SUMMARY Canonical Wnt signaling requires inhibition of Glycogen Synthase Kinase 3 (GSK3) activity, but the molecular mechanism by which this is achieved remains unclear. Here we report that Wnt signaling triggers the sequestration of GSK3 from the cytosol into multivesicular bodies (MVBs), so that this enzyme becomes separated from its many cytosolic substrates. Endocytosed Wnt co-localized with GSK3 in acidic vesicles positive for endosomal markers. After Wnt addition, endogenous GSK3 activity decreased in the cytosol, and GSK3 became protected from protease treatment inside membrane-bounded organelles. Cryoimmuno electron microscopy showed that these corresponded to multivesicular bodies. Two proteins essential for MVB formation, HRS/Vps27 and Vps4, were required for Wnt signaling. The sequestration of GSK3 extended the half-life of many other proteins in addition to β-Catenin, including an artificial Wnt-regulated reporter protein containing GSK3 phosphorylation sites. We conclude that multivesicular endosomes are essential components of the Wnt signal transduction pathway.

SummaryA Xenopus gene whose expression can be activated by the organizer-specific homeobox genes goosecoid and Xnot2 was isolated by differential screening. The chordin gene encodes a novel protein of 941 amino acids that has a signal sequence and four Cys-rich domains. The expression of chordin starts in Spemann's organizer subsequent to that of goosecoid, and its induction by activin requires de novo protein synthesis. Microinjection of chordin mRNA induces twinned axes and can completely rescue axial development in ventralized embryos. This molecule is a potent dorsalizing factor that is expressed at the right time and in the right place to regulate cell-cell interactions in the organizing centers of head, trunk, and tail development.

Chordin (Chd) is an abundant protein secreted by Spemann organizer tissue during gastrulation. Chd antagonizes signaling by mature bone morphogenetic proteins (BMPs) by blocking binding to their receptors. Recombinant Xenopus Chd binds to BMP-4 with high affinity (KD, 3 x 10(-10) M), binding specifically to BMPs but not to activin or TGF-beta1. Chd protein is able to dorsalize mesoderm and to neuralize ectoderm in Xenopus gastrula explants at 1 nM. We propose that the noncell-autonomous effects of Spemann's organizer on dorsoventral patterning are executed in part by diffusible signals that directly bind to and neutralize ventral BMPs during gastrulation.

We review the current status of research in dorsal-ventral (D-V) patterning in vertebrates. Emphasis is placed on recent work on Xenopus, which provides a paradigm for vertebrate development based on a rich heritage of experimental embryology. D-V patterning starts much earlier than previously thought, under the influence of a dorsal nuclear β-Catenin signal. At mid-blastula two signaling centers are present on the dorsal side: The prospective neuroectoderm expresses bone morphogenetic protein (BMP) antagonists, and the future dorsal endoderm secretes Nodal-related mesoderminducing factors. When dorsal mesoderm is formed at gastrula, a cocktail of growth factor antagonists is secreted by the Spemann organizer and further patterns the embryo. A ventral gastrula signaling center opposes the actions of the dorsal organizer, and another set of secreted antagonists is produced ventrally under the control of BMP4. The early dorsal β-Catenin signal inhibits BMP expression at the transcriptional level and promotes expression of secreted BMP antagonists in the prospective central nervous system (CNS). In the absence of mesoderm, expression of Chordin and Noggin in ectoderm is required for anterior CNS formation. FGF (fibroblast growth factor) and IGF (insulinlike growth factor) signals are also potent neural inducers. Neural induction by anti-BMPs such as Chordin requires mitogen-activated protein kinase (MAPK) activation mediated by FGF and IGF. These multiple signals can be integrated at the level of Smad1. Phosphorylation by BMP receptor stimulates Smad1 transcriptional activity, whereas phosphorylation by MAPK has the opposite effect. Neural tissue is formed only at very low levels of activity of BMP-transducing Smads, which require the combination of both low BMP levels and high MAPK signals. Many of the molecular players that regulate D-V patterning via regulation of BMP signaling have been conserved between Drosophila and the vertebrates.

BMP receptors determine the intensity of BMP signals via Smad1 C-terminal phosphorylations. Here we show that a finely controlled cell biological pathway terminates this activity. The duration of the activated pSmad1(Cter) signal was regulated by sequential Smad1 linker region phosphorylations at conserved MAPK and GSK3 sites required for its polyubiquitinylation and transport to the centrosome. Proteasomal degradation of activated Smad1 and total polyubiquitinated proteins took place in the centrosome. Inhibitors of the Erk, p38, and JNK MAPKs, as well as GSK3 inhibitors, prolonged the duration of a pulse of BMP7. Wnt signaling decreased pSmad1(GSK3) antigen levels and redistributed it from the centrosome to cytoplasmic LRP6 signalosomes. In Xenopus embryos, it was found that Wnts induce epidermis and that this required an active BMP-Smad pathway. Epistatic experiments suggested that the dorsoventral (BMP) and anteroposterior (Wnt/GSK3) patterning gradients are integrated at the level of Smad1 phosphorylations during embryonic pattern formation.

Frzb-1 is a secreted protein containing a domain similar to the putative Wnt-binding region of the frizzled family of transmembrane receptors. Frzb-1 is widely expressed in adult mammalian tissues. In the Xenopus gastrula, it is expressed and regulated as a typical Spemann organizer component. Injection of frzb-1 mRNA blocks expression of XMyoD mRNA and leads to embryos with enlarged heads and shortened trunks. Frzb-1 antagonizes the effects of Xwnt-8 ectopic expression in a non-cell-autonomous manner. Cultured cells transfected with a membrane-tethered form of Wnt-1 bind epitope-tagged Frzb-1 in the 10(-10) M range. The results strengthen the view that the Spemann organizer is a source of secreted inhibitory factors.

An abundant cDNA enriched in Spemann's organizer, cerberus, was isolated by differential screening. It encodes a secreted protein that is expressed in the anterior endomesoderm. Microinjection of cerberus mRNA into Xenopus embryos induces ectopic heads, and duplicated hearts and livers. The results suggest a role for a molecule expressed in the anterior endoderm in the induction of head structures in the vertebrate embryo.

A central question in biology is how a small number of cell-cell signaling pathways are integrated during development, leading to the differentiation of many cell types. The induction of the central nervous system (CNS) has been a primary focus of embryological research for many years (Spemann 1938;Harland 2000;Wilson and Edlund 2001;Stern 2002). In Xenopus, bone morphogenetic protein (BMP) antagonists such as Noggin, Chordin, and Follistatin inhibit signaling by BMP receptors, causing neural induction through a default pathway (Weinstein and Hemmati-Brivanlou 1999;De Robertis et al. 2000). However, active signals such as fibroblast growth factors (FGFs) and insulin-like growth factors (IGFs) are also potent neural inducers (Hongo et al. 1999;Hardcastle et al. 2000;Streit et al. 2000;Wilson et al. 2000;Pera et al. 2001; RichardParpaillon et al. 2002). Here we show that Chordin, FGF8, and IGF2 have similar phenotypic effects on CNS development and synergize with each other. In loss-of-function experiments, FGF and IGF signaling are required for neurogenesis by Chordin. Microinjection of phosphorylation-deficient mutants of Smad1 suggests that antagonism of BMP receptor activity is not the sole regulator of neural induction in the embryo. In Xenopus, FGF8 and IGF2 induce phosphorylation of the linker region of Smad1 via mitogen-activated protein kinase (MAPK), which further inhibits Smad1 activity (Kretzschmar et al. 1997). The results suggest a common molecular mechanism for the action of diverse neural inducers in vertebrate development, in which multiple signaling inputs lead to inhibition of the BMP pathway through the regulation of Smad phosphorylation. Results and DiscussionThe unexpected finding that IGF mRNA can induce neural differentiation (Pera et al. 2001) prompted us to compare the phenotypic effects of various neural inducers. First we compared the effects of the BMP antagonist Chordin (Chd), IGF2, or FGF8 on neural plate formation. Careful titration of microinjected mRNAs revealed that each agent was able to expand the neural plate (Fig. 1A-D). At lower doses, Chd, IGF2, and FGF8 induced ectopic sensory neurons, marked by N-tubulin, in the epidermis (Fig. 1E-H). Interestingly, genetic studies in zebrafish have shown that differentiation of these lateral-most sensory neurons is controlled by a gradient of BMP signals in the ectoderm (Nguyen et al. 2000).To determine whether these signaling pathways synergized in vivo, we microinjected recombinant proteins into the blastocoele cavity of the embryo ( Fig. 1I-P; Cooke and Smith 1989). Co-injection of Chd and IGF2 proteins cooperated, leading to dorsalized embryos with large cement glands, shortened axes, and expanded dorso-anterior structures (Fig. 1L). A synergism was also observed between Chd and FGF8, resulting in the induction of abundant ectopic neurons in the epidermis (Fig. 1P). Although IGF2 and FGF8 have similar effects, we noted differences as well. IGF2 induces prominent eye and cement gland structures (Pera et al. 2001), whereas FGF8 induce...