RESUMEN
Measurements of gene expression or signal transduction activity are conventionally performed using methods that require either the destruction or live imaging of a biological sample within the timeframe of interest. Here we demonstrate an alternative paradigm in which such biological activities are stably recorded to the genome. Enhancer-driven genomic recording of transcriptional activity in multiplex (ENGRAM) is based on the signal-dependent production of prime editing guide RNAs that mediate the insertion of signal-specific barcodes (symbols) into a genomically encoded recording unit. We show how this strategy can be used for multiplex recording of the cell-type-specific activities of dozens to hundreds of cis-regulatory elements with high fidelity, sensitivity and reproducibility. Leveraging signal transduction pathway-responsive cis-regulatory elements, we also demonstrate time- and concentration-dependent genomic recording of WNT, NF-κB and Tet-On activities. By coupling ENGRAM to sequential genome editing via DNA Typewriter1, we stably record information about the temporal dynamics of two orthogonal signalling pathways to genomic DNA. Finally we apply ENGRAM to integratively record the transient activity of nearly 100 transcription factor consensus motifs across daily windows spanning the differentiation of mouse embryonic stem cells into gastruloids, an in vitro model of early mammalian development. Although these are proof-of-concept experiments and much work remains to fully realize the possibilities, the symbolic recording of biological signals or states within cells, to the genome and over time, has broad potential to complement contemporary paradigms for how we make measurements in biological systems.
Asunto(s)
ADN , Edición Génica , FN-kappa B , Transducción de Señal , Animales , Ratones , ADN/genética , ADN/metabolismo , Transducción de Señal/genética , FN-kappa B/metabolismo , Elementos de Facilitación Genéticos/genética , Humanos , ARN Guía de Sistemas CRISPR-Cas/genética , ARN Guía de Sistemas CRISPR-Cas/metabolismo , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Diferenciación Celular/genética , Reproducibilidad de los Resultados , Células Madre Embrionarias de Ratones/metabolismo , Células Madre Embrionarias de Ratones/citología , Genómica , Factores de Tiempo , Secuencias Reguladoras de Ácidos Nucleicos/genética , Vía de Señalización Wnt/genética , Transcripción GenéticaRESUMEN
The house mouse (Mus musculus) is an exceptional model system, combining genetic tractability with close evolutionary affinity to humans1,2. Mouse gestation lasts only 3 weeks, during which the genome orchestrates the astonishing transformation of a single-cell zygote into a free-living pup composed of more than 500 million cells. Here, to establish a global framework for exploring mammalian development, we applied optimized single-cell combinatorial indexing3 to profile the transcriptional states of 12.4 million nuclei from 83 embryos, precisely staged at 2- to 6-hour intervals spanning late gastrulation (embryonic day 8) to birth (postnatal day 0). From these data, we annotate hundreds of cell types and explore the ontogenesis of the posterior embryo during somitogenesis and of kidney, mesenchyme, retina and early neurons. We leverage the temporal resolution and sampling depth of these whole-embryo snapshots, together with published data4-8 from earlier timepoints, to construct a rooted tree of cell-type relationships that spans the entirety of prenatal development, from zygote to birth. Throughout this tree, we systematically nominate genes encoding transcription factors and other proteins as candidate drivers of the in vivo differentiation of hundreds of cell types. Remarkably, the most marked temporal shifts in cell states are observed within one hour of birth and presumably underlie the massive physiological adaptations that must accompany the successful transition of a mammalian fetus to life outside the womb.
Asunto(s)
Animales Recién Nacidos , Embrión de Mamíferos , Desarrollo Embrionario , Gástrula , Análisis de la Célula Individual , Imagen de Lapso de Tiempo , Animales , Femenino , Ratones , Embarazo , Animales Recién Nacidos/embriología , Animales Recién Nacidos/genética , Diferenciación Celular/genética , Embrión de Mamíferos/citología , Embrión de Mamíferos/embriología , Desarrollo Embrionario/genética , Gástrula/citología , Gástrula/embriología , Gastrulación/genética , Riñón/citología , Riñón/embriología , Mesodermo/citología , Mesodermo/enzimología , Neuronas/citología , Neuronas/metabolismo , Retina/citología , Retina/embriología , Somitos/citología , Somitos/embriología , Factores de Tiempo , Factores de Transcripción/genética , Transcripción Genética , Especificidad de Órganos/genéticaRESUMEN
The human embryo undergoes morphogenetic transformations following implantation into the uterus, but our knowledge of this crucial stage is limited by the inability to observe the embryo in vivo. Models of the embryo derived from stem cells are important tools for interrogating developmental events and tissue-tissue crosstalk during these stages1. Here we establish a model of the human post-implantation embryo, a human embryoid, comprising embryonic and extraembryonic tissues. We combine two types of extraembryonic-like cell generated by overexpression of transcription factors with wild-type embryonic stem cells and promote their self-organization into structures that mimic several aspects of the post-implantation human embryo. These self-organized aggregates contain a pluripotent epiblast-like domain surrounded by extraembryonic-like tissues. Our functional studies demonstrate that the epiblast-like domain robustly differentiates into amnion, extraembryonic mesenchyme and primordial germ cell-like cells in response to bone morphogenetic protein cues. In addition, we identify an inhibitory role for SOX17 in the specification of anterior hypoblast-like cells2. Modulation of the subpopulations in the hypoblast-like compartment demonstrates that extraembryonic-like cells influence epiblast-like domain differentiation, highlighting functional tissue-tissue crosstalk. In conclusion, we present a modular, tractable, integrated3 model of the human embryo that will enable us to probe key questions of human post-implantation development, a critical window during which substantial numbers of pregnancies fail.
Asunto(s)
Implantación del Embrión , Embrión de Mamíferos , Desarrollo Embrionario , Modelos Biológicos , Células Madre Pluripotentes , Femenino , Humanos , Embarazo , Proteínas Morfogenéticas Óseas , Diferenciación Celular , Embrión de Mamíferos/citología , Embrión de Mamíferos/embriología , Cuerpos Embrioides/citología , Estratos Germinativos/citología , Estratos Germinativos/embriología , Células Madre Embrionarias Humanas/citología , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Células Madre Pluripotentes/citologíaRESUMEN
Sex chromosome disorders severely compromise gametogenesis in both males and females. In oogenesis, the presence of an additional Y chromosome or the loss of an X chromosome disturbs the robust production of oocytes1-5. Here we efficiently converted the XY chromosome set to XX without an additional Y chromosome in mouse pluripotent stem (PS) cells. In addition, this chromosomal alteration successfully eradicated trisomy 16, a model of Down's syndrome, in PS cells. Artificially produced euploid XX PS cells differentiated into mature oocytes in culture with similar efficiency to native XX PS cells. Using this method, we differentiated induced pluripotent stem cells from the tail of a sexually mature male mouse into fully potent oocytes, which gave rise to offspring after fertilization. This study provides insights that could ameliorate infertility caused by sex chromosome or autosomal disorders, and opens the possibility of bipaternal reproduction.
Asunto(s)
Ingeniería Genética , Técnicas In Vitro , Oocitos , Cromosoma X , Animales , Femenino , Masculino , Ratones , Oocitos/metabolismo , Oocitos/fisiología , Cromosoma X/genética , Cromosoma Y/genética , Células Madre Pluripotentes/metabolismo , Síndrome de Down/genética , Síndrome de Down/terapia , Fertilización , Infertilidad/terapia , Homosexualidad Masculina , Trastornos de los Cromosomas Sexuales/complicaciones , Trastornos de los Cromosomas Sexuales/genética , Trastornos de los Cromosomas Sexuales/terapia , Ingeniería Genética/métodosRESUMEN
DNA is naturally well suited to serve as a digital medium for in vivo molecular recording. However, contemporary DNA-based memory devices are constrained in terms of the number of distinct 'symbols' that can be concurrently recorded and/or by a failure to capture the order in which events occur1. Here we describe DNA Typewriter, a general system for in vivo molecular recording that overcomes these and other limitations. For DNA Typewriter, the blank recording medium ('DNA Tape') consists of a tandem array of partial CRISPR-Cas9 target sites, with all but the first site truncated at their 5' ends and therefore inactive. Short insertional edits serve as symbols that record the identity of the prime editing guide RNA2 mediating the edit while also shifting the position of the 'type guide' by one unit along the DNA Tape, that is, sequential genome editing. In this proof of concept of DNA Typewriter, we demonstrate recording and decoding of thousands of symbols, complex event histories and short text messages; evaluate the performance of dozens of orthogonal tapes; and construct 'long tape' potentially capable of recording as many as 20 serial events. Finally, we leverage DNA Typewriter in conjunction with single-cell RNA-seq to reconstruct a monophyletic lineage of 3,257 cells and find that the Poisson-like accumulation of sequential edits to multicopy DNA tape can be maintained across at least 20 generations and 25 days of in vitro clonal expansion.
Asunto(s)
ADN , Edición Génica , Genoma , Sistemas CRISPR-Cas/genética , ADN/genética , Edición Génica/métodos , Genoma/genética , ARN Guía de Kinetoplastida/genética , RNA-Seq , Análisis de la Célula Individual , Factores de TiempoRESUMEN
During female germline development, oocytes become a highly specialized cell type and form a maternal cytoplasmic store of crucial factors. Oocyte growth is triggered at the transition from primordial to primary follicle and is accompanied by dynamic changes in gene expression1, but the gene regulatory network that controls oocyte growth remains unknown. Here we identify a set of transcription factors that are sufficient to trigger oocyte growth. By investigation of the changes in gene expression and functional screening using an in vitro mouse oocyte development system, we identified eight transcription factors, each of which was essential for the transition from primordial to primary follicle. Notably, enforced expression of these transcription factors swiftly converted pluripotent stem cells into oocyte-like cells that were competent for fertilization and subsequent cleavage. These transcription-factor-induced oocyte-like cells were formed without specification of primordial germ cells, epigenetic reprogramming or meiosis, and demonstrate that oocyte growth and lineage-specific de novo DNA methylation are separable from the preceding epigenetic reprogramming in primordial germ cells. This study identifies a core set of transcription factors for orchestrating oocyte growth, and provides an alternative source of ooplasm, which is a unique material for reproductive biology and medicine.
Asunto(s)
Oocitos/metabolismo , Oogénesis/genética , Factores de Transcripción/metabolismo , Animales , Linaje de la Célula , Epigénesis Genética , Femenino , Fertilización , Meiosis , Metilación , Ratones , Oocitos/citología , Folículo Ovárico/citología , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismoRESUMEN
In mammals, primordial germ cells (PGCs), the origin of the germ line, are specified from the epiblast at the posterior region where gastrulation simultaneously occurs, yet the functional relationship between PGC specification and gastrulation remains unclear. Here, we show that OVOL2, a transcription factor conserved across the animal kingdom, balances these major developmental processes by repressing the epithelial-to-mesenchymal transition (EMT) that drives gastrulation and the upregulation of genes associated with PGC specification. Ovol2a, a splice variant encoding a repressor domain, directly regulates EMT-related genes and, consequently, induces re-acquisition of potential pluripotency during PGC specification, whereas Ovol2b, another splice variant missing the repressor domain, directly upregulates genes associated with PGC specification. Taken together, these results elucidate the molecular mechanism underlying allocation of the germ line among epiblast cells differentiating into somatic cells through gastrulation. This article has an associated 'The people behind the papers' interview.
Asunto(s)
Desarrollo Embrionario/genética , Gastrulación/genética , Células Germinativas/metabolismo , Factores de Transcripción/metabolismo , Animales , Cadherinas/genética , Cadherinas/metabolismo , Linaje de la Célula , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Transición Epitelial-Mesenquimal/genética , Femenino , Células Germinativas/citología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Factor 1 de Unión al Dominio 1 de Regulación Positiva/genética , Factor 1 de Unión al Dominio 1 de Regulación Positiva/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Factores de Transcripción/deficiencia , Factores de Transcripción/genética , Regulación hacia Arriba , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/genética , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/metabolismoRESUMEN
A set of sex chromosomes is required for gametogenesis in both males and females, as represented by sex chromosome disorders causing agametic phenotypes. Although studies using model animals have investigated the functional requirement of sex chromosomes, involvement of these chromosomes in gametogenesis remains elusive. Here, we elicit a germ cell-intrinsic effect of sex chromosomes on oogenesis, using a novel culture system in which oocytes were induced from embryonic stem cells (ESCs) harboring XX, XO or XY. In the culture system, oogenesis using XO and XY ESCs was severely disturbed, with XY ESCs being more strongly affected. The culture system revealed multiple defects in the oogenesis of XO and XY ESCs, such as delayed meiotic entry and progression, and mispairing of the homologous chromosomes. Interestingly, Eif2s3y, a Y-linked gene that promotes proliferation of spermatogonia, had an inhibitory effect on oogenesis. This led us to the concept that male and female gametogenesis appear to be in mutual conflict at an early stage. This study provides a deeper understanding of oogenesis under a sex-reversal condition.
Asunto(s)
Células Germinativas/metabolismo , Oocitos/metabolismo , Cromosoma X , Cromosoma Y , Animales , Diferenciación Celular/fisiología , Células Madre Embrionarias/metabolismo , Femenino , Células Germinativas/citología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos ICR , Ratones Endogámicos , Oocitos/citología , Oocitos/ultraestructura , OogénesisRESUMEN
The female germ line undergoes a unique sequence of differentiation processes that confers totipotency to the egg. The reconstitution of these events in vitro using pluripotent stem cells is a key achievement in reproductive biology and regenerative medicine. Here we report successful reconstitution in vitro of the entire process of oogenesis from mouse pluripotent stem cells. Fully potent mature oocytes were generated in culture from embryonic stem cells and from induced pluripotent stem cells derived from both embryonic fibroblasts and adult tail tip fibroblasts. Moreover, pluripotent stem cell lines were re-derived from the eggs that were generated in vitro, thereby reconstituting the full female germline cycle in a dish. This culture system will provide a platform for elucidating the molecular mechanisms underlying totipotency and the production of oocytes of other mammalian species in culture.
Asunto(s)
Oocitos/citología , Oogénesis/fisiología , Células Madre Pluripotentes/citología , Animales , Línea Celular , Embrión de Mamíferos/citología , Embrión de Mamíferos/embriología , Femenino , Fertilización , Técnicas In Vitro , Masculino , Meiosis , Ratones , Células Madre Embrionarias de Ratones/citología , Oocitos/metabolismo , Oogénesis/genética , Transcriptoma/genéticaRESUMEN
In mammals, most immature oocytes remain dormant in the primordial follicles to ensure the longevity of female reproductive life. A precise understanding of mechanisms underlying the dormancy is important for reproductive biology and medicine. In this study, by comparing mouse oogenesis in vivo and in vitro, the latter of which bypasses the primordial follicle stage, we defined the gene-expression profile representing the dormant state of oocytes. Overexpression of constitutively active FOXO3 partially reproduced the dormant state in vitro. Based on further gene-expression analysis, we found that a hypoxic condition efficiently induced the dormant state in vitro. The effect of hypoxia was severely diminished by disruption of the Foxo3 gene and inhibition of hypoxia-inducible factors. Our findings provide insights into the importance of environmental conditions and their effectors for establishing the dormant state.
Asunto(s)
Proteína Forkhead Box O3/fisiología , Hipoxia/metabolismo , Oocitos/metabolismo , Oogénesis , Animales , Proteína Forkhead Box O3/metabolismo , Ratones , Oocitos/fisiología , TranscriptomaRESUMEN
Specific gene expression in granulosa cells is key for the function of ovary, but the molecular mechanism of transcriptional activation is not well studied. Here we investigated the regulatory mechanism of the mouse stearoyl-CoA desaturase 2 (Scd2) gene encoding an enzyme for lipid metabolism. Northern blot and in situ hybridization indicated that the mouse Scd2 mRNA was highly expressed in ovarian granulosa cells. We found four conserved noncoding sequences (CNSs) and two long noncoding RNAs (lncRNAs) transcribed from regions upstream of the Scd2 gene as candidates of regulatory elements/factors. These lncRNAs were predominantly transcribed in the opposite direction to Scd2 and localized in nuclei and showed the correlation with Scd2 expression, raising the possibility of their transcriptional regulatory roles. Indeed, knockdown of both lncRNAs, lncRNA-sc1 and lncRNA-sc2, significantly decreased the Scd2 mRNA level in primary granulosa cells. Then, we investigated the histone modification pattern at this locus by a chromatin immunoprecipitation assay, and two CNSs, CNS1 and CNS2, were found to be marked with high levels of histone H3K9/K27 acetylation in primary granulosa cells. By a reporter gene assay, both CNS1 and CNS2 interdependently exhibited enhancer activity for the Scd2 promoter in primary granulosa cells. These data suggest that the mouse Scd2 gene is activated by two lncRNAs and interdependent enhancers in ovarian granulosa cells, which provides a new insight into transcriptional activation in granulosa cells.
Asunto(s)
Regulación de la Expresión Génica , Células de la Granulosa/metabolismo , ARN Largo no Codificante , Estearoil-CoA Desaturasa/genética , Activación Transcripcional , Animales , Inmunoprecipitación de Cromatina , Secuencia Conservada , Elementos de Facilitación Genéticos , Femenino , Perfilación de la Expresión Génica , Genes Reporteros , Células de la Granulosa/citología , Histonas/metabolismo , Humanos , Ratones , Ratones Endogámicos C57BL , Oligonucleótidos Antisentido/metabolismo , Ovario/metabolismo , Regiones Promotoras GenéticasRESUMEN
In mice, zygotic activation occurs for a wide variety of genes, mainly at the 2-cell stage. Long noncoding RNAs (lncRNAs) are increasingly being recognized as modulators of gene expression. In this study, directional RNA-seq of MII oocytes and 2-cell embryos identified more than 1000 divergently transcribed lncRNA/mRNA gene pairs. Expression of these bidirectional promoter-associated noncoding RNAs (pancRNAs) was strongly associated with the upregulation of their cognate genes. Conversely, knockdown of three abundant pancRNAs led to reduced mRNA expression, accompanied by sustained DNA methylation even in the presence of enzymes responsible for DNA demethylation. In particular, microinjection of siRNA against the abundant pancRNA partner of interleukin 17d (Il17d) mRNA at the 1-cell stage caused embryonic lethality, which was rescued by supplying IL17D protein in vitro at the 4-cell stage. Thus, this novel class of lncRNAs can modulate the transcription machinery in cis to activate zygotic genes and is important for preimplantation development.
Asunto(s)
ARN Largo no Codificante/genética , Animales , Blastocisto/metabolismo , Blastocisto/fisiología , Metilación de ADN/genética , Minería de Datos , Epigénesis Genética/genética , Femenino , Masculino , Ratones , Oocitos/metabolismo , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , Reacción en Cadena de la Polimerasa , Embarazo , Espermatozoides/citologíaRESUMEN
Primordial germ cells (PGCs) are the earliest precursors of the gametes. During normal development, PGCs only give rise to oocytes or spermatozoa. However, PGCs can acquire pluripotency in vitro by forming embryonic germ (EG) cells and in vivo during teratocarcinogenesis. Classic embryological experiments directly assessed the potency of PGCs by injection into the pre-implantation embryo. As no contribution to embryos or adult mice was observed, PGCs have been described as unipotent. Here, we demonstrate that PGCs injected into 8-cell embryos can initially survive, divide, and contribute to the developing inner cell mass. Apoptosis-deficient PGCs exhibit improved survival in isolated epiblasts and can form naive pluripotent embryonic stem cell lines. However, contribution to the post-implantation embryo is limited, with no functional incorporation observed. In contrast, PGC-like cells show an extensive contribution to mid-gestation chimeras. We thus propose that PGC formation in vivo establishes a latent form of pluripotency that restricts chimera contribution.
Asunto(s)
Células Germinativas , Células Madre Pluripotentes , Masculino , Ratones , Animales , Células Germinativas/metabolismo , Células Madre Embrionarias/metabolismo , Células Madre Pluripotentes/metabolismo , Espermatozoides , Estratos Germinativos , Diferenciación CelularRESUMEN
Gastruloids are a powerful in vitro model of early human development. However, although elongated and composed of all three germ layers, human gastruloids do not morphologically resemble post-implantation human embryos. Here we show that an early pulse of retinoic acid (RA), together with later Matrigel, robustly induces human gastruloids with posterior embryo-like morphological structures, including a neural tube flanked by segmented somites and diverse cell types, including neural crest, neural progenitors, renal progenitors and myocytes. Through in silico staging based on single-cell RNA sequencing, we find that human RA-gastruloids progress further than other human or mouse embryo models, aligning to E9.5 mouse and CS11 cynomolgus monkey embryos. We leverage chemical and genetic perturbations of RA-gastruloids to confirm that WNT and BMP signalling regulate somite formation and neural tube length in the human context, while transcription factors TBX6 and PAX3 underpin presomitic mesoderm and neural crest, respectively. Looking forward, RA-gastruloids are a robust, scalable model for decoding early human embryogenesis.
RESUMEN
Embryonic organoids are emerging as powerful models for studying early mammalian development. For example, stem cell-derived 'gastruloids' form elongating structures containing all three germ layers1-4. However, although elongated, human gastruloids do not morphologically resemble post-implantation embryos. Here we show that a specific, discontinuous regimen of retinoic acid (RA) robustly induces human gastruloids with embryo-like morphological structures, including a neural tube and segmented somites. Single cell RNA-seq (sc-RNA-seq) further reveals that these human 'RA-gastruloids' contain more advanced cell types than conventional gastruloids, including neural crest cells, renal progenitor cells, skeletal muscle cells, and, rarely, neural progenitor cells. We apply a new approach to computationally stage human RA-gastruloids relative to somite-resolved mouse embryos, early human embryos and other gastruloid models, and find that the developmental stage of human RA-gastruloids is comparable to that of E9.5 mouse embryos, although some cell types show greater or lesser progression. We chemically perturb WNT and BMP signaling in human RA-gastruloids and find that these signaling pathways regulate somite patterning and neural tube length, respectively, while genetic perturbation of the transcription factors PAX3 and TBX6 markedly compromises the formation of neural crest and somites/renal cells, respectively. Human RA-gastruloids complement other embryonic organoids in serving as a simple, robust and screenable model for decoding early human embryogenesis.
RESUMEN
The house mouse, Mus musculus, is an exceptional model system, combining genetic tractability with close homology to human biology. Gestation in mouse development lasts just under three weeks, a period during which its genome orchestrates the astonishing transformation of a single cell zygote into a free-living pup composed of >500 million cells. Towards a global framework for exploring mammalian development, we applied single cell combinatorial indexing (sci-*) to profile the transcriptional states of 12.4 million nuclei from 83 precisely staged embryos spanning late gastrulation (embryonic day 8 or E8) to birth (postnatal day 0 or P0), with 2-hr temporal resolution during somitogenesis, 6-hr resolution through to birth, and 20-min resolution during the immediate postpartum period. From these data (E8 to P0), we annotate dozens of trajectories and hundreds of cell types and perform deeper analyses of the unfolding of the posterior embryo during somitogenesis as well as the ontogenesis of the kidney, mesenchyme, retina, and early neurons. Finally, we leverage the depth and temporal resolution of these whole embryo snapshots, together with other published data, to construct and curate a rooted tree of cell type relationships that spans mouse development from zygote to pup. Throughout this tree, we systematically nominate sets of transcription factors (TFs) and other genes as candidate drivers of the in vivo differentiation of hundreds of mammalian cell types. Remarkably, the most dramatic shifts in transcriptional state are observed in a restricted set of cell types in the hours immediately following birth, and presumably underlie the massive changes in physiology that must accompany the successful transition of a placental mammal to extrauterine life.
RESUMEN
In vitro gametogenesis, the process of generating gametes from pluripotent cells in culture, is a powerful tool for improving our understanding of germ cell development and an alternative source of gametes. Here, we induced primordial germ cell-like cells (PGCLCs) from pluripotent stem cells of the northern white rhinoceros (NWR), a species for which only two females remain, and southern white rhinoceros (SWR), the closest species to the NWR. PGCLC differentiation from SWR embryonic stem cells is highly reliant on bone morphogenetic protein and WNT signals. Genetic analysis revealed that SRY-box transcription factor 17 (SOX17) is essential for SWR-PGCLC induction. Under the defined condition, NWR induced pluripotent stem cells differentiated into PGCLCs. We also identified cell surface markers, CD9 and Integrin subunit alpha 6 (ITGA6), that enabled us to isolate PGCLCs without genetic alteration in pluripotent stem cells. This study provides a first step toward the production of NWR gametes in culture and understanding of the basic mechanism of primordial germ cell specification in a large animal.
Asunto(s)
Células Madre Pluripotentes Inducidas , Células Madre Pluripotentes , Animales , Femenino , Células Germinativas , Células Madre Embrionarias/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Diferenciación CelularRESUMEN
The most immature oocytes remain dormant in primordial follicles in the ovary, ensuring the longevity of female reproductive life. Despite its biological and clinical importance, knowledge of mechanisms regulating the dormant state remains limited. Here, we show that mechanical stress plays a key role in maintaining the dormant state of the oocytes in primordial follicles in mice. Transcriptional and histological analyses revealed that oocytes were compressed by surrounding granulosa cells with extracellular matrix. This environmental state is functionally crucial, as oocytes became activated upon loosening the structure and the dormancy was restored by additional compression with exogenous pressure. The nuclei of oocytes in primordial follicles rotated in response to the mechanical stress. Pausing the rotation triggered activation of oocytes through nuclear export of forkhead box O3 (FOXO3). These results provide insights into the mechanisms by which oocytes are kept dormant to sustain female reproductive life.
Asunto(s)
Núcleo Celular/fisiología , Oocitos/fisiología , Animales , Núcleo Celular/metabolismo , Matriz Extracelular/metabolismo , Femenino , Proteína Forkhead Box O3/metabolismo , Ratones , Oocitos/metabolismo , Técnicas de Cultivo de Órganos/métodos , Folículo Ovárico/metabolismo , Folículo Ovárico/fisiología , Rotación , Transducción de Señal/fisiología , Estrés MecánicoRESUMEN
Minimal sets of transcription factors can directly reprogram somatic cells into neurons. However, epigenetic remodeling during neuronal reprogramming has not been well reconciled with transcriptional regulation. Here we show that NeuroD1 achieves direct neuronal conversion from mouse microglia both in vitro and in vivo. Exogenous NeuroD1 initially occupies closed chromatin regions associated with bivalent trimethylation of histone H3 at lysine 4 (H3K4me3) and H3K27me3 marks in microglia to induce neuronal gene expression. These regions are resolved to a monovalent H3K4me3 mark at later stages of reprogramming to establish the neuronal identity. Furthermore, the transcriptional repressors Scrt1 and Meis2 are induced as NeuroD1 target genes, resulting in a decrease in the expression of microglial genes. In parallel, the microglial epigenetic signature in promoter and enhancer regions is erased. These findings reveal NeuroD1 pioneering activity accompanied by global epigenetic remodeling for two sequential events: onset of neuronal property acquisition and loss of the microglial identity during reprogramming.