Fig. 5: Over expression of an O-class isoform results in both ISC maintenance and differentiation.

Representative images of control midguts (A) and midguts overexpressing Grh.RL in progenitor cells (B). Scale Bar 40 μm. C In comparison to control midguts (n = 13) there is an increase in the proportion of esg+ cells in midguts over expressing Grh.RL (n = 13). (Mean ± SEM, Unpaired Student's T-test with Welsh’s Correction, ****p < 0.0001). D An increase in the mitotic index as measured by the number of pH3+ cells are observed in midguts ectopically expressing Grh.RL (n = 16). This increase in mitotic index is rescued by the co-expression of the apoptotic inhibitor p35 with Grh.RL (n = 9) with its mitotic index returning to levels seen in control midguts (n = 16) and midguts only expressing p35 (n = 6). (Mean ± SEM, One-Way ANOVA with Tukey’s Test, **p = 0.0027, ***p = 0.0001, ****p < 0.0001). E- F Confocal images of control midguts and midguts ectopically expressing Grh.RL in only ISCs (green). Ectopically expressing Grh.RL in ISCs does not appear induce differentiation with the YFP+ ISCs remaining at a similar size to YFP+ cells in control midguts. Scale Bar 40 μm. G The proportion of ISCs in control midguts (n = 20) and midguts ectopically expressing Grh.RL (n = 17) remain at similar level. (Mean ± SEM, unpaired Student's T-test with Welsh’s Correction, p = 0.0749). H In comparison to controls (n = 11 midguts), an increase in GFP+ EBs (express Su(H) EB marker) is observed in midguts ectopically expressing Grh.RL (n = 4). (Mean ± SEM, Unpaired Student's T-test with Welsh’s Correction, ****p < 0.0001). I, J Representative images of control midguts (I) and midguts overexpressing Grh.RL (J) in EBs (driven from Su(H)Gal4). In control midguts, ISCs are solely marked by Dl with mitotically active ISCs marked by pH3 (arrowheads) whereas overexpression of Grh.RL in EBs induces EB expression of Dl and EB mitotic division (arrows). Scale Bar 40μm. Dotted boxes outline magnified regions. K Comparison of the mitotic index in control midguts (n = 19) and midguts overexpressing Grh.RL (n = 14) in EBs. An increase in the mitotic index is observed in midguts overexpressing Grh.RL in EBs. (Mean ± SEM, Unpaired Student's T-test, ****p < 0.0001). L Quantification of the number of EBs undergoing mitotic division. (Mean ± SEM, Unpaired Student's T-test, ****p < 0.0001). M, N Confocal images of control midguts and midguts overexpressing Grh.RL in EBs immunostained with EC marker, Pdm-1. Whereas GFP+ EBs in controls rarely express Pdm-1, an increase in Su(H)^TS > UAS-Grh.RL cells are positive for Pdm-1 is observed. Scale Bar 40 μm. O The proportion of EBs expressing Pdm-1 increases in Su(H)^TS > UAS-Grh.RL midguts (n = 4) in comparison to control midguts (n = 11). (Mean ± SEM, Unpaired Student's T-test, ****p < 0.0001).