RESUMEN
We have defined a network of interacting Drosophila cell surface proteins in which a 21-member IgSF subfamily, the Dprs, binds to a nine-member subfamily, the DIPs. The structural basis of the Dpr-DIP interaction code appears to be dictated by shape complementarity within the Dpr-DIP binding interface. Each of the six dpr and DIP genes examined here is expressed by a unique subset of larval and pupal neurons. In the neuromuscular system, interactions between Dpr11 and DIP-γ affect presynaptic terminal development, trophic factor responses, and neurotransmission. In the visual system, dpr11 is selectively expressed by R7 photoreceptors that use Rh4 opsin (yR7s). Their primary synaptic targets, Dm8 amacrine neurons, express DIP-γ. In dpr11 or DIP-γ mutants, yR7 terminals extend beyond their normal termination zones in layer M6 of the medulla. DIP-γ is also required for Dm8 survival or differentiation. Our findings suggest that Dpr-DIP interactions are important determinants of synaptic connectivity.
Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Inmunoglobulinas/metabolismo , Proteínas de la Membrana/metabolismo , Neuronas/metabolismo , Sinapsis , Secuencia de Aminoácidos , Animales , Drosophila/crecimiento & desarrollo , Proteínas de Drosophila/química , Larva/metabolismo , Modelos Moleculares , Familia de Multigenes , Mapas de Interacción de Proteínas , Alineación de SecuenciaRESUMEN
Information processing relies on precise patterns of synapses between neurons. The cellular recognition mechanisms regulating this specificity are poorly understood. In the medulla of the Drosophila visual system, different neurons form synaptic connections in different layers. Here, we sought to identify candidate cell recognition molecules underlying this specificity. Using RNA sequencing (RNA-seq), we show that neurons with different synaptic specificities express unique combinations of mRNAs encoding hundreds of cell surface and secreted proteins. Using RNA-seq and protein tagging, we demonstrate that 21 paralogs of the Dpr family, a subclass of immunoglobulin (Ig)-domain containing proteins, are expressed in unique combinations in homologous neurons with different layer-specific synaptic connections. Dpr interacting proteins (DIPs), comprising nine paralogs of another subclass of Ig-containing proteins, are expressed in a complementary layer-specific fashion in a subset of synaptic partners. We propose that pairs of Dpr/DIP paralogs contribute to layer-specific patterns of synaptic connectivity.
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Proteínas de Drosophila/metabolismo , Inmunoglobulinas/metabolismo , Neuronas/metabolismo , Receptores Inmunológicos/metabolismo , Sinapsis , Animales , Drosophila , Citometría de Flujo , Análisis de Secuencia de ARN , Visión OcularRESUMEN
WD40 repeat-containing proteins form a large family of proteins present in all eukaryotes. Here, we identified five pediatric probands with de novo variants in WDR37, which encodes a member of the WD40 repeat protein family. Two probands shared one variant and the others have variants in nearby amino acids outside the WD40 repeats. The probands exhibited shared phenotypes of epilepsy, colobomas, facial dysmorphology reminiscent of CHARGE syndrome, developmental delay and intellectual disability, and cerebellar hypoplasia. The WDR37 protein is highly conserved in vertebrate and invertebrate model organisms and is currently not associated with a human disease. We generated a null allele of the single Drosophila ortholog to gain functional insights and replaced the coding region of the fly gene CG12333/wdr37 with GAL4. These flies are homozygous viable but display severe bang sensitivity, a phenotype associated with seizures in flies. Additionally, the mutant flies fall when climbing the walls of the vials, suggesting a defect in grip strength, and repeat the cycle of climbing and falling. Similar to wall clinging defect, mutant males often lose grip of the female abdomen during copulation. These phenotypes are rescued by using the GAL4 in the CG12333/wdr37 locus to drive the UAS-human reference WDR37 cDNA. The two variants found in three human subjects failed to rescue these phenotypes, suggesting that these alleles severely affect the function of this protein. Taken together, our data suggest that variants in WDR37 underlie a novel syndromic neurological disorder.
Asunto(s)
Trastorno Dismórfico Corporal/patología , Cerebelo/anomalías , Coloboma/patología , Discapacidades del Desarrollo/patología , Epilepsia/patología , Discapacidad Intelectual/patología , Mutación , Malformaciones del Sistema Nervioso/patología , Repeticiones WD40/genética , Adulto , Secuencia de Aminoácidos , Animales , Trastorno Dismórfico Corporal/genética , Cerebelo/patología , Niño , Coloboma/genética , Discapacidades del Desarrollo/genética , Drosophila melanogaster/genética , Drosophila melanogaster/crecimiento & desarrollo , Epilepsia/genética , Femenino , Humanos , Lactante , Recién Nacido , Discapacidad Intelectual/genética , Masculino , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismo , Malformaciones del Sistema Nervioso/genética , Fenotipo , Homología de Secuencia , Adulto JovenRESUMEN
In multipolar vertebrate neurons, action potentials (APs) initiate close to the soma, at the axonal initial segment. Invertebrate neurons are typically unipolar with dendrites integrating directly into the axon. Where APs are initiated in the axons of invertebrate neurons is unclear. Voltage-gated sodium (NaV) channels are a functional hallmark of the axonal initial segment in vertebrates. We used an intronic Minos-Mediated Integration Cassette to determine the endogenous gene expression and subcellular localization of the sole NaV channel in both male and female Drosophila, para Despite being the only NaV channel in the fly, we show that only 23 ± 1% of neurons in the embryonic and larval CNS express para, while in the adult CNS para is broadly expressed. We generated a single-cell transcriptomic atlas of the whole third instar larval brain to identify para expressing neurons and show that it positively correlates with markers of differentiated, actively firing neurons. Therefore, only 23 ± 1% of larval neurons may be capable of firing NaV-dependent APs. We then show that Para is enriched in an axonal segment, distal to the site of dendritic integration into the axon, which we named the distal axonal segment (DAS). The DAS is present in multiple neuron classes in both the third instar larval and adult CNS. Whole cell patch clamp electrophysiological recordings of adult CNS fly neurons are consistent with the interpretation that Nav-dependent APs originate in the DAS. Identification of the distal NaV localization in fly neurons will enable more accurate interpretation of electrophysiological recordings in invertebrates.SIGNIFICANCE STATEMENT The site of action potential (AP) initiation in invertebrates is unknown. We tagged the sole voltage-gated sodium (NaV) channel in the fly, para, and identified that Para is enriched at a distal axonal segment. The distal axonal segment is located distal to where dendrites impinge on axons and is the likely site of AP initiation. Understanding where APs are initiated improves our ability to model neuronal activity and our interpretation of electrophysiological data. Additionally, para is only expressed in 23 ± 1% of third instar larval neurons but is broadly expressed in adults. Single-cell RNA sequencing of the third instar larval brain shows that para expression correlates with the expression of active, differentiated neuronal markers. Therefore, only 23 ± 1% of third instar larval neurons may be able to actively fire NaV-dependent APs.
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Segmento Inicial del Axón/metabolismo , Proteínas de Drosophila/biosíntesis , Drosophila/metabolismo , Neuronas/metabolismo , Canales de Sodio/biosíntesis , Canales de Sodio Activados por Voltaje/biosíntesis , Potenciales de Acción/fisiología , Animales , Axones/fisiología , Dendritas/metabolismo , Proteínas de Drosophila/genética , Fenómenos Electrofisiológicos , Electrorretinografía , Expresión Génica/genética , Larva , Unión Neuromuscular/metabolismo , Unión Neuromuscular/fisiología , Técnicas de Placa-Clamp , Canales de Sodio/genética , Transcriptoma , Canales de Sodio Activados por Voltaje/genéticaRESUMEN
Interferon regulatory factor 2 binding protein-like (IRF2BPL) encodes a member of the IRF2BP family of transcriptional regulators. Currently the biological function of this gene is obscure, and the gene has not been associated with a Mendelian disease. Here we describe seven individuals who carry damaging heterozygous variants in IRF2BPL and are affected with neurological symptoms. Five individuals who carry IRF2BPL nonsense variants resulting in a premature stop codon display severe neurodevelopmental regression, hypotonia, progressive ataxia, seizures, and a lack of coordination. Two additional individuals, both with missense variants, display global developmental delay and seizures and a relatively milder phenotype than those with nonsense alleles. The IRF2BPL bioinformatics signature based on population genomics is consistent with a gene that is intolerant to variation. We show that the fruit-fly IRF2BPL ortholog, called pits (protein interacting with Ttk69 and Sin3A), is broadly detected, including in the nervous system. Complete loss of pits is lethal early in development, whereas partial knockdown with RNA interference in neurons leads to neurodegeneration, revealing a requirement for this gene in proper neuronal function and maintenance. The identified IRF2BPL nonsense variants behave as severe loss-of-function alleles in this model organism, and ectopic expression of the missense variants leads to a range of phenotypes. Taken together, our results show that IRF2BPL and pits are required in the nervous system in humans and flies, and their loss leads to a range of neurological phenotypes in both species.
RESUMEN
Memory is initially labile and gradually consolidated over time through new protein synthesis into a long-lasting stable form. Studies of odor-shock associative learning in Drosophila have established the mushroom body (MB) as a key brain structure involved in olfactory long-term memory (LTM) formation. Exactly how early neural activity encoded in thousands of MB neurons is consolidated into protein-synthesis-dependent LTM remains unclear. Here, several independent lines of evidence indicate that changes in two MB vertical lobe V3 (MB-V3) extrinsic neurons are required and contribute to an extended neural network involved in olfactory LTM: (i) inhibiting protein synthesis in MB-V3 neurons impairs LTM; (ii) MB-V3 neurons show enhanced neural activity after spaced but not massed training; (iii) MB-V3 dendrites, synapsing with hundreds of MB α/ß neurons, exhibit dramatic structural plasticity after removal of olfactory inputs; (iv) neurotransmission from MB-V3 neurons is necessary for LTM retrieval; and (v) RNAi-mediated down-regulation of oo18 RNA-binding protein (involved in local regulation of protein translation) in MB-V3 neurons impairs LTM. Our results suggest a model of long-term memory formation that includes a systems-level consolidation process, wherein an early, labile olfactory memory represented by neural activity in a sparse subset of MB neurons is converted into a stable LTM through protein synthesis in dendrites of MB-V3 neurons synapsed onto MB α lobes.
Asunto(s)
Proteínas de Drosophila/fisiología , Drosophila melanogaster/fisiología , Regulación de la Expresión Génica , Memoria a Largo Plazo/fisiología , Cuerpos Pedunculados/fisiología , Proteínas de Unión al ARN/fisiología , Animales , Cruzamientos Genéticos , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/metabolismo , Modelos Neurológicos , Cuerpos Pedunculados/metabolismo , Neuronas/metabolismo , Proteínas de Unión al ARN/metabolismo , Transmisión Sináptica , Factores de Transcripción/metabolismo , Factores de Escisión y Poliadenilación de ARNm/metabolismoRESUMEN
Pavlovian olfactory learning in Drosophila produces two genetically distinct forms of intermediate-term memories: anesthesia-sensitive memory, which requires the amnesiac gene, and anesthesia-resistant memory (ARM), which requires the radish gene. Here, we report that ARM is specifically enhanced or inhibited in flies with elevated or reduced serotonin (5HT) levels, respectively. The requirement for 5HT was additive with the memory defect of the amnesiac mutation but was occluded by the radish mutation. This result suggests that 5HT and Radish protein act on the same pathway for ARM formation. Three supporting lines of evidence indicate that ARM formation requires 5HT released from only two dorsal paired medial (DPM) neurons onto the mushroom bodies (MBs), the olfactory learning and memory center in Drosophila: (i) DPM neurons were 5HT-antibody immunopositive; (ii) temporal inhibition of 5HT synthesis or release from DPM neurons, but not from other serotonergic neurons, impaired ARM formation; (iii) knocking down the expression of d5HT1A serotonin receptors in α/ß MB neurons, which are innervated by DPM neurons, inhibited ARM formation. Thus, in addition to the Amnesiac peptide required for anesthesia-sensitive memory formation, the two DPM neurons also release 5HT acting on MB neurons for ARM formation.
Asunto(s)
Anestesia/efectos adversos , Resistencia a Medicamentos/genética , Memoria/efectos de los fármacos , Cuerpos Pedunculados/fisiología , Serotonina/fisiología , Animales , Drosophila , Resistencia a Medicamentos/fisiología , Aprendizaje , Memoria/fisiología , MutaciónRESUMEN
Octopamine is essential for egg-laying in Drosophila melanogaster, but the neuronal pathways and receptors by which it regulates visceral muscles in the reproductive tract are not known. We find that the two octopamine receptors that have been previously implicated in egg-laying-OAMB and Octß2R-are expressed in octopaminergic and glutamatergic neurons that project to the reproductive tract, peripheral ppk(+) neurons within the reproductive tract and epithelial cells that line the lumen of the oviducts. Further optogenetic and mutational analyses indicate that octopamine regulates both oviduct contraction and relaxation via Octß2 and OAMB respectively. Interactions with glutamatergic pathways modify the effects of octopamine. Octopaminergic activation of Octß2R on glutamatergic processes provides a possible mechanism by which octopamine initiates lateral oviduct contractions. We speculate that aminergic pathways in the oviposition circuit may be comparable to some of the mechanisms that regulate visceral muscle contractility in mammals.
RESUMEN
Individuals with autism spectrum disorder (ASD) exhibit an increased burden of de novo mutations (DNMs) in a broadening range of genes. While these studies have implicated hundreds of genes in ASD pathogenesis, which DNMs cause functional consequences in vivo remains unclear. We functionally test the effects of ASD missense DNMs using Drosophila through "humanization" rescue and overexpression-based strategies. We examine 79 ASD variants in 74 genes identified in the Simons Simplex Collection and find 38% of them to cause functional alterations. Moreover, we identify GLRA2 as the cause of a spectrum of neurodevelopmental phenotypes beyond ASD in 13 previously undiagnosed subjects. Functional characterization of variants in ASD candidate genes points to conserved neurobiological mechanisms and facilitates gene discovery for rare neurodevelopmental diseases.
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Trastorno del Espectro Autista , Trastorno Autístico , Drosophila , Trastornos del Neurodesarrollo , Receptores de Glicina , Animales , Trastorno del Espectro Autista/genética , Trastorno del Espectro Autista/patología , Trastorno Autístico/genética , Drosophila/genética , Predisposición Genética a la Enfermedad , Humanos , Trastornos del Neurodesarrollo/genética , Receptores de Glicina/genéticaRESUMEN
Drosophila reproductive behaviors are directed by fruitless neurons. A reanalysis of genomic studies shows that genes encoding dpr and DIP immunoglobulin superfamily (IgSF) members are expressed in fru P1 neurons. We find that each fru P1 and dpr/DIP (fru P1 â© dpr/DIP) overlapping expression pattern is similar in both sexes, but there are dimorphisms in neuronal morphology and cell number. Behavioral studies of fru P1 â© dpr/DIP perturbation genotypes indicate that the mushroom body functions together with the lateral protocerebral complex to direct courtship behavior. A single-cell RNA-seq analysis of fru P1 neurons shows that many DIPs have high expression in a small set of neurons, whereas the dprs are often expressed in a larger set of neurons at intermediate levels, with a myriad of dpr/DIP expression combinations. Functionally, we find that perturbations of sex hierarchy genes and of DIP-ε change the sex-specific morphologies of fru P1 â© DIP-α neurons.
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Moléculas de Adhesión Celular/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Factores de Transcripción/metabolismo , Animales , Moléculas de Adhesión Celular/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/fisiología , Femenino , Masculino , Proteínas del Tejido Nervioso/genética , Neuronas/citología , Neuronas/metabolismo , Análisis de Secuencia de ARN , Caracteres Sexuales , Conducta Sexual Animal , Factores de Transcripción/genéticaRESUMEN
In Drosophila, long-term memory (LTM) requires the cAMP-dependent transcription factor CREBB, expressed in the mushroom bodies (MB) and phosphorylated by PKA. To identify other kinases required for memory formation, we integrated Trojan exons encoding T2A-GAL4 into genes encoding putative kinases and selected for genes expressed in MB. These lines were screened for learning/memory deficits using UAS-RNAi knockdown based on an olfactory aversive conditioning assay. We identified a novel, conserved kinase, Meng-Po (MP, CG11221, SBK1 in human), the loss of which severely affects 3 hr memory and 24 hr LTM, but not learning. Remarkably, memory is lost upon removal of the MP protein in adult MB but restored upon its reintroduction. Overexpression of MP in MB significantly increases LTM in wild-type flies showing that MP is a limiting factor for LTM. We show that PKA phosphorylates MP and that both proteins synergize in a feedforward loop to control CREBB levels and LTM. key words: Drosophila, Mushroom bodies, SBK1, deGradFP, T2A-GAL4, MiMIC.
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Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila/fisiología , Memoria a Largo Plazo , Proteínas Quinasas/metabolismo , Transactivadores/metabolismo , Animales , Condicionamiento Clásico , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Técnicas de Silenciamiento del Gen , Prueba de Complementación Genética , Percepción OlfatoriaRESUMEN
We generated two new genetic tools to efficiently tag genes in Drosophila. The first, Double Header (DH) utilizes intronic MiMIC/CRIMIC insertions to generate artificial exons for GFP mediated protein trapping or T2A-GAL4 gene trapping in vivo based on Cre recombinase to avoid embryo injections. DH significantly increases integration efficiency compared to previous strategies and faithfully reports the expression pattern of genes and proteins. The second technique targets genes lacking coding introns using a two-step cassette exchange. First, we replace the endogenous gene with an excisable compact dominant marker using CRISPR making a null allele. Second, the insertion is replaced with a protein::tag cassette. This sequential manipulation allows the generation of numerous tagged alleles or insertion of other DNA fragments that facilitates multiple downstream applications. Both techniques allow precise gene manipulation and facilitate detection of gene expression, protein localization and assessment of protein function, as well as numerous other applications.
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Drosophila/genética , Entomología/métodos , Marcación de Gen/métodos , Animales , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Genes Reporteros , Integrasas/metabolismo , Mutagénesis InsercionalRESUMEN
Mutations in PLA2G6 (PARK14) cause neurodegenerative disorders in humans, including autosomal recessive neuroaxonal dystrophy and early-onset parkinsonism. We show that loss of iPLA2-VIA, the fly homolog of PLA2G6, reduces lifespan, impairs synaptic transmission, and causes neurodegeneration. Phospholipases typically hydrolyze glycerol phospholipids, but loss of iPLA2-VIA does not affect the phospholipid composition of brain tissue but rather causes an elevation in ceramides. Reducing ceramides with drugs, including myriocin or desipramine, alleviates lysosomal stress and suppresses neurodegeneration. iPLA2-VIA binds the retromer subunits Vps35 and Vps26 and enhances retromer function to promote protein and lipid recycling. Loss of iPLA2-VIA impairs retromer function, leading to a progressive increase in ceramide. This induces a positive feedback loop that affects membrane fluidity and impairs retromer function and neuronal function. Similar defects are observed upon loss of vps26 or vps35 or overexpression of α-synuclein, indicating that these defects may be common in Parkinson disease.
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Ceramidas/metabolismo , Proteínas de Drosophila/metabolismo , Fosfolipasas A2 Grupo VI/metabolismo , Fosfolipasas A2 Grupo X/metabolismo , Enfermedad de Parkinson/metabolismo , Proteínas de Transporte Vesicular/metabolismo , alfa-Sinucleína/metabolismo , Animales , Encéfalo/metabolismo , Línea Celular Tumoral , Drosophila/genética , Proteínas de Drosophila/química , Retroalimentación Fisiológica , Femenino , Fosfolipasas A2 Grupo VI/genética , Células HeLa , Humanos , Lisosomas/metabolismo , Masculino , Fluidez de la Membrana , Mutación , Neuronas/metabolismo , Proteínas Nucleares/química , Proteínas de Unión al ARN/química , Esfingolípidos/metabolismoRESUMEN
Drosophila Dpr (21 paralogs) and DIP proteins (11 paralogs) are cell recognition molecules of the immunoglobulin superfamily (IgSF) that form a complex protein interaction network. DIP and Dpr proteins are expressed in a synaptic layer-specific fashion in the visual system. How interactions between these proteins regulate layer-specific synaptic circuitry is not known. Here we establish that DIP-α and its interacting partners Dpr6 and Dpr10 regulate multiple processes, including arborization within layers, synapse number, layer specificity, and cell survival. We demonstrate that heterophilic binding between Dpr6/10 and DIP-α and homophilic binding between DIP-α proteins promote interactions between processes in vivo. Knockin mutants disrupting the DIP/Dpr binding interface reveal a role for these proteins during normal development, while ectopic expression studies support an instructive role for interactions between DIPs and Dprs in circuit development. These studies support an important role for the DIP/Dpr protein interaction network in regulating cell-type-specific connectivity patterns.
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Proteínas de Drosophila/metabolismo , Regulación del Desarrollo de la Expresión Génica/genética , Neurópilo/metabolismo , Factores de Transcripción/metabolismo , Animales , Animales Modificados Genéticamente , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/fisiología , Drosophila , Proteínas de Drosophila/genética , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Humanos , Bulbo Raquídeo/citología , Bulbo Raquídeo/crecimiento & desarrollo , Mutación/genética , Mapas de Interacción de Proteínas , Resonancia por Plasmón de Superficie , Factores de Transcripción/genética , Transfección , Vías Visuales/metabolismoRESUMEN
Nuclei are actively positioned and anchored to the cytoskeleton via the LINC (Linker of Nucleoskeleton and Cytoskeleton) complex. We identified mutations in the Parkin-like E3 ubiquitin ligase Ariadne-1 (Ari-1) that affect the localization and distribution of LINC complex members in Drosophila. ari-1 mutants exhibit nuclear clustering and morphology defects in larval muscles. We show that Ari-1 mono-ubiquitinates the core LINC complex member Koi. Surprisingly, we discovered functional redundancy between Parkin and Ari-1: increasing Parkin expression rescues ari-1 mutant phenotypes and vice versa. We further show that rare variants in the human homolog of ari-1 (ARIH1) are associated with thoracic aortic aneurysms and dissections, conditions resulting from smooth muscle cell (SMC) dysfunction. Human ARIH1 rescues fly ari-1 mutant phenotypes, whereas human variants found in patients fail to do so. In addition, SMCs obtained from patients display aberrant nuclear morphology. Hence, ARIH1 is critical in anchoring myonuclei to the cytoskeleton.
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Aneurisma de la Aorta/patología , Proteínas Portadoras/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Mutación , Miocitos del Músculo Liso/patología , Ubiquitina-Proteína Ligasas/metabolismo , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Animales , Aneurisma de la Aorta/genética , Aneurisma de la Aorta/metabolismo , Proteínas Portadoras/genética , Núcleo Celular/genética , Núcleo Celular/metabolismo , Núcleo Celular/patología , Preescolar , Citoesqueleto , Proteínas de Drosophila/genética , Drosophila melanogaster/crecimiento & desarrollo , Femenino , Humanos , Masculino , Persona de Mediana Edad , Miocitos del Músculo Liso/metabolismo , Linaje , Fenotipo , Ubiquitina-Proteína Ligasas/genética , Adulto JovenRESUMEN
We generated a library of ~1000 Drosophila stocks in which we inserted a construct in the intron of genes allowing expression of GAL4 under control of endogenous promoters while arresting transcription with a polyadenylation signal 3' of the GAL4. This allows numerous applications. First, ~90% of insertions in essential genes cause a severe loss-of-function phenotype, an effective way to mutagenize genes. Interestingly, 12/14 chromosomes engineered through CRISPR do not carry second-site lethal mutations. Second, 26/36 (70%) of lethal insertions tested are rescued with a single UAS-cDNA construct. Third, loss-of-function phenotypes associated with many GAL4 insertions can be reverted by excision with UAS-flippase. Fourth, GAL4 driven UAS-GFP/RFP reports tissue and cell-type specificity of gene expression with high sensitivity. We report the expression of hundreds of genes not previously reported. Finally, inserted cassettes can be replaced with GFP or any DNA. These stocks comprise a powerful resource for assessing gene function.
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Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Biblioteca de Genes , Factores de Transcripción/genética , Animales , Animales Modificados Genéticamente , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citología , Drosophila melanogaster/metabolismo , Perfilación de la Expresión Génica , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Mutagénesis Insercional , Especificidad de Órganos/genética , Factores de Transcripción/metabolismoRESUMEN
Previously, we described a large collection of Minos-Mediated Integration Cassettes (MiMICs) that contain two phiC31 recombinase target sites and allow the generation of a new exon that encodes a protein tag when the MiMIC is inserted in a codon intron (Nagarkar-Jaiswal et al., 2015). These modified genes permit numerous applications including assessment of protein expression pattern, identification of protein interaction partners by immunoprecipitation followed by mass spec, and reversible removal of the tagged protein in any tissue. At present, these conversions remain time and labor-intensive as they require embryos to be injected with plasmid DNA containing the exon tag. In this study, we describe a simple and reliable genetic strategy to tag genes/proteins that contain MiMIC insertions using an integrated exon encoding GFP flanked by FRT sequences. We document the efficiency and tag 60 mostly uncharacterized genes.
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Marcación de Gen/métodos , Coloración y Etiquetado/métodos , Animales , Fusión Artificial Génica , Drosophila , Genes Reporteros , Vectores Genéticos , Proteínas Fluorescentes Verdes/análisis , Proteínas Fluorescentes Verdes/genética , Mutagénesis Insercional , Plásmidos , Recombinación Genética , Transposasas/metabolismoRESUMEN
Here, we document a collection of â¼7434 MiMIC (Minos Mediated Integration Cassette) insertions of which 2854 are inserted in coding introns. They allowed us to create a library of 400 GFP-tagged genes. We show that 72% of internally tagged proteins are functional, and that more than 90% can be imaged in unfixed tissues. Moreover, the tagged mRNAs can be knocked down by RNAi against GFP (iGFPi), and the tagged proteins can be efficiently knocked down by deGradFP technology. The phenotypes associated with RNA and protein knockdown typically correspond to severe loss of function or null mutant phenotypes. Finally, we demonstrate reversible, spatial, and temporal knockdown of tagged proteins in larvae and adult flies. This new strategy and collection of strains allows unprecedented in vivo manipulations in flies for many genes. These strategies will likely extend to vertebrates.