RESUMO
During Hedgehog (Hh) signal transduction in development and disease, the atypical G protein-coupled receptor (GPCR) SMOOTHENED (SMO) communicates with GLI transcription factors by binding the protein kinase A catalytic subunit (PKA-C) and physically blocking its enzymatic activity. Here, we show that GPCR kinase 2 (GRK2) orchestrates this process during endogenous mouse and zebrafish Hh pathway activation in the primary cilium. Upon SMO activation, GRK2 rapidly relocalizes from the ciliary base to the shaft, triggering SMO phosphorylation and PKA-C interaction. Reconstitution studies reveal that GRK2 phosphorylation enables active SMO to bind PKA-C directly. Lastly, the SMO-GRK2-PKA pathway underlies Hh signal transduction in a range of cellular and in vivo models. Thus, GRK2 phosphorylation of ciliary SMO and the ensuing PKA-C binding and inactivation are critical initiating events for the intracellular steps in Hh signaling. More broadly, our study suggests an expanded role for GRKs in enabling direct GPCR interactions with diverse intracellular effectors.
Assuntos
Cílios , Proteínas Quinases Dependentes de AMP Cíclico , Quinase 2 de Receptor Acoplado a Proteína G , Proteínas Hedgehog , Transdução de Sinais , Receptor Smoothened , Peixe-Zebra , Animais , Cílios/metabolismo , Receptor Smoothened/metabolismo , Receptor Smoothened/genética , Proteínas Hedgehog/metabolismo , Quinase 2 de Receptor Acoplado a Proteína G/metabolismo , Camundongos , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Peixe-Zebra/metabolismo , Fosforilação , Proteínas de Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Células NIH 3T3RESUMO
Background: Uveal coloboma, a developmental eye defect, is caused by failed development of the optic fissure, a ventral structure in the optic stalk and cup where axons exit the eye and vasculature enters. The Hedgehog (Hh) signaling pathway regulates optic fissure development: loss-of-function mutations in the Hh receptor ptch2 produce overactive Hh signaling and can result in coloboma. We previously proposed a model where overactive Hh signaling disrupts optic fissure formation by upregulating transcriptional targets acting both cell- and non-cell-autonomously. Here, we examine the Netrin family of secreted ligands as candidate Hh target genes. Results: We find multiple Netrin ligands upregulated in the zebrafish ptch2 mutant during optic fissure development. Using a gain-of-function approach to overexpress Netrin in a spatiotemporally specific manner, we find that netrin1a or netrin1b overexpression is sufficient to cause coloboma and disrupt wild-type optic fissure formation. We used loss-of-function alleles, CRISPR/Cas9 mutagenesis, and morpholino knockdown to test if loss of Netrin can rescue coloboma in the ptch2 mutant: loss of netrin genes does not rescue the ptch2 mutant phenotype. Conclusion: These results suggest that Netrin is sufficient but not required to disrupt optic fissure formation downstream of overactive Hh signaling in the ptch2 mutant.
RESUMO
Here we present the Multisite Assembly of Gateway Induced Clones (MAGIC) system, which harnesses site-specific recombination-based cloning via Gateway technology for rapid, modular assembly of between 1 and 3 "Entry" vector components, all into a fourth, standard high copy "Destination" plasmid backbone. The MAGIC toolkit spans a range of in vitro and in vivo uses, from directing tunable gene expression, to driving simultaneous expression of microRNAs and fluorescent reporters, to enabling site-specific recombinase-dependent gene expression. All MAGIC system components are directly compatible with existing multisite gateway Tol2 systems currently used in zebrafish, as well as existing eukaryotic cell culture expression Destination plasmids, and available mammalian lentiviral and adenoviral Destination vectors, allowing rapid cross-species experimentation. Moreover, herein we describe novel vectors with flanking piggyBac transposon elements for stable genomic integration in vitro or in vivo when used with piggyBac transposase. Collectively, the MAGIC system facilitates transgenesis in cultured mammalian cells, electroporated mouse and chick embryos, as well as in injected zebrafish embryos, enabling the rapid generation of innovative DNA constructs for biological research due to a shared, common plasmid platform.
RESUMO
During Hedgehog (Hh) signal transduction in development and disease, the atypical G protein-coupled receptor (GPCR) SMOOTHENED (SMO) communicates with GLI transcription factors by binding the protein kinase A catalytic subunit (PKA-C) and physically blocking its enzymatic activity. Here we show that GPCR kinase 2 (GRK2) orchestrates this process during endogenous Hh pathway activation in the primary cilium. Upon SMO activation, GRK2 rapidly relocalizes from the ciliary base to the shaft, triggering SMO phosphorylation and PKA-C interaction. Reconstitution studies reveal that GRK2 phosphorylation enables active SMO to bind PKA-C directly. Lastly, the SMO-GRK2-PKA pathway underlies Hh signal transduction in a range of cellular and in vivo models. Thus, GRK2 phosphorylation of ciliary SMO, and the ensuing PKA-C binding and inactivation, are critical initiating events for the intracellular steps in Hh signaling. More broadly, our study suggests an expanded role for GRKs in enabling direct GPCR interactions with diverse intracellular effectors.
RESUMO
Proper eye structure is essential for visual function: Multiple essential eye tissues must take shape and assemble into a precise three-dimensional configuration. Accordingly, alterations to eye structure can lead to pathological conditions of visual impairment. Changes in eye shape can also be adaptive over evolutionary time. Eye structure is first established during development with the formation of the optic cup, which contains the neural retina, retinal pigment epithelium, and lens. This crucial yet deceptively simple hemispherical structure lays the foundation for all later elaborations of the eye. Building on descriptions of the embryonic eye that started with hand drawings and micrographs, the field is beginning to identify mechanisms driving dynamic changes in three-dimensional cell and tissue shape. A combination of molecular genetics, imaging, and pharmacological approaches is defining connections among transcription factors, signaling pathways, and the intracellular machinery governing the emergence of this crucial structure.
Assuntos
Vertebrados , Baixa Visão , Animais , Retina , Epitélio Pigmentado da Retina , MorfogêneseRESUMO
Transgenesis is an essential technique for any genetic model. Tol2-based transgenesis paired with Gateway-compatible vector collections has transformed zebrafish transgenesis with an accessible modular system. Here, we establish several next-generation transgenesis tools for zebrafish and other species to expand and enhance transgenic applications. To facilitate gene regulatory element testing, we generated Gateway middle entry vectors harboring the small mouse beta-globin minimal promoter coupled to several fluorophores, CreERT2 and Gal4. To extend the color spectrum for transgenic applications, we established middle entry vectors encoding the bright, blue-fluorescent protein mCerulean and mApple as an alternative red fluorophore. We present a series of p2A peptide-based 3' vectors with different fluorophores and subcellular localizations to co-label cells expressing proteins of interest. Finally, we established Tol2 destination vectors carrying the zebrafish exorh promoter driving different fluorophores as a pineal gland-specific transgenesis marker that is active before hatching and through adulthood. exorh-based reporters and transgenesis markers also drive specific pineal gland expression in the eye-less cavefish (Astyanax). Together, our vectors provide versatile reagents for transgenesis applications in zebrafish, cavefish and other models.
Assuntos
Técnicas de Transferência de Genes , Peixe-Zebra , Animais , Camundongos , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Animais Geneticamente Modificados , Plasmídeos/genética , Regiões Promotoras Genéticas/genética , Elementos de DNA Transponíveis/genéticaRESUMO
Cilia are essential for the development and function of many different tissues. Although cilia machinery is crucial in the eye for photoreceptor development and function, a role for cilia in early eye development and morphogenesis is still somewhat unclear: many zebrafish cilia mutants retain cilia at early stages due to maternal deposition of cilia components. An eye phenotype has been described in the mouse Arl13 mutant, however, zebrafish arl13b is maternally deposited, and an early role for cilia proteins has not been tested in zebrafish eye development. Here we use the zebrafish dzip1 mutant, which exhibits a loss of cilia throughout stages of early eye development, to examine eye development and morphogenesis. We find that in dzip1 mutants, initial formation of the optic cup proceeds normally, however, the optic fissure subsequently fails to close and embryos develop the structural eye malformation ocular coloboma. Further, neural crest cells, which are implicated in optic fissure closure, do not populate the optic fissure correctly, suggesting that their inappropriate localization may be the underlying cause of coloboma. Overall, our results indicate a role for dzip1 in proper neural crest localization in the optic fissure and optic fissure closure.
Assuntos
Proteínas de Transporte/metabolismo , Coloboma , Disco Óptico , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Coloboma/genética , Olho/metabolismo , Mesoderma/metabolismo , Camundongos , Disco Óptico/metabolismo , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
Morphogenesis, the formation of three-dimensional organ structures, requires precise coupling of genetic regulation and complex cell behaviors. The genetic networks governing many morphogenetic systems, including that of the embryonic eye, are poorly understood. In zebrafish, several forward genetic screens have sought to identify factors regulating eye development. These screens often look for eye defects at stages after the optic cup is formed and when retinal neurogenesis is under way. This approach can make it difficult to identify mutants specific for morphogenesis, as opposed to neurogenesis. To this end, we carried out a forward genetic, small-scale haploid mutagenesis screen in zebrafish (Danio rerio) to identify factors that govern optic cup morphogenesis. We screened â¼100 genomes and isolated shutdown corner (sco), a mutant that exhibits multiple tissue defects and harbors a â¼10-Mb deletion that encompasses 89 annotated genes. Using a combination of live imaging and antibody staining, we found cell proliferation, cell death, and tissue patterning defects in the sco optic cup. We also observed other phenotypes, including paralysis, neuromuscular defects, and ocular vasculature defects. To date, the largest deletion mutants reported in zebrafish are engineered using CRISPR-Cas9 and are less than 300 kb. Because of the number of genes within the deletion interval, shutdown corner [Df(Chr05:sco)z207] could be a useful resource to the zebrafish community, as it may be helpful for gene mapping, understanding genetic interactions, or studying many genes lost in the mutant.
Assuntos
Proteínas de Peixe-Zebra , Peixe-Zebra , Animais , Haploidia , Morfogênese/genética , Mutagênese/genética , Mutação , Neurogênese/genética , Retina , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genéticaRESUMO
BACKGROUND: Pax2 is required for optic fissure development in many organisms, including humans and zebrafish. Zebrafish loss-of-function mutations in pax2a display coloboma, yet the etiology of the morphogenetic defects is unclear. Further, pax2 is duplicated in zebrafish, and a role for pax2b in optic fissure development has not been examined. RESULTS: Using a combination of imaging and molecular genetics, we interrogated a potential role for pax2b and examined how loss of pax2 affects optic fissure development. Although optic fissure formation appears normal in pax2 mutants, an endothelial-specific subset of periocular mesenchyme (POM) fails to initially localize within the optic fissure, yet both neural crest and endothelial-derived POM ectopically accumulate at later stages in pax2a and pax2a; pax2b mutants. Apoptosis is not up-regulated within the optic fissure in pax2 mutants, yet cell death is increased in tissues outside of the optic fissure, and when apoptosis is inhibited, coloboma is partially rescued. In contrast to pax2a, loss of pax2b does not appear to affect optic fissure morphogenesis. CONCLUSIONS: Our results suggest that pax2a, but not pax2b, supports cell survival outside of the optic fissure and POM abundance within it to facilitate optic fissure closure.
Assuntos
Proteínas de Peixe-Zebra , Peixe-Zebra , Animais , Sobrevivência Celular/genética , Olho , Mesoderma/metabolismo , Morfogênese/genética , Fator de Transcrição PAX2/genética , Fator de Transcrição PAX2/metabolismo , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
Visual system function requires the establishment of precise tissue and organ structures. In the vertebrate eye, structural defects are a common cause of visual impairment, yet mechanisms of eye morphogenesis are still poorly understood. The basic organization of the embryonic eye is conserved throughout vertebrates, thus live imaging of zebrafish embryos has become a powerful approach to directly observe eye development at real time under normal and pathological conditions. Dynamic cell processes including movements, morphologies, interactions, division, and death can be visualized in the embryo. We have developed methods for uniform labeling of subcellular structures and timelapse confocal microscopy of early eye development in zebrafish. This protocol outlines the method of generating capped mRNA for injection into the 1-cell zebrafish embryo, mounting embryos at optic vesicle stage (~12 hours post fertilization, hpf), and performing multi-dimensional timelapse imaging of optic cup morphogenesis on a laser scanning confocal microscope, such that multiple datasets are acquired sequentially in the same imaging session. Such an approach yields data that can be used for a variety of purposes, including cell tracking, volume measurements, three-dimensional (3D) rendering, and visualization. Our approaches allow us to pinpoint the cellular and molecular mechanisms driving optic cup development, in both wild type and genetic mutant conditions. These methods can be employed directly by other groups or adapted to visualize many additional aspects of zebrafish eye development.
Assuntos
Olho , Peixe-Zebra , Animais , Morfogênese , Organogênese , Proteínas de Peixe-ZebraRESUMO
The basic structure of the eye, which is crucial for visual function, is established during the embryonic process of optic cup morphogenesis. Molecular pathways of specification and patterning are integrated with spatially distinct cell and tissue shape changes to generate the eye, with discrete domains and structural features: retina and retinal pigment epithelium enwrap the lens, and the optic fissure occupies the ventral surface of the eye and optic stalk. Interest in the underlying cell biology of eye morphogenesis has led to a growing body of work, combining molecular genetics and imaging to quantify cellular processes such as adhesion and actomyosin activity. These studies reveal that intrinsic machinery and spatiotemporally specific extrinsic inputs collaborate to control dynamics of cell movements and morphologies. Here we consider recent advances in our understanding of eye morphogenesis, with a focus on the mechanics of eye formation throughout vertebrate systems, including insights and potential opportunities using organoids, which may provide a tractable system to test hypotheses from embryonic models.
Assuntos
Olho/embriologia , Disco Óptico/embriologia , Actomiosina/metabolismo , Animais , Movimento Celular , Olho/metabolismo , Olho/patologia , Humanos , Cristalino/embriologia , Morfogênese/genética , Morfogênese/fisiologia , Disco Óptico/metabolismo , Organogênese/genética , Organogênese/fisiologia , Retina/embriologia , Epitélio Pigmentado da Retina/citologia , Transdução de Sinais , Vertebrados/fisiologiaRESUMO
Efficient precision genome engineering requires high frequency and specificity of integration at the genomic target site. Here, we describe a set of resources to streamline reporter gene knock-ins in zebrafish and demonstrate the broader utility of the method in mammalian cells. Our approach uses short homology of 24-48 bp to drive targeted integration of DNA reporter cassettes by homology-mediated end joining (HMEJ) at high frequency at a double strand break in the targeted gene. Our vector series, pGTag (plasmids for Gene Tagging), contains reporters flanked by a universal CRISPR sgRNA sequence which enables in vivo liberation of the homology arms. We observed high rates of germline transmission (22-100%) for targeted knock-ins at eight zebrafish loci and efficient integration at safe harbor loci in porcine and human cells. Our system provides a straightforward and cost-effective approach for high efficiency gene targeting applications in CRISPR and TALEN compatible systems.
Assuntos
Proteínas Associadas a CRISPR/genética , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Técnicas de Introdução de Genes , Genes Reporter , Proteínas de Fluorescência Verde/genética , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição/genética , Peixe-Zebra/genética , Animais , Animais Geneticamente Modificados , Proteínas Associadas a CRISPR/metabolismo , Fibroblastos/metabolismo , Regulação da Expressão Gênica , Proteínas de Fluorescência Verde/metabolismo , Humanos , Células K562 , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , RNA Guia de Cinetoplastídeos/genética , RNA Guia de Cinetoplastídeos/metabolismo , Reparo de DNA por Recombinação , Homologia de Sequência do Ácido Nucleico , Sus scrofa , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição/metabolismoRESUMO
Organogenesis requires precise interactions between a developing tissue and its environment. In vertebrates, the developing eye is surrounded by a complex extracellular matrix as well as multiple mesenchymal cell populations. Disruptions to either the matrix or periocular mesenchyme can cause defects in early eye development, yet in many cases the underlying mechanism is unknown. Here, using multidimensional imaging and computational analyses in zebrafish, we establish that cell movements in the developing optic cup require neural crest. Ultrastructural analysis reveals that basement membrane formation around the developing eye is also dependent on neural crest, but only specifically around the retinal pigment epithelium. Neural crest cells produce the extracellular matrix protein nidogen: impairing nidogen function disrupts eye development, and, strikingly, expression of nidogen in the absence of neural crest partially restores optic cup morphogenesis. These results demonstrate that eye formation is regulated in part by extrinsic control of extracellular matrix assembly.This article has an associated 'The people behind the papers' interview.
Assuntos
Membrana Basal/embriologia , Olho/embriologia , Crista Neural/embriologia , Alelos , Animais , Sistemas CRISPR-Cas , Proteínas de Ligação ao Cálcio/fisiologia , Movimento Celular , Eletroforese Capilar , Matriz Extracelular/fisiologia , Proteínas da Matriz Extracelular/fisiologia , Fatores de Transcrição Forkhead/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Genótipo , Mesoderma/embriologia , Microscopia Eletrônica de Transmissão , Morfogênese , Mutação , Crista Neural/citologia , Organogênese , Retina/embriologia , Epitélio Pigmentado da Retina/embriologia , Transdução de Sinais , Fator de Transcrição AP-2/fisiologia , Peixe-Zebra , Proteínas de Peixe-Zebra/fisiologiaRESUMO
Epithelial morphogenesis, a fundamental aspect of development, generates 3-dimensional tissue structures crucial for organ function. Underlying morphogenetic mechanisms are, in many cases, poorly understood, but mutations that perturb organ development can affect epithelial cell shape and orientation - difficult features to quantify in three dimensions. The basic structure of the eye is established via epithelial morphogenesis: in the embryonic optic cup, the retinal progenitor epithelium enwraps the lens. We previously found that loss of the extracellular matrix protein laminin-alpha1 (lama1) led to mislocalization of apical polarity markers and apparent misorientation of retinal progenitors. We sought to visualize and quantify this phenotype, and determine whether loss of the apical polarity determinant pard3 might rescue the phenotype. To this end, we developed LongAxis, a MATLAB-based program optimized for the retinal progenitor neuroepithelium. LongAxis facilitates 3-dimensional cell segmentation, visualization, and quantification of cell orientation and morphology. Using LongAxis, we find that retinal progenitors in the lama1-/- optic cup are misoriented and slightly less elongated. In the lama1;MZpard3 double mutant, cells are still misoriented, but larger. Therefore, loss of pard3 does not rescue loss of lama1, and in fact uncovers a novel cell size phenotype. LongAxis enables population-level visualization and quantification of retinal progenitor cell orientation and morphology. These results underscore the importance of visualizing and quantifying cell orientation and shape in three dimensions within the retina.
Assuntos
Forma Celular , Células Epiteliais , Processamento de Imagem Assistida por Computador , Retina , Software , Peixe-Zebra/embriologia , Animais , Animais Geneticamente Modificados , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Laminina/genética , Laminina/metabolismo , Retina/citologia , Retina/embriologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
Epithelial wound healing requires a complex orchestration of cellular rearrangements and movements to restore tissue architecture and function after injury. While it is well known that mechanical forces can affect tissue morphogenesis and patterning, how the biophysical cues generated after injury influence cellular behaviors during tissue repair is not well understood. Using time-lapse confocal imaging of epithelial tissues in living zebrafish larvae, we provide evidence that localized increases in cellular crowding during wound closure promote the extrusion of nonapoptotic cells via mechanically regulated stretch-activated ion channels (SACs). Directed cell migration toward the injury site promoted rapid changes in cell number and generated shifts in tension at cellular interfaces over long spatial distances. Perturbation of SAC activity resulted in failed extrusion and increased proliferation in crowded areas of the tissue. Together, we conclude that localized cell number plays a key role in dictating cellular behaviors that facilitate wound closure and tissue repair.
Assuntos
Epitélio/patologia , Cicatrização , Actinas/metabolismo , Animais , Contagem de Células , Proliferação de Células , Canais Iônicos/metabolismo , Miosinas/metabolismo , Peixe-ZebraRESUMO
Establishment of precise three-dimensional tissue structure is vital for organ function. In the visual system, optic fissure and stalk morphogenesis is a crucial yet poorly understood process, disruptions of which can lead to coloboma, a birth defect causing visual impairment. Here, we use four-dimensional imaging, cell tracking, and molecular genetics in zebrafish to define the cell movements underlying normal optic fissure and stalk formation. We determine how these events are disrupted in a coloboma model in which the Hedgehog (Hh) receptor ptch2 is lost, resulting in overactive Hh signaling. In the ptch2 mutant, cells exhibit defective motile behaviors and morphology. Cells that should contribute to the fissure do not arrive at their correct position, and instead contribute to an ectopically large optic stalk. Our results suggest that overactive Hh signaling, through overexpression of downstream transcriptional targets, impairs cell motility underlying optic fissure and stalk formation, via non-cell-autonomous and cell-autonomous mechanisms. More broadly, our cell motility and morphology analyses provide a new framework for studying other coloboma-causing mutations that disrupt optic fissure or stalk formation.
Assuntos
Movimento Celular , Olho/citologia , Olho/crescimento & desenvolvimento , Proteínas Hedgehog/metabolismo , Morfogênese , Transdução de Sinais , Peixe-Zebra/crescimento & desenvolvimento , Peixe-Zebra/metabolismo , Animais , Olho/anatomia & histologia , Modelos Biológicos , Mutação/genética , Transcrição Gênica , Proteínas de Peixe-Zebra/metabolismoRESUMO
The vertebrate eye forms via a complex set of morphogenetic events. The optic vesicle evaginates and undergoes transformative shape changes to form the optic cup, in which neural retina and retinal pigmented epithelium enwrap the lens. It has long been known that a complex, glycoprotein-rich extracellular matrix layer surrounds the developing optic cup throughout the process, yet the functions of the matrix and its specific molecular components have remained unclear. Previous work established a role for laminin extracellular matrix in particular steps of eye development, including optic vesicle evagination, lens differentiation, and retinal ganglion cell polarization, yet it is unknown what role laminin might play in the early process of optic cup formation subsequent to the initial step of optic vesicle evagination. Here, we use the zebrafish lama1 mutant (lama1(UW1)) to determine the function of laminin during optic cup morphogenesis. Using live imaging, we find, surprisingly, that loss of laminin leads to divergent effects on focal adhesion assembly in a spatiotemporally-specific manner, and that laminin is required for multiple steps of optic cup morphogenesis, including optic stalk constriction, invagination, and formation of a spherical lens. Laminin is not required for single cell behaviors and changes in cell shape. Rather, in lama1(UW1) mutants, loss of epithelial polarity and altered adhesion lead to defective tissue architecture and formation of a disorganized retina. These results demonstrate that the laminin extracellular matrix plays multiple critical roles regulating adhesion and polarity to establish and maintain tissue structure during optic cup morphogenesis.
Assuntos
Proteínas do Olho/fisiologia , Laminina/fisiologia , Cristalino/embriologia , Retina/embriologia , Proteínas de Peixe-Zebra/fisiologia , Peixe-Zebra/embriologia , Animais , Movimento Celular , Polaridade Celular , Matriz Extracelular/fisiologia , Proteínas do Olho/genética , Adesões Focais , Laminina/deficiência , Laminina/genética , Proteínas Luminescentes/análise , Proteínas Luminescentes/genética , Microscopia Confocal , Organogênese , Retina/citologia , Células Ganglionares da Retina/citologia , Epitélio Pigmentado da Retina/citologia , Epitélio Pigmentado da Retina/embriologia , Imagem com Lapso de Tempo , Proteínas de Peixe-Zebra/deficiência , Proteínas de Peixe-Zebra/genéticaRESUMO
BACKGROUND: The application of the Gal4/UAS system to enhancer and gene trapping screens in zebrafish has greatly increased the ability to label and manipulate cell populations in multiple tissues, including the central nervous system (CNS). However the ability to select existing lines for specific applications has been limited by the lack of detailed expression analysis. RESULTS: We describe a Gal4 enhancer trap screen in which we used advanced image analysis, including three-dimensional confocal reconstructions and documentation of expression patterns at multiple developmental time points. In all, we have created and annotated 98 lines exhibiting a wide range of expression patterns, most of which include CNS expression. Expression was also observed in nonneural tissues such as muscle, skin epithelium, vasculature, and neural crest derivatives. All lines and data are publicly available from the Zebrafish International Research Center (ZIRC) from the Zebrafish Model Organism Database (ZFIN). CONCLUSIONS: Our detailed documentation of expression patterns, combined with the public availability of images and fish lines, provides a valuable resource for researchers wishing to study CNS development and function in zebrafish. Our data also suggest that many existing enhancer trap lines may have previously uncharacterized expression in multiple tissues and cell types.
Assuntos
Animais Geneticamente Modificados/genética , Sistema Nervoso Central/metabolismo , Proteínas de Ligação a DNA/genética , Elementos Facilitadores Genéticos , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica no Desenvolvimento , Genes Reporter , Imageamento Tridimensional/métodos , Proteínas do Tecido Nervoso/genética , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/genética , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/genética , Animais , Animais Geneticamente Modificados/embriologia , Sistema Nervoso Central/embriologia , Elementos de DNA Transponíveis , Bases de Dados Factuais , Genes Sintéticos , Proteínas Luminescentes/análise , Proteínas Luminescentes/genética , Mutagênese Insercional , Proteínas do Tecido Nervoso/biossíntese , Neurônios/metabolismo , Especificidade de Órgãos , Peixe-Zebra/embriologia , Proteínas de Peixe-Zebra/biossíntese , Proteína Vermelha FluorescenteRESUMO
The vertebrate eye acquires its basic form during the process of optic cup morphogenesis, during which the optic vesicle emerges from the brain neuroepithelium and, through a series of cell and tissue movements, transforms itself into the multilayered optic cup, containing neural retina (comprised of retinal progenitors), retinal pigmented epithelium, and the lens, which is derived from the overlying ectoderm. While great strides have been made to understand the developmental signals controlling specification, patterning, and differentiation of the optic cup, only in recent years have the cellular and molecular bases of optic cup morphogenesis begun to be unraveled. One critical component of the morphogenetic process is the extracellular matrix: the complex, glycoprotein-rich layer that surrounds the optic vesicle and lens. Though the extracellular matrix has long been visualized by classical histological techniques and postulated to play various roles in optic cup development, its functional role was uncertain. This is now beginning to change, as live imaging techniques, quantitative image analyses, molecular genetics and in vitro models yield new insights into the process of optic cup morphogenesis and the specific influences of particular extracellular matrix components and their associated signaling pathways.
Assuntos
Matriz Extracelular/fisiologia , Olho/embriologia , Morfogênese , Vertebrados/embriologia , Animais , Humanos , Cristalino/citologia , Cristalino/embriologia , Retina/embriologia , Epitélio Pigmentado da Retina/embriologia , Epitélio Pigmentado da Retina/fisiologiaRESUMO
Optic cup morphogenesis (OCM) generates the basic structure of the vertebrate eye. Although it is commonly depicted as a series of epithelial sheet folding events, this does not represent an empirically supported model. Here, we combine four-dimensional imaging with custom cell tracking software and photoactivatable fluorophore labeling to determine the cellular dynamics underlying OCM in zebrafish. Although cell division contributes to growth, we find it dispensable for eye formation. OCM depends instead on a complex set of cell movements coordinated between the prospective neural retina, retinal pigmented epithelium (RPE) and lens. Optic vesicle evagination persists for longer than expected; cells move in a pinwheel pattern during optic vesicle elongation and retinal precursors involute around the rim of the invaginating optic cup. We identify unanticipated movements, particularly of central and peripheral retina, RPE and lens. From cell tracking data, we generate retina, RPE and lens subdomain fate maps, which reveal novel adjacencies that might determine corresponding developmental signaling events. Finally, we find that similar movements also occur during chick eye morphogenesis, suggesting that the underlying choreography is conserved among vertebrates.