RESUMEN
Tissue-specific transcription regulators emerged as key developmental control genes, which operate in the context of complex gene regulatory networks (GRNs) to coordinate progressive cell fate specification and tissue morphogenesis. We discuss how GRNs control the individual cell behaviors underlying complex morphogenetic events. Cell behaviors classically range from mesenchymal cell motility to cell shape changes in epithelial sheets. These behaviors emerge from the tissue-specific, multiscale integration of the local activities of universal and pleiotropic effectors, which underlie modular subcellular processes including cytoskeletal dynamics, cell-cell and cell-matrix adhesion, signaling, polarity, and vesicle trafficking. Extrinsic cues and intrinsic cell competence determine the subcellular spatiotemporal patterns of effector activities. GRNs influence most subcellular activities by controlling only a fraction of the effector-coding genes, which we argue is enriched in effectors involved in reading and processing the extrinsic cues to contextualize intrinsic subcellular processes and canalize developmental cell behaviors. The properties of the transcription-cell behavior interface have profound implications for evolution and disease.
Asunto(s)
Células/metabolismo , Regulación del Desarrollo de la Expresión Génica , Transcripción Genética , Animales , Redes Reguladoras de Genes , Genómica , Humanos , Especificidad de Órganos/genéticaRESUMEN
Transcription control is a major determinant of cell fate decisions in somatic tissues. By contrast, early germline fate specification in numerous vertebrate and invertebrate species relies extensively on RNA-level regulation, exerted on asymmetrically inherited maternal supplies, with little-to-no zygotic transcription. However delayed, a maternal-to-zygotic transition is nevertheless poised to complete the deployment of pre-gametic programs in the germline. Here, we focus on early germline specification in the tunicate Ciona to study zygotic genome activation. We first demonstrate that a peculiar cellular remodeling event excludes localized postplasmic Pem-1 mRNA, which encodes the general inhibitor of transcription. Subsequently, zygotic transcription begins in Pem-1-negative primordial germ cells (PGCs), as revealed by histochemical detection of elongating RNA Polymerase II, and nascent Mef2 transcripts. In addition, we uncover a provisional antagonism between JAK and MEK/BMPRI/GSK3 signaling, which controls the onset of zygotic gene expression, following cellular remodeling of PGCs. We propose a 2-step model for the onset of zygotic transcription in the Ciona germline and discuss the significance of germ plasm dislocation and remodeling in the context of developmental fate specification.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Células Germinativas , Quinasas Janus , Cigoto , Animales , Ciona/genética , Ciona/metabolismo , Ciona intestinalis/genética , Ciona intestinalis/embriología , Células Germinativas/metabolismo , Quinasas Janus/antagonistas & inhibidores , Quinasas Janus/metabolismo , Transducción de Señal , Transcripción Genética , Cigoto/metabolismoRESUMEN
During embryonic development, cell-fate specification gives rise to dedicated lineages that underlie tissue formation. In olfactores, which comprise tunicates and vertebrates, the cardiopharyngeal field is formed by multipotent progenitors of both cardiac and branchiomeric muscles. The ascidian Ciona is a powerful model to study cardiopharyngeal fate specification with cellular resolution, as only two bilateral pairs of multipotent cardiopharyngeal progenitors give rise to the heart and to the pharyngeal muscles (also known as atrial siphon muscles, ASM). These progenitors are multilineage primed, in as much as they express a combination of early ASM- and heart-specific transcripts that become restricted to their corresponding precursors, following oriented and asymmetric divisions. Here, we identify the primed gene ring finger 149 related (Rnf149-r), which later becomes restricted to the heart progenitors, but appears to regulate pharyngeal muscle fate specification in the cardiopharyngeal lineage. CRISPR/Cas9-mediated loss of Rnf149-r function impairs atrial siphon muscle morphogenesis, and downregulates Tbx1/10 and Ebf, two key determinants of pharyngeal muscle fate, while upregulating heart-specific gene expression. These phenotypes are reminiscent of the loss of FGF/MAPK signaling in the cardiopharyngeal lineage, and an integrated analysis of lineage-specific bulk RNA-seq profiling of loss-of-function perturbations has identified a significant overlap between candidate FGF/MAPK and Rnf149-r target genes. However, functional interaction assays suggest that Rnf149-r does not directly modulate the activity of the FGF/MAPK/Ets1/2 pathway. Instead, we propose that Rnf149-r acts both in parallel to the FGF/MAPK signaling on shared targets, as well as on FGF/MAPK-independent targets through (a) separate pathway(s).
Asunto(s)
Fibrilación Atrial , Ciona intestinalis , Animales , Fibrilación Atrial/genética , Ciona intestinalis/genética , Músculos Faríngeos , Corazón , Diferenciación Celular/genética , Regulación del Desarrollo de la Expresión Génica , Linaje de la Célula/genéticaRESUMEN
The analysis of single-cell genomics data presents several statistical challenges, and extensive efforts have been made to produce methods for the analysis of this data that impute missing values, address sampling issues and quantify and correct for noise. In spite of such efforts, no consensus on best practices has been established and all current approaches vary substantially based on the available data and empirical tests. The k-Nearest Neighbor Graph (kNN-G) is often used to infer the identities of, and relationships between, cells and is the basis of many widely used dimensionality-reduction and projection methods. The kNN-G has also been the basis for imputation methods using, e.g., neighbor averaging and graph diffusion. However, due to the lack of an agreed-upon optimal objective function for choosing hyperparameters, these methods tend to oversmooth data, thereby resulting in a loss of information with regard to cell identity and the specific gene-to-gene patterns underlying regulatory mechanisms. In this paper, we investigate the tuning of kNN- and diffusion-based denoising methods with a novel non-stochastic method for optimally preserving biologically relevant informative variance in single-cell data. The framework, Denoising Expression data with a Weighted Affinity Kernel and Self-Supervision (DEWÄKSS), uses a self-supervised technique to tune its parameters. We demonstrate that denoising with optimal parameters selected by our objective function (i) is robust to preprocessing methods using data from established benchmarks, (ii) disentangles cellular identity and maintains robust clusters over dimension-reduction methods, (iii) maintains variance along several expression dimensions, unlike previous heuristic-based methods that tend to oversmooth data variance, and (iv) rarely involves diffusion but rather uses a fixed weighted kNN graph for denoising. Together, these findings provide a new understanding of kNN- and diffusion-based denoising methods. Code and example data for DEWÄKSS is available at https://gitlab.com/Xparx/dewakss/-/tree/Tjarnberg2020branch.
Asunto(s)
Algoritmos , Genómica/métodos , Análisis de la Célula Individual/métodos , Aprendizaje Automático Supervisado , Animales , Línea Celular , Bases de Datos Genéticas , Humanos , Ratones , RNA-Seq , Saccharomyces cerevisiaeRESUMEN
Many species in the tunicate family Molgulidae have independently lost their swimming larval form and instead develop as tailless, immotile larvae. These larvae do not develop structures that are essential for swimming such as the notochord, otolith, and tail muscles. However, little is known about neural development in these nonswimming larvae. Here, we studied the patterning of the Motor Ganglion (MG) of Molgula occulta, a nonswimming species. We found that spatial patterns of MG neuron regulators in this species are conserved, compared with species with swimming larvae, suggesting that the gene networks regulating their expression are intact despite the loss of swimming. However, expression of the key motor neuron regulatory gene Ebf (Collier/Olf/EBF) was reduced in the developing MG of M. occulta when compared with molgulid species with swimming larvae. This was corroborated by measuring allele-specific expression of Ebf in hybrid embryos from crosses of M. occulta with the swimming species M. oculata. Heterologous reporter construct assays in the model tunicate species Ciona robusta revealed a specific cis-regulatory sequence change that reduces expression of Ebf in the MG, but not in other cells. Taken together, these data suggest that MG neurons are still specified in M. occulta larvae, but their differentiation might be impaired due to reduction of Ebf expression levels.
Asunto(s)
Urocordados , Animales , Evolución Biológica , Larva/genética , Neuronas Motoras , Notocorda , Urocordados/genéticaRESUMEN
The neural crest is an evolutionary novelty that fostered the emergence of vertebrate anatomical innovations such as the cranium and jaws. During embryonic development, multipotent neural crest cells are specified at the lateral borders of the neural plate before delaminating, migrating and differentiating into various cell types. In invertebrate chordates (cephalochordates and tunicates), neural plate border cells express conserved factors such as Msx, Snail and Pax3/7 and generate melanin-containing pigment cells, a derivative of the neural crest in vertebrates. However, invertebrate neural plate border cells have not been shown to generate homologues of other neural crest derivatives. Thus, proposed models of neural crest evolution postulate vertebrate-specific elaborations on an ancestral neural plate border program, through acquisition of migratory capabilities and the potential to generate several cell types. Here we show that a particular neuronal cell type in the tadpole larva of the tunicate Ciona intestinalis, the bipolar tail neuron, shares a set of features with neural-crest-derived spinal ganglia neurons in vertebrates. Bipolar tail neuron precursors derive from caudal neural plate border cells, delaminate and migrate along the paraxial mesoderm on either side of the neural tube, eventually differentiating into afferent neurons that form synaptic contacts with both epidermal sensory cells and motor neurons. We propose that the neural plate borders of the chordate ancestor already produced migratory peripheral neurons and pigment cells, and that the neural crest evolved through the acquisition of a multipotent progenitor regulatory state upstream of multiple, pre-existing neural plate border cell differentiation programs.
Asunto(s)
Ciona intestinalis/citología , Placa Neural/citología , Células-Madre Neurales/citología , Animales , Movimiento Celular , Polaridad Celular , Ganglios Espinales/citología , Larva/citología , Mesodermo/citología , Células Madre Multipotentes/citología , Cresta Neural/citología , Neurogénesis , Neuronas/citología , Sinapsis , Cola (estructura animal)/citología , VertebradosRESUMEN
It has been more than 30 years since the publication of the new head hypothesis, which proposed that the vertebrate head is an evolutionary novelty resulting from the emergence of neural crest and cranial placodes. Neural crest generates the skull and associated connective tissues, whereas placodes produce sensory organs. However, neither crest nor placodes produce head muscles, which are a crucial component of the complex vertebrate head. We discuss emerging evidence for a surprising link between the evolution of head muscles and chambered hearts - both systems arise from a common pool of mesoderm progenitor cells within the cardiopharyngeal field of vertebrate embryos. We consider the origin of this field in non-vertebrate chordates and its evolution in vertebrates.
Asunto(s)
Evolución Biológica , Región Branquial/embriología , Cabeza/anatomía & histología , Cabeza/embriología , Corazón/anatomía & histología , Corazón/embriología , Vertebrados/anatomía & histología , Vertebrados/embriología , Animales , Región Branquial/anatomía & histología , Región Branquial/citología , Mesodermo/citología , Modelos Biológicos , Músculos/anatomía & histología , Músculos/citología , Músculos/embriología , Cresta Neural/citologíaRESUMEN
In vertebrate embryos, the cardiopharyngeal mesoderm gives rise to both cardiac and branchiomeric head muscles. The canonical Wnt signaling pathway regulates many aspects of cardiomyocyte specification, and modulates a balance between skeletal and cardiac myogenesis during vertebrate head muscle development. However, the role of Wnt signaling during ascidian cardiopharyngeal development remains elusive. Here, we documented the expression of Wnt pathway components during cardiopharyngeal development in Ciona, and generated tools to investigate potential roles for Wnt signaling, and its transcriptional effector Tcf, on heart vs. pharyngeal muscle fate specification. Neither focused functional analyses nor lineage-specific transcriptome profiling uncovered a significant role for Tcf during early cardiac vs. pharyngeal muscle fate choice. By contrast, Wnt gene expression patterns of Frizzled4 and Lrp4/8 and CRISPR/Cas9-mediated Tcf knock-down suggested a later requirement for Wnt signaling during heart morphogenesis and/or cardiomyocyte differentiation. This study provides a provisional set of reagents to study Wnt signaling function in Ciona, and promising insights for future analyses of Wnt functions during heart organogenesis.
Asunto(s)
Ciona intestinalis/embriología , Ciona intestinalis/genética , Corazón/embriología , Factores de Transcripción TCF/metabolismo , Proteínas Wnt/metabolismo , Animales , Tipificación del Cuerpo/genética , Linaje de la Célula/genética , Desarrollo Embrionario/genética , Regulación del Desarrollo de la Expresión Génica , Organogénesis/genética , Faringe/embriología , Factores de Transcripción TCF/genética , Transcriptoma/genética , Regulación hacia Arriba/genética , Proteínas Wnt/genética , Vía de Señalización Wnt/genéticaRESUMEN
Through a myriad of pigments stored in different cells, animal pigmentation represents a crucial process to face disparate environmental and ecological challenges. In vertebrates, the small GTPase Rab32 and Rab38 have a conserved role in the transport of key melanogenic enzymes, as tyrosinase (tyr) and tyrosinase-related protein (tyrp), to the melanosomes in formation. We provide a survey on Rab32/38 evolution and its regulatory logics during pigment cell formation in Ciona robusta. Our phylogeny supports the existence of a single Rab32/38 gene in tunicates, which is probably the unique transporter for tyrosinase family members in this clade. Different deletions allow us to identify the minimal cis-regulatory element able to recapitulate the endogenous gene expression during pigment cell development in C. robusta. In this conserved region, we identified two putative binding sites for the transcription factor Mitf, which is known for its role as regulator of pigmentation in vertebrates. Mutational analysis revealed that both Mitf binding sites are essential for the activity of this regulatory region and we demonstrated that Mitf misexpression is able to induce ectopic activation of the Rab32/38 regulatory region in vivo. Our results strongly indicate that Mitf is involved in the regulation of Rab32/38 activity during Ciona pigment cell development.
Asunto(s)
Biomarcadores/metabolismo , Ciona intestinalis/citología , Ciona intestinalis/genética , Regulación de la Expresión Génica , Pigmentación/genética , Transcripción Genética , Proteínas de Unión al GTP rab/genética , Animales , Secuencia de Bases , Sitios de Unión , Evolución Molecular , Factor de Transcripción Asociado a Microftalmía/metabolismo , Notocorda/metabolismo , Filogenia , Unión Proteica , Secuencias Reguladoras de Ácidos Nucleicos/genética , Proteínas de Unión al GTP rab/metabolismoRESUMEN
A fundamental assumption, common to the vast majority of high-throughput transcriptome analyses, is that the expression of most genes is unchanged among samples and that total cellular RNA remains constant. As the number of analyzed experimental systems increases however, different independent studies demonstrate that this assumption is often violated. We present a calibration method using RNA spike-ins that allows for the measurement of absolute cellular abundance of RNA molecules. We apply the method to pooled RNA from cell populations of known sizes. For each transcript, we compute a nominal abundance that can be converted to absolute by dividing by a scale factor determined in separate experiments: the yield coefficient of the transcript relative to that of a reference spike-in measured with the same protocol. The method is derived by maximum likelihood theory in the context of a complete statistical model for sequencing counts contributed by cellular RNA and spike-ins. The counts are based on a sample from a fixed number of cells to which a fixed population of spike-in molecules has been added. We illustrate and evaluate the method with applications to two global expression data sets, one from the model eukaryote Saccharomyces cerevisiae, proliferating at different growth rates, and differentiating cardiopharyngeal cell lineages in the chordate Ciona robusta. We tested the method in a technical replicate dilution study, and in a k-fold validation study.
Asunto(s)
Funciones de Verosimilitud , Modelos Estadísticos , Análisis de Secuencia de ARN/normas , Animales , Calibración , Ciona/embriología , Ciona/genética , Expresión Génica , Genes Fúngicos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/normas , ARN de Hongos/genética , Saccharomyces cerevisiae/genéticaRESUMEN
ANISEED (www.aniseed.cnrs.fr) is the main model organism database for tunicates, the sister-group of vertebrates. This release gives access to annotated genomes, gene expression patterns, and anatomical descriptions for nine ascidian species. It provides increased integration with external molecular and taxonomy databases, better support for epigenomics datasets, in particular RNA-seq, ChIP-seq and SELEX-seq, and features novel interactive interfaces for existing and novel datatypes. In particular, the cross-species navigation and comparison is enhanced through a novel taxonomy section describing each represented species and through the implementation of interactive phylogenetic gene trees for 60% of tunicate genes. The gene expression section displays the results of RNA-seq experiments for the three major model species of solitary ascidians. Gene expression is controlled by the binding of transcription factors to cis-regulatory sequences. A high-resolution description of the DNA-binding specificity for 131 Ciona robusta (formerly C. intestinalis type A) transcription factors by SELEX-seq is provided and used to map candidate binding sites across the Ciona robusta and Phallusia mammillata genomes. Finally, use of a WashU Epigenome browser enhances genome navigation, while a Genomicus server was set up to explore microsynteny relationships within tunicates and with vertebrates, Amphioxus, echinoderms and hemichordates.
Asunto(s)
Bases de Datos Genéticas , Conjuntos de Datos como Asunto , Genoma , Urocordados/genética , Animales , Evolución Biológica , Ciona intestinalis/genética , ADN/metabolismo , Minería de Datos , Evolución Molecular , Expresión Génica , Ontología de Genes , Internet , Anotación de Secuencia Molecular , Filogenia , Unión Proteica , Especificidad de la Especie , Factores de Transcripción/metabolismo , Transcripción Genética , Vertebrados/genética , Navegador WebRESUMEN
Skeletal muscles arise from diverse embryonic origins in vertebrates, yet converge on extensively shared regulatory programs that require muscle regulatory factor (MRF)-family genes. Myogenesis in the tail of the simple chordate Ciona exhibits a similar reliance on its single MRF-family gene, and diverse mechanisms activate Ci-Mrf Here, we show that myogenesis in the atrial siphon muscles (ASMs) and oral siphon muscles (OSMs), which control the exhalant and inhalant siphons, respectively, also requires Mrf We characterize the ontogeny of OSM progenitors and compare the molecular basis of Mrf activation in OSM versus ASM. In both muscle types, Ebf and Tbx1/10 are expressed and function upstream of Mrf However, we demonstrate that regulatory relationships between Tbx1/10, Ebf and Mrf differ between the OSM and ASM lineages. We propose that Tbx1, Ebf and Mrf homologs form an ancient conserved regulatory state for pharyngeal muscle specification, whereas their regulatory relationships might be more evolutionarily variable.
Asunto(s)
Ciona intestinalis/metabolismo , Proteínas de Dominio T Box/metabolismo , Factores de Transcripción/metabolismo , Animales , Ciona intestinalis/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Regulación del Desarrollo de la Expresión Génica/genética , Regulación del Desarrollo de la Expresión Génica/fisiología , Hibridación in Situ , Desarrollo de Músculos/genética , Desarrollo de Músculos/fisiología , Proteínas de Dominio T Box/genética , Factores de Transcripción/genéticaRESUMEN
The CRISPR/Cas9 system has emerged as an important tool for various genome engineering applications. A current obstacle to high throughput applications of CRISPR/Cas9 is the imprecise prediction of highly active single guide RNAs (sgRNAs). We previously implemented the CRISPR/Cas9 system to induce tissue-specific mutations in the tunicate Ciona. In the present study, we designed and tested 83 single guide RNA (sgRNA) vectors targeting 23 genes expressed in the cardiopharyngeal progenitors and surrounding tissues of Ciona embryo. Using high-throughput sequencing of mutagenized alleles, we identified guide sequences that correlate with sgRNA mutagenesis activity and used this information for the rational design of all possible sgRNAs targeting the Ciona transcriptome. We also describe a one-step cloning-free protocol for the assembly of sgRNA expression cassettes. These cassettes can be directly electroporated as unpurified PCR products into Ciona embryos for sgRNA expression in vivo, resulting in high frequency of CRISPR/Cas9-mediated mutagenesis in somatic cells of electroporated embryos. We found a strong correlation between the frequency of an Ebf loss-of-function phenotype and the mutagenesis efficacies of individual Ebf-targeting sgRNAs tested using this method. We anticipate that our approach can be scaled up to systematically design and deliver highly efficient sgRNAs for the tissue-specific investigation of gene functions in Ciona.
Asunto(s)
Sistemas CRISPR-Cas , Ciona intestinalis/genética , Mutagénesis , ARN Guía de Kinetoplastida/genética , Animales , Secuencia de Bases , Ciona intestinalis/embriología , Embrión no Mamífero/embriología , Embrión no Mamífero/metabolismo , Edición Génica/métodos , Regulación del Desarrollo de la Expresión Génica , Marcación de Gen/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Mutación , Reproducibilidad de los Resultados , Homología de Secuencia de Ácido Nucleico , Transcriptoma/genéticaRESUMEN
Collectively migrating cells maintain group polarity and interpret external cues to reach their destination. The cardiogenic progenitors (also known as trunk ventral cells, TVCs) of the ascidian Ciona intestinalis provide a simple chordate model with which to study collective migration. Bilateral pairs of associated TVCs undergo a stereotyped polarized migration away from the tail towards the ventral trunk, arguably constituting the simplest possible example of directed collective migration. To identify tissues contributing to TVC polarity and migration, we quantified the contact between TVCs and surrounding tissues, and blocked the secretory pathway in a tissue-specific manner. Even though TVCs normally migrate as an invariably determined leader-trailer polarized pair of adherent cells, they are capable of migrating individually, albeit a shorter distance and with altered morphology. The mesenchyme contacts newborn TVCs and contributes to robust specification of the trailer but appears to have only minor effects on directed migration. The notochord does not contact the TVCs but contributes to the onset of migration. The trunk endoderm first contacts the leader TVC, then 'encases' both migrating cells and provides the inputs maintaining leader-trailer polarity. Migrating TVCs adhere to the epidermis and need this contact for their cohesion. These phenomenological studies reveal that inherently motile cardiopharyngeal progenitors are channeled into stereotyped behaviors by interactions with surrounding tissues.
Asunto(s)
Comunicación Celular/fisiología , Movimiento Celular/fisiología , Polaridad Celular/fisiología , Corazón/embriología , Células Madre/fisiología , Animales , Ciona intestinalis , Clonación Molecular , Procesamiento de Imagen Asistido por Computador , Inmunohistoquímica , Hibridación in Situ , Mesodermo/fisiología , Microscopía Confocal , Miocardio/citologíaRESUMEN
Genome-wide studies in Ciona often require highly purified cell populations. In this methods chapter, we introduce multi-channel combinatorial fluorescence activated cells sorting (FACS) and magnetic-activated cell sorting (MACS) as two sensitive and efficient tools for isolating lineage-specific cell populations from dissociated Ciona embryos and larvae. We present isolation of trunk ventral cell (TVC) progeny as the test case most commonly used in our laboratory. These approaches may also be applied to purify other cell populations with the proper combination of tissue-specific reporters.
Asunto(s)
Ciona intestinalis/embriología , Citometría de Flujo/métodos , Genes Reporteros , Separación Inmunomagnética/métodos , Proteínas Luminiscentes/análisis , Animales , Linaje de la Célula , Ciona intestinalis/citología , Ciona intestinalis/genética , Técnicas de Cultivo de Embriones , Embrión no Mamífero/química , Embrión no Mamífero/citología , Elementos de Facilitación Genéticos , Citometría de Flujo/instrumentación , Separación Inmunomagnética/instrumentación , Mosaicismo , ARN/aislamiento & purificaciónRESUMEN
Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 has emerged as a revolutionary tool for fast and efficient targeted gene knockouts and genome editing in almost any organism. The laboratory model tunicate Ciona is no exception. Here, we describe our latest protocol for the design, implementation, and evaluation of successful CRISPR/Cas9-mediated gene knockouts in somatic cells of electroporated Ciona embryos. Using commercially available reagents, publicly accessible plasmids, and free web-based software applications, any Ciona researcher can easily knock out any gene of interest in their favorite embryonic cell lineage.
Asunto(s)
Ciona intestinalis/genética , Técnicas de Inactivación de Genes/métodos , Animales , Sistemas CRISPR-Cas , Ciona intestinalis/embriología , Electroporación , Embrión no Mamífero/citología , Embrión no Mamífero/metabolismo , Mutación INDEL , Mutagénesis , Plásmidos , ARN Guía de Kinetoplastida/administración & dosificación , ARN Guía de Kinetoplastida/genéticaRESUMEN
The CRISPR/Cas9 system has ushered in a new era of targeted genetic manipulations. Here, we report the use of CRISPR/Cas9 to induce double-stranded breaks in the genome of the sea squirt Ciona intestinalis. We use electroporation to deliver CRISPR/Cas9 components for tissue-specific disruption of the Ebf (Collier/Olf/EBF) gene in hundreds of synchronized Ciona embryos. Phenotyping of transfected embryos in the 'F0' generation revealed that endogenous Ebf function is required for specification of Islet-expressing motor ganglion neurons and atrial siphon muscles. We demonstrate that CRISPR/Cas9 is sufficiently effective and specific to generate large numbers of embryos carrying mutations in a targeted gene of interest, which should allow for rapid screening of gene function in Ciona.
Asunto(s)
Ciona intestinalis/embriología , Ciona intestinalis/metabolismo , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/fisiología , Genoma/genética , Animales , Ciona intestinalis/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genéticaRESUMEN
Understanding gene regulatory networks is critical to understanding cellular differentiation and response to external stimuli. Methods for global network inference have been developed and applied to a variety of species. Most approaches consider the problem of network inference independently in each species, despite evidence that gene regulation can be conserved even in distantly related species. Further, network inference is often confined to single data-types (single platforms) and single cell types. We introduce a method for multi-source network inference that allows simultaneous estimation of gene regulatory networks in multiple species or biological processes through the introduction of priors based on known gene relationships such as orthology incorporated using fused regression. This approach improves network inference performance even when orthology mapping and conservation are incomplete. We refine this method by presenting an algorithm that extracts the true conserved subnetwork from a larger set of potentially conserved interactions and demonstrate the utility of our method in cross species network inference. Last, we demonstrate our method's utility in learning from data collected on different experimental platforms.
Asunto(s)
Biología Computacional/métodos , Regulación Bacteriana de la Expresión Génica/genética , Redes Reguladoras de Genes/genética , Modelos Genéticos , Algoritmos , Bacillus/genética , Bacillus/metabolismo , Simulación por Computador , Perfilación de la Expresión Génica , Análisis de RegresiónRESUMEN
ERK1/2 MAP kinase exhibits a highly dynamic activation pattern in developing embryos, which largely depends on fibroblast growth factor (FGF) signals. In ascidian embryos, FGF-dependent activation of ERK1/2 occurs differentially between sister cells during marginal zone and neural lineage patterning. Selective attenuation of FGF signals by localised ephrin/Eph signals accounts for this differential ERK activation, which controls the binary fate choice of each sibling cell pair. Here, we show that p120 Ras GTPase-activating protein (p120RasGAP) is a crucial mediator of these ephrin/Eph signals. First, inhibition of p120RasGAP has a similar effect to inhibition of ephrin/Eph function during marginal zone and neural patterning. Second, p120RasGAP acts epistatically to ephrin/Eph signals. Third, p120RasGAP physically associates with Eph3 in an ephrin-dependent manner. This study provides the first in vivo evidence that the functional association between Eph and RasGAP controls the spatial extent of FGF-activated ERK.
Asunto(s)
Diferenciación Celular/fisiología , Ciona intestinalis/embriología , Sistema de Señalización de MAP Quinasas/fisiología , Proteína Activadora de GTPasa p120/metabolismo , Animales , Western Blotting , Linaje de la Célula , Cartilla de ADN/genética , Electroporación , Embrión no Mamífero/metabolismo , Factores de Crecimiento de Fibroblastos/metabolismo , Inmunohistoquímica , Hibridación in SituRESUMEN
How does a fertilized egg decode its own genome to eventually develop into a mature animal? Each developing cell must activate a battery of genes in a timely manner and according to the function it will ultimately perform, but how? During development of the notochord--a structure akin to the vertebrate spine--in a simple marine invertebrate, an essential protein called Brachyury binds to specific sites in its target genes. A study just published in PLOS Biology reports that if the target gene contains multiple Brachyury-binding sites it will be activated early in development but if it contains only one site it will be activated later. Genes that contain no binding site can still be activated by Brachyury, but only indirectly by an earlier Brachyury-dependent gene product, so later than the directly activated genes. Thus, this study shows how several genes can interpret the presence of a single factor differently to become active at distinct times in development.