Fig. 3: CG cells exhibit multiple cell cycle strategies.

a G1 (green), S (magenta) and G2/M (grey) phases of the cell cycle along CG network detected with Fly-FUCCI. FUCCI sensors are labelled in magenta (CycB) and green (E2F1). Scale bar: 25 μm. b Quantification of cell cycle phase distribution in CG by Fly-FUCCI at ALH0 (n = 11), ALH24 (n = 15), ALH48 (n = 23), ALH72 (n = 13) and ALH96 (n = 6) (at 25 °C). n, number of CNS analysed. Stacked bars represent the percentage of cells in each phase. Error bars correspond to SEM. c Quantification of nuclear volume in CG at ALH0 (n = 95), ALH24 (n = 189), ALH48 (n = 140), ALH72 (n = 70) and ALH96 (n = 108) (at 25 °C). n, number of CG nuclei. Results are presented as box and whisker plots. Data statistics: Kruskal–Wallis with a Dunn’s multiple comparison test. d Fluorescence in situ hybridisation (FISH) using probes for chromosomes 2 (Chr2, cyan) and 3 (Chr3, red) in CNS expressing NLS-LacZ (yellow) to mark the CG nuclei. 2n (upper) and >2n (bottom) nuclei are shown. Scale bar: 5 μm. e Quantification of FISH signals in CG nuclei at ALH0 (n = 95), ALH24 (n = 189), ALH48 (n = 140), ALH72 (n = 70) and ALH96 (n = 108). n, number of CG cells analysed. Results are presented as box and whisker plots. Data statistics: two-way ANOVA with a Dunnett’s multiple comparison test. f Still images of a time-lapse movie (Supplementary Movie 2) of a CG expressing Hist::RFP (grey) undergoing endomitosis. Scale bar: 5 μm. g Representative image of a larval VNC expressing Hist::RFP in CG (magenta) and stained with phospho-histone H3 antibody (pHistone-3, green) to visualise mitotic CG nuclei (grey). Scale bar: 20 μm. Higher magnification of separate channels from the region inside the dashed rectangle are shown on the right. h CG mitotic index quantification in larval CNS at ALH0 (n = 15), ALH24 (n = 26), ALH48 (n = 27), ALH72 (n = 13) and ALH96 (n = 13) (at 25 °C). n, number of CNS analysed. Results are presented as box and whisker plots. Data statistics: ordinary one-way ANOVA with a Tukey’s multiple comparison test. i Still images of a time-lapse movie (Supplementary Movie 3) of mitotic CG expressing Hist::RFP (grey). Scale bar: 5 μm. j Expression of mRFP::scra (magenta) in CG to monitor contractile ring and midbody formation. CG membranes and nuclei are labelled with Nrv2::GFP (green) and Hist::IFP (blue) respectively. Arrows indicate midbodies/contractile ring. Scale bar: 10 μm. Higher magnifications of mRFP::scra and Nrv2::GFP separate channels from the region demarcated by the dashed rectangle are shown on the right. k Quantification of the number of midbodies per 100 CG cells in larval VNCs at ALH24 (n = 4), ALH48 (n = 8), ALH72 (n = 4) and ALH96 (n = 4) (at 25 °C). n, number of VNCs analysed. Results are presented as box and whisker plots. Data statistics: ordinary one-way ANOVA with a Tukey’s multiple comparison test. l Representative image of a larval VNC expressing Hist::RFP in CG (magenta) and stained with Drosophila cleaved caspase 1 (Dcp-1, green) to visualise apoptotic CG nuclei (grey). Scale bar: 20 μm. m Quantification of the number of CG nuclei in larval VNCs in control CNS and in CNS where CG-specific downregulation of doubled-parked (dup RNAi) was induced. ALH24 (ctrl, n = 8; dup RNAi n = 9), ALH48 (ctrl, n = 9; dup RNAi n = 12) and ALH72 (ctrl, n = 10; dup RNAi n = 12) (at 29 °C). n, number of VNCs analysed. Results are presented as box and whisker plots. Data statistics: unpaired t-tests were performed for each timepoint. n Quantification of the proportion of Dcp-1⁺ CG nuclei over the whole CG population in larval VNCs in control CNS and in CNS where CG-specific downregulation of doubled-parked (dup RNAi) was induced. ALH24 (ctrl, n = 8; dup RNAi n = 9), ALH48 (ctrl, n = 9; dup RNAi n = 12) and ALH72 (ctrl, n = 10; dup RNAi n = 12) (at 29 °C). n, number of VNCs analysed. Results are presented as box and whisker plots. Data statistics: unpaired two-tailed t-tests were performed for each timepoint. Source data are provided as a Source Data file.