The Drosophila Gene CheB42a Is a Novel Modifier of Deg/ENaC Channel Function

Degenerin/epithelial Na+ channels (DEG/ENaC) represent a diverse family of voltage-insensitive cation channels whose functions include Na+ transport across epithelia, mechanosensation, nociception, salt sensing, modification of neurotransmission, and detecting the neurotransmitter FMRFamide. We previously showed that the Drosophila melanogaster Deg/ENaC gene lounge lizard (llz) is co-transcribed in an operon-like locus with another gene of unknown function, CheB42a. Because operons often encode proteins in the same biochemical or physiological pathway, we hypothesized that CHEB42A and LLZ might function together. Consistent with this hypothesis, we found both genes expressed in cells previously implicated in sensory functions during male courtship. Furthermore, when coexpressed, LLZ coprecipitated with CHEB42A, suggesting that the two proteins form a complex. Although LLZ expressed either alone or with CHEB42A did not generate ion channel currents, CHEB42A increased current amplitude of another DEG/ENaC protein whose ligand (protons) is known, acid-sensing ion channel 1a (ASIC1a). We also found that CHEB42A was cleaved to generate a secreted protein, suggesting that CHEB42A may play an important role in the extracellular space. These data suggest that CHEB42A is a modulatory subunit for sensory-related Deg/ENaC signaling. These results are consistent with operon-like transcription of CheB42a and llz and explain the similar contributions of these genes to courtship behavior

ppk23-Dependent Chemosensory Functions Contribute to Courtship Behavior in Drosophila melanogaster

Insects utilize diverse families of ion channels to respond to environmental cues and control mating, feeding, and the response to threats. Although degenerin/epithelial sodium channels (DEG/ENaC) represent one of the largest families of ion channels in Drosophila melanogaster, the physiological functions of these proteins are still poorly understood. We found that the DEG/ENaC channel ppk23 is expressed in a subpopulation of sexually dimorphic gustatory-like chemosensory bristles that are distinct from those expressing feeding-related gustatory receptors. Disrupting ppk23 or inhibiting activity of ppk23-expressing neurons did not alter gustatory responses. Instead, blocking ppk23-positive neurons or mutating the ppk23 gene delayed the initiation and reduced the intensity of male courtship. Furthermore, mutations in ppk23 altered the behavioral response of males to the female-specific aphrodisiac pheromone 7(Z), 11(Z)-Heptacosadiene. Together, these data indicate that ppk23 and the cells expressing it play an important role in the peripheral sensory system that determines sexual behavior in Drosophila

Feminization of pheromone-sensing neurons affects mating decisions in Drosophila males

Summary The response of individual animals to mating signals depends on the sexual identity of the individual and the genetics of the mating targets, which represent the mating social context (social environment). However, how social signals are sensed and integrated during mating decisions remains a mystery. One of the models for understanding mating behaviors in molecular and cellular terms is the male courtship ritual in the fruit fly (Drosophila melanogaster). We have recently shown that a subset of gustatory receptor neurons (GRNs) that are enriched in the male appendages and express the ion channel ppk23 play a major role in the initiation and maintenance of male courtship via the perception of cuticular contact pheromones, and are likely to represent the main chemosensory pathway that influences mating decisions by males. Here we show that genetic feminization of ppk23-expressing GRNs in male flies resulted in a significant increase in male–male sexual attraction without an apparent impact on sexual attraction to females. Furthermore, we show that this increase in male–male sexual attraction is sensory specific, which can be modulated by variable social contexts. Finally, we show that feminization of ppk23-expressing sensory neurons lead to major transcriptional shifts, which may explain the altered interpretation of the social environment by feminized males. Together, these data indicate that the sexual cellular identity of pheromone sensing GRNs plays a major role in how individual flies interpret their social environment in the context of mating decisions

<i>ppk23</i> expression is reduced in appendages of the <i>Poxn</i> mutant.

<p>(A) Real-time quantitative RT-PCR analysis of total RNA extracted from adult appendages (legs and wings) from a mixed sex population. Analysis compared <i>Poxn</i>^M22-B5, which do not have external chemosensory bristles, and <i>CyO</i> balanced siblings, which develop normal sensory bristles <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002587#pgen.1002587-Boll1" target="_blank">[54]</a>. For illustrative purposes, data are represented as relative expression fold differences in <i>Poxn</i> flies relative to controls <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002587#pgen.1002587-BenShahar3" target="_blank">[51]</a>. Each data point includes the relative expression of a gene in <i>Poxn</i> homozygous flies relative to balanced <i>Poxn/CyO</i> flies, which develop normal sensory system. <i>CheB42a</i> gene was used as positive controls for chemosensory specific genes expressed in appendages <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002587#pgen.1002587-BenShahar1" target="_blank">[16]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002587#pgen.1002587-BenShahar2" target="_blank">[17]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002587#pgen.1002587-Xu1" target="_blank">[55]</a>. Statistical analyses were performed on the ΔCt data as previously described <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002587#pgen.1002587-BenShahar1" target="_blank">[16]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002587#pgen.1002587-BenShahar2" target="_blank">[17]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002587#pgen.1002587-BenShahar3" target="_blank">[51]</a>. *, <i>p</i><0.05; **, <i>p</i><0.01 (n = 4 per genotype, one-tail paired <i>t</i>-test) (B) Northern blot analysis of <i>ppk23</i> spatial expression patterns. Lower panel shows ribosomal bands on the RNA gel, indicating equal sample loading. Using RT-PCR coupled with 5′ and 3′ RACE analyses on RNA extracted from male appendages, we were able to identify only a single <i>ppk23</i> transcript (black arrow; <i>ppk23</i>-RX; see also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002587#pgen.1002587.s001" target="_blank">Figure S1A</a>). Numbers on right represent RNA size markers (bp). (C) <i>ppk23</i> promoter activity in forelegs of male and female flies (reporter was nuclear GFP). Scale bar represents 50 µm. (D) Quantitative data from (C) showing significantly fewer <i>ppk23</i>-positive cells in the female foreleg (Independent sample <i>t</i>-test; *, p<0.05; n = 10 for each bar).</p

<i>ppk23</i> is expressed in adult chemosensory receptor neurons.

<p>(A) <i>ppk23-GAL4 x UAS-CD8::GFP</i> flies show expression in chemosensory neurons in male forelegs. Left panel is a z-stack of confocal GFP images. Right panel is a merge of the left panel with differential interference contrast (DIC) image. Solid arrows point to the base of bract-less chemosensory bristles. Note the sensory cilia projecting to the base of the bristle. Open arrowhead points to a bract associated with a mechanosensory bristle. Scale bar represent 20 µm. (B) <i>ppk23-GAL4 x UAS-CD8::GFP</i> flies show expression in sensory neurons projecting to chemosensory bristles in wings. Images are as in (A) and were obtained from the same animal. Solid arrowheads point to the base of chemosensory bristles. The open arrowhead points to the line of pure mechanosensory bristles found at the most outer rim of the wing. Scale bar represents 20 µm.</p

<i>ppk23</i>-expressing cells are required for male courtship behavior.

<p>(A) Expression of tetanus toxin light chain (TNT) in <i>ppk23</i> cells resulted in a high proportion of non-courting males over a 10 min observation time (<i>Chi-square test</i>, <i>p<0.001</i>). Flies expressing TNT in <i>ppk23</i> cells showed increased courtship latency (B; <i>Kruskal-Wallis rank sum test</i>, <i>p<0.001</i>) and reduced courtship index relative to control crosses (<i>ppk23</i>>UAS-IMP-TNT) or parental strains (<i>ppk23-Gal4</i>, <i>UAS-TNT-E</i> and <i>UAS-IMP-TNT-V1A</i> [inactive TNT]) (C; <i>Kruskal-Wallis rank sum test</i>, <i>p<0.001</i>). n = 29–36 males per each genotype. Error bars denote the standard error of the means. Letters above bars represent the significantly different groups after <i>post hoc</i> analyses.</p

The behavioral response to the aphrodisiac pheromone 7,11-HD is reduced in <i>ppk23^PB</i> flies.

<p>(A–B) Larger percent of wild type CS flies exhibited courtship behavior in response to male or female targets laced with 125 ng 7,11-HD (<i>Chi-squared test</i>; only significant contrasts are shown; *, <i>p<0.05</i>; <i>**, p<0,01</i>; ***, <i>p</i><0.001, n = 20–30 for each genotype per treatment). (C–D) Courtship latency was not affected by the <i>ppk23</i> mutation in flies that did court pheromone-laced dummies. (E–F) Significant effects of <i>ppk23</i> mutation on courtship index were observed in males that courted pheromone-laced dummies (<i>Two samples t-test</i> or <i>two samples wilcoxon test</i>, *, <i>p<0.05</i>; <i>**, p<0,01</i>; ***, <i>p</i><0.001, n = 20–30 for each genotype per treatment).</p

Axonal projections of <i>ppk23</i>-expressing cells are sexually dimorphic.

<p>(A) and (B) The axonal projections of <i>ppk23</i>-positive cells are sexually dimorphic. Left panel is a male (A) and right panel is a female (B). Panels represent brain (top) and thoracic ganglion (bottom) confocal z-stacks that were dissected from a single animal. Note the lack of midline crossings by foreleg <i>ppk23</i>-positive axons in females (white arrows). Scale bars represent 100 µm. Identical dimorphic axonal projection patterns were observed in at least five individuals per sex. (C) <i>ppk23</i> expression co-localizes with <i>fru</i> expression in the forelegs of males. Red marked <i>ppk23</i>-expressing cells and green marked the <i>fru</i> positive cells. The majority of <i>ppk23</i>-expressing cells also show <i>fru</i> expression. Scale bars represent 20 µm. Genotype imaged: <i>w; ppk23-GAL4>UAS-RFP/lexAop-rCD2::GFP; fruP1.LexA/+</i>.</p

<i>ppk23</i> is required for normal male courtship behavior.

<p>(A)Significantly higher proportion of <i>ppk23^PB</i> males did not show any courtship behavior in a 10-min observation time under dark (“red light”) conditions towards females (<i>Chi-square test</i>; ***, <i>p<0.001</i>). (B) <i>ppk23^PB</i> males that did court showed increased courtship latency only towards females (<i>Two samples wilcoxon test</i>; *, <i>p<0.05</i>) and (C) reduced courtship index (<i>Two samples wilcoxon test</i>; ***, <i>p<0.001</i>). (D) Under white light conditions, proportion of courting <i>ppk23^PB</i> males was not different than wild types. <i>Chi-square test</i>, <i>N.S.</i>). (E) No courtship latency differences were detected either (<i>Two samples wilcoxon test</i>, <i>N.S.</i>). (F) In contrast, <i>ppk23^PB</i> males exhibited reduced courtship index even under white light conditions (<i>Two samples wilcoxon test</i>; ***, <i>p<0.001</i>). There were no effects of the mutation on any of the measured courtship parameters towards males under white light conditions. (G–I) <i>piggyBac</i> excision rescued the effect of <i>ppk23^PB</i> mutation on male courtship behaviors in all measured parameters under red light conditions (G; Chi-square test, <i>p</i><0.001; H; <i>Kruskal-Wallis rank sum test, p<0.05; I; Kruskal-Wallis rank sum test, p<0.05</i>). n = 24–38 for each genotype and each condition. <i>N.S.</i> indicates no significant difference. Error bars denote the standard errors of the means.</p