RESUMEN
Cooperative DNA binding of transcription factors (TFs) integrates the cellular context to support cell specification during development. Naive mouse embryonic stem cells are derived from early development and can sustain their pluripotent identity indefinitely. Here, we ask whether TFs associated with pluripotency evolved to directly support this state or if the state emerges from their combinatorial action. NANOG and ESRRB are key pluripotency factors that co-bind DNA. We find that when both factors are expressed, ESRRB supports pluripotency. However, when NANOG is absent, ESRRB supports a bistable culture of cells with an embryo-like primitive endoderm identity ancillary to pluripotency. The stoichiometry between NANOG and ESRRB allows quantitative titration of this differentiation, and in silico modeling of bipartite ESRRB activity suggests it safeguards plasticity in differentiation. Thus, the concerted activity of cooperative TFs can transform their effect to sustain intermediate cell identities and allow ex vivo expansion of immortal stem cells. A record of this paper's transparent peer review process is included in the supplemental information.
Asunto(s)
Células Madre Embrionarias de Ratones , Factores de Transcripción , Animales , Ratones , Diferenciación Celular , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Mammalian development is precisely controlled by cell differentiation. Identifying new regulators and investigating their interactions provide insight into genetic networks defining pre-implantation development. We established a knockout mouse model of Dis3, an exosome associated ribonuclease. Homozygous Dis3 null embryos arrest at the morula stage of development. Using single-embryo RNA sequencing (RNA-seq), we observed persistence of Pou6f1 mRNA in homozygous null Dis3 embryos and that the cognate protein represses transcription of Nanog and Cdx2. The resultant defects in cell differentiation disrupt the morula-to-blastocyst transition and are embryonic lethal. Microinjection of Dis3 mRNA into zygotes rescues the phenotype. Point mutations of Dis3 ribonuclease in individual blastomeres prevents their incorporation into embryos. To overcome the paucity of embryos, we derived homozygous Dis3 null mouse embryonic stem cells to identify additional gene targets of POU6F1. Our findings delineate a regulatory pathway of DIS3-POU6F1 in pre-implantation mammalian embryogenesis.
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Diferenciación Celular , Desarrollo Embrionario , Ribonucleasas , Animales , Ratones , Blastocisto/metabolismo , Complejo Multienzimático de Ribonucleasas del Exosoma/metabolismo , Mamíferos/metabolismo , Ribonucleasas/metabolismo , ARN Mensajero/metabolismoRESUMEN
The zona pellucida plays a crucial role in the process of fertilization to early embryonic development, including cellular arrangement and communication between blastomeres. However, little is known regarding the role of the zona pellucida in pre- and post-implantation embryonic development associated with gene expression. We investigated the effect of zona pellucida removal on pre- and post-implantation development of mouse embryos. After zona pellucida removal of two-cell stage embryos was performed by acid Tyrode's solution, which is commonly used for zona pellucida treatment, compaction occurred earlier in zona pellucida-free than zona pellucida-intact embryos. In addition, the expression of differentiation-related genes in the inner cell mass and trophectoderm was significantly altered in zona pellucida-free blastocyst compared with zona pellucida-intact embryos. After embryo transfer, the rate of implantation and live fetuses was lower in zona pellucida-free embryos than in control embryos, whereas the fetal weight at E17.5 was not different. However, placental weight significantly increased in zona pellucida-free embryos. RNA-sequencing analysis of the placenta showed that a total of 473 differentially expressed genes significantly influenced the biological process. The present study suggests that zona pellucida removal by acid Tyrode's solution at the two-cell stage not only disturbs the expression pattern of inner cell mass-/trophectoderm-related genes but affects the post-implantation development of mouse embryos. Overall, this study provides deeper insight into the role of the zona pellucida during early embryonic development and the viability of post-implantation development.
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Placenta , Zona Pelúcida , Femenino , Ratones , Embarazo , Animales , Blastocisto , Desarrollo Embrionario , Expresión GénicaRESUMEN
Not that long ago, the human genome was discovered to be mainly non-coding, that is comprised of DNA sequences that do not code for proteins. The initial paradigm that non-coding is also non-functional was soon overturned and today the work to uncover the functions of non-coding DNA and RNA in human early embryogenesis has commenced. Early human development is characterized by large-scale changes in genomic activity and the transcriptome that are partly driven by the coordinated activation and repression of repetitive DNA elements scattered across the genome. Here we provide examples of recent novel discoveries of non-coding DNA and RNA interactions and mechanisms that ensure accurate non-coding activity during human maternal-to-zygotic transition and lineage segregation. These include studies on small and long non-coding RNAs, transposable element regulation, and RNA tailing in human oocytes and early embryos. High-throughput approaches to dissect the non-coding regulatory networks governing early human development are a foundation for functional studies of specific genomic elements and molecules that has only begun and will provide a wider understanding of early human embryogenesis and causes of infertility.
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Elementos Transponibles de ADN , ARN Largo no Codificante , Embrión de Mamíferos , Genoma Humano/genética , Genómica , Humanos , ARN Largo no Codificante/genéticaRESUMEN
The activation of the embryonic genome marks the first major wave of transcription in the developing organism. Zygotic genome activation (ZGA) in mouse 2-cell embryos and 8-cell embryos in humans is crucial for development. Here, we report the discovery of human 8-cell-like cells (8CLCs) among naive embryonic stem cells, which transcriptionally resemble the 8-cell human embryo. They express ZGA markers, including ZSCAN4 and LEUTX, and transposable elements, such as HERVL and MLT2A1. 8CLCs show reduced SOX2 levels and can be identified using TPRX1 and H3.Y marker proteins in vitro. Overexpression of the transcription factor DUX4 and spliceosome inhibition increase human ZGA-like transcription. Excitingly, the 8CLC markers TPRX1 and H3.Y are also expressed in ZGA-stage 8-cell human embryos and may thus be relevant in vivo. 8CLCs provide a unique opportunity to characterize human ZGA-like transcription and might provide critical insights into early events in embryogenesis in humans.
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Regulación del Desarrollo de la Expresión Génica , Cigoto , Animales , Desarrollo Embrionario/genética , Genoma Humano , Humanos , Ratones , Factores de Transcripción/metabolismo , Cigoto/metabolismoRESUMEN
How maternal factors in oocytes initiate zygotic genome activation (ZGA) remains elusive in mammals, partly due to the challenge of de novo identification of key factors using scarce materials. Two-cell (2C)-like cells have been widely used as an in vitro model in order to understand mouse ZGA and totipotency because of their expression of a group of two-cell embryo-specific genes and their simplicity for genetic manipulation. Recent studies indicate that DPPA2 and DPPA4 are required for establishing the 2C-like state in mouse embryonic stem cells in a DUX-dependent manner. These results suggest that DPPA2 and DPPA4 are essential maternal factors that regulate Dux and ZGA in embryos. By analyzing maternal knockout and maternal-zygotic knockout embryos, we unexpectedly found that DPPA2 and DPPA4 are dispensable for Dux activation, ZGA and pre-implantation development. Our study suggests that 2C-like cells do not fully recapitulate two-cell embryos in terms of regulation of two-cell embryo-specific genes, and, therefore, caution should be taken when studying ZGA and totipotency using 2C-like cells as the model system.
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Desarrollo Embrionario/genética , Células Madre Embrionarias de Ratones/citología , Proteínas Nucleares/genética , Factores de Transcripción/genética , Animales , Regulación del Desarrollo de la Expresión Génica/genética , Genoma/genética , Herencia Materna/genética , Ratones , Ratones Noqueados , Células Madre Embrionarias de Ratones/metabolismo , Oocitos/crecimiento & desarrollo , Cigoto/crecimiento & desarrollo , Cigoto/metabolismoRESUMEN
Macroscopic, phenomenological models are useful as concise framings of our understandings in fields from statistical physics to finance to biology. Constructing a phenomenological model for development would provide a framework for understanding the complicated, regulatory nature of oogenesis and embryogenesis. Here, we use a data-driven approach to infer quantitative, precise models of human oocyte maturation and pre-implantation embryo development, by analysing clinical in-vitro fertilization (IVF) data on 7399 IVF cycles resulting in 57 827 embryos. Surprisingly, we find that both oocyte maturation and early embryo development are quantitatively described by simple models with minimal interactions. This simplicity suggests that oogenesis and embryogenesis are composed of modular processes that are relatively siloed from one another. In particular, our analysis provides strong evidence that (i) pre-antral follicles produce anti-Müllerian hormone independently of effects from other follicles, (ii) oocytes mature to metaphase-II independently of the woman's age, her BMI and other factors, (iii) early embryo development is memoryless for the variables assessed here, in that the probability of an embryo transitioning from its current developmental stage to the next is independent of its previous stage. Our results both provide insight into the fundamentals of oogenesis and embryogenesis and have implications for the clinical IVF.
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Desarrollo Embrionario , Oocitos , Femenino , Fertilización In Vitro , Humanos , Oogénesis , Folículo OváricoRESUMEN
Elevated molecular stress in women is known to have negative impacts on the reproductive development of oocytes and the embryos prior to implantation. In recent years, the prevalence of cannabis use among women of reproductive age has risen due to its ability to relieve psychological stress and nausea, which are mediated by its psychoactive component, ∆-9-tetrahydrocannabinol (THC). Although cannabis is the most popular recreational drug of the 21st century, much is unknown about its influence on molecular stress in reproductive tissues. The current literature has demonstrated that THC causes dose- and time-dependent alterations in glucocorticoid signaling, which have the potential to compromise morphology, development, and quality of oocytes and embryos. However, there are inconsistencies across studies regarding the mechanisms for THC-dependent changes in stress hormones and how either compounds may drive or arrest development. Factors such as variability between animal models, physiologically relevant doses, and undiscovered downstream gene targets of both glucocorticoids and THC could account for such inconsistencies. This review evaluates the results of studies which have investigated the effects of glucocorticoids on reproductive development and how THC may alter stress signaling in relevant tissues.
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Dronabinol/farmacocinética , Desarrollo Embrionario/efectos de los fármacos , Glucocorticoides/metabolismo , Animales , Cannabis/química , Humanos , Náusea/tratamiento farmacológico , Náusea/metabolismo , Transducción de Señal/efectos de los fármacos , Estrés Psicológico/tratamiento farmacológico , Estrés Psicológico/metabolismoRESUMEN
Naive human embryonic stem cells (hESCs) have been isolated that more closely resemble the pre-implantation epiblast compared to conventional "primed" hESCs, but the signaling principles underlying these discrete stem cell states remain incompletely understood. Here, we describe the results from a high-throughput screen using â¼3,000 well-annotated compounds to identify essential signaling requirements for naive human pluripotency. We report that MEK1/2 inhibitors can be replaced during maintenance of naive human pluripotency by inhibitors targeting either upstream (FGFR, RAF) or downstream (ERK1/2) kinases. Naive hESCs maintained under these alternative conditions display elevated levels of ERK phosphorylation but retain genome-wide DNA hypomethylation and a transcriptional identity of the pre-implantation epiblast. In contrast, dual inhibition of MEK and ERK promotes efficient primed-to-naive resetting in combination with PKC, ROCK, and TNKS inhibitors and activin A. This work demonstrates that induction and maintenance of naive human pluripotency are governed by distinct signaling requirements.
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Ensayos Analíticos de Alto Rendimiento , Células Madre Pluripotentes/metabolismo , Transducción de Señal , Activinas/farmacología , Células Cultivadas , Implantación del Embrión/efectos de los fármacos , Humanos , Modelos Biológicos , Células Madre Pluripotentes/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Quinasas raf/antagonistas & inhibidores , Quinasas raf/metabolismoRESUMEN
STUDY QUESTION: What is the role of POU class 5 homeobox 1 (POU5F1) in human preimplantation development and how does it compare with the mouse model? SUMMARY ANSWER: POU5F1 is required for successful development of mouse and human embryos to the blastocyst stage as knockout embryos exhibited a significantly lower blastocyst formation rate, accompanied by lack of inner cell mass (ICM) formation. WHAT IS KNOWN ALREADY: Clustered regularly interspaced short palindromic repeats-CRISPR associated genes (CRISPR-Cas9) has previously been used to examine the role of POU5F1 during human preimplantation development. The reported POU5F1-targeted blastocysts always retained POU5F1 expression in at least one cell, because of incomplete CRISPR-Cas9 editing. The question remains of whether the inability to obtain fully edited POU5F1-targeted blastocysts in human results from incomplete editing or the actual inability of these embryos to reach the blastocyst stage. STUDY DESIGN, SIZE, DURATION: The efficiency of CRISPR-Cas9 to induce targeted gene mutations was first optimized in the mouse model. Two CRISPR-Cas9 delivery methods were compared in the B6D2F1 strain: S-phase injection (zygote stage) (n = 135) versus metaphase II-phase (M-phase) injection (oocyte stage) (n = 23). Four control groups were included: non-injected media-control zygotes (n = 43)/oocytes (n = 48); sham-injected zygotes (n = 45)/oocytes (n = 47); Cas9-protein injected zygotes (n = 23); and Cas9 protein and scrambled guide RNA (gRNA)-injected zygotes (n = 27). Immunofluorescence analysis was performed in Pou5f1-targeted zygotes (n = 37), media control zygotes (n = 19), and sham-injected zygotes (n = 15). To assess the capacity of Pou5f1-null embryos to develop further in vitro, additional groups of Pou5f1-targeted zygotes (n = 29) and media control zygotes (n = 30) were cultured to postimplantation stages (8.5 dpf). Aiming to identify differences in developmental capacity of Pou5f1-null embryos attributed to strain variation, zygotes from a second mouse strain-B6CBA (n = 52) were targeted. Overall, the optimized methodology was applied in human oocytes following IVM (metaphase II stage) (n = 101). The control group consisted of intracytoplasmically sperm injected (ICSI) IVM oocytes (n = 33). Immunofluorescence analysis was performed in human CRISPR-injected (n = 10) and media control (n = 9) human embryos. PARTICIPANTS/MATERIALS, SETTING, METHODS: A gRNA-Cas9 protein mixture targeting exon 2 of Pou5f1/POU5F1 was microinjected in mouse oocytes/zygotes or human IVM oocytes. Reconstructed embryos were cultured for 4 days (mouse) or 6.5 days (human) in sequential culture media. An additional group of mouse-targeted zygotes was cultured to postimplantation stages. Embryonic development was assessed daily, with detailed scoring at late blastocyst stage. Genomic editing was assessed by immunofluorescence analysis and next-generation sequencing. MAIN RESULTS AND THE ROLE OF CHANCE: Genomic analysis in mouse revealed very high editing efficiencies with 95% of the S-Phase and 100% of the M-Phase embryos containing genetic modifications, of which 89.47% in the S-Phase and 84.21% in the M-Phase group were fully edited. The developmental capacity was significantly compromised as only 46.88% embryos in the S-Phase and 19.05% in the M-Phase group reached the blastocyst stage, compared to 86.36% in control M-Phase and 90.24% in control S-Phase groups, respectively. Immunofluorescence analysis confirmed the loss of Pou5f1 expression and downregulation of the primitive marker SRY-Box transcription factor (Sox17). Our experiments confirmed the requirement of Pou5f1 expression for blastocyst development in the second B6CBA strain. Altogether, our data obtained in mouse reveal that Pou5f1 expression is essential for development to the blastocyst stage. M-Phase injection in human IVM oocytes (n = 101) similarly resulted in 88.37% of the POU5F1-targeted embryos being successfully edited. The developmental capacity of generated embryos was compromised from the eight-cell stage onwards. Only 4.55% of the microinjected embryos reached the late blastocyst stage and the embryos exhibited complete absence of ICM and an irregular trophectoderm cell layer. Loss of POU5F1 expression resulted in absence of SOX17 expression, as in mouse. Interestingly, genetic mosaicism was eliminated in a subset of targeted human embryos (9 out of 38), three of which developed into blastocysts. LIMITATIONS, REASONS FOR CAUTION: One of the major hurdles of CRISPR-Cas9 germline genome editing is the occurrence of mosaicism, which may complicate phenotypic analysis and interpretation of developmental behavior of the injected embryos. Furthermore, in this study, spare IVM human oocytes were used, which may not recapitulate the developmental behavior of in vivo matured oocytes. WIDER IMPLICATIONS OF THE FINDINGS: Comparison of developmental competency following CRISPR-Cas-mediated gene targeting in mouse and human may be influenced by the selected mouse strain. Gene targeting by CRISPR-Cas9 is subject to variable targeting efficiencies. Therefore, striving to reduce mosaicism can provide novel molecular insights into mouse and human embryogenesis. STUDY FUNDING/COMPETING INTEREST(S): The research was funded by the Ghent University Hospital and Ghent University and supported by the FWO-Vlaanderen (Flemish fund for scientific research, Grant no. G051516N), and Hercules funding (FWO.HMZ.2016.00.02.01). The authors declare no competing interests. TRIAL REGISTRATION NUMBER: N/A.
Asunto(s)
Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Técnicas de Maduración In Vitro de los Oocitos , Animales , Blastocisto , Sistemas CRISPR-Cas , Desarrollo Embrionario/genética , Femenino , Genes Homeobox , Humanos , Masculino , Ratones , Factor 3 de Transcripción de Unión a Octámeros/genética , EmbarazoRESUMEN
Pre-implantation mammalian development unites extreme plasticity with a robust outcome: the formation of a blastocyst, an organised multi-layered structure ready for implantation. The process of blastocyst formation is one of the best-known examples of self-organisation. The first three cell lineages in mammalian development specify and arrange themselves during the morphogenic process based on cell-cell interactions. Despite decades of research, the unifying principles driving early mammalian development are still not fully defined. Here, we discuss the role of physical forces, and molecular and cellular mechanisms, in driving self-organisation and lineage formation that are shared between eutherian mammals.
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Embrión de Mamíferos/metabolismo , Animales , Blastocisto/metabolismo , Diferenciación Celular , Linaje de la Célula , Desarrollo Embrionario , Regulación del Desarrollo de la Expresión Génica , Estratos Germinativos/metabolismo , Humanos , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Maternal mRNAs deposited in the egg during oogenesis are critical during the development of early embryo, before the activation of the embryonic genome. However, there is little known about the dynamic expression of maternally expressed genes in mammals. In this study, we made buffalo parthenogenesis as our research model to analyse maternal transcription profiles of pre-implantation embryo in buffalo using RNA sequencing. In total, 3,567 unique genes were detected to be differentially expressed among all constant stages during early embryo development (FPKM > 0). Interestingly, a total of 10,442 new genes were discovered in this study, and gene ontology analysis of the new differentially expressed genes indicated that the new genes have a wide cellular localization and are involved in many molecular functions and biological processes. Moreover, we identified eight clusters that were only highly expressed in a particular developmental stage and enriched a number of GO terms and KEGG pathways that were related to specific stage. Furthermore, we identified 1,530 hub genes (or key members) from the maternally expressed gene networks, and these hub genes were involved in 11 stage-specific modules. After visualization using Cytoscape 3.2.1 software, we obtained complex interaction network of hub genes, indicating the highly efficient cooperation between genes during the development in buffalo embryos. Further research of these genes will greatly deepen our understanding of embryo development in buffalo. Collectively, this research lays the foundation for future studies on the maternal genome function, buffalo nuclear transfer and parthenogenetic embryonic stem cells.
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Búfalos/embriología , Búfalos/genética , Perfilación de la Expresión Génica , Animales , Búfalos/metabolismo , Embrión de Mamíferos/metabolismo , Desarrollo Embrionario/genética , Femenino , Regulación del Desarrollo de la Expresión Génica , Técnicas de Maduración In Vitro de los Oocitos/veterinaria , Partenogénesis/genética , Análisis de Secuencia de ARNRESUMEN
Pre-implantation development is a dynamic, complex and precisely regulated process that is critical for mammalian development. There is currently no description of the role of the long noncoding RNAs (lncRNAs) during the pre-implantation stages in the goat. The in vivo transcriptomes of oocytes (n = 3) and pre-implantation stages (n=19) at seven developmental stages in the goat were analyzed by RNA sequencing (RNA-Seq). The major zygotic gene activation (ZGA) event was found to occur between the 8- and 16-cell stages in the pre-implantation stages. We identified 5,160 differentially expressed lncRNAs (DELs) in developmental stage comparisons and functional analyses of the major and minor ZGAs. Fourteen lncRNA modules were found corresponding to specific pre-implantation developmental stages by weighted gene co-expression network analysis (WGCNA). A comprehensive analysis of the lncRNAs at each developmental transition of high correlation modules was done. We also identified lncRNA-mRNA networks and hub-lncRNAs for the high correlation modules at each stage. The extensive association of lncRNA target genes with other embryonic genes suggests an important regulatory role for lncRNAs in embryonic development. These data will facilitate further exploration of the role of lncRNAs in the developmental transformation in the pre-implantation stage.
RESUMEN
Mammalian embryo cloning by nuclear transfer has a low success rate. This is hypothesized to correlate with a high variability of early developmental steps that segregate outer cells, which are fated to extra-embryonic tissues, from inner cells, which give rise to the embryo proper. Exploring the cell lineage of wild-type embryos and clones, imaged in toto until hatching, highlights the respective contributions of cell proliferation, death and asymmetric divisions to phenotypic variability. Preferential cell death of inner cells in clones, probably pertaining to the epigenetic plasticity of the transferred nucleus, is identified as a major difference with effects on the proportion of inner cell. In wild type and clones, similar patterns of outer cell asymmetric divisions are shown to be essential to the robust proportion of inner cells observed in wild type. Asymmetric inner cell division, which is not described in mice, is identified as a regulator of the proportion of inner cells and likely gives rise to resilient clones.
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División Celular Asimétrica , Masa Celular Interna del Blastocisto/citología , Clonación de Organismos/métodos , Animales , Recuento de Células , Muerte Celular , Diferenciación Celular , Linaje de la Célula , Proliferación Celular , Simulación por Computador , Desarrollo Embrionario , Femenino , Proteínas Fluorescentes Verdes/genética , Imagenología Tridimensional , Masculino , Microscopía de Fluorescencia por Excitación Multifotónica , Técnicas de Transferencia Nuclear , Embarazo , ConejosRESUMEN
The technology to derive embryonic and induced pluripotent stem cells from early embryonic stages and adult somatic cells, respectively, emerged as a powerful resource to enable the establishment of new in vitro models, which recapitulate early developmental processes and disease. Additionally, pluripotent stem cells (PSCs) represent an invaluable source of relevant differentiated cell types with immense potential for regenerative medicine and cell replacement therapies. Pluripotent stem cells support self-renewal, potency and proliferation for extensive periods of culture in vitro. However, the core pathways that rule each of these cellular features specific to PSCs only recently began to be clarified. The Wnt signaling pathway is pivotal during early embryogenesis and is central for the induction and maintenance of the pluripotency of PSCs. Signaling by the Wnt family of ligands is conveyed intracellularly by the stabilization of ß-catenin in the cytoplasm and in the nucleus, where it elicits the transcriptional activity of T-cell factor (TCF)/lymphoid enhancer factor (LEF) family of transcription factors. Interestingly, in PSCs, the Wnt/ß-catenin-TCF/LEF axis has several unrelated and sometimes opposite cellular functions such as self-renewal, stemness, lineage commitment and cell cycle regulation. In addition, tight control of the Wnt signaling pathway enhances reprogramming of somatic cells to induced pluripotency. Several recent research efforts emphasize the pleiotropic functions of the Wnt signaling pathway in the pluripotent state. Nonetheless, conflicting results and unanswered questions still linger. In this review, we will focus on the diverse functions of the canonical Wnt signaling pathway on the developmental processes preceding embryo implantation, as well as on its roles in pluripotent stem cell biology such as self-renewal and cell cycle regulation and somatic cell reprogramming.
RESUMEN
Regulation of chromatin composition through post-translational modifications of histones contributes to transcriptional regulation and is essential for many cellular processes, including differentiation and development. KDM4A (JMJD2A) is a lysine demethylase with specificity towards di- and tri-methylated lysine 9 and lysine 36 of histone H3 (H3K9me2/me3 and H3K36me2/me3). Here, we report that Kdm4a as a maternal factor plays a key role in embryo survival and is vital for female fertility. Kdm4a-/- female mice ovulate normally with comparable fertilization but poor implantation rates, and cannot support healthy transplanted embryos to term. This is due to a role for Kdm4a in uterine function, where its loss causes reduced expression of key genes involved in ion transport, nutrient supply and cytokine signalling, which impact embryo survival. In addition, a significant proportion of Kdm4a-deficient oocytes displays a poor intrinsic ability to develop into blastocysts. These embryos cannot compete with healthy embryos for implantation in vivo, highlighting Kdm4a as a maternal effect gene. Thus, our study dissects an important dual role for maternal Kdm4a in determining faithful early embryonic development and the implantation process.
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Implantación del Embrión , Histona Demetilasas/metabolismo , Animales , Citocinas/metabolismo , Implantación del Embrión/genética , Embrión de Mamíferos/metabolismo , Femenino , Regulación del Desarrollo de la Expresión Génica , Genitales Femeninos/metabolismo , Infertilidad Femenina/genética , Infertilidad Femenina/patología , Ratones Endogámicos C57BL , Ratones Noqueados , Embarazo , Transducción de Señal , Útero/metabolismo , Cigoto/metabolismoRESUMEN
Germ cell development involves major reprogramming of the epigenome to prime the zygote for totipotency. Histone 3 lysine 4 (H3K4) methylations are universal epigenetic marks mediated in mammals by six H3K4 methyltransferases related to fly Trithorax, including two yeast Set1 orthologs: Setd1a and Setd1b. Whereas Setd1a plays no role in oogenesis, we report that Setd1b deficiency causes female sterility in mice. Oocyte-specific Gdf9-iCre conditional knockout (Setd1bGdf9 cKO) ovaries develop through all stages; however, follicular loss accumulated with age and unfertilized metaphase II (MII) oocytes exhibited irregularities of the zona pellucida and meiotic spindle. Most Setd1bGdf9 cKO zygotes remained in the pronuclear stage and displayed polyspermy in the perivitelline space. Expression profiling of Setd1bGdf9 cKO MII oocytes revealed (1) that Setd1b promotes the expression of the major oocyte transcription factors including Obox1, 2, 5, 7, Meis2 and Sall4; and (2) twice as many mRNAs were upregulated than downregulated, suggesting that Setd1b also promotes the expression of negative regulators of oocyte development with multiple Zfp-KRAB factors implicated. Together, these findings indicate that Setd1b serves as maternal effect gene through regulation of the oocyte gene expression program.
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N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Oogénesis/genética , Oogénesis/fisiología , Animales , Blastocisto/citología , Blastocisto/metabolismo , Epigénesis Genética , Femenino , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Factor 9 de Diferenciación de Crecimiento/deficiencia , Factor 9 de Diferenciación de Crecimiento/genética , Factor 9 de Diferenciación de Crecimiento/metabolismo , N-Metiltransferasa de Histona-Lisina/deficiencia , Masculino , Herencia Materna , Ratones , Ratones Noqueados , Ratones Transgénicos , Oocitos/citología , Oocitos/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Análisis de la Célula Individual , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Zona Pelúcida/metabolismo , Zona Pelúcida/patología , Cigoto/citología , Cigoto/metabolismoRESUMEN
Zygote cytokinesis produces two symmetric blastomeres, which contain one copy of each parental genome. Contrary to this dogma, we recently discovered that mammalian zygotes can spontaneously segregate entire parental genomes into different blastomeres and coined this novel form of genome segregation heterogoneic division. The molecular mechanisms underlying the emergence of blastomeres with different parental genomes during the first mitotic cycle remain to be elucidated. Here, we speculate on which parental genome asymmetries could provide a mechanistic foundation for these remarkable zygote divisions. In reviewing the field and considering our findings, we revisit the architecture of the first zygotic metaphase by invoking asymmetric interactions between the mitotic spindle and the parental kinetochores. We also speculate on how asynchronous parental cell cycles can be a source of heterogoneic zygote divisions through the formation of parental genome private spindles.
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Blastómeros , Segregación Cromosómica , Mitosis , Cigoto/fisiología , Animales , Genoma , Humanos , Huso AcromáticoRESUMEN
BACKGROUND: Instead of testing predefined hypotheses, the goal of exploratory data analysis (EDA) is to find what data can tell us. Following this strategy, we re-analyzed a large body of genomic data to study the complex gene regulation in mouse pre-implantation development (PD). RESULTS: Starting with a single-cell RNA-seq dataset consisting of 259 mouse embryonic cells derived from zygote to blastocyst stages, we reconstructed the temporal and spatial gene expression pattern during PD. The dynamics of gene expression can be partially explained by the enrichment of transposable elements in gene promoters and the similarity of expression profiles with those of corresponding transposons. Long Terminal Repeats (LTRs) are associated with transient, strong induction of many nearby genes at the 2-4 cell stages, probably by providing binding sites for Obox and other homeobox factors. B1 and B2 SINEs (Short Interspersed Nuclear Elements) are correlated with the upregulation of thousands of nearby genes during zygotic genome activation. Such enhancer-like effects are also found for human Alu and bovine tRNA SINEs. SINEs also seem to be predictive of gene expression in embryonic stem cells (ESCs), raising the possibility that they may also be involved in regulating pluripotency. We also identified many potential transcription factors underlying PD and discussed the evolutionary necessity of transposons in enhancing genetic diversity, especially for species with longer generation time. CONCLUSIONS: Together with other recent studies, our results provide further evidence that many transposable elements may play a role in establishing the expression landscape in early embryos. It also demonstrates that exploratory bioinformatics investigation can pinpoint developmental pathways for further study, and serve as a strategy to generate novel insights from big genomic data.
Asunto(s)
Biología Computacional , ADN Intergénico , Desarrollo Embrionario/genética , Regulación del Desarrollo de la Expresión Génica , Animales , Secuencia de Bases , Análisis por Conglomerados , Biología Computacional/métodos , Islas de CpG , Metilación de ADN , Elementos Transponibles de ADN , Células Madre Embrionarias/metabolismo , Perfilación de la Expresión Génica , Genoma , Genómica/métodos , Ratones , Motivos de Nucleótidos , Especificidad de Órganos/genética , Regiones Promotoras Genéticas , Retroelementos , Transcriptoma , Cigoto/metabolismoRESUMEN
The cellular plasticity of pluripotent stem cells is thought to be sustained by genomic regions that display both active and repressive chromatin properties. These regions exhibit low levels of gene expression, yet the mechanisms controlling these levels remain unknown. Here, we describe Elongin BC as a binding factor at the promoters of bivalent sites. Biochemical and genome-wide analyses show that Elongin BC is associated with Polycomb Repressive Complex 2 (PRC2) in pluripotent stem cells. Elongin BC is recruited to chromatin by the PRC2-associated factor EPOP (Elongin BC and Polycomb Repressive Complex 2 Associated Protein, also termed C17orf96, esPRC2p48, E130012A19Rik), a protein expressed in the inner cell mass of the mouse blastocyst. Both EPOP and Elongin BC are required to maintain low levels of expression at PRC2 genomic targets. Our results indicate that keeping the balance between activating and repressive cues is a more general feature of chromatin in pluripotent stem cells than previously appreciated.