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. 2009 Jul 15;122(Pt 14):2360-70.
doi: 10.1242/jcs.041806. Epub 2009 Jun 16.

The Hippo pathway regulates apical-domain size independently of its growth-control function

Alice Genevet  1 , Cédric Polesello, Ken Blight, Francesca Robertson, Lucy M Collinson, Franck Pichaud, Nicolas Tapon
Affiliations

The Hippo pathway regulates apical-domain size independently of its growth-control function

Alice Genevet et al. J Cell Sci. .

Abstract

The Hippo pathway, identified in Drosophila and conserved in vertebrates, regulates tissue growth by promoting cell cycle exit and apoptosis. In addition to their well-characterised overproliferation phenotype, adult Drosophila epithelial cells mutant for the kinases Hippo and Warts have hypertrophic apical domains. Here we examine the molecular basis of this apical hypertrophy and its impact on cell proliferation. In the wing imaginal disc epithelium, we observe increased staining for members of the apical polarity complexes aPKC and Crumbs as well as adherens junction components when Hippo activity is compromised, while basolateral markers are not affected. This increase in apical proteins is correlated with a hypertrophy of the apical domain and adherens junctions. The cell surface localisation of the Notch receptor is also increased in mutant clones, opening the possibility that aberrant receptor signalling may participate in overgrowth of hpo-deficient tissue. Interestingly, however, although the polarity determinant Crumbs is required for the accumulation of apical proteins, this does not appear to significantly contribute to the overproliferation defect elicited by loss of Hippo signalling. Therefore, Hippo signalling controls growth and apical domain size by distinct mechanisms.

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Figures

Fig. 1.
Fig. 1.
hpo loss of function induces an increase in apical polarity proteins in wing imaginal cells. (A-H′) Transverse sections of third instar wing discs containing clones of hpo mutant cells (marked by absence of GFP). Apical is to the top in this and all subsequent XZ sections. Scale bar: 10 μm. (A′-H′) Merged images of A-H with GFP images (green). (I,I′) Transverse section of a third instar wing disc containing clones of hpo5.1 mutant cells (marked by absence of β-gal) stained for aPKC (green) and DE-cad (red). (I′) Merged image of I with β-gal (blue). Scale bar: 5 μm. hpo mutant cells have an increased apical staining of the following apical determinants: Cad99c (A,A′), aPKC (B,B′), Crb (C,C′), P-Moe (D,D′), Arm (E,E′) and DE-cad (F,F′). By contrast, they do not show an increase of the septate junction protein Dlg (G,G′) or the basal marker Dystroglycan (DG; H,H′). Despite their respective increase, the proteins aPKC and DE-cad remain in non-overlapping membrane domains (I,I′).
Fig. 2.
Fig. 2.
Inactivation of Hpo pathway members induces apical protein accumulation. (A-F′) Transverse sections of wing discs. Scale bar: 10 μm. (A″,B″) Merged images of A,B, A′,B′, respectively and of GFP. C′-F′ Merged images of C-F with GFP (green). Grey or red: stainings for aPKC (C-D′,F,F′) or for DE-cad (E,E′). (A-A″) Cells mutant for mer;ex show an increase in the apical markers aPKC (grey in A, blue in A″) but not of the lateral marker Dlg (grey in A′, red in A″). (B-B″) Similarly, wts mutant cells show an increase in aPKC (grey in B, blue in B″) but not in Dlg (grey in B′, red in B″). (C,C′) Mimicking Hpo pathway inactivation by overexpressing yki also leads to an increase in apical aPKC (cells marked by GFP). (D-F′) Activating the Hpo pathway, by overexpressing the caspase inhibitor p35 and either ex (D-E′) or hpo (F,F′), leads to a reduction in apically localised aPKC (D,D′,F,F′) or DE-cad (E,E′) (cells marked by GFP).
Fig. 3.
Fig. 3.
Correlative transmission electron microscopy of yki-overexpressing wing imaginal cells reveals apical membrane hypertrophy. (A) Transmitted light and GFP merged image of a third instar wing disc overexpressing Yki under the engrailed promoter. The disc was attached to an etched grid coverslip to stabilise its shape. (B) Same disc as in A now resin-embedded and prepared for TEM. The disc retains its gross morphology, allowing us, by comparing A and B, to distinguish the yki-overexpressing cells (green in A) and the WT cells. The dotted red line indicates the transverse section shown in C. (C) Semi-thin transverse section of the en>>yki disc, viewed in transmitted light. The different features of the discs, such as the peripodial membrane and the pouch, are distinguishable. By comparing A,B and C, it is possible to locate the position of WT and yki-overexpressing cells. The red rectangles indicate the position of the higher magnification images shown in D and E. (D,E) High magnification micrographs of control cells (D) and of yki-overexpressing cells (E). Scale bar: 0.5 μm. The AJs are indicated by red brackets. The white dashed lines indicate the width at the AJs (W). The yellow dashed lines give a measure of apical domain length (L). The product PLW of L×W gives an indication of relative apical domain size. (F) Box and whisker plot of AJ measurements. The P-value from a Mann-Whitney test is indicated. (G) Box and whisker plot of relative apical domain size, assessed by calculating the PLW product. The P-value from a Mann-Whitney test is indicated.
Fig. 4.
Fig. 4.
Transcriptional regulation of polarity genes in loss-of-function clones for the Hpo pathway. (A) Graph of mRNA levels of different polarity genes measured by qRT-PCR in yki-overexpressing and WT discs. (B-D′) XY sections of third instar wing discs. Scale bar: 20 μm. C″ is a merged image of C (red) and C′ (green). D′ is a merged image of D with that of GFP (green). Grey or red: anti-β-galactosidase staining. Compared with WT cells (A), hpo mutant cells (B,B′), do not show an overall change in β-gal staining (monitoring crb-lacZ activity). A weak increase could occasionally be observed close to the dorsoventral boundary (arrow). By contrast, wts cells (C,C′) show a marked increase in shg-lacZ staining. (E) Alignment between the Hippo response element (HRE) identified in the DIAP1 locus (Wu et al., 2008) and a putative HRE found in the first intron of the shg gene (positions 369-395). The Scalloped binding site is indicated. Conserved nucleotides are shown in red.
Fig. 5.
Fig. 5.
Downregulation of Notch activity in Hpo signalling-deficient cells. (A-D′) XY sections of wing imaginal discs. (A′,B′) Merged images of A and B with GFP (green). (D″) The merged images of D (red) and D′ (green). Scale bar: 20 μm. mer;ex (A,A′) and hpo (B,B′) mutant cells do not show an increase in Cut staining at the dorsoventral boundary. Compared with WT cells (C), wts mutant cells (D,D′) show a decrease in β-galactosidase staining monitoring the activity of the Notch reporter Gre(H)-lacZ in the pouch, and also more weakly at the dorsoventral boundary.
Fig. 6.
Fig. 6.
The Hpo pathway controls proliferation independently of cell polarity. (A-C) Transverse sections of wing imaginal discs containing mutant clones of different genotypes (marked by absence of GFP) and stained for aPKC. Scale bar: 10 μm. Cells mutated for wts show an increase in aPKC staining (A), whereas cells mutant for crb show less apical aPKC (C). As for crb mutant cells, crb/wts cells have less aPKC on their apical surface (B). (D) Quantification of the apical determinant accumulation in hpo or wts mutant cells, as well as its rescue in crb/wts mutant cells, compared with WT cells. Histogram of the ratio of staining intensities for aPKC, NICD and Dlg between clones of WT, hpo, wts or crb/wts cells, and non-mutant cells of the same wing disc. P-values from Mann-Whitney tests are shown on the graph. (E,F) Quantification of the proliferative advantage of wts, crb and crb/wts mutant cells compared with WT cells. (E) Sample of wing discs used to measure and compare the clonal (absence of GFP) and twin spot areas (two copies of GFP); DAPI (blue) is used to visualise disc outlines. (F) Graph of rCT ratio between clonal and twin spot areas, for each genotype. Blank clones are clones of WT cells. P-values from Mann-Whitney tests are shown on the graph.
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