RESUMEN
The mechanism by which transcription factor (TF) network instructs cell-type-specific transcriptional programs to drive primitive endoderm (PrE) progenitors to commit to parietal endoderm (PE) versus visceral endoderm (VE) cell fates remains poorly understood. To address the question, we analyzed the single-cell transcriptional signatures defining PrE, PE, and VE cell states during the onset of the PE-VE lineage bifurcation. By coupling with the epigenomic comparison of active enhancers unique to PE and VE cells, we identified GATA6, SOX17, and FOXA2 as central regulators for the lineage divergence. Transcriptomic analysis of cXEN cells, an in vitro model for PE cells, after the acute depletion of GATA6 or SOX17 demonstrated that these factors induce Mycn, imparting the self-renewal properties of PE cells. Concurrently, they suppress the VE gene program, including key genes like Hnf4a and Ttr, among others. We proceeded with RNA-seq analysis on cXEN cells with FOXA2 knockout, in conjunction with GATA6 or SOX17 depletion. We found FOXA2 acts as a potent suppressor of Mycn while simultaneously activating the VE gene program. The antagonistic gene regulatory activities of GATA6/SOX17 and FOXA2 in promoting alternative cell fates, and their physical co-bindings at the enhancers provide molecular insights to the plasticity of the PrE lineage. Finally, we show that the external cue, BMP signaling, promotes the VE cell fate by activation of VE TFs and repression of PE TFs including GATA6 and SOX17. These data reveal a putative core gene regulatory module that underpins PE and VE cell fate choice.
Asunto(s)
Endodermo , Redes Reguladoras de Genes , Proteína Proto-Oncogénica N-Myc/genética , Diferenciación Celular/genética , Factores de Transcripción/genética , Regulación del Desarrollo de la Expresión Génica/genéticaRESUMEN
Histone acetylation is a pivotal epigenetic modification that controls chromatin structure and regulates gene expression. It plays an essential role in modulating zygotic transcription and cell lineage specification of developing embryos. While the outcomes of many inductive signals have been described to require enzymatic activities of histone acetyltransferases and deacetylases (HDACs), the mechanisms by which HDACs confine the utilization of the zygotic genome remain to be elucidated. Here, we show that histone deacetylase 1 (Hdac1) progressively binds to the zygotic genome from mid-blastula and onward. The recruitment of Hdac1 to the genome at blastula is instructed maternally. Cis-regulatory modules (CRMs) bound by Hdac1 possess epigenetic signatures underlying distinct functions. We highlight a dual function model of Hdac1 where Hdac1 not only represses gene expression by sustaining a histone hypoacetylation state on inactive chromatin, but also maintains gene expression through participating in dynamic histone acetylation-deacetylation cycles on active chromatin. As a result, Hdac1 maintains differential histone acetylation states of bound CRMs between different germ layers and reinforces the transcriptional program underlying cell lineage identities, both in time and space. Taken together, our study reveals a comprehensive role for Hdac1 during early vertebrate embryogenesis.
Asunto(s)
Histona Desacetilasa 1 , Histonas , Histonas/metabolismo , Histona Desacetilasa 1/genética , Histona Desacetilasa 1/metabolismo , Cromatina/metabolismo , Blastocisto/metabolismo , Histona Desacetilasas/genética , Histona Desacetilasas/metabolismo , Desarrollo Embrionario/genética , Acetilación , Histona Desacetilasa 2/metabolismoRESUMEN
How the embryonic genome regulates accessibility to transcription factors is one of the major questions in understanding the spatial and temporal dynamics of gene expression during embryogenesis. Epigenomic analyses of embryonic chromatin provide molecular insights into cell-specific gene activities and genomic architectures. In recent years, significant advances have been made to elucidate the dynamic changes behind the activation of the zygotic genome in various model organisms. Here we provide an overview of the recent epigenomic studies pertaining to early Xenopus development.
Asunto(s)
Cromatina , Epigenómica , Animales , Xenopus laevis/genética , Cromatina/metabolismo , Desarrollo Embrionario/genética , Cigoto/metabolismo , Regulación del Desarrollo de la Expresión GénicaRESUMEN
Early embryonic cell fates are specified through coordinated integration of transcription factor activities and epigenetic states of the genome. Foxh1 is a key maternal transcription factor controlling the mesendodermal gene regulatory program. Proteomic interactome analyses using FOXH1 as a bait in mouse embryonic stem cells revealed that FOXH1 interacts with PRC2 subunits and HDAC1. Foxh1 physically interacts with Hdac1, and confers transcriptional repression of mesendodermal genes in Xenopus ectoderm. Our findings reveal a central role of Foxh1 in coordinating the chromatin states of the Xenopus embryonic genome.
Asunto(s)
Cromatina , Factores de Transcripción Forkhead , Regulación del Desarrollo de la Expresión Génica , Proteínas de Xenopus , Animales , Cromatina/genética , Factores de Transcripción Forkhead/genética , Ratones , Proteómica , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Xenopus laevis/genéticaRESUMEN
Mesendodermal specification is one of the earliest events in embryogenesis, where cells first acquire distinct identities. Cell differentiation is a highly regulated process that involves the function of numerous transcription factors (TFs) and signaling molecules, which can be described with gene regulatory networks (GRNs). Cell differentiation GRNs are difficult to build because existing mechanistic methods are low throughput, and high-throughput methods tend to be non-mechanistic. Additionally, integrating highly dimensional data composed of more than two data types is challenging. Here, we use linked self-organizing maps to combine chromatin immunoprecipitation sequencing (ChIP-seq)/ATAC-seq with temporal, spatial, and perturbation RNA sequencing (RNA-seq) data from Xenopus tropicalis mesendoderm development to build a high-resolution genome scale mechanistic GRN. We recover both known and previously unsuspected TF-DNA/TF-TF interactions validated through reporter assays. Our analysis provides insights into transcriptional regulation of early cell fate decisions and provides a general approach to building GRNs using highly dimensional multi-omic datasets.
Asunto(s)
Endodermo/embriología , Redes Reguladoras de Genes , Genómica , Mesodermo/embriología , Xenopus/embriología , Xenopus/genética , Animales , Cromatina/metabolismo , Secuencia de Consenso/genética , ADN/metabolismo , Gastrulación/genética , Regulación del Desarrollo de la Expresión Génica , Unión Proteica , ARN/metabolismo , Factores de Transcripción/metabolismo , Transcripción GenéticaRESUMEN
Heavy metals are both integral parts of cells and environmental toxicants, and their deregulation is associated with severe cellular dysfunction and various diseases. Here we show that the Hippo pathway plays a critical role in regulating heavy metal homeostasis. Hippo signalling deficiency promotes the transcription of heavy metal response genes and protects cells from heavy metal-induced toxicity, a process independent of its classic downstream effectors YAP and TAZ. Mechanistically, the Hippo pathway kinase LATS phosphorylates and inhibits MTF1, an essential transcription factor in the heavy metal response, resulting in the loss of heavy metal response gene transcription and cellular protection. Moreover, LATS activity is inhibited following heavy metal treatment, where accumulated zinc directly binds and inhibits LATS. Together, our study reveals an interplay between the Hippo pathway and heavy metals, providing insights into this growth-related pathway in tissue homeostasis and stress response.
Asunto(s)
Cadmio/metabolismo , Proteínas de Unión al ADN/metabolismo , Vía de Señalización Hippo/fisiología , Proteínas Serina-Treonina Quinasas/metabolismo , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Zinc/metabolismo , Cadmio/toxicidad , Línea Celular Tumoral , Regulación de la Expresión Génica/genética , Células HEK293 , Células HeLa , Homeostasis/genética , Humanos , Inactivación Metabólica/fisiología , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , Estrés Fisiológico/fisiología , Transcripción Genética/genética , Proteínas Supresoras de Tumor/genética , Zinc/toxicidad , Factor de Transcripción MTF-1RESUMEN
The fertilized frog egg contains all the materials needed to initiate development of a new organism, including stored RNAs and proteins deposited during oogenesis, thus the earliest stages of development do not require transcription. The onset of transcription from the zygotic genome marks the first genetic switch activating the gene regulatory network that programs embryonic development. Zygotic genome activation occurs after an initial phase of transcriptional quiescence that continues until the midblastula stage, a period called the midblastula transition, which was first identified in Xenopus. Activation of transcription is programmed by maternally supplied factors and is regulated at multiple levels. A similar switch exists in most animals and is of great interest both to developmental biologists and to those interested in understanding nuclear reprogramming. Here we review in detail our knowledge on this major switch in transcription in Xenopus and place recent discoveries in the context of a decades old problem.
Asunto(s)
Genoma/genética , Xenopus laevis/embriología , Xenopus laevis/genética , Cigoto/metabolismo , Animales , Oogénesis , Cigoto/citologíaRESUMEN
During early mammalian embryo development, a small number of cells make robust fate decisions at particular spatial locations in a tight time window to form inner cell mass (ICM), and later epiblast (Epi) and primitive endoderm (PE). While recent single-cell transcriptomics data allows scrutinization of heterogeneity of individual cells, consistent spatial and temporal mechanisms the early embryo utilize to robustly form the Epi/PE layers from ICM remain elusive. Here we build a multiscale three-dimensional model for mammalian embryo to recapitulate the observed patterning process from zygote to late blastocyst. By integrating the spatiotemporal information reconstructed from multiple single-cell transcriptomic datasets, the data-informed modeling analysis suggests two major processes critical to the formation of Epi/PE layers: a selective cell-cell adhesion mechanism (via EphA4/EphrinB2) for fate-location coordination and a temporal attenuation mechanism of cell signaling (via Fgf). Spatial imaging data and distinct subsets of single-cell gene expression data are then used to validate the predictions. Together, our study provides a multiscale framework that incorporates single-cell gene expression datasets to analyze gene regulations, cell-cell communications, and physical interactions among cells in complex geometries at single-cell resolution, with direct application to late-stage development of embryogenesis.
Asunto(s)
Desarrollo Embrionario/genética , Estratos Germinativos , Modelos Biológicos , Transcriptoma/genética , Animales , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Embrión de Mamíferos/fisiología , Estratos Germinativos/citología , Estratos Germinativos/metabolismo , Estratos Germinativos/fisiología , Ratones , Análisis de la Célula IndividualRESUMEN
Although Wnt/ß-catenin signaling is generally conserved and well understood, the regulatory mechanisms controlling context-specific direct Wnt target gene expression in development and disease are still unclear. The onset of zygotic gene transcription in early embryogenesis represents an ideal, accessible experimental system to investigate context-specific direct Wnt target gene regulation. We combine transcriptomics using RNA-seq with genome-wide ß-catenin association using ChIP-seq to identify stage-specific direct Wnt target genes. We propose coherent feedforward regulation involving two distinct classes of direct maternal Wnt target genes, which differ both in expression and persistence of ß-catenin association. We discover that genomic ß-catenin association overlaps with Foxh1-associated regulatory sequences and demonstrate that direct maternal Wnt target gene expression requires Foxh1 function and Nodal/Tgfß signaling. Our results support a new paradigm for direct Wnt target gene co-regulation with context-specific mechanisms that will inform future studies of embryonic development and more widely stem cell-mediated homeostasis and human disease.
RESUMEN
For decades, the early development of the Xenopus embryo has been an essential model system to study the gene regulatory mechanisms that govern cellular specification. At the top of the hierarchy of gene regulatory networks, maternally deposited transcription factors initiate this process and regulate the expression of zygotic genes that give rise to three distinctive germ layer cell types (ectoderm, mesoderm, and endoderm), and subsequent generation of organ precursors. The onset of germ layer specification is also closely coupled with changes associated with chromatin modifications. This review will examine the timing of maternal transcription factors initiating the zygotic genome activation, the epigenetic landscape of embryonic chromatin, and the network structure that governs the process.
Asunto(s)
Cromatina/genética , Embrión no Mamífero/metabolismo , Regulación del Desarrollo de la Expresión Génica , Redes Reguladoras de Genes , Herencia Materna/genética , Factores de Transcripción/genética , Proteínas de Xenopus/genética , Xenopus/genética , Animales , Cromatina/metabolismo , Embrión no Mamífero/citología , Embrión no Mamífero/embriología , Factores de Transcripción/metabolismo , Xenopus/clasificación , Xenopus/embriología , Proteínas de Xenopus/metabolismo , Xenopus laevis/embriología , Xenopus laevis/genéticaRESUMEN
High-throughput sequencing methods have created exciting opportunities to explore the regulatory landscape of the entire genome. Here we introduce methods to characterize the genomic locations of bound proteins, open chromatin, and sites of DNA-DNA contact in Xenopus embryos. These methods include chromatin immunoprecipitation followed by sequencing (ChIP-seq), a combination of DNase I digestion and sequencing (DNase-seq), the assay for transposase-accessible chromatin and sequencing (ATAC-seq), and the use of proximity-based DNA ligation followed by sequencing (Hi-C).
Asunto(s)
Cromatina/genética , Embrión no Mamífero/metabolismo , Genómica/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Xenopus/genética , Animales , Cromatina/metabolismo , Inmunoprecipitación de Cromatina/métodos , ADN/genética , ADN/metabolismo , Embrión no Mamífero/embriología , Genoma/genética , Xenopus/embriologíaRESUMEN
The general field of quantitative biology has advanced significantly on the back of recent improvements in both sequencing technology and proteomics methods. The development of high-throughput, short-read sequencing has revolutionized RNA-based expression studies, while improvements in proteomics methods have enabled quantitative studies to attain better resolution. Here we introduce methods to undertake global analyses of gene expression through RNA and protein quantification in Xenopus embryos and tissues.
Asunto(s)
Embrión no Mamífero/metabolismo , Perfilación de la Expresión Génica/métodos , Regulación del Desarrollo de la Expresión Génica , Proteómica/métodos , Xenopus laevis/genética , Xenopus laevis/metabolismo , Animales , Cromatografía Liquida/métodos , Embrión no Mamífero/embriología , Proteoma/genética , Proteoma/metabolismo , Análisis de Secuencia de ARN/métodos , Espectrometría de Masas en Tándem/métodos , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Xenopus laevis/embriologíaRESUMEN
Development of quantitative, safe and rapid techniques for assessing embryo quality provides significant advances in Assisted Reproductive Technologies (ART). Instead of assessing the embryo quality by the standard morphologic evaluation, we apply the phasor-FLIM (Fluorescence Lifetime Imaging Microscopy) method to capture endogenous fluorescent biomarkers of pre-implantation embryos as a non-morphological caliber for embryo quality. Here, we identify, under hypoxic and non-hypoxic conditions, the unique spectroscopic trajectories at different stages of mouse pre-implantation development, which is referred to as the developmental, or "D-trajectory", that consists of fluorescence lifetime from different stages of mouse pre-implantation embryos. The D-trajectory correlates with intrinsic fluorescent species from a distinctive energy metabolism and oxidized lipids, as seen with Third Harmonic Generation (THG) that changes over time. In addition, we have defined a non-morphological Embryo Viability Index (EVI) to distinguish pre-implantation embryo quality using the Distance Analysis (DA), a machine learning algorithm to process the fluorescence lifetime distribution patterns. We show, under our experimental conditions, that the phasor-FLIM approach provides a much-needed non-invasive quantitative technology for identifying healthy embryos at the early compaction stage with 86% accuracy. The DA and phasor-FLIM method may provide the opportunity to improve implantation success rates for in vitro fertilization clinics.
Asunto(s)
Blastocisto/citología , Blastocisto/fisiología , Microscopía Fluorescente/métodos , Animales , Técnicas de Cultivo de Embriones , Implantación del Embrión , Desarrollo Embrionario , Femenino , Glucólisis , Rayos Láser , Aprendizaje Automático , Ratones Endogámicos C57BL , Fosforilación Oxidativa , Especies Reactivas de Oxígeno/metabolismo , Estrés Fisiológico , Imagen de Lapso de Tiempo/métodosRESUMEN
Elucidation of the sequence of events underlying the dynamic interaction between transcription factors and chromatin states is essential. Maternal transcription factors function at the top of the regulatory hierarchy to specify the primary germ layers at the onset of zygotic genome activation (ZGA). We focus on the formation of endoderm progenitor cells and examine the interactions between maternal transcription factors and chromatin state changes underlying the cell specification process. Endoderm-specific factors Otx1 and Vegt together with Foxh1 orchestrate endoderm formation by coordinated binding to select regulatory regions. These interactions occur before the deposition of enhancer histone marks around the regulatory regions, and these TFs recruit RNA polymerase II, regulate enhancer activity, and establish super-enhancers associated with important endodermal genes. Therefore, maternal transcription factors Otx1, Vegt, and Foxh1 combinatorially regulate the activity of super-enhancers, which in turn activate key lineage-specifying genes during ZGA.
Asunto(s)
Factores de Transcripción Forkhead/metabolismo , Genoma , Factores de Transcripción Otx/metabolismo , Proteínas de Dominio T Box/metabolismo , Proteínas de Xenopus/metabolismo , Cigoto/metabolismo , Animales , Sitios de Unión , Cromatina/metabolismo , Endodermo/metabolismo , Elementos de Facilitación Genéticos , Femenino , Factores de Transcripción Forkhead/genética , Histonas/genética , Histonas/metabolismo , Masculino , Morfolinos/metabolismo , Factores de Transcripción Otx/antagonistas & inhibidores , Factores de Transcripción Otx/genética , ARN Polimerasa II/metabolismo , Proteínas de Dominio T Box/genética , Transcriptoma , Xenopus/metabolismo , Proteínas de Xenopus/antagonistas & inhibidores , Proteínas de Xenopus/genéticaRESUMEN
It has recently been reported that a common side effect of translation-blocking morpholino antisense oligonucleotides is the induction of a set of innate immune response genes in Xenopus embryos and that splicing-blocking morpholinos lead to unexpected off-target mis-splicing events. Here, we present an analysis of all publicly available Xenopus RNA sequencing (RNA-seq) data in a reexamination of the effects of translation-blocking morpholinos on the innate immune response. Our analysis does not support the authors' general conclusion, which was based on a limited number of RNA-seq datasets. Moreover, the strong induction of an immune response appears to be specific to the tbxt/tbxt2 morpholinos. The more comprehensive study presented here indicates that using morpholinos for targeted gene knockdowns remains of considerable value for the rapid identification of gene function.
Asunto(s)
Inmunidad Innata/inmunología , Morfolinos/inmunología , Morfolinos/metabolismo , Animales , Desarrollo Embrionario/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Inmunidad Innata/fisiología , Oligonucleótidos Antisentido/genética , Empalme del ARN , Proteínas de Dominio T Box/genética , Proteínas de Dominio T Box/metabolismo , Transcriptoma/genética , Xenopus/embriología , Xenopus/genética , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Xenopus laevis/embriología , Xenopus laevis/genéticaRESUMEN
Transcriptional regulatory elements are typically found in relatively nucleosome-free genomic regions, often referred to as "open chromatin." Deoxyribonuclease I (DNase I) can digest nucleosome-depleted DNA (presumably bound by transcription factors), but DNA in nucleosomes or higher-order chromatin fibers is less accessible to the nuclease. The DNase-seq method uses high-throughput sequencing to permit the interrogation of DNase hypersensitive sites (DHSs) across the entire genome and does not require prior knowledge of histone modifications, transcription factor binding sites, or high quality antibodies to identify potentially active regions of chromatin. Here, discontinuous iodixanol gradients are used as a gentle preparation of the nuclei from Xenopus embryos. Short DNase I digestion times are followed by size selection of digested genomic DNA, yielding DHS fragments. These DNA fragments are subjected to real-time quantitative polymerase chain reaction (qPCR) and sequencing library construction. A library generation method and pipeline for analyzing DNase-seq data are also described.
Asunto(s)
Cromatina/metabolismo , Desoxirribonucleasa I/metabolismo , Xenopus/embriología , Animales , Embrión no Mamífero/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos , Transcripción GenéticaRESUMEN
The interplay between transcription factors and chromatin dictates gene regulatory network activity. Germ layer specification is tightly coupled with zygotic gene activation and, in most metazoans, is dependent upon maternal factors. We explore the dynamic genome-wide interactions of Foxh1, a maternal transcription factor that mediates Nodal/TGF-ß signaling, with cis-regulatory modules (CRMs) during mesendodermal specification. Foxh1 marks CRMs during cleavage stages and recruits the co-repressor Tle/Groucho in the early blastula. We highlight a population of CRMs that are continuously occupied by Foxh1 and show that they are marked by H3K4me1, Ep300, and Fox/Sox/Smad motifs, suggesting interplay between these factors in gene regulation. We also propose a molecular "hand-off" between maternal Foxh1 and zygotic Foxa at these CRMs to maintain enhancer activation. Our findings suggest that Foxh1 functions at the top of a hierarchy of interactions by marking developmental genes for activation, beginning with the onset of zygotic gene expression.
Asunto(s)
Endodermo/metabolismo , Factores de Transcripción Forkhead/metabolismo , Regulación del Desarrollo de la Expresión Génica , Mesodermo/metabolismo , Factores de Transcripción/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus/embriología , Xenopus/genética , Animales , Blástula/metabolismo , Fase de Segmentación del Huevo/metabolismo , Proteínas Co-Represoras/metabolismo , Embrión no Mamífero/metabolismo , Endodermo/embriología , Elementos de Facilitación Genéticos/genética , Factores de Transcripción Forkhead/genética , Genoma , Histonas/metabolismo , Lisina/metabolismo , Mesodermo/embriología , Metilación , Proteína Nodal/metabolismo , Unión Proteica/genética , ARN Polimerasa II/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos/genética , Análisis de Secuencia de ARN , Transducción de Señal/genética , Transcripción Genética , Xenopus/metabolismo , Proteínas de Xenopus/genéticaRESUMEN
Germ layer formation is among the earliest differentiation events in metazoan embryos. In triploblasts, three germ layers are formed, among which the endoderm gives rise to the epithelial lining of the gut tube and associated organs including the liver, pancreas and lungs. In frogs (Xenopus), where early germ layer formation has been studied extensively, the process of endoderm specification involves the interplay of dozens of transcription factors. Here, we review the interactions between these factors, summarized in a transcriptional gene regulatory network (GRN). We highlight regulatory connections conserved between frog, fish, mouse, and human endodermal lineages. Especially prominent is the conserved role and regulatory targets of the Nodal signaling pathway and the T-box transcription factors, Vegt and Eomes. Additionally, we highlight network topologies and motifs, and speculate on their possible roles in development.
Asunto(s)
Endodermo/embriología , Redes Reguladoras de Genes/genética , Factores de Transcripción/metabolismo , Proteínas de Xenopus/genética , Xenopus/genética , Animales , Diferenciación CelularRESUMEN
Advances in RNA sequencing technologies have led to the surprising discovery that a vast number of transcripts emanate from regions of the genome that are not part of coding genes. Although some of the smaller ncRNAs such as microRNAs have well-characterized functions, the majority of long ncRNA (lncRNA) functions remain poorly understood. Understanding the significance of lncRNAs is an important challenge facing biology today. A powerful approach to uncovering the function of lncRNAs is to explore temporal and spatial expression profiling. This may be particularly useful for classes of lncRNAs that have developmentally important roles as the expression of such lncRNAs will be expected to be both spatially and temporally regulated during development. Here, we take advantage of our ultra-high frequency (temporal) sampling of Xenopus embryos to analyze gene expression trajectories of lncRNA transcripts over the first 3 days of development. We computationally identify 5689 potential single- and multi-exon lncRNAs. These lncRNAs demonstrate clear dynamic expression patterns. A subset of them displays highly correlative temporal expression profiles with respect to those of the neighboring genes. We also identified spatially localized lncRNAs in the gastrula stage embryo. These results suggest that lncRNAs have regulatory roles during early embryonic development.