Polyploidy in the adult Drosophila brain

  1. Shyama Nandakumar
  2. Olga Grushko
  3. Laura A Buttitta  Is a corresponding author
  1. Molecular, Cellular, and Developmental Biology, University of Michigan, United States
8 figures, 1 table and 5 additional files

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, …

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 …

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 …

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 …

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 …

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 …

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 …

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
DesignationSource or
reference
IdentifiersAdditional
information
Genetic reagent (D. melanogaster)w1118Bloomington Drosophila Stock CenterBDSC 5905isogenic
Genetic reagent (D. melanogaster)Canton-SO. Shafer labn/aWT
Genetic reagent (D. melanogaster)Oregon-RC. Collins labn/aWT
Genetic reagent (D. americana)Drosophila americanaP. Wittkopp labn/aNon melanogaster Drosophila
Genetic reagent (D. mauritiana)Drosophila mauritianaP. Wittkopp labn/aNon melanogaster Drosophila
Genetic reagent (D. melanogaster)glass60JO. Shafer labn/aMutant for glass
Genetic reagent (D. melanogaster)w;nSyb-GAL4/CyoM. Dus labn/apan-neuronal
Genetic reagent (D. melanogaster)w;+;nSyb-GAL4M. Dus labn/apan-neuronal
Genetic reagent (D. melanogaster)w;UAS-nGFP;Repo-GAL4, tubulin GAL80TSButtitta lab stocksn/apan-glial
Genetic reagent (D. melanogaster)w;UAS-nGFPButtitta lab stocksn/aUAS nuclear GFP
Genetic reagent (D. melanogaster)w;+;UAS-nGFPButtitta lab stocksn/aUAS nuclear GFP
Genetic reagent (D. melanogaster)w;Moody-GAL4C. Collins lab via Klambt Labn/aSub-perineurial glia
Genetic reagent (D. melanogaster)y,w;mz19-mCD8::GFPBloomington Drosophila Stock CenterBDSC 23300Antennal lobe projection neuron
Genetic reagent (D. melanogaster)w1118;ELAV-GAL4,UAS-nGFPBloomington Drosophila Stock CenterBDSC 49226pan-neuronal
Genetic reagent (D. melanogaster)y,w;breathless-GAL4DGRC Kyoto105276Trachea
Genetic reagent (D. melanogaster)w-;GAD1-GAL4/SM6O. Shafer labn/aGABAergic
Genetic reagent (D. melanogaster)w-;OK371-GAL4,UASn-GFPButtitta lab stocksn/aGlutamatergic
Genetic reagent (D. melanogaster)w;ChaT-GAL4O. Shafer labn/aCholinergic
Genetic reagent (D. melanogaster)w1118;+;GMR-12C11-GAL4Bloomington Drosophila Stock CenterBDSC 76324Tm3a
Genetic reagent (D. melanogaster)w1118;+;GMR-
42H01-GAL4
Bloomington Drosophila Stock CenterBDSC 48150Dm9
Genetic reagent (D. melanogaster)w1118;+;GMR-23G11-GAL4Bloomington Drosophila Stock CenterBDSC 49043Dm4
Genetic reagent (D. melanogaster)w1118;+;GMR-30B06-GAL4Bloomington Drosophila Stock CenterBDSC 47529Dm10
Genetic reagent (D. melanogaster)w1118;+;GMR-26H07-GAL4Bloomington Drosophila Stock CenterBDSC 49204Dm2
Genetic reagent (D. melanogaster)y;w;NP3233-GAL4/CyoDGRC Kyoto113173Astrocyte-like
Genetic reagent (D. melanogaster)y;w;NP2222-GAL4/CyoDGRC Kyoto112830Cortex glia
Genetic reagent (D. melanogaster)w;mz97-GAL4C. Collins lab via Klambt Labn/aWrapping glia
Genetic reagent (D. melanogaster)y,w,UAS-mCD8::RFP,LexAop2-mCD8::GFP; CoinFLP-LexA::GAD.GAL4Bloomington Drosophila Stock CenterBDSC 59270 and 59271CoinFLP
Genetic reagent (D. melanogaster)y,w,hs-FLP;LexAop-RFPnls; UAS-GFPnlsButtitta lab stocksn/ahs-FLP used with with CoinFLP nuclear GFP and RFP
Genetic reagent (D. melanogaster)ey-FLPBloomington Drosophila Stock CenterBDSC 5576ey-FLP
Genetic reagent (D. melanogaster)y,sev,w;UAS-cdc6RNAiBloomington Drosophila Stock CenterBDSC 55734cdc6KD
Genetic reagent (D. melanogaster)w; UAS-gemininRNAiBloomington Drosophila Stock CenterBDSC 30929 and 50720gemininKD
Genetic reagent (D. melanogaster)w1118;GUS-p53Bloomington Drosophila Stock CenterBDSC 6584UAS-p53WT
Genetic reagent (D. melanogaster)y,w1118; UAS-p53 259NBloomington Drosophila Stock CenterBDSC 6582UAS-p53DN
Antibodyanti-ELAV
(rat monoclonal)
Developmental Studies Hybridoma BankRat-ELAV-7E8A101: 100
Antibodyanti-pH2AV
(mouse monoclonal)
Developmental Studies Hybridoma BankUNC93-5.2.11: 100
Antibodyanti-Repo
(mouse monoclonal)
Developmental Studies Hybridoma Bank8D121: 100
Antibodyanti-Lamin
(mouse monoclonal)
Developmental Studies Hybridoma BankADL67.101: 100
AntibodyAlexa Fluor 568 anti-mouse
(goat polyclonal)
ThermoFisherA110311: 1000
AntibodyAlexa Fluor 568 anti-rat (goat polyclonal)ThermoFisherA110771: 1000
AntibodyAlexa Fluor 488 anti-mouse
(goat polyclonal)
ThermoFisherA110291: 1000
AntibodyAlexa Fluor 488 anti-rat
(donkey polyclonal)
ThermoFisherA212081: 1000
OtherDAPISigma-AldrichD95421: 1000
OtherDye-cycle violetThermoFisherV350032: 1000
OtherSytox GreenThermoFisherS70202: 1000
OtherPropidium IodideSigma-AldrichP41702.25: 1000

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