Genes analyzed in this study. (A) We analyze three components of the BRM complex in this study. In this schematic representation, Drosophila genes are labeled in blue and their human homologs are labeled in black. Shapes, sizes, and locations have no significance. Additional complex components are not shown. (B) We analyze three components of the TIP60 complex in this study. Symbols are as in (A). (C) Bap55^-/- in this study denotes the LL05955 allele, which contains a piggyBac insertion in the coding sequence (CDS) [22]. (D) brm^-/- denotes the brm² allele, an ethylmethanesulfonate (EMS) mutant that is a protein null [27]. (E) Snr1^-/- denotes the Snr1^6C allele, which is a deletion removing much of the Snr1 gene and extending into the next gene, HDAC3. HDAC3 mutants have been previously shown to have no phenotype in PNs [8]. Yet in this study we also expressed UAS-HDAC3-3xFLAG in the Snr1^6C mutant PNs to account for any phenotypes due to HDAC3 deletion. We additionally analyzed the 01319 allele, a P-element insertion in the Snr1 coding sequence, which gave the same phenotypes as the Snr1^6C allele (data not shown). (F) dom^-/- denotes the LL05537 allele, which contains a piggyBac insertion in an intron upstream of the translational start. The piggyBac element contains splice acceptor sites and stop codons in all three coding frames [22], which likely disrupts all dom isoforms. (G) E(Pc)^-/- denotes the E(Pc)¹ allele, an EMS mutant in which E(Pc)¹/+ heterozygous flies exhibit only 10 to 21% of the mRNA of wild-type flies. In principle, a null would be expected to have 50% [35]. Scale bars are provided for each panel.

Bap55 regulates projection neuron dendrite targeting. (A) Wild-type (WT) DL1 PN dendrites target specifically to the posterior glomerulus DL1 (yellow dashed circle in A₂) and never target to the anterior glomerulus DA4l (yellow dashed circle in A₁). White arrowhead denotes cell body in all images. (B) WT DL1 axons show an L-shaped pattern in the lateral horn (LH), with branches in the mushroom body calyx (MBC; both outlined with white dashed circles). (C) Bap55^-/- PN dendrites fully mistarget to the anterior glomerulus DA4l (C₁), abandoning the posterior glomerulus DL1 (C₂). (D) Bap55^-/- PN axons show the stereotypical L-shaped pattern in the LH with branches in the MBC. (E) WT GH146 anterodorsal neuroblast clones are characterized by their cell body location dorsal to the antennal lobe (white arrowhead in all images). The dendrites target to stereotyped glomeruli in the antennal lobe. (F) WT GH146 lateral neuroblast clones are characterized by their cell body location lateral to the antennal lobe with dendrites targeting to stereotyped glomeruli. (G) WT GH146 ventral neuroblast clones are characterized by their cell body location ventral to the antennal lobe with dendrites targeting across the antennal lobe. (H, I) Bap55^-/- GH146-labeled anterodorsal (H) and lateral (I) neuroblast clones maintain their cell body location dorsal and lateral to the antennal lobe, respectively. Yet their dendrites do not target proper glomeruli. (J) Bap55^-/- GH146-labeled ventral neuroblast clones maintain their cell body location ventral to the antennal lobe, yet their dendrites target a small area of the antennal lobe. Green marks mCD8-GFP-labeled PNs generated by MARCM and labeled using GH146-GAL4. (A, C) show partial confocal stacks; (B, D, E-J) show full confocal stacks; magenta is the presynaptic marker nc82; extraneous magenta punctate staining outside of the antennal lobe in some panels is dsRed background from the piggyBac and/or GH146-GAL4 insertions. Scale bars: 20 μm in (A) (for A, C, E-J) and (B) (for B, D).

Bap55 mutants cause derepression of a PN-GAL4. (A) WT Mz19 anterodorsal neuroblast clones label PNs targeting to the VA1d and DC3 glomeruli. The DC3 glomerulus is difficult to visualize in confocal stacks. (B) WT Mz19 lateral neuroblast clones label a single class of PNs targeting to the DA1 glomerulus. (C) WT Mz19 never labels ventral PN neuroblast clones. No labeling is observed in the antennal lobe. (D) Bap55^-/- Mz19 anterodorsal neuroblast clones exhibit ectopic labeling of PNs targeting many glomeruli in the anterior antennal lobe. (E) Bap55^-/- Mz19 lateral neuroblast clones exhibit ectopic labeling of local interneurons, which target throughout the antennal lobe. (F) Bap55^-/- Mz19 clones ectopically label ventral PNs, which are never labeled in WT, and that target to very few glomeruli in the antennal lobe. (G, H) WT Mz19 never labels a single anterodorsal PN when clones are generated just after larval hatching. (G₁, G₂) show no labeling in the antennal lobe. (H) shows no axon labeling in the lateral horn. (I, J) Bap55^-/- Mz19 exhibits ectopic labeling of single anterodorsal PNs, which target to the anterodorsal glomerulus DA4l (I₁), while avoiding the posterior glomerulus DL1 (I₂). The axons form an L-shaped pattern in the lateral horn, with branches in the mushroom body calyx (J). Green marks mCD8-GFP-labeled PNs generated by MARCM and labeled using Mz19-GAL4. (G, I) show partial confocal stacks; (A-F, H, J) show full confocal stacks; magenta is the presynaptic marker nc82; symbols are as in Figure 2. Scale bars: 20 μm in (A) (for A-G, I) and (H) (for H, J).

Bap55 acts postmitotically in PNs and dendrite mistargeting can be suppressed by human BAF53a and b. (A, B) Postmitotic expression of UAS-Bap55 can rescue the Bap55^-/- dendrite mistargeting phenotype. The PN no longer targets to the anterior glomerulus DA4l (A₁), but to the posterior glomerulus DL1 (A₂). The L-shaped axon pattern is not altered (B). (C, D) In a small number of cases, postmitotic expression of UAS-Bap55 in a Bap55^-/- single cell causes a de novo phenotype. The dendrites target to the anterior and medial glomerulus DM6 (C₁), abandoning both DA4l (C₁) and DL1 (C₂). In these cases, it also causes a de novo axon phenotype of mistargeting ventrally to the lateral horn (white arrow in (D)). (E, F) Postmitotic expression of human UAS-Baf53a also rescues the Bap55^-/- phenotype (E) and does not alter the L-shaped axon pattern (F). (G, H) Postmitotic expression of human UAS-Baf53b also rescues the Bap55^-/- phenotype (G) and does not alter the L-shaped axon pattern (H). (I) Quantification of mistargeting phenotypes for (A, C, E, G). Green marks mCD8-GFP-labeled PNs generated by MARCM and labeled using GH146-GAL4. (A, C, E, G) show partial confocal stacks; (B, D, F, H) show full confocal stacks; magenta is the presynaptic marker nc82; symbols are as in Figure 2. Scale bars: 20 μm in (A) (for A, C, E, G) and (B) (for B, D, F, H).

Other BRM complex component mutants do not exhibit the same phenotypes as Bap55 mutants. (A) Dendrites of brm^-/- DL1 PNs mistarget to non-stereotyped areas of the antennal lobe. (B, C) brm^-/- anterodorsal (B) and lateral (C) neuroblast clone PNs exhibit perturbed cell morphology, weak labeling, and dendrite mistargeting, with small meandering projections to incorrect glomeruli. (D) brm^-/- ventral neuroblast clone PNs exhibit perturbed cell morphology, weak labeling, and a lack of innervation throughout the antennal lobe. (E) Dendrites of Snr1^-/- DL1 PNs mistarget to non-stereotyped areas of the antennal lobe. (F, G) Snr1^-/- anterodorsal (F) and lateral (G) neuroblast clone PNs exhibit perturbed cell morphology, weak labeling, and dendrite mistargeting, with small meandering projections to incorrect glomeruli. (H) Snr1^-/- ventral neuroblast clone PNs exhibit perturbed cell morphology, weak labeling, and a lack of innervation throughout the antennal lobe. Green marks mCD8-GFP-labeled PNs generated by MARCM and labeled using GH146-GAL4. All panels show full confocal stacks; magenta is the presynaptic marker nc82; symbols are as in Figure 2. Scale bar: 20 μm in (A) (for A-H).

dom mutants yield similar phenotypes to Bap55 mutants. (A, B) dom^-/- DL1 PNs mistarget their dendrites to the anterior glomerulus DA4l (A₁). dom^-/- DL1 PNs retain part of their dendritic mass in the posterior glomerulus DL1 (A₂). They maintain their L-shaped axon pattern in the lateral horn and branches in the mushroom body calyx (B). (C, D) Expression of UAS-Bap55 in a dom^-/- DL1 PN cannot suppress the dendrite mistargeting phenotype. Dendrites maintain mass in both DA4l (C₁) and DL1 (C₂). The axon also maintains its L-shaped pattern (D). (E) Quantification of mistargeting phenotypes for (A, C). Green marks mCD8-GFP-labeled PNs generated by MARCM and labeled using GH146-GAL4. (A, C) show partial confocal stacks; (B, D) show full confocal stacks; magenta is the presynaptic marker nc82; symbols are as in Figure 2. Scale bars: 20 μm in (A) (for A, C) and (B) (for B, D).

E(Pc) mutants yield similar phenotypes to Bap55 mutants. (A, B) E(Pc)^-/- DL1 PNs mistarget their dendrites to the anterior glomerulus DA4l (A₁), avoiding the posterior glomerulus DL1 (A₂), and maintaining the stereotypical L-shaped axon targeting the lateral horn with branches in the mushroom body calyx (B). (C, D) Postmitotic expression of UAS-Bap55 causes a de novo phenotype in E(Pc)^-/- DL1 PNs. The dendrites split between DA4l and the anterior medial glomerulus DM6 (C₁), and the axon mistargets ventrally to the lateral horn (white arrow in (D)). (E) Quantification of mistargeting phenotypes for (A, C). Green marks mCD8-GFP-labeled PNs generated by MARCM and labeled using GH146-GAL4. (A, C) show partial confocal stacks; (B, D) show full confocal stacks; magenta is the presynaptic marker nc82; symbols are as in Figure 2. Scale bars: 20 μm in (A) (for A, C) and (B) (for B, D).

dom and E(Pc) mutants cause derepression of a PN-GAL4. (A, B) dom^-/- GH146-labeled anterodorsal (A) and lateral (B) neuroblast clones show a mild reduction in cell number and disorganization of dendrite targeting. (C) dom^-/- GH146-labeled ventral neuroblast clones show a mild reduction in dendrite elaboration. (D, E) dom^-/- Mz19-labeled anterodorsal (D) and lateral (E) neuroblast clones ectopically mark a large number of PNs targeting to many glomeruli. (F) dom^-/- Mz19-labeled neuroblast clones ectopically mark ventral cells targeting to a number of glomeruli. (G, H) dom^-/- Mz19 ectopically labels single cell clones, which target dendrites to both DA4l (G₁) and DL1 (G₂), with an L-shaped axon pattern in the lateral horn (H). (I-P) Equivalent experiments to (A-H) using E(Pc) mutants. E(Pc)^-/- GH146 and MZ19 clones show similar phenotypes to dom^-/- clones. Green marks mCD8-GFP-labeled PNs generated by MARCM and labeled using GH146-GAL4 or Mz19-GAL4. (G, O) show partial confocal stacks; (A-F, H-N, P) show full confocal stacks; magenta is the presynaptic marker nc82; symbols are as in Figure 2. Scale bars: 20 μm in (A) (for A-G, I-O) and (H) (for H, P).