Dickkopf-1 Regulates Gastrulation Movements by Coordinated Modulation of Wnt/␤Catenin and Wnt/PCP Activities, Through Interaction with the Dally-Like Homolog Knypek

Dickkopf-1 regulates gastrulation movements by coordinated modulation of Wnt/␤catenin and Wnt/PCP activities, through interaction with the Dally-like homolog Knypek

Luca Caneparo,1 Ya-Lin Huang,3,4 Nicole Staudt,1,4 Masasumi Tada,2,4 Reiner Ahrendt,1 Olga Kazanskaya,3 Christof Niehrs,3 and Corinne Houart1,5 1Medical Research Council Centre for Developmental Neurobiology, King’s College London, SE1 1UL London, United Kingdom;2Anatomy and Developmental Biology Department, University College London, WC1E 6BT London, United Kingdom; 3Division of Molecular Embryology, German Cancer Research Center, D-69120 Heidelberg, Germany

Dickkopf-1 (Dkk1) is a secreted protein that negatively modulates the Wnt/␤catenin pathway. Lack of Dkk1 function affects head formation in frog and mice, supporting the idea that Dkk1 acts as a “head inducer” during gastrulation. We show here that lack of Dkk1 function accelerates internalization and rostral progression of the mesendoderm and that gain of function slows down both internalization and convergence extension, indicating a novel role for Dkk1 in modulating these movements. The motility phenotype found in the morphants is not observed in embryos in which the Wnt/␤catenin pathway is overactivated, and that dominant-negative Wnt proteins are not able to rescue the gastrulation movement defect induced by absence of Dkk1. These data strongly suggest that Dkk1 is acting in a ␤catenin independent fashion when modulating gastrulation movements. We demonstrate that the glypican 4/6 homolog Knypek (Kny) binds to Dkk1 and that they are able to functionally interact in vivo. Moreover, Dkk1 regulation of gastrulation movements is kny dependent. Kny is a component of the Wnt/planar cell polarity (PCP) pathway. We found that indeed Dkk1 is able to activate this pathway in both Xenopus and zebrafish. Furthermore, concomitant alteration of the ␤catenin and PCP activities is able to mimic the morphant accelerated cell motility phenotype. Our data therefore indicate that Dkk1 regulates gastrulation movement through interaction with LRP5/6 and Kny and coordinated modulations of Wnt/␤catenin and Wnt/PCP pathways. [Keywords: Dickkopf-1; HSPG; Wnt/PCP; gastrulation movements] Supplemental material is available at http://www.genesdev.org. Received August 15, 2006; revised version accepted December 22, 2006.

The anterior brain is first defined as a specific portion of ing antagonists to, these signals (Wilson and Houart rostral neural ectoderm expressing a set of early neural 2004). markers such as hesx1/anf1 and otx2. This initial terri- Among the most studied posteriorizing signals are the tory is progressively refined into specific presumptive secreted Wnt molecules (Erter et al. 2001; Kiecker and midbrain and forebrain areas (Wilson and Houart 2004). Niehrs 2001a; Lekven et al. 2001). Wnts are secreted by The anterior–posterior (AP) patterning of the CNS is the mesendoderm at the marginal zone, as gastrulation thought to arise, in early gastrula, through the action of proceeds and gastrula embryos with increased Wnt ac- posteriorizing signals, coming from the newly formed tivity fail to develop rostral neural identity and show a mesoderm, on an “anterior” neural ectoderm (Stern posterior transformation of the anterior neural plate 2001). The anterior neural plate therefore needs to be (Kim et al. 2000; Kiecker and Niehrs 2001b; Lekven et al. protected from “posteriorizing” influence to maintain 2001). A variety of molecules, acting as secreted antago- rostral neural identities such as forebrain and midbrain. nists of the Wnt pathway, have been identified in the This is achieved by both moving away from, and express- Spemann Organizer. Most of them are related to the extracellular domain of the Wnt receptor Frizzled and act 4These authors contributed equally to this work. by direct binding to Wnt proteins (Leyns et al. 1997; 5Corresponding author. E-MAIL [email protected]; FAX 20-78486550. Wang et al. 1997; Hsieh et al. 1999; Kawano and Kypta Article is online at http://www.genesdev.org/cgi/doi/10.1101/gad.406007. 2003).

Distinct from these, the Dickkopf family of secreted to defects in gastrulation movements. Indeed, lack of molecules (Dkk) (Glinka et al. 1998; Kawano and Kypta Dkk1 function accelerates internalization and rostral 2003) influences the reception of Wnt signals by binding progression of the mesendoderm, while gain of function to the Frizzled coreceptors LRP5/6 trans-membrane pro- slows down both internalization and convergence exten- teins (Mao et al. 2001) and Kremen (Mao et al. 2002). sion. These findings point to a role for Dkk1 in modu- Among them, Dkk1 is shown to have a strict inhibitory lating these movements. Surprisingly, we show that up- effect on Frizzled receptors (Kazanskaya et al. 2000). It is regulation of the ␤catenin pathway is not inducing the first expressed in the forming mesendoderm of the late gastrulation movement defects observed in the Dkk1 zebrafish, Xenopus, and mouse blastula and then local- morphants, strongly suggesting that Dkk1 is able to in- ized specifically in the nascent Spemann Organizer of teract with another signaling pathway. We therefore the early gastrula (Kazanskaya et al. 2000; Shinya et al. tested the possible interaction of Dkk1 with known 2000). Moreover, both in zebrafish and mouse, dkk1 is regulators of gastrulation movements. We demonstrate expressed in early extra-embryonic tissue (yolk syncytial that Dkk1 is able to bind in vivo to the glypican4/6 layer [YSL] and anterior visceral endoderm [AVE]). member Knypek (Kny, a Dally-like homolog). We find In frog and fish, Dkk1 overexpression is able to ante- that Kny and Dkk1 are able to potentiate each other’s riorize neural tissue (Kazanskaya et al. 2000; Shinya et activity when coexpressed in early embryos. Comple- al. 2000). It can also induce a secondary head if coex- mentarily, propagation of the secreted Dkk1 is greatly pressed with BMP antagonists in ventral blastomeres of reduced in kny mutants. These data demonstrate that Xenopus early blastula embryos. These data led to the propagation and range of action of Dkk1 are likely to be conclusion that Dkk1 acts as a “head inducer” through dependent on interaction with the heparan sulfate pro- inhibition of the Wnt/␤catenin posteriorizing activity in teoglycan (HSPG) Kny. Potentiation of Kny function by early gastrula embryos (Niehrs et al. 2001). Requirement Dkk1 and Kny requirement for acceleration of gastrula- for Dkk1 function in head formation has been further tion movements in the Dkk1 morphants together supported by the characterization of mouse embryos strongly suggest that Dkk1 cooperates with Kny in regu- lacking Dkk1 gene function. Indeed, the genetic knock- lating gastrulation movements. We therefore tested out of the mouse dkk1 leads to the formation of headless whether Dkk1 was able to modulate the Wnt/planar cell embryos due to lack of maintenance of anterior neural polarity (PCP) pathway. Our results show that Dkk1 identity during gastrulation (Mukhopadhyay et al. 2001). overexpression mimics up-regulation of the Wnt/PCP Interestingly, failure in establishing the forebrain terri- pathway both in Xenopus and zebrafish and dramatically tories in these embryos is not due to requirement of increases JNK phosphorylation, strongly suggesting that Dkk1 in the AVE, as mosaic embryos with a dkk1−/− Dkk1 is positively modulating the Wnt/PCP pathway. AVE but containing a functional dkk1 gene in all other However, decrease of Wnt/PCP activity in the Dkk1 tissues develop normally (Mukhopadhyay et al. 2001). morphants is not the only cause of the gastrulation Thus, Dkk1 has been undoubtedly shown to be required movement phenotype observed, as loss of PCP function for establishment of the forebrain in vertebrates, and its in vertebrates is not associated with acceleration of biochemical properties have been reasonably well under- movements. In fact, we find that increase of Wnt/ stood (van Tilbeurgh et al. 1999). However, the mecha- ␤catenin activity accompanied by mild down-regulation nism by which Dkk1 acts on the neural ectoderm to of the Wnt/PCP pathway is a condition for which accel- support forebrain formation is yet to be unraveled. eration of internalization is often observed. We therefore Late in gastrulation, local inhibition of Wnt signaling propose that Dkk1 modulates gastrulation movements is also required to maintain telencephalon and eye iden- by coordinated modulation of the Wnt//␤catenin and tities inside the vertebrate anterior neural plate (Heisen- PCP pathways, through interaction with both Kny and berg et al. 2001; Houart et al. 2002; Kim et al. 2002; the LRP/Kremen complex. All together, our results led Lagutin et al. 2003). One of Dkk1’s possible functions us to propose a model by which Dkk1, via endocytosis of may therefore be to ensure the expression of specific se- LRP5/6, may transform the biochemical properties of the creted Frizzled-Related Proteins (sFRPs) such as tlc in- Frizzled receptors and/or an interacting cytoplasmic side the anterior neural border (ANB). We found that component from Wnt/␤catenin to a Wnt/PCP conforma- such is the case, as lack of Dkk1 dramatically reduces tion, thereby up-regulating the latter pathway while in- the expression of tlc (L. Caneparo, R. Ahrendt, J. Peres, hibiting the “canonical” one. M. Kapsimali, and C. Houart, in prep.). In this study, we address the role of Dkk1 in the Results mesendoderm during gastrulation. Dkk1 is thought to Dkk1 modulates gastrulation movements act through vertical signaling from the anterior mesendoderm (Kiecker and Niehrs 2001b; Niehrs et al. 2001). To study the possible role of Dkk1 in induction of the We show that in zebrafish embryos with little or no anterior neural border signaling center (Houart et al. Dkk1 activity, very little change in the molecular iden- 1998), we used specific antisense morpholino oligo- tity of the internalized axial tissue is observed, suggest- nucleotides (Nasevicius and Ekker 2000) that nearly ing that Dkk1 protein may also act on planar signaling completely inhibit Dkk1 translation in injected ze- events prior to internalization. More importantly, our brafish embryos (Fig. 1G–J). The morphant embryos data show that gain and loss of Dkk1 function both lead show a severe reduction of forebrain territories (Fig. 1A–

466 GENES & DEVELOPMENT Dkk1 binds Kny and regulates gastrulation movements

F), as predicted by previous studies of Dkk1 loss of func- been made looking at the expression of a set of axial tion in Xenopus and mouse (Glinka et al. 1998; Muk- markers and suggested acceleration in axial mesendo- hopadhyay et al. 2001). It has been strongly suggested derm progression. In order to address this possibility, we that vertical signals emanating from the anterior axial followed the movement of the axial mesendoderm in mesendoderm/prechordal plate are required for the es- live transgenic embryos containing the insertion of a tablishment of the anterior neural territory (Kazanskaya gene coding for the green fluorescent protein (GFP) under et al. 2000; Kiecker and Niehrs 2001b) and that Dkk1 is the control of the gsc promoter (n = 48/55) (Fig. 2Q–Y). In one of the key players in this signaling function. How- wild-type embryos, the rostral tip of the internalized ever, in the absence of dkk1 in both fish (L. Caneparo, R. mesendoderm is progressively moving toward the ani- Ahrendt, J. Peres, M. Kapsimali, and C. Houart, in prep.) mal pole during gastrulation and reaches the pole by and mouse (Mukhopadhyay et al. 2001), otx2 is properly 90% epiboly. In dkk1MO embryos, the rostral axial induced in the anterior neural territory, suggesting that mesendoderm reaches the animal pole by 75% epiboly some initial patterning events occur in the absence of Dkk1. (Fig. 2U,V), 45–60 min earlier than in wild-type em- In Dkk1 antibody injected Xenopus embryos hhex and bryos raised at 28°C. We concurrently observed that Blimp1, two anterior endoderm markers and gsc and shh dkk1MO embryos show a slightly narrower anterior in prechordal plate are down-regulated (Kazanskaya et al. neural plate by the end of gastrulation, although no 2000). In the mouse dkk1/noggin double mutants, Hesx1 defect in BMP signaling has been detected rostrally expression is down-regulated in the presumptive pre- (n = 31/31, Fig. 2N,O), and no anterior neural plate size chordal plate (del Barco et al. 2003). These results indi- difference is visible earlier in gastrulation (n = 35/35) cate that the most rostral population of axial mesendo- (Fig. 2K,L). Finally, the rostral limit of expression of derm is impaired in the absence of dkk1. In order to snail1a (data not shown) and sprouty4 (Fig. 2I,J) in non- assess whether the zebrafish Dkk1 morphant (dkk1MO) axial mesendoderm is also shifted anteriorly in mor- embryos also present some axial mesendodermal defects, phant embryos, due to change in cell identity and/or cell we analyzed the expression pattern of a set of axial mark- motility. Both patterning and motility phenotype ob- ers (Fig. 2A–H; data not shown). No change in expression served in the morphants can be corrected by coinjection has been found (between 41 and 70 injected embryos of both dkk1MO and dkk1 full-length transcripts lacking tested for seven axial markers at 60%–65%, 80%–90% the MO target sequence (Supplementary Fig. S1). These epiboly and bud stages) except for a significant reduction observations suggest that although epiboly movements of the anterior prechordal plate marker gsc (n = 17/58) are not visibly perturbed in dkk1MO morphants, some (Fig. 2G,H), confirming the defect observed in Xenopus gastrulation movements are accelerated in these em- and mouse. bryos. In the course of this analysis, we noticed a striking In order to test this further, we assessed directionality difference in position of the rostral-most limit of the and speed of mesendodermal movements in Dkk1 mor- axial mesendoderm (Fig. 2A–F). This observation has phant embryos. We first assessed movements of lateral

Figure 1. dkk1MO efficiently represses dkk1 translation, which induces severe reduction of the forebrain. (A–F) Loss of Dkk1 function represses formation of eye and telencephalon. Lateral views, anterior to the left, of heads of wild-type (A,C,E) and dkk1MO- injected (B,D,F) embryos. (G–JЈ) Rescue of dkk1GFP overexpression by Dkk1MO. (G–J) Lateral view of live 80% epiboly (under UV illumination, GЈ–JЈ) and prim5 (G,J) embryos after one-cell-stage injection of 50 ng/µL dkk1GFP alone (G,GЈ) or together with 0.4 mg/mL (H,HЈ), 1 mg/mL (I,IЈ), and 1.5 mg/mL (J,JЈ) Dkk1MO. With the exception of C and D, the embryos shown in this figure are alive. Bar, 100 µm.

GENES & DEVELOPMENT 467 Caneparo et al.

Figure 2. Dkk1 regulates gastrulation movements. Lateral (A,B,E,F,I–Y), dorsal (C,D), and animal pole (G,H) views of control (A,C,E,G,I,K,N,Q–S), Dkk1MO-injected (B,D,F,H,J,L,O,T–V), and dkk1RNA-injected (M,P,W–Y) gastrula embryos. A–J and Q–Y show progression of the mesendoderm by markers in fixed embryos (A–J) and by observation of the GFP-expressing axial mesendoderm in live embryos (Q–Y). The black arrowhead indicates the position of the rostral-most mesendoderm. The red frame highlights the stages at which the difference in mesendoderm progression is obvious in the live Dkk1 morphants. (K–P) Lateral views of, in blue, neural plate (K–M) and epidermal (N–P) territories in wild-type (K,N), dkk1MO (L,O), and dkk1RNA (M,P) embryos. Red arrowhead shows the caudally shifted anterior edge of the neural territory. mesoderm by transplanting in the germ ring, on either cells). Expectedly, wild-type and dkk1MO clones move side of the shield (at 35° from it), rhodamin-labeled wild- in very similar ways if transplanted in wild-type hosts type and fluorescein-tagged dkk1MO cells in a dkk1MO (n = 25/25) (Fig. 3D), as secreted Dkk1 from host cells shield stage host embryo. We followed their progression almost certainly compensates for the absence of protein by confocal time-lapse analysis (see Materials and Meth- in the dkk1MO grafted cells. Similarly, if wild-type and ods; n = 56, Fig. 3A–C). In most cases, dkk1MO cells dkk1MO clones are grafted adjacent inside the shield of move faster toward the animal pole than the wild-type dkk1MO host embryos (Fig. 3E,F), most cells are moving clones (n = 48/56). A difference in cell behavior is visible together (defective cells receiving Dkk1 from secreted from the onset of gastrulation as dkk1MO cells move wild-type neighbors), with some dkk1MO cells running under the epiblast more frequently than wild-type cells in front and wild-type cells trailing behind (n = 9/16 and (Fig. 3B, graph in I). This acceleration does not seem to be 7/16 without a significant difference in position between dependent on the clone size (seen for wild-type and the two cell types). dkk1MO clones of either five, 15, or 25 transplanted To quantify further the changes observed in the mor-

468 GENES & DEVELOPMENT Dkk1 binds Kny and regulates gastrulation movements

Figure 3. More frequent mesendoderm internalization and faster rostral progression in the Dkk1 morphants and slowed-down internalization and CE defects in Dkk1 gain of function. (A–H) Dorsal (B–F), animal pole (A), and lateral (G,H) views of dkk1MO (A–C,E,F) or wild-type (D,G,H) embryos in which wild-type (red in live embryos and brown in fixed specimen) and dkk1MO (green in live embryos and dark blue in fixed specimen) cells have been transplanted inside the germ ring at onset of gastrulation. A–C show the same embryo at progressively later stages of gastrulation. Note that H shows transplanted dkk1RNA-expressing cells in dark blue. Dashed line indicates the midline. (I) Timing of internalization of 15 wild-type (blue) and dkk1MO (red) cells transplanted in the margin of early gastrula hosts. Measure of transplants in the lateral margin are represented by full dots and transplants in the axial margin (shield) are represented by empty dots. X represents time and Y represents the number of cells. (J) Measure of the AP length of the transplanted cell population at bud stage in microns (Y axis). Wild-type clones are shown in blue, dkk1MO are shown in red, and dkk1RNA are shown in green. (K) Measure of the distance of the leading transplanted cell from the embryo margin at 95% epiboly in microns (Y axis). Wild-type clones are shown in blue, dkk1MO are shown in red, and dkk1RNA are shown in green. (L) Expression of dkk1 in the marginal mesendoderm. Animal pole view of a shield stage wild-type embryo. (S) Shield (Spemann Organizer). Note the absence of transcript in the ventral margin and the graded level of expression from dorsal (S, shield) to lateral. (M,N) Cell shape and cohesion in the lateral (M) and dorsal (N) mesendoderm (arrows) as it internalizes in a live shield stage embryo. Inset in M shows a close-up of the internalizing lateral mesendoderm cell, emphasizing the mesenchymal shape of the lateral mesendoderm cells. phant gastrulae, we followed the progression of 15 cells nalized cells/minute (Fig. 3I), the AP length of the clone transplanted into the germ ring of a morphant host and at 90% epiboly (Fig. 3J), and the distance of the rostral- took three sets of measurements: the number of inter- most cell from the posterior margin at 95%–100%

GENES & DEVELOPMENT 469 Caneparo et al. epiboly (Fig. 3K). In our experimental conditions, wild- these data strongly suggest that the influence of Dkk1 on type transplanted cells internalize at an average of 0.55 gastrulation movements is at least in part Wnt/␤catenin cell per minute in lateral and axial mesoderm (Fig. 3I, independent. However, this pathway seems to regulate blue dots/circles), while morphant cells move inward at some aspects of cell cohesion, as we observed that cells an average of 0.85 cell per minute in lateral mesoderm from clones lacking both mbl and tcf3 function were losing and 1.8 cell per minute in the axial mesendoderm (Fig. touch with each other faster than wild type after transplan- 3I, red dots/circles). The AP length of morphant clones at tation (clone shape at 75%–80% epiboly) (Fig. 4G,H). 90% epiboly is slightly bigger than wild type, strongly To quantify further the changes in cell movements suggesting that convergence/extension (CE) movements imposed by gain of Wnt/␤catenin activity, we used our are not impaired in the absence of Dkk1 function (Fig. transplantation approach again and transplanted 15–20 3J). However, the position of the leading cell inside the wnt8 mRNA-injected donor cells into the margin (35° clones is significantly shifted anteriorly in morphants from the shield) of early shield stage wild-type host em- compared with wild type (Fig. 3K), indicating that rostral bryos and compared with wild-type transplants in the progression is increased when Dkk1 is lacking. Indi- same conditions (either transplanted on the other side of rectly, this finding (similar clone shape and length but the same hosts or in different host embryos). A series of shifted rostrally) suggests that more cells are internal- wnt8 doses has been tested (Fig. 4K–N). The wnt8-ex- ized in the morphants. Lack of Dkk1 function therefore pressing cells internalized as well as the wild-type cells, favors mesendodermal internalization and rostral pro- except for the cells expressing the highest dose, for gression, indicating that Dkk1 protein normally nega- which delay in progression and CE defect begins to be tively regulates this type of movement. observed (Fig. 4N, graph in O,P), reminiscent of the de- We next assessed the effect the overexpression of dkk1 fects observed in Xenopus (Ku¨ hl et al. 2001). In most has on cell movement, using the same approaches. A lag cases, the clones are more dispersed (Fig. 4L–N). Finally, in progression of the axial mesendoderm is observed (Fig. a very small number of wnt8-expressing cells (from set of 2W–Y), and a caudal displacement of the anterior limit of 3 pg of transcripts/donor) are very rarely detected slightly the neural plate (Fig. 2M,P, arrowheads) typical of em- further away from the margin than in wild type (n =1/ bryos defective in extension movements. Clonal analysis 74) (data not shown). These results indicate that Wnt/ of cell motility shows that dkk1 overexpressing clones ␤catenin activity is not readily able to induce accelera- present an increase in cell dispersion and a slowing down tion of mesendoderm internalization and progression, of internalization, rostral progression, and convergence but confirm the less cohesive nature of cells exposed to (n = 39/39) (Fig. 3,H; green graph in J,K). All together, high level of Wnt/␤catenin signaling. This observation these data indicate that Dkk1 modulates gastrulation nicely correlates with the distinct cell adhesion proper- movements. ties found in the lateral (noncohesive) (Fig. 3M) and dorsal (cohesive) (Fig. 3N) mesendoderm, which are exposed to low and high levels of Dkk1, respectively (Fig. 3L). Gain of Wnt/␤catenin function does not lead Dkk1 is therefore likely to regulate cohesion through to acceleration of mesendoderm internalization modulation of the Wnt/␤catenin pathway. As Dkk1 regulates negatively the Wnt/␤catenin pathway, we tested whether the acceleration of gastrulation The glypican4/6 Knypek is able to bind to Dkk1 movements in the morphant embryos is the conse- and potentiates its activity in vivo quence of up-regulation of this pathway. We first ad- dressed whether mesendodermal progression was af- In vertebrates, a subset of gastrulation movements are fected in the mbl mutant embryos, lacking axin1 func- regulated by the Wnt/PCP pathway (for reviews, see tion (Heisenberg et al. 2001) and therefore unable to Tada et al. 2002; Wallingford 2005). Both Wnt5 and degrade ␤catenin. We find no visible difference, either by Wnt11 are signaling molecules required for proper con- measuring the extent of the lefty1 expression in axial vergence–extension (Heisenberg et al. 2000; Kilian et al. mesendoderm or when monitoring cell movements of 2003) through activation of pathways including the JNK labeled mesendoderm (data not shown). To ascertain and Ca++. One possible way Dkk1 may regulate cell that the absence of modulation of movement was not movements is through modulation of the PCP pathway. caused by a relatively mild increase in ␤catenin activity Such a modulation may be indirect, through repression in mbl, we tested whether any change was detectable in of the Wnt/␤catenin (Kühl et al. 2001). Alternatively, it embryos lacking both mbl and tcf3 function (injection of may be direct, by interaction with PCP pathway compo- tcf3 and tcf3b morpholino [Dorsky et al. 2003] in mbl) nents. Since Dkk1 binds heparan (Fedi et al. 1999), we (Fig. 4). In these embryos, we have been unable to ob- tested if a related molecule, Knypek, a member of the serve any increase in cell movement. Conversely, we HSPG family, required for Wnt/PCP activity (Topczewski observed a slight reduction of axial mesendoderm pro- et al. 2001), is able to interact with Dkk1. gression in 29% of the double mbl−/−; tcf3MO embryos We tested whether Dkk1 and Knypek are able to bind (n = 8/28) (Fig. 4D). Finally, ⌬NWnt8 overexpression, ob- each other, using immunoprecipitation techniques. We tained by RNA injection at the one-cell stage (25 pg/ tested membrane colocalization and binding between embryo, n = 37) (Fig. 4J) is unable to rescue the accelera- these two molecules in a cell culture system, by trans- tion observed in the Dkk1 morphants. All together, fection of an expression vector containing a Flag-tagged

470 GENES & DEVELOPMENT Dkk1 binds Kny and regulates gastrulation movements

Figure 4. Gain of Wnt/␤catenin activity does not phenocopy the gastrulation movement defect seen in the Dkk1 morphants. (A–F) Lateral views of live prim5 (A,C,E) or dorsal views of lefty1 exression in fixed 90%–95% epiboly (B,D,F), wild-type (A,B), and mbl mutant injected with tcf3 + 3bMO (C,D), and dkk1MO (E,F) embryos. (G,H) Clone shape of transplanted wild type (G)ormbl mutant injected with tcf3 + 3bMO (H) in the host embryo at 75%–80% epiboly. (I,J) Lateral views of 75%–80% epiboly gscGFP transgenic live embryo (I)ordkk1MO coinjected with 20 pg of dominant-negative wnt8 RNA (J), showing the GFP-expressing axial mesendoderm in green under UV illumination. (K–N) Lateral views of 90% epiboly embryos showing the distribution of the wnt8-expressing cells (blue) transplanted at shield stage. The dose injected per embryo is indicated in the bottom right corner. (O) Timing of internalization of 25 wild-type (blue) and wnt8-expressing (10 pg, pink; 25 pg red; 40 pg purple) cells transplanted in the margin of early gastrula hosts. X represents time and Y represents the number of cells. (P) Measure of the distance of the leading transplanted cell from the embryo margin at 100% epiboly in microns (Y axis). Wild-type clones are shown in blue and wnt8-expressing clones are shown in pink (10 pg), red (25 pg), and purple (40 pg). knypek c-DNA sequence (kny-Flg) (Topczewski et al. tracts from cells cotransfected with both dkk1GFP and 2001) and/or another containing a dkk1GFP fusion mol- kny-Flg DNA (or from a mixed culture of dkk1GFP-ex- ecule, coding for an active fluorescent Dkk1 protein (see pressing and kny-Flg-expressing cells), Kny-Flg proteins Fig. 1G,GЈ). We assessed localization of the two proteins are detected in the anti-GFP precipitates (Fig. 5A), un- in these transfected cells. Protein extracts from trans- equivocally showing that Dkk1 is able to bind to Knypek fected cells were precipitated with either an anti-Flag or in cell culture conditions. By the same technique, we an anti-GFP antibody. The precipitates were run on also show this binding in gastrulae extracts (Fig. 7J, be- acrylamide gel and Western blot immunostained either low), opening the possibility that Dkk1 may directly with GFP or Flag antibody. The blots show that in ex- modulate Knypek-dependent signaling events in vivo.

GENES & DEVELOPMENT 471 Caneparo et al.

Figure 5. Knypek binds to Dkk1, is required for its propagation, and potentiates its effects. (A) Western blot of an immunoprecipitation assay. Protein extracts from cells transfected with different combinations of DNA (+) are either run untreated (last three lanes) or first immunoprecipitated with an anti-GFP antibody (first five lanes), then run on a gel. The gel is then transferred on filter and stained with an anti-Flag antibody (detecting the Flag-tagged Knypek protein) or an anti- GFP antibody (detecting the GFP [1] or dkkGFP fusion [2] proteins). (Lane 4) Red arrowshows the presence, after GFP immunoprecipitation, of Kny-Flag proteins in extracts from cells expressing both Dkk1GFP and KnyFlag. (B–EЈ) Lateral views, rostral to the left, of prim5 live wild-type (B), kny RNA-injected (C), dkk1RNA-injected (D), and kny + dkk1RNA-injected (E,EЈ) embryos. In E, the quantity of RNA injected is identical to the that used for the single injections in C and D. The phenotype shown in E and EЈ are found in 31% and 64% of the injected embryos, respectively. (F–I) Ani- mal pole views (insets) and lateral views of live or fixed (insets) wild type (F,G) and kny homozygous (H,I) injected with control (F,H)ordkk1 morpholino (G,I). Insets show expression patterns of hgg1 (rostral mesendoderm, hg) and brachyury (in the notochord, n). Arrowhead shows rostral end of the body axis. (J–M) Visualization of the dkk1GFP molecules (in green) secreted by the transplanted dkk1GFP-expressing wild-type cells (yellow) in wild-type (J,K) and kny−/− (L,M) hosts. K and M are high- magnification views of the distribution of secreted proteins away from the graft (or its absence at a distance from the clone in M). Note that the cell nuclei are visible as dark discs in unlabeled cells. The wild- type dkk1GFP-expressing cells are very in- tensely fluorescent when placed in the kny−/− mutant, masking the nucleus in these cells. (N–P) Lateral views, rostral to the left, of wild-type (N)orkny−/− (O,P) embryos, untreated (inset in N,O) or in which cells from a dkk1 RNA-injected donor embryo (in brown) have been transplanted in the shield at 50% epiboly (N,P). Note the shorter tail and big eye and brain in N.(P) No change in eye size is ever observed in transplanted kny−/− embryos; the brain size is generally comparable to untransplanted mutants. (Q–Z) Lateral views of live embryos (Q–V) or dorsal views of fixed gastrulae (W–Z). All embryos come from a kny+/− cross injected with either 1.8 pg of kny transcripts (Q–S,W,X) or 1.8 pg of kny and2pgofdkk1 transcripts (T–V,Y,Z). The number of embryos showing each illustrated phenotype is given in the bottom right corner of each picture. In W–Z, hgg1 expression shows rostral mesendoderm (hg) and brachyury, the notochord (n).

The defects in cell movements observed in Dkk1 gain (n = 31/31 fixed kny−/− embryos and n = 15/15 kny−/− live or loss of function may therefore be due to modulation of embryos) (Fig. 5F–I). This suggests that either Dkk1 in- a Knypek-dependent signaling activity. We tested this fluence on motility may depend on its direct binding to hypothesis by assessing whether embryos carrying a null Knypek or that Knypek is functionally downstream from mutation in the kny gene are losing the ability to re- Dkk1 in the same developmental process. spond to the absence of Dkk1 function. If Dkk1 acts To attempt to discriminate between these two possi- through a kny independent way, Dkk1 lack of function bilities, we tested whether Kny could increase Dkk1 ac- may rescue part of the movement defects observed in the tivity when misexpressed in wild-type embryos. We in- kny−/− embryos. We observed that the embryos lacking jected kny or dkk1 transcripts alone or together in wild- both Dkk1 and Knypek show delay in movements iden- type embryos, choosing a quantity of transcripts that tical to the ones observed in single kny−/− embryos gives no (for kny) (Fig. 5C) or weak (for dkk1) (Fig. 5D)

472 GENES & DEVELOPMENT Dkk1 binds Kny and regulates gastrulation movements phenotype when injected alone. Embryos injected with Kny control cell motility together through regulation of the same or half the dose of both transcripts (Supplemen- the same pathway(s). tary Table 1), develop an enormous head and the short All together, the results in this section show that (1) body axis typical of a severe dkk1 overexpression (Fig. Dkk1 is able to bind to Kny and requires Kny for its 5E,EЈ; Supplementary Table 1). These results indicate propagation, (2) propagation is necessary for Dkk1 func- that Dkk1 and Kny can act together in the same devel- tion, and (3) Dkk1 and Kny can act together to regulate opmental process. Finally, we analyzed the localization gastrulation movements. of Dkk1 protein in wild-type and kny−/− embryos, using the dkk1GFP fusion molecule described above. We Dkk1 promotes the activity of the Wnt/PCP pathway transplanted 20–30 cells from dkk1GFP-injected wild- type donor embryos into shield stage wild-type embryos As Dkk1 is able to bind to Knypek and enhance its func- or progenies of kny−/+ fish. We then followed the expres- tion, it may directly regulate the Wnt/PCP pathway. We sion of the fusion proteins under the confocal micro- first tested whether Dkk1 negatively modulates Wnt/ scope. kny−/− hosts are identified based on their pheno- PCP activity. To address this possibility, we examined if type at 24 h post-fertilization (hpf). We first observed the acceleration of rostral progression observed in the that propagation of the Dkk1GFP proteins is extremely Dkk1 morphants may rescue the gastrulation phenotype fast (an average of 21 cell diameters after 45 min) in observed in slb embryos, homozygous for a null muta- wild-type embryos (data not shown). Moreover, very in- tion in the wnt11 gene (Heisenberg et al. 2000). CE de- tense small and highly dynamic fluorescent dots are vis- fects were not rescued in slb−/− embryos (obtained from ible inside the host cells, strongly suggesting an endo- slb−/− homozygous parents) injected with dkk1MO cytic pathway (Fig. 5K; also suggested by Mao et al. (n = 41) (Fig. 6A–CЈ; data not shown). However, we ob- 2002). More importantly, we found extensive propaga- served some change in the AP extent of the prechordal tion of the secreted molecule in 23/24 of the wild-type territory marked by gsc (51% of double lack of function embryos while no propagation has been detected in 8/9 embryos) (Fig. 6C,CЈ). This deformation of the pre- kny−/− analyzed (Fig. 5J–M). In the absence of propaga- chordal territory points to a slight increase of the CE tion, the fluorescence is detected weakly around the defect in slb embryos and suggests that Dkk1 may acti- cells and highly inside the cytoplasm. Kny is therefore vate the Wnt/PCP pathway. We therefore set out to ad- required for propagation of Dkk1. dress this, first, at the biochemical level in Xenopus,in To assess whether this propagation is necessary for which Dkk1, has been thoroughly studied and biochemi- Dkk1 function, we asked if propagation of the Dkk1 cal assays are routinely done. We first found that, as in molecules was required for its well-described anterioriz- fish, Dkk1 overexpression is inducing CE defects in ing function. We therefore transplanted donor dkk1-ex- Xenopus (Fig. 6D–G). Animal caps, from embryos in- pressing cells (from dkk1 mRNA-injected donors em- jected with activin mRNA, are able to form mesoderm bryos) into the shield (Spemann Organizer) of early gas- and characteristically elongate due to CE movements. trula wild-type or kny hosts. We observed that grafted After injection of both activin and wnt8, animal cap dkk1-expressing cells are able to induce a “big head/ elongation is slightly enhanced compared with activin short trunk” phenotype in wild type (n = 19/21) (Fig. 5N). alone. Contrasting with this observation, caps injected Introduction of Dkk1-overexpressing cells in the wild- with activin and dkk1 are unable to elongate, and this is type shield is therefore able to affect the general AP pat- accompanied by induction of gsc at the expense of the terning such that transplanted embryos always show re- mesodermal marker Xbra (Fig. 6M). This loss of elonga- duction of the trunk and the giant eyes typical of the tion is reminiscent of the phenotype induced by gain or Dkk1-injected individuals. We were unable to observe an loss of Wnt/PCP activity. Indeed, the cells of explants of increase of the eye and overall brain in kny embryos dorsal blastopore lip (DMZ) in culture normally elongate (n = 0/23) (Fig. 5P), but we sometimes observed a slight and orient along the medio-lateral axis (Fig. 6H,L). Cells increase in the size of the telencephalon (n = 6/23), prob- in explants from Wnt3a-injected embryos show similar ably due to the effect of Dkk1 from donor cells when in or greater orientation relative to the medio-lateral axis close vicinity of the forebrain. (Fig. 6J,L), while dkk1 reduces elongation and loss of ori- Finally, we directly assess whether Dkk1 is able to entation relative to the medio-lateral axis (Fig. 6K,L). cooperate with Kny in regulating gastrulation move- This cell shape change phenocopies the loss of polariza- ments. To this aim, we tested whether Dkk1 is able to tion observed in explants after overexpression of Wnt11 help Kny in rescuing the kny mutant phenotype. We and its receptor Fz7 (Fig. 6I,L). Our data in frog and fish injected, at the one-cell stage, the progeny of kny+/− par- therefore strongly suggest that Dkk1 is able to promote ents with a suboptimal quantity of kny full-length RNA Wnt/PCP activity. To test this possibility at a molecular alone or accompanied by a low dose (alone not inducing level, we quantified Wnt/PCP activation by measuring a phenotype) of dkk1 transcript. To our surprise, the the level of phosphorylation of one of its major transduc- coinjected embryos showed a robust rescue (Fig. 5Q–Z), ers, JNK (Boutros et al. 1998). We compared the level of strongly suggesting that Kny and Dkk1 act similarly on phosphorylated JNK in embryos injected with a HA epi- the same gastrulation movements. As Dkk1 alone is un- tope-tagged JNK mRNA alone (control) or together with able to rescue kny mutant embryos (n = 32) (data not either wnt11 or dkk1 mRNA or both. After specific pre- shown), the “double” rescue indicates that Dkk1 and cipitation of HA-JNK, its level of activation was mea-

GENES & DEVELOPMENT 473 Caneparo et al.

Figure 6. Dkk1 up-regulates the Wnt/PCP pathway. (A–CЈ) Lateral (A–C) and dorsal (AЈ–BЈ) views of bud stage wild type (A,AЈ), slb (B,BЈ), and slb injected with dkk1MO (C,CЈ) embryos, expressing hgg (in the hatching gland, hg), ntl (in the notochord, n), and dlx3b (in the neural plate border, nb). (D–G) Dkk1 blocks activin-induced animal cap elongation. Xenopus embryos were uninjected (D)or injected animally at the four- to eight-cell stage with activin (E), activin and wnt8 (F), or activin and dkk1. Animal caps were dissected from stage 9 embryos and cultured until sibling embryos had reached stage 17. (H–L) Dkk1 disrupts cell polarity. Xenopus embryos were injected at the two- to four-cell stage equatorially with mRFP mRNA, and either preprolactin (ctl) or the indicated mRNAs (top right corner). Explants of the dorsal upper blastopore lip were cut at stage 10.5, and cells were imaged by confocal microscopy. The yellow line indicates orientation of the medio-lateral axis. (L, left panel) Cell elongation was determined by the mean length-to-width ratio (LWR). (Right panel) Cell orientation was calculated as the percentage of cells with their long axis tilted >20° relative to the medio-lateral axis. (M)RT–PCR analysis of animal caps injected as in D–G for the indicated genes. (N) Dkk1 activates JNK. HA epitope-tagged JNK mRNA was injected either alone or in combination with mRNA of the indicated genes into two- to four-cell-stage embryos. HA-JNK was immunoprecipitated from extracts of stage 11 embryos and JNK phosphorylation was monitored using a phospho-specific antibody on Western blot. (ni) Noninjected control. (O–R) Dorsal views of bud stage wild-type (O,Q) and mbl (P,R) embryos uninjected (O,P) or injected with 30 pg of dkk1 RNA at the one-cell stage (Q,R), expressing hgg (in the hatching gland, hg), ntl (in the notochord, n), and dlx3b (in the neural plate border, nb). sured by detection of its phosphorylated form, using a Dkk1 mRNA in mbl is inducing a CE defect comparable phospho-specific antibody. Wnt11 or Dkk1 expression to the one seen in wild type (Fig. 6O–R). This last result alone is able to enhance JNK phosphorylation, and ex- therefore shows that Dkk1 can act on CE movements in pression of both of them increases dramatically this embryos with a constitutively active Wnt/␤catenin phosphorylation, while Wnt8 blocks phosporylation (Fig. pathway. Furthermore, Dkk1 activation of the Wnt/PCP 6N). These results demonstrate that Dkk1 directly or pathway is not due to indirect up-regulation of wnt11 indirectly activates the Wnt/PCP pathway. expression, as no increase of wnt11 transcripts is de- The interaction found between Kny and Dkk1 sug- tected after dkk1 RNA injection (Supplementary Fig. gests the possibility of a direct regulation of the Wnt/ S2A–C). PCP pathway. However, it may stimulate Wnt/PCP in- As Dkk1 is able to up-regulate the PCP pathway, we directly since canonical Wnt signaling is able to block tested whether it could rescue the CE defect in slb/ the PCP pathway in vitro (Fig. 6N). A direct interaction wnt11 mutant embryos. Injection of various doses of with the Wnt/PCP is, however, very likely. Indeed, not dkk1 RNA in slb homozygous embryos did not lead to a only is moderate DNwnt8 overexpression unable to res- general rescue (Supplementary Fig. S3). However, high cue the gastrulation movement phenotype in the Dkk1 dose of transcripts is able to rescue the shape of the an- morphants (Fig. 4J), but we also found that injection of terior-most axial mesendoderm although not other as-

474 GENES & DEVELOPMENT Dkk1 binds Kny and regulates gastrulation movements pects of the CE defect in slb (Supplementary Fig. S3D– defects. These are more pronounced in the double-in- EЉ). This observation shows that Dkk1 is able to partially jected embryos, highlighting again the cooperation of correct a subset of the cell movement defect induced by Dkk1 and Kny in modulation of gastrulation movements loss of Wnt11, namely the rostral progression of the an- (Fig. 7 FЉ,HЉ). More importantly, mild expression of LRP6 terior mesendoderm. rescues this CE defect (Fig. 7IЉ). This rescue is not due to direct competition between LRP and Kny for binding to Dkk1. Indeed, our coimmunoprecipitation assay on pro- Dkk1 may act as a switch between the ␤catenin and tein extracts of these embryos (Fig. 7J) shows that LRP6 PCP pathways activated by the Wnt/Fz complex and Kny do not compete for binding to Dkk1 (Fig. 7J, cf. One of our first observations is still unresolved by the third and fourth lanes). This finding therefore strongly results so far. Indeed, the ability of Dkk1 to bind to Kny suggests that Kny is endocytosed with Dkk1 in embryos and activate the Wnt/PCP pathway is not explaining the overexpressing LRP6 and, more importantly, that Dkk1 acceleration of gastrulation movements observed in the is able to act concomitantly on both Kny and LRP pro- Dkk1 morphant embryos. As our data indicate that teins, thereby modulating both patterning and cell mo- Dkk1 simultaneously represses the Wnt/␤catenin and tility. activates the Wnt/PCP pathway, we tested whether acceleration of gastrulation movements in the Dkk1 mor- Discussion phants may be generated by concomitant up-regulation Dkk1 is a Wnt/␤catenin antagonist acting through bind- of the Wnt/␤catenin pathway and down-regulation of the ing to Kremen and the Frizzled coreceptors LRP5/6 (Mao Wnt/PCP activity. We injected one- to two-cell-stage do- et al. 2001, 2002). It plays a crucial role in early neural nor embryos with either wnt8 RNA, a low dose of dsh- patterning, as its activity is required for maintenance of DEP+ (coding for a version of Dishevelled unable to ac- forebrain identity in the anterior neural plate in both frog tivate the PCP pathway) (Axelrod et al. 1998; Heisenberg and mouse (Mukhopadhyay et al. 2001). Our study un- et al. 2000), or both transcripts together. We then trans- covered a novel involvement for Dkk1 in modulation of planted 15–20 cells in the margin of shield stage wild- gastrulation movements through binding to the Glypi- type host embryos and watched internalization of the can4-like Knypek and direct regulation of both Wnt/ donor cells (Fig. 7A–D). The cells coming from the ␤catenin and PCP signaling pathways. double-injected donors do indeed progress faster than the wild-type controls in a majority of the cases (25 of 43) Dkk1 is modulating gastrulation movements (Fig. 7C,D). Single wnt8-expressing cells display the dis- perse distribution described above (n = 22) (Fig. 7B), When we first observed the changes in cell motility of while the DSH-DEP+ cells show difficulty in progression the internalizing mesendoderm in the Dkk1 morphants, and in CE (n = 25) (Fig. 7A; data not shown). Cells in Dkk1 had not yet been associated with regulation of cell which the Wnt/␤catenin activity is up-regulated and the movement. Since then, a very recent study has shown Wnt/PCP reduced are therefore able to mimic the cellu- that the rostral movement of the AVE just preceding lar behavior observed in embryos deprived of Dkk1 ac- gastrulation in mouse requires Dkk1 (Kimura-Yoshida et tivity. al. 2005). Moreover, this study also shows that Wnt/ In light of all our results, we therefore propose that ␤catenin activity represses such movement and proposes Dkk1 acts at the level of the Wnt receptors, concomi- a role of attractant for Dkk1 and repellent for the Wnt tantly repressing Wnt/␤catenin and increasing Wnt/PCP ligands. However, when looking at gastrulation move- reception, via interaction with both LRP5/6 and Kny ments, the three different approaches taken in our study (Fig. 7E). Such a model suggests that LRP5/6 and Kny to up-regulate the Wnt/␤catenin pathway in early gas- may either compete for binding to Dkk1 or differentially trula embryos all fail to mimic the acceleration in mo- regulate localization of Dkk1. We tested these possibili- tility observed in the Dkk1 morphants, pointing here to ties by monitoring Dkk1GFP subcellular localization a novel Wnt/␤catenin-independent function for Dkk1. and binding to Kny-Flg in embryos in the presence or Moreover, none of our transplant experiments show cell absence of exogenous LRP6. We found that, in gastrula behavior compatible with an attractant/repellent model. embryos expressing both Dkk1GFP and KnyFlg, the dis- Interestingly, the cell behavior described in the mouse tribution of Dkk1GFP is shifted from being found both AVE in the presence or absence of Dkk1 activity is also in cytoplasmic and extracellular location (n = 16; Fig. compatible with a fluctuation of the level of both Wnt/ 7FЈ) to almost exclusively extracellular (n = 15) (Fig. ␤catenin and Wnt/PCP activity, a possibility not tested 7HЈ). This distribution is completely reversed in em- by the study, as the authors did not question the nature bryos that also mildly overexpress LRP6 (1.5 pg of RNA of the pathway modulated by Dkk1. Future analysis of injected at the one-cell stage), where the dkkGFP pro- the molecular pathways required for AVE movements in teins are mostly found in the cytoplasm (n = 19) (Fig. 7IЈ). mice is needed to elucidate whether Dkk1 may act on This result shows that Kny alone promotes extracellular the same set of signaling pathways while regulating the localization of Dkk1, while LRP6 drives its endocytosis. movements of the AVE and those of the mesendoderm. We also monitored gastrulation movements in the same A puzzling observation is that acceleration of gastru- four experimental conditions. As expected, injection of lation movements has only been reported once before dkk1 alone or in combination with kny both induce CE this study, in zebrafish embryos lacking Lefty function

GENES & DEVELOPMENT 475 Caneparo et al.

Figure 7. Dkk1 may act as a switch between the ␤catenin and PCP pathways activated by the Wnt/Fz complex. (A–C) Lateral views of 90% epiboly embryos showing the distribution of the dsh-DEP+ (A), wnt8 (B), and dsh-DEP + wnt8-expressing cells (blue) transplanted in wild-type hosts at shield stage. The dose injected per donor embryo is 5 pg for dsh-DEP+ and 12 pg for wnt8.(D) Seventy-five percent epiboly wild-type host embryo showing the rostral progression of wild-type (brown) and wnt8 + dshDEP+ (5 pg + 12 pg, blue) transplanted cells. The dashed line indicates the midline. (E) Model of the proposed mechanism by which Dkk1 may both down- regulate the Wnt/␤catenin and up-regulate the Wnt/PCP pathways. (F–IЈ) Live embryos injected with 8 pg of dkk1GFP (F,FЈ), 1.8 pg of kny-flg (G,GЈ),4pgofdkkGFP + 1.8 pg kny-flg (H,HЈ),and4pgofdkkGFP + 1.8pg of kny-flg + 1.5 pg of LRP6 (I,IЈ). Confocal images have been taken at 60% epiboly (FЈ–IЈ) of the GFP localization and the embryos have been left to develop until 48 hpf (F–I) to check activity of the transcripts injected. (FЉ–IЉ) Dorsal view, anterior to the top, of embryos injected with dkk1GFP (FЉ), kny-flg (GЉ), dkkGFP + kny-flg (HЉ), and dkkGFP + kny-flg + LRP6 (IЉ) at the same doses as in F–IЈ, showing expression of hgg (in the hatching gland, hg), ntl (in the notochord, n), and dlx3b (in the neural plate border). (J) Western blot of the immunoprecipitation assay done on extracts of 30 late-gastrula injected embryos for each condition tested. Protein extracts from the four combinations of injection (+) are either run untreated (last four lanes) or first immunoprecipitated with an anti-Flg antibody (first four lanes), then run on a gel. The gel is then transferred on filter and incubated with an anti-GFP antibody (detecting the GFP-tagged Dkk1 proteins) or an anti-Flg antibody (detecting the Flg-tagged Kny proteins. Lanes 3 and 4 show the presence, after Flg immuno-precipitation, of Dkk1-GFP proteins in extracts from embryos expressing both Dkk1GFP and KnyFlag, regardless of LRP6 overexpression. Note that a same set of injected embryos was split into one half for phenotype analysis (F–I) and the other for the coimmunoprecipitation assays (J), thereby controlling that the proteins tested for binding were active in the injected embryos.

476 GENES & DEVELOPMENT Dkk1 binds Kny and regulates gastrulation movements

(Feldman et al. 2002), an antagonist of the Nodal signal- indirectly regulating Fgf expression and, through it, ing pathway. Nodal, like the Wnt/␤catenin pathway, is modulating cell movement inside the forming limb bud. able to inhibit head organizer activity (Piccolo et al. However, temporal and spatial expression of Wnt and Fgf 1999). As Nodal is able to repress the expression of signals in the limb bud suggests the intriguing possibil- Dkk1, the phenotype observed in the zebrafish Lefty ity that Dkk1 may act over the Fgf pathway in a Wnt- morphant embryos may therefore be caused by loss of independent fashion. Dkk1 activity. Another possible option is that Dkk1 Glypicans, HSPG (Kramer and Yost 2003), control the negatively modulates the Nodal pathway, this latter distribution of numerous other signaling molecules such positively regulating internalization and rostral progres- as Hedgehog, Wingless, and TGF␤ members during de- sion. Future thorough biochemical assays will be needed velopment in Drosophila (Lin and Perrimon 2002; Kreu- to test the interaction of Dkk1 with receptors of the ger et al. 2004), Xenopus (Galli et al. 2003), and zebrafish main signaling pathways active during gastrulation. (Myers et al. 2002). HSPGs are now perceived as major modulators of morphogen gradients. In Drosophila, the The Wnt/␤catenin pathway modulates cell cohesion glypicans Dally and Dally-like are essential to the formation of the morphogenetic gradient of Dpp in the wing Although not accelerating rostral mesendoderm progres- disc (Belenkaya et al. 2004). In this system, the glypicans sion, gain of Wnt/␤catenin activity does influence some are strictly required for the movement of the Dpp pro- aspects of cell–cell interaction and motility, as we ob- teins away from their source. Interestingly, a Drosophila served that mesendoderm cells, exposed to high Wnt/ Wnt antagonist, called Notum (Giraldez et al. 2002), ␤catenin activity, internalize and progress rostrally at a negatively modulates the Wnt signaling pathway by spe- similar speed as wild type cells but are less cohesive. cifically inducing cleavage of Dally-like from the mem- This echoes results obtained in tumors showing that an brane (Kreuger et al. 2004). Dkk1 may thus be acting on increase in Wnt/␤catenin activity induces a loss in cell the Fgf and Wnt pathways by modulating interaction be- adhesion by reducing the pool of ␤catenin proteins nor- tween glypicans and signaling effectors of these path- mally used at the cell membrane in the E-cadherin/ ways. If so, the next challenge will be to unravel the ␣catenin/␤catenin complex of the adherens junction mechanisms by which Dkk1 is able to interact with (Brembeck et al. 2006) and provides indirect evidence glypican-dependent pathways supporting the idea of integration of patterning and modification in cell adhesion. Both ␤catenin and APC Dkk1 activates the Wnt/PCP pathway regulate concomitantly gene expression and cell adhe- We show here that Dkk1 is activating the Wnt/PCP sion specifically in the context of cancer and metastasis pathway. The molecular interaction between Kny and (for review, see Willert and Jones 2006). The very recent Dkk1, the lack of rescue of the gastrulation movement study done in mice AVE shows that the Wnt/␤-catenin defect by DNwnt8 overexpression, the ability of Dkk1 activity represses, directly or indirectly, the rostral overexpression to induce CE defects in mutant embryos movement of this tissue (Kimura-Yoshida et al. 2005). in which the Wnt/␤catenin pathway is constitutively ac- This may suggest a possible need for adherens junction- tive, and the capacity of Dkk1 to cooperate with Kny in regulated cohesion for the AVE to migrate properly. rescue of the kny mutants all suggest a direct interaction between Dkk1 and this pathway. This direct interaction Dkk1 binds to Glypican 4/6 is not likely to act at the level of the ligands. Indeed, Our study identifies a new binding partner for Dkk1: the although Dkk1 is able to potentiate the rescue of the kny Glypican4 homolog Knypek. The fact that Dkk1 is able mutants by Kny, it is unable to help Wnt11 in the rescue to bind to the glypican molecule Knypek opens the pos- of the slb/wnt11 mutants (data not shown). In light of sibility that Dkk1 may modulate directly a set of signals our data, we therefore propose a mechanism by which requiring Glypican4 for their activity. Glypican4 is cru- Dkk1 concomitantly represses Wnt/␤catenin and acti- cial for reception of the Fgf signaling both in cell culture vates Wnt/PCP pathways through a shift in the intracel- and in vivo (Richard et al. 2000; Zhang et al. 2001; Galli lular property of the Fz receptor (Fig. 7). Dissociation of et al. 2003). Interestingly, we observed a dramatic in- the LRP/Fz complex induced by Dkk1 may change the crease of Fgf signaling in the germ ring of Dkk1 mor- receptor such that it now has more affinity to the Wnt phant gastrula embryos (data not shown). Lack or gain of ligands defined as PCP activators (Wnt2, Wnt4, and Dkk1 function in mice limb buds has a spectacular effect Wnt5) and/or is now able to impose a switch in Dsh over Fgf signaling inside the forming limb, which may activity. Two very recent studies provided evidence for account for most of the limb defects subsequently ob- the presence of such a switch between the two pathways. served (Mukhopadhyay et al. 2001). In the limb, expres- Casein kinase 1 ␧ (Strutt et al. 2006) and metastasis- sion of Wnt signals precedes the induction of Fgf signal- associated kinase (MAK) (Kibardin et al. 2006) are both ing and is required for such induction (Tickle and Mun- able to transform the activity of the Wnt/␤catenin path- sterberg 2001). Fgf signaling in the AER is required for way into a Wnt/PCP function by inducing change in cell movement inside the developing limb (Li and Mu- pathway specificity at the level of Dsh. Dkk1 may there- neoka 1999) and the gene snail is expressed in the bud in fore act as a catalyzer of a PCP activity of Dsh by inhib- response to Fgf (Isaac et al. 2000). In this context, Dkk1 iting LRP5/6 and providing Kny molecules in the vicin- may directly modulate the level of Wnt activity, thereby ity of the LRP-depleted receptor complex.

GENES & DEVELOPMENT 477 Caneparo et al.

Dkk1 establishes a connection between patterning Whole-mount in situ hybridization and and cell movements immunohistochemistry Although patterning and gastrulation movements have In situ hybridization and immunohistochemistry were per- been suggested to be fairly independent events (Myers et formed as previously described (Macdonald et al. 1994). For immunohistochemistry, the following antibodies were used: anti- al. 2002), some recent findings reopen the possibility of a fluorescein-AP (Roche), anti-biotin (Vectastain), anti-GFP (Tor- complex relationship between these two key mecha- reyPines Biolabs), and anti-Flag (Sigma). nisms in development of the vertebrate nervous system. The ability of a modulator such as Dkk1 protein to act simultaneously on patterning and gastrulation move- RNA injections and MO experiments ments via coordinated modulation of both Wnt/␤catenin Capped RNAs were transcribed with SP6 RNA polymerase and PCP pathways provides a means to achieve coordi- using the mMessage mMachine Kit (Ambion). dkk1GFP RNA nation between cell movements and fate specification. was injected at 50 ng/µL. MO antisense oligonucleotides One interesting possibility is that Dkk1 may partly regu- (GeneTools) were designed against 25 bases around the AUG of late patterning by controlling the progression of the cells the zebrafish dkk1 and tcf3 transcripts (against dkk1,5Ј-AATTG Ј Ј carrying the patterning signals. The gradient of Fgf pro- TAGGATGTATTCCCTGGGTG-3 and 5 -TAGAGAGCATG Ј Ј tein inside the presomitic mesoderm is formed by the GCGATGTGCATCAT-3 ; against tcf3,5-CTCCGTTTAACT GAGGCATGTTGGC-3Ј). Injections of MOs were done at con- progressive degradation of an initial synthesis inside a centrations between 2 and 0.4 mg/mL. Routine controls in- moving involuting population (Dubrulle and Pourquie volved injection of four nucleotide-modified versions of the MO 2004). If this mechanism may concern not only the Fgf tested. We tested whether the phenotype observed in the mor- but also the Wnt/␤catenin pathway, one can predict that phants was due to specific loss of Dkk1 function by rescue any change in the speed at which a signaling population assay, injecting dkk1MO together with a dkk1 transcript lack- progresses will have a direct impact on the patterning ing the MO target sequence (Supplementary Fig. S1). We also that signal regulates. So, as lack of Dkk1 accelerates ros- assessed the specificity of the MO phenotypes by testing tral progression, under the developing neural plate, of the whether the phenotype observed when overexpressing dkk1 Wnt-expressing internalizing mesendoderm, it therefore RNA could be rescued by dkk1MO. Two MOs have been used, also exposes prematurely part of the neural epithelium both rescuing dkk1 RNA overexpression and giving the same phenotype when injected alone. We also showed that dkk1MOs to the underlying Wnt/␤catenin and Fgf signals. It has inhibit in vivo translation and activity of the GFP-tagged ver- been shown recently that not only does the spatial sion of Dkk1 proteins (Fig. 1). Overexpression of dkk1GFP alone graded distribution of a morphogen establish positional leads to an increase in anterior brain size when injected in one- information but, as importantly, timing of exposure to two-cell-stage embryos, mimicking the activity of early act- to the morphogen shapes this information (Ahn and ing Wnt antagonists such as Cerberus, Dkk1, and Frzb1 Joyner 2004; Harfe et al. 2004). Modification of the (Yamaguchi 2001). Coinjection with dkk1MO decreases fluores- timing at which a given neural area is exposed to meso- cence and led to concentration-dependent rescue of the derm vertical signaling may therefore be sufficient to dkk1GFP overexpression phenotype (Fig. 1A–H). When perturb significantly the patterning decision of this re- dkk1GFP RNA was coinjected with a high amount of dkk1MO, gion. the embryos began to show a decrease in telencephalon and eye size typical of dkk1MO-injected embryos (Fig. 1I–J) indicating In conclusion, this work unraveled the involvement of that the dkk1MO was reducing both exogenous and endogenous Dkk1 in regulation of cell movements, via binding to the Dkk1 proteins. HSPG Knypek, driving coordinated modulation of the Wnt/␤catenin and PCP signaling pathways. Modulation of movements may be a crucial part of Dkk1’s influence Injections and transplantations on patterning, not only during gastrulation but also dur- Injections of RNA or MOs were done at the one-cell stage, with ing limb development, forebrain organization, and or- the exception of Figure 1O,P, where the MO was injected in the ganogenesis. This report therefore opens new avenues in newly formed YSL at the 1000-cell stage. For transplantations, the understanding of the mechanisms governing spatial donor embryos were injected with dkk1 or wnt11or wnt8 or and temporal regulation of signaling centers during de- GFP-RNA or fluorescein-tagged MOs or the tracer rhodamine velopment. biotinylated dextran shortly after fertilization. Donor cells were taken from germ ring late blastula embryos and transplanted to the germ ring of early gastrula hosts. For reproducibility, it was crucial to keep the initial position of the clones identical. We Materials and methods chose to place them at 35°–45° from the shield, inside the embryo margin. Transplantations were performed on embryos mounted in 4% methyl-cellulose in embryo medium and were Constructs viewed with a fixed-stage Nikon Optiphot microscope. Cells The construct for the in vitro synthesis of dkk1GFP mRNA was were moved by suction using a mineral oil-filled glass micropi- generated by cloning the zebrafish dkk1 cDNA into the pette attached to a 50-µL Hamilton syringe (Houart et al. 1998). pcDNA3.1/CT-GFP vector (LifeTechnology). The Kny-Flag expression construct is a gift from Lila Solnica-Krezel (Vanderbilt Immunoprecipitation and blotting University, Nashville, TN) and Jacek Topczewski (Northwest- ern University, Chicago, IL) (Topczewski et al. 2001). The LRP6 HEG cells were used as cellular hosts to test molecular inter- expression construct is provided by Tamai et al. (2000). action between Dkk1 and Knypek. The cells were plated at a

478 GENES & DEVELOPMENT Dkk1 binds Kny and regulates gastrulation movements concentration of 2 × 106 to 3 × 106 cells in a 10-cm-diameter tcf3 genes cooperate to pattern the zebrafish brain. Develop- dish. The cells were transfected the next day by adding a total ment 130: 1937–1947. volume of 500 µL containing 25 µg of DNA, 50 µL of CaCl2,, and Dubrulle, J. and Pourquie, O. 2004. fgf8 mRNA decay estab- 2.5 M in BES buffer, and incubation for 16 h at 37°Cin5%CO2. lishes a gradient that couples axial elongation to patterning The cells were then left to recover in DMEM culture medium in the vertebrate embryo. Nature 427: 419–422. for 48 h. The efficiency of transfection was determined by analy- Erter, C.E., Wilm, T.P., Basler, N., Wright, C.V., and Solnica- sis of the GFP fluorescence under UV light (generally ∼90%– Krezel, L. 2001. Wnt8 is required in lateral mesendodermal 95%). Cells were lysed and 1:300 GFP polyclonal antibody was precursors for neural posteriorization in vivo. Development added to the lysate for immunoprecipitation overnight at 4°C. 128: 3571–3583. The immunoprecipitate was purified on A-Sepharose and Fedi, P., Bafico, A., Nieto Soria, A., Burgess, W.H., Miki, T., G-Sepharose columns. Interaction was also tested in gastrula Bottaro, D.P., Kraus, M.H., and Aaronson, S.A. 1999. Isola- embryos. For each condition tested, extracts of 30 embryos in- tion and biochemical characterization of the human Dkk-1 jected at the one-cell stage were used for the control extracts homologue, a novel inhibitor of mammalian Wnt signaling. (1/10 of the total volume) and both anti-GFP and anti-Flg im- J. Biol. Chem. 274: 19465–19472. munoblotting of the anti-Flg (Sigma) immunoprecipitation (4.5/10 Feldman, B., Concha, M.L., Saude, L., Parsons, M.J., Adams, of total extract each). R.J., Wilson, S.W., and Stemple, D.L. 2002. Lefty antagonism The samples were run on polyacrylamide gels and transferred of Squint is essential for normal gastrulation. Curr. Biol. to nitrocellulose filters. The filters were incubated overnight 12: 2129–2135. with the anti-Flag (Sigma) and anti-GFP (TorreyPines Biolabs) at Galli, A., Roure, A., Zeller, R., and Dono, R. 2003. Glypican 4 4°C. After washes and incubation with secondary antibody, the modulates FGF signalling and regulates dorsoventral fore- filter was developed on photographic film (Kodak), using a brain patterning in Xenopus embryos. Development 130: chemo-luminescence detection kit (Perkin Elmer). 4919–4929. Giraldez, A.J., Copley, R.R., and Cohen, S.M. 2002. HSPG modi- Time-lapse analysis fication by the secreted enzyme Notum shapes the Wingless morphogen gradient. Dev. Cell 2: 667–676. The movements of cells transplanted in the germ ring of shield Glinka, A., Wu, W., Delius, H., Monaghan, A.P., Blumenstock, stage host embryos were followed by time-lapse imaging using C., and Niehrs, C. 1998. Dickkopf-1 is a member of a new the Nikon C1 confocal microscope. 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