The apical-basal cell polarity determinant Crumbs regulates Hippo signaling in Drosophila

Abstract

Defects in apical-basal cell polarity and abnormal expression of cell polarity determinants are often associated with cancer in vertebrates. In Drosophila, abnormal expression of apical-basal determinants can cause neoplastic phenotypes, including loss of cell polarity and overproliferation. However, the pathways through which apical-basal polarity determinants affect growth are poorly understood. Here, we investigated the mechanism by which the apical determinant Crumbs (Crb) affects growth in Drosophila imaginal discs. Overexpression of Crb causes severe overproliferation, and we found that loss of Crb similarly results in overgrowth of imaginal discs. Crb gain and loss of function caused defects in Hippo signaling, a key signaling pathway that controls tissue growth in Drosophila and mammals. Manipulation of Crb levels caused the up-regulation of Hippo target genes, genetically interacted with known Hippo pathway components, and required Yorkie, a transcriptional coactivator that acts downstream in the Hippo pathway, for target gene induction and overgrowth. Interestingly, Crb regulates growth and cell polarity through different motifs in its intracellular domain. A juxtamembrane FERM domain-binding motif is responsible for growth regulation and induction of Hippo target gene expression, whereas Crb uses a PDZ-binding motif to form a complex with other polarity factors. The Hippo pathway component Expanded, an apically localized adaptor protein, is mislocalized in both crb mutant cells and Crb overexpressing tissues, whereas the other Hippo pathway components, Fat and Merlin, are unaffected. Taken together, our data show that Crb regulates growth through Hippo signaling, and thus identify Crb as a previously undescribed upstream input into the Hippo pathway.

Fig. 1.

Crb overexpression causes overgrowth and up-regulation of Hippo pathway target genes. (A) WT wing. (B) Wing that overexpressed Crb during development under the control of C765-Gal4. (C) Overlay of the images in A (red) and B (blue) shows that the Crb-expressing wing is overgrown. Confocal images of wing imaginal discs of third instar larvae expressing GFP under the control of the dpp-Gal4 driver (D and F) or larvae overexpressing Crb in addition to GFP(E and G). (D and E) Discs are stained for BrdU incorporation to mark cells in S-phase (red in D and E, gray in D′ and E′). (F and G) Discs are stained to reveal the expression of the Hippo pathway reporter ex-lacZ (red in F and G, gray in F′ and G′). For disc panels, anterior is to the left and ventral is up.

Fig. 2.

Crb is required for proper tissue size, cell-cycle arrest, and Hippo target gene expression. (A and B) Scanning electron micrographs of a WT fly head and a head with crb^11A22 mutant clones, which is overgrown. The crb^11A22 mutant heads were generated via ey-FLP–mediated mitotic recombination against a Minute chromosome, which results in heads that are nearly entirely mutant for crb. (C and D) Wings of flies with WT and crb^11A22 mutant clones that were induced in Minute backgrounds by ubx-Flp, which results in wings that are composed nearly entirely of clonal tissue. The crb mutant wing is overgrown. (E) Eye imaginal disc from a third instar larva containing crb^11A22 mutant clones that are marked by the absence of GFP expression (green). This disc is labeled for BrdU incorporation (red in E, gray in E′). WT cells arrest cell proliferation posterior to the second mitotic wave (arrows), but crb^11A22 mutant cells show ectopic cell proliferation (arrowheads). Anterior is up. (F) Hinge region of a third instar wing disc containing crb^11A22 mutant clones marked by the absence of GFP expression (green). Discs are stained to reveal the expression of the Hippo pathway reporter ex-lacZ (red in F, gray in F′). The ex-lacZ levels are elevated in mutant non-GFP cells (arrowheads point to a mutant area). For disc panels, anterior is to the left and ventral is up.

Fig. 3.

Crb genetically interacts with Hippo pathway components and requires Yki for overgrowth and regulation of Hippo target genes. (A and B) Confocal images of pupal retinae with crb^11A22 mutant clones marked by the absence of GFP expression (green) stained with antibodies to detect Dlg to visualize cell outlines (red in A and B, gray in A′ and B′). The retina in A is otherwise WT, whereas the retina in B also has knockdown of Mer by a GMR-Gal4-driven UAS-mer^RNAi construct. The crb mutant clones are normal in A but show extra interommatidial cells in B (arrowheads). (C–H) Adult wings of the indicated genotypes. Wing-specific overexpression of crb^RNAi or D using nub-Gal4 did not cause significant overgrowth (D and E), but coexpression caused synergistic overgrowth effects (F). (G and H) Overexpression of Crb^intra caused overgrowth, which was suppressed by removing one copy of yki. Confocal images of wing imaginal discs that expressed Crb^intra (I), yki^RNAi (J), and Crb^intra and yki^RNAi (K) in the posterior compartment driven by hh-Gal4 and marked by coexpression of GFP. These discs are also stained for β-Gal to reveal the expression of the Hippo reporter ex-lacZ. Crb^intra expression caused overgrowth and induction of ex-lacZ, whereas yki^RNAi expression caused reduced ex-lacZ expression and compartment size. Coexpression of yki^RNAi with Crb^intra suppressed the induction of ex-lacZ and overgrowth. Arrowheads point to the compartment boundaries.

Fig. 4.

JM of the Crb intracellular domain mediates the regulation of growth and the Hippo pathway. (A–D) Confocal images of third instar wing discs overexpressing different mutant versions of Crb^intra driven by the dpp-Gal4 driver. The expression domain is marked by coexpression of GFP (green). Discs are stained for the expression of ex-lacZ (red in A–D, gray in A′–D′). Overexpression of the WT version of Crb^intra and the Crb^intraΔPBM mutant is able to drive growth and ex-lacZ induction, but mutation of the JM domain abolishes these effects. (E) Alignment of the intracellular domain sequence of Drosophila Crb (Dm) with that of the three human Crb homologs (Hs Crb1–3). Conserved residues are red, and the extent of the JM and PBM is indicated with blue bars. (F) Quantification of the overgrowth phenotypes shown in A–D. Labeling indicates overexpressed protein. FL, Crb full-length. For disc panels, anterior is to the left and ventral is up.

Fig. 5.

Crb is required for Ex membrane localization. (A–C, E, and F) crb^11A22 mutant clones in wing imaginal discs that are marked by the absence of GFP expression (green). (A) crb mutant cells lose Ex from the apical membrane (A′ shows an apical section) and accumulate Ex in more basal and intracellular locations (A′′ shows a basal section). (B) Z-section through a crb mutant clone shows the mislocalization of Ex in crb mutant cells. (C) Higher magnification of Ex localization at crb clone borders. Ex is localized in finger-like patterns at clone borders (arrowheads), which indicates that loss of Crb leads to loss of Ex from the corresponding membrane of abutting neighboring cells. Adherens junctions are labeled by Arm localization, which is not affected (blue in C′′, gray in C′′′). (D) Ex is lost in ex^e1 mutant clones but not in the neighboring WT cells (arrowhead). (E and F) crb mutant cells lose Crb and Patj from the apical membranes (red in E, F, and F′′; gray in E′ and F′). As for Ex, Crb and Patj are localized in finger-like patterns at clone borders (arrowheads). Cells in F are marked by E-cad staining (blue in F′′, gray in F′′′). (G) Expression of Crb in the posterior compartment by hh-Gal4 causes reduction of Ex at the apical membrane and basal redistribution of Ex. (H) Mutation of the JM abrogates the effects of Crb^intra expression on Ex localization. (I and J) Cross-sections through the discs shown in G and H. Arrowheads point to clone borders.