A Screen for Genes Expressed in the Olfactory Organs of Drosophila melanogaster Identifies Genes Involved in Olfactory Behaviour

BACKGROUND: For insects the sense of smell and associated olfactory-driven behaviours are essential for survival. Insects detect odorants with families of olfactory receptor proteins that are very different to those of mammals, and there are likely to be other unique genes and genetic pathways involved in the function and development of the insect olfactory system. METHODOLOGY/PRINCIPAL FINDINGS: We have performed a genetic screen of a set of 505 Drosophila melanogaster gene trap insertion lines to identify novel genes expressed in the adult olfactory organs. We identified 16 lines with expression in the olfactory organs, many of which exhibited expression of the trapped genes in olfactory receptor neurons. Phenotypic analysis showed that six of the lines have decreased olfactory responses in a behavioural assay, and for one of these we showed that precise excision of the P element reverts the phenotype to wild type, confirming a role for the trapped gene in olfaction. To confirm the identity of the genes trapped in the lines we performed molecular analysis of some of the insertion sites. While for many lines the reported insertion sites were correct, we also demonstrated that for a number of lines the reported location of the element was incorrect, and in three lines there were in fact two pGT element insertions. CONCLUSIONS/SIGNIFICANCE: We identified 16 new genes expressed in the Drosophila olfactory organs, the majority in neurons, and for several of the gene trap lines demonstrated a defect in olfactory-driven behaviour. Further characterisation of these genes and their roles in olfactory system function and development will increase our understanding of how the insect olfactory system has evolved to perform the same essential function to that of mammals, but using very different molecular genetic mechanisms

RBF1 promotes chromatin condensation through a conserved interaction with the Condensin II protein dCAP-D3

The Drosophila retinoblastoma family of proteins (RBF1 and RBF2) and their mammalian homologs (pRB, p130, and p107) are best known for their regulation of the G1/S transition via the repression of E2F-dependent transcription. However, RB family members also possess additional functions. Here, we report that rbf1 mutant larvae have extensive defects in chromatin condensation during mitosis. We describe a novel interaction between RBF1 and dCAP-D3, a non-SMC component of the Condensin II complex that links RBF1 to the regulation of chromosome structure. RBF1 physically interacts with dCAP-D3, RBF1 and dCAP-D3 partially colocalize on polytene chromosomes, and RBF1 is required for efficient association of dCAP-D3 with chromatin. dCap-D3 mutants also exhibit chromatin condensation defects, and mutant alleles of dCap-D3 suppress cellular and developmental phenotypes induced by the overexpression of RBF1. Interestingly, this interaction is conserved between flies and humans. The re-expression of pRB into a pRB-deficient human tumor cell line promotes chromatin association of hCAP-D3 in a manner that depends on the LXCXE-binding cleft of pRB. These results uncover an unexpected link between pRB/RBF1 and chromatin condensation, providing a mechanism by which the functional inactivation of RB family members in human tumor cells may contribute to genome instability

Geminin and Brahma act antagonistically to regulate EGFR-Ras-MAPK signaling in Drosophila

Geminin was identified in Xenopus as a dual function protein involved in the regulation of DNA replication and neural differentiation. In Xenopus, Geminin acts to antagonize the Brahma (Brm) chromatin-remodeling protein, Brg1, during neural differentiation. Here, we investigate the interaction of Geminin with the Brm complex during Drosophila development. We demonstrate that Drosophila Geminin (Gem) interacts antagonistically with the Brm–BAP complex during wing development. Moreover, we show in vivo during wing development and biochemically that Brm acts to promote EGFR–Ras–MAPK signaling, as indicated by its effects on pERK levels, while Gem opposes this. Furthermore, gem and brm alleles modulate the wing phenotype of a Raf gain-of-function mutant and the eye phenotype of a EGFR gain-of-function mutant. Western analysis revealed that Gem over-expression in a background compromised for Brm function reduces Mek (MAPKK/Sor) protein levels, consistent with the decrease in ERK activation observed. Taken together, our results show that Gem and Brm act antagonistically to modulate the EGFR–Ras–MAPK signaling pathway, by affecting Mek levels during Drosophila development

Olfactory Trap Response Index of pGT lines.

<p>Defects in olfactory behaviour were tested using an olfactory trap assay. Only seven pGT lines could be tested reproducibly for olfactory trap behaviour because of high mortality rates. The response index (RI) of flies entering traps was recorded at 20-hour intervals over 60 hours and the average at 60 hours is shown. A. Females. B. Males. The pGT lines are represented in numerical order. The error bars represent SEM; n = 10 for all lines. * p<0.05 t-test.</p

GFP expression patterns observed in ‘olfactory positive’ pGT lines.

<p><i>Note</i>. Mouth - Mouthparts including proboscis, labellum and/or cibarial organs. Leg - Tips/distal parts or joints of legs. Wing - Wing margin or joints of wings. Brain - Majority of lines had staining in the mushroom bodies or uniformly in the brain, some lines also had staining in the optic lobes.</p>a<p>These lines showed inconsistent Elav co-localisation patterns and expression is also possibly in accessory cells.</p

Geotaxis Response Index is normal in pGT lines.

<p>Negative geotactic ability was tested to investigate CNS and locomotor function of the lines that exhibited abnormal olfactory behaviour. All lines tested showed negative geotactic behaviour to at least control levels, with one line (BG01746) showing a small increase (* ANOVA, t-test, p<0.007). The pGT lines are represented in numerical order. The error bars represent SEM; n = 5–19.</p

Molecular analysis of pGT lines and predicted candidate genes.

<p><i>Note</i>.</p>a<p>Number of pGT inserts identified by Southern blot analysis.</p>b<p>BDGP prediction as taken from Flybase.</p>c<p>Cytological location of BDGP predicted gene.</p>d<p>Candidate gene identified from 3′RACE experiments.</p>e<p>Cytological location of gene identified by RACE.</p>f<p>Cytological location as determined by polytene chromosome <i>in situ</i> hybridisation with a <i>Gal4</i> probe.</p>g<p>As the Southern blot indicates two inserts there may be a second unidentified candidate gene for this line.</p>h<p>For BG02836 a Southern blot suggested one insert but polytene chromosome <i>in situ</i> hybridisation gave two signals, one at ∼18D1 and one at ∼67E thus there may be two inserts. N.D. – not determined.</p

Precise excision of the pGT element in BG00076 and BG00973 restores olfactory behaviour.

<p>Comparison of response indices of wild type flies (wt), pGT insertion mutants (BG00076 or BG00973) and two precise excision lines for each (ex1, ex2). Asterisks for excision lines indicate significantly higher responses than pGT mutants (ANOVA, t-test, p<0.01). (A–B) For BG00076 mutant responses are rescued in both ex1 and ex2 in females (A) and males (B). (C–D) For BG00973 mutant responses are rescued in both ex1 and ex2 in females (C) but only in ex1 in males (D). The error bars represent SEM; n = 10 for all lines.</p

Torso-like functions independently of Torso to regulate Drosophila growth and developmental timing

Activation of the Drosophila receptor tyrosine kinase Torso (Tor) only at the termini of the embryo is achieved by the localized expression of the maternal gene Torso-like (Tsl). Tor has a second function in the prothoracic gland as the receptor for prothoracicotropic hormone (PTTH) that initiates metamorphosis. Consistent with the function of Tor in this tissue, Tsl also localizes to the prothoracic gland and influences developmental timing. Despite these commonalities, in our studies of Tsl we unexpectedly found that tsl and tor have opposing effects on body size; tsl null mutants are smaller than normal, rather than larger as would be expected if the PTTH/Tor pathway was disrupted. We further found that whereas both genes regulate developmental timing, tsl does so independently of tor. Although tsl null mutants exhibit a similar length delay in time to pupariation to tor mutants, in tsl:tor double mutants this delay is strikingly enhanced. Thus, loss of tsl is additive rather than epistatic to loss of tor. We also find that phenotypes generated by ectopic PTTH expression are independent of tsl. Finally, we show that a modified form of tsl that can rescue developmental timing cannot rescue terminal patterning, indicating that Tsl can function via distinct mechanisms in different contexts. We conclude that Tsl is not just a specialized cue for Torso signaling but also acts independently of PTTH/Tor in the control of body size and the timing of developmental progression. These data highlight surprisingly diverse developmental functions for this sole Drosophila member of the perforin-like superfamily