RESUMEN
The trafficking of intracellular vesicles is essential for a number of cellular processes and defects in this process have been implicated in a wide range of human diseases. We identify the zebrafish mutant lbk as a novel model for such disorders. lbk displays hypopigmentation of skin melanocytes and the retinal pigment epithelium (RPE), an absence of iridophore reflections, defects in internal organs (liver, intestine) as well as functional defects in vision and the innate immune system (macrophages). Positional cloning, an allele screen, rescue experiments and morpholino knock-down reveal a mutation in the zebrafish orthologue of the vam6/vps39 gene. Vam6p is part of the HOPS complex, which is essential for vesicle tethering and fusion. Affected cells in the lbk RPE, liver, intestine and macrophages display increased numbers and enlarged intracellular vesicles. Physiological and behavioural analyses reveal severe defects in visual ability in lbk mutants. The present study provides the first phenotypic description of a lack of vam6 gene function in a multicellular organism. lbk shares many of the characteristics of human diseases and suggests a novel disease gene for pathologies associated with defective vesicle transport, including the arthrogryposis-renal dysfunction-cholestasis (ARC) syndrome, the Hermansky-Pudlak syndrome, the Chediak-Higashi syndrome and the Griscelli syndrome.
Asunto(s)
Endosomas/metabolismo , Endosomas/patología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Atrofia de Múltiples Sistemas/patología , Mutación/genética , Vesículas Transportadoras/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Proteínas de Pez Cebra/metabolismo , Pez Cebra/genética , Secuencia de Aminoácidos , Animales , Transporte Biológico/efectos de los fármacos , Mapeo Cromosómico , Endosomas/efectos de los fármacos , Tracto Gastrointestinal/efectos de los fármacos , Tracto Gastrointestinal/patología , Tracto Gastrointestinal/ultraestructura , Hepatomegalia/patología , Humanos , Inmunidad Innata/efectos de los fármacos , Péptidos y Proteínas de Señalización Intracelular/química , Péptidos y Proteínas de Señalización Intracelular/genética , Larva/efectos de los fármacos , Larva/microbiología , Hígado/efectos de los fármacos , Hígado/patología , Hígado/ultraestructura , Datos de Secuencia Molecular , Oligonucleótidos Antisentido/farmacología , Fenotipo , Epitelio Pigmentado Ocular/efectos de los fármacos , Epitelio Pigmentado Ocular/patología , Epitelio Pigmentado Ocular/ultraestructura , Pigmentación/efectos de los fármacos , Vesículas Transportadoras/efectos de los fármacos , Proteínas de Transporte Vesicular/química , Proteínas de Transporte Vesicular/genética , Visión Ocular/efectos de los fármacos , Pez Cebra/embriología , Pez Cebra/inmunología , Proteínas de Pez Cebra/química , Proteínas de Pez Cebra/genéticaRESUMEN
PURPOSE: To characterize morphologic and physiological alterations in the retina of three laminin mutant zebrafish, bashful (bal, lama1), grumpy (gup, lamb1), and sleepy (sly, lamc1), which were identified in forward genetic screens and were found to be impaired in visual functions. METHODS: Mutant larvae were observed for defects in visual behavior by testing their optokinetic response (OKR). In addition, electroretinograms (ERG) were measured and retinal morphology was examined by standard histology, immunocytochemistry, TUNEL assay, and electron microscopy. RESULTS: Both, gup and sly showed no OKR at any light intensity tested, whereas bal embryos showed some remaining OKR behavior at more than 40% of contrast. Consistent with the OKR result, gup and sly did not show an ERG response at any light intensity tested, whereas bal mutants exhibited small a- and b-waves at high light intensities. All three laminin mutants showed altered ganglion cell layers, optic nerve fasciculations, and lens defects. Again, bal showed the least severe morphologic phenotype with no additional defects. In contrast, both, gup and sly, showed severe photoreceptor outer segment shortening and synapse alteration (floating ribbons) as well as increased cell death. CONCLUSIONS: Lamb1 and lamc1 chains play an important role in the morphogenesis of photoreceptors and their synapses. In contrast, lama1 is not involved in outer retina development.
Asunto(s)
Diferenciación Celular , Embrión no Mamífero/patología , Laminina/genética , Retina/embriología , Proteínas de Pez Cebra/genética , Pez Cebra/embriología , Animales , Electrorretinografía , Técnica del Anticuerpo Fluorescente Indirecta , Etiquetado Corte-Fin in Situ , Cristalino/embriología , Cristalino/patología , Microscopía Fluorescente , Morfogénesis , Mutación , Nistagmo Optoquinético , Nervio Óptico/embriología , Nervio Óptico/patología , Retina/patología , Células Ganglionares de la Retina/patología , Pez Cebra/genéticaRESUMEN
PURPOSE: To characterize retinal morphology and visual system function in the zebrafish mutant fade out (fad) and to establish the mutant as a lower vertebrate model for Hermansky-Pudlak syndrome (HPS). METHODS: Retinal morphology of fad larvae was examined between 3 and 9 days postfertilization (dpf) by standard histology, transmission electron microscopy, and immunohistochemistry examination. Apoptotic cells were visualized by TdT-mediated dUTP nick-end labeling (TUNEL) staining. Visual system function was probed by electroretinography and behavioral assessment by optokinetic response measurements. Blood clotting was evaluated by time to occlusion testing of blood vessels as an arterial thrombosis assay. The chromosomal location of fad was determined by simple sequence-length polymorphism mapping. Genomic fragments of candidate genes were cloned by standard molecular techniques and mapped to the zebrafish genome by radiation hybrid mapping. RESULTS: Mutant fad larvae are hypopigmented and show structural defects in the outer retina. Melanosomes of these larvae in the retinal pigment epithelium are hypopigmented, generally smaller, and progressively reduced in number compared to nonmutant larvae. Progressive microvilli protrusions into the photoreceptor cell layer are not detectable, and photoreceptor outer segments get shorter and are misaligned. Photoreceptors subsequently undergo apoptosis, with a peak of cell death at 6 dpf. Electrical responses of the retina and visual performance are severely reduced. Blood clotting is prolonged in mutant fad larvae. Genomic mapping of fad reveals distinct genomic positions of the mutant gene from known human HPS genes. CONCLUSIONS: The fad mutant shows syndromic defects in pigmentation, outer retinal structure and function, and blood clotting. This syndrome is characteristic of Hermansky-Pudlak syndrome (HPS), making fad a novel genetic model of HPS. The gene does not cosegregate with the known human HPS genes, suggesting a novel molecular cause of HPS.
Asunto(s)
Síndrome de Hermanski-Pudlak/genética , Hipopigmentación/genética , Melanosomas/genética , Modelos Genéticos , Mutación , Enfermedades de la Retina/genética , Pez Cebra/genética , Animales , Apoptosis , Electrorretinografía , Técnica del Anticuerpo Fluorescente Indirecta , Genes Recesivos , Ligamiento Genético , Glutamato-Amoníaco Ligasa/metabolismo , Síndrome de Hermanski-Pudlak/patología , Etiquetado Corte-Fin in Situ , Melanosomas/patología , Nistagmo Optoquinético/fisiología , Células Fotorreceptoras de Vertebrados/fisiología , Células Fotorreceptoras de Vertebrados/ultraestructura , Epitelio Pigmentado Ocular/patología , Mapeo de Híbrido por Radiación , Enfermedades de la Retina/patología , Proteína 25 Asociada a Sinaptosomas/metabolismo , Tiempo de Coagulación de la Sangre TotalRESUMEN
The first synapse in the vertebrate visual system is the photoreceptor synapse between rod and cone photoreceptors and the second-order bipolar cells. Although mutations in the nyctalopin gene (NYX) in humans lead to congenital stationary night blindness (CSNB1), affecting synaptic transmission between both types of photoreceptors and ON-bipolar cells, the function of nyctalopin in cone-dominant animal models has not been studied. Because the larval zebrafish retina is cone-dominant, we isolated the zebrafish nyx ortholog and raised a polyclonal antibody against the protein. Nyctalopin is expressed postsynaptically in both synaptic layers of the retina. Functional disruption via morpholino antisense injection leads to characteristic defects in the electroretinogram and defects in visual contrast sensitivity. We therefore demonstrated that nyctalopin plays a similar role in retinal synapse function in the cone pathway as in the rod pathway, thereby creating a genetic model for CSNB1 and its effects on cone vision.
Asunto(s)
Proteoglicanos/metabolismo , Retina/citología , Células Fotorreceptoras Retinianas Conos/metabolismo , Transmisión Sináptica/fisiología , Animales , Clonación Molecular/métodos , Electrorretinografía/métodos , Humanos , Inmunohistoquímica/métodos , Hibridación in Situ/métodos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Morfolinas/antagonistas & inhibidores , Morfolinas/química , Nistagmo Optoquinético/efectos de los fármacos , Nistagmo Optoquinético/fisiología , Oligodesoxirribonucleótidos Antisentido/farmacología , Proteoglicanos/genética , Células Fotorreceptoras Retinianas Conos/efectos de los fármacos , Análisis de Secuencia de Proteína , Transfección/métodos , Pez Cebra , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
Addiction is a complex maladaptive behavior involving alterations in several neurotransmitter networks. In mammals, psychostimulants trigger elevated extracellular levels of dopamine, which can be modulated by central cholinergic transmission. Which elements of the cholinergic system might be targeted for drug addiction therapies remains unknown. The rewarding properties of drugs of abuse are central for the development of addictive behavior and are most commonly measured by means of the conditioned place preference (CPP) paradigm. We demonstrate here that adult zebrafish show robust CPP induced by the psychostimulant D-amphetamine. We further show that this behavior is dramatically reduced upon genetic impairment of acetylcholinesterase (AChE) function in ache/+ mutants, without involvement of concomitant defects in exploratory activity, learning, and visual performance. Our observations demonstrate that the cholinergic system modulates drug-induced reward in zebrafish, and identify genetically AChE as a promising target for systemic therapies against addiction to psychostimulants. More generally, they validate the zebrafish model to study the effect of developmental mutations on the molecular neurobiology of addiction in vertebrates.
Asunto(s)
Acetilcolina/metabolismo , Acetilcolinesterasa/metabolismo , Trastornos Relacionados con Anfetaminas/metabolismo , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Dextroanfetamina/efectos adversos , Pez Cebra/metabolismo , Acetilcolinesterasa/genética , Trastornos Relacionados con Anfetaminas/genética , Trastornos Relacionados con Anfetaminas/fisiopatología , Animales , Evolución Biológica , Encéfalo/fisiopatología , Química Encefálica/efectos de los fármacos , Química Encefálica/genética , Estimulantes del Sistema Nervioso Central/efectos adversos , Fibras Colinérgicas/efectos de los fármacos , Fibras Colinérgicas/metabolismo , Inhibidores de la Colinesterasa/farmacología , Inhibidores de la Colinesterasa/uso terapéutico , Condicionamiento Psicológico/efectos de los fármacos , Condicionamiento Psicológico/fisiología , Modelos Animales de Enfermedad , Dopamina/metabolismo , Femenino , Masculino , Mutación/genética , Recompensa , Pez Cebra/genéticaRESUMEN
Phosphorylation of rhodopsin by rhodopsin kinase GRK1 is an important desensitization mechanism in scotopic vision. For cone vision GRK1 is not essential. However, cone opsin is phosphorylated following light stimulation. In cone-dominant animals as well as in humans, but not in rodents, GRK7, a cone-specific homolog of GRK1, has been identified in cone outer segments. To investigate the function of GRK7 in vivo, we cloned two orthologs of grk7 in zebrafish and knocked down gene expression of grk7a in zebrafish larvae by morpholino antisense nucleotides. Photoresponse recovery in Grk7a-deficient larvae was delayed in electroretinographic measurements, and temporal contrast sensitivity was reduced, particularly under bright-light conditions. These results show that function of a cone-specific kinase is essential for cone vision in the zebrafish retina and argue that pigment bleaching and spontaneous decay alone are not sufficient for light adaptation and rapid cone response inactivation.
Asunto(s)
Adaptación a la Oscuridad/genética , Larva/genética , Proteínas Serina-Treonina Quinasas/fisiología , Células Fotorreceptoras Retinianas Conos/fisiología , Pez Cebra/fisiología , Animales , Animales Modificados Genéticamente , Arrestina/metabolismo , Western Blotting , Clonación Molecular/métodos , Sensibilidad de Contraste/genética , Sensibilidad de Contraste/fisiología , Electrorretinografía/métodos , Potenciales Evocados/genética , Potenciales Evocados/efectos de la radiación , Ojo/metabolismo , Ojo/patología , Quinasas de Receptores Acoplados a Proteína-G , Regulación del Desarrollo de la Expresión Génica , Inmunohistoquímica/métodos , Hibridación in Situ/métodos , Larva/fisiología , Estimulación Luminosa/métodos , Glándula Pineal/embriología , Glándula Pineal/metabolismo , Proteínas Serina-Treonina Quinasas/deficiencia , Psicofísica , Células Fotorreceptoras Retinianas Conos/embriología , Células Fotorreceptoras Retinianas Conos/metabolismo , Rodopsina/metabolismo , Factores de Tiempo , Pez Cebra/genética , Proteínas de Pez CebraRESUMEN
In the sensory receptors of both the eye and the ear, specialized apical structures have evolved to detect environmental stimuli such as light and sound. Despite the morphological divergence of these specialized structures and differing transduction mechanisms, the receptors appear to rely in part on a shared group of genes for function. For example, mutations in Usher (USH) genes cause a syndrome of visual and acoustic-vestibular deficits in humans. Several of the affected genes have been identified, including the USH1F gene, which encodes protocadherin 15 (PCDH15). Pcdh15 mutant mice also have both auditory and vestibular defects, although visual defects are not evident. Here we show that zebrafish have two closely related pcdh15 genes that are required for receptor-cell function and morphology in the eye or ear. Mutations in pcdh15a cause deafness and vestibular dysfunction, presumably because hair bundles of inner-ear receptors are splayed. Vision, however, is not affected in pcdh15a mutants. By contrast, reduction of pcdh15b activity using antisense morpholino oligonucleotides causes a visual defect. Optokinetic and electroretinogram responses are reduced in pcdh15b morpholino-injected larvae. In electron micrographs, morphant photoreceptor outer segments are improperly arranged, positioned perpendicular to the retinal pigment epithelium and are clumped together. Our results suggest that both cadherins act within their respective transduction organelles: Pcdh15a is necessary for integrity of the stereociliary bundle, whereas Pcdh15b is required for alignment and interdigitation of photoreceptor outer segments with the pigment epithelium. We conclude that after a duplication of pcdh15, one gene retained an essential function in the ear and the other in the eye.
Asunto(s)
Cadherinas/genética , Cadherinas/metabolismo , Proteínas de Peces/metabolismo , Genes Duplicados/genética , Audición/fisiología , Visión Ocular/fisiología , Proteínas de Pez Cebra/metabolismo , Pez Cebra/genética , Animales , Proteínas Relacionadas con las Cadherinas , Electrofisiología , Proteínas de Peces/genética , Duplicación de Gen , Regulación del Desarrollo de la Expresión Génica , Audición/genética , Microscopía Electrónica , Mutación/genética , Fenotipo , Células Fotorreceptoras/citología , Células Fotorreceptoras/embriología , Células Fotorreceptoras/metabolismo , Células Fotorreceptoras/ultraestructura , Retina/embriología , Retina/metabolismo , Retina/ultraestructura , Sensibilidad y Especificidad , Visión Ocular/genética , Pez Cebra/metabolismo , Pez Cebra/fisiología , Proteínas de Pez Cebra/genéticaRESUMEN
Electroretinographic (ERG) method records a sum field potential of the retina in response to light. It mainly arises in the outer retina and is used as a non-invasive measure in both animal experiments and the clinic. Since it is a comprehensive method to assess outer retinal function, it is becoming increasingly useful in genetic studies of vision. Here we present a simple in-house built setup to measure ERGs of aquatic vertebrates. We have used this setup to efficiently and reliably measure intact larvae of zebrafish (Danio rerio), Medaka fish (Oryzias latipes), and Xenopus laevis tadpoles. By slight modification of the setup, we were also able to measure adult zebrafish and Medaka, demonstrating the general versatility of the setup. We picked these organisms since they are increasingly used to study visual function with genetic means. This setup is easily built and will be particularly useful for laboratories setting up ERG measurements as a complement to their genetic studies.