Systematic determination of patterns of gene expression during Drosophila embryogenesis
TL;DR: Analyzing gene-expression patterns by in situ hybridization to whole-mount embryos provides an extremely rich dataset that can be used to identify genes involved in developmental processes that have been missed by traditional genetic analysis.
Abstract: Background: Cell-fate specification and tissue differentiation during development are largely achieved by the regulation of gene transcription. Results: As a first step to creating a comprehensive atlas of gene-expression patterns during Drosophila embryogenesis, we examined 2,179 genes by in situ hybridization to fixed Drosophila embryos. Of the genes assayed, 63.7% displayed dynamic expression patterns that were documented with 25,690 digital photomicrographs of individual embryos. The photomicrographs were annotated using controlled vocabularies for anatomical structures that are organized into a developmental hierarchy. We also generated a detailed time course of gene expression during embryogenesis using microarrays to provide an independent corroboration of the in situ hybridization results. All image, annotation and microarray data are stored in publicly available database. We found that the RNA transcripts of about 1% of genes show clear subcellular localization. Nearly all the annotated expression patterns are distinct. We present an approach for organizing the data by hierarchical clustering of annotation terms that allows us to group tissues that express similar sets of genes as well as genes displaying similar expression patterns. Conclusions: Analyzing gene-expression patterns by in situ hybridization to whole-mount embryos provides an extremely rich dataset that can be used to identify genes involved in developmental processes that have been missed by traditional genetic analysis. Systematic analysis of rigorously annotated patterns of gene expression will complement and extend the types of analyses carried out using expression microarrays.
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TL;DR: The generation and validation of a genome-wide library of Drosophila melanogaster RNAi transgenes, enabling the conditional inactivation of gene function in specific tissues of the intact organism and opening up the prospect of systematically analysing gene functions in any tissue and at any stage of the Drosophile lifespan.
Abstract: Forward genetic screens in model organisms have provided important insights into numerous aspects of development, physiology and pathology. With the availability of complete genome sequences and the introduction of RNA-mediated gene interference (RNAi), systematic reverse genetic screens are now also possible. Until now, such genome-wide RNAi screens have mostly been restricted to cultured cells and ubiquitous gene inactivation in Caenorhabditis elegans. This powerful approach has not yet been applied in a tissue-specific manner. Here we report the generation and validation of a genome-wide library of Drosophila melanogaster RNAi transgenes, enabling the conditional inactivation of gene function in specific tissues of the intact organism. Our RNAi transgenes consist of short gene fragments cloned as inverted repeats and expressed using the binary GAL4/UAS system. We generated 22,270 transgenic lines, covering 88% of the predicted protein-coding genes in the Drosophila genome. Molecular and phenotypic assays indicate that the majority of these transgenes are functional. Our transgenic RNAi library thus opens up the prospect of systematically analysing gene functions in any tissue and at any stage of the Drosophila lifespan.
2,721 citations
TL;DR: How properties of enhancer sequences and chromatin are used to predict enhancers in genome-wide studies are discussed and recently developed high-throughput methods that allow the direct testing and identification of enhancers on the basis of their activity are covered.
Abstract: Cellular development, morphology and function are governed by precise patterns of gene expression. These are established by the coordinated action of genomic regulatory elements known as enhancers or cis-regulatory modules. More than 30 years after the initial discovery of enhancers, many of their properties have been elucidated; however, despite major efforts, we only have an incomplete picture of enhancers in animal genomes. In this Review, we discuss how properties of enhancer sequences and chromatin are used to predict enhancers in genome-wide studies. We also cover recently developed high-throughput methods that allow the direct testing and identification of enhancers on the basis of their activity. Finally, we discuss recent technological advances and current challenges in the field of regulatory genomics.
1,163 citations
TL;DR: It is reported that a large set of genes involved in basic cellular processes avoid microRNA regulation due to short 3'UTRs that are specifically depleted of microRNA binding sites, ensuring tissue identity and supporting cell-lineage decisions.
1,119 citations
Cites methods from "Systematic determination of pattern..."
...We used an extensive collection of annotated in situ gene expression patterns for Drosophila embryogenesis (Tomancak et al., 2002)....
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TL;DR: A high-resolution fluorescent in situ hybridization procedure was developed and employed to comprehensively evaluate mRNA localization dynamics during early Drosophila embryogenesis, indicating major roles for mRNA localization in nucleating localized cellular machineries.
942 citations
Cites background or methods from "Systematic determination of pattern..."
...…RNA localization database resource, containing annotation terms and representative images of all transcripts detected, has been established using previously described web-based tools (Tomancak et al., 2002) and can be accessed through a searchable web-browser at: http://fly-fish.ccbr. utoronto.ca....
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...In addition, an RNA localization database resource, containing annotation terms and representative images of all transcripts detected, has been established using previously described web-based tools (Tomancak et al., 2002) and can be accessed through a searchable web-browser at: http://fly-fish....
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...Previous in situ screening efforts in Drosophila have established speculative estimates of the proportion of localized mRNAs, ranging from one to ten percent (Dubowy and Macdonald, 1998; Tomancak et al., 2002)....
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...Data Annotation and Database Setup All of the image and annotation data were organized within a MySQL database using Perl-based annotation tools adapted from a previous study (Tomancak et al., 2002)....
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...…maternal Bsg25D transcripts (A), and for zygotically expressed CG4500 and Trn-SR transcripts (B), detected using optimized FISH (mRNAs in green/nuclei in red), or standard alkaline phosphatase-based detection ([A] left panel, image obtained from the BDGP in situ database, Tomancak et al., 2002)....
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TL;DR: The results suggest that the D. melanogaster genome contains >50,000 enhancers and that multiple enhancers drive distinct subsets of expression of a gene in each tissue and developmental stage.
Abstract: We demonstrate the feasibility of generating thousands of transgenic Drosophila melanogaster lines in which the expression of an exogenous gene is reproducibly directed to distinct small subsets of cells in the adult brain. We expect the expression patterns produced by the collection of 5,000 lines that we are currently generating to encompass all neurons in the brain in a variety of intersecting patterns. Overlapping 3-kb DNA fragments from the flanking noncoding and intronic regions of genes thought to have patterned expression in the adult brain were inserted into a defined genomic location by site-specific recombination. These fragments were then assayed for their ability to function as transcriptional enhancers in conjunction with a synthetic core promoter designed to work with a wide variety of enhancer types. An analysis of 44 fragments from four genes found that >80% drive expression patterns in the brain; the observed patterns were, on average, comprised of 50,000 enhancers and that multiple enhancers drive distinct subsets of expression of a gene in each tissue and developmental stage. We expect that these lines will be valuable tools for neuroanatomy as well as for the elucidation of neuronal circuits and information flow in the fly brain.
927 citations
References
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TL;DR: The goal of the Gene Ontology Consortium is to produce a dynamic, controlled vocabulary that can be applied to all eukaryotes even as knowledge of gene and protein roles in cells is accumulating and changing.
Abstract: Genomic sequencing has made it clear that a large fraction of the genes specifying the core biological functions are shared by all eukaryotes. Knowledge of the biological role of such shared proteins in one organism can often be transferred to other organisms. The goal of the Gene Ontology Consortium is to produce a dynamic, controlled vocabulary that can be applied to all eukaryotes even as knowledge of gene and protein roles in cells is accumulating and changing. To this end, three independent ontologies accessible on the World-Wide Web (http://www.geneontology.org) are being constructed: biological process, molecular function and cellular component.
35,225 citations
TL;DR: A system of cluster analysis for genome-wide expression data from DNA microarray hybridization is described that uses standard statistical algorithms to arrange genes according to similarity in pattern of gene expression, finding in the budding yeast Saccharomyces cerevisiae that clustering gene expression data groups together efficiently genes of known similar function.
Abstract: A system of cluster analysis for genome-wide expression data from DNA microarray hybridization is de- scribed that uses standard statistical algorithms to arrange genes according to similarity in pattern of gene expression. The output is displayed graphically, conveying the clustering and the underlying expression data simultaneously in a form intuitive for biologists. We have found in the budding yeast Saccharomyces cerevisiae that clustering gene expression data groups together efficiently genes of known similar function, and we find a similar tendency in human data. Thus patterns seen in genome-wide expression experiments can be inter- preted as indications of the status of cellular processes. Also, coexpression of genes of known function with poorly charac- terized or novel genes may provide a simple means of gaining leads to the functions of many genes for which information is not available currently.
16,371 citations
TL;DR: A high-capacity system was developed to monitor the expression of many genes in parallel by means of simultaneous, two-color fluorescence hybridization, which enabled detection of rare transcripts in probe mixtures derived from 2 micrograms of total cellular messenger RNA.
Abstract: A high-capacity system was developed to monitor the expression of many genes in parallel. Microarrays prepared by high-speed robotic printing of complementary DNAs on glass were used for quantitative expression measurements of the corresponding genes. Because of the small format and high density of the arrays, hybridization volumes of 2 microliters could be used that enabled detection of rare transcripts in probe mixtures derived from 2 micrograms of total cellular messenger RNA. Differential expression measurements of 45 Arabidopsis genes were made by means of simultaneous, two-color fluorescence hybridization.
10,287 citations
"Systematic determination of pattern..." refers background or methods in this paper
...Moreover, the quantitative comparison of expression levels for a given gene, or among different genes, in multiple experiments is complicated by differential hybridization kinetics and cross-hybridization properties of each target-probe pair [12,13]....
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...Existing microarray datasets, such as that reported in [21], were generated using spotted cDNA arrays [13] derived from the same DGC clone set [14-16] that we used for the in situ probes, and were therefore unsuitable as independent controls....
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TL;DR: A comprehensive catalog of yeast genes whose transcript levels vary periodically within the cell cycle is created, and it is found that the mRNA levels of more than half of these 800 genes respond to one or both of these cyclins.
Abstract: We sought to create a comprehensive catalog of yeast genes whose transcript levels vary periodically within the cell cycle. To this end, we used DNA microarrays and samples from yeast cultures sync...
5,176 citations
"Systematic determination of pattern..." refers background in this paper
...Approaches using DNA microarrays have been successful in studying genome-wide transcriptional regulation during animal development [9-11], but suffer from several limitations....
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TL;DR: In this article, the authors proposed a method for monitoring the expression levels of a multiplicity of genes by hybridizing a nucleic acid sample to a high density array of oligonucleotide probes and quantifying the hybridized nucleic acids in the array.
Abstract: This invention provides methods of monitoring the expression levels of a multiplicity of genes. The methods involve hybridizing a nucleic acid sample to a high density array of oligonucleotide probes where the high density array contains oligonucleotide probes complementary to subsequences of target nucleic acids in the nucleic acid sample. In one embodiment, the method involves providing a pool of target nucleic acids comprising RNA transcripts of one or more target genes, or nucleic acids derived from the RNA transcripts, hybridizing said pool of nucleic acids to an array of oligonucleotide probes immobilized on surface, where the array comprising more than 100 different oligonucleotides and each different oligonucleotide is localized in a predetermined region of the surface, the density of the different oligonucleotides is greater than about 60 different oligonucleotides per 1 cm2, and the oligonucleotide probes are complementary to the RNA transcripts or nucleic acids derived from the RNA transcripts; and quantifying the hybridized nucleic acids in the array.
4,382 citations