Catenin Signaling Pathway, but Not by Vg1, Activin Or Noggin

Development 124, 453-460 (1997) 453 Printed in Great Britain © The Company of Biologists Limited 1997 DEV9489

Induction of the primary dorsalizing center in Xenopus by the Wnt/GSK/β- catenin 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 10021, USA *Author for correspondence

SUMMARY

The molecular nature of the primary dorsalizing inducing sensitive to inhibition of β-catenin activity and only Wnt event in Xenopus is controversial and several secreted could induce Siamois expression. Therefore, Wnt is able to factors have been proposed as potential candidates: Wnts, induce a bonaﬁde Nieuwkoop Center, while Vg1, Activin Vg1, Activin and Noggin. Recent studies, however, have and Noggin probably induce dorsal structures by a provided new insight into the activity of the dorsalizing different mechanism. region, called the Nieuwkoop Center. (1) The activity of this To order the steps in the Nieuwkoop Center signaling dorsalizing center involves an entire signal transduction cascade, we have tested the relationship between β-catenin pathway that requires maternal β-catenin (Heasman, J., and GSK, a serine-threonine kinase that has been impli- Crawford, A., Goldstone, K., Garner-Hamrick, P., cated in axis formation in a step downstream of Wnt. We Gumbiner, B., McCrea, P., Kintner, C., Noro, C. Y. and found that GSK acts upstream of β-catenin, similar to the Wylie, C. (1994) Cell 79, 791-803). (2) A transcription factor order of these components in the Wingless pathway in with potent dorsalizing activity, Siamois, is expressed Drosophila. We have also examined the relationship within the Nieuwkoop Center (Lemaire, P., Garrett, N. and between the Wnt/β-catenin pathway and Siamois. We show Gurdon, J. B. (1995) Cell 81, 85-94). We have used these that β-catenin induces expression of Siamois and that the two properties of the Nieuwkoop Center to evaluate the free signaling pool of β-catenin is required for normal dorsalizing activity of the four secreted factors Wnt8, Vg1, expression of endogenous Siamois. We conclude that Activin and Noggin. The requirement for β-catenin was the sequence of steps in the signaling pathway is tested by coexpressing a cadherin, which sequesters β- Wnt | GSK | β-catenin→Siamois. 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, Key words: axis formation, dorsoventral patterning, Nieuwkoop 1105-1114). Induction of Siamois expression was detected Center, Spemann Organizer, growth factor, signal transduction, by RT-PCR. Of the four growth factors, only Wnt was Siamois, Xenopus, Wnt, β-catenin, Activin, Noggin

INTRODUCTION Organizer is restricted to the equatorial region, and (2) the cells of the Spemann Organizer themselves become part of the Determination of the dorsoventral axis in Xenopus eggs occurs induced dorsal mesoderm, independently of their origin, while at fertilization by an asymmetric redistribution of an unknown cells expressing Nieuwkoop Center-like activities are not determinant within the vegetal pole (Gerhart et al., 1989). The recruited into axial structures, but maintain their original fates resulting dorsalizing region, called the Nieuwkoop Center, then (endoderm in the case of the vegetal pole) (Smith and Harland, induces the overlying mesodermal cells to become the 1991). According to these criteria, several members of the Wnt Spemann Organizer, which orchestrates gastrulation and the family, as well as Vg1, Activin and Noggin (Christian et al., patterning of axial structures. Various secreted factors have 1991; Smith and Harland, 1991; Sokol et al., 1991; Ku and been shown to have dorsalizing activity, as detected by their Melton, 1993; Thomsen and Melton, 1993; Thomsen et al., abilities to induce a secondary axis when expressed ventrally 1990; Steinbeisser et al., 1993; Smith and Harland, 1992), and to rescue an axis in UV-treated, ventralized embryos. appear to mimic the activity of the primary dorsalizing center, Although such phenotypes are characteristic of both i.e. the Nieuwkoop Center. Despite their common abilities to Nieuwkoop Center and Spemann Organizer activities, these act as dorsalizers, these growth factors have quite different two signaling centers can be distinguished according to two properties. For instance, Vg1 and Activin, both members of the criteria: (1) the activity of the Nieuwkoop Center is maximal TGFβ family, are also mesodermal inducers (Thomsen et al., in the lower vegetal region, while the activity of the Spemann 1990; Green et al., 1992; Dale et al., 1993; Thomsen and 454 F. Fagotto, K. Guger and B. M. Gumbiner

Melton, 1993; Kessler and Melton, 1995), while Noggin is a cadherins (Fagotto et al., 1996), and (2) the ability to activate potent neuralizer (Lamb et al., 1993). Some Wnts (Wnt1, Siamois expression. 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 MATERIALS AND METHODS inducing events, for example patterning of the ventral mesoderm during gastrulation (Wnt8, Christian and Moon, Wnt8 and Noggin expression plasmids have been kindly provided by 1993) and patterning of the central nervous system (Wnt1, Dr R. Harland, UCBerkeley, and Activin and BMP2-Vg1 plasmids by McMahon and Bradley, 1990; Thomas and Cappechi, 1990). Dr D. Melton, Harvard. Siamois (Lemaire et al., 1995) was cloned by Several intracellular signaling molecules, β-catenin, RT-PCR using RNA from stage 10G Xenopus embryos and inserted Glycogen Synthase Kinase-3 (GSK-3) and Dishevelled, have into the pCS2+MT expression vector (Rupp et al., 1994; Turner and Weintraub, 1994). mRNAs were synthesized in vitro using the SP6 also been recently implicated in the activity of the Nieuwkoop RNA polymerase (Promega Corp., Madison, WI) and were dissolved Center (Heasman et al., 1994; Funayama et al., 1995; Guger at the appropriate concentration in DPEC-treated water. and Gumbiner, 1995; He et al., 1995; Pierce and Kimelman, For cadherin coinjection experiments, mRNAs were injected into 1995; Sokol et al., 1995). Interestingly, the Drosophila homo- the vegetal region of a ventral blastomere at the 4- to 8-cell stage logues of these three molecules (Armadillo, Zeste-white-3 and (Fagotto et al., 1996). For this set of experiments, the amount of Disheveled, respectively) are components of a well-character- mRNA for each of the various factors was titrated down to the smallest ized signaling pathway activated by Wingless, a Drosophila amount required for maximal activity, measured as maximal Wnt homologue (Peifer, 1995). One of these intracellular frequency and completeness of secondary axis formation. The amount proteins, β-catenin, has been shown to be required both for of mRNA injected (in pg) was: Wnt8: 10-20; Noggin: 200; Activin: → normal axis formation in Xenopus and for the dorsalizing 7.5-15; BMP2-Vg1: 100-200; GSK-3-K R: 1000-3000; Siamois: activity of Wnt (Heasman et al., 1994). β-catenin is a cyto- 50-100. Higher amounts of Wnt8 cause dorsalization, and thus reduction and disappearance of a body axis. Higher amounts of plasmic protein that associates with cadherin cell-adhesion Activin resulted in uninterpretable phenotypes, probably caused by molecules (Kemler, 1993), but also has an independent intra- generalized mesodermal induction over most of the embryo. 4-5 ng cellular signaling activity (Fagotto et al., 1996). of C-cadherin mRNA were used for coinjections. Embryos were In Drosophila, the signaling activity of the β-catenin allowed to develop at room temperature in 0.1× MMR (Kay and Peng, homologue, Armadillo, is negatively regulated by a 1991) and axis duplication was scored at the neurula stage. The serine/threonine kinase, Zeste-white-3/Shaggy (Peifer, 1995) number of embryos scored in Fig. 1 (pooled from several experiments) and Wingless activates the signaling pathway by antagonizing were (+,−) C-cadherin respectively: Wnt8: (104,95); Noggin: (95,84); Zeste-white-3. Similarly, in Xenopus embryos, the homologue Activin: (68,65); BMP2-Vg1: (76,85); GSK-3-K→R: (46,44); of Zeste-white-3, GSK-3, has ventralizing activity, and a Siamois: (119,125). BMP2-Vg1 is a chimeric molecule that is → processed in the embryo into the active, secreted fragment of Vg1 dominant negative mutant form, GSK-3K R, induces axis → β duplication (He et al., 1995; Pierce and Kimelman, 1995). (Thomsen and Melton, 1993). GSK-3-K R is a mutant of GSK-3 without kinase activity (Pierce and Kimelman, 1995). Overexpression of GSK can inhibit axis induction by Wnt, For histological studies, tailbud-early tadpole stages (stage 25-29) indicating that GSK acts downstream of Wnt, similar to Zeste- were fixed in Dent’s fixative and embedded in gelatin as described white-3 acting downstream of Wingless. (Fagotto and Gumbiner, 1994). Serial 10 µm frozen transverse Another intracellular molecule with strong dorsalizing sections of the entire embryos were collected. Sections were immuno- activity is Siamois, a novel Hox-type transcription factor labeled (Fagotto and Gumbiner, 1994) using the rabbit anti-N-CAM expressed in the region of the Nieuwkoop Center (Lemaire et polyclonal antibody R016 (affinity purified IgG, 2 µg/ml, generous al., 1995). Its potent activity, its transient expression at the gift of Dr U. Rutishauser) and the mouse monoclonal antibody Tor70, beginning of the midblastula transition, and its restriction to a notochord marker (ascites fluid, 1/5000 dilution, Bolce et al., 1992). the dorsal-vegetal region of the late blastula, strongly suggest Both antibodies were detected with FITC-labeled secondary antibodies (each at 50 µg/ml, Molecular Probes, Inc., Eugene, OR, USA). that Siamois is an important component of the Nieuwkoop µ Center. Nuclei were then stained with DAPI (20 g/ml, Molecular Probes, Inc., Eugene, OR, USA) and the yolk was counterstained with 0.1% The Nieuwkoop Center appears not to be simply the site of Eriochrome Black (Aldrich Chemical C0., Milwaukee, WI, USA). secretion of a dorsalizing inducer, but to involve an entire intra- Triple exposure microphotographs were taken under an Axioplan cellular signal transduction pathway. The established require- microscope (Karl Zeiss, Inc., Thornwood, NY, USA) using a 20× ment for β-catenin and the strong evidence of the involvement objective and filter blocks for DAPI, fluoresceine and rhodamine (for of GSK suggest that this pathway is similar to the Drosophila background counterstain). Wingless→Armadillo pathway. However, the order of these For RT-PCR experiments, mRNA was extracted from whole components along the pathway has not been established in embryos (Fig. 7) or dissected dorsal and ventral halves at early Xenopus and the endogenous activator of this pathway has not gastrula stage (stage 10-10G). Animal caps were dissected at stage 8 been identified. The possibility remains that growth factors and incubated for further 2 hours (stage 10) before RNA extraction. other than Wnts are able to activate the pathway. Finally, the mRNA was extracted either using the proteinase K treatment protocol relationship between the novel transcription factor Siamois and (Wilson and Melton, 1994) or the Ultraspec RNA isolation system (Biotecx laboratories, Inc., Houston, TX, USA). Various specific these signaling pathways has not yet been investigated. We mRNAs were detected by RT-PCR as described (Wilson and Melton, have therefore evaluated the dorsalizing activity of four 1994). Primers were: for Siamois: 5′: CGC GGA TCC ATG GCC TAT candidate growth factors, Wnt8, Vg1, Activin and Noggin, GAG GCT GAA ATG GAG and 3′: GCT CTA GAG AAG TCA GTT according to the two newly established properties of the TGG GTA GGG CT (Fig. 6) or 5′: TTG GGA GAC AGA CAT GA Nieuwkoop Center: (1) requirement for β-catenin activity, and 3′: GCT CTA GAG AAG TCA GTT TGG GTA GGG CT (Fig. which can be experimentally blocked by co-expressing 7); for Goosecoid: 5: ACA ACT GGA AGC ACT GGA and 3: TCT Primary dorsalizing induction in Xenopus 455

TAT TCC AGA GGA ACC; for Chordin: 5 ATG CAG TGT CCC (Fig. 3C-F). Similar to previous reports, Noggin, BMP2-Vg1 CCC ATC and 3: GCA GTG CAT AAC TCC GAA; for Noggin: 5: and Activin only induced partial secondary axes, but not ATG GAT CAT TCC CAG TGC and 3: TCT GTG CTT TTT GCT complete axes (Figs 1B-D, 2C-F). These partial axes always CTG; for Xnr3: 5: ATG GCA TTT CTG AAC CTG and 3TCT ACT lacked anterior structures and, in most cases, a differentiated GTC ACA CTG TGA; for EF1: 5: CAG ATT GGT GCT GGA TAT notochord was not detected (Fig. 3C,E,F, see legend of Fig. 3). GC AND 3: AC TGC CTT GAT GAC TCC TAG. However, the partial duplicated axes were otherwise well struc- tured, with a neural tube, dorsal mesoderm (muscle) and an intestinal lumen (Fig. 3C-F). Note that, in the cases in which RESULTS incomplete axis induction was generated by Wnt expression, the secondary axes also often lacked notochord (not shown). Dependence of axis induction by Wnt, Noggin, Vg1 Another indication that the dorsalizing activities of Noggin, and Activin on β-catenin signaling Vg1 and Activin were not sensitive to cadherin inhibition was Since β-catenin is required for normal axis formation the appearance of a second blastoporal lip. This lip, which is (Heasman et al., 1994), we tested which of the putative dor- particularly prominent in BMP2-Vg1 and Activin-expressing salizing inducers Wnt8, Noggin, Vg1 or Activin require active embryos, was still present in embryos co-expressing C- β-catenin signaling. We have previously shown that signaling cadherin (not shown). In contrast, no secondary lip was by β-catenin can be speciﬁcally inactivated by coexpressing observed in embryos co-expressing Wnt and C-cadherin (not cadherins (Fagotto et al., 1996). Indeed, C-cadherin inhibits β- shown). We conclude that Wnt8 is the only one out of the four catenin-induced axis duplication by sequestering β-catenin at growth factors tested that requires β-catenin signaling for axis- the plasma membrane and thus depleting the cytosolic pool of inducing activity. β-catenin that is active in intracellular signal transduction. We therefore used cadherin coexpression as a simple method to inhibit this AB 100 100 step in the endogenous dorsalizing signaling Wnt8 Noggin pathway. mRNAs encoding Wnt8, Noggin, 80 Wnt8 + C-cadherin 80 Noggin + C-cadherin BMP2-Vg1 and Activin were injected into the 60 60 ventral side of early cleaving embryos, either alone or coinjected with C-cadherin mRNA 40 40 (see Materials and Methods for ratios). BMP2- 20 20 % total embryos (0) % total embryos (0) Vg1 is a chimeric molecule that is processed 0 0 into an active, secreted fragment of Vg1 when complete partial vestigial normal complete partial vestigial sec. axis sec. axis normal expressed in Xenopus embryos (Thomsen and C axis D axis 100 100 Melton, 1993). Axis duplication was scored at BMP2-Vg1 Activin the neurula stage (Fig. 1). Also, the histology 80 BMP2-Vg1 + C-cadherin 80 Activin + C-cadherin of secondary axial structures was analyzed 60 60 from serial frozen sections of late tailbud-early tadpole stages (Fig. 3). 40 40 20 20

C-cadherin coexpression strongly inhibited % total embryos

(0) (0) % total embryos (0)(0) Wnt8 axis-inducing activity. While expression 0 0 of Wnt8 alone induced a complete secondary complete partial vestigial normal complete partial vestigial normal Esec. axis axis sec. axis axis axis at high frequency (Figs 1A, 2A, 3A), most F 100 100 embryos expressing both Wnt8 and C-cadherin GSK-3K->R Siamois mRNA developed normally (Figs 1A, 2B, 3B). 80 GSK-3K->R + C-cadherin 80 Siamois + C-cadherin At most, a short protrusion or small pigmented 60 60 area was observed (vestigial axis), indicative of 40 a weak remnant of dorsalizing activity. Thus, 40 20 20 axis induction by Wnt is sensitive to depletion % total embryos (0) (0) % total embryos of cytoplasmic β-catenin, similar to the results 0 0 complete partial complete partial vestigial normal vestigial normal obtained by Heasman et al. (1994) using sec. axis axis sec. axis axis antisense technology. In contrast, C-cadherin coexpression was Fig. 1. Effect of C-cadherin co-expression on axis duplication by Wnt8, Noggin, found to have no detectable effect on axis BMP2-Vg1, Activin, GSK-3-K→R and Siamois. Embryos were scored in four duplication induced by Noggin, BMP2-Vg1 or categories: complete secondary axis (with cement gland), partial secondary axis (i.e. Activin. C-cadherin coexpression did not any secondary axis lacking the cement gland), vestigial axis (very small posterior protrusion or pigmented spot or line) and normal (only one axis). Axis duplication by affect the frequency of axis duplication → induced by these three growth factors (Fig. 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 1B-D). Moreover, the secondary axes induced C-cadherin coexpression. Axis induction by Siamois (F) was only marginally in the presence of C-cadherin were similar to affected: the most anterior structures were often lacking, but the secondary axis were the axes induced by the growth factors alone, still very long, consistently more complete than axis induced by Noggin or BMP2- by both criteria of their external morphology Vg1. The frequency of Siamois-induced axis duplication (complete and partial axis (Fig. 2C-F) and of their internal organization combined) was unchanged in the presence of C-cadherin (70-75%). 456 F. Fagotto, K. Guger and B. M. Gumbiner

We have further confirmed these observations by evaluating Spemann Organizer by Wnt, but not by the other growth the effect of C-cadherin coexpression on the capacity of the factors. various dorsalizing factors to induce the expression of Goosecoid, a marker of the Spemann Organizer (Cho et al., β-catenin is required for the dorsalizing activity of 1991). mRNA encoding the various factors were injected in the GSK-3K→R but not the dorsalizing activity of ventral side, either alone or with C-cadherin. Embryos were Siamois dissected at early gastrula stage (10G) into dorsal and ventral GSK has been implicated in the Wnt signaling pathway leading halves and the expression of Goosecoid was analyzed by RT- to axis induction. GSK-3K→R, a dominant negative mutant PCR. Dorsal halves embryos were used as controls for the form of Xenopus GSK-3, induces axis duplication when normal expression of Goosecoid in the endogenous Spemann expressed in the ventral side of early cleaving embryos (He et Organizer and uninjected ventral halves as negative controls. al., 1995; Pierce and Kimelman, 1995). To order the steps in All four growth factors were able to induce Goosecoid this pathway, we asked whether axis duplication by GSK- expression when injected alone. Coexpression of C-cadherin 3K→R requires free β-catenin. Axis duplication was indeed strongly inhibited the induction of Goosecoid by Wnt8, but not strongly inhibited by C-cadherin co-expression (Fig. 1E). This the induction by Noggin, BMP2-Vg1 or Activin (Fig. 4). finding indicates that GSK acts upstream of β-catenin, similar Therefore, free b-catenin is required for the induction of the to the relationship between Zeste-white-3 and Armadillo in Drosophila. We also tested whether β-catenin was required for axis duplication by Siamois, another factor with Nieuwkoop Center-like dorsalizing activity. In contrast to Wnt8, β-catenin or GSK-3K→R, the axis-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 axis-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 Fig. 2. Examples of axis duplication by various dorsalizing growth determine which of the known dorsalizing factors require β- factors and dependence on β-catenin signaling activity. (A) catenin signaling in the Nieuwkoop Center. A second specific Complete axis duplication, with two cement glands, by Wnt8. characteristic of the Nieuwkoop Center is the expression of the (B) Complete inhibition of Wnt-mediated axis duplication by transcription factor Siamois. We therefore asked which of the coexpression of C-cadherin. (C,E) Partial secondary axes induced dorsalizing factors can induce Siamois expression. mRNAs by Noggin and BMP2-Vg1, respectively. (D,F) No inhibition of axis encoding β-catenin and the four growth factors Wnt8, Noggin, duplication obtained when C-cadherin mRNA is coinjected with BMP2-Vg1 and Activin were injected ventrally, and the Noggin or BMP2-Vg1 mRNA. (G) Complete axis duplication by Siamois. (H) Axis duplication by Siamois is largely unaffected by embryos were dissected at early gastrula stage (the normal C-cadherin coinjections. Although the formation of the most peak of Siamois expression) into dorsal and ventral halves. anterior structures is inhibited, secondary axes are still very long, Siamois expression was analyzed by RT-PCR (Fig. 5A). Dorsal which is in contrast to the strong inhibition of Wnt-induced axis halves were used as internal controls for Siamois expression in duplication (compare to B). the endogenous Nieuwkoop Center and ventral halves from Primary dorsalizing induction in Xenopus 457 uninjected embryos were used as negative controls. Both β- by increasing the levels of C-cadherin, either globally in catenin and Wnt8 induced strong expression of Siamois in the oocytes (Heasman et al., 1994), or specifically in the vegetal ventral halves. However, none of the other secreted factors dorsal side of early embryos (Fagotto et al., 1996), similar to (Noggin, BMP2-Vg1, Activin) induced Siamois, even though the effect resulting from the depletion of maternal β-catenin all of them activated expression of Goosecoid, a marker for the (Heasman et al., 1994). Therefore Siamois expression was Spemann Organizer, which is the overlying induced dorsal analyzed in embryos where β-catenin signaling in the endogen- mesoderm (Cho et al., 1991). Similar injections were made in ous Nieuwkoop Center was blocked by overexpression of C- the animal hemisphere, and animal caps were removed by dis- cadherin. When 5 ng C-cadherin mRNA were injected into the section and analyzed for Siamois expression. Although the two dorsal blastomeres of 4- to 8-cell-stage embryos, Siamois animal hemisphere is normally not involved in the dorsalizing expression was greatly decreased compared to control unin- process, it is a very useful tissue for analysis of mesoderm- jected embryos (Fig. 6). Siamois expression was fully rescued inducing and dorsalizing activities. Again, both Wnt8 and β- in embryos in which β-catenin mRNA (1.5 ng) was coinjected catenin activated Siamois expression, but neither Noggin nor with C-cadherin mRNA, confirming that the inhibition of BMP2-Vg1 induced its expression (Fig. 5B). Therefore, Siamois expression by C-cadherin was specifically due to the although all factors analyzed were able to induce Goosecoid in inhibition of β-catenin signaling. Thus, Siamois expression the Spemann Organizer, only Wnt and β-catenin could induce depends on β-catenin signaling in the Nieuwkoop Center. expression of Siamois, a marker for the Nieuwkoop Center. Siamois induces the expression of markers of the β-catenin requirement for Siamois expression in the Spemann Organizer endogenous dorsalizing center The selective activation of Siamois in the Nieuwkoop Center The experiments described above suggest that Siamois is a by the Wnt/β-catenin pathway and the strong dorsalizing specific target of the Wnt-β-catenin pathway. We therefore activity of Siamois suggest that this pathway may account for asked whether the normal expression of Siamois in the endoge- most of the Nieuwkoop Center activity. To determine whether nous Nieuwkoop Center is dependent upon β-catenin the activity of Siamois is sufficient to induce a Spemann signaling. Normal axis formation can be significantly inhibited 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 C- cadherin 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 Tor70, 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 C- cadherin coinjection resulted in embryos with a normal morphology having a single axis. (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 axis posteriorly. Differentiated notochords detected by Tor70 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). 458 F. Fagotto, K. Guger and B. M. Gumbiner

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 axis induction could be interpreted as resulting from differences in Fig. 4. Effect of C-cadherin co-expression on the induction of the strength of the induction of injected constructs, rather than Goosecoid, a marker of the Spemann Organizer, by Wnt8, Noggin, from fundamental qualitative differences between the effects of BMP2-Vg1, Activin, GSK-3-K→R and Siamois. mRNAs coding for various factors. In the case of BMP2-Vg1, for example, it has the various dorsalizing factors were injected vegetally into one been argued that this exogenously expressed protein is poorly ventral blastomere of 4- to 8-cell-stage embryos, alone (−) or secreted. In fact, another Vg1 chimeric molecule, which is more coinjected with 5 ng C-cadherin mRNA (+C). Ventral halves were efficiently processed, has been reported to cause a more 1 dissected at stage 10 /2 and analyzed for Goosecoid expression by complete axis duplication (Kessler and Melton, 1995). Also, it RT-PCR. Dorsal (D) and ventral (V) halves from uninjected embryos has been reported that BMP2-Vg1 (Thomsen and Melton, 1993) were used respectively as positive and negative controls for the normal expression of Goosecoid. All dorsalizing factors were found and Noggin (Smith and Harland, 1992) can rescue complete axis to ectopically induce Goosecoid when expressed alone. Coexpression in UV-treated embryos, which has been interpreted to mean that of C-cadherin strongly inhibited Goosecoid expression induced by they possess full axis-inducing activity. We have not observed Wnt8 and GSK-K→R, but not by Noggin, BMP2-Vg1, Activin or any compelling qualitative difference in the nature of the Siamois. EF1 is an ubiquitous mRNA used as loading control. 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, whether other markers, Chordin, Xnr3 and Noggin, were notochord or muscle, matches quite faithfully the degree of expressed under similar conditions (Fig. 7). We found that complete induction as assessed by external morphology. Siamois was able to induce ectopic expression of all the Therefore, independent criteria are necessary to characterize the markers tested, suggesting that Siamois is sufficient, at least properties of the various candidate dorsalizing inducers. by the presently available criteria, to induce an entire Spemann Our ﬁndings show that the dorsalizing activities of Vg1, Organizer. A B

DISCUSSION

Several kinds of growth factors are able to induce a secondary body axis 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 Fig. 5. Siamois expression in response to various dorsalizing factors. (A) Ectopic Heasman et al. (1994) is the requirement for β-catenin expression of factors in the vegetal-ventral region. mRNAs were injected in the normal process of dorsalization and axis devel- vegetally into the two ventral blastomeres of 4- to 8-cell-stage embryos. Total opment. We confirm their findings that axis induction amounts of injected mRNAs are indicated. Dorsal (D) and ventral (V) halves by Wnts requires β-catenin, and go on to show that were dissected at stage 10 and analyzed for Siamois and Goosecoid expression none of the other growth factors tested, Noggin, Vg1 by RT-PCR. β-catenin and Wnt8 induce high Siamois expression (compared to or Activin, require β-catenin for their axis-inducing normal expression in the dorsal side). No expression was observed in ventral activities. A second criterion is the induction of the halves from embryos injected with Noggin, Activin or BMP2-Vg1. All these expression of Siamois, a Hox protein with potent axis- factors induced Goosecoid expression in the same embryos. (B) Ectopic expression in the animal hemisphere. mRNAs were injected into the animal pole inducing activity that is normally expressed in the of the four blastomeres of 4- to 8-cell-stage embryos. Total amounts of injected endogenous Nieuwkoop Center (Lemaire et al., mRNAs are indicated. Animal caps were dissected from injected embryos at 1995). Only Wnts, but not Noggin, Vg1 or Activin, stage 8 and were analyzed at stage 10 for Siamois expression. β-catenin and can induce the expression of Siamois when expressed Wnt8 ectopic expression induced Siamois expression in the animal hemisphere, ectopically in the early embryo. Therefore, a Wnt/ β- but Noggin and BMP2-Vg1 did not. Primary dorsalizing induction in Xenopus 459

Wnt

GSK-3 Nieuwkoop Center β-catenin

Siamois

Vg1 X?Activin Fig. 6. Inhibition of the normal expression of endogenous Siamois by Noggin overexpression of C-cadherin in the Nieuwkoop Center. 5 ng C- β cadherin mRNA (C) or 5 ng C-cadherin and 1.5 ng -catenin Spemann mRNAs (C+β) were injected vegetally into both dorsal blastomeres Organizer Goosecoid 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 Fig. 8. Model for the dorsalizing inducting pathway in Xenopus. A complete inhibition of Siamois expression, which could be rescued to Wnt, or Wnt-related factor, initiates a signal transduction pathway in high levels when excess β-catenin was coexpressed with C-cadherin. 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 Activin and Noggin do not require β-catenin and do not induce factor or factors ‘X’, which induces the overlying mesodermal cells to Siamois expression. Therefore, the dorsalizing activity of Vg1, become the Spemann Organizer, expressing Goosecoid and other dorsal mesoderm markers. Vg1, Activin and Noggin can induce a Activin and Noggin probably derives from direct induction of β components of the Spemann Organizer, at sites either down- Spemann Organizer, but they do not activate the -catenin pathway β and Siamois expression. It is possible, however, that one or more of stream of Siamois or parallel with the Wnt/ -catenin/Siamois them could act at the step ‘X’, mediating the induction of the Spemann pathway (Fig. 8). One possibility is that they act at the second Organizer by the Nieuwkoop Center (dotted arrow). Alternatively, Vg1 inducing step, mimicking endogenous factors secreted by the and Activin may act in parallel to the Nieuwkoop Center activity, for Nieuwkoop Center to induce the overlying Spemann Organizer instance by participating in the mesodermal induction, in which case tissue (Figs 4, 5). Another possibility is that these or related they would normally act synergistically with the dorsalizing signal to factors normally act synergistically with the signal emanating induce the Spemann Organizer in the dorsal marginal zone. from the Nieuwkoop Center (Watabe et al., 1995), but may partially bypass the requirement for the Nieuwkoop Center signal steps when expressed at high levels. This would be con- sistent with a model in which the Spemann Organizer normally 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) normal 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, Fig. 7. Ectopic activation of markers of the Spemann Organizer by which dramatically blocks signaling by Wnt and GSK-K→R. Siamois. Siamois mRNA was injected vegetally in one ventral We conclude that Wnt, GSK and β-catenin act in sequence to blastomere at the 4- to 8-cell stage. Embryos were dissected at stage induce Siamois expression and axis formation. 10-10G (early gastrula) into dorsal and ventral halves and markers of Although a Wnt-activatable β-catenin signaling pathway the Spemann Organizer were analyzed by RT-PCR. Gsc, Goosecoid; Xnr3, Xenopus nodal-related-3. Ventral halves from uninjected appears to be required for the activity of the Nieuwkoop Center, embryos (V) were used as negative controls, and dorsal halves (D) as the endogenous secreted factor that initiates this signaling positive controls. Ventral ectopic expression of Siamois (Sia) induced cascade has yet to be identified. The known maternally expression of all the markers tested, often to levels similar to the expressed Wnts are uncertain candidates, because they either levels found in the dorsal side. have only weak or partial dorsalizing activity (Wnt5a, Moon et 460 F. Fagotto, K. Guger and B. M. Gumbiner al., 1993, and Wnt11, Ku and Melton, 1993), or do not appear Heasman, J., Crawford, A., Goldstone, K., Garner-Hamrick, P., Gumbiner, to be properly localized (Wnt8b, Cui et al., 1995). Furthermore, B., McCrea, P., Kintner, C., Noro, C. Y. and Wylie, C. (1994). β it has been observed that Siamois expression can occur, at least Overexpression of cadherins and underexpression of -catenin inhibit dorsal mesoderm induction in early Xenopus embryos. Cell 79, 791-803. partially, even when cells are kept dispersed (Lemaire et al., Kay, B. K. and Peng, H. B. (1991). Xenopus laevis: Practical Uses in Cell and 1995). However, since Wnts are known to remain associated Molecular Biology. In Methods in Cell Biology. Vol. 36. New York: with the cell surface of the secreting cell (Bradley and Brown, Academic Press, Inc. 1990; Papkoff and Schrywer, 1990), it is quite possible that Kemler, R. (1993). From cadherins to catenins: cytoplasmic protein interactions and regulation of cell adhesion. Trends in Genetics 9, 317-321. autocrine induction could still take place in isolated cells. It is Kessler, D. S. and Melton, D. A. (1995). Induction of mesoderm by soluble, also conceivable that dorsalization normally may be initiated mature Vg1 protein. Development 121, 2155-2164. cell autonomously by activation of an intracellular component Kimelman, D., Christian, J. L. and Moon, R. T. (1992). Synergistic of the signaling pathway upstream of β-catenin, such as Dishev- principles of development: overlapping patterning system in Xenopus elled or GSK. Nevertheless, it appears that if a secreted growth mesoderm induction. Development 116, 1-9. Ku, M. and Melton, D. A. (1993). 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