RESUMO
Male Lepidoptera produce two distinct sperm types; each ejaculate contains both eupyrene sperm, which can fertilize the egg, and apyrene sperm, which are not fertilization competent. These sperm have distinct morphologies, unique functions and different proteomes. Their production is highly regulated, however, very few genes with specific roles in the production of one or other morph have been described. We present the first comparative transcriptomics study of precursors of eupyrene and apyrene sperm to identify genes potentially implicated in regulating or enacting the distinct differentiation programmes. Differentially expressed genes included genes with potential roles in transcriptional regulation, cell cycle and sperm morphology. We identified gene duplications generating paralogues with functions restricted to one or other morph. However, phylogenetic analysis also revealed evolutionary flexibility in expression patterns of duplicated genes between different lepidopteran species. An improved understanding of lepidopteran reproduction will be vital in targeting prevalent pests in agriculture, and on the flip side, ensuring the fertility and thus survival of pollinator populations in response to environmental stress.
Assuntos
Mariposas , Filogenia , Espermatozoides , Animais , Masculino , Mariposas/genética , Espermatozoides/metabolismo , Transcriptoma , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Duplicação Gênica , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismoRESUMO
The endosymbiotic bacteria Wolbachia can invade insect populations by modifying host reproduction through cytoplasmic incompatibility (CI), an effect that results in embryonic lethality when Wolbachia-carrying males mate with Wolbachia-free females. Here we describe a transgenic system for recreating CI in the major arbovirus vector Aedes aegypti using CI factor (cif) genes from wAlbB, a Wolbachia strain currently being deployed to reduce dengue transmission. CI-like sterility is induced when cifA and cifB are co-expressed in testes; this sterility is rescued by maternal cifA expression, thereby reproducing the pattern of Wolbachia-induced CI. Expression of cifB alone is associated with extensive DNA damage and disrupted spermatogenesis. The strength of rescue by maternal cifA expression is dependent on the comparative levels of cifA/cifB expression in males. These findings are consistent with CifB acting as a toxin and CifA as an antitoxin, with CifA attenuating CifB toxicity in both the male germline and in developing embryos. These findings provide important insights into the interactions between cif genes and their mechanism of activity and provide a foundation for the building of a cif gene-based drive system in Ae. aegypti.
Assuntos
Aedes , Infertilidade , Wolbachia , Animais , Masculino , Feminino , Mosquitos Vetores/genética , Animais Geneticamente ModificadosRESUMO
Proper differentiation of sperm from germline stem cells, essential for production of the next generation, requires dramatic changes in gene expression that drive remodeling of almost all cellular components, from chromatin to organelles to cell shape itself. Here, we provide a single nucleus and single cell RNA-seq resource covering all of spermatogenesis in Drosophila starting from in-depth analysis of adult testis single nucleus RNA-seq (snRNA-seq) data from the Fly Cell Atlas (FCA) study. With over 44,000 nuclei and 6000 cells analyzed, the data provide identification of rare cell types, mapping of intermediate steps in differentiation, and the potential to identify new factors impacting fertility or controlling differentiation of germline and supporting somatic cells. We justify assignment of key germline and somatic cell types using combinations of known markers, in situ hybridization, and analysis of extant protein traps. Comparison of single cell and single nucleus datasets proved particularly revealing of dynamic developmental transitions in germline differentiation. To complement the web-based portals for data analysis hosted by the FCA, we provide datasets compatible with commonly used software such as Seurat and Monocle. The foundation provided here will enable communities studying spermatogenesis to interrogate the datasets to identify candidate genes to test for function in vivo.
Assuntos
Células-Tronco Adultas , Testículo , Animais , Masculino , Testículo/metabolismo , Drosophila , RNA-Seq , SêmenRESUMO
For more than 100 years, the fruit fly Drosophila melanogaster has been one of the most studied model organisms. Here, we present a single-cell atlas of the adult fly, Tabula Drosophilae, that includes 580,000 nuclei from 15 individually dissected sexed tissues as well as the entire head and body, annotated to >250 distinct cell types. We provide an in-depth analysis of cell type-related gene signatures and transcription factor markers, as well as sexual dimorphism, across the whole animal. Analysis of common cell types between tissues, such as blood and muscle cells, reveals rare cell types and tissue-specific subtypes. This atlas provides a valuable resource for the Drosophila community and serves as a reference to study genetic perturbations and disease models at single-cell resolution.
Assuntos
Drosophila melanogaster/citologia , Drosophila melanogaster/genética , Transcriptoma , Animais , Núcleo Celular/metabolismo , Bases de Dados Genéticas , Proteínas de Drosophila/genética , Drosophila melanogaster/fisiologia , Feminino , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Genes de Insetos , Masculino , RNA-Seq , Caracteres Sexuais , Análise de Célula Única , Fatores de Transcrição/genéticaRESUMO
The mRNA export pathway is responsible for the transport of mRNAs from the nucleus to the cytoplasm, and thus is essential for protein production and normal cellular functions. A partial loss of function allele of the mRNA export factor Nxt1 in Drosophila shows reduced viability and sterility. A previous study has shown that the male fertility defect is due to a defect in transcription and RNA stability, indicating the potential for this pathway to be implicated in processes beyond the known mRNA transport function. Here we investigate the reduced viability of Nxt1 partial loss of function mutants, and describe a defect in growth and maintenance of the larval muscles, leading to muscle degeneration. RNA-seq revealed reduced expression of a set of mRNAs, particularly from genes with long introns in Nxt1 mutant carcass. We detected differential expression of circRNA, and significantly fewer distinct circRNAs expressed in the mutants. Despite the widespread defects in gene expression, muscle degeneration was rescued by increased expression of the costamere component tn (abba) in muscles. This is the first report of a role for the RNA export pathway gene Nxt1 in the maintenance of muscle integrity. Our data also links the mRNA export pathway to a specific role in the expression of mRNA and circRNA from common precursor genes, in vivo.
Assuntos
Proteínas de Drosophila/fisiologia , Drosophila , Músculos/fisiologia , Proteínas de Transporte Nucleocitoplasmático/fisiologia , Transporte Ativo do Núcleo Celular , Animais , Drosophila/fisiologia , Masculino , RNARESUMO
BACKGROUND: During Drosophila spermatogenesis, testis-specific meiotic arrest complex (tMAC) and testis-specific TBP-associated factors (tTAF) contribute to activation of hundreds of genes required for meiosis and spermiogenesis. Intriguingly, tMAC is paralogous to the broadly expressed complex Myb-MuvB (MMB)/dREAM and Mip40 protein is shared by both complexes. tMAC acts as a gene activator in spermatocytes, while MMB/dREAM was shown to repress gene activity in many cell types. RESULTS: Our study addresses the intricate interplay between tMAC, tTAF, and MMB/dREAM during spermatogenesis. We used cell type-specific DamID to build the DNA-binding profiles of Cookie monster (tMAC), Cannonball (tTAF), and Mip40 (MMB/dREAM and tMAC) proteins in male germline cells. Incorporating the whole transcriptome analysis, we characterized the regulatory effects of these proteins and identified their gene targets. This analysis revealed that tTAFs complex is involved in activation of achi, vis, and topi meiosis arrest genes, implying that tTAFs may indirectly contribute to the regulation of Achi, Vis, and Topi targets. To understand the relationship between tMAC and MMB/dREAM, we performed Mip40 DamID in tTAF- and tMAC-deficient mutants demonstrating meiosis arrest phenotype. DamID profiles of Mip40 were highly dynamic across the stages of spermatogenesis and demonstrated a strong dependence on tMAC in spermatocytes. Integrative analysis of our data indicated that MMB/dREAM represses genes that are not expressed in spermatogenesis, whereas tMAC recruits Mip40 for subsequent gene activation in spermatocytes. CONCLUSIONS: Discovered interdependencies allow to formulate a renewed model for tMAC and tTAFs action in Drosophila spermatogenesis demonstrating how tissue-specific genes are regulated.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila/genética , Perfilação da Expressão Gênica/métodos , Proteínas Nucleares/metabolismo , Espermatogênese , Fatores de Transcrição/metabolismo , Animais , Proteínas de Transporte/genética , Proteínas de Drosophila/genética , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Masculino , Meiose , Especificidade de Órgãos , Testículo/química , Testículo/fisiologia , Ativação TranscricionalRESUMO
BACKGROUND: Synthetic biology approaches are promising new strategies for control of pest insects that transmit disease and cause agricultural damage. These strategies require characterised modular components that can direct appropriate expression of effector sequences, with components conserved across species being particularly useful. The goal of this study was to identify genes from which new potential components could be derived for manipulation of the male germline in two major pest species, the mosquito Aedes aegypti and the tephritid fruit fly Ceratitis capitata. RESULTS: Using RNA-seq data from staged testis samples, we identified several candidate genes with testis-specific expression and suitable expression timing for use of their regulatory regions in synthetic control constructs. We also developed a novel computational pipeline to identify candidate genes with testis-specific splicing from this data; use of alternative splicing is another method for restricting expression in synthetic systems. Some of the genes identified display testis-specific expression or splicing that is conserved across species; these are particularly promising candidates for construct development. CONCLUSIONS: In this study we have identified a set of genes with testis-specific expression or splicing. In addition to their interest from a basic biology perspective, these findings provide a basis from which to develop synthetic systems to control important pest insects via manipulation of the male germline.
Assuntos
Aedes/genética , Ceratitis capitata/genética , Genes de Insetos , Engenharia Genética , Células Germinativas/metabolismo , Animais , Feminino , Regulação da Expressão Gênica , Masculino , Especificidade de Órgãos/genética , Splicing de RNA , Análise de Sequência de RNA , Biologia Sintética/métodos , Testículo/metabolismoRESUMO
Although we now have a wealth of information on the transcription patterns of all the genes in the Drosophila genome, much less is known about the properties of the encoded proteins. To provide information on the expression patterns and subcellular localisations of many proteins in parallel, we have performed a large-scale protein trap screen using a hybrid piggyBac vector carrying an artificial exon encoding yellow fluorescent protein (YFP) and protein affinity tags. From screening 41 million embryos, we recovered 616 verified independent YFP-positive lines representing protein traps in 374 genes, two-thirds of which had not been tagged in previous P element protein trap screens. Over 20 different research groups then characterized the expression patterns of the tagged proteins in a variety of tissues and at several developmental stages. In parallel, we purified many of the tagged proteins from embryos using the affinity tags and identified co-purifying proteins by mass spectrometry. The fly stocks are publicly available through the Kyoto Drosophila Genetics Resource Center. All our data are available via an open access database (Flannotator), which provides comprehensive information on the expression patterns, subcellular localisations and in vivo interaction partners of the trapped proteins. Our resource substantially increases the number of available protein traps in Drosophila and identifies new markers for cellular organelles and structures.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/fisiologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Membrana/metabolismo , Animais , Proteínas de Bactérias/química , Cruzamentos Genéticos , Éxons , Feminino , Técnicas Genéticas , Genoma , Proteínas Luminescentes/química , Masculino , Ovário/metabolismo , Fatores Sexuais , Testículo/metabolismo , Transcrição GênicaRESUMO
The highly conserved, Nxf/Nxt (TAP/p15) RNA nuclear export pathway is important for export of most mRNAs from the nucleus, by interacting with mRNAs and promoting their passage through nuclear pores. Nxt1 is essential for viability; using a partial loss of function allele, we reveal a role for this gene in tissue specific transcription. We show that many Drosophila melanogaster testis-specific mRNAs require Nxt1 for their accumulation. The transcripts that require Nxt1 also depend on a testis-specific transcription complex, tMAC. We show that loss of Nxt1 leads to reduced transcription of tMAC targets. A reporter transcript from a tMAC-dependent promoter is under-expressed in Nxt1 mutants, however the same transcript accumulates in mutants if driven by a tMAC-independent promoter. Thus, in Drosophila primary spermatocytes, the transcription factor used to activate expression of a transcript, rather than the RNA sequence itself or the core transcription machinery, determines whether this expression requires Nxt1. We additionally find that transcripts from intron-less genes are more sensitive to loss of Nxt1 function than those from intron-containing genes and propose a mechanism in which transcript processing feeds back to increase activity of a tissue specific transcription complex.
Assuntos
Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Proteínas de Transporte Nucleocitoplasmático/genética , RNA Mensageiro/genética , Transcrição Gênica , Transporte Ativo do Núcleo Celular/genética , Animais , Sobrevivência Celular/genética , Íntrons/genética , Masculino , Poro Nuclear/genética , Especificidade de Órgãos/genética , RNA Mensageiro/metabolismo , Espermatócitos/citologia , Espermatócitos/crescimento & desenvolvimento , Testículo/crescimento & desenvolvimento , Testículo/metabolismoRESUMO
In this chapter we will concentrate on the transcriptional and translational regulations that govern the development and differentiation of male germline cells. Our focus will be on the processes that occur during differentiation, that distinguish the differentiating population of cells from their stem cell parents. We discuss how these defining features are established as cells transit from a stem cell character to that of a fully committed differentiating cell. The focus will be on how GSCs differentiate, via spermatogonia, to spermatocytes. We will achieve this by first describing the transcriptional activity in the differentiating spermatocytes, cataloguing the known transcriptional regulators in these cells and then investigating how the transcription programme is set up by processes in the progentior cells. This process is particularly interesting to study from a stem cell perspective as the male GSCs are unipotent, so lineage decisions in differentiating progeny of stem cells, which occurs in many other stem cell systems, do not impinge on the behaviour of these cells.
Assuntos
Drosophila melanogaster/genética , Regulação da Expressão Gênica no Desenvolvimento , Biossíntese de Proteínas , Espermatócitos/metabolismo , Espermatogônias/metabolismo , Células-Tronco/metabolismo , Transcrição Gênica , Animais , Diferenciação Celular , Proliferação de Células , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/metabolismo , Epigênese Genética , Masculino , Transdução de Sinais , Espermatócitos/citologia , Espermatogônias/citologia , Células-Tronco/citologia , Testículo/citologia , Testículo/crescimento & desenvolvimento , Testículo/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
The Drosophila testis has numerous advantages for the study of basic cellular processes, as production of sperm requires a highly orchestrated and complex combination of morphological changes and developmentally regulated transitions. Experimental genetics using Drosophila melanogaster has advanced dramatically with the advent of systems for ectopic expression of genetic elements in specific cells. However the genetic tools used in Drosophila research have rarely been generated with the testes in mind, and the utility of relatively few systems has been documented for this tissue. Here I will summarize ectopic expression systems that are known to work for the testis, and provide advice for selection of the most appropriate expression system in specific experimental situations.
RESUMO
Spermatogenesis is a complex and ordered differentiation process in which the spermatogonial stem cell population gives rise to primary spermatocytes that undergo two successive meiotic divisions followed by a major biochemical and structural reorganization of the haploid cells to generate mature elongate spermatids. The transcriptional regulatory programs that orchestrate this process have been intensively studied in model organisms such as Drosophila melanogaster and mouse. Genetic and biochemical approaches have identified the factors involved and revealed mechanisms of action that are unique to male germ cells. In a well-studied example, cofactors and pathways distinct from those used in somatic tissues mediate the action of CREM in male germ cells. But perhaps the most striking feature concerns the paralogs of somatically expressed transcription factors and of components of the general transcription machinery that act in distinct regulatory mechanisms in both Drosophila and murine spermatogenesis.
Assuntos
Drosophila melanogaster/genética , Regulação da Expressão Gênica no Desenvolvimento , Células Germinativas/metabolismo , Animais , Modulador de Elemento de Resposta do AMP Cíclico/genética , Modulador de Elemento de Resposta do AMP Cíclico/fisiologia , Proteínas de Drosophila/genética , Proteínas de Drosophila/fisiologia , Haploidia , Masculino , Meiose/genética , Camundongos , Modelos Genéticos , Espermatogênese/genética , Fator de Transcrição TFIID/química , Fator de Transcrição TFIID/fisiologiaRESUMO
A conserved multi-subunit complex (MybMuvB, MMB), regulates transcriptional activity of many different target genes in Drosophila somatic cells. A paralogous complex, tMAC, controls expression of at least 1500 genes in the male germline, and is essential for sperm production. The roles of specific subunits of tMAC, MMB or orthologous complexes in regulating target gene expression are not understood. MMB and orthologous complexes have Lin-52 as a subunit, but Lin-52 did not co-purify with tMAC. We identified wake-up-call (wuc), a lin-52 paralogue, via a physical interaction with the tMAC lin-9-related subunit Aly, and find that Wuc co-localises with known tMAC subunits. We show that wuc, like aly, is required for spermatogenesis. However, despite phenotypic similarities, the role of wuc is very different from that of previously characterised tMAC mutants. Unlike aly, loss of wuc results in only relatively mild defects in testis-specific gene expression. Strikingly, wuc loss of function partially rescues expression of target genes in aly mutant testes. We propose that wuc represses testis-specific gene expression, that this repression is counteracted by aly, and that aly and a testis-specific TF(II)D complex work together to promote high transcriptional activity of spermiogenic genes specifically in primary spermatocytes.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Proteínas Repressoras/metabolismo , Espermatogênese/genética , Testículo/metabolismo , Animais , Western Blotting , Drosophila melanogaster/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Hibridização In Situ , Masculino , Análise em Microsséries , Microscopia de Fluorescência , Microscopia de Contraste de Fase , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Espermatogênese/fisiologia , Técnicas do Sistema de Duplo-HíbridoRESUMO
Sexual reproduction depends on the production of haploid gametes, and their fusion to form diploid zygotes. Here, we discuss sperm production and function in a molecular and functional evolutionary context, drawing predominantly from studies in model organisms (mice, Drosophila, Caenorhabditis elegans). We consider the mechanisms involved in establishing and maintaining a germline stem cell population in testes, as well as the factors that regulate their contribution to the pool of differentiating cells. These processes involve considerable interaction between the germline and the soma, and we focus on regulatory signalling events in a variety of organisms. The male germline has a unique transcriptional profile, including expression of many testis-specific genes. The evolutionary pressures associated with gene duplication and acquisition of testis function are discussed in the context of genome organization and transcriptional regulation. Post-meiotic differentiation of spermatids involves very dramatic changes in cell shape and acquisition of highly specialized features. We discuss the variety of sperm motility mechanisms and how various reproductive strategies are associated with the diversity of sperm forms found in animals.
Assuntos
Evolução Biológica , Espermatogênese/genética , Testículo/fisiologia , Animais , Diferenciação Celular/genética , Duplicação Gênica , Regulação da Expressão Gênica no Desenvolvimento , Masculino , Camundongos , Transcrição GênicaRESUMO
MOTIVATION: Integrating heterogeneous data across distributed sources is a major requirement for in silico bioinformatics supporting translational research. For example, genome-scale data on patterns of gene expression in the fruit fly Drosophila melanogaster are widely used in functional genomic studies in many organisms to inform candidate gene selection and validate experimental results. However, current data integration solutions tend to be heavy weight, and require significant initial and ongoing investment of effort. Development of a common Web-based data integration infrastructure (a.k.a. data web), using Semantic Web standards, promises to alleviate these difficulties, but little is known about the feasibility, costs, risks or practical means of migrating to such an infrastructure. RESULTS: We describe the development of OpenFlyData, a proof-of-concept system integrating gene expression data on D. melanogaster, combining Semantic Web standards with light-weight approaches to Web programming based on Web 2.0 design patterns. To support researchers designing and validating functional genomic studies, OpenFlyData includes user-facing search applications providing intuitive access to and comparison of gene expression data from FlyAtlas, the BDGP in situ database, and FlyTED, using data from FlyBase to expand and disambiguate gene names. OpenFlyData's services are also openly accessible, and are available for reuse by other bioinformaticians and application developers. Semi-automated methods and tools were developed to support labour- and knowledge-intensive tasks involved in deploying SPARQL services. These include methods for generating ontologies and relational-to-RDF mappings for relational databases, which we illustrate using the FlyBase Chado database schema; and methods for mapping gene identifiers between databases. The advantages of using Semantic Web standards for biomedical data integration are discussed, as are open issues. In particular, although the performance of open source SPARQL implementations is sufficient to query gene expression data directly from user-facing applications such as Web-based data fusions (a.k.a. mashups), we found open SPARQL endpoints to be vulnerable to denial-of-service-type problems, which must be mitigated to ensure reliability of services based on this standard. These results are relevant to data integration activities in translational bioinformatics. AVAILABILITY: The gene expression search applications and SPARQL endpoints developed for OpenFlyData are deployed at http://openflydata.org. FlyUI, a library of JavaScript widgets providing re-usable user-interface components for Drosophila gene expression data, is available at http://flyui.googlecode.com. Software and ontologies to support transformation of data from FlyBase, FlyAtlas, BDGP and FlyTED to RDF are available at http://openflydata.googlecode.com. SPARQLite, an implementation of the SPARQL protocol, is available at http://sparqlite.googlecode.com. All software is provided under the GPL version 3 open source license.
Assuntos
Sistemas de Gerenciamento de Base de Dados , Bases de Dados Genéticas , Drosophila melanogaster/genética , Internet , Animais , Drosophila melanogaster/fisiologia , Expressão Gênica , Perfilação da Expressão Gênica , Hibridização de Ácido Nucleico , Análise de Sequência com Séries de Oligonucleotídeos , Interface Usuário-ComputadorRESUMO
Dengue and dengue hemorrhagic fever are increasing public health problems with an estimated 50-100 million new infections each year. Aedes aegypti is the major vector of dengue viruses in its range and control of this mosquito would reduce significantly human morbidity and mortality. Present mosquito control methods are not sufficiently effective and new approaches are needed urgently. A "sterile-male-release" strategy based on the release of mosquitoes carrying a conditional dominant lethal gene is an attractive new control methodology. Transgenic strains of Aedes aegypti were engineered to have a repressible female-specific flightless phenotype using either two separate transgenes or a single transgene, based on the use of a female-specific indirect flight muscle promoter from the Aedes aegypti Actin-4 gene. These strains eliminate the need for sterilization by irradiation, permit male-only release ("genetic sexing"), and enable the release of eggs instead of adults. Furthermore, these strains are expected to facilitate area-wide control or elimination of dengue if adopted as part of an integrated pest management strategy.
Assuntos
Aedes/genética , Insetos Vetores/genética , Controle de Mosquitos/métodos , Actinas/genética , Aedes/virologia , Animais , Animais Geneticamente Modificados , Cruzamentos Genéticos , Vírus da Dengue/crescimento & desenvolvimento , Feminino , Voo Animal , Humanos , Proteínas de Insetos/genética , Insetos Vetores/virologia , Masculino , Fenótipo , Regiões Promotoras Genéticas/genética , Dengue Grave/prevenção & controle , Dengue Grave/transmissãoRESUMO
The differentiation of sperm from morphologically unremarkable cells into highly specialised free-living, motile cells requires the co-ordinated action of a very large number of gene products. The expression of these products must be regulated in a developmental context to ensure normal cellular differentiation. Many genes essential for spermatogenesis are not used elsewhere in the animal, or are expressed elsewhere, but using a different transcription regulation module. Spermatogenesis is thus a good system for elucidating the principles of tissue-specific gene expression, as well as being interesting in its own right. Here, I discuss the regulation of gene expression during spermatogenesis in Drosophila, focussing on the processes underlying the expression of testis-specific genes in the male germline.
Assuntos
Drosophila/genética , Regulação da Expressão Gênica no Desenvolvimento , Espermatogênese/genética , Animais , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Células Germinativas/metabolismo , Masculino , Especificidade de Órgãos , Testículo/anatomia & histologia , Testículo/metabolismoRESUMO
FlyTED, the Drosophila Testis Gene Expression Database, is a biological research database for gene expression images from the testis of the fruit fly Drosophila melanogaster. It currently contains 2762 mRNA in situ hybridization images and ancillary metadata revealing the patterns of gene expression of 817 Drosophila genes in testes of wild type flies and of seven meiotic arrest mutant strains in which spermatogenesis is defective. This database has been built by adapting a widely used digital library repository software system, EPrints (http://eprints.org/software/), and provides both web-based search and browse interfaces, and programmatic access via an SQL dump, OAI-PMH and SPARQL. FlyTED is available at http://www.fly-ted.org/.
Assuntos
Biologia Computacional/métodos , Bases de Dados Genéticas , Bases de Dados de Ácidos Nucleicos , Drosophila melanogaster/metabolismo , Regulação da Expressão Gênica , Testículo/metabolismo , Animais , Biologia Computacional/tendências , Bases de Dados de Proteínas , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Genes de Insetos , Armazenamento e Recuperação da Informação/métodos , Internet , Masculino , Meiose , SoftwareRESUMO
We describe a whole-mount RNA in situ hybridization (ISH) method optimized for detection of the cellular and subcellular distributions of specific mRNA within Drosophila testes and male genital tract. Digoxygenin (dig)-labeled antisense RNA probes are in vitro transcribed from a template synthesized by (RT)-PCR; the probe length is reduced by hydrolysis. Testes and male genital tracts are dissected from adult flies, fixed and processed for hybridization. Both probe and fixed testes can be stored before use. Extensive post-hybridization washing reduces the background. Detection is through alkaline phosphatase-conjugated anti-dig antibodies followed by a color reaction. This protocol is suitable for low-medium throughput applications with parallel processing of 2-48 samples, and takes 4-5 d to complete. We have used this protocol, which is similar to other RNA ISH protocols, but optimized for whole-mount Drosophila testes, to document the expression of about 1,000 genes in Drosophila melanogaster male genital tract.
Assuntos
Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Perfilação da Expressão Gênica/métodos , Hibridização In Situ/métodos , Testículo/metabolismo , Animais , Digoxigenina/análise , Drosophila melanogaster/metabolismo , Proteínas de Fluorescência Verde/análise , Masculino , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The discovery of a set of highly conserved genes implicated in patterning during animal development represents one of the most striking findings from the field of evolutionary developmental biology. Existence of these "developmental toolkit" genes in diverse taxa, however, does not necessarily imply that they always perform the same functions. Here, we demonstrate functional evolution in a major toolkit gene. hedgehog (hh) encodes a protein that undergoes autocatalytic cleavage, releasing a signaling molecule involved in major developmental processes, notably neural patterning. We find that the hh gene of a colonial pterobranch hemichordate, Rhabdopleura compacta, is expressed in a dramatically different pattern to its ortholog in a harrimaniid enteropneust hemichordate, Saccoglossus kowalevskii. These represent two of the three major hemichordate lineages, the third being the indirect developing ptychoderid enteropneusts. We also show that the normally well-conserved amino acid sequence of the autoproteolytic cleavage site has a derived change in S. kowalevskii. Using ectopic expression in Drosophila, we find that this amino acid substitution reduces the efficiency of Hh autocatalytic cleavage and its signaling function. We conclude that the Hh sequence and expression in S. kowalevskii represent the derived state for deuterostomes, and we argue that functional evolution accompanied secondary reduction of the central nervous system in harrimaniids.