Polyploidy in the adult Drosophila brain
- Molecular, Cellular, and Developmental Biology, University of Michigan, United States
Figures
Figure 1 with 2 supplements
Polyploid cells accumulate in the adult Drosophila brain.
(A,B) Percentage of cells in individual brains exhibiting polyploidy in w1118 (A) and Canton-S (B) male and female whole brains. Age in days indicates days post-eclosion. Box plots showing range, …
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Figure 1—source data 1
Source Data for Figure 1A.
- https://cdn.elifesciences.org/articles/54385/elife-54385-fig1-data1-v1.csv
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Figure 1—source data 2
Source Data for Figure 1B.
- https://cdn.elifesciences.org/articles/54385/elife-54385-fig1-data2-v1.csv
Figure 1—figure supplement 1
Examples of flow cytometry and S-phase labeling.
(A–C) Flow Cytometry gating strategy to ensure doublet discrimination. (D) The small population of mz19-GAL4, UAS nGFP labeled neurons are detectable by flow cytometry assay. (E) Dot plots showing …
Figure 1—figure supplement 2
Polyploidy in Optic lobes is not light or photoreceptor dependent.
(A) 14d Canton-S (CS) males reared in 12 h L/D cycles do not show significantly different polyploidy compared to age-matched CS males reared in 24 hr darkness from eclosion. Error bars = mean ± SEM; …
Figure 2 with 1 supplement
Identification of various neuronal and glial cell types that become polyploid in the adult brain.
Representative flow cytometry dot plots showing polyploid neuronal (A) and glial (B) cells in 2 week old male brain (A) Neuronal nuclei are labeled using nsyb-GAL4, UAS-nGFP, neuronal cells are …
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Figure 2—source data 1
Source Data for Figure 2C.
- https://cdn.elifesciences.org/articles/54385/elife-54385-fig2-data1-v1.csv
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Figure 2—source data 2
Source Data for Figure 2D.
- https://cdn.elifesciences.org/articles/54385/elife-54385-fig2-data2-v1.csv
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Figure 2—source data 3
Source Data for Figure 2E.
- https://cdn.elifesciences.org/articles/54385/elife-54385-fig2-data3-v1.csv
Figure 2—figure supplement 1
Trachea and Sub-perineurial glia comprise less than 5% of all polyploid cells.
(A,B) Micrographs showing expression pattern of moody-GAL4 (A) and (Breathless) btl-GAL4 (B) in the adult brain to label SPGs and tracheal cells respectively. (C) Flow cytometry based quantification …
Figure 3
Very few polyploid cells arise from cell fusion in the adult brain.
(A) Schematic of ‘early labeling’ using CoinFLP to identify potential cell fusion events. Early CoinFLP labeling will label diploid cells either with GFP or RFP. Any double-labeled cells in an …
Figure 4 with 2 supplements
Cells in the Optic Lobes undergo cell cycle re-entry to become polyploid.
(A) Schematic showing labeling protocol for inducing a ‘late-FLP’ in brains where polyploidy is expected to identify polyploid cells in situ. A proportion of cells with multiple copies of the genome …
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Figure 4—source data 1
Source Data for Figure 4C.
- https://cdn.elifesciences.org/articles/54385/elife-54385-fig4-data1-v1.csv
Figure 4—figure supplement 1
CoinFLP double positive cells are polyploid.
Heat-shock induced CoinFLP was used to stochastically label cells in the adult brain with UAS-GFPNLS (magenta), LexAop-RFPNLS (green), or both 24 hr prior to dissection. Optic lobes at 14 days (A–G) …
Figure 4—figure supplement 2
CoinFLP ‘double-labeled’ cells have polyploid DNA content.
(A) DAPI integrated intensity was quantified for 132 nuclei from CoinFLP- labeled 14 day optic lobes using FIJI. Nuclear boundaries were guided by lamin staining for Dapi quantifications from at …
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Figure 4—figure supplement 2—source data 1
Source Data for Figure 4—figure supplement 2 panel A.
- https://cdn.elifesciences.org/articles/54385/elife-54385-fig4-figsupp2-data1-v1.csv
Figure 5 with 2 supplements
DNA damage accumulates with age in the Optic Lobes Changes in gene expression in 21d OL compared to 2d OL shown by GO term analysis.
Padj = adjusted P value. Upregulated GO terms shown in solid purple (A), downregulated GO terms shown in grey bars outlined with purple (B’). Representative images showing pH2AV foci in 1 (C,C’) and …
Figure 5—figure supplement 1
Supplemental RNAseq GO analysis.
(A) Most significantly upregulated GO terms in 7d OL compared to 2d OL. Most significantly upregulated (B) and downregulated (C) GO terms in 21d CB compared to 2d CB. Most significantly upregulated …
Figure 5—figure supplement 2
Validation of RNAseq data Validation of our RNA sequencing dataset was performed by comparing our dataset to other published datasets.
Aggregated data based on (A) age (2d vs 21d, n = 6 replicates), sex (B, n = 9 replicates) or brain region (C n = 9 replicates) was compared to previously published datasets. Bar graphs show Mean log …
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Figure 5—figure supplement 2—source data 1
The list of comparisons and references for validation of RNAseq dataset.
- https://cdn.elifesciences.org/articles/54385/elife-54385-fig5-figsupp2-data1-v1.docx
Figure 6 with 1 supplement
Oxidative stress and DNA damage results in increased polyploidy during early adulthood and polyploid cells are protected from cell death.
(A) Polyploidy in neurons is not p53 dependent. Percentage of polyploidy under each condition was measured in individual 7d male brains (n = 5). (B) w1118 males treated with 2 mM paraquat (PQ) from …
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Figure 6—source data 1
Source Data for Figure 6D.
- https://cdn.elifesciences.org/articles/54385/elife-54385-fig6-data1-v1.csv
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Figure 6—source data 2
Source Data for Figure 6F.
- https://cdn.elifesciences.org/articles/54385/elife-54385-fig6-data2-v1.csv
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Figure 6—source data 3
Source Data for Figure 6H.
- https://cdn.elifesciences.org/articles/54385/elife-54385-fig6-data3-v1.csv
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Figure 6—source data 4
Source Data for Figure 6I.
- https://cdn.elifesciences.org/articles/54385/elife-54385-fig6-data4-v1.csv
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Figure 6—source data 5
Source Data for Figure 6J.
- https://cdn.elifesciences.org/articles/54385/elife-54385-fig6-data5-v1.csv
Figure 6—figure supplement 1
Supplemental Cell death and DNA damage data.
(A) One day and 7d Canton-S brains stained with both Sytox-Green (green) and Propidium Iodide (red) show similar labeling with both cell death markers. (B) Percentage of cells incorporating …
Author response image 1
Author response image 2
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Genetic reagent (D. melanogaster) | w1118 | Bloomington Drosophila Stock Center | BDSC 5905 | isogenic |
| Genetic reagent (D. melanogaster) | Canton-S | O. Shafer lab | n/a | WT |
| Genetic reagent (D. melanogaster) | Oregon-R | C. Collins lab | n/a | WT |
| Genetic reagent (D. americana) | Drosophila americana | P. Wittkopp lab | n/a | Non melanogaster Drosophila |
| Genetic reagent (D. mauritiana) | Drosophila mauritiana | P. Wittkopp lab | n/a | Non melanogaster Drosophila |
| Genetic reagent (D. melanogaster) | glass60J | O. Shafer lab | n/a | Mutant for glass |
| Genetic reagent (D. melanogaster) | w;nSyb-GAL4/Cyo | M. Dus lab | n/a | pan-neuronal |
| Genetic reagent (D. melanogaster) | w;+;nSyb-GAL4 | M. Dus lab | n/a | pan-neuronal |
| Genetic reagent (D. melanogaster) | w;UAS-nGFP;Repo-GAL4, tubulin GAL80TS | Buttitta lab stocks | n/a | pan-glial |
| Genetic reagent (D. melanogaster) | w;UAS-nGFP | Buttitta lab stocks | n/a | UAS nuclear GFP |
| Genetic reagent (D. melanogaster) | w;+;UAS-nGFP | Buttitta lab stocks | n/a | UAS nuclear GFP |
| Genetic reagent (D. melanogaster) | w;Moody-GAL4 | C. Collins lab via Klambt Lab | n/a | Sub-perineurial glia |
| Genetic reagent (D. melanogaster) | y,w;mz19-mCD8::GFP | Bloomington Drosophila Stock Center | BDSC 23300 | Antennal lobe projection neuron |
| Genetic reagent (D. melanogaster) | w1118;ELAV-GAL4,UAS-nGFP | Bloomington Drosophila Stock Center | BDSC 49226 | pan-neuronal |
| Genetic reagent (D. melanogaster) | y,w;breathless-GAL4 | DGRC Kyoto | 105276 | Trachea |
| Genetic reagent (D. melanogaster) | w-;GAD1-GAL4/SM6 | O. Shafer lab | n/a | GABAergic |
| Genetic reagent (D. melanogaster) | w-;OK371-GAL4,UASn-GFP | Buttitta lab stocks | n/a | Glutamatergic |
| Genetic reagent (D. melanogaster) | w;ChaT-GAL4 | O. Shafer lab | n/a | Cholinergic |
| Genetic reagent (D. melanogaster) | w1118;+;GMR-12C11-GAL4 | Bloomington Drosophila Stock Center | BDSC 76324 | Tm3a |
| Genetic reagent (D. melanogaster) | w1118;+;GMR- 42H01-GAL4 | Bloomington Drosophila Stock Center | BDSC 48150 | Dm9 |
| Genetic reagent (D. melanogaster) | w1118;+;GMR-23G11-GAL4 | Bloomington Drosophila Stock Center | BDSC 49043 | Dm4 |
| Genetic reagent (D. melanogaster) | w1118;+;GMR-30B06-GAL4 | Bloomington Drosophila Stock Center | BDSC 47529 | Dm10 |
| Genetic reagent (D. melanogaster) | w1118;+;GMR-26H07-GAL4 | Bloomington Drosophila Stock Center | BDSC 49204 | Dm2 |
| Genetic reagent (D. melanogaster) | y;w;NP3233-GAL4/Cyo | DGRC Kyoto | 113173 | Astrocyte-like |
| Genetic reagent (D. melanogaster) | y;w;NP2222-GAL4/Cyo | DGRC Kyoto | 112830 | Cortex glia |
| Genetic reagent (D. melanogaster) | w;mz97-GAL4 | C. Collins lab via Klambt Lab | n/a | Wrapping glia |
| Genetic reagent (D. melanogaster) | y,w,UAS-mCD8::RFP,LexAop2-mCD8::GFP; CoinFLP-LexA::GAD.GAL4 | Bloomington Drosophila Stock Center | BDSC 59270 and 59271 | CoinFLP |
| Genetic reagent (D. melanogaster) | y,w,hs-FLP;LexAop-RFPnls; UAS-GFPnls | Buttitta lab stocks | n/a | hs-FLP used with with CoinFLP nuclear GFP and RFP |
| Genetic reagent (D. melanogaster) | ey-FLP | Bloomington Drosophila Stock Center | BDSC 5576 | ey-FLP |
| Genetic reagent (D. melanogaster) | y,sev,w;UAS-cdc6RNAi | Bloomington Drosophila Stock Center | BDSC 55734 | cdc6KD |
| Genetic reagent (D. melanogaster) | w; UAS-gemininRNAi | Bloomington Drosophila Stock Center | BDSC 30929 and 50720 | gemininKD |
| Genetic reagent (D. melanogaster) | w1118;GUS-p53 | Bloomington Drosophila Stock Center | BDSC 6584 | UAS-p53WT |
| Genetic reagent (D. melanogaster) | y,w1118; UAS-p53 259N | Bloomington Drosophila Stock Center | BDSC 6582 | UAS-p53DN |
| Antibody | anti-ELAV (rat monoclonal) | Developmental Studies Hybridoma Bank | Rat-ELAV-7E8A10 | 1: 100 |
| Antibody | anti-pH2AV (mouse monoclonal) | Developmental Studies Hybridoma Bank | UNC93-5.2.1 | 1: 100 |
| Antibody | anti-Repo (mouse monoclonal) | Developmental Studies Hybridoma Bank | 8D12 | 1: 100 |
| Antibody | anti-Lamin (mouse monoclonal) | Developmental Studies Hybridoma Bank | ADL67.10 | 1: 100 |
| Antibody | Alexa Fluor 568 anti-mouse (goat polyclonal) | ThermoFisher | A11031 | 1: 1000 |
| Antibody | Alexa Fluor 568 anti-rat (goat polyclonal) | ThermoFisher | A11077 | 1: 1000 |
| Antibody | Alexa Fluor 488 anti-mouse (goat polyclonal) | ThermoFisher | A11029 | 1: 1000 |
| Antibody | Alexa Fluor 488 anti-rat (donkey polyclonal) | ThermoFisher | A21208 | 1: 1000 |
| Other | DAPI | Sigma-Aldrich | D9542 | 1: 1000 |
| Other | Dye-cycle violet | ThermoFisher | V35003 | 2: 1000 |
| Other | Sytox Green | ThermoFisher | S7020 | 2: 1000 |
| Other | Propidium Iodide | Sigma-Aldrich | P4170 | 2.25: 1000 |
Additional files
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Supplementary file 1
A list of antibodies and stains used.
- https://cdn.elifesciences.org/articles/54385/elife-54385-supp1-v1.docx
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Supplementary file 2
A list of Drosophila stocks used.
- https://cdn.elifesciences.org/articles/54385/elife-54385-supp2-v1.docx
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Supplementary file 3
Proportions of cell types polyploid in the whole brain.
- https://cdn.elifesciences.org/articles/54385/elife-54385-supp3-v1.docx
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Supplementary file 4
Proportions of polyploid cell types in the OL.
- https://cdn.elifesciences.org/articles/54385/elife-54385-supp4-v1.docx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/54385/elife-54385-transrepform-v1.docx
