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. 2021 Feb 25;10:e62312. doi: 10.7554/eLife.62312

Figure 2. G-TRACE-Lineage characterization of Non-stop targeted young ECs and, aged ECs.

(A) Schematic diagram of EC-G-TRACE-lineage tracing system adapted from Flint Brodsly et al., 2019. PPC** (RFP-GFP+), are EC that are no-longer differentiated. PPC* (RFP-GFP-) are miss-differentiated progenitors. (B–Q) Confocal microscopy of midguts expressing the indicated transgenes, under the control of MyoIAts G-TRACE system using the indicated antibodies. DAPI (blue) marks DNA. Arrows point to cells shown in the insets with individual far-RFP, RFP and GFP channels. scale bar is 10 μM. (B–G) G-TRACE of EC in control young midgut expressing either UAS-LacZ (B, D, F), or UAS-Non-stop- RNAi (C, E, G). Arrows point to cells shown in the insets with individual far-RFP, RFP and GFP channels. (H–I) Expression of Delta (purple) on the surface of the indicated PPCs. Numbered cells are shown in (I) with individual channels dashed circle outlines the nucleus. (J) G-TRACE-based quantification of PPC types (wildtype, PPC* PPC**) observed in control midguts or where Non-stop was targeted ECs. (K–Q) Confocal microscopy of midguts expressing MyoIAts> G-TRACE system using the indicated antibodies. (B, L, N, P) G-TRACE of EC in young, and (K, M, O, Q) old midguts. (R) Quantification of indicated PPCs expressing Odd-Skipped, and Delta similar to experiments shown in L-O (**** = p<0.0001).

Figure 2—source data 1. Quantification data for Figure 2J and R,.

Figure 2.

Figure 2—figure supplement 1. Non-stop is required for expression of EC transcription factors, and epistatic studies with Hey (A–H).

Figure 2—figure supplement 1.

(A–H)Confocal images of the midgut tissue and the indicated transgenes expressed in EC using the MyoIA-Gal4/Gal80ts. DAPI marks DNA and Scale bar is 10 μM. (A, B) anti-Pdm1 (C, D) anti-Caudal Arrows point to cells shown in the inset. Scale bar is 10 μM. (E–F) Co-expression of Non-stop does not suppress the loss of identity and ectopic Delta expression observed upon loss of Hey in ECs. (G, H) Co-expression of Hey does not suppress the loss of identity and ectopic Delta expression observed upon loss of Non-stop in ECs.
Figure 2—figure supplement 2. Miss-regulation of enhancers activity and nuclear Lamins in aged enterocytes.

Figure 2—figure supplement 2.

(A–N) Confocal images with the indicated antibodies of adult Drosophila midgut epithelium expressing the indicated transgenes, DAPI marks DNA, and scale bar is 10 μm. (A–H) transgenic lines expressing UAS-GFP under the control of the EC- specific promoter MyoIA-GAL4/Gal80ts system. (I–P) Transgenic lines expressing G-TRACE system under the control of the enterocyte specific promoter MyoIAts system. The expression of M5-4::LacZ stem cells enhancer of esg gene is shown in A, B, I, and J. The protein level and distribution of the indicated protein is shown; LamC expression (C, D, K, L). Lamin Dm0 (LamDm0), E, F, M, N., and Otefin (Ote) protein level in control young ECs or old (G, H).
Figure 2—figure supplement 3. Hallmarks of aging in the Drosophila midgut (A–L).

Figure 2—figure supplement 3.

(A–L) Confocal images of indirect immunofluorescence staining with the indicated antibodies of adult Drosophila midgut intestinal epithelium expressing termed MyoIAts. Scale bar is 10 μm. (A, C, E, G, I, K): Young mid-guts (four days old adults). (B, D, F, H, J, L) Four weeks old guts. DAPI marks DNA and arrows indicates cells shown in the insets. SSK; Snake Skin; Arm, Armadillo; Dlg, Disc large.