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. 2018 Mar 8;145(5):dev159467.
doi: 10.1242/dev.159467.

Mechanical Strain Regulates the Hippo Pathway in Drosophila

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Free PMC article

Mechanical Strain Regulates the Hippo Pathway in Drosophila

Georgina C Fletcher et al. Development. .
Free PMC article

Abstract

Animal cells are thought to sense mechanical forces via the transcriptional co-activators YAP (or YAP1) and TAZ (or WWTR1), the sole Drosophila homolog of which is named Yorkie (Yki). In mammalian cells in culture, artificial mechanical forces induce nuclear translocation of YAP and TAZ. Here, we show that physiological mechanical strain can also drive nuclear localisation of Yki and activation of Yki target genes in the Drosophila follicular epithelium. Mechanical strain activates Yki by stretching the apical domain, reducing the concentration of apical Crumbs, Expanded, Kibra and Merlin, and reducing apical Hippo kinase dimerisation. Overexpressing Hippo kinase to induce ectopic activation in the cytoplasm is sufficient to prevent Yki nuclear localisation even in flattened follicle cells. Conversely, blocking Hippo signalling in warts clones causes Yki nuclear localisation even in columnar follicle cells. We find no evidence for involvement of other pathways, such as Src42A kinase, in regulation of Yki. Finally, our results in follicle cells appear generally applicable to other tissues, as nuclear translocation of Yki is also readily detectable in other flattened epithelial cells such as the peripodial epithelium of the wing imaginal disc, where it promotes cell flattening.

Keywords: Cell shape; Drosophila; Hippo pathway; Mechanosensing; Yorkie.

Conflict of interest statement

Competing interestsThe authors declare no competing or financial interests.

Figures

Fig. 1.
Fig. 1.
Yki target gene expression is induced in stretch cells and repressed in columnar cells. (A) Drosophila egg chambers at stages 8, 9 and 10 of oogenesis. The largest cell is the oocyte, which is fed by the polyploid nurse cells. Surrounding the germline cells are the somatic follicular epithelial cells (green, GFP+). At stage 8, all follicle cells are cuboidal. At stage 9, anterior follicle cells become stretched while posterior follicle cells remain cuboidal or columnar. By stage 10, anterior follicle cells are completely stretched flat, whereas posterior columnar cells surround the oocyte. Note also migration of border cells during stage 9. (B) The level of endogenously tagged Myosin-II:GFP does not change during formation of stretch cells or columnar cells. The actomyosin cortex labelling reveals the degree of flattening of stretch cells versus columnar cells. (C) A Yki reporter gene (expanded.lacZ) is upregulated in stretch cells and downregulated in columnar cells. The two polar cells also show increased ex.lacZ expression. DAPI is shown in blue and phalloidin is shown in red. Arrows and arrowheads indicate the regions enlarged in the panels below.
Fig. 2.
Fig. 2.
Canonical upstream Hippo pathway components concentrate apically in columnar cells and are diluted in stretch cells to control Hippo kinase dimerisation and signalling. (A-E) The upstream Hippo pathway components, β-Heavy Spec (A), Crb (B), Kib (C), Ex (D) and Mer (E) strongly label the apical membrane domain of columnar follicle cells but become less concentrated in stretch cells. (F) Schematic diagram showing dilution of Kib, Ex and Mer proteins upon stretching of the apical domain. Green represents the apical domain. Purple/orange represents the apical-junctional ring of Crb-Ex and Mer-Kib complexes. Blue represents Yki localisation. (G-I) Measurement of Hippo kinase dimerisation with a split-Venus reporter reveals strong apical membrane signal in columnar cells and weak membrane signal in stretch cells. Both columnar and stretch cells have some cytoplasmic background Venus signal. G is a cross-section. H is a surface view. I contains magnified views of G and H. (J-L) Yki reporter gene expression is inversely correlated with Hippo kinase activation at the plasma membrane (compare with G-I). J is a cross-section. K is a surface view. L contains magnified views of G and H. (M) Schematic diagram showing dilution of upstream Hippo pathway complexes, reduced Hpo-Wts phosphorylation, reduced Yki phosphorylation, and increased Yki nuclear localisation and transcriptional activation upon stretching of the apical membrane domain. Measurement of Hpo dimerisation via the split-Venus BiFC reporter is diagrammed. Arrows indicate stretch cells.
Fig. 3.
Fig. 3.
Yki translocates to the nucleus in stretch cells and to the cytoplasm of columnar cells, inversely correlating with Hippo dimerisation at the apical plasma membrane. (A) Endogenous Yki with a GFP tag was visualised in the views shown at different stages of oogenesis. At stage 8, Yki:GFP is predominantly cytoplasmic in the cuboidal epithelium, but at the anterior where the cells start to flatten Yki:GFP is found in the nucleus. During stage 9, Yki:GFP can clearly be found in the nucleus of stretch cells and in the cytoplasm of columnar cells. This pattern is even more pronounced at stage 10. (B) The HippoKD-Venus dimerisation reporter was visualised in the views shown over different stages of oogenesis. At stage 8, a weak Hippo dimerisation signal is observed with an apical signal apparent in the posterior epithelium. At stages 9 and 10, a clear apical and lateral signal can be seen in columnar cells, whereas in stretch cells there is a faint cytoplasmic signal.
Fig. 4.
Fig. 4.
Dicephalic mutants disrupt stretch cell flattening, but not cell fate, and reduce nuclear Yki:GFP. (A) Control and (B) Dicephalic (dic1/dic1) mutant stage 10 egg chambers (Eya staining is in red, DAPI staining is in blue) show that the less stretched cells in the dic mutant have less nuclear Yki:GFP than the control. (C) Quantification of the ratio of nuclear to cytoplasmic Yki:GFP (n=12). Data are mean±s.d. The difference is statistically significant (P<0.001).
Fig. 5.
Fig. 5.
High-resolution imaging of Yki translocation and Hippo pathway component dilution as the apical domain becomes diluted in stretch cells compared with columnar cells. Top-down views of stretch cells and columnar cells at progressive stages of oogenesis: (A) Yki:GFP, (B) βH-Spectrin:YFP, (C) Crb:GFP, (D) Kib:GFP, (E) HpoKD-Venus dimers and (F) Sav-HpoKD-Venus dimers.
Fig. 6.
Fig. 6.
Canonical Hippo signalling is necessary and sufficient to control the nuclear localisation of Yki:GFP in follicle cells, whereas a Src family kinase inhibitor has no effect. (A) Endogenously tagged Yki:GFP localises to the nucleus in stretch cells and to the cytoplasm in columnar cells. (B) Overexpression of Hippo kinase (Hpo) with GR1.Gal4 in follicle cells causes Yki:GFP to relocalise to the cytoplasm in all cells. (C) Overexpression of Ex with GR1.Gal4 in follicle cells causes Yki:GFP to relocalise to the cytoplasm in all cells. (D) Mutation of Wts in clones marked by the absence of nuclear RFP (red) causes nuclear localisation of Yki:GFP in columnar cells. Boxed area is magnified in the panel below. (E) DMSO-treated control shows normal localisation of Yki:GFP in stretch cells and columnar cells. (F) Inhibition of Src family kinases with dasatinib has no effect on the localisation of Yki:GFP in either stretch cells or columnar cells. Arrows indicate the regions enlarged in the panels below.
Fig. 7.
Fig. 7.
Nuclear localisation of Yki:GFP occurs in mechanically stretched cells throughout Drosophila development. (A) In stage 10 egg chambers, Yki:GFP accumulates in the nucleus in extremely flat follicle cells in the anterior half of the egg chamber (stretch cells), and is exclusively cytoplasmic in columnar posterior follicle cells (main body follicle cells). (B) In stage 11 egg chambers, Yki:GFP accumulates in the nuclei of stretch cells and in the progressively flatter posterior main body follicle cells (white arrows). However, it remains cytoplasmic in the non-stretched follicle cells in the middle. (C) In stage 13 egg chambers, Yki:GFP localises in the nuclei of main body follicle cells at the posterior (white arrows), and in the precursors of the dorsal appendages at the anterior tip of the egg chamber, which are also flattening. Non-stretched centripetal cells accumulate Yki:GFP in the cytoplasm (dashed arrows). (D) Control stage 14 egg chamber. Nurse cell nuclei have disappeared, only the nuclei belonging to the muscle sheath that cover the egg are present around the dorsal appendages. (E) Stage 14 TJ.G4, UAS.Yki-RNAi egg chamber in which the nurse cell nuclei have failed to be degraded. (F) Bright-field image of a control egg mature egg. (G) Bright-field image of a TJ.G4 UAS.Yki-RNAi mature egg showing reduced extension in the anterior-posterior axis and shorter dorsal appendages.
Fig. 8.
Fig. 8.
Nuclear localisation of Yki occurs in the peripodial epithelium of the developing wing and is required for cell flattening and tissue expansion. (A) In third instar (L3) wing discs, Yki:GFP accumulates in the nuclei of the extremely stretched cells that are the peripodial membrane, but is mostly cytoplasmic in the highly columnar wing epithelial cells. (B) Control L3 wing discs stained with DAPI (blue). Wing cross-sections (left) and high magnifications of peripodial membrane cells (right). (C) Ubx.Gal4, UAS.Yki-RNAi L3 wing discs stained with DAPI (blue) showing reduced flattening of the peripodial membrane and a smaller wing disc area, as wing epithelial cells have been displaced to the peripodial membrane region to compensate for the reduction of tissue area. Wing cross-sections (left), and high magnifications of peripodial membrane cells (right). (D) Distribution of Mer, Kib and Ex in peripodial epithelial cells versus the underlying columnar epithelia of the third instar wing imaginal disc. Note the strong dilution of these complexes in the peripodial epithelial cells.

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References

    1. Badouel C., Gardano L., Amin N., Garg A., Rosenfeld R., Le Bihan T. and McNeill H. (2009a). The FERM-domain protein Expanded regulates Hippo pathway activity via direct interactions with the transcriptional activator Yorkie. Dev. Cell 16, 411-420. 10.1016/j.devcel.2009.01.010 - DOI - PubMed
    1. Badouel C., Garg A. and McNeill H. (2009b). Herding Hippos: regulating growth in flies and man. Curr. Opin. Cell Biol. 21, 837-843. 10.1016/j.ceb.2009.09.010 - DOI - PubMed
    1. Baumgartner R., Poernbacher I., Buser N., Hafen E. and Stocker H. (2010). The WW domain protein Kibra acts upstream of Hippo in Drosophila. Dev. Cell 18, 309-316. 10.1016/j.devcel.2009.12.013 - DOI - PubMed
    1. Benham-Pyle B. W., Pruitt B. L. and Nelson W. J. (2015). Cell adhesion. Mechanical strain induces E-cadherin-dependent Yap1 and beta-catenin activation to drive cell cycle entry. Science 348, 1024-1027. 10.1126/science.aaa4559 - DOI - PMC - PubMed
    1. Chen C.-L., Gajewski K. M., Hamaratoglu F., Bossuyt W., Sansores-Garcia L., Tao C. and Halder G. (2010). The apical-basal cell polarity determinant Crumbs regulates Hippo signaling in Drosophila. Proc. Natl. Acad. Sci. USA 107, 15810-15815. 10.1073/pnas.1004060107 - DOI - PMC - PubMed

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