Michael M. Halford

Vascular endothelial growth factor D is dispensable for development of the lymphatic system

ABSTRACT Vascular endothelial growth factor receptor 3 (Vegfr-3) is a tyrosine kinase that is expressed on the lymphatic endothelium and that signals for the growth of the lymphatic vessels (lymphangiogenesis). Vegf-d, a secreted glycoprotein, is one of two known activating ligands for Vegfr-3, the other being Vegf-c. Vegf-d stimulates lymphangiogenesis in tissues and tumors; however, its role in embryonic development was previously unknown. Here we report the generation and analysis of mutant mice deficient for Vegf-d. Vegf-d-deficient mice were healthy and fertile, had normal body mass, and displayed no pathologic changes consistent with a defect in lymphatic function. The lungs, sites of strong Vegf-d gene expression during embryogenesis in wild-type mice, were normal in Vegf-d-deficient mice with respect to tissue mass and morphology, except that the abundance of the lymphatics adjacent to bronchioles was slightly reduced. Dye uptake experiments indicated that large lymphatics under the skin were present in normal locations and were functional. Smaller dermal lymphatics were similar in number, location, and function to those in wild-type controls. The lack of a profound lymphatic phenotype in Vegf-d-deficient mice suggests that Vegf-d does not play a major role in lymphatic development or that Vegf-c or another, as-yet-unknown activating Vegfr-3 ligand can compensate for Vegf-d during development.

The Wnt Receptor Ryk Is Required for Wnt5a-Mediated Axon Guidance on the Contralateral Side of the Corpus Callosum

Ryk (receptor related to tyrosine kinase) has been shown to be a novel Wnt receptor in both Caenorhabditis elegans and Drosophila melanogaster. Recently, Ryk–Wnt interactions were shown to guide corticospinal axons down the embryonic mouse spinal cord. Here we show that, 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. We also show that 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. Our analysis of Ryk function further advances our understanding of the molecular mechanisms underlying the formation of this important commissure.

Ryk-deficient mice exhibit craniofacial defects associated with perturbed Eph receptor crosstalk

Secondary palate formation is a complex process that is frequently disturbed in mammals, resulting in the birth defect cleft palate. Gene targeting has identified components of cytokine/growth factor signalling systems such as Tgf-α/Egfr, Eph receptors B2 and B3 (Ephb2 and Ephb3, respectively), Tgf-β2, Tgf-β3 and activin-βA (ref. 3) as regulators of secondary palate development. Here we demonstrate that the mouse orphan receptor ‘related to tyrosine kinases’ (Ryk) is essential for normal development and morphogenesis of craniofacial structures including the secondary palate. Ryk belongs to a subclass of catalytically inactive, but otherwise distantly related, receptor protein tyrosine kinases (RTKs). Mice homozygous for a null allele of Ryk have a distinctive craniofacial appearance, shortened limbs and postnatal mortality due to feeding and respiratory complications associated with a complete cleft of the secondary palate. Consistent with cleft palate phenocopy in Ephb2/Ephb3-deficient mice and the role of a Drosophila melanogaster Ryk orthologue, Derailed, in the transduction of repulsive axon pathfinding cues, our biochemical data implicate Ryk in signalling mediated by Eph receptors and the cell-junction–associated Af-6 (also known as Afadin). Our findings highlight the importance of signal crosstalk between members of different RTK subfamilies.

Mutagenesis and selection of PDZ domains that bind new protein targets

PDZ domains are a recently characterized protein–recognition module. In most cases, PDZ domains bind to the C–terminal end of target proteins and are thought thereby to link these target proteins into functional signaling networks. We report the isolation of artificial PDZ domains selected via a mutagenesis screen in vivo, each recognizing a different C–terminal peptide. We demonstrate that the PDZ domains isolated can bind selectively to their target peptides in vitro and in vivo. Two of the target peptides chosen are the C–terminal ends of two cellular transmembrane proteins with which no known PDZ domains have been reported to interact. By targeting these artificial PDZ domains to the nucleus, interacting target peptides were efficiently transported to the same subcellular localization. One of the isolated PDZ domains was tested and shown to be efficiently directed to the plasma membrane when cotransfected with the full–length transmembrane protein in mammalian cells. Thus, artificial PDZ domains can be engineered and used to target intracellular proteins to different subcellular compartments.

The Wnt coreceptor Ryk regulates Wnt/planar cell polarity by modulating the degradation of the core planar cell polarity component Vangl2

Background: Wnt5a regulates planar cell polarity (PCP) in development and signals through Ryk in axon guidance. Results: Ryk interacts with Vangl2 genetically and biochemically and mediates Wnt5a signaling in controlling PCP. Conclusion: Ryk regulates PCP by controlling the degradation of Vangl2. Significance: Revealing regulatory mechanisms underlying PCP is essential for understanding morphogenesis and human birth defects. The Wnt signaling pathways control many critical developmental and adult physiological processes. In vertebrates, one fundamentally important function of Wnts is to provide directional information by regulating the evolutionarily conserved planar cell polarity (PCP) pathway during embryonic morphogenesis. However, despite the critical roles of Wnts and PCP in vertebrate development and disease, little is known about the molecular mechanisms underlying Wnt regulation of PCP. Here, we have found that the receptor-like tyrosine kinase (Ryk), a Wnt5a-binding protein required in axon guidance, regulates PCP signaling. We show that Ryk interacts with Vangl2 genetically and biochemically, and such interaction is potentiated by Wnt5a. Loss of Ryk in a Vangl2+/− background results in classic PCP defects, including open neural tube, misalignment of sensory hair cells in the inner ear, and shortened long bones in the limbs. Complete loss of both Ryk and Vangl2 results in more severe phenotypes that resemble the Wnt5a−/− mutant in many aspects such as shortened anterior-posterior body axis, limb, and frontonasal process. Our data identify the Wnt5a-binding protein Ryk as a general regulator of the mammalian Wnt/PCP signaling pathway. We show that Ryk transduces Wnt5a signaling by forming a complex with Vangl2 and that Ryk regulates PCP by at least in part promoting Vangl2 stability. As human mutations in WNT5A and VANGL2 are found to cause Robinow syndrome and neural tube defects, respectively, our results further suggest that human mutations in RYK may also be involved in these diseases.

The Wnt Receptor Ryk Plays a Role in Mammalian Planar Cell Polarity Signaling

Background: Ryk acts as a Wnt receptor in several processes, including mouse neuronal development. Results: Ryk interacts with Wnt11 in zebrafish convergent extension and with Vangl2 in mouse cochlear development and neural tube closure. Conclusion: Ryk is required for Wnt/planar cell polarity signaling during mammalian development and signals via Vangl2 and RhoA. Significance: This study extends our knowledge of signaling downstream of Ryk. Wnts are essential for a wide range of developmental processes, including cell growth, division, and differentiation. Some of these processes signal via the planar cell polarity (PCP) pathway, which is a β-catenin-independent Wnt signaling pathway. Previous studies have shown that Ryk, a member of the receptor tyrosine kinase family, can bind to Wnts. Ryk is required for normal axon guidance and neuronal differentiation during development. Here, we demonstrate that mammalian Ryk interacts with the Wnt/PCP pathway. In vitro analysis showed that the Wnt inhibitory factor domain of Ryk was necessary for Wnt binding. Detailed analysis of two vertebrate model organisms showed Ryk phenotypes consistent with PCP signaling. In zebrafish, gene knockdown using morpholinos revealed a genetic interaction between Ryk and Wnt11 during the PCP pathway-regulated process of embryo convergent extension. Ryk-deficient mouse embryos displayed disrupted polarity of stereociliary hair cells in the cochlea, a characteristic of disturbed PCP signaling. This PCP defect was also observed in mouse embryos that were double heterozygotes for Ryk and Looptail (containing a mutation in the core Wnt/PCP pathway gene Vangl2) but not in either of the single heterozygotes, suggesting a genetic interaction between Ryk and Vangl2. Co-immunoprecipitation studies demonstrated that RYK and VANGL2 proteins form a complex, whereas RYK also activated RhoA, a downstream effector of PCP signaling. Overall, our data suggest an important role for Ryk in Wnt/planar cell polarity signaling during vertebrate development via the Vangl2 signaling pathway, as demonstrated in the mouse cochlea.

Revelations of the RYK receptor.

Significant progress has been made over the last decade in elucidating the mechanisms employed by receptor protein tyrosine kinases (RTKs) in transducing extracellular signals critical for the regulation of diverse cellular activities. Nevertheless, revealing the biological significance of a subset of the RTKs that contain catalytically inactive protein tyrosine kinase domains has proven more elusive. ErbB3 has served as the prototype for models of catalytically inactive RTK function, performing the role of signal diversification in heterodimeric receptor complexes with other ErbB subfamily members. The receptor related to tyrosine kinases (RYK) is unique amongst the catalytically inactive RTKs. Based on structural or functional properties of the extracellular domain, RYK cannot be classified into an existing RTK subfamily. Recent genetic analyses of mouse Ryk and its Drosophila orthologue derailed have defined a role for this novel subfamily of receptors in the control of craniofacial development and neuronal pathway selection, respectively. Recent biochemical data lead us to propose a model that involves RYK in signal crosstalk and scaffold assembly with Eph receptors. This model is consistent with the established roles of Eph receptors and ephrins in craniofacial and nervous system morphogenesis. BioEssays 23:34–45, 2001.

GENOMIC STRUCTURE AND EXPRESSION OF THE MOUSE GROWTH FACTOR RECEPTOR RELATED TO TYROSINE KINASES (RYK)

We report the genomic organization of the mouse orphan receptor related to tyrosinekinases (Ryk), a structurally unclassified member of the growth factor receptor family. The mouse RYK protein is encoded by 15 exons distributed over a minimum of 81 kilobases. Genomic DNA sequences encoding a variant protein tyrosine kinase ATP-binding motif characteristic of RYK are unexpectedly found in two separate exons. A feature of the gene is an unmethylated CpG island spanning exon 1 and flanking sequences, including a TATA box-containing putative promoter and single transcription start site. Immunohistochemical examination of RYK protein distribution revealed widespread but developmentally regulated expression, which was spatially restricted within particular adult organs. Quantitative reduction of Southern blotting stringency for the detection ofRyk-related sequences provided evidence for a retroprocessed mouse pseudogene and a more distantly related gene paralogue. Extensive cross-species reactivity of a mouseRyk kinase subdomain probe and the cloning of aRyk orthologue from Caenorhabditis elegansdemonstrate that Ryk and its relatives encode widely conserved members of a novel receptor tyrosine kinase subfamily.

The Wnt Receptor Ryk Reduces Neuronal and Cell Survival Capacity by Repressing FOXO Activity During the Early Phases of Mutant Huntingtin Pathogenicity

A study of Huntingtons disease reveals that neurons might fail to cope with maintaining their function during the pre-symptomatic, pathogenic phases of HD, possibly due to the early repression of key longevity-promoting transcription factors by abnormal developmental signaling.

A Fully Human Inhibitory Monoclonal Antibody to the Wnt Receptor RYK

RYK is an unusual member of the receptor tyrosine kinase (RTK) family that is classified as a putative pseudokinase. RYK regulates fundamental biological processes including cell differentiation, migration and target selection, axon outgrowth and pathfinding by transducing signals across the plasma membrane in response to the high affinity binding of Wnt family ligands to its extracellular Wnt inhibitory factor (WIF) domain. Here we report the generation and initial characterization of a fully human inhibitory monoclonal antibody to the human RYK WIF domain. From a naïve human single chain fragment variable (scFv) phage display library, we identified anti-RYK WIF domain–specific scFvs then screened for those that could compete with Wnt3a for binding. Production of a fully human IgG1κ from an inhibitory scFv yielded a monoclonal antibody that inhibits Wnt5a-responsive RYK function in a neurite outgrowth assay. This antibody will have immediate applications for modulating RYK function in a range of settings including development and adult homeostasis, with significant potential for therapeutic use in human pathologies.