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. 2009 May 26;106(21):8579-84.
doi: 10.1073/pnas.0811691106. Epub 2009 May 13.

Scribble participates in Hippo signaling and is required for normal zebrafish pronephros development

Kassiani Skouloudaki  1 , Michael Puetz, Matias Simons, Jean-Remy Courbard, Christopher Boehlke, Björn Hartleben, Christina Engel, Marcus J Moeller, Christoph Englert, Frank Bollig, Tobias Schäfer, Haribaskar Ramachandran, Marek Mlodzik, Tobias B Huber, E Wolfgang Kuehn, Emily Kim, Albrecht Kramer-Zucker, Gerd Walz
Affiliations

Scribble participates in Hippo signaling and is required for normal zebrafish pronephros development

Kassiani Skouloudaki et al. Proc Natl Acad Sci U S A. .

Abstract

Spatial organization of cells and their appendages is controlled by the planar cell polarity pathway, a signaling cascade initiated by the protocadherin Fat in Drosophila. Vertebrates express 4 Fat molecules, Fat1-4. We found that depletion of Fat1 caused cyst formation in the zebrafish pronephros. Knockdown of the PDZ domain containing the adaptor protein Scribble intensified the cyst-promoting phenotype of Fat1 depletion, suggesting that Fat1 and Scribble act in overlapping signaling cascades during zebrafish pronephros development. Supporting the genetic interaction with Fat1, Scribble recognized the PDZ-binding site of Fat1. Depletion of Yes-associated protein 1 (YAP1), a transcriptional co-activator inhibited by Hippo signaling, ameliorated the cyst formation in Fat1-deficient zebrafish, whereas Scribble inhibited the YAP1-induced cyst formation. Thus, reduced Hippo signaling and subsequent YAP1 disinhibition seem to play a role in the development of pronephric cysts after depletion of Fat1 or Scribble. We hypothesize that Hippo signaling is required for normal pronephros development in zebrafish and that Scribble is a candidate link between Fat and the Hippo signaling cascade in vertebrates.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Zebrafish Fat1 genetically interacts with zebrafish Scribble. (A) MO-induced knockdown of zFat1 was compared with the knockdown of zebrafish Prickle 2 (zPk2), zebrafish Daam1 (zDaam1), zebrafish Protocadherin 8 (zPcdh8), zebrafish Fuzzy (zfuzzy), and zScrib. White arrowheads indicate examples of cyst formation. (B) Pronephric cyst formation, caused by the depletion of these molecules, was scored at 55 h post fertilization (h.p.f), using the transgenic zebrafish line Wt1b:gfp. A reproducible degree of cyst formation (30%–40%) was noted in zFat1-depleted embryos without significant reduction in larval survival. (C) Epistasis assays between components of the PCP pathway revealed a strong synergism between zFat1 MO (0.125 pmol) and zScrib MO (0.125 pmol) injections. Transverse sections at the level of glomerulus and proximal tubules revealed bilateral pronephric cyst formation adjacent to the glomerulus in combined knockdown of Fat1 and Scribble in zebrafish embryos. (D) The presence of pronephric cysts was scored at 50–55 h.p.f. in zebrafish embryos injected with low MO concentrations. Note that pronephric cysts are hardly detectable after single injections of either 0.125 pmol zFat1 MO or 0.125 pmol zScrib MO, whereas the combination causes pronephric cysts in > 10% of microinjected embryos. (*, P < 0.05; **, P < 0.0001).
Fig. 2.
Fig. 2.
Molecular interaction between Fat1 and Scribble. (A) Human full-length Scribble was co-expressed with the cytoplasmic tail of mFat1 fused to the sIg7 tag (sIg7.mFat1-cyt) or with control proteins (sIg7.TRPC4 C-terminal and sIg7.nephrin-cyt) in HEK 293T cells. After immunoprecipitation with protein A-Sepharose beads, eGFP-tagged human Scribble was present in immunoprecipitates immobilized by sIg7.mFat1-cyt but not by sIg7.TRPC4 C-terminal or sIg7.nephrin-cyt. Equal expression of Scribble in cellular lysates was confirmed by antibody against GFP. (B) Scribble interacts with the C-terminal PDZ-binding site of Fat1. Wild-type Fat1, but not a truncated version lacking the last 3 amino acids (amino acids 4596–4598), interacted with Scribble. Expression levels of Scribble and the sIg7-tagged proteins are shown in the lower panels. (C) Fat1 interacts with the PDZ domain containing the C-terminal part of Scribble. HEK293T cells were transfected with sIg7.Fat1 and a Flag-tagged Scribble truncation containing either the amino-terminal half (hScrib N), the C-terminal half (hScrib C), or the 4 PDZ domains of Scribble (hScrib PDZ). Fat1 immobilized the C-terminal half and the PDZ domain-containing part but not the amino-terminal half of Scribble. The middle panel shows the expression of the Scribble truncations. The bottom panel shows the precipitated sIg7.mFat1-cyt. (D) The C-terminal domain of Fat1 interacted with a recombinant GST fusion protein containing the third and forth or the second, third, and forth PDZ domain of Scribble. Fat1 (sIg7.mFat1-cyt) expressed in HEK 293T cells was incubated with GST or GST fusion proteins as indicated. The Par3 PDZ domains 1 and 2, used as a control, did not bind to Fat1. (E) Drosophila Fat interacts with Scribble. An mFat1 fusion protein containing the sIg tag, a short part of extracellular domain, and the transmembrane and C-terminal domain of Fat1 and an identical Drosophila Fat fusion protein precipitated Scribble but not the control protein sIgTM.nephrin. (F) Fat1, but not Fat4, interacts with Scribble. mFat4 cytoplasmic tail (mFat4-cyt) was fused to the sIg7 tag. Neither sIg7.Fat4-cyt nor the control protein sIg7.nephrin-cyt interacted with Scribble. (G) Endogenous Fat and Scribble co-localize in NRK-52E cells. Confluent NRK-52E cells were labeled with rabbit anti-Fat1 antiserum and goat anti-Scribble antiserum. Both Fat1 and Scribble co-localized at the plasma membrane of NRK-52E cells. (H) Co-localization of endogenous Fat1 and Scribble was confirmed by confocal microscopy. Both Fat1 and Scribble co-localized at cell–cell contacts of NRK-52E cells. The red line depicts the site of z-section (Top Row). Z-reconstruction (x-z direction) of a z-stack (15 planes, z-distance 0.2 μm), showing co-localization of Fat1 and Scribble (Bottom row). (Scale bars: G, 20 μm; H, 5 μm.)
Fig. 3.
Fig. 3.
Control of YAP1 expression is essential for normal zebrafish pronephros development. (A) Knockdown of zYAP1 by MO (zYAP1 MO) results in pronephric cysts. Lateral view of embryos injected with zYAP1 MO (0.5 pmol, 1.25 pmol, and 2.5 pmol) at 55 h.p.f. A curled tail and shortening of the body (arrows) are observed in the zYAP1 morphants (Upper). At higher concentration, zYAP1 MO injections led to cyst formation (Lower). (B) Cyst formation caused by zFat1 MO was partially rescued by the simultaneous knockdown of zYAP1. (C) Overexpression of human YAP1 (hYAP1) recapitulated the loss-of-function phenotypes (cyst formation) caused by zFat1 knockdown (zFat1 MO). (D) Over-expression of human YAP1 resulted in pronephric cysts in zebrafish embryos. This result was reversed by co-injection of human Scribble RNA (hScrib). (Numbers in parentheses indicate the total number of embryos used in each experiment.)
Fig. 4.
Fig. 4.
Scribble inhibits YAP1 activation. (A) Wild-typeYAP1 activity (Black Bars) is repressed by Scribble (Scrib), whereas the YAP1S127A phosphorylation mutant (Gray Bars) reverses the Scribble-mediated inhibition. The 5xUAS-luciferase reporter, GAL4-TEAD4, β-galactosidase, and V5.YAP1 were simultaneously transfected into HEK 293T cells with plasmids as indicated. Luciferase activity was measured and normalized to β-galactosidase activity. Lats2 was used as a positive control for active Hippo signaling. (B) The combined knockdown of zScrib and zFat1 activated the YAP1-dependent Survivin reporter construct (pLuc-Survivin 1430) in zebrafish embryos when compared with single MO injections combined with a control MO. (C) HEK 293T cells expressing V5-YAP1 (Left), V5-YAP1 plus Scribble (Middle), or Lats2 (Right) were analyzed by subcellular fractionation. Nuclear YAP1 decreased in response to active Hippo signaling. Nuclear Lamin and cytosolic tubulin were used to control for the quality of the fractionation. C, cytoplasmic; N, nuclear. (D) Scribble and Lats2 are present in the cytosolic fraction. (E) HEK 293T cells were transfected with GFP-YAP1, HA-14–3-3β, Fat1, or Scribble, as indicated. GFP-YAP1 was immunoprecipitated with anti-GFP, and immobilized 14–3-3β was stained with anti-HA antibodies. Expression of the 2 proteins mFat1 and Scribble led to the interaction of YAP1 with 14–3-3β. (F) HeLa cells were transfected with GFP-YAP1 or GFP-YAP1 plus V5-Scribble or Flag-Lats2 and were stained with anti-V5, anti-Scribble and anti-Flag antibodies. Nuclear YAP1 signal is detectable in cells expressing GFP-YAP1 alone, whereas cells expressing Scribble or Lats2 show a predominant cytoplasmic staining of YAP1.
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