RESUMO
Morphology is a defining feature of neuronal identity. Like neurons, glia display diverse morphologies, both across and within glial classes, but are also known to be morphologically plastic. Here, we explored the relationship between glial morphology and transcriptional signature using the Drosophila central nervous system (CNS), where glia are categorised into 5 main classes (outer and inner surface glia, cortex glia, ensheathing glia, and astrocytes), which show within-class morphological diversity. We analysed and validated single-cell RNA sequencing data of Drosophila glia in 2 well-characterised tissues from distinct developmental stages, containing distinct circuit types: the embryonic ventral nerve cord (VNC) (motor) and the adult optic lobes (sensory). Our analysis identified a new morphologically and transcriptionally distinct surface glial population in the VNC. However, many glial morphological categories could not be distinguished transcriptionally, and indeed, embryonic and adult astrocytes were transcriptionally analogous despite differences in developmental stage and circuit type. While we did detect extensive within-class transcriptomic diversity for optic lobe glia, this could be explained entirely by glial residence in the most superficial neuropil (lamina) and an associated enrichment for immune-related gene expression. In summary, we generated a single-cell transcriptomic atlas of glia in Drosophila, and our extensive in vivo validation revealed that glia exhibit more diversity at the morphological level than was detectable at the transcriptional level. This atlas will serve as a resource for the community to probe glial diversity and function.
Assuntos
Proteínas de Drosophila , Drosophila , Animais , Drosophila/metabolismo , Neuroglia/metabolismo , Neurônios/metabolismo , Neurópilo/metabolismo , Astrócitos/metabolismo , Proteínas de Drosophila/metabolismoRESUMO
Generating cellular heterogeneity is crucial to the development of complex organs. Organ-fate selector genes and signalling pathways generate cellular diversity by subdividing and patterning naïve tissues to assign them regional identities. The Drosophila eye-antennal imaginal disc is a well-characterised system in which to study regional specification; it is first divided into antennal and eye fates and subsequently retinal differentiation occurs within only the eye field. During development, signalling pathways and selector genes compete with and mutually antagonise each other to subdivide the tissue. Wingless (Wg) signalling is the main inhibitor of retinal differentiation; it does so by promoting antennal/head-fate via selector factors and by antagonising Hedgehog (Hh), the principal differentiation-initiating signal. Wg signalling must be suppressed by JAK/STAT at the disc posterior in order to initiate retinal differentiation. Ras/MEK/MAPK signalling has also been implicated in initiating retinal differentiation but its mode of action is not known. We find that compromising Ras/MEK/MAPK signalling in the early larval disc results in expanded antennal/head cuticle at the expense of the compound eye. These phenotypes correspond both to perturbations in selector factor expression, and to de-repressed wg. Indeed, STAT activity is reduced due to decreased mobility of the ligand Unpaired (Upd) along with a corresponding loss in Dally-like protein (Dlp), a heparan sulphate proteoglycan (HSPG) that aids Upd diffusion. Strikingly, blocking HSPG biogenesis phenocopies compromised Ras/MEK/MAPK, while restoring HSPG expression rescues the adult phenotype significantly. This study identifies a novel mode by which the Ras/MEK/MAPK pathway regulates regional-fate specification via HSPGs during development.
Assuntos
Proteoglicanas de Heparan Sulfato/química , MAP Quinase Quinase 1/metabolismo , Retina/embriologia , Proteínas ras/metabolismo , Animais , Linhagem da Célula , Cruzamentos Genéticos , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Larva/metabolismo , Ligantes , Sistema de Sinalização das MAP Quinases , Fenótipo , Proteínas Quinases/metabolismo , Interferência de RNA , Transdução de Sinais , Proteína Wnt1/metabolismoRESUMO
During neurogenesis, conserved tissue-specific proneural factors establish a cell's competence to take on neural fate from within a field of unspecified cells. Proneural genes encode basic helix-loop-helix transcription factors that promote the expression of 'core' and subtype-specific target genes. Target genes include both pan-neuronal genes and genes that aid in the process of refinement, known as lateral inhibition. In this process, proneural gene expression is increased in the neural progenitor while simultaneously down-regulated in the surrounding cells, in a Notch signalling-dependent manner. Here, we identify nemo (nmo) as a target of members of both Drosophila Atonal and Achaete-Scute proneural factor families and find that mammalian proneural homologs induce Nemo-like-kinase (Nlk) expression in cell culture. We find that nmo loss of function leads to reduced expression of Notch targets and to perturbations in Notch-mediated lateral inhibition. Furthermore, Notch hyperactivity can compensate for nmo loss in the Drosophila eye. Thus nmo promotes Notch-mediated lateral inhibition downstream of proneural factors during neurogenesis.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila/embriologia , Olho/embriologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurogênese/fisiologia , Transdução de Sinais/fisiologia , Animais , Animais Geneticamente Modificados , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Drosophila/genética , Proteínas de Drosophila/genética , Ensaio de Desvio de Mobilidade Eletroforética , Imuno-Histoquímica , Imunoprecipitação , Proteínas Quinases Ativadas por Mitógeno/genética , Receptores Notch/metabolismoRESUMO
Ants are some of the most abundant and familiar animals on Earth, and they play vital roles in most terrestrial ecosystems. Although all ants are eusocial, and display a variety of complex and fascinating behaviors, few genomic resources exist for them. Here, we report the draft genome sequence of a particularly widespread and well-studied species, the invasive Argentine ant (Linepithema humile), which was accomplished using a combination of 454 (Roche) and Illumina sequencing and community-based funding rather than federal grant support. Manual annotation of >1,000 genes from a variety of different gene families and functional classes reveals unique features of the Argentine ant's biology, as well as similarities to Apis mellifera and Nasonia vitripennis. Distinctive features of the Argentine ant genome include remarkable expansions of gustatory (116 genes) and odorant receptors (367 genes), an abundance of cytochrome P450 genes (>110), lineage-specific expansions of yellow/major royal jelly proteins and desaturases, and complete CpG DNA methylation and RNAi toolkits. The Argentine ant genome contains fewer immune genes than Drosophila and Tribolium, which may reflect the prominent role played by behavioral and chemical suppression of pathogens. Analysis of the ratio of observed to expected CpG nucleotides for genes in the reproductive development and apoptosis pathways suggests higher levels of methylation than in the genome overall. The resources provided by this genome sequence will offer an abundance of tools for researchers seeking to illuminate the fascinating biology of this emerging model organism.
Assuntos
Formigas/genética , Genoma de Inseto/genética , Genômica/métodos , Filogenia , Animais , Formigas/fisiologia , Sequência de Bases , California , Metilação de DNA , Biblioteca Gênica , Genética Populacional , Hierarquia Social , Dados de Sequência Molecular , Polimorfismo de Nucleotídeo Único/genética , Receptores Odorantes/genética , Análise de Sequência de DNARESUMO
We report the draft genome sequence of the red harvester ant, Pogonomyrmex barbatus. The genome was sequenced using 454 pyrosequencing, and the current assembly and annotation were completed in less than 1 y. Analyses of conserved gene groups (more than 1,200 manually annotated genes to date) suggest a high-quality assembly and annotation comparable to recently sequenced insect genomes using Sanger sequencing. The red harvester ant is a model for studying reproductive division of labor, phenotypic plasticity, and sociogenomics. Although the genome of P. barbatus is similar to other sequenced hymenopterans (Apis mellifera and Nasonia vitripennis) in GC content and compositional organization, and possesses a complete CpG methylation toolkit, its predicted genomic CpG content differs markedly from the other hymenopterans. Gene networks involved in generating key differences between the queen and worker castes (e.g., wings and ovaries) show signatures of increased methylation and suggest that ants and bees may have independently co-opted the same gene regulatory mechanisms for reproductive division of labor. Gene family expansions (e.g., 344 functional odorant receptors) and pseudogene accumulation in chemoreception and P450 genes compared with A. mellifera and N. vitripennis are consistent with major life-history changes during the adaptive radiation of Pogonomyrmex spp., perhaps in parallel with the development of the North American deserts.
Assuntos
Formigas/genética , Redes Reguladoras de Genes/genética , Genoma de Inseto/genética , Genômica/métodos , Filogenia , Animais , Formigas/fisiologia , Sequência de Bases , Clima Desértico , Hierarquia Social , Dados de Sequência Molecular , América do Norte , Fenótipo , Polimorfismo de Nucleotídeo Único/genética , Receptores Odorantes/genética , Análise de Sequência de DNARESUMO
Glia play a crucial role in providing metabolic support to neurons across different species. To do so, glial cells isolate distinct neuronal compartments from systemic signals and selectively transport specific metabolites and ions to support neuronal development and facilitate neuronal function. Because of their function as barriers, glial cells occupy privileged positions within the nervous system and have also evolved to serve as signaling intermediaries in various contexts. The fruit fly, Drosophila melanogaster, has significantly contributed to our understanding of glial barrier development and function. In this review, we will explore the formation of the glial sheath, blood-brain barrier, and nerve barrier, as well as the significance of glia-extracellular matrix interactions in barrier formation. Additionally, we will delve into the role of glia as signaling intermediaries in regulating nervous system development, function, and response to injury.
Assuntos
Proteínas de Drosophila , Drosophila melanogaster , Animais , Drosophila melanogaster/metabolismo , Neuroglia/metabolismo , Neurônios/metabolismo , Drosophila/metabolismo , Proteínas de Drosophila/metabolismoRESUMO
Defining the origin of neuronal diversity is a major challenge in developmental neurobiology. The Drosophila visual system is an excellent paradigm to study how cellular diversity is generated. Photoreceptors from the eye disc grow their axons into the optic lobe and secrete Hedgehog (Hh) to induce the lamina, such that for every unit eye there is a corresponding lamina unit made up of post-mitotic precursors stacked into columns. Each differentiated column contains five lamina neuron types (L1-L5), making it the simplest neuropil in the optic lobe, yet how this diversity is generated was unknown. Here, we found that Hh pathway activity is graded along the distal-proximal axis of lamina columns, and further determined that this gradient in pathway activity arises from a gradient of Hh ligand. We manipulated Hh pathway activity cell autonomously in lamina precursors and non-cell autonomously by inactivating the Hh ligand and by knocking it down in photoreceptors. These manipulations showed that different thresholds of activity specify unique cell identities, with more proximal cell types specified in response to progressively lower Hh levels. Thus, our data establish that Hh acts as a morphogen to pattern the lamina. Although this is the first such report during Drosophila nervous system development, our work uncovers a remarkable similarity with the vertebrate neural tube, which is patterned by Sonic Hh. Altogether, we show that differentiating neurons can regulate the neuronal diversity of their distant target fields through morphogen gradients.
Assuntos
Proteínas de Drosophila , Drosophila , Animais , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Ligantes , Neurônios/fisiologia , Transdução de Sinais/fisiologiaRESUMO
Extracellular signal-regulated kinase (ERK) lies downstream of a core signalling cascade that controls all aspects of development and adult homeostasis. Recent developments have led to new tools to image and manipulate the pathway. However, visualising ERK activity in vivo with high temporal resolution remains a challenge in Drosophila. We adapted a kinase translocation reporter (KTR) for use in Drosophila, which shuttles out of the nucleus when phosphorylated by ERK. We show that ERK-KTR faithfully reports endogenous ERK signalling activity in developing and adult tissues, and that it responds to genetic perturbations upstream of ERK. Using ERK-KTR in time-lapse imaging, we made two novel observations: firstly, sustained hyperactivation of ERK by expression of dominant-active epidermal growth factor receptor raised the overall level but did not alter the kinetics of ERK activity; secondly, the direction of migration of retinal basal glia correlated with their ERK activity levels, suggesting an explanation for the heterogeneity in ERK activity observed in fixed tissue. Our results show that KTR technology can be applied in Drosophila to monitor ERK activity in real-time and suggest that this modular tool can be further adapted to study other kinases. This article has an associated First Person interview with the first author of the paper.
Assuntos
Drosophila , Sistema de Sinalização das MAP Quinases , Animais , Drosophila/genética , Drosophila/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fosforilação , Transdução de SinaisRESUMO
Neural circuit formation and function require that diverse neurons are specified in appropriate numbers. Known strategies for controlling neuronal numbers involve regulating either cell proliferation or survival. We used the Drosophila visual system to probe how neuronal numbers are set. Photoreceptors from the eye-disc induce their target field, the lamina, such that for every unit eye there is a corresponding lamina unit (column). Although each column initially contains ~6 post-mitotic lamina precursors, only 5 differentiate into neurons, called L1-L5; the 'extra' precursor, which is invariantly positioned above the L5 neuron in each column, undergoes apoptosis. Here, we showed that a glial population called the outer chiasm giant glia (xgO), which resides below the lamina, secretes multiple ligands to induce L5 differentiation in response to epidermal growth factor (EGF) from photoreceptors. By forcing neuronal differentiation in the lamina, we uncovered that though fated to die, the 'extra' precursor is specified as an L5. Therefore, two precursors are specified as L5s but only one differentiates during normal development. We found that the row of precursors nearest to xgO differentiate into L5s and, in turn, antagonise differentiation signalling to prevent the 'extra' precursors from differentiating, resulting in their death. Thus, an intricate interplay of glial signals and feedback from differentiating neurons defines an invariant and stereotyped pattern of neuronal differentiation and programmed cell death to ensure that lamina columns each contain exactly one L5 neuron.
Assuntos
Proteínas de Drosophila , Fator de Crescimento Epidérmico , Diferenciação Celular/fisiologia , Proteínas de Drosophila/metabolismo , Fator de Crescimento Epidérmico/metabolismo , Ligantes , Neuroglia/metabolismo , Neurônios/fisiologiaRESUMO
Many genetic networks are astonishingly robust to quantitative variation, allowing these networks to continue functioning in the face of mutation and environmental perturbation. However, the evolution of such robustness remains poorly understood for real genetic networks. Here we explore whether and how ploidy and recombination affect the evolution of robustness in a detailed computational model of the segment polarity network. We introduce a novel computational method that predicts the quantitative values of biochemical parameters from bit sequences representing genotype, allowing our model to bridge genotype to phenotype. Using this, we simulate 2,000 generations of evolution in a population of individuals under stabilizing and truncation selection, selecting for individuals that could sharpen the initial pattern of engrailed and wingless expression. Robustness was measured by simulating a mutation in the network and measuring the effect on the engrailed and wingless patterns; higher robustness corresponded to insensitivity of this pattern to perturbation. We compared robustness in diploid and haploid populations, with either asexual or sexual reproduction. In all cases, robustness increased, and the greatest increase was in diploid sexual populations; diploidy and sex synergized to evolve greater robustness than either acting alone. Diploidy conferred increased robustness by allowing most deleterious mutations to be rescued by a working allele. Sex (recombination) conferred a robustness advantage through "survival of the compatible": those alleles that can work with a wide variety of genetically diverse partners persist, and this selects for robust alleles.
Assuntos
Ploidias , Recombinação Genética , Animais , Efeito Fundador , Genética Populacional , MutaçãoRESUMO
Glial cells are an essential component of the nervous system of vertebrates and invertebrates. In the human brain, glia are as numerous as neurons, yet the importance of glia to nearly every aspect of nervous system development has only been expounded over the last several decades. Glia are now known to regulate neural specification, synaptogenesis, synapse function, and even broad circuit function. Given their ubiquity, it is not surprising that the contribution of glia to neuronal disease pathogenesis is a growing area of research. In this review, we will summarize the accumulated evidence of glial participation in several distinct phases of nervous system development and organization-neural specification, circuit wiring, and circuit function. Finally, we will highlight how these early developmental roles of glia contribute to nervous system dysfunction in neurodevelopmental and neurodegenerative disorders.
RESUMO
Time-lapse imaging is an essential tool to study dynamic biological processes that cannot be discerned from fixed samples alone. However, imaging cell- and tissue-level processes in intact animals poses numerous challenges if the organism is opaque and/or motile. Explant cultures of intact tissues circumvent some of these challenges, but sample drift remains a considerable obstacle. We employed a simple yet effective technique to immobilize tissues in medium-bathed agarose. We applied this technique to study multiple Drosophila tissues from first-instar larvae to adult stages in various orientations and with no evidence of anisotropic pressure or stress damage. Using this method, we were able to image fine features for up to 18 h and make novel observations. Specifically, we report that fibers characteristic of quiescent neuroblasts are inherited by their basal daughters during reactivation; that the lamina in the developing visual system is assembled roughly 2-3 columns at a time; that lamina glia positions are dynamic during development; and that the nuclear envelopes of adult testis cyst stem cells do not break down completely during mitosis. In all, we demonstrate that our protocol is well-suited for tissue immobilization and long-term live imaging, enabling new insights into tissue and cell dynamics in Drosophila.
RESUMO
Various regions of the developing brain coordinate their construction so that the correct types and numbers of cells are generated to build a functional network. We previously discovered that wrapping glia in the Drosophila visual system are essential for coordinating retinal and lamina development. We showed that wrapping glia, which ensheath photoreceptor axons, respond to an epidermal growth factor cue from photoreceptors by secreting insulins. Wrapping glial insulins activate the mitogen-activated protein kinase (MAPK) pathway downstream of insulin receptor in lamina precursors to induce neuronal differentiation. The signaling relay via wrapping glia introduces a delay that allows the lamina to assemble the correct stoichiometry and physical alignment of precursors before differentiating and imposes a stereotyped spatiotemporal pattern that is relevant for specifying the individual lamina neuron fates. Here, we further describe how wrapping glia morphogenesis correlates with the timing of lamina neuron differentiation by 2-photon live imaging. We also show that although MAPK activity in lamina precursors drives neuronal differentiation, the upstream receptor driving MAPK activation in lamina precursors and the ligand secreted by wrapping glia to trigger it differentially affect lamina neuron differentiation. These results highlight differences in MAPK signaling properties and confirm that communication between photoreceptors, wrapping glia, and lamina precursors must be precisely controlled to build a complex neural network.
RESUMO
During development a limited number of progenitors generate diverse cell types that comprise the nervous system. Neuronal diversity, which arises largely at the level of neural stem cells, is critical for brain function. Often these cells exhibit temporal patterning: they sequentially produce neurons of distinct cell fates as a consequence of intrinsic and/or extrinsic cues. Here, we review recent advances in temporal patterning during neuronal specification, focusing on conserved players and mechanisms in invertebrate and vertebrate models. These studies underscore temporal patterning as an evolutionarily conserved strategy to generate neuronal diversity. Understanding the general principles governing temporal patterning and the molecular players involved will improve our ability to direct neural progenitors towards specific neuronal fates for brain repair.
Assuntos
Encéfalo/citologia , Encéfalo/embriologia , Animais , Evolução Biológica , Invertebrados/embriologia , Células-Tronco Neurais/citologia , Neurônios/citologia , Fatores de Tempo , Vertebrados/embriologiaRESUMO
Neuronal birth and specification must be coordinated across the developing brain to generate the neurons that constitute neural circuits. We used the Drosophila visual system to investigate how development is coordinated to establish retinotopy, a feature of all visual systems. Photoreceptors achieve retinotopy by inducing their target field in the optic lobe, the lamina neurons, with a secreted differentiation cue, epidermal growth factor (EGF). We find that communication between photoreceptors and lamina cells requires a signaling relay through glia. In response to photoreceptor-EGF, glia produce insulin-like peptides, which induce lamina neuronal differentiation. Our study identifies a role for glia in coordinating neuronal development across distinct brain regions, thus reconciling the timing of column assembly with that of delayed differentiation, as well as the spatiotemporal pattern of lamina neuron differentiation.
Assuntos
Drosophila melanogaster/embriologia , Neurogênese , Neuroglia/citologia , Lobo Óptico de Animais não Mamíferos/embriologia , Células Fotorreceptoras de Invertebrados/citologia , Animais , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/genética , Receptores ErbB/genética , Receptores ErbB/metabolismo , Insulina/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Mutação , Lobo Óptico de Animais não Mamíferos/citologia , Receptores de Peptídeos de Invertebrados/genética , Receptores de Peptídeos de Invertebrados/metabolismo , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Transdução de SinaisRESUMO
How neuronal and glial fates are specified from neural precursor cells is an important question for developmental neurobiologists. We address this question in the Drosophila optic lobe, composed of the lamina, medulla, and lobula complex. We show that two gliogenic regions posterior to the prospective lamina also produce lamina wide-field (Lawf) neurons, which share common progenitors with lamina glia. These progenitors express neither canonical neuroblast nor lamina precursor cell markers. They bifurcate into two sub-lineages in response to Notch signaling, generating lamina glia or Lawf neurons, respectively. The newly born glia and Lawfs then migrate tangentially over substantial distances to reach their target tissue. Thus, Lawf neurogenesis, which includes a common origin with glia, as well as neuronal migration, resembles several aspects of vertebrate neurogenesis.
RESUMO
During morphogenesis, extracellular signals trigger actomyosin contractility in subpopulations of cells to coordinate changes in cell shape. To illuminate the link between signaling-mediated tissue patterning and cytoskeletal remodeling, we study the progression of the morphogenetic furrow (MF), the wave of apical constriction that traverses the Drosophila eye imaginal disc preceding photoreceptor neurogenesis. Apical constriction depends on actomyosin contractility downstream of the Hedgehog (Hh) and bone morphogenetic protein (BMP) pathways. We identify a role for integrin adhesion receptors in MF progression. We show that Hh and BMP regulate integrin expression, the loss of which disrupts apical constriction and slows furrow progression; conversely, elevated integrins accelerate furrow progression. We present evidence that integrins regulate MF progression by promoting microtubule stabilization, since reducing microtubule stability rescues integrin-mediated furrow acceleration. Thus, integrins act as a genetic link between tissue-level signaling events and morphological change at the cellular level, leading to morphogenesis and neurogenesis in the eye.