Figure 5. α’β’ neurons are specified by low Imp levels at L3.

(A-A’) Representative image of wildtype mushroom body neurons labeled by mb-Gal4 driving UAS-CD8::GFP (green, white-dashed outline) during the wandering L3 stage. Mamo (gray) is used as a marker for α’β’ neurons. (B-B’) When mb-Gal4 is used to drive UAS-Imp-RNAi, Mamo is not expressed. (C-C’) Similarly, Mamo expression is lost when overexpressing Imp (UAS-Imp-overexpression (OE)). (D-D’). Expressing UAS-Syp-RNAi also leads to the loss of Mamo. (E) Expressing UAS-Syp-overexpression (OE) does not affect Mamo. Scale bar: 5 µm.

Figure 5—figure supplement 1. Low Imp levels are required for α’β’ specification.

(A-E) Representative images of adult mushroom body lobes labeled by mb-Gal4 driving UAS-CD8::GFP (green) in wildtype (A), UAS-Imp-RNAi (B), UAS-Imp-OE (C), UAS-Syp-RNAi (D) or UAS-Syp-OE (E). When visible, γ axons are outlined in red, α’β’ axons are outlined in magenta, and αβ axons are outlined in cyan. Trio (magenta) labels γ and α’β’ axons. (A) In wildtype, all three axonal types are present. (B) With Imp-RNAi, the majority of γ axons (red outline) are lost and α’β’ axons are completely missing. (C) Imp-OE leads to mushroom body axons projecting almost entirely into the γ lobe (red outline). Some αβ axons (cyan outline) are present but are missing the vertical α projection. α’β’ axons are completely lost. (D) Syp-RNAi is similar but more severe than Imp-OE as mushroom body axons project entirely into the γ lobe (red outline). (E) Syp-OE does not abolish α’β’ axons (magenta outline). αβ axons (cyan outline) are also present. However, the vertical α’ and α lobes are both missing. (F-J) Representative images of single z slices. Strong Trio (magenta) labels α’β’ neurons while weak Trio labels γ neurons. Strong Mamo (gray) also labels α’β’ neurons while weak Mamo labels γ neurons. Arrows points to α’β’ neurons inside the clone. F. In wildtype clones driven by mb-Gal4, GFP⁺ cells (green) contain strong Trio⁺ and Mamo⁺ cells. (G-H) These cells are lost in babo clones (G) but rescued in clones expressing UAS-babo (H). (I-J) Expressing UAS-Imp-RNAi (I) or UAS-Syp (J) does not rescue the loss of α’β’ neurons. (K-O). Representative maximum-projection images of adult mushroom body lobes from mb-Gal4 MARCM clones induced at L1, focused on the α’ and β’ lobes. Clonally related neurons are GFP⁺ (green). All mushroom body axons, both clonal and non-clonal, are marked by Trio (magenta). Outlines mark GFP⁺ axons, where γ axons are outlined in red, α’β’ axons are outlined in magenta, and αβ axons are outlined in cyan. A gray box outlines the Inset panel. (K) In wildtype, GFP⁺ axons are observed in the α’β’ lobes (magenta outline). (L) In babo mutant clones, γ neurons do not remodel (red outline) and α’β’ neurons are missing. (M) These phenotypes are rescued by expressing UAS-babo inside mutant clones, as GFP⁺ axons colocalize within the Trio labeled α’β’ lobes (magenta outline). (N-O) Neither reducing Imp with UAS-Imp-RNAi or decreasing the Imp to Syp ratio by expressing UAS-Syp rescues the loss of α’β’ neurons. (P) Quantification of MARCM clones represented in K-O. Plotted is the percentage of strong Mamo⁺ and GFP⁺ cells (clonal cells) versus all Mamo⁺ cells (clonal and non-clonal cells) within a single mushroom body. The number of α’β’ neurons is quantified in wildtype (n = 7, replotted from data in Figure 1H), babo (n = 8, replotted from data in Figure 1H), babo, UAS-babo (n = 6), babo, UAS-imp-RNAi (n = 7), and babo, UAS-Syp (n = 7), In wildtype, 25.5 ± 0.7% of the total strong Mamo expressing cells (α’β’ neurons) are within a clone while they only represent 2.2 ± 0.4% in babo clones. Expressing UAS-babo rescues to 21.1 ± 2.4%. In contrast, expression of UAS-Imp-RNAi (0.17 ± 0.17%) or UAS-Syp (1.8 ± 0.5%) is not statistically different from babo. Significance values were determined using a Tukey test. ***p<0.001, ns: not significant.