Table 1.
GFP expression patterns observed in ‘olfactory positive’ pGT lines.
Figure 1.
GFP expression patterns of pGT lines in olfactory organs.
Cryostatic antennal sections were double stained with α-GFP (green) and α-ELAV (red) and then the images overlain. The GFP reporter used is mcd8:GFP which localises to membranes. For most lines expression is neuronal as indicated by co-localization of GFP and ELAV. Note that when expressed in olfactory neurons mcd8:GFP is localised to membranes of cell bodies encircling ELAV staining of the cell nucleus, and also extends into dendrites (sensillum shafts) and axons. In BG00842, BG01140 and BG02759 α-GFP labels larger cells at the base of sensilla while α-ELAV labels the neuronal nuclei located more deeply, indicating the expression of GFP is in accessory cells. In BG02184 expression is seen in both neurons and accessory cells. Examples of cells showing GFP fluorescence but negative for ELAV are indicated with arrows. These sections are representative of 10–20 examined for each line.
Table 2.
Molecular analysis of pGT lines and predicted candidate genes.
Figure 2.
Olfactory Trap Response Index of pGT lines.
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.
Figure 3.
Geotaxis Response Index is normal in pGT lines.
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.
Figure 4.
Precise excision of the pGT element in BG00076 and BG00973 restores olfactory behaviour.
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.
Figure 5.
The pGT insert in BG00076 decreases Skip expression level.
Quantitative real-time PCR analysis of Skip expression in whole adult flies. The amount of Skip mRNA from CS5 and BG00076 flies was normalized to cyclin K and is indicated in arbitrary units. Values are shown as the mean ± the SEM and are averaged from four separate biological experiments (each replicated in quadruplicate). There is an approximately five fold decrease of Skip transcription in line BG00076. *** p<0.001 t-test.