Non-apoptotic caspase activation preserves Drosophila intestinal progenitor cells in quiescence (2020)
Figure 5

Figure 5. Dronc activation limits Notch-signalling in the intestine.

A. Representative ReDDM labeling of a Drosophila Dronc heterozygous (+/-) intestine overexpressing an RNAi against Notch for 3 days; note the lack of fully differentiated Histone-RFP cells as EC (red) without esg expression (green, GFP). DAPI staining labels cell nuclei in panels A, B, D and E. All of the experiments described in the figure were performed in Oxford medium following an experimental regime that protects the epithelial integrity. Genotype: w1118 UAS-Notch-RNAi (Joaquin Navascués); esg-Gal4 UAS-CD8-GFP / + ; TubG80ts UAS-Histone-RFP DroncKO / +

B. Representative ReDDM labelling of a Drosophila Dronc mutant homozygous (-/-) intestine overexpressing an RNAi against Notch for 3 post temperature shift at 29 oC; note the lack of differentiated Histone-RFP cells as EC (red) without esg expression (green, GFP), as well as the increase of GFP-positive cells (compare A to B). Genotype: w1118 UAS-Notch-RNAi (Joaquin Navascués); esg-Gal4 UAS-CD8-GFP / + ; TubG80ts UAS-Histone-RFP DroncKO / UAS-Flippase (BL8209) FRT Dronc-GFP-APEX FRT suntag-HA-Cherry

C. Quantification of the number of Histone-RFP cells normalised to DAPI (proxy of progenitor cell proliferation obtained from the experiments shown in A and B panels); note the statically significant increase in Histone-RFP positive cells in Dronc homozygous mutant intestines compared to controls (**; P =0.0080) (Quantifications were made using N ≥ 2 biological replicates; unpaired two-tailed t test, +/- n = 8, -/- n = 15). Error bars represent Standard Deviation of the mean. Genotypes:

+/-: w1118 UAS-Notch-RNAi; esg-Gal4 UAS-CD8-GFP / + ; TubG80ts UAS-Histone-RFP DroncKO / +

-/-: w1118 UAS-Notch-RNAi (Joaquin Navascués); esg-Gal4 UAS-CD8-GFP / + ; TubG80ts UAS-Histone-RFP DroncKO / UAS-Flippase (BL8209) FRT Dronc-GFP-APEX FRT suntag-HA-Cherry

D. Drosophila Dronc heterozygous intestine overexpressing the Notch intracellular domain for 7 days post temperature shift at 29 oC; intestinal esg-positive progenitor cells (green (GFP) and red (Histone-RFP). Genotype: w1118; esg-Gal4 UAS-CD8-GFP / UAS-Notchintra (Joaquin Navascués); TubG80ts UAS-Histone-RFP DroncKO / +

E. Drosophila Dronc homozygous intestine overexpressing the Notch intracellular domain for 7d post temperature shift at 29 oC; notice that the Dronc deficiency accelerates the elimination of intestinal progenitor cells induced by Notch overactivation (compare D and E). The white arrows indicate the position of insets 500μm from the posterior region. Note the complete loss of esg labelled cells in this region. Genotype: w1118; esg-Gal4 UAS-CD8-GFP / UAS-Notchintra; TubG80ts UAS-Histone-RFP DroncKO / UAS-Flippase (BL8209) FRT Dronc-GFP-APEX FRT suntag-HA-Cherry

F. Relative number of esg-positive cells to DAPI in either heterozygous or homozygous Dronc mutant esg cells overexpressing Notch-Intra; note the significant reduction of esg-expressing cells (****; P < 0.0001) (Quantifications were made using N ≥ 2 biological replicates; Mann-Whitney test, +/- n = 17, -/- n = 17). Error bars represent Standard Deviation of the mean. Genotypes:

+/-: w1118; esg-Gal4 UAS-CD8-GFP / UAS-Notchintra ; TubG80ts UAS-Histone-RFP DroncKO / +

-/-: w1118; esg-Gal4 UAS-CD8-GFP / UAS-Notchintra; TubG80ts UAS-Histone-RFP DroncKO / UAS-Flippase (BL8209) FRT Dronc-GFP-APEX FRT suntag-HA-Cherry

G. Representative image of a Drosophila Dronc heterozygous intestines 7 days after transfer to 29 oC. esg expression in green (GFP) labels all intestinal progenitors cells, whilst Su(H)-lacZ (red) distinguishes a subpopulation of EBs within the esg-expressing cells. Genotype: w1118, Su(H)GBE-LacZ; esg-Gal4 UAS-CD8-GFP / + ; TubG80ts UAS-Histone-RFP DroncKO / +

H. Representative image of a Drosophila Dronc KO intestine 7 days after transfer to 29 oC. Note, the transcription of Su(H) in large cells GFP (-), suggestive of an aberrant upregulation of Notch-signalling within fully differentiated ECs. Genotype: w1118, Su(H)GBE-LacZ; esg-Gal4 UAS-CD8-GFP / + ; TubG80ts UAS-Histone-RFP DroncKO / UAS-Flippase (BL8209) FRT Dronc-GFP-APEX FRT suntag-HA-Cherry

I. Representative image of a 7d Drosophila Dronc heterozygous intestines following a 16hr treatment with paraquat. Note the absence of large Su(H) positive, GFP (-) cells compared to (H). The white arrows indicate the enlarged area depicted in the insets. Genotypes:

+/-: w1118, Su(H)GBE-LacZ; esg-Gal4 UAS-CD8-GFP / + ; TubG80ts UAS-Histone-RFP DroncKO / +

-/-: w1118, Su(H)GBE-LacZ; esg-Gal4 UAS-CD8-GFP / + ; TubG80ts UAS-Histone-RFP DroncKO / UAS-Flippase (BL8209) FRT Dronc-GFP-APEX FRT suntag-HA-Cherry

Data information: (D-H) The white arrows indicate the enlarged area depicted in the insets.

EXPANDED VIEW FIGURE LEGENDS