RESUMO
The study of cellular and developmental processes in physiologically relevant three-dimensional (3D) systems facilitates an understanding of mechanisms underlying cell fate, disease and injury. While cutting-edge microscopy technologies permit the routine acquisition of 3D datasets, there is currently a limited number of open-source software packages to analyse such images. Here, we describe General Image Analysis of Nuclei-based Images (GIANI; https://djpbarry.github.io/Giani), new software for the analysis of 3D images. The design primarily facilitates segmentation of nuclei and cells, followed by quantification of morphology and protein expression. GIANI enables routine and reproducible batch-processing of large numbers of images, and comes with scripting and command line tools. We demonstrate the utility of GIANI by quantifying cell morphology and protein expression in confocal images of mouse early embryos and by segmenting nuclei from light-sheet microscopy images of the flour beetle embryo. We also validate the performance of the software using simulated data. More generally, we anticipate that GIANI will be a useful tool for researchers in a variety of biomedical fields.
Assuntos
Imageamento Tridimensional , Microscopia , Algoritmos , Animais , Núcleo Celular , Processamento de Imagem Assistida por Computador/métodos , Imageamento Tridimensional/métodos , Camundongos , SoftwareRESUMO
BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers mainly due to spatial obstacles to complete resection, early metastasis and therapy resistance. The molecular events accompanying PDAC progression remain poorly understood. SOX9 is required for maintaining the pancreatic ductal identity and it is involved in the initiation of pancreatic cancer. In addition, SOX9 is a transcription factor linked to stem cell activity and is commonly overexpressed in solid cancers. It cooperates with Snail/Slug to induce epithelial-mesenchymal transition (EMT) during neural development and in diseases such as organ fibrosis or different types of cancer. METHODS: We investigated the roles of SOX9 in pancreatic tumor cell plasticity, metastatic dissemination and chemoresistance using pancreatic cancer cell lines as well as mouse embryo fibroblasts. In addition, we characterized the clinical relevance of SOX9 in pancreatic cancer using human biopsies. RESULTS: Gain- and loss-of-function of SOX9 in PDAC cells revealed that high levels of SOX9 increased migration and invasion, and promoted EMT and metastatic dissemination, whilst SOX9 silencing resulted in metastasis inhibition, along with a phenotypic reversion to epithelial features and loss of stemness potential. In both contexts, EMT factors were not altered. Moreover, high levels of SOX9 promoted resistance to gemcitabine. In contrast, overexpression of SOX9 was sufficient to promote metastatic potential in K-Ras transformed MEFs, triggering EMT associated with Snail/Slug activity. In clinical samples, SOX9 expression was analyzed in 198 PDAC cases by immunohistochemistry and in 53 patient derived xenografts (PDXs). SOX9 was overexpressed in primary adenocarcinomas and particularly in metastases. Notably, SOX9 expression correlated with high vimentin and low E-cadherin expression. CONCLUSIONS: Our results indicate that SOX9 facilitates PDAC progression and metastasis by triggering stemness and EMT.
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We dissect genetically a gene regulatory network that involves the transcription factors Tbx4, Pitx1 and Isl1 acting cooperatively to establish the hindlimb bud, and identify key differences in the pathways that initiate formation of the hindlimb and forelimb. Using live image analysis of murine limb mesenchyme cells undergoing chondrogenesis in micromass culture, we distinguish a series of changes in cellular behaviours and cohesiveness that are required for chondrogenic precursors to undergo differentiation. Furthermore, we provide evidence that the proximal hindlimb defects observed in Tbx4 mutant mice result from a failure in the early differentiation step of chondroprogenitors into chondrocytes, providing an explanation for the origins of proximally biased limb defects.
Assuntos
Membro Posterior/anormalidades , Botões de Extremidades/metabolismo , Proteínas com Domínio T/metabolismo , Animais , Células Cultivadas , Condrócitos/citologia , Condrócitos/metabolismo , Condrogênese , Proteínas com Homeodomínio LIM/genética , Proteínas com Homeodomínio LIM/metabolismo , Botões de Extremidades/citologia , Botões de Extremidades/crescimento & desenvolvimento , Células-Tronco Mesenquimais/metabolismo , Camundongos , Fatores de Transcrição Box Pareados/genética , Fatores de Transcrição Box Pareados/metabolismo , Proteínas com Domínio T/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
The heart develops from 2 sources of mesoderm progenitors, the first and second heart field (FHF and SHF). Using a single-cell transcriptomic assay combined with genetic lineage tracing and live imaging, we find the FHF and SHF are subdivided into distinct pools of progenitors in gastrulating mouse embryos at earlier stages than previously thought. Each subpopulation has a distinct origin in the primitive streak. The first progenitors to leave the primitive streak contribute to the left ventricle, shortly after right ventricle progenitor emigrate, followed by the outflow tract and atrial progenitors. Moreover, a subset of atrial progenitors are gradually incorporated in posterior locations of the FHF. Although cells allocated to the outflow tract and atrium leave the primitive streak at a similar stage, they arise from different regions. Outflow tract cells originate from distal locations in the primitive streak while atrial progenitors are positioned more proximally. Moreover, single-cell RNA sequencing demonstrates that the primitive streak cells contributing to the ventricles have a distinct molecular signature from those forming the outflow tract and atrium. We conclude that cardiac progenitors are prepatterned within the primitive streak and this prefigures their allocation to distinct anatomical structures of the heart. Together, our data provide a new molecular and spatial map of mammalian cardiac progenitors that will support future studies of heart development, function, and disease.
Assuntos
Linhagem da Célula/genética , Coração/embriologia , Linha Primitiva/embriologia , Animais , Linhagem da Célula/fisiologia , Feminino , Gástrula , Expressão Gênica/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Coração/fisiologia , Átrios do Coração/embriologia , Ventrículos do Coração/embriologia , Masculino , Mesoderma , Camundongos , Camundongos Endogâmicos C57BL , Morfogênese , Linha Primitiva/fisiologia , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodosRESUMO
Although the factors regulating muscle cell differentiation are well described, we know very little about how differentiating muscle fibers are organized into individual muscle tissue bundles. Disruption of these processes leads to muscle hypoplasia or dysplasia, and replicating these events is vital in tissue engineering approaches. We describe the progressive cellular events that orchestrate the formation of individual limb muscle bundles and directly demonstrate the role of the connective tissue cells that surround muscle precursors in controlling these events. We show how disruption of gene activity within or genetic ablation of connective tissue cells impacts muscle precursors causing disruption of muscle bundle formation and subsequent muscle dysplasia and hypoplasia. We identify several markers of the populations of connective tissue cells that surround muscle precursors and provide a model for how matrix-modifying proteoglycans secreted by these cells may influence muscle bundle formation by effects on the local extracellular matrix (ECM) environment.
Assuntos
Células do Tecido Conjuntivo/citologia , Extremidades/fisiologia , Desenvolvimento Muscular , Músculo Esquelético/fisiologia , Animais , Padronização Corporal , Agregação Celular , Deleção de Genes , Integrases/metabolismo , Camundongos Transgênicos , Morfogênese , Células Musculares/citologia , Fibras Musculares Esqueléticas/citologia , Proteínas com Domínio T/metabolismo , Tendões/citologia , Fatores de Transcrição/metabolismoRESUMO
Perturbation of CaMKIIß expression has been associated with multiple neuropsychiatric diseases, highlighting CaMKIIß as a gene of interest. Yet, in contrast to CaMKIIα, the specific functions of CaMKIIß in the brain remain poorly explored. Here, we reveal a novel function for this CaMKII isoform in vivo during neuronal development. By using in utero electroporation, we show that CaMKIIß is an important regulator of radial migration of projection neurons during cerebral cortex development. Knockdown of CaMKIIß causes accelerated migration of nascent pyramidal neurons, whereas overexpression of CaMKIIß inhibits migration, demonstrating that precise regulation of CaMKIIß expression is required for correct neuronal migration. More precisely, CaMKIIß controls the multipolar-bipolar transition in the intermediate zone and locomotion in the cortical plate through its actin-binding and -bundling activities. In addition, our data indicate that a fine-tuned balance between CaMKIIß and cofilin activities is necessary to ensure proper migration of cortical neurons. Thus, our findings define a novel isoform-specific function for CaMKIIß, demonstrating that CaMKIIß has a major biological function in the developing brain.
Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/fisiologia , Movimento Celular/fisiologia , Córtex Cerebral/fisiologia , Neurogênese/fisiologia , Animais , Encéfalo/embriologia , Encéfalo/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Córtex Cerebral/metabolismo , Embrião de Mamíferos/fisiologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento/genética , Camundongos , Proteínas dos Microfilamentos/genética , Transtornos do Neurodesenvolvimento/genética , Neurogênese/genética , Neurônios/metabolismo , Cultura Primária de Células , Isoformas de Proteínas/metabolismo , Células Piramidais/metabolismoRESUMO
The enteric nervous system (ENS) is essential for digestive function and gut homeostasis. Here we show that the amorphous neuroglia networks of the mouse ENS are composed of overlapping clonal units founded by postmigratory neural crest-derived progenitors. The spatial configuration of ENS clones depends on proliferation-driven local interactions of ENS progenitors with lineally unrelated neuroectodermal cells, the ordered colonization of the serosa-mucosa axis by clonal descendants, and gut expansion. Single-cell transcriptomics and mutagenesis analysis delineated dynamic molecular states of ENS progenitors and identified RET as a regulator of neurogenic commitment. Clonally related enteric neurons exhibit synchronous activity in response to network stimulation. Thus, lineage relationships underpin the organization of the peripheral nervous system.
Assuntos
Linhagem da Célula , Sistema Nervoso Entérico/citologia , Animais , Linhagem da Célula/genética , Proliferação de Células , Células Clonais/citologia , Sistema Nervoso Entérico/metabolismo , Mucosa Intestinal/citologia , Camundongos , Mosaicismo , Mutagênese , Crista Neural/citologia , Neurogênese , Neuroglia/fisiologia , Neurônios/citologia , Análise de Sequência de RNA , Transdução de Sinais , Análise de Célula Única , Células-Tronco/citologia , TranscriptomaRESUMO
The hostile environment of the microscope stage poses numerous challenges to successful imaging of morphogenesis in live tissues. This review aims to highlight some of the main practical considerations to take into account when embarking on a project to image cell behaviour in the context of cells' normal surroundings. Scrutiny of these activities is likely to be the most informative approach to understanding mechanical morphogenesis but is often confounded by the substantial technical difficulties involved in imaging samples over extended periods of time. Repeated observation of cells in live tissue requires that strategies be adopted to prioritize the stability of the sample, ensuring that it remains viable and develops normally while being held in a manner accessible to microscopic examination. Key considerations when creating reliable protocols for time-lapse imaging may be broken down into three main criteria; labelling, mounting and image acquisition. Choices and compromises made here, however, will directly influence image quality, and even small refinements can substantially improve what information may be extracted from images. Live imaging of tissue is difficult but paying close attention to the basics along with a little innovation is likely to be well rewarded.This article is part of the themed issue 'Systems morphodynamics: understanding the development of tissue hardware'.
Assuntos
Microscopia/métodos , Morfogênese , Imagem com Lapso de Tempo/métodos , Microscopia/instrumentação , Imagem com Lapso de Tempo/instrumentaçãoRESUMO
Hirschsprung disease (HSCR) is characterized by absence of enteric neurons from the distal colon and severe intestinal dysmotility. To understand the pathophysiology and genetics of HSCR we developed a unique zebrafish model that allows combined genetic, developmental and in vivo physiological studies. We show that ret mutant zebrafish exhibit cellular, physiological and genetic features of HSCR, including absence of intestinal neurons, reduced peristalsis, and varying phenotype expressivity in the heterozygous state. We perform live imaging experiments using a UAS-GAL4 binary genetic system to drive fluorescent protein expression in ENS progenitors. We demonstrate that ENS progenitors migrate at reduced speed in ret heterozygous embryos, without changes in proliferation or survival, establishing this as a principal pathogenic mechanism for distal aganglionosis. We show, using live imaging of actual intestinal movements, that intestinal motility is severely compromised in ret mutants, and partially impaired in ret heterozygous larvae, and establish a clear correlation between neuron position and organised intestinal motility. We exploited the partially penetrant ret heterozygous phenotype as a sensitised background to test the influence of a candidate modifier gene. We generated mapk10 loss-of-function mutants, which show reduced numbers of enteric neurons. Significantly, we show that introduction of mapk10 mutations into ret heterozygotes enhanced the ENS deficit, supporting MAPK10 as a HSCR susceptibility locus. Our studies demonstrate that ret heterozygous zebrafish is a sensitized model, with many significant advantages over existing murine models, to explore the pathophysiology and complex genetics of HSCR.
Assuntos
Sistema Nervoso Entérico/metabolismo , Doença de Hirschsprung/genética , Proteína Quinase 10 Ativada por Mitógeno/genética , Proteínas Proto-Oncogênicas c-ret/genética , Peixe-Zebra/genética , Animais , Colo/inervação , Colo/patologia , Modelos Animais de Doenças , Sistema Nervoso Entérico/patologia , Doença de Hirschsprung/metabolismo , Doença de Hirschsprung/patologia , Humanos , Mutação , Neurônios/metabolismo , Neurônios/patologia , Fenótipo , Proteínas Proto-Oncogênicas c-ret/metabolismoRESUMO
Malaria is caused by an obligate intracellular protozoan parasite that replicates within and destroys erythrocytes. Asexual blood stages of the causative agent of the most virulent form of human malaria, Plasmodium falciparum, can be cultivated indefinitely in vitro in human erythrocytes, facilitating experimental analysis of parasite cell biology, biochemistry and genetics. However, efforts to improve understanding of the basic biology of this important pathogen and to develop urgently required new antimalarial drugs and vaccines, suffer from a paucity of basic research tools. This includes a simple means of quantifying the effects of drugs, antibodies and gene modifications on parasite fitness and replication rates. Here we describe the development and validation of an extremely simple, robust plaque assay that can be used to visualise parasite replication and resulting host erythrocyte destruction at the level of clonal parasite populations. We demonstrate applications of the plaque assay by using it for the phenotypic characterisation of two P. falciparum conditional mutants displaying reduced fitness in vitro.
Assuntos
Técnica de Placa Hemolítica/métodos , Malária Falciparum/parasitologia , Parasitos/isolamento & purificação , Plasmodium falciparum/isolamento & purificação , Animais , Eritrócitos/parasitologia , Humanos , Estágios do Ciclo de Vida , Proteína 1 de Superfície de Merozoito/metabolismo , Mutação/genética , Fenótipo , Plasmodium falciparum/crescimento & desenvolvimentoRESUMO
The proneural factor Ascl1 controls multiple steps of neurogenesis in the embryonic brain, including progenitor division and neuronal migration. Here we show that Cenpj, also known as CPAP, a microcephaly gene, is a transcriptional target of Ascl1 in the embryonic cerebral cortex. We have characterized the role of Cenpj during cortical development by in utero electroporation knockdown and found that silencing Cenpj in the ventricular zone disrupts centrosome biogenesis and randomizes the cleavage plane orientation of radial glia progenitors. Moreover, we show that downregulation of Cenpj in post-mitotic neurons increases stable microtubules and leads to slower neuronal migration, abnormal centrosome position and aberrant neuronal morphology. Moreover, rescue experiments shows that Cenpj mediates the role of Ascl1 in centrosome biogenesis in progenitor cells and in microtubule dynamics in migrating neurons. These data provide insights into genetic pathways controlling cortical development and primary microcephaly observed in humans with mutations in Cenpj.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Córtex Cerebral/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Células-Tronco Neurais/metabolismo , Neurogênese/genética , Neurônios/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Divisão Celular , Movimento Celular , Centrossomo/metabolismo , Centrossomo/ultraestrutura , Córtex Cerebral/citologia , Eletroporação , Embrião de Mamíferos , Regulação da Expressão Gênica no Desenvolvimento , Injeções Intraventriculares , Camundongos , Camundongos Transgênicos , Microtomia , Proteínas Associadas aos Microtúbulos/antagonistas & inibidores , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Células-Tronco Neurais/ultraestrutura , Neurônios/ultraestrutura , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Técnicas de Cultura de TecidosRESUMO
Down Syndrome (DS) is a highly prevalent developmental disorder, affecting 1/700 births. Intellectual disability, which affects learning and memory, is present in all cases and is reflected by below average IQ. We sought to determine whether defective morphology and connectivity in neurons of the cerebral cortex may underlie the cognitive deficits that have been described in two mouse models of DS, the Tc1 and Ts1Rhr mouse lines. We utilised in utero electroporation to label a cohort of future upper layer projection neurons in the cerebral cortex of developing mouse embryos with GFP, and then examined neuronal positioning and morphology in early adulthood, which revealed no alterations in cortical layer position or morphology in either Tc1 or Ts1Rhr mouse cortex. The number of dendrites, as well as dendrite length and branching was normal in both DS models, compared with wildtype controls. The sites of projection neuron synaptic inputs, dendritic spines, were analysed in Tc1 and Ts1Rhr cortex at three weeks and three months after birth, and significant changes in spine morphology were observed in both mouse lines. Ts1Rhr mice had significantly fewer thin spines at three weeks of age. At three months of age Tc1 mice had significantly fewer mushroom spines--the morphology associated with established synaptic inputs and learning and memory. The decrease in mushroom spines was accompanied by a significant increase in the number of stubby spines. This data suggests that dendritic spine abnormalities may be a more important contributor to cognitive deficits in DS models, rather than overall neuronal architecture defects.
Assuntos
Córtex Cerebral/patologia , Espinhas Dendríticas/patologia , Síndrome de Down/patologia , Animais , Espinhas Dendríticas/metabolismo , Modelos Animais de Doenças , Feminino , Proteínas de Fluorescência Verde/metabolismo , Camundongos , FenótipoRESUMO
In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system (1-5). To date this tool has been widely used to study the regulation of cellular proliferation, differentiation and neuronal migration especially in the developing cerebral cortex (6-8). Here we detail our protocol to electroporate in utero the cerebral cortex and the hippocampus and provide evidence that this approach can be used to study dendrites and spines in these two cerebral regions. Visualization and manipulation of neurons in primary cultures have contributed to a better understanding of the processes involved in dendrite, spine and synapse development. However neurons growing in vitro are not exposed to all the physiological cues that can affect dendrite and/or spine formation and maintenance during normal development. Our knowledge of dendrite and spine structures in vivo in wild-type or mutant mice comes mostly from observations using the Golgi-Cox method( 9). However, Golgi staining is considered to be unpredictable. Indeed, groups of nerve cells and fiber tracts are labeled randomly, with particular areas often appearing completely stained while adjacent areas are devoid of staining. Recent studies have shown that IUE of fluorescent constructs represents an attractive alternative method to study dendrites, spines as well as synapses in mutant / wild-type mice (10-11) (Figure 1A). Moreover in comparison to the generation of mouse knockouts, IUE represents a rapid approach to perform gain and loss of function studies in specific population of cells during a specific time window. In addition, IUE has been successfully used with inducible gene expression or inducible RNAi approaches to refine the temporal control over the expression of a gene or shRNA (12). These advantages of IUE have thus opened new dimensions to study the effect of gene expression/suppression on dendrites and spines not only in specific cerebral structures (Figure 1B) but also at a specific time point of development (Figure 1C). Finally, IUE provides a useful tool to identify functional interactions between genes involved in dendrite, spine and/or synapse development. Indeed, in contrast to other gene transfer methods such as virus, it is straightforward to combine multiple RNAi or transgenes in the same population of cells. In summary, IUE is a powerful method that has already contributed to the characterization of molecular mechanisms underlying brain function and disease and it should also be useful in the study of dendrites and spines.
Assuntos
Córtex Cerebral/fisiologia , Dendritos/fisiologia , Eletroporação/métodos , Hipocampo/fisiologia , Animais , Córtex Cerebral/química , Córtex Cerebral/ultraestrutura , Dendritos/genética , Dendritos/ultraestrutura , Embrião de Mamíferos , Hipocampo/química , Hipocampo/ultraestrutura , CamundongosRESUMO
The plaque assay is a standard technique for measuring influenza virus infectivity and inhibition of virus replication. Counting plaque numbers and quantifying virus infection of cells in multiwell plates quickly, accurately and automatically remain a challenge. Visual inspection relies upon experience, is subjective, often time consuming, and has less reproducibility than automated methods. In this paper, a simple, high throughput imaging-based alternative is proposed which uses a flatbed scanner and image processing software to quantify the infected cell population and plaque formation. Quantitation results were evaluated with reference to visual counting and achieved better than 80% agreement. The method was shown to be particularly advantageous in titration of the number of plaques and infected cells when influenza viruses produce a heterogeneous population of small plaques. It was also shown to be insensitive to the densities of plaques in determination of neutralization titres and IC(50)s of drug susceptibility. In comparison to other available techniques, this approach is cost-effective, relatively accurate, and readily available.
Assuntos
Ensaios de Triagem em Larga Escala/métodos , Processamento de Imagem Assistida por Computador/métodos , Ensaio de Placa Viral/métodos , Virologia/métodos , Animais , Humanos , Orthomyxoviridae/isolamento & purificaçãoRESUMO
Little is known of the intracellular machinery that controls the motility of newborn neurons. We have previously shown that the proneural protein Neurog2 promotes the migration of nascent cortical neurons by inducing the expression of the atypical Rho GTPase Rnd2. Here, we show that another proneural factor, Ascl1, promotes neuronal migration in the cortex through direct regulation of a second Rnd family member, Rnd3. Both Rnd2 and Rnd3 promote neuronal migration by inhibiting RhoA signaling, but they control distinct steps of the migratory process, multipolar to bipolar transition in the intermediate zone and locomotion in the cortical plate, respectively. Interestingly, these divergent functions directly result from the distinct subcellular distributions of the two Rnd proteins. Because Rnd proteins also regulate progenitor divisions and neurite outgrowth, we propose that proneural factors, through spatiotemporal regulation of Rnd proteins, integrate the process of neuronal migration with other events in the neurogenic program.
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Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Movimento Celular/fisiologia , Córtex Cerebral/metabolismo , Neurônios/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Proteína rhoA de Ligação ao GTP/antagonistas & inibidores , Análise de Variância , Animais , Western Blotting , Contagem de Células , Córtex Cerebral/citologia , Transferência Ressonante de Energia de Fluorescência , Imuno-Histoquímica , Hibridização In Situ , Camundongos , Neurônios/fisiologia , Interferência de RNA , Transdução de Sinais/fisiologiaRESUMO
The classical view of neural plate development held that it arises from the ectoderm, after its separation from the mesodermal and endodermal lineages. However, recent cell-lineage-tracing experiments indicate that the caudal neural plate and paraxial mesoderm are generated from common bipotential axial stem cells originating from the caudal lateral epiblast. Tbx6 null mutant mouse embryos which produce ectopic neural tubes at the expense of paraxial mesoderm must provide a clue to the regulatory mechanism underlying this neural versus mesodermal fate choice. Here we demonstrate that Tbx6-dependent regulation of Sox2 determines the fate of axial stem cells. In wild-type embryos, enhancer N1 of the neural primordial gene Sox2 is activated in the caudal lateral epiblast, and the cells staying in the superficial layer sustain N1 activity and activate Sox2 expression in the neural plate. In contrast, the cells destined to become mesoderm activate Tbx6 and turn off enhancer N1 before migrating into the paraxial mesoderm compartment. In Tbx6 mutant embryos, however, enhancer N1 activity persists in the paraxial mesoderm compartment, eliciting ectopic Sox2 activation and transforming the paraxial mesoderm into neural tubes. An enhancer-N1-specific deletion mutation introduced into Tbx6 mutant embryos prevented this Sox2 activation in the mesodermal compartment and subsequent development of ectopic neural tubes, indicating that Tbx6 regulates Sox2 via enhancer N1. Tbx6-dependent repression of Wnt3a in the paraxial mesodermal compartment is implicated in this regulatory process. Paraxial mesoderm-specific misexpression of a Sox2 transgene in wild-type embryos resulted in ectopic neural tube development. Thus, Tbx6 represses Sox2 by inactivating enhancer N1 to inhibit neural development, and this is an essential step for the specification of paraxial mesoderm from the axial stem cells.
Assuntos
Linhagem da Célula , Mesoderma/citologia , Células-Tronco Neurais/citologia , Tubo Neural/citologia , Fatores de Transcrição SOXB1/metabolismo , Células-Tronco/citologia , Fatores de Transcrição/metabolismo , Animais , Animais Geneticamente Modificados , Sequência de Bases , Coristoma/embriologia , Coristoma/metabolismo , Embrião de Mamíferos/citologia , Embrião de Mamíferos/embriologia , Embrião de Mamíferos/metabolismo , Elementos Facilitadores Genéticos/genética , Regulação da Expressão Gênica no Desenvolvimento , Mesoderma/embriologia , Mesoderma/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Dados de Sequência Molecular , Placa Neural/citologia , Placa Neural/embriologia , Placa Neural/metabolismo , Tubo Neural/embriologia , Tubo Neural/metabolismo , Fatores de Transcrição SOXB1/genética , Proteínas com Domínio T , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética , Proteínas Wnt/antagonistas & inibidores , Proteínas Wnt/metabolismo , Proteína Wnt3 , Proteína Wnt3ARESUMO
To facilitate studies of neural network architecture and formation, we generated three Drosophila melanogaster variants of the mouse Brainbow-2 system, called Flybow. Sequences encoding different membrane-tethered fluorescent proteins were arranged in pairs within cassettes flanked by recombination sites. Flybow combines the Gal4-upstream activating sequence binary system to regulate transgene expression and an inducible modified Flp-FRT system to drive inversions and excisions of cassettes. This provides spatial and temporal control over the stochastic expression of one of two or four reporters within one sample. Using the visual system, the embryonic nervous system and the wing imaginal disc, we show that Flybow in conjunction with specific Gal4 drivers can be used to visualize cell morphology with high resolution. Finally, we demonstrate that this labeling approach is compatible with available Flp-FRT-based techniques, such as mosaic analysis with a repressible cell marker; this could further support the genetic analysis of neural circuit assembly and function.
Assuntos
Drosophila melanogaster/citologia , Proteínas Luminescentes/análise , Rede Nervosa/citologia , Neurônios/citologia , Coloração e Rotulagem/métodos , Animais , Sequência de Bases , Membrana Celular/química , Membrana Celular/metabolismo , Proteínas de Ligação a DNA/genética , Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Proteínas Luminescentes/genética , Camundongos , Dados de Sequência Molecular , Rede Nervosa/embriologia , Neuroglia/química , Neuroglia/citologia , Neuroglia/metabolismo , Neurônios/química , Neurônios/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/genéticaRESUMO
BACKGROUND: Present technology uses mostly chimeric proteins as regulators and hormones or antibiotics as signals to induce spatial and temporal gene expression. METHODOLOGY/PRINCIPAL FINDINGS: Here, we show that a chromosomally integrated yeast 'Leu3p-alpha-IotaRhoMu' system constitutes a ligand-inducible regulatory "off-on" genetic switch with an extensively dynamic action area. We find that Leu3p acts as an active transcriptional repressor in the absence and as an activator in the presence of alpha-isopropylmalate (alpha-IotaRhoMu) in primary fibroblasts isolated from double transgenic mouse embryos bearing ubiquitously expressing Leu3p and a Leu3p regulated GFP reporter. In the absence of the branched amino acid biosynthetic pathway in animals, metabolically stable alpha-IPM presents an EC(50) equal to 0.8837 mM and fast "OFF-ON" kinetics (t(50)ON = 43 min, t(50)OFF = 2.18 h), it enters the cells via passive diffusion, while it is non-toxic to mammalian cells and to fertilized mouse eggs cultured ex vivo. CONCLUSIONS/SIGNIFICANCE: Our results demonstrate that the 'Leu3p-alpha-IotaRhoMu' constitutes a simpler and safer system for inducible gene expression in biomedical applications.
Assuntos
Cromossomos de Mamíferos/metabolismo , Engenharia Genética/métodos , Malatos/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Transativadores/genética , Transativadores/metabolismo , Animais , Sequência de Bases , Feminino , Fibroblastos/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Gravidez , Saccharomyces cerevisiae/genéticaRESUMO
Nuclear architecture and chromatin reorganization have recently been shown to orchestrate gene expression and act as key players in developmental pathways. To investigate how regulatory elements in the mouse CD8 gene locus are arranged in space and in relation to each other, three-dimensional fluorescence in situ hybridization and chromosome conformation capture techniques were employed to monitor the repositioning of the locus in relation to its subchromosomal territory and to identify long-range interactions between the different elements during development. Our data demonstrate that CD8 gene expression in murine lymphocytes is accompanied by the relocation of the locus outside its subchromosomal territory. Similar observations in the CD4 locus point to a rather general phenomenon during T cell development. Furthermore, we show that this relocation of the CD8 gene locus is associated with a clustering of regulatory elements forming a tight active chromatin hub in CD8-expressing cells. In contrast, in nonexpressing cells, the gene remains close to the main body of its chromosomal domain and the regulatory elements appear not to interact with each other.