RESUMO
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.
Assuntos
Endoderma , Redes Reguladoras de Genes , Proteína Proto-Oncogênica N-Myc/genética , Diferenciação Celular/genética , Fatores de Transcrição/genética , Regulação da Expressão Gênica no Desenvolvimento/genéticaRESUMO
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.
Assuntos
Cromatina , Epigenômica , Animais , Xenopus laevis/genética , Cromatina/metabolismo , Desenvolvimento Embrionário/genética , Zigoto/metabolismo , Regulação da Expressão Gênica no DesenvolvimentoRESUMO
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.
Assuntos
Cromatina , Fatores de Transcrição Forkhead , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Xenopus , Animais , Cromatina/genética , Fatores de Transcrição Forkhead/genética , Camundongos , Proteômica , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Xenopus laevis/genéticaRESUMO
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.
Assuntos
Desenvolvimento Embrionário/genética , Camadas Germinativas , Modelos Biológicos , Transcriptoma/genética , Animais , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Embrião de Mamíferos/fisiologia , Camadas Germinativas/citologia , Camadas Germinativas/metabolismo , Camadas Germinativas/fisiologia , Camundongos , Análise de Célula ÚnicaRESUMO
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.
Assuntos
Endoderma/embriologia , Redes Reguladoras de Genes/genética , Fatores de Transcrição/metabolismo , Proteínas de Xenopus/genética , Xenopus/genética , Animais , Diferenciação CelularRESUMO
CRISPR/Cas9 genome editing is revolutionizing genetic loss-of-function analysis but technical limitations remain that slow progress when creating mutant lines. First, in conventional genetic breeding schemes, mosaic founder animals carrying mutant alleles are outcrossed to produce F1 heterozygotes. Phenotypic analysis occurs in the F2 generation following F1 intercrosses. Thus, mutant analyses will require multi-generational studies. Second, when targeting essential genes, efficient mutagenesis of founders is often lethal, preventing the acquisition of mature animals. Reducing mutagenesis levels may improve founder survival, but results in lower, more variable rates of germline transmission. Therefore, an efficient approach to study lethal mutations would be useful. To overcome these shortfalls, we introduce 'leapfrogging', a method combining efficient CRISPR mutagenesis with transplantation of mutated primordial germ cells into a wild-type host. Tested using Xenopus tropicalis, we show that founders containing transplants transmit mutant alleles with high efficiency. F1 offspring from intercrosses between F0 animals that carry embryonic lethal alleles recapitulate loss-of-function phenotypes, circumventing an entire generation of breeding. We anticipate that leapfrogging will be transferable to other species.
Assuntos
Sistemas CRISPR-Cas/fisiologia , Células Germinativas/metabolismo , Mutação/genética , Animais , Anuros , Blástula/citologia , Blástula/metabolismo , Sistemas CRISPR-Cas/genética , Embrião não Mamífero , Feminino , Células Germinativas/citologia , Masculino , Mutagênese , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição/genética , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição/metabolismo , XenopusRESUMO
Gene regulatory networks (GRNs) involve highly combinatorial interactions between transcription factors and short sequence motifs in cis-regulatory modules of target genes to control cellular phenotypes. The GRNs specifying most cell types are largely unknown and are the subject of wide interest. A catalog of transcription factors is a valuable tool toward obtaining a deeper understanding of the role of these critical effectors in any biological setting. Here we present a comprehensive catalog of the transcription factors for the diploid frog Xenopus tropicalis. We identify 1235 genes encoding DNA-binding transcription factors, comparable to the numbers found in typical mammalian species. In detail, the repertoire of X. tropicalis transcription factor genes is nearly identical to human and mouse, with the exception of zinc finger family members, and a small number of species/lineage-specific gene duplications and losses relative to the mammalian repertoires. We applied this resource to the identification of transcription factors differentially expressed in the early gastrula stage embryo. We find transcription factor enrichment in Spemann's organizer, the ventral mesoderm, ectoderm and endoderm, and report 218 TFs that show regionalized expression patterns at this stage. Many of these have not been previously reported as expressed in the early embryo, suggesting thus far unappreciated roles for many transcription factors in the GRNs regulating early development. We expect our transcription factor catalog will facilitate myriad studies using Xenopus as a model system to understand basic biology and human disease.
Assuntos
Gástrula/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Fatores de Transcrição/biossíntese , Proteínas de Xenopus/biossíntese , Xenopus/metabolismo , Animais , Sequência de Bases , Embrião não Mamífero/metabolismo , Humanos , Camundongos , Especificidade da Espécie , Fatores de Transcrição/genética , Xenopus/embriologia , Xenopus/genética , Proteínas de Xenopus/genéticaRESUMO
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.
Assuntos
RNA Longo não Codificante/genética , Xenopus/genética , Animais , Embrião não Mamífero/metabolismo , Éxons/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Modelos Genéticos , RNA Longo não Codificante/biossíntese , RNA Longo não Codificante/isolamento & purificação , Transcriptoma , Xenopus/embriologiaRESUMO
A critical event in mammalian embryo development is construction of an inner cell mass surrounded by a trophoectoderm (a shell of cells that later form extraembryonic structures). We utilize multi-scale, stochastic modeling to investigate the design principles responsible for robust establishment of these structures. This investigation makes three predictions, each supported by our quantitative imaging. First, stochasticity in the expression of critical genes promotes cell plasticity and has a critical role in accurately organizing the developing mouse blastocyst. Second, asymmetry in the levels of noise variation (expression fluctuation) of Cdx2 and Oct4 provides a means to gain the benefits of noise-mediated plasticity while ameliorating the potentially detrimental effects of stochasticity. Finally, by controlling the timing and pace of cell fate specification, the embryo temporally modulates plasticity and creates a time window during which each cell can continually read its environment and adjusts its fate. These results suggest noise has a crucial role in maintaining cellular plasticity and organizing the blastocyst.
Assuntos
Blastocisto/citologia , Blastocisto/fisiologia , Diferenciação Celular/fisiologia , Desenvolvimento Embrionário/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Modelos Biológicos , Animais , Plasticidade Celular/fisiologia , Células Cultivadas , Simulação por Computador , Humanos , Camundongos , Modelos Estatísticos , Razão Sinal-Ruído , Processos EstocásticosRESUMO
Nodal/TGFß signaling regulates diverse biological responses. By combining RNA-seq on Foxh1 and Nodal signaling loss-of-function embryos with ChIP-seq of Foxh1 and Smad2/3, we report a comprehensive genome-wide interaction between Foxh1 and Smad2/3 in mediating Nodal signaling during vertebrate mesendoderm development. This study significantly increases the total number of Nodal target genes regulated by Foxh1 and Smad2/3, and reinforces the notion that Foxh1-Smad2/3-mediated Nodal signaling directly coordinates the expression of a cohort of genes involved in the control of gene transcription, signaling pathway modulation and tissue morphogenesis during gastrulation. We also show that Foxh1 may function independently of Nodal signaling, in addition to its role as a transcription factor mediating Nodal signaling via Smad2/3. Finally, we propose an evolutionarily conserved interaction between Foxh1 and PouV, a mechanism observed in Pou5f1-mediated regulation of pluripotency in human embryonic stem and epiblast cells.
Assuntos
Endoderma/embriologia , Fatores de Transcrição Forkhead/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Mesoderma/embriologia , Fator de Crescimento Transformador beta/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus/embriologia , Animais , Western Blotting , Imunoprecipitação da Cromatina , Biologia Computacional , Fatores de Transcrição Forkhead/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Técnicas de Silenciamento de Genes , Humanos , Imunoprecipitação , Morfolinos/genética , Proteína Nodal/genética , Proteína Nodal/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Proteína Smad2/metabolismo , Proteína Smad3/metabolismo , Estatísticas não Paramétricas , Proteínas de Xenopus/genéticaRESUMO
The mechanisms regulating cell division during development of the mouse pre-implantation embryo are poorly understood. We have investigated whether bone morphogenetic protein (BMP) signaling is involved in controlling cell cycle during mouse pre-implantation development. We mapped and quantitated the dynamic activities of BMP signaling through high-resolution immunofluorescence imaging combined with a 3D segmentation method. Immunostaining for phosphorylated Smad1/5/8 shows that BMP signaling is activated in mouse embryos as early as the 4-cell stage, and becomes spatially restricted by late blastocyst stage. Perturbation of BMP signaling in preimplantation mouse embryos, whether by treatment with a small molecule inhibitor, with Noggin protein, or by overexpression of a dominant-negative BMP receptor, indicates that BMPs regulate cell cleavage up to the morula stage. These results indicate that BMP signaling is active during mouse pre-implantation development and is required for cell cleavage in preimplantation mouse embryos.
Assuntos
Blastocisto/fisiologia , Proteínas Morfogenéticas Ósseas/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Animais , Proteínas de Transporte/metabolismo , Divisão Celular , Células Cultivadas , Desenvolvimento Embrionário/genética , Células-Tronco Embrionárias/citologia , Feminino , Genes Dominantes , Camundongos , Camundongos Transgênicos , Microscopia Confocal , Microscopia de Fluorescência , Fosforilação , Transdução de Sinais , Proteína Smad1/fisiologia , Proteína Smad5/fisiologia , Proteína Smad8/fisiologia , Fatores de TempoRESUMO
BACKGROUND: Analysis of single cells in their native environment is a powerful method to address key questions in developmental systems biology. Confocal microscopy imaging of intact tissues, followed by automatic image segmentation, provides a means to conduct cytometric studies while at the same time preserving crucial information about the spatial organization of the tissue and morphological features of the cells. This technique is rapidly evolving but is still not in widespread use among research groups that do not specialize in technique development, perhaps in part for lack of tools that automate repetitive tasks while allowing experts to make the best use of their time in injecting their domain-specific knowledge. RESULTS: Here we focus on a well-established stem cell model system, the C. elegans gonad, as well as on two other model systems widely used to study cell fate specification and morphogenesis: the pre-implantation mouse embryo and the developing mouse olfactory epithelium. We report a pipeline that integrates machine-learning-based cell detection, fast human-in-the-loop curation of these detections, and running of active contours seeded from detections to segment cells. The procedure can be bootstrapped by a small number of manual detections, and outperforms alternative pieces of software we benchmarked on C. elegans gonad datasets. Using cell segmentations to quantify fluorescence contents, we report previously-uncharacterized cell behaviors in the model systems we used. We further show how cell morphological features can be used to identify cell cycle phase; this provides a basis for future tools that will streamline cell cycle experiments by minimizing the need for exogenous cell cycle phase labels. CONCLUSIONS: High-throughput 3D segmentation makes it possible to extract rich information from images that are routinely acquired by biologists, and provides insights - in particular with respect to the cell cycle - that would be difficult to derive otherwise.
Assuntos
Caenorhabditis elegans/crescimento & desenvolvimento , Ensaios de Triagem em Larga Escala , Processamento de Imagem Assistida por Computador/métodos , Imageamento Tridimensional/métodos , Mucosa Olfatória/citologia , Análise de Célula Única/métodos , Software , Algoritmos , Animais , Blastocisto/citologia , Blastocisto/metabolismo , Caenorhabditis elegans/metabolismo , Ciclo Celular/fisiologia , Células Cultivadas , Biologia Computacional/métodos , Feminino , Gônadas/citologia , Gônadas/metabolismo , Humanos , Masculino , Camundongos , Microscopia Confocal/métodos , Mucosa Olfatória/metabolismoRESUMO
Bone morphogenetic protein (BMP) signaling plays an essential role in early tooth development, evidenced by disruption of BMP signaling leading to an early arrested tooth development. Despite being a central mediator of BMP canonical signaling pathway, inactivation of Smad4 in dental mesenchyme does not result in early developmental defects. In the current study, we investigated the mechanism of receptor-activated Smads (R-Smads) and Smad4 in the regulation of the odontogenic gene Msx1 expression in the dental mesenchyme. We showed that the canonical BMP signaling is not operating in the early developing tooth, as assessed by failed activation of the BRE-Gal transgenic allele and the absence of phospho-(p)Smad1/5/8-Smad4 complexes. The absence of pSmad1/5/8-Smad4 complex appeared to be the consequence of saturation of Smad4 by pSmad2/3 in the dental mesenchyme as knockdown of Smad2/3 or overexpression of Smad4 led to the formation of pSmad1/5/8-Smad4 complexes and activation of canonical BMP signaling in dental mesenchymal cells. We showed that Smad1/5 but not Smad4 are required for BMP-induced expression of Msx1 in dental mesenchymal cells. We further presented evidence that in the absence of Smad4, BMPs are still able to induce pSmad1/5/8 nuclear translocation and their binding to the Msx1 promoter directly in dental mesenchymal cells. Our results demonstrate the functional operation of an atypical canonical BMP signaling (Smad4-independent and Smad1/5/8-dependent) pathway in the dental mesenchyme during early odontogenesis, which may have general implication in the development of other organs.
Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Fator de Transcrição MSX1/metabolismo , Odontogênese/fisiologia , Transdução de Sinais , Transporte Ativo do Núcleo Celular , Alelos , Animais , Núcleo Celular/metabolismo , Éxons , Genes Homeobox , Mesoderma/metabolismo , Camundongos , Camundongos Transgênicos , Interferência de RNA , Proteína Smad1/metabolismo , Proteína Smad4/metabolismo , Proteína Smad5/metabolismo , Dente/embriologia , Fator de Crescimento Transformador beta1/metabolismo , TransgenesRESUMO
Bone morphogenetic proteins (BMPs) play crucial roles in craniofacial development but little is known about their interactions with other signals, such as Endothelin 1 (Edn1) and Jagged/Notch, which pattern the dorsal-ventral (DV) axis of the pharyngeal arches. Here, we use transgenic zebrafish to monitor and perturb BMP signaling during arch formation. With a BMP-responsive transgene, Tg(Bre:GFP), we show active BMP signaling in neural crest (NC)-derived skeletal precursors of the ventral arches, and in surrounding epithelia. Loss-of-function studies using a heat shock-inducible, dominant-negative BMP receptor 1a [Tg(hs70I:dnBmpr1a-GFP)] to bypass early roles show that BMP signaling is required for ventral arch development just after NC migration, the same stages at which we detect Tg(Bre:GFP). Inhibition of BMP signaling at these stages reduces expression of the ventral signal Edn1, as well as ventral-specific genes such as hand2 and dlx6a in the arches, and expands expression of the dorsal signal jag1b. This results in a loss or reduction of ventral and intermediate skeletal elements and a mis-shapen dorsal arch skeleton. Conversely, ectopic BMP causes dorsal expansion of ventral-specific gene expression and corresponding reductions/transformations of dorsal cartilages. Soon after NC migration, BMP is required to induce Edn1 and overexpression of either signal partially rescues ventral skeletal defects in embryos deficient for the other. However, once arch primordia are established the effects of BMPs become restricted to more ventral and anterior (palate) domains, which do not depend on Edn1. This suggests that BMPs act upstream and in parallel to Edn1 to promote ventral fates in the arches during early DV patterning, but later acquire distinct roles that further subdivide the identities of NC cells to pattern the craniofacial skeleton.
Assuntos
Padronização Corporal/fisiologia , Proteínas Morfogenéticas Ósseas/metabolismo , Região Branquial/embriologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Transdução de Sinais/fisiologia , Peixe-Zebra/embriologia , Animais , Animais Geneticamente Modificados , Movimento Celular/fisiologia , Endotelina-1/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Crista Neural/embriologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
Gene inactivation is an important tool for correlation of phenotypic and genomic data, allowing researchers to infer normal gene function based on the phenotype when the gene is impaired. New and better approaches are needed to overcome the shortfalls of existing methods for any significant acceleration of scientific progress. We have adapted the CRISPR/Cas system for use in Xenopus tropicalis and report on the efficient creation of mutations in the gene encoding the enzyme tyrosinase, which is responsible for oculocutaneous albinism. Biallelic mutation of this gene was detected in the F0 generation, suggesting targeting efficiencies similar to that of TALENs. We also find that off-target mutagenesis seems to be negligible, and therefore, CRISPR/Cas may be a useful system for creating genome modifications in this important model organism.
Assuntos
Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Monofenol Mono-Oxigenase/genética , Proteínas de Xenopus/genética , Xenopus/embriologia , Xenopus/genética , Albinismo/genética , Alelos , Animais , Sequência de Bases , Embrião não Mamífero/metabolismo , Feminino , Dosagem de Genes , Técnicas de Inativação de Genes , Genoma , Mutação INDEL , Monofenol Mono-Oxigenase/metabolismo , Fenótipo , Proteínas de Xenopus/metabolismoRESUMO
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.
Assuntos
Histona Desacetilase 1 , Histonas , Histonas/metabolismo , Histona Desacetilase 1/genética , Histona Desacetilase 1/metabolismo , Cromatina/metabolismo , Blastocisto/metabolismo , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Desenvolvimento Embrionário/genética , Acetilação , Histona Desacetilase 2/metabolismoRESUMO
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.
Assuntos
Cádmio/metabolismo , Proteínas de Ligação a DNA/metabolismo , Via de Sinalização Hippo/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Zinco/metabolismo , Cádmio/toxicidade , Linhagem Celular Tumoral , Regulação da Expressão Gênica/genética , Células HEK293 , Células HeLa , Homeostase/genética , Humanos , Inativação Metabólica/fisiologia , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Estresse Fisiológico/fisiologia , Transcrição Gênica/genética , Proteínas Supressoras de Tumor/genética , Zinco/toxicidade , Fator MTF-1 de TranscriçãoRESUMO
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.
Assuntos
Endoderma/embriologia , Redes Reguladoras de Genes , Genômica , Mesoderma/embriologia , Xenopus/embriologia , Xenopus/genética , Animais , Cromatina/metabolismo , Sequência Consenso/genética , DNA/metabolismo , Gastrulação/genética , Regulação da Expressão Gênica no Desenvolvimento , Ligação Proteica , RNA/metabolismo , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
The basal chordate amphioxus resembles vertebrates in having a dorsal, hollow nerve cord, a notochord and somites. However, it lacks extensive gene duplications, and its embryos are small and gastrulate by simple invagination. Here we demonstrate that Nodal/Vg1 signaling acts from early cleavage through the gastrula stage to specify and maintain dorsal/anterior development while, starting at the early gastrula stage, BMP signaling promotes ventral/posterior identity. Knockdown and gain-of-function experiments show that these pathways act in opposition to one another. Signaling by these pathways is modulated by dorsally and/or anteriorly expressed genes including Chordin, Cerberus, and Blimp1. Overexpression and/or reporter assays in Xenopus demonstrate that the functions of these proteins are conserved between amphioxus and vertebrates. Thus, a fundamental genetic mechanism for axial patterning involving opposing Nodal and BMP signaling is present in amphioxus and probably also in the common ancestor of amphioxus and vertebrates or even earlier in deuterostome evolution.