RESUMO
The proper functioning of the nervous system is dependent on the establishment and maintenance of intricate networks of neurons that form functional neural circuits. Once neural circuits are assembled during development, a distinct set of molecular programs is likely required to maintain their connectivity throughout the lifetime of the organism. Here, we demonstrate that Fasciclin 3 (Fas3), an axon guidance cell adhesion protein, is necessary for the maintenance of the olfactory circuit in adult Drosophila. We utilized the TARGET system to spatiotemporally knockdown Fas3 in selected populations of adult neurons. Our findings show that Fas3 knockdown results in the death of olfactory circuit neurons and reduced survival of adults. We also demonstrated that Fas3 knockdown activates caspase-3-mediated cell death in olfactory local interneurons, which can be rescued by overexpressing baculovirus p35, an anti-apoptotic protein. This work adds to the growing set of evidence indicating a crucial role for axon guidance proteins in the maintenance of neuronal circuits in adults.
Assuntos
Proteínas de Drosophila , Drosophila melanogaster , Interneurônios , Animais , Caspase 3/metabolismo , Caspase 3/genética , Drosophila melanogaster/metabolismo , Drosophila melanogaster/genética , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Técnicas de Silenciamento de Genes , Interneurônios/metabolismoRESUMO
Axon guidance cues direct the growth and steering of neuronal growth cones, thus guiding the axons to their targets during development. Nonetheless, after axons have reached their targets and established functional circuits, many mature neurons continue to express these developmental cues. The role of axon guidance cues in the adult nervous system has not been fully elucidated. Using the expression pattern data available on FlyBase, we found that more than 96% of the guidance genes that are expressed in the Drosophila melanogaster embryo continue to be expressed in adults. We utilized the GeneSwitch and TARGET systems to spatiotemporally knockdown the expression of these guidance genes selectively in the adult neurons, once the development was completed. We performed an RNA interference (RNAi) screen against 44 guidance genes in the adult Drosophila nervous system and identified 14 genes that are required for adult survival and normal motility. Additionally, we show that adult expression of Semaphorins and Plexins in motor neurons is necessary for neuronal survival, indicating that guidance genes have critical functions in the mature nervous system.
Assuntos
Drosophila melanogaster , Semaforinas , Animais , Drosophila melanogaster/metabolismo , Semaforinas/genética , Neurônios Motores/metabolismo , Axônios/metabolismo , Drosophila/metabolismoRESUMO
Retrograde bone morphogenetic protein (BMP) signaling at the Drosophila neuromuscular junction (NMJ) has served as a paradigm to study TGF-ß-dependent synaptic function and maturation. Yet, how retrograde BMP signaling transcriptionally regulates these functions remains unresolved. Here, we uncover a gene network, enriched for neurotransmission-related genes, that is controlled by retrograde BMP signaling in motor neurons through two Smad-binding cis-regulatory motifs, the BMP-activating (BMP-AE) and silencer (BMP-SE) elements. Unpredictably, both motifs mediate direct gene activation, with no involvement of the BMP derepression pathway regulators Schnurri and Brinker. Genome editing of candidate BMP-SE and BMP-AE within the locus of the active zone gene bruchpilot, and a novel Ly6 gene witty, demonstrated the role of these motifs in upregulating genes required for the maturation of pre- and post-synaptic NMJ compartments. Our findings uncover how Smad-dependent transcriptional mechanisms specific to motor neurons directly orchestrate a gene network required for synaptic maturation by retrograde BMP signaling.
Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Proteínas de Drosophila , Drosophila/metabolismo , Redes Reguladoras de Genes , Junção Neuromuscular/metabolismo , Animais , Animais Geneticamente Modificados , Drosophila/genética , Proteínas de Drosophila/metabolismo , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismoRESUMO
All viruses must usurp host ribosomes for viral protein synthesis. Dicistroviruses utilize an intergenic region internal ribosome entry site (IGR IRES) to directly recruit ribosomes and mediate translation initiation from a non-AUG start codon. The IGR IRES adopts a three-pseudoknot structure that comprises a ribosome binding domain of pseudoknot II and III (PKII and PKIII), and a tRNA-like anticodon domain (PKI) connected via a short, one to three nucleotide hinge region. Recent cryo-EM structural analysis of the dicistrovirus Taura syndrome virus (TSV) IGR IRES bound to the ribosome suggests that the hinge region may facilitate translocation of the IRES from the ribosomal A to P site. In this study, we provide mechanistic and functional insights into the role of the hinge region in IGR IRES translation. Using the honeybee dicistrovirus, Israeli acute paralysis virus (IAPV), as a model, we demonstrate that mutations of the hinge region resulted in decreased IRES-dependent translation in vitro. Toeprinting primer extension analysis of mutant IRESs bound to purified ribosomes and in rabbit reticulocyte lysates showed defects in the initial ribosome positioning on the IRES. Finally, using a hybrid dicistrovirus clone, mutations in the hinge region of the IAPV IRES resulted in decreased viral yield. Our work reveals an unexpected role of the hinge region of the dicistrovirus IGR IRES coordinating the two independently folded domains of the IRES to properly position the ribosome to start translation. IMPORTANCE Viruses must use the host cell machinery to direct viral protein expression for productive infection. One such mechanism is an internal ribosome entry site that can directly recruit host cell machinery. In this study, we have identified a novel sequence in an IRES that provides insight into the mechanism of viral gene expression. Specifically, this novel sequence promotes viral IRES activity by directly guiding the host cell machinery to start gene expression at a specific site.
Assuntos
Dicistroviridae , Sítios Internos de Entrada Ribossomal , Viroses , Vírus , Animais , Dicistroviridae/genética , Dicistroviridae/metabolismo , Sítios Internos de Entrada Ribossomal/genética , Mutação , Biossíntese de Proteínas , Coelhos , Ribossomos/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismo , Viroses/metabolismo , Viroses/virologia , Vírus/genéticaRESUMO
Gene variant discovery is becoming routine, but it remains difficult to usefully interpret the functional consequence or disease relevance of most variants. To fill this interpretation gap, experimental assays of variant function are becoming common place. Yet, it remains challenging to make these assays reproducible, scalable to high numbers of variants, and capable of assessing defined gene-disease mechanism for clinical interpretation aligned to the ClinGen Sequence Variant Interpretation (SVI) Working Group guidelines for 'well-established assays'. Drosophila melanogaster offers great potential as an assay platform, but was untested for high numbers of human variants adherent to these guidelines. Here, we wished to test the utility of Drosophila as a platform for scalable well-established assays. We took a genetic interaction approach to test the function of ~100 human PTEN variants in cancer-relevant suppression of PI3K/AKT signaling in cellular growth and proliferation. We validated the assay using biochemically characterized PTEN mutants as well as 23 total known pathogenic and benign PTEN variants, all of which the assay correctly assigned into predicted functional categories. Additionally, function calls for these variants correlated very well with our recent published data from a human cell line. Finally, using these pathogenic and benign variants to calibrate the assay, we could set readout thresholds for clinical interpretation of the pathogenicity of 70 other PTEN variants. Overall, we demonstrate that Drosophila offers a powerful assay platform for clinical variant interpretation, that can be used in conjunction with other well-established assays, to increase confidence in the accurate assessment of variant function and pathogenicity.
Assuntos
Proliferação de Células , Drosophila melanogaster/genética , PTEN Fosfo-Hidrolase/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Humanos , Transdução de SinaisRESUMO
Retrograde BMP signaling and canonical pMad/Medea-mediated transcription regulate diverse target genes across subsets of Drosophila efferent neurons, to differentiate neuropeptidergic neurons and promote motor neuron terminal maturation. How a common BMP signal regulates diverse target genes across many neuronal subsets remains largely unresolved, although available evidence implicates subset-specific transcription factor codes rather than differences in BMP signaling. Here we examine the cis-regulatory mechanisms restricting BMP-induced FMRFa neuropeptide expression to Tv4-neurons. We find that pMad/Medea bind at an atypical, low affinity motif in the FMRFa enhancer. Converting this motif to high affinity caused ectopic enhancer activity and eliminated Tv4-neuron expression. In silico searches identified additional motif instances functional in other efferent neurons, implicating broader functions for this motif in BMP-dependent enhancer activity. Thus, differential interpretation of a common BMP signal, conferred by low affinity pMad/Medea binding motifs, can contribute to the specification of BMP target genes in efferent neuron subsets.
Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Drosophila melanogaster/metabolismo , Neurônios/metabolismo , Elementos de Resposta , Animais , Proteínas Morfogenéticas Ósseas/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Regulação da Expressão Gênica , Transdução de Sinais , Proteína Smad4/genética , Proteína Smad4/metabolismoRESUMO
Functional variomics provides the foundation for personalized medicine by linking genetic variation to disease expression, outcome and treatment, yet its utility is dependent on appropriate assays to evaluate mutation impact on protein function. To fully assess the effects of 106 missense and nonsense variants of PTEN associated with autism spectrum disorder, somatic cancer and PTEN hamartoma syndrome (PHTS), we take a deep phenotypic profiling approach using 18 assays in 5 model systems spanning diverse cellular environments ranging from molecular function to neuronal morphogenesis and behavior. Variants inducing instability occur across the protein, resulting in partial-to-complete loss-of-function (LoF), which is well correlated across models. However, assays are selectively sensitive to variants located in substrate binding and catalytic domains, which exhibit complete LoF or dominant negativity independent of effects on stability. Our results indicate that full characterization of variant impact requires assays sensitive to instability and a range of protein functions.
Assuntos
Doença/genética , Modelos Genéticos , Mutação de Sentido Incorreto/genética , PTEN Fosfo-Hidrolase/genética , Animais , Comportamento Animal , Caenorhabditis elegans/fisiologia , Células Cultivadas , Dendritos/fisiologia , Drosophila/genética , Drosophila/crescimento & desenvolvimento , Ensaios Enzimáticos , Células HEK293 , Humanos , Neoplasias/genética , Sistema Nervoso/crescimento & desenvolvimento , Fosforilação , Estabilidade Proteica , Proteínas Proto-Oncogênicas c-akt/metabolismo , Células Piramidais/metabolismo , Ratos Sprague-Dawley , Saccharomyces cerevisiae/metabolismoRESUMO
Retrograde Bone Morphogenetic Protein (BMP) signaling in neurons is essential for the differentiation and synaptic function of many neuronal subtypes. BMP signaling regulates these processes via Smad transcription factor activity, yet the scope and nature of Smad-dependent gene regulation in neurons are mostly unknown. Here, we applied a computational approach to predict Smad-binding cis-regulatory BMP-Activating Elements (BMP-AEs) in Drosophila, followed by transgenic in vivo reporter analysis to test their neuronal subtype enhancer activity in the larval central nervous system (CNS). We identified 34 BMP-AE-containing genomic fragments that are responsive to BMP signaling in neurons, and showed that the embedded BMP-AEs are required for this activity. RNA-seq analysis identified BMP-responsive genes in the CNS and revealed that BMP-AEs selectively enrich near BMP-activated genes. These data suggest that functional BMP-AEs control nearby BMP-activated genes, which we validated experimentally. Finally, we demonstrated that the BMP-AE motif mediates a conserved Smad-responsive function in the Drosophila and vertebrate CNS. Our results provide evidence that BMP signaling controls neuronal function by directly coordinating the expression of a battery of genes through widespread deployment of a conserved Smad-responsive cis-regulatory motif.
Assuntos
Proteínas Morfogenéticas Ósseas/fisiologia , Proteínas de Drosophila/fisiologia , Neurônios/metabolismo , Elementos de Resposta , Transdução de Sinais , Ativação Transcricional , Animais , Antígenos Ly/genética , Antígenos Ly/metabolismo , Embrião de Galinha , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Evolução Molecular , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Proteínas Smad/metabolismo , Proteína Smad4/metabolismo , Fatores de Transcrição/metabolismoRESUMO
RNA structures can interact with the ribosome to alter translational reading frame maintenance and promote recoding that result in alternative protein products. Here, we show that the internal ribosome entry site (IRES) from the dicistrovirus Cricket paralysis virus drives translation of the 0-frame viral polyprotein and an overlapping +1 open reading frame, called ORFx, via a novel mechanism whereby a subset of ribosomes recruited to the IRES bypasses 37 nucleotides downstream to resume translation at the +1-frame 13th non-AUG codon. A mutant of CrPV containing a stop codon in the +1 frame ORFx sequence, yet synonymous in the 0-frame, is attenuated compared to wild-type virus in a Drosophila infection model, indicating the importance of +1 ORFx expression in promoting viral pathogenesis. This work demonstrates a novel programmed IRES-mediated recoding strategy to increase viral coding capacity and impact virus infection, highlighting the diversity of RNA-driven translation initiation mechanisms in eukaryotes.
Assuntos
Dicistroviridae/genética , Regulação Viral da Expressão Gênica , Interações Hospedeiro-Patógeno/genética , Sítios Internos de Entrada Ribossomal , Biossíntese de Proteínas , Proteínas Virais/genética , Animais , Pareamento de Bases , Sequência de Bases , Linhagem Celular , Dicistroviridae/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/metabolismo , Drosophila melanogaster/virologia , Conformação de Ácido Nucleico , Fases de Leitura Aberta , Poliproteínas/genética , Poliproteínas/metabolismo , Ribossomos/genética , Ribossomos/metabolismo , Transdução de Sinais , Proteínas Virais/metabolismoRESUMO
Female-specific Ilp7 neuropeptide-expressing motoneurons (FS-Ilp7 motoneurons) are required in Drosophila for oviduct function in egg laying. Here, we uncover cellular and genetic mechanisms underlying their female-specific generation. We demonstrate that programmed cell death (PCD) eliminates FS-Ilp7 motoneurons in males, and that this requires male-specific splicing of the sex-determination gene fruitless (fru) into the FruMC isoform. However, in females, fru alleles that only generate FruM isoforms failed to kill FS-Ilp7 motoneurons. This blockade of FruM-dependent PCD was not attributable to doublesex gene function but to a non-canonical role for transformer (tra), a gene encoding the RNA splicing activator that regulates female-specific splicing of fru and dsx transcripts. In both sexes, we show that Tra prevents PCD even when the FruM isoform is expressed. In addition, we found that FruMC eliminated FS-Ilp7 motoneurons in both sexes, but only when Tra was absent. Thus, FruMC-dependent PCD eliminates female-specific neurons in males, and Tra plays a double-assurance function in females to establish and reinforce the decision to generate female-specific neurons.
Assuntos
Proteínas de Drosophila/metabolismo , Neurônios Motores/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neuropeptídeos/metabolismo , Proteínas Nucleares/metabolismo , Caracteres Sexuais , Fatores de Transcrição/metabolismo , Animais , Morte Celular/genética , Proteínas de Drosophila/genética , Drosophila melanogaster , Feminino , Masculino , Neurônios Motores/citologia , Proteínas do Tecido Nervoso/genética , Neuropeptídeos/genética , Proteínas Nucleares/genética , Fatores de Transcrição/genéticaRESUMO
Oriented cell division is one mechanism progenitor cells use during development and to maintain tissue homeostasis. Common to most cell types is the asymmetric establishment and regulation of cortical NuMA-dynein complexes that position the mitotic spindle. Here, we discover that HMMR acts at centrosomes in a PLK1-dependent pathway that locates active Ran and modulates the cortical localization of NuMA-dynein complexes to correct mispositioned spindles. This pathway was discovered through the creation and analysis of Hmmr-knockout mice, which suffer neonatal lethality with defective neural development and pleiotropic phenotypes in multiple tissues. HMMR over-expression in immortalized cancer cells induces phenotypes consistent with an increase in active Ran including defects in spindle orientation. These data identify an essential role for HMMR in the PLK1-dependent regulatory pathway that orients progenitor cell division and supports neural development.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proliferação de Células , Proteínas da Matriz Extracelular/metabolismo , Receptores de Hialuronatos/metabolismo , Células-Tronco Neurais/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Fuso Acromático/metabolismo , Animais , Encéfalo/embriologia , Dineínas/metabolismo , Camundongos Knockout , Proteínas Nucleares/metabolismo , Proteína ran de Ligação ao GTP/metabolismo , Quinase 1 Polo-LikeRESUMO
Hyperpolarization-activated cyclic nucleotide-gated "HCN" channels, which underlie the hyperpolarization-activated current (Ih), have been proposed to play diverse roles in neurons. The presynaptic HCN channel is thought to both promote and inhibit neurotransmitter release from synapses, depending upon its interactions with other presynaptic ion channels. In larvae of Drosophila melanogaster, inhibition of the presynaptic HCN channel by the drug ZD7288 reduces the enhancement of neurotransmitter release at motor terminals by serotonin but this drug has no effect on basal neurotransmitter release, implying that the channel does not contribute to firing under basal conditions. Here, we show that genetic disruption of the sole HCN gene (Ih) reduces the amplitude of the evoked response at the neuromuscular junction (NMJ) of third instar larvae by decreasing the number of released vesicles. The anatomy of the (NMJ) is not notably affected by disruption of the Ih gene. We propose that the presynaptic HCN channel is active under basal conditions and promotes neurotransmission at larval motor terminals. Finally, we demonstrate that Ih partial loss-of-function mutant adult flies have impaired locomotion, and, thus, we hypothesize that the presynaptic HCN channel at the (NMJ) may contribute to coordinated movement.
RESUMO
Stress granules (SGs) are cytosolic ribonucleoprotein aggregates that are induced during cellular stress. Several viruses modulate SG formation, suggesting that SGs have an impact on virus infection. However, the mechanisms and impact of modulating SG assembly in infected cells are not completely understood. In this study, we identify the dicistrovirus cricket paralysis virus 1A (CrPV-1A) protein that functions to inhibit SG assembly during infection. Moreover, besides inhibiting RNA interference, CrPV-1A also inhibits host transcription, which indirectly modulates SG assembly. Thus, CrPV-1A is a multifunctional protein. We identify a key R146A residue that is responsible for these effects, and mutant CrPV(R146A) virus infection is attenuated in Drosophila melanogaster S2 cells and adult fruit flies and results in increased SG formation. Treatment of CrPV(R146A)-infected cells with actinomycin D, which represses transcription, restores SG assembly suppression and viral yield. In summary, CrPV-1A modulates several cellular processes to generate a cellular environment that promotes viral translation and replication.IMPORTANCE RNA viruses encode a limited set of viral proteins to modulate an array of cellular processes in order to facilitate viral replication and inhibit antiviral defenses. In this study, we identified a viral protein, called CrPV-1A, within the dicistrovirus cricket paralysis virus that can inhibit host transcription, modulate viral translation, and block a cellular process called stress granule assembly. We also identified a specific amino acid within CrPV-1A that is important for these cellular processes and that mutant viruses containing mutations of CrPV-1A attenuate virus infection. We also demonstrate that the CrPV-1A protein can also modulate cellular processes in human cells, suggesting that the mode of action of CrPV-1A is conserved. We propose that CrPV-1A is a multifunctional, versatile protein that creates a cellular environment in virus-infected cells that permits productive virus infection.
Assuntos
Grânulos Citoplasmáticos/fisiologia , Vírus de Insetos/fisiologia , Proteínas Virais/fisiologia , Animais , Drosophila melanogaster , Feminino , Inativação Gênica , Células HeLa , Humanos , Masculino , Transcrição Gênica , Replicação ViralRESUMO
Neuronal differentiation often requires target-derived signals from the cells they innervate. These signals typically activate neural subtype-specific genes, but the gene regulatory mechanisms remain largely unknown. Highly restricted expression of the FMRFa neuropeptide in Drosophila Tv4 neurons requires target-derived BMP signaling and a transcription factor code that includes Apterous. Using integrase transgenesis of enhancer reporters, we functionally dissected the Tv4-enhancer of FMRFa within its native cellular context. We identified two essential but discrete cis-elements, a BMP-response element (BMP-RE) that binds BMP-activated pMad, and a homeodomain-response element (HD-RE) that binds Apterous. These cis-elements have low activity and must be combined for Tv4-enhancer activity. Such combinatorial activity is often a mechanism for restricting expression to the intersection of cis-element spatiotemporal activities. However, concatemers of the HD-RE and BMP-RE cis-elements were found to independently generate the same spatiotemporal expression as the Tv4-enhancer. Thus, the Tv4-enhancer atypically combines two low-activity cis-elements that confer the same output from distinct inputs. The activation of target-dependent genes is assumed to 'wait' for target contact. We tested this directly, and unexpectedly found that premature BMP activity could not induce early FMRFa expression; also, we show that the BMP-insensitive HD-RE cis-element is activated at the time of target contact. This led us to uncover a role for the nuclear receptor, seven up (svp), as a repressor of FMRFa induction prior to target contact. Svp is normally downregulated immediately prior to target contact, and we found that maintaining Svp expression prevents cis-element activation, whereas reducing svp gene dosage prematurely activates cis-element activity. We conclude that the target-dependent FMRFa gene is repressed prior to target contact, and that target-derived BMP signaling directly activates FMRFa gene expression through an atypical gene regulatory mechanism.
Assuntos
Drosophila/genética , FMRFamida/genética , Redes Reguladoras de Genes , Neurônios/metabolismo , Elementos de Resposta , Sequência de Aminoácidos , Animais , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , FMRFamida/metabolismo , Proteínas com Homeodomínio LIM/genética , Proteínas com Homeodomínio LIM/metabolismo , Dados de Sequência Molecular , Receptores de Esteroides/genética , Receptores de Esteroides/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Aggregation of highly phosphorylated tau is a hallmark of Alzheimer's disease and other tauopathies. Nevertheless, animal models demonstrate that tau-mediated dysfunction/toxicity may not require large tau aggregates but instead may be caused by soluble hyper-phosphorylated tau or by small tau oligomers. Challenging this widely held view, we use multiple techniques to show that insoluble tau oligomers form in conditions where tau-mediated dysfunction is rescued in vivo. This shows that tau oligomers are not necessarily always toxic. Furthermore, their formation correlates with increased tau levels, caused intriguingly, by either pharmacological or genetic inhibition of tau kinase glycogen-synthase-kinase-3beta (GSK-3ß). Moreover, contrary to common belief, these tau oligomers were neither highly phosphorylated, and nor did they contain beta-pleated sheet structure. This may explain their lack of toxicity. Our study makes the novel observation that tau also forms non-toxic insoluble oligomers in vivo in addition to toxic oligomers, which have been reported by others. Whether these are inert or actively protective remains to be established. Nevertheless, this has wide implications for emerging therapeutic strategies such as those that target dissolution of tau oligomers as they may be ineffective or even counterproductive unless they act on the relevant toxic oligomeric tau species.
Assuntos
Neurônios/patologia , Multimerização Proteica , Proteínas tau/metabolismo , Animais , Drosophila melanogaster/metabolismo , Drosophila melanogaster/ultraestrutura , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Humanos , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/ultraestrutura , Fenótipo , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Multimerização Proteica/efeitos dos fármacos , Estrutura Secundária de Proteína , Solubilidade , Análise Espectral Raman , Proteínas tau/químicaRESUMO
The broad range of tissue and cellular diversity of animals is generated to a large extent by the hierarchical deployment of sequence-specific transcription factors and co-factors (collectively referred to as TF's herein) during development. Our understanding of these developmental processes has been facilitated by the recognition that the activities of many TF's can be meaningfully described by a few functional categories that usefully convey a sense for how the TF's function, and also provides a sense for the regulatory organization of the developmental processes in which they participate. Here, we draw on examples from studies in Caenorhabditis elegans, Drosophila melanogaster, and vertebrates to discuss how the terms spatial selector, temporal selector, tissue/cell type selector, terminal selector and combinatorial code may be usefully applied to categorize the activities of TF's at critical steps of nervous system construction. While we believe that these functional categories are useful for understanding the organizational principles by which TF's direct nervous system construction, we however caution against the assumption that a TF's function can be solely or fully defined by any single functional category. Indeed, most TF's play diverse roles within different functional categories, and their roles can blur the lines we draw between these categories. Regardless, it is our belief that the concepts discussed here are helpful in clarifying the regulatory complexities of nervous system development, and hope they prove useful when interpreting mutant phenotypes, designing future experiments, and programming specific neuronal cell types for use in therapies.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Neurogênese/genética , Ativação Transcricional , Animais , Caenorhabditis elegans/embriologia , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismoRESUMO
The Drosophila Bithorax complex (BX-C) Hox cluster contains a bidirectionally transcribed miRNA locus, and a deletion mutant (Δmir) lays no eggs and is completely sterile. We show these miRNAs are expressed and active in distinct spatial registers along the anterior-posterior axis in the CNS. Δmir larvae derepress a network of direct homeobox gene targets in the posterior ventral nerve cord (VNC), including BX-C genes and their TALE cofactors. These are phenotypically critical targets, because sterility of Δmir mutants was substantially rescued by heterozygosity of these genes. The posterior VNC contains Ilp7+ oviduct motoneurons, whose innervation and morphology are defective in Δmir females, and substantially rescued by heterozygosity of Δmir targets, especially within the BX-C. Collectively, we reveal (1) critical roles for Hox miRNAs that determine segment-specific expression of homeotic genes, which are not masked by transcriptional regulation; and (2) that BX-C miRNAs are essential for neural patterning and reproductive behavior.
Assuntos
Sistema Nervoso Central/metabolismo , Drosophila melanogaster/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Larva/metabolismo , MicroRNAs/genética , Oviductos/metabolismo , Animais , Sequência de Bases , Padronização Corporal , Sistema Nervoso Central/citologia , Proteínas de Drosophila/genética , Drosophila melanogaster/crescimento & desenvolvimento , Feminino , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Genes Homeobox/fisiologia , Processamento de Imagem Assistida por Computador , Técnicas Imunoenzimáticas , Hibridização In Situ , Larva/crescimento & desenvolvimento , Dados de Sequência Molecular , Neurônios Motores/citologia , Neurônios Motores/metabolismo , Família Multigênica , Oviductos/citologia , Homologia de Sequência do Ácido Nucleico , Comportamento Sexual Animal , Fatores de Transcrição/genéticaRESUMO
In Drosophila melanogaster, much of our understanding of sexually dimorphic neuronal development and function comes from the study of male behavior, leaving female behavior less well understood. Here, we identify a post-embryonic population of Insulin-like peptide 7 (Ilp7)-expressing neurons in the posterior ventral nerve cord that innervate the reproductive tracts and exhibit a female bias in their function. They form two distinct dorsal and ventral subsets in females, but only a single dorsal subset in males, signifying a rare example of a female-specific neuronal subset. Female post-embryonic Ilp7 neurons are glutamatergic motoneurons innervating the oviduct and are required for female fertility. In males, they are serotonergic/glutamatergic neuromodulatory neurons innervating the seminal vesicle but are not required for male fertility. In both sexes, these neurons express the sex-differentially spliced fruitless-P1 transcript but not doublesex. The male fruitless-P1 isoform (fruM) was necessary and sufficient for serotonin expression in the shared dorsal Ilp7 subset, but although it was necessary for eliminating female-specific Ilp7 neurons in males, it was not sufficient for their elimination in females. By contrast, sex-specific RNA-splicing by female-specific transformer is necessary for female-type Ilp7 neurons in females and is sufficient for their induction in males. Thus, the emergence of female-biased post-embryonic Ilp7 neurons is mediated in a subset-specific manner by a tra- and fru-dependent mechanism in the shared dorsal subset, and a tra-dependent, fru-independent mechanism in the female-specific subset. These studies provide an important counterpoint to studies of the development and function of male-biased neuronal dimorphism in Drosophila.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/embriologia , Embrião não Mamífero/citologia , Neurônios/metabolismo , Neuropeptídeos/metabolismo , Caracteres Sexuais , Envelhecimento , Animais , Drosophila melanogaster/fisiologia , Embrião não Mamífero/metabolismo , Feminino , Fertilidade , Masculino , Neurônios Motores/citologia , Neurônios Motores/metabolismo , Neurônios/citologia , Fenótipo , Glândulas Seminais/citologia , Glândulas Seminais/metabolismo , Neurônios Serotoninérgicos/citologia , Neurônios Serotoninérgicos/metabolismo , Sistema Urogenital/citologia , Sistema Urogenital/inervaçãoRESUMO
During metamorphosis in holometabolous insects, the nervous system undergoes dramatic remodeling as it transitions from its larval to its adult form. Many neurons are generated through post-embryonic neurogenesis to have adult-specific roles, but perhaps more striking is the dramatic remodeling that occurs to transition neurons from functioning in the larval to the adult nervous system. These neurons exhibit a remarkable degree of plasticity during this transition; many subsets undergo programmed cell death, others remodel their axonal and dendritic arbors extensively, whereas others undergo trans-differentiation to alter their terminal differentiation gene expression profiles. Yet other neurons appear to be developmentally frozen in an immature state throughout larval life, to be awakened at metamorphosis by a process we term temporally-tuned differentiation. These multiple forms of remodeling arise from subtype-specific responses to a single metamorphic trigger, ecdysone. Here, we discuss recent progress in Drosophila melanogaster that is shedding light on how subtype-specific programs of neuronal remodeling are generated during metamorphosis.