Fig. 3

HDAC4^WT and mutants disrupt mushroom body development. A Immunohistochemistry with anti-Fas2 on whole mount brains expressing HDAC4^WT with elav-GAL4. Brains displaying representative phenotypes are shown. Scale bar = 75 μm. α, β and γ lobes of the mushroom body are labeled in white in a control brain in which elav-GAL4 was crossed to w(CS10) (top left image). Thin lobes are indicated with arrowheads, β lobe fusion is indicated with arrows and missing lobes are indicated with asterisks. B–D Quantification of mushroom body phenotypes. Key shown in B applies to all four graphs. The percentage of brains displaying each phenotype is shown. B Quantification of phenotypes from brains expressing each of the mutants. To assist interpretation of the results, the cartoon above the graph indicates the relative approximate level of expression of the HDAC4^WT transgene by the intensity of the green shading. The proportion of brains displaying defects was significantly different between HDAC4^WT and HDAC4^ΔMEF2, ****p < 0.00001, and HDAC4^WT and HDAC4^ΔNLS, *p < 0.05, Fishers exact test. MB = mushroom body. C tubP-GAL80ts; OK107-GAL4 females were crossed to males carrying each UAS-HDAC4 transgene and to the w(CS10) control and raised at 30 °C for expression in the mushroom body of progeny throughout development. The proportion of brains displaying defects was significantly different between HDAC4^WT and HDAC4^ΔMEF2, ****p < 0.0001, Fishers exact test. D Flies carrying HDAC4::EGFP; tubP-GAL80ts; OK107-GAL4 and each UAS-HDAC4 transgene or the w(CS10) control were raised at 27 °C for expression in the mushroom body throughout development in a WT (left graph) or HDAC4-depleted (right graph, + UAS-deGradFP) background. Light green in the cartoon indicates HDAC4::EGFP expression throughout the brain. Left graph. In the absence of deGradFP, there was no difference in the percentage of brains displaying defects between HDAC4^WT and any of the mutants except for HDAC4^ΔMEF2, ***p < 0.001, Fisher’s exact test. Right graph. When endogenous HDAC4 was depleted with deGradFP and replaced with HDAC4^WT or mutants, there was no significant difference between HDAC4 knockdown + HDAC4^WT, and HDAC4 knockdown alone (w[CS10] + deGradFP), indicating the overall level of HDAC4 expression was close to wild-type. However, in the deGradFP background, expression of HDAC4^3SA (***p < 0.001) and HDAC4^ΔANK (*p < 0.05) resulted in a significantly increased proportion of brains displaying defects compared to HDAC4^WT, Fisher’s exact test