Fig. 1: grh O-isoforms maintain ISC identity.

A Schematic of grh mRNA transcripts. The single grh gene is alternatively spliced to produce two classes of transcripts based on the splicing of exons 4 and 5 (red box). Transcripts with exons 4 and 5 are classed as O-isoforms while those without these exons are classed as N-isoforms. Transcripts denoted with an apostrophe lack 30 amino acids in exon 12 (red dotted box). grh.RP (grh.N) and grh.RL (grh.O’) are shown as representatives of the two classes. Positions of mutations are indicated by triangles and the grh.Gal4 insertion by a black and white bar. Sequences that were utilized for RNAi constructs are indicated by bars. B–I Representative images of GFP (green) marked control, grh^S2140, grh^IM and grh³⁷⁰ MARCM clones at 5PDCI and 10DPCI. Scale Bar 40 µm. J Quantification of the mean number of clones per midgut at 5 and 10 days post clone induction (DPCI). MARCM control (5DPCI: n = 18 midguts, 10DPCI n = 26 midguts) and grh null (grh^S2140 – 5DPCI: n = 7 midguts, 10DPCI n = 23 midguts; grh^IM – 5DPCI: n = 6 midguts 10DPCI: n = 28 midguts) O-specific mutant grh³⁷⁰ (5DPCI: n = 10 midguts, 10DPCI: n = 25 midguts) clones are lost at 10DPCI. (Mean ± SEM, One-way ANOVA with Dunnett’s test, ns= not significant, ****p < 0.0001). K Proportion of MARCM clones at 10DPCI containing at least a single Dl+ ISC in control (n = 14), grh^S2140 (n = 8), grh^IM (n = 14) and grh³⁷⁰ (n = 18) midguts. In comparison to control clones, there is a significant decrease in the fraction of grh³⁷⁰ clones containing ISCs. (Mean ± SEM, One-Way ANOVA with Tukey’s test, ns= not significant, ****p < 0.0001). L grh³⁷⁰ MARCM clones affect ISC proliferation. Control clones are larger in size compared to grh mutant clones at 10DPCI. Clonal size was calculated by counting the number of cells within each clone in control and grh mutants. Control – n = 770 clones, 26 midguts. grh^S2140 – n = 526 clones, 15 midguts. grh^IM- n = 554 clones, 21 midguts. grh³⁷⁰ – n = 269 clones, 25 midguts. (Mean ± SEM, One-Way ANOVA with Tukey’s Test, ***p = 0.0002, ****p < 0.0001). M grh³⁷⁰ MARCM clones affect ISC differentiation. Quantification of the cellular composition of MARCM clones immunostained with Prospero and Pdm-1. The number of ECs (GFP+ Pdm-1+), EE cells (GFP+ Prospero+) and ISC/EBs (GFP+) for control and grh mutant clones was quantified for each genotype and percentage values calculated. (χ² Test, control vs. grh^S2140 p = 0.8325, control vs grh^IM p = 0.8650 and control vs. grh³⁷⁰ ****p < 0.0001).