Fig. 5

The MEF2 binding site is required for HDAC4-induced defects. A Myc immunohistochemistry on brains expressing the indicated Myc-tagged HDAC4 mutants in the mushroom body. Left panel: frontal projection through the mushroom body. Scale bar = 100 μm. Right panel: optical Sect. (0.5 μm) through the Kenyon cell bodies. Representative images are shown, scale bar = 10 μm. B Quantification of mushroom body phenotypes following expression of HDAC4^WT and mutants with elav-GAL4. The percentage of brains displaying each phenotype is shown. The proportion of brains displaying defects was significantly different between HDAC4^3SA and HDAC4^3SAΔMEF2 (****p < 0.0001); between HDAC4^ΔNLS and HDAC4^ΔNLSΔMEF2 (****p < 0.0001) and between HDAC4^ΔANK and HDAC4^ΔANKΔMEF2 (***p < 0.001), Fisher’s exact test. C Myc (green) and MEF2 (magenta) immunohistochemistry on brains expressing HDAC4^ΔNLS as described in A. Optical Sects. (0.5 μm) through the Kenyon cell bodies are shown. HDAC4^ΔNLS is present in cytoplasmic haloes and in the calyx but MEF2 is only present in nuclei and does not overlap with HDAC4^ΔNLS outside the nucleus. Scale bar = 10 μm. D OK107-GAL4-driven expression of HDAC4^WT and HDAC4^ΔMEF2 was induced in the adult brain with GAL80ts by raising the temperature from 18 to 30 °C at 3 days post-eclosion for 72 h, followed by anti-MEF2 immunohistochemistry. Maximum projections through the Kenyon cell layer are shown. Scale bar = 50 μm. E Quantification of MEF2 intensity, n = 4 brains/genotype (ANOVA, F_(2,9) = 22.61, p = 0.0003, Tukey’s post hoc test, **p < 0.01). F HA-tagged wild-type MEF2 (MEF2^WT) was expressed with elav-GAL4. Frontal projections through the mushroom body of brains expressing MEF2^WT stained with Fas2 are shown in comparison to the control (elav-GAL4 crossed to w[CS10]). Overexpression of MEF2 results in defects in axon elongation and guidance. Scale bar = 50 μm. G Quantification of mushroom body phenotypes following Fas2 staining as shown in F and Table 4. The key is the same as shown in B, ****p < 0.0001, Fisher’s exact test. H–J The indicated HDAC4 and MEF2 transgenes were expressed to approximately half maximal GAL80ts-induced levels by raising flies at 25 °C. H Co-expression of HDAC4^WT and MEF2^WT resulted in a significantly higher proportion of brains with β lobe fusion than HDAC4^WT alone, **p < 0.01, Fisher’s exact test. There was no significant difference between HDAC4^ΔMEF2 and HDAC4^ΔMEF2 co-expressed with MEF2^WT, p = 0.1468, Fisher’s exact test. I When co-expressed with MEF2^WT, HDAC4^WT was more nuclear as observed by the increased colocalization with MEF2 (white), and the reduced cytoplasmic halos (green) that were observed when HDAC4^WT was individually expressed. Scale bar = 20 μm. J Left image: A frontal projection through the mushroom body lobes shows that HDAC4^ΔNES is almost completely restricted to nuclei. The intensity of the image has been increased to show the outline of the brain and location of the mushroom body lobes, which were not visible at the same confocal gain settings used to visualize HDAC4^WT in the lobes. Scale bar = 100 μm. Right images: HDAC4^ΔNES colocalizes in with MEF2 in Kenyon cell nuclei. Scale bar = 20 μm. K Expression of HDAC4^ΔNES resulted in a significantly higher proportion of brains with β lobe fusion than HDAC4^WT, Fisher’s exact test, *p < 0.05