Scribble participates in Hippo signaling and is required for normal zebrafish pronephros development Kassiani Skouloudakia, Michael Puetza, Matias Simonsb, Jean-Remy Courbardb, Christopher Boehlkea, Bjo¨ rn Hartlebena, Christina Engela, Marcus J. Moellerc, Christoph Englertd, Frank Bolligd, Tobias Scha¨ fera, Haribaskar Ramachandrana, Marek Mlodzikb, Tobias B. Hubera, E. Wolfgang Kuehna, Emily Kima, Albrecht Kramer-Zuckera, and Gerd Walza,e,1 aRenal Division, University Hospital Freiburg, 79106 Freiburg, Germany; bDepartment of Developmental and Regenerative Biology, Mount Sinai School of Medicine, New York, NY 10029; cDivision of Nephrology, Rheinisch-Westfa¨lische Technische Hochschule Aachen, 52074 Aachen, Germany; dLeibniz Institute for Age Research-Fritz Lipmann Institute, 07745 Jena, Germany; and eCenter for Biological Signaling Studies (bioss), 79104 Freiburg, Germany Edited by Kathryn V. Anderson, Sloan–Kettering Institute, New York, NY, and approved March 31, 2009 (received for review November 20, 2008) Spatial organization of cells and their appendages is controlled by [reviewed in (8)], Fat is required to recruit Expanded to the plasma the planar cell polarity pathway, a signaling cascade initiated by membrane, facilitating the assembly of a protein complex that the protocadherin Fat in Drosophila. Vertebrates express 4 Fat contains the scaffold protein Salvador and the Ste20 family kinase molecules, Fat1–4. We found that depletion of Fat1 caused cyst Hippo. Hippo phosphorylates and activates the Dbf2-related kinase formation in the zebrafish pronephros. Knockdown of the PDZ Warts; Warts in turn, complexed with Mob1-related protein Mats, domain containing the adaptor protein Scribble intensified the phosphorylates Yorkie. Phosphorylation of Yorkie, a transcrip- cyst-promoting phenotype of Fat1 depletion, suggesting that Fat1 tional co-activator, prevents its nuclear translocation and activation and Scribble act in overlapping signaling cascades during zebrafish of Yorkie target genes such as cyclin E and diap1 (9, 10). Upstream pronephros development. Supporting the genetic interaction with signals that activate Fat and the molecular links between Fat and Fat1, Scribble recognized the PDZ-binding site of Fat1. Depletion of the Expanded/Hippo/Warts cascade remain ill defined. Localiza- Yes-associated protein 1 (YAP1), a transcriptional co-activator tion and stabilization of Expanded at apical cell junctions depends inhibited by Hippo signaling, ameliorated the cyst formation in on Fat, because loss of Fat leads to reduced Expanded levels and Fat1-deficient zebrafish, whereas Scribble inhibited the YAP1- mislocalization (9, 11). However, studies have failed to demonstrate induced cyst formation. Thus, reduced Hippo signaling and subse- a direct interaction between Expanded and Fat or between Ex- quent YAP1 disinhibition seem to play a role in the development panded and other components of the Hippo pathway. of pronephric cysts after depletion of Fat1 or Scribble. We hypoth- Based on sequence similarities and domain architecture, Dro- esize that Hippo signaling is required for normal pronephros sophila Fat is related most closely to vertebrate Fat4. Fat4 development in zebrafish and that Scribble is a candidate link deficiency in mice causes developmental abnormalities of the between Fat and the Hippo signaling cascade in vertebrates. inner ear, neural tube, and kidney (12). Disruption of oriented cell division and defective elongation of kidney tubules result in Fat ͉ planar cell polarity ͉ polycystic kidney disease cystic kidneys, suggesting that Fat4 controls PCP signaling during renal development. Because the zebrafish embryo is amenable to utations in more than 20 seemingly unrelated human genes rapid genetic manipulation, we targeted zebrafish Fat1 (zFat1) Mcause polycystic kidney disease. In many patients, mutations and zebrafish Fat4 (zFat4) by antisense morpholino oligonucle- of these genes are associated with a plethora of extrarenal abnor- otides (MO) to compare their roles during zebrafish pronephros malities, ranging from polydactyly and CNS defects to obesity and development. Here we show that knockdown of zFat1, but not blindness (1). Virtually all gene products localize to the primary, of zFat4, caused extensive pronephric cysts. Epistasis assays non-motile cilium, a microtubular organelle attached to most body revealed a strong genetic interaction between Fat1 and Scribble. cells. Hence, it has been postulated that a dysfunction of the cilium Scribble recognized the PDZ-binding site that decorates verte- is responsible for the diverse manifestations, including kidney cysts. brate Fat1 but is absent in Fat4. Surprisingly, depletion of Multiple pathways use the cilium as a signaling platform or are zebrafish Yes-associated protein 1 (zYAP1) ameliorated the modulated by ciliary signals (2). One such pathway, the Wnt changes caused by Fat1 and Scribble knockdown, indicating that signaling cascade, plays an important role in kidney development a dysregulation of Hippo signaling contributes to the formation (3). Although the ␤-catenin–dependent branch of this pathway is of pronephric cysts caused by the absence of Fat1 and Scribble. required to convert metanephric mesenchyme into tubular epithe- Results lium, the ␤-catenin–independent planar cell polarity (PCP) path- way seems to promote nephron differentiation and maturation Knockdown of Zebrafish Fat1, but Not of Zebrafish Fat4, Promotes during later developmental stages (4, 5). The PCP pathway origi- Profound Cystogenesis. Because knockout of Fat4 in mice results in cystic kidney disease (12), we targeted zFat4 with MO. nally was identified as a signaling cascade that organizes cells and Ͼ their appendages in the Drosophila wing and eye but is increasingly Substantial ( 10%) cyst formation was detectable only at 3.75 pmol zFat4 MO (Fig. S1). At comparable efficacy (Fig. S2), recognized as playing a critical role in vertebrate organogenesis (6). BIOLOGY Drosophila genetics has delineated 3 classes of PCP proteins, the upstream PCP proteins (Fat, Dachsous, 4-jointed), the PCP core DEVELOPMENTAL Author contributions: K.S., M.M., T.B.H., and G.W. designed research; K.S., M.P., M.S., proteins (Frizzled, Dishevelled, Flamingo, Strabismus, Prickle, Di- J.-R.C., C.B., B.H., and C. Engel performed research; K.S., M.J.M., C. Englert, F.B., T.S., and ego), and downstream PCP effector proteins (Inturned, Fuzzy, H.R. contributed new reagents/analytic tools; K.S., M.P., E.W.K., and A.K.-Z. analyzed data; RhoA). Mutations of Drosophila Fat are associated with reversed and E.K. and G.W. wrote the paper. dorsal–ventral polarity, abnormal distal-to-proximal wing develop- The authors declare no conflict of interest. ment, and hyperplastic overgrowth of all larval imaginal discs This article is a PNAS Direct Submission. [reviewed in (7)]. These phenotypes define 2 distinct branches of 1To whom correspondence should be addressed. E-mail: [email protected]. Drosophila Fat signaling: the Fat polarity pathway and the Fat This article contains supporting information online at www.pnas.org/cgi/content/full/ tumor suppressor/Hippo pathway. In Drosophila Hippo signaling 0811691106/DCSupplemental. www.pnas.org͞cgi͞doi͞10.1073͞pnas.0811691106 PNAS ͉ May 26, 2009 ͉ vol. 106 ͉ no. 21 ͉ 8579–8584 Downloaded by guest on September 29, 2021 C-terminal amino acids of both zFat1 and mouse Fat1 (mFat1) constitute a class I PDZ-binding site (HTEV). Accordingly, we tested whether the cytoplasmic tail of Fat1 interacts with Scrib- ble by co-expressing full-length Scribble with the C-terminal cytoplasmic (cyt) domain of mFat1 fused to a secreted Ig truncation and the transmembrane domain of CD7 (sIg.7). Scribble was immunoprecipitated by the sIg7.mFat1-cyt con- struct (Fig. 2A). Deletional analysis further delineated the PDZ domains of Scribble and the PDZ-binding site of mFat1 as essential interaction domains (Fig. 2 B and C). To provide further evidence of a direct interaction between Fat and Scrib- ble, we generated recombinant GST-fusion proteins of the Scribble PDZ domains and precipitated sIg7.mFat1 from lysates of transiently transfected HEK 293T cells. sIg7.mFat1-cyt was precipitated by the PDZ2–4 and PDZ3–4 of Scribble but not by the PDZ domains of Par-3 or GST alone (Fig. 2D). These results demonstrate that mFat1 interacts with the PDZ domains of Scribble, presumably PDZ domains 3 and/or 4. Unlike Drosoph- ila Fat-like protein, Drosophila Fat also contains a C-terminal PDZ-binding site (class III; EEYV). To assess whether the PDZ-binding site of Drosophila Fat interacts with Scribble, we replaced the cytoplasmic and transmembrane domain of sIg7.mFat1-cyt with the corresponding Drosophila sequences (sIg.TM.dFat). This construct, which partially rescued the cystic phenotype caused by the knockdown of zFat1 (Fig. S3), immu- noprecipitated co-transfected human Scribble, confirming that both Drosophila Fat and mammalian Fat1 interact with Scribble Fig. 1. Zebrafish Fat1 genetically interacts with zebrafish Scribble. (A) (Fig. 2E). However, the interaction between Scribble and Dro- MO-induced knockdown of zFat1 was compared with the knockdown of sophila Fat was not contingent on the C-terminal PDZ-binding zebrafish Prickle 2 (zPk2), zebrafish Daam1 (zDaam1), zebrafish Protocad- site (Fig. S4), suggesting that Scribble recognizes internal ligands herin 8 (zPcdh8), zebrafish Fuzzy (zfuzzy), and zScrib. White arrowheads of Drosophila Fat instead of or in addition to the C-terminal indicate