Figure 4. Lar and Sns act in different neurons to control mushroom body (MB) dorsal and medial lobe development.

(A–F’) Lar>GFP and Sns>GFP expression in the larval brain. Brains were triple-stained for Lar>GFP (green), FasII (red), and anti-HRP (blue). Anti-FasII labels the MB neuropil; anti-HRP labels neuronal membranes. (A) Lar>GFP expression in Kenyon cells (KCs) (green, arrows). (B, B’) Projection of confocal slices through the entire larval MB showing Lar expression in the MB neuropil. (C, C’) Single optical slice showing Lar expression in the medial (m) and dorsal (d) lobes of the MB. (D) There is no Sns>GFP expression in KCs. (E, E’) Projection of confocal slices through the entire MB showing no overlap between Sns>GFP and the MB neuropil labeled by FasII. (F, F’) Single optical slice through the MB showing no Sns>GFP expression in the MB neuropil. (G–L) Third-instar larval MBs visualized with FasII staining. 3D reconstructions of confocal stacks using Imaris software are shown. (G) and (H) have normal MBs. (I) has missing dorsal lobes and medial lobe fusion (arrow). (J) has a medial lobe fusion phenotype (arrow). (K) has missing dorsal lobes. (L) has missing dorsal lobes and medial lobe fusion (arrow). (M–N’) Quantification of MB phenotypes in heterozygote controls (blue), Lar mutants (red), and Lar/sns transhets (red). In (M) and (N), the percentages of normal MBs are shown; in (M’) and (N’), the percentages of MBs with the phenotype are shown. (M, M’) Medial lobe fusion phenotype, (N, N’) Dorsal lobe branching defect. Data were analyzed using Fisher’s exact test, and each genotype was compared to every other genotype. ****p<0.0001; ***p<0.001; *p<0.05. Scale bar, 20 µm. See Figure 4—figure supplement 1 for single-slice analysis in Lar/sns transhets and Lar and Sns RNAi-mediated MB phenotypes.

Figure 4—figure supplement 1. Medial lobe fusion in the larval mushroom body in Lar/sns transhets and upon RNAi-mediated Lar and Sns knockdown.

(A–F’) Single optical slices through the medial lobes of control (A–C’) and genetic transhet (D–F’) animals showing normal unfused medial lobes in controls (close-up of medial lobes in A’–C’) and fused medial lobes in transhets (D’–F’). Anti-FasII (red) is used to label the mushroom body neuropil. (G–I) 3D rendering of confocal stacks of FasII-stained larval brains from RNAi knockdown experiments. (J–K’) Quantification of medial lobe fusion and dorsal lobe branching defects upon RNAi-mediated knockdown of Lar and Sns. GAL4 only control in blue and Lar or sns RNAi genotypes in red. Data were analyzed using Fisher’s exact test, and each genotype was compared to every other genotype. ****p<0.0001; *p<0.05. Scale bar, 20 µm.

Figure 4—figure supplement 2. Larval medial lobe fusion in Lar^13.2/Sns^Df transhets, neuronal Sns and Kirre knockdown, and mushroom body (MB)-specific Lar and Sns RNAi knockdown.

(A–F) Single optical slices through the medial lobes of larval MBs stained with FasII antibody to visualize the MB neuropil. Asterisks denote normal, unfused medial lobes. Arrowheads show fused medial lobes with medial lobe axons crossing the midline. (G, H) Quantification of medial lobe fusion phenotype in Lar^13.2/sns^Df transhets, neuronal Sns and Kirre knockdown, and cis RNAi for Lar and Sns in MB neurons (OK107-GAL4). Data were analyzed using Fisher’s exact test, and each genotype was compared to every other genotype. *p<0.05.