Tsukushi functions as a Wnt signaling inhibitor by competing with Wnt2b for binding to transmembrane protein Frizzled4 Kunimasa Ohtaa,b,1,2, Ayako Itoa,c,1, Sei Kuriyamaa,d,3, Giuseppe Lupoe,f, Mitsuko Kosakag,4, Shin-ichi Ohnumah, Shinichi Nakagawai, and Hideaki Tanakaa,c,d aDepartment of Developmental Neurobiology, Graduate School of Medical Sciences, Kumamoto University, Kumamoto 860-8556, Japan; bPrecursory Research for Embryonic Science and Technology, Japan Science and Technology Agency, Saitama 332-0012, Japan; cGlobal Center of Excellence, Kumamoto University, Kumamoto 860-8556, Japan; d21st Century Center of Excellence, Kumamoto University, Kumamoto 860-8556, Japan; eDepartment of Biology and Biotechnology “C. Darwin,” University of Rome “La Sapienza,” 00185 Rome, Italy; fIstituto Pasteur–Fondazione Cenci Bolognetti, 00185, Rome, Italy; gRIKEN Center for Developmental Biology, Kobe 650-0047, Japan; hInstitute of Ophthalmology, University College London, London EC1V 9EL, United Kingdom; and IRIKEN Advanced Science Institute, Nakagawa RNA Biology Laboratory, Saitama 351-0198, Japan Edited* by Lynn T. Landmesser, Case Western Reserve University, Cleveland, OH, and approved July 8, 2011 (received for review January 11, 2011) The Wnt signaling pathway is essential for the development of We previously described the isolation of Tsukushi (TSK) protein diverse tissues during embryogenesis. Signal transduction is acti- isoforms (13), soluble molecules belonging to the small leucine- vated by the binding of Wnt proteins to the type I receptor low- rich proteoglycan (SLRP) family (14), and showed that they work density lipoprotein receptor–related protein 5/6 and the seven-pass as extracellular modulators of pivotal signaling cascades during transmembrane protein Frizzled (Fzd), which contains a Wnt- early embryonic development in chicks and frogs (13, 15–17). We binding site in the form of a cysteine-rich domain. Known extracel- also observed that TSK rescues Xenopus embryos from the lular antagonists of the Wnt signaling pathway can be subdivided hyperdorsalized effects induced by Wnts (Fig. S1). In this study, we into two broad classes depending on whether they bind primarily to show that TSK functions as a novel Wnt signaling inhibitor by Wntortolow-densitylipoproteinreceptor–related protein 5/6. We competing with Wnt2b for binding to Fzd4. Our biochemical show that the secreted protein Tsukushi (TSK) functions as a Wnt analysis demonstrates direct binding between TSK and Fzd4 with −10 signaling inhibitor by binding directly to the cysteine-rich domain of an affinity of 2.3 × 10 M. Using overexpression assays in chicken − Fzd4 with an affinity of 2.3 × 10 10 M and competing with Wnt2b. In embryonic retinal cells, we found that TSK inhibits Wnt2b activity the developing chick eye, TSK is expressed in the ciliary/iris epithe- both in vitro and in vivo and represses Wnt2b-dependent induction lium, whereas Wnt2b is expressed in the adjacent anterior rim of the of peripheral eye character. Conversely, TSK inactivation results in optic vesicle, where it controls the differentiation of peripheral eye expansion of the CB in mice. Consequently, TSK is an important structures, such as the ciliary body and iris. TSK overexpression ef- component of the molecular pathways controlling retinal and pe- fectively antagonizes Wnt2b signaling in chicken embryonic retinal ripheral eye development. fi cells both in vivo and in vitro and represses Wnt-dependent speci - Results cation of peripheral eye fates. Conversely, targeted inactivation of the TSK gene in mice causes expansion of the ciliary body and up- Expression of TSK at the Peripheral Chick Retina. There is substantial regulation of Wnt2b and Fzd4 expression in the developing periph- evidence that the Wnt signaling pathway controls the development eral eye. Thus, we uncover a crucial role for TSK as a Wnt signaling of peripheral eye structures in several animal models (8, 12, 18– inhibitor that regulates peripheral eye formation. 20). TSK is expressed in the peripheral region of the developing chick eye at E6 (Fig. S2 A and B). As shown in Fig. S2C,Wnt2b eye development | signaling modulator | small leucine-rich proteoglycan expression is localized to the anteriormost tip of the optic cup, whereas chick TSKB (C-TSKB) (15) is expressed in the adjacent ciliary/iris epithelium (Fig. S2B). Comparing C-TSKB expression nt signaling is involved in multiple developmental events with the expression pattern of collagen IX (Fig. S2F), a marker of during embryogenesis and it has also been implicated in W the neighboring ciliary/iris epithelium (8, 21), shows that C-TSKB adult tissue homeostasis (1, 2). Wnt proteins act on target cells by binding to the Frizzled (Fzd)/low-density lipoprotein receptor– expression does not extend into the ciliary epithelium region. Other Wnt ligands, such as Wnt3a and Wnt6, are not transcribed related protein (LRP) complex at the cell surface, which trans- G duces Wnt signals into the target cells (3, 4). Extracellular antag- in the developing peripheral eye (Fig. S2 ). Fzd1, 3, and 4 are onists of the Wnt signaling pathway can be subdivided into two expressed in both the ciliary and the iris epithelia, whereas Fzd5 is D E G broad classes: the secreted Fzd-related protein class and the not expressed in either of these areas (Fig. S2 , ,and ). These Dikkopf class (5). The members of the secreted Fzd-related pro- expression patterns suggest that Wnt2b and C-TSKB could func- tein class bind directly to Wnts and alter their ability to bind to the Wnt receptor complex, whereas members of the Dikkopf family inhibit Wnt signaling by binding to the LRP5/LRP6 component of Author contributions: K.O., A.I., G.L., S.-i.O., and H.T. designed research; K.O., A.I., and the Wnt receptor complex. A Wnt inhibitor that specifically binds S.K. performed research; M.K. and S.N. contributed new reagents/analytic tools; K.O., A.I., to the Fzd receptor has not yet been identified, however. and S.K. analyzed data; and K.O. and G.L. wrote the paper. The peripheral rim of the optic cup is a unique region of the The authors declare no conflict of interest. developing eye that forms two of the peripheral support tissues, *This Direct Submission article had a prearranged editor. the ciliary body (CB) and the iris (6). These tissues are composed 1K.O. and A.I. contributed equally to this work. of a nonpigmented inner layer, which is continuous with the retina, 2To whom correspondence should be addressed. E-mail: [email protected]. and a pigmented outer layer, which is continuous with the retinal jp. pigmented epithelium (7). Cho and Cepko (8) performed gain-of- 3Present address: Department of Molecular Medicine and Biochemistry, Akita University function analyses in vivo and found that retinal cells exposed to Graduate School of Medicine and Faculty of Medicine, Akita 010-8543, Japan. high levels of Wnt signaling are induced to acquire CB and iris cell 4Present address: Okayama University Graduate School of Medicine, Dentistry and Phar- fates. Thus, Wnt signaling is involved in the differentiation of maceutical Sciences, Okayama 700-8558, Japan. peripheral eye structures, but the molecular interactions involved This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. are not yet clearly understood (8–12). 1073/pnas.1100513108/-/DCSupplemental. 14962e14967 | PNAS | September 6, 2011 | vol. 108 | no. 36 www.pnas.org/cgi/doi/10.1073/pnas.1100513108 Downloaded by guest on September 25, 2021 tionally interact with each other and regulate Fzd-dependent sig- Wnt2b in the chick optic vesicle and observed the effects on the naling in the peripheral eye. proliferation of retinal explants and the expression of the CB marker collagen IX, both of which are stimulated by Wnt2b (22). TSK Inhibits Wnt2b Activity in Vitro. To address whether TSK We first electroporated the optic vesicles of E1.5 chicken embryos functions as a Wnt inhibitor, we co-overexpressed C-TSKB with with a Wnt2b-expressing provirus as described previously (22). We then dissected explants of the electroporated retinal tissues (250 × 250 μm2 surface area) from the central region of E5.5 retinas and cultured them in vitro for 8 d (Fig. 1A). Confirming the results of A 2days 4days layer formation control Wnt2b Wnt2b + C-TSKB B DF GFP C EG BMP7 HKN GFP ILO collagen IX JMP DAPI NEUROSCIENCE Q m) μ 180 * * 150 * 120 90 60 30 Thickness of retinal layer ( 0 Wnt2b C-TSKB control-GFP Wnt2b + C-TSKB Wnt2b + C-TSKB-LRR6Wnt2b + C-TSKB-LRR4Wnt2b + C-TSKB-LRR3Wnt2b + C-TSKB-LRR2 Fig. 1. TSK inhibits Wnt activity in vitro. (A) Schematic of the experimental Fig. 2. TSK inhibits Wnt activity in vivo. (A) Schematic of the experimental procedure. (B–G) BrdU incorporation was increased in the explants expressing procedure. (B–P) Histological sections of central retinal regions from chicken Wnt2b. (B and C) DAPI. (D and E)GFP.(F and G) BrdU. (Scale bar: 100 μm.) (H– embryos electroporated with the indicated DNA constructs plus RCAS:GFP at J) Retinal explants of control (H), Wnt2b- (I), and Wnt2b+C-TSKB–expressing E1.5 and incubated for 6 d in vivo. (B, D, F, H, K,andN) GFP expression. (C, E, chick retinas (J), which were dissected out at E5.5 and cultured for 8 d in vitro and G) Expression of BMP7 detected by in situ hybridization. (I, L, and O)Ex- after electroporation at E1.5. The arrow in H points to an explant immediately pression of collagen IX detected by immunohistochemistry. White arrowheads after dissection. RCAS:GFP was cotransfected to visualize electroporated cells. indicate the immunoreactivity of anti-collagen IX antibody on the choroid. (K–P) Expression of collagen IX in the explants expressing Wnt2b. (K, M,and (J, M,andP) DAPI. Note that the thinning and folding of the retinal epithelium O) DAPI.