ROR and RYK extracellular region structures suggest that receptor tyrosine kinases have distinct WNT-recognition modes.

WNTs play key roles in development and disease, signaling through Frizzled (FZD) seven-pass transmembrane receptors and numerous co-receptors including ROR and RYK family receptor tyrosine kinases (RTKs). We describe crystal structures and WNT-binding characteristics of extracellular regions from the Drosophila ROR and RYK orthologs Nrk (neurospecific receptor tyrosine kinase) and Derailed-2 (Drl-2), which bind WNTs though a FZD-related cysteine-rich domain (CRD) and WNT-inhibitory factor (WIF) domain respectively. Our crystal structures suggest that neither Nrk nor Drl-2 can accommodate the acyl chain typically attached to WNTs. The Nrk CRD contains a deeply buried bound fatty acid, unlikely to be exchangeable. The Drl-2 WIF domain lacks the lipid-binding site seen in WIF-1. We also find that recombinant DWnt-5 can bind Drosophila ROR and RYK orthologs despite lacking an acyl chain. Alongside analyses of WNT/receptor interaction sites, our structures provide further insight into how WNTs may recruit RTK co-receptors into signaling complexes.

Coordinated changes in the expression of Wnt pathway genes following human and rat peripheral nerve injury.

A human neuroma-in continuity (NIC), formed following a peripheral nerve lesion, impedes functional recovery. The molecular mechanisms that underlie the formation of a NIC are poorly understood. Here we show that the expression of multiple genes of the Wnt family, including Wnt5a, is changed in NIC tissue from patients that underwent reconstructive surgery. The role of Wnt ligands in NIC pathology and nerve regeneration is of interest because Wnt ligands are implicated in tissue regeneration, fibrosis, axon repulsion and guidance. The observations in NIC prompted us to investigate the expression of Wnt ligands in the injured rat sciatic nerve and in the dorsal root ganglia (DRG). In the injured nerve, four gene clusters were identified with temporal expression profiles corresponding to particular phases of the regeneration process. In the DRG up- and down regulation of certain Wnt receptors suggests that nerve injury has an impact on the responsiveness of injured sensory neurons to Wnt ligands in the nerve. Immunohistochemistry showed that Schwann cells in the NIC and in the injured nerve are the source of Wnt5a, whereas the Wnt5a receptor Ryk is expressed by axons traversing the NIC. Taken together, these observations suggest a central role for Wnt signalling in peripheral nerve regeneration.

The non-canonical Wnt receptor Ryk regulates hematopoietic stem cell repopulation in part by controlling proliferation and apoptosis.

The development of blood and immune cells requires strict control by various signaling pathways in order to regulate self-renewal, differentiation and apoptosis in stem and progenitor cells. Recent evidence indicates critical roles for the canonical and non-canonical Wnt pathways in hematopoiesis. The non-canonical Wnt pathway is important for establishment of cell polarity and cell migration and regulates apoptosis in the thymus. We here investigate the role of the non-canonical Wnt receptor Ryk in hematopoiesis and lymphoid development. We show that there are dynamic changes in Ryk expression during development and in different hematopoietic tissues. Functionally, Ryk regulates NK cell development in a temporal fashion. Moreover, Ryk-deficient mice show diminished, but not absent self-renewal of hematopoietic stem cells (HSC), via effects on mildly increased proliferation and apoptosis. Thus, Ryk deficiency in HSCs from fetal liver reduces their quiescence, leading to proliferation-induced apoptosis and decreased self-renewal.

Guidance of Drosophila Mushroom Body Axons Depends upon DRL-Wnt Receptor Cleavage in the Brain Dorsomedial Lineage Precursors.

In vivo axon pathfinding mechanisms in the neuron-dense brain remain relatively poorly characterized. We study the Drosophila mushroom body (MB) axons, whose α and ß branches connect to different brain areas. We show that the Ryk family WNT5 receptor, DRL (derailed), which is expressed in the dorsomedial lineages, brain structure precursors adjacent to the MBs, is required for MB α branch axon guidance. DRL acts to capture and present WNT5 to MB axons rather than transduce a WNT5 signal. DRL's ectodomain must be cleaved and shed to guide α axons. DRL-2, another Ryk, is expressed within MB axons and functions as a repulsive WNT5 signaling receptor. Finally, our biochemical data support the existence of a ternary complex composed of the cleaved DRL ectodomain, WNT5, and DRL-2. Thus, the interaction of MB-extrinsic and -intrinsic Ryks via their common ligand acts to guide MB α axons.

Wnt5 and drl/ryk gradients pattern the Drosophila olfactory dendritic map.

During development, dendrites migrate to their correct locations in response to environmental cues. The mechanisms of dendritic guidance are poorly understood. Recent work has shown that the Drosophila olfactory map is initially formed by the spatial segregation of the projection neuron (PN) dendrites in the developing antennal lobe (AL). We report here that between 16 and 30 h after puparium formation, the PN dendrites undergo dramatic rotational reordering to achieve their final glomerular positions. During this period, a novel set of AL-extrinsic neurons express high levels of the Wnt5 protein and are tightly associated with the dorsolateral edge of the AL. Wnt5 forms a dorsolateral-high to ventromedial-low pattern in the antennal lobe neuropil. Loss of Wnt5 prevents the ventral targeting of the dendrites, whereas Wnt5 overexpression disrupts dendritic patterning. We find that Drl/Ryk, a known Wnt5 receptor, is expressed in a dorsolateral-to-ventromedial (DL > VM) gradient by the PN dendrites. Loss of Drl in the PNs results in the aberrant ventromedial targeting of the dendrites, a defect that is suppressed by reduction in Wnt5 gene dosage. Conversely, overexpression of Drl in the PNs results in the dorsolateral targeting of their dendrites, an effect that requires Drl's cytoplasmic domain. We propose that Wnt5 acts as a repulsive guidance cue for the PN dendrites, whereas Drl signaling in the dendrites inhibits Wnt5 signaling. In this way, the precise expression patterns of Wnt5 and Drl orient the PN dendrites allowing them to target their final glomerular positions.

Homodimerization of the Wnt receptor DERAILED recruits the Src family kinase SRC64B.

Ryk pseudokinase receptors act as important transducers of Wnt signals, particularly in the nervous system. Little is known, however, of their interactions at the cell surface. Here, we show that a Drosophila Ryk family member, DERAILED (DRL), forms cell surface homodimers and can also heterodimerize with the two other fly Ryks, DERAILED-2 and DOUGHNUT ON 2. DERAILED homodimerization levels increase significantly in the presence of its ligand, WNT5. In addition, DERAILED displays ligand-independent dimerization mediated by a motif in its transmembrane domain. Increased dimerization of DRL upon WNT5 binding or upon the replacement of DERAILED's extracellular domain with the immunoglobulin Fc domain results in an increased recruitment of the Src family kinase SRC64B, a previously identified downstream pathway effector. Formation of the SRC64B/DERAILED complex requires SRC64B's SH2 domain and DERAILED's PDZ-binding motif. Mutations in DERAILED's inactive tyrosine kinase-homologous domain also disrupt the formation of DERAILED/SRC64B complexes, indicating that its conformation is likely important in facilitating its interaction with SRC64B. Finally, we show that DERAILED's function during embryonic axon guidance requires its Wnt-binding domain, a putative juxtamembrane extracellular tetrabasic cleavage site, and the PDZ-binding domain, indicating that DERAILED's activation involves a complex set of events including both dimerization and proteolytic processing.

WNT5 interacts with the Ryk receptors doughnut and derailed to mediate muscle attachment site selection in Drosophila melanogaster.

In recent years a number of the genes that regulate muscle formation and maintenance in higher organisms have been identified. Studies employing invertebrate and vertebrate model organisms have revealed that many of the genes required for early mesoderm specification are highly conserved throughout evolution. Less is known about the molecules that mediate the steps subsequent to myogenesis, e. g. myotube guidance and attachment to tendon cells. We use the stereotypic pattern of the Drosophila embryonic body wall musculature in genetic approaches to identify novel factors required for muscle attachment site selection. Here, we show that Wnt5 is needed in this process. The lateral transverse muscles frequently overshoot their target attachment sites and stably attach at novel epidermal sites in Wnt5 mutant embryos. Restoration of WNT5 expression in either the muscle or the tendon cell rescues the mutant phenotype. Surprisingly, the novel attachment sites in Wnt5 mutants frequently do not express the Stripe (SR) protein which has been shown to be required for terminal tendon cell differentiation. A muscle bypass phenotype was previously reported for embryos lacking the WNT5 receptor Derailed (DRL). drl and Wnt5 mutant embryos also exhibit axon path finding errors. DRL belongs to the conserved Ryk receptor tyrosine kinase family which includes two other Drosophila orthologs, the Doughnut on 2 (DNT) and Derailed-2 (DRL-2) proteins. We generated a mutant allele of dnt and find that dnt, but not Drl-2, mutant embryos also show a muscle bypass phenotype. Genetic interaction experiments indicate that drl and dnt act together, likely as WNT5 receptors, to control muscle attachment site selection. These results extend previous findings that at least some of the molecular pathways that guide axons towards their targets are also employed for guidance of muscle fibers to their appropriate attachment sites.

Src family kinases are required for WNT5 signaling through the Derailed/RYK receptor in the Drosophila embryonic central nervous system.

Members of the RYK/Derailed family have recently been shown to regulate axon guidance in both Drosophila and mammals by acting as Wnt receptors. Little is known about how the kinase activity-deficient RYKs transduce Wnt signals. Here, we show that the non-receptor Src family tyrosine kinases, SRC64B and SRC42A, are involved in WNT5-mediated signaling through Derailed in the Drosophila embryonic central nervous system. Analysis of animals lacking SRC64B and SRC42A reveals defects in commissure formation similar to those observed in Wnt5 and derailed mutants. Reductions in SRC64B expression levels suppress a Wnt5/derailed-dependent dominant gain-of-function phenotype, and increased levels of either SRC64B or SRC42A enhance Wnt5/derailed-mediated axon commissure switching. Derailed and SRC64B form a complex, which contains catalytically active SRC64B, the formation or stability of which requires SRC64B kinase activity. Furthermore, Derailed is phosphorylated in a SRC64B-dependent manner and coexpression of Derailed and SRC64B results in the activation of SRC64B. The mammalian orthologs of Derailed and SRC64B also form complexes, suggesting that Src roles in RYK signaling are conserved. Finally, we show that coexpression of WNT5 and Derailed has no apparent effect upon TCF/LEF-dependent transcription, suggesting that the WNT5/Derailed signaling pathway is unlikely to directly regulate canonical Wnt pathway targets. Together, these findings indicate that the Src family kinases play novel roles in WNT5/Derailed-mediated signaling.

Derailed regulates development of the Drosophila neuromuscular junction.

Neural function is dependent upon the proper formation and development of synapses. We show here that Wnt5 regulates the growth of the Drosophila neuromuscular junction (NMJ) by signaling through the Derailed receptor. Mutations in both wnt5 and drl result in a significant reduction in the number of synaptic boutons. Cell-type specific rescue experiments show that wnt5 functions in the presynaptic motor neuron while drl likely functions in the postsynaptic muscle cell. Epistatic analyses indicate that drl acts downstream of wnt5 to promote synaptic growth. Structure-function analyses of the Drl protein indicate that normal synaptic growth requires the extracellular Wnt inhibitory factor domain and the intracellular domain, which includes an atypical kinase. Our findings reveal a novel signaling mechanism that regulates morphology of the Drosophila NMJ.

Antagonistic roles of Wnt5 and the Drl receptor in patterning the Drosophila antennal lobe.

Numerous studies have shown that ingrowing olfactory axons exert powerful inductive influences on olfactory map development. From an overexpression screen, we have identified wnt5 as a potent organizer of the olfactory map in Drosophila melanogaster. Loss of wnt5 resulted in severe derangement of the glomerular pattern, whereas overexpression of wnt5 resulted in the formation of ectopic midline glomeruli. Cell type-specific cDNA rescue and mosaic experiments showed that wnt5 functions in olfactory neurons. Mutation of the derailed (drl) gene, encoding a receptor for Wnt5, resulted in derangement of the glomerular map, ectopic midline glomeruli and the accumulation of Wnt5 at the midline. We show here that drl functions in glial cells, where it acts upstream of wnt5 to modulate its function in glomerular patterning. Our findings establish wnt5 as an anterograde signal that is expressed by olfactory axons and demonstrate a previously unappreciated, yet powerful, role for glia in patterning the Drosophila olfactory map.

A signaling network for patterning of neuronal connectivity in the Drosophila brain.

The precise number and pattern of axonal connections generated during brain development regulates animal behavior. Therefore, understanding how developmental signals interact to regulate axonal extension and retraction to achieve precise neuronal connectivity is a fundamental goal of neurobiology. We investigated this question in the developing adult brain of Drosophila and find that it is regulated by crosstalk between Wnt, fibroblast growth factor (FGF) receptor, and Jun N-terminal kinase (JNK) signaling, but independent of neuronal activity. The Rac1 GTPase integrates a Wnt-Frizzled-Disheveled axon-stabilizing signal and a Branchless (FGF)-Breathless (FGF receptor) axon-retracting signal to modulate JNK activity. JNK activity is necessary and sufficient for axon extension, whereas the antagonistic Wnt and FGF signals act to balance the extension and retraction required for the generation of the precise wiring pattern.

The Drosophila Wnt5 protein mediates selective axon fasciculation in the embryonic central nervous system.

The decision of whether and where to cross the midline, an evolutionarily conserved line of bilateral symmetry in the central nervous system, is the first task for many newly extending axons. We show that Wnt5, a member of the conserved Wnt secreted glycoprotein family, is required for the formation of the anterior of the two midline-crossing commissures present in each Drosophila hemisegment. Initial path finding of pioneering neurons across the midline in both commissures is normal in wnt5 mutant embryos; however, the subsequent separation of the early midline-crossing axons into two distinct commissures does not occur. The majority of the follower axons that normally cross the midline in the anterior commissure fail to do so, remaining tightly associated near their cell bodies, or projecting inappropriately across the midline in between the commissures. The lateral and intermediate longitudinal pathways also fail to form correctly, similarly reflecting earlier failures in pathway defasciculation. Panneural expression of Wnt5 in a wnt5 mutant background rescues both the commissural and longitudinal defects. We show that the Wnt5 protein is predominantly present on posterior commissural axons and at a low level on the anterior commissure and longitudinal projections. Finally, we demonstrate that transcriptional repression of wnt5 in AC neurons by the recently described Wnt5 receptor, Derailed, contributes to this largely posterior commissural localization of Wnt5 protein.

Genomewide analysis of the Drosophila tetraspanins reveals a subset with similar function in the formation of the embryonic synapse.

Tetraspanins encode a large conserved family of proteins that span the membrane four times and are expressed in a variety of eukaryotic tissues. They are part of membrane complexes that are involved in such diverse processes as intracellular signaling, cellular motility, metastasis, and tumor suppression. The single fly tetraspanin characterized to date, late bloomer (lbm), is expressed on the axons, terminal arbors, and growth cones of motoneurons. In embryos lacking Lbm protein, motoneurons reach their muscle targets, but initially fail to form synaptic terminals. During larval stages, however, functional contacts are formed. The newly available genomic sequence of Drosophila melanogaster indicates the existence of 34 additional members of the tetraspanin family in the fly. To address the possibility that other tetraspanins with functions that might compensate for a lack of lbm exist, we determined the expression domains of the Drosophila tetraspanin gene family members by RNA in situ analysis. We found two other tetraspanins also expressed in motoneurons and subsequently generated a small chromosomal deletion that removes all three motoneuron-specific tetraspanins. The deletion results in a significant enhancement in the lbm phenotype, indicating that the two additional motoneuron-expressed tetraspanins can, at least in part, compensate for the absence of lbm during the formation of the embryonic synapse.