Figure 1. α’β’ neurons are not generated from babo mutant neuroblasts.

(A) Summary of intrinsic temporal patterning mechanism operating during mushroom body development. During early larval stages, mushroom body neuroblasts express high levels of Imp (red) and Chinmo (red) in neurons to specify γ identity for ~85 neuroblast divisions (red-dashed box). From mid-L3 to metamorphosis, when Imp and Syp (cyan) are both at low levels, the same neuroblast divides ~40 times to produce α’β’ neurons (magenta-dashed box). Low Chinmo regulates the expression Mamo, a terminal selector of α’β’ identity. From the beginning of metamorphosis throughout pupal development, high Syp leads to αβ neurons (cyan-dashed outline). (B) Known molecular markers can distinguish between the three mushroom body neuronal types in the adult. (C) Mushroom body projections originating from neurons born from four neuroblasts (numbered 1 to 4) per hemisphere fasciculate into a single bundle (peduncle) before branching into the five mushroom body lobes. The first-born γ neurons (red) remodel during development to project into a single, medial lobe in the adult. This lobe is the most anterior of the medial lobes. Axons from α’β’ neurons (magenta) bifurcate to project into the vertical and medial α’ and β’ lobes. The β’ lobe is posterior to the γ lobe. The last-born αβ neurons (cyan) also bifurcate their axons into the vertical projecting α lobe and medial projecting β lobe. The α lobe is positioned adjacent and medial to the α’ lobe. The β lobe is the most posterior medial lobe. (D-E) Representative max projections showing adult axons of clonally related neurons born from L1 stage in wildtype and babo 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. (D) In wildtype, GFP⁺ axons (green, outlined in red, magenta and cyan) are visible in all observable mushroom body lobes. (E) In babo mutant clones, γ neurons (red outline) remain unpruned. GFP⁺ axons are missing inside the Trio⁺ α’ lobe, indicating the absence of α’β’ neurons. (F-G) Representative, single z-slices from the adult cell body region of clones induced at L1 in wildtype and babo conditions. UAS-CD8::GFP is driven by mb-Gal4. (F) Wildtype clones show the presence of strongly expressing Trio (magenta) and Mamo (blue, gray in single channel) neurons, indicative of α’β’ identity. (G) In babo mutant clones, cells strongly expressing Trio and Mamo are not present. (H) 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) and babo (n = 8) 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, consistent with our expectation since each mushroom body is made from four neuroblasts. In babo clones, only 2.2 ± 0.4% of α’β’ neurons are within clones. (H’) There are no significant differences between the average clone sizes (wildtype:533.6 ± 33.3; babo:551.3 ± 17.6). (I) Quantification of γ neurons marked by γ-Gal4 (R71G10-Gal4) in MARCM clones. Plotted is the total number of γ neurons marked by GFP and Trio in wildtype (n = 10) and babo mutant (n = 12) clones. In wildtype, the average number of γ neurons is 154.3 ± 11.4. In babo mutants, the average is 178.4 ± 11.9. (J) Quantification of α’β’ neurons marked by α’β’-Gal4 (R41C07-Gal4) in MARCM clones. Plotted is the total number of α’β’ neurons marked by GFP and strong Trio in wildtype (n = 4) and babo mutant (n = 8) clones. In wildtype, the average number of α’β’ neurons is 81.5 ± 3.4. In babo mutants, the average is 2.1 ± 0.5. (K) Quantification of αβ neurons marked by αβ-Gal4 (R44E04-Gal4) in MARCM clones. Plotted is the total number of GFP⁺ cells in wildtype (n = 7) and babo mutant (n = 8) clones. In wildtype, the average number is 276 ± 9.1. In babo mutants, the average number is 228.9 ± 13.2. A two-sample, two-tailed t-test was performed. ***p<0.001, **p<0.01, ns: not significant. Scale bars: D, 20 µm; F, 5 µm.

Figure 1—figure supplement 1. α’β’ neurons are lost from the adult neuropil in activin signaling mutant clones.

(A-H) Representative images of adult mushroom body lobes from MARCM screen in which clones were induced at L1 stage. Clonally related neurons are GFP⁺ (green). UAS-CD8::GFP is driven by mb-Gal4. All mushroom body axons, both clonal and non-clonal, are marked by FasII (γ and αβ axonal marker, 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) In wildtype clones, all three mushroom body neuronal types are present. α’β’ axons are GFP⁺ and FasII^-. (B) In babo mutants, γ neurons do not remodel (red outline in vertical lobe). α’β’ neurons are also missing, as there are no GFP⁺, FasII^- axons. (C) UAS-EcR-DN expressing clones are missing α’β’ axons and γ neurons do not remodel. (D-G) No loss of neuronal diversity is observed. (H) svp mutant clones (GFP⁺, green) contain all three mushroom body neuronal types. (I) Smad2 mutant clones (GFP, green) have unpruned γ axons (red arrow) and no α’β’ axons (Trio⁺, magenta). (J-J’’’) Representative adult wildtype clone labelled with R41C07-Gal4 (α’β’-Gal4). A single z-slice showing that strong Trio and strong Mamo label α’β’ neuronal cell bodies. (K-L) Representative, single z-slice showing strong Trio⁺ and Mamo⁺ cells inside and outside a GFP⁺ clone in wildtype (K) but not in babo (L) clones. Images represent the same sections as in Figure 1F–G. (M-M’’) Representative images showing the presence of α’β’ neurons based on strong Mamo (gray) and Trio (magenta) expression in adult mushroom body neurons labeled by mb-Gal4 driving UAS-CD8::GFP. (N-N’’’) Images highlighting the vertical mushroom body axons. α’ axons are Trio⁺ and GFP⁺. FasII (gray) labels α axons. (O-O’’) The majority of α’β’ neurons are still present following expression of UAS-babo-RNAi based on strong Mamo and Trio expression. (P-P’’’) The vertical lobes indicate that γ neurons do not remodel (FasII⁺/Trio⁺/GFP⁺) and that α’β’ neurons are still present (α’ lobe that is FasII^-/Trio⁺/GFP⁺) following UAS-babo-RNAi expression. (Q) Quantification of the number of α’β’ neurons following expression of UAS-babo-RNAi (329 ± 10.4, n = 6) compared to wildtype controls (379 ± 11, n = 6). A two-sample, two-tailed t-test was performed. **p<0.01. Scale bars: A, 10 µm; J,K,M,N,P, 5 µm.

Figure 1—figure supplement 2. γ neuron numbers likely increase, while αβ numbers decrease, in babo mutant clones.

(A-A’’) The loss of α’β’ neurons in babo mutant clones marked by mb-Gal4 (GFP, green) is not rescued by blocking cell death. γ neurons (red outline) also retain their larval branching. (B-E) Representative wildtype and babo mutant clones made using R71G10-Gal4 (γ-Gal4) driving UAS-CD8::GFP. Note the presence of some GFP⁺ axons in the αβ lobes (cyan outline). Arrowheads in the cell body region point to αβ neurons based on the absence of Trio expression. Only cells that were both GFP⁺ and Trio⁺ were used for quantification. (F-I) Representative wildtype and babo mutant clones made using R41C07-Gal4 (α’β’-Gal4) driving UAS-CD8::GFP. Note the presence of some GFP⁺ axons in the αβ lobes (cyan outline). Arrowheads in the cell body region point to αβ neurons based on the absence of Trio expression. In babo mutants, the majority of neurons remaining are αβ (GFP⁺ and Trio^-). The ‘*” indicates the presence of an α’β’ neuron cell body based on strong Trio expression. Only cells that were both GFP⁺ and Trio⁺ were used for quantification. (J-M) Representative wildtype and babo mutant clones made using R44E04-Gal4 (αβ-Gal4) driving UAS-CD8::GFP.