Marketing MIX

It is well known that expression of human tau in the fly eye causes a degenerative „rough eye‟ phenotype when visualised externally with SEM (Wittmann et al., 2001; Shulman and Feany, 2003; Steinhilb et al., 2007a). In order to test the hypothesis that human BRSK2 would interact with and phosphorylate human tau in Drosophila, UAS-0N4R tau and UAS-BRSK2 (B-WT, B-KD or B-NP) were expressed in the eye (Figure 4.5).

This manipulation caused a drastic exacerbation of the tau phenotype, demonstrating for the first time, that human 0N4R tau and BRSK2 are able to interact in-vivo in Drosophila. Further to this, it is reasonable to conclude that there is an endogenous

Drosophila kinase capable of phosphorylating and activating BRSK2. This indicates

that intact signalling cascades, composed of both human and Drosophila genes and proteins can be observed in the fly.

Expression of B-KD or B-NP with 0N4R tau had no effect on the previously observed degenerative tau phenotype, demonstrating that the exacerbation seen upon B-WT co-expression with 0N4R tau was not simply due to expression of exogenous proteins. Further to this, quantification of western blots for BRSK2 expression (shown in Chapter 3, Figure 3.7) demonstrates that the lack of exacerbation in the flies expressing 0N4R tau with B-NP or B-KD was not due to a lack of BRSK2 expression.

Figure 4.6 shows a cumulative distribution plot produced for the flies of the genotypes in Figure 4.5. As in Figure 4.3, expression of B-WT (green line) had little

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or no effect on the ommatidial structure of the eye when compared to GMR (black line). Expression of 0N4R tau caused a degenerative phenotype which can be seen by the shift of the tau curve (red line) further towards 1. However, co-expression of B-WT with 0N4R tau caused a dramatic exacerbation of the 0N4R tau phenotype as can be seen in the large shift to the right of the blue line. This was not the case with B-KD as this line overlies that of the 0N4R tau flies. B-NP appeared to exhibit an intermediate phenotype which was not visually evident from the SEMs.

Figure 4.7 shows the images from the two extremes of the cumulative distribution plot for flies expressing tau, T + B-WT, T + B-NP and T + B-KD; the images with the lowest and highest DCs for each genotype are shown (the images for GMR and B-WT flies were shown in Figure 4.4 and so have not been repeated here). Tau flies had a DC range that was higher than that of the GMR flies (0.31-0.55 and 0.22-0.29 respectively) and correlated with the appearance of a tau-induced degenerative phenotype. Co-expression of B-WT with Tau gave a range of 0.40-0.99. This was visible in the images – the eye with the lowest DC showed signs of B-WT enhancement of Tau, with a smaller eye size and a slightly altered shape. The eye with the highest DC was very degenerated and had almost no ommatidial structure, hence a DC of 0.99. Tau + B-NP and Tau + B-KD flies had ranges of 0.33-0.64 and 0.29-0.72 respectively. These ranges were lower than that of the Tau + B-WT flies and this could be seen in the images; flies of these two genotypes looked more like the Tau flies.

Semi-thin 2.5µm resin sections of eyes from flies of the genotypes shown in Figure 4.5 were produced in order to investigate the underlying cellular morphology of the

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eyes. Figure 4.8 shows that expression of the GMR driver caused no phenotype; hexagonally packed ommatidia are visible, each one containing seven rhabdomeres (R8 is not visible). Expression of B-WT did not alter this structured phenotype, whereas expression of 0N4R tau caused marked disruption to the cellular structure. This was manifested as holes where some cells had died at an early stage of development (arrow), merged ommatidia (*) and an incorrect number of rhabdomeres (arrowhead). This phenotype was exacerbated upon co-expression of B-WT, with very few detectable ommatidia and an obvious increase in disorganisation. The effects caused by B-WT were not visible in the sections from eyes co-expressing 0N4R tau and B-NP or B-KD.

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Figure 4.5: SEM images of flies co-expressing human 0N4R tau with human BRSK2 isoforms.

Expression of 0N4R human tau under the GMR::GAL4 driver caused a degenerative eye phenotype (Tau). This was dramatically enhanced upon co-expression of human B-WT (Tau + B-WT). Enhancement was not seen in eyes expressing NP or KD isoforms of human BRSK2 (Tau + B-NP and Tau + B-KD). Scale bar 100µm applies to all panels.

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Figure 4.6: Cumulative distribution plot of ommatidial distortion measures (DCs) produced by QED software for flies co-expressing human 0N4R tau and BRSK2 isoforms.

Quantitative analysis of flies co-expressing 0N4R tau and human BRSK2 isoforms under the control of the GMR-GAL4 driver showed that expression of B-WT enhanced the 0N4R tau induced ommatidial distortion (T + B-WT). Enhancement was not seen with B-KD (T + B-KD), whereas B-NP produced an intermediate phenotype (T + B-NP). GMR n=11, Tau n=20, B-WT n=21, T + B-WT n=17, T + B- KD n=17 and T + B-NP n=12.

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Figure 4.7: Scanning electron micrographs of flies co-expressing 0N4R tau and BRSK2 isoforms with the lowest and highest DCs.

Tau flies had a DC range that was 0.31-0.55, values which were higher than those of the GMR flies (GMR and B-WT flies were shown in Figure 4.3). Co-expression of B-WT with Tau gave a range of 0.40-0.99 indicating a more severe phenotype. Tau + B-NP and Tau + B-KD flies had ranges of 0.33-0.64 and 0.29-0.72 respectively. These ranges were lower than that of the Tau + B-WT flies – this is clear upon visual examination of the images. DCs are shown on each image. Scale bar 100µm applies to all panels.

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Figure 4.8: Semi-thin 2.5µm resin sections of eyes co-expressing human 0N4R tau with human BRSK2 isoforms.

GMR and B-WT eyes had a very regular sub-cellular structure consisting of hexagonally packed ommatidia, each containing 7 rhabdomeres (R8 was not visible). Expression of 0N4R human tau under the GMR-GAL4 driver caused a degenerative eye phenotype manifested as an incorrect number of rhabdomeres (arrowhead), merged ommatidia (*) and absent cells (arrow). This was enhanced upon co- expression of human B-WT with very little sub-cellular structure visible in the sections. Enhancement was not seen in eyes co-expressing B-NP or B-KD. Scale bar 25µm applies to all panels.

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