Supplemental Figure Legends



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Supplemental Figure Legends

Fig. S1. Two FlyLight-GAL4 lines and SPARC-GAL4 label antennal lobe ensheathing glia. Confocal sections of adult antennal lobe. (A1, B1, C1, D1) Ensheathing glia processes are labeled by GMR56F03-GAL4 (at 25℃) (A1, C1), GMR10E12-GAL4 (at 18℃) (B1), or SPARC-GAL4 (D1) driven (>) UAS-mCD8GFP. Ensheathing glia nuclei are marked by UAS-nuclear-LacZ. (A2, B2) Green shows glia nuclei stained by the Repo antibody. (A3, B3) The channel for Repo staining is overlaid on the nuclear-LacZ channel. (C–D) Co-labeling of ensheathing glia (mCD8GFP and nuclear-lacZ) and astrocyte marker GAT, visualized by antibody staining (white). GAT staining is overlaid on the nuclear-LacZ channel (C2, D2) to show that GAT does not label GAL4+ cells, with few exceptions (arrows). (E) Co-labeling of ensheathing glia (SPARC-QF>QUAS-mtdT and QUAS-nuclear-lacZ, green and magenta) and astrocytes (Alrm-GAL4>mCD8GFP, white). The GFP channel is overlaid on the nuclear-LacZ channel (E2) to show that the group of glia marked by SPARC-QF are not astrocytes. Scale bars are 10 µm.



Fig. S2. P35 suppresses the reduction of ensheathing glia number caused by htl knockdown. (A–B) Projections of confocal sections along the Z-axis for wild type (A) and UAS-Htl RNAi (VDRC 27180) together with UAS-P35 driven by SPARC-GAL4. Red: ensheathing glia cell bodies marked by SPARC-GAL4>UAS-nuclear-LacZ. Blue: Ncad antibody stains for antennal lobe neuropil. (C) Quantification of ensheathing glia cell number in each antennal lobe. (D–E) Confocal sections for the antennal lobe at 96 h APF of wild type (D) and UAS-Htl RNAi (VDRC 27180) together with UAS-P35 driven by SPARC-GAL4 (E). Ensheathing glia processes are labeled by SPARC-GAL4>UAS-mCD8GFP. Neuropil compartments are stained with Ncad antibody. (F–H) Quantification of the intensity of ensheathing process within the antennal lobe (F), relative standard deviation of Ncad staining (G), and the correlation coefficient for the intensities of ensheathing glia processes and neuropil staining (H). Scale bars are 10 µm. Error bars represent SD. **, p<0.01; ****, p<0.0001; ns, p>0.05.

Fig. S3. Additional MARCM clone examples and analysis. (A–C) Green shows wild-type ensheathing glia processes labeled by GMR10E12-GAL4>UAS-mCD8GFP. Blue shows ensheathing glia cell bodies marked by UAS-nuclear-LacZ. Magenta shows neuropil staining by the Ncad antibody. Scale bar is 10 µm. D, dorsal; L, lateral. An ensheathing glia located on the dorsal surface of the antennal lobe (A3) extends processes ventrally and semi-circles the DA3 glomerulus (A2). An ensheathing glia located on the medial surface of the antennal lobe (B3) extends processes laterally to access glomeruli in the center of the antennal lobe (B2, B3). An ensheathing glia located on the most anterior surface of the antennal lobe (C2) extends processes into posterior antennal lobe (C4). (D) Quantification of the total intensity of processes from each ensheathing glia (wild-type and HtlAb42/Ab42) labeled by MARCM. Error bars represent SD. **, p<0.01.

Fig. S4. Orb0449-GAL4 labels local interneurons in the antennal lobe. (A) Green shows orb0449-GAL4>UAS-mCD8GFP. (A1) A projection of confocal sections along the Z-axis at 24 h APF. Around 23 local interneurons are labeled. (A2) A confocal section of antennal lobe at 96 h APF. A total of ~55 local interneurons are labeled over all confocal sections of the antennal lobe. (B) ey-FLP intersects with orb0449-GAL4 together with UAS-FRT-stop-FRT-mCD8GFP to show that orb0449-GAL4 is inactive in the majority of ORNs, except two classes that project to the medial side of the antennal lobe (arrow). (C) GH146-FLP intersection shows a rare case in which an interneuron is labeled (1 cell out of 10 antennal lobes), whereas around other antennal lobes 0 cell is labeled. Magenta shows neuropil staining by the Ncad antibody. Scale bar is 10 µm.

Fig. S5. Loss of Thisbe from ORNs and PNs does not cause ensheathing glia or glomerulus defects. (A) ey-FLP MARCM combined with cell lethal strategy creates ths mutant in nearly all ORNs. Ensheathing glia processes are labeled by GMR56F03-GAL4>UAS-mCD8GFP. (B) GH146-GAL4 drives RNAi against ths. Ensheathing glia processes are labeled by SPARC- QF>QUAS-mtdT. Magenta shows neuropil staining by the Ncad antibody. Scale bar is 10 µm. (C-F) Quantification of ensheathing glia process intensities (C, E), and relative standard deviation of Ncad staining intensities (D, F). Error bars represent SD. ns, p>0.05.


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