Crumbs is required to achieve proper organ size control during Drosophila head development

Abstract

Crumbs (Crb) is a conserved apical polarity determinant required for zonula adherens specification and remodelling during Drosophila development. Interestingly, crb function in maintaining apicobasal polarity appears largely dispensable in primary epithelia such as the imaginal discs. Here, we show that crb function is not required for maintaining epithelial integrity during the morphogenesis of the Drosophila head and eye. However, although crb mutant heads are properly developed, they are also significantly larger than their wild-type counterparts. We demonstrate that in the eye, this is caused by an increase in cell proliferation that can be attributed to an increase in ligand-dependent Notch (N) signalling. Moreover, we show that in crb mutant cells, ectopic N activity correlates with an increase in N and Delta endocytosis. These data indicate a role for Crb in modulating endocytosis at the apical epithelial plasma membrane, which we demonstrate is independent of Crb function in apicobasal polarity. Overall, our work reveals a novel function for Crb in limiting ligand-dependent transactivation of the N receptor at the epithelial cell membrane.

Fig. 1.

crb function is required for proper head, eye and wing size development. (A,A′) Wild-type (WT) Drosophila WT head (A). crb^11A22 whole mutant head (A′). Dashed arrows indicate the width of head capsule. Inset shows close up of facet lenses. (B,B′) WT eye (B). crb^11A22 whole mutant eye (B′). Dashed arrow and solid lines indicate eye height. (C) WT eye imaginal disc stained with ELAV (green) and Phalloidin (red). Numbers indicate row number. (D) Mean number of ommatidia per row as indicated in C in WT and crb^11A22 discs. WT, n=11; crb^11A22, n=9. (E) Left, WT wing; right, enGAL4 driving crbRNAi³⁹¹⁷⁸ in the posterior compartment of the wing (arrow). (F-F″″) WT head (F). (F′-F″′) crb^11A22 whole mutant heads expressing crb transgenes under the control of the eyGAL4 promoter. (F′) crb^–/–. (F″) UAS-crbExtTMGFP. (F″′) UAS-crbExt. (F″″) UAS-crbTMGFP. (G) Mean ratio of the head width of the genotypes indicated compared with that of the WT. Scale bars: 250 μm in A,A′,B,B′,F-F″″; 20 μm in insets; 50 μm in C.

Fig. 2.

crb regulates cell proliferation during Drosophila eye development. (A) Control clones generated using a WT FRT82. (B) crb^11A22 clones. (C) Quantification of the total size of the crb^11A22 mutant tissue and WT homozygous tissue. n=10 discs per genotype. ^***, t-test: P<0.001. (D) Quantification of the total size of the FRT82 control tissue and WT tissue. n=8 discs per genotype. t-test: P>0.05. (E) crb^11A22 clones in the eye imaginal disc marked by lack of GFP staining (green) and stained for cleaved Caspase 3 (red). (E′) Cleaved Caspase 3. (F,G) Anterior compartments of the eye imaginal disc stained for phospho-Histone H3 (green) and with Phalloidin (red). WT (F) and crb^11A22 (G) whole mutant eye discs. (H) Quantification of the mitotic index in WT and crb^11A22 mutant discs. n=8 discs per genotype. ^***, t-test: P<0.001. (C,D,H) Error bars represent the s.e. Scale bars: 50 μm in A,B; 10 μm in E-G.

Fig. 3.

crb modifies N function in controlling Drosophila eye size. (A-C″) White solid arrow represents the WT eye height for comparison and white dashed arrow represents the actual eye height. Inset shows a close up of facet lenses. (A) WT eye. (A′) N^264-39 heterozygous eye. (A″) N^264-39 and crb^11A22 heterozygous eye. (B) WT eye. (B′) Dl^revF10 heterozygous eye. (B″) Dl^revF10 and crb^11A22 heterozygous eye. (C) WT eye. (C′) aph1^D35 heterozygous eye. (C″) aph1^D35 and crb^11A22 heterozygous eye. (D) Mean eye size represented by the eye area. n=6 eyes per genotype. ^***, t-test: P<0.001. Error bars represent the s.e. Scale bars: 200 μm in A-C″; 20 μm in insets.

Fig. 4.

The crb overgrowth phenotype is dependent on N signalling. (A) crb^11A22 clones in the eye imaginal disc marked by lack of GFP staining (green) and expressing mβlacZ (red). (A′) Close up of boxed area in A. (A″) Fluorescence intensity along the dashed line in A′. (B) Normalized mβlacZ fluorescence intensity per cell in WT and crb mutant clones. n=15 cells per genotype. ^*, t-test: P<0.05. (C) FRT82 control clones. (D) FRT82 control clones expressing mam^DN. (E) crb^11A22 clones. (F) crb^11A22 clones expressing mam^DN. Clones are positively labelled with GFP (green). (G) Clone size area. n=10 discs per genotype. ^***, t-test: P<0.001. (B,G) Error bars represent the s.e. Scale bars: 5 μm in A; 50 μm in C-F.

Fig. 5.

crb loss of function leads to increased N and Dl endocytosis. (A) Electron micrograph of the stalk membrane region from a crb^11A22 mutant photoreceptor. Arrowheads indicate coated pit-like structures. (B) The mean number of NECD- and Hrs-positive vesicles in WT and crb^11A22 mutant discs. WT, n=7; crb, n=6; ^***, t-test: P<0.001. (C) The mean number of NICD- and Hrs-positive vesicles in WT and crb^11A22 mutant discs. WT, n=5; crb, n=6; ^***, t-test: P<0.001. (D) The mean number of Dl- and Hrs-positive vesicles in WT and crb^11A22 eye discs. WT, n=5; crb, n=6; ^***, t-test: P<0.001. (E-F″) Eye discs after 20 minute dextran uptake. Anterior compartment, apical left. (E) WT disc. Merge of NECD (green) and dextran (red). Arrowheads indicate NECD- and dextran-positive vesicles. (E′) NECD. (E″) Dextran. (F) crb^11A22 mutant disc. Merge of NECD (green) and dextran (red). Arrowheads indicate NECD- and dextran-positive vesicles. (F′) NECD. (F″) Dextran. (G) Proportion of dextran vesicles positive for NECD in WT and crb^11A22 mutant discs. WT, n=5; crb, n=7; ^***, t-test: P<0.001. (H) Proportion of dextran vesicles positive for NICD in WT and crb^11A22 mutant discs. WT, n=5; crb, n=4; ^***, t-test: P<0.001. (I,J) Anterior compartment of the eye disc stained for Hrs (green), apical left. (I) WT disc. (J) crb^11A22 mutant disc. (K) Number of Hrs-positive vesicles in WT and crb^11A22 mutant discs. WT, n=8; crb, n=8; t-test: P>0.05. n, number of discs. Vesicles were counted in multiple sample areas per disc. Error bars represent the s.e. Scale bars: 0.5 μm in A; 5 μm in E-F″; 2 μm in I,J.

Fig. 6.

crb limits Dl-dependent N transactivation and S2 cleavage. (A,B) crb^11A22 mutant clones marked by lack of GFP (green) as outlined by the white dotted line in the follicular epithelium (FE) of the egg chamber. At stage 5, no Hnt (red) expression in either the crb^11A22 clones or WT tissue is detected. tsg101 clones (B) marked by lack of GFP (green) lead to ectopic Hnt expression (red) at stage 5. (C) Number of Dl- and NICD-positive vesicles in WT and crb^11A22 eye discs. WT, n=6; crb, n=8; t-test: P>0.05. (D) Number of Dl- and NECD-positive vesicles in WT and crb^11A22 eye discs. WT, n=5; crb, n=4; ^***, t-test: P<0.001. (E) The effect of S2 cleavage inhibition upon the number of dextran vesicles positive for NECD and Dl in WT and crb^11A22 mutant discs. Left-hand bars, control with no S2 cleavage inhibitor; right-hand bars, 5 mM OP. WT, n=6; crb, n=6; ^***, t-test: P<0.001. n, number of discs. Vesicles were counted in multiple sample areas per disc. Error bars represent the s.e. Scale bars: 25 μm.