ABSTRACT
MEF2 (myocyte enhancer factor 2) transcription factors are found in the brain and muscle of insects and vertebrates and are essential for the differentiation of multiple cell types. We show that in the fruitfly Drosophila, MEF2 is essential for normal development of wing veins, and for mushroom body formation in the brain. In embryos mutant for D-mef2, there was a striking reduction in the number of mushroom body neurons and their axon bundles were not detectable. D-MEF2 expression coincided with the formation of embryonic mushroom bodies and, in larvae, expression onset was confirmed to be in post-mitotic neurons. With a D-mef2 point mutation that disrupts nuclear localization, we find that D-MEF2 is restricted to a subset of Kenyon cells that project to the α/β, and γ axonal lobes of the mushroom bodies, but not to those forming the α’/β’ lobes. Our findings that ancestral mef2 is specifically important in dopamine-receptive neurons has broad implications for its function in mammalian neurocircuits.
INTRODUCTION
Cellular identity is established during development by mechanisms that can be maintained for an animal’s lifetime and are conserved across evolution (Arendt, 2008; Arlotta & Hobert, 2015). Gene duplications can lead to functional variations among family members, thereby driving increased cell-type diversity (Arendt, 2008) and evolutionary pressure to maintain replicates (Assis & Bachtrog, 2013). To understand the most basic functions of a gene family it is expedient to evaluate the founding member. The MEF2 family of transcription factors has been assigned a myriad functions ranging from the differentiation of multiple cell lineages during development, to cellular stress response regulation and neuronal plasticity in adulthood. D-mef2 is the single Drosophila homolog of the vertebrate mef2a, b, c and d family and as such can provide insight to conserved functions of this family.
Drosophila mef2 and vertebrate mef2 members exhibit considerable diversity in their transcriptional activation domains, but over 80% identity in the N-terminal sequences that encode the dimerization and DNA binding MEF and MADS domains (named for the evolutionarily conserved founding members MCM1, AGAMOUS, DEFICIENS, SRF) (Molkentin et al., 1996; Potthoff & Olson, 2007). Correspondingly, the DNA sequences bound by MEF2 are evolutionarily conserved and MEF2 has been shown to activate transcription of orthologous gene sets in muscle, brain and immune systems of both flies and mice (Bour et al., 1995; Lilly et al., 1995; Ranganayakulu et al., 1995; Lin et al., 1997; Potthoff & Olson, 2007). The tissue specificity of MEF2’s actions is strongly influenced by the expression pattern of cofactors. Depending on which transcription factors MEF2 interacts with, immortalized cells in culture can be induced to display variable cell phenotypes: MEF2 and myogenin activate each other’s expression to initiate differentiation into skeletal muscle, MEF2 and Nkx2-5 activate each other’s expression to induce cardiac muscle formation, and MEF2 and MASH-1 activate each other’s expression to yield a neuronal phenotype (Skerjanc et al., 1998; Ridgeway et al., 2000; Skerjanc & Wilton, 2000). The specification and maintenance of cellular identity comprise major functions for MEF2. In both flies and mice, MEF2 is critical for the differentiation of multiple muscle cell lineages (Lilly et al., 1995; Lin et al., 1997; Potthoff & Olson, 2007). However, in mammalian neurons, a complex array of functions have been found for mef2 family members in both development and neuroplasticity (Mao et al., 1999; Okamoto et al., 2000; Okamoto et al., 2002; Flavell et al., 2006; Shalizi et al., 2006; Li et al., 2008; Ryan et al., 2013; Okamoto et al., 2014; Chen et al., 2016). The existence of different MEF2 family members within individual cells must be coordinated, and can even be antagonistic (Desjardins & Naya, 2017). Studies of a single ortholog, like the one in Drosophila, might serve to simplify this complexity and enhance our understanding of MEF2 by elucidating its most conserved functions.
D-mef2 is expressed in Drosophila Kenyon neurons that make up the mushroom bodies (Schulz et al., 1996), brain structures known for their functions in learning and memory (for review see Busto et al., 2010 and Cognigni et al., 2017). Mushroom bodies (MBs) are comprised of bilateral clusters of cell bodies that extend single neurites anteriorly to form a dendritic calyx and a fasciculated peduncle that branches to form axonal lobes. In adult Drosophila, each of the three major axon projection patterns, the branched α/β and α’/β’ lobes, or the γ axonal lobe (Crittenden et al., 1998; Tanaka et al., 2008) are formed from four neuroblasts that divide throughout embryonic, larval and pupal development (Lee et al., 1999). The axonal lobes are segregated into domains according to their interconnections with distinct types of dopaminergic neurons and cholinergic mushroom body output neurons (Aso et al., 2014). Numerous genes required for normal olfactory learning are preferentially expressed in the MBs, often in subsets of axonal lobes that likely reflects their distinct functions (McGuire et al., 2001; Yu et al., 2006; Krashes et al., 2007; Akalal et al., 2010; DasGupta et al., 2014; Lim et al., 2017). The role of D-mef2 in MB development and function remains untested. Here, we examine the expression of D-MEF2 in developing mushroom bodies and among subsets of Kenyon cells in the adult fly, and evaluate mushroom body formation and overt phenotypes in D-mef2 mutant alleles.
MATERIALS AND METHODS
Drosophila genetics
Fly stocks were raised at room temperature on standard sucrose and cornmeal media. The nine enhancer detector lines described were generated in a screen for mushroom body expression (Han et al., 1996). The EMS, DEB, and γ ray mutants shown in Table 1 were identified in a screen for lethal genes at the cytological location 46C-F (Goldstein et al., 2001). The parental chromosome for these lines was adh cn pr and they were maintained balanced over CyO.
Molecular biology
Bacteriophage clones surrounding the enhancer detector insertion site in line 2487 were isolated from a Canton-S genomic library. The map constructed of the 46C region was expanded by 12 kb from coordinate 20 kb to 32 kb (Fig. 1) relative to the previously published maps (Bour et al., 1995; Lilly et al., 1995). The expansion was due to a stretch of repetitive DNA suggesting the likely insertion of a transposable element. Genomic DNA fragments adjacent to the insertions in lines 429, 883, 919, 2487, 3046, and 3775 were obtained by Hind III or Xhol plasmid rescue, according to previously described methods (Pirrotta, 1986). The insertion sites in lines 1484, 1828, and 2109 were determined by Southern blotting experiments.
Histology
β–galactosidase histochemistry and RNA in situ hybridization experiments were performed on frontal cryosections of the Drosophila head as previously described (Skoulakis & Davis, 1996). RNA probes were generated from the 5’ and the 3’ end of a D-mef2 cDNA and used in separate experiments to validate results. Immunohistochemistry with chromogenic substrates was performed on paraffin and plastic sections of adult, larval, and embryonic Drosophila as previously described (Crittenden et al., 1998). For immunofluorescence, FITC-or CY3-conjugated anti-rabbit and anti-mouse antibodies (Sigma) were used. Slides were mounted in Vectashield (Vector Laboratories). Antisera were raised against a fusion protein that contained both the MADS box and MEF domain of D-MEF2 and were provided by Drs. H. Nguyen and E. Olson.
Cell counting experiments
We counted MB cells in embryos after immunofluorescent staining. Mushroom body cells were apparent in approximately 15 - 1 μm serial sections of each brain hemisphere. Each cell was visible in an average of 3.5 serial sections. Therefore, to estimate the number of MB cells per brain hemisphere, we divided the total number of cells counted by 3.5. Statistical comparisons between homozygous and heterozygous control embryos were made using unpaired, 2-tailed Student’s t-tests.
Cell death
Acridine orange staining was performed as previously described (Abrams et al., 1993). Homozygous D-mef2 mutants were distinguished from sibling controls based on the absence of somatic muscles and the presence of bloated gut morphology.
BrdU labeling
The treatment of larvae with BrdU to label dividing cells followed the protocol of Truman and Bate (Truman & Bate, 1988). For immunohistochemical detection of BrdU, paraffin sections of larvae were additionally treated with 2N HCl. For injections of embryos with BrdU, embryos were collected from timed egg lays on grape juice agar plates at 25 0C. They were dechorionated according to standard procedures, aligned on double-sided tape, and covered with halocarbon oil. The posterior ends of the animals were then injected with 10 mg/ml of BrdU in insect Ringer’s solution (182 mM KCl, 46 mM NaCl, 3 mM CaCl2, 10 mM Tris-HCl, pH 7.2). The embryos were allowed to develop for three hours under halocarbon oil at 25 0C and then fixed for 20 minutes in 50% Carnoy’s fixative in heptane with shaking. Finally, the embryos were sectioned in paraffin and prepared for immunohistochemistry following the protocol for larvae.
RESULTS
Enhancer detector lines identify distal D-mef2 regulatory regions
From approximately 100 enhancer detector lines that were selected for mushroom body expression of the β–galactosidase reporter (Han et al., 1996), we identified nine with insertions in cytological region 46C3 (Fig. 1). We mapped the transposon insertion sites by isolating plasmid rescue clones and using restriction mapping and DNA hybridization to compare to the 46C locus map (O’Brien et al., 1994; Bour et al., 1995; Lilly et al., 1995). All nine lines harbored reporter gene insertions within a 3.5 kb region (Fig. 1) that is approximately 35 kb (including repetitive sequences) upstream of D-mef2.
The group of insertions more proximal to D-mef2 (Fig. 1) presented preferential β–galactosidase activity in the MB and antennal lobes (Fig. 2). Additional expression throughout the cortex of the central brain and the optic lobes was exhibited by more distal insertions (Fig. 2). We compared the reporter β–galactosidase expression pattern to that of D-mef2 mRNA and D-MEF2 protein in adult brain sections and found concordant enrichment in Kenyon cells and antennal lobe neurons (Fig. 3 A – F). These data suggest that reporter expression in the 46C enhancer detector lines is under the control of D-mef2 MB and antennal lobe enhancers. A genomic fragment that is 17 kb proximal to the enhancer detector elements (Fig. 1) has been reported to possess MB enhancer activity (Schulz et al., 1996). However, the deficiency Df(2R)P544, which was derived from enhancer detector line 2487 and lacks DNA sequence between D-mef2 and the 2487 insertion site (Fig. 1), retained preferential β–galactosidase expression in the MB (not shown), suggesting that there are at least two mushroom body enhancer sequences at 46C.
D-mef2 is required for normal wing development
Additional evidence that the enhancer detector lines are inserted within D-mef2 regulatory sequences came from our discovery of a D-mef2 wing phenotype. Enhancer detector line 919 showed strong expression and complete penetrance of disrupted wing morphology ranging from incomplete or ectopic cross-veins to bubbled wings (Fig. 4 A, B). A similar phenotype, at lower penetrance and expressivity, was observed in lines with insertions clustered more proximally to D-mef2. The wing phenotype was not observed in lines with P-element insertions more distal to D-mef2.
We observed a similar wing veination phenotype (Fig. 4 C) in flies with mutations that were determined to disrupt D-mef2 function based on lack of complementation for viability with the deficiencies Df(2R)X1 and Df(2R)P520 (Bour et al., 1995; Goldstein et al., 2001). Six of these lines were generated by EMS (D-mef222-21, D-mef222-24, D-mef225-34, Dmef226-6, D-mef226-7, and D-mef226-49), three by DEB (D-mef230-5, D-mef244-5, and D-mef248-7), and two by γ ray mutagenesis (D-mef266-65 and D-mef278-11). By inter se complementation tests for viability (Table 1), we found that some alleles were strong (0% viability in combination), some medium (1 – 40% viability in any combination), and others weak (> 40% viability in any combination). In transheterozygous escapers, we frequently observed ectopic wing veination (e. g. Fig. 4 C).
To confirm that D-mef2 dysfunction is responsible for the wing phenotype in the enhancer detector lines, we performed complementation tests with the null allele D-mef222-21, which harbors a nonsense mutation in the 6th codon of D-mef2 (Bour et al., 1995). We observed wing blistering or abnormal veination in 74% of the transheterozygotes with line 919 (Fig. 4 D) and in 58% of transheterozygotes with line 429. Heterozygosity for P element insertion or D-mef222-21 alone did not cause a wing phenotype. Our results suggest that the D-mef2-proximal P-element insertions disrupt a wing enhancer, and establish a role for this protein in wing development.
D-MEF2 is expressed in mushroom body neurons that send axonal projections into the α/β and γ lobes
We noticed that several clusters of mushroom body neurons lacked D-MEF2 immunoreactivity as determined by double-labeling (not shown) of wild-type animals with anti-LEONARDO (LEO), an immunomarker that exhibits global mushroom body expression (Skoulakis & Davis, 1996). D-MEF2-positive Kenyon cell subtypes were identified according to their axonal projection patterns, by evaluation of D-mef226-49 mutants that we discovered to harbor cytoplasmic D-MEF2. In D-mef226-49 mutants, anti-D-MEF2 decorated the axons of the α/β and γ lobe-projecting neurons but was absent from the α’/β’ lobes (Fig. 5 A – D). Although line D-mef226-49 was almost completely embryonic lethal, the few adult homozygous escapers (e. g. Fig. 5 A – D) showed grossly normal morphology and D-MEF2-immunoreactivity.
In horizontal brain sections from heterozygous D-mef226-49 mutants, D-MEF2 immunoreactivity was apparent in all four bundles of the posterior peduncle (Fig. 6 A, B), each of which is formed from the progeny of a single mushroom body neuroblast (Lee et al., 1999). Thus, D-mef2 is expressed in the descendants of all four mushroom body neuroblasts, but only those that project axons into the α/β branched lobes and into the γ lobes.
In the antennal lobe of D-mef226-49 flies, cytoplasmic D-MEF2 appeared restricted to the glomeruli and was not observed in projections of antennal lobe neurons or the antennal glomerular tract (Fig. 6 B), consistent with D-MEF2 expression in antennal interneurons. In the mutants, cytoplasmic D-MEF2 immunoreactivity was also detected in branches of the antennal nerve that extend into the antenno-mechanosensory center and into the antennal lobe (not shown), neurons that arise from the 2nd and 3rd antennal segments, respectively (Power, 1946). Correspondingly, nuclei within both antennal segments exhibited wildtype D-MEF2 immunoreactivity, a pattern also shown by the β-galactosidase expression in the enhancer detector lines (not shown). Other cells with D-MEF2 immunoreactivity in the head included muscles, photoreceptor cells, most cells of the lamina, and cells distributed throughout the medulla, lobula, and lobula plate.
D-MEF2 is expressed in subsets of embryonic and larval mushroom body neurons
To explore the onset of D-mef2 expression in the mushroom bodies, we surveyed expression from early stages of development. At embryonic stage 15, D-MEF2 was detectable in one or two cells in the dorso-posterior brain where mushroom body cells reside (Fig. 7 A). By stage 17, the number of brain cells expressing D-MEF2 in the mushroom body region had grown (Fig. 7 B), which is consistent with expression in a neuronal subtype that is dividing in late embryogenesis. Cell division appeared to be mostly restricted to the mushroom body region in late stage embryogenesis as evidenced by BrdU incorporation (Fig. 8). Moreover, in D-mef226-49 embryos, which display cytoplasmic D-MEF2 immunoreactivity, there was neuropil labeling in the central brain that resembled the mushroom body peduncle and dorsally oriented lobe (Fig. 7C). Doublelabeling experiments with antibodies against D-MEF2 and against the Kenyon cell markers DACHSHUND (DAC) (Kurusu et al., 2000; Martini & Davis, 2005) and against EYELESS (Kurusu et al., 2000; Noveen et al., 2000; Kunz et al., 2012) showed only a partial overlap with D-MEF2 (not shown). We concluded that D-MEF2 is expressed in a subset of newly born Kenyon cells, from stage 15 to stage 17 of embryogenesis.
At the first instar larval stage, D-MEF2 expression was confirmed to be in the post-mitotic Kenyon cells but not in the neuroblasts or ganglion mother precursor cells (Fig. 9 A, B). Weak D-MEF2 expression was also visible in cells surrounding, but not within, the single dividing neuroblast in the anterior brain (Fig. 9 A) that is known to give rise to a variety of antennal lobe cell types (Ito & Hotta, 1992; Stocker et al., 1997; Lai et al., 2008). In short, D-MEF2 was found in post-mitotic Kenyon cells and antennal lobe cells, but not in neuroblasts or ganglion mother cells of the developing brain.
D-mef2 is required for embryonic mushroom body formation
Considering that D-mef2 was expressed in embryonic mushroom bodies, we tested for mushroom body malformation in severe D-mef2 mutants that die as late stage embryos. We examined two different homozygous lethal lines as embryos, the null mutant D-mef222-21, and D-mef226-6, which carries a point mutation that disrupts the DNA binding domain of the protein, but retains its expression (Nguyen et al., 2002). Although the heterozygous control and homozygous mutant embryos formed cuticle at the same time, gut distension was a prominent D-mef2 mutant phenotype (Ranganayakulu et al., 1995) in the homozygotes. Homozygotes were further distinguished by the absence of muscle immunolabeling for D-MEF2 in line D-mef222-21 and myosin heavy chain in line D-mef226-6 (Bour et al., 1995; Lilly et al., 1995).
We used the mushroom body immunomarker DAC to count mushroom body neurons in consecutive sagittal sections through stage 16 homozygous and heterozygous D-mef222-21 animals (Fig. 10 A). Anti-DAC immunoreactivity was observed in an estimated average of 63 cells per dorso-posterior brain hemisphere in the heterozygotes, compared to only 36 cells per hemisphere in the null homozygotes (Fig. 10 B), representing a 43% reduction in the number of DAC-positive mushroom body neurons. This loss was not consequent of failed neuroblast formation, as in the process of cell counting we observed four neuroblasts in each hemisphere of the D-mef222-21 homozygotes, and we also confirmed that these neuroblasts do incorporate BrdU upon injection 19 hours after egg laying (not shown).
We made similar cell counts in stage 16 homozygous and heterozygous embryos from line D-mef226-6. The control heterozygotes had an average of 80 DAC positive mushroom body neurons, whereas the homozygous mutants had an average of 68 (Fig. 10 B), representing a 15% reduction. We also counted the number of D-MEF2-positive neurons in this mutant. An average of 37 cells were counted per dorso-posterior hemisphere in the controls, whereas only 7 were found on average in the homozygous mutants (Fig. 10 B), an 81% reduction. Double-labeling experiments in controls showed that DAC was expressed in a greater proportion of mushroom body neurons than D-MEF2 (not shown). Therefore, a more substantial effect on D-MEF2-expressing Kenyon cells than on DAC-expressing neurons would be expected if the phenotype is cell autonomous as suggested by our observations. The difference in the number of DAC positive cells between the D-mef222-21 and D-mef226-6 heterozygous animals is likely to be due to a slight difference in the ages of the animals between experiments; however, since the heterozygous and homozygous animals within each genotype were aged and collected together, our primary findings were not compromised.
The inability of D-mef2 mutants to undergo complete mushroom body formation was further indicated by their loss of mushroom body neuropil. We assessed neuropil immunolabeling with the embryonic mushroom body markers FAS2 and the protein kinase A subunit DCO (Skoulakis et al., 1993; Crittenden et al., 1998; Cheng et al., 2001). In stage 17 heterozygous D-mef222-21 embryos, the immunostained peduncle and lobes (Fig. 11 A, C, E) appeared similar to what we had observed with these and other markers previously (Crittenden et al., 1998). In contrast, neither anti-FAS2 nor anti-DCO labeled mushroom body structures in any sections from homozygous D-mef222-21 embryos processed on the same slides as controls (Fig. 11 B, D, F). It is unlikely that the failure to see immunostaining is due to a role for D-MEF2 in regulating the expression of both of these markers because ectopic D-MEF2 expression using the GAL4/UAS system with 5 different drivers failed to reveal ectopic expression of DCO or FAS2 (not shown). Furthermore, no reduction in DCO or FAS2 expression was evident in the brains of adult hypomorphic D-mef2 mutants (not shown). In summary, we found that severe hypomorphic and null D-mef2 mutants have reduced numbers of differentiated mushroom body neurons and a failure of mushroom body formation in embryogenesis.
DISCUSSION
D-mef2 functions in wing veination
P element insertions upstream of D-mef2 led to our discovery of a wing veination function for D-mef2. The 46C enhancer detector lines did not show overt myogenesis or mushroom body development problems but did show ectopic wing veination or bubbling that is non-complementary with D-mef2 mutations and that appears identical to what we found in viable transheterozygous D-mef2 point mutants. Overexpression of D-mef2 was found, in a large-scale screen of transcription factors, to induce wing blistering (Schertel et al., 2015) but it was not investigated further. Screens for wing veination phenotypes have identified over 300 genes with enrichment for members of the Notch, EGFR and Dpp (TGF-β homolog) signaling pathways, considered essential for intercellular communication (Molnar et al., 2006; Bilousov et al., 2014). MEF2 can be linked to the regulation of these pathways – for example, Tkv (thick veins), which encodes a Dpp receptor, is repressed by D-MEF2 during Drosophila egg formation (Mantrova et al., 1999). Indeed, disruptions in Dpp and tkv expression can result in anterior cross-vein and blistering phenotypes that are similar to what we found in D-mef2 hypomorphs (de Celis, 1997). Another member of the Dpp-Tkv pathway is p38 mitogen-activated protein kinase, which phosphorylates and activates mammalian MEF2 (Mao et al., 1999) and in its dominant negative form causes ectopic wing veination in flies (Adachi-Yamada et al., 1999). Collectively with our results, these data suggest that the abnormal vein formation in hypomorphic D-mef2 mutants is caused by a failure in the Dpp-Tkv pathway.
Nuclear retention signal for D-MEF2
Mammalian MEF2 contains several sequences near the C-terminus that are required for its nuclear localization, but these sequences are not conserved in Drosophila and the D-MEF2 nuclear localization sequence has not been identified (Yu, 1996; Borghi et al., 2001). We identified a mutant, D-mef226-49, in which D-MEF2 fails to be retained in the nucleus. The mutation in line D-mef226-49 was previously described as a missense point mutation that converts amino acid 148 from Thr to Ala (Lovato et al., 2009). From a BLAST® comparison to mouse MEF2 it appeared that this Thr is conserved in MEF2A but not in other MEF2 family members. This region of the protein is evolutionarily conserved and is termed the HJURP-C domain (Holliday junction regulator protein family C-terminal repeat). The HJURP-C domain is present in MEF2A, MEF2C and MEF2D but is lacking in MEF2B. The function of the HJURP-C domain is poorly understood but our results suggest that it is required for nuclear localization of D-MEF2.
Mushroom body expression pattern of D-mef2
Previous reports have shown that mushroom body neurons begin to differentiate at stage 14 and continue to be born until shortly before pupal eclosion (Ito & Hotta, 1992). Our embryonic expression studies indicated that D-MEF2 becomes detectable in the mushroom body neurons as early as stage 15. In the embryo and larva, D-MEF2 immunoreactivity was in post-mitotic Kenyon cells and antennal lobe neurons, but not in neuroblasts or ganglion mother cells, consistent with the developmental expression profile in the honeybee Apis mellifera (Farris et al., 1999). Likewise in mammals the initiation of mef2 expression in cortical neurons coincides with their exit from the cell cycle (Lyons et al., 1995; Mao et al., 1999). Thus, the expression profile of mef2 is consistent with a role in specification of neuronal cell identity in both insects and mammals during development.
Mushroom body neurons that give rise to the different lobes are generated sequentially from the four dorsal posterior neuroblasts and are interdependent for pathfinding and survival (Kurusu et al., 2002; Martini & Davis, 2005). In adults, we found D-MEF2 expression in all four tracts of the posterior peduncle, indicating D-MEF2 expression in descendants of all four mushroom body neuroblasts. However, based on double-labeling experiments with other Kenyon cell markers, D-MEF2 is expressed in only a subset of mushroom body neurons in the embryonic and adult stages. The cytoplasmic mislocalization of D-MEF2 in line D-mef226-49 served to show that D-MEF2 is expressed at high levels in Kenyon cells that form medially and vertically-extending lobes in the embryo and in adult, α/β- and γ-lobe forming neurons, but not in the α’/β’ neurons. Mutant cytoplasmic D-MEF2 showed that the antennal lobe expression appeared to be confined to interneurons whereas projection neurons were found in the antennal segments that house olfactory receptors, hygroreceptors, thermoreceptors and the sound-sensing Johnston’s organ (Stocker, 1994). These structures are serially linked in the pathway for odor perception (Power, 1946): odor detection occurs in olfactory neurons of the third antennal segment, which synapse onto projection neurons in the antennal lobe glomeruli that in turn send sensory information to the mushroom body calyces. Mammalian MEF2 expression is maintained into adulthood and is important for neuronal plasticity (Flavell et al., 2006; Sivachenko et al., 2013; Chen et al., 2016; Chen et al., 2017). Together with the D-MEF2 expression pattern that we found, these data suggest that D-MEF2 is important for plasticity differences between the mushroom body lobes (Yu et al., 2006) in olfactory learning.
MEF2 interacts physically with myogenic and neurogenic factors to potentiate cell-type specific gene transcription (Molkentin et al., 1995; Black et al., 1996; Mao & Nadal-Ginard, 1996). The D-MEF2 mushroom body lobe expression pattern expression gives clues to possible transcriptional interactors for D-MEF2. Examples of mushroom body markers with similar Kenyon cell subtype distribution to D-MEF2 include FOXP (DasGupta et al., 2014), HDAC4 (Fitzsimons et al., 2013), DRK (Crittenden et al., 1998; Kotoula et al., 2017), and FAS2 (Crittenden et al., 1998; Cheng et al., 2001). MEF2 interactions with several of these molecules have already been established. In mammals, HDAC4 (histone deacetylase 4) is known to bind to MEF2 to repress transcription and Drosophila HDAC4 is important for muscle development, circadian rhythmicity and mushroom body function (Zhao et al., 2005; Fogg et al., 2014). An interaction between D-MEF2 and FAS2 (the fly ortholog of NCAM) in cell-cell communication or adhesion is suggested by our finding that D-mef2 hypomorphs exhibit an ectopic veination phenotype similar to that reported for fas2 loss of function mutant clones (Mao & Freeman, 2009). This is supported by experiments on clock neurons where FAS2 and D-MEF2 work together to control their circadian fasciculation and defasciculation that in turn regulate motor output (Blanchard et al., 2010; Sivachenko et al., 2013). A function for D-MEF2 in defasciculation raises a possible parallel to MEF2’s role in synapse elimination in cultured mouse neurons (Flavell et al., 2006). FOXP (forkhead box transcription factors) proteins are also known to function in synapse elimination. Mammalian FOXP2 has been shown to co-localize with MEF2C early in development but to subsequently suppress MEF2C expression in the striatum (Chen et al., 2016), a dopamine rich forebrain region that is important for motor learning and that has cellular organization that been directly compared to the mushroom bodies (Strausfeld & Herth, 2013; Crittenden & Graybiel, 2017). These results are consistent with distinct cellular functions for MEF2 in development, and later in learning. Disruption of FOXP in the α/β mushroom body neurons results in motor problems and delayed decision-making in an associative olfactory-discrimination task (DasGupta et al., 2014; Lawton et al., 2014) but whether this involves D-MEF2 remains untested.
D-mef2 function in mushroom body formation
Deletion of murine mef2 family members impairs normal development of lymphocytes, bone, endothelial cells, photoreceptor cells and neurons (Mao et al., 1999; Potthoff & Olson, 2007; Andzelm et al., 2015; Latchney et al., 2015). MEF2 activity is required for the survival of primary cerebellar granule neurons that are differentiating in culture, as well as P19 embryonal carcinoma cells that have been induced to undergo neurogenesis (Mao & Nadal-Ginard, 1996; Okamoto et al., 2000). The survival of both cell types is dependent upon p38 mitogen-activated protein kinase, which phosphorylates and thereby activates MEF2 (Mao & Nadal-Ginard, 1996; Han et al., 1997; Okamoto et al., 2000). We have now shown that D-mef2 is essential for the differentiation of mushroom body neurons.
Constitutive loss of D-mef2 led to a complete or nearly complete failure in mushroom body formation in all of the homozygous D-mef2 mutant embryos that we examined. In the homozygous lethal line D-mef226-6, there was a 15% reduction in DAC-positive mushroom body cells and an 81% reduction of D-MEF2 positive mushroom body neurons. Thus, the hypomorphic mutation in D-mef2 had a more profound impact on D-MEF2-positive Kenyon cells than on the surrounding D-MEF2-negative/DAC-positive Kenyon cells. We could not detect any mushroom body neuropil in the D-mef2 null embryos with the immunomarkers anti-DCO and anti-FAS2, indicating either that the remaining DAC-positive Kenyon cells failed to extend processes or that they were too sparse to detect. Modifiers of the phenotype are suggested by the fact that escaper transheterozygous flies showed grossly normal mushroom body morphology as adults. FAS2 mutations were found to disrupt mushroom body development in one study but not in another (Cheng et al., 2001; Kurusu et al., 2002), further highlighting such phenotypic variability in mushroom body development.
We considered three possible explanations for the reduced mushroom body cell number in D-mef2 mutants. First, the mushroom body neurons may die prematurely. Second, the mushroom body neuroblasts may fail to proliferate normally. Third, the neurons may not differentiate properly, owing either to a fate change or to a block in the differentiation program. To test whether the primary cause of reduced mushroom body cell numbers in D-mef2 mutants was cell death, we employed the vital dye acridine orange. Acridine orange was applied to homozygous D-mef226-6 animals at stages of 14, 15, and 16, periods preceding and including the time at which mutants exhibited a clear reduction in the number of mushroom body neurons. At stage 14, a tight cluster of cells in the dorso-posterior brain was stained with acridine orange in both heterozygous and homozygous D-mef226-6 animals. By stage 15 and 16 this staining had subsided however, leaving fewer labeled cells that were scattered throughout the CNS (not shown). Although we observed acridine orange staining in the muscle cells of D-mef226-6 homozygous embryos as previously reported (Ranganayakulu et al., 1995), we did not detect an increase in cell death within the brains of the mutants compared to controls. Therefore, we did not find evidence of apoptotic cell death in the mushroom body neurons of D-mef2 mutants. Nor was the phenotype caused by the failure of neuroblasts to form: all four mushroom body neuroblasts were apparent at stage 17 in D-mef222-21 animals as determined by counting experiments. Moreover, the neuroblasts did not express D-mef2 and did incorporate BrdU, although we cannot rule out that BrdU incorporation was slowed. In conclusion, we propose that the reduction in the number of mushroom body neurons in D-mef2 mutants may best be explained by a failure of these cells to form or differentiate properly, which is consistent with the absence of mushroom body processes and the neuronal markers FAS2 and DCO.
ACKNOWLEDGEMENTS
We thank S. Ahmed and B. Schroeder for technical assistance. We thank Profs. Olson, Nguyen and Lilly for providing anti-D-MEF2 antisera. We are grateful to Prof. K.-A. Han for critical reading of the manuscript. This work was supported by a predoctoral NIMH grant to J. R. C. and NINDS grant 1R35NS097224 to R. L. D.