Domain structure and biochemical interactions of Diversin. (A) Schematic representation of the domain structure of mouse Diversin and of various deletion constructs. Numbers represent amino acids of Diversin. (B) Coimmunoprecipitation analyses in transfected Neuro2A cells, showing that the central domain of Diversin (white in A) interacts with CKIε (top). The C-terminal domain (black in A) confers interaction with Conductin (middle). Similar interactions were seen with Axin. Expression of the indicated proteins was verified by Western blotting (bottom). (C,D) Yeast two-hybrid and coimmunoprecipitation analyses showing that Diversin interacts with the GSK3β-binding domain of Conductin and Axin. For constructs see Behrens et al. (1998). (+) Growth of yeast on selection medium, (−) absence of growth. The conductin ΔGSK-bd is as in C (conductin lacking amino acids 343–396). (E) Coimmunoprecipitation analyses of Diversin, Conductin, and GSK3β complexes. Full-size Conductin is able to dimerize through the DIX-domain (Hsu et al. 1999; data not shown) and allows the formation of a triple complex that contains Diversin, Conductin, and GSK3β (a). Conductin1–465 and the GSK3β-binding domain of Conductin (Conductin GSK-bd) cannot dimerize and triple complexes with Diversin and GSK3β are not observed (b,c). The conductin GSK-bd is as in C (amino acids 343–465; Behrens et al. 1998). (d) Note that amounts of Conductin GSK-bd similar to those in c are able to efficiently coprecipitate GSK3β. Schematic representations of the interactions in (a–d) are shown at right. For immunoprecipitation experiments, 6 × 10⁵ cells per 10-cm culture dish were transfected with 5 μg of the indicated cDNAs (in the pCDNA vector, Invitrogen) and cultured for 48 h, followed by lysis and immunoprecipitation (Behrens et al. 1998). Polyacrylamide gel electophoresis and Western blot were performed with 1/5 of the immunoprecipitate from 1-ml lysate, 1/50 of the lysate was used as expression control. Concentrations of the commercial antibodies were as suggested by the manufacturers.

Diversin recruits CKIε to the β-catenin degradation complex. (A) Diversin promotes association of endogenous CKIε with Conductin. The Conductin-binding domain of Diversin did not mediate this interaction. Note that the Diversin constructs (lanes a,f) are as in Fig. 1A. Immunoprecipitations were performed as described in Fig. 1. A scheme of Diversin linking CKIε to the Axin/Conductin-GSK3β complex is shown at right. (B) Hybrid molecules that contain the catalytic domain of CKIε and the carboxy-termini of murine or zebrafish Diversin inhibit Dishevelled-induced Lef/Tcf-dependent transcription. cDNAs that encode the individual domains have no effect. The catalytic activity of CKIε is essential, as shown by a kinase-inactive fusion construct or addition of 50 μM CKI-7, a specific CKI inhibitor. The indicated cDNAs (3 μg) were transfected into 293 cells either with Dishevelled (1.5 μg) cDNA (gray bars) or transfected alone (white bars). Reporter assays are as described in Fig. 2, hybrid constructs are specified in Materials and Methods. (C) Diversin cooperates with Frat-1 in displacing GSK3β from Axin/Conductin complex. Expression of Frat-1 (1.5 μg) in 293 cells activates Lef/Tcf-dependent transcription. Cotransfection of Diversin cDNA (3 μg) enhances this further. The C-terminal, but not the central domain of Diversin is responsible for this activity. Reporter assays were performed as in Fig. 2. Gray bars represent the TOP and white bars the control FOP reporters. A cooperative effect of Diversin and Frat-1 was also observed in axis duplication of Xenopus embryos. For this, Diversin (3 ng) and Frat-1 mRNA (1 ng) were injected ventrally into the 4–8-cell embryo. Note that limited amounts of Frat-1 mRNA were injected to visualize cooperation. A schematic model of the action of Diversin in displacing GSK3β from the Axin/Conductin complex, which leads to enhanced signaling of β-catenin, is presented at right.

Diversin activates the JNK branch of the Wnt-signaling pathway. (A) Coimmunoprecipitation analyses of CKIε, Diego and Diversin, which show that both Diego and Diversin interact with CKIε. Note that CKIε was transfected here. Immunoprecipitations were performed as described in Fig. 1. Note equal expression of Diego and Diversin. (B) Transfection of 1- and 3-μg Diversin cDNA activate transcription from a JNK-dependent luciferase reporter gene. Diversin (1–3 μg) also enhances Dishevelled (1.5 μg) and Wnt11 (1.5 μg)-induced JNK activity. Wnt-1 (1.5 and 3 μg) do not activate JNK-dependent, but activate Lef/Tcf-dependent transcription (inset). The indicated cDNAs were transfected into 293 cells, and MEKK (3 μg) served as control. (C) Diversin affects gastrulation movements in zebrafish embryos. Diversin induces failure of convergence and extension (CE), or affects dorso-ventral patterning, depending on the amounts of injected mRNA. (D,E) Zebrafish embryos injected with low amounts (0.1 ng) of Diversin and standard amounts (0.4 ng) of Diego mRNA show a shortened body axis and a curled tail, typical for defects in gastrulation movements. (F–I) In situ hybridization of flattened embryos injected with either Diversin MO (1.5 ng), Diversin mRNA (0.1 ng), or Diego mRNA (0.4 ng), respectively, at the 5–10 somite stage. Embryos are stained for myoD to visualize somites. Note the undulation of the body axis, the compression of the anterior-posterior axis, and the shortening of the intersomitic distances in the injected embryos. In situ hybridization with krox20 expressed in the hindbrain (see the two stripes at left) shows that the embryos are not ventralized.

Diversin inhibits Wnt signaling in cell culture and in Xenopus embryos. (A) Diversin promotes degradation of β-catenin in 293 cells, as analyzed by Western blot analysis of cytoplasmic β-catenin. Cells were mildly lysed using 0.1% Triton-X 100, and amounts of cell lysates were normalized using Shp2. (Top, left) Transfection of increasing amounts (2 and 5 μg) of Diversin cDNA destabilized endogenous β-catenin and blocked β-catenin that is induced by transfection of 1.5 μg of Dishevelled cDNA. (Bottom, left) Diversin inhibits the stabilization of β-catenin induced by transfection of increasing amounts (0.25–2 μg) of Dishevelled cDNA. (Right) The activity of Diversin is blocked by addition of 20 μM MG132, and is observable up to 16 h after transient transfection. Dishevelled (1.5 μg) and Diversin (3 μg) were transfected, and expression was monitored by Western blotting (data not shown). The proteasome inhibitor MG132 was added 2 h before cell lysis. (B,C) Diversin blocks Dishevelled- and Wnt-induced transcription of a Lef/Tcf-dependent luciferase reporter gene in a dose-dependent manner. A total of 1–3 μg of Diversin cDNA and 1.5 μg of Dishevelled or Wnt3a cDNA were cotransfected in 293 cells with the TOP (gray bars) or the control FOP reporter (white bars). (D) Analysis of the functionally important domains of Diversin in Lef/Tcf-dependent transcriptional assays. Full-length Diversin and a fragment able to interact with CKIε and Axin/Conductin inhibit transcription. Expression of similar levels of Diversin and Diversin fragments were verified by Western blot analysis. Reporter assays were performed as in B and C. (E) Epistasis analysis of Diversin in Xenopus embryos. Ventral injection of 1 ng of Dishevelled, CKIε, (dn)GSK3β, or β-catenin mRNA induced secondary body axes (percent axis duplication shown). Coinjection of 3 ng of Diversin mRNA blocked axis duplication by Dishevelled and CKIε, but not dn-GSK3β or β-catenin.

Diversin affects the formation of the dorsal organizer in zebrafish embryos. (A–D) In situ hybridization of diversin in zebrafish embryos at different developmental stages. Diversin is expressed ubiquitously (B) in the blastomeres at the 4–8-cell stage and (C) in shield-stage embryos. Pronounced expression in the yolk-blastoderm interphase is indicated by arrows. (D) Specific expression of diversin in ganglial layers of the retina, otic vesicles, the roof plate of the brain, and in the forebrain-midbrain (arrowhead) and midbrain-hindbrain boundaries (arrow) at 72 h post fertilization (hpf). A shows the sense control. (E–P) Down-regulation of Diversin by the injection of antisense morpholinos leads to a dorsalization of zebrafish embryos and the appearance of an enlarged or ectopically localized dorsal organizer. (E) Wild-type embryo at 30 hpf (dorsal up, arrowhead indicates ventral tail fin). (F,G) Embryos at 30 hpf after injection with 4–6 ng of Diversin antisense morpholino oligonucleotides (divMO) that display class 3 (F, arrowhead indicates missing ventral tail fin) and class 4 dorsalization (G, arrowhead indicates wound-up trunk). (H,I,K,L) In situ hybridization of gsc at the shield stage (animal view, dorsal to the right; arrowheads indicate the size of the gsc expression domain; arrow indicates ectopic gsc expression). (H) Uninjected control; (I,K) embryo injected with Diversin MOs; (L) embryo coinjected with Diversin MOs and mouse Diversin mRNA. (M,N) In situ hybridization of tbx6 at 70% epiboly (lateral view, anterior up, dorsal to the right). (M) Control; (N) embryo injected with Diversin MOs; note the expansion of the tbx6 expression domain. (O,P) In situ hybridization of pax2.1 at the 5-somite stage (lateral view, anterior to the left, dorsal up; arrows indicate the midbrain-hindbrain boundary region). (O) Control; (P) embryo injected with Diversin MOs; note the ventral expansion of the pax2.1 expression domain. (Q–Z) Overexpression of Diversin ventralizes zebrafish embryos. (Q,R) Strongly ventralized embryos (Q, class 3, and R, class 4) at 30 hpf after injection with full-length Diversin mRNA; (S,T) In situ hybridization of embryos at shield stage for gsc (arrowheads in S). (S) Uninjected control. (T) Embryo injected with Diversin mRNA loses gsc expression at the dorsal side. (U,V) In situ hybridization of embryos at 70% epiboly stained for eve1 in the ventral mesoderm (arrowheads) and gsc in the prechordal plate (arrow). (U) Uninjected control. (V) Embryo injected with Diversin mRNA shows an expansion of the eve1 expression domain and looses gsc expression at the dorsal side. (W–Z) Diversin acts downstream of Wnt and upstream of β-catenin, as shown on injected embryos by in situ hybridization with gsc that marks the dorsal organizer. (W) Embryo injected with Wnt8 mRNA; (X) embryo coinjected with Diversin and Wnt8 mRNA; (Y) embryo injected with β-catenin mRNA; (Z) embryo coinjected with Diversin and β-catenin mRNA. Arrowheads indicate lateral borders of gsc expression domains, arrow indicates ectopic gsc expression. All embryos are shown as lateral views (dorsal to the right and animal pole up).

Schematic model of the action of Diversin in β-catenin degradation. (A) Diversin recruits CKIε to the Axin/Conductin complex, and this leads to priming phosphorylation of β-catenin on Ser 45. Dimeric Axin/Conductin allows simultaneous binding of Diversin/CKIε and GSK3β in the complex. (B) GSK3β mediates subsequent phosphorylation of β-catenin on Thr 41, Ser 37, and Ser 33. Phosphorylated Ser 37 and Ser 33 are binding sites for the E3ubiquitin ligase β-TrCP (Amit et al. 2002), which promotes degradation of β-catenin.