Steroid Hormone Ecdysone Signaling Specifies Mushroom Body Neuron Sequential Fate via Chinmo  Giovanni Marchetti, Gaia Tavosanis  Current Biology  Volume 27, Issue 19, Pages 3017-3024.e4 (October 2017) DOI: 10.1016/j.cub.2017.08.037 Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 1 Ecdysone Signaling Controls MB Neuronal Fate (A, B, F, G, K, L, P, and Q) Adult MB lobes from control (A, F, K, and P) and OK107>EcR-DN (B, G, L, and Q) brains stained with anti-FasII antibody (magenta). Insets in (A) and (B), single FasII signal. Green, GAL4-OK107-driven mCD8-GFP (mGFP) in (A)–(D); LexA-γ-MB-driven mGFP in (F)–(I); LexA-α′β′-MB-driven mGFP in (K)–(N); LexA-αβ core-MB-driven mGFP in (P)–(S). (C, D, H, I, M, N, R, and S) Cell bodies of adult MB neurons from control (C, H, M, and R) and OK107>EcR-DN (D, I, N, and S) brains. (E, J, O, and T) Boxplot comparing normalized numbers of MB neuron cell bodies in control and OK107>EcR-DN conditions. In this and all following boxplots, the boxes show median and interquartile range; the whiskers show the minima/maxima. Cell bodies were identified based on mGFP signal. Values were normalized to 100 for controls. ∗∗∗p < 0.001; N.S., not significant (two-tailed t test). See also Figures S1 and S2. Current Biology 2017 27, 3017-3024.e4DOI: (10.1016/j.cub.2017.08.037) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 2 Chinmo Positively Regulates EcR-B1 Expression in Early-Born MB Neurons (A–B′) WL3 MB cell bodies from control (A and A′) and OK107>EcR-DN (B and B′) brains stained with anti-Chinmo antibody (magenta). (C) Quantification of Chinmo-positive, OK107-positive MB cells for both control and OK107>EcR-DN WL3 brains. Statistical comparison to the control: N.S., not significant (two-tailed t test). (D and E) α′β′ MB cell bodies from WL3 (D) and 18H APF (E) brains labeled with mGFP (green) using the α′β′-specific GAL4-c305 driver and stained with anti-EcR-B1 antibody (magenta). Arrows indicate examples of α′β′ MB neurons expressing EcR-B1 isoform, which are magnified in the insets. (F–G′) Control (F and F′) and chinmo1 (G and G′) cell bodies from WL3 MARCM MB neuroblast clones labeled with mGFP (green) using the GAL4-OK107 driver and stained with anti-EcR-B1 antibody (magenta). (H) Quantification of EcR-B1-positive cell clones for both control and chinmo1 WL3 MB neuroblast clones. Statistical comparison to the control: ∗∗∗p < 0.001 (two-tailed t test). Error bars represent SD. (I and J) MB lobes at 18H APF from control (I) and chinmo1 (J) neuroblast clones labeled with mGFP (green) using the GAL4-201Y driver. (K) MB lobe from chinmo1 neuroblast clone at 18H APF expressing wild-type EcR-B1 isoform. (L) Quantification of axonal pruning defects in control, chinmo1, and chinmo1; UAS-EcR-B1 MB neuroblast clones. Pruning defects were estimated by the presence of dorsal and medial larva γ MB axonal lobes at 18H APF. Statistical comparison to the control: ∗∗∗p < 0.001; N.S., not significant (Fisher’s exact test). (M) Quantification of Chinmo and EcR transcript levels in control (actin5C>LacZ-RNAi) and in actin5C>Chinmo-RNAi WL3 larvae. Statistical comparison to the LacZ RNAi: ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001 (one-way ANOVA test). Error bars represent SD. See also Figure S3. Current Biology 2017 27, 3017-3024.e4DOI: (10.1016/j.cub.2017.08.037) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 3 EcR-B1 Isoform Mediates Chinmo-Dependent γ and α′β′ MB Neuron Production (A–C) Adult MB lobes from control (A), chinmo1 (B), and chinmo1; UAS-EcR-B1 (C) neuroblast MARCM clones labeled with mGFP (green) using the GAL4-201Y driver and stained with anti-FasII antibody (magenta). (D) Quantification of chinmo1 neuronal identity defects in γ MB neurons in control, chinmo1, and chinmo1; UAS-EcR-B1 MB neuroblast clones. (E–G) Adult MB lobes from control (E), chinmo1 (F), and chinmo1; UAS-EcR-B1 (G) neuroblast MARCM clones labeled with mGFP (green) using the GAL4-OK107 driver and stained with anti-FasII antibody (magenta). (H) Quantification of chinmo1 neuronal identity defects in α′β′ MB neurons in control, chinmo1, and chinmo1; UAS-EcR-B1 MB neuroblast clones. Fate defects were estimated by the absence of adult γ or α′β′ lobes. Statistical comparison to the control: ∗p < 0.05; ∗∗∗p < 0.001; N.S., not significant (Fisher’s exact test). (I) Percentages of different subtypes of MB neurons among control, chinmo1, and chinmo1; UAS-EcR-B1 single-cell clones that were induced at two different developmental stages. The reduction of γ and α′β′ MB neuron production observed for chinmo1 mutant was significantly rescued in chinmo1; UAS-EcR-B1 clones. Current Biology 2017 27, 3017-3024.e4DOI: (10.1016/j.cub.2017.08.037) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 4 Ecdysone Signaling Induces MicroRNA let-7-Dependent Negative Feedback Loop to Control chinmo Expression during Transition from α′β′ to αβ MB Neurons (A) Quantitative analysis of the number of MB pioneer αβ neurons after removing one copy of chinmo and/or EcR gene. MB pioneer αβ neurons were labeled by GAL4-c708a [13]. Statistical comparison to the control (+/+) or to chinmo1/+: ∗p < 0.05 and ∗∗p < 0.01; N.S., not significant (one-way ANOVA test). (B–C′) Adult MB cell bodies from control (B and B′) and OK107>EcR-DN (C and C′) brains stained with anti-Chinmo antibody (magenta). (D–E′) 18H APF MB cell bodies from control (D and D′) and OK107>EcR-DN (E and E′) brains carrying the let-7-Cp12.5kb::LacZ reporter and stained with anti-βGal antibody (magenta). (F–G′) WL3 MB cell bodies from control (F and F′) and OK107>UAS-let-7-C (G, G′) brains stained with anti-EcR-B1 antibody (magenta). Green, GAL4-OK107-driven mGFP in (B)–(G′). (H) Model of sequential MB neuron specification. Chinmo positively regulates the expression of EcR-B1 to promote transition from early-born to late-born MB neurons. The α′β′ to αβ MB neurons identity switch requires induction of let-7 [5, 9] by EcR-B1 receptor to inhibit Chinmo expression. See also Figures S3 and S4. Current Biology 2017 27, 3017-3024.e4DOI: (10.1016/j.cub.2017.08.037) Copyright © 2017 Elsevier Ltd Terms and Conditions