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. 2020 Nov 1;21(12):e48892. doi: 10.15252/embr.201948892

Figure 3. Phenotypic analysis of Dronc deficiency in escargot‐ and Delta‐expressing cells.

Figure 3

  1. A 7d intestine Dronc heterozygous (+/−) intestine in which intestinal stem cells express GFP under the regulation of Dl‐Gal4. All of the experiments described in the figure were performed in Oxford medium following an experimental regime that protects the epithelial integrity (flies transferred every 2 days into vials with fresh food). DAPI (blue) labels the DNA in the entire Figure. Genotype: w 1118;;delta‐Gal4 UAS‐GFP TubG80ts UAS‐Histone‐RFP Dronc KO/+ (recombinant chromosome made for this study; the parental line delta‐Gal4 UAS‐GFP TubG80ts UAS‐Histone‐RFP was obtained from Maria Dominguez).
  2. A 7d intestine in which the Intestinal Stem Cells have become Dronc homozygous mutant (−/−) upon expressing UAS‐Flp under the regulation of Dl‐Gal4. Dl‐expressing cells are labelled with UAS‐GFP (green); there are no morphological differences between (A) and (B). Genotype: w 1118;;delta‐Gal4 UAS‐GFP TubG80ts UAS‐Histone‐RFP Dronc KO/ UAS‐Flippase (BL8209) FRT Dronc‐GFP‐APEX FRT suntag‐HA‐Cherry.
  3. Relative number of GFP + Delta‐expressing cells normalised to DAPI; there is no significant increase in intestinal stem cell number between heterozygous and homozygous Dronc mutant conditions (ns; P = 0.9231) (Quantifications were made using N ≥ 2 biological replicates; unpaired two‐tailed t‐test, +/− n = 22, −/− n = 13). Error bars represent standard deviation of the mean. Genotypes: +/−:w 1118;;delta‐Gal4 UAS‐GFP TubG80ts UAS‐Histone‐RFP Dronc KO/ UAS‐Flippase (BL8209) FRT Dronc‐GFP‐APEX FRT suntag‐HA‐Cherry; −/−: w 1118;;delta‐Gal4 UAS‐GFP TubG80ts UAS‐Histone‐RFP Dronc KO/UAS‐Flippase (BL8209) FRT Dronc‐GFP‐APEX FRT suntag‐HA‐Cherry.
  4. Average Delta cell size (μm2); notice that the cell size does not change between heterozygous and homozygous Dronc mutant ISCs (ns; P = 0.9694) (Quantifications were made using N ≥ 2 biological replicates; unpaired two‐tailed t‐test, +/− n = 42, −/− n = 21). Error bars represent standard deviation of the mean. Genotypes: +/−:w 1118;;delta‐Gal4 UAS‐GFP TubG80ts UAS‐Histone‐RFP Dronc KO/UAS‐Flippase (BL8209) FRT Dronc‐GFP‐APEX FRT suntag‐HA‐Cherry; −/−: w 1118;;delta‐Gal4 UAS‐GFP TubG80ts UAS‐Histone‐RFP Dronc KO/UAS‐Flippase (BL8209) FRT Dronc‐GFP‐APEX FRT suntag‐HA‐Cherry.
  5. A 7d intestine Dronc heterozygous (+/−) intestine in which intestinal stem cells express GFP under the regulation of Su(H)‐Gal4. Genotype: w 1118; Su(H)‐Gal4 UAS‐GFP; TubG80ts UAS‐Histone‐RFP Dronc KO/+.
  6. A 7d intestine in which the Enteroblasts have become Dronc homozygous mutant (−/−) upon expressing UAS‐Flp under the regulation of Su(H)‐Gal4. Su(H)‐Gal4‐expressing cells are labelled with UAS‐GFP (green) and UAS‐Histone‐RFP; note the increase in cell size when compared to (E) and the absence of Histone‐RFP cells without GFP signal. Genotype: w 1118; Su(H)‐Gal4 UAS‐GFP; TubG80ts UAS‐Histone‐RFP Dronc KO/ UAS‐Flippase (BL8209) FRT Dronc‐GFP‐APEX FRT suntag‐HA‐Cherry.
  7. Relative number of Su(H)‐Gal4‐expressing cells normalised to DAPI; there is no significant increase in intestinal stem cell number between heterozygous and homozygous Dronc mutant conditions (n.s.; P = 0.5099) (Quantifications were made using N ≥ 2 biological replicates; Mann–Whitney test, +/− n = 30, −/− n = 29). Error bars represent standard deviation of the mean. Genotypes: +/−: w 1118; Su(H)‐Gal4 UAS‐GFP; TubG80ts UAS‐Histone‐RFP Dronc KO/+; −/−: w 1118; Su(H)‐Gal4 UAS‐GFP; TubG80ts UAS‐Histone‐RFP Dronc KO/UAS‐Flippase (BL8209) FRT Dronc‐GFP‐APEX FRT suntag‐HA‐Cherry.
  8. Average Su(H) cell size (μm2), there is a significant increase in Su(H) Dronc null cells (−/−) when compared to the heterozygous control condition (**P = 0.0064) (Quantifications were made using N ≥ 2 biological replicates; Mann–Whitney test, +/− n = 49, −/− n = 33). Error bars represent standard deviation of the mean. Genotypes: +/−: w 1118; Su(H)‐Gal4 UAS‐GFP; TubG80ts UAS‐Histone‐RFP Dronc KO/+; −/−: w 1118; Su(H)‐Gal4 UAS‐GFP; TubG80ts UAS‐Histone‐RFP Dronc KO/UAS‐Flippase (BL8209) FRT Dronc‐GFP‐APEX FRT suntag‐HA‐Cherry.