The Drosophila adult central brains are post-mitotic

(A) (Left panels) Representative confocal images of control (w1118) adult central brains ubiquitously expressing the Fly-FUCCI cell cycle reporters, which label cells in distinct phases of the cell cycle. GFP-tagged E2F1 amino acid 1-230 fragment (EGFP-E2F1, green) marks cells in G1/G0 phase, while RFP-tagged Cyclin B amino acid 1-266 fragment (mRFP-NLS-CycB, red) marks cells in S/G2/M phases. Brains were counterstained with DAPI (blue) for DNA and phosphorylated histone H3 (pH3, grey) to label mitotic cells. Upper panels show anterior views, and lower panels show posterior views. In the adult central brain, nearly all cells are GFP-positive and lack RFP signals, confirming that they reside in the G1/G0 phase. No pH3-positive mitotic cells were detected, indicating that neurogenesis does not occur under normal conditions. A small number of cells near the antennal lobes exhibit weak mRFP-NLS-CycB signals, suggesting rare G2-phase cells. Scale bars: 50 μm. (Right) Schematic diagram of the Fly-FUCCI system, illustrating its ability to label cell cycle states based on the differential degradation of GFP-E2F1 and RFP-CycB. (B) (Top) tSNE plot depicting the clustering of scRNAseq data from adult Drosophila brains (Davie et al., 2018). Cells are classified into 17 distinct clusters, with different colours representing different neuronal, glial, and neuroblast populations. (Bottom) Dot plot showing the expression of 112 selected CCR genes across the identified clusters. The colour intensity represents the average expression level, with darker red indicating higher expression, and dot size indicating the proportion of cells within each cluster expressing the respective gene. (C) Dot plot showing the expression patterns of CCR genes across 29 cell clusters identified from the single-cell RNA sequencing dataset of Drosophila larval brains (Avalos et al., 2019). Colour intensity and dot size are as described in (B). These analyses reveal that most positive CCRs are highly expressed in neuroblast clusters in larval brains but are minimally expressed in adult neuronal and glial clusters, supporting the postmitotic state of the adult brain.

Neuroblast-specific Kr depletion and KrIf-1 mutation causes mushroom body neuroblast retention and prolonged neurogenesis in adult brains.

(A) Representative images of EdU-labeled proliferative clones in adult central brains. Adult flies of control Kr wild type (Kr+/Kr+, upper left panel), heterozygous KrIf-1 mutant (KrIf-1/Kr+, upper right panel), UAS control insc>lacZ (lower left panel), and KrRNAi insc>KrIR#1 (lower right panel) were subjected to EdU incorporation assays and stained with DAPI (blue) and EdU (red). In both control brains, no EdU-positive clones were detected. In contrast, insc>KrIR#1 and KrIf-1 mutant brains exhibited multiple EdU-positive clones per hemisphere, predominantly in the dorsoposterior region. Scale bars: 100 µm. (B) Quantification of EdU-positive clones in adult brains aged 4–6 days, 12–14 days, and 19–21 days post-eclosion. Scatter dot plots represent the number of EdU-positive clones per hemisphere. The means and standard deviations are depicted by thick and thin red bars, respectively, with actual values annotated above. Statistical significance was determined using the Mann-Whitney U test (*:p ≤ 0.05, **:p ≤ 0.01, ***: p ≤ 0.001, ****: p < 0.0001, ns = non-significant). ‘n’ represents the number of brain hemispheres analysed per condition. (C) Kr wild type (left) and KrIf-1 mutant (right) adult brains expressing mCD8::GFP (green) under the NB-specific insc-Gal4 driver were stained with DAPI (blue), pH3 (red), and Mira (gray). Mitotic and interphase NBs, identified by co-expression of Mira and GFP, were detected in the dorsoposterior regions of KrIf-1 mutant brains but not in controls. Scale bars: 50 µm. (D) insc>KrIR#2 adult brains co-expressing mCD8::GFP were stained with DAPI (blue), EdU (red), and the pan-neuronal marker Elav (gray). EdU incorporation was observed in Elav-positive neurons, indicating continued neurogenesis in adult brains upon Kr depletion. Scale bars: 50 µm. (E) Wild-type adult brain expressing mCD8::GFP (green) under the mushroom body (MB)-specific OK107-Gal4 driver to visualise the location and the structure of the MBs, stained with DAPI (blue). Posterior and anterior views are shown. The MB cell body region is located dorsoposteriorly, while MB lobes are visible in the anterior view. Scale bars: 100 µm. (F) KrIf-1 mutant adult brains expressing mCD8::GFP driven by the MB-specific driver mb247-Gal4 (green), stained with DAPI (blue), pH3 (red), and Mira (grey). Mitotic cells in the MB cell body region exhibited cortical Mira localisation, characteristic of dividing NBs. Scale bars: 50 µm. (G) Representative images of KrIf-1 mutant brains expressing mCD8::GFP under OK107-Gal4 after EdU labelling. EdU incorporation was observed in MBNBs, weakly marked by OK107>GFP expression and Mira staining. Scale bars: 10 µm.

Kr is expressed in MBNBs and regulates their elimination during the pupal stage

(A) Representative images of the MB cell body region in wild-type pupal brains (54–60 h APF) expressing the Kr::GFP(Bac) reporter and stained with two Kr-specific antibodies (rat anti-Kr: red, rabbit anti-Kr: cyan) and DAPI (grey). MBNBs, identified by their cell size and position within the MB cell body region, exhibit weak Kr signals detected by both antibodies and Kr::GFP (green). Scale bar: 20 µm. (B) Quantification of Kr expression in MBNBs during pupal development. (Left) Scatter dot plots showing normalised Kr signal intensities inside and outside individual MBNBs at different pupal stages. (Right) Kr signal enrichment in MBNBs, calculated as the ratio of intra-MBNB signal intensity to its surrounding area. The means and standard deviations are shown by thick and thin red bars, respectively. ‘n’ represents the number of brain hemispheres analysed per condition. Statistical analyses were performed using two-way ANOVA with Šídák’s post-hoc test (left panel) and one-way ANOVA with Tukey’s post-hoc test (right panel). Significance levels: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p < 0.0001, ns = non-significant. See Materials and methods for details on quantification. (C) (Left) Experimental design for the temporal knockdown of Kr during different developmental stages using insc-Gal4, alongside pTub-Gal80ts, and Kr RNAi (KrIR#2). A table summarises nine experimental conditions with temperature shifts: flies raised at 29°C (pale red boxes) induced Kr RNAi, while those at 19°C (pale blue boxes) suppressed RNAi by Gal80ts. ‘n’ indicates the number of brain hemispheres used for EdU-positive clone counting in each condition. (Right) Quantification of EdU-positive clones in adult brains from different experimental conditions. Scatter dot plots show the number of EdU-positive clones per hemisphere. Greyed boxes and red bars represent the means and standard deviations, respectively, with actual values annotated above. n’ represents the number of hemispheres analysed per condition. Pair-wise Mann-Whitney U tests was used for statistical analysis. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p < 0.0001. (D) Representative images of posterior adult brain regions from Group 2 (control) and Group 5 (Kr RNAi during pupal stage) conditions in (C), stained for DAPI (blue), Mira (green), and EdU (red). Persistent EdU-positive clones were observed in Kr RNAi conditions. Scale bar: 100 µm.

Kr regulates Imp expression to promote MBNB proliferation and survival.

(A) Representative confocal images of dorsoposterior regions of wild type, KrIf-1 mutant, and insc>KrIR adult brains. EdU labelling (red) marks proliferating cells, and Imp expression (green) was visualised using an Imp-specific antibody. DAPI (blue) stains nuclei. Compared to controls, Imp-expressing regions around the MB cell body region appear expanded in KrIf-1 and insc>KrIR brains. Scale bars: 50 µm. (B) Quantification of Imp-expressing areas within MB cell body regions. (Left) Representative 3D reconstructions of adult brain dorsoposterior surfaces for the indicated genotypes, with Imp (red), DAPI (blue), and OK107-Gal4-driven GFP (green) marking MB lineages. Scale bars: 100 µm. (Right) Scatter dot plots showing Imp+ area ratios to MB cell body regions on the dorsoposterior brain surface, quantified per hemisphere. Thick and thin red bars indicate means and SDs, respectively. ‘n’ represents the number of hemispheres analysed per condition. Mann-Whitney tests were performed for statistical significance. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, **p < 0.0001, ns = non-significant. (C–E) Imp and Syp expression in persisting MBNBs in Kr RNAi and KrIf-1 mutant adult brains. (C) Representative image of active MBNBs in insc>KrIR#2 brains, immunostained for Imp (upper panels) and Syp (lower panels). Merged images show Imp/Syp (green), EdU (red), and DAPI (grey), with individual Imp or Syp channels in grey. Arrowheads indicate mitotic MBNBs, identified by EdU signals and cell morphology. Scale bars: 20 µm. (D) Representative confocal images of KrIf-1 mutant adult brains, showing co-expression of Imp (green) and Syp (blue) in active (EdU+) MBNBs. Arrowheads indicate mitotic MBNBs. Single-channel Imp and Syp signals are shown in grey. Scale bars: 20 µm. (E) Counting of mitotic MBNBs expressing Imp and/or Syp in KrIf-1 and insc>KrIR adult brains. Upper and lower tables show the number of Imp+ and Syp+ mitotic MBNBs, respectively. Almost all retained MBNBs co-expressed Imp and Syp at high levels, an atypical phenotype for NB progression. (F) Imp depletion suppresses MBNB persistence in Kr RNAi brains. (Left) Representative confocal images of adult brains from insc>KrIR#2, mCherryIR (upper panels) and insc>KrIR#2, ImpIR (lower panels), immunostained for pH3 (red), insc>GFP (green), Mira (grey), and DAPI (blue). While mitotic MBNBs persisted in insc>KrIR#2, mCherryIR brains, they were absent in insc>KrIR#2, ImpIR brains. Scale bars: 100 µm. (Right) Scatter dot plots of mitotic MBNB (pH3+) counts per hemisphere, with means and SDs depicted by thick and thin red bars, respectively. ‘n’ represents the number of hemispheres analysed. Mann-Whitney test: p = 0.0006 (****). insc>ImpIR alone was lethal, preventing adult brain analysis.

Kr antagonises Kr-h1 to promote MBNB cell cycle exit and prevent neuroblast overgrowth.

(A) Kr-h1 co-depletion suppresses MBNB persistence in Kr RNAi adult brains. (Left) Representative confocal images of insc>Kr-h1IR, insc>KrIR#2, mCherryIR, and insc>KrIR#2, Kr-h1IR adult brains, immunostained for EdU (red), insc>GFP (green), Mira (grey), and DAPI (blue). While Kr-h1 RNAi alone did not affect MBNB elimination, the number of persisting EdU-labelled MBNB clones observed in insc>KrIR#2, mCherryIR brains was significantly reduced when Kr-h1 was co-depleted in NBs. Scale bars: 100 µm. (Right) Quantification of EdU-positive MBNB clones per hemisphere in indicated genotypes. Scatter dot plots display individual data points, with means and SDs represented by thick and thin red bars, respectively. ‘n’ represents the number of hemispheres analysed. Kruskal-Wallis test followed by Dunn’s post-hoc test. **Significance levels: ** *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, **p < 0.0001, ns = non-significant. (B, C) Kr-h1 overexpression promotes MBNB proliferation and blocks differentiation, leading to a neuroblastoma-like phenotype. (B) Representative posterior views of adult brains overexpressing Kr-h1 in NBs (insc>Kr-h1::flag), immunostained for DAPI (blue), insc>GFP (green), EdU (red), and Mira (grey), with magnified views of the MB cell body regions (right panels). Most EdU-positive clones were composed of cells co-expressing Mira and insc>GFP, indicating their NB-like status and a failure in differentiation. Scale bars: 100 µm. (C) Kr-h1 overexpression inhibits neuronal differentiation. Confocal images of insc>Kr-h1::flag adult brains stained for DAPI (blue), insc>GFP (green), EdU (red), and Elav (grey). Unlike control brains, EdU-labelled clones in Kr-h1-overexpressing brains contained very few Elav-positive neurons, indicating a block in neuronal differentiation. Scale bars: 100 µm.

Kr regulates MB development by promoting MBNB elimination and facilitating neuronal fate transitions.

(A) Representative confocal images of anterior views of adult brains from OK107, insc>mCherryIR, GFP (control) and OK107, insc>KrIR#2, GFP flies, with MB lobe structures visualised by OK107>GFP (green). No overt structural abnormalities were observed in MB lobes upon Kr depletion. Scale bars: 50 μm. (B) Representative images of MB α/β lobe morphology in wild-type and insc>KrIR#2, GFP adult brains, visualised by Fasciclin II (FasII) staining (red). Kr depletion led to thinner and more curved α/β lobes compared to controls. Scale bars: 50 μm. (C) Effects of Kr overexpression in MB lineages on MB development. Anterior views of 3D-reconstructed confocal images of adult brains from control (no UAS gene), OK107>KrIR#2, KrIf-1 mutant and OK107>Kr::V5 flies, with MBs visualised by OK107>GFP (green). While control, Kr knockdown and KrIf-1 mutant adult brains exhibited normal MB morphology, Kr overexpression (OK107>Kr::V5) led tto high lethality. In the few surviving adult flies, there was severe disorganisation of MB lobes, particularly with pronounced reduction in α/β and α’/β’ lobes and extensive disarray in γ lobes. Scale bars: 50 μm. (D) A proposed model for Kr function in MBNB regulation. During early developmental stages, specifically from mid-L3 to late L3, elevated levels of Imp and Kr-h1 are key drivers of MBNB proliferation and early neuronal fate specification. As development transitions into the pupal stage, Kr assumes a critical role by repressing Imp and counteracting Kr-h1. This dual action by Kr facilitates cell cycle exit and ensures proper transitions in MB neuronal fate. Further complexity in MBNB regulation is introduced by extracellular signaling pathways. The Activin/Babo pathway contributes to the suppression of Imp, enhancing the transition control exerted by Kr. Concurrently, delayed ecdysone signaling, mediated through the Kr-h1-E93 pathway, plays a pivotal role in facilitating MBNB elimination. Kr, functioning as a transcription factor, may endow MBNBs with the specific competence necessary to integrate and coordinate these extracellular signals, which are essential for regulating MBNB proliferation and fate transitions. The absence of Kr disrupts this finely tuned regulatory mechanism, leading to continued MBNB proliferation and neurogenesis in adult brains. This disruption may also delay the transition of MB neural fates, underscoring Kr’s integral role in maintaining developmental timelines and structural integrity within the MBs.

Expression of CCR genes across all 84 cell clusters in the adult Drosophila brain.

(Top) tSNE plot depicting 84 cell clusters identified in the adult Drosophila brain (Davie et al., 2018). (Bottom) A dot plot illustrating the expression levels of 112 CCRs across 84 cell clusters. Each dot represents the average expression level of a CCR gene within a cluster, with colour intensity indicating expression level (darker red corresponds to higher expression), and dot size representing the proportion of cells within the cluster expressing the gene. The data show that positive regulators of the cell cycle, including Cdk1, Cdk2, CycB, and Polo, are largely absent from neuronal and glial clusters but are enriched in the small population of neuroblast-like cells. In contrast, negative regulators, such as fzr, Wee1, and rux, remain broadly expressed across neuronal clusters, reinforcing the molecular mechanisms that maintain the postmitotic state of the adult brain.

Forced expression of positive CCRs induces mitotic entry in mature neurons.

(A) Overexpression of the positive CCR combination Dp-E2F1 and CycE-Cdk2 in dopaminergic neurons using TH-Gal4 (left panels) and in mushroom body neurons using mb247-Gal4 (right panels) induces mitotic entry in young adult flies (∼2 days post-eclosion at 19°C). Flies were incubated at 29°C for 10 days to sustain CCR gene expression. pH3 immunostaining reveals mitotic neurons in these brains, whereas no pH3-positive neurons were observed in controls. However, when gene induction was performed in older flies (10 days post-eclosion at 29°C), no pH3-positive neurons were detected (n ≥ 10), suggesting that neurons become increasingly refractory to cell cycle entry with age. Scale bars: 50 μm. (B) Overexpression of Dp-E2F1 and CycE-Cdk2 in dopaminergic neurons during the late pupal stage (∼2 days before eclosion) allowed some adult flies to emerge; however, these flies exhibited premature lethality, dying within 3–5 days post-eclosion. Immunostaining of dissected brains 5 days after gene induction revealed pH3-positive neurons, with nearly half of the examined flies showing positive signals (12 out of 27 brains examined). Some pH3-positive neurons also exhibited apoptotic marker Dcp-1 (grey), indicating that forced mitotic re-entry may induce cell death. Overexpression of the same CCR combination in the mushroom body using mb247-Gal4 resulted in 100% lethality before eclosion, preventing adult emergence. (C) Survival analysis of flies overexpressing positive CCRs in dopaminergic neurons. Kaplan-Meier survival curves comparing adult flies overexpressing Dp-E2F1 and CycE-Cdk2 in dopaminergic neurons (TH>E2F-Dp, CycE-Cdk2, mCD8::GFP, gal80ts) with control flies expressing lacZ (TH>lacZ, mCD8::GFP, gal80ts). Gene expression was induced by shifting 2-day-old adult flies from 19°C to 29°C. Flies overexpressing CCRs exhibited significantly shorter lifespans compared to controls (P < 0.0001, Log-rank Mantel-Cox test). Pale blue and pale pink lines represent individual survival curves from three experimental replicates, while dark blue and red thicker lines indicate the mean survival for the experimental and control groups, respectively. Error bars indicate SD. These findings indicate that forced mitotic re-entry leads to reduced longevity, likely due to apoptosis or mitotic catastrophe in neurons.

Kr expression in MBNBs during embryonic and larval stages

(A, B) Representative confocal images of the embryonic central nervous system (CNS) (Stage 12) in wild-type embryos stained with rabbit anti-Kr antibody (red), OK107>GFP (green), DAPI (blue), and Mira (grey). (Left panel) Zoomed-out confocal image showing the overall CNS structure. (Right panels) Magnified views of MBNBs, showing merged coloured images and individual grey-scale channels. Yellow asterisks indicate MBNBs identified by OK107>GFP and Mira co-expression. (B) Arrows indicate a neuron within the MB lineage expressing Kr. Although Kr was broadly expressed in the embryonic CNS, it was absent from MBNBs. Scale bars: 20 µm. (C-E) Kr expression in third-instar larval brains. (C) A confocal image of the MB cell body region stained for OK107>GFP (green), Mira (cyan), and Kr (red) using a rat anti-Kr antibody, with the individual Kr channel shown in grey on the right. Asterisks mark MBNBs, which exhibit weak Kr signals. (D) Kr::GFP(Bac) reporter expression in the MB cell body region, showing Kr::GFP (green), DAPI (grey), and Mira (red), with the individual GFP channel shown in grey on the right. Asterisks mark MBNBs. Strong Kr::GFP signals were detected in single cells adjacent to MBNBs, likely representing GMCs or early-born neurons, which might inherit Kr::GFP from MBNBs. (E) A confocal image of the MB calyx area stained for OK107>GFP (green), Mira (cyan), and Kr (red), with the individual Kr channel shown in grey. Kr-expressing neurons near the MB calyx are indicated by arrowheads. Scale bars: 20 µm.

Syp expression in persisting MBNBs of Kr RNAi and KrIf-1 mutant adult brains.

(A) Representative confocal images of wild type, KrIf-1 mutant, and insc>KrIR adult brains, showing dorsoposterior MB cell body regions. EdU (red) labels proliferating cells, Syp expression (green) is visualised with Syp-specific antibodies, and nuclei are stained with DAPI (blue). Unlike Imp, Syp-expressing regions remained largely unchanged despite Kr depletion. Scale bars: 50 µm. (B) Imp depletion suppresses the expansion of mitotic MBNBs in Kr RNAi adult brains. (Left) Representative confocal images of control insc>mCherryIR (upper panels), insc>KrIR#2, mCherryIR (middle panels), and insc>KrIR#2, ImpIR (lower panels) brains, immunostained for Imp (red), insc>GFP (green), and DAPI (blue). Induction of Imp RNAi under insc-Gal4 effectively depleted Imp expression in brain tissue including MB lineages. Scale bars: 50 µm.