RESUMEN
NADPH oxidase (Nox)-derived reactive oxygen species (ROS) have been linked to neuronal polarity, axonal outgrowth, cerebellar development, regeneration of sensory axons, and neuroplasticity. However, the specific roles that individual Nox isoforms play during nervous system development in vivo remain unclear. To address this problem, we investigated the role of Nox activity in the development of retinotectal connections in zebrafish embryos. Zebrafish broadly express four nox genes (nox1, nox2/cybb, nox5, and duox) throughout the CNS during early development. Application of a pan-Nox inhibitor, celastrol, during the time of optic nerve (ON) outgrowth resulted in significant expansion of the ganglion cell layer (GCL), thinning of the ON, and a decrease in retinal axons reaching the optic tectum (OT). With the exception of GCL expansion, these effects were partially ameliorated by the addition of H2O2, a key ROS involved in Nox signaling. To address isoform-specific Nox functions, we used CRISPR/Cas9 to generate mutations in each zebrafish nox gene. We found that nox2/cybb chimeric mutants displayed ON thinning and decreased OT innervation. Furthermore, nox2/cybb homozygous mutants (nox2/cybb-/-) showed significant GCL expansion and mistargeted retinal axons in the OT. Neurite outgrowth from cultured zebrafish retinal ganglion cells was reduced by Nox inhibitors, suggesting a cell-autonomous role for Nox in these neurons. Collectively, our results show that Nox2/Cybb is important for retinotectal development in zebrafish.SIGNIFICANCE STATEMENT Most isoforms of NADPH oxidase (Nox) only produce reactive oxygen species (ROS) when activated by an upstream signal, making them ideal candidates for ROS signaling. Nox enzymes are present in neurons and their activity has been shown to be important for neuronal development and function largely by in vitro studies. However, whether Nox is involved in the development of axons and formation of neuronal connections in vivo has remained unclear. Using mutant zebrafish embryos, this study shows that a specific Nox isoform, Nox2/Cybb, is important for the establishment of axonal connections between retinal ganglion cells and the optic tectum.
Asunto(s)
NADPH Oxidasa 2/metabolismo , Neurogénesis/fisiología , Lóbulo Óptico de Animales no Mamíferos/embriología , Retina/embriología , Vías Visuales/embriología , Animales , Embrión no Mamífero , Lóbulo Óptico de Animales no Mamíferos/metabolismo , Retina/metabolismo , Vías Visuales/metabolismo , Pez CebraRESUMEN
Zebrafish are a popular vertebrate model in drug discovery. They produce a large number of small and rapidly-developing embryos. These embryos display rich visual-behaviors that can be used to screen drugs for treating retinal degeneration (RD). RD comprises blinding diseases such as Retinitis Pigmentosa, which affects 1 in 4000 people. This disease has no definitive cure, emphasizing an urgency to identify new drugs. In this review, we will discuss advantages, challenges, and research developments in using zebrafish behaviors to screen drugs in vivo. We will specifically discuss a visual-motor response that can potentially expedite discovery of new RD drugs.
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Evaluación Preclínica de Medicamentos/métodos , Retina/efectos de los fármacos , Degeneración Retiniana/tratamiento farmacológico , Pez Cebra/fisiología , Animales , Modelos Animales de Enfermedad , Retina/patología , Degeneración Retiniana/patología , Visión Ocular/efectos de los fármacosRESUMEN
The chromatin remodeler CHD5 plays a critical role in tumor suppression and neurogenesis in mammals. CHD5 contributes to gene expression during neurogenesis, but there is still much to learn regarding how this class of remodelers contributes to differentiation and development. CHD5 remodelers are vertebrate-specific, raising the prospect that CHD5 plays one or more conserved roles in this phylum. Expression of chd5 in adult fish closely mirrors expression of CHD5 in adult mammals. Knockdown of Chd5 during embryogenesis suggests new roles for CHD5 remodelers based on resulting defects in craniofacial development including reduced head and eye size as well as reduced cartilage formation in the head. In addition, knockdown of Chd5 results in altered expression of neural markers in the developing brain and eye as well as a profound defect in differentiation of dopaminergic amacrine cells. Recombinant zebrafish Chd5 protein exhibits nucleosome remodeling activity in vitro, suggesting that it is the loss of this activity that contributes to the observed phenotypes. Our studies indicate that zebrafish is an appropriate model for functional characterization of CHD5 remodelers in vertebrates and highlight the potential of this model for generating novel insights into the role of this vital class of remodelers.
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ADN Helicasas/fisiología , Desarrollo Embrionario/genética , Cabeza/embriología , Proteínas de Pez Cebra/fisiología , Pez Cebra/embriología , Adenosina Trifosfatasas/genética , Animales , Animales Modificados Genéticamente , Encéfalo/embriología , Encéfalo/metabolismo , Cartílago/embriología , Cartílago/metabolismo , Diferenciación Celular/genética , Ensamble y Desensamble de Cromatina/genética , ADN Helicasas/genética , Neuronas Dopaminérgicas/fisiología , Embrión no Mamífero , Ojo/embriología , Ojo/metabolismo , Neurogénesis/genética , Pez Cebra/genética , Proteínas de Pez Cebra/genéticaRESUMEN
BACKGROUND: Early growth response 1 (egr1) is a transcription factor (TF) for controlling the differentiation of Parvalbumin (Parv) -expressing amacrine cells (ACs) in zebrafish. However, the downstream factors of this process have not been identified. The purpose of this study was to investigate the role of p35, a neuronal-specific activator of cyclin-dependent kinase 5 (Cdk5) and a known in vitro target of egr1, in the differentiation of these ACs. RESULTS: In the p35-knockdown retinas, Parv+ but not islet1+ ACs were specifically reduced. This phenotype was highly similar to that in the Egr1-knockdown retinas. Furthermore, p35 expression was reduced in the Egr1-knockdown retinas, particularly in the AC region; while egr1 was only modestly reduced in this region in the p35-knockdown retinas. CONCLUSIONS: p35 likely acts downstream of egr1 to control the differentiation of Parv+ ACs.
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Células Amacrinas/fisiología , Diferenciación Celular/fisiología , Proteína 1 de la Respuesta de Crecimiento Precoz/metabolismo , Regulación del Desarrollo de la Expresión Génica/genética , Proteínas del Tejido Nervioso/metabolismo , Retina/embriología , Pez Cebra/embriología , Animales , Diferenciación Celular/genética , Cartilla de ADN/genética , Técnicas de Silenciamiento del Gen , Procesamiento de Imagen Asistido por Computador , Inmunohistoquímica , Hibridación in Situ , Microscopía Fluorescente , Morfolinos/genética , Parvalbúminas/metabolismo , Retina/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
PURPOSE: The purpose of this study was to develop a framework for analyzing retinal pigment epithelium (RPE) expression profiles from zebrafish eye mutants. METHODS: The fish model we used was SWI/SNF-related, matrix associated, actin dependent regulator of chromatin, subfamily a, member 4 (smarca4), a retinal dystrophic mutant with a previously described retinal phenotype and expression profiles. Histological and Affymetrix GeneChip analyses were conducted to characterize the RPE defects and underlying differential expression, respectively. RESULTS: Histological analysis revealed that smarca4 RPE was formed, but its differentiation was abnormal. In particular, ultrastructural analysis of smarca4 RPE by transmission electron microscopy demonstrated several defects in melanogenesis. The nature of these defects also suggests that the cytoskeletal dynamics, which are tightly linked with melanogenesis, were impaired in smarca4 RPE. To compare the expression profile of normal wild-type (WT) and smarca4 RPE, the gene expression profiles of microdissected retinas and RPE-attached retinas were measured with Affymetrix GeneChip analysis. The RPE expression values were then estimated from these samples by subtracting the retinal expression values from the expression values of the RPE-attached retinas. A factorial analysis was conducted using the expression values of the RPE, retinal, and whole-embryo samples. Specific rules (contrasts) were built using the coefficients of the resulting fitted models to select for three groups of genes: 1) smarca4-regulated RPE genes, 2) smarca4-regulated retinal genes, and 3) smarca4-regulated RPE genes that are not differentially expressed in the retina. Interestingly, the third group consists of 39 genes that are highly related to cytoskeletal dynamics, melanogenesis, and paracrine and intracellular signal transduction. CONCLUSIONS: Our analytical framework provides an experimental approach to identify differentially-regulated genes in the retina and the RPE of zebrafish mutants in which both of these tissues are affected by the underlying mutation. Specifically, we have used the method to identify a group of 39 genes that can potentially explain the melanogenesis defect in the smarca4 RPE. In addition, several genes in this group are secreted signaling molecules. Thus, this observation further implicates that the smarca4 RPE might play a role in the retinal dystrophic phenotype in smarca4.
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Proteínas Adaptadoras Transductoras de Señales/genética , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Mutación/genética , Epitelio Pigmentado de la Retina/patología , Proteínas de Pez Cebra/genética , Pez Cebra/embriología , Pez Cebra/genética , Animales , Diferenciación Celular/genética , Melanosomas/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Epitelio Pigmentado de la Retina/embriología , Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/ultraestructura , Transducción de Señal/genéticaRESUMEN
BACKGROUND: Zebrafish are vertebrates with a high potential of regeneration after injury in the central nervous system. Therefore, they have emerged as a useful model system for studying traumatic spinal cord injuries. NEW METHOD: Using larval zebrafish, we have developed a robust paradigm to model the effects of anterior spinal cord injury, which correspond to the debilitating injuries of the cervical and thoracic regions in humans. Our new paradigm consists of a more anterior injury location compared to previous studies, a modified behavioral assessment using the visual motor response, and a new data analysis code. RESULTS: Our approach enables a spinal cord injury closer to the hindbrain with more functional impact compared to previous studies using a more posterior injury location. Results reported in this work reveal recovery over seven days following spinal cord injury. COMPARING WITH EXISTING METHODS: The present work describes a modified paradigm for the in vivo study of spinal cord regeneration after injury using larval zebrafish, including an anterior injury location, a robust behavioral assessment, and a new data analysis software. CONCLUSIONS: Our findings lay the foundation for applying this paradigm to study the effects of drugs, nutrition, and other treatments to improve the regeneration process.
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Modelos Animales de Enfermedad , Larva , Traumatismos de la Médula Espinal , Pez Cebra , Animales , Traumatismos de la Médula Espinal/fisiopatología , Recuperación de la Función/fisiología , Regeneración de la Medula Espinal/fisiología , Médula Espinal/fisiopatología , Regeneración Nerviosa/fisiologíaRESUMEN
BACKGROUND: In a recent genomic study, Leung et al. used a factorial microarray analysis to identify Smarca4 (Brg1)-regulated genes in micro-dissected zebrafish retinas. Two hundred and fifty nine genes were grouped in three-way ANOVA models which carried the most specific retinal change. To validate the microarray results and to elucidate cellular expression patterns of the significant genes for further characterization, 32 known genes were randomly selected from this group. In situ hybridization of these genes was performed on the same types of samples (wild-type (WT) and smarca4a50/a50 (yng) mutant) at the same stages (36 and 52 hours post-fertilization (hpf)) as in the microarray study. RESULTS: Thirty out of 32 riboprobes showed a positive in situ staining signal. Twenty seven out of these 30 genes were originally further classified as Smarca4-regulated retinal genes, while the remaining three as retinal-specific expression independent of Smarca4 regulation. It was found that 90.32% of the significant microarray comparisons that were used to identify Smarca4-regulated retinal genes had a corresponding qualitative expression change in the in situ hybridization comparisons. This is highly concordant with the theoretical true discovery rate of 95%. Hierarchical clustering was used to investigate the similarity of the cellular expression patterns of 25 out of the 27 Smarca4-regulated retinal genes that had a sufficiently high expression signal for an unambiguous identification of retinal expression domains. Three broad groups of expression pattern were identified; including 1) photoreceptor layer/outer nuclear layer specific expression at 52 hpf, 2) ganglion cell layer (GCL) and/or inner nuclear layer (INL) specific expression at both 36 & 52 hpf, and 3) GCL and/or INL specific expression at 52 hpf only. Some of these genes have recently been demonstrated to play key roles in retinal cell-type specification, differentiation and lamination. For the remaining three retinal-specific genes that are independent of Smarca4 regulation, they all had a subtle expression difference between WT and smarca4a50/a50 retinas as detected by in situ hybridization. This subtle expression difference was also detected by the original microarray analysis. However, the difference was lower than the fold change cut-off used in that study and hence these genes were not inferred as Smarca4-regulated retinal genes. CONCLUSIONS: This study has successfully investigated the expression pattern of 32 genes identified from the original factorial microarray analysis. The results have demonstrated that the true discovery rate for identifying Smarca4-regulated retinal genes is 90.3%. Hence, the significant genes from the microarray study are good candidates for cell-type specific markers and will aid further investigation of retinal differentiation.
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ADN Helicasas/fisiología , Regulación de la Expresión Génica , Retina/metabolismo , Proteínas de Pez Cebra/fisiología , Pez Cebra/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Animales , ADN Helicasas/genética , ADN Helicasas/metabolismo , Embrión no Mamífero/citología , Embrión no Mamífero/metabolismo , Análisis por Micromatrices , Retina/citología , Retina/embriología , Células Ganglionares de la Retina/citología , Células Ganglionares de la Retina/metabolismo , Pez Cebra/embriología , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
In a zebrafish recessive mutant young (yng), retinal cells are specified to distinct cell classes, but they fail to morphologically differentiate. A null mutation in a brahma-related gene 1 (brg1) is responsible for this phenotype. To identify retina-specific Brg1-regulated genes that control cellular differentiation, we conducted a factorial microarray analysis. Gene expression profiles were compared from wild-type and yng retinas and stage-matched whole embryos at 36 and 52 hours postfertilization (hpf). From our analysis, three categories of genes were identified: (i) Brg1-regulated retinal differentiation genes (731 probesets), (ii) retina-specific genes independent of Brg1 regulation (3,038 probesets), and (iii) Brg1-regulated genes outside the retina (107 probesets). Biological significance was confirmed by further analysis of components of the Cdk5 signaling pathway and Irx transcription factor family, representing genes identified in category 1. This study highlights the utility of factorial microarray analysis to efficiently identify relevant regulatory pathways influenced by both specific gene products and normal developmental events.
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Regulación del Desarrollo de la Expresión Génica , Análisis por Micromatrices , Retina/embriología , Pez Cebra/embriología , Pez Cebra/genética , Proteínas Adaptadoras Transductoras de Señales , Animales , Ciclo Celular , Quinasa 5 Dependiente de la Ciclina/metabolismo , ADN Helicasas/genética , ADN Helicasas/metabolismo , Genes del Desarrollo , Modelos Genéticos , Organogénesis , Reproducibilidad de los Resultados , Retina/citología , Retina/enzimología , Transducción de Señal , Transcripción Genética , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
Retinitis Pigmentosa (RP) is a mostly incurable inherited retinal degeneration affecting approximately 1 in 4000 individuals globally. The goal of this work was to identify drugs that can help patients suffering from the disease. To accomplish this, we screened drugs on a zebrafish autosomal dominant RP model. This model expresses a truncated human rhodopsin transgene (Q344X) causing significant rod degeneration by 7 days post-fertilization (dpf). Consequently, the larvae displayed a deficit in visual motor response (VMR) under scotopic condition. The diminished VMR was leveraged to screen an ENZO SCREEN-WELL REDOX library since oxidative stress is postulated to play a role in RP progression. Our screening identified a beta-blocker, carvedilol, that ameliorated the deficient VMR of the RP larvae and increased their rod number. Carvedilol may directly on rods as it affected the adrenergic pathway in the photoreceptor-like human Y79 cell line. Since carvedilol is an FDA-approved drug, our findings suggest that carvedilol can potentially be repurposed to treat autosomal dominant RP patients.
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Animales Modificados Genéticamente , Conducta Animal/efectos de los fármacos , Enfermedades Genéticas Congénitas , Retinitis Pigmentosa , Rodopsina , Visión Ocular , Pez Cebra , Animales , Animales Modificados Genéticamente/genética , Animales Modificados Genéticamente/metabolismo , Línea Celular , Evaluación Preclínica de Medicamentos , Enfermedades Genéticas Congénitas/tratamiento farmacológico , Enfermedades Genéticas Congénitas/genética , Enfermedades Genéticas Congénitas/metabolismo , Humanos , Mutación , Células Fotorreceptoras Retinianas Bastones , Retinitis Pigmentosa/tratamiento farmacológico , Retinitis Pigmentosa/genética , Retinitis Pigmentosa/metabolismo , Rodopsina/genética , Rodopsina/metabolismo , Transgenes , Visión Ocular/efectos de los fármacos , Visión Ocular/inmunología , Pez Cebra/genética , Pez Cebra/metabolismoRESUMEN
Purpose: Although zebrafish rods begin to develop as early as 2 days postfertilization (dpf), they are not deemed anatomically mature and functional until 15 to 21 dpf. A recent study detected a small electroretinogram (ERG) from rods in a cone mutant called no optokinetic response f (nof) at 5 dpf, suggesting that young rods are functional. Whether they can mediate behavioral responses in larvae is unknown. Methods: We first confirmed rod function by measuring nof ERGs under photopic and scotopic illumination at 6 dpf. We evaluated the role of rods in visual behaviors using two different assays: the visual-motor response (VMR) and optokinetic response (OKR). We measured responses from wild-type (WT) larvae and nof mutants under photopic and scotopic illuminations at 6 dpf. Results: Nof mutants lacked a photopic ERG. However, after prolonged dark adaptation, they displayed scotopic ERGs. Compared with WT larvae, the nof mutants displayed reduced VMRs. The VMR difference during light onset gradually diminished with decreased illumination and became nearly identical at lower light intensities. Additionally, light-adapted nof mutants did not display an OKR, whereas dark-adapted nof mutants displayed scotopic OKRs. Conclusions: Because the nof mutants lacked a photopic ERG but displayed scotopic ERGs after dark adaptation, the mutants clearly had functional rods. WT larvae and the nof mutants displayed comparable scotopic light-On VMRs and scotopic OKRs after dark adaptation, suggesting that these responses were driven primarily by rods. Together, these observations indicate that rods contribute to zebrafish visual behaviors as early as 6 dpf.
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Células Fotorreceptoras Retinianas Bastones/fisiología , Visión Ocular/fisiología , Pez Cebra/fisiología , Animales , Visión de Colores/fisiología , Electrorretinografía , Técnicas de Genotipaje , Larva , Visión Nocturna/fisiología , Nistagmo Optoquinético/fisiologíaRESUMEN
Current medications inadequately treat the symptoms of chronic pain experienced by over 50 million people in the United States, and may come with substantial adverse effects signifying the need to find novel treatments. One novel therapeutic target is the Transient Receptor Potential A1 channel (TRPA1), an ion channel that mediates nociception through calcium influx of sensory neurons. Drug discovery still relies heavily on animal models, including zebrafish, a species in which TRPA1 activation produces hyperlocomotion. Here, we investigated if this hyperlocomotion follows zebrafish TRPA1 pharmacology and evaluated the strengths and limitations of using TRPA1-mediated hyperlocomotion as potential preclinical screening tool for drug discovery. To support face validity of the model, we pharmacologically characterized mouse and zebrafish TRPA1 in transfected HEK293 cells using calcium assays as well as in vivo. TRPA1 agonists and antagonists respectively activated or blocked TRPA1 activity in HEK293 cells, mice, and zebrafish in a dose-dependent manner. However, our results revealed complexities including partial agonist activity of TRPA1 antagonists, bidirectional locomotor activity, receptor desensitization, and off-target effects. We propose that TRPA1-mediated hyperlocomotion in zebrafish larvae has the potential to be used as in vivo screening tool for novel anti-nociceptive drugs but requires careful evaluation of the TRPA1 pharmacology.
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Descubrimiento de Drogas , Locomoción/efectos de los fármacos , Dolor Nociceptivo/genética , Canal Catiónico TRPA1/genética , Proteínas de Pez Cebra/genética , Animales , Células HEK293 , Humanos , Locomoción/genética , Masculino , Ratones , Nocicepción/efectos de los fármacos , Dolor Nociceptivo/tratamiento farmacológico , Dolor Nociceptivo/patología , Canal Catiónico TRPA1/antagonistas & inhibidores , Pez Cebra/genética , Pez Cebra/crecimiento & desarrollo , Proteínas de Pez Cebra/antagonistas & inhibidoresRESUMEN
Many contemporary neuroscience experiments utilize high-throughput approaches to simultaneously collect behavioural data from many animals. The resulting data are often complex in structure and are subjected to systematic biases, which require new approaches for analysis and normalization. This study addressed the normalization need by establishing an approach based on linear-regression modeling. The model was established using a dataset of visual motor response (VMR) obtained from several strains of wild-type (WT) zebrafish collected at multiple stages of development. The VMR is a locomotor response triggered by drastic light change, and is commonly measured repeatedly from multiple larvae arrayed in 96-well plates. This assay is subjected to several systematic variations. For example, the light emitted by the machine varies slightly from well to well. In addition to the light-intensity variation, biological replication also created batch-batch variation. These systematic variations may result in differences in the VMR and must be normalized. Our normalization approach explicitly modeled the effect of these systematic variations on VMR. It also normalized the activity profiles of different conditions to a common baseline. Our approach is versatile, as it can incorporate different normalization needs as separate factors. The versatility was demonstrated by an integrated normalization of three factors: light-intensity variation, batch-batch variation and baseline. After normalization, new biological insights were revealed from the data. For example, we found larvae of TL strain at 6 days post-fertilization (dpf) responded to light onset much stronger than the 9-dpf larvae, whereas previous analysis without normalization shows that their responses were relatively comparable. By removing systematic variations, our model-based normalization can facilitate downstream statistical comparisons and aid detecting true biological differences in high-throughput studies of neurobehaviour.
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Conducta Animal/fisiología , Bases de Datos Factuales , Actividad Motora/fisiología , Pez Cebra/fisiología , AnimalesRESUMEN
Ocular coloboma is a developmental structural defect of the eye that often occurs as complex ocular anomalies. However, its genetic etiology remains largely unexplored. Here we report the identification of mutation (c.331C>T, p.R111C) in the IPO13 gene in a consanguineous family with ocular coloboma, microphthalmia, and cataract by a combination of whole-exome sequencing and homozygosity mapping. IPO13 encodes an importin-B family protein and has been proven to be associated with the pathogenesis of coloboma and microphthalmia. We found that Ipo13 was expressed in the cornea, sclera, lens, and retina in mice. Additionally, the mRNA expression level of Ipo13 decreased significantly in the patient compared with its expression in a healthy individual. Morpholino-oligonucleotide-induced knockdown of ipo13 in zebrafish caused dose-dependent microphthalmia and coloboma, which is highly similar to the ocular phenotypes in the patient. Moreover, both visual motor response and optokinetic response were impaired severely. Notably, these ocular phenotypes in ipo13-deficient zebrafish could be rescued remarkably by full-length ipo13 mRNA, suggesting that the phenotypes observed in zebrafish were due to insufficient ipo13 function. Altogether, our findings demonstrate, for the first time, a new role of IPO13 in eye morphogenesis and that loss of function of IPO13 could lead to ocular coloboma, microphthalmia, and cataract in humans and zebrafish.
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Catarata/genética , Coloboma/genética , Carioferinas/genética , Microftalmía/genética , Mutación Puntual , Pez Cebra/genética , Adulto , Animales , Modelos Animales de Enfermedad , Técnicas de Silenciamiento del Gen , Humanos , Masculino , Ratones , Modelos Moleculares , TranscriptomaRESUMEN
Microarray technology is a powerful approach for genomics research. The multi-step, data-intensive nature of this technology has created an unprecedented informatics and analytical challenge. It is important to understand the crucial steps that can affect the outcome of the analysis. In this review, we provide an overview of the contemporary trend on various main analysis steps in the microarray data analysis process, which includes experimental design, data standardization, image acquisition and analysis, normalization, statistical significance inference, exploratory data analysis, class prediction and pathway analysis, as well as various considerations relevant to their implementation.
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ADN Complementario , Interpretación Estadística de Datos , Análisis de Secuencia por Matrices de Oligonucleótidos , Análisis de Secuencia de ADN/métodos , Animales , HumanosRESUMEN
PURPOSE: Eye development and photoreceptor maintenance is dependent on the retinal pigment epithelium (RPE), a thin layer of cells that underlies the neural retina. Despite its importance, development of RPE has not been studied by a genomic approach. In this study, a microarray expression-profiling methodology was established for studying RPE development. METHODS: The intact retina with RPE attached was dissected from developing embryos, and differentially expressed genes in RPE were inferred by comparing the dissected tissues with retinas without RPE, in microarray and statistical analyses. RESULTS: Of the probesets used, 8810 were significantly expressed in RPE at 52 hours postfertilization (hpf), of which 1443 may have biologically meaningful expression levels. Further, 78 and 988 probesets were found to be significantly over- or underexpressed in RPE, respectively, compared with retina. Also, 79.2% (38/48) of the known overexpressed probesets were independently validated as RPE-related transcripts. CONCLUSIONS: The results strongly suggest that this methodology can obtain in vivo RPE-specific gene expression from the zebrafish embryos and identify novel RPE markers.
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Embrión no Mamífero/metabolismo , Regulación del Desarrollo de la Expresión Génica/fisiología , Epitelio Pigmentado Ocular/embriología , Proteínas de Pez Cebra/genética , Pez Cebra/embriología , Animales , Proteínas del Ojo/genética , Perfilación de la Expresión Génica/métodos , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , ARN/aislamiento & purificación , Retina/embriologíaRESUMEN
Retinal degeneration often affects the whole retina even though the disease-causing gene is specifically expressed in the light-sensitive photoreceptors. The molecular basis of the retinal defect can potentially be determined by gene-expression profiling of the whole retina. In this study, we measured the gene-expression profile of retinas microdissected from a zebrafish pde6cw59 (pde6c) mutant. This retinal-degeneration model not only displays cone degeneration caused by a cone-specific mutation, but also other secondary cellular changes starting from 4 days postfertilization (dpf). To capture the underlying molecular changes, we subjected pde6c and wild-type (WT) retinas at 5 dpf/ 120 h postfertilization (hpf) to RNA sequencing (RNA-Seq) on the Illumina HiSeq 2,000 platform. We also validated the RNA-Seq results by Reverse Transcription Quantitative Polymerase Chain Reaction (RT-qPCR) of seven phototransduction genes. Our analyses indicate that the RNA-Seq dataset was of high quality, and effectively captured the molecular changes in the whole pde6c retina. This dataset will facilitate the characterization of the molecular defects in the pde6c retina at the initial stage of retinal degeneration.
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Fosfodiesterasas de Nucleótidos Cíclicos Tipo 6/genética , Retina/metabolismo , Degeneración Retiniana/genética , Proteínas de Pez Cebra/genética , Animales , Análisis por Micromatrices , Transcriptoma , Pez CebraRESUMEN
Upon a drastic change in environmental illumination, zebrafish larvae display a rapid locomotor response. This response can be simultaneously tracked from larvae arranged in multi-well plates. The resulting data have provided new insights into neuro-behaviour. The features of these data, however, present a challenge to traditional statistical tests. For example, many larvae display little or no movement. Thus, the larval responses have many zero values and are imbalanced. These responses are also measured repeatedly from the same well, which results in correlated observations. These analytical issues were addressed in this study by the generalized linear mixed model (GLMM). This approach deals with binary responses and characterizes the correlation of observations in the same group. It was used to analyze a previously reported dataset. Before applying the GLMM, the activity values were transformed to binary responses (movement vs. no movement) to reduce data imbalance. Moreover, the GLMM estimated the variations among the effects of different well locations, which would eliminate the location effects when two biological groups or conditions were compared. By addressing the data-imbalance and location-correlation issues, the GLMM effectively quantified true biological effects on zebrafish locomotor response.
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Conducta Animal , Modelos Lineales , Locomoción , Actividad Motora , Pez Cebra/fisiología , Algoritmos , Análisis de Varianza , Animales , Larva , Estadios del Ciclo de Vida , Modelos Estadísticos , Especificidad de la Especie , Factores de TiempoRESUMEN
Nicotinamide dinucleotide phosphate oxidases (NOX) control various cellular signaling cascades. In the nervous system, there is recent evidence that NOX-derived reactive oxygen species (ROS) regulate neurite outgrowth, regeneration, and stem cell proliferation; however, a comprehensive NOX gene expression analysis is missing for all major model systems. Zebrafish embryos provide an excellent model system to study neurodevelopment and regeneration because they develop quickly and are well suited for in vivo imaging and molecular approaches. Although the sequences of five NOX genes (nox1, nox2/cybb, nox4, nox5, and duox) have been identified in the zebrafish genome, nothing is known about their expression pattern. Here, we used quantitative polymerase chain reaction combined with in situ hybridization to develop a catalog of nox1, nox2/cybb, nox5, and duox expression in zebrafish during early nervous system development from 12 to 48 hours post fertilization. We found that expression levels of nox1, nox5, and duox are dynamic during the first 2 days of development, whereas nox2/cybb levels remain remarkably stable. By sectioning in situ hybridized embryos, we found a pattern of broad and overlapping NOX isoform expression at 1 and 1.5 days post fertilization. After 2 days of development, a few brain regions displayed increased NOX expression levels. Collectively, these results represent the first comprehensive analysis of NOX gene expression in the zebrafish and will provide a basis for future studies aimed at determining the functions of NOX enzymes in neurodevelopment and regeneration. J. Comp. Neurol. 524:2130-2141, 2016. © 2015 Wiley Periodicals, Inc.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica/fisiología , NADPH Oxidasas/metabolismo , Pez Cebra , Animales , Embrión no Mamífero , NADPH Oxidasas/genética , ARN Mensajero/metabolismo , Pez Cebra/embriología , Pez Cebra/crecimiento & desarrollo , Pez Cebra/metabolismoRESUMEN
BACKGROUND: The mouse double minute 1 (Mdm1) gene was first reported and cloned in mouse tumor cell lines as an oncogene candidate. Later, it was found that mutation of Mdm1 might cause age-related retinal degeneration 2 in mice by genetic linkage analysis. Additionally, the MDM1 protein was found to be expressed in the centrosomes, cilia, and the nucleus of multiciliated tracheal epithelial cells in mice. These observations suggest that MDM1 may have some basal functions in cell physiology. However, the evolutionary history of this gene and its expression during embryonic development remain largely unexplored. RESULTS: Using molecular phylogenetic analysis, we found that the MDM1 gene encoded an evolutionarily conserved protein across all metazoans. We also found that the MDM1 gene was in a conserved synteny in vertebrates. In almost all the species that were analyzed, there was only one MDM1 gene based on current genome annotations. Since vertebrate genomes underwent two to three rounds of whole-genome duplications around the origin of the vertebrates, it is interesting that only one MDM1 ohnolog was retained. This observation implies that other MDM1 ohnologs were lost after the whole-genome duplications. Furthermore, using whole-mount in situ hybridization, we found that mdm1 was expressed in the forebrain, nephric ducts, and tail buds during zebrafish early embryonic development. CONCLUSION: MDM1 is an evolutionary conserved gene, and its homologous genes can be traced back to basal metazoan lineages. In vertebrates, the MDM1 gene is in a conserved synteny and there is only one MDM1 ohnolog suggesting it is a "duplication-resistant" gene. Its expression patterns in early zebrafish embryos indicate that mdm1 may play important roles in the development of the central nervous system, kidneys, and hematopoietic system.
RESUMEN
Gene co-option, usually after gene duplication, in the evolution of development is found to contribute to vertebrate morphological innovations, including the endothelium-based vascular system. Recently, a zebrafish kank gene was found expressed in the vascular vessel primordium, suggesting KANK genes are a component of the developmental tool kit for the vertebrate vascular system. However, how the KANK gene family is involved in vascular vessel development during evolution remains largely unknown. First, we analyzed the molecular evolution of the KANK genes in metazoan, and found that KANK1, KANK2, KANK3 and KANK4 emerged in the lineage of vertebrate, consistent with the two rounds of vertebrate whole-genome duplications (WGD). Moreover, KANK genes were further duplicated in teleosts through the bony-fish specific WGD, while only kank1 and kank4 duplicates were retained in some of the examined fish species. We also found all zebrafish kank genes, except kank1b, are primarily expressed during embryonic vascular development. Compared to invertebrate KANK gene expression in the central nervous system, the vascular expression of zebrafish kank genes suggested KANK genes were co-opted for vertebrate vascular development. Given the cellular roles of KANK genes, our results suggest that this co-option may facilitate the evolutionary origin of vertebrate vascular vessels.