JNK Signaling and Antagonized by Aida

JNK Signaling and Antagonized by Aida

Developmental Cell Article A b-Catenin-Independent Dorsalization Pathway Activated by Axin/JNK Signaling and Antagonized by Aida Yanning Rui,1,3,4 Zh...

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Developmental Cell

Article A b-Catenin-Independent Dorsalization Pathway Activated by Axin/JNK Signaling and Antagonized by Aida Yanning Rui,1,3,4 Zhen Xu,2,3,4 Bo Xiong,2,4 Ying Cao,2,4 Shuyong Lin,1 Min Zhang,2 Siu-Chiu Chan,3 Wen Luo,1 Ying Han,1 Zailian Lu,1 Zhiyun Ye,1 Hai-Meng Zhou,2 Jiahuai Han,1 Anming Meng,2,* and Sheng-Cai Lin1,3,* 1

Key Laboratory of Ministry of Education for Cell Biology, School of Life Sciences, Xiamen University, Fujian 361005, Xiamen, China Protein Science Laboratory of the Ministry of Education, Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing 100084, China 3 Department of Biochemistry, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, China 4 These authors contributed equally to this work. *Correspondence: [email protected] (S.-C.L.), [email protected] (A.M.) DOI 10.1016/j.devcel.2007.07.006 2

SUMMARY

Axin is a scaffold protein that controls multiple important pathways, including the canonical Wnt pathway and JNK signaling. Here we have identified an Axin-interacting protein, Aida, which blocks Axin-mediated JNK activation by disrupting Axin homodimerization. During investigation of in vivo functions of Axin/JNK signaling and aida in development, it was found that Axin, besides ventralizing activity by facilitating b-catenin degradation, possesses a dorsalizing activity that is mediated by Axin-induced JNK activation. This dorsalizing activity is repressed when aida is overexpressed in zebrafish embryos. Whereas Aida-MO injection leads to dorsalized embryos, JNK-MO and MKK4-MO can ventralize embryos. The anti-dorsalization activity of aida is conferred by its ability to block Axin-mediated JNK activity. We further demonstrate that dorsoventral patterning regulated by Axin/JNK signaling is independent of maternal or zygotic Wnt signaling. We have thus identified a dorsalization pathway that is exerted by Axin/JNK signaling and its inhibitor Aida during vertebrate embryogenesis. INTRODUCTION Specification of the dorsoventral axis is one of the first steps during development of vertebrate embryos. Stabilization and translocation of intracellular b-catenin into nuclei of dorsal blastomeres are essential for the formation of the dorsal organizer (Harland and Gerhart, 1997; Kelly et al., 2000; Weaver and Kimelman, 2004), which is activated by maternally supplied, dorsally enriched Wnt11 (Tao et al., 2005). On the ventral side of the embryo, intracellular b-catenin is degraded to prevent formation of an embryonic axis on this side. Axin plays an important role

in the establishment of the dorsoventral axis: mouse Fused mutants that lack the major Axin mRNA have duplicated axes; overexpression of Axin on the dorsal side inhibits dorsal axis formation in Xenopus (Zeng et al., 1997). Subsequent studies have indicated that Axin is required for forming b-catenin degradation complexes, which consist of many factors, including b-catenin, Axin, GSK3b, APC, and casein kinase, in ventral blastomeres of the embryos (Kikuchi, 1999). Alternatively, Axin serves as a cytoplasmic anchor for b-catenin. In response to Wnt signaling, LRPmediated Axin degradation leads to nuclear signaling by b-catenin independently of GSK3b activity (Tolwinski et al., 2003). Conductin, the other member in the Axin gene family, has also been shown to repress Wnt signaling (Behrens et al., 1998). Over the past few years, Axin has been shown to be a multidomain scaffold protein, and it plays important roles in many other pathways, including TGF-b, the c-Jun Nterminal kinase, and p53 signaling pathways (Furuhashi et al., 2001; Liu et al., 2006; Rui et al., 2004; Salahshor and Woodgett, 2005; Zhang et al., 1999), in addition to a role in downregulating the b-catenin-mediated canonical Wnt signaling pathway (Peifer and Polakis, 2000). Our previous work has identified an Axin-mediated JNK pathway in which Axin forms complexes with MEKK1 or MEKK4 and activates JNK via MKK4/7 (Luo et al., 2003; Zhang et al., 1999). The domains of Axin that are required for JNK activation are distinct from those required for b-catenin degradation. However, developmental implications of Axin-induced JNK activation have not been demonstrated. Interestingly, Zeng et al. (1997) reported that a mutant form of Axin, which lacks the APC-binding domain, or the RGS domain, but retains domains for JNK activation, induced an ectopic axis upon overexpression in ventral cells of Xenopus embryos. Shimizu et al. (2000) found that in zebrafish, overexpression of full-length axin resulted in expanded expression of dorsal markers goosecoid (gsc, an organizer-specific gene) and chordin (chd, a specific antagonist of bone morphogenetic proteins [Bmp]) in some embryos and reduced expression of the same markers in others at the shield stage. These findings raise a possibility that Axin may have an intrinsic dorsalizing

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activity exerted by the JNK pathway. If so, the question to follow is what factor or factors control the intrinsic dorsalizing activity of Axin. In order to differentiate the biological consequences of different sets of Axin domains, we have generated a series of deletion mutants. Among them, AxinDMID is defective in JNK activation and dominantly inhibits JNK activation induced by wild-type Axin. In addition, Axin-mediated JNK activation is highly regulated; e.g., it is negatively regulated by several key Wnt pathway components, including GSK-3b, casein kinases, and DIX-domain proteins Dvl or Ccd1 (Wong et al., 2004; Zhang et al., 2000, 2001, 2002). We therefore continued to characterize Axin-interacting factors identified by yeast two-hybrid screening (Rui et al., 2002). As a result, we identified Aida as a factor that interacts with Axin and inhibits Axin-mediated JNK activation by disrupting Axin homodimerization. Our current study establishes that Axin-mediated JNK activation plays a critical role in dorsoventral patterning of vertebrate embryos, which is negatively regulated by Aida. Our results further indicate that Axin controls dorsoventral patterning as a result of two opposing effects: ventralizing by downregulating b-catenin signaling and dorsalizing by activating JNK. RESULTS Identification of an Axin-Interacting Protein As an attempt to identify factors that modulate Axin JNK activation, we previously carried out a yeast two-hybrid screen and identified multiple factors (Rui et al., 2002). Among them is an unreported gene that exists as a mouse EST clone in GenBank with the accession number BC004835. It encodes a putative protein of 305 aa, with no region similar to any known structure, indicating that BC004835 may represent a novel protein with unique functions. It is highly conserved from zebrafish to human (see Figure S1 in the Supplemental Data available with this article online). Based on its biochemical nature and in vivo function (see data below), we have designated this protein as Aida for Axin interaction partner and dorsalization antagonist. To test for Axin interaction with Aida in mammalian cells, we carried out immunoprecipitation of endogenous cellular proteins using rabbit polyclonal antibody raised against the full-length Aida (for characterization of the antibody, see Figure S2). After immunoprecipitation with anti-Axin antibody or anti-Aida antibody, the immunoprecipitates were separately subjected to immunoblotting with the different antibodies. Axin was detected in the Aida immunoprecipitate and vice versa (Figure 1A). In addition, we performed a GST pull-down assay using GST fusion proteins and total cell lysates (TCLs) of human embryonic kidney 293T (HEK293T) cells expressing either HA-Axin or HA-Aida. HA-Axin was specifically pulled down by GST-Aida and HA-Aida was retained by GST-Axin, but not by GST alone (Figure 1B). We also transfected HA-Axin and Myc-Aida into HEK293T cells and carried out coimmunoprecipitation. Similar results were obtained (Figure 1C). Importantly, the immunostain-

ing experiment also indicated that Aida is colocalized with Axin in the COS-7 cells (Figure 1D). Expression of Aida alone appears to be evenly distributed throughout the entire cell. However, when coexpressed with Axin, Aida forms punctated speckles that overlap those of Axin. The above experiments demonstrate that Axin strongly interacts with Aida both in vitro and in vivo. To map for the interfaces of Aida-Axin interaction, we created a series of Aida deletion mutants and different Axin deletion mutants. It was revealed that the region of aa 730 to 753 in Axin is responsible for interaction with Aida (Figure 1E), and that the region of aa 153 to 220 in Aida forms the interaction interface with Axin (Figure 1F). Aida Inhibits JNK Activation by Disrupting Homodimerization of Axin To understand the significance of Aida interaction with Axin, we tested the effect of Aida on cellular function of Axin as judged by a JNK assay. Aida, but not the Axinbinding defective mutant AidaDAxin (MD2 as indicated in Figure 1F), strongly repressed the JNK activity induced by Axin (Figures 2A and 2B). Aida could not abolish the JNK activation by AxinDAida (M6 as indicated in Figure 1E), an Axin mutant that lacks the domain for interaction with Aida (Figure 2C) but retains the ability to interact with MEKK and activates JNK. This indicates that Aida has to undergo physical interactions with Axin in its inhibition of JNK activation. We isolated the zebrafish aida gene and tested if it inhibits JNK activation by zebrafish Axin. Results showed that zebrafish Aida also strongly attenuated JNK activation by wild-type zebrafish Axin (Figure 2D). Consistent with an inhibitory role of Aida in JNK activation is the observation that knockdown of endogenous Aida in HEK293T cells by siRNA (for pSUPER-Aida efficiency, see Figure S3) led to an increase of AP-1 transcriptional activity monitored by an AP-1 luciferase reporter (Wong et al., 2004) (Figure 2E). To rule in or out the possibility that Aida may affect Axinmediated b-catenin degradation, we performed a LEF-1 luciferase assay in HEK293T cells transfected with Aida alone or both Aida and Axin. Results showed that Aida alone had no effect on Wnt signaling, nor did it have an effect on Axin-mediated downregulation of Wnt signaling (Figure 2F), indicating that Aida may not play a role in Wnt signaling. Thus, Aida negatively regulates the Axininduced JNK pathway, but not that of canonical Wnt signaling. We further characterized the molecular mechanism underlying the inhibition of Axin-mediated JNK activation by Aida. As previously mentioned, Axin-mediated JNK activation is highly conformation dependent, in that binding of GSK-3b, casein kinases, Ccd1, or Dishevelled renders Axin incapable of association with MEKK, and in that homodimerization of Axin is strictly required for JNK activation (Wong et al., 2004; Zhang et al., 2000, 2001, 2002). We tested if Aida would prevent complex formation between Axin and MEKK1 or MEKK4, and found that Aida did not have such an effect (Figures S4A–S4B). When assayed for an effect on Axin homodimerization, Aida, but not

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Figure 1. Identification of Aida as an Axin Interaction Partner (A) Endogenous Axin and Aida interact with each other. Axin and Aida in untransfected C2C12 cells were immunoprecipitated with rabbit anti-Axin and anti-Aida, respectively. Rabbit IgG was used as control. Detection of Axin and Aida in the immunoprecipitates and total cell lysates (TCLs) was carried out using anti-Axin and anti-Aida antibodies, respectively. (B) GST pull-down assay to evaluate Axin interaction with Aida in vitro. The ability of GST-Axin to retain Aida present in the cell lysate from HEK293T cells transfected with pCMV-HA-Aida was analyzed by western blotting with anti-HA (upper panel). GST-Aida specifically pulled down HA-Axin in the cell lysate (lower panel). In either case, GST alone did not interact with Aida or Axin. Input represented one-sixth of lysate used for GST pull-down. (C) Axin and Aida interact with each other when overexpressed in HEK293T cells. HA-Axin and Myc-Aida were expressed in HEK293T cells singly or in combination. Reciprocal coimmunoprecipitation was performed by using anti-HA or anti-myc antibody followed by immunoblotting using anti-Myc or anti-HA antibody as indicated. (D) Colocalization of Axin and Aida in mammalian cells. COS-7 cells were transiently transfected with myc-Aida and Axin and stained for Axin (red) and Aida (green) using rhodamine conjugated mouse antibody and FITC conjugated rabbit antibody, respectively. (E) Aida interacts with a specific C-terminal domain of Axin. Schematic diagrams depict different Axin deletion mutants used in the domain-mapping experiments. Different HA-tagged Axin deletion constructs were transiently transfected into HEK293T cells together with Myc-tagged Aida. Cell lysates were immunoprecipitated with anti-Myc antibody, followed by immunoblotting using anti-HA for Axin proteins and anti-Myc for Aida. (F) Determination of Axin binding sites in Aida. Shown on the top are schematic diagrams of different Aida deletion mutants used in the domainmapping experiments. Same experiments were performed as described in (E).

AidaDAxin, was found to drastically interfere with Axin homodimerization (Figures 2G and 2H), indicating that the physical interaction of Aida with Axin disrupts Axin homodimerization. In parallel, we also found that Aida did not affect Axin interaction with Dishevelled protein (Figure S4C), consistent with the above results that Aida is not involved in Wnt signaling either alone or in combination with Axin. Axin-Injected Embryos Display Both Dorsalized and Ventralized Phenotypes Axin is a multidomain protein, and it interacts with diverse factors that participate in multiple signaling pathways. To understand the biological function of Axin-mediated JNK activation in vivo, we used zebrafish as a model system.

We first generated the full-length coding sequence of zebrafish axin, and created zebrafish axinDMID, which is a deletion mutant corresponding to the mouse AxinDMID (Zhang et al., 1999). In vitro analyses confirmed that wild-type zebrafish axin and axinDMID behaved similarly to their corresponding mouse Axin constructs, with wildtype axin capable of activating JNK activity (Figure 3A) and axinDMID acting as a dominant-negative form to block JNK activation in HEK293T cells (Figure 3B). We then injected mRNA of zebrafish axin into one-cell embryos (300 pg per embryo) and observed the effect of its ectopic expression on embryonic development. Among the injected embryos (n = 168) at 24 hr postfertilization (hpf) (Figure 3C), we observed two populations of abnormal embryos that exhibited dorsalized (embryos

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Figure 2. Aida Inhibits Axin-Meditated JNK Activation by Disrupting Homodimerization of Axin (A) Aida inhibits Axin-mediated JNK activation. HEK293T cells were transfected with FLAG-tagged JNK together with other plasmids as indicated. Following immunoprecipitation of FLAG-JNK, JNK kinase activities were assayed using GST-c-Jun as substrate. The amount of the kinase and the kinase activity in each immunoprecipitate were quantified by immunoblotting using anti-JNK antibody and anti-phospho-c-Jun antibody, respectively. (B) AidaDAxin that cannot bind to Axin fails to attenuate Axin-mediated JNK activation. (C) AxinDAida-mediated JNK activation could not be attenuated by Aida. (D) Zebrafish Aida inhibits Axin-induced JNK activation. An experiment similar to (A) was performed using zebrafish axin and aida instead of their mouse counterparts. The immunokinase assay was performed as described above. (E) The siRNA against Aida enhances AP-1 activity. HEK293T cells were transfected with AP-1 luciferase reporter together with empty pSUPER vector, pSUPER-Aida, or control RNAi. At 32 hr posttransfection, relative luciferase activity was measured. (F) Aida has no effect on Wnt signaling as assessed with LEF-1 luciferase reporter. HEK293T cells were transfected with 0.5 mg of LEF-1 luciferase reporter plasmid together with 1.5 mg of Axin, Aida alone, or both. Aida did not affect basal, wnt-1-induced, or Axin-downregulated reporter expression. (G) Aida disrupts Axin homodimerization as determined by using two differently tagged Axins, HA-Axin and FLAG-Axin. HEK293T cells were transfected with HA-Axin and FLAG-Axin together with increasing amounts of Aida (0 mg, 1 mg, and 3 mg). When HA-tagged Axin was immunoprecipitated by anti-HA, the amount of coprecipitated FLAG-tagged Axin was negatively correlated with the increasing amounts of cotransfected Aida (left panel); similarly, decreasing amounts of HA-tagged Axin were coprecipitated with FLAG-Axin by the anti-FLAG antibody in the presence of increasing amounts of Aida (middle panel). The right panel shows similar expression levels of HA- and FLAG-tagged Axin among the three samples, and it shows that Aida levels were proportionally increased when increasing amounts of DNA were transfected. (H) AidaDAxin has no effect on Axin homodimerization. Coimmunoprecipitated HA-Axin levels in anti-FLAG immunoprecipitates were not changed in the presence of increasing amounts of AidaDAxin.

D1–D3) or ventralized (V1 and V2) phenotypes, with their percentages being 38% and 41%, respectively. The remaining 21% of embryos appeared to be normal (Figure 3D). Among the dorsalized embryos, 82% (type D3) showed altered morphology of the posterior trunk

and tail, including smaller posterior trunk, loss of the ventral tail fin, and a reduction of the yolk extension; the remaining 18% displayed more dramatic phenotypes (types D1 and D2) (Figure 3C). When the injection dose increased to 500 pg, more embryos had the D1-type phenotype,

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Figure 3. Axin Contains Intrinsic Dorsalizing Activity (A) Zebrafish axin and axinDMID behave similarly to their mouse counterparts in JNK activation. HEK293T cells were transfected with FLAG-JNK together with other plasmids as indicated. Immunokinase assays were performed as described above. (B) Zebrafish axinDMID is a dominant-negative form for axin-mediated JNK activation. HEK293T cells were transfected and followed by immunokinase assay at 36 hr posttransfection. (C) Injection of zebrafish axin mRNA induces opposing phenotypic changes in embryos at 24 hpf. axin mRNA was injected into one-cell embryos at a dose of 300 pg. Shown are lateral views of live embryos at 24 hpf. D1, D2, and D3 display characteristic dorsalized phenotypes at 24 hpf. D2 and D3 phenotypes of injected embryos showed shortened tail with the ventral tail fin missing. D1 phenotype was the most severe one. V1 and V2 phenotypes displayed loss of the head and notochord together with enlargement of the blood island. (D) Statistical data for (C). (E) Expression of marker genes in axin-injected embryos. Full-length axin mRNA (300 pg) was injected into one-cell embryos and followed by in situ hybridization at the shield stage. Embryos for chd and eve1 were animal pole views with dorsal to the right; embryos for gsc were dorsal views with animal pole to the top; and embryos for bmp2 were lateral views with dorsal to the right. (F) Statistical data for (E). (G) chd expression changes in an injection-position-dependent manner. Wild-type axin mRNA (250 pg) was coinjected with GFP mRNA (50 pg), and double in situ hybridization was performed at the shield stage for GFP (blue) and chd (red). The blue areas expressing GFP indicated the injection position. Note that when injected mRNAs were distributed into areas proximal to the dorsal side as judged at the shield stage, chd expression was weaker (shorter distance between arrows), whereas ventral distribution gave rise to enhanced expression of chd. (H) The percentage of embryos with reduced chd expression and dorsal distribution of injected GFP mRNA, and the percentage of embryos with enhanced chd expression and ventral distribution of GFP mRNA, were diagramed.

suggesting that the morphological changes are dosage dependent. The ventralized embryos showed a loss of the head and the notochord, and had an enlarged blood island (V1 and V2 types, Figure 3C), which is consistent with the well-established view that Axin negatively regulates canonical Wnt signaling in dorsal development of vertebrate embryos (De Robertis and Kuroda, 2004; Shimizu et al., 2000; Zeng et al., 1997). These observations raised a provocative possibility that native Axin may in fact have a dual role in dorsoventral patterning.

We then carried out in situ analysis to see if the axininduced opposing phenotypes were accompanied by altered expression of dorsal and ventral marker genes. We selected two dorsal mesoderm markers, gsc and chd, and two ventral markers, bmp2 and eve1 (Cao et al., 2004; Herbomel et al., 1999; Kishimoto et al., 1997; Stachel et al., 1993; Zhao et al., 2003). Injection of axin mRNA gave rise to mixed populations with opposite changes in marker gene expression (Figure 3E): 30%– 37% of the embryos expressed increased levels of dorsal markers and decreased ventral markers, 25%–27% were

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unchanged, and the remaining 36%–45% of embryos showed opposite changes in the expression levels of the dorsal and ventral markers (Figure 3F). Similar phenomena were observed previously by Shimizu et al. (2000). Loss-of-function study of Axin in Xenopus shows that ventral activity of Axin is a regional effect (Kofron et al., 2001). We suspected that axin-induced different phenotypes were also determined by differential distribution of the injected mRNA in embryos. To verify that, we coinjected wild-type axin mRNA with GFP mRNA, and determined the correlation between locations of injected mRNAs and changes of the marker pattern by double in situ hybridization with antisense fluorescein RNA probe for chd (red) and digoxigenin probe for GFP (blue) (Figure 3G). Marked expansion of chd expression was found in 78% of the embryos with exogenous mRNAs located proximal to the ventral areas. Conversely, 71% of dorsally injected embryos showed reduction in chd expression (Figures 3G and 3H). These results suggest that Axin exerts both dorsalizing and ventralizing activities depending on the injection positions. JNK Signaling Is Required in Axin-Induced Dorsalization In a separate experiment, we injected one-cell embryos with mRNA of axinDMID, which lacks the MEKK1-interacting domain and exhibits a dominant-negative effect on Axin activation of JNK. Unlike overexpression of full-length axin (Figure 3C), axinDMID overexpression led to embryonic ventralization only, with omission of the head and the notochord at 24 hpf, as well as decreased expression of the dorsal markers and expanded expression of the ventral markers during early gastrulation (Figures 4A and 4B). This observation implies that the ventralizing aspect of axin function is independent of its JNK activation. Consideration of its inability to activate JNK with the loss of its dorsalizing activity raises a possibility that JNK activity of Axin is responsible for embryonic dorsalization induced by overexpression of full-length axin. We then employed more direct ways to ascertain whether JNK activity triggered by Axin indeed exerts a dorsalizing effect during embryogenesis. MKK4-KM is a dominant-negative mutant of MKK4, an upstream activating kinase of JNK (Derijard et al., 1995). In the presence of zebrafish MKK4-KM or MKK4-RNAi, JNK could not be activated by Axin (Figure 4C). Likewise, zebrafish JNK-APF in which the TPY dual activation motif of the JNK/MAP kinase is altered to APF was not activated by Axin (Figure 4D). MKK4-KM or JNK-APF mRNA was injected, alone or in combination with axin mRNA, into one-cell embryos, and the injected embryos were subsequently examined for their phenotypic changes and subjected to in situ hybridization analysis for alteration of marker expression (Figures 4E–4H). As observed above, injection of axin alone gave rise to two opposing populations of dorsalized and ventralized embryos (46% and 35%, respectively), in addition to 19% of normal embryos (Figure 4E). When MKK4-KM mRNA was coinjected, the percentage of ventralized embryos at 24 hpf greatly increased from 35%

to 70%, with a concomitant decrease in the proportion of dorsalized embryos (from 46% to 17%). A similar effect was reflected by changes in marker gene expression at the shield stages (Figure 4F). When JNK-APF mRNA was coinjected with axin mRNA, the ventralized population also increased (Figures 4G and 4H). These results suggest that axin-induced dorsalization requires MKK4 and JNK. JNK Cascade Components Are Involved in Proper Dorsal Development We carried out additional experiments to test if the JNK signaling cascade plays a positive role in dorsalization. While overexpression of MKK4-ED, a constitutively active form of MKK4, induced dorsalization, MKK4 morpholino oligonucleotides (MKK4-MO) injection caused ventralization as analyzed by both in situ hybridization and quantitative RT-PCR experiments on the expression of the dorsal and ventral markers (Figure 5A and Figure S7). The MKK4-MO-induced ventralization could be rescued by coinjection of MKK4-ED mRNA (Figures 5A and 5C and Figure S7). In parallel, we found that JNK overexpression dorsalized embryos, and that JNK knockdown with JNKMO ventralized embryos (Figures 5B and 5D and Figure S7). The requirement of MKK4 and JNK in dorsalization was also tested by coinjection of axin mRNA with MKK4-MO or JNK-MO. MKK4-MO and JNK-MO each drastically reduced axin-induced dorsalization in injected embryos, as indicated by changes in marker gene expression (Figures 5E and 5F). These data demonstrate that MKK4 and JNK functionally mediate the intrinsic dorsalizing activity of Axin. Aida Is a Ventralizing Factor Because Aida strongly inhibits Axin-mediated JNK activation, which is closely linked to embryonic dorsoventral patterning, we wondered whether Aida might modulate the dorsalizing activity of Axin. We first determined the zebrafish aida expression pattern by several assays. First, RT-PCR analysis showed that aida transcripts are present maternally and zygotically (Figure S5A). Second, wholemount in situ hybridization indicated that aida is expressed strongly and ubiquitously throughout early developmental stages. Expression of aida at 24 hpf persists in most tissues with the strongest expression in brain and somites (Figure S5C). Finally, northern blotting analysis also revealed that human AIDA is expressed in all of the examined adult tissues with the highest level in heart and skeletal muscle (Figure S5B). To explore the role of zebrafish aida in embryonic development, we first took a gain-of-function and loss-offunction approach. Embryos injected with 300 pg of aida mRNA showed a slight reduction in expression of the dorsal markers gsc and chd, concomitant with increased expression of the ventral markers bmp2, eve1, and gata2 at the shield stage (Figure 6A). In contrast, aida knockdown by injecting aida-MO resulted in increased expression of the dorsal markers and reduced expression of the ventral markers (Figure 6A; see Figure 6B for the statistical data of Figure 6A; and see

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Figure 4. Components of JNK Signaling Are Required for Axin-Induced Dorsalization (A) Injection with 50 pg of axinDMID mRNA caused embryonic ventralization only. The top panel on the left shows an illustration of axinDMID structure. Live ventralized embryos at 24 hpf lack the head and the notochord, but possess an enlarged blood island (indicated by an arrow). The right panel shows that axinDMID overexpression inhibited gsc and chd expression (dorsal views) but expanded bmp2 and drl expression (animal pole views with dorsal to the right) at the shield stage. (B) Statistical data for axinDMID overexpression. Upward and downward arrows indicated increased and decreased expression, respectively. (C and D) MKK4 siRNA, MKK4-KM (C), and JNK-APF (D) abolish axin-mediated JNK activation. FLAG-tagged JNK was cotransfected with different constructs as indicated on the top. JNK activities from different transfections were determined as described in Figure 2. (E–H) Percentages of dorsalized and ventralized embryos, judged by morphological changes at 24 hpf (E and G) or by marker changes at the shield stage (F and H). Injection dosages: axin mRNA, 200 pg; MKK4-KM mRNA, 300 pg; JNK-APF mRNA, 300 pg.

real-time RT-PCR data in Figure S7). The effect of aidaMO was rescued by coinjection of aida mRNA, demonstrating that aida-MO has a specific targeting effect. When injected with 300 pg of aida mRNA, 32% (n = 175) of embryos at 24 hpf had an expanded blood island (arrow), a shorter tail, and closer eyes (arrow head); 26% showed a reduced head (arrow head) and an expanded blood island; and 4% lacked a patterned head or the notochord and exhibited great expansion of the blood island and ventral fin (Figure 6C). Conversely, 72% of embryos (n = 128) injected with 10 ng of aida-MO showed shorter, thinner yolk extension with a reduced blood island (Figure 6D, arrow head). The effect of aida overexpression and knockdown on the size of the blood island was further confirmed by examination of the hematopoietic marker gata1 at 24 hpf (Figure S6, arrows). Taken together, these results suggest that Aida acts as a ventralizing factor during embryogenesis.

We noted that aida knockdown only resulted in weakly dorsalized phenotypes. This could be explained by the possible fact that maternal Aida protein compensates the reduction caused by aida knockdown. Therefore, we used antibody injection to test the maternal Aida protein function. Injection of Aida antibody (300 pg) could lead to the ovoid phenotype and broader somites at the tailbud stage or during early somitogenesis (Figure 6F), which were efficiently rescued by injection of zebrafish Aida protein (Figure 6F). Preincubation of Aida antibody with Aida protein could diminish the dorsalizing effect, further demonstrating the specificity of Aida antibody in vivo. In situ analysis for chd, gsc, and eve1 showed that Aida antibody injection led to elevated expression of the dorsal markers chd and gsc, but reduced expression of the ventral marker eve1, which could be rescued by coinjection (300 pg) of Aida protein (Figure 6E and Figure S7). These results further confirmed that Aida has ventralizing activity during fish embryonic development.

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Figure 5. JNK Is Involved in Dorsoventral Patterning (A) Expression patterns of marker genes in wild-type embryos or embryos injected with 300 pg of MKK4-ED mRNA, 10 ng of MKK4-MO, or both. Embryos for chd and eve1 are animal pole views with dorsal oriented toward the right; embryos for gsc are in dorsal views with animal pole to the top; and embryos for bmp2 and gata2 are in lateral views with dorsal to the right. (B) Expression patterns of marker genes in wild-type embryos or embryos injected with 300 pg of JNK mRNA, 10 ng of JNK-MO, or both. Experiments are performed as described in (A). (C and D) Statistical data for (A) and (B), respectively. (E) Percentages of embryos with expression changes of marker genes in shield stage caused by axin mRNA injection or coinjection of axin and MKK4MO. Approximately 200 pg of axin mRNA was injected into one-cell embryos alone or together with 10 ng of MKK4-MO. In situ hybridization analysis of different marker genes in injected embryos was performed. Percentages of embryos with elevated, normal, or reduced expression levels of the marker genes were calculated and diagramed. (F) Ratios of dorsalized and ventralized embryos derived from the same experiments as those detailed for (E), except that JNK-MO was used instead of MKK4-MO.

aida Antagonizes Dorsalizing Activity of Axin To address the functional linkage of aida to axin-induced phenotypic changes in vivo, we injected mRNA of aida,

aidaDAxin, and axin either singly or in combination into fertilized eggs of zebrafish, followed by examination for morphological and molecular changes. Coinjection of

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Figure 6. aida Induces Ventralization and Inhibits axin-Induced Dorsalization in Zebrafish Embryos (A) Effect of aida overexpression or knockdown on expression of the dorsal and ventral markers. Embryos were injected with 300 pg of aida mRNA or 10 ng of aida-MO and examined for marker expression at the shield stage by in situ hybridization. Ectopic expression of aida repressed expression of gsc and chd, but expanded the expression of bmp2, eve1, and gata2. Knockdown of aida by MO injection caused reversed effects, which could be attenuated by coinjection with 100 pg of aida mRNA.

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Figure 7. Coinjection of MKK4-MO or JNK-MO Could Cancel the Dorsalizing Effect of aida-MO (A) Expression patterns of marker genes in wild-type embryos or embryos injected with 10 ng of aida-MO, 10 ng of MKK4-MO, 10 ng JNK-MO, or the aforementioned in combination. (B) Statistical data for (A). Injection of 300 pg of aida mRNA induced ventralization, while injection of 10 ng of aida-MO had the opposite effect. The dorsalized phenotype could be rescued by coinjection of aida mRNA.

axin mRNA with aida mRNA, but not with aidaDAxin, led to a dramatic increase in the percentage of ventralized embryos at 24 hpf, from 39% to 67% (Figure 6G). Examination of marker expression in injected embryos also showed that aida, but not aidaDaxin, attenuated axininduced dorsalization (Figure 6H). On the other hand, coinjection of axin mRNA with aida-MO led to an increase in the percentage of dorsalized embryos, from 34% to 48%, and a decrease in ventralized population from 42% to 37% at 24 hpf (Figure 6I). Changes in expression of chd, gsc, bmp2, or eve1 also showed that coinjection of aida-MO with axin mRNA could yield a slightly higher percentage of dorsalized embryos compared with axin injection alone (Figure 6J). To further establish whether aida acts to block axin-mediated JNK signaling through phys-

ical association, we injected axinDaida, aida-MO, and aida mRNA singly or in combination into fish embryos. As shown in Figure 6K, at 24 hpf axinDaida overexpression yielded both ventralized and dorsalized embryos; importantly, the percentage of dorsalized embryos was slightly higher than that of the ventralized embryos. Coinjection of aida-MO or aida mRNA with axinDaida had little effect on the ratio of dorsalized and ventralized embryos. Expression patterns of the marker genes in these injected embryos were consistent with the morphological distributions (Figure 6L), showing that aida blocks axin-induced dorsalization through direct interaction. The above results suggested that the effect of aida on dorsoventral patterning is exerted through its ability to inhibit Axin-induced JNK activation. To verify this, we

(B) Statistical data for (A). (C) aida-induced ventralization of various degrees at 24 hpf. (Ca) Uninjected embryos. (Cb–Cd) Embryos were injected with 300 pg of aida mRNA. Of the injected embryos, 32% showed an increased blood island (Cb, indicated by an arrow), short tail, and closer eyes (Cb, indicated by arrowhead); 26% showed an increased blood island (Cc, indicated by arrowhead), reduced head, and eye fusion; and 4% showed complete loss of head and notochord (Cd). (D) aida-MO causes dorsalization in embryos at 24 hpf. Embryos were injected with 10 ng of aida-MO. Arrowhead marks a reduced blood island. (E) Depletion of aida protein by antibody injection leads to formation of dorsalized embryos. One-cell embryos were similarly injected with affinitypurified anti-Aida polyclonal antibody or with the antibody preincubated with recombinant aida protein, and in situ analysis of the expression of chd and gsc in 30% epiboly embryos, and eve1 in shield-stage embryos, was performed. (F) One-cell embryos were injected as in (D). Photos were taken at the six-somite stage. (G) Statistical data for aida inhibition of axin-induced dorsalization. Injection with 300 pg of aida mRNA, but not with 300 pg of aidaDAxin mRNA, decreased the percentage of 24 hpf embryos that were dorsalized by injection of wild-type axin. (H) Ratios of embryos with normal, expanded, or reduced expression of various markers indicated, after injection of axin mRNA, coinjection of axin with aida, or aidaDaxin mRNA. (I) aida-MO increases the ratio of dorsalized embryos induced by axin. axin mRNA (300 pg) and 10 ng of aida-MO were injected either alone or together. The ratios of different embryos were calculated based on morphological observations of embryos at 24 hpf. (J) Ratios of different embryos determined by in situ analysis of marker genes at the shield stage after injection of axin mRNA (300 pg) or coinjection of axin with aida-MO (10 ng). (K) Statistical data for the effect of aida mRNA or aida-MO on axinDaida-induced dorsalization. Injection with 300 pg of aida mRNA or 10 ng of aida-MO had no significant effect on the percentage of 24 hpf embryos that were dorsalized by injection of axinDaida. (L) Ratios of embryos with normal, expanded, or reduced expression of various markers indicated, after injection of axinDaida mRNA, coinjection of axinDaida with aida mRNA (300 pg), or coinjection of axinDaida with aida-MO (10 ng).

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coinjected aida-MO together with MKK4-MO or JNK-MO. Results showed that the coinjection of MKK4-MO or JNKMO could cancel the dorsalizing effect of aida-MO, indicating that MKK and JNK act downstream of aida (Figure 7A; see Figure 7B for the statistical data). The fact that endogenous or overexpressed Aida was not able to alter the expression patterning of the marker genes in a JNK-MO or MKK4-MO background strongly suggests that all aida (and by implication, dorsalizing Axin) activity acts via JNK. RT-PCR analysis further confirmed the changes in marker gene expression (Figure S7). Collectively, these data suggest that aida could suppress the intrinsic dorsalizing activity of Axin through inhibiting Axin-mediated JNK activation. Axin/JNK Signaling-Regulated Dorsoventral Patterning Is Independent of Wnt Signaling We next asked if Axin-mediated JNK signaling exerts its dorsalizing activity by interfering with Wnt/b-catenin signaling in the dorsal organizer. In zebrafish, maternal b-catenin2 is essential for the formation of the dorsal organizer (Bellipanni et al., 2006). Consistent with this report, bcatenin2 knocknown with b-cat2-MO resulted in reduction or elimination of expression of the dorsal markers gsc and chd with expanded expression of the ventral markers bmp2 and eve1 in most of the embryos (87%–90%, n > 29) (Figure 8A). When b-cat2-MO and full-length axin mRNA were coinjected, however, a large proportion (60%– 66%, n > 27) of embryos showed normal expression levels for those marker genes. This can be interpreted to mean that b-cat2-MO-induced ventralized phenotypes are rescued to certain degrees by the intrinsic dorsalizing activity of ectopic Axin. This result also suggests that intrinsic dorsalizing activity of Axin is independent of b-catenin during embryogenesis. In contrast, coinjection with bcat2-MO and axinDMID caused similar or more severe ventralization compared with the effect of b-cat2-MO alone, implying that the ventralizing activity, but not the dorsalizing activity, of Axin relies on the availability of bcatenin. Moreover, we injected JNK mRNA and JNK-MO separately into fish embryos and determined the effect on the expression of bozozok, which is a direct downstream marker for maternal Wnt signaling. As shown in Figure 8B,

neither overexpression nor knockdown of JNK had any discernible effect on bozozok expression, indicating that JNK signaling may not interfere with maternal Wnt signaling in vivo. We also addressed whether aida affects bozozok expression by injecting aida mRNA, aida-MO, or purified anti-Aida antibody. Results showed that bozozok expression was not perturbed by any of these injections (Figure 8B). We then explored whether JNK signaling plays a role in zygotic Wnt signaling by injecting JNK mRNA, JNK-MO, Wnt8-MO, or in combinations as indicated followed by in situ analysis with a tbx6 probe (Figure 8C). Whereas Wnt8-MO abolished tbx6 expression, JNK mRNA or JNK-MO injection did not alter the expression of tbx6. These data all conform to the conjecture that JNK signaling both maternally and zygotically independent of Wnt signaling. Moreover, we also transfected JNK, MKK4, MKK4-KM, JNK-APF, and MKK4-RNAi alone or in combination, along with Axin as a positive control, into mammalian cells, and found that activation or inhibition of JNK activity has no effect on LEF-1 reporter activity, nor did it have any effect on b-catenin protein level, suggesting that JNK is not involved in canonical Wnt signaling (Figure 8D). DISCUSSION Since its initial identification as a negative regulator of Wnt signaling, Axin has emerged as a master scaffold in multiple other pathways, including the JNK signaling pathway, TGF-b signaling, and p53 activation cascade (De Robertis and Kuroda, 2004; Luo et al., 2003; Rui et al., 2004; Salahshor and Woodgett, 2005). In this study, we have unexpectedly found that Axin possesses an intrinsic activity to induce dorsalization of zebrafish embryos. Injection of wild-type axin into one-cell embryos gave rise to two populations of embryos with altered dorsoventral patterning, one dorsalized and the other ventralized, which is against the accepted view of Axin as a ventralizing factor in the control of dorsoventral patterning (De Robertis and Kuroda, 2004). It was previously shown that an Axin mutant that lacks the APC-binding domain, AxinDRGS, induces formation of an ectopic axis in Xenopus, suggesting a possibility that this deletion mutant of Axin enhances Wnt signaling by acting as a dominant-negative form (Zeng et al.,

Figure 8. Wnt Signaling-Independent Regulation of Dorsoventral Patterning by the Axin/JNK Signaling Cascade (A) Genetic interaction between b-catenin and axin. Ten nanograms of b-cat2-MO was injected into one-cell embryos alone or together with 300 pg axin mRNA or 150 pg axinDMID mRNA. In situ hybridization of marker genes was performed at the shield stage. (B) JNK mRNA, JNK-MO, aida mRNA, aida-MO, or aida antibody was injected into one-cell embryos, and expression of bozozok was analyzed in sphere-stage embryos. (C) JNK mRNA, JNK-MO, and wnt8-MO were injected singly or in combination as indicated. In situ analysis of tbx6 expression was performed at the shield stage. (D) JNK signaling has no effect on Wnt signaling as assessed with LEF-1 luciferase reporter and b-catenin stability assay. HEK293T cells were transfected with 0.5 mg of LEF-1 luciferase reporter plasmid together with 1.5 mg of MKK4, JNK, MKK4-KM, JNK-APF, or MKK4-RNAi alone or in combination for the LEF-1 luciferase activity. None of the plasmids affected basal or wnt-1-induced reporter expression. For the b-catenin stability assay, HEK293T cells were transfected with 4 mg of MKK4, JNK, MKK4-KM, JNK-APF, or MKK4-RNAi alone or in combination together with 0.5 mg of GFP, 1 mg of Myc-b-catenin, and 1.5 mg of wnt-1. Results showed that JNK signaling did not affect the b-catenin protein level. (E) A model for dual activities of Axin in dorsal development of vertebrate embryos. In the canonical Wnt pathway, b-catenin promotes dorsalization. Axin reduces b-catenin levels by serving as a scaffold for the assembly of b-catenin degradation complex, and hence attenuates dorsalization. Axin also exerts an opposing role, i.e., promotion of dorsalization, by means of activation of JNK. Aida, by virtue of repressing Axin-induced JNK activation through disrupting Axin homodimerization, attenuates Axin-enhanced dorsalization.

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1997). However, AxinDRGS-induced axis duplication occurs only when it is overexpressed on the ventral side. This is consistent with our finding that wild-type axin-induced dorsalization in fish embryos was also caused by ventral injection. In fact, enhanced expression of dorsal markers gsc and chd was previously noticed in axin-injected fish embryos (Shimizu et al., 2000). Our results establish that Axin, a central scaffold for the assembly of bcatenin degradation complex, in fact plays two opposing roles in dorsoventral patterning during zebrafish early development. JNK signaling has been implicated in numerous biological processes ranging from stress responses to formation of planar cell polarity during development (Boutros et al., 1998; Weston and Davis, 2002; Xia and Karin, 2004). Little information is available as to whether JNK activity is involved in dorsoventral patterning. One study showed that loss of function of Puckered, a phosphatase that antagonizes JNK signaling, could suppress armadillo defects in Drosophila, and that activation of JNK signaling could result in weak dorsalization (McEwen et al., 2000). Here, we have unequivocally demonstrated that JNK signaling plays a necessary role in promoting dorsal development in fish embryos. There exists a close correlation between JNK-activating ability and dorsalization function. The Axin mutant axinDMID, which fails to activate JNK, does not have a dorsalizing effect, suggesting that JNK activation by Axin underlies this induction of dorsalization in fish embryos. Overexpression of JNK or MKK4 leads to embryonic dorsalization. Knockdown of JNK or MKK4 leads to embryonic ventralization and is sufficient to block axin-induced dorsalization. These lines of evidence all point to the conclusion that JNK is required for proper dorsal development. Importantly, we have demonstrated that axin induces embryo dorsalization independently of b-catenin. It is clear that maternal b-catenin is a dorsalizing factor (Heasman et al., 1994). This protein is stabilized on the dorsal side, whereas it is degraded on the ventral side. Thus, specification of the dorsal signaling center involves accumulation of b-catenin, the nuclear effector of Wnt signaling, in dorsal nuclei (Schier, 2001). An important question here is whether b-catenin is involved in Axin-induced dorsalization. As previously shown, knockdown of b-catenin2, which is essential for the formation of the dorsal organizer, causes exclusively ventralized phenotypes (Bellipanni et al., 2006). However, the ventralizing effect of b-catenin2 knockdown was effectively blocked by overexpression of wild-type axin. Consistently, axinDMID, which is unable to activate JNK but is fully capable of repressing Wnt signaling, resulted in unique ventralized phenotypes, suggesting that axin-induced dorsalization is independent of maternal Wnt/b-catenin signaling. Consistent with this conjecture are our additional results obtained with in situ analysis of bozozok and tbx6 expression upon overexpression or knockdown of aida or JNK, which are, respectively, maternal and zygotic downstream target genes of Wnt signaling. We found that these markers were not affected under those conditions. However, it has oc-

curred to us that Liao et al. (2006) found in Xenopus that JNK activity prevents nuclear b-catenin accumulation, and consequentially ventralizes embryos. One simplest explanation is that JNK may play different roles in the two different organisms. In fact, while high maternal JNK activity is detected from oocyte maturation to gastrulation in Xenopus, JNK is weakly expressed at early developmental stages, but shows higher expression levels at late gastrula stages through segmentation (Bagowski et al., 2001; Krens et al., 2006). It is unclear how the intrinsic dorsalizing activity of axin is repressed in embryos, particularly on the ventral side during early development. It is highly possible that some ventral determinants are expressed and more enriched on the ventral side, as previously suggested (Kofron et al., 2001). In this study, we have identified Aida as an antagonist of Axin-mediated JNK activation. Aida interacts with Axin and prevents Axin from the homodimerization that is a prerequisite for Axin to activate JNK, since only wild-type Aida, but not AidaDAxin, can attenuate Axinmediated JNK activation and dorsalization. The observation that knockdown of aida induces dorsalized phenotypes during embryonic development also conforms to a requirement of JNK signaling in dorsalization. It is important to point out that Aida does not appear to affect the canonical Wnt pathway. We further suggest that Aida is one possible candidate of the proposed ventral determinants that fine-tunes the intrinsic activity of Axin in the regulation of dorsoventral patterning. In sum, we have identified a bcatenin-independent dorsalization pathway that is positively regulated by Axin/JNK signaling and antagonized by Aida (Figure 8E). EXPERIMENTAL PROCEDURES Yeast Two-Hybrid Screen and Construction of Zebrafish aida Plasmids A yeast two-hybrid screen using the C-terminal fragment of mouse Axin as bait was performed as described (Rui et al., 2002), and in this study, mouse Aida was characterized. Zebrafish aida cDNA was isolated from an embryonic cDNA library by RT-PCR and subcloned into pBluescript to make antisense probe for in situ hybridization. The coding sequence of aida was cloned into vector pXT7 for mRNA synthesis. Preparation of Rabbit Polyclonal Antibody against Aida The DNA fragment encoding full-length mouse Aida was inserted into pGEX4T and transformed into E. coli BL21 bacterial cells. The GSTAida fusion protein was induced with 1 mM IPTG and purified using glutathione beads. Rabbits were immunized with the purified GSTAida fusion protein (500 mg each in the complete Freund’s adjuvant, Sigma) followed by three repetitions of boosting (250 mg each in the incomplete Freund’s adjuvant, Sigma). The Aida antibody was then purified with the Aida protein using an affinity purification method as previously described (Lin and Morrison-Bogorad, 1991) and was tested for specificity by western blotting of lysates of HEK293T cells transfected with or without Aida, and by immunoprecipitation (Figure S1). Transient Transfection and Immunokinase Assays HEK293T cells were maintained in DMEM medium supplemented with 10% fetal bovine serum, 100 IU penicillin, 100 mg/ml streptomycin, and 2 mM glutamine. Transfections were performed in 60 mm dishes using Dosper according to the manufacturer’s instructions (Roche). The total

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amount of transfected DNA was adjusted to 4 mg with the empty vector pCMV5 where necessary. Cells were harvested at 36 hr posttransfection and lysed in a lysis buffer (20 mM Tris-HCl [pH 7.4], 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM b-glycerolphosphate, 1 mM sodium orthovanadate, 1 mg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride). FLAG-tagged JNK was immunoprecipitated using mouse monoclonal anti-FLAG M2 beads (Sigma), and the kinase activities were determined as described previously using 1 mg of GST-c-Jun (aa 1–79; Stratagene) as substrate. Fold activation of the kinases was determined by an imaging analyzer (Molecular Dynamics model 425E) and normalized to their expression levels. Coimmunoprecipitation and Western Blot Analysis Transiently transfected HEK293T cells in 60 mm dishes were lysed in a lysis buffer, sonicated three times for 5 s each, and centrifuged at 13,200 rpm for 30 min at 4 C. HA-tagged or Myc-tagged proteins were immunoprecipitated from the cell lysate with anti-HA (F-7), antiMyc (9E10), or anti-JNK (Santa Cruz Biotechnology, Inc.) antibodies and Protein A/G Plus-agarose beads (Santa Cruz Biotechnology, Inc.) as indicated. Immunoprecipitates or TCLs were analyzed by western blotting. The boiled samples were separated on 10% SDSpolyacrylamide gels and transferred to Immobilon-P membranes (Millipore). After blocking with 5% nonfat milk in Tris-buffered saline with 0.1% Tween 20 for 1 hr, the membranes were probed with anti-HA, anti-Myc (9E10), anti-p-c-Jun, or anti-FLAG M2 (Sigma) antibodies. Bound antibodies were visualized by enhanced chemiluminescence (Amersham Pharmacia Biotech) using horseradish peroxidase-conjugated antibodies. GST Pull-Down Assay GST-Axin and GST-aida were expressed in BL21 bacterial cells and purified using glutathione-agarose beads (Sigma). Expression was induced with 1 mM IPTG for 4 hr at 37 C. About 4 mg of GST fusion protein bound to agarose beads was added to each total lysate from HEK293T cells and incubated for 3 hr with gentle rotation. The beads were washed three times with cell lysis buffer, and the proteins were eluted with 25 ml of 2XSDS sample buffer. LEF-1 Luciferase Reporter Assay HEK293T cells were transfected with 0.5 mg of pGL3-fos-7LEF-luciferase (provided by L. Williams), 0.1 mg of pCMV-b-galactosidase, 1 mg of b-catenin, and 1.5 mg of vector or of each of the Axin and Aida constructs as indicated. Luciferase activities were measured as described previously (Rui et al., 2002). Data were presented as means from three separate experiments performed in triplicate. Immunofluorescence Staining COS-7 cells grown on coverslips in 6-well plates were transfected with Myc-Axin and HA-Aida. After 24 hr, cells were washed with PBS twice and fixed in 4% PF buffer for 30 min following by blocking in 4% BSA buffer for 30 min. Cells were then incubated for 1 hr with primary rabbit anti-HA and mouse anti-Myc antibodies at room temperature. After being washed five times with PBS, cells were incubated with FITC conjugated goat anti-rabbit IgG and Texas red conjugated goat antimouse IgG for 1 hr. Cells were visualized under a fluorescence microscope (Olympus). In Vitro Synthesis of mRNA Linearized plasmids were used as templates for in vitro transcription using an appropriate Cap-Scribe Kit (Roche). The synthesized mRNA was purified using the RNAeasy Mini Kit (QIAGEN) after treatment with RNase-free DNase and dissolved in nuclease-free water. Microinjection The MO and mRNA were diluted to an appropriate concentration prior to injection. RNAs and MOs were injected into the yolk or cytoplasm between the one- and two-cell stages, as described before (Zhao

et al., 2003). GFP mRNA or related control MO was injected as control to confirm specific effects. Data obtained from independent injections were pooled. Whole-Mount In Situ Hybridization Digoxigenin-UTP-labeled or fluorescein-UTP-labeled antisense RNA probes were generated by in vitro transcription. Whole-mount in situ hybridizations were performed essentially following the standard protocol with minor modifications. Supplemental Data Supplemental Data include seven figures, Supplemental Experimental Procedures, and Supplemental References and can be found with this article online at http://www.developmentalcell.com/cgi/content/full/ 13/2/268/DC1/. ACKNOWLEDGMENTS We thank Dr. T. Hirano (Osaka University, Japan) for the zebrafish Axin cDNA and Dr. D.M. Virshup for comments. This work was supported by grants from National Science Foundation (30528014, to S.C.L), National Basic Research Program of China (2006CB503900, to S.C.L; 2006CB943401 and 2005CB522502, to A.M.M.), the Fujian and Xiamen Councils of Science and Technology (2002F002 and 20055004, to S.C.L.), and the Hong Kong Research Grants Council (6416/05M, to S.C.L.). Received: January 1, 2007 Revised: April 16, 2007 Accepted: July 12, 2007 Published: August 6, 2007 REFERENCES Bagowski, C.P., Myers, J.W., and Ferrell, J.E., Jr. (2001). The classical progesterone receptor associates with p42 MAPK and is involved in phosphatidylinositol 3-kinase signaling in Xenopus oocytes. J. Biol. Chem. 276, 37708–37714. Behrens, J., Jerchow, B.A., Wurtele, M., Grimm, J., Asbrand, C., Wirtz, R., Kuhl, M., Wedlich, D., and Birchmeier, W. (1998). Functional interaction of an axin homolog, conductin, with beta-catenin, APC, and GSK3beta. Science 280, 596–599. Bellipanni, G., Varga, M., Maegawa, S., Imai, Y., Kelly, C., Myers, A.P., Chu, F., Talbot, W.S., and Weinberg, E.S. (2006). Essential and opposing roles of zebrafish beta-catenins in the formation of dorsal axial structures and neurectoderm. Development 133, 1299–1309. Boutros, M., Paricio, N., Strutt, D.I., and Mlodzik, M. (1998). Dishevelled activates JNK and discriminates between JNK pathways in planar polarity and wingless signaling. Cell 94, 109–118. Cao, Y., Zhao, J., Sun, Z., Zhao, Z., Postlethwait, J., and Meng, A. (2004). fgf17b, a novel member of Fgf family, helps patterning zebrafish embryos. Dev. Biol. 271, 130–143. De Robertis, E.M., and Kuroda, H. (2004). Dorsal-ventral patterning and neural induction in Xenopus embryos. Annu. Rev. Cell Dev. Biol. 20, 285–308. Derijard, B., Raingeaud, J., Barrett, T., Wu, I.H., Han, J., Ulevitch, R.J., and Davis, R.J. (1995). Independent human MAP-kinase signal transduction pathways defined by MEK and MKK isoforms. Science 267, 682–685. Furuhashi, M., Yagi, K., Yamamoto, H., Furukawa, Y., Shimada, S., Nakamura, Y., Kikuchi, A., Miyazono, K., and Kato, M. (2001). Axin facilitates Smad3 activation in the transforming growth factor beta signaling pathway. Mol. Cell. Biol. 21, 5132–5141. Harland, R., and Gerhart, J. (1997). Formation and function of Spemann’s organizer. Annu. Rev. Cell Dev. Biol. 13, 611–667.

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