Figure 5: Aneuploid Nbs undergo premature differentiation in Pros-dependent manner.

WT and SakOE,mad2 brain pictures stained with Dpn (purple, middle panel), Pros (green, right panel), phalloidin is in yellow and DNA in blue. The yellow dashed line labels the CB. The large nuclei seen in the bottom inset might have resulted from cytokinesis defects, as described in Fig. 1. Scale bar, 50 μm. (b,c) Graph bars showing the percentage of Dpn⁺ and Dpn⁺, Pros⁺ cells in WT (525 cells/12 lobes), SakOE,mad2 (324 cells/16 lobes) and bub3 (103 cells/11 lobes) in (b) and the percentage of Dpn⁺ and Dpn⁺, Elav⁺ cells in WT (192 cells/4 lobes), SakOE,mad2 (277 cells/ 16 lobes) and bub3 (118 cells/10 lobes) in (c). Statistical significance determined using Fisher’s exact test: in b- **(P=0.0012 SakOE,mad2 and 0.0034 bub3), in c- *(P=0.024) and **(P=0.0012). (d) WT and SakOE,mad2 brain pictures stained with Dpn (purple, middle panel) and Elav (green, right panel), phalloidin is in yellow and DNA in blue. The yellow dashed line labels the CB. Cells with large nuclei showing Elav accumulation can be seen (insets) in SakOE,mad2 brains, but never in the WT. Scale bar= 50 μm. (e) Dot plot showing the nuclear width of Elav⁺ nuclei in WT (189 cells/6 lobes), SakOE,mad2 (216 cells/11 lobes) and bub3 (433 cells/5 lobes). The line represents the mean and the error bars the s.d. Statistical significance determined using an unpaired t-test. ***(P<0.0001). (f) WT, bub3 and bub3, Pros^RNAi brain pictures stained with Dpn (purple and bottom panels), phalloidin is in green and DNA in blue. Scale bar, 50 μm. Since decrease in Pros levels causes reversion of differentiating daughters to Nb like cells⁵⁸, small nuclei expressing the stem cell marker Dpn are present (inset). (g) Dot plot showing the number of Dpn⁺ nuclei with diameter >8 μm, to distinguish between previous GMCs and Nbs, in WT (red circles, 14 lobes), bub3 (full circles, 32 lobes) and bub3, Pros^RNAi (empty circles nine lobes). The line represents the mean and the error bars the s.d. Statistical significance determined using an unpaired t-test. **(P=0.0048) and ****(P<0.0001).