Tools for neuroanatomy and neurogenetics in Drosophila. Proc Natl Acad Sci

Janelia Farm Research Campus, Howard Hughes Medical Institute, Ashburn VA 20147, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 07/2008; 105(28):9715-20. DOI: 10.1073/pnas.0803697105
Source: PubMed


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 <100 cells. Our 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. 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.

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Available from: Kenneth H Wan
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    • "5 PCR additions are used to add minimal promoter elements, an intron to stabilize mRNAs (Zieler & Huynh, 2002), the 20 N randomer tag, and Illumina paired-end sequence upstream of an arbitrary ORF, in this case GFP. The synthetic minimal promoter used was designed to contain several core motifs and has been shown to function with a wide range of enhancers (Pfeiffer et al., 2008). The two fragments are then ligated together to create the final construct library pictured in Fig. 1A. "
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    ABSTRACT: Here we present a genome-wide method for de novo identification of enhancer regions. This approach enables massively parallel empirical investigation of DNA sequences that mediate transcriptional activation and provides a platform for discovery of regulatory modules capable of driving context-specific gene expression. The method links fragmented genomic DNA to the transcription of randomer molecule identifiers and measures the functional enhancer activity of the library by massively parallel sequencing. We transfected a Drosophila melanogaster library into S2 cells in normoxia and hypoxia, and assayed 4,599,881 genomic DNA fragments in parallel. The locations of the enhancer regions strongly correlate with genes up-regulated after hypoxia and previously described enhancers. Novel enhancer regions were identified and integrated with RNAseq data and transcription factor motifs to describe the hypoxic response on a genome-wide basis as a complex regulatory network involving multiple stress-response pathways. This work provides a novel method for high-throughput assay of enhancer activity and the genome-scale identification of 31 hypoxia-activated enhancers in Drosophila .
    Full-text · Article · Dec 2015 · PeerJ
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    • "For a complete list of fly stocks see Supplemental Information. For EL-AD and CQ2-lexA, molecular constructs and transgenic flies were generated Neuron 88, 1–16, October 21, 2015 ª2015 Elsevier Inc. 13 Please cite this article in press as: Heckscher et al., Even-Skipped + Interneurons Are Core Components of a Sensorimotor Circuit that Maintains Left- Right Symmetric Muscle Contraction Amplitude, Neuron (2015), using standard methods as previously described (Pfeiffer et al., 2008, 2010). "
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    ABSTRACT: Bilaterally symmetric motor patterns-those in which left-right pairs of muscles contract synchronously and with equal amplitude (such as breathing, smiling, whisking, and locomotion)-are widespread throughout the animal kingdom. Yet, surprisingly little is known about the underlying neural circuits. We performed a thermogenetic screen to identify neurons required for bilaterally symmetric locomotion in Drosophila larvae and identified the evolutionarily conserved Even-skipped(+) interneurons (Eve/Evx). Activation or ablation of Eve(+) interneurons disrupted bilaterally symmetric muscle contraction amplitude, without affecting the timing of motor output. Eve(+) interneurons are not rhythmically active and thus function independently of the locomotor CPG. GCaMP6 calcium imaging of Eve(+) interneurons in freely moving larvae showed left-right asymmetric activation that correlated with larval behavior. TEM reconstruction of Eve(+) interneuron inputs and outputs showed that the Eve(+) interneurons are at the core of a sensorimotor circuit capable of detecting and modifying body wall muscle contraction.
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    • "Projections from the VLP to other brain regions remain uncharacterized (but see Yu et al. (2010)). While genetic tools exist that label many AMMC or VLP neurons (Pfeiffer et al., 2008), only a handful have been functionally characterized (Lai et al., 2012; Tootoonian et al., 2012; Vaughan et al., 2014). Here we sample a larger population of AMMC and VLP neurons and generate computational models that effectively recapitulate responses to naturalistic stimuli. "
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    ABSTRACT: Brains are optimized for processing ethologically relevant sensory signals. However, few studies have characterized the neural coding mechanisms that underlie the transformation from natural sensory information to behavior. Here, we focus on acoustic communication in Drosophila melanogaster and use computational modeling to link natural courtship song, neuronal codes, and female behavioral responses to song. We show that melanogaster females are sensitive to long timescale song structure (on the order of tens of seconds). From intracellular recordings, we generate models that recapitulate neural responses to acoustic stimuli. We link these neural codes with female behavior by generating model neural responses to natural courtship song. Using a simple decoder, we predict female behavioral responses to the same song stimuli with high accuracy. Our modeling approach reveals how long timescale song features are represented by the Drosophila brain and how neural representations can be decoded to generate behavioral selectivity for acoustic communication signals.
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