Tools for neuroanatomy and neurogenetics in
            <i>Drosophila</i>

Pfeiffer, Barret D.; Jenett, Arnim; Hammonds, Ann S.; Ngo, Teri T.B.; Misra, Sima; Murphy, Christine; Scully, Audra L.; Carlson, Joseph W.; Wan, Kenneth H.; Laverty, Todd R.; Mungall, Christopher J.; Svirskas, Robert; Kadonaga, James T.; Doe, Chris Q.; Eisen, Michael B.; Celniker, S; Rubin, Gerald M.

doi:10.1073/pnas.0803697105

Cited by 917 publications

(983 citation statements)

References 19 publications

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“…Because these drivers label many neurons in the DOG, we sought a sparser line in the GAL4 collection at Janelia Farm (26). We found that the R11F02 driver reliably and specifically labels the three thermosensory neurons with minimal expression in the rest of the animal (Fig.…”

Section: Resultsmentioning

confidence: 99%

Sensory determinants of behavioral dynamics in Drosophila thermotaxis

Klein

Afonso

Vonner

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

147

178

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Complex animal behaviors are built from dynamical relationships between sensory inputs, neuronal activity, and motor outputs in patterns with strategic value. Connecting these patterns illuminates how nervous systems compute behavior. Here, we study Drosophila larva navigation up temperature gradients toward preferred temperatures (positive thermotaxis). By tracking the movements of animals responding to fixed spatial temperature gradients or random temperature fluctuations, we calculate the sensitivity and dynamics of the conversion of thermosensory inputs into motor responses. We discover three thermosensory neurons in each dorsal organ ganglion (DOG) that are required for positive thermotaxis. Random optogenetic stimulation of the DOG thermosensory neurons evokes behavioral patterns that mimic the response to temperature variations. In vivo calcium and voltage imaging reveals that the DOG thermosensory neurons exhibit activity patterns with sensitivity and dynamics matched to the behavioral response. Temporal processing of temperature variations carried out by the DOG thermosensory neurons emerges in distinct motor responses during thermotaxis. N avigation toward environmental conditions that improve survival and fitness is of near-universal importance in motile biological organisms. Quantitative analysis of such animal behaviors to defined sensory inputs is a powerful approach to elucidate how behavior is encoded in underlying neurons and circuits. The advantage of studying navigation in small, optically transparent, genetically modifiable animals like Caenorhabditis elegans (1) or Drosophila larvae (2) is the opportunity to dissect sensory, neuronal, and behavioral dynamics in live animals by using optical neurophysiology and optogenetics throughout the nervous system.The Drosophila melanogaster larva navigates gradients of many sensory cues, including light, temperature, odors, and tastes, but with fewer neurons in its sensory periphery and brain than the adult. Moreover, the simpler body plan and crawling movements of the larva facilitate the precise quantification of behavioral dynamics. Poikilotherms like C. elegans or Drosophila use sensitive thermosensory mechanisms to navigate moderate temperature ranges, thereby enabling them to use their environments to regulate their own body temperatures (3, 4). Here, we study sensory and behavioral dynamics during positive thermotaxis (i.e., cool avoidance) by the Drosophila larva. Tracking the movements of Drosophila exploring temperature, olfactory, or gaseous gradients has shown that their navigation is generated by a sequence of two alternating motor programs: runs involving peristaltic forward movement that are interrupted by turns involving probing side-to-side head sweeps until the initiation of a new run (5-8). Larvae negotiating temperature gradients stochastically transition between runs and turns by strategies that cause runs pointed in favorable directions to be more frequent and longer than runs pointed in unfavorable directions. These transitions b...

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Section: Resultsmentioning

confidence: 99%

Sensory determinants of behavioral dynamics in Drosophila thermotaxis

Klein

Afonso

Vonner

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

147

178

View full text Add to dashboard Cite

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“…Constructs were injected using standard transformation techniques (37), with w 1118 as the recipient strain. sens enhancer-reporter constructs were cloned through a pCR8/GW/TOPO intermediate into pGreenFriend, a variant of pBPGUw (38) in which the GAL4 coding sequence was removed via a FseI/HindIII double digest and replaced with an FseI/HindIII-linkered eGFP coding sequence amplified from pH-Stinger (35). These constructs were integrated into the attP2 docking site (39) using a germ-line ΦC31 integrase source (40).…”

Section: Methodsmentioning

confidence: 99%

Neural precursor-specific expression of multiple Drosophila genes is driven by dual enhancer modules with overlapping function

Miller

Rebeiz

Atanasov

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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Transcriptional cis-regulatory modules (CRMs), or enhancers, are responsible for directing gene expression in specific territories and cell types during development. In some instances, the same gene may be served by two or more enhancers with similar specificities. Here we show that the utilization of dual, or "shadow", enhancers is a common feature of genes that are active specifically in neural precursor (NP) cells in Drosophila. By genome-wide computational discovery of statistically significant clusters of binding motifs for both proneural activator (P) proteins and basic helix-loop-helix (bHLH) repressor (R) factors (a "P+R" regulatory code), we have identified NP-specific enhancer modules associated with multiple genes expressed in this cell type. These CRMs are distinct from those previously identified for the corresponding gene, establishing the existence of a dual-enhancer arrangement in which both modules reside close to the gene they serve. Using wild-type and mutant reporter gene constructs in vivo, we show that P sites in these modules mediate activation by proneural factors in "proneural cluster" territories, whereas R sites mediate repression by bHLH repressors, which serves to restrict expression specifically to NP cells. To our knowledge, our results identify the first direct targets of these bHLH repressors. Finally, using genomic rescue constructs for neuralized (neur), we demonstrate that each of the gene's two NP-specific enhancers is sufficient to rescue neur function in the lateral inhibition process by which adult sensory organ precursor (SOP) cells are specified, but that deletion of both enhancers results in failure of this event.neural precursors | dual enhancer modules | cis-regulatory code | proneural proteins | bHLH repressors S pecification of neural precursor (NP) cell fates is a core step in the process of neural development, and there has long been intense interest in its mechanistic basis. Among the most heavily investigated questions in this arena is how NP-specific expression of key regulatory factors associated with the NP fate is achieved. For example, multiple prior studies have used computational methods to identify NP-specific cis-regulatory modules (CRMs) genome-wide, in an attempt to define common transcription factor inputs that might underlie NP-specific gene expression (1-3).Proneural transcriptional activators of the basic helix-loophelix (bHLH) family are the top-level regulators of NP specification. They confer on cells in ectodermal tissue the potential to adopt the NP fate; loss of proneural gene function results in loss of all NPs and thus loss of expression of all NP-specific regulatory factors. However, proneural factors are not expressed only in NPs; rather, they are initially expressed in small groups of cells called proneural clusters (PNCs). At this stage of the process, most or all cells in the cluster have the potential to become an NP. This is prevented by "lateral inhibition", in which the single NP that will ultimately arise from the PNC inhibits al...

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“…For each of these regions, we cloned wild-type and mutant sequences in which we disrupted all TAGteam motifs by point mutations upstream of a Gal4 reporter gene and inserted them into the Drosophila melanogaster genome by sitespecific integration (Pfeiffer et al 2008). We found that all four wild-type sequences drove reporter expression in diverse dorsoventral patterns in the early embryo (Fig.…”

Section: Early Embryonic Enhancers Depend On Vielfaltig's Tagteam Motifmentioning

confidence: 99%

“…All were cloned into a pBPGUw vector, verified by Sanger sequencing, and integrated into an attP2 landing site on chromosome 3 (Pfeiffer et al 2008). Finally, transgenic flies were confirmed by PCR and Sanger sequencing.…”

Section: Cloning and Transgenesismentioning

confidence: 99%

“…An antisense Gal4 RNA probe generated using the primers described in Pfeiffer et al (2008) was used to visualize the expression of the Gal4 reporter gene in transgenic embryos. Briefly, embryos were collected, fixed, and hybridized with the digoxigenin-labeled antisense RNA probe followed by staining with anti-DIG FAB fragments coupled to alkaline phosphatase (Roche cat.…”

Section: Whole-mount In Situ Hybridizationmentioning

confidence: 99%

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Uncovering cis-regulatory sequence requirements for context-specific transcription factor binding

et al. 2012

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The regulation of gene expression is mediated at the transcriptional level by enhancer regions that are bound by sequence-specific transcription factors (TFs). Recent studies have shown that the in vivo binding sites of single TFs differ between developmental or cellular contexts. How this context-specific binding is encoded in the cis-regulatory DNA sequence has, however, remained unclear. We computationally dissect context-specific TF binding sites in Drosophila, Caenorhabditis elegans, mouse, and human and find distinct combinations of sequence motifs for partner factors, which are predictive and reveal specific motif requirements of individual binding sites. We predict that TF binding in the early Drosophila embryo depends on motifs for the early zygotic TFs Vielfaltig (also known as Zelda) and Tramtrack. We validate experimentally that the activity of Twist-bound enhancers and Twist binding itself depend on Vielfaltig motifs, suggesting that Vielfaltig is more generally important for early transcription. Our finding that the motif content can predict contextspecific binding and that the predictions work across different Drosophila species suggests that characteristic motif combinations are shared between sites, revealing context-specific motif codes (cis-regulatory signatures), which appear to be conserved during evolution. Taken together, this study establishes a novel approach to derive predictive cis-regulatory motif requirements for individual TF binding sites and enhancers. Importantly, the method is generally applicable across different cell types and organisms to elucidate cis-regulatory sequence determinants and the corresponding trans-acting factors from the increasing number of tissue-and cell-type-specific TF binding studies.

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Tools for neuroanatomy and neurogenetics in Drosophila

Cited by 917 publications

References 19 publications

Sensory determinants of behavioral dynamics in Drosophila thermotaxis

Sensory determinants of behavioral dynamics in Drosophila thermotaxis

Neural precursor-specific expression of multiple Drosophila genes is driven by dual enhancer modules with overlapping function

Uncovering cis-regulatory sequence requirements for context-specific transcription factor binding

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