Crumbs limits oxidase-dependent signaling to maintain epithelial integrity and prevent photoreceptor cell death

Abstract

Drosophila melanogaster Crumbs (Crb) and its mammalian orthologues (CRB1-3) share evolutionarily conserved but poorly defined roles in regulating epithelial polarity and, in photoreceptor cells, morphogenesis and stability. Elucidating the molecular mechanisms of Crb function is vital, as mutations in the human CRB1 gene cause retinal dystrophies. Here, we report that Crb restricts Rac1-NADPH oxidase-dependent superoxide production in epithelia and photoreceptor cells. Reduction of superoxide levels rescued epithelial defects in crb mutant embryos, demonstrating that limitation of superoxide production is a crucial function of Crb and that NADPH oxidase and superoxide contribute to the molecular network regulating epithelial tissue organization. We further show that reduction of Rac1 or NADPH oxidase activity or quenching of reactive oxygen species prevented degeneration of Crb-deficient retinas. Thus, Crb fulfills a protective role during light exposure by limiting oxidative damage resulting from Rac1-NADPH oxidase complex activity. Collectively, our results elucidate an important mechanism by which Crb functions in epithelial organization and the prevention of retinal degeneration.

Figure 1.

Crb controls ROS production through the inhibition of Rac1 and NADPH oxidase in epithelial tissues. (A) Monitoring of ROS production using lucigenin assay in wild-type embryos, Crb overexpressing embryos (Crb Over.), crb^11A22 mutant embryos (null allele), Rac1^V12-expressing embryos, and in embryos expressing Rac1^V12 and overexpressing Sod1 (Sod1 over.). Error bars represent the 95% confidence level. (B) crb homozygous mutant clones were produced in adult crb/+ female flies (mutant clones are GFP negative). Ovaries were then dissected, incubated in the presence of the ROS-sensing DHE probe, and mounted for confocal microscopy analysis. (C) Crb was clonally overexpressed in the follicular epithelium, and ROS production was assessed using DHE and confocal microscopy. Crb overexpressing clones are positively marked with GFP. (D) Determination of ROS levels using DHE in crb mutant clones in which Rac1 was knocked down. (E and F) After induction of crb homozygous mutant clones in crb/+ female flies (mutant clones are GFP-negative), ovaries were incubated in the presence of the Rac1 activation inhibitor NSC23766 (E) or the NADPH oxidase complex inhibitor apocynin (F). Then, ROS levels were determined using DHE and confocal microscopy. Bar, 10 µm.

Figure 2.

Repression of superoxide production by Crb is crucial for epithelial integrity. (A–L) Panels show a portion of the lateral epidermis of late-stage embryos immunostained for Fasciclin 3 (Fas3; A, C, E, G, I, and K) or a cuticle preparation (B, D, F, H, J, and L) for the following genotypes: wild-type (A and B), crb^11A22 (null allele; C and D), daGAL4/UAS-Sod1 (overexpression of Sod1 in a wild-type background; E and F), crb^11A22, daGAL4/crb^11A22, UAS-Sod1 (overexpression of Sod1 in a crb^11A22 mutant background; G and H), UAS-p35; daGAL4 (expression of BacA/p35 in a wild-type background; I and J), or UAS-p35; crb^11A22/crb^11A22, daGAL4 (expression of BacA/p35 in a crb^11A22 mutant background; K and L). (M and N) Panels show Crb staining (M) or costaining of Crb and Dlg (N; red and green, respectively) in the ventral epidermis of a stage 16 Rac heterozygous mutant embryo (ptcGAL4/+; Rac1^J11, Rac2^Δ, Mtl^Δ/+). (O and P) Crb staining or staining of Crb (red) and Dlg (green) in the ventral epidermis of a stage 16 embryo knockdown for Nox and heterozygous for Rac (ptcGAL4/UAS-shRNA Nox; Rac1^J11, Rac2^Δ, Mtl^Δ/+). Bars: (A, C, E, G, I, and K) 10 µm; (B, D, F, H, J, and L) 100 µm; (M–P) 10 µm.

Figure 3.

Crb represses Rac1 activation and ROS production in photoreceptor cells. (A) Heads from wild-type flies or flies carrying crb mutant retinas were homogenized. A portion of each homogenate was kept to monitor Rac1 expression levels (input), and GST-PAK^CRIB was used to pull down active Rac1, which was detected by Western blotting. Native GST was used as a negative control. Western blotting controlled the amount of GST or GST-PAK^CRIB used in each experiment. (B) Lucigenin assays were performed to compare ROS production in control and crb mutant retinas. (C) Comparison of ROS production in retinas after knockdown of Crb or concomitant knockdown of Crb and Rac1 or Crb and Nox. Error bars represent the 95% confidence level.

Figure 4.

Crb controls Rac1 and NADPH oxidase activity and ROS production to maintain retinal integrity. (A–I) Heads from flies maintained under constant illumination (light; A and C–I) or kept in total darkness (dark; B) were fixed, sectioned, and stained for light microscopy analysis. Panels show cross section of a wild-type retina (A), crb mutant (crb−/−) retinas (B and C), crb mutant retina of a fly fed with NSC23766 while submitted to light stress (D), crb mutant retina of a fly raised on a diet containing apocynin (E), crb mutant retina of a fly fed with glutathione (F), crb mutant retina knocked down for Rac1 (G), crb mutant retina in which Nox was knocked down (H), and a Crb knockdown retina in which Sod1 was overexpressed (Sod1 over.; I). Bar, 5 µm. (J) Rhabdomeres were counted on retinal cross sections to quantify the number of surviving photoreceptor cells after 7 d of constant illumination. Reduction of Rac1 activity using NSC23766 or knockdown (Rac1 KD) significantly increased the number of surviving photoreceptor cells in crb mutant retinas. Chemical inhibition of NADPH oxidase using apocynin or knockdown of Nox (Nox KD) had a similar effect. Finally, ROS quenching with glutathione or superoxide detoxification by Sod1 overexpression improved cell fitness in crb mutant or Crb knockdown retinas. Error bars represent the 95% confidence level.