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Search Results: 1 - 10 of 3225 matches for " Pavel Tomancak "
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Mapping the complexity of transcription control in higher eukaryotes
Pavel Tomancak, Uwe Ohler
Genome Biology , 2010, DOI: 10.1186/gb-2010-11-4-115
Abstract: The complexity of life does not correlate with an increased size of the list of parts (the genes) from which organisms are built, but rather with an increased complexity in how these parts are regulated and combined into networks to specify the correct tissue-specific expression of genes. Analyses of yeast had shown a fairly simple hierarchical regulatory architecture, in which master regulators drive expression of many genes and any given gene is typically regulated by at most a handful of transcription factors (TFs) [1]. Some studies in animals, including studies of the early development of Drosophila, suggested a straightforward extension of the concept of a small number of highly specific TFs that define expression domains. Recent studies, including one by Adryan and Teichmann in this issue of Genome Biology [2], put the idea to the test by evaluating large genomic datasets, and their conclusions challenge this hypothesis.Adryan and Teichmann's study is based on datasets obtained by two popular methods for analyzing gene expression [3,4]. Transcriptional profiling using microarrays requires substantial amounts of biological material and is thus typically used on intact multicellular specimens or cultured cell lines. RNA in situ hybridization is used to visualize spatial and temporal gene expression, but is limited for several reasons: some classes of eukaryotic genes, such as microRNAs, are difficult to study in this way; many tissues, such as brains, cannot be permeabilized enough to deliver the probe throughout the sample; temporal resolution is limited; and there is a lack of reliable quantification methods. Systematic RNA in situ surveys are therefore routinely combined with microarray analysis to counter the drawbacks of the two methods [4].Drosophila embryonic development is particularly amenable to analysis by both in situ hybridization and microarray analysis. Large numbers of approximately staged embryos enable the isolation of sufficient amounts of RNA
BigDataViewer: Interactive Visualization and Image Processing for Terabyte Data Sets
Tobias Pietzsch,Stephan Saalfeld,Stephan Preibisch,Pavel Tomancak
Computer Science , 2014,
Abstract: The increasingly popular light sheet microscopy techniques generate very large 3D time-lapse recordings of living biological specimen. The necessity to make large volumetric datasets available for interactive visualization and analysis has been widely recognized. However, existing solutions build on dedicated servers to generate virtual slices that are transferred to the client applications, practically leading to insufficient frame rates (less than 10 frames per second) for truly interactive experience. An easily accessible open source solution for interactive arbitrary virtual re-slicing of very large volumes and time series of volumes has yet been missing. We fill this gap with BigDataViewer, a Fiji plugin to interactively navigate and visualize large image sequences from both local and remote data sources.
An automated workflow for parallel processing of large multiview SPIM recordings
Christopher Schmied,Peter Steinbach,Tobias Pietzsch,Stephan Preibisch,Pavel Tomancak
Quantitative Biology , 2015,
Abstract: Multiview light sheet fluorescence microscopy (LSFM) allows to image developing organisms in 3D at unprecedented temporal resolution over long periods of time. The resulting massive amounts of raw image data requires extensive processing interactively via dedicated graphical user interface (GUI) applications. The consecutive processing steps can be easily automated and the individual time points can be processed independently, which lends itself to trivial parallelization on a high performance cluster (HPC). Here we introduce an automated workflow for processing large multiview, multi-channel, multi-illumination time-lapse LSFM data on a single workstation or in parallel on a HPC. The pipeline relies on snakemake to resolve dependencies among consecutive processing steps and can be easily adapted to any cluster environment for processing LSFM data in a fraction of the time required to collect it.
In Vivo RNAi Rescue in Drosophila melanogaster with Genomic Transgenes from Drosophila pseudoobscura
Christoph C. H. Langer,Radoslaw K. Ejsmont,Cornelia Sch?nbauer,Frank Schnorrer,Pavel Tomancak
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0008928
Abstract: Systematic, large-scale RNA interference (RNAi) approaches are very valuable to systematically investigate biological processes in cell culture or in tissues of organisms such as Drosophila. A notorious pitfall of all RNAi technologies are potential false positives caused by unspecific knock-down of genes other than the intended target gene. The ultimate proof for RNAi specificity is a rescue by a construct immune to RNAi, typically originating from a related species.
An Integrated Micro- and Macroarchitectural Analysis of the Drosophila Brain by Computer-Assisted Serial Section Electron Microscopy
Albert Cardona,Stephan Saalfeld,Stephan Preibisch,Benjamin Schmid,Anchi Cheng,Jim Pulokas,Pavel Tomancak,Volker Hartenstein
PLOS Biology , 2012, DOI: 10.1371/journal.pbio.1000502
Abstract: The analysis of microcircuitry (the connectivity at the level of individual neuronal processes and synapses), which is indispensable for our understanding of brain function, is based on serial transmission electron microscopy (TEM) or one of its modern variants. Due to technical limitations, most previous studies that used serial TEM recorded relatively small stacks of individual neurons. As a result, our knowledge of microcircuitry in any nervous system is very limited. We applied the software package TrakEM2 to reconstruct neuronal microcircuitry from TEM sections of a small brain, the early larval brain of Drosophila melanogaster. TrakEM2 enables us to embed the analysis of the TEM image volumes at the microcircuit level into a light microscopically derived neuro-anatomical framework, by registering confocal stacks containing sparsely labeled neural structures with the TEM image volume. We imaged two sets of serial TEM sections of the Drosophila first instar larval brain neuropile and one ventral nerve cord segment, and here report our first results pertaining to Drosophila brain microcircuitry. Terminal neurites fall into a small number of generic classes termed globular, varicose, axiform, and dendritiform. Globular and varicose neurites have large diameter segments that carry almost exclusively presynaptic sites. Dendritiform neurites are thin, highly branched processes that are almost exclusively postsynaptic. Due to the high branching density of dendritiform fibers and the fact that synapses are polyadic, neurites are highly interconnected even within small neuropile volumes. We describe the network motifs most frequently encountered in the Drosophila neuropile. Our study introduces an approach towards a comprehensive anatomical reconstruction of neuronal microcircuitry and delivers microcircuitry comparisons between vertebrate and insect neuropile.
Motif composition, conservation and condition-specificity of single and alternative transcription start sites in the Drosophila genome
Elizabeth A Rach, Hsiang-Yu Yuan, William H Majoros, Pavel Tomancak, Uwe Ohler
Genome Biology , 2009, DOI: 10.1186/gb-2009-10-7-r73
Abstract: To identify TSSs in Drosophila melanogaster, we applied a hierarchical clustering strategy on available 5' expressed sequence tags (ESTs) and identified a high quality set of 5,665 TSSs for approximately 4,000 genes. We distinguished two initiation patterns: 'peaked' TSSs, and 'broad' TSS cluster groups. Peaked promoters were found to contain location-specific sequence elements; conversely, broad promoters were associated with non-location-specific elements. In alignments across other Drosophila genomes, conservation levels of sequence elements exceeded 90% within the melanogaster subgroup, but dropped considerably for distal species. Elements in broad promoters had lower levels of conservation than those in peaked promoters. When characterizing the distributions of ESTs, 64% of TSSs showed distinct associations to one out of eight different spatiotemporal conditions. Available whole-genome tiling array time series data revealed different temporal patterns of embryonic activity across the majority of genes with distinct alternative promoters. Many genes with maternally inherited transcripts were found to have alternative promoters utilized later in development. Core promoters of maternally inherited transcripts showed differences in motif composition compared to zygotically active promoters.Our study provides a comprehensive map of Drosophila TSSs and the conditions under which they are utilized. Distinct differences in motif associations with initiation pattern and spatiotemporal utilization illustrate the complex regulatory code of transcription initiation.Transcription is a crucial part of gene expression that involves complex interactions of cis-regulatory sequence elements and trans-factors. It is mediated in large part through the binding of transcription factors (TFs) to DNA sequence motifs. The majority of eukaryotic genes (protein-coding genes and many regulatory RNAs) are transcribed by RNA polymerase II (RNA pol II), an enzyme that contains various subuni
Computational identification of Drosophila microRNA genes
Eric C Lai, Pavel Tomancak, Robert W Williams, Gerald M Rubin
Genome Biology , 2003, DOI: 10.1186/gb-2003-4-7-r42
Abstract: We developed an informatic procedure called 'miRseeker', which analyzed the completed euchromatic sequences of Drosophila melanogaster and D. pseudoobscura for conserved sequences that adopt an extended stem-loop structure and display a pattern of nucleotide divergence characteristic of known miRNAs. The sensitivity of this computational procedure was demonstrated by the presence of 75% (18/24) of previously identified Drosophila miRNAs within the top 124 candidates. In total, we identified 48 novel miRNA candidates that were strongly conserved in more distant insect, nematode, or vertebrate genomes. We verified expression for a total of 24 novel miRNA genes, including 20 of 27 candidates conserved in a third species and 4 of 11 high-scoring, Drosophila-specific candidates. Our analyses lead us to estimate that drosophilid genomes contain around 110 miRNA genes.Our computational strategy succeeded in identifying bona fide miRNA genes and suggests that miRNAs constitute nearly 1% of predicted protein-coding genes in Drosophila, a percentage similar to the percentage of miRNAs recently attributed to other metazoan genomes.Although the analysis of sequenced genomes to date has focused most heavily on the protein-coding set of genes, all genomes also contain a constellation of non-coding RNA genes. With the exception of certain classes of RNAs with strongly conserved sequences and/or structures, such as ribosomal and transfer RNAs, identification of most non-coding RNAs has historically been a relatively serendipitous affair. Only very recently have there been concerted efforts to identify such genes systematically, using both experimental and computational approaches [1].Our collective ignorance of the totality of non-coding RNA genes was laid bare by recent work on microRNAs (miRNAs), an abundant family of 21-22 nucleotide non-coding RNAs [2,3]. The founding members of this family, lin-4 and let-7, were identified through forward analysis of extant Caenorhabditis eleg
Transcriptional control in embryonic Drosophila midline guidance assessed through a whole genome approach
Tiago R Magalh?es, Jessica Palmer, Pavel Tomancak, Katherine S Pollard
BMC Neuroscience , 2007, DOI: 10.1186/1471-2202-8-59
Abstract: Using hopach, a novel clustering method which is well suited to microarray data analysis, we identified groups of genes with similar expression patterns across guidance mutants and transgenics. We then systematically characterized the resulting clusters with respect to their relevance to axon guidance using two complementary controlled vocabularies: the Gene Ontology (GO) and anatomical annotations of the Atlas of Pattern of Gene Expression (APoGE) in situ hybridization database. The analysis indicates that regulation of gene expression does play a role in the process of axon guidance in Drosophila. We also find a strong link between axon guidance and hemocyte migration, a result that agrees with mounting evidence that axon guidance molecules are co-opted in vertebrate vascularization. Cell cyclin activity in the context of axon guidance is also suggested from our array data. RNA and protein expression patterns of cell cyclins in axon guidance mutants and transgenics support this possible link.This study provides important insights into the regulation of axon guidance in vivo.The process by which axons cross the midline during development of the Central Nervous System (CNS) in Drosophila is of great interest and has been the focus of much scientific research [1,2]. After neuroblast delamination and cell fate decisions, axons undergo a journey that will eventually wire them to the appropriate targets. This journey includes a series of steps, one of which is the decision to cross (or not cross) the midline. Four key regulators of midline crossing are known: slit, robo, robo2 and comm. Growth cones in which the robo and robo2 receptors are present at the membrane sense the presence of the slit ligand and do not cross the midline [3,4]. Conversely, growth cones without robo and robo2 at the membrane are not able to respond to the repulsive cue of slit and do cross the midline. robo levels at the growth cone membrane are regulated by comm.A major question in midline cros
An Excess of Gene Expression Divergence on the X Chromosome in Drosophila Embryos: Implications for the Faster-X Hypothesis
Melek A. Kayserili equal contributor,Dave T. Gerrard equal contributor,Pavel Tomancak ,Alex T. Kalinka
PLOS Genetics , 2012, DOI: 10.1371/journal.pgen.1003200
Abstract: The X chromosome is present as a single copy in the heterogametic sex, and this hemizygosity is expected to drive unusual patterns of evolution on the X relative to the autosomes. For example, the hemizgosity of the X may lead to a lower chromosomal effective population size compared to the autosomes, suggesting that the X might be more strongly affected by genetic drift. However, the X may also experience stronger positive selection than the autosomes, because recessive beneficial mutations will be more visible to selection on the X where they will spend less time being masked by the dominant, less beneficial allele—a proposal known as the faster-X hypothesis. Thus, empirical studies demonstrating increased genetic divergence on the X chromosome could be indicative of either adaptive or non-adaptive evolution. We measured gene expression in Drosophila species and in D. melanogaster inbred strains for both embryos and adults. In the embryos we found that expression divergence is on average more than 20% higher for genes on the X chromosome relative to the autosomes; but in contrast, in the inbred strains, gene expression variation is significantly lower on the X chromosome. Furthermore, expression divergence of genes on Muller's D element is significantly greater along the branch leading to the obscura sub-group, in which this element segregates as a neo-X chromosome. In the adults, divergence is greatest on the X chromosome for males, but not for females, yet in both sexes inbred strains harbour the lowest level of gene expression variation on the X chromosome. We consider different explanations for our results and conclude that they are most consistent within the framework of the faster-X hypothesis.
An Integrated Micro- and Macroarchitectural Analysis of the Drosophila Brain by Computer-Assisted Serial Section Electron Microscopy
Albert Cardona,Stephan Saalfeld,Stephan Preibisch,Benjamin Schmid,Anchi Cheng,Jim Pulokas,Pavel Tomancak,Volker Hartenstein
PLOS Biology , 2010, DOI: 10.1371/journal.pbio.1000502
Abstract: The analysis of microcircuitry (the connectivity at the level of individual neuronal processes and synapses), which is indispensable for our understanding of brain function, is based on serial transmission electron microscopy (TEM) or one of its modern variants. Due to technical limitations, most previous studies that used serial TEM recorded relatively small stacks of individual neurons. As a result, our knowledge of microcircuitry in any nervous system is very limited. We applied the software package TrakEM2 to reconstruct neuronal microcircuitry from TEM sections of a small brain, the early larval brain of Drosophila melanogaster. TrakEM2 enables us to embed the analysis of the TEM image volumes at the microcircuit level into a light microscopically derived neuro-anatomical framework, by registering confocal stacks containing sparsely labeled neural structures with the TEM image volume. We imaged two sets of serial TEM sections of the Drosophila first instar larval brain neuropile and one ventral nerve cord segment, and here report our first results pertaining to Drosophila brain microcircuitry. Terminal neurites fall into a small number of generic classes termed globular, varicose, axiform, and dendritiform. Globular and varicose neurites have large diameter segments that carry almost exclusively presynaptic sites. Dendritiform neurites are thin, highly branched processes that are almost exclusively postsynaptic. Due to the high branching density of dendritiform fibers and the fact that synapses are polyadic, neurites are highly interconnected even within small neuropile volumes. We describe the network motifs most frequently encountered in the Drosophila neuropile. Our study introduces an approach towards a comprehensive anatomical reconstruction of neuronal microcircuitry and delivers microcircuitry comparisons between vertebrate and insect neuropile.
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