RESUMEN
Oncogenic mutations are abundant in the tissues of healthy individuals, but rarely form tumours1-3. Yet, the underlying protection mechanisms are largely unknown. To resolve these mechanisms in mouse mammary tissue, we use lineage tracing to map the fate of wild-type and Brca1-/-;Trp53-/- cells, and find that both follow a similar pattern of loss and spread within ducts. Clonal analysis reveals that ducts consist of small repetitive units of self-renewing cells that give rise to short-lived descendants. This offers a first layer of protection as any descendants, including oncogenic mutant cells, are constantly lost, thereby limiting the spread of mutations to a single stem cell-descendant unit. Local tissue remodelling during consecutive oestrous cycles leads to the cooperative and stochastic loss and replacement of self-renewing cells. This process provides a second layer of protection, leading to the elimination of most mutant clones while enabling the minority that by chance survive to expand beyond the stem cell-descendant unit. This leads to fields of mutant cells spanning large parts of the epithelial network, predisposing it for transformation. Eventually, clone expansion becomes restrained by the geometry of the ducts, providing a third layer of protection. Together, these mechanisms act to eliminate most cells that acquire somatic mutations at the expense of driving the accelerated expansion of a minority of cells, which can colonize large areas, leading to field cancerization.
Asunto(s)
Proteína BRCA1 , Linaje de la Célula , Transformación Celular Neoplásica , Glándulas Mamarias Animales , Mutación , Proteína p53 Supresora de Tumor , Animales , Ratones , Femenino , Glándulas Mamarias Animales/citología , Glándulas Mamarias Animales/patología , Glándulas Mamarias Animales/metabolismo , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Linaje de la Célula/genética , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Transformación Celular Neoplásica/genética , Células Clonales/metabolismo , Células Clonales/citología , Carcinogénesis/genética , Carcinogénesis/patología , Autorrenovación de las Células/genéticaRESUMEN
Age is a risk factor for hematologic malignancies. Attributes of the aging hematopoietic system include increased myelopoiesis, impaired adaptive immunity, and a functional decline of the hematopoietic stem cells (HSCs) that maintain hematopoiesis. Changes in the composition of diverse HSC subsets have been suggested to be responsible for age-related alterations, however, the underlying regulatory mechanisms are incompletely understood in the context of HSC heterogeneity. In this study, we investigated how distinct HSC subsets, separated by CD49b, functionally and molecularly change their behavior with age. We demonstrate that the lineage differentiation of both lymphoid-biased and myeloid-biased HSC subsets progressively shifts to a higher myeloid cellular output during aging. In parallel, we show that HSCs selectively undergo age-dependent gene expression and gene regulatory changes in a progressive manner, which is initiated already in the juvenile stage. Overall, our studies suggest that aging intrinsically alters both cellular and molecular properties of HSCs.
Asunto(s)
Envejecimiento , Células Madre Hematopoyéticas , Ratones Endogámicos C57BL , Células Madre Hematopoyéticas/metabolismo , Células Madre Hematopoyéticas/citología , Animales , Envejecimiento/genética , Envejecimiento/fisiología , Ratones , Diferenciación Celular , Linaje de la Célula/genética , Hematopoyesis/genética , Células Mieloides/metabolismo , Células Mieloides/citología , Masculino , Regulación de la Expresión Génica , FemeninoRESUMEN
Cancer is a highly heterogeneous disease, where phenotypically distinct subpopulations coexist and can be primed to different fates. Both genetic and epigenetic factors may drive cancer evolution, however little is known about whether and how such a process is pre-encoded in cancer clones. Using single-cell multi-omic lineage tracing and phenotypic assays, we investigate the predictive features of either tumour initiation or drug tolerance within the same cancer population. Clones primed to tumour initiation in vivo display two distinct transcriptional states at baseline. Remarkably, these states share a distinctive DNA accessibility profile, highlighting an epigenetic basis for tumour initiation. The drug tolerant niche is also largely pre-encoded, but only partially overlaps the tumour-initiating one and evolves following two genetically and transcriptionally distinct trajectories. Our study highlights coexisting genetic, epigenetic and transcriptional determinants of cancer evolution, unravelling the molecular complexity of pre-encoded tumour phenotypes.
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Epigénesis Genética , Regulación Neoplásica de la Expresión Génica , Neoplasias , Humanos , Neoplasias/genética , Animales , Análisis de la Célula Individual/métodos , Ratones , Linaje de la Célula/genética , Línea Celular Tumoral , Transcripción Genética , Fenotipo , MultiómicaRESUMEN
Arterial endothelial cells (AECs) are the founder cells for intraembryonic haematopoiesis. Here, we report a method for the efficient generation of human haemogenic DLL4+ AECs from pluripotent stem cells (PSC). Time-series single-cell RNA-sequencing reveals the dynamic evolution of haematopoiesis and lymphopoiesis, generating cell types with counterparts present in early human embryos, including stages marked by the pre-haematopoietic stem cell genes MECOM/EVI1, MLLT3 and SPINK2. DLL4+ AECs robustly support lymphoid differentiation, without the requirement for exogenous NOTCH ligands. Using this system, we find IL7 acts as a morphogenic factor determining the fate choice between the T and innate lymphoid lineages and also plays a role in regulating the relative expression level of RAG1. Moreover, we document a developmental pathway by which human RAG1+ lymphoid precursors give rise to the natural killer cell lineage. Our study describes an efficient method for producing lymphoid progenitors, providing insights into their endothelial and haematopoietic ontogeny, and establishing a platform to investigate the development of the human blood system.
Asunto(s)
Hematopoyesis , Linfopoyesis , Humanos , Hematopoyesis/genética , Linfopoyesis/genética , Células Endoteliales/metabolismo , Células Endoteliales/citología , Diferenciación Celular , Linaje de la Célula/genética , Interleucina-7/metabolismo , Interleucina-7/genética , Células Madre Pluripotentes/metabolismo , Células Madre Pluripotentes/citología , Proteínas de Unión al Calcio/metabolismo , Proteínas de Unión al Calcio/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Células Asesinas Naturales/metabolismo , Células Asesinas Naturales/citología , Hemangioblastos/metabolismo , Hemangioblastos/citología , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Proteínas de Homeodominio/metabolismo , Proteínas de Homeodominio/genética , Análisis de la Célula Individual/métodos , Receptores Notch/metabolismo , Receptores Notch/genéticaRESUMEN
Lymphocyte receptors independently evolved in both jawed and jawless vertebrates with similar adaptive immune responses. However, the diversity of functional subtypes and molecular architecture in jawless vertebrate lymphocytes, comparable to jawed species, is not well defined. Here, we profile the gills, intestines, and blood of the lamprey, Lampetra morii, with single-cell RNA sequencing, using a full-length transcriptome as a reference. Our findings reveal higher tissue-specific heterogeneity among T-like cells in contrast to B-like cells. Notably, we identify a unique T-like cell subtype expressing a homolog of the nonlymphoid hematopoietic growth factor receptor, MPL-like (MPL-L). These MPL-L+ T-like cells exhibit features distinct from T cells of jawed vertebrates, particularly in their elevated expression of hematopoietic genes. We further discovered that MPL-L+ VLRA+ T-like cells are widely present in the typhlosole, gill, liver, kidney, and skin of lamprey and they proliferate in response to both a T cell mitogen and recombinant human thrombopoietin. These findings provide new insights into the adaptive immune response in jawless vertebrates, shedding new light on the evolution of adaptive immunity.
Asunto(s)
Inmunidad Adaptativa , Linaje de la Célula , Lampreas , Animales , Lampreas/inmunología , Lampreas/genética , Inmunidad Adaptativa/genética , Linaje de la Célula/genética , Evolución Biológica , Transcriptoma , Linfocitos T/inmunología , Branquias/inmunología , Branquias/metabolismo , Linfocitos/inmunología , Análisis de la Célula Individual , HumanosRESUMEN
Multiple embryonic origins give rise to forebrain oligodendrocytes (OLs), yet controversies and uncertainty exist regarding their differential contributions. We established intersectional and subtractional strategies to genetically fate map OLs produced by medial ganglionic eminence/preoptic area (MGE/POA), lateral/caudal ganglionic eminences (LGE/CGE), and dorsal pallium in the mouse brain. We found that, contrary to the canonical view, LGE/CGE-derived OLs make minimum contributions to the neocortex and corpus callosum, but dominate piriform cortex and anterior commissure. Additionally, MGE/POA-derived OLs, instead of being entirely eliminated, make small but sustained contribution to cortex with a distribution pattern distinctive from those derived from the dorsal origin. Our study provides a revised and more comprehensive view of cortical and white matter OL origins, and established valuable new tools and strategies for future OL studies.
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Oligodendroglía , Prosencéfalo , Animales , Oligodendroglía/metabolismo , Oligodendroglía/citología , Prosencéfalo/embriología , Prosencéfalo/citología , Ratones , Linaje de la Célula/genéticaRESUMEN
Long known as the site of ribosome biogenesis, the nucleolus is increasingly recognized for its role in shaping three-dimensional (3D) genome organization. Still, the mechanisms governing the targeting of selected regions of the genome to nucleolus-associated domains (NADs) remain enigmatic. Here, we reveal the essential role of ZNF274, a SCAN-bearing member of the Krüppel-associated box (KRAB)-containing zinc finger protein (KZFP) family, in sequestering lineage-specific gene clusters within NADs. Ablation of ZNF274 triggers transcriptional activation across entire genomic neighborhoods-encompassing, among others, protocadherin and KZFP-encoding genes-with loss of repressive chromatin marks, altered the 3D genome architecture and de novo CTCF binding. Mechanistically, ZNF274 anchors target DNA sequences at the nucleolus and facilitates their compartmentalization via a previously uncharted function of the SCAN domain. Our findings illuminate the mechanisms underlying NAD organization and suggest that perinucleolar entrapment into repressive hubs constrains the activation of tandemly arrayed genes to enable selective expression and modulate cell differentiation programs during development.
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Nucléolo Celular , Familia de Multigenes , Nucléolo Celular/metabolismo , Nucléolo Celular/genética , Animales , Humanos , Ratones , Factor de Unión a CCCTC/metabolismo , Factor de Unión a CCCTC/genética , Cromatina/metabolismo , Cromatina/genética , Linaje de la Célula/genética , Dedos de Zinc/genética , Diferenciación Celular/genética , Unión ProteicaRESUMEN
LMNA-related dilated cardiomyopathy (DCM) is an autosomal-dominant genetic condition with cardiomyocyte and conduction system dysfunction often resulting in heart failure or sudden death. The condition is caused by mutation in the Lamin A/C (LMNA) gene encoding Type-A nuclear lamin proteins involved in nuclear integrity, epigenetic regulation of gene expression, and differentiation. The molecular mechanisms of the disease are not completely understood, and there are no definitive treatments to reverse progression or prevent mortality. We investigated possible mechanisms of LMNA-related DCM using induced pluripotent stem cells derived from a family with a heterozygous LMNA c.357-2A>G splice-site mutation. We differentiated one LMNA-mutant iPSC line derived from an affected female (Patient) and two non-mutant iPSC lines derived from her unaffected sister (Control) and conducted single-cell RNA sequencing for 12 samples (four from Patients and eight from Controls) across seven time points: Day 0, 2, 4, 9, 16, 19, and 30. Our bioinformatics workflow identified 125,554 cells in raw data and 110,521 (88%) high-quality cells in sequentially processed data. Unsupervised clustering, cell annotation, and trajectory inference found complex heterogeneity: ten main cell types; many possible subtypes; and lineage bifurcation for cardiac progenitors to cardiomyocytes (CMs) and epicardium-derived cells (EPDCs). Data integration and comparative analyses of Patient and Control cells found cell type and lineage-specific differentially expressed genes (DEGs) with enrichment, supporting pathway dysregulation. Top DEGs and enriched pathways included 10 ZNF genes and RNA polymerase II transcription in pluripotent cells (PP); BMP4 and TGF Beta/BMP signaling, sarcomere gene subsets and cardiogenesis, CDH2 and EMT in CMs; LMNA and epigenetic regulation, as well as DDIT4 and mTORC1 signaling in EPDCs. Top DEGs also included XIST and other X-linked genes, six imprinted genes (SNRPN, PWAR6, NDN, PEG10, MEG3, MEG8), and enriched gene sets related to metabolism, proliferation, and homeostasis. We confirmed Lamin A/C haploinsufficiency by allelic expression and Western blot. Our complex Patient-derived iPSC model for Lamin A/C haploinsufficiency in PP, CM, and EPDC provided support for dysregulation of genes and pathways, many previously associated with Lamin A/C defects, such as epigenetic gene expression, signaling, and differentiation. Our findings support disruption of epigenomic developmental programs, as proposed in other LMNA disease models. We recognized other factors influencing epigenetics and differentiation; thus, our approach needs improvement to further investigate this mechanism in an iPSC-derived model.
Asunto(s)
Cardiomiopatía Dilatada , Diferenciación Celular , Haploinsuficiencia , Células Madre Pluripotentes Inducidas , Lamina Tipo A , Miocitos Cardíacos , Transcriptoma , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/patología , Humanos , Cardiomiopatía Dilatada/genética , Cardiomiopatía Dilatada/patología , Cardiomiopatía Dilatada/metabolismo , Lamina Tipo A/genética , Lamina Tipo A/metabolismo , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Diferenciación Celular/genética , Haploinsuficiencia/genética , Femenino , Transcriptoma/genética , Pericardio/patología , Pericardio/metabolismo , Linaje de la Célula/genética , Análisis de la Célula Individual , Regulación de la Expresión Génica , Mutación/genética , AdultoRESUMEN
Alternative cleavage and polyadenylation (APA) often results in production of mRNA isoforms with either longer or shorter 3' UTRs from the same genetic locus, potentially impacting mRNA translation, localization, and stability. Developmentally regulated APA can thus make major contributions to cell type-specific gene expression programs as cells differentiate. During Drosophila spermatogenesis, â¼500 genes undergo APA when proliferating spermatogonia differentiate into spermatocytes, producing transcripts with shortened 3' UTRs, leading to profound stage-specific changes in the proteins expressed. The molecular mechanisms that specify usage of upstream polyadenylation sites in spermatocytes are thus key to understanding the changes in cell state. Here, we show that upregulation of PCF11 and Cbc, the two components of cleavage factor II (CFII), orchestrates APA during Drosophila spermatogenesis. Knockdown of PCF11 or cbc in spermatocytes caused dysregulation of APA, with many transcripts normally cleaved at a proximal site in spermatocytes now cleaved at their distal site, as in spermatogonia. Forced overexpression of CFII components in spermatogonia switched cleavage of some transcripts to the proximal site normally used in spermatocytes. Our findings reveal a developmental mechanism where changes in expression of specific cleavage factors can direct cell type-specific APA at selected genes.
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Linaje de la Célula , Poliadenilación , Espermatocitos , Espermatogénesis , Animales , Poliadenilación/genética , Masculino , Espermatogénesis/genética , Espermatocitos/metabolismo , Espermatocitos/citología , Linaje de la Célula/genética , Regulación del Desarrollo de la Expresión Génica/genética , Células Madre Adultas/metabolismo , Células Madre Adultas/citología , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/citología , Drosophila melanogaster/metabolismo , Espermatogonias/citología , Espermatogonias/metabolismo , Factores de Escisión y Poliadenilación de ARNm/metabolismo , Factores de Escisión y Poliadenilación de ARNm/genéticaRESUMEN
Cell fate specification occurs along invariant species-specific trajectories that define the animal body plan. This process is controlled by gene regulatory networks that regulate the expression of the limited set of transcription factors encoded in animal genomes. Here we globally assess the spatial expression of ~90% of expressed transcription factors during sea urchin development from embryo to larva to determine the activity of gene regulatory networks and their regulatory states during cell fate specification. We show that >200 embryonically expressed transcription factors together define >70 cell fates that recapitulate the morphological and functional organization of this organism. Most cell fate-specific regulatory states consist of ~15-40 transcription factors with similarity particularly among functionally related cell types regardless of developmental origin. Temporally, regulatory states change continuously during development, indicating that progressive changes in regulatory circuit activity determine cell fate specification. We conclude that the combinatorial expression of transcription factors provides molecular definitions that suffice for the unique specification of cell states in time and space during embryogenesis.
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Embrión no Mamífero , Desarrollo Embrionario , Regulación del Desarrollo de la Expresión Génica , Redes Reguladoras de Genes , Factores de Transcripción , Animales , Desarrollo Embrionario/genética , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Embrión no Mamífero/metabolismo , Linaje de la Célula/genética , Erizos de Mar/embriología , Erizos de Mar/genética , Erizos de Mar/metabolismo , Diferenciación Celular/genética , Larva/metabolismo , Larva/genética , Larva/crecimiento & desarrolloRESUMEN
Proneural transcription factors establish molecular cascades to orchestrate neuronal diversity. One such transcription factor, Atonal homolog 1 (Atoh1), gives rise to cerebellar excitatory neurons and over 30 distinct nuclei in the brainstem critical for hearing, breathing, and balance. Although Atoh1 lineage neurons have been qualitatively described, the transcriptional programs that drive their fate decisions and the full extent of their diversity remain unknown. Here, we analyzed single-cell RNA sequencing and ATOH1 DNA binding in Atoh1 lineage neurons of the developing mouse hindbrain. This high-resolution dataset identified markers for specific brainstem nuclei and demonstrated that transcriptionally heterogeneous progenitors require ATOH1 for proper migration. Moreover, we identified a sizable population of proliferating unipolar brush cell progenitors in the mouse Atoh1 lineage, previously described in humans as the origin of one medulloblastoma subtype. Collectively, our data provide insights into the developing mouse hindbrain and markers for functional assessment of understudied neuronal populations.
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Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Linaje de la Célula , Neuronas , Rombencéfalo , Análisis de la Célula Individual , Transcriptoma , Animales , Rombencéfalo/metabolismo , Rombencéfalo/citología , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Ratones , Neuronas/metabolismo , Neuronas/citología , Linaje de la Célula/genética , Análisis de la Célula Individual/métodos , Transcriptoma/genética , Diferenciación Celular , Regulación del Desarrollo de la Expresión Génica , Neurogénesis/genética , Movimiento CelularRESUMEN
The rate at which cells enter the T cell pathway depends not only on the immigration of hematopoietic precursors into the strong Notch signaling environment of the thymus but also on the kinetics with which each individual precursor cell reaches T-lineage commitment once it arrives. Notch triggers a complex, multistep gene regulatory network in the cells in which the steps are stereotyped but the transition speeds between steps are variable. Progenitor-associated transcription factors delay T-lineage differentiation even while Notch-induced transcription factors within the same cells push differentiation forward. Progress depends on regulator cross-repression, on breaching chromatin barriers, and on shifting, competitive collaborations between stage-specific and stably expressed transcription factors, as reviewed here.
Asunto(s)
Diferenciación Celular , Redes Reguladoras de Genes , Receptores Notch , Linfocitos T , Linfocitos T/metabolismo , Linfocitos T/citología , Linfocitos T/inmunología , Animales , Diferenciación Celular/genética , Humanos , Receptores Notch/metabolismo , Receptores Notch/genética , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Transducción de Señal , Linaje de la Célula/genética , Transcripción GenéticaRESUMEN
Variability between human pluripotent stem cell (hPSC) lines remains a challenge and opportunity in biomedicine. In this study, hPSC lines from multiple donors were differentiated toward neuroectoderm and mesendoderm lineages. We revealed dynamic transcriptomic patterns that delineate the emergence of these lineages, which were conserved across lines, along with individual line-specific transcriptional signatures that were invariant throughout differentiation. These transcriptomic signatures predicted an antagonism between SOX21-driven forebrain fates and retinoic acid-induced hindbrain fates. Replicate lines and paired adult tissue demonstrated the stability of these line-specific transcriptomic traits. We show that this transcriptomic variation in lineage bias had both genetic and epigenetic origins, aligned with the anterior-to-posterior structure of early mammalian development, and was present across a large collection of hPSC lines. These findings contribute to developing systematic analyses of PSCs to define the origin and consequences of variation in the early events orchestrating individual human development.
Asunto(s)
Diferenciación Celular , Linaje de la Célula , Células Madre Pluripotentes , Transcriptoma , Humanos , Células Madre Pluripotentes/metabolismo , Células Madre Pluripotentes/citología , Diferenciación Celular/genética , Linaje de la Célula/genética , Línea Celular , Tretinoina/farmacología , Tretinoina/metabolismo , Regulación del Desarrollo de la Expresión Génica , Epigénesis GenéticaRESUMEN
Single-cell RNA sequencing (scRNA-seq) enables comprehensive characterization of the micro-evolutionary processes of B cells during an adaptive immune response, capturing features of somatic hypermutation (SHM) and class switch recombination (CSR). Existing phylogenetic approaches for reconstructing B cell evolution have primarily focused on the SHM process alone. Here, we present tree inference of B cell clonal lineages (TRIBAL), an algorithm designed to optimally reconstruct the evolutionary history of B cell clonal lineages undergoing both SHM and CSR from scRNA-seq data. Through simulations, we demonstrate that TRIBAL produces more comprehensive and accurate B cell lineage trees compared to existing methods. Using real-world datasets, TRIBAL successfully recapitulates expected biological trends in a model affinity maturation system while reconstructing evolutionary histories with more parsimonious class switching than state-of-the-art methods. Thus, TRIBAL significantly improves B cell lineage tracing, useful for modeling vaccine responses, disease progression, and the identification of therapeutic antibodies.
Asunto(s)
Algoritmos , Linfocitos B , Linaje de la Célula , Análisis de la Célula Individual , Linfocitos B/inmunología , Análisis de la Célula Individual/métodos , Linaje de la Célula/genética , Humanos , Filogenia , Hipermutación Somática de Inmunoglobulina/genética , Cambio de Clase de Inmunoglobulina/genética , Análisis de Secuencia de ARN/métodosRESUMEN
Alveolar type 2 epithelial (AT2) cells synthesize surfactant protein C (SPC) and repair an injured alveolar epithelium. A mutated surfactant protein C gene (SftpcL184Q, Gene ID: 6440) in newborns has been associated with respiratory distress syndrome and pulmonary fibrosis. However, the underlying mechanisms causing Sftpc gene mutations to regulate AT2 lineage remain unclear. We utilized three-dimensional (3D) feeder-free AT2 organoids in vitro to simulate the alveolar epithelium and compared AT2 lineage characteristics between WT (C57BL/6) and SftpcL184Q mutant mice using colony formation assays, immunofluorescence, flow cytometry, qRT-PCR, and Western blot assays. The AT2 numbers were reduced significantly in SftpcL184Q mice. Organoid numbers and colony-forming efficiency were significantly attenuated in the 3D cultures of primary SftpcL184Q AT2 cells compared to those of WT mice. Podoplanin (PDPN, Alveolar type 1 cell (AT1) marker) expression and transient cell count was significantly increased in SftpcL184Q organoids compared to in the WT mice. The expression levels of CD74, heat shock protein 90 (HSP90), and ribosomal protein S3A1 (RPS3A1) were not significantly different between WT and SftpcL184Q AT2 cells. This study demonstrated that humanized SftpcL184Q mutation regulates AT2 lineage intrinsically. This regulation is independent of CD74, HSP90, and RPS3A1 pathways.
Asunto(s)
Células Epiteliales Alveolares , Proteína C Asociada a Surfactante Pulmonar , Animales , Humanos , Ratones , Células Epiteliales Alveolares/metabolismo , Células Epiteliales Alveolares/citología , Diferenciación Celular/genética , Linaje de la Célula/genética , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Ratones Endogámicos C57BL , Mutación , Organoides/metabolismo , Organoides/citología , Proteína C Asociada a Surfactante Pulmonar/genética , Proteína C Asociada a Surfactante Pulmonar/metabolismo , Masculino , FemeninoRESUMEN
Human induced pluripotent stem cell (hiPSC) to cardiomyocyte (CM) differentiation has reshaped approaches to studying cardiac development and disease. In this study, we employed a genome-wide CRISPR screen in a hiPSC to CM differentiation system and reveal here that BRD4, a member of the bromodomain and extraterminal (BET) family, regulates CM differentiation. Chemical inhibition of BET proteins in mouse embryonic stem cell (mESC)-derived or hiPSC-derived cardiac progenitor cells (CPCs) results in decreased CM differentiation and persistence of cells expressing progenitor markers. In vivo, BRD4 deletion in second heart field (SHF) CPCs results in embryonic or early postnatal lethality, with mutants demonstrating myocardial hypoplasia and an increase in CPCs. Single-cell transcriptomics identified a subpopulation of SHF CPCs that is sensitive to BRD4 loss and associated with attenuated CM lineage-specific gene programs. These results highlight a previously unrecognized role for BRD4 in CM fate determination during development and a heterogenous requirement for BRD4 among SHF CPCs.
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Sistemas CRISPR-Cas , Diferenciación Celular , Células Madre Pluripotentes Inducidas , Miocitos Cardíacos , Factores de Transcripción , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/citología , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Animales , Diferenciación Celular/genética , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/citología , Humanos , Sistemas CRISPR-Cas/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Ratones , Células Madre Embrionarias de Ratones/metabolismo , Células Madre Embrionarias de Ratones/citología , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Regulación del Desarrollo de la Expresión Génica , Linaje de la Célula/genética , Células Cultivadas , Análisis de la Célula Individual , Proteínas que Contienen BromodominioRESUMEN
We designed a simulation program that mimics the CRISPR-Cas9 editing on evolving barcode and double strand break repair procedure along with cell divisions. Emerging barcode mutations tend to build upon previously existing mutations, occurring sequentially with each generation. This process results in a unique mutation profile in each cell. We sample the barcodes in leaf cells and reconstruct the lineage, comparing it to the original lineage tree to test algorithm accuracy under different parameter settings. Our computational simulations validate the reasonable assumptions deduced from experimental observations, emphasizing that factors such as sampling size, barcode length, multiple barcodes, indel probabilities, and Cas9 activity are critical for accurate and successful lineage tracing. Among the many factors we found that sampling size and indel probabilities are two major ones that affect lineage tracing accuracy. Large segment deletions in early generations could greatly impact lineage accuracy. These simulation results offer insightful recommendations for enhancing the design and analysis of Cas9-mediated molecular barcodes in actual experiments.
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Sistemas CRISPR-Cas , Simulación por Computador , Edición Génica , Edición Génica/métodos , Algoritmos , Código de Barras del ADN Taxonómico/métodos , Linaje de la Célula/genética , Mutación INDEL , MutaciónRESUMEN
Exploring the complexity of the epithelial-to-mesenchymal transition (EMT) unveils a diversity of potential cell fates; however, the exact timing and mechanisms by which early cell states diverge into distinct EMT trajectories remain unclear. Studying these EMT trajectories through single-cell RNA sequencing is challenging due to the necessity of sacrificing cells for each measurement. In this study, we employed optimal-transport analysis to reconstruct the past trajectories of different cell fates during TGF-beta-induced EMT in the MCF10A cell line. Our analysis revealed three distinct trajectories leading to low EMT, partial EMT, and high EMT states. Cells along the partial EMT trajectory showed substantial variations in the EMT signature and exhibited pronounced stemness. Throughout this EMT trajectory, we observed a consistent downregulation of the EED and EZH2 genes. This finding was validated by recent inhibitor screens of EMT regulators and CRISPR screen studies. Moreover, we applied our analysis of early-phase differential gene expression to gene sets associated with stemness and proliferation, pinpointing ITGB4, LAMA3, and LAMB3 as genes differentially expressed in the initial stages of the partial versus high EMT trajectories. We also found that CENPF, CKS1B, and MKI67 showed significant upregulation in the high EMT trajectory. While the first group of genes aligns with findings from previous studies, our work uniquely pinpoints the precise timing of these upregulations. Finally, the identification of the latter group of genes sheds light on potential cell cycle targets for modulating EMT trajectories.
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Transición Epitelial-Mesenquimal , Análisis de la Célula Individual , Transición Epitelial-Mesenquimal/genética , Humanos , Análisis de la Célula Individual/métodos , Linaje de la Célula/genética , Factor de Crecimiento Transformador beta/metabolismo , Proteína Potenciadora del Homólogo Zeste 2/metabolismo , Proteína Potenciadora del Homólogo Zeste 2/genéticaRESUMEN
In animals, stem cell populations of varying potency facilitate regeneration and tissue homeostasis. Notably, germline stem cells in both vertebrates and invertebrates express highly conserved RNA binding proteins, such as nanos, vasa, and piwi. In highly regenerative animals, these genes are also expressed in somatic stem cells, which led to the proposal that they had an ancestral role in all stem cells. In cnidarians, multi- and pluripotent interstitial stem cells have only been identified in hydrozoans. Therefore, it is currently unclear if cnidarian stem cell systems share a common evolutionary origin. We, therefore, aimed to characterize conserved stem cell marker genes in the sea anemone Nematostella vectensis. Through transgenic reporter genes and single-cell transcriptomics, we identify cell populations expressing the germline-associated markers piwi1 and nanos2 in the soma and germline, and gene knockout shows that Nanos2 is indispensable for germline formation. This suggests that nanos and piwi genes have a conserved role in somatic and germline stem cells in cnidarians.
Asunto(s)
Células Germinativas , Proteínas de Unión al ARN , Anémonas de Mar , Animales , Anémonas de Mar/genética , Anémonas de Mar/metabolismo , Células Germinativas/metabolismo , Células Germinativas/citología , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Linaje de la Célula/genética , Células Madre/metabolismo , Células Madre/citología , Proteínas Argonautas/metabolismo , Proteínas Argonautas/genéticaRESUMEN
The development of highly specialized blood cells from hematopoietic stem cells (HSCs) in the bone marrow (BM) is dependent upon a stringently orchestrated network of stage- and lineage-restricted transcription factors (TFs). Thus, the same stem cell can give rise to various types of differentiated blood cells. One of the key regulators of B-lymphocyte development is early B-cell factor 1 (EBF1). This TF belongs to a small, but evolutionary conserved, family of proteins that harbor a Zn-coordinating motif and an IPT/TIG (immunoglobulin-like, plexins, transcription factors/transcription factor immunoglobulin) domain, creating a unique DNA-binding domain (DBD). EBF proteins play critical roles in diverse developmental processes, including body segmentation in the Drosophila melanogaster embryo, and retina formation in mice. While several EBF family members are expressed in neuronal cells, adipocytes, and BM stroma cells, only B-lymphoid cells express EBF1. In the absence of EBF1, hematopoietic progenitor cells (HPCs) fail to activate the B-lineage program. This has been attributed to the ability of EBF1 to act as a pioneering factor with the ability to remodel chromatin, thereby creating a B-lymphoid-specific epigenetic landscape. Conditional inactivation of the Ebf1 gene in B-lineage cells has revealed additional functions of this protein in relation to the control of proliferation and apoptosis. This may explain why EBF1 is frequently targeted by mutations in human leukemia cases. This chapter provides an overview of the biochemical and functional properties of the EBF family proteins, with a focus on the roles of EBF1 in normal and malignant B-lymphocyte development.