Background : Ror2 is an orphan receptor, belonging to the Ror family of receptor tyrosine kinases. Although Ror2 has been shown to play crucial roles in developmental morphogenesis, the precise signalling events that Ror2 mediates remain elusive. Since Ror2 possesses an extracellular cysteine-rich domain (CRD) that resembles the Wnt-binding sites of the Frizzled (Fz) proteins, it is conceivable that Ror2 interacts with members of the Wnt family.

The secretion and extracellular transport of Wnt protein are thought to be well-regulated processes. Wnt is known to be acylated with palmitic acid at a conserved cysteine residue (Cys77 in murine Wnt-3a), and this residue appears to be required for the control of extracellular transport. Here, we show that murine Wnt-3a is also acylated at a conserved serine residue (Ser209). Of note, we demonstrated that this residue is modified with a monounsaturated fatty acid, palmitoleic acid. Wnt-3a defective in acylation at Ser209 is not secreted from cells in culture or in Xenopus embryos, but it is retained in the endoplasmic reticulum (ER). Furthermore, Porcupine, a protein with structural similarities to membrane-bound O-acyltransferases, is required for Ser209-dependent acylation, as well as for Wnt-3a transport from the ER for secretion. These results strongly suggest that Wnt protein requires a particular lipid modification for proper intracellular transport during the secretory process.

Recent genetic studies in Drosophila identified a novel noncanonical Wnt pathway, the planar cell polarity (PCP) pathway, that signals via JNK to control epithelial cell polarity in Drosophila. Most recently, a pathway regulating convergent extension movements during gastrulation in vertebrate embryos has been shown to be a vertebrate equivalent of the PCP pathway. However, it is not known whether the JNK pathway functions in this non-canonical Wnt pathway to regulate convergent extension movements in vertebrates. In addition, it is not known whether JNK is in fact activated by Wnt stimulation. Here we show that Wnt5a is capable of activating JNK in cultured cells, and present evidence that the JNK pathway mediates the action of Wnt5a to regulate convergent extension movements in Xenopus. Our results thus demonstrate that the non-canonical Wnt/JNK pathway is conserved in both vertebrate and invertebrate and define that JNK has an activity to regulate morphogenetic cell movements.

The identity and patterning of ventral cell types in the vertebrate central nervous system depends on cell interactions. For example, induction of a specialized population of ventral midline cells, the floor plate, appears to require contact-mediated signalling by the underlying notochord, whereas diffusible signals from the notochord and floor plate can induce ventrolaterally positioned motor neurons. Sonic hedgehog (Shh), a vertebrate hedgehog-family member, is processed to generate two peptides (M(r) 19K and 26/27K) which are secreted by both of these organizing centres. Moreover, experiments in a variety of vertebrate embryos, and in neural explants in vitro, indicate that Shh can mediate floor-plate induction. Here we have applied recombinant Shh peptides to neural explants in serum-free conditions. High concentrations of Shh bound to a matrix induce floor plate and motor neurons, and addition of Shh to the medium leads to dose-dependent induction of motor neurons. All inducing activity resides in a highly conserved amino-terminal peptide (M(r) 19K). Moreover, antibodies that specifically recognize this peptide block induction of motor neurons by the notochord. We propose that Shh acts as a morphogen to induce distinct ventral cell types in the vertebrate central nervous system.

Microphthalmia-associated transcription factor (Mitf) plays a critical role in the development of neural crest-derived melanocytes. Here, we show that exogenously added Wnt-3a protein, an intercellular signaling molecule, up-regulates the expression of endogenous melanocyte-specific Mitf (Mitf-M) mRNA in cultured melanocytes. The melanocyte-specific promoter of the human MITF gene (MITF-M promoter) contains a functional LEF-1-binding site, which is bound in vitro by LEF-1 and confers the preferential expression on a reporter gene in melanocytes and melanoma cells, as judged by the transient transfection assays. Moreover, the LEF-1-binding site is required for the transactivation of a reporter gene by LEF-1, beta-catenin, or their combination. Exogenously added Wnt-3a protein also transactivates the MITF-M promoter via the LEF-1-binding site; this activation was abolished when a dominant-negative form of LEF-1 was coexpressed. These results suggest that Wnt-3a signaling recruits beta-catenin and LEF-1 to the LEF-1-binding site of the MITF-M promoter. Therefore, the present study identifies Mitf-M/MITF-M as a direct target of Wnt signaling.

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Background: Wnt-3a is an intercellular signalling molecule that is involved in a variety of morphogenetic events. However, the molecular mechanisms underlying Wnt-3a signalling are poorly understood. We have sought to establish in vitro systems to assay the activity of this protein and investigate its biological roles.

When Axin, a negative regulator of the Wnt signaling pathway, was expressed in COS cells, it coeluted with glycogen synthase kinase-3b (GSK-3b), b-catenin, and adenomatous polyposis coli protein (APC) in a high molecular weight fraction on gel ®ltration column chromatography. In this fraction, GSK-3b, b-catenin, and APC were co-precipitated with Axin. Although bcatenin was detected in the high molecular weight fraction in L cells on gel ®ltration column chromatography, addition of conditioned medium expressing Wnt3a to the cells increased b-catenin in the low molecular weight fraction. However, Wnt-3a-dependent accumulation of b-catenin was greatly inhibited in L cells stably expressing Axin. Axin also suppressed Wnt-3a-dependent activation of Tcf-4 which binds to b-catenin and acts as a transcription factor. These results suggest that Axin forms a complex with GSK-3b, b-catenin, and APC, resulting in the stimulation of the degradation of bcatenin and that Wnt-3a induces the dissociation of bcatenin from the Axin complex and accumulates bcatenin.