Induction of the primary dorsalizing center in Xenopus by the Wnt/GSK/βcatenin signaling pathway, but not by Vg1, Activin or Noggin

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1 Development 124, (1997) Printed in Great Britain The Company of Biologists Limited 1997 DEV Induction of the primary dorsalizing center in Xenopus by the Wnt/GSK/βcatenin signaling pathway, but not by Vg1, Activin or Noggin François Fagotto, Kathleen Guger and Barry M. Gumbiner* Cellular Biochemistry and Biophysics Program, Memorial Sloan-Kettering Cancer Center, New York, 1275 York Avenue, Box 564, New York, NY 121, USA *Author for correspondence SUMMARY The molecular nature of the primary dorsalizing inducing event in Xenopus is controversial and several secreted factors have been proposed as potential candidates: Wnts, Vg1, Activin and Noggin. Recent studies, however, have provided new insight into the activity of the dorsalizing region, called the Nieuwkoop Center. (1) The activity of this dorsalizing center involves an entire signal transduction pathway that requires maternal β-catenin (Heasman, J., Crawford, A., Goldstone, K., Garner-Hamrick, P., Gumbiner, B., McCrea, P., Kintner, C., Noro, C. Y. and Wylie, C. (1994) Cell 79, ). (2) A transcription factor with potent dorsalizing activity, Siamois, is expressed within the Nieuwkoop Center (Lemaire, P., Garrett, N. and Gurdon, J. B. (1995) Cell 81, 85-94). We have used these two properties of the Nieuwkoop Center to evaluate the dorsalizing activity of the four secreted factors Wnt8, Vg1, Activin and Noggin. The requirement for β-catenin was tested by coexpressing a cadherin, which sequesters β- catenin at the cell membrane and specifically blocks its intracellular signaling activity (Fagotto, F., Funayama, N., Glück, U. and Gumbiner, B. M. (1996) J. Cell Biol. 132, ). Induction of Siamois expression was detected by RT-PCR. Of the four growth factors, only Wnt was sensitive to inhibition of β-catenin activity and only Wnt could induce Siamois expression. Therefore, Wnt is able to induce a bonafide Nieuwkoop Center, while Vg1, Activin and Noggin probably induce dorsal structures by a different mechanism. To order the steps in the Nieuwkoop Center signaling cascade, we have tested the relationship between β-catenin and GSK, a serine-threonine kinase that has been implicated in formation in a step downstream of Wnt. We found that GSK acts upstream of β-catenin, similar to the order of these components in the Wingless pathway in Drosophila. We have also examined the relationship between the Wnt/β-catenin pathway and Siamois. We show that β-catenin induces expression of Siamois and that the free signaling pool of β-catenin is required for expression of endogenous Siamois. We conclude that the sequence of steps in the signaling pathway is Wnt GSK β-catenin Siamois. Key words: formation, dorsoventral patterning, Nieuwkoop Center, Spemann Organizer, growth factor, signal transduction, Siamois, Xenopus, Wnt, β-catenin, Activin, Noggin INTRODUCTION Determination of the dorsoventral in Xenopus eggs occurs at fertilization by an asymmetric redistribution of an unknown determinant within the vegetal pole (Gerhart et al., 1989). The resulting dorsalizing region, called the Nieuwkoop Center, then induces the overlying mesodermal cells to become the Spemann Organizer, which orchestrates gastrulation and the patterning of axial structures. Various secreted factors have been shown to have dorsalizing activity, as detected by their abilities to induce a secondary when expressed ventrally and to rescue an in UV-treated, ventralized embryos. Although such phenotypes are characteristic of both Nieuwkoop Center and Spemann Organizer activities, these two signaling centers can be distinguished according to two criteria: (1) the activity of the Nieuwkoop Center is maximal in the lower vegetal region, while the activity of the Spemann Organizer is restricted to the equatorial region, and (2) the cells of the Spemann Organizer themselves become part of the induced dorsal mesoderm, independently of their origin, while cells expressing Nieuwkoop Center-like activities are not recruited into axial structures, but maintain their original fates (endoderm in the case of the vegetal pole) (Smith and Harland, 1991). According to these criteria, several members of the Wnt family, as well as Vg1, Activin and Noggin (Christian et al., 1991; Smith and Harland, 1991; Sokol et al., 1991; Ku and Melton, 1993; Thomsen and Melton, 1993; Thomsen et al., 199; Steinbeisser et al., 1993; Smith and Harland, 1992), appear to mimic the activity of the primary dorsalizing center, i.e. the Nieuwkoop Center. Despite their common abilities to act as dorsalizers, these growth factors have quite different properties. For instance, Vg1 and Activin, both members of the TGFβ family, are also mesodermal inducers (Thomsen et al., 199; Green et al., 1992; Dale et al., 1993; Thomsen and

2 454 F. Fagotto, K. Guger and B. M. Gumbiner Melton, 1993; Kessler and Melton, 1995), while Noggin is a potent neuralizer (Lamb et al., 1993). Some Wnts (Wnt1, Wnt8, Wnt8b) have strong dorsalizing activity, but none can induce mesoderm (Moon, 1993). On the other hand, Wnts are probably also involved later during development in various inducing events, for example patterning of the ventral mesoderm during gastrulation (Wnt8, Christian and Moon, 1993) and patterning of the central nervous system (Wnt1, McMahon and Bradley, 199; Thomas and Cappechi, 199). Several intracellular signaling molecules, β-catenin, Glycogen Synthase Kinase-3 (GSK-3) and Dishevelled, have also been recently implicated in the activity of the Nieuwkoop Center (Heasman et al., 1994; Funayama et al., 1995; Guger and Gumbiner, 1995; He et al., 1995; Pierce and Kimelman, 1995; Sokol et al., 1995). Interestingly, the Drosophila homologues of these three molecules (Armadillo, Zeste-white-3 and Disheveled, respectively) are components of a well-characterized signaling pathway activated by Wingless, a Drosophila Wnt homologue (Peifer, 1995). One of these intracellular proteins, β-catenin, has been shown to be required both for formation in Xenopus and for the dorsalizing activity of Wnt (Heasman et al., 1994). β-catenin is a cytoplasmic protein that associates with cadherin cell-adhesion molecules (Kemler, 1993), but also has an independent intracellular signaling activity (Fagotto et al., 1996). In Drosophila, the signaling activity of the β-catenin homologue, Armadillo, is negatively regulated by a serine/threonine kinase, Zeste-white-3/Shaggy (Peifer, 1995) and Wingless activates the signaling pathway by antagonizing Zeste-white-3. Similarly, in Xenopus embryos, the homologue of Zeste-white-3, GSK-3, has ventralizing activity, and a dominant negative mutant form, GSK-3K R, induces duplication (He et al., 1995; Pierce and Kimelman, 1995). Overexpression of GSK can inhibit induction by Wnt, indicating that GSK acts downstream of Wnt, similar to Zestewhite-3 acting downstream of Wingless. Another intracellular molecule with strong dorsalizing activity is Siamois, a novel Hox-type transcription factor expressed in the region of the Nieuwkoop Center (Lemaire et al., 1995). Its potent activity, its transient expression at the beginning of the midblastula transition, and its restriction to the dorsal-vegetal region of the late blastula, strongly suggest that Siamois is an important component of the Nieuwkoop Center. The Nieuwkoop Center appears not to be simply the site of secretion of a dorsalizing inducer, but to involve an entire intracellular signal transduction pathway. The established requirement for β-catenin and the strong evidence of the involvement of GSK suggest that this pathway is similar to the Drosophila Wingless Armadillo pathway. However, the order of these components along the pathway has not been established in Xenopus and the endogenous activator of this pathway has not been identified. The possibility remains that growth factors other than Wnts are able to activate the pathway. Finally, the relationship between the novel transcription factor Siamois and these signaling pathways has not yet been investigated. We have therefore evaluated the dorsalizing activity of four candidate growth factors, Wnt8, Vg1, Activin and Noggin, according to the two newly established properties of the Nieuwkoop Center: (1) requirement for β-catenin activity, which can be experimentally blocked by co-expressing cadherins (Fagotto et al., 1996), and (2) the ability to activate Siamois expression. MATERIALS AND METHODS Wnt8 and Noggin expression plasmids have been kindly provided by Dr R. Harland, UCBerkeley, and Activin and BMP2-Vg1 plasmids by Dr D. Melton, Harvard. Siamois (Lemaire et al., 1995) was cloned by RT-PCR using RNA from stage 1G Xenopus embryos and inserted into the pcs2+mt expression vector (Rupp et al., 1994; Turner and Weintraub, 1994). mrnas were synthesized in vitro using the SP6 RNA polymerase (Promega Corp., Madison, WI) and were dissolved at the appropriate concentration in DPEC-treated water. For cadherin coinjection experiments, mrnas were injected into the vegetal region of a ventral blastomere at the 4- to 8-cell stage (Fagotto et al., 1996). For this set of experiments, the amount of mrna for each of the various factors was titrated down to the smallest amount required for maximal activity, measured as maximal frequency and completeness of secondary formation. The amount of mrna injected (in pg) was: Wnt8: 1-; Noggin: ; Activin: ; BMP2-Vg1: 1-; GSK-3-K R: 1-3; Siamois: 5-1. Higher amounts of Wnt8 cause dorsalization, and thus reduction and disappearance of a body. Higher amounts of Activin resulted in uninterpretable phenotypes, probably caused by generalized mesodermal induction over most of the embryo. 4-5 ng of C-cadherin mrna were used for coinjections. Embryos were allowed to develop at room temperature in.1 MMR (Kay and Peng, 1991) and duplication was scored at the neurula stage. The number of embryos scored in Fig. 1 (pooled from several experiments) were (+, ) C-cadherin respectively: Wnt8: (14,95); Noggin: (95,84); Activin: (68,65); BMP2-Vg1: (76,85); GSK-3-K R: (46,44); Siamois: (119,125). BMP2-Vg1 is a chimeric molecule that is processed in the embryo into the active, secreted fragment of Vg1 (Thomsen and Melton, 1993). GSK-3-K R is a mutant of GSK-3β without kinase activity (Pierce and Kimelman, 1995). For histological studies, tailbud-early tadpole stages (stage 25-29) were fixed in Dent s fixative and embedded in gelatin as described (Fagotto and Gumbiner, 1994). Serial 1 µm frozen transverse sections of the entire embryos were collected. Sections were immunolabeled (Fagotto and Gumbiner, 1994) using the rabbit anti-n-cam polyclonal antibody R16 (affinity purified IgG, 2 µg/ml, generous gift of Dr U. Rutishauser) and the mouse monoclonal antibody Tor7, a notochord marker (ascites fluid, 1/5 dilution, Bolce et al., 1992). Both antibodies were detected with FITC-labeled secondary antibodies (each at 5 µg/ml, Molecular Probes, Inc., Eugene, OR, USA). Nuclei were then stained with DAPI ( µg/ml, Molecular Probes, Inc., Eugene, OR, USA) and the yolk was counterstained with.1% Eriochrome Black (Aldrich Chemical C., Milwaukee, WI, USA). Triple exposure microphotographs were taken under an Axioplan microscope (Karl Zeiss, Inc., Thornwood, NY, USA) using a objective and filter blocks for DAPI, fluoresceine and rhodamine (for background counterstain). For RT-PCR experiments, mrna was extracted from whole embryos (Fig. 7) or dissected dorsal and ventral halves at early gastrula stage (stage 1-1G). Animal caps were dissected at stage 8 and incubated for further 2 hours (stage 1) before RNA extraction. mrna was extracted either using the proteinase K treatment protocol (Wilson and Melton, 1994) or the Ultraspec RNA isolation system (Biotecx laboratories, Inc., Houston, TX, USA). Various specific mrnas were detected by RT-PCR as described (Wilson and Melton, 1994). Primers were: for Siamois: 5 : CGC GGA TCC ATG GCC TAT GAG GCT GAA ATG GAG and 3 : GCT CTA GAG AAG TCA GTT TGG GTA GGG CT (Fig. 6) or 5 : TTG GGA GAC AGA CAT GA and 3 : GCT CTA GAG AAG TCA GTT TGG GTA GGG CT (Fig. 7); for Goosecoid: 5: ACA ACT GGA AGC ACT GGA and 3: TCT

3 Primary dorsalizing induction in Xenopus 455 TAT TCC AGA GGA ACC; for Chordin: 5 ATG CAG TGT CCC CCC ATC and 3: GCA GTG CAT AAC TCC GAA; for Noggin: 5: ATG GAT CAT TCC CAG TGC and 3: TCT GTG CTT TTT GCT CTG; for Xnr3: 5: ATG GCA TTT CTG AAC CTG and 3TCT ACT GTC ACA CTG TGA; for EF1: 5: CAG ATT GGT GCT GGA TAT GC AND 3: AC TGC CTT GAT GAC TCC TAG. RESULTS Dependence of induction by Wnt, Noggin, Vg1 and Activin on β-catenin signaling Since β-catenin is required for formation (Heasman et al., 1994), we tested which of the putative dorsalizing inducers Wnt8, Noggin, Vg1 or Activin require active β-catenin signaling. We have previously shown that signaling by β-catenin can be specifically inactivated by coexpressing cadherins (Fagotto et al., 1996). Indeed, C-cadherin inhibits β- catenin-induced duplication by sequestering β-catenin at the plasma membrane and thus depleting the cytosolic pool of β-catenin that is active in intracellular signal transduction. We therefore used cadherin coexpression as a simple method to inhibit this step in the endogenous dorsalizing signaling pathway. mrnas encoding Wnt8, Noggin, BMP2-Vg1 and Activin were injected into the ventral side of early cleaving embryos, either alone or coinjected with C-cadherin mrna (see Materials and Methods for ratios). BMP2- Vg1 is a chimeric molecule that is processed into an active, secreted fragment of Vg1 when expressed in Xenopus embryos (Thomsen and Melton, 1993). Axis duplication was scored at the neurula stage (Fig. 1). Also, the histology of secondary axial structures was analyzed from serial frozen sections of late tailbud-early tadpole stages (Fig. 3). C-cadherin coexpression strongly inhibited Wnt8 -inducing activity. While expression of Wnt8 alone induced a complete secondary at high frequency (Figs 1A, 2A, 3A), most embryos expressing both Wnt8 and C-cadherin mrna developed ly (Figs 1A, 2B, 3B). At most, a short protrusion or small pigmented area was observed ( ), indicative of a weak remnant of dorsalizing activity. Thus, induction by Wnt is sensitive to depletion of cytoplasmic β-catenin, similar to the results obtained by Heasman et al. (1994) using antisense technology. In contrast, C-cadherin coexpression was found to have no detectable effect on duplication induced by Noggin, BMP2-Vg1 or Activin. C-cadherin coexpression did not affect the frequency of duplication induced by these three growth factors (Fig. 1B-D). Moreover, the secondary axes induced in the presence of C-cadherin were similar to the axes induced by the growth factors alone, by both criteria of their external morphology (Fig. 2C-F) and of their internal organization A C E (Fig. 3C-F). Similar to previous reports, Noggin, BMP2-Vg1 and Activin only induced partial secondary axes, but not complete axes (Figs 1B-D, 2C-F). These partial axes always lacked anterior structures and, in most cases, a differentiated notochord was not detected (Fig. 3C,E,F, see legend of Fig. 3). However, the partial duplicated axes were otherwise well structured, with a neural tube, dorsal mesoderm (muscle) and an intestinal lumen (Fig. 3C-F). Note that, in the cases in which incomplete induction was generated by Wnt expression, the secondary axes also often lacked notochord (not shown). Another indication that the dorsalizing activities of Noggin, Vg1 and Activin were not sensitive to cadherin inhibition was the appearance of a second blastoporal lip. This lip, which is particularly prominent in BMP2-Vg1 and Activin-expressing embryos, was still present in embryos co-expressing C- cadherin (not shown). In contrast, no secondary lip was observed in embryos co-expressing Wnt and C-cadherin (not shown). We conclude that Wnt8 is the only one out of the four growth factors tested that requires β-catenin signaling for inducing activity. Wnt8 Wnt8 + C-cadherin () BMP2-Vg1 BMP2-Vg1 + C-cadherin () () GSK-3K->R GSK-3K->R + C-cadherin () () B D F Noggin Noggin + C-cadherin () Activin Activin + C-cadherin ()() Siamois Siamois + C-cadherin Fig. 1. Effect of C-cadherin co-expression on duplication by Wnt8, Noggin, BMP2-Vg1, Activin, GSK-3-K R and Siamois. Embryos were scored in four categories: complete secondary (with cement gland), partial secondary (i.e. any secondary lacking the cement gland), (very small posterior protrusion or pigmented spot or line) and (only one ). Axis duplication by Wnt8 (A) and GSK-3-K R (E) was strongly inhibited by C-cadherin expression. Axis duplication by Noggin (B), BMP2-Vg1 (C) and Activin (D) was unaffected by C-cadherin coexpression. Axis induction by Siamois (F) was only marginally affected: the most anterior structures were often lacking, but the secondary were still very long, consistently more complete than induced by Noggin or BMP2- Vg1. The frequency of Siamois-induced duplication (complete and partial combined) was unchanged in the presence of C-cadherin (7-75%).

4 456 F. Fagotto, K. Guger and B. M. Gumbiner We have further confirmed these observations by evaluating the effect of C-cadherin coexpression on the capacity of the various dorsalizing factors to induce the expression of Goosecoid, a marker of the Spemann Organizer (Cho et al., 1991). mrna encoding the various factors were injected in the ventral side, either alone or with C-cadherin. Embryos were dissected at early gastrula stage (1G) into dorsal and ventral halves and the expression of Goosecoid was analyzed by RT- PCR. Dorsal halves embryos were used as controls for the expression of Goosecoid in the endogenous Spemann Organizer and uninjected ventral halves as negative controls. All four growth factors were able to induce Goosecoid expression when injected alone. Coexpression of C-cadherin strongly inhibited the induction of Goosecoid by Wnt8, but not the induction by Noggin, BMP2-Vg1 or Activin (Fig. 4). Therefore, free b-catenin is required for the induction of the Fig. 2. Examples of duplication by various dorsalizing growth factors and dependence on β-catenin signaling activity. (A) Complete duplication, with two cement glands, by Wnt8. (B) Complete inhibition of Wnt-mediated duplication by coexpression of C-cadherin. (C,E) Partial secondary axes induced by Noggin and BMP2-Vg1, respectively. (D,F) No inhibition of duplication obtained when C-cadherin mrna is coinjected with Noggin or BMP2-Vg1 mrna. (G) Complete duplication by Siamois. (H) Axis duplication by Siamois is largely unaffected by C-cadherin coinjections. Although the formation of the most anterior structures is inhibited, secondary axes are still very long, which is in contrast to the strong inhibition of Wnt-induced duplication (compare to B). Spemann Organizer by Wnt, but not by the other growth factors. β-catenin is required for the dorsalizing activity of GSK-3K R but not the dorsalizing activity of Siamois GSK has been implicated in the Wnt signaling pathway leading to induction. GSK-3K R, a dominant negative mutant form of Xenopus GSK-3, induces duplication when expressed in the ventral side of early cleaving embryos (He et al., 1995; Pierce and Kimelman, 1995). To order the steps in this pathway, we asked whether duplication by GSK- 3K R requires free β-catenin. Axis duplication was indeed strongly inhibited by C-cadherin co-expression (Fig. 1E). This finding indicates that GSK acts upstream of β-catenin, similar to the relationship between Zeste-white-3 and Armadillo in Drosophila. We also tested whether β-catenin was required for duplication by Siamois, another factor with Nieuwkoop Center-like dorsalizing activity. In contrast to Wnt8, β-catenin or GSK-3K R, the -inducing activity of Siamois was barely affected by C-cadherin overexpression (Figs 1F, 2G,H, 3G,H). The only effect of C-cadherin coexpression was a partial reduction in anterior structures, while secondary axes were otherwise reproducibly long and complete, including the presence of well-differentiated notochords (Fig. 3H). In fact, these secondary axes were still more complete than the majority of the secondary axes generated by expressing Noggin or BMP2-Vg1. The effect of C-cadherin expression on Siamois activity was therefore very marginal compared to the striking inhibition of Wnt, GSK-3K R or β-catenin-induced secondary axes. The small effect observed might be due to a β-catenin-independent effect of C-cadherin overexpression, for example, a slight perturbation of gastrulation. Alternatively, β- catenin may also be involved in a later signaling step during formation of head structures. Nevertheless, the -inducing activity of Siamois does not seem to depend on free β-catenin, suggesting that it acts downstream in the pathway, or in parallel to it. Similar results were obtained by analyzing the expression of Goosecoid in the ventral side of injected embryos. Induction of Goosecoid expression by GSK-3K R was strongly reduced in the presence of C-cadherin, while Siamois induced similar levels of Goosecoid transcripts in the presence or the absence of C-cadherin (Fig. 4). Induction of Siamois expression by a subset of dorsalizing molecules The C-cadherin coexpression experiments have allowed us to determine which of the known dorsalizing factors require β- catenin signaling in the Nieuwkoop Center. A second specific characteristic of the Nieuwkoop Center is the expression of the transcription factor Siamois. We therefore asked which of the dorsalizing factors can induce Siamois expression. mrnas encoding β-catenin and the four growth factors Wnt8, Noggin, BMP2-Vg1 and Activin were injected ventrally, and the embryos were dissected at early gastrula stage (the peak of Siamois expression) into dorsal and ventral halves. Siamois expression was analyzed by RT-PCR (Fig. 5A). Dorsal halves were used as internal controls for Siamois expression in the endogenous Nieuwkoop Center and ventral halves from

5 Primary dorsalizing induction in Xenopus uninjected embryos were used as negative controls. Both βcatenin and Wnt8 induced strong expression of Siamois in the ventral halves. However, none of the other secreted factors (Noggin, BMP2-Vg1, Activin) induced Siamois, even though all of them activated expression of Goosecoid, a marker for the Spemann Organizer, which is the overlying induced dorsal mesoderm (Cho et al., 1991). Similar injections were made in the animal hemisphere, and animal caps were removed by dissection and analyzed for Siamois expression. Although the animal hemisphere is ly not involved in the dorsalizing process, it is a very useful tissue for analysis of mesoderminducing and dorsalizing activities. Again, both Wnt8 and βcatenin activated Siamois expression, but neither Noggin nor BMP2-Vg1 induced its expression (Fig. 5B). Therefore, although all factors analyzed were able to induce Goosecoid in the Spemann Organizer, only Wnt and β-catenin could induce expression of Siamois, a marker for the Nieuwkoop Center. β-catenin requirement for Siamois expression in the endogenous dorsalizing center The experiments described above suggest that Siamois is a specific target of the Wnt-β-catenin pathway. We therefore asked whether the expression of Siamois in the endogenous Nieuwkoop Center is dependent upon β-catenin signaling. Normal formation can be significantly inhibited 457 by increasing the levels of C-cadherin, either globally in oocytes (Heasman et al., 1994), or specifically in the vegetal dorsal side of early embryos (Fagotto et al., 1996), similar to the effect resulting from the depletion of maternal β-catenin (Heasman et al., 1994). Therefore Siamois expression was analyzed in embryos where β-catenin signaling in the endogenous Nieuwkoop Center was blocked by overexpression of Ccadherin. When 5 ng C-cadherin mrna were injected into the two dorsal blastomeres of 4- to 8-cell-stage embryos, Siamois expression was greatly decreased compared to control uninjected embryos (Fig. 6). Siamois expression was fully rescued in embryos in which β-catenin mrna (1.5 ng) was coinjected with C-cadherin mrna, confirming that the inhibition of Siamois expression by C-cadherin was specifically due to the inhibition of β-catenin signaling. Thus, Siamois expression depends on β-catenin signaling in the Nieuwkoop Center. Siamois induces the expression of markers of the Spemann Organizer The selective activation of Siamois in the Nieuwkoop Center by the Wnt/β-catenin pathway and the strong dorsalizing activity of Siamois suggest that this pathway may account for most of the Nieuwkoop Center activity. To determine whether the activity of Siamois is sufficient to induce a Spemann Organizer in the overlying mesoderm, we analyzed the expression of several markers of the Organizer. In Fig. 4, we have shown that ectopic expression of Siamois in the ventral side can induce the expression of Goosecoid. We further tested Fig. 3. Internal organization of axial structures in embryos injected with various dorsalizing molecules in the presence and absence of Ccadherin coexpression. Injected embryos fixed at tailbud-early tadpole stages were cut throughout in serial transverse frozen sections. Both the neural tube (nt) and notochord (no) were immunolabeled, respectively, with an anti-n-cam antibody and Tor7, an antibody specific for the extracellular matrix of the notochord. Both were detected with FITC-labeled secondary antibodies (green). Nuclei were stained with DAPI (blue) and the yolk counterstained with Eriochrome Black (red). (A) Embryos injected with Wnt8 developed with high frequency two complete axes, with two neural tubes (nt), two notochords (no) flanked by somitic mesoderm (muscle, ms). Two separate intestinal lumens (in) were often observed. Similar structures were observed when partial secondary axes resulted from Wnt8 injections, except that the notochord was generally missing (not shown). (B) Wnt8 and Ccadherin coinjection resulted in embryos with a morphology having a single. (C-F) The general organization of the secondary axes (stars) induced by Noggin and BMP2-Vg1 was indistinguishable in the presence or the absence of C-cadherin. These partial axes displayed well differentiated axial structures such as neural tube, muscle and dorsal fin, and a secondary digestive tract was often observed in the anterior part, eventually merging with the tract of the primary posteriorly. Differentiated notochords detected by Tor7 staining were found only in a few cases, generally in the most posterior region of the embryo. One case of secondary notochord is shown in D (Noggin + C-cadherin). In this case, the notochord extended more anteriorly than in the other cases observed. In C, ot indicates a tangential section through the periphery of an otic vesicle stained with N-CAM. (G,H) Cross-sections through embryos expressing exogenous Siamois or Siamois + C-cadherin showed identical histological features, with well organized twin axes, with two neural tubes, two notochords extending quite anteriorly, and two intestinal lumen (which in the embryo shown in H have merged more anteriorly).

6 458 F. Fagotto, K. Guger and B. M. Gumbiner Fig. 4. Effect of C-cadherin co-expression on the induction of Goosecoid, a marker of the Spemann Organizer, by Wnt8, Noggin, BMP2-Vg1, Activin, GSK-3-K R and Siamois. mrnas coding for the various dorsalizing factors were injected vegetally into one ventral blastomere of 4- to 8-cell-stage embryos, alone ( ) or coinjected with 5 ng C-cadherin mrna (+C). Ventral halves were dissected at stage 1 1 / 2 and analyzed for Goosecoid expression by RT-PCR. Dorsal (D) and ventral (V) halves from uninjected embryos were used respectively as positive and negative controls for the expression of Goosecoid. All dorsalizing factors were found to ectopically induce Goosecoid when expressed alone. Coexpression of C-cadherin strongly inhibited Goosecoid expression induced by Wnt8 and GSK-K R, but not by Noggin, BMP2-Vg1, Activin or Siamois. EF1 is an ubiquitous mrna used as loading control. whether other markers, Chordin, Xnr3 and Noggin, were expressed under similar conditions (Fig. 7). We found that Siamois was able to induce ectopic expression of all the markers tested, suggesting that Siamois is sufficient, at least by the presently available criteria, to induce an entire Spemann Organizer. A catenin pathway leading to the induction of Siamois expression is the only known signal transduction pathway that can account for the characteristics of the Nieuwkoop Center. Although Vg1, Activin and Noggin have dorsalizing activities, our results suggest that these growth factors do not act by inducing the primary dorsalizing Nieuwkoop Center. Indeed, the role of these three factors as endogenous dorsalizers had been previously questioned, mainly because they could rarely induce complete secondary axes (see e.g. Dale et al., 1993; Lemaire et al., 1995). However, due to lack of more precise criteria, the issue has remained controversial. Complete versus partial induction could be interpreted as resulting from differences in the strength of the induction of injected constructs, rather than from fundamental qualitative differences between the effects of various factors. In the case of BMP2-Vg1, for example, it has been argued that this exogenously expressed protein is poorly secreted. In fact, another Vg1 chimeric molecule, which is more efficiently processed, has been reported to cause a more complete duplication (Kessler and Melton, 1995). Also, it has been reported that BMP2-Vg1 (Thomsen and Melton, 1993) and Noggin (Smith and Harland, 1992) can rescue complete in UV-treated embryos, which has been interpreted to mean that they possess full -inducing activity. We have not observed any compelling qualitative difference in the nature of the secondary axes induced by the various dorsalizing factors (Fig. 3). Indeed, the degree of internal organization, according to the presence of differentiated axial structures such as neural tube, notochord or muscle, matches quite faithfully the degree of complete induction as assessed by external morphology. Therefore, independent criteria are necessary to characterize the properties of the various candidate dorsalizing inducers. Our findings show that the dorsalizing activities of Vg1, B DISCUSSION Several kinds of growth factors are able to induce a secondary body in Xenopus embryos. However, they may do so by different mechanisms. We have used two independent objective criteria to evaluate which factors and/or signaling pathways can account for the primary dorsal induction events of the Nieuwkoop Center, the earliest dorsalizing region in the Xenopus embryo arising from cortical rotation of the egg after fertilization. Of the four growth factors tested, only a pathway triggered by Wnts satisfies these criteria. The first criterion, established by Heasman et al. (1994) is the requirement for β-catenin in the process of dorsalization and development. We confirm their findings that induction by Wnts requires β-catenin, and go on to show that none of the other growth factors tested, Noggin, Vg1 or Activin, require β-catenin for their -inducing activities. A second criterion is the induction of the expression of Siamois, a Hox protein with potent inducing activity that is ly expressed in the endogenous Nieuwkoop Center (Lemaire et al., 1995). Only Wnts, but not Noggin, Vg1 or Activin, can induce the expression of Siamois when expressed ectopically in the early embryo. Therefore, a Wnt/ β- Fig. 5. Siamois expression in response to various dorsalizing factors. (A) Ectopic expression of factors in the vegetal-ventral region. mrnas were injected vegetally into the two ventral blastomeres of 4- to 8-cell-stage embryos. Total amounts of injected mrnas are indicated. Dorsal (D) and ventral (V) halves were dissected at stage 1 and analyzed for Siamois and Goosecoid expression by RT-PCR. β-catenin and Wnt8 induce high Siamois expression (compared to expression in the dorsal side). No expression was observed in ventral halves from embryos injected with Noggin, Activin or BMP2-Vg1. All these factors induced Goosecoid expression in the same embryos. (B) Ectopic expression in the animal hemisphere. mrnas were injected into the animal pole of the four blastomeres of 4- to 8-cell-stage embryos. Total amounts of injected mrnas are indicated. Animal caps were dissected from injected embryos at stage 8 and were analyzed at stage 1 for Siamois expression. β-catenin and Wnt8 ectopic expression induced Siamois expression in the animal hemisphere, but Noggin and BMP2-Vg1 did not.

7 Primary dorsalizing induction in Xenopus 459 Wnt Nieuwkoop Center GSK-3 β-catenin Siamois Fig. 6. Inhibition of the expression of endogenous Siamois by overexpression of C-cadherin in the Nieuwkoop Center. 5 ng C- cadherin mrna (C) or 5 ng C-cadherin and 1.5 ng β-catenin mrnas (C+β) were injected vegetally into both dorsal blastomeres of 4- to 8-cell-stage embryos. Siamois expression was analyzed at early gastrula stages by RT-PCR. Uninjected embryos were used as controls (ctr). C-cadherin overexpression caused a dramatic, often complete inhibition of Siamois expression, which could be rescued to high levels when excess β-catenin was coexpressed with C-cadherin. Activin and Noggin do not require β-catenin and do not induce Siamois expression. Therefore, the dorsalizing activity of Vg1, Activin and Noggin probably derives from direct induction of components of the Spemann Organizer, at sites either downstream of Siamois or parallel with the Wnt/β-catenin/Siamois pathway (Fig. 8). One possibility is that they act at the second inducing step, mimicking endogenous factors secreted by the Nieuwkoop Center to induce the overlying Spemann Organizer tissue (Figs 4, 5). Another possibility is that these or related factors ly act synergistically with the signal emanating from the Nieuwkoop Center (Watabe et al., 1995), but may partially bypass the requirement for the Nieuwkoop Center Fig. 7. Ectopic activation of markers of the Spemann Organizer by Siamois. Siamois mrna was injected vegetally in one ventral blastomere at the 4- to 8-cell stage. Embryos were dissected at stage 1-1G (early gastrula) into dorsal and ventral halves and markers of the Spemann Organizer were analyzed by RT-PCR. Gsc, Goosecoid; Xnr3, Xenopus nodal-related-3. Ventral halves from uninjected embryos (V) were used as negative controls, and dorsal halves (D) as positive controls. Ventral ectopic expression of Siamois (Sia) induced expression of all the markers tested, often to levels similar to the levels found in the dorsal side. Spemann Organizer X? Goosecoid Vg1 Activin Noggin Fig. 8. Model for the dorsalizing inducting pathway in Xenopus. A Wnt, or Wnt-related factor, initiates a signal transduction pathway in the Nieuwkoop Center, involving sequentially the inhibition of GSK-3 and the consequent stimulation of β-catenin signaling, leading to Siamois expression. The Nieuwkoop Center then secretes an unknown factor or factors X, which induces the overlying mesodermal cells to become the Spemann Organizer, expressing Goosecoid and other dorsal mesoderm markers. Vg1, Activin and Noggin can induce a Spemann Organizer, but they do not activate the β-catenin pathway and Siamois expression. It is possible, however, that one or more of them could act at the step X, mediating the induction of the Spemann Organizer by the Nieuwkoop Center (dotted arrow). Alternatively, Vg1 and Activin may act in parallel to the Nieuwkoop Center activity, for instance by participating in the mesodermal induction, in which case they would ly act synergistically with the dorsalizing signal to induce the Spemann Organizer in the dorsal marginal zone. signal steps when expressed at high levels. This would be consistent with a model in which the Spemann Organizer ly would be determined by the co-ordination of a mesodermal inducing signal (by Vg1, Activin or a related molecule) and a dorsalizing signal secreted by the Nieuwkoop Center (Kimelman et al., 1992). Our results suggest that the dorsalizing center in Xenopus is established by a Wnt GSK β-catenin signaling cascade leading to the induction of the transcription factor Siamois. We have confirmed that Wnt signaling requires β-catenin and we have shown that GSK acts upstream of β-catenin in this pathway. This is similar to Zeste-white-3 being upstream of Armadillo in Drosophila, and confirms the existence of a conserved Wnt/Wingless-activated pathway. Furthermore, we have established that Siamois is a downstream target of the Wnt/β-catenin pathway by the following criteria: (1) Siamois can be ectopically activated by Wnt and β-catenin; (2) expression of Siamois in the endogenous Nieuwkoop Center requires free β-catenin, because it is inhibited by C-cadherin expression and (3) the dorsalizing activity of Siamois is largely unaffected by inhibition of β-catenin signaling by C-cadherin, which dramatically blocks signaling by Wnt and GSK-K R. We conclude that Wnt, GSK and β-catenin act in sequence to induce Siamois expression and formation. Although a Wnt-activatable β-catenin signaling pathway appears to be required for the activity of the Nieuwkoop Center, the endogenous secreted factor that initiates this signaling cascade has yet to be identified. The known maternally expressed Wnts are uncertain candidates, because they either have only weak or partial dorsalizing activity (Wnt5a, Moon et

8 46 F. Fagotto, K. Guger and B. M. Gumbiner al., 1993, and Wnt11, Ku and Melton, 1993), or do not appear to be properly localized (Wnt8b, Cui et al., 1995). Furthermore, it has been observed that Siamois expression can occur, at least partially, even when cells are kept dispersed (Lemaire et al., 1995). However, since Wnts are known to remain associated with the cell surface of the secreting cell (Bradley and Brown, 199; Papkoff and Schrywer, 199), it is quite possible that autocrine induction could still take place in isolated cells. It is also conceivable that dorsalization ly may be initiated cell autonomously by activation of an intracellular component of the signaling pathway upstream of β-catenin, such as Dishevelled or GSK. Nevertheless, it appears that if a secreted growth factor is actually involved in the formation of the Nieuwkoop Center, it is likely to be some member of the Wnt family. We are grateful to Dr R. Harland and Dr D. 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