The correct attachment of a subset of muscles in the Drosophila embryo requires the expression and function of the RYK subfamily receptor tyrosine kinase gene derailed (drl). A second RYK homolog, doughnut (dnt), has been isolated from Drosophila. The Dnt protein exhibits 60% amino acid identity to Drl, and is structurally as similar to the mammalian RYK (for related to tyrosine kinase) proteins as is Drl, indicating an ancient duplication event. dnt is expressed in dynamic patterns in the embryonic epidermis; it is found at high levels in epithelia adjacent to cells that are invaginating the interior of the embryo, including ventral furrow, cephalic furrow, fore- and hind-gut, optic lobe and tracheal pits. dnt is capable of a partial rescue of the muscle attachment defect of drl-/- embryos, indicating that it encodes a receptor with a related and significantly overlapping biochemical function (Oates, 1998).

Wnt proteins are intercellular signals that regulate various aspects of animal development. In C. elegans, mutations in lin-17, a Frizzled-class Wnt receptor, and in lin-18 affect cell fate patterning in the P7.p vulval lineage. lin-18 encodes a member of the Ryk/Derailed family of tyrosine kinase-related receptors, found to function as Wnt receptors. Members of this family have nonactive kinase domains. The LIN-18 kinase domain is dispensable for LIN-18 function, while the Wnt binding WIF domain is required. Wnt proteins LIN-44, MOM-2, and CWN-2 redundantly regulate P7.p patterning. Genetic interactions indicate that LIN-17 and LIN-18 function independently of each other in parallel pathways, and different ligands display different receptor specificities. Thus, two independent Wnt signaling pathways, one employing a Ryk receptor and the other a Frizzled receptor, function in parallel to regulate cell fate patterning in the C. elegans vulva (Inoue, 2004).

Since lin-44(null) enhances lin-18(null) but not lin-17(null), lin-44 must function in parallel to lin-18. Similarly, since mom-2(null) enhances lin-17(null), mom-2 must function in parallel to lin-17. Based on these results, it is proposed that LIN-44 preferentially functions as the ligand for LIN-17/Frizzled and MOM-2 preferentially functions as the ligand for LIN-18/Ryk. Since lin-44 and mom-2 single mutant phenotypes are weaker than those of lin-17 and lin-18, each receptor likely transduces additional signals (including LIN-44/LIN-18 and MOM-2/LIN-17 combinations as well as CWN-2). A weak enhancement of lin-18(e620) by mom-2(RNAi) supports this possibility. The results do not rule out the possibility that LIN-44 or MOM-2 signals through a third pathway. However, the complete reversal of the P7.p orientation observed in the lin-17; lin-18 double mutant suggests that the two receptors account for most of the P7.p orienting activity. LIN-17 and LIN-44 are also required for other fate specifications in C. elegans, suggesting that LIN-17 acts as a LIN-44 receptor in multiple tissues. Sequence analysis suggests that CWN-2 is the ortholog of Wnt5, the ligand for Derailed in Drosophila. Therefore, the involvement of CWN-2 is consistent with it functioning as a LIN-18 ligand, although it was not possible to resolve the receptor specificity for this ligand. The orthology relationship of MOM-2 is not clear. MOM-2/Wnt and MOM-5/Frizzled are required for endoderm induction. However, no evidence of MOM-5 involvement in P7.p orientation was found, and LIN-18 is not required for endoderm induction (Inoue, 2004).

The C. elegans vulva is comprised of highly similar anterior and posterior halves that are arranged in a mirror symmetric pattern. The cell lineages that form each half of the vulva are identical, except that they occur in opposite orientations with respect to the anterior/posterior axis. Most vulval cell divisions produce sister cells that have asymmetric levels of POP-1 and that the asymmetry has opposite orientations in the two halves of the vulva. lin-17 (Frizzled type Wnt receptor) and lin-18 (Ryk/Derailed family) regulate the pattern of POP-1 localization and cell type specification in the posterior half of the vulva. In the absence of lin-17 and lin-18, posterior lineages are reversed and resemble anterior lineages. These experiments suggest that Wnt signaling pathways reorient cell lineages in the posterior half of the vulva from a default orientation displayed in the anterior half of the vulva (Deshpande, 2005).

The orientation of asymmetric cell division contributes to the organization of cells within a tissue or organ. For example, mirror-image symmetry of the C. elegans vulva is achieved by the opposite division orientation of the vulval precursor cells (VPCs) flanking the axis of symmetry. This study characterized the molecular mechanisms contributing to this division pattern. Wnts MOM-2 and LIN-44 are expressed at the axis of symmetry and orient the VPCs toward the center. These Wnts act via Fz/LIN-17 and Ryk/LIN-18, which control beta-catenin localization and activate gene transcription. In addition, VPCs on both sides of the axis of symmetry possess a uniform underlying 'ground' polarity, established by the instructive activity of Wnt/EGL-20. EGL-20 establishes ground polarity via a novel type of signaling involving the Ror receptor tyrosine kinase CAM-1 and the planar cell polarity component Van Gogh/VANG-1. Thus, tissue polarity is determined by the integration of multiple Wnt pathways (Green, 2008).

These results describe the contributions of multiple Wnt pathways to the orientation of cell polarity in the C. elegans vulval epithelium. Because no factor required for the posterior orientation of P5.p or P7.p had previously been identified, this orientation was thought to be signaling independent or 'default'. However, when a new approach was used to reduce Wnt levels in a spatiotemporally controlled manner (overexpression of Ror/CAM-1, a Wnt sink), the VPCs displayed instead a randomized orientation, which is likely to be the true default. The posterior orientation seen in the absence of Fz/lin-17 and Ryk/lin-18 depends on the instructive activity of Wnt/EGL-20. This polarity is referred to as 'ground' polarity. In response to centrally located Wnt/MOM-2 (and possibly Wnt/LIN-44), the receptors Fz/LIN-17 and Ryk/LIN-18 orient P5.p and P7.p toward the center. This reorientation of P7.p, 'refined' polarity, provides the mirror-image symmetry required for a functional organ (Green, 2008).

That P7.p is oriented toward the center in wild-type worms suggests that Wnts LIN-44 and MOM-2 have a greater ability to affect P7.p orientation than does EGL-20. Although the posterior-anterior EGL-20 gradient reaches the VPCs, EGL-20 levels may be much lower here than the levels of Wnts secreted from the nearby AC. Indeed, it was found that local expression of egl-20 in the AC can overcome the effects of distally expressed egl-20. lin-44 is expressed in the tail in addition to the AC but has not been shown to have long-range activity. It is thus possible that this posterior source of lin-44 does not affect P7.p orientation and that LIN-44, in addition to MOM-2, acts as a central cue (Green, 2008).

LIN-17 and LIN-18 were previously reported to reorient P7.p and to reverse the AP pattern of nuclear TCF/POP-1 levels in P7.p daughters. This study extended knowledge of the signaling downstream of Fz/LIN-17 and Ryk/LIN-18 by showing that these receptors control the asymmetric localization of two β-catenins, SYS-1 and BAR-1, the first evidence that Ryk proteins regulate β-catenin. Although asymmetric localization of SYS-1 suggests involvement of the Wnt/β-catenin asymmetry pathway, disruption of pathway components either did not cause a P-Rvl phenotype (lit-1(rf)) or caused only a weakly penetrant P-Rvl phenotype [pop-1(RNAi), sys-1(rf), and wrm-1(rf)], making the function of the Wnt/β-catenin asymmetry pathway in refined polarity unclear. LIN-17 and LIN-18 were also shown to activate transcription in the proximal VPC daughters. Yet, this transcription is not required for P7.p reorientation, since transcriptional states observed by POPTOP, a reporter of Wnt target genes, do not always correspond with the morphological phenotype. Therefore, refined polarity may be largely independent of BAR-1 or the Wnt/β-catenin asymmetry pathway and instead be analagous to the spindle reorientation of the EMS cell during C. elegans embryogenesis, in which Wnt signaling affects the cytoskeleton independent of Wnt's effect on gene expression (Green, 2008).

What then, is the purpose of the Wnt/β-catenin asymmetry pathway in the VPCs? The weakly penetrant A-Rvl phenotype seen in wrm-1(rf) and lin-17(lf); lit-1(lf) worms, combined with the observation that EGL-20 regulates SYS-1 asymmetry, suggests that the Wnt/β-catenin asymmetry pathway functions in ground polarity. Therefore, both ground and refined polarity may converge on regulation of these components, although they are not absolutely required for refined polarity. Because the localization of Wnt/β-catenin asymmetry pathway components in ground polarity matches the reiterative pattern seen in most other asymmetric cell divisions in C. elegans, it is hypothesized that localization of these components is initially established as part of a global anterior-posterior polarity. It is likely that LIN-17 and LIN-18 overcome ground polarity by inhibiting the Wnt/β-catenin asymmetry pathway, a scenario consistent with the ability of lit-1(rf) to suppress lin-17(lf) and lin-18(lf) mutations (Green, 2008).

Remarkably, it is only by peeling back the layer of refined polarity that ground polarity can be observed and manipulated. By doing so, it was found that Wnt/EGL-20, expressed from a distant posterior source, imparts uniform AP polarity to the field of VPCs via a new pathway involving Van Gogh/vang-1, a core PCP pathway component. It is noteworthy that Fz is also a core PCP pathway component, yet it does not seem to be involved in EGL-20 signaling via VANG-1. This is not incompatible with other descriptions of PCP. For example, in the Drosophila wing, Van Gogh and Fz antagonize each other and cause wing hairs to orient in opposite directions. The molecular mechanism by which VANG-1 functions in ground polarity is unknown; however, regulation of SYS-1 by VANG-1 provides evidence that the pathway involving egl-20 and vang-1is associated with the Wnt/β-catenin asymmetry pathway (Green, 2008).

A major difference between VPC orientation in C. elegans and PCP in Drosophila is that no Wnt has been directly implicated in Drosophila PCP. Therefore, VPC orientation may be more similar to some forms of PCP in vertebrates. For example, Wnts are believed to act as permissive polarizing factors during vertebrate convergent extension. Also, VPC orientation is strikingly similar to hair cell orientation in the utricular epithelia of the mammalian inner ear, wherein hair cells flanking the axis of symmetry are oriented in opposite directions. In this system, both medial and lateral hair cells possess a uniform underlying polarity as evidenced by asymmetric localization of Prickle, a core PCP pathway component, to the medial side of cells in both populations. Van Gogh is required for proper Prickle asymmetry, perhaps similarly to the role of vang-1 in ground polarity of the VPCs. It is not understood how the position of the utricular axis of symmetry is determined, but the similarities between these two systems suggest that it may represent a local source of Wnt (Green, 2008).

By moving the source of EGL-20 from the posterior to the anterior side of P7.p and thereby reversing P7.p orientation, this study showed that EGL-20 acts as a directional cue. Although it is not presently clear if the pathway involving egl-20 and vang-1 is mechanistically similar to the PCP pathway described in Drosophila and vertebrates, the result nonetheless provides a long-sought example of a Wnt that acts instructively via a PCP pathway component. Detailed description of the subcellular localization of Van Gogh/VANG-1 and other PCP pathway components in the VPCs will be required to make meaningful comparisons between VPC orientation and established models of PCP (Green, 2008).

In addition to vang-1, a role of Ror/cam-1 in ground polarity was identified. The results provide the first evidence that Ror proteins interpret directional Wnt signals, as well as the first evidence that they interact with Van Gogh. Although a Xenopus Ror homolog, Xror2, was previously described to function in PCP during convergent extension, a recent report indicates that the involvement of Xror2 in convergent extension (CE) is actually via a different pathway. In response to Wnt5a, Xror2 activates JNK by a mechanism requiring Xror2 kinase activity. In contrast to Wnt5a/Xror2 signaling, Ror/CAM-1 function in ground polarity does not require JNK. Therefore, the ground polarity pathway involving Wnt/EGL-20, Ror/CAM-1, and Van Gogh/VANG-1 may be a new type of Wnt signaling (Green, 2008).

Using C. elegans vulva development as a model, this study showed that multiple coexisting Wnt pathways with distinct ligand specificities and signaling mechanisms act in concert to regulate the polarity of individual cells during their assembly into complex structures (Green, 2008).

By using the polymerase chain reaction with degenerate oligonucleotides based on highly conserved motifs held in common between all members of the protein tyrosine kinase (PTK) family, a PTK-related sequence was isolated from murine peritoneal macrophage cDNA. Full-length clones have been isolated that encompass the entire coding region of the mRNA, and the predicted amino acid sequence indicates that the protein encoded has the structure of a growth factor receptor PTK (RTK). This molecule has been dubbed RYK (for related to tyrosine kinase). The RYK-encoded protein bears a transmembrane domain, with a relatively small (183 amino acid) extracellular domain, containing five potential N-linked glycosylation sites. The intracellular domain of RYK is unique among the broader family of RTKs and has several unusual sequence idiosyncrasies in some of the most highly conserved elements of the PTK domain. These sequence differences call into question the potential catalytic activity of the RYK protein (Hovens, 1992).

A cDNA encoding the human homolog of mouse RYK (related to receptor tyrosine kinases) has been cloned from an interleukin 1 (IL-1)-stimulated human hepatoma cDNA library by cross-species hybridization using the mouse RYK cDNA as a probe. The sequence of the 3067-bp cDNA clone encoding human RYK predicts a transmembrane protein with a cytoplasmic domain that contains the consensus sequences (subdomains I-XI) of the protein tyrosine kinase (PTK) family. The highly conserved motif -D-F-G- (subdomain VII) of the catalytic domain of other receptor-type tyrosine kinases is altered to -D-N-A- in human RYK. In addition, a number of other changes are found in the ATP binding site (subdomains I and II) and the motif [-I-H-R-D-L-A-A-R-N-] found in subdomain VI. Comparison of the human and mouse RYK sequences shows a 92% conservation at the nucleotide level and 97% at the amino acid level. There was no significant homology between the extracellular domain of RYK and the other families of receptor tyrosine kinases described to date. RYK therefore appears to define a new subclass of receptor-type tyrosine kinases whose structure has remained highly conserved across species (Stacker, 1993).

Degenerate primers designed from conserved tyrosine kinase domains were used to identify and clone a novel receptor-like molecule (designated Nbtk-1) from an NB41 mouse neuroblastoma cell line. Nbtk-1 is related to the met proto-oncogene family of tyrosine kinase receptors. Transcripts of approximately 2.1 and 2.6 kb have been found in mouse cell lines and one transcript of approximately 3 kb in human cell lines and in a wide range of primary human tumors, such as neuroblastomas, primitive neuroectodermal tumors (PNETs), Wilms' tumors, and melanomas and in the corresponding normal human tissues. These observations suggest that Nbtk-1 may have important roles in normal and tumor cell growth (Maminta, 1992).

The gene encoding the murine RYK growth factor receptor protein tyrosine kinase has been mapped by genetic linkage analysis with recombinant inbred strains of mouse. Two distinct Ryk loci (Ryk-1 and Ryk-2) have been identified. Ryk-1 maps to Chromosome (Chr) 9, whereas Ryk-2 maps to Chr 12. A similar arrangement of RYK-related loci has previously been determined in the human. Synteny has already been established between murine Chr 9 in the region of Ryk-1, and human chromosome 3q11-12, the location of the human RYK-1 gene. However, the Ryk-2/RYK-2 loci on murine Chr 12 and human Chr 17p13.3 define a new region of synteny (Gough, 1995).

By screening for expressed sequences with conserved tyrosine kinase catalytic domains, an attempt was made to isolate novel receptor tyrosine kinases that may play roles in hematopoietic development. Among the known tyrosine kinases identified in this screen, a gene was found with characteristics of a receptor tyrosine kinase but unusual motifs in the catalytic domain. This gene is identical to ryk as described independently by other investigators. Chromosomal fluorescence in situ hybridization localization of human ryk was clarified by using monochromosomal hybrids, a process that places the gene as a single locus in 3q22. Although Northern analysis reveals widespread expression in adult mouse tissues, ryk expression is not ubiquitous. Expression increases in bone marrow cells from mice treated with 5-fluorouracil. Northern analysis on cell lines indicates expression in CD3-, CD4-, CD8- T cells (at a low level), pre-T cells, thymic epithelial cells, and mature myeloid cells, but not myeloid precursors or B cell precursors. Expression analysis with the use of RT-PCR on mouse bone marrow cells separated on the basis of cell surface markers (B220, CD4, CD8, Gr-1, Mac-1) reveals that this receptor is expressed in differentiated cells (Lin+) but is not expressed in the precursor cells (Lin-). Flow cytometric analysis with a monospecific anti-Ryk antibody demonstrates that Ryk+ cells constitute 36.7% and Lin+/Ryk+ cells constitute 33.7% of low density bone marrow cells, whereas Ryk+ cells represent only 0.3% of the Lin- population. It is concluded that during hematopoietic development, ryk expression is regulated by lineage commitment and stage of maturation (Simoneaux, 1995).

Protein tyrosine kinases play an important role in cellular metabolism as key components of signal transduction pathways. An unusual receptor tyrosine kinase, H-RYK, has been isolated from a complimentary DNA library of SKOV-3, an epithelial ovarian cancer cell line, using a polymerase chain reaction-mediated approach. The primary structure of the predicted amino acid sequence of the protein shows a novel NH2-terminal region. The catalytic region shows homology to other tyrosine kinases, the closest homology being with v-sea (39%). A significant alteration in the catalytic domain is that the highly conserved 'DFG' triplet in subdomain VII is altered to 'DNA'. The gene was mapped to chromosome 3q22. Northern analysis has determined that a single transcript of 3.0 kb is expressed in heart, brain, lung, placenta, liver, muscle, kidney, and pancreas, with maximal expression in skeletal muscle. In situ hybridization analysis on human tissues demonstrates localization of message in the epithelial and stromal compartment of tissues such as brain, lung, colon, kidney, and breast. There is minimal to absent expression of H-RYK on the surface epithelium of ovaries. In benign and borderline tumors of the ovary, there is expression in the stromal compartment. However, in malignant tumors there is increased expression predominantly confined to the epithelium. Polyclonal antisera raised against synthetic peptides recognize a 100-kD protein in ovarian cancer cells and other cell lines. In contrast to other receptor tyrosine kinases, the receptor does not phosphorylate in an in vitro kinase assay. The expression of this unusual receptor tyrosine kinase in epithelial ovarian cancer suggests that it may be involved in tumor progression; this is an area in need of further investigation (Wang, 1996).

Receptor tyrosine kinase RYK is a mammalian homolog of Drosophila Derailed, which is involved in learning and memory and in axon guidance. A rat ryk gene has been cloned and its expression pattern in the central nervous system characterized. Northern blot analysis of the whole brain reveals that the RYK mRNA is abundant during the period from 13 to 18 embryonic days (E13-18); it decreases by E20. In the postnatal brain, the RYK signal is higher in postnatal one week (P1W) cerebrum and in P2W cerebellum than in later stages. In situ hybridization reveals that RYK is expressed throughout the central nervous system, mainly in the ventricular zone on E11 and E13. On E18 and E20, the remarkable level of RYK mRNA is detected in the ventricular zone as well as in the cortical plate of the forebrain. These two regions overlap the immunoreactive areas of nestin and MAP2, a neural stem cell marker and a mature neural marker, respectively. Moreover, the double-labeling analysis shows that the same cells express both RYK and nestin in the ventricular zone. In the postnatal brain, RYK is predominantly expressed in neurons of various regions. These observations suggest that RYK plays a contributory role as a multifunctional molecule in the differentiation and maturation of neuronal cells in the central nervous system (Kamitori, 1999).

The Ryk receptor belongs to the atypical receptor tyrosine kinase family. It is a new member of the family of Wnt receptor proteins. However, the molecular mechanisms by which the Ryk receptor functions remain unknown. Mammalian Ryk, unlike the Drosophila Ryk homolog Derailed, functions as a coreceptor along with Frizzled for Wnt ligands. Ryk also binds to Dishevelled, through which it activates the canonical Wnt pathway, providing a link between Wnt and Dishevelled. Transgenic mice expressing Ryk siRNA exhibit defects in axon guidance, and Ryk is required for neurite outgrowth induced by Wnt-3a and in the activation of T cell factor (TCF) induced by Wnt-1. Thus, Ryk appears to play a crucial role in Wnt-mediated signaling (Lu, 2004).

Ryk siRNA mice have defects in axon guidance of craniofacial motor nerves, ophthalmic nerves, and other nerves, suggesting an essential role of Ryk in axon guidance. Although there is no obvious deficiency in dorsal root ganglion neurite outgrowth in Ryk siRNA transgenic mice, dorsal root ganglion explants isolated from Ryk siRNA mice exhibit defects in neurite outgrowth in response to Wnt-3a stimulation. The lack of deficiency in DRG neurite outgrowth in Ryk siRNA mice is probably because NGF and other growth factors are also involved in inducing neurite outgrowth in vivo. The fact that the Wnt-3a-induces neurite outgrowth of dorsal root ganglion explants is inhibited in Ryk siRNA mice provides strong evidence that there is a functional interaction between Wnt and Ryk in neurite outgrowth (Lu, 2004).

Ryk is a transmembrane receptor tyrosine kinase (RTK). It functions as a receptor of Wnt proteins required for cell-fate determination, axon guidance, and neurite outgrowth in different organisms; however, the molecular mechanism of Ryk signaling is unknown. This study shows that Ryk is cleaved, permitting the intracellular C-terminal fragment of Ryk to translocate to the nucleus in response to Wnt3 stimulation. The cleaved intracellular domain of Ryk is required for Wnt3-induced neuronal differentiation in vitro and in vivo. These results demonstrate an unexpected mechanism of signal transduction for Ryk as a Wnt receptor, in which the intracellular domain itself functions as the transducing molecule to bring extracellular signals from the cell surface into the nucleus, to regulate neural progenitor cell differentiation (Lyu, 2008).

Guidance cues along the longitudinal axis of the CNS are poorly understood. Wnt proteins attract ascending somatosensory axons to project from the spinal cord to the brain. Wnt proteins repel corticospinal tract (CST) axons in the opposite direction. Several Wnt genes were found to be expressed in the mouse spinal cord gray matter, cupping the dorsal funiculus, in an anterior-to-posterior decreasing gradient along the cervical and thoracic cord. Wnts repel CST axons in collagen gel assays through a conserved high-affinity receptor, Ryk, which is expressed in CST axons. Neonatal spinal cord secretes diffusible repellent(s) in an anterior-posterior graded fashion, with anterior cord being stronger, and the repulsive activity is blocked by antibodies to Ryk (anti-Ryk). Intrathecal injection of anti-Ryk blocks the posterior growth of CST axons. Therefore, Wnt proteins may have a general role in anterior-posterior guidance of multiple classes of axons (Liu, 2005).

Mammalian RYK is a receptor related to tyrosine kinase without detectable catalytic activity. Rat RYK is dominantly expressed in neural progenitor cells and mature neurons in the developing central nervous system. Mouse RYK has been found to bind to EphB2/B3 receptors, which have diverse functions during development. RYK, EphB2, EphB3, ephrinB1, and ephrinB2 are expressed in embryonic brain. In vitro analysis using COS-7 cells revealed binding between rat RYK and EphB3, and the RYK deletion mutant without extracellular leucine-rich motifs lacks this binding ability. To investigate the function of RYK in vivo, embryonic cortical slice cultures were analyzed after electroporation of expression plasmids for RYK or its deletion mutants. The results show that overexpression of RYK suppresses cell migration from the ventricular zone to the pial surface, however, overexpression of the RYK deletion mutant without leucine-rich motifs has no effect on cell migration. These results suggest that RYK regulates cell migration during mammalian cortical development through the binding to Eph receptors (Kamitori, 2005).

Ryk is novel Wnt receptor in both Caenorhabditis elegans and Drosophila melanogaster. Ryk-Wnt interactions have been shown to guide corticospinal axons down the embryonic mouse spinal cord. In Ryk-deficient mice, cortical axons project aberrantly across the major forebrain commissure, the corpus callosum. Many mouse mutants have been described in which loss-of-function mutations result in the inability of callosal axons to cross the midline, thereby forming Probst bundles on the ipsilateral side. In contrast, loss of Ryk does not interfere with the ability of callosal axons to cross the midline but impedes their escape from the midline into the contralateral side. Therefore, Ryk^-/- mice display a novel callosal guidance phenotype. Wnt5a acts as a chemorepulsive ligand for Ryk, driving callosal axons toward the contralateral hemisphere after crossing the midline. In addition, whereas callosal axons do cross the midline in Ryk^-/- embryos, they are defasciculated on the ipsilateral side, indicating that Ryk also promotes fasciculation of axons before midline crossing. In summary, this study expands the emerging role for Wnts in axon guidance and identifies Ryk as a key guidance receptor in the establishment of the corpus callosum. This analysis of Ryk function further advances understanding of the molecular mechanisms underlying the formation of this important commissure (Keeble, 2006).

Computational modelling has suggested that at least two counteracting forces are required for establishing topographic maps. Ephrin-family proteins are required for both anterior-posterior and medial-lateral topographic mapping, but the opposing forces have not been well characterized. Wnt-family proteins are recently discovered axon guidance cues. Wnt3 is expressed in a medial-lateral decreasing gradient in chick optic tectum and mouse superior colliculus. Retinal ganglion cell (RGC) axons from different dorsal-ventral positions show graded and biphasic response to Wnt3 in a concentration-dependent manner. Wnt3 repulsion is mediated by Ryk, expressed in a ventral-to-dorsal decreasing gradient, whereas attraction of dorsal axons at lower Wnt3 concentrations is mediated by Frizzled(s). Overexpression of Wnt3 in the lateral tectum repels the termination zones of dorsal RGC axons in vivo. Expression of a dominant-negative Ryk in dorsal RGC axons causes a medial shift of the termination zones, promoting medially directed interstitial branches and eliminating laterally directed branches. Therefore, a classical morphogen, Wnt3, acting as an axon guidance molecule, plays a role in retinotectal mapping along the medial-lateral axis, counterbalancing the medial-directed EphrinB1-EphB activity (Schmitt, 2006).