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

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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
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