RESUMO
Developmental studies have revealed the importance of the transcription factor Hand2 in cardiac development. Hand2 promotes cardiac progenitor differentiation and epithelial maturation, while repressing other tissue types. The mechanisms underlying the promotion of cardiac fates are far better understood than those underlying the repression of alternative fates. Here, we assess Hand2-dependent changes in gene expression and chromatin remodeling in cardiac progenitors of zebrafish embryos. Cell-type specific transcriptome analysis shows a dual function for Hand2 in activation of cardiac differentiation genes and repression of pronephric pathways. We identify functional cis- regulatory elements whose chromatin accessibility are increased in hand2 mutant cells. These regulatory elements associate with non-cardiac gene expression, and drive reporter gene expression in tissues associated with Hand2-repressed genes. We find that functional Hand2 is sufficient to reduce non-cardiac reporter expression in cardiac lineages. Taken together, our data support a model of Hand2-dependent coordination of transcriptional programs, not only through transcriptional activation of cardiac and epithelial maturation genes, but also through repressive chromatin remodeling at the DNA regulatory elements of non-cardiac genes.
RESUMO
The expansion of fluorescence bioimaging toward more complex systems and geometries requires analytical tools capable of spanning widely varying timescales and length scales, cleanly separating multiple fluorescent labels and distinguishing these labels from background autofluorescence. Here we meet these challenging objectives for multispectral fluorescence microscopy, combining hyperspectral phasors and linear unmixing to create Hybrid Unmixing (HyU). HyU is efficient and robust, capable of quantitative signal separation even at low illumination levels. In dynamic imaging of developing zebrafish embryos and in mouse tissue, HyU was able to cleanly and efficiently unmix multiple fluorescent labels, even in demanding volumetric timelapse imaging settings. HyU permits high dynamic range imaging, allowing simultaneous imaging of bright exogenous labels and dim endogenous labels. This enables coincident studies of tagged components, cellular behaviors and cellular metabolism within the same specimen, providing more accurate insights into the orchestrated complexity of biological systems.
Assuntos
Peixe-Zebra , Animais , Camundongos , Microscopia de Fluorescência/métodosRESUMO
Visualizing cell shapes and interactions of differentiating cells is instrumental for understanding organ development and repair. Across species, strategies for stochastic multicolour labelling have greatly facilitated in vivo cell tracking and mapping neuronal connectivity. Yet integrating multi-fluorophore information into the context of developing zebrafish tissues is challenging given their cytoplasmic localization and spectral incompatibility with common fluorescent markers. Inspired by Drosophila Raeppli, we developed FRaeppli (Fish-Raeppli) by expressing bright membrane- or nuclear-targeted fluorescent proteins for efficient cell shape analysis and tracking. High spatiotemporal activation flexibility is provided by the Gal4/UAS system together with Cre/lox and/or PhiC31 integrase. The distinct spectra of the FRaeppli fluorescent proteins allow simultaneous imaging with GFP and infrared subcellular reporters or tissue landmarks. We demonstrate the suitability of FRaeppli for live imaging of complex internal organs, such as the liver, and have tailored hyperspectral protocols for time-efficient acquisition. Combining FRaeppli with polarity markers revealed previously unknown canalicular topologies between differentiating hepatocytes, reminiscent of the mammalian liver, suggesting common developmental mechanisms. The multispectral FRaeppli toolbox thus enables the comprehensive analysis of intricate cellular morphologies, topologies and lineages at single-cell resolution in zebrafish.
Assuntos
Integrases , Peixe-Zebra , Animais , Animais Geneticamente Modificados , Proteínas de Fluorescência Verde/metabolismo , Integrases/metabolismo , Mamíferos/metabolismo , Neurônios/metabolismo , Peixe-Zebra/metabolismoRESUMO
Hyperspectral fluorescence imaging is gaining popularity for it enables multiplexing of spatio-temporal dynamics across scales for molecules, cells and tissues with multiple fluorescent labels. This is made possible by adding the dimension of wavelength to the dataset. The resulting datasets are high in information density and often require lengthy analyses to separate the overlapping fluorescent spectra. Understanding and visualizing these large multi-dimensional datasets during acquisition and pre-processing can be challenging. Here we present Spectrally Encoded Enhanced Representations (SEER), an approach for improved and computationally efficient simultaneous color visualization of multiple spectral components of hyperspectral fluorescence images. Exploiting the mathematical properties of the phasor method, we transform the wavelength space into information-rich color maps for RGB display visualization. We present multiple biological fluorescent samples and highlight SEER's enhancement of specific and subtle spectral differences, providing a fast, intuitive and mathematical way to interpret hyperspectral images during collection, pre-processing and analysis.
Assuntos
Processamento de Imagem Assistida por Computador/métodos , Espectrometria de Fluorescência/métodos , Algoritmos , Animais , Animais Geneticamente Modificados , Cor , Embrião não Mamífero , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Processamento de Imagem Assistida por Computador/normas , Camundongos Endogâmicos C57BL , Microscopia Confocal/métodos , Razão Sinal-Ruído , Peixe-Zebra/embriologia , Peixe-Zebra/genéticaRESUMO
Canonical roles for macrophages in mediating the fibrotic response after a heart attack include extracellular matrix turnover and activation of cardiac fibroblasts to initiate collagen deposition. Here we reveal that macrophages directly contribute collagen to the forming post-injury scar. Unbiased transcriptomics shows an upregulation of collagens in both zebrafish and mouse macrophages following heart injury. Adoptive transfer of macrophages, from either collagen-tagged zebrafish or adult mouse GFPtpz-collagen donors, enhances scar formation via cell autonomous production of collagen. In zebrafish, the majority of tagged collagen localises proximal to the injury, within the overlying epicardial region, suggesting a possible distinction between macrophage-deposited collagen and that predominantly laid-down by myofibroblasts. Macrophage-specific targeting of col4a3bpa and cognate col4a1 in zebrafish significantly reduces scarring in cryoinjured hosts. Our findings contrast with the current model of scarring, whereby collagen deposition is exclusively attributed to myofibroblasts, and implicate macrophages as direct contributors to fibrosis during heart repair.
Assuntos
Cicatriz/metabolismo , Cicatriz/patologia , Colágeno/metabolismo , Coração/fisiopatologia , Macrófagos/patologia , Cicatrização , Peixe-Zebra/fisiologia , Transferência Adotiva , Animais , Embrião de Mamíferos/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Regulação da Expressão Gênica , Proteínas de Fluorescência Verde/metabolismo , Macrófagos/metabolismo , Camundongos , Monócitos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Baço/patologia , Transcrição Gênica , Transcriptoma/genética , Peixe-Zebra/embriologia , Proteínas de Peixe-Zebra/metabolismoRESUMO
Interrogation of gene regulatory circuits in complex organisms requires precise and robust methods to label cell-types for profiling of target proteins in a tissue-specific fashion as well as data analysis to understand interconnections within the circuits. There are several strategies for obtaining cell-type and subcellular specific genome-wide data. We have developed a methodology, termed "biotagging" that uses tissue-specific, genetically encoded components to biotinylate target proteins, enabling in depth genome-wide profiling in zebrafish. We have refined protocols to use the biotagging approach that led to enhanced isolation of coding and non-coding RNAs from ribosomes and nuclei of genetically defined cell-types. The ability to study both the actively translated and transcribed transcriptome in the same cell population, coupled to genomic accessibility assays has enabled the study of cell-type specific gene regulatory circuits in zebrafish due to the high signal-to-noise achieved via its stringent purification protocol. Here, we provide detailed methods to isolate, profile and analyze cell-type specific polyribosome and nuclear transcriptome in zebrafish.
Assuntos
Biotinilação/métodos , Perfilação da Expressão Gênica/métodos , Coloração e Rotulagem/métodos , Peixe-Zebra/genética , Animais , Fracionamento Celular , Redes Reguladoras de Genes/genética , Polirribossomos/genética , Polirribossomos/metabolismo , RNA/isolamento & purificação , RNA/metabolismo , Transcriptoma/genética , Peixe-Zebra/metabolismoRESUMO
The essential organization of microtubules within neurons has been described; however, less is known about how neuronal actin is arranged and the functional implications of its arrangement. Here, we describe, in live cells, an actin-based structure in the proximal axon that selectively prevents some proteins from entering the axon while allowing the passage of others. Concentrated patches of actin in proximal axons are present shortly after axonal specification in rat and zebrafish neurons imaged live, and they mark positions where anterogradely traveling vesicles carrying dendritic proteins halt and reverse. Patches colocalize with the ARP2/3 complex, and when ARP2/3-mediated nucleation is blocked, a dendritic protein mislocalizes to the axon. Patches are highly dynamic, with few persisting longer than 30 min. In neurons in culture and in vivo, actin appears to form a contiguous, semipermeable barrier, despite its apparently sparse distribution, preventing axonal localization of constitutively active myosin Va but not myosin VI.
Assuntos
Actinas/metabolismo , Neurônios/metabolismo , Citoesqueleto de Actina/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Animais , Axônios/metabolismo , Sobrevivência Celular/fisiologia , Dendritos/metabolismo , Microtúbulos/metabolismo , Miosinas/metabolismo , RatosRESUMO
Branching morphogenesis underlies organogenesis in vertebrates and invertebrates, yet is incompletely understood. Here, we show that the sarco-endoplasmic reticulum Ca2+ reuptake pump (SERCA) directs budding across germ layers and species. Clonal knockdown demonstrated a cell-autonomous role for SERCA in Drosophila air sac budding. Live imaging of Drosophila tracheogenesis revealed elevated Ca2+ levels in migratory tip cells as they form branches. SERCA blockade abolished this Ca2+ differential, aborting both cell migration and new branching. Activating protein kinase C (PKC) rescued Ca2+ in tip cells and restored cell migration and branching. Likewise, inhibiting SERCA abolished mammalian epithelial budding, PKC activation rescued budding, while morphogens did not. Mesoderm (zebrafish angiogenesis) and ectoderm (Drosophila nervous system) behaved similarly, suggesting a conserved requirement for cell-autonomous Ca2+ signaling, established by SERCA, in iterative budding.
RESUMO
Interrogation of gene regulatory circuits in complex organisms requires precise tools for the selection of individual cell types and robust methods for biochemical profiling of target proteins. We have developed a versatile, tissue-specific binary in vivo biotinylation system in zebrafish termed biotagging that uses genetically encoded components to biotinylate target proteins, enabling in-depth genome-wide analyses of their molecular interactions. Using tissue-specific drivers and cell-compartment-specific effector lines, we demonstrate the specificity of the biotagging toolkit at the biochemical, cellular, and transcriptional levels. We use biotagging to characterize the in vivo transcriptional landscape of migratory neural crest and myocardial cells in different cellular compartments (ribosomes and nucleus). These analyses reveal a comprehensive network of coding and non-coding RNAs and cis-regulatory modules, demonstrating that tissue-specific identity is embedded in the nuclear transcriptomes. By eliminating background inherent to complex embryonic environments, biotagging allows analyses of molecular interactions at high resolution.
Assuntos
Crista Neural/crescimento & desenvolvimento , Fatores de Transcrição/biossíntese , Transcriptoma/genética , Peixe-Zebra/genética , Animais , Compartimento Celular/genética , Linhagem da Célula/genética , Sequência Conservada/genética , Regulação da Expressão Gênica no Desenvolvimento , Redes Reguladoras de Genes/genética , Especificidade de Órgãos/genética , Fatores de Transcrição/genética , Peixe-Zebra/crescimento & desenvolvimentoRESUMO
Optical coherence microscopy (OCM) has unique advantages of non-invasive 3D imaging without the need of exogenous labels for studying biological samples. However, the imaging depth of this technique is limited by the tradeoff between the depth of focus (DOF) and high lateral resolution in Gaussian optics. To overcome this limitation, we have developed an extended-focus OCM (xf-OCM) imaging system using quasi-Bessel beam illumination to extend the DOF to â¼100â µm, about 3-fold greater than standard OCM. High lateral resolution of 1.6â µm ensured detailed identification of structures within live animal samples. The insensitivity to spherical aberrations strengthened the capability of our xf-OCM system in 3D biological imaging.
Assuntos
Imageamento Tridimensional/métodos , Microscopia/métodos , Animais , Desenho de Equipamento , Microscopia/instrumentação , Controle de Qualidade , Peixe-ZebraRESUMO
Time-lapse imaging of multiple labels is challenging for biological imaging as noise, photobleaching and phototoxicity compromise signal quality, while throughput can be limited by processing time. Here, we report software called Hyper-Spectral Phasors (HySP) for denoising and unmixing multiple spectrally overlapping fluorophores in a low signal-to-noise regime with fast analysis. We show that HySP enables unmixing of seven signals in time-lapse imaging of living zebrafish embryos.
Assuntos
Software , Imagem com Lapso de Tempo/métodos , Animais , Animais Geneticamente Modificados , Cor , Embrião não Mamífero , Análise de Fourier , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Processamento de Imagem Assistida por Computador , Peixe-Zebra/embriologia , Peixe-Zebra/genéticaRESUMO
A phase variance optical coherence microscope (pvOCM) has been created to image blood flow in the microvasculature of zebrafish embryos, without the use of exogenous labels. The pvOCM imaging system has axial and lateral resolutions of 2.8 ?? ? m in tissue and imaging depth of more than 100 ?? ? m . Images of 2 to 5 days postfertilization zebrafish embryos identified the detailed anatomical structure based on OCM intensity contrast. Phase variance contrast offered visualization of blood flow in the arteries, veins, and capillaries. The pvOCM images of the vasculature were confirmed by direct comparisons with fluorescence microscopy images of transgenic embryos in which the vascular endothelium is labeled with green fluorescent protein. The ability of pvOCM to capture activities of regional blood flow permits it to reveal functional information that is of great utility for the study of vascular development.
Assuntos
Vasos Sanguíneos/diagnóstico por imagem , Processamento de Imagem Assistida por Computador/métodos , Tomografia de Coerência Óptica/métodos , Algoritmos , Animais , Vasos Sanguíneos/crescimento & desenvolvimento , Embrião não Mamífero/irrigação sanguínea , Embrião não Mamífero/diagnóstico por imagem , Desenho de Equipamento , Peixe-Zebra/crescimento & desenvolvimentoRESUMO
Mycobacterium tuberculosis infection in humans triggers formation of granulomas, which are tightly organized immune cell aggregates that are the central structure of tuberculosis. Infected and uninfected macrophages interdigitate, assuming an altered, flattened appearance. Although pathologists have described these changes for over a century, the molecular and cellular programs underlying this transition are unclear. Here, using the zebrafish-Mycobacterium marinum model, we found that mycobacterial granuloma formation is accompanied by macrophage induction of canonical epithelial molecules and structures. We identified fundamental macrophage reprogramming events that parallel E-cadherin-dependent mesenchymal-epithelial transitions. Macrophage-specific disruption of E-cadherin function resulted in disordered granuloma formation, enhanced immune cell access, decreased bacterial burden, and increased host survival, suggesting that the granuloma can also serve a bacteria-protective role. Granuloma macrophages in humans with tuberculosis were similarly transformed. Thus, during mycobacterial infection, granuloma macrophages are broadly reprogrammed by epithelial modules, and this reprogramming alters the trajectory of infection and the associated immune response.
Assuntos
Epitélio/imunologia , Macrófagos/imunologia , Mycobacterium marinum/imunologia , Animais , Caderinas/imunologia , Epitélio/microbiologia , Granuloma/imunologia , Granuloma/microbiologia , Macrófagos/microbiologia , Mycobacterium tuberculosis/imunologia , Peixe-ZebraRESUMO
Although neuronal activity can be modulated using a variety of techniques, there are currently few methods for controlling neuronal connectivity. We introduce a tool (GFE3) that mediates the fast, specific and reversible elimination of inhibitory synaptic inputs onto genetically determined neurons. GFE3 is a fusion between an E3 ligase, which mediates the ubiquitination and rapid degradation of proteins, and a recombinant, antibody-like protein (FingR) that binds to gephyrin. Expression of GFE3 leads to a strong and specific reduction of gephyrin in culture or in vivo and to a substantial decrease in phasic inhibition onto cells that express GFE3. By temporarily expressing GFE3 we showed that inhibitory synapses regrow following ablation. Thus, we have created a simple, reversible method for modulating inhibitory synaptic input onto genetically determined cells.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Membrana/metabolismo , Neurônios/metabolismo , Técnicas de Patch-Clamp/métodos , Sinapses/fisiologia , Transmissão Sináptica/fisiologia , Ubiquitina-Proteína Ligases/metabolismo , Animais , Células Cultivadas , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Feminino , Hipocampo , Masculino , Transtornos Motores/metabolismo , Transtornos Motores/patologia , Neurônios/citologia , Ratos , Ratos Sprague-Dawley , Coluna Vertebral/citologia , Coluna Vertebral/metabolismo , Ubiquitinação , Peixe-ZebraRESUMO
The development of the vertebrate head requires cell-cell and tissue-tissue interactions between derivatives of the three germ layers to coordinate morphogenetic movements in four dimensions (4D: x, y, z, t). The high spatial and temporal resolution offered by optical microscopy has made it the main imaging modularity for capturing the molecular and cellular dynamics of developmental processes. In this chapter, we highlight the challenges and new opportunities provided by emerging technologies that enable dynamic, high-information-content imaging of craniofacial development. We discuss the challenges of varying spatial and temporal scales encountered from the biological and technological perspectives. We identify molecular and fluorescence imaging technology that can provide solutions to some of the challenges. Application of the techniques described within this chapter combined with considerations of the biological and technical challenges will aid in formulating the best image-based studies to extend our understanding of the genetic and environmental influences underlying craniofacial anomalies.
Assuntos
Padronização Corporal/fisiologia , Comunicação Celular/fisiologia , Diagnóstico por Imagem/métodos , Crânio/embriologia , Animais , Padronização Corporal/genética , Comunicação Celular/genética , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Hibridização In Situ/métodos , Cinética , Microscopia Confocal/métodos , Crânio/citologia , Crânio/metabolismoRESUMO
Targeted protein degradation is a powerful tool in determining the function of specific proteins or protein complexes. We fused nanobodies to SPOP, an adaptor protein of the Cullin-RING E3 ubiquitin ligase complex, resulting in rapid ubiquitination and subsequent proteasome-dependent degradation of specific nuclear proteins in mammalian cells and zebrafish embryos. This approach is easily modifiable, as substrate specificity is conferred by an antibody domain that can be adapted to target virtually any protein.
Assuntos
Proteínas Nucleares/metabolismo , Anticorpos de Domínio Único/metabolismo , Complexos Ubiquitina-Proteína Ligase/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Expressão Gênica , Genes Reporter , Proteína HMGA2/genética , Proteína HMGA2/metabolismo , Proteínas Nucleares/genética , Ligação Proteica , Proteólise , Interferência de RNA , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Anticorpos de Domínio Único/imunologia , Complexos Ubiquitina-Proteína Ligase/imunologia , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/imunologia , Ubiquitinação , Peixe-ZebraRESUMO
Dystrophin (Dmd) is a structural protein that links the extracellular matrix to actin filaments in muscle fibers and is required for the maintenance of muscles integrity. Mutations in Dmd lead to muscular dystrophies in humans and other vertebrates. Here, we report the characterization of a zebrafish gene trap line that fluorescently labels the endogenous Dmd protein (Dmd-citrine, Gt(dmd-citrine) ct90a). We show that the Dmd-citrine line recapitulates endogenous dmd transcript expression and Dmd protein localization. Using this Dmd-citrine line, we follow Dmd localization to the myosepta in real-time using time-lapse microscopy, and find that the accumulation of Dmd protein at the transverse myosepta coincides with the onset of myotome formation, a critical stage in muscle maturation. We observed that Dmd protein localizes specifically to the myosepta prior to dmd mRNA localization. Additionally, we demonstrate that the Dmd-citrine line can be used to assess muscular dystrophy following both genetic and physical disruptions of the muscle.
Assuntos
Proteínas de Bactérias/genética , Proteínas Luminescentes/genética , Proteínas de Membrana/genética , Proteínas Musculares/genética , Músculo Esquelético/metabolismo , RNA Mensageiro/genética , Proteínas Recombinantes de Fusão/genética , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/genética , Animais , Proteínas de Bactérias/metabolismo , Embrião não Mamífero , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Luminescentes/metabolismo , Proteínas de Membrana/metabolismo , Microscopia de Fluorescência , Morfogênese/genética , Morfolinos , Proteínas Musculares/metabolismo , Músculo Esquelético/citologia , Músculo Esquelético/embriologia , Plasmídeos/química , Plasmídeos/metabolismo , RNA Mensageiro/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Imagem com Lapso de Tempo , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/metabolismoRESUMO
We present an imaging and image reconstruction pipeline that captures the dynamic three-dimensional beating motion of the live embryonic zebrafish heart at subcellular resolution. Live, intact zebrafish embryos were imaged using 2-photon light sheet microscopy, which offers deep and fast imaging at 70 frames per second, and the individual optical sections were assembled into a full 4D reconstruction of the beating heart using an optimized retrospective image registration algorithm. This imaging and reconstruction platform permitted us to visualize protein expression patterns at endogenous concentrations in zebrafish gene trap lines.
RESUMO
Enhancer and gene trapping methods are highly effective means for the identification and functional analysis of transcriptionally active genes. With recent advances in fluorescent proteins and transposon based integration technologies, a growing family of trapping approaches has been developed in zebrafish, offering powerful tools to both visualize and functionally dissect gene networks during development. Coupled with the intrinsic advantages of the zebrafish model system, creative genetic engineering of trap vectors has enabled high-resolution molecular imaging and genetic manipulations. This review highlights the different enhancer and gene trap approaches that have been developed in zebrafish and offers insights into the strengths, limitations and experimental strategies for their application to enrich our knowledge of gene function and the cellular processes they control.
Assuntos
Elementos de DNA Transponíveis/genética , Elementos Facilitadores Genéticos , Imagem Molecular/métodos , Mutagênese , Animais , Técnicas Genéticas , Vetores Genéticos , Proteínas de Fluorescência Verde/metabolismo , Fatores de Transcrição/metabolismo , Transcriptoma , Peixe-ZebraRESUMO
The pharyngeal pouches, which form by budding of the foregut endoderm, are essential for segmentation of the vertebrate face. To date, the cellular mechanism and segmental nature of such budding have remained elusive. Here, we find that Wnt11r and Wnt4a from the head mesoderm and ectoderm, respectively, play distinct roles in the segmental formation of pouches in zebrafish. Time-lapse microscopy, combined with mutant and tissue-specific transgenic experiments, reveal requirements of Wnt signaling in two phases of endodermal epithelial transitions. Initially, Wnt11r and Rac1 destabilize the endodermal epithelium to promote the lateral movement of pouch-forming cells. Next, Wnt4a and Cdc42 signaling induce the rearrangement of maturing pouch cells into bilayers through junctional localization of the Alcama immunoglobulin-domain protein, which functions to restabilize adherens junctions. We propose that this dynamic control of epithelial morphology by Wnt signaling may be a common theme for the budding of organ anlagen from the endoderm.