RESUMEN
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.
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Pez Cebra , Animales , Ratones , Microscopía Fluorescente/métodosRESUMEN
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.
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Integrasas , Pez Cebra , Animales , Animales Modificados Genéticamente , Proteínas Fluorescentes Verdes/metabolismo , Integrasas/metabolismo , Mamíferos/metabolismo , Neuronas/metabolismo , Pez Cebra/metabolismoRESUMEN
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.
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Epitelio/inmunología , Macrófagos/inmunología , Mycobacterium marinum/inmunología , Animales , Cadherinas/inmunología , Epitelio/microbiología , Granuloma/inmunología , Granuloma/microbiología , Macrófagos/microbiología , Mycobacterium tuberculosis/inmunología , Pez CebraRESUMEN
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.
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Programas Informáticos , Imagen de Lapso de Tiempo/métodos , Animales , Animales Modificados Genéticamente , Color , Embrión no Mamífero , Análisis de Fourier , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Procesamiento de Imagen Asistido por Computador , Pez Cebra/embriología , Pez Cebra/genéticaRESUMEN
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.
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Proteínas Portadoras/metabolismo , Proteínas de la Membrana/metabolismo , Neuronas/metabolismo , Técnicas de Placa-Clamp/métodos , Sinapsis/fisiología , Transmisión Sináptica/fisiología , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Células Cultivadas , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Femenino , Hipocampo , Masculino , Trastornos Motores/metabolismo , Trastornos Motores/patología , Neuronas/citología , Ratas , Ratas Sprague-Dawley , Columna Vertebral/citología , Columna Vertebral/metabolismo , Ubiquitinación , Pez CebraRESUMEN
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.
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Biotinilación/métodos , Perfilación de la Expresión Génica/métodos , Coloración y Etiquetado/métodos , Pez Cebra/genética , Animales , Fraccionamiento Celular , Redes Reguladoras de Genes/genética , Polirribosomas/genética , Polirribosomas/metabolismo , ARN/aislamiento & purificación , ARN/metabolismo , Transcriptoma/genética , Pez Cebra/metabolismoRESUMEN
We report a multifunctional gene-trapping approach, which generates full-length Citrine fusions with endogenous proteins and conditional mutants from a single integration event of the FlipTrap vector. We identified 170 FlipTrap zebrafish lines with diverse tissue-specific expression patterns and distinct subcellular localizations of fusion proteins generated by the integration of an internal citrine exon. Cre-mediated conditional mutagenesis is enabled by heterotypic lox sites that delete Citrine and "flip" in its place mCherry with a polyadenylation signal, resulting in a truncated fusion protein. Inducing recombination with Cerulean-Cre results in fusion proteins that often mislocalize, exhibit mutant phenotypes, and dramatically knock down wild-type transcript levels. FRT sites in the vector enable targeted genetic manipulation of the trapped loci in the presence of Flp recombinase. Thus, the FlipTrap captures the functional proteome, enabling the visualization of full-length fluorescent fusion proteins and interrogation of function by conditional mutagenesis and targeted genetic manipulation.
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Proteoma , Proteómica/métodos , Animales , Proteínas Bacterianas/genética , Bases de Datos de Proteínas , Embrión no Mamífero , Vectores Genéticos , Internet , Proteínas Luminiscentes/genética , Anotación de Secuencia Molecular , Mutación , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Pez CebraRESUMEN
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.
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Elementos Transponibles de ADN/genética , Elementos de Facilitación Genéticos , Imagen Molecular/métodos , Mutagénesis , Animales , Técnicas Genéticas , Vectores Genéticos , Proteínas Fluorescentes Verdes/metabolismo , Factores de Transcripción/metabolismo , Transcriptoma , Pez CebraRESUMEN
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.
RESUMEN
Yeast Periodic tryptophan protein 2 gene (Pwp2) is involved in ribosome biogenesis and has been implicated in regulation of the cell cycle in yeast. Here, we report a zebrafish protein-trap line that produces fluorescently tagged Periodic tryptophan protein 2 gene homologue (Pwp2h) protein, which can be dynamically tracked in living fish at subcellular resolution. We identified both full-length zebrafish Pwp2h and a short variant. The expression results show that Pwp2h is present in numerous sites in the early developing embryo, but later is restricted to highly proliferative regions, including the forebrain ventricular zone and endoderm-derived organs in the early larval stage. At the subcellular level, Pwp2h protein appears to be localized to the region of the nucleolus consistent with its presumed function in ribosomal RNA synthesis. This Pwp2h protein trap line offers a powerful tool to study the link between ribosome biogenesis and cell cycle progression during vertebrate development.
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Proteínas de Ciclo Celular/genética , Proteínas de Pez Cebra/genética , Pez Cebra/embriología , Pez Cebra/genética , Secuencia de Aminoácidos , Animales , Animales Modificados Genéticamente , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Proteínas de Ciclo Celular/metabolismo , Rastreo Celular/métodos , Embrión no Mamífero , Desarrollo Embrionario/genética , Regulación del Desarrollo de la Expresión Génica , Imagenología Tridimensional , Datos de Secuencia Molecular , Transportadores de Anión Orgánico/genética , Transportadores de Anión Orgánico/metabolismo , Homología de Secuencia , Grabación en Video/métodos , Pez Cebra/metabolismo , Proteínas de Pez Cebra/metabolismoRESUMEN
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.
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Procesamiento de Imagen Asistido por Computador/métodos , Espectrometría de Fluorescencia/métodos , Algoritmos , Animales , Animales Modificados Genéticamente , Color , Embrión no Mamífero , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Procesamiento de Imagen Asistido por Computador/normas , Ratones Endogámicos C57BL , Microscopía Confocal/métodos , Relación Señal-Ruido , Pez Cebra/embriología , Pez Cebra/genéticaRESUMEN
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.
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Cicatriz/metabolismo , Cicatriz/patología , Colágeno/metabolismo , Corazón/fisiopatología , Macrófagos/patología , Cicatrización de Heridas , Pez Cebra/fisiología , Traslado Adoptivo , Animales , Embrión de Mamíferos/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Regulación de la Expresión Génica , Proteínas Fluorescentes Verdes/metabolismo , Macrófagos/metabolismo , Ratones , Monocitos/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Bazo/patología , Transcripción Genética , Transcriptoma/genética , Pez Cebra/embriología , Proteínas de Pez Cebra/metabolismoRESUMEN
Myocardial differentiation is initiated by the activation of terminal-differentiation gene expression within a subset of cells in the anterior lateral plate mesoderm. We have previously shown that shortly after this activation, myocardial cells undergo epithelial maturation [1], suggesting that myocardial differentiation encompasses both molecular and cellular changes. To address the question of how the molecular programs driving myocardial gene expression and the formation of the myocardial epithelium are integrated, we analyzed the role of two essential myocardial terminal-differentiation factors, Hand2 and Gata5, in myocardial epithelia formation. hand2 and gata5 mutants exhibit a much-reduced number of myocardial cells and defects in myocardial gene expression [2,3]. We find that the few myocardial precursors that are present in hand2 mutants do not polarize. In contrast, embryos with reduced Gata5 function exhibit polarized myocardial epithelia despite a similar reduction in myocardial precursor number, indicating that proper cell number is not required for epithelial formation. Taken thogether, these results indicate that Hand2 is uniquely required for myocardial polarization, a previously unappreciated role for this critical transcription factor. Furthermore, these results demonstrate that two independent processes, the polarizaton of myocardial precursors and the allocation of proper cell number, contribute to myocardial development.
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Diferenciación Celular/fisiología , Polaridad Celular/fisiología , Regulación del Desarrollo de la Expresión Génica , Morfogénesis , Miocitos Cardíacos/fisiología , Factores de Transcripción/metabolismo , Pez Cebra/embriología , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Proteínas de Unión al ADN/metabolismo , Epitelio/embriología , Factor de Transcripción GATA5 , Hibridación in Situ , Microscopía Confocal , Microscopía Fluorescente , Oligonucleótidos Antisentido , Factores de Transcripción/fisiología , Proteínas de Pez CebraRESUMEN
In situ hybridization techniques typically employ chromogenic staining by enzymatic amplification to detect domains of gene expression. We demonstrate the previously unreported near infrared (NIR) fluorescence of the dark purple stain formed from the commonly used chromogens, nitro blue tetrazolium (NBT) and 5-bromo-4-chloro-3-indolyl phosphate (BCIP). The solid reaction product has significant fluorescence that enables the use of confocal microscopy to generate high-resolution three-dimensional (3-D) imaging of gene expression.
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Hibridación Fluorescente in Situ/métodos , Indoles/química , Nitroazul de Tetrazolio/química , Coloración y Etiquetado , Animales , Miosinas Cardíacas/genética , Miosinas Cardíacas/metabolismo , Embrión no Mamífero , Lampreas/embriología , Microscopía Confocal , Pez Cebra/embriologíaRESUMEN
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.
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Imagenología Tridimensional/métodos , Microscopía/métodos , Animales , Diseño de Equipo , Microscopía/instrumentación , Control de Calidad , Pez CebraRESUMEN
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.
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Actinas/metabolismo , Neuronas/metabolismo , Citoesqueleto de Actina/metabolismo , Complejo 2-3 Proteico Relacionado con la Actina/metabolismo , Animales , Axones/metabolismo , Supervivencia Celular/fisiología , Dendritas/metabolismo , Microtúbulos/metabolismo , Miosinas/metabolismo , RatasRESUMEN
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.
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Cresta Neural/crecimiento & desarrollo , Factores de Transcripción/biosíntesis , Transcriptoma/genética , Pez Cebra/genética , Animales , Compartimento Celular/genética , Linaje de la Célula/genética , Secuencia Conservada/genética , Regulación del Desarrollo de la Expresión Génica , Redes Reguladoras de Genes/genética , Especificidad de Órganos/genética , Factores de Transcripción/genética , Pez Cebra/crecimiento & desarrolloRESUMEN
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.
RESUMEN
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.
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Vasos Sanguíneos/diagnóstico por imagen , Procesamiento de Imagen Asistido por Computador/métodos , Tomografía de Coherencia Óptica/métodos , Algoritmos , Animales , Vasos Sanguíneos/crecimiento & desarrollo , Embrión no Mamífero/irrigación sanguínea , Embrión no Mamífero/diagnóstico por imagen , Diseño de Equipo , Pez Cebra/crecimiento & desarrolloRESUMEN
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.