Journal: bioRxiv

Article Title: Steroid hormone-dependent glial-neuronal interaction promotes brain development during Drosophila metamorphosis

doi: 10.64898/2025.12.30.696973

Figure Lengend Snippet: (A) Schematic illustration of mushroom body (MB) remodeling during metamorphosis. α and β indicate the newly extending axonal lobes of a subset of Kenyon cells (KCs) called α/β neurons, whereas γ indicates the larval-specific axonal lobe of KC γ neurons. Cell bodies of γ neurons (γ-KCs) are located in the dark grey area. (B) Images of the MB at 24 h after puparium formation (24 hAPF) of control animals, as well as those expressing EcI-RNAi , EcR-RNAi , or a dominant-negative form of EcR ( EcR-DN ) in glial cells. Repo-GAL4, UAS-dicer2 was used to induce transgene expression in glial cells. The MB lobes were visualized by anti-Fasciclin-II (Fas-II) antibody staining (magenta). Scale bars represent 25 μm. (C) EcR expression in the MB at 0 hAPF and 12 hAPF. EcR localization was visualized by anti-EcR antibody (red) in w 1118 . Nuclei were stained with Hoechst 33342 (blue). Allows indicate locations of KCs. Scale bars represent 100 μm. (D) Ligand-dependent EcR activity in the brain at 0 hAPF and 12 hAPF. The MB lobes were labeled by OK107-GAL4 -driven expression of mCD8-GFP (green), whereas ligand-dependent EcR activity was visualized by the ecdysone response element-driven LacZ reporter ( EcRE-LacZ ; yellow). Dashed circles indicate locations of KCs. Scale bars represent 100 μm. (E) Images of the MB of pupae at 24 hAPF and adult males 3 days after eclosion. OK107-GAL4 was used to induce EcR-RNAi , EcI-RNAi , or E23 overexpression in the MB. The MB lobes were visualized by anti-Fas-II antibody staining (magenta). At the adult stage, the γ lobes were specifically labeled (green) by expressing mCD8-GFP under the control of the γ lobe-specific driver R22B09-LexA . Scale bars represent 25 μm. (F) Images of the pupal CNS and MB at 24 hAPF and adult males 3 days after eclosion. OK107-GAL4 was used to induce EcI-RNAi in the MB of pupae in the EcI2 , EcI3 , and EcI4 triple mutant background. Transheterozygotes of EcI2 , EcI3 , and EcI4 null mutants and their deficiency strains were used. Neuronal axons were stained with anti-N-cadherin (N-Cad) antibody (green), nuclei were stained with Hoechst 33342 (blue), and the MB lobes were visualized by anti-Fas-II antibody staining (magenta). Scale bars represent 100 μm for the CNS images and 25 μm for the MB images.

Article Snippet: Primary antibodies used were rat anti-N-cadherin (clone: DN-Ex #8, 1:50, Developmental Studies Hybridoma Bank), mouse anti-Fasciclin II (clone: 1D4, 1:500, Developmental Studies Hybridoma Bank), mouse anti-β-Galactosidase (LacZ) (clone: 40-1a, 1:500, Developmental Studies Hybridoma Bank), mouse anti-EcR (clone: Ag10.2, 1:200, Developmental Studies Hybridoma Bank), mouse anti-EcR-B1 (clone: AD4.4, 1:10, Developmental Studies Hybridoma Bank), mouse anti-GFP (clone: GFP-20, 1:1000, Sigma) and chicken anti-GFP (1:1000, Abcam).

Techniques: Control, Expressing, Dominant Negative Mutation, Staining, Activity Assay, Labeling, Over Expression, Mutagenesis