RESUMEN
Precise, repeatable genetic access to specific neurons via GAL4/UAS and related methods is a key advantage of Drosophila neuroscience. Neuronal targeting is typically documented using light microscopy of full GAL4 expression patterns, which generally lack the single-cell resolution required for reliable cell type identification. Here, we use stochastic GAL4 labeling with the MultiColor FlpOut approach to generate cellular resolution confocal images at large scale. We are releasing aligned images of 74,000 such adult central nervous systems. An anticipated use of this resource is to bridge the gap between neurons identified by electron or light microscopy. Identifying individual neurons that make up each GAL4 expression pattern improves the prediction of split-GAL4 combinations targeting particular neurons. To this end, we have made the images searchable on the NeuronBridge website. We demonstrate the potential of NeuronBridge to rapidly and effectively identify neuron matches based on morphology across imaging modalities and datasets.
Asunto(s)
Proteínas de Drosophila , Neurociencias , Animales , Drosophila/metabolismo , Neuronas/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Sistema Nervioso Central/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
The use of genetically encoded 'self-labeling tags' with chemical fluorophore ligands enables rapid labeling of specific cells in neural tissue. To improve the chemical tagging of neurons, we synthesized and evaluated new fluorophore ligands based on Cy, Janelia Fluor, Alexa Fluor, and ATTO dyes and tested these with recently improved Drosophila melanogaster transgenes. We found that tissue clearing and mounting in DPX substantially improves signal quality when combined with specific non-cyanine fluorophores. We compared and combined this labeling technique with standard immunohistochemistry in the Drosophila brain.
Asunto(s)
Drosophila melanogaster/citología , Colorantes Fluorescentes , Inmunohistoquímica , Neuronas/citología , Coloración y Etiquetado , Animales , Animales Modificados Genéticamente , Encéfalo/citología , Femenino , Colorantes Fluorescentes/síntesis química , Colorantes Fluorescentes/química , Microscopía Confocal , Estructura MolecularRESUMEN
PURPOSE: Age-related macular degeneration (AMD) is a devastating disease characterized by central vision impairment in individuals with advanced age. Neovascular AMD is a form of end-stage disease in which choroidal vessel outgrowth occurs beneath the retina. While many hypotheses have been raised as to what triggers the formation of pathological choroidal neovascular membranes, the exact mechanism for their initiation remains unresolved. Polymorphisms in the FLT1 gene have previously been associated with neovascular AMD risk, including the rs9943922 single nucleotide polymorphism (SNP). Here, we aimed to determine the association between the high-risk FLT1 genotype and FLT1 protein levels in human retina or retinal pigment epithelium (RPE)/choroid tissue. METHODS: Retina and RPE/choroid tissue from 10 human donor eyes was selected from a collection of eyes genotyped for the rs9943922 SNP. Differences in soluble and membrane bound FLT1 protein levels were assessed for retina versus RPE/choroid donor tissue using ELISA and Western blotting analyses. Genotype-associated changes in FLT1 protein levels were also evaluated. RESULTS: We found soluble FLT1 levels in the RPE/choroid tissue to be approximately three times higher than that of the retina (p < 0.001), while both samples have similar levels of the membrane bound form. When tissue with the rs9943922 SNP was compared with controls, no significant genotypic differences in FLT1 protein levels were observed. CONCLUSIONS: Based on these data, we conclude that the rs9943922 SNP in the FLT1 gene does not result in a large difference in FLT1 protein levels, regardless of whether it is the soluble or the membrane bound form.
Asunto(s)
Coroides/metabolismo , Polimorfismo de Nucleótido Simple , Retina/metabolismo , Epitelio Pigmentado de la Retina/metabolismo , Receptor 1 de Factores de Crecimiento Endotelial Vascular/genética , Receptor 1 de Factores de Crecimiento Endotelial Vascular/metabolismo , Degeneración Macular Húmeda/metabolismo , Anciano , Anciano de 80 o más Años , Western Blotting , Ensayo de Inmunoadsorción Enzimática , Femenino , Técnicas de Genotipaje , Humanos , Masculino , Persona de Mediana Edad , Reacción en Cadena en Tiempo Real de la Polimerasa , Donantes de Tejidos , Degeneración Macular Húmeda/genéticaRESUMEN
Retinitis pigmentosa (RP) is a highly heterogeneous group of disorders characterized by degeneration of the retinal photoreceptor cells and progressive loss of vision. While hundreds of mutations in more than 100 genes have been reported to cause RP, discovering the causative mutations in many patients remains a significant challenge. Exome sequencing in an individual affected with non-syndromic RP revealed two plausibly disease-causing variants in TRNT1, a gene encoding a nucleotidyltransferase critical for tRNA processing. A total of 727 additional unrelated individuals with molecularly uncharacterized RP were completely screened for TRNT1 coding sequence variants, and a second family was identified with two members who exhibited a phenotype that was remarkably similar to the index patient. Inactivating mutations in TRNT1 have been previously shown to cause a severe congenital syndrome of sideroblastic anemia, B-cell immunodeficiency, recurrent fevers and developmental delay (SIFD). Complete blood counts of all three of our patients revealed red blood cell microcytosis and anisocytosis with only mild anemia. Characterization of TRNT1 in patient-derived cell lines revealed reduced but detectable TRNT1 protein, consistent with partial function. Suppression of trnt1 expression in zebrafish recapitulated several features of the human SIFD syndrome, including anemia and sensory organ defects. When levels of trnt1 were titrated, visual dysfunction was found in the absence of other phenotypes. The visual defects in the trnt1-knockdown zebrafish were ameliorated by the addition of exogenous human TRNT1 RNA. Our findings indicate that hypomorphic TRNT1 mutations can cause a recessive disease that is almost entirely limited to the retina.
Asunto(s)
Nucleotidiltransferasas/genética , Retinitis Pigmentosa/genética , Adolescente , Animales , Proteínas Portadoras , Células Cultivadas , Exoma , Expresión Génica , Humanos , Masculino , Mutación , Nucleótidos/metabolismo , Perilipina-1 , Fosfoproteínas , Empalme del ARN , Análisis de Secuencia de ADN , Adulto Joven , Pez CebraRESUMEN
We identified the neurons comprising the Drosophila mushroom body (MB), an associative center in invertebrate brains, and provide a comprehensive map describing their potential connections. Each of the 21 MB output neuron (MBON) types elaborates segregated dendritic arbors along the parallel axons of â¼2000 Kenyon cells, forming 15 compartments that collectively tile the MB lobes. MBON axons project to five discrete neuropils outside of the MB and three MBON types form a feedforward network in the lobes. Each of the 20 dopaminergic neuron (DAN) types projects axons to one, or at most two, of the MBON compartments. Convergence of DAN axons on compartmentalized Kenyon cell-MBON synapses creates a highly ordered unit that can support learning to impose valence on sensory representations. The elucidation of the complement of neurons of the MB provides a comprehensive anatomical substrate from which one can infer a functional logic of associative olfactory learning and memory.
Asunto(s)
Aprendizaje por Asociación , Drosophila melanogaster/citología , Drosophila melanogaster/fisiología , Lógica , Cuerpos Pedunculados/citología , Cuerpos Pedunculados/inervación , Células Receptoras Sensoriales/fisiología , Animales , Encéfalo/anatomía & histología , Encéfalo/fisiología , Compartimento Celular , Forma de la Célula , Dendritas/metabolismo , Neuronas Dopaminérgicas/citología , Neuronas Dopaminérgicas/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Modelos Neurológicos , Neurotransmisores/metabolismo , Vías Olfatorias/anatomía & histología , Vías Olfatorias/fisiología , Olfato/fisiologíaRESUMEN
Animals discriminate stimuli, learn their predictive value and use this knowledge to modify their behavior. In Drosophila, the mushroom body (MB) plays a key role in these processes. Sensory stimuli are sparsely represented by â¼2000 Kenyon cells, which converge onto 34 output neurons (MBONs) of 21 types. We studied the role of MBONs in several associative learning tasks and in sleep regulation, revealing the extent to which information flow is segregated into distinct channels and suggesting possible roles for the multi-layered MBON network. We also show that optogenetic activation of MBONs can, depending on cell type, induce repulsion or attraction in flies. The behavioral effects of MBON perturbation are combinatorial, suggesting that the MBON ensemble collectively represents valence. We propose that local, stimulus-specific dopaminergic modulation selectively alters the balance within the MBON network for those stimuli. Our results suggest that valence encoded by the MBON ensemble biases memory-based action selection.
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Conducta de Elección , Drosophila melanogaster/citología , Drosophila melanogaster/fisiología , Memoria , Cuerpos Pedunculados/citología , Cuerpos Pedunculados/inervación , Neuronas/fisiología , Animales , Conducta Apetitiva/efectos de la radiación , Aprendizaje por Asociación/efectos de la radiación , Reacción de Prevención/efectos de la radiación , Conducta Animal/efectos de la radiación , Conducta de Elección/efectos de la radiación , Luz , Memoria/efectos de la radiación , Modelos Neurológicos , Cuerpos Pedunculados/efectos de la radiación , Neuronas/efectos de la radiación , Odorantes , Sueño/efectos de la radiación , Factores de Tiempo , Visión OcularRESUMEN
Age-related macular degeneration (AMD) is a common disease that can result in severe visual impairment. Abnormal regulation of the complement system has been implicated in its pathogenesis, and CFH polymorphisms contribute substantially to risk. How these polymorphisms exert their effects is poorly understood. We performed enzyme-linked immunosorbent assay (ELISA) analysis on young, aged, and AMD choroids to determine the abundance of the membrane attack complex (MAC) and performed immunofluorescence studies on eyes from 117 donors to evaluate the MAC in aging, early AMD, and advanced AMD. Morphometric studies were performed on eyes with high- or low-risk CFH genotypes. ELISA confirmed that MAC increases significantly with aging and with AMD. MAC was localized to Bruch's membrane and the choriocapillaris and was detectable at low levels as early as 5 years of age. Hard drusen were labeled with anti-MAC antibody, but large or confluent drusen and basal deposits were generally unlabeled. Labeling of retinal pigment epithelium was observed in some cases of advanced AMD, but not in early disease. Eyes homozygous for the high-risk CFH genotype had thinner choroids than low-risk homozygotes (P < 0.05). These findings suggest that increased complement activation in AMD and in high-risk genotypes can lead to loss of endothelial cells in early AMD. Treatments to protect the choriocapillaris in early AMD are needed.
Asunto(s)
Envejecimiento/patología , Coroides/patología , Factor H de Complemento/genética , Complejo de Ataque a Membrana del Sistema Complemento/genética , Degeneración Macular/patología , Adulto , Anciano , Anciano de 80 o más Años , Envejecimiento/genética , Envejecimiento/metabolismo , Preescolar , Coroides/metabolismo , Factor H de Complemento/metabolismo , Complejo de Ataque a Membrana del Sistema Complemento/metabolismo , Femenino , Humanos , Lactante , Recién Nacido , Degeneración Macular/genética , Degeneración Macular/metabolismo , Masculino , Persona de Mediana Edad , Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/patología , Adulto JovenRESUMEN
Mutations in ABCA4 cause Stargardt disease and other blinding autosomal recessive retinal disorders. However, sequencing of the complete coding sequence in patients with clinical features of Stargardt disease sometimes fails to detect one or both mutations. For example, among 208 individuals with clear clinical evidence of ABCA4 disease ascertained at a single institution, 28 had only one disease-causing allele identified in the exons and splice junctions of the primary retinal transcript of the gene. Haplotype analysis of these 28 probands revealed 3 haplotypes shared among ten families, suggesting that 18 of the 28 missing alleles were rare enough to be present only once in the cohort. We hypothesized that mutations near rare alternate splice junctions in ABCA4 might cause disease by increasing the probability of mis-splicing at these sites. Next-generation sequencing of RNA extracted from human donor eyes revealed more than a dozen alternate exons that are occasionally incorporated into the ABCA4 transcript in normal human retina. We sequenced the genomic DNA containing 15 of these minor exons in the 28 one-allele subjects and observed five instances of two different variations in the splice signals of exon 36.1 that were not present in normal individuals (P < 10(-6)). Analysis of RNA obtained from the keratinocytes of patients with these mutations revealed the predicted alternate transcript. This study illustrates the utility of RNA sequence analysis of human donor tissue and patient-derived cell lines to identify mutations that would be undetectable by exome sequencing.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/genética , Empalme Alternativo/genética , Retina/patología , Adulto , Anciano de 80 o más Años , Alelos , Exoma/genética , Exones/genética , Femenino , Haplotipos , Humanos , Degeneración Macular/genética , Degeneración Macular/fisiopatología , Masculino , Mutación , Linaje , Sitios de Empalme de ARN/genética , Enfermedad de StargardtRESUMEN
Retinitis pigmentosa (RP) is a genetically heterogeneous heritable disease characterized by apoptotic death of photoreceptor cells. We used exome sequencing to identify a homozygous Alu insertion in exon 9 of male germ cell-associated kinase (MAK) as the cause of disease in an isolated individual with RP. Screening of 1,798 unrelated RP patients identified 20 additional probands homozygous for this insertion (1.2%). All 21 affected probands are of Jewish ancestry. MAK encodes a kinase involved in the regulation of photoreceptor-connecting cilium length. Immunohistochemistry of human donor tissue revealed that MAK is expressed in the inner segments, cell bodies, and axons of rod and cone photoreceptors. Several isoforms of MAK that result from alternative splicing were identified. Induced pluripotent stem cells were derived from the skin of the proband and a patient with non-MAK-associated RP (RP control). In the RP control individual, we found that a transcript lacking exon 9 was predominant in undifferentiated cells, whereas a transcript bearing exon 9 and a previously unrecognized exon 12 predominated in cells that were differentiated into retinal precursors. However, in the proband with the Alu insertion, the developmental switch to the MAK transcript bearing exons 9 and 12 did not occur. In addition to showing the use of induced pluripotent stem cells to efficiently evaluate the pathogenicity of specific mutations in relatively inaccessible tissues like retina, this study reveals algorithmic and molecular obstacles to the discovery of pathogenic insertions and suggests specific changes in strategy that can be implemented to more fully harness the power of sequencing technologies.
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Cilios/genética , Exones/genética , Células Madre Pluripotentes Inducidas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Retinitis Pigmentosa/enzimología , Retinitis Pigmentosa/genética , Análisis de Secuencia de ADN , Elementos Alu/genética , Secuencia de Aminoácidos , Biomarcadores/metabolismo , Diferenciación Celular , Genealogía y Heráldica , Humanos , Isoenzimas/metabolismo , Judíos/genética , Datos de Secuencia Molecular , Mutagénesis Insercional/genética , Especificidad de Órganos , Mutación Puntual/genética , Proteínas Serina-Treonina Quinasas/química , Células Fotorreceptoras Retinianas Conos/enzimología , Células Fotorreceptoras Retinianas Conos/patología , Degeneración Retiniana/complicaciones , Degeneración Retiniana/enzimología , Células Fotorreceptoras Retinianas Bastones/enzimología , Células Fotorreceptoras Retinianas Bastones/patología , Retinitis Pigmentosa/complicacionesAsunto(s)
Síndrome de Exfoliación/genética , Variación Genética , Glaucoma de Ángulo Abierto/genética , Hiperhomocisteinemia/genética , Metilenotetrahidrofolato Reductasa (NADPH2)/genética , Adulto , Amplificación de Genes , Frecuencia de los Genes , Genotipo , Humanos , Presión Intraocular , Iowa , Reacción en Cadena de la PolimerasaRESUMEN
In the hawkmoth, Manduca sexta, thoracic leg motoneurons survive the degeneration of the larval leg muscles to innervate new muscles of the adult legs. The same motoneurons, therefore, participate in the very different modes of terrestrial locomotion that are used by larvae (crawling) and adults (walking). Consequently, changes in locomotor behavior may reflect changes in both the CNS and periphery. The present study was undertaken to determine whether motor patterns produced by the isolated CNS of adult Manduca, in the absence of sensory feedback, would resemble adult specific patterns of coordination. Pilocarpine, which evokes a fictive crawling motor pattern from the isolated larval CNS, also evoked robust patterned activity from leg motoneurons in the isolated adult CNS. As in the larva, levator and depressor motoneurons innervating the same leg were active in antiphase. Unlike fictive crawling, however, bursts of activity in levator or depressor motoneurons of one leg alternated with bursts in the homologous motoneurons innervating the opposite leg of the same segment and the leg on the same side in the adjacent segment. The most common mode of intersegmental activity generated by the isolated adult CNS resembled an alternating tripod gait, which is displayed, albeit infrequently, during walking in intact adult Manduca. A detailed analysis revealed specific differences between the patterned motor activity that is evoked from the isolated adult CNS and activity patterns observed during walking in intact animals, perhaps indicating an important role for sensory feedback. Nevertheless, the basic similarity to adult walking and clear distinctions from the larval fictive crawling pattern suggest that changes within the CNS contribute to alterations in locomotor activity during metamorphosis.