Figure 6: Intestine stem cells undergo premature differentiation upon bub3 depletion.

(a) Intestinal stem cell (ISC)s division scheme: ISC expresses Escargot (Esg) and Delta (Dl), divides asymmetrically to self-renew and to generate an enteroblast (Esg⁺) that generate enterocytes (ECs) (Pdm1⁺) that undergo polyploidization and enteroendocrine cells (EEs), diploid (Pros⁺). (b) Fluorescent in situ hybridization in Esg⁺ (green) clones. Wild-type ECs appear homogeneous in size and the FISH probes appear clustered in a large dot. bub3^RNAi inset shows a nucleus with abnormal morphology, never seen in WT and with several unclustered signals that allow the distinction between ECs (>2 n, that would still be Esg⁺) and non-euploid Esg⁺ cells. Scale bar, 10 μm. (c) Fluorescent in situ hybridization quantification (n=99 and 246 WT and bub3^RNAi Esg⁺ cells, respectively). Statistical significance determined using Fisher’s exact test (Fet). *(P=0.012). (d) WT or bub3^RNAi GFP+ MARCM clones 12 days after heat shock (AHS) (in yellow), Dl (cytoplasmic red) marks SCs and Pros (nuclear red) marks EEs. Scale bar, 20 μm. (e) Quantification of number of cells/clone in WT and bub3^RNAi (n=31 and 45 clones, respectively). The line represents the mean and the error bars the s.d. Statistical significance determined using an unpaired t-test. ***(P<0.0001). (f) Quantification of Delta⁺ cells in WT and bub3^RNAi (n=31 and 45 clones, respectively). The line represents the mean and the error bars the s.d. Statistical significance determined using unpaired t-test. ***(P<0.0001). (g) Percentage of WT and bub3^RNAi (n=31 and 45 clones, respectively) without Dl⁺ cells. Statistical significance determined using Fet ***(P<0.0001). (h) Percentage of single cell clones in WT and bub3^RNAi (n=32 and 55 clones, respectively). Statistical significance determined using Fet. **(P=0.029). (i) WT and bub3^RNAi midguts expressing Esg GFP (green). DNA is in blue. Scale bars, 20 μm. (j) WT or bub3^RNAi midguts basal side with Dl (labelling ISCs, in white) and GFP. Wild-type Esg⁺ cells remain basally located and some express vesicular Dl, ISC marker (yellow arrows). Scale bars, 20 μm. (k) Apical side of the same WT or bub3^RNAi midguts than in (j) with GFP and Pdm1. Esg⁺, Pdm1⁺ cells are only observed in bub3^RNAi midguts (magenta arrows). Scale bars, 20 μm. (l) Percentage of Esg⁺ cells with nuclear size >7 μm in WT (n=40) and bub3^RNAi (n=54) midguts. Statistical significance determined using Fet ***(P<0.0001).