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. 2008 Sep 30;105(39):14897-902.
doi: 10.1073/pnas.0805201105. Epub 2008 Sep 22.

Boundaries of Dachsous Cadherin activity modulate the Hippo signaling pathway to induce cell proliferation

Maria Willecke  1 , Fisun Hamaratoglu, Leticia Sansores-Garcia, Chunyao Tao, Georg Halder
Affiliations

Boundaries of Dachsous Cadherin activity modulate the Hippo signaling pathway to induce cell proliferation

Maria Willecke et al. Proc Natl Acad Sci U S A. .

Abstract

The conserved Hippo tumor suppressor pathway is a key signaling pathway that controls organ size in Drosophila. To date a signal transduction cascade from the Cadherin Fat at the plasma membrane into the nucleus has been discovered. However, how the Hippo pathway is regulated by extracellular signals is poorly understood. Fat not only regulates growth but also planar cell polarity, for which it interacts with the Dachsous (Ds) Cadherin, and Four-jointed (Fj), a transmembrane kinase that modulates the interaction between Ds and Fat. Ds and Fj are expressed in gradients and manipulation of their expression causes abnormal growth. However, how Ds and Fj regulate growth and whether they act through the Hippo pathway is not known. Here, we report that Ds and Fj regulate Hippo signaling to control growth. Interestingly, we found that Ds/Fj regulate the Hippo pathway through a remarkable logic. Induction of Hippo target genes is not proportional to the amount of Ds or Fj presented to a cell, as would be expected if Ds and Fj acted as traditional ligands. Rather, Hippo target genes are up-regulated when neighboring cells express different amounts of Ds or Fj. Consistent with a model that differences in Ds/Fj levels between cells regulate the Hippo pathway, we found that artificial Ds/Fj boundaries induce extra cell proliferation, whereas flattening the endogenous Ds and Fj gradients results in growth defects. The Ds/Fj signaling system thus defines a cell-to-cell signaling mechanism that regulates the Hippo pathway, thereby contributing to the control of organ size.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Boundaries of Ds and Fj activity induce Hippo target genes. Third instar imaginal discs, which contain clones of cells either overexpressing or mutant for ds or fj. Clones induced the expression of β-Gal expression from lacZ enhancer trap insertions into the Hippo target genes ex (A–C, E, and F) and fj (D). β-Gal induction was observed specifically at clone borders and induction was seen on both sides of clone borders except for dsUA071 mutant clones, which induced fj-lacZ expression only in cells outside mutant clones. Cells overexpressing Ds (A–C) or Fj (F) were positively marked by coexpression of GFP. Clones mutant for dsUAO71 (D) and fjd1 (E) were marked by the absence of GFP. A, B, D, and E show eye imaginal discs and C and F show wing discs. B is a close-up of a clone shown in A. (F) Wing disc with clones overexpressing Fj in the hinge region marked by GFP expression. For all stainings, GFP is shown in green and β-Gal antibody stainings are shown in red. A′–F′ show β-Gal antibody stainings in gray. Anterior is to the left in all discs and arrowheads point to clone borders.
Fig. 2.
Fig. 2.
Boundaries of Ds and Fj activity regulate Ex stability and localization. Ex protein was reduced at the plasma membrane along the borders of clones of cells overexpressing Ds in a wing (A) and an eye disc (B) and along the borders of fjd1 mutant clones (C). (D) Ds overexpressing clones did not affect Mer levels (wing disc). Ds overexpressing clones did not affect Ex in a fatfd/422 (E) or a dGC13 homozygous mutant wing disc (F). (Left) GFP in green and antibody staining in red. (Right) Antibody staining in gray. Arrowheads point to clone boundaries. Anterior is to the left in all discs.
Fig. 3.
Fig. 3.
Ds is required for sensing of the boundary signal. (A) Overexpression of Ds in a wild-type background induced fj-lacZ expression inside and outside of clone borders. (B) Overexpression of Ds in dsUA071 homozygous mutant eye discs also induced fj-lacZ expression, but only in cells within the clone. (C and D) Overexpression of a version of Ds that lacks the intracellular domain, Ds(ΔICD), was sufficient to induce fj-lacZ expression in wild-type (C), but not dsUA071 homozygous mutant discs (D). For all stainings: β-Gal expression is shown in red and overexpression clones are marked by coexpression of GFP (green) (Left); anti-β-Gal staining is shown in gray (Center); schematic representations of the four experiments illustrating the requirement of the Ds intracellular domain for the sensing of the boundary signal (Right). These schematics depict the pattern of Hippo target gene induction (brown) as a result of clones overexpressing different versions of Ds.
Fig. 4.
Fig. 4.
Discontinuities of Ds/Fj activities drive cell proliferation and tissue growth. Eye imaginal discs containing a Ds overexpressing clone marked by GFP expression (A) and fjd1 mutant clones marked by the absence of GFP expression (B and C). These discs were labeled for BrdU incorporation (red in A–C and gray in A′–C′). Wild-type cells arrest in G1 in the morphogenetic furrow (asterisks) and nondifferentiating cells go through one synchronous S phase in the second mitotic wave (arrows). Ds expressing and fjd1 mutant clones posterior to the second mitotic wave show ectopic cell proliferation along clone borders in mutant and wild-type cells (arrowheads). (The second mitotic wave in A is out of focus.) (D) Midpupal retina carrying fjd1 mutant clones marked by the absence of GFP expression stained with Discs large (Dlg) antibodies to mark cell outlines. Extra interommatidial cells were produced at clone borders (arrowheads). (E–G) Images of wings from adults of the indicated genotypes. (F) dpp-Gal4-driven overexpression of Ds caused an enlargement of the distance between veins 3 and 4 (arrowhead). (G) tub-Gal4-driven overexpression of Ds and Fj caused small wings. (H) Quantification of the area between veins 3 and 4 of flies shown in E and F, as well as additional genotypes including overexpression of Fj. The areas were normalized to sibling wild-type control flies. (I) Quantification of the wing area of flies that overexpressed Ds and Fj under the tub-Gal4 driver shown in G as well as additional genotypes that expressed Ds and Fj in ds, fj double mutants, and Ds and Fj by themselves. The wing areas were normalized to sibling wild-type control flies.
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References

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