Fig. 2

Reduced Ank2 expression disrupts neuronal development. (A-F) Immunohistochemistry with anti-Fas2 on whole mount brains reveals morphological defects of the mushroom body resulting from knockdown of Ank2. Ank2 RNAi1 was driven by elav-GAL4 and Ank2 RNAi2 was driven by the mushroom body driver OK107-GAL4, as elav-GAL4-driven expression was lethal. All images are frontal confocal projections through the mushroom body and examples of the range of phenotypes are shown. Scale bar = 50 μm. (A) Wild-type mushroom body stained with anti-Fas2 to highlight the α, β and γ lobes. (B) Thin α lobe (arrowhead) and a prematurely terminated α lobe (arrow). (C) β lobe fusion (arrow). (D) Missing α lobe (arrow). (E) Both α lobes are missing (arrows). (F) None of the lobes have elongated and axon stalling is observed whereby the axons grow in a ball-like structure. (G-I) Immunohistochemistry on whole mount brains labelled with GFP to detect Lifeact driven by 3A-GAL4 in LPTCs. All images are confocal projections through the optic lobe of the brain. (G) The dendritic arbor of the six neurons comprising the vertical system of LPTCs in a wild-type brain is visualized with anti-GFP. Scale bar = 50 μm. (H) Cartoon trace of the confocal micrograph showing the dendritic branching of each of the vertical system neurons. VS1 to VS6 = vertical system neurons type 1 to 6. The branches of each VS neuron type are labelled in a different color, with the main branch, major projecting branches and branchlets shown. (I) A manual trace of the main branch and major projecting branches was generated using SNT Tracer (Image J) to calculate the total main and major projecting branch length for each brain (n = 18 to 20 per genotype). (J,K) Knockdown of Ank2 (RNAi1) results in defects in branch extension. Arrowhead in J points to a short VS4 main branch and the arrowhead in K points to a short VS5 projecting branch. (L) Total main and major projecting branch length was reduced by knockdown of Ank2 (student’s t-test t₍₃₆₎ = 2.27, p < 0.05)