Figure 6. Ecdysone signaling is not necessary for α’β’ specification.

(A-B) Representative max projections showing adult axons of clonally related neurons born from L1 stage in wildtype and UAS-EcR-DN conditions. UAS-CD8::GFP is driven by mb-Gal4 (OK107-Gal4). 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 white box outlines the Inset panel. Trio (magenta) is used to label all γ and α’β’ axons for comparison to GFP⁺ axons. (A) In wildtype, GFP⁺ axons are visible in all mushroom body lobes. (B) α’β’ axons are lost, and γ neurons do not remodel, in UAS-EcR-DN expressing clones. (C-D) Representative, single z-slices from the adult cell body region of clones induced at L1 in wildtype and UAS-EcR-DN conditions. UAS-CD8::GFP is driven by mb-Gal4. (C) Wildtype clones show the presence of strongly expressing Trio (magenta) and Mamo (blue, gray in single channel) neurons, indicative of α’β’ identity. (D) In UAS-EcR-DN clones, strong Trio and Mamo cells are not present. (E) Quantification of MARCM clones marked by mb-Gal4, which labels all mushroom body neuronal types. The number of α’β’ neurons are quantified in wildtype (n = 7, replotted from data in Figure 1H) and UAS-EcR-DN (n = 6) conditions. 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. In wildtype, 25.5 ± 0.7% of the total strong Mamo expressing cells (α’β’ neurons) are within clones. In UAS-EcR-DN clones, only 3.4 ± 0.6% of α’β’ neurons are within clones. (F) usp mutant clones contain α’β’ neurons. FasII (magenta) is used to label γ and αβ lobes. Red arrow indicates unpruned γ neurons. (G) Representative image of an UAS-EcR-DN expressing neuroblast marked by UAS-CD8::GFP driven by mb-Gal4 (red box) ventral to a wildtype neuroblast (green-dashed box) from the same wandering L3 stage brain, immunostained for Imp (blue, gray in single channel) and Syp (magenta). (H) Close-up view of wildtype neuroblast (green-dashed box in G). (I) Close-up view of UAS-EcR-DN neuroblast (red box in G). (J) Quantification of the Imp to Syp ratio in UAS-EcR-DN neuroblasts (n = 4 from four different brains) compared to wildtype (n = 27 from the same four brains as UAS-EcR-DN neuroblasts). (K) A representative adult mushroom body clone (green) induced at L1 expressing UAS-EcR-DN driven by mb-Gal4. α’β’ neurons (GFP⁺ (green), FasII- (magenta)) are not observed and γ neurons do not remodel (GFP⁺, FasII⁺, red outline). (L) A representative adult wildtype clone induced at L1 driven by NB + mb2-Gal4. All three neuron types are present, including α’β’ neurons (GFP⁺, FasII^-, magenta outline). (M) α’β’ neurons are also present when UAS-EcR-DN is driven by NB + mb2-Gal4 although γ neurons do not remodel. (N) Quantification of MARCM clones in which UAS-EcR-DN is driven by mb-Gal4 (n = 6, replotted from data in E) or NB + mb2-Gal4 (n = 6) compared to wildtype (n = 7, replotted from data in Figure 1H). In UAS-EcR-DN clones driven by NB + mb2-Gal4, 24.6 ± 2.1% of α’β’ neurons are within a clone, similar to wildtype. O. At L3, Mamo (gray) is expressed in young mushroom body neurons (α’β’) while EcR (magenta) can only be detected in more mature neurons (mainly γ at this stage). Note that there is no overlap between Mamo and EcR. (P) Expressing UAS-EcR-DN with mb-Gal4 (green, white outline) leads to the loss of Mamo expression (gray) inside the clone but not in surrounding wildtype mushroom body neurons. (Q) In contrast, expressing UAS-EcR-RNAi drivenE by mb-Gal4 abolishes EcR expression but does not affect Mamo. For E and J a two-sample, two-tailed t-test was performed. For N, a Tukey test was performed. ***p<0.001, ns: not significant. Scale bars: A, 20 µm; G, 10 µm; P, 5 µm.

Figure 6—figure supplement 1. Ecdysone signaling is not necessary for α’β’ specification.

(A) Representative, single z-slice showing strong Trio⁺ and Mamo⁺ cells both inside and outside a GFP⁺ clone. (B) Expressing UAS-EcR-DN leads to the loss of α’β’ neurons. Images in A and B represent the same sections as in Figure 6C–D. (C) usp³ mutant clones contain α’β’ neurons, similar to usp² mutant clones. (D-D’) Arbitrary fluorescent intensity values (scaled to 100) of Imp and Syp in wildtype (n = 27) and UAS-EcR-DN (n = 4) neuroblasts. Neither Imp nor Syp intensity values are different compared to wildtype. (E-E’) Insc-Gal4 + R13F02-Gal4 (NB-Gal4 + mb2-Gal4) driving expression of UAS-CD8::GFP at L3 stage. Strong GFP (green) is detected in mushroom body neuroblasts (white-dashed circle) but not in newborn neurons (arrows) positioned adjacent to mushroom body neuroblasts. GFP is strongly expressed in more distally positioned, mature neurons marked by EcR (magenta). (F-F’) OK107-Gal4 (mb-Gal4) driving expression of UAS-CD8::GFP at L3. GFP (green) is detected in mushroom body neuroblasts (white-dashed circles), newborn neurons (arrows), and mature neurons marked by EcR expression (magenta). (G-G’’) Mushroom body neuroblasts (white-dashed circles) marked by Dpn (gray) do not express EcR based on antibody staining (magenta) or an EcR-GFP (green) at the wandering L3 stage. (H-H’’) Mushroom body neurons (white-dashed outline) positioned ventrally to the mushroom body neuroblasts in G do express EcR but not in young neurons (black regions inside white-dashed line). (I) α’β’ neurons are not rescued in babo clones by expressing UAS-EcR. (J) Quantification of phenotype presented in (I) The number of α’β’ neurons is quantified in wildtype (replotted from data in Figure 1H), babo (replotted from data in Figure 1H), babo, UAS-babo (replotted from Figure 5—figure supplement 1), and babo, UAS-EcR (n = 8, 4.4 ± 0.4%). Significance values were determined using a Tukey test. ***p<0.001, ns: not significant.