mef2 mRNA and protein are enriched in adult mushroom body and antennal lobe neurons. Frontal sections through the adult brain showing the corresponding localization of β–galactosidase activity, the mef2 transcript, and MEF2 protein. Arrows in panels A,C and E designate the MB cells. Arrows in B,D and F designate cells dorsal and lateral to the antennal lobe glomeruli. (A,B) β–galactosidase activity in cryosections from line 2487. (C,D) Immunohistochemistry showing MEF2 protein distribution in paraffin-embedded sections. (E,F) RNA in situ hybridization on cryosections showing mef2 transcript distribution. More than five flies were used for each experiment.

MEF2 is expressed in mushroom body neurons that project to the α, β and γ lobes, but not the α’ and β’ lobes. (A) A frontal paraffin-embedded section through the central brain of a wild-type animal with an illustrative drawing over the lobes on one side. MB lobes were identified by gross anatomy and immunomarkers. LEO and FASII were detected by rabbit and mouse primary antisera, respectively, which were visualized with corresponding secondary antibodies coupled to red fluorophore (for LEO) or green fluorophore (for FASII). Regions with co-expression of LEO and FASII appear yellow. LEO is present in all five lobes and FASII is in the α/β lobe branches but not the α’/β’ lobes. The spur (s) and the posterior tips of the γ lobes are defined by light FASII immunoreactivity. (B–D) In the mef2^26-49 line (homozygote shown here), frontal paraffin-embedded brain sections from (B) posterior to (D) anterior are co-immunolabeled for cytoplasmic MEF2 (green) and LEO (red). Co-labeling is apparent in the α/β and γ lobes (yellow) whereas the α’/β’ lobes are not co-labeled for MEF2. More than five flies were found to have a similar pattern of expression.

MEF2 is expressed in Kenyon cell descendants from all four mushroom body neuroblasts. (A) A cartoon of the adult MB in a sagittal plane, with anterior to the right. The black horizontal line represents the approximate plane of the section shown in B. (B) A near-horizontal section from a heterozygous mef2^26-49 adult shows immunoreactivity (brown) in all four axon bundles of the posterior pedunculus. In the mirrored image, the four axon bundles arising from the Kenyon cells are numbered and the antennal lobe (AL), fan-shaped body (FB), ellipsoid body (EB), and MB lobes (β and γ) are outlined. More than five flies were confirmed to have similar results.

MEF2 is expressed in embryonic mushroom bodies. (A) A horizontal section through a stage 15 wild-type embryo embedded in plastic and immunostained for MEF2 (alkaline phosphatase-coupled secondary antibody, blue) and FASII (horseradish peroxidase-coupled secondary antibody, brown). MEF2 expression is abundant in somatic and visceral muscle cell nuclei and is also visible in bilaterally symmetrical cells of the dorso-posterior brain where MB neurons are localized (arrow and magnified in inset). FASII labels axon tracts throughout the developing nervous system whereas MEF2 is localized to cell nuclei. (B) A wholemount of the central nervous system dissected from a late stage 17 wild-type embryo and immunolabeled for MEF2 and FASII (both detected with horseradish peroxidase-coupled secondary antibody substrate, brown). MEF2 and FASII immunoreactivity is distinguished by the respective localization to nuclei and axons. MEF2 expression is highly enriched in the MB nuclei (arrow). The brain is slightly turned so that both hemispheres are equally visible. (C) A sagittal paraffin section through a late stage 17 embryo that is heterozygous for the mef2^26-49 mutation in which MEF2 is mislocalized to the cytoplasm. MEF2 immunoreactivity is apparent in the pedunculus and vertical lobe (arrow) and MB nuclei (arrows in dorso-posterior brain). Anterior is to the left in A–C. At least three embryos showed similar results for each experiment.

MEF2 is expressed in mushroom body neurons, but not their neuroblast or ganglion mother cell precursors. (A) A sagittal paraffin-embedded section through the brain of a first instar larva fed BrdU immediately after hatching and then immunolabeled for BrdU (brown) and MEF2 (blue); anterior is to the left. Anti-BrdU labels the nuclei of MB neuroblasts (MB Nb) and antennal lobe neuroblast (AL Nb) and their daughter cells, including a putative ganglion mother cell (GMC, arrowhead). Highly specific anti-MEF2 labeling is apparent in cell nuclei surrounding the MB neuroblast, and more weakly staining cell nuclei are visible near the antennal lobe neuroblast (left-most arrow). (B) A first instar larval brain section immunolabeled only for MEF2 shows the absence of MEF2 in neuroblasts and a putative ganglion mother cell. At least three larvae were evaluated for each experiment.

mef2 mutant embryos have a paucity of mushroom body neurons. Sections from stage 17 embryos that are (A–C) heterozygous balanced mef2^22-21 controls or (D–F) homozygous mef2^22-21 mutants. Section orientations are (A,D) sagittal through the entire central nervous system of paraffin-embedded tissue, (B,E) horizontal through the brain of paraffin-embedded tissue and (C,F) sagittal through the brain of plastic-embedded tissue, all with anterior to the left. (A,B,D,E) Anti-DC0 decorates the central nervous system neuropil in both genotypes (green, with highest intensity false-colored in red) but the MB lobes are visible only in controls (vertically-extending lobe at arrow in A and medially-extending lobe at arrow in B). Anti-MEF2 (also in green) labels only the cell nuclei of muscles and MB neurons in controls, not the MB axonal lobes, and was included for genotyping purposes. (C,F) Anti-FASII labels the cervical connectives the MB pedunculus, and vertical MB lobe of controls (arrow in C) but not homozygous mutants (F). (G) Example of a sagittal paraffin-embedded section through a mef2^22-21 embryo immunostained for DAC (black) that was used to count MB neurons located in the dorso-posterior brain (magnified in inset). The MB neuroblast is not labeled for DAC (arrowhead). (H) Counts of MB neurons that were immunolabeled for DAC or MEF2. mef2^22-21 and mef2^26-6 homozygous mutant embryos had significantly fewer MB neurons than their age-matched heterozygous balancer-chromosome controls (*P<0.05 for each pair-wise comparison by Student's unpaired two-tailed t-test). The number of brain hemispheres evaluated is indicated on each column. Error bars show standard errors of the mean.