Figure 3. Atg9 is required for adult Drosophila midgut morphogenesis.

(A) Atg9 mutant midguts are shortened and display enlargement in the posterior region (arrow). Scale bar: 500 μm. Quantification of adult midgut length and posterior midgut width of control and Atg9 mutant flies. n = 10, **p<0.01. (B) Phalloidin staining of midgut visceral muscles revealed that loss of Atg9 leads to disruption of actin filaments. Scale bar: 20 μm. (C–D) Optical sections of control and the Atg9 mutant midgut epithelium layer stained with anti-Dlg showing that Atg9 mutants display abnormally enlarged cells with apical protrusions (arrowheads) into the lumen. Scale bar: 20 μm. (E) Quantification of cell size (shown in panel C) in control and Atg9 mutant posterior midgut. n ≥ 25, *p<0.05, **p<0.01. (F) Quantification of phospho-Histone3 positive (PH3⁺) cells per midgut of control and Atg9 mutant flies at 5 days and 30 days of age. n ≥ 8. (G) Quantification of total midgut cell numbers, posterior midgut ISC (Delta⁺) and EE (Pros⁺) cell numbers of 5-day-old control and Atg9 mutant adults. n ≥ 10. Data are mean ±s.e.m. ns, not statistically significant.

Figure 3—figure supplement 1. Loss of intestinal integrity in aged Atg9 mutant flies.

(A) Representative images of Smurf flies. Smurf-, the blue dye is restricted to the proboscis and digestive tract. Smurf+, the dye is seen throughout the body due to intestinal barrier dysfunction. (B) Quantification of Smurf flies showing loss of intestinal integrity in 30-day-old Atg9 mutants compared with control and Atg9 genomic rescue flies. n > 100, data are mean ±s.e.m., ***p<0.001.