To evaluate a possible involvement of ppk25 in male response to pheromones, ppk25 expression was tested in pooled adult appendages that are highly enriched for gustatory (legs and wings) and olfactory (third antennal segment) sensory hairs, as well as in body parts that have much fewer chemosensory cells relative to their total mass: heads (without third antennal segment) and bodies (without heads or appendages). mRNA was isolated from all three types of body parts and analyzed by Northern blot using a full-length ppk25 cDNA probe. Remarkably, hybridization is by far the strongest to mRNAs from the appendages fraction, yielding a set of bands between 2.1 and 2.4 kb in size, consistent with the predicted ppk25 transcript. Upon longer exposure of the filter, mRNAs of identical sizes, but much lower abundance, are detected in both head and body fractions. Probing the same filter with CheB42a sequences reveals a much smaller mRNA of ~700 nt that is present only in the appendages fraction (Lin, 2005).

To determine which appendages express ppk25 mRNA, quantitative real-time RT-PCR was used on total RNA extracted from different types of male appendages. This analysis confirms that, in adults, ppk25 mRNA is most abundant in appendages and also shows that ppk25 expression is approximately three times higher in male than female appendages. Interestingly, however, ppk25 mRNA is present at equivalent levels in male legs and wings, appendages that carry many gustatory sensilla, and in the third antennal segment, the main olfactory organ of the fly. Finally, ppk25 mRNA is not detectable in larvae or at early pupal stages (light pupae), but first appears at late pupal stages (dark pupae), and persists for at least 3 days after eclosion, by which time males are sexually mature (Lin, 2005).

Together, these data show that ppk25 expression is highest in olfactory and gustatory appendages of sexually mature males, a distribution consistent with a role in response to female pheromones. In addition, two observations argue that, despite their proximity, CheB42a and ppk25 are indeed two separate genes that are independently transcribed into two separate mRNAs: (1) no evidence was found of any transcript containing both CheB42a and ppk25 sequences; (2) the two mRNAs have related, but not identical, tissue distributions. Both transcripts are present at highest levels in male appendages. However, whereas CheB42a expression is only detectable in male front legs, ppk25 mRNA is expressed equally in male legs and antennae and at lower, but significant levels in female appendages as well as bodies and heads of either sex (Lin, 2005).

Effects of Mutation or Deletion

To test the possible involvement of CheB42a and ppk25 in male response to female pheromones, three homozygous viable deletions were generated in the region by imprecise excision of a P element inserted ~1 kb upstream of CheB42a. All three deletions remove part or all of the CheB42a gene, leading to the complete absence of CheB42a-mRNA and CheB42a protein. In contrast, these three deletions have very different effects on ppk25. Males homozygous for Delta5-68, a deletion removing all sequences between the P insertion site and roughly the middle of the CheB42a gene, have normal or even slightly increased levels of ppk25 mRNA in their appendages. The Delta5-2 deletion completely removes the CheB42a gene and terminates only 59 bp before the ppk25 ATG initiation codon. Although this deletion preserves the predicted ppk25 coding region in its entirety, it significantly impairs transcription of ppk25, such that ppk25 mRNA in male appendages is undetectable by Northern blot. Finally, in addition to deleting all sequences between the P-element insertion site and the midpoint of ppk25, the Delta5-22 deletion retains part of the original transposon, resulting in a series of hybrid transcripts that originate in P-element sequences but retain the 3' half of the normal ppk25 mRNA. Characterization of the corresponding cDNAs indicates that these aberrant transcripts are unlikely to produce any Ppk25-related polypeptide and suggest that Delta5-22 is a null mutant for ppk25 (Lin, 2005).

Is the function of either CheB42a or ppk25 required for male response to female pheromones? When placed in the presence of a female, a Drosophila male quickly initiates a striking series of stereotyped steps that include following the female, tapping her with his front legs, generating a courtship song by vibrating one of his wings, licking her genitalia, attempting copulation, and copulating. Both visual and chemosensory perception of the female stimulate male courtship behavior. Therefore, the response was tested of males carrying deletions in the CheB42a]ppk25 region to females under infrared lights, which Drosophila cannot detect, to enhance the contribution of pheromone detection to male behavior. For each male, a courtship index is calculated, which represents the fraction of the total observation time spent performing any courtship behavior multiplied by 100 (Lin, 2005).

Males homozygous for the Delta5-68 deletion display normal levels of overall courtship. In contrast, males homozygous for either Delta5-2 or Delta5-22 have a much reduced courtship index relative to the G7 controls, suggesting unexpectedly that males require ppk25, but not CheB42a, to achieve normal overall levels of courtship behavior in response to a female. In addition, introduction of a transgenic copy of the genomic region that spans both CheB42a and ppk25 genes rescues the courtship behavior of Delta5-22 homozygous males, whereas an almost identical transgene that lacks ppk25 does not. This result indicates that the courtship deficit of Delta5-22 homozygous males is indeed caused by the loss of ppk25. Importantly, ppk25 is not required for two behaviors unrelated to courtship: walking and preening. In fact, males homozygous for Delta5-22 walk more than controls or those carrying a transgenic ppk25, whereas Delta5-2 homozygous males display normal levels of this behavior. To further test whether Delta5-2 and Delta5-22 cause generalized brain dysfunction, two other complex behavioral responses to sensory stimuli were tested. Neither the typical climbing response of Drosophila to mechanosensory detection of gravity nor stimulation of food intake by gustatory detection of sucrose is affected by any of the deletions in the region. Together, these results suggest that ppk25 is required specifically for male response to females (Lin, 2005).

Analysis of the deletion lines suggests that the ppk25 gene is required for normal male response to females. As a further test of this possibility, males with an independent mutation in ppk25 (Parks, 2004; Thibault, 2004) were tested. In this ppk25 mutant, a transposable element is inserted in the second intron of the ppk25 gene, resulting in what is referred to as the ppk25^PB allele. The presence of 4 kb of extraneous sequences, including a termination site from the miniwhite gene, make it unlikely that this modified ppk25 intron 2 can be spliced properly to produce functional ppk25 mRNA. Instead, transcription from the normal ppk25 promoter can be expected to result in an mRNA that retains exons 1 and 2 followed by part of intron 2, which contains multiple in-frame stop codons. Alternatively, the 5' splice junction of intron 2 may be spliced aberrantly to a cryptic 3' splice site within Piggyback sequences. The protein product of ppk25^PB should therefore be limited to the first transmembrane domain and part of the extracellular domain of Ppk25, perhaps fused to Piggyback sequences. Interestingly, for several other DEG/ENaC genes, similarly truncated or fused proteins that retain the first transmembrane domain have dominant-negative properties likely caused by the formation of nonfunctional complexes with other DEG/ENaC subunits or other interacting proteins (Lin, 2005).

To test the effect of the ppk25^PB allele on male response to females, males that carry the following four mutations were generated: (1) ppk25^PB, (2) CPB a similar Piggyback insertion in an unrelated site on the second chromosome in an otherwise isogenic background to ppk25^PB, (3) Delta42E, a deletion of the ppk25 genomic region spanning ~100 kb and 20 genes, or (4) C^Delta, a deletion of similar size in an unrelated area of the second chromosome. Remarkably, none of the 28 ppk25^PB/Delta42E males that were tested displayed any detectable courtship behavior during the 10-min observation period under infrared lights, a highly significant decrease relative to control males (compare the courtship index for ppk25^PB/Delta42E and CPB/Delta42E males). This result confirms that ppk25 is required for male response to females. In addition, because ppk25^PB homozygous males have normal levels of CheB42a mRNA, the result indicates that the requirement for ppk25 is independent of CheB42a. Finally, the complete loss of male response to females in ppk25^PB/Delta42E males is a significantly more severe phenotype than the reduced courtship observed for Delta5-22 homozygous males, suggesting that ppk25^PB is indeed a dominant-negative allele. This conclusion is validated by the significantly reduced levels of courtship behavior exhibited by males that carry a single copy of ppk25^PB in the presence of a wild-type ppk25 gene compared to males that only carry one wild-type copy of ppk25 (Lin, 2005).

The deficient male response to females observed for ppk25 loss-of-function and dominant-negative alleles under infrared light could be due either to a lack of sensory detection of females or to a more general inability to perform courtship behaviors, regardless of sensory stimulus. To distinguish between these two possibilities, the effect of visible light on the response of ppk25^PB mutant males was tested. The two types of males compared in this experiment carry a single copy of either the wild-type ppk25 gene or the dominant-negative ppk25^PB allele in an otherwise isogenic background that includes the Delta5-22 deletion. Under infrared light and in the absence of any wild-type ppk25, a single copy of the dominant-negative ppk25^PB allele results in the complete loss of male response to females under infrared light, but no decrease in walking or preening. In sharp contrast, in the presence of visible light, males of the same genotype perform all of the normal steps of courtship, albeit at a significantly reduced rate. This result suggests that the complete inability of males carrying the dominant-negative ppk25^PB allele to respond to females under infrared lights is due to a lack of sensory input rather than an inability to perform courtship behaviors (Lin, 2005).

Furthermore, this experiment provides an indirect test of whether the dominant-negative ppk25^PB mutation blocks pheromone perception through olfaction, gustation, or both chemical senses. Both visual and olfactory inputs can initiate courtship behavior. In contrast, gustatory perception of pheromones may only be required for efficient performance of subsequent steps. Because the lag to initiation of courtship behavior and the number of courtship bouts per second displayed by ppk25^PB/Delta5-22 males are similar to controls in the presence of visible light, the lack of response of the same males under infrared lights likely results at least in part from their inability to initiate courtship in response to pheromones as would be expected for an olfactory defect. In contrast, despite the presence of visible lights, the average length of a courtship bout for mutant males is less than half that of controls, suggesting that the ppk25^PB mutation also affects a subsequent step, perhaps gustatory detection of pheromones. ppk25^PB's dominant negative properties in the absence of wild-type ppk25 are most likely due to interactions between the truncated protein and other factors involved in pheromone perception. However, the decreased levels of courtship behavior displayed by males homozygous for the Delta5-2 or Delta5-22 deletions also result from a combination of increased lags to courtship, decreased numbers of bouts initiated per seconds, and shorter bout lengths. Together, these results suggest that ppk25 itself is required for both initiation and maintenance of courtship bouts in response to female pheromones (Lin, 2005).

Reference names in red indicate recommended papers.

Bray, S. and Amrein, H. (2003). A putative Drosophila pheromone receptor expressed in male-specific taste neurons is required for efficient courtship. Neuron 39: 1019-1029. 12971900

Lin, H., Mann, K. J., Starostina, E., Kinser, R. D. and Pikielny, C. W. (2005). A Drosophila DEG/ENaC channel subunit is required for male response to female pheromones. Proc. Natl. Acad. Sci. 102(36): 12831-6. 16129837

Lin, W., Finger, T. E., Rossier, B. C. and Kinnamon, S. C. (1999). Epithelial Na+ channel subunits in rat taste cells: localization and regulation by aldosterone. J. Comp. Neurol. 405: 406-420. 10076935

Lin, W., Ogura, T. & Kinnamon, S. C. (2002). Acid-activated cation currents in rat vallate taste receptor cells. J. Neurophysiol. 88: 133-141. 12091539

Liu, L., Leonard, A. S., Motto, D. G., Feller, M. A., Price, M. P., Johnson, W. A. and Welsh, M. J. (2003). Contribution of Drosophila DEG/ENaC genes to salt taste. Neuron 39: 133-146. 12848938

Mano, I. and Driscoll, M. (1999). DEG/ENaC channels: a touchy superfamily that watches its salt. Bioessays 21(7): 568-78. 10472184

O'Hagan, R., Chalfie, M., Goodman, M. B. (2005). The MEC-4 DEG/ENaC channel of Caenorhabditis elegans touch receptor neurons transduces mechanical signals. Nat. Neurosci. 8(1): 43-50. 15580270

Parks, A. L., et al. (2004). Systematic generation of high-resolution deletion coverage of the Drosophila melanogaster genome. Nat. Genet. 36: 288-292. 14981519

Thibault, S. T., et al. (2004). A complementary transposon tool kit for Drosophila melanogaster using P and piggyBac. Nat. Genet. 36: 283-287. 14981521

Xu, A., Park, S. K., D'Mello, S., Kim, E., Wang, Q. and Pikielny, C. W. (2002). Novel genes expressed in subsets of chemosensory sensilla on the front legs of male Drosophila melanogaster. Cell Tissue Res. 307: 381-392. 11904775

date revised: 12 November 2005

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