Combinatorial Activation and Repression by Seven Transcription Factors Specify Drosophila Odorant Receptor Expression

A systematic analysis reveals a regulatory network controlling selective odorant receptor expression and neuronal diversity in Drosophila

A Genome-wide Drosophila Screen for Heat Nociception Identifies α2δ3 as an Evolutionarily Conserved Pain Gene

Worldwide, acute and chronic pain affects 20% of the adult population and represents an enormous financial and emotional burden. Using genome-wide neuronal-specific RNAi knock-down in Drosophila, we report a global screen for an innate behavior and identify hundreds of novel genes implicated in heat nociception, including the α2δ-family calcium channel subunit straightjacket (stj). Mice mutant for the stj ortholog CACNA2D3 (α2δ3) also exhibit impaired behavioral heat pain sensitivity. In addition, in humans, α2δ3 SNP variants associate with reduced sensitivity to acute noxious heat and chronic back pain. Functional imaging in α2δ3 mutant mice revealed impaired transmission of thermal pain evoked signals from the thalamus to higher order pain centers. Intriguingly, in α2δ3 mutant mice thermal pain and tactile stimulation triggered strong cross-activation or synesthesia of brain regions involved in vision, olfaction, and hearing

A cilia-bound unconventional secretory pathway for Drosophila odorant receptors

Background: Post-translational transport is a vital process which ensures that each protein reaches its site of function. Though most do so via an ordered ER-to-Golgi route, an increasing number of proteins are now shown to bypass this conventional secretory pathway. Results: In the Drosophila olfactory sensory neurons (OSNs), odorant receptors (ORs) are trafficked from the ER towards the cilia. Here, we show that Or22a, a receptor of various esters and alcoholic compounds, reaches the cilia partially through unconventional means. Or22a frequently present as puncta at the somatic cell body exit and within the dendrite prior to the cilia base. These rarely coincide with markers of either the intermediary ER-Golgi-intermediate-compartment (ERGIC) or Golgi structures. ERGIC and Golgi also displayed axonal localization biases, a further indication that at least some measure of OR transport may occur independently of their involvement. Additionally, neither the loss of several COPII genes involved in anterograde trafficking nor ERGIC itself affected puncta formation or Or22a transport to the cilium. Instead, we observed the consistent colocalization of Or22a puncta with Grasp65, the sole Drosophila homolog of mammalian GRASP55/Grh1, a marker of the unconventional pathway. The numbers of both Or22a and Grasp65-positive puncta were furthermore increased upon nutritional starvation, a condition known to enhance Golgi-bypassing secretory activity. Conclusions: Our results demonstrate an alternative route of Or22a transport, thus expanding the repertoire of unconventional secretion mechanisms in neurons

Cis-Regulatory Mechanisms for Robust Olfactory Sensory Neuron Class-restricted Odorant Receptor Gene Expression in Drosophila

Odor perception requires that each olfactory sensory neuron (OSN) class continuously express a single odorant receptor (OR) regardless of changes in the environment. However, little is known about the control of the robust, class-specific OR expression involved. Here, we investigate the cis-regulatory mechanisms and components that generate robust and OSN class-specific OR expression in Drosophila. Our results demonstrate that the spatial restriction of expression to a single OSN class is directed by clusters of transcription-factor DNA binding motifs. Our dissection of motif clusters of differing complexity demonstrates that structural components such as motif overlap and motif order integrate transcription factor combinations and chromatin status to form a spatially restricted pattern. We further demonstrate that changes in metabolism or temperature perturb the function of complex clusters. We show that the cooperative regulation between motifs around and within the cluster generates robust, class-specific OR expression.Funding Agencies|Swedish Foundation for Strategic Research [F06-0013]; Swedish Research Council [522-2006-6364 / K2007-66P-20436-01-04]</p

Odor response adaptation in Drosophila : a continuous individualization process

Olfactory perception is very individualized in humans and also in Drosophila. The process that individualize olfaction is adaptation that across multiple time scales and mechanisms shape perception and olfactory-guided behaviors. Olfactory adaptation occurs both in the central nervous system and in the periphery. Central adaptation occurs at the level of the circuits that process olfactory inputs from the periphery where it can integrate inputs from other senses, metabolic states, and stress. We will here focus on the periphery and how the fast, slow, and persistent (lifelong) adaptation mechanisms in the olfactory sensory neurons individualize the Drosophila olfactory system

Spatial expression pattern is dictated by the structure of the <i>Or59b</i> cluster.

<p>GFP expression (green) produced by (A) the <i>Or59b</i> cluster, (B) the cluster with the E-box displaced 125bps, (C) with the E-box 10bps upstream the cluster, (D-E) the Ebox 5 or 10 bp downstream the cluster. Synaptic neuropil regions are labeled with the presynaptic marker nc82 (magenta). A schematic representation of different rearrangements is shown under each figure. Schematic interpretations of the results are presented in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005051#pgen.1005051.s005" target="_blank">S5 Fig.</a></p

One motif cluster combines the TF regulation of <i>Or59b</i> gene expression.

<p>(A) Diagram of the 1000-bp upstream region of the <i>Or59b</i> gene showing the locations of the Pou (blue), Acj6^Hox (orange), Pdm3^Hox (red) and E-box (green) motifs. The gray box marks the cluster of Hox/pou/E-box motifs. Below, the 36-bp <i>Or59b</i> cluster sequence is presented. (B-D) A whole-mount brain shows GFP expression driven by the <i>Or59b</i> cluster (green) and the synaptic neuropil marked by nc82 (magenta). The marked region defines the whole brain and the antennal lobes. (C) GFP expression from the <i>Or59b</i> reporter and <i>Or59b</i> cluster in the antenna and in the antennal lobe, where it marks axonal projections to the DM4 and VC4 glomeruli. (D) Loss of expression produced by the <i>Or59b</i> cluster is observed in the <i>Acj6-</i>, <i>Fer1-</i> and <i>Pdm3-IRs</i> but not in <i>Atro-IR</i> or <i>UAS-Atro</i> overexpression lines. Control flies were crossed to <i>Peb-Gal4</i>.</p

Heterochromatin modulation of the <i>Or59b</i> cluster.

<p>(A) GFP expression (green) driven by the <i>Or59b</i> reporter or <i>Or59b</i> cluster in <i>su(var)3–9</i>^<i>06</i> heterozygote flies. Synaptic neuropil regions are labeled with the presynaptic marker nc82 (magenta). Control flies were crossed to <i>w</i>^<i>1118</i>. (B-E) GFP expression (green) driven by mutated <i>Or59b</i> cluster versions in <i>su(var)3–9</i>^<i>06</i> heterozygote flies. Note that the loss of GFP expression driven by the <i>Or59b</i> cluster with a mutated Pou motif or with a distant E-box is rescued in <i>su(var)3–9</i>^<i>06</i> heterozygote flies. (F) GFP expression (green) driven by an <i>Or59b</i> cluster with an additional E-box in <i>su(var)3–9</i>^<i>06</i> heterozygote flies. Note that the E-box rescues the produced <i>su(var)3–9</i>^<i>06</i> expression phenotypes. (G) Model depicting the function of the cluster in the regulation of <i>Or59b</i> expression. Our results propose that the Hox/Pou motif regulates the heterochromatin state and allows bHLH proteins to bind the E-box, which induces expression. The E-box and Pou motif sequences overlap to generate unstable binding, and a steady state is generated that drive expression in the Ab2a and Ab7b OSN classes. Cooperative interactions between E-boxes stabilize expression in the face of environmental perturbations. Schematic models of the results are presented in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005051#pgen.1005051.s005" target="_blank">S5</a>–<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005051#pgen.1005051.s006" target="_blank">S6</a> Figs.</p

Cooperative regulation between the cluster and surrounding motifs produces robust class-specific expression.

<p>(A) GFP expression (green) driven by the <i>2×Or59b</i> cluster at 14°C and 24°C. Note the three expression phenotypes produced by the <i>2×Or59b</i> cluster at 14°C. Synaptic neuropil regions are labeled with the presynaptic marker nc82 (magenta). (B) GFP expression (green) produced by the <i>Or85a</i> cluster at 14°C, 24°C or following 3 days of starvation. Note that GFP expression is equally strong in different lines at 14°C. (C) The <i>Or59b</i> cluster with an additional E-box produces robust expression at 14°C and 24°C. (D) The fractions of the brains showing stable or bimodal expression of GFP according to the genotype and temperature. Note that only the <i>Or59b</i> cluster and 2×<i>Or59b</i> cluster are unstable at 14°C. (E) Quantification of GFP positive cells in the antenna. <i>Or59b</i> cluster shows a varied number of GFP positive cells at 14°C compared to 24°C. <i>Or59b</i> and <i>Or59b</i> cluster with an additional E-box show stable expression in both temperatures. Schematic interpretations of the results are presented in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005051#pgen.1005051.s006" target="_blank">S6 Fig.</a></p