RESUMEN
BACKGROUND: Rare cell subtypes can profoundly impact the course of human health and disease, yet their presence within a sample is often missed with bulk molecular analysis. Single-cell analysis tools such as FACS, FISH-FC and single-cell barcode-based sequencing can investigate cellular heterogeneity; however, they have significant limitations that impede their ability to identify and transcriptionally characterize many rare cell subpopulations. RESULTS: PCR-activated cell sorting (PACS) is a novel cytometry method that uses single-cell TaqMan PCR reactions performed in microfluidic droplets to identify and isolate cell subtypes with high-throughput. Here, we extend this method and demonstrate that PACS enables high-dimensional molecular profiling on TaqMan-targeted cells. Using a random priming RNA-Seq strategy, we obtained high-fidelity transcriptome measurements following PACS sorting of prostate cancer cells from a heterogeneous population. The sequencing data revealed prostate cancer gene expression profiles that were obscured in the unsorted populations. Single-cell expression analysis with PACS was subsequently used to confirm a number of the differentially expressed genes identified with RNA sequencing. CONCLUSIONS: PACS requires minimal sample processing, uses readily available TaqMan assays and can isolate cell subtypes with high sensitivity. We have now validated a method for performing next-generation sequencing on mRNA obtained from PACS isolated cells. This capability makes PACS well suited for transcriptional profiling of rare cells from complex populations to obtain maximal biological insight into cell states and behaviors.
Asunto(s)
Perfilación de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento , Reacción en Cadena de la Polimerasa , Transcriptoma , Línea Celular , Análisis por Conglomerados , Perfilación de la Expresión Génica/métodos , Regulación de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Reacción en Cadena de la Polimerasa Multiplex , Reacción en Cadena en Tiempo Real de la Polimerasa , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Análisis de la Célula Individual/métodos , Flujo de TrabajoRESUMEN
We present a new approach to genotyping based on multiplexed shotgun sequencing that can identify recombination breakpoints in a large number of individuals simultaneously at a resolution sufficient for most mapping purposes, such as quantitative trait locus (QTL) mapping and mapping of induced mutations. We first describe a simple library construction protocol that uses just 10 ng of genomic DNA per individual and makes the approach accessible to any laboratory with standard molecular biology equipment. Sequencing this library results in a large number of sequence reads widely distributed across the genomes of multiplexed bar-coded individuals. We develop a Hidden Markov Model to estimate ancestry at all genomic locations in all individuals using these data. We demonstrate the utility of the approach by mapping a dominant marker allele in D. simulans to within 105 kb of its true position using 96 F1-backcross individuals genotyped in a single lane on an Illumina Genome Analyzer. We further demonstrate the utility of our method by genetically mapping more than 400 previously unassembled D. simulans contigs to linkage groups and by evaluating the quality of targeted introgression lines. At this level of multiplexing and divergence between strains, our method allows estimation of recombination breakpoints to a median of 38-kb intervals. Our analysis suggests that higher levels of multiplexing and/or use of strains with lower levels of divergence are practicable.
Asunto(s)
Mapeo Cromosómico/métodos , Tipificación Molecular/métodos , Análisis de Secuencia de ADN/métodos , Animales , Puntos de Rotura del Cromosoma , Biología Computacional , Drosophila/genética , Femenino , Genes Dominantes/genética , Marcadores Genéticos , Genotipo , Masculino , Fenotipo , Sitios de Carácter Cuantitativo/genética , Recombinación Genética/genética , Proyectos de InvestigaciónRESUMEN
A defining characteristic of neuronal cell type is the growth of axons and dendrites into specific layers and columns of the brain. Although differences in cell surface receptors and adhesion molecules are known to cause differences in synaptic specificity, differences in downstream signaling mechanisms that determine cell type-appropriate targeting patterns are unknown. Using a forward genetic screen in Drosophila, we identify the GTPase effector Genghis khan (Gek) as playing a crucial role in the ability of a subset of photoreceptor (R cell) axons to innervate appropriate target columns. In particular, single-cell mosaic analyses demonstrate that R cell growth cones lacking Gek function grow to the appropriate ganglion, but frequently fail to innervate the correct target column. Further studies reveal that R cell axons lacking the activity of the small GTPase Cdc42 display similar defects, providing evidence that these proteins regulate a common set of processes. Gek is expressed in all R cells, and a detailed structure-function analysis reveals a set of regulatory domains with activities that restrict Gek function to the growth cone. Although Gek does not normally regulate layer-specific targeting, ectopic expression of Gek is sufficient to alter the targeting choices made by another R cell type, the targeting of which is normally Gek independent. Thus, specific regulation of cytoskeletal responses to targeting cues is necessary for cell type-appropriate synaptic specificity.
Asunto(s)
Proteínas de Drosophila/fisiología , Drosophila/genética , Ojo/inervación , Proteínas Serina-Treonina Quinasas/fisiología , Visión Ocular/genética , Vías Visuales/fisiología , Animales , Animales Modificados Genéticamente , Axones/metabolismo , Axones/fisiología , Citoesqueleto/metabolismo , Dendritas/metabolismo , Drosophila/crecimiento & desarrollo , Drosophila/fisiología , Proteínas de Drosophila/genética , Estudios de Asociación Genética , Conos de Crecimiento/metabolismo , Conos de Crecimiento/fisiología , Modelos Biológicos , Neuronas Aferentes/metabolismo , Neuronas Aferentes/fisiología , Células Fotorreceptoras de Invertebrados/metabolismo , Células Fotorreceptoras de Invertebrados/fisiología , Proteínas Serina-Treonina Quinasas/genética , Sensibilidad y Especificidad , Transmisión Sináptica/genética , Transmisión Sináptica/fisiología , Vías Visuales/metabolismoRESUMEN
Structural plasticity in the brain often necessitates dramatic remodeling of neuronal processes, with attendant reorganization of the cytoskeleton and membranes. Although cytoskeletal restructuring has been studied extensively, how lipids might orchestrate structural plasticity remains unclear. We show that specific glial cells in Drosophila produce glucocerebrosidase (GBA) to locally catabolize sphingolipids. Sphingolipid accumulation drives lysosomal dysfunction, causing gba1b mutants to harbor protein aggregates that cycle across circadian time and are regulated by neural activity, the circadian clock, and sleep. Although the vast majority of membrane lipids are stable across the day, a specific subset that is highly enriched in sphingolipids cycles daily in a gba1b-dependent fashion. Remarkably, both sphingolipid biosynthesis and degradation are required for the diurnal remodeling of circadian clock neurites, which grow and shrink across the day. Thus, dynamic sphingolipid regulation by glia enables diurnal circuit remodeling and proper circadian behavior.
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Relojes Circadianos , Proteínas de Drosophila , Animales , Relojes Circadianos/fisiología , Ritmo Circadiano/fisiología , Drosophila/metabolismo , Proteínas de Drosophila/metabolismo , Glucosilceramidasa , Lípidos de la Membrana , Neuroglía/metabolismo , Agregado de Proteínas , Esfingolípidos/metabolismoRESUMEN
Phosphomannomutase 2 Deficiency (PMM2-CDG) is the most common monogenic congenital disorder of glycosylation (CDG) affecting at least 800 patients globally. PMM2 orthologs are present in model organisms, including the budding yeast Saccharomyces cerevisiae gene SEC53 Here we describe conserved genotype-phenotype relationships across yeast and human patients between five PMM2 loss-of-function missense mutations and their orthologous SEC53 mutations. These alleles range in severity from folding defective (hypomorph) to dimerization defective (severe hypomorph) to catalytic dead (null). We included the first and second most common missense mutations - R141H, F119L respectively- and the most common compound heterozygote genotype - PMM2R141H/F119L - observed in PMM2-CDG patients. Each mutation described is expressed in haploid as well as homozygous and heterozygous diploid yeast cells at varying protein expression levels as either SEC53 protein variants or PMM2 protein variants. We developed a 384-well-plate, growth-based assay for use in a screen of the 2,560-compound Microsource Spectrum library of approved drugs, experimental drugs, tool compounds and natural products. We identified three compounds that suppress growth defects of SEC53 variants, F126L and V238M, based on the biochemical defect of the allele, protein abundance or ploidy. The rare PMM2 E139K protein variant is fully functional in yeast cells, suggesting that its pathogenicity in humans is due to the underlying DNA mutation that results in skipping of exon 5 and a nonfunctional truncated protein. Together, these results demonstrate that yeast models can be used to characterize known and novel PMM2 patient alleles in quantitative growth and enzymatic activity assays, and used as patient avatars for PMM2-CDG drug screens yielding compounds that could be rapidly cross-validated in zebrafish, rodent and human organoid models.
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Trastornos Congénitos de Glicosilación/genética , Mutación con Pérdida de Función , Fosfotransferasas (Fosfomutasas)/deficiencia , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Humanos , Mutación Missense , Fosfotransferasas (Fosfomutasas)/genética , Fosfotransferasas (Fosfomutasas)/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
N-glycanase 1 (NGLY1) deficiency is an ultra-rare and complex monogenic glycosylation disorder that affects fewer than 40 patients globally. NGLY1 deficiency has been studied in model organisms such as yeast, worms, flies and mice. Proteasomal and mitochondrial homeostasis gene networks are controlled by the evolutionarily conserved transcriptional regulator NRF1, whose activity requires deglycosylation by NGLY1. Hypersensitivity to the proteasome inhibitor bortezomib is a common phenotype observed in whole-animal and cellular models of NGLY1 deficiency. Here, we describe unbiased phenotypic drug screens to identify FDA-approved drugs that are generally recognized as safe natural products, and novel chemical entities, that rescue growth and development of NGLY1-deficient worm and fly larvae treated with a toxic dose of bortezomib. We used image-based larval size and number assays for use in screens of a 2560-member drug-repurposing library and a 20,240-member lead-discovery library. A total of 91 validated hit compounds from primary invertebrate screens were tested in a human cell line in an NRF2 activity assay. NRF2 is a transcriptional regulator that regulates cellular redox homeostasis, and it can compensate for loss of NRF1. Plant-based polyphenols make up the largest class of hit compounds and NRF2 inducers. Catecholamines and catecholamine receptor activators make up the second largest class of hits. Steroidal and non-steroidal anti-inflammatory drugs make up the third largest class. Only one compound was active in all assays and species: the atypical antipsychotic and dopamine receptor agonist aripiprazole. Worm and fly models of NGLY1 deficiency validate therapeutic rationales for activation of NRF2 and anti-inflammatory pathways based on results in mice and human cell models, and suggest a novel therapeutic rationale for boosting catecholamine levels and/or signaling in the brain.
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Catecolaminas/fisiología , Trastornos Congénitos de Glicosilación/etiología , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos/métodos , Inflamación/prevención & control , Factor 2 Relacionado con NF-E2/fisiología , Péptido-N4-(N-acetil-beta-glucosaminil) Asparagina Amidasa/deficiencia , Animales , Bortezomib/farmacología , Dípteros , Descubrimiento de Drogas , Humanos , Proteína 1 Asociada A ECH Tipo Kelch/fisiología , Nematodos , Transducción de Señal/fisiologíaRESUMEN
N-glycanase 1 (NGLY1) Deficiency is a rare monogenic multi-system disorder first described in 2014. NGLY1 is evolutionarily conserved in model organisms. Here we conducted a natural history study and chemical-modifier screen on the Drosophila melanogaster NGLY1 homolog, Pngl We generated a new fly model of NGLY1 Deficiency, engineered with a nonsense mutation in Pngl at codon 420 that results in a truncation of the C-terminal carbohydrate-binding PAW domain. Homozygous mutant animals exhibit global development delay, pupal lethality and small body size as adults. We developed a 96-well-plate, image-based, quantitative assay of Drosophila larval size for use in a screen of the 2,650-member Microsource Spectrum compound library of FDA approved drugs, bioactive tool compounds, and natural products. We found that the cholesterol-derived ecdysteroid molting hormone 20-hydroxyecdysone (20E) partially rescued the global developmental delay in mutant homozygotes. Targeted expression of a human NGLY1 transgene to tissues involved in ecdysteroidogenesis, e.g., prothoracic gland, also partially rescues global developmental delay in mutant homozygotes. Finally, the proteasome inhibitor bortezomib is a potent enhancer of global developmental delay in our fly model, evidence of a defective proteasome "bounce-back" response that is also observed in nematode and cellular models of NGLY1 Deficiency. Together, these results demonstrate the therapeutic relevance of a new fly model of NGLY1 Deficiency for drug discovery and gene modifier screens.
Asunto(s)
Trastornos Congénitos de Glicosilación/genética , Drosophila/genética , Estudios de Asociación Genética , Trastornos del Neurodesarrollo/genética , Péptido-N4-(N-acetil-beta-glucosaminil) Asparagina Amidasa/deficiencia , Alelos , Animales , Animales Modificados Genéticamente , Trastornos Congénitos de Glicosilación/metabolismo , Modelos Animales de Enfermedad , Drosophila/metabolismo , Femenino , Genes Letales , Genotipo , Humanos , Larva , Mutación , Trastornos del Neurodesarrollo/metabolismo , Péptido-N4-(N-acetil-beta-glucosaminil) Asparagina Amidasa/genética , Péptido-N4-(N-acetil-beta-glucosaminil) Asparagina Amidasa/metabolismo , Fenotipo , Complejo de la Endopetidasa Proteasomal/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Inhibidores de Proteasoma/farmacologíaRESUMEN
Species of the Drosophila melanogaster species subgroup, including the species D. simulans, D. mauritiana, D. yakuba, and D. santomea, have long served as model systems for studying evolution. However, studies in these species have been limited by a paucity of genetic and transgenic reagents. Here, we describe a collection of transgenic and genetic strains generated to facilitate genetic studies within and between these species. We have generated many strains of each species containing mapped piggyBac transposons including an enhanced yellow fluorescent protein (EYFP) gene expressed in the eyes and a ÏC31 attP site-specific integration site. We have tested a subset of these lines for integration efficiency and reporter gene expression levels. We have also generated a smaller collection of other lines expressing other genetically encoded fluorescent molecules in the eyes and a number of other transgenic reagents that will be useful for functional studies in these species. In addition, we have mapped the insertion locations of 58 transposable elements in D. virilis that will be useful for genetic mapping studies.
Asunto(s)
Drosophila/genética , Alelos , Animales , Animales Modificados Genéticamente , Elementos Transponibles de ADN/genética , Drosophila simulans/genética , Ojo/metabolismo , Regulación de la Expresión Génica , Genómica , Proteínas Fluorescentes Verdes/metabolismo , Mutagénesis Insercional/genética , Especificidad de la Especie , TransgenesRESUMEN
Pheromones, chemical signals that convey social information, mediate many insect social behaviors, including navigation and aggregation. Several studies have suggested that behavior during the immature larval stages of Drosophila development is influenced by pheromones, but none of these compounds or the pheromone-receptor neurons that sense them have been identified. Here we report a larval pheromone-signaling pathway. We found that larvae produce two novel long-chain fatty acids that are attractive to other larvae. We identified a single larval chemosensory neuron that detects these molecules. Two members of the pickpocket family of DEG/ENaC channel subunits (ppk23 and ppk29) are required to respond to these pheromones. This pheromone system is evolving quickly, since the larval exudates of D. simulans, the sister species of D. melanogaster, are not attractive to other larvae. Our results define a new pheromone signaling system in Drosophila that shares characteristics with pheromone systems in a wide diversity of insects.
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Evolución Biológica , Drosophila/efectos de los fármacos , Drosophila/fisiología , Feromonas/farmacología , Conducta Social , Animales , Señales (Psicología) , Proteínas de Drosophila/metabolismo , Ácidos Grasos Monoinsaturados/farmacología , Larva/efectos de los fármacos , Larva/fisiología , Células Receptoras Sensoriales/efectos de los fármacos , Células Receptoras Sensoriales/fisiología , Transducción de Señal/efectos de los fármacosRESUMEN
Mitochondrial dysfunction is a hallmark of many neurodegenerative diseases, yet its precise role in disease pathology remains unclear. To examine this link directly, we subtly perturbed electron transport chain function in the Drosophila retina, creating a model of Leigh Syndrome, an early-onset neurodegenerative disorder. Using mutations that affect mitochondrial complex II, we demonstrate that mild disruptions of mitochondrial function have no effect on the initial stages of photoreceptor development, but cause degeneration of their synapses and cell bodies in late pupal and adult animals. In this model, synapse loss is caused by reactive oxygen species (ROS) production, not energy depletion, as ATP levels are normal in mutant photoreceptors, and both pharmacological and targeted genetic manipulations that reduce ROS levels prevent synapse degeneration. Intriguingly, these manipulations of ROS uncouple synaptic effects from degenerative changes in the cell body, suggesting that mitochondrial dysfunction activates two genetically separable processes, one that induces morphological changes in the cell body, and another that causes synapse loss. Finally, by blocking mitochondrial trafficking into the axon using a mutation affecting a mitochondrial transport complex, we find that ROS action restricted to the cell body is sufficient to cause synaptic degeneration, demonstrating that ROS need not act locally at the synapse. Thus, alterations in electron transport chain function explain many of the neurodegenerative changes seen in both early- and late-onset disorders.
Asunto(s)
Enfermedades del Sistema Nervioso Central/metabolismo , Drosophila melanogaster/metabolismo , Enfermedades Mitocondriales/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Sinapsis/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Antioxidantes/farmacología , Enfermedades del Sistema Nervioso Central/genética , Enfermedades del Sistema Nervioso Central/patología , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Drosophila melanogaster/genética , Complejo II de Transporte de Electrones/genética , Complejo II de Transporte de Electrones/metabolismo , Regulación del Desarrollo de la Expresión Génica , Enfermedades Mitocondriales/genética , Enfermedades Mitocondriales/patología , Mutación/genética , Sinapsis/efectos de los fármacosRESUMEN
The development of the Drosophila visual system provides a framework for investigating how circuits assemble. A sequence of reciprocal interactions amongst photoreceptors, target neurons and glia creates a precise pattern of connections while reducing the complexity of the targeting process. Both afferent-afferent and afferent-target interactions are required for photoreceptor (R cell) axons to select appropriate synaptic partners. With the identification of some critical cell adhesion and signaling molecules, the logic by which axons make choices amongst alternate synaptic partners is becoming clear. These studies also provide an opportunity to examine the molecular basis of neural circuit evolution.