Tsukushi Functions As a Wnt Signaling Inhibitor by Competing with Wnt2b for Binding to Transmembrane Protein Frizzled4

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

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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. (L, N,andP) Collagen IX. White arrowheads indicate the immuno- and the ectopic expression of BMP7 and collagen IX caused by Wnt2b over- reactivity of anti-collagen IX antibody on the choroid. (Scale bar: 180 μm.) (Q) expression were suppressed by C-TSKB in vivo. (Q)Quantification of the Quantification of cell numbers in explants electroporated with the indicated thickness of the retinal layer in retinas electroporated with the indicated DNA DNA constructs. Note that at least four LRR domains are necessary and suf- constructs. Note that at least four LRR domains are necessary and sufficient to ficient to inhibit the effects of Wnt2b on cell proliferation in vitro. *P < 0.001 inhibit the effects of Wnt2b in vivo. *P < 0.001 (vs. Wnt2b) based on a two- (vs. Wnt2b) based on a two-tailed Student’s t test. Error bars represent the SD. tailed Student’s t test. Error bars represent the SD. (Scale bar: 100 μm.)

Ohta et al. PNAS | September 6, 2011 | vol. 108 | no. 36 | 14963 Downloaded by guest on September 25, 2021 B A B b K C 2 tn S T AK BKST A B - KS KS B W C S KS + +x T T T -C - -C -C x x T-C+ C e e e- -5 - +DRC-4 + +D +D 5 5PR P PRL D DR DR RL RC R RC-5d b2 b L 2 C- C- C t tn -4 - 4d 5 5 nW d d d dz W zF z z z z Pull down: Wnt2b/C-TSKB WB: -Flag F F F F F Pull down: C-TSKB WB: -Fc Input: Wnt2b WB: -Fc Pull down: C-TSKA/C-TSKB WB: -Flag Input: C-TSKB WB: -Myc Input: C-TSKB WB: -Fc Input: C-TSKA/C-TSKB WB: -Fc

Input: Wnt2b WB: -Fc Input: LRP5-ex WB: -Flag Input: Fzd4/Fzd5 WB: -Flag

D E BKST-C F BK G ST-C+

+ b b2 b b 2 2 2 t tnW+D tnW tn nW D W R + + + C -4 D D DR dzF+K R RC-4d R C C C -4 - -4 4 dzF d d zF z z KST-M F F S Fzd4-CRD +Fzd4-CRD C-TSKB-LRR4 +Fzd4-CRD C-TSKB-LRR3 +Fzd4-CRD C-TSKB-LRR2 +Fzd4-CRD C-TSKB-LRR1 T-M Pull down: Wnt2b WB: -Flag Pull down: Wnt2b WB: -Flag Pull down: C-TSKB-LRRs WB: -Myc

Pull down: Fzd4-CRD WB: -V5 Input: C-TSKB WB: -Myc Input: C-TSKB WB: -Myc Input: C-TSKB-LRRs WB: -Fc Input: Fzd4-CRD WB: -Fc Input: Wnt2b WB: -Fc Input: Wnt2b WB: -Fc

Input: M-TSK WB: -V5 Input: Fzd4-CRD WB: -Flag Input: Fzd4-CRD WB: -Flag Input: Fzd4-CRD WB: -Myc

Fig. 3. TSK binds to Fzd4 directly. (A) Coprecipitation of C-TSKB-Myc-His and Wnt2b-Fc. (B) Coprecipitation of Wnt2b-Fc or C-TSKB-Fc and LRP5-ex-Flag. (C) Coprecipitation of C-TSKA-Fc or C-TSKB-Fc and Fzd4-CRD-Myc-Flag or Fzd5-CRD-Myc-Flag. (D) Coprecipitation of puriﬁed M-TSK-V5-His and Fzd4-CRD-Fc. (E) Competition assay of Wnt2b-Fc and C-TSKB-Myc-His for binding to Fzd4-CRD-Myc-Flag. (F) Competition assay of C-TSKB-Fc and Norrin-3Myc-AP for binding to Fzd4-CRD-Myc-Flag. (G) A single LRR domain is sufﬁcient to bind to Fzd4-CRD.

previous studies (22), Wnt2b-expressing retinal explants generated C-TSKB on Wnt signaling could be observed in vivo as well (Fig. large folded sheets of tissue (Fig. 1I), with high numbers of cells 2A). Developing optic vesicles were infected with RCAS:Wnt2b at incorporating BrdU (Fig. 1 B–G). Conversely, the coexpression of E1.5 and examined at E7.5. The infected eyes showed thinning and RCAS:Wnt2b with RCAS:C-TSKB resulted in explants of similar folding of the retinal epithelium at the level of the central retina, size as the control explants (Fig. 1 H and J). Wnt2b activity also up-regulation of the CB markers BMP7 and collagen IX (Fig. 2 D, induced ectopic collagen IX expression in retinal explants (Fig. 1 E, K, L,andM) (8, 21), and down-regulation of β-tubulin III and M and N), which was prevented in explants coexpressing Wnt2b pax6 expression (Fig. S4 C and D). The average thickness of the and C-TSKB (Fig. 1 O and P). Cell number quantifications (Fig. RCAS:Wnt2b-infected thinner retina was 40 ± 19 μm(n =11) 1Q) showed that the number of cells in explants coexpressing (Fig. 2Q). In the presence of both Wnt2b and C-TSKB, the Wnt2b and C-TSKB (7.2 ± 0.4 × 103; n = 30) was similar to that of infected retinal tissue failed to show any abnormalities (Fig. 2 F, G, control explants (4.3 ± 1.3 × 103; n = 30) and C-TSKB–expressing N, O,andP) and was similar to the control retina (Fig. 2 B, C, H, I, explants (4.6 ± 1.7 × 103; n = 30), whereas it was exponentially and J). The average thickness of control-, RCAS:C-TSKB- and increased in Wnt2b-expressing explants (1.2 ± 0.1 × 105; n =30) RCAS:Wnt2b+RCAS:C-TSKB-infected retinas was 142 ± 21 μm (Fig. 1Q). These effects were not due to changes in cell death (Fig. (n = 8), 147 ± 6 μm(n =3),and151± 26 μm(n = 7), respectively S3 A and B). (Fig. 2Q). We next sought to identify specific subdomains of TSK involved We performed the same experiments using mutant forms of in the inhibition of Wnt activity. For this purpose, we generated Fc- C-TSK. The average thickness of C-TSKB-LRR2-Fc–, C-TSKB- fusion constructs encoding between one and four leucine-rich re- LRR3-Fc–, C-TSKB-LRR4-Fc–, and C-TSKB-LRR6-Fc–infected peat (LRR) domains from the N terminus (C-TSKB-LRR1-4-Fc), retinas was 72 ± 28 μm(n =4),65± 33 μm(n = 4), 132 ± 23 μm and evaluated their inhibitory function on Wnt2b activity using (n = 4), and 143 ± 22 μm(n = 4), respectively (Fig. 2Q). This similar electroporation assays. Only C-TSKB-LRR4-Fc was able to indicates that, similar to the in vitro assay shown in Fig. 1, at least completely inhibit Wnt activity in vitro (C-TSKB-LRR4-Fc: 7.3 ± four LRR domains are necessary and sufficient to inhibit the 0.6 × 103, n = 22; C-TSKB-LRR3-Fc: 3.0 ± 0.7 × 104, n = 32; effects of Wnt2b in vivo (Fig. 2Q). In the same assay, the infection C-TSKB-LRR2-Fc: 1.4 ± 0.6 × 105, n = 20; C-TSKB-LRR1-Fc: 1.7 ± of RCAS:Wnt2b with RCAS:LINGO-1-ex, RCAS:Akhirin, or 0.4 × 105, n = 20) (Fig. 1Q). Thus, it appears that at least four LRR RCAS:Equarin failed to inhibit Wnt2b activity (Fig. S4E). Con- domains of the C-TSKB protein are necessary for robust inhibition sequently, TSK specifically and efficiently abrogates Wnt2b ac- of Wnt signaling. To examine the specificity of the inhibition of tivity both in vivo and in vitro. Wnt2b activity by C-TSKB, we coelectroporated embryos with Wnt2b and either the extracellular domain of LINGO-1 (23), TSK Binds to Fzd4 Directly. To investigate the possibility of a bio- containing 12 LRR domains (LINGO-1-ex), or the secreted pro- chemical interaction between TSK and Wnt signaling molecules, teins Akhirin (24) or Equarin (25), which are not related to the we performed coprecipitation assays by transfecting tagged con- SLRP family. None of these proteins inhibited Wnt activity structs into COS-7 cells (Fig. S5A). Wnt2b-Fc pulled down both (Fig. S3C). Flag-tagged Fzd4–cysteine-rich domain [CRD; the site of high- affinity Wnt binding (26)] and Flag-tagged Fzd5-CRD (Fig. S5B), TSK Inhibits Wnt2b Activity in Vivo. Confirming previous studies (8), in addition to Flag-tagged LRP5-ex (extracellular domain of LRP5) overexpression of Wnt2b in the optic vesicle in vivo induced (Fig. 3B). When Myc-His–tagged C-TSKB was coprecipitated with the expression of peripheral eye markers (Fig. 2 C, E, I,andL) Fc-tagged Wnt2b, precipitation with nickel-chelating resins did not and a significant reduction in the number of proliferating cells pull down Wnt2b (Fig. 3A). When Fc-tagged C-TSKB was copre- in Wnt2b-infected regions (Fig. S4 A and B). We used this cipitated with Flag-tagged LRP5-ex, Flag-tagged Fzd4-CRD, or overexpression assay to examine whether the inhibitory activity of Flag-tagged Fzd5-CRD, precipitation with protein G resins pulled

14964 | www.pnas.org/cgi/doi/10.1073/pnas.1100513108 Ohta et al. Downloaded by guest on September 25, 2021 − − down only Fzd4-CRD (Fig. 3 B and C). Another isoform of the TSK 2.3 × 10 10 M and 1.4 × 10 9 M, respectively (Fig. 4 I–K). More- gene, C-TSKA, which differs in the structure of the C terminus over, in the presence of Wnt3a protein, the binding of TSK-AP from C-TSKB (15), also pulled down Fzd4-CRD (Fig. 3C). protein to Fzd4 was inhibited (Fig. S6). Taken together, these To exclude the possibility that the detected binding between observations strongly suggest that TSK inhibits Wnt signal trans- C-TSKB and Fzd4 is due to nonspecific interactions in the en- duction by interacting directly with Fzd4 at the cell surface and doplasmic reticulum of transfected cells, we also performed preventing Wnt2b from binding Fzd4 and thereby stimulating coprecipitation assays with purified proteins. As shown in Fig. receptor activity. 3D, binding between the affinity-purified V5-His–tagged mouse TSK (M-TSK) protein (Fig. S5C) and purified Fc-tagged mouse Loss of TSK Results in the Expansion of CB in Vivo. To address the −/− Fzd4-CRD (M-Fzd4-CRD) protein (Fig. S5D) was detectable requirement for TSK function in vivo, we generated TSK mice as well. by inserting a lacZ/Neo cassette into the TSK coding exon (29). +/− To confirm that TSK binds to the CRD domain of the Fzd4 We examined the expression of the TSK gene in adult TSK eyes receptor, we performed competitive binding assays with Fzd4- and found β-gal activity in the retinal layers, CB, and lens epi- CRD, C-TSKB, Wnt2b, and Norrin, a highly specific ligand for thelium (Fig. S7 A and B). The expression of TSK-driven β-gal was Fzd4 (27). Fig. 3E shows that the binding of Myc-Flag–tagged observed in the prospective CB and continued until adulthood Fzd4-CRD with Fc-tagged Wnt2b was inhibited by the addition (Fig. 5 A–F). The CB is composed of folds of bilayered epithelium of the Myc-His–tagged C-TSKB protein, whereas Akhirin and with an inner nonpigmented layer and an outer pigmented layer Equarin did not interfere with the interaction of Wnt2b and (30); β-gal activity was present predominantly in the inner non- Fzd4 (Fig. S5 E and F). Furthermore, the binding of Myc-Flag– pigmented layer (Fig. S7 C–E). tagged Fzd4-CRD with Fc-tagged C-TSKB was inhibited by To elucidate the role of TSK in CB formation, we performed −/− the Myc–alkaline phosphatase (AP)-tagged Norrin protein a morphological analysis of adult eyes from TSK and WT mice (Fig. 3F). by observing the CB structure from the vitreous side. Fig. 5 G and We next sought to identify the specific subdomains of TSK in- H shows the CB structure in the dorsal and the ventral regions of volved in Fzd4 binding. We evaluated the binding of different C- a WT eye. We found that the ventral CB (Fig. 5H) was larger than terminally truncated constructs of C-TSKB containing a variable the dorsal CB (Fig. 5G). Both the ventral and dorsal CB appeared −/− number of LRR domains to Fzd4-CRD. Interestingly, Fzd4-CRD to be expanded in TSK eyes compared with WT eyes; however, bound to all mutant forms of C-TSKB (Fig. 3G), indicating that their dorsoventral difference was maintained in the mutant eyes a single LRR is sufficient for the binding of C-TSKB to Fzd4-CRD. (Fig. 5 I and J). This effect was quantified by measuring the −/− maximal CB length in the dorsal and ventral regions of TSK Fzd4 Is a TSK Receptor. Using immunocytochemistry and immu- (n = 12 animals) and WT eyes (n = 10 animals) (Fig. 5 K and L). noblotting, we examined the nuclear translocation of β-catenin Measurements of the CB area in histological sections confirmed − − after Wnt signaling stimulation in L cells expressing Fzd4 (Fig. 4F) the expansion of the CB in TSK / eyes (n = 10 animals) (Fig. 5 O– in the presence or absence of TSK (Fig. 4 A–E). As described Q) compared with WT eyes (n = 8 animals) (Fig. 5 M, N,andQ), previously (28), the application of Wnt3a protein to L cells in- Furthermore, immunohistochemical analyses failed to show any −/− duced the translocation of β-catenin into the nuclei (Fig. 4 A and difference between adult WT and TSK retinal layering within E), which was not detected when L cells were pretreated with C- the inner retina (Fig. S8 A–L). To exclude the possibility that the TSKB protein (Fig. 4 C and E). This finding is most readily adjacent lens tissue, which normally expresses TSK, may be in- −/− NEUROSCIENCE explained by the hypothesis that Fzd4 functions as a receptor for volved in the abnormal CB development of TSK mice, we ex- TSK and that TSK inhibits Wnt signaling by competing with Wnt amined the morphology and mitotic activity of the lens and found −/− ligands for receptor binding at the cell surface. To further confirm no significant changes in TSK mice (Fig. S8 M–Q). Bone mor- this possibility, we expressed Fzd4 or Fzd5 proteins in COS-7 cells phogenetic protein (BMP) signaling is required for the de- and assayed their binding to TSK-AP, a fusion protein of TSK velopment of the CB, and we previously showed that TSK can conjugated with AP. Only Fzd4-expressing COS-7 cells showed work as a BMP antagonist during early embryonic development specific binding to TSK-AP compared with control COS-7 cells (13). However, the expression of TGF-β signaling components in −/− (Fig. 4 G and H). We calculated the dissociation constants (Kd)for the CB was not obviously changed in TSK mice compared with TSK-AP binding to Fzd4 and Fzd5 and generated affinity values of WT animals (Fig. S9A).

Wnt-3a Wnt-3a + C-TSKB TSK x Fzd4 F I J 120 ) Kd=2.3 x 10-10M 3

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.. r human IgG 0 667 0 0 (nM) 0 0 0 10 20 30 40 50 F /

052010 30 40 0dn 0 0 429 1430 (nM) 1.0 C-TSKB-Fc uo Total (OD/hr x 103) B β -catenin COS-7-Fzd4 0 03142 Bound (pM)

Fig. 4. TSK is a Fzd4 ligand. (A–E) Immunocytochemical (A and C), DAPI staining (B and D), and immunoblotting (E) analyses of β-catenin translocation into the nucleus in the presence (C–E) or absence (A, B, and E) of C-TSKB protein. (F) L cells express Fzd4. (G and H) Binding of C-TSKB::AP to control COS-7 cells (G) and Fzd4-expressing COS-7 cells (H). (I–K) Saturation binding (I) and Scatchard analysis of C-TSKB::AP binding to Fzd4- expressing (J) or Fzd5- expressing (K) COS-7 cells. (Scale bar: 20 μm.)

Ohta et al. PNAS | September 6, 2011 | vol. 108 | no. 36 | 14965 Downloaded by guest on September 25, 2021 We next examined the expression of typical CB markers, Msx1 ABC LE −/− and Otx1, at E12.5 and detected their ectopic induction in TSK Re R–U Re mice retina (Fig. 5 ). Normal development of peripheral eye RPE structures is also regulated by Wnt signaling (10–12, 31), with RPE RPE Wnt2b and Fzd4 acting as the primary Wnt ligand and receptor in DELE F CB Re this context. In situ hybridization analysis of the expression pattern LE Re of Wnt2b and Fzd4 at E15.5 eye showed increased and expanded Re −/− RPE expression of both these genes in the peripheral eye of TSK V–Y RPE mice compared with WT mice (Fig. 5 ). Notably, Wnt2b RPE overexpression in chick retinal cells up-regulated Fzd4 expression both in vitro and in vivo, suggesting that Fzd4 up-regulation in − − K μm TSK / eyes is due to enhanced Wnt signaling (Fig. S9C). Fur- WT TSK -/- 250 * 200 thermore, overexpression of TSK in chick peripheral retina down- G I 150 regulated the expression of Lef1, which can be used as readout for dorsal length 100 50 Wnt signaling activation (10) (Fig. S9B). Thus, TSK inactivation leads to up-regulation of key Wnt signaling components in the WT -/- μm developing peripheral eye, similar to what was seen previously H J L 250 * after overexpression of an active form of β-catenin (10–12), ventral 200 150 whereas TSK overexpression has the opposite effects, suggesting

length 100 that TSK is both necessary and sufﬁcient to modulate Wnt sig- 50 naling levels during peripheral eye development. WT -/- WT TSK -/- Discussion M O The SLRP family encompasses 17 genes, which are subgrouped Q into ﬁve discrete classes based on common structural and func- dorsal mm2 tional properties (14). TSK belongs to the group of class IV SLRPs, 2.0 * which includes structurally related chondroadherin (32) and nyc- 1.6 talopin (33), but it shares functional properties with class I SLRPs N P 1.2 (34), including their role as BMP inhibitors (13, 15). We have

area / eye 0.8 ventral previously reported that TSK interacts with BMP ligands and the 0.4 BMP antagonist chordin and functions as an inhibitor of BMP WT -/- signaling during gastrula and neurula stages. In this context, TSK- dependent BMP inhibition is required for germ layer specification, WT TSK -/- organizer formation, and early ectodermal patterning. However, the BMP antagonistic activity is stronger for the C-TSKA isoform R S than for C-TSKB, which is the homolog to Xenopus and M-TSK fi Msx1 (15). Although BMP signaling has been implicated in the speci - cation of peripheral eye structures, in this context TSK does not seem to have a critical function as a BMP inhibitor, as shown by the T U normal expression of BMP signaling components in TSK mutant eyes. Instead, we found that TSK regulates β-catenin–dependent Otx1 Wnt signaling, which plays a crucial role in peripheral eye development. In the peripheral eye, this pathway is activated mainly by the Wnt2b ligand and transduced mainly through the Fzd4 re- WT TSK -/- Z ceptor. We show that TSK directly binds to the CRD region of Fzd4 Mouse and competes with Wnt2b for binding to the same domain, thereby V W Wnt2b preventing Wnt activation of β-catenin–dependent signaling. Wnt2b TSK Our assays in chick retinal cells showed that TSK over- TSK + Fzd4 expression can effectively inhibit Wnt signaling both in vivo and X Y Chick in vitro, and in particular, TSK can negate the peripheral eye- Fzd4 Wnt2b inducing effects of Wnt2b overexpression in both conditions. Fzd4 TSK + Fzd4 Activation of Wnt signaling also has been shown to affect proliferation of retinal cells; however, there is considerable con- − − Fig. 5. Expansion of CB in TSK / mouse eye. (A–F) Detection of β-gal ac- troversy in the literature with respect to the neurogenic and − tivity in the developing eye of TSK+/ mice at E11.5 (A), E12.5 (B), E14.5 (C), proliferative effects of the Wnt pathway in the chick retina, with E15.5 (D), postnatal day 0 (P0) (E), and adult (F). TSK is expressed in the assays performed in vitro (35) and in vivo (8) leading to appar- anteriormost tip of the developing optic cup and the mature CB. RPE, retinal ently conflicting results. Kubo et al. (35) demonstrated an in- pigmented epithelium; Re, retina; LE, lens epithelium. (Scale bars: A,40μm; crease in the proliferation of Wnt-2b–expressing retinal explants B,50μm; C,70μm; D,80μm; E, 120 μm; F, 200 μm.) (G–J) CB structure of the −/− in vitro, whereas Cho and Cepko (8) showed that the level of dorsal (G and I) and ventral (H and J) areas of the eye of an adult TSK (I proliferation in Wnt-2b–infected central retinal areas in vivo was and J) and WT mouse (G and H). Quantification of the length of the CB in WT and TSK−/− mice. (K) Dorsal area. (L) Ventral area. *P < 0.01 based on a two- tailed Student t test. (M–P) H&E-stained sections of the CB area from rep- − − resentative eyes of TSK / mice (O and P) compared with WT (M and N). (Q) − − Quantification of the size of the CB area in WT and TSK / mice. *P < 0.01, that the expression of Wnt2b (arrows) and Fzd4 in TSK−/− mice (W and Y)is two-tailed Student t test. Error bars represent the SD. (Scale bars: G–J, 100 expanded in both the pigmented epithelium and the nonpigmented epi- μm; M–P,50μm.) (R–U) Expression of Msx1 (R and S) and Otx1 (T and U)in thelium compared with WT mice (V and X). (Scale bars: 50 μm.) (Z) During the E12.5 peripheral retina. Note that the expression of Msx1 and Otx1 in early retinal development, Wnt2b (yellow) expressed in the peripheral pig- − − TSK / mice (bracket in S and U) is induced in the nonpigmented epithelium mented epithelium (mouse) and the tip of retina (chick) acts on the non- compared with WT mice (R and T). (Scale bars: 50 μm.) (V–Y) Expression of pigmented cells expressing Fzd4 and TSK (green) to cause proliferation. TSK Wnt2b (V and W) and Fzd4 (X and Y) in the E15.5 peripheral retina. Note regulates this effect by inhibiting Wnt2b activity.

14966 | www.pnas.org/cgi/doi/10.1073/pnas.1100513108 Ohta et al. Downloaded by guest on September 25, 2021 lower than that in the normal control central retina. In this work, function is also required to restrict their activities at the tran- we performed the same in vivo and in vitro assays and obtained scriptional level. Taken together, our results support a model in results similar to those reported previously. which TSK expression in the peripheral eye modulates Wnt2b These discrepancies might be explained by the need for addi- signaling in this region by quenching Fzd4 activation toward more tional factors besides Wnt to stimulate proliferation in vivo, or central areas, thereby regulating the size of peripheral eye struc- possibly that some other unknown aspects of the in vitro culture tures and especially the CB (Fig. 5Z). environment enhance proliferation in response to Wnt signaling. These data also may reflect experimental differences in the timing, Materials and Methods duration, and intensity of Wnt signaling, or regional and temporal differences in the outcome of Wnt signaling in the developing eye. Detailed information on TSK KO mice, measurement of the length and area of CB, The fact that TSK is able to efficiently abrogate Wnt2b activity in in situ hybridization, immunohistochemistry, chick electroporation and retinal both in vitro and in vivo assays indicates that the differential re- explant culture, immunoprecipitation assays, and AP binding assays is provided in sponse of retinal cells to Wnt2b in vitro and in vivo is likely SI Materials and Methods. modulated downstream from Fzd4 receptor activation. Although the currently available data indicate that Wnt sig- ACKNOWLEDGMENTS. We thank Shinji Takada, Ritsuko Takada, Yoshiyuki Mochida, Toshihiro Inoue, Fumi Kubo, Jeremy Nathans, Isao Matsuo, Michio naling is involved in the proliferation of retinal cells in a context- Yoshida, and the Developmental Studies Hybridoma Bank for reagents; Bill dependent manner, further work is needed to elucidate its precise Harris, Constance L. Cepko, Seo-Hee Cho, Shin-Ichi Aizawa, Tetsuya Taga, role in eye development. Nevertheless, the fact that TSK was able Tohru Nakano, Kohji Nishida, Douglas S. Campbell, and Yohei Shinmyo for to negate the effects of Wnt2b overexpression in both in vitro and critical comments; Kumiko Hori and Mihoko IImori for help with the chick in vivo experimental paradigms strongly supports the idea that embryos; and all members of our laboratories for their valuable help. This work was supported by the Precursory Research for Embryonic Science and TSK can function as a Wnt antagonist in the developing eye and Technology program of the Japan Science and Technology Agency (K.O.), the especially in the context of peripheral eye differentiation, which Takeda Science Foundation (K.O.), the Brain Science Foundation (K.O.), and was promoted by Wnt2b and repressed by TSK regardless of the Grants-in-Aid for Scientific Research on Molecular Brain Science from the experimental conditions applied. The analysis of TSK knockout Ministry of Education Culture, Sports, Science, and Technology of Japan (to mice confirmed the requirement for endogenous TSK function in K.O. and H.T.), the Italian Ministry of Education, University, and Research “ ” – peripheral eye development. Consistent with overexpression program Rientro dei Cervelli and a start-up grant from Istituto Pasteur Fondazione Cenci Bolognetti (to G.L.), a Cancer Research UK Senior Cancer assays, TSK mutant eyes showed an enlarged CB. Furthermore, Research Fellowship (to S.-i.O.), a Kumamoto University 21st Century Center the expression of Wnt2b and Fzd4 was enhanced and expanded in of Excellence research grant (to S.K. and H.T.), and a Kumamoto University the developing peripheral eye epithelium, indicating that TSK Global Center of Excellence research grant (to A.I. and H.T.).

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