Figure 3. Tau and Shot are required for the maintenance of synaptic markers in cultured neurons and the ageing adult fly brain.

(A) Primary neurons at 3 DIV and 26 DIV cultured from embryos that were wildtype or jointly expressing UAS-tau^RNAi and UAS-shot^RNAi in all neurons driven by the pan-neuronal driver elav-Gal4 (tau^RNAi shot^RNAi). Neurons are stained with anti-tubulin and anti-Syt; at 26 DIV, tau^RNAi shot^RNAineurons display a reduction in the number of Syt puncta when compared to wildtype. (B) Quantification of the experiments in A, shown as the number of Syt puncta per neuron at 3 DIV, 18 DIV and 26 DIV, normalised to wildtype controls (the number of assessed neurons is indicated in each bar; ***P_MW<0.001; ns, not significant P_MW>0.05). (C) A region of Drosophila adult brains including the medulla (delimited by dashed lines) where Syt-GFP is expressed in dorsal cluster neurons using atonal-Gal4, in the absence (control) or together with tau^RNAi and shot^RNAi (tau^RNAi shot^RNAi). Brains are stained with anti-GFP at 2–5 days (young) and 24–29 days (old) after eclosion. Note that GFP-labelled synapses (arrowheads) are decreased in old brains upon shot and tau knock-down. (D) Quantification of the experiments in C, showing the normalised number of Syt-GFP-labelled puncta in old specimen per mean number of puncta in young specimens for the following phenotypes: ato-Gal4 UAS-syt-GFP alone (control), co-expressing UAS-tauRNAi (tau^RNAi), UAS-shot^RNAi(shot^RNAi), or both knock-down constructs (tau^RNAi shot^RNAi; the number of analysed brains is indicated in each bar, ***P_MW<0.001; ns, not significant P_MW>0.05). (E) Brain regions as in C, of animals expressing the membrane marker myr-tdTomato driven by ato-Gal4 revealing the morphology of the projections of dorsal cluster neurons within the medulla ; brains were from adults at 2–5 days (young) and 24–29 days (old) after eclosure, expressing myr-tdTomato either alone (control) or together with tau^RNAi and shot^RNAi(tau^RNAi shot^RNAi). (F) Quantification of the experiments in E, displayed as number of branches per axon projecting into the medulla (the number of axons analysed is indicated in each bar; ns, not significant P_MW>0.05). Scale bar: 10 µm in A and 40 µm in C and E. A statistics summary of the data shown here is available in Figure 3—source data 1.

DOI: http://dx.doi.org/10.7554/eLife.14694.011

Figure 3—figure supplement 1. Delayed effect of RNAi mediated knock-down of Shot and Tau.

(A) Primary neurons at 3 DIV and 25 DIV cultured from embryos that were wildtype or jointly expressing UAS-tau^RNAi and UAS-shot^RNAiin all neurons driven by the pan-neuronal driver elav-Gal4 (tau^RNAi shot^RNAi). Neurons are stained with antibodies against Tau, Shot and Tubulin (red, green and blue respectively); images on the right show: a selected axon segment taken from the main image (top) followed by grey scale images of the separated channels for Tau (2^nd from top), Shot (3^rd from top) and Tubulin (bottom). At 25 DIV, tau^RNAi shot^RNAineurons display a reduction in both Tau and Shot when compared to wildtype. (B) Quantification of the experiments in A, shown as mean intensity of Tau or Shot signal per neuron at 3 DIV and 25 DIV, normalised to wildtype controls (30–39 neurons were assessed per genotype; ***P_MW<0.001; **P_MW<0.01; ns, not significant P_MW>0.05). Comparative data for shot³ and tau^MR22 homozygous mutant neurons are given as control, indicating low Tau background staining and incomplete knock-down of Tau at 3 DIV, but high Shot background suggesting strong or complete Shot knock-down at 25 DIV. A statistics summary of the data shown here is available in Figure 3—figure supplement 1—source data 1.

DOI: http://dx.doi.org/10.7554/eLife.14694.013

Figure 3—figure supplement 2. Schematic drawings of brain areas analysed in this study.

Dorso-fronto-lateral view onto a schematic adult CNS composed of the brain and ventral nerve cord (vNC). Beige areas indicate some synaptic areas, in particular the ventral nerve cord neuropile (Np) and the optic lobes (OL) composed of lamina (1), medulla (2), lobula (3) and lobula plate (4). DCN neurons project to the optic lobe of the contralateral brain half where they branch out in a layered fashion. The blue stippled frame and image inset embox the area (rotated 90 degrees counterclockwise) shown in Figures 3E and 8E without and with synaptic markers (yellow dots), respectively.

DOI: http://dx.doi.org/10.7554/eLife.14694.015

Figure 3—figure supplement 3. Loss of function mutations in shot and tau induce morphological changes.

(A–D) Representative examples of the shape of primary neurons at 2 DIV obtained from embryos that were wildtype (wt, A), shot³ tau^MR22 (shot^-/- tau^-/-B), shot^-/- tau^-/- with elav-Gal4 driven expression of UAS-unc-104 (shot^-/- tau^-/- UAS-unc-104, C) and shot³ tau^MR22 wnd² (shot^-/- tau^-/- wnd^-/- D). (E–F) Quantification of morphological parameters of experiment in (A–D), including the length of axons (E) and the number of branches (F), (the assessed numbers of neurons are indicated in each bar, ***P_MW<0.001; ns, not significant P_MW>0.05). Note that wnd² (wnd^-/-) restored the number of branches in shot-tau mutant neurons, suggesting that JNK not only mediates synapse regulation but also morphogenetic processes downstream of the Shot-Tau deficiency. A statistics summary of the data shown here is available in Figure 3—figure supplement 3—source data 1.

DOI: http://dx.doi.org/10.7554/eLife.14694.016

Figure 3—figure supplement 4. RNAi-mediated knock-down of Shot and Tau has no effect on axonal length and branch number.

(A–B) Representative examples of the shape of primary neurons at 3, 18 and 26 DIV obtained from embryos that were wildtype (wt, A) or jointly expressing UAS-tau^RNAi and UAS-shot^RNAi in all neurons driven by the pan-neuronal driver elav-Gal4 (tau^RNAi shot^RNAi, B). (C–D) Quantification of morphological parameters of experiment in (A–B), including the length of axons (C) and the number of branches (D), (the assessed numbers of neurons are indicated in each bar, ns, not significant P_MW>0.05). A statistics summary of the data shown here is available in Figure 3—figure supplement 4—source data 1.

DOI: http://dx.doi.org/10.7554/eLife.14694.017