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
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
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
Epithelia provide a crucial protective barrier for our organs and are also the sites where the majority of carcinomas form. Most studies on epithelia and carcinomas use cell culture or organisms where high-resolution live imaging is inaccessible without invasive techniques. Here, we introduce the developing zebrafish epidermis as an excellent in vivo model system for studying a living epithelium. We developed tools to fluorescently tag specific epithelial cell types and express genes in a mosaic fashion using five Gal4 lines identified from an enhancer trap screen. When crossed to a variety of UAS effector lines, we can now track, ablate or monitor single cells at sub-cellular resolution. Using photo-cleavable morpholino oligonucleotides that target gal4, we can also express genes in a mosaic fashion at specific times during development. Together, this system provides an excellent in vivo alternative to tissue culture cells, without the intrinsic concerns of culture conditions or transformation, and enables the investigation of distinct cell types within living epithelial tissues.
Assuntos
Técnicas Citológicas/métodos , Células Epidérmicas , Peixe-Zebra/metabolismo , Animais , Morte Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Cruzamentos Genéticos , Proteínas de Ligação a DNA/metabolismo , Elementos Facilitadores Genéticos/genética , Epiderme/efeitos dos fármacos , Epiderme/ultraestrutura , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Feminino , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Imageamento Tridimensional , Masculino , Morfolinos/farmacologia , Fatores de Tempo , Fatores de Transcrição/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/genética , 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
Attractive growth cone turning requires Igf2bp1-dependent local translation of ß-actin mRNA in response to external cues in vitro. While in vivo studies have shown that Igf2bp1 is required for cell migration and axon terminal branching, a requirement for Igf2bp1 function during axon outgrowth has not been demonstrated. Using a timelapse assay in the zebrafish retinotectal system, we demonstrate that the ß-actin 3'UTR is sufficient to target local translation of the photoconvertible fluorescent protein Kaede in growth cones of pathfinding retinal ganglion cells (RGCs) in vivo. Igf2bp1 knockdown reduced RGC axonal outgrowth and tectal coverage and retinal cell survival. RGC-specific expression of a phosphomimetic Igf2bp1 reduced the density of axonal projections in the optic tract while sparing RGCs, demonstrating for the first time that Igf2bp1 is required during axon outgrowth in vivo. Therefore, regulation of local translation mediated by Igf2bp proteins may be required at all stages of axon development.
Assuntos
Axônios/fisiologia , Proteínas de Ligação a RNA/fisiologia , Células Ganglionares da Retina/fisiologia , Proteínas de Peixe-Zebra/fisiologia , Actinas/fisiologia , Animais , Técnicas de Silenciamento de Genes , Peixe-Zebra/crescimento & desenvolvimento , Peixe-Zebra/fisiologiaRESUMO
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
Mirror movements are involuntary movements on one side of the body that occur simultaneously with intentional movements on the contralateral side. Humans with heterozygous mutations in the axon guidance receptor DCC display such mirror movements, where unilateral stimulation results in inappropriate bilateral motor output. Currently, it is unclear whether mirror movements are caused by incomplete midline crossing and reduced commissural connectivity of DCC-dependent descending pathways or by aberrant ectopic ipsilateral axonal projections of normally commissural neurons. Here, we show that in response to unilateral tactile stimuli, zebrafish dcc mutant larvae perform involuntary turns on the inappropriate body side. We show that these mirror movement-like deficits are associated with axonal guidance defects of two identified groups of commissural reticulospinal hindbrain neurons. Moreover, we demonstrate that in dcc mutants, axons of these identified neurons frequently fail to cross the midline and instead project ipsilaterally. Whereas laser ablation of these neurons in wild-type animals does not affect turning movements, their ablation in dcc mutants restores turning movements. Thus, our results demonstrate that in dcc mutants, turns on the inappropriate side of the body are caused by aberrant ipsilateral axonal projections, and suggest that aberrant ipsilateral connectivity of a very small number of descending axons is sufficient to induce incorrect movement patterns.
Assuntos
Genes DCC/genética , Genes DCC/fisiologia , Mutação/fisiologia , Neurônios/fisiologia , Reflexo de Sobressalto/fisiologia , Rombencéfalo/fisiologia , Peixe-Zebra/fisiologia , Animais , Axônios/fisiologia , Comportamento Animal/fisiologia , Mapeamento Cromossômico , DNA Complementar/biossíntese , DNA Complementar/genética , Imunofluorescência , Deleção de Genes , Genótipo , Interneurônios/fisiologia , Larva , Mutação de Sentido Incorreto/genética , Mutação de Sentido Incorreto/fisiologia , Vias Neurais/fisiologia , Fenótipo , Rombencéfalo/citologia , Rombencéfalo/metabolismo , Natação/fisiologia , Tato/fisiologiaRESUMO
Proximal spinal muscular atrophy (SMA) is the most common inherited motor neuropathy and the leading hereditary cause of infant mortality. Currently there is no effective treatment for the disease, reflecting a need for pharmacologic interventions that restore performance of dysfunctional motor neurons or suppress the consequences of their dysfunction. In a series of assays relevant to motor neuron biology, we explored the activities of a collection of tetrahydroindoles that were reported to alter the metabolism of amyloid precursor protein (APP). In Drosophila larvae the compounds suppressed aberrant larval locomotion due to mutations in the Khc and Klc genes, which respectively encode the heavy and light chains of kinesin-1. A representative compound of this class also suppressed the appearance of axonal swellings (alternatively termed axonal spheroids or neuritic beads) in the segmental nerves of the kinesin-deficient Drosophila larvae. Given the importance of kinesin-dependent transport for extension and maintenance of axons and their growth cones, three members of the class were tested for neurotrophic effects on isolated rat spinal motor neurons. Each compound stimulated neurite outgrowth. In addition, consistent with SMA being an axonopathy of motor neurons, the three axonotrophic compounds rescued motor axon development in a zebrafish model of SMA. The results introduce a collection of small molecules as pharmacologic suppressors of SMA-associated phenotypes and nominate specific members of the collection for development as candidate SMA therapeutics. More generally, the results reinforce the perception of SMA as an axonopathy and suggest novel approaches to treating the disease.
Assuntos
Axônios/efeitos dos fármacos , Drosophila melanogaster/metabolismo , Indóis/farmacologia , Cinesinas/deficiência , Neurônios Motores/efeitos dos fármacos , Atrofia Muscular Espinal/patologia , Peixe-Zebra , Secretases da Proteína Precursora do Amiloide/metabolismo , Peptídeos beta-Amiloides/biossíntese , Animais , Axônios/metabolismo , Modelos Animais de Doenças , Drosophila melanogaster/efeitos dos fármacos , Feminino , Indóis/química , Indóis/uso terapêutico , Larva/efeitos dos fármacos , Larva/metabolismo , Locomoção/efeitos dos fármacos , Masculino , Neurônios Motores/metabolismo , Atrofia Muscular Espinal/tratamento farmacológico , Atrofia Muscular Espinal/fisiopatologia , Neuritos/efeitos dos fármacos , Neuritos/metabolismo , Fragmentos de Peptídeos/biossíntese , Medula Espinal/patologiaRESUMO
Proper arrangement of axonal projections into topographic maps is crucial for brain function, especially in sensory systems. An important mechanism for map formation is pretarget axon sorting, in which topographic ordering of axons appears in tracts before axons reach their target, but this process remains poorly understood. Here, we show that selective axon degeneration is used as a correction mechanism to eliminate missorted axons in the optic tract during retinotectal development in zebrafish. Retinal axons are not precisely ordered during initial pathfinding but become corrected later, with missorted axons selectively fragmenting and degenerating. We further show that heparan sulfate is required non-cell-autonomously to correct missorted axons and that restoring its synthesis at late stages in a deficient mutant is sufficient to restore topographic sorting. These findings uncover a function for developmental axon degeneration in ordering axonal projections and identify heparan sulfate as a key regulator of that process.
Assuntos
Movimento Celular/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Proteoglicanas/metabolismo , Vias Visuais/fisiologia , Adenilil Imidodifosfato/farmacologia , Animais , Animais Geneticamente Modificados , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Corantes/metabolismo , Embrião não Mamífero , Lateralidade Funcional/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Heparitina Sulfato/metabolismo , Técnicas In Vitro , Microscopia Confocal , Morfolinos/farmacologia , Mutação/genética , Degeneração Neural/genética , Degeneração Neural/patologia , Degeneração Neural/cirurgia , Proteoglicanas/genética , Retina/citologia , Células Ganglionares da Retina/transplante , Fatores de Tempo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Vias Visuais/embriologia , Peixe-Zebra , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
Proteoglycans (PGs) modulate numerous signaling pathways during development through binding of their glycosaminoglycan (GAG) side chains to various signaling molecules, including fibroblast growth factors (FGFs). A majority of PGs possess two or more GAG side chains, suggesting that GAG multivalency is imperative for biological functions in vivo. However, only a few studies have examined the biological significance of GAG multivalency. In this report, we utilized a library of bis- and tris-xylosides that produce two and three GAG chains on the same scaffold, respectively, thus mimicking PGs, to examine the importance of GAG valency and chain type in regulating FGF/FGFR interactions in vivo in zebrafish. A number of bis- and tris-xylosides, but not mono-xylosides, caused an elongation phenotype upon their injection into embryos. In situ hybridization showed that elongated embryos have elevated expression of the FGF target gene mkp3 but unchanged expression of reporters for other pathways, indicating that FGF/FGFR signaling was specifically hyperactivated. In support of this observation, elongation can be reversed by the tyrosine kinase inhibitor SU5402, mRNA for the FGFR antagonist sprouty4, or FGF8 morpholino. Endogenous GAGs seem to be unaffected after xyloside treatment, suggesting that this is a gain-of-function phenotype. Furthermore, expression of a multivalent but not a monovalent GAG containing syndecan-1 proteoglycan recapitulates the elongation phenotype observed with the bivalent xylosides. On the basis of these in vivo findings, we propose a new model for GAG/FGF/FGFR interactions in which dimerized GAG chains can activate FGF-mediated signal transduction pathways.
Assuntos
Fatores de Crescimento de Fibroblastos/metabolismo , Glicosaminoglicanos/metabolismo , Peixe-Zebra/embriologia , Animais , Animais Geneticamente Modificados , Sequência de Bases , Dimerização , Embrião não Mamífero/efeitos dos fármacos , Embrião não Mamífero/metabolismo , Fatores de Crescimento de Fibroblastos/genética , Regulação da Expressão Gênica no Desenvolvimento , Glicosaminoglicanos/química , Glicosaminoglicanos/farmacologia , Glicosídeos/química , Hibridização In Situ , Dados de Sequência Molecular , Inibidores de Proteínas Quinases/farmacologia , Pirróis/farmacologia , Receptores de Fatores de Crescimento de Fibroblastos/antagonistas & inibidores , Receptores de Fatores de Crescimento de Fibroblastos/genética , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Transdução de Sinais , Sindecana-1/metabolismo , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
Previous studies have raised the possibility that Wnt signaling may regulate both neural progenitor maintenance and neuronal differentiation within a single population. Here we investigate the role of Wnt/ß-catenin activity in the zebrafish hypothalamus and find that the pathway is first required for the proliferation of unspecified hypothalamic progenitors in the embryo. At later stages, including adulthood, sequential activation and inhibition of Wnt activity is required for the differentiation of neural progenitors and negatively regulates radial glia differentiation. The presence of Wnt activity is conserved in hypothalamic progenitors of the adult mouse, where it plays a conserved role in inhibiting the differentiation of radial glia. This study establishes the vertebrate hypothalamus as a model for Wnt-regulated postembryonic neural progenitor differentiation and defines specific roles for Wnt signaling in neurogenesis.
Assuntos
Hipotálamo/citologia , Neurogênese , Células-Tronco/citologia , Proteínas Wnt/metabolismo , Via de Sinalização Wnt , Peixe-Zebra/crescimento & desenvolvimento , Animais , Hipotálamo/metabolismo , Camundongos , Neuroglia/citologia , Neuroglia/metabolismo , Células-Tronco/metabolismo , Peixe-Zebra/embriologiaRESUMO
foxP2, a forkhead-domain transcription factor, is critical for speech and language development in humans, but its role in the establishment of CNS connectivity is unclear. While in vitro studies have identified axon guidance molecules as targets of foxP2 regulation, and cell culture assays suggest a role for foxP2 in neurite outgrowth, in vivo studies have been lacking regarding a role for foxP2 in axon pathfinding. We used a modified zinc finger nuclease methodology to generate mutations in the zebrafish foxP2 gene. Using PCR-based high resolution melt curve analysis (HRMA) of G0 founder animals, we screened and identified three mutants carrying nonsense mutations in the 2(nd) coding exon: a 17 base-pair (bp) deletion, an 8bp deletion, and a 4bp insertion. Sequence analysis of cDNA confirmed that these were frameshift mutations with predicted early protein truncations. Homozygous mutant fish were viable and fertile, with unchanged body morphology, and no apparent differences in CNS apoptosis, proliferation, or patterning at embryonic stages. There was a reduction in expression of the known foxP2 target gene cntnap2 that was rescued by injection of wild-type foxP2 transcript. When we examined axon pathfinding using a pan-axonal marker or transgenic lines, including a foxP2-neuron-specific enhancer, we did not observe any axon guidance errors. Our findings suggest that foxP2 is not necessary for axon pathfinding during development.
Assuntos
Axônios/metabolismo , Encéfalo/metabolismo , Fatores de Transcrição Forkhead/genética , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/genética , Animais , Animais Geneticamente Modificados , Fatores de Transcrição Forkhead/metabolismo , Mutação , Neuritos/metabolismo , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Dedos de Zinco/genéticaRESUMO
Dorsal retinal fate is established early in eye development, via expression of spatially restricted dorsal-specific transcription factors in the optic vesicle; yet the events leading to initiation of dorsal fate are not clear. We hypothesized that induction of dorsal fate would require an extraocular signal arising from a neighboring tissue to pattern the prospective dorsal retina, however no such signal has been identified. We used the zebrafish embryo to determine the source, timing, and identity of the dorsal retina-inducing signal. Extensive cell movements occur during zebrafish optic vesicle morphogenesis, however the location of prospective dorsal cells within the early optic vesicle and their spatial relationship to early dorsal markers is currently unknown. Our mRNA expression and fate mapping analyses demonstrate that the dorsolateral optic vesicle is the earliest region to express dorsal specific markers, and cells from this domain contribute to the dorsal retinal pole at 24 hpf. We show that three bmp genes marking dorsal retina at 25 hpf are also expressed extraocularly before retinal patterning begins. We identified gdf6a as a dorsal initiation signal acting from the extraocular non-neural ectoderm during optic vesicle evagination. We find that bmp2b is involved in dorsal retina initiation, acting upstream of gdf6a. Together, this work has identified the nature and source of extraocular signals required to pattern the dorsal retina.
Assuntos
Proteína Morfogenética Óssea 2/metabolismo , Ectoderma/fisiologia , Olho/embriologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Fator 6 de Diferenciação de Crescimento/metabolismo , Morfogênese/fisiologia , Retina/embriologia , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Animais , Diferenciação Celular/fisiologia , Primers do DNA/genética , Ectoderma/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Genótipo , Hibridização In Situ , Reação em Cadeia da Polimerase , Pirazóis , Pirimidinas , Retina/citologia , Peixe-Zebra/genéticaRESUMO
BACKGROUND: Growth cone navigation across the vertebrate midline is critical in the establishment of nervous system connectivity. While midline crossing is achieved through coordinated signaling of attractive and repulsive cues, this has never been demonstrated at the single cell level. Further, though growth cone responsiveness to guidance cues changes after crossing the midline, it is unclear whether midline crossing itself is required for subsequent guidance decisions in vivo. In the zebrafish, spinal commissures are initially formed by a pioneer neuron called CoPA (Commissural Primary Ascending). Unlike in other vertebrate models, CoPA navigates the midline alone, allowing for single-cell analysis of axon guidance mechanisms. RESULTS: We provide evidence that CoPA expresses the known axon guidance receptors dcc, robo3 and robo2. Using loss of function mutants and gene knockdown, we show that the functions of these genes are evolutionarily conserved in teleosts and that they are used consecutively by CoPA neurons. We also reveal novel roles for robo2 and robo3 in maintaining commissure structure. When midline crossing is prevented in robo3 mutants and dcc gene knockdown, ipsilaterally projecting neurons respond to postcrossing guidance cues. Furthermore, DCC inhibits Robo2 function before midline crossing to allow a midline approach and crossing. CONCLUSIONS: Our results demonstrate that midline crossing is not required for subsequent guidance decisions by pioneer axons and that this is due, in part, to DCC inhibition of Robo2 function prior to midline crossing.
Assuntos
Axônios/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Neurogênese/fisiologia , Neurônios/citologia , Medula Espinal/citologia , Animais , Animais Geneticamente Modificados , Axônios/efeitos dos fármacos , Receptor DCC , Embrião não Mamífero , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Proteínas de Fluorescência Verde/genética , Microscopia Confocal , Morfolinas/farmacologia , Mutação/genética , Neurogênese/efeitos dos fármacos , Neurogênese/genética , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Receptores Imunológicos/genética , Receptores Imunológicos/metabolismo , Análise de Célula Única , Medula Espinal/embriologia , Peixe-Zebra , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
Successful axon pathfinding requires both correct patterning of tissues, which will later harbor axonal tracts, and precise localization of axon guidance cues along these tracts at the time of axon outgrowth. Retinal ganglion cell (RGC) axons grow towards the optic disc in the central retina, where they turn to exit the eye through the optic nerve. Normal patterning of the optic disc and stalk and the expression of guidance cues at this choice point are necessary for the exit of RGC axons out of the eye. Sonic hedgehog (Shh) has been implicated in both patterning of ocular tissue and direct guidance of RGC axons. Here, we examine the precise spatial and temporal requirement for Hedgehog (Hh) signaling for intraretinal axon pathfinding and show that Shh acts to pattern the optic stalk in zebrafish but does not guide RGC axons inside the eye directly. We further reveal an interaction between the Hh and chemokine pathways for axon guidance and show that cxcl12a functions downstream of Shh and depends on Shh for its expression at the optic disc. Together, our results support a model in which Shh acts in RGC axon pathfinding indirectly by regulating axon guidance cues at the optic disc through patterning of the optic stalk.
Assuntos
Axônios/metabolismo , Quimiocinas/metabolismo , Proteínas Hedgehog/metabolismo , Nervo Óptico/metabolismo , Retina/metabolismo , Animais , Proteínas Hedgehog/genética , Disco Óptico/citologia , Disco Óptico/metabolismo , Nervo Óptico/citologia , Retina/citologia , Células Ganglionares da Retina/citologia , Células Ganglionares da Retina/metabolismo , Vias Visuais/citologia , Vias Visuais/metabolismo , Peixe-ZebraRESUMO
For an epithelium to provide a protective barrier, it must maintain homeostatic cell numbers by matching the number of dividing cells with the number of dying cells. Although compensatory cell division can be triggered by dying cells, it is unknown how cell death might relieve overcrowding due to proliferation. When we trigger apoptosis in epithelia, dying cells are extruded to preserve a functional barrier. Extrusion occurs by cells destined to die signalling to surrounding epithelial cells to contract an actomyosin ring that squeezes the dying cell out. However, it is not clear what drives cell death during normal homeostasis. Here we show in human, canine and zebrafish cells that overcrowding due to proliferation and migration induces extrusion of live cells to control epithelial cell numbers. Extrusion of live cells occurs at sites where the highest crowding occurs in vivo and can be induced by experimentally overcrowding monolayers in vitro. Like apoptotic cell extrusion, live cell extrusion resulting from overcrowding also requires sphingosine 1-phosphate signalling and Rho-kinase-dependent myosin contraction, but is distinguished by signalling through stretch-activated channels. Moreover, disruption of a stretch-activated channel, Piezo1, in zebrafish prevents extrusion and leads to the formation of epithelial cell masses. Our findings reveal that during homeostatic turnover, growth and division of epithelial cells on a confined substratum cause overcrowding that leads to their extrusion and consequent death owing to the loss of survival factors. These results suggest that live cell extrusion could be a tumour-suppressive mechanism that prevents the accumulation of excess epithelial cells.
Assuntos
Células Epiteliais/citologia , Homeostase , Nadadeiras de Animais/anatomia & histologia , Nadadeiras de Animais/citologia , Nadadeiras de Animais/embriologia , Animais , Apoptose , Contagem de Células , Morte Celular , Linhagem Celular , Movimento Celular , Proliferação de Células , Sobrevivência Celular , Colo/citologia , Cães , Embrião não Mamífero/citologia , Embrião não Mamífero/embriologia , Células Epidérmicas , Epiderme/embriologia , Humanos , Canais Iônicos/deficiência , Canais Iônicos/genética , Canais Iônicos/metabolismo , Lisofosfolipídeos/metabolismo , Modelos Biológicos , Neoplasias/patologia , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Peixe-Zebra/anatomia & histologia , Peixe-Zebra/embriologia , Proteínas de Peixe-Zebra/deficiência , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
2D image space methods are processing methods applied after the volumetric data are projected and rendered into the 2D image space, such as 2D filtering, tone mapping and compositing. In the application domain of volume visualization, most 2D image space methods can be carried out more efficiently than their 3D counterparts. Most importantly, 2D image space methods can be used to enhance volume visualization quality when applied together with volume rendering methods. In this paper, we present and discuss the applications of a series of 2D image space methods as enhancements to confocal microscopy visualizations, including 2D tone mapping, 2D compositing, and 2D color mapping. These methods are easily integrated with our existing confocal visualization tool, FluoRender, and the outcome is a full-featured visualization system that meets neurobiologists' demands for qualitative analysis of confocal microscopy data.
RESUMO
Extracting neural structures with their fine details from confocal volumes is essential to quantitative analysis in neurobiology research. Despite the abundance of various segmentation methods and tools, for complex neural structures, both manual and semi-automatic methods are ine ective either in full 3D or when user interactions are restricted to 2D slices. Novel interaction techniques and fast algorithms are demanded by neurobiologists to interactively and intuitively extract neural structures from confocal data. In this paper, we present such an algorithm-technique combination, which lets users interactively select desired structures from visualization results instead of 2D slices. By integrating the segmentation functions with a confocal visualization tool neurobiologists can easily extract complex neural structures within their typical visualization workflow.
RESUMO
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.