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. 2020 Nov 18;10:20023. doi: 10.1038/s41598-020-76891-9

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© The Author(s) 2020

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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Spen regulates Notch signaling in Drosophila adult glial cells. (A) Schematic of the Notch reporter construct NRE-GFP used to monitor Notch activity in Drosophila brain. The EGFP coding sequence (green) is under the control of a promoter containing multiple Su(H) binding sites (dark gray). (B) Simplified schematic of Drosophila adult brain, showing mushroom bodies (MB) and antennal lobes (AL), which are known to have high Notch activity (green). OL, optic lobes. (C) Immunofluorescence confocal micrographs of whole-mount brains from control flies (Eaat1 glial driver alone: Eaat1-GAL4/ +;NRE-GFP/ +) or flies with glia-specific spen^RNAi (Eaat1-GAL4/UAS-spen^RNAi;NRE-GFP/ +). Flies also harbored the nuclear marker mCherry-NLS expressed under the control of the Eaat1 driver (magenta). GFP fluorescence was detected in MB and AL lobes in close proximity with Eaat1 + glial cells. The NRE-GFP signal is reduced in Eaat1 > spen^RNAi (Eaat1-GAL4/UAS-spen^RNAi; NRE-GFP/ +) flies compared with controls. Scale bar, 50 μm. (D) Quantification of NRE-GFP fluorescence in flies with driver alone Eaat1 > + (Eaat1-GAL4/ +;NRE-GFP/ +), UAS-spen^RNAi alone (UAS-spen^RNAi/ +;NRE-GFP/ +), or spen^RNAi Eaat1 > spen^RNAi (Eaat1-GAL4/UAS-spen^RNAi; NRE-GFP/ +). N = 18 flies per genotype from 3 independent experiments. (E) NRE-GFP fluorescence was quantified in Eaat1 > + (Eaat1-GAL4/ +;NRE-GFP/ +), UAS-spen^RNAi (UAS-spen^RNAi, UAS-Notch^intra;NRE-GFP/), or Notch intracellular domain-overexpressing (Eaat1-GAL4/UAS-Notch^intra; NRE-GFP/ +) flies alone or together with spen^RNAi (Eaat1-GAL4/ UAS-spen^RNAi, UAS-Notch^intra;NRE-GFP/ +). N = 18 flies per genotype from 3 independent experiments. P < 0.001 by the nonparametric Kruskal–Wallis test. (F) Density of Repo-positive glial cells in control (Eaat1-GAL4/ +) or spen^RNAi flies (Eaat1-GAL4/UAS-spen^RNAi). Boxes show the median and upper, and lower quartiles, and the whiskers represent 1.5 times the interquartile range. Repo-positive glial cells were quantified over 12 confocal slices across one 12 µm-thick brain section from each of Drosophila brains. Circles represent individual data points. (G) Simplified proposed scheme showing the Notch activator complex in adult glial cells, which includes the Notch intracellular domain (NICD), Suppressor of hairless (Su(H)), mastermind (Mam), and Spen. The Notch repressor complex includes Su(H), Hairless, and dCtBP (Drosophila C-terminal Binding Protein). Adapted from³⁸.