be targeted for improving this process. These data show that iTEC programming consists of discrete gene expression changes that differ early and late in the process, and that iTECs upregulate markers of both cortical and medullary TEC (cTEC and mTEC) lineages. We demonstrate that promoting proliferation enhances iTEC generation, and that Notch inhibition allows the induction of mTEC differentiation. Finally, we show that MHCII expression is the major difference between iTECs and fetal TECs. MHCII expression was improved by co-culturing iTECs with fetal double-positive T-cells. This study supports future efforts to improve iTEC generation for both research and translational uses.
Asunto(s)
Diferenciación Celular , Células Epiteliales , Fibroblastos , Factores de Transcripción Forkhead , Timo , Animales , Células Epiteliales/metabolismo , Células Epiteliales/citología , Timo/citología , Timo/metabolismo , Timo/embriología , Fibroblastos/metabolismo , Fibroblastos/citología , Factores de Transcripción Forkhead/metabolismo , Factores de Transcripción Forkhead/genética , Ratones , Proliferación Celular , Transdiferenciación Celular , Linfocitos T/citología , Linfocitos T/metabolismo , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Técnicas de Cocultivo , Receptores Notch/metabolismoRESUMEN
Hematopoietic stem cells (HSCs) develop from hemogenic endothelial cells (HECs) in vivo during mouse embryogenesis. When cultured in vitro, cells from the embryo phenotypically defined as pre-HSC-I and pre-HSC-II have the potential to differentiate into HSCs. However, minimal factors required for HSC induction from HECs have not yet been determined. In this study, we demonstrated that stem cell factor (SCF) and thrombopoietin (TPO) induced engrafting HSCs from embryonic day (E) 11.5 pre-HSC-I in a serum-free and feeder-free culture condition. In contrast, E10.5 pre-HSC-I and HECs required an endothelial cell layer in addition to SCF and TPO to differentiate into HSCs. A single-cell RNA sequencing analysis of E10.5 to 11.5 dorsal aortae with surrounding tissues and fetal livers detected TPO expression confined in hepatoblasts, while SCF was expressed in various tissues, including endothelial cells and hepatoblasts. Our results suggest a transition of signal requirement during HSC development from HECs. The differentiation of E10.5 HECs to E11.5 pre-HSC-I in the aorta-gonad-mesonephros region depends on SCF and endothelial cell-derived factors. Subsequently, SCF and TPO drive the differentiation of E11.5 pre-HSC-I to pre-HSC-II/HSCs in the fetal liver. The culture system established in this study provides a beneficial tool for exploring the molecular mechanisms underlying the development of HSCs from HECs.
Asunto(s)
Diferenciación Celular , Hemangioblastos , Células Madre Hematopoyéticas , Factor de Células Madre , Trombopoyetina , Animales , Células Madre Hematopoyéticas/metabolismo , Células Madre Hematopoyéticas/citología , Ratones , Trombopoyetina/metabolismo , Factor de Células Madre/metabolismo , Hemangioblastos/metabolismo , Hemangioblastos/citología , Células Endoteliales/metabolismo , Células Endoteliales/citología , Transducción de Señal , Hematopoyesis/fisiología , Desarrollo Embrionario , Embrión de Mamíferos/metabolismo , Embrión de Mamíferos/citología , Hígado/embriología , Hígado/metabolismo , Hígado/citologíaRESUMEN
Cellular Potts models are broadly applied across developmental biology and cancer research. We overcome limitations of the traditional approach, which reinterprets a modified Metropolis sampling as ad hoc dynamics, by introducing a physical timescale through Poissonian kinetics and by applying principles of stochastic thermodynamics to separate thermal and relaxation effects from athermal noise and nonconservative forces. Our method accurately describes cell-sorting dynamics in mouse-embryo development and identifies the distinct contributions of nonequilibrium processes, e.g., cell growth and active fluctuations.
Asunto(s)
Modelos Biológicos , Procesos Estocásticos , Animales , Ratones , Cinética , Termodinámica , Desarrollo Embrionario/fisiología , Embrión de Mamíferos/citologíaRESUMEN
Despite a distinct developmental origin, extraembryonic cells in mice contribute to gut endoderm and converge to transcriptionally resemble their embryonic counterparts. Notably, all extraembryonic progenitors share a non-canonical epigenome, raising several pertinent questions, including whether this landscape is reset to match the embryonic regulation and if extraembryonic cells persist into later development. Here we developed a two-colour lineage-tracing strategy to track and isolate extraembryonic cells over time. We find that extraembryonic gut cells display substantial memory of their developmental origin including retention of the original DNA methylation landscape and resulting transcriptional signatures. Furthermore, we show that extraembryonic gut cells undergo programmed cell death and neighbouring embryonic cells clear their remnants via non-professional phagocytosis. By midgestation, we no longer detect extraembryonic cells in the wild-type gut, whereas they persist and differentiate further in p53-mutant embryos. Our study provides key insights into the molecular and developmental fate of extraembryonic cells inside the embryo.
Asunto(s)
Apoptosis , Linaje de la Célula , Metilación de ADN , Endodermo , Regulación del Desarrollo de la Expresión Génica , Animales , Endodermo/citología , Endodermo/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Proteína p53 Supresora de Tumor/genética , Fagocitosis , Ratones Endogámicos C57BL , Ratones , Diferenciación Celular , Femenino , Desarrollo Embrionario , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Ratones Transgénicos , Tracto Gastrointestinal/citología , Tracto Gastrointestinal/embriología , Tracto Gastrointestinal/metabolismoRESUMEN
Monochorionic twinning of human embryos increases the risk of complications during pregnancy. The rarity of such twinning events, combined with ethical constraints in human embryo research, makes investigating the mechanisms behind twinning practically infeasible. As a result, there is a significant knowledge gap regarding the origins and early phenotypic presentation of monochorionic twin embryos. In this study, a microthermoformed-based microwell screening platform is used to identify conditions that efficiently induce monochorionic twins in human stem cell-based blastocyst models, termed "twin blastoids". These twin blastoids contain a cystic GATA3+ trophectoderm-like epithelium encasing two distinct inner cell masses (ICMs). Morphological and morphokinetic analyses reveal that twinning occurs during the cavitation phase via splitting of the OCT4+ pluripotent core. Notably, each ICM in twin blastoids contains its own NR2F2+ polar trophectoderm-like region, ready for implantation. This is functionally tested in a microfluidic chip-based implantation assay with epithelial endometrium cells. Under defined flow regimes, twin blastoids show increased adhesion capacity compared to singleton blastoids, suggestive of increased implantation potential. In conclusion, the development of technology enabling large-scale formation of twin blastoids, coupled with high-sensitivity readout capabilities, presents an unprecedented opportunity for systematically exploring monochorionic twin formation and its impact on embryonic development.
Asunto(s)
Gemelización Monocigótica , Humanos , Femenino , Embarazo , Blastocisto/citología , Embrión de Mamíferos/citología , Corion/citología , Bioingeniería/métodos , Modelos Biológicos , Implantación del EmbriónRESUMEN
Recently, several studies using cultures of human embryos together with single-cell RNA-seq analyses have revealed differences between humans and mice, necessitating the study of human embryos1-8. Despite the importance of human embryology, ethical and legal restrictions have limited post-implantation-stage studies. Thus, recent efforts have focused on developing in vitro self-organizing models using human stem cells9-17. Here, we report genetic and non-genetic approaches to generate authentic hypoblast cells (naive hPSC-derived hypoblast-like cells (nHyCs))-known to give rise to one of the two extraembryonic tissues essential for embryonic development-from naive human pluripotent stem cells (hPSCs). Our nHyCs spontaneously assemble with naive hPSCs to form a three-dimensional bilaminar structure (bilaminoids) with a pro-amniotic-like cavity. In the presence of additional naive hPSC-derived analogues of the second extraembryonic tissue, the trophectoderm, the efficiency of bilaminoid formation increases from 20% to 40%, and the epiblast within the bilaminoids continues to develop in response to trophectoderm-secreted IL-6. Furthermore, we show that bilaminoids robustly recapitulate the patterning of the anterior-posterior axis and the formation of cells reflecting the pregastrula stage, the emergence of which can be shaped by genetically manipulating the DKK1/OTX2 hypoblast-like domain. We have therefore successfully modelled and identified the mechanisms by which the two extraembryonic tissues efficiently guide the stage-specific growth and progression of the epiblast as it establishes the post-implantation landmarks of human embryogenesis.
Asunto(s)
Desarrollo Embrionario , Estratos Germinativos , Células Madre Pluripotentes , Humanos , Diferenciación Celular , Implantación del Embrión , Embrión de Mamíferos/citología , Embrión de Mamíferos/embriología , Embrión de Mamíferos/metabolismo , Desarrollo Embrionario/genética , Desarrollo Embrionario/fisiología , Estratos Germinativos/citología , Estratos Germinativos/embriología , Estratos Germinativos/metabolismo , Células Madre Pluripotentes/citología , Interleucina-6/metabolismo , Gástrula/citología , Gástrula/embriología , Amnios/citología , Amnios/embriología , Amnios/metabolismo , Ectodermo/citología , Ectodermo/embriología , Ectodermo/metabolismo , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Factores de Transcripción Otx/genética , Factores de Transcripción Otx/metabolismoAsunto(s)
Quimera , Investigaciones con Embriones , Embrión de Mamíferos , Haplorrinos , Animales , Humanos , Quimera/embriología , Modelos Animales de Enfermedad , Investigaciones con Embriones/ética , Embrión de Mamíferos/citología , Embrión de Mamíferos/embriología , Haplorrinos/embriología , Trasplante de Células MadreRESUMEN
It has been over three decades since Drs. Herzenberg and Herzenberg proposed the layered immune system hypothesis, suggesting that different types of stem cells with distinct hematopoietic potential produce specific immune cells. This layering of immune system development is now supported by recent studies showing the presence of fetal-derived immune cells that function in adults. It has been shown that various immune cells arise at different embryonic ages via multiple waves of hematopoiesis from special endothelial cells (ECs), referred to as hemogenic ECs. However, it remains unknown whether these fetal-derived immune cells are produced by hematopoietic stem cells (HSCs) during the fetal to neonatal period. To address this question, many advanced tools have been used, including lineage-tracing mouse models, cellular barcoding techniques, clonal assays, and transplantation assays at the single-cell level. In this review, we will review the history of the search for the origins of HSCs, B-1a progenitors, and mast cells in the mouse embryo. HSCs can produce both B-1a and mast cells within a very limited time window, and this ability declines after embryonic day (E) 14.5. Furthermore, the latest data have revealed that HSC-independent adaptive immune cells exist in adult mice, which implies more complicated developmental pathways of immune cells. We propose revised road maps of immune cell development.
Asunto(s)
Sistema Inmunológico , Sistema Inmunológico/citología , Sistema Inmunológico/crecimiento & desarrollo , Humanos , Animales , Hematopoyesis , Embrión de Mamíferos/citología , Células Madre Hematopoyéticas/citología , Linfocitos/citología , Linaje de la CélulaRESUMEN
Haematopoietic stem cells (HSCs) arise in the embryo from the arterial endothelium through a process known as the endothelial-to-haematopoietic transition (EHT)1-4. This process generates hundreds of blood progenitors, of which a fraction go on to become definitive HSCs. It is generally thought that most adult blood is derived from those HSCs, but to what extent other progenitors contribute to adult haematopoiesis is not known. Here we use in situ barcoding and classical fate mapping to assess the developmental and clonal origins of adult blood in mice. Our analysis uncovers an early wave of progenitor specification-independent of traditional HSCs-that begins soon after EHT. These embryonic multipotent progenitors (eMPPs) predominantly drive haematopoiesis in the young adult, have a decreasing yet lifelong contribution over time and are the predominant source of lymphoid output. Putative eMPPs are specified within intra-arterial haematopoietic clusters and represent one fate of the earliest haematopoietic progenitors. Altogether, our results reveal functional heterogeneity during the definitive wave that leads to distinct sources of adult blood.
Asunto(s)
Envejecimiento , Linaje de la Célula , Embrión de Mamíferos , Hematopoyesis , Células Madre Hematopoyéticas , Animales , Embrión de Mamíferos/citología , Células Madre Hematopoyéticas/citología , Ratones , Células Madre Multipotentes/citologíaRESUMEN
Embryo transfer is a crucial step in IVF cycle, with increasing trend during the last decade of transferring a single embryo, preferably at the blastocyst stage. Despite increasing evidence supporting Day 5 blastocyst-stage transfer, the optimal day of embryo transfer remains controversial. The crucial questions are therefore, whether the mechanisms responsible to embryos arrest are embryo aneuploidy or others, and whether those embryos arrested in-vitro between the cleavage to the blastocyst stage would survive in-vivo if transferred on the cleavage-stage. We therefore aim to explore whether aneuploidy can directly contribute to embryo development to the blastocyst stage. Thirty Day-5 embryos, that their Day-3 blastomere biopsy revealed a single-gene defect, were donated by 10 couples undergoing preimplantation genetic testing treatment at our center. Affected high quality Day-3 embryos were cultured to Day-5, and were classified to those that developed to the blastocyst-stage and those that were arrested. Each embryo underwent whole genome amplification. Eighteen (60%) embryos were arrested, did not develop to the blastocyst stage and 12 (40%) have developed to the blastocyst stage. Nineteen embryos (63.3%) were found to be euploid. Of them, 12 (66.6%) were arrested embryos and 7 (58.3%) were those that developed to the blastocyst-stage. These figures were not statistically different (p = 0.644). Our observation demonstrated that the mechanism responsible to embryos arrest in vitro is not embryo aneuploidy, but rather other, such as culture conditions. If further studies will confirm that Day-5 blastocyst transfer might cause losses of embryos that would have been survived in vivo, cleavage-stage embryo transfer would be the preferred timing. This might reduce the cycle cancellations due to failure of embryo to develop to the blastocyst stage and will provide the best cumulative live birth-rate per started cycle.
Asunto(s)
Blastocisto/metabolismo , Fase de Segmentación del Huevo/metabolismo , Embrión de Mamíferos/metabolismo , Desarrollo Embrionario/genética , Trofoblastos/metabolismo , Adulto , Aneuploidia , Blastocisto/citología , Blastómeros/citología , Blastómeros/metabolismo , Células Cultivadas , Fase de Segmentación del Huevo/citología , Hibridación Genómica Comparativa/métodos , Transferencia de Embrión , Embrión de Mamíferos/citología , Femenino , Fertilización In Vitro , Pruebas Genéticas/métodos , Humanos , Nacimiento Vivo , Embarazo , Índice de Embarazo , Trofoblastos/citologíaRESUMEN
After fertilization, the quiescent zygote experiences a burst of genome activation that initiates a short-lived totipotent state. Understanding the process of totipotency in human cells would have broad applications. However, in contrast to in mice1,2, demonstration of the time of zygotic genome activation or the eight-cell (8C) stage in in vitro cultured human cells has not yet been reported, and the study of embryos is limited by ethical and practical considerations. Here we describe a transgene-free, rapid and controllable method for producing 8C-like cells (8CLCs) from human pluripotent stem cells. Single-cell analysis identified key molecular events and gene networks associated with this conversion. Loss-of-function experiments identified fundamental roles for DPPA3, a master regulator of DNA methylation in oocytes3, and TPRX1, a eutherian totipotent cell homeobox (ETCHbox) family transcription factor that is absent in mice4. DPPA3 induces DNA demethylation throughout the 8CLC conversion process, whereas TPRX1 is a key executor of 8CLC gene networks. We further demonstrate that 8CLCs can produce embryonic and extraembryonic lineages in vitro or in vivo in the form of blastoids5 and complex teratomas. Our approach provides a resource to uncover the molecular process of early human embryogenesis.
Asunto(s)
Embrión de Mamíferos , Desarrollo Embrionario , Células Madre Pluripotentes , Cigoto , Humanos , Proteínas Cromosómicas no Histona/genética , Embrión de Mamíferos/citología , Proteínas de Homeodominio/genética , Células Madre Pluripotentes/citología , Factores de Transcripción/genética , Cigoto/citologíaRESUMEN
The tracheal epithelium is a primary target for pulmonary diseases as it provides a conduit for air flow between the environment and the lung lobes. The cellular and molecular mechanisms underlying airway epithelial cell proliferation and differentiation remain poorly understood. Hedgehog (HH) signaling orchestrates communication between epithelial and mesenchymal cells in the lung, where it modulates stromal cell proliferation, differentiation and signaling back to the epithelium. Here, we reveal a previously unreported autocrine function of HH signaling in airway epithelial cells. Epithelial cell depletion of the ligand sonic hedgehog (SHH) or its effector smoothened (SMO) causes defects in both epithelial cell proliferation and differentiation. In cultured primary human airway epithelial cells, HH signaling inhibition also hampers cell proliferation and differentiation. Epithelial HH function is mediated, at least in part, through transcriptional activation, as HH signaling inhibition leads to downregulation of cell type-specific transcription factor genes in both the mouse trachea and human airway epithelial cells. These results provide new insights into the role of HH signaling in epithelial cell proliferation and differentiation during airway development.
Asunto(s)
Comunicación Autocrina/fisiología , Diferenciación Celular , Proliferación Celular , Proteínas Hedgehog/metabolismo , Transducción de Señal/genética , Animales , Células Cultivadas , Regulación hacia Abajo , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Células Epiteliales/citología , Células Epiteliales/metabolismo , Proteínas Hedgehog/deficiencia , Proteínas Hedgehog/genética , Humanos , Pulmón/crecimiento & desarrollo , Pulmón/metabolismo , Pulmón/patología , Ratones , Ratones Noqueados , Receptor Smoothened/deficiencia , Receptor Smoothened/genética , Receptor Smoothened/metabolismo , Tráquea/citología , Tráquea/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Mitochondria are energy-generating organelles and mitochondrial biogenesis is stimulated to meet energy requirements in response to extracellular stimuli, including exercise. However, the mechanisms underlying mitochondrial biogenesis remain unknown. Here, we demonstrate that transcriptional coactivator with PDZ-binding motif (TAZ) stimulates mitochondrial biogenesis in skeletal muscle. In muscle-specific TAZ-knockout (mKO) mice, mitochondrial biogenesis, respiratory metabolism, and exercise ability were decreased compared to wild-type mice. Mechanistically, TAZ stimulates the translation of mitochondrial transcription factor A via Ras homolog enriched in brain (Rheb)/Rheb like 1 (Rhebl1)-mTOR axis. TAZ stimulates Rhebl1 expression via TEA domain family transcription factor. Rhebl1 introduction by adeno-associated virus or mTOR activation recovered mitochondrial biogenesis in mKO muscle. Physiologically, mKO mice did not stimulate exercise-induced mitochondrial biogenesis. Collectively, our results suggested that TAZ is a novel stimulator for mitochondrial biogenesis and exercise-induced muscle adaptation.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas de Unión al ADN/genética , Mitocondrias Musculares/genética , Proteínas Mitocondriales/genética , Biogénesis de Organelos , Condicionamiento Físico Animal , Factores de Transcripción/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Línea Celular , Células Cultivadas , ADN Mitocondrial/genética , ADN Mitocondrial/metabolismo , Proteínas de Unión al ADN/metabolismo , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Fibroblastos/citología , Fibroblastos/metabolismo , Células HEK293 , Humanos , Ratones Noqueados , Mitocondrias Musculares/metabolismo , Proteínas Mitocondriales/metabolismo , Músculo Esquelético/citología , Músculo Esquelético/metabolismo , Mioblastos/citología , Mioblastos/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Hemogenic endothelial (HE) cells in the dorsal aorta undergo an endothelial-to-hematopoietic transition (EHT) to form multipotent progenitors, lympho-myeloid biased progenitors (LMPs), pre-hematopoietic stem cells (pre-HSCs) and adult-repopulating HSCs. These briefly accumulate in intra-arterial hematopoietic clusters (IAHCs) before being released into the circulation. It is generally assumed that the number of IAHC cells correlates with the number of HSCs. Here, we show that changes in the number of IAHC cells, LMPs and HSCs can be uncoupled. Mutations impairing MyD88-dependent toll-like receptor (TLR) signaling decreased the number of IAHC cells and LMPs, but increased the number of HSCs in the aorta-gonad-mesonephros region of mouse embryos. TLR4-deficient embryos generated normal numbers of HE cells, but IAHC cell proliferation decreased. Loss of MyD88-dependent TLR signaling in innate immune myeloid cells had no effect on IAHC cell numbers. Instead, TLR4 deletion in endothelial cells (ECs) recapitulated the phenotype observed with germline deletion, demonstrating that MyD88-dependent TLR signaling in ECs and/or in IAHCs regulates the numbers of LMPs and HSCs.
Asunto(s)
Embrión de Mamíferos/metabolismo , Células Madre Hematopoyéticas/metabolismo , Factor 88 de Diferenciación Mieloide/metabolismo , Transducción de Señal , Animales , Diferenciación Celular , Subunidad alfa 2 del Factor de Unión al Sitio Principal/metabolismo , Embrión de Mamíferos/citología , Células Endoteliales/citología , Células Endoteliales/metabolismo , Hemangioblastos/citología , Hemangioblastos/metabolismo , Células Madre Hematopoyéticas/citología , Inmunidad Innata , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Células Mieloides/citología , Células Mieloides/metabolismo , Factor 88 de Diferenciación Mieloide/deficiencia , Factor 88 de Diferenciación Mieloide/genética , Receptor Toll-Like 4/deficiencia , Receptor Toll-Like 4/genética , Receptor Toll-Like 4/metabolismo , Receptores Toll-Like/metabolismoRESUMEN
The gene activity underlying cell differentiation is regulated by a diverse set of transcription factors (TFs), histone modifications, chromatin structures and more. Although definitive hematopoietic stem cells (HSCs) are known to emerge via endothelial-to-hematopoietic transition (EHT), how the multi-layered epigenome is sequentially unfolded in a small portion of endothelial cells (ECs) transitioning into the hematopoietic fate remains elusive. With optimized low-input itChIP-seq and Hi-C assays, we performed multi-omics dissection of the HSC ontogeny trajectory across early arterial ECs (eAECs), hemogenic endothelial cells (HECs), pre-HSCs and long-term HSCs (LT-HSCs) in mouse embryos. Interestingly, HSC regulatory regions are already pre-configurated with active histone modifications as early as eAECs, preceding chromatin looping dynamics within topologically associating domains. Chromatin looping structures between enhancers and promoters only become gradually strengthened over time. Notably, RUNX1, a master TF for hematopoiesis, enriched at half of these loops is observed early from eAECs through pre-HSCs but its enrichment further increases in HSCs. RUNX1 and co-TFs together constitute a central, progressively intensified enhancer-promoter interactions. Thus, our study provides a framework to decipher how temporal epigenomic configurations fulfill cell lineage specification during development.
Asunto(s)
Cromatina/química , Embrión de Mamíferos/citología , Células Madre Hematopoyéticas/citología , Código de Histonas , Animales , Análisis por Conglomerados , Subunidad alfa 2 del Factor de Unión al Sitio Principal/metabolismo , Elementos de Facilitación Genéticos/genética , Genoma , Ratones Endogámicos C57BL , Anotación de Secuencia Molecular , Regiones Promotoras Genéticas/genética , Factores de Transcripción/metabolismoRESUMEN
LIN28 is an RNA binding protein with important roles in early embryo development, stem cell differentiation/reprogramming, tumorigenesis and metabolism. Previous studies have focused mainly on its role in the cytosol where it interacts with Let-7 microRNA precursors or mRNAs, and few have addressed LIN28's role within the nucleus. Here, we show that LIN28 displays dynamic temporal and spatial expression during murine embryo development. Maternal LIN28 expression drops upon exit from the 2-cell stage, and zygotic LIN28 protein is induced at the forming nucleolus during 4-cell to blastocyst stage development, to become dominantly expressed in the cytosol after implantation. In cultured pluripotent stem cells (PSCs), loss of LIN28 led to nucleolar stress and activation of a 2-cell/4-cell-like transcriptional program characterized by the expression of endogenous retrovirus genes. Mechanistically, LIN28 binds to small nucleolar RNAs and rRNA to maintain nucleolar integrity, and its loss leads to nucleolar phase separation defects, ribosomal stress and activation of P53 which in turn binds to and activates 2C transcription factor Dux. LIN28 also resides in a complex containing the nucleolar factor Nucleolin (NCL) and the transcriptional repressor TRIM28, and LIN28 loss leads to reduced occupancy of the NCL/TRIM28 complex on the Dux and rDNA loci, and thus de-repressed Dux and reduced rRNA expression. Lin28 knockout cells with nucleolar stress are more likely to assume a slowly cycling, translationally inert and anabolically inactive state, which is a part of previously unappreciated 2C-like transcriptional program. These findings elucidate novel roles for nucleolar LIN28 in PSCs, and a new mechanism linking 2C program and nucleolar functions in PSCs and early embryo development.
Asunto(s)
Células Madre Pluripotentes , Proteínas de Unión al ARN/metabolismo , Animales , Diferenciación Celular , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Desarrollo Embrionario , Ratones , Células Madre Pluripotentes/metabolismo , ARN Mensajero/genética , ARN Ribosómico , Factores de Transcripción/metabolismo , Cigoto/metabolismoRESUMEN
In vitro generation and expansion of hematopoietic stem cells (HSCs) holds great promise for the treatment of any ailment that relies on bone marrow or blood transplantation. To achieve this, it is essential to resolve the molecular and cellular pathways that govern HSC formation in the embryo. HSCs first emerge in the aorta-gonad-mesonephros (AGM) region, where a rare subset of endothelial cells, hemogenic endothelium (HE), undergoes an endothelial-to-hematopoietic transition (EHT). Here, we present full-length single-cell RNA sequencing (scRNA-seq) of the EHT process with a focus on HE and dorsal aorta niche cells. By using Runx1b and Gfi1/1b transgenic reporter mouse models to isolate HE, we uncovered that the pre-HE to HE continuum is specifically marked by angiotensin-I converting enzyme (ACE) expression. We established that HE cells begin to enter the cell cycle near the time of EHT initiation when their morphology still resembles endothelial cells. We further demonstrated that RUNX1 AGM niche cells consist of vascular smooth muscle cells and PDGFRa+ mesenchymal cells and can functionally support hematopoiesis. Overall, our study provides new insights into HE differentiation toward HSC and the role of AGM RUNX1+ niche cells in this process. Our expansive scRNA-seq datasets represents a powerful resource to investigate these processes further.
Asunto(s)
Embrión de Mamíferos/embriología , Hemangioblastos/citología , Hematopoyesis , Células Madre Hematopoyéticas/citología , Animales , Diferenciación Celular , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Hemangioblastos/metabolismo , Células Madre Hematopoyéticas/metabolismo , Mesonefro/citología , Mesonefro/embriología , Mesonefro/metabolismo , Ratones , Análisis de la Célula Individual , Transcriptoma , Pez CebraRESUMEN
SHARPIN, together with RNF31/HOIP and RBCK1/HOIL1, form the linear ubiquitin chain assembly complex (LUBAC) E3 ligase that catalyzes M1-linked polyubiquitination. Mutations in RNF31/HOIP and RBCK/HOIL1 in humans and Sharpin in mice lead to autoinflammation and immunodeficiency, but the mechanism underlying the immune dysregulation remains unclear. We now show that the phenotype of the Sharpincpdm/cpdm mice is dependent on CYLD, a deubiquitinase previously shown to mediate removal of K63-linked polyubiquitin chains. Dermatitis, disrupted splenic architecture, and loss of Peyer's patches in the Sharpincpdm/cpdm mice were fully reversed in Sharpincpdm/cpdm Cyld-/- mice. We observed enhanced association of RIPK1 with the death-signaling Complex II following TNF stimulation in Sharpincpdm/cpdm cells, a finding dependent on CYLD since we observed reversal in Sharpincpdm/cpdm Cyld-/- cells. Enhanced RIPK1 recruitment to Complex II in Sharpincpdm/cpdm cells correlated with impaired phosphorylation of CYLD at serine 418, a modification reported to inhibit its enzymatic activity. The dermatitis in the Sharpincpdm/cpdm mice was also ameliorated by the conditional deletion of Cyld using LysM-cre or Cx3cr1-cre indicating that CYLD-dependent death of myeloid cells is inflammatory. Our studies reveal that under physiological conditions, TNF- and RIPK1-dependent cell death is suppressed by the linear ubiquitin-dependent inhibition of CYLD. The Sharpincpdm/cpdm phenotype illustrates the pathological consequences when CYLD inhibition fails.
Asunto(s)
Enzima Desubiquitinante CYLD/metabolismo , Fibroblastos/metabolismo , Inflamación/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Animales , Muerte Celular , Enzima Desubiquitinante CYLD/genética , Embrión de Mamíferos/citología , Femenino , Regulación de la Expresión Génica/inmunología , Regulación de la Expresión Génica/fisiología , Péptidos y Proteínas de Señalización Intracelular/genética , Masculino , Ratones , Ratones Noqueados , Células Mieloides , Fosforilación , Enfermedades de la Piel , UbiquitinaciónRESUMEN
Intestinal mesenchymal cells encompass multiple subsets, whose origins, functions, and pathophysiological importance are still not clear. Here, we used the Col6a1Cre mouse, which targets distinct fibroblast subsets and perivascular cells that can be further distinguished by the combination of the CD201, PDGFRα and αSMA markers. Developmental studies revealed that the Col6a1Cre mouse also targets mesenchymal aggregates that are crucial for intestinal morphogenesis and patterning, suggesting an ontogenic relationship between them and homeostatic PDGFRαhi telocytes. Cell depletion experiments in adulthood showed that Col6a1+/CD201+ mesenchymal cells regulate homeostatic enteroendocrine cell differentiation and epithelial proliferation. During acute colitis, they expressed an inflammatory and extracellular matrix remodelling gene signature, but they also retained their properties and topology. Notably, both in homeostasis and tissue regeneration, they were dispensable for normal organ architecture, while CD34+ mesenchymal cells expanded, localised at the top of the crypts, and showed increased expression of villous-associated morphogenetic factors, providing thus evidence for the plasticity potential of intestinal mesenchymal cells. Our results provide a comprehensive analysis of the identities, origin, and functional significance of distinct mesenchymal populations in the intestine.
Asunto(s)
Colágeno Tipo VI/metabolismo , Receptor de Proteína C Endotelial/metabolismo , Intestinos/metabolismo , Animales , Diferenciación Celular , Linaje de la Célula , Plasticidad de la Célula , Proliferación Celular , Colitis/inducido químicamente , Colitis/patología , Colágeno Tipo VI/deficiencia , Colágeno Tipo VI/genética , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Matriz Extracelular/metabolismo , Fibroblastos/citología , Fibroblastos/metabolismo , Intestinos/citología , Intestinos/fisiología , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Ratones , Ratones Noqueados , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/metabolismo , RegeneraciónRESUMEN
Despite four decades of effort, robust propagation of pluripotent stem cells from livestock animals remains challenging. The requirements for self-renewal are unclear and the relationship of cultured stem cells to pluripotent cells resident in the embryo uncertain. Here, we avoided using feeder cells or serum factors to provide a defined culture microenvironment. We show that the combination of activin A, fibroblast growth factor and the Wnt inhibitor XAV939 (AFX) supports establishment and continuous expansion of pluripotent stem cell lines from porcine, ovine and bovine embryos. Germ layer differentiation was evident in teratomas and readily induced in vitro. Global transcriptome analyses highlighted commonality in transcription factor expression across the three species, while global comparison with porcine embryo stages showed proximity to bilaminar disc epiblast. Clonal genetic manipulation and gene targeting were exemplified in porcine stem cells. We further demonstrated that genetically modified AFX stem cells gave rise to cloned porcine foetuses by nuclear transfer. In summary, for major livestock mammals, pluripotent stem cells related to the formative embryonic disc are reliably established using a common and defined signalling environment. This article has an associated 'The people behind the papers' interview.