RESUMO
Many genes are known to regulate retinal regeneration after widespread tissue damage. Conversely, genes controlling regeneration after limited cell loss, as per degenerative diseases, are undefined. As stem/progenitor cell responses scale to injury levels, understanding how the extent and specificity of cell loss impact regenerative processes is important. Here, transgenic zebrafish enabling selective retinal ganglion cell (RGC) ablation were used to identify genes that regulate RGC regeneration. A single cell multiomics-informed screen of 100 genes identified seven knockouts that inhibited and 11 that promoted RGC regeneration. Surprisingly, 35 out of 36 genes known and/or implicated as being required for regeneration after widespread retinal damage were not required for RGC regeneration. The loss of seven even enhanced regeneration kinetics, including the proneural factors neurog1, olig2 and ascl1a. Mechanistic analyses revealed that ascl1a disruption increased the propensity of progenitor cells to produce RGCs, i.e. increased 'fate bias'. These data demonstrate plasticity in the mechanism through which Müller glia convert to a stem-like state and context specificity in how genes function during regeneration. Increased understanding of how the regeneration of disease-relevant cell types is specifically controlled will support the development of disease-tailored regenerative therapeutics.
Assuntos
Animais Geneticamente Modificados , Células Ganglionares da Retina , Proteínas de Peixe-Zebra , Peixe-Zebra , Animais , Peixe-Zebra/genética , Células Ganglionares da Retina/metabolismo , Células Ganglionares da Retina/citologia , Células Ganglionares da Retina/fisiologia , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo , Regeneração Nervosa/genética , Regeneração Nervosa/fisiologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Sistemas CRISPR-Cas/genética , Regeneração/genética , Regeneração/fisiologia , Retina/metabolismo , Retina/citologia , Células-Tronco/metabolismo , Células-Tronco/citologia , Fatores de TranscriçãoRESUMO
Retinal Müller glia (MG) can act as stem-like cells to generate new neurons in both zebrafish and mice. In zebrafish, retinal regeneration is innate and robust, resulting in the replacement of lost neurons and restoration of visual function. In mice, exogenous stimulation of MG is required to reveal a dormant and, to date, limited regenerative capacity. Zebrafish studies have been key in revealing factors that promote regenerative responses in the mammalian eye. Increased understanding of how the regenerative potential of MG is regulated in zebrafish may therefore aid efforts to promote retinal repair therapeutically. Developmental signaling pathways are known to coordinate regeneration following widespread retinal cell loss. In contrast, less is known about how regeneration is regulated in the context of retinal degenerative disease, i.e., following the loss of specific retinal cell types. To address this knowledge gap, we compared transcriptomic responses underlying regeneration following targeted loss of rod photoreceptors or bipolar cells. In total, 2,531 differentially expressed genes (DEGs) were identified, with the majority being paradigm specific, including during early MG activation phases, suggesting the nature of the injury/cell loss informs the regenerative process from initiation onward. For example, early modulation of Notch signaling was implicated in the rod but not bipolar cell ablation paradigm and components of JAK/STAT signaling were implicated in both paradigms. To examine candidate gene roles in rod cell regeneration, including several immune-related factors, CRISPR/Cas9 was used to create G0 mutant larvae (i.e., "crispants"). Rod cell regeneration was inhibited in stat3 crispants, while mutating stat5a/b, c7b and txn accelerated rod regeneration kinetics. These data support emerging evidence that discrete responses follow from selective retinal cell loss and that the immune system plays a key role in regulating "fate-biased" regenerative processes.
Assuntos
Transcriptoma , Peixe-Zebra , Animais , Camundongos , Peixe-Zebra/genética , Animais Geneticamente Modificados , Transcriptoma/genética , Retina/metabolismo , Neurônios , Proliferação de Células , MamíferosRESUMO
Transgenic expression of bacterial nitroreductase (NTR) enzymes sensitizes eukaryotic cells to prodrugs such as metronidazole (MTZ), enabling selective cell-ablation paradigms that have expanded studies of cell function and regeneration in vertebrates. However, first-generation NTRs required confoundingly toxic prodrug treatments to achieve effective cell ablation, and some cell types have proven resistant. Here we used rational engineering and cross-species screening to develop an NTR variant, NTR 2.0, which exhibits ~100-fold improvement in MTZ-mediated cell-specific ablation efficacy, eliminating the need for near-toxic prodrug treatment regimens. NTR 2.0 therefore enables sustained cell-loss paradigms and ablation of previously resistant cell types. These properties permit enhanced interrogations of cell function, extended challenges to the regenerative capacities of discrete stem cell niches, and novel modeling of chronic degenerative diseases. Accordingly, we have created a series of bipartite transgenic reporter/effector resources to facilitate dissemination of NTR 2.0 to the research community.
Assuntos
Metronidazol/farmacologia , Nitrorredutases/metabolismo , Pró-Fármacos/química , Animais , Animais Geneticamente Modificados , Células CHO , Cricetulus , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Metronidazol/farmacocinética , Nitrorredutases/química , Nitrorredutases/genética , Pró-Fármacos/farmacologia , Engenharia de Proteínas/métodos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Retina/citologia , Retina/efeitos dos fármacos , Vibrio/enzimologia , Peixe-Zebra/genéticaRESUMO
The advent of stem cell-derived retinal organoids has brought forth unprecedented opportunities for developmental and physiological studies, while presenting new therapeutic promise for retinal degenerative diseases. From a translational perspective, organoid systems provide exciting new prospects for drug discovery, offering the possibility to perform compound screening in a three-dimensional (3D) human tissue context that resembles the native histoarchitecture and to some extent recapitulates cellular interactions. However, inherent variability issues and a general lack of robust quantitative technologies for analyzing organoids on a large scale pose severe limitations for their use in translational applications. To address this need, we have developed a screening platform that enables accurate quantification of fluorescent reporters in complex human iPSC-derived retinal organoids. This platform incorporates a fluorescence microplate reader that allows xyz-dimensional detection and fine-tuned wavelength selection. We have established optimal parameters for fluorescent reporter signal detection, devised methods to compensate for organoid size variability, evaluated performance and sensitivity parameters, and validated this technology for functional applications.
Assuntos
Técnicas Genéticas , Células-Tronco Pluripotentes Induzidas/citologia , Organoides/fisiologia , Retina/fisiologia , Técnicas de Cultura de Células , Diferenciação Celular , Linhagem Celular , Corantes Fluorescentes , Genes Reporter , Humanos , Microscopia de Fluorescência , Estresse Oxidativo , Transplante de Células-Tronco , Transgenes , Pesquisa Translacional BiomédicaRESUMO
Müller glia (MG) function as inducible retinal stem cells in zebrafish, completely repairing the eye after damage. The innate immune system has recently been shown to promote tissue regeneration in which classic wound-healing responses predominate. However, regulatory roles for leukocytes during cellular regeneration-i.e., selective cell-loss paradigms akin to degenerative disease-are less well defined. To investigate possible roles innate immune cells play during retinal cell regeneration, we used intravital microscopy to visualize neutrophil, macrophage, and retinal microglia responses to induced rod photoreceptor apoptosis. Neutrophils displayed no reactivity to rod cell loss. Peripheral macrophage cells responded to rod cell loss, as evidenced by morphological transitions and increased migration, but did not enter the retina. Retinal microglia displayed multiple hallmarks of immune cell activation: increased migration, translocation to the photoreceptor cell layer, proliferation, and phagocytosis of dying cells. To test function during rod cell regeneration, we coablated microglia and rod cells or applied immune suppression and quantified the kinetics of (i) rod cell clearance, (ii) MG/progenitor cell proliferation, and (iii) rod cell replacement. Coablation and immune suppressants applied before cell loss caused delays in MG/progenitor proliferation rates and slowed the rate of rod cell replacement. Conversely, immune suppressants applied after cell loss had been initiated led to accelerated photoreceptor regeneration kinetics, possibly by promoting rapid resolution of an acute immune response. Our findings suggest that microglia control MG responsiveness to photoreceptor loss and support the development of immune-targeted therapeutic strategies for reversing cell loss associated with degenerative retinal conditions.
Assuntos
Células Ependimogliais/imunologia , Imunidade Inata , Imunomodulação , Regeneração/imunologia , Células Fotorreceptoras Retinianas Bastonetes/fisiologia , Peixe-Zebra/imunologia , Animais , Células Ependimogliais/patologia , Humanos , Células Fotorreceptoras Retinianas Bastonetes/patologiaRESUMO
BACKGROUND: We have investigated a simple strategy for enhancing transgene expression specificity by leveraging genetic silencer elements. The approach serves to restrict transgene expression to a tissue of interest - the nervous system in the example provided here - thereby promoting specific/exclusive targeting of discrete cellular subtypes. Recent innovations are bringing us closer to understanding how the brain is organized, how neural circuits function, and how neurons can be regenerated. Fluorescent proteins enable mapping of the 'connectome', optogenetic tools allow excitable cells to be short-circuited or hyperactivated, and targeted ablation of neuronal subtypes facilitates investigations of circuit function and neuronal regeneration. Optimally, such toolsets need to be expressed solely within the cell types of interest as off-site expression makes establishing causal relationships difficult. To address this, we have exploited a gene 'silencing' system that promotes neuronal specificity by repressing expression in non-neural tissues. This methodology solves non-specific background issues that plague large-scale enhancer trap efforts and may provide a means of leveraging promoters/enhancers that otherwise express too broadly to be of value for in vivo manipulations. RESULTS: We show that a conserved neuron-restrictive silencer element (NRSE) can function to restrict transgene expression to the nervous system. The neuron-restrictive silencing factor/repressor element 1 silencing transcription factor (NRSF/REST) transcriptional repressor binds NRSE/repressor element 1 (RE1) sites and silences gene expression in non-neuronal cells. Inserting NRSE sites into transgenes strongly biased expression to neural tissues. NRSE sequences were effective in restricting expression of bipartite Gal4-based 'driver' transgenes within the context of an enhancer trap and when associated with a defined promoter and enhancer. However, NRSE sequences did not serve to restrict expression of an upstream activating sequence (UAS)-based reporter/effector transgene when associated solely with the UAS element. Morpholino knockdown assays showed that NRSF/REST expression is required for NRSE-based transgene silencing. CONCLUSIONS: Our findings demonstrate that the addition of NRSE sequences to transgenes can provide useful new tools for functional studies of the nervous system. However, the general approach may be more broadly applicable; tissue-specific silencer elements are operable in tissues other than the nervous system, suggesting this approach can be similarly applied to other paradigms. Thus, creating synthetic associations between endogenous regulatory elements and tissue-specific silencers may facilitate targeting of cellular subtypes for which defined promoters/enhancers are lacking.
Assuntos
Neurônios/metabolismo , Proteínas Repressoras/metabolismo , Animais , Animais Geneticamente Modificados , Regulação da Expressão Gênica/fisiologia , Técnicas de Transferência de Genes , Engenharia Genética/métodos , Plasmídeos , Regiões Promotoras Genéticas , Proteínas Repressoras/genética , Transgenes , Peixe-ZebraRESUMO
Retinal Müller glia function as injury-induced stem-like cells in zebrafish but not mammals. However, insights gleaned from zebrafish have been applied to stimulate nascent regenerative responses in the mammalian retina. For instance, microglia/macrophages regulate Müller glia stem cell activity in the chick, zebrafish, and mouse. We previously showed that post-injury immunosuppression by the glucocorticoid dexamethasone accelerated retinal regeneration kinetics in zebrafish. Similarly, microglia ablation enhances regenerative outcomes in the mouse retina. Targeted immunomodulation of microglia reactivity may therefore enhance the regenerative potential of Müller glia for therapeutic purposes. Here, we investigated potential mechanisms by which post-injury dexamethasone accelerates retinal regeneration kinetics, and the effects of dendrimer-based targeting of dexamethasone to reactive microglia. Intravital time-lapse imaging revealed that post-injury dexamethasone inhibited microglia reactivity. The dendrimer-conjugated formulation: (1) decreased dexamethasone-associated systemic toxicity, (2) targeted dexamethasone to reactive microglia, and (3) improved the regeneration enhancing effects of immunosuppression by increasing stem/progenitor proliferation rates. Lastly, we show that the gene rnf2 is required for the enhanced regeneration effect of D-Dex. These data support the use of dendrimer-based targeting of reactive immune cells to reduce toxicity and enhance the regeneration promoting effects of immunosuppressants in the retina.
Assuntos
Dendrímeros , Peixe-Zebra , Animais , Camundongos , Microglia , Dendrímeros/farmacologia , Retina/fisiologia , Terapia de Imunossupressão , Dexametasona/farmacologia , MamíferosRESUMO
Functional screening of environmental DNA (eDNA) libraries is a potentially powerful approach to discover enzymatic "unknown unknowns", but is usually heavily biased toward the tiny subset of genes preferentially transcribed and translated by the screening strain. We have overcome this by preparing an eDNA library via partial digest with restriction enzyme FatI (cuts CATG), causing a substantial proportion of ATG start codons to be precisely aligned with strong plasmid-encoded promoter and ribosome-binding sequences. Whereas we were unable to select nitroreductases from standard metagenome libraries, our FatI strategy yielded 21 nitroreductases spanning eight different enzyme families, each conferring resistance to the nitro-antibiotic niclosamide and sensitivity to the nitro-prodrug metronidazole. We showed expression could be improved by co-expressing rare tRNAs and encoded proteins purified directly using an embedded His6-tag. In a transgenic zebrafish model of metronidazole-mediated targeted cell ablation, our lead MhqN-family nitroreductase proved ~5-fold more effective than the canonical nitroreductase NfsB.
RESUMO
Functional screening of environmental DNA (eDNA) libraries is a potentially powerful approach to discover enzymatic "unknown unknowns", but is usually heavily biased toward the tiny subset of genes preferentially transcribed and translated by the screening strain. We have overcome this by preparing an eDNA library via partial digest with restriction enzyme FatI (cuts CATG), causing a substantial proportion of ATG start codons to be precisely aligned with strong plasmid-encoded promoter and ribosome-binding sequences. Whereas we were unable to select nitroreductases from standard metagenome libraries, our FatI strategy yielded 21 nitroreductases spanning eight different enzyme families, each conferring resistance to the nitro-antibiotic niclosamide and sensitivity to the nitro-prodrug metronidazole. We showed expression could be improved by co-expressing rare tRNAs and encoded proteins purified directly using an embedded His6-tag. In a transgenic zebrafish model of metronidazole-mediated targeted cell ablation, our lead MhqN-family nitroreductase proved â¼5-fold more effective than the canonical nitroreductase NfsB.
Assuntos
Metronidazol , Peixe-Zebra , Animais , Metronidazol/farmacologia , Metronidazol/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Metagenoma , Clonagem Molecular , Nitrorredutases/genéticaRESUMO
Many genes are known to regulate retinal regeneration following widespread tissue damage. Conversely, genes controlling regeneration following limited retinal cell loss, akin to disease conditions, are undefined. Combining a novel retinal ganglion cell (RGC) ablation-based glaucoma model, single cell omics, and rapid CRISPR/Cas9-based knockout methods to screen 100 genes, we identified 18 effectors of RGC regeneration kinetics. Surprisingly, 32 of 33 previously known/implicated regulators of retinal tissue regeneration were not required for RGC replacement; 7 knockouts accelerated regeneration, including sox2, olig2, and ascl1a . Mechanistic analyses revealed loss of ascl1a increased "fate bias", the propensity of progenitors to produce RGCs. These data demonstrate plasticity and context-specificity in how genes function to control regeneration, insights that could help to advance disease-tailored therapeutics for replacing lost retinal cells. One sentence summary: We discovered eighteen genes that regulate the regeneration of retinal ganglion cells in zebrafish.
RESUMO
Retinitis pigmentosa (RP) and associated inherited retinal diseases (IRDs) are caused by rod photoreceptor degeneration, necessitating therapeutics promoting rod photoreceptor survival. To address this, we tested compounds for neuroprotective effects in multiple zebrafish and mouse RP models, reasoning drugs effective across species and/or independent of disease mutation may translate better clinically. We first performed a large-scale phenotypic drug screen for compounds promoting rod cell survival in a larval zebrafish model of inducible RP. We tested 2934 compounds, mostly human-approved drugs, across six concentrations, resulting in 113 compounds being identified as hits. Secondary tests of 42 high-priority hits confirmed eleven lead candidates. Leads were then evaluated in a series of mouse RP models in an effort to identify compounds effective across species and RP models, that is, potential pan-disease therapeutics. Nine of 11 leads exhibited neuroprotective effects in mouse primary photoreceptor cultures, and three promoted photoreceptor survival in mouse rd1 retinal explants. Both shared and complementary mechanisms of action were implicated across leads. Shared target tests implicated parp1-dependent cell death in our zebrafish RP model. Complementation tests revealed enhanced and additive/synergistic neuroprotective effects of paired drug combinations in mouse photoreceptor cultures and zebrafish, respectively. These results highlight the value of cross-species/multi-model phenotypic drug discovery and suggest combinatorial drug therapies may provide enhanced therapeutic benefits for RP patients.
Photoreceptors are the cells responsible for vision. They are part of the retina: the light-sensing tissue at the back of the eye. They come in two types: rods and cones. Rods specialise in night vision, while cones specialise in daytime colour vision. The death of these cells can cause a disease, called retinitis pigmentosa, that leads to vision loss. Symptoms often start in childhood with a gradual loss of night vision. Later on, loss of cone photoreceptors can lead to total blindness. Unfortunately, there are no treatments available that protect photoreceptor cells from dying. Research has identified drugs that can protect photoreceptors in animal models, but these drugs have failed in humans. The classic way to look for new treatments is to find drugs that target molecules implicated in a disease, and then test them to see if they are effective. Unfortunately, many drugs identified in this way fail in later stages of testing, either because they are ineffective, or because they have unacceptable side effects. One way to reverse this trend is to first test whether a drug is effective at curing a disease in animals, and later determining what it does at a molecular level. This could reveal whether drugs can protect photoreceptors before research to discover their molecular targets begins. Tests like this across different species could maximise the chances of finding a drug that works in humans, because if a drug works in several species, it is more likely to have shared target molecules across species. Applying this reasoning, Zhang et al. tested around 3,000 drug candidates for treating retinitis pigmentosa in a strain of zebrafish that undergoes photoreceptor degeneration similar to the human disease. Most of these drug candidates already have approval for use in humans, meaning that if they were found to be effective for treating retinitis pigmentosa, they could be fast-tracked for use in people. Zhang et al. found three compounds that helped photoreceptors survive both in zebrafish and in retinas grown in the laboratory derived from a mouse strain with degeneration similar to retinitis pigmentosa. Tests to find out how these three compounds worked at the molecular level revealed that they interfered with a protein that can trigger cell death. The tests also found other promising compounds, many of which offered increased protection when combined in pairs. Worldwide there are between 1.5 and 2.5 million people with retinitis pigmentosa. With this disease, loss of vision happens slowly, so identifying drugs that could slow or stop the process could help many people. These results suggest that placing animal testing earlier in the drug discovery process could complement traditional target-based methods. The compounds identified here, and the information about how they work, could expand potential treatment research. The next step in this research is to test whether the drugs identified by Zhang et al. protect mammals other than mice from the degeneration seen in retinitis pigmentosa.
Assuntos
Fármacos Neuroprotetores/farmacologia , Retinose Pigmentar/tratamento farmacológico , Animais , Animais Geneticamente Modificados , Células Cultivadas/efeitos dos fármacos , Nucleotídeo Cíclico Fosfodiesterase do Tipo 6/genética , Nucleotídeo Cíclico Fosfodiesterase do Tipo 6/metabolismo , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Regulação da Expressão Gênica/efeitos dos fármacos , Camundongos , Mutação , Poli(ADP-Ribose) Polimerase-1/genética , Poli(ADP-Ribose) Polimerase-1/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/efeitos dos fármacos , Peixe-ZebraRESUMO
Several key attributes of zebrafish make them an ideal model system for the discovery and development of regeneration promoting therapeutics; most notably their robust capacity for self-repair which extends to the central nervous system. Further, by enabling large-scale drug discovery directly in living vertebrate disease models, zebrafish circumvent critical bottlenecks which have driven drug development costs up. This review summarizes currently available zebrafish phenotypic screening platforms, HTS-ready neurodegenerative disease modeling strategies, zebrafish small molecule screens which have succeeded in identifying regeneration promoting compounds and explores how intravital imaging in zebrafish can facilitate comprehensive analysis of nanocarrier biodistribution and pharmacokinetics. Finally, we discuss the benefits and challenges attending the combination of zebrafish and nanoparticle-based drug optimization, highlighting inspiring proof-of-concept studies and looking toward implementation across the drug development community.
Assuntos
Nanomedicina , Nanopartículas/química , Doenças Neurodegenerativas/tratamento farmacológico , Fármacos Neuroprotetores/uso terapêutico , Animais , Sistemas de Liberação de Medicamentos , Descoberta de Drogas , Humanos , Fármacos Neuroprotetores/química , Medicina Regenerativa , Peixe-ZebraRESUMO
Thousands of genes have been implicated in retinal regeneration, but only a few have been shown to impact the regenerative capacity of Müller glia-an adult retinal stem cell with untapped therapeutic potential. Similarly, among nearly 300 genetic loci associated with human retinal disease, the majority remain untested in animal models. To address the large-scale nature of these problems, we are applying CRISPR/Cas9-based genome modification strategies in zebrafish to target over 300 genes implicated in retinal regeneration or degeneration. Our intent is to enable large-scale reverse genetic screens by applying a multiplexed gene disruption strategy that markedly increases the efficiency of the screening process. To facilitate large-scale phenotyping, we incorporate an automated reporter quantification-based assay to identify cellular degeneration and regeneration-deficient phenotypes in transgenic fish. Multiplexed gene targeting strategies can address mismatches in scale between "big data" bioinformatics and wet lab experimental capacities, a critical shortfall limiting comprehensive functional analyses of factors implicated in ever-expanding multiomics datasets. This report details the progress we have made to date with a multiplexed CRISPR/Cas9-based gene targeting strategy and discusses how the methodologies applied can further our understanding of the genes that predispose to retinal degenerative disease and which control the regenerative capacity of retinal Müller glia cells.
RESUMO
Transgenic expression of bacterial nitroreductase (NTR) facilitates chemically-inducible targeted cell ablation. In zebrafish, the NTR system enables studies of cell function and cellular regeneration. Metronidazole (MTZ) has become the most commonly used prodrug substrate for eliciting cell loss in NTR-expressing transgenic zebrafish due to the cell-specific nature of its cytotoxic derivatives. Unfortunately, MTZ treatments required for effective cell ablation border toxic effects, and, thus, likely incur undesirable nonspecific effects. Here, we tested whether a triple mutant variant of NTR, previously shown to display improved activity in bacterial assays, can solve this issue by promoting cell ablation in zebrafish using reduced prodrug treatment regimens. We generated several complementary transgenic zebrafish lines expressing either wild-type or mutant NTR (mutNTR) in specific neural cell types, and assayed prodrug-induced cell ablation kinetics using confocal time series imaging and plate reader-based quantification of fluorescent reporters expressed in targeted cell types. The results show that cell ablation can be achieved in mutNTR expressing transgenic lines with markedly shortened prodrug exposure times and/or at lower prodrug concentrations. The mutNTR variant characterized here can circumvent problematic nonspecific/toxic effects arising from low prodrug conversion efficiency, thus increasing the effectiveness and versatility of this selective cell ablation methodology.
Assuntos
Técnicas de Ablação/métodos , Proteínas de Escherichia coli/genética , Escherichia coli/enzimologia , Metronidazol/metabolismo , Neurônios/fisiologia , Nitrorredutases/genética , Pró-Fármacos/metabolismo , Regeneração/efeitos dos fármacos , Peixe-Zebra/fisiologia , Animais , Animais Geneticamente Modificados/genética , Animais Geneticamente Modificados/fisiologia , Morte Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Larva/genética , Larva/fisiologia , Metronidazol/farmacologia , Nitrorredutases/metabolismo , Pró-Fármacos/farmacologia , Fatores de Tempo , Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimentoRESUMO
Due to several inherent advantages, zebrafish are being utilized in increasingly sophisticated screens to assess the physiological effects of chemical compounds directly in living vertebrate organisms. Diverse screening platforms showcase these advantages. Morphological assays encompassing basic qualitative observations to automated imaging, manipulation, and data-processing systems provide whole organism to subcellular levels of detail. Behavioral screens extend chemical screening to the level of complex systems. In addition, zebrafish-based disease models provide a means of identifying new potential therapeutic strategies. Automated systems for handling/sorting, high-resolution imaging and quantitative data collection have significantly increased throughput in recent years. These advances will make it easier to capture multiple streams of information from a given sample and facilitate integration of zebrafish at the earliest stages of the drug-discovery process, providing potential solutions to current drug-development bottlenecks. Here we outline advances that have been made within the growing field of zebrafish chemical screening.
Assuntos
Descoberta de Drogas/métodos , Peixe-Zebra/fisiologia , Animais , Comportamento Animal/efeitos dos fármacos , Ensaios de Triagem em Larga Escala/métodos , Proteínas Luminescentes/análise , Peixe-Zebra/anatomia & histologiaRESUMO
Reporter-based assays underlie many high-throughput screening (HTS) platforms, but most are limited to in vitro applications. Here, we report a simple whole-organism HTS method for quantifying changes in reporter intensity in individual zebrafish over time termed, Automated Reporter Quantification in vivo (ARQiv). ARQiv differs from current "high-content" (e.g., confocal imaging-based) whole-organism screening technologies by providing a purely quantitative data acquisition approach that affords marked improvements in throughput. ARQiv uses a fluorescence microplate reader with specific detection functionalities necessary for robust quantification of reporter signals in vivo. This approach is: 1) Rapid; achieving true HTS capacities (i.e., >50,000 units per day), 2) Reproducible; attaining HTS-compatible assay quality (i.e., Z'-factors of ≥0.5), and 3) Flexible; amenable to nearly any reporter-based assay in zebrafish embryos, larvae, or juveniles. ARQiv is used here to quantify changes in: 1) Cell number; loss and regeneration of two different fluorescently tagged cell types (pancreatic beta cells and rod photoreceptors), 2) Cell signaling; relative activity of a transgenic Notch-signaling reporter, and 3) Cell metabolism; accumulation of reactive oxygen species. In summary, ARQiv is a versatile and readily accessible approach facilitating evaluation of genetic and/or chemical manipulations in living zebrafish that complements current "high-content" whole-organism screening methods by providing a first-tier in vivo HTS drug discovery platform.
Assuntos
Dosagem de Genes , Genes Reporter , Ensaios de Triagem em Larga Escala/métodos , Processamento de Imagem Assistida por Computador/métodos , Peixe-Zebra , Animais , Animais Geneticamente Modificados , Automação/métodos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Embrião não Mamífero , Dosagem de Genes/fisiologia , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Concentração Osmolar , Reprodutibilidade dos Testes , Estudos de Validação como Assunto , Peixe-Zebra/embriologia , Peixe-Zebra/genéticaRESUMO
The use of real-time reporters has accelerated our understanding of gene expression in vivo. This study examined the feasibility of a luciferase-based reporter to image spatiotemporal changes in fetal gene expression in utero. We chose to monitor Period1 (Per1) because it is expressed broadly in the body and plays a role in circadian rhythmicity. Using rats carrying a Per1::luc transgene, we repetitively imaged fetuses in utero throughout gestation. We found that bioluminescence was specific to transgenic pups, increased dramatically on embryonic day 10 (10 days after successful mating), and continued to increase logarithmically until birth. Diurnal fluctuations in Per1 expression were apparent several days prior to birth. These results demonstrate the feasibility of in utero imaging of mammalian gene expression, tracking of fetal gene expression from the same litter, and early detection of mammalian clock gene expression. We conclude that luciferase-based reporters can provide a sensitive, noninvasive measure of in utero gene expression.
Assuntos
Proteínas do Olho/genética , Genes Reporter , Medições Luminescentes/métodos , Útero/química , Animais , Animais Geneticamente Modificados , Feminino , Expressão Gênica , Luciferases/análise , Luciferases/genética , Proteínas Circadianas Period , Ratos , Útero/metabolismoRESUMO
Circadian pacemakers drive many daily molecular, physiological and behavioural rhythms. We investigated whether the main olfactory bulb is a functional circadian pacemaker in rats. Long-term, multielectrode recordings revealed that individual, cultured bulb neurons expressed near 24-h oscillations in firing rate. Real-time recordings of Period1 gene activity showed that a population of cells within the bulb expressed synchronized rhythmicity starting on embryonic day 19. This rhythmicity was intrinsic to the mitral, and not the granule, cell layer, entrainable to physiological temperature cycles and temperature compensated in its period. However, removal of the olfactory bulbs had no effect on running wheel behaviour. These results indicate that individual mitral/tufted cells are competent circadian pacemakers which normally synchronize to each other. The daily rhythms in gene expression and firing rate intrinsic to the olfactory bulb are not required for circadian patterns of locomotion, indicating that they are involved in rhythms outside the canonical circadian system.
Assuntos
Ritmo Circadiano/fisiologia , Neurônios/metabolismo , Bulbo Olfatório/metabolismo , Animais , Animais Geneticamente Modificados , Animais Recém-Nascidos , Proteínas de Ciclo Celular , Feminino , Regulação da Expressão Gênica/fisiologia , Masculino , Atividade Motora/fisiologia , Proteínas Nucleares/biossíntese , Proteínas Nucleares/genética , Proteínas Circadianas Period , Gravidez , Ratos , Ratos WistarRESUMO
Notch receptors and the amyloid precursor protein are type I membrane proteins that are proteolytically cleaved within their transmembrane domains by a presenilin (PS)-dependent gamma-secretase activity. In both proteins, two peptide bonds are hydrolyzed: one near the inner leaflet and the other in the middle of the transmembrane domain. Under saturating conditions the substrates compete with each other for proteolysis, but not for binding to PS. At least some Alzheimer's disease-causing PS mutations reside in proteins possessing low catalytic activity. We demonstrate (i) that differentially tagged PS molecules coimmunoprecipitate, and (ii) that PS N-terminal fragment dimers exist by using a photoaffinity probe based on a transition state analog gamma-secretase inhibitor. We propose that gamma-secretase contains a PS dimer in its catalytic core, that binding of substrate is at a site separate from the active site, and that substrate is cleaved at the interface of two PS molecules.