Neuropil glia proliferation in normal conditions and upon crush injury in adults

A, Schematic representation of Drosophila adult Central Nervous System (CNS) and Ventral Nerve Cord (VNC), indicating the MtN, where the injury is performed. B, Representative confocal image showing dividing cells (EdU+) with ALG identity (Repo+ and Pros+) and glial identity different from ALG (Repo+, Pros-). C, Number of ALG or EG cells in G2-M per MtN, in normal conditions and 24 h after injury. Paired t-test, *P<0.05, **P<0.01. C’-C’’, Representative images of ALG in G2-M showing their position within the injured area (C’) and a magnification showing the detail of GFP+RFP+ cells (C’’). D, Diagram of how the Twin-Spot MARCM tool works. D’, Representative confocal image of ALG clones generated with Twin-Spot MARCM. E, Number of VNCs with/without clones in controls and Injured VNCs in young (1 day old) and mature (7 day old) animals. Chi-square binomial test, **P<0.01, ****P<0.0001. F-G, Representative confocal images of a two-color ALG clone (F) and a single-color ALG clone (G). H, Percentage of ALG single-color clones in injured controls and Injured VNCs where apoptosis was inhibited in ALG. Unpaired t-test **P<0.01. Genotypes: wild type (B); AlrmGal4>UAS-Fly-FUCCI (C-C’); R56F03Gal4>UAS-Fly-FUCCI (C); HsFLP; UAS-GFP, UAS-RFPRNAi /UAS-RFP, UAS-GFPRNAi; tubGal80ts:alrmGal4; (D’-H): HsFLP; UAS-GFP, UAS-RFPRNAi /UAS-RFP, UAS-GFPRNAi; tubGal80ts:alrmGal4/UAS-p35 (H). Scale bars: 50 μm (B, C, E); 15 μm (C’’, F,G)

Neuropil glia transdifferentiate to two different cell types

A-B. Representative confocal slices showing transdifferentiated EG cells with the G-TRACE tool. A, Medial slice showing NP-like cells position within the MtN. B, Zoom slice (merge and separated channels) showing NP-like cells (GPF+, RFP-) have glial identity (repo+). A’, Ventral slice showing VC cells position within the MtN. B’, Zoom slice (merge and separated channels) showing VC cells (GPF+, RFP-) are not glia (repo-). C, Representative ventral slice showing transdifferentiated CV cells visualised with G-TRACE toll driven by the pan-glial factor Repo-Gal4. D, Number of NP-like cells originated from ALG or EG in not injured and injured MtM. Mann Whitney test ns p>0.05, **P<0.01. E, Percentage of NP-like cells compared with the total number of NP glia that originate them. Scale bars: 50 μm (A, A’, C); 15 μm (B,B’). Genotypes: tubGal80ts, R56F03Gal4>UAS G-TRACE (A, B, D, E); tubGal80ts, repoGal4>UAS G-TRACE (C); tubGal80ts, AlrmGal4>UAS G-TRACE (D, E).

EG transdifferentiation into ALG requires prospero

A. Representative image of NP-like cells originated from EG stained with ALG marker Prospero. Top left: Merge image with DAPI. Top right: Merge image without DAPI. Bottom left: GFP signal. Bottom right: RFP signal and pros staining. B, Number of NP-like cells originated from EG in controls animals where pros was inhibited in EG. Unpaired t-test *P<0.05. Scale bar: 15 μm (A). Genotypes: tubGal80ts, R56F03Gal4>UAS G-TRACE (A, B)

EG and ALG transdifferentiate into neurons upon injury through two different mechanisms

A-A’.Transdifferentiated VC cells (GFP+,RFP-) raised from ALG (A) and EG (A’) are elav+. From left to right: Merge with DAPI, merge without DAPI and green and red channels merged. B, Number of transdifferentiated glia (VC cells, GFP+, RFP-) in controls and injured VNCs. STATS. C, Number of transdifferentiated VC cells (GFP+, RFP-) originates from ALG or EG in controls and injured mature animals. D, Number of ALG or EG that remain ALG or EG in controls and injured VNCs. Statistics (B, C, D): Paired t-test ***p<0.001, **p<0.01, ns p>0.05. Scale bar: 15 μm (A). Genotypes: tubGal80ts, R56F03Gal4>UAS G-TRACE and tubGal80ts, AlrmGal4>UAS G-TRACE

New neurons do not co-localize with markers for mature neurons.

Representative confocal images of VC cells (GFP+, RFP+) stained with DAPI (grey) and different antibodies in magenta, anti-ChAT (A), GluRIIA (B) and GABA (C). Dotted white circles indicate the VC cells. Scale bar: 15 μm (A). Genotypes: tubGal80ts, repoGal4>UAS G-TRACE.