RESUMO
One of the key issues in studying transcriptional regulation during development is how to employ genome-wide assays that reveals sites of open chromatin and transcription factor binding to efficiently identify biologically relevant genes and enhancers. Analysis of Drosophila CNS midline cell development provides a useful system for studying transcriptional regulation at the genomic level due to a large, well-characterized set of midline-expressed genes and in vivo validated enhancers. In this study, FAIRE-seq on FACS-purified midline cells was performed and the midline FAIRE data were compared with whole-embryo FAIRE data. We find that regions of the genome with a strong midline FAIRE peak and weak whole-embryo FAIRE peak overlap with known midline enhancers and provide a useful predictive tool for enhancer identification. In a complementary analysis, we compared a large dataset of fragments that drive midline expression in vivo with the FAIRE data. Midline enhancer fragments with a midline FAIRE peak tend to be near midline-expressed genes, whereas midline enhancers without a midline FAIRE peak were often distant from midline-expressed genes and unlikely to drive midline transcription in vivo.
Assuntos
Cromatina/metabolismo , Proteínas de Drosophila/metabolismo , Animais , Animais Geneticamente Modificados , Cromatina/genética , Imunoprecipitação da Cromatina , Drosophila , Proteínas de Drosophila/genética , Elementos Facilitadores Genéticos/genética , Citometria de Fluxo , Regulação da Expressão Gênica no Desenvolvimento/genéticaRESUMO
A major limitation in understanding embryonic development is the lack of cell type-specific markers. Existing gene expression and marker atlases provide valuable tools, but they typically have one or more limitations: a lack of single-cell resolution; an inability to register multiple expression patterns to determine their precise relationship; an inability to be upgraded by users; an inability to compare novel patterns with the database patterns; and a lack of three-dimensional images. Here, we develop new 'atlas-builder' software that overcomes each of these limitations. A newly generated atlas is three-dimensional, allows the precise registration of an infinite number of cell type-specific markers, is searchable and is open-ended. Our software can be used to create an atlas of any tissue in any organism that contains stereotyped cell positions. We used the software to generate an 'eNeuro' atlas of the Drosophila embryonic CNS containing eight transcription factors that mark the major CNS cell types (motor neurons, glia, neurosecretory cells and interneurons). We found neuronal, but not glial, nuclei occupied stereotyped locations. We added 75 new Gal4 markers to the atlas to identify over 50% of all interneurons in the ventral CNS, and these lines allowed functional access to those interneurons for the first time. We expect the atlas-builder software to benefit a large proportion of the developmental biology community, and the eNeuro atlas to serve as a publicly accessible hub for integrating neuronal attributes - cell lineage, gene expression patterns, axon/dendrite projections, neurotransmitters--and linking them to individual neurons.
Assuntos
Sistema Nervoso Central/citologia , Bases de Dados Genéticas , Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Animais , Axônios/metabolismo , Linhagem da Célula , Biologia Computacional , Dendritos/metabolismo , Proteínas de Drosophila/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Marcadores Genéticos , Interneurônios/metabolismo , Camundongos , Neurônios/metabolismo , Neurotransmissores , Ratos , SoftwareRESUMO
Transcriptional enhancers integrate information derived from transcription factor binding to control gene expression. One key question concerns the extent of trans- and cis-regulatory variation in how co-expressed genes are controlled. The Drosophila CNS midline cells constitute a group of neurons and glia in which expression changes can be readily characterized during specification and differentiation. Using a transgenic approach, we compare the cis-regulation of multiple genes expressed in the Drosophila CNS midline primordium cells, and show that while the expression patterns may appear alike, the target genes are not equivalent in how these common expression patterns are achieved. Some genes utilize a single enhancer that promotes expression in all midline cells, while others utilize multiple enhancers with distinct spatial, temporal, and quantitative contributions. Two regulators, Single-minded and Notch, play key roles in controlling early midline gene expression. While Single-minded is expected to control expression of most, if not all, midline primordium-expressed genes, the role of Notch in directly controlling midline transcription is unknown. Midline primordium expression of the rhomboid gene is dependent on cell signaling by the Notch signaling pathway. Mutational analysis of a rhomboid enhancer reveals at least 5 distinct types of functional cis-control elements, including a binding site for the Notch effector, Suppressor of Hairless. The results suggest a model in which Notch/Suppressor of Hairless levels are insufficient to activate rhomboid expression by itself, but does so in conjunction with additional factors, some of which, including Single-minded, provide midline specificity to Notch activation. Similarly, a midline glial enhancer from the argos gene, which is dependent on EGF/Spitz signaling, is directly regulated by contributions from both Pointed, the EGF transcriptional effector, and Single-minded. In contrast, midline primordium expression of other genes shows a strong dependence on Single-minded and varying combinations of additional transcription factors. Thus, Single-minded directly regulates midline primordium-expressed genes, but in some cases plays a primary role in directing target gene midline expression, and in others provides midline specificity to cell signaling inputs.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Sistema Nervoso Central/citologia , Sistema Nervoso Central/crescimento & desenvolvimento , Proteínas de Drosophila/metabolismo , Drosophila/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Neuroglia/metabolismo , Neurônios/metabolismo , Proteínas Nucleares/metabolismo , Animais , Sequência de Bases , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Sítios de Ligação/genética , Sistema Nervoso Central/metabolismo , Biologia Computacional , Drosophila/metabolismo , Proteínas de Drosophila/genética , Elementos Facilitadores Genéticos/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Processamento de Imagem Assistida por Computador , Hibridização In Situ , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Microscopia Confocal , Dados de Sequência Molecular , Proteínas Nucleares/genética , Receptores Notch/genética , Receptores Notch/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Análise de Sequência de DNARESUMO
Dopaminergic neurons play important roles in animal behavior, including motivation, reward and locomotion. The Drosophila dopaminergic H-cell interneuron is an attractive system for studying the genetics of neural development because analysis is focused on a single neuronal cell type. Here we provide a mechanistic understanding of how MP3, the precursor to the H-cell, forms and acquires its identity. We show that the gooseberry/gooseberry-neuro (gsb/gsb-n) transcription factor genes act to specify MP3 cell fate. It is proposed that single-minded commits neuroectodermal cells to a midline fate, followed by a series of signaling events that result in the formation of a single gsb(+)/gsb-n(+) MP3 cell per segment. The wingless signaling pathway establishes a midline anterior domain by activating expression of the forkhead transcription factors sloppy paired 1 and sloppy paired 2. This is followed by hedgehog signaling that activates gsb/gsb-n expression in a subgroup of anterior cells. Finally, Notch signaling results in the selection of a single MP3, with the remaining cells becoming midline glia. In MP3, gsb/gsb-n direct H-cell development, in large part by activating expression of the lethal of scute and tailup H-cell regulatory genes. Thus, a series of signaling and transcriptional events result in the specification of a unique dopaminergic precursor cell. Additional genetic experiments indicate that the molecular mechanisms that govern MP3/H-cell development might also direct the development of non-midline dopaminergic neurons.
Assuntos
Neurônios Dopaminérgicos/citologia , Neurônios Dopaminérgicos/metabolismo , Proteínas de Drosophila/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Drosophila , Proteínas de Drosophila/genética , Embrião não Mamífero/citologia , Embrião não Mamífero/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Hibridização In Situ , Neurogênese/genética , Neurogênese/fisiologia , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Transativadores/genética , Transativadores/metabolismo , Proteína Wnt1/genética , Proteína Wnt1/metabolismoRESUMO
Dopaminergic neurons play important behavioral roles in locomotion, reward and aggression. The Drosophila H-cell is a dopaminergic neuron that resides at the midline of the ventral nerve cord. Both the H-cell and the glutamatergic H-cell sib are the asymmetric progeny of the MP3 midline precursor cell. H-cell sib cell fate is dependent on Notch signaling, whereas H-cell fate is Notch independent. Genetic analysis of genes that could potentially regulate H-cell fate revealed that the lethal of scute [l(1)sc], tailup and SoxNeuro transcription factor genes act together to control H-cell gene expression. The l(1)sc bHLH gene is required for all H-cell-specific gene transcription, whereas tailup acts in parallel to l(1)sc and controls genes involved in dopamine metabolism. SoxNeuro functions downstream of l(1)sc and controls expression of a peptide neurotransmitter receptor gene. The role of l(1)sc may be more widespread, as a l(1)sc mutant shows reductions in gene expression in non-midline dopaminergic neurons. In addition, l(1)sc mutant embryos possess defects in the formation of MP4-6 midline precursor and the median neuroblast stem cell, revealing a proneural role for l(1)sc in midline cells. The Notch-dependent progeny of MP4-6 are the mVUM motoneurons, and these cells also require l(1)sc for mVUM-specific gene expression. Thus, l(1)sc plays an important regulatory role in both neurogenesis and specifying dopaminergic neuron and motoneuron identities.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Neurônios Motores/fisiologia , Neurogênese , Neurônios/fisiologia , Fatores de Transcrição/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Sistema Nervoso Central/embriologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Dopamina/metabolismo , Drosophila/citologia , Drosophila/genética , Proteínas de Drosophila/genética , Hibridização In Situ , Neurônios Motores/citologia , Neurônios/citologia , Receptores de Neurotransmissores/biossíntese , Receptores de Neurotransmissores/genética , Receptores Notch/metabolismo , Fatores de Transcrição SOX/genética , Fatores de Transcrição SOX/metabolismo , Transdução de Sinais , Fatores de Transcrição/genéticaRESUMO
The Drosophila CNS contains a variety of glia, including highly specialized glia that reside at the CNS midline and functionally resemble the midline floor plate glia of the vertebrate spinal cord. Both insect and vertebrate midline glia play important roles in ensheathing axons that cross the midline and secreting signals that control a variety of developmental processes. The Drosophila midline glia consist of two spatially and functionally distinct populations. The anterior midline glia (AMG) are ensheathing glia that migrate, surround and send processes into the axon commissures. By contrast, the posterior midline glia (PMG) are non-ensheathing glia. Together, the Notch and hedgehog signaling pathways generate AMG and PMG from midline neural precursors. Notch signaling is required for midline glial formation and for transcription of a core set of midline glial-expressed genes. The Hedgehog morphogen is secreted from ectodermal cells adjacent to the CNS midline and directs a subset of midline glia to become PMG. Two transcription factor genes, runt and engrailed, play important roles in AMG and PMG development. The runt gene is expressed in AMG, represses engrailed and maintains AMG gene expression. The engrailed gene is expressed in PMG, represses runt and maintains PMG gene expression. In addition, engrailed can direct midline glia to a PMG-like non-ensheathing fate. Thus, two signaling pathways and runt-engrailed mutual repression initiate and maintain two distinct populations of midline glia that differ functionally in gene expression, glial migration, axon ensheathment, process extension and patterns of apoptosis.
Assuntos
Movimento Celular/fisiologia , Proteínas de Homeodomínio/metabolismo , Neuroglia/metabolismo , Transdução de Sinais/fisiologia , Fatores de Transcrição/metabolismo , Animais , Proteínas de Ligação a DNA/metabolismo , Drosophila , Proteínas de Drosophila/metabolismo , Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Hedgehog/metabolismo , Imuno-Histoquímica , Hibridização In Situ , Neuroglia/citologia , Proteínas Nucleares/metabolismo , Receptores Notch/metabolismoRESUMO
UNLABELLED: Many algorithms analyze enhancers for overrepresentation of known and novel motifs, with the goal of identifying binding sites for direct regulators of gene expression. Twine is a Java GUI with multiple graphical representations ('Views') of enhancer alignments that displays motifs, as IUPAC consensus sequences or position frequency matrices, in the context of phylogenetic conservation to facilitate cis-regulatory element discovery. Thresholds of phylogenetic conservation and motif stringency can be altered dynamically to facilitate detailed analysis of enhancer architecture. Views can be exported to vector graphics programs to generate high-quality figures for publication. Twine can be extended via Java plugins to manipulate alignments and analyze sequences. AVAILABILITY: Twine is freely available as a compiled Java .jar package or Java source code at http://labs.bio.unc.edu/crews/twine/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Assuntos
Elementos Facilitadores Genéticos , Software , Animais , Sítios de Ligação , Gráficos por Computador , Motivos de Nucleotídeos , Filogenia , Alinhamento de Sequência , Análise de Sequência de DNA , Fatores de Transcrição/metabolismoRESUMO
One of the key aspects of neuronal differentiation is the array of neurotransmitters and neurotransmitter receptors that each neuron possesses. One important goal of developmental neuroscience is to understand how these differentiated properties are established during development. In this paper, we use fluorescence activated cell sorting and RNA-seq to determine the transcriptome of the Drosophila CNS midline cells, which consist of a small number of well-characterized neurons and glia. These data revealed that midline cells express 9 neuropeptide precursor genes, 13 neuropeptide receptor genes, and 31 small-molecule neurotransmitter receptor genes. In situ hybridization and high-resolution confocal analyses were carried-out to determine the midline cell identity for these neuropeptides and the neuropeptide receptors. The results revealed a surprising level of diversity. Neuropeptide genes are expressed in a variety of midline cell types, including motoneurons, GABAergic interneurons, and midline glia. These data revealed previously unknown functional differences among the highly-related iVUM neurons. There also exist segmental differences in expression for the same neuronal sub-type. Similar experiments on midline-expressed neuropeptide receptor genes reveal considerable diversity in synaptic inputs. Multiple receptor types were expressed in midline interneurons and motoneurons, and, in one case, link feeding behavior to gut peristalsis and locomotion. There were also segmental differences, variations between the 3 iVUMs, and three hormone receptor genes were broadly expressed in most midline cells. The Drosophila Castor transcription factor is present at high levels in iVUM5, which is both GABAergic and expresses the short neuropeptide F precursor gene. Genetic and misexpression experiments indicated that castor specifically controls expression of the short neuropeptide F precursor gene, but does not affect iVUM cell fate or expression of Gad1. This indicates a novel function for castor in regulating neuropeptide gene expression.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Drosophila/genética , Drosophila/genética , Neurônios/citologia , Neuropeptídeos/metabolismo , Animais , Diferenciação Celular , Sistema Nervoso Central/citologia , Sistema Nervoso Central/metabolismo , Proteínas de Ligação a DNA/metabolismo , Drosophila/embriologia , Drosophila/metabolismo , Proteínas de Drosophila/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Interneurônios/metabolismo , Neurônios/metabolismoRESUMO
The Drosophila CNS midline glia (MG) are multifunctional cells that ensheath and provide trophic support to commissural axons, and direct embryonic development by employing a variety of signaling molecules. These glia consist of two functionally distinct populations: the anterior MG (AMG) and posterior MG (PMG). Only the AMG ensheath axon commissures, whereas the function of the non-ensheathing PMG is unknown. The Drosophila MG have proven to be an excellent system for studying glial proliferation, cell fate, apoptosis, and axon-glial interactions. However, insight into how AMG migrate and acquire their specific positions within the axon-glial scaffold has been lacking. In this paper, we use time-lapse imaging, single-cell analysis, and embryo staining to comprehensively describe the proliferation, migration, and apoptosis of the Drosophila MG. We identified 3 groups of MG that differed in the trajectories of their initial inward migration: AMG that migrate inward and to the anterior before undergoing apoptosis, AMG that migrate inward and to the posterior to ensheath commissural axons, and PMG that migrate inward and to the anterior to contact the commissural axons before undergoing apoptosis. In a second phase of their migration, the surviving AMG stereotypically migrated posteriorly to specific positions surrounding the commissures, and their final position was correlated with their location prior to migration. Most noteworthy are AMG that migrated between the commissures from a ventral to a dorsal position. Single-cell analysis indicated that individual AMG possessed wide-ranging and elaborate membrane extensions that partially ensheathed both commissures. These results provide a strong foundation for future genetic experiments to identify mutants affecting MG development, particularly in guidance cues that may direct migration. Drosophila MG are homologous in structure and function to the glial-like cells that populate the vertebrate CNS floorplate, and study of Drosophila MG will provide useful insights into floorplate development and function.
Assuntos
Movimento Celular , Drosophila melanogaster/citologia , Drosophila melanogaster/embriologia , Neuroglia/citologia , Imagem com Lapso de Tempo/métodos , Animais , Apoptose , Axônios/metabolismo , Divisão Celular , Embrião não Mamífero/citologia , Embrião não Mamífero/metabolismo , Desenvolvimento Embrionário , Membranas/embriologia , Membranas/metabolismo , Modelos BiológicosRESUMO
The Drosophila Zelda transcription factor plays an important role in regulating transcription at the embryonic maternal-to-zygotic transition. However, expression of zelda continues throughout embryogenesis in cells including the developing CNS and trachea, but little is known about its post-blastoderm functions. In this paper, it is shown that zelda directly controls CNS midline and tracheal expression of the link (CG13333) gene, as well as link blastoderm expression. The link gene contains a 5' enhancer with multiple Zelda TAGteam binding sites that in vivo mutational studies show are required for link transcription. The link enhancer also has a binding site for the Single-minded:Tango and Trachealess:Tango bHLH-PAS proteins that also influences link midline and tracheal expression. These results provide an example of how a transcription factor (Single-minded or Trachealess) can interact with distinct co-regulatory proteins (Zelda or Sox/POU-homeodomain proteins) to control a similar pattern of expression of different target genes in a mechanistically different manner. While zelda and single-minded midline expression is well-conserved in Drosophila, midline expression of link is not well-conserved. Phylogenetic analysis of link expression suggests that ~60 million years ago, midline expression was nearly or completely absent, and first appeared in the melanogaster group (including D. melanogaster, D. yakuba, and D. erecta) >13 million years ago. The differences in expression are due, in part, to sequence polymorphisms in the link enhancer and likely due to altered binding of multiple transcription factors. Less than 6 million years ago, a second change occurred that resulted in high levels of expression in D. melanogaster. This change may be due to alterations in a putative Zelda binding site. Within the CNS, the zelda gene is alternatively spliced beginning at mid-embryogenesis into transcripts that encode a Zelda isoform missing three zinc fingers from the DNA binding domain. This may result in a protein with altered, possibly non-functional, DNA-binding properties. In summary, Zelda collaborates with bHLH-PAS proteins to directly regulate midline and tracheal expression of an evolutionary dynamic enhancer in the post-blastoderm embryo.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Proteínas Nucleares/genética , Fatores de Transcrição/genética , Animais , Drosophila melanogaster/embriologia , Embrião não Mamífero/citologia , Embrião não Mamífero/embriologia , Embrião não Mamífero/fisiologia , Elementos Facilitadores Genéticos , Regulação da Expressão Gênica no Desenvolvimento , Genes de Insetos , Sistema Nervoso/citologia , Sistema Nervoso/embriologia , FilogeniaRESUMO
BACKGROUND: The Drosophila CNS midline cells are an excellent model system to study neuronal and glial development because of their diversity of cell types and the relative ease in identifying and studying the function of midline-expressed genes. In situ hybridization experiments generated a large dataset of midline gene expression patterns. To help synthesize these data and make them available to the scientific community, we developed a web-accessible database. DESCRIPTION: MidExDB (Drosophila CNS Midline Gene Expression Database) is comprised of images and data from our in situ hybridization experiments that examined midline gene expression. Multiple search tools are available to allow each type of data to be viewed and compared. Descriptions of each midline cell type and their development are included as background information. CONCLUSION: MidExDB integrates large-scale gene expression data with the ability to identify individual cell types providing the foundation for detailed genetic, molecular, and biochemical studies of CNS midline cell neuronal and glial development and function. This information has general relevance for the study of nervous system development in other organisms, and also provides insight into transcriptional regulation.
Assuntos
Sistema Nervoso Central/metabolismo , Bases de Dados Factuais , Drosophila/crescimento & desenvolvimento , Drosophila/metabolismo , Animais , Drosophila/embriologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Hibridização In Situ , InternetRESUMO
The Drosophila dysfusion basic-helix-loop-helix-PAS transcription factor gene is expressed in specialized fusion cells that reside at the tips of migrating tracheal branches. dysfusion mutants were isolated, and genetic analysis of live embryos revealed that mutant tracheal branches migrate to close proximity but fail to recognize and adhere to each other. Misexpression of dysfusion throughout the trachea further indicated that dysfusion has the ability to both inhibit cell migration and promote ectopic tracheal fusion. Nineteen genes whose expression either increases or decreases in fusion cells during development were analyzed in dysfusion mutant embryos. dysfusion upregulates the levels of four genes, including the shotgun cell adhesion protein gene and the zona pellucida family transmembrane protein gene, CG13196. Misexpression experiments with CG13196 result in ectopic tracheal fusion events, suggesting that it also encodes a cell adhesion protein. Another target gene of dysfusion is members only, which inhibits protein nuclear export and influences tracheal fusion. dysfusion also indirectly downregulates protein levels of Trachealess, an important regulator of tracheal development. These results indicate that fusion cells undergo dynamic changes in gene expression as they switch from migratory to fusion modes and that dysfusion regulates a discrete, but important, set of these genes.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/citologia , Drosophila melanogaster/genética , Traqueia/citologia , Transcrição Gênica , Alelos , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , Adesão Celular , Fusão Celular , Sobrevivência Celular , Regulação para Baixo/genética , Proteínas de Drosophila/química , Embrião não Mamífero/citologia , Genes de Insetos/genética , Mutagênese , Mutação/genéticaRESUMO
The Drosophila embryonic central nervous system (CNS) is a complex organ consisting of â¼15,000 neurons and glia that is generated in â¼1 day of development. For the past 40 years, Drosophila developmental neuroscientists have described each step of CNS development in precise molecular genetic detail. This has led to an understanding of how an intricate nervous system emerges from a single cell. These studies have also provided important, new concepts in developmental biology, and provided an essential model for understanding similar processes in other organisms. In this article, the key genes that guide Drosophila CNS development and how they function is reviewed. Features of CNS development covered in this review are neurogenesis, gliogenesis, cell fate specification, and differentiation.
Assuntos
Diferenciação Celular , Linhagem da Célula , Sistema Nervoso Central/embriologia , Drosophila melanogaster/citologia , Drosophila melanogaster/embriologia , Embrião não Mamífero/citologia , Neurogênese , AnimaisRESUMO
The Drosophila larval tracheal system consists of a highly branched tubular organ that becomes interconnected by migration-fusion events during embryonic development. Fusion cells at the tip of each branch guide migration, adhere, and then undergo extensive remodeling as the tracheal lumen extends between the two branches. The Drosophila dead end gene is expressed in fusion cells, and encodes an Arf-like3 GTPase. Analyses of dead end RNAi and mutant embryos reveal that the lumen fails to connect between the two branches. Expression of a constitutively active form of Dead end in S2 cells reveals that it influences the state of actin polymerization, and is present on particles that traffic along actin/microtubule-containing processes. Imaging experiments in vivo reveal that Dead end-containing vesicles are associated with recycling endosomes and the exocyst, and control exocyst localization in fusion cells. These results indicate that the Dead end GTPase plays an important role in trafficking membrane components involved in tracheal fusion cell morphogenesis and lumenal development.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster , GTP Fosfo-Hidrolases/metabolismo , Morfogênese , Fatores de Ribosilação do ADP/genética , Fatores de Ribosilação do ADP/metabolismo , Actinas/metabolismo , Sequência de Aminoácidos , Animais , Fusão Celular , Movimento Celular , Forma Celular , Células Cultivadas , Citoesqueleto/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/anatomia & histologia , Drosophila melanogaster/embriologia , Drosophila melanogaster/metabolismo , Endossomos/metabolismo , GTP Fosfo-Hidrolases/genética , Humanos , Microtúbulos/metabolismo , Dados de Sequência Molecular , Mutação , Interferência de RNA , Alinhamento de SequênciaRESUMO
The development of the mature insect trachea requires a complex series of cellular events, including tracheal cell specification, cell migration, tubule branching, and tubule fusion. Here we describe the identification of the Drosophila melanogaster dysfusion gene, which encodes a novel basic helix-loop-helix (bHLH)-PAS protein conserved between Caenorhabditis elegans, insects, and humans, and controls tracheal fusion events. The Dysfusion protein functions as a heterodimer with the Tango bHLH-PAS protein in vivo to form a putative DNA-binding complex. The dysfusion gene is expressed in a variety of embryonic cell types, including tracheal-fusion, leading-edge, foregut atrium cells, nervous system, hindgut, and anal pad cells. RNAi experiments indicate that dysfusion is required for dorsal branch, lateral trunk, and ganglionic branch fusion but not for fusion of the dorsal trunk. The escargot gene, which is also expressed in fusion cells and is required for tracheal fusion, precedes dysfusion expression. Analysis of escargot mutants indicates a complex pattern of dysfusion regulation, such that dysfusion expression is dependent on escargot in the dorsal and ganglionic branches but not the dorsal trunk. Early in tracheal development, the Trachealess bHLH-PAS protein is present at uniformly high levels in all tracheal cells, but since the levels of Dysfusion rise in wild-type fusion cells, the levels of Trachealess in fusion cells decline. The downregulation of Trachealess is dependent on dysfusion function. These results suggest the possibility that competitive interactions between basic helix-loop-helix-PAS proteins (Dysfusion, Trachealess, and possibly Similar) may be important for the proper development of the trachea.
Assuntos
Drosophila melanogaster/genética , Traqueia/embriologia , Transativadores/genética , Fatores de Transcrição/genética , Sequência de Aminoácidos , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Proteínas de Ligação a DNA/metabolismo , Regulação para Baixo , Proteínas de Drosophila/genética , Hibridização In Situ , Microscopia de Fluorescência , Dados de Sequência Molecular , Filogenia , Estrutura Terciária de Proteína , Interferência de RNA , RNA Mensageiro/metabolismo , Homologia de Sequência de AminoácidosRESUMO
Here, we describe the embryonic central nervous system expression of 5,000 GAL4 lines made using molecularly defined cis-regulatory DNA inserted into a single attP genomic location. We document and annotate the patterns in early embryos when neurogenesis is at its peak, and in older embryos where there is maximal neuronal diversity and the first neural circuits are established. We note expression in other tissues, such as the lateral body wall (muscle, sensory neurons, and trachea) and viscera. Companion papers report on the adult brain and larval imaginal discs, and the integrated data sets are available online (http://www.janelia.org/gal4-gen1). This collection of embryonically expressed GAL4 lines will be valuable for determining neuronal morphology and function. The 1,862 lines expressed in small subsets of neurons (<20/segment) will be especially valuable for characterizing interneuronal diversity and function, because although interneurons comprise the majority of all central nervous system neurons, their gene expression profile and function remain virtually unexplored.
Assuntos
Sistema Nervoso Central/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Fatores de Transcrição/metabolismo , Animais , Animais Geneticamente Modificados , Sistema Nervoso Central/crescimento & desenvolvimento , Bases de Dados Factuais , Drosophila/genética , Proteínas de Drosophila/genética , Embrião não Mamífero/metabolismo , Feminino , Expressão Gênica , Genes Reporter , Internet , Masculino , Elementos Reguladores de Transcrição , Fatores de Transcrição/genéticaRESUMO
Developmental regulatory proteins are commonly utilized in multiple cell types throughout development. The Drosophila single-minded (sim) gene acts as master regulator of embryonic CNS midline cell development and transcription. However, it is also expressed in the brain during larval development. In this paper, we demonstrate that sim is expressed in three clusters of anterior central brain neurons: DAMv1/2, BAmas1/2, and TRdm and in three clusters of posterior central brain neurons: a subset of DPM neurons, and two previously unidentified clusters, which we term PLSC and PSC. In addition, sim is expressed in the lamina and medulla of the optic lobes. MARCM studies confirm that sim is expressed at high levels in neurons but is low or absent in neuroblasts (NBs) and ganglion mother cell (GMC) precursors. In the anterior brain, sim(+) neurons are detected in 1st and 2nd instar larvae but rapidly increase in number during the 3rd instar stage. To understand the regulation of sim brain transcription, 12 fragments encompassing 5'-flanking, intronic, and 3'-flanking regions were tested for the presence of enhancers that drive brain expression of a reporter gene. Three of these fragments drove expression in sim(+) brain cells, including all sim(+) neuronal clusters in the central brain and optic lobes. One fragment upstream of sim is autoregulatory and is expressed in all sim(+) brain cells. One intronic fragment drives expression in only the PSC and laminar neurons. Another downstream intronic fragment drives expression in all sim(+) brain neurons, except the PSC and lamina. Thus, together these two enhancers drive expression in all sim(+) brain neurons. Sequence analysis of existing sim mutant alleles identified three likely null alleles to utilize in MARCM experiments to examine sim brain function. Mutant clones of DAMv1/2 neurons revealed a consistent axonal fasciculation defect. Thus, unlike the embryonic roles of sim that control CNS midline neuron and glial formation and differentiation, postembryonic sim, instead, controls aspects of axon guidance in the brain. This resembles the roles of vertebrate sim that have an early role in neuronal migration and a later role in axonogenesis.
Assuntos
Encéfalo/embriologia , Proteínas de Drosophila/biossíntese , Embrião não Mamífero/embriologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Proteínas do Tecido Nervoso/biossíntese , Transcrição Gênica/fisiologia , Animais , Encéfalo/citologia , Drosophila melanogaster , Embrião não Mamífero/citologia , Larva/citologia , Larva/metabolismo , Neurônios/citologia , Neurônios/metabolismoRESUMO
The glia that reside at the midline of the Drosophila CNS are an important embryonic signaling center and also wrap the axons that cross the CNS. The development of the midline glia (MG) is characterized by migration, ensheathment, subdivision of axon commissures, apoptosis, and the extension of glial processes. All of these events are characterized by cell-cell contact between MG and adjacent neurons. Cell adhesion and signaling proteins that mediate different aspects of MG development and MG-neuron interactions have been identified. This provides a foundation for ultimately obtaining an integrated picture of how the MG assemble into a characteristic axonal support structure in the CNS.
Assuntos
Axônios/metabolismo , Comunicação Celular , Sistema Nervoso Central/citologia , Drosophila melanogaster/citologia , Neuroglia/citologia , Animais , Adesão Celular , Movimento Celular , Proteínas de Drosophila/metabolismo , Transdução de Sinais , VertebradosRESUMO
Drosophila tracheal fusion cells play multiple important roles in guiding and facilitating tracheal branch fusion. Mechanistic understanding of how fusion cells function during development requires deciphering their transcriptional circuitry. In this paper, three genes with distinct patterns of fusion cell expression were dissected by transgenic analysis to identify the cis-regulatory modules that mediate their transcription. Bioinformatic analysis involving phylogenetic comparisons coupled with mutational experiments were employed. The dysfusion bHLH-PAS gene was shown to have two fusion cell cis-regulatory modules; one driving initial expression and another autoregulatory module to enhance later transcription. Mutational dissection of the early module identified at least four distinct inputs, and included putative binding sites for ETS and POU-homeodomain proteins. The ETS transcription factor Pointed mediates the transcriptional output of the branchless/breathless signaling pathway, suggesting that this pathway directly controls dysfusion expression. Fusion cell cis-regulatory modules of CG13196 and CG15252 require two Dysfusion:Tango binding sites, but additional sequences modulate the breadth of activation in different fusion cell classes. These results begin to decode the regulatory circuitry that guides transcriptional activation of genes required for fusion cell morphogenesis.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Proteínas de Drosophila/genética , Traqueia/citologia , Transcrição Gênica/genética , Animais , Animais Geneticamente Modificados , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Biologia Computacional , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Drosophila , Proteínas de Drosophila/metabolismo , Elementos Facilitadores Genéticos , Imunofluorescência , Regulação da Expressão Gênica no Desenvolvimento/genética , Mutação , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Receptores de Fatores de Crescimento de Fibroblastos/genética , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Transdução de Sinais/genética , Traqueia/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Glia play crucial roles in ensheathing axons, a process that requires an intricate series of glia-neuron interactions. The membrane-anchored protein Wrapper is present in Drosophila midline glia and is required for ensheathment of commissural axons. By contrast, Neurexin IV is present on the membranes of neurons and commissural axons, and is highly concentrated at their interfaces with midline glia. Analysis of Neurexin IV and wrapper mutant embryos revealed identical defects in glial migration, ensheathment and glial subdivision of the commissures. Mutant and misexpression experiments indicated that Neurexin IV membrane localization is dependent on interactions with Wrapper. Cell culture aggregation assays and biochemical experiments demonstrated the ability of Neurexin IV to promote cell adhesion by binding to Wrapper. These results show that neuronal-expressed Neurexin IV and midline glial-expressed Wrapper act as heterophilic adhesion molecules that mediate multiple cellular events involved in glia-neuron interactions.