InteractiveFly: GeneBrief

derailed : Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References


Gene name - derailed

Synonyms -

Cytological map position - 37D2--37D2

Function - receptor

Keywords - axon guidance, brain, CNS, receptor for Wnt5

Symbol - drl

FlyBase ID:FBgn0015380

Genetic map position - 2-

Classification - transmembrane protein-tyrosine kinase (inactive), Wnt inhibitory factor (WIF) domain

Cellular location - cell surface



NCBI link: Entrez Gene

drl orthologs: Biolitmine
Recent literature
Long, H., Yoshikawa, S. and Thomas, J. B. (2016). Equivalent activities of repulsive axon guidance receptors. J Neurosci 36: 1140-1150. PubMed ID: 26818503
Summary:
Receptors on the growth cone at the leading edge of elongating axons play critical guidance roles by recognizing cues via their extracellular domains and transducing signals via their intracellular domains, resulting in changes in direction of growth. An important concept to have emerged in the axon guidance field is the importance of repulsion as a major guidance mechanism. Given the number and variety of different repulsive receptors, it is generally thought that there are likely to be qualitative differences in the signals they transduce. However, the nature of these possible differences is unknown. By creating chimeras using the extracellular and intracellular domains of three different Drosophila repulsive receptors, Unc5, Roundabout (Robo), and Derailed (Drl) and expressing them in defined cells within the embryonic nervous system, the responses elicited by their intracellular domains were examined systematically. Surprisingly, no qualitative differences were found in growth cone response or axon growth, suggesting that, despite their highly diverged sequences, each intracellular domain elicits repulsion via a common pathway. In terms of the signaling pathway(s) used by the repulsive receptors, mutations in the guanine nucleotide exchange factor Trio strongly enhance the repulsive activity of all three intracellular domains, suggesting that repulsion by Unc5, Robo, and Drl, and perhaps repulsion in general, involves Trio activity.
Hing, H., Reger, N., Snyder, J. and Fradkin, L. G. (2020). Interplay between axonal Wnt5-Vang and dendritic Wnt5-Drl/Ryk signaling controls glomerular patterning in the Drosophila antennal lobe. PLoS Genet 16(5): e1008767. PubMed ID: 32357156
Summary:
Despite the importance of dendritic targeting in neural circuit assembly, the mechanisms by which it is controlled still remain incompletely understood. Previously work showed that in the developing Drosophila antennal lobe, the Wnt5 protein forms a gradient that directs the ~45° rotation of a cluster of projection neuron (PN) dendrites, including the adjacent DA1 and VA1d dendrites. The Van Gogh (Vang) transmembrane planar cell polarity (PCP) protein is required for the rotation of the DA1/VA1d dendritic pair. Cell type-specific rescue and mosaic analyses showed that Vang functions in the olfactory receptor neurons (ORNs), suggesting a codependence of ORN axonal and PN dendritic targeting. Loss of Vang suppressed the repulsion of the VA1d dendrites by Wnt5, indicating that Wnt5 signals through Vang to direct the rotation of the DA1 and VA1d glomeruli. The Derailed (Drl)/Ryk atypical receptor tyrosine kinase is also required for the rotation of the DA1/VA1d dendritic pair. Antibody staining showed that Drl/Ryk is much more highly expressed by the DA1 dendrites than the adjacent VA1d dendrites. Mosaic and epistatic analyses showed that Drl/Ryk specifically functions in the DA1 dendrites in which it antagonizes the Wnt5-Vang repulsion and mediates the migration of the DA1 glomerulus towards Wnt5. Thus, the nascent DA1 and VA1d glomeruli appear to exhibit Drl/Ryk-dependent biphasic responses to Wnt5. This work shows that the final patterning of the fly olfactory map is the result of an interplay between ORN axons and PN dendrites, wherein converging pre- and postsynaptic processes contribute key Wnt5 signaling components, allowing Wnt5 to orient the rotation of nascent synapses through a PCP mechanism.
BIOLOGICAL OVERVIEW

derailed mutants exhibit axon guidance phenotypes related to central nervous system development and also exhibit muscle attachment defects as well as structural brain defects in the mushroom body (MB) and the central complex (CX), a highly ordered structure located at the center of the brain, at the junction between the two protocerebral hemispheres. The mushroom body originates as a structure of four clonal units, each of which contains a virtually identical set of neurons and glial cells. The mature MB is composed of a pair of neuropiles. Each neuropile consists of about 2500 Kenyon cells. (Kenyon cells (KCs) were first described by F. C. Kenyon in 1896). KC cell bodies lie in the dorsoposterior cortex of the protocerebrum, well removed from the anterior protocerebrum. They send dendritic processes ventrally to form the calyx neuropil, where the dendritic fibers receive afferent olfactory signals from the antennal lobe interneurons and other interneurons. From the calyx, the fibers extend to the anterior via the pedunculus to reach the anterior protocerebrum, where they form the alpha, alpha', beta and gamma lobes. It is in the protocerebral lobes that olfactory learning takes place (Moreau-Fauvarque, 1998 and references).

The derailed receptor tyrosine kinase (RTK) gene has been independently identified in a screen for embryonic nervous system axonal guidance defects. The mushroom body defect in derailed mutants can be rescued by targeted expression of drl in the developing MB (Moreau-Fauvarque, 1998). These results suggest that, analogous to its role within the embryonic nervous system, the Drl RTK is involved in neuronal pathway selection during adult brain development. The results also imply that the defects in MB and/or CX are responsible for the learning and memory deficit of derailed mutants. This overview will describe the structural defects in the embryonic neurons in the ventral nerve cord of derailed mutants, and the defects in the central complex and the mushroom body.

Advances in the genetics of Drosophila have provided a powerful tool for extending understanding of the MB organization and development. The P(GAL4) enhancer-trap technique has been used to selectively visualize the axonal processes of KCs and to reveal a previously unknown structural complexity within the MB. KCs are heterogeneous with respect to gene expression patterns; different GAL4 lines display reporter expression patterns in specific, distinct and various subsets of KCs, and consequently identify anatomical subdivisions within the MB (Yang, 1995 and Ito, 1997). For example, particular subsets of KCs, defined by independent gene expression characteristics, have discrete projection patterns within the peduncle that segregate to characteristic regions of the lobes. KC axons appear to be organized concentrically in the peduncle, with four symmetrically arranged discrete fiber bundles surrounded by one or more outer rings. The axons forming the quartet of fiber tracts internal to the peduncle project to the alpha and beta lobes and the axons forming the circumference of the peduncle project to the gamma lobe, the alpha' lobe and the spur. This anatomical complexity of the adult MB could account for the diverse behavioral and computational properties of the MB (Moreau-Fauvarque, 1998 and references).

Before detailing the phenotypic effects of derailed mutants, we must digress here for a few words about a learning and memory mutant called linotte (Dura, 1993 and Bolwig, 1995). linotte codes for a novel protein that is not to be confused with derailed in spite of the fact that FlyBase still lists linotte as a synonym for derailed. The reason for this confusion lies in the fact that linotte and derailed are adjacent to each other on the second chromosome. The original lio1 P element mutation is inserted 800 bp downstream of the linotte transcription unit, perhaps in the 5' untranslated region of derailed. Hence, one might expect the lio1 mutation to create some sort of derailed phenotype. Dura's group has generated an excision-deletion of derailed by mobilizing lio1. This deletion allele has been termed lio2; lio2 molecularly deletes derailed but not linotte and the anatomical defects of lio2 are more severe than those of lio1. It is likely that while linotte mutants are responsible exclusively for learning and memory defects, derailed mutation is responsible for more severe anatomical defects (T. Tully, personal communication to T. Brody, editor, The Interactive Fly, 1999).

In strong lio2 mutants, which display a more severe memory deficit than the lio1 mutant, fibers of the beta lobes appear completely fused across the midline. The beta lobes are also deformed and thickened. This phenotype is displayed by all lio2 mutants. The gamma lobes also present fusion defects, but the phenotype is more variable and is more difficult to observe on paraffin frontal sections, due to the complex and bulbous shape of these lobes. A partial fusion of these lobes is often observed; the most posterior fibers that are dorsal and parallel to the fibers of the beta lobes are usually fused across the midline; the anterior fibers, which constitute the bulbous part of the gamma lobes are usually not fused. For the strongest phenotypes, both gamma and beta lobes are completely fused and they are also so deformed that it is not possible to distinguish them. Finally, the alpha and alpha' lobes are also affected: for the strongest phenotypes, a complete loss of the fibers of the alpha and alpha' lobes is observed. In other cases, it seems that only the fibers from the alpha' lobes remain. Brain phenotype defines an allelic series of linotte mutations, with lio1 classified as a hypomorph (Moreau-Fauvarque, 1998).

In wild-type flies, the alpha and beta lobes probably constitute the two projection domains of axons of the median fascicle within the peduncle (Ito, 1997 and Yang, 1995). When these axons reach the anterior part of the brain, they face a typical 'choice point' problem: they must change and split their direction of growth, one axon projecting upward into the alpha lobe and one projecting toward the midline of the beta lobe. In drl mutants, at the choice point these axons fail to find the route toward the alpha lobe when they reach the anterior part of the brain. The only projection of growth in these mutants is in the beta/gamma direction. In wild-type flies, when axons of the beta lobe arrive at the midline, they normally stop at the level of the median bundle. In drl mutants, instead of stopping, they continue to grow, and so the axons of the beta lobes cross one another. The beta lobes are fused across the midline and appear defasciculated. It is not known to what extent they interpenetrate. It seems likely that the alpha and beta lobe phenotypes are correlated in drl mutants (Moreau-Fauvarque, 1998).

The interpretation of the CX phenotype in terms of axonal guidance is less obvious, and thus remains largely hypothetical. The CX proper is composed of four neuropilar regions: the protocerebral bridge, the fan-shaped body, the ellipsoid body and the noduli. These four substructures are composed of tangential 'large-field' neurons forming parallel strata within each substructure and are interconnected by complex sets of columnar 'small-field' interneurons forming regular projection patterns. These give the CX the appearance of a repetitive and modular architecture. Two general functions have been proposed for the CX on the basis of neuroanatomical considerations, data from surgery, and electrical stimulation and data from genetic dissection: (1) control of behavioral activity, involving its initiation and regulation, and (2) integration of data between the right and left hemispheres of the brain. In derailed mutants, the fan-shaped body is distorted on its dorsal part and appear flattened. At the junction between the two hemispheres and just dorsal to the fan-shaped body, fibers from the dorsal protocerebrum of each hemisphere abnormally cross the midline. These defects may correspond to an altered projection pattern of fibers forming the superior part of the fan-shaped body, as for instance, fibers from the Horizontal Fiber System or the Vertical Fiber System. Alternatively, this defect may be a consequence of abnormal projections across the midline of fibers just dorsal to the fan-shaped body. The circuitry of the CX is much more complex and has been less studied than the circuitry in the MB; therefore, no simple rule can describe the neuroarchitecture of the CX in terms of fiber projections (Moreau-Fauvarque, 1998).

The original CNS axon pathfinding derailed mutations were identified in a screen for genes expressed in restricted subsets of developing neurons that choose common pathways. Incorporated into the P element vector was a tau-lacZ reporter gene whose product is a fusion between the microtubule-associated Tau protein and beta-galactosidase. This reporter efficiently labels the axon projections of expressing neurons, presumably owing to the microtubule binding properties of Tau protein. In embryos heterozygous for the P element insertion defining the derailed mutant, Tau-beta-gal expression is restricted to a cluster of approximately 20 interneurons per hemisegment. Expression commences postmitotically in the neurons as axons begin to elongate and continues throughout embryogenesis. In heterozygotes for the tau-lacZ P element insert, these interneurons project in a highly stereotyped fashion. Most if not all of the drl neurons extend their axons across the midline within the anterior commissure. On reaching the contralateral longitudinal connective, their growth cones turn to the anterior and choose to follow one or the other of two discrete pathways located close together at the medial edge of the connective. Once the drl neurons have reached the adjacent anterior segment, they fasciculate with their homologs, forming two continuous axon fascicles, termed DD and DV, in each connective (Callahan, 1996).

In drl homozygous mutant embryos, the drl neurons cross the midline and reach the contralateral longitudinal connective, but they project to the anterior along inappropriate paths. Instead of choosing their normal medial pathways and fasciculating with one another, drl neurons project without any apparent preference for pathways within the connectives and thus fail to form the distinctive DD and DV bundles. In many segments the drl neurons can be seen crossing one another. drl neurons also appear less organized within the anterior commissure, and in approximately 10% of segments drl axons can be detected crossing abnormally in the posterior commissure. Although the drl fascicles are dramatically affected in drl mutants, the overall structure of the nervous system and the formation of non-drl axon bundles are indistinguishable from the wild type. Thus neuronal pathfinding defects in drl mutants are not widespread (Callahan, 1995).

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, wnt5 was identified as a potent organizer of the olfactory map in Drosophila. Loss of wnt5 results in severe derangement of the glomerular pattern, whereas overexpression of wnt5 results 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. drl functions in glial cells, where it acts upstream of wnt5 to modulate its function in glomerular patterning. These 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 (Yao, 2007).

The mechanisms by which ingrowing axons sort into precise maps, such as those found in the olfactory glomeruli or the somatosensory barrels, are poorly understood. Deafferentation and transplantation experiments revealed that ingrowing axons are important for specifying the maps in the initially homogenous structures. However, little is known about how the ingrowing axons carry out these feats. This report shows that ingrowing ORN axons express Wnt5, which contributes to organizing the glomerular pattern of the Drosophila olfactory system. The Drl receptor tyrosine kinase acts in glial cells to modulate Wnt5 signaling. This previously unknown interaction between ORN axons and glia reveals an important function of ORN axon-glia interactions in regulating the precise neural circuitry of the Drosophila antennal lobes (Yao, 2007).

The wnt5 mutant has characteristic disruptions of the olfactory map. Many dorsomedial glomeruli are displaced ventrally (resulting in heart-shaped antennal lobes) and the antennal commissure fails to form. In contrast to the loss-of-function defects, overexpression of wnt5 leads to the displacement of glomeruli into the midline. Examination of the ORN axons in the wnt5 mutant showed that they take circuitous paths to their targets and frequently misproject to dorsal regions of the brain. Consistent with a role for wnt5 in antennal lobe development, the antennal lobe defects appears during the pupal stage, when ORN axon targeting and glomerular development occur. Genetic mosaic and cell type-specific rescue experiments indicated that wnt5 is required in the ORNs. Antibody stainings indicated that the Wnt5 protein is enriched on the dendrites of the projection neurons, where it presumably accumulates subsequent to its secretion by ORNs. In addition to the projection neuron dendrites, Wnt5 also accumulates in the antennal commissure in the drl2 mutant. It is proposed that Wnt5 is a signal by which ingrowing ORN axons direct the development of their target field (Yao, 2007).

Mutation of the drl gene also produces disruptions of the olfactory map. However, unlike the stereotyped shifts of glomeruli seen in the wnt5 mutant, the glomeruli were randomly positioned in or missing from one antennal lobe in the drl mutant. Furthermore, there was a strong tendency for glomeruli to form at the midline. As in the wnt5 mutant, ORN axons take indirect routes to their targets. That drl functions in development is supported by the observation that antennal lobe defects are visible at 40 hAPF, the time when ORN axon targeting and glomerular development take place (Yao, 2007).

Antibody staining showed that the Drl protein is highly expressed by the projection neurons and TIFR glia, cells that are intimately associated with the ingrowing ORN axons. In the projection neurons, Drl is enriched in the dendrites of nascent glomeruli, four of which also appeared to accumulate Wnt5. The TIFR is a donut-shaped mid-sagittal structure located between the antennal lobes. Histological studies showed that TIFR glial processes are closely associated with ORN axons that project across the midline. Several observations indicated that drl functions in the TIFR to regulate wnt5 function. First, removal of drl from single projection neuron clones does not disrupt the development and morphology of the projection neurons. Second, neuronal expression of drl in the drl2 mutant background does not rescue the mutant phenotype. Third, expression of UAS-drl under the control of Repo-Gal4 strongly rescues the drl mutant phenotype, suggesting that drl functions in glial cells. Although roles for Drl in the projection neurons cannot be ruled out, collectively, the observations suggest that drl functions predominantly in glial cells to regulate antennal lobe development (Yao, 2007).

The phenotypic similarities between the drl loss-of-function and the wnt5-overexpressing mutants raise the intriguing possibility that the two genes act antagonistically in antennal lobe development. Indeed, expression of a weak wnt5 transgene in the ORNs, which has no effect in the wild type, triggers the formation of ectopic glomeruli in the drl2 mutant. Thus, wnt5 and drl function in opposition to each other in antennal lobe development. To ascertain the relative positions of wnt5 and drl in this signaling pathway, animals carrying null mutations in both genes were generated. The wnt5400;drl2 double mutants was found to have the characteristic wnt5 phenotype. The wnt5 gene is therefore epistatic to the drl gene, indicating that wnt5 functions downstream of drl in antennal lobe development. This conclusion is also supported by the observation that, although the removal of a copy of the wnt5 gene strongly suppresses the drl homozygous mutant phenotype, the removal of a copy of the drl gene has no effect on the wnt5 homozygous mutant phenotype. The genetic data that drl downregulates wnt5 function is further supported by the observation that the Wnt5 protein significantly accumulates in the commissure in the absence of Drl. Taken together, these genetic and histological data indicate that drl acts to inhibit the activity of wnt5 during antennal lobe development (Yao, 2007).

To probe the molecular mechanisms by which Drl regulates antennal lobe development, the various domains of Drl were mutated. It was observed that neither disruption of the kinase activity nor deletion of the intracellular domain significantly impaired rescue by the drl transgene. In contrast, deletion of the extracellular WIF domain completely abolishes Drl's ability to rescue the mutant phenotype. These results suggest that Drl regulates antennal lobe patterning predominantly through its extracellular WIF domain. How might Drl inhibit the function of Wnt5? One possibility is that Drl inhibits Wnt5 function simply by promoting Wnt5's sequestration or endocytosis, thus limiting its interaction with another as yet unidentified receptor. This receptor might be one of the other Drosophila receptor tyrosine kinases or a member of the Frizzled family, one of which, frizzled 2 (fz2), interacts genetically with wnt5 to stabilize axons of the Drosophila visual system. Alternatively, Drl may directly interact with another receptor and Wnt5, as has been observed previously for its mammalian ortholog Ryk and members of the Wnt and Frizzled families (Lu, 2004). This interaction could inhibit or alter the signal transduced from the membrane. However, no requirement was detected for Drl's cytoplasmic domain, suggesting that transduction of the Wnt5 signal by Drl alone is unlikely to have a major role in patterning the antennal lobes (Yao, 2007).

How do glial cells interact with the ORN axons to specify the olfactory map? The data suggest that the ingrowing ORN axons contribute to antennal lobe patterning through secretion of Wnt5 and that glial cells locally regulate Wnt5 actions through Drl. The following working model is proposed for how Wnt5-Drl signaling might regulate glomerular patterning. Ingrowing ORN axons express Wnt5, which is important for the precise organization of the glomeruli and pathfinding of the ORN axons, such as those crossing the midline or projecting to the dorsomedial region of the antennal lobes. Normal antennal lobe development requires that the Wnt5 signal be locally attenuated by the TIFR glial cell-expressed Drl protein. In the wnt5 mutant, the lack of Wnt5 signaling results in the failure of ORN axons to cross the midline and the establishment of glomeruli in more ventral positions. In the drl mutant, Wnt5 accumulates at the midline and presumably inappropriately signals through another receptor, resulting in aberrant ORN axon targeting to the midline and the formation of ectopic glomeruli at the dorsomedial corner of the antennal lobe and at the midline. Further studies will hopefully help to unravel the precise mechanisms by which Wnt5 and Drl act together to specify the patterning of the Drosophila olfactory map (Yao, 2007).


REGULATION

The derailed (drl) gene encodes a receptor tyrosine kinase (RTK) that governs aspects of axon guidance and muscle-epidermal interactions in the Drosophila embryo. To determine the types of neurons that express drl, a series of drl promoter fusions to axon-targeted reporters have been studied. Described here are intronic enhancers that drive reporter expression in four distinct subtypes of embryonic neurons, all of which project axons in the anterior commissure of the developing nervous system. The first population is defined by drlR, which drives reporter expression in 10 medial interneurons per hemisegment clustered ventral to the anterior commissure. These neurons extend ipsilateral axons in the anterior commissure and ramify local processes near the junction of the anterior commissure and the longitudinal connective. The second population is defined by the expression of drlT, and is composed of 20 lateral interneurons in each hemisegment that project contralaterally in the anterior commissure. A subset of these axons turns anteriorly upon reaching the contralateral connective, and form a single longitudinal fascicle. The third population is defined by drlU expression in a single bilaterally-paired interneuron present in segment A3-A8 that crosses the midline in the anterior commissure and extends anteriorly in the longitudinal connective. The final population is defined by drlZ expression in two VUM motorneurons that bifurcate in the anterior commissure and exit in the intersegmental nerve. The combined expression of these four neuronal populations, appears to give the pattern of CNS expression observed in a drl enhancer trap line (Bonkowsky, 1999).

Enhancers driving expression in the drl-expressing muscles and epidermal attachment cells are also described. These enhancers define the classes of neurons projecting in the anterior commissure and can be used to precisely define axon pathfinding errors in drl and other mutants. drl neurons are a heterogeneous population of neurons that ultimately choose different pathways and synapse with different target cells. However, they all share the common property of projecting axons in the anterior commissure, as well as expressing drl, suggesting a role for drl in axon guidance through the anterior commissure. Isolation of separate enhancers for the drl and their corresponding epidermal attachment cells provides the opportunity to analyze the reciprocal interactions during muscle attachment site selection. Finally, expression of drl in each of the four subclasses of anterior commissural neurons is controlled by separate transcriptional regulatory sequences. This cell-type specific modular nature of drl transcriptional regulation makes it an attractive system for understanding higher order cis-regulation of gene expression in the developing nervous system (Bonkowsky, 1999).

Nerfin-1 is a nuclear regulator of axon guidance required for a subset of early pathfinding events in the developing Drosophila CNS. Nerfin-1 belongs to a highly conserved subfamily of Zn-finger proteins with cognates identified in nematodes and man. The neural precursor gene prospero is essential for nerfin-1 expression. Unlike nerfin-1 mRNA, which is expressed in many neural precursor cells, the encoded Nerfin-1 protein is only detected in the nuclei of neuronal precursors that will divide just once and then transiently in their nascent neurons. Although nerfin-1 null embryos have no discernible alterations in neural lineage development or in neuronal or glial identities, CNS pioneering neurons require nerfin-1 function for early axon guidance decisions. Furthermore, nerfin-1 is required for the proper development of commissural and connective axon fascicles. Nerfin-1 is essential for the proper expression of robo2, wnt5, derailed, G-oα47A, Lar, and futsch<, genes whose encoded proteins participate in these early navigational events (Kuzin, 2005).

Given the axon guidance defects in nerfin-1null embryos and the fact that Nerfin-1 is a Zn-finger nuclear protein, it was hypothesized that Nerfin-1 may be required for the correct expression of genes involved in axon guidance. Accordingly, the embryonic expression profiles of over 35 genes that have been shown to play important roles in axon guidance were examined. Included in the candidate screen were genes encoding transcription factors, RNA-binding proteins, cell surface receptor proteins, their ligands, signal transduction proteins, and components of the cytoskeleton. Homozygous nerfin-1null embryos were identified by the absence of Nerfin-1 immunoreactivity. Whole-mount in situ hybridization and/or protein immunostaining for altered spatial or temporal expression in nerfin-1null embryos identified six genes that require nerfin-1 function to achieve full wild-type expression levels (Kuzin, 2005).

Two genes involved in anterior vs. posterior commissure choice, those encoding the receptor tyrosine kinase Derailed, and its ligand Wnt5, both required nerfin-1 for full expression. In the absence of nerfin-1, ventral cord expression levels of Robo and Robo3 were unaffected; however, Robo2 expression levels were significantly reduced. Expression of Slit, the ligand for Robo receptors, and Commissureless, a factor responsible for clearing Robo receptors from commissural axons, was unaffected in nerfin-1null embryos (Kuzin, 2005).

Loss of nerfin-1 function also significantly delayed and/or reduced the early expression of the neuron-specific microtubule-associated MAP1B-like gene futsch. futsch expression is normally activated in newborn neurons starting at stage 11; however, in nerfin-1null embryos expression is first detected only at the stage 13. Not until embryonic stage 15 did the level of futsch expression in mutant embryos approach that of wild type. Reduced mRNA steady state levels for the genes encoding Leukocyte-antigen-related-like (Lar), another receptor tyrosine kinase, and G-oα47A gene, which encodes an alpha subunit of heterotrimeric G proteins, were also detected in nerfin-1null embryos. The reduced level of gene expression in mutant embryos was nervous system specific. For example, G-oα47A gene expression in mesodermal derived tissues was not altered in nerfin-1null embryos (Kuzin, 2005).

Intra-axonal patterning: intrinsic compartmentalization of the axonal membrane in Drosophila neurons

In the developing nervous system, distribution of membrane molecules, particularly axon guidance receptors, is often restricted to specific segments of axons. Such localization of membrane molecules can be important for the formation and function of neural networks; however, how this patterning within axons is achieved remains elusive. This study shows that Drosophila neurons in culture establish intra-axonal patterns in a cell-autonomous manner; several membrane molecules localize to either proximal or distal axon segments without cell-cell contacts. This distinct patterning of membrane proteins is not explained by a simple temporal control of expression, and likely involves spatially controlled vesicular targeting or retrieval. Mobility of transmembrane molecules is restricted at the boundary of intra-axonal segments, indicating that the axonal membrane is compartmentalized by a barrier mechanism. It is proposed that this intra-axonal compartmentalization is an intrinsic property of Drosophila neurons that provides a basis for the structural and functional development of the nervous system (Katsuki, 2009).

This study describes a patterning phenomenon that takes place within single axonal processes as a cell-intrinsic event. This patterning involves compartmentalization of the axonal membrane with a diffusion barrier located at a medial point of the axon. The data suggest that this patterning ability is a fundamental property of Drosophila neurons, because the compartment-specific localization of GFP-tagged receptors can be observed in the majority (>90%) of neurons. In the CNS of Drosophila, more than 90% of neurons project their axons to the contralateral side of the nervous system, and the width of the commissural segment or precrossing segment of those neurons is 20-40 μm, which parallels the length of the proximal compartment observed in vitro. This raises the possibility that the intrinsic patterning ability of neurons may serve as the basis of generating the intra-axonal localization of guidance molecules in vivo (Katsuki, 2009).

In addition to these intrinsic abilities of neurons, the results suggest that extrinsic factors may also contribute to the intra-axonal patterning, because not all ROBO receptors examined in this study recapitulated the localization patterns observed in vivo. All three members of ROBO family receptors are localized to distal axon in vivo. Whereas ROBO2 and ROBO3 retained the ability to localize distally when isolated in culture, ROBO was uniformly distributed along axons under such conditions. Localization of ROBO may require extrinsic signals that are absent in the culture system. One of the candidate extrinsic factors are the midline cells, which lie on an axonal region where ROBO expression is downregulated in vivo. It is also possible that the location of the compartment boundary determined by the intrinsic mechanisms is refined by extrinsic signals. It would be interesting to test whether contact with midline cells in culture can induce distal localization of ROBO, or alter the position of the boundary (Katsuki, 2009).

It has been commonly suggested that axon guidance receptors are targeted to the growth cone, and intra-axonal localization patterns of guidance receptors reflect temporal profiles of receptor expression at the growth cone during axonal extension. This study demonstrates that intra-axonal localization patterns that are evident in the culture condition can form regardless of the timing of receptor expression. Although this result does not rule out the involvement of temporal control of expression during axon navigation in vivo, it suggests that critical mechanisms for the intra-axonal localization of receptors described in this study are compartment-specific trafficking pathways. One such trafficking mechanism could involve local translation or targeted membrane transport, which can specifically deposit membrane proteins to either the proximal or distal membrane compartment. It is also possible that membrane proteins are selectively retrieved from one compartment through endocytic pathways (Katsuki, 2009).

A time course experiment in shits1 mutant backgrounds suggests that Derailed (DRL) is preferentially targeted to the proximal compartment. It was also shown that the correct intra-axonal localization of DRL requires Dynamin-dependent endocytosis; however, at present it cannot be distinguished whether or not the endocytosis of DRL is compartment specific. Because fluorescence recovery after photobleaching (FRAP) experiments on CD8-GFP suggest that the barrier between the proximal and distal axon compartments does not completely block the movement of membrane proteins between the compartments, it is possible that the Dynamin-dependent endocytosis is required to remove DRL that leaks into the distal compartment, serving to maintain the pattern generated by targeting. Alternatively, endocytosis itself may be compartment specific, contributing to the establishment of the pattern (Katsuki, 2009).

In contrast to DRL, ROBO3 does not appear to require shibire function for its localization, demonstrating the presence of differential trafficking mechanisms for DRL and ROBO3. Due to this shi-independence of ROBO3, it is not possible to conclusively demonstrate the presence of preferential targeting of ROBO3 by performing a time course experiment. Even if there is preferential targeting, it is likely that ROBO3 also needs to be removed from the incorrect compartment, because ROBO3 shows a level of lateral mobility on the axon similar to that of DRL. Since ROBO3 localization is largely independent of Dynamin function, such a retrieval pathway must be based on Dynamin-independent mechanisms. While the complementary localization patterns of DRL and ROBO3 suggests that intra-axonal compartments are fundamental units for localization of multiple molecules, molecular mechanisms for generating or maintaining their compartmental localization could be diverse (Katsuki, 2009).

Another critical issue raised in the previous studies in vivo is how the intra-axonal localization of guidance receptors is maintained over time. If the guidance receptors are freely diffusible on the axonal membrane, they may spread along the axon, leading to a uniform distribution. FRAP experiments in cultured neurons revealed that localized receptors (ROBO3-EGFP and DRL-EGFP) are indeed mobile within the intra-axonal compartment. Although the mobility of these localized receptors across the compartment boundary was not directly measurable, the mobility of several transmembrane proteins (ROBO-EGFP and CD8-GFP) and lipid-anchored protein (GFPgpi) that distribute along the entire axon length was significantly restricted at the compartment boundary. This restriction is likely due to the diffusion barrier that spans over a 10 μm axon length around the boundary. It is proposed that this barrier is a part of the mechanisms that maintain the pattern of compartment-specific membrane proteins, as shown in different subcellular regions such as the tight junction of epithelial cells, the posterior ring of sperm, the cleavage furrow of dividing yeast and mammalian cells, and the initial segment of mammalian neurons. No significant barrier effect on GAP-GFP, which resides in the inner leaflet of the plasma membrane, was detected. It was also observed that vesicles containing membrane proteins pass through the barrier region. Thus, a model is favored in which the barrier becomes effective only after the molecules are inserted into the axonal membrane. It would be important to test whether or not a diffusion barrier exists in vivo, and whether or not it plays a role in the development of the nervous system (Katsuki, 2009).

An important but yet poorly explored question is the role of the guidance receptors localized on axon shafts. A straightforward explanation can be offered based on non-cell-autonomous functions of guidance receptors or membrane proteins in general; they may 'label' axon pathways through specific adhesion (fasciculation), or through presenting their ligands, thereby providing instructive spatial cues for the navigation of other axons. For example, Fasciclin cell-adhesion molecules have been suggested to provide pathway labels for guiding other growth cones. Drosophila Netrin receptor Frazzled/DCC relocates its ligand Netrin to strategic positions in the nervous system, thereby generating guidance information for a longitudinal pioneer neuron. Other studies reported that guidance receptors can also play non-cell-autonomous roles in cell migration and synaptogenesis. Thus, spatial patterns of molecules on axon shafts likely have direct roles in neuronal circuit formation (Katsuki, 2009).

Lastly, it is proposed that the compartmentalization of the axonal membrane could be a common basis for the structure and function of the nervous system. In the Drosophila ventral nerve cord, formation of the longitudinal axon tracts depends on the expression of ROBO receptors. On the other hand, longitudinal axon tracts are considered as the site for synapse formation, because synaptic proteins such as synaptotagmin and synapsin accumulate on the longitudinal tracts. This study found that in cultured neurons both ROBO receptors and synaptic proteins localize to the distal axon compartment. This may suggest that the spatial distribution of guidance molecules and synaptic proteins can be collectively governed by the compartmentalization of the axonal membrane. Future work to identify the molecular basis of the compartmentalization, and to establish the link between cellular identity and this intracellular pattern, will aid in determining how intra-axonal patterning contributes to tissue organization (Katsuki, 2009).

Protein Interactions

Wnt-mediated axon guidance via the Drosophila Derailed receptor

In nervous systems with bilateral symmetry, many neurons project axons across the midline to the opposite side. In each segment of the Drosophila embryonic nervous system, axons that display this projection pattern choose one of two distinct tracts: the anterior or posterior commissure. Commissure choice is controlled by Derailed, an atypical receptor tyrosine kinase expressed on axons projecting in the anterior commissure. Derailed keeps these axons out of the posterior commissure by acting as a receptor for Wnt5, a member of the Wnt family of secreted signalling molecules. These results reveal an unexpected role in axon guidance for a Wnt family member, and show that the Derailed receptor is an essential component of Wnt signalling in these guidance events (Yoshikawa, 2003).

The growth cones of developing neurons are guided to their targets by attractive and repulsive cues in the extracellular environment. Specific receptors on the growth cones recognize these cues and transduce signals that ultimately lead to changes in direction of growth. The best understood of these cues and their axonal receptors are involved in guidance of the large number of axons that project across the midline to the opposite side of the central nervous system (CNS). Distinct groups of cells at the midline divide the two halves of the CNS and have a critical role in axon guidance. These cells, termed midline glia in Drosophila, secrete diffusible factors, the Netrins, capable of attracting contralaterally projecting axons. They also secrete a repellent factor Slit, which together with its receptor Roundabout (Robo) and an intracellular sorting factor that modulates the delivery of Robo to the cell surface, controls whether or not axons will cross the midline (Yoshikawa, 2003).

Once axons commit to crossing the midline, they do not do so randomly. Instead, they follow particular tracts. In each segment of the Drosophila embryonic ventral nerve cord, crossing axons choose one of two commissural tracts, either the anterior or posterior commissure (AC or PC, respectively), which connect the two sides. This choice of commissure is controlled in part by the Derailed (Drl) guidance receptor. Drl is expressed on the growth cones and axons of all neurons that project through the AC, and seems to act as a receptor for a repellent factor in the PC. In drl mutants, AC axons abnormally cross in the PC of many segments. Conversely, misexpression of Drl in PC neurons switches their axonal projections to the AC. Thus, Drl is both necessary and sufficient for axons to cross the midline in the AC. The behavioral phenotypes of drl mutants suggest that at least some of the neurons that require Drl for their guidance fail to make synaptic connections essential for coordinated locomotion and learning and memory (Yoshikawa, 2003).

Drl is a member of the RYK subfamily of atypical receptor tyrosine kinases (RTKs). All members of this subfamily have unusual, but highly conserved amino acid substitutions in their kinase domains plus relatively short extracellular domains devoid of motifs commonly found in other RTKs. Consistent with the unusual amino acid substitutions, the kinase domain of RYK family members appears to lack catalytic activity. However, whereas catalytic activity of Drl has been shown to be dispensable, its cytoplasmic domain is required to dictate commissure choice, suggesting that Drl transduces a signal within growth cones in an unconventional manner, perhaps together with another catalytically active kinase20. The extracellular domain of each RYK family member contains a Wnt inhibitory factor (WIF) domain. The WIF domain of other molecules has been shown to bind to and inhibit the function of members of the Wnt family of secreted signalling molecules, raising the possibility that members of the RYK receptor family, including Drl, bind to Wnt proteins. The Wnt family is large, consisting of seven members in Drosophila and 19 in humans, and is involved in a diverse array of developmental events. Wnt proteins have well-established roles in early cell fate decisions and embryonic patterning, but have also been implicated in synaptic remodelling and terminal arborization within the developing CNS, as well as in regulating planar cell polarity by virtue of the phenotypes of mutations in Frizzled (Fz), one of the Drosophila members of the Fz family of Wnt receptors (Yoshikawa, 2003).

To identify components of the Drl signalling pathway, a genetic screen was carried out for mutations that suppress the ability of Drl to switch axons to the AC when misexpressed by PC neurons. A set of chromosomal deletions covering approximately 80% of the Drosophila genome was screened. One of the deletions that showed strong dominant suppression of the PC-to-AC switching activity of Drl is Df(1)N19, a deletion that removes the X chromosome interval 17A1 to 18A2. By testing a series of overlapping deletions within the Df(1)N19 region, the interval was narrowed to 17B. One of the genes in this interval is wnt5 (also called Dwnt3), a member of the Wnt gene family in Drosophila. wnt5 is a single-exon gene encoding an unusually large Wnt protein of 1,004 amino acids with a unique amino-terminal domain that seems to be proteolytically cleaved, followed by the Wnt domain common to all members of the family (Yoshikawa, 2003).

Given the possibility that Drl might interact with Wnt proteins by means of its WIF domain, mutations were generated specifically in the wnt5 gene to test whether reduction of Wnt5 itself is responsible for the suppression observed with the larger chromosomal deletions. A P element transposon, BG00642, was identified from the Berkeley Drosophila Genome Project inserted in the 5'-untranslated region (5' UTR) of wnt5, and mobilized to generate deletions of the wnt5 coding region. A deletion, wnt5D7, was recovered that removes the first 261 amino acids of the Wnt5 protein but does not affect either adjacent gene. In addition, a larger deletion was recovered, wnt5D84, that removes the entire wnt5 coding region plus part of the 3' end of the adjacent gene encoding a member of a family of gamma-glutamyl transferases. Both wnt5D7 and wnt5D84 abolish Wnt5 expression: the phenotypes of wnt5D7 and wnt5D84 homozygotes, as well as wnt5D7/wnt5D84 individuals, are indistinguishable. Thus, wnt5D7 acts as a null allele. wnt5D7 and wnt5D84 homozygotes are viable and fertile, but similar to drl mutants, adults are uncoordinated (Yoshikawa, 2003).

Tests were performed to see whether mutations in wnt5 could suppress the ability of Drl to switch axons to the AC. Using the Gal4/UAS transactivation system, Drl was misexpressed in PC neurons with eagle-GAL4 (eg-GAL4). This driver expresses Gal4 in a sufficiently small subset of neurons so that their axonal projections could be followed unambiguously with a UAS-tau-myc-green fluorescent protein (GFP) axon-targeted reporter transgene. eg-GAL4 drives expression in two small clusters of Eg interneurons in each hemisegment, both of which project axons across the midline (Yoshikawa, 2003).

One of the clusters projects in the PC and the other in the AC. At the midline, the axons from homologous clusters on either side of each segment fasciculate with one another, forming two distinct axon bundles, one within each of the commissures. When forced to misexpress Drl using a UAS-drl transgene, Eg PC neurons switch their projections to the AC in all segments, whereas Eg AC neurons are unaffected. In 92% of segments, every Eg PC axon was switched to the AC, whereas in 8% of segments some axons remained within the PC. Misexpression of Drl using the same transgenes, but in a wnt5/+ heterozygous background, resulted in significantly fewer PC-to-AC switched axons: 34% of segments had all axons switched; 34% had some switched and 32% had none switched. Notably, when Drl is misexpressed in a wnt5 hemizygous or homozygous mutant background, its ability to switch Eg PC neurons to the AC is completely abolished. Thus, Drl requires Wnt5 to switch axons to a different commissure, suggesting that Wnt5 is an essential component of the Drl signalling pathway (Yoshikawa, 2003).

In situ hybridization of wnt5 probes to wild-type embryos revealed that wnt5 messenger RNA expression in the CNS commences at stage 12, a point in development when differentiating neurons begin to extend axons, and continues throughout embryogenesis. High levels of wnt5 were detected in subsets of neurons restricted to the posterior half of each segment and low levels in neurons located more anteriorly in the segment. The neurons expressing high levels of wnt5 lie adjacent to and ventral to the PC in each segment. Although the precise identity of these neurons is unknown, their proximity to the PC and the fact that most of the CNS neurons project axons across the midline suggest that many, perhaps all, of the wnt5-expressing neurons project axons through the PC (Yoshikawa, 2003).

To examine the extracellular distribution of Wnt5 protein, live embryos were stained with an antibody raised against a unique region of the protein N-terminal to the Wnt domain. Staining was detected on the major axonal tracts within the CNS, with the highest levels on the two commissures, a pattern similar to that described previously (Fradkin, 1995). Staining is abolished in wnt5 mutants, demonstrating the specificity of the antibody. Given that wnt5 mRNA is expressed predominantly by subsets of neurons located posteriorly within the segment, Wnt5 apparently either diffuses to the AC or is picked up by AC growth cones and axons as they project toward the midline. Whether the proteolytic processing of Wnt5 observed in cultured cells (Fradkin, 1995) is involved in its distribution in vivo is not known, since the anti-Wnt5 antibody recognizes both the unprocessed and processed forms of the protein [relative molecular mass (Mr) of 140K and 80K, respectively]. Similarly, it is unknown whether all of the Wnt5 recognized by the antibody is biologically active, since processing may be required for activity (Yoshikawa, 2003).

If Wnt5 were a component of the Drl signalling pathway, then loss-of- function mutations in wnt5 might be expected to exhibit drl-like mutant phenotypes. Using an antibody that labels all CNS axons, it was found that wnt5 mutant embryos, similar to drl mutants, have disorganized commissures. In many segments commissures appear irregular, and there are often abnormal axonal projections between the AC and PC. To determine whether these abnormal projections arise from AC axons projecting to the PC or vice versa, subsets of AC and PC axons were labelled in wnt5 mutants with marker lines used for analysing drl mutants. In all cases it was found that AC axons either wander from the AC into the PC or project entirely through the PC, whereas projections of PC axons appear unaltered. These defects are similar to those seen in drl mutants. For example, in wnt5 mutants assayed with a P{tau-lacZ} marker for AC axons, 80% of segments displayed abnormal projections of AC axons into the PC. Similar results were found using Sema2b-tau-myc, another marker for a subset of AC neurons. In contrast to AC axons, in no segments did PC axons, as assayed with a P{tau-lacZ} marker for the PC, project abnormally into the AC. Thus, Wnt5, similar to Drl, is required for proper projection of AC axons across the midline of the CNS (Yoshikawa, 2003).

It has been proposed that Drl functions to keep axons in the AC by acting as a guidance receptor for a repellent ligand in the PC. The high levels of expression by neurons associated with the PC is consistent with Wnt5 acting as such a repulsive ligand. To examine whether Wnt5 is capable of repelling Drl-expressing axons, it was misexpressed at the midline and the effect on crossing axons was assayed. Misexpression of Wnt5 in midline glia using the sim-GAL4 driver caused a marked reduction or complete loss of AC in 43% of segments, but had no discernible effect on the PC. The affected AC axons appeared to either stall or project ipsilaterally within the longitudinal connectives. This phenotype is interpreted as repulsion of the Drl-expressing AC axons from the ectopic source of Wnt5 at the midline. To determine whether this loss of the AC is dependent on Drl, Wnt5 was misexpressed using the identical combination of transgenes, but in a drl homozygous mutant background. Elimination of Drl completely suppresses the loss of the AC, restoring it in every segment, although as expected, abnormal axonal projections between the AC and PC are detected due to the loss of Drl. Thus, in the absence of Drl, axons are insensitive to Wnt5, a feature that may explain the tight spatial regulation of the Drl receptor during development. Drl is normally expressed on growth cones and axons as they project through the AC, but is rapidly downregulated once these growth cones leave the commissure and begin to project in the longitudinal connectives on the contralateral side. This downregulation of Drl may be required to allow further extension of the AC growth cones as they traverse regions of repellent Wnt5 in the connectives, similar to the downregulation of Robo allowing axons to traverse the Slit-expressing midline (Yoshikawa, 2003).

These results suggest that Drl is a receptor for Wnt5. To test for binding of Drl to Wnt5, an examination was made of the in vivo binding of Drl-Fc, a soluble probe consisting of the Drl extracellular domain epitope-tagged with the human immunoglobulin-g (IgG) Fc fragment. In wild-type embryos, Drl-Fc binding was detected at the PC and to regions at the intersection of the PC and the longitudinal connectives. In wnt5 mutant embryos, binding of Drl-Fc is abolished. Conversely, when Wnt5 is misexpressed by heat-shocking late-stage embryos carrying a heat shock-wnt5 (hswnt5) transgene, followed by incubation with Drl-Fc, binding is markedly expanded to include all axon tracts in the CNS. The observation that Drl-Fc labels only the PC in wild-type embryos, whereas Wnt5 is present on both commissures, suggests that Wnt5 in the AC is bound to endogenous Drl present on the AC growth cones and axons, and that this interaction may block Drl-Fc access to Wnt5. Consistent with this, in drl mutants Drl-Fc labelled both the AC as well as the PC (Yoshikawa, 2003).

SDS-polyacrylamide gel electrophoresis (PAGE), immunoblotted with the anti-Wnt5 antibody was used to further examine the interaction between Wnt5 and Drl. Drl-Fc is able to co-precipitate both the unprocessed and the proteolytically processed forms of Wnt5, as evidenced by the presence of 140K and 80K bands from wild-type extracts, but not from wnt5 mutant extracts. Consistent with the lack of Drl-Fc binding to misexpressed Wg in vivo, Drl-Fc did not co-precipitate Wg, as assayed by immunoblotting with an anti-Wg antibody. Although both forms of Wnt5 present in the extracts are capable of binding to Drl-Fc, it is not known, in vivo, whether both actually have access to the Drl receptor. For example, the 140K form may not be efficiently secreted, as has been observed in cultured cells (Fradkin, 1995). However, regardless of the in vivo distribution of the two forms of Wnt5, this result, together with the genetic evidence, indicates that Wnt5 is the ligand for Drl (Yoshikawa, 2003).

The fact that Wnt proteins are known to signal through Fz receptors raises the possibility that Drl might not be acting as a 'classical' guidance receptor, but as a co-receptor for Wnt5, modulating its signalling through one or more of the Fz proteins in a manner similar to that proposed for Arrow/LRP6. For example, binding of Wnt5 by Drl might modify Wnt5 signalling through Fz and/or Fz2, the two Drosophila Fz family members expressed by embryonic CNS neurons. However, in contrast to wnt5, neither fz;fz2 double mutants, nor mutations in dishevelled (a downstream Fz signalling component) show any effect on Drl-mediated axon switching, and drl/+;fz fz2/+ trans-heterozygous embryos do not show defects in midline crossing. Furthermore, interfering with Fz-mediated Wnt signalling by pan-neuronally expressing a dominant-negative form of Fz2 (GPI-Dfz2) causes no defects in midline crossing (Yoshikawa, 2003).

Although these results do not rule out signalling through Fz proteins, they do advance the idea that Wnt5 might be signalling through the Drl receptor, a possibility consistent with the finding that misexpression of Drl lacking its intracellular domain fails to switch any Eg axons, even when misexpressed at high levels from multiple transgenes. In either event, whether Drl transduces the Wnt5 signal or modulates Wnt5 signalling through Fz proteins, it is an essential component of Wnt5 signalling in the guidance of axons across the midline (Yoshikawa, 2003).

In both drl and wnt5 mutants many axons still project appropriately in the AC, suggesting that Wnt5 and Drl are part of a larger multi-component system to ensure proper sorting of axons as they cross the midline. For example, it seems probable that there are additional attractive cues for AC axons, and that once they are attracted to the AC, Drl functions to prevent them from entering the PC. In addition, there may be a similar mechanism for the guidance of PC axons, whose choice of commissure is unaffected in both drl and wnt5 mutant embryos. Possibilities for additional molecules involved in commissure choice include other members of the Drl and Wnt families, some of which are expressed in the developing CNS. These results in Drosophila suggest that a similar receptor-ligand interaction between RYK and Wnt family members might be functioning in mammalian CNS development. Although nervous system phenotypes have not yet been described for the mouse knockouts of RYK and Wnt5a, the two mutants, although differing in severity, do display qualitatively similar skeletal defects, suggesting the possibility of an interaction. Within the mammalian CNS, Wnt proteins have been implicated in the guidance of commissural axons along the anterior-posterior axis of the spinal cord after they cross the midline. It will be of interest to test whether RYK has a role in these guidance events (Yoshikawa, 2003).

There are at least three distinct guidance mechanisms involved in midline crossing of contralaterally projecting axons within the Drosophila CNS. As in vertebrates, growing axons are attracted to the midline by diffusible cues such as Netrins acting through their receptor Frazzled/Dcc. Once there, the choice of whether or not to cross is controlled by Slit through its receptor Robo. Finally, as shown here, their choice of commissure is controlled by Wnt5 by means of its receptor Drl (Yoshikawa, 2003).

Different Wnt signals act through the Frizzled and RYK receptors during Drosophila salivary gland migration

Guided cell migration is necessary for the proper function and development of many tissues, one of which is the Drosophila embryonic salivary gland. Two distinct Wnt signaling pathways regulate salivary gland migration. Early in migration, the salivary gland responds to a WNT4-Frizzled signal for proper positioning within the embryo. Disruption of this signal, through mutations in Wnt4, frizzled or frizzled 2, results in misguided salivary glands that curve ventrally. Furthermore, disruption of downstream components of the canonical Wnt pathway, such as dishevelled or Tcf, also results in ventrally curved salivary glands. Analysis of a second Wnt signal, which acts through the atypical Wnt receptor Derailed, indicates a requirement for Wnt5 signaling late in salivary gland migration. WNT5 is expressed in the central nervous system and acts as a repulsive signal, needed to keep the migrating salivary gland on course. The receptor for WNT5, Derailed, is expressed in the actively migrating tip of the salivary glands. In embryos mutant for derailed or Wnt5, salivary gland migration is disrupted; the tip of the gland migrates abnormally toward the central nervous system. These results suggest that both the Wnt4-frizzled pathway and a separate Wnt5-derailed pathway are needed for proper salivary gland migration (Harris, 2007).

Salivary gland migration can be separated into three phases. In the first phase, the salivary glands invaginate into the embryo at a 45° angle, moving dorsally until they reach the visceral mesoderm. fkh, RhoGEF2 and 18 wheeler have been shown to regulate apical constriction of the salivary gland cells during this invagination process. In addition, hkb and faint sausage are needed for proper positioning of the site of invagination. No guidance cues have been identified for this first phase of migration; it may be that the patterns of constriction and cell movements at the surface of the embryo are sufficient to direct the invaginating tube (Harris, 2007).

During the second phase of migration, as the salivary gland moves posteriorly within the embryo, two guidance cues, Netrin and Slit, guide salivary gland migration along the visceral mesoderm. Netrin, which is expressed in the CNS and the visceral mesoderm, works to maintain salivary gland positioning on the visceral mesoderm. At the same time, Slit acts as a repellent from the CNS to keep the salivary glands parallel to the CNS. A third guidance signal, WNT4, which acts through FZ or FZ2 receptors, is also required in the second phase of salivary gland migration. Loss of Wnt4, fz or fz2 in the embryo results in salivary glands that are curved in a ventromedial direction. This curving affects a large portion of the salivary gland and may result from the fact that the fz and fz2 receptors, in contrast to drl, are expressed throughout the salivary gland. Furthermore, dominant-negative transgenes that disrupt the function of DSH or TCF cause the same phenotype, suggesting that transcription induced by the canonical Wnt signaling pathway is needed to maintain the proper migratory path of the salivary glands on the circular visceral mesoderm (CVM). The migration along the CVM takes more than 2 hours for completion, which would leave adequate time for a transcriptional response (Harris, 2007).

Although Wnt4 and slit are both required for the second phase of migration, and their mutants show similar, though distinguishable, phenotypes, they are thought to act independently. While most slit-mutant embryos have medially curving salivary glands, embryos lacking Wnt4 have salivary glands that curved in a distinctly different, ventromedial, direction. Embryos doubly mutant for Wnt4 and slit show predominantly one or the other phenotype and neither phenotype increases in severity. These results suggest, though they do not prove, that Wnt4 and slit act in distinct pathways (Harris, 2007).

After the entire salivary gland has invaginated, migrated posteriorly within the embryo and lies parallel to the anteroposterior axis of the embryo, the distal ends of the salivary glands come into contact with the LVM. drl and Wnt5 are required for this late phase of salivary gland positioning. Loss of either drl in the salivary gland or Wnt5 in the CNS results in the distal tip of the salivary gland being misguided to a more ventromedial position. This change in the shape of the salivary gland is seen only after the salivary glands are no longer in contact with the CVM (after stage 13). Thus it is proposed that drl is required during the third phase of salivary gland migration, as the salivary gland detaches from the CVM and contacts the LVM (Harris, 2007).

The striking expression of drl at the tip of the salivary gland makes the leading cells uniquely different from the rest of the salivary gland cells. These cells project lamellipodia upon reaching the visceral mesoderm and beginning their posterior migration. They may act to both guide and pull the rest of the gland during migration. Cells at the tip of a migrating organ are frequently specialized to guide migration. For example, the coordinated migration of the tracheal branches in Drosophila is achieved by induction of distinct tracheal cell fates within the migrating tips. This is illustrated by the fact that FGF (Branchless) signaling becomes restricted to the tips of the tracheal branches soon after they begin to extend. The migration and growth of Drosophila Malpighian tubules provide another clear example of specialized cells needed at the tip of a migrating tissue. One cell is singled out to become the tip cell, which directs the growth of the Malpighian tubules as well as organizes the mitotic response and migration of the other cells forming each tubule. In other systems, such as Dictyostelium slugs, cells at the tip of a migrating group are required and solely able to guide migration. These results establish that the leading cells of the migrating salivary glands have a specialized role to play in proper salivary gland positioning. First they are required to initiate invagination within the embryo, then they actively participate in migration along the CVM, and finally they ensure that the distal tip of the gland will remain associated with the LVM at the end of the migratory phase (Harris, 2007).

Despite the fact that it has been firmly established that Wnt5 and drl are important for the final placement of salivary glands, the signaling pathways downstream are not well defined. Because salivary-gland expression of full-length drl can rescue the drl-mutant phenotype, but drl lacking the intracellular domain cannot, it is thought that the intracellular domain of DRL is important for signaling. Similarly, misexpression of full-length drl can misguide axons in the ventral nerve cord, but misexpression of drl lacking its intracellular domain cannot (Yoshikawa, 2003). The genetic interactions found in this study between drl and Src64 support recent findings suggesting that Src64 acts downstream of drl in the ventral nerve cord. In addition, the other Drosophila Src kinase, Src42, may be required at two stages, during salivary gland migration along the CVM and downstream of WNT5-DRL signaling as the gland moves onto the longitudinal visceral mesoderm (Harris, 2007).

Another intriguing finding of this study is the involvement of the two remaining Drosophila RYKs, Drl-2 and dnt, in salivary gland development. The phenotypes of Drl-2 and dnt mutants are less penetrant than drl mutants, but they are qualitatively very similar. Furthermore, embryos doubly heterozygous for drl and Drl-2 have salivary glands that resemble those seen in drl mutant embryos. These three RYKs appear to act in a partially redundant fashion in the salivary glands, since none of the single gene mutations leads to completely penetrant phenotypes. However, no increase was seen in penetrance of the drl phenotype in embryos lacking both drl and Drl-2. In addition, it was not possible to detect transcripts for either Drl-2 or dnt in the salivary gland. While it is possible that dnt and Drl-2 are expressed at very low levels in the salivary gland, they might be acting non-autonomously (Harris, 2007).

An interesting dilemma in understanding RYK signaling is how inactive kinases propagate a signal into the cell. Recent mammalian studies have suggested that RYKs may associate with another catalytically active receptor, such as FZ or EPH, at the membrane. In the mouse, the extracellular WIF domain of RYK interacts with FZD8, and it has been proposed that the two proteins may form a ternary complex with WNT1 to initiate signaling. However, data from flies and nematodes support the argument that DRL and its C. elegans homolog LIN-18 act independently of FZ. Genetic studies of cell specification in the nematode vulva suggest that LIN-18 acts in a parallel and separate pathway from the LIN-17/FZ receptor. Similarly, reduction of fz and fz2 gene activity in flies has no effect on a DRL misexpression phenotype in the ventral nerve cord (Yoshikawa, 2003). This study has shown that double mutants for the Wnt4 and Wnt5 ligands and for the fz and drl receptors both show strong enhancements in comparison to the single mutants, reinforcing the conclusion that these two ligands are activating different pathways. In addition, the functions of these two pathways can be separated by phenotype. The Wnt4-fz/fz2 phenotype becomes evident earlier and affects a larger portion of the salivary gland than the Wnt5-drl phenotype. Taken together, these results demonstrate that there are two independent Wnt pathways regulating salivary gland positioning. The early WNT4 signal appears to activate the canonical Wnt pathway, whereas there is a later requirement for WNT5 signaling through DRL and the Src kinases (Harris, 2007).

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. This study used the stereotypic pattern of the Drosophila embryonic body wall musculature in genetic approaches to identify novel factors required for muscle attachment site selection. Wnt5 is shown to be 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. A mutant allele of dnt was generated and it was found 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 (Lahaye, 2012).

The development of the intricate muscle pattern of higher organisms requires the coordinate expression of numerous cellular factors regulating the specific fate, differentiation, orientation and attachment of the individual muscle fibers. The first steps of muscle formation likely occur autonomously, but guidance of myofibers towards and attachment to their appropriate tendon cells are, at least in part, controlled by secreted and transmembrane proteins emanating from both the target cell and the approaching muscle fiber. This study has shown that, in Drosophila, the secreted WNT5 protein and the Ryk transmembrane receptor family members, DRL and DNT, are essential for guidance of a subset of embryonic body wall muscle fibers to their tendon cells (Lahaye, 2012).

There are three Ryk orthologs in Drosophila, drl, dnt and Drl-2. 36%, 8%, 0% of hemisegments display a lateral transverse muscles (LTM) muscle bypass phenotype when drl, dnt or Drl-2 is absent, respectively. Homozygosity for relatively small deficiencies that uncover both drl and dnt results in the bypass phenotype in virtually all hemisegments (96%). Embryos which completely lack DRL and are heterozygous for a mutant allele of dnt display intermediate penetrance of the phenotype (50%). Embryos lacking DNT and are heterozygous for drl have bypassing muscles in 8% of their hemisegments. These results suggest that the Ryk family members, dnt and drl, coordinately regulate the attachment of the LTM muscle fibers to tendon cells with drl being the dominant player. The decrease in penetrance in the animals lacking both copies of drl and one copy of dnt (50%), relative to those completely lacking both genes (96%), indicates that dnt can at least partially compensate for the absence of drl. Consistent with this is the reported ability of the expression of dnt in the LTMs to partially rescue the drl mutant bypass phenotype (Oates, 1998; Lahaye, 2012 and references therein).

Does WNT5 signal through DNT and DRL? Genetic studies indicate that this is likely the case. Female embryos simultaneously heterozygous for Wnt5 and a deficiency which uncovers both drl and dnt display the bypass phenotype while those heterozygous for either Wnt5 or the deficiency alone do not. Furthermore, male Wnt5 mutant hemizygotes, display increased penetrance when single copies of drl and dnt are removed. Thus, it is concluded that Wnt5 genetically interacts with drl and dnt, likely indicating that the WNT5 protein acts as a ligand for these two Ryk family members during muscle attachment site selection (Lahaye, 2012).

DRL is specifically expressed at the muscle tips of fibers 21-23 while they are in the process of extending towards their attachment sites. The protein is also expressed early in development from 6 hours AEL (stage 10) onwards in reiterated stripes in the epidermis and at stage 12 in clusters of epidermal tendon precursor cells, partially overlapping with the SR expression domain. Rescue of the drl mutant LTM bypass phenotype was only achieved when DRL was restored in the muscle and not the attachment sites. At present, the role of the early expression of drl in the tendon precursor cells is not clear (Lahaye, 2012).

dnt mRNA is also expressed in stripes in the epidermis associated with invaginating cells (Oates, 1998; Savant-Bhonsale, 1999). This transcript is also present at a low level in many embryonic tissues including the somatic musculature. Like DRL, DNT is likely required in the muscle fiber since transgenic expression of dnt in the LTMs rescues the drl phenotype. DRL-2 is expressed most predominantly in the central nervous system, suggesting that it was unlikely to have a role in LTM guidance, as was shown in this study. While almost all hemisegments display overshooting LTMs in the absence of DRL and DNT, only one or two of the three LTM fibers, usually muscles 21 and/or 23, exhibit this phenotype. This result indicates that other non Ryk-dependent mechanisms are required to guide these three muscles to their attachment sites. Alternatively, these two muscles may experience fewer physical barriers blocking their ventral extension beyond muscle 12. In addition, the overshooting of the appropriate tendon cells by these muscles is only observed at the ventral and not the dorsal attachment sites, indicating that guidance mechanisms differ for the two ends of the muscle (Lahaye, 2012).

WNT5 has an important role in guidance of embryonic central nervous system commissural axons and acts as a ligand for DRL in these tissues. When LTM trajectories were investigated in Wnt5 mutant embryos it was found that one or more LTMs overshoot their normal tendon cells in only 17% of the hemisegments compared with 36% in the drl mutant. This result suggests that there are likely other DRL ligands in addition to WNT5. Possible other candidates include the other six wnt genes present in Drosophila, wg, Wnt2, Wnt4, Wnt6, Wnt8 and Wnt10 [reviewed at 'The Wnt Home page' (www.stanford.edu/group/nusselab/cgi-bin/wnt)]. Segmentation defects during early embryogenesis in wg mutants and the lack of available mutants for Wnt6 and Wnt10 precludes further analyses of muscle pattern formation in the absence of these genes. Furthermore, Wnt8 is not detectably expressed in the somatic mesoderm. Since both Wnt2 and Wnt4 had been previously implicated in diverse stages of muscle formation and function, this study analyzed LTM trajectories in a Wnt2/Wnt4 double mutant. No bypassing LTMs were observed in the double mutant embryos, nor in the singly homozygous mutants, indicating that these two Wnt genes are not likely involved in regulating LTM attachment. WNT10 is the most probable alternative ligand for DRL and DNT in muscle since its mRNA is expressed in the developing somatic mesoderm (Janson, 2001), however evaluation of its potential roles awaits the generation of a mutant allele (Lahaye, 2012).

In which cells is WNT5 expressed and required? This study found that Wnt5 mRNA and protein are expressed at low levels in all somatic muscles while they are extending, in mature attachment sites and also during early development in a subset of the tendon cell precursors and in the epidermis. Furthermore, rescue of the bypass phenotype is seen when a Wnt5 transgene is expressed in either of these two tissues. Since WNT5 is a secreted factor and rescue of the Wnt5 phenotype is observed with restoration in either the muscle or the tendon cells, it is difficult to conclude unambiguously in which tissue it is needed. Restoring expression of WNT5 in muscle fiber 12 only does not rescue the bypass phenotype. This result suggests that it is not simply sufficient to have a high source of WNT5 in the muscle close to the original attachment sites for appropriate inhibition of LTM extension. It is more likely, that WNT5, which is widely expressed in the epidermis and musculature, is modified in some way to become locally activated as a specific LTM repulsive guidance cue. Support for this hypothesis comes from previous observations that Wnt5 is proteolytically-processed (Fradkin, 1995). Furthermore, WNT5 expressed by anterior commissural midline glial cells, but not in all neurons, blocks anterior commissure formation (Fradkin, 1995) due to the repulsion of DRL+ axons, indicating that elevated local expression of WNT5 can have different outcomes depending on the cell types which express it. Finally, although WNT5 is observed to be widely expressed in the larval/adult brain, it acts specifically to guide mushroom body α-lobe axons indicating that an apparently ubiquitously-expressed ligand can act as a directional cue. Alternatively, WNT5 may be sequestered from some regions of the extending muscle fiber by so-called 'extrinsic receptors' which results in a directional cue received by the leading edge of the muscle (Lahaye, 2012).

There is mounting evidence that the final differentiation of the Drosophila tendon cell, in particular the secretion of an elaborate extracellular matrix, is tightly coupled to the arrival of the muscle fiber. The resulting myotendinous junction is essential for force transmission and counteraction of muscle contraction by tendon cells. Similar junctions exist in vertebrates where tendons attach the muscles to the bone. In Drosophila, it consists of hemi-adherens junction formed by the association of integrin receptor heterodimers on the muscle tip and the tendon cell with the intercalating ECM proteins (Schweitzer, 2010) such as Laminin and TSP secreted from the tendon cells and Tiggrin from the muscle cell. The myotendinous junction is not functional when integrin, TSP or laminin are absent resulting in dissociation of fibers from their attachment sites which leads to lethality. The signals allowing recognition of the appropriate tendon cell, arrest of muscle fiber extension and the formation of the myotendinous junction remain unclear. However, genetic phenotypic analyses indicate that changes in local integrin receptor accumulation on muscle tips and differential responses to TSP presented on the tendons might slow down and stop muscle migration prior to the initiation of myotendinous junction formation (Schweitzer, 2010). A functional myotendinous junction is formed at the novel attachment site of Wnt5 and drl mutants as evidenced by the observation that βPS integrin accumulates at this site. βPS integrin expression was not observed at the original attachment site indicating that the interaction of the muscle tip with the bypassed site, if it occurs at all, is not of sufficient duration to initiate attachment site maturation (Lahaye, 2012).

The observation that the initial outgrowth and guidance of the LTMs are normal in Wnt5 and drl mutants suggests that these proteins act during the recognition of the target cell and not earlier during muscle extension. Wnt/Ryk signaling may be required for induction of a localized 'stop' signal for the LTM at its normal attachment site. In this scenario DRL and DNT present on muscle fibers would bind activated WNT5 secreted from their normal attachment sites. This interaction might then result in the transcription of genes encoding extracellular matrix proteins in the muscle fiber which are required to increase adhesiveness between the muscle and tendon cell, slowing down the fibers extension. When either WNT5 or DRL/DNT is absent this signal is not appropriately received by the approaching fiber and it overshoots its target and attaches relatively randomly to a more distant epidermal cell (Lahaye, 2012).

In the Drosophila embryonic CNS, DRL acts as a repulsive guidance receptor on growth cones of anterior commissural axons to steer them away from the posterior commissural axons which express WNT5. It seemed thus possible that DRL/DNT also acts in the muscle as a repulsive receptor upon binding of WNT5. However, no clear muscle guidance defects were observed when WNT5 was ectopically expressed on either specific muscle fibers or in the tendon cells. As mentioned above, it is possible that WNT5 has to be locally modified and activated or differentially sequestered to function as a guidance cue in this tissue (Lahaye, 2012).

It was found that that the novel attachment site for the overshooting muscle in embryos and larvae is an epidermal cell and not another muscle. The normal LTM attachment site that is not recognized by the bypassing muscle is present in Wnt5 and drl mutants as visualized by its ability to express Stripe, a transcription factor that is both necessary and sufficient to drive tendon cell fate. Therefore, this tendon cell follows important early stages of normal tendon cell differentiation, but does not bind the fiber (Lahaye, 2012).

In contrast, only 35% of the ectopic tendon cells express Stripe suggesting that Stripe expression is not obligatorily required for formation of a stable myotendinous junction. At present, it is not known whether the novel attachment site expressed SR earlier in development or whether, despite its stability against contraction-induced damage, the ectopic myotendinous junction is different in some manner from the normal junction as to not allow maintenance of Stripe expression. The FAS2 protein that is normally expressed at the muscle tip and the tendon cell to which it attaches, is present at both the original and the novel attachment sites in drl and Wnt5 mutant larvae. This result indicates that the muscle 'filopodia' likely transiently interact with its normal tendon cell target but does not cease extension. This further supports the notion that Wnt/Ryk signaling may increase the stability of muscle/tendon cell interactions (Lahaye, 2012).

It is too early to evaluate whether the molecular mechanisms of muscle attachment site selection are conserved between vertebrates and invertebrates because of the paucity of knowledge about the molecules required for tendon differentiation and its connections to muscle and skeletal tissues in vertebrates. Components of Integrin-mediated adhesion complexes, e. g., talin 1 and talin 2 and several laminin integrin receptors were, however, recently shown to be essential for the formation of the vertebrate myotendinous junction, as has been observed for their orthologs in Drosophila. In the coming years, as more becomes known about the mechanisms that mediate the connections between muscles and tendons, it will be apparent whether other aspects of muscle guidance and target site selection are also conserved (Lahaye, 2012).


DEVELOPMENTAL BIOLOGY

Embryonic

With regard to Drl expression in the CNS, Drl is localized to the growth cones and axons of the drl neurons as they cross within the anterior commissure. Interestingly, after the neurons have turned toward the anterior, Drl cannot be detected on the portion of the axons within the connectives, and remains restricted to the axon segments within the anterior commissure. This suggests that Drl does not physically participate in the fasciculation of drl neurons within the DD and DV bundles. Instead, the results suggest the existence of multiple, discrete steps in neuronal pathway recognition, with Drl playing a key controlling role as the following suggests: once activated by its ligand, Drl is thought to control pathway selection by modulating the function of specific cell adhesion molecules that mediate proper axons fasciculation (Callahan, 1996)

In addition to its expression within the CNS, drl is expressed in both the mesoderm and epidermis during embryogenesis. Using antibodies directed against its extracellular domain, Drl protein can be detected in a subset of somatic muscles as they grow and form attachments to the epidermis. Each hemisegment in abdominal segments A2 through A7 contains 30 muscles with predictable orientations and attachments to the epidermis. Beginning at hour 10 of development, low levels of Drl are detected in the precursors for muscles 21-23 as they begin to elongate dorsoventrally along the epidermis. Expression increases as these muscles continue to grow toward their attachment sites, and is still present by hour 13 as assayed by in situ hybridization or antibody staining. During the period of attachment site selection, Drl appears enriched near the tips of muscles 21-23, especially at the locations where they contact their ventral attachment sites. By hour 15, after attachment events have been completed, drl transcripts and protein are no longer detectable (Callahan, 1996).

Drl protein is first detected at approximately hour 6, appearing in stripes 3-4 cells wide in each segment. During segmental groove formation, Drl is restricted to anterior cells of each segment near the segmental grooves. At hour 9.5, expression begins to expand posteriorly from the grooves at lateral positions, resulting in broad patches of Drl-expressing epidermal cells. These lateral Drl patches overlie the differentiating muscle precursors 21-23. By hour 11, the lateral Drl patches become more restricted, forming two smaller clusters of Drl-expressing epidermal cells located near the dorsal and ventral attachment sites for muscles 21-23. This pattern subsequently becomes more refined, such that by hour 12.5, each cluster has become restricted to approximately 15 cells that abut and partially overlap the epidermal attachment cell clusters for muscles 21-23, as revealed by double-labeling for Drl and sr expression. As in muscles 21- 23, Drl ceases to be expressed in the epidermis by hour 15. Despite this pattern of drl epidermal expression, drl null mutants do not show any defects in segmental groove formation or in differentiation of the epidermis (Callahan, 1996) .

Larvae and Adults

In third instar larvae, two areas of Drl RTK expression are revealed in the CNS: the optic ganglion primordium, and a small cluster of cells at the inter-hemispheric region. In the adult brain, a lacZ reporter gene gives a cytoplasmic expression, which allows investigators to follow expression patterns in all the subcellular compartments that express GAL4. A preferential expression of the reporter gene is observed in the mushroom body (MB) and central complex (CX). There is also a strong expression in the optic lobes and a weaker expression in many other substructures in the brain. In the CX, expression is observed at the level of the fan-shaped body in the superior arch. There is also a strong expression in the cell body cluster, which is in contact with the top of the fan-shaped body at the junction between the two hemispheres. In the MB, staining is observed in the entire volume of the MB, from the KC body layer to the tips of the lobes. At the level of the lobes, intense staining is observed in the alpha and beta lobes. Staining is restricted to the circumferential elements of the lobes. There is also a weaker staining in a sub-component of the gamma lobes that corresponds to the most posterior fibers of the gamma lobe. These fibers lie dorsal and parallel to the beta lobe (Moreau-Fauvarque, 1998).

Weak Derailed expression appears in young third instar larvae, which increases until pupariation. Drl is then detected in the optic lobe anlage and in fibrous elements of the interhemispheric area, near the commissure. In the central area of the larval brain, the following dynamic pattern of expression is observed. The protein is first detected at 75 hours after egg laying (young third instar larvae) in two anterior projections that grow in multiple directions. At 85 hours, these anterior projections cross the commissure and diverge in two branches. This expression is clearly visible in late larvae. At 95 hours Drl is also detected more posteriorly in symmetrical fibrous elements that cross the commissure and also diverge in two brances. The position of these latter elements is compatible with the idea that they contact axons of mushroom body Kenyon cells. Expression declines in the early pupa, and in the adult brain no clear expression can be detected. This expression pattern suggests that Drl plays an important role at the late larval/early pupal stage (Simon, 1998).

A complete deletion of Drl activity causes specific structural defects in the adult brain. Gal4 enhancer-trap lines used as cell markers reveal that in drl mutants central brain axons behave as if they are abnormally attracted by the midbrain area. Defects are documented in the mushroom bodies and the central complex. Optic lobes also appear abnormal. The Drl protein is also expressed in third instar larvae in a few cells at the junction of the cerebral hemispheres. These glial cells form a newly described ring structure, showing an invariable fibrous organization. The ring -- which has been named transient interhemispheric fibrous ring or TIFR -- is located in a sagittal section in the middle of the brain. The TIFR appears to be highly organized, with entangled fibers creating a constant pattern of loops and hooks. The four large nuclei revealed with a beta-galactosidase reporter in a drl reporter are localized on the TIFR fibrous path. The TIFR is present in late larvae. It seems to be broken up in midpupae since no such structure has been described in the adult. In the wild-type this ring disappears at midpupation. These results indicate that the Drl putative kinase plays a major role in the modeling of the adult brain by controlling the fate of the transient interhemispheric ring (Simon, 1998).


EFFECTS OF MUTATION

In drl mutants, drl muscles attach at abnormal locations within the epidermis. These muscle attachment defects are not the result of gross alterations of the epidermis nor loss of epidermal attachment cell precursors. In contrast, analogous to its role in axon pathway selection within the nervous system, Drl participates in a mechanism required for muscle attachment site recognition. Epidermal cells destined to become muscle attachment cells express stripe before muscle attachment events, these epidermal cells to which muscles 21-23 attach were identified by their expression of stripe during myogenesis. At hour 10, a stripe marker begins to be expressed at positions where muscles will eventually insert. This includes sites at segmental boundaries where longitudinal muscles will attach, ventral rows of cells parallel to the segmental grooves where ventral muscles will attach, and in small clusters of cells located laterally between segment boundaries where the 21-23 group will attach. In hour 11.5 embryos, muscles 21-23 have extended dorsoventrally and can be seen forming both their dorsal and ventral attachment sites within the lateral clusters of stripe-expressing epidermal cells. By hour 14, the stripe clusters have become restricted to 6-8 cells, and the ventral attachments for muscles 21-23 lie adjacent to one another within the cluster. Normally, muscles 21-23 extend similar distances ventrally and attach adjacent to the dorsal border of muscle 12, always inserting within the stripe expressing epidermal cluster. In drl mutants, muscles 21-23 are present in their normal locations and elongate dorsoventrally, but have ventral attachment site defects. Muscle morphology was examined in drl null mutants. In 20% of hemisegments of drl mutants, one or more muscles of the 21-23 group pass over their normal ventral attachment sites and appear to attach far more ventrally, beyond muscle 13. This dramatic phenotype is termed the 'bypass' phenotype. An additional 10% of hemisegments have more subtle attachment defects where at least one muscle fails to attach within the ventral stripe-expressing cluster, but does not extend beyond muscle 13. Importantly, both the numbers and locations of the stripe-expressing epidermal cells in drl mutants are indistinguishable from drl + embryos. This indicates that the muscle phenotype seen in drl mutants is not the result of a loss of epidermal attachment cell clusters, but instead results from the inability of the muscles and their ventral tendon cells to coordinate functional attachments. Targeted expression of Drl to muscles is shown to rescue the attachment defects (Callahan, 1996).

These results are consistent with a role for drl in the muscle-epidermal interactions that underlie proper attachment site selection. One possibility is that the Drl RTK participates directly in recognition events between muscles 21-23 and their ventral attachment cells. Drl itself may mediate specific contacts between muscles 21-23 and their appropriate attachment site targets. Alternatively, Drl may be more indirectly involved in muscle-epidermal recognition events. For instance, Drl function may be required in muscles 21-23 before attachment site recognition events, serving to regulate or modify other gene products that are more directly involved in recognition processes. Such a possible modulatory role for Drl in muscle-epidermal recognition would be similar to the proposed role for Drl in neuronal pathway recognition events (Callahan, 1995). A further possibility is that Drl is not required for muscle-epidermal recognition events, but instead is required for the subsequent cytoskeletal changes that must accompany successful attachment. In this scenario, drl mutant muscles would fail to attach at their appropriate sites because they are unable to physically anchor at any location. This possibility seems less likely considering that those muscles displaying a mutant phenotype do appear eventually to attach to the epidermis, albeit at incorrect ventral locations (Callahan, 1996).

The fact that muscle defects are not 100% penetrant in drl null mutants illustrates that there are additional genes involved in muscle 21-23 attachment site selection. At present, it is not known what relationship these additional genes have to drl. One possibility is that the products of these genes are somehow modified by activation of Drl, yet are not completely dependent on Drl for their function. Alternatively, the fidelity of muscle 21-23 attachment site selection may arise from the combinatorial functioning of several distinct recognition processes, of which Drl controls only one. Interestingly, misexpression of Drl in muscles that normally do not express Drl does not cause mistargeting to the 21-23 attachment sites. This may be due to a lack of competence on the part of inappropriate muscles to respond to the Drl signal, or may reflect a restricted localization of the Drl ligand. Misexpression of a constitutively active form of Drl may help to resolve this point. The finding that Drl is expressed both in muscles and at epidermal locations along which the muscles grow and attach suggests that there may be both epidermal and mesodermal components to Drl function. For example, Drl could participate in a homotypic interaction mediating some form of recognition during muscle growth over the epidermis. The results argue against this possibility, since the muscle attachment phenotype could be rescued by targeted expression of Drl in muscles. These results strongly suggest that Drl function in muscles 21-23 is sufficient for proper attachment site selection (Callahan, 1996).

Mutations in Drosophila neurotactin, a gene that encodes a cell adhesion protein widely expressed during neural development, have been isolated and characterized. The lack of widespread axonal defects in the CNS of neurotactin mutants suggests that the function of Nrt in CNS morphogenesis might be largely replaced by functionally related molecules. If so, embryos lacking Nrt as well as one of these other molecules may display synergistic mutant phenotypes. To test this possibility, embryos lacking function of both nrt and one of several genes encoding neural CAMs were examined. Embryos of some double mutant combinations of neurotactin and other genes encoding adhesion/signaling molecules, including neuroglian, derailed, and kekkon1, display phenotypic synergy. This result provides evidence for functional cooperativity in vivo between the adhesion and signaling pathways controlled by neurotactin and the other three genes (Speicher, 1998).

linotte (more properly termed derailed) is a new autosomal gene in Drosophila involved with learning and memory. The linotte mutant was derived from a PlacW transposon mutagenesis and was screened for three-hour memory deficits after classical conditioning of an olfactory avoidance response. Sensory and motor systems (olfactory acuity and shock reactivity) required for the classical conditioning experiments are normal in mutant linotte flies--indicating that the mutation disrupts learning/memory specifically. A chromosomal deficiency of the 37D region, where the linotte P insert is localized in situ, fails to complement linotte's memory defect, and flies from two lines homozygous for independent PlacW excisions show normal memory--indicating that the P insertion is responsible for the mutant phenotype. Additional behavior-genetic data suggest that the linotte gene is non-vital (Dura, 1993).

The Drosophila transmembrane protein Derailed (Drl) is expressed in a glial transient interhemispheric fibrous ring (TIFR), which is hypothesized to serve as scaffold for adult brain formation during metamorphosis. (Interactive Fly editor's note: although this paper refers to drl as linotte, this review refers to the gene as drl, following FlyBase convention). TIFR specific enhancers have been isolated from the Drl locus and it has been shown that only four interhemispheric cells give rise to this complex fibrous structure. drl controls the TIFR differentiation, and the major role played by this structure in central brain metamorphosis has been confirmed because TIFR destruction by apoptosis leads to a pronounced adult phenotype. drl interhemispheric expression is specifically affected in a no bridge mutant context, confirming that nob plays a key role in adult brain development via the TIFR (Hitier, 2000).

To identify postembryonic drl enhancers, restriction fragments spanning the drl locus were fused to a reporter encoding either a nuclear form of beta-galactosidase (PX constructs), a cytoplasmic beta-galactosidase (PZ construct) or the transcription factor Gal4. The PX423 construct leads to no obvious enhancer activity in the CNS. The PX421 construct shows enhancer activity restricted to the midline part of the VNC. The PX422 construct, corresponding to the 5' part of the first intron, exhibits enhancer activity in the primordium of the optic lobes and in four cells at the interhemispheric junction (IJ). The PX432 construct corresponding to the 5' part of the 422 fragment, exhibits strong enhancer activity in the primordium of the optic lobes, the CNS, the lateral parts of the brain and in the four interhemispheric cells. The interhemispheric junction specific enhancer was further restricted to 1.3 kb since the PZ442 construct leads to a TIFR staining. Interestingly, the 425 fragment, which lies 5' from the drl transcription start, also shows specific expression in the four IJ cells. Thus two independent TIFR specific enhancers have been isolated. The alternative use of nuclear or cytoplasmic reporter in combination with TIFR specific enhancers demonstrates that only four glial cells generate the complex fibrous structure of the TIFR. The fragments used are partially redundant with the fragments isolated for drl embryonic enhancer activity analysis (Bonkowsky, 1999). The 425 and the 442 fragment are respectively comprised in the drlU and drlT fragments, showing enhancer activity in midline crossing neurons at the embryonic stage. This observation suggests that the same enhancer elements might control expression at the midline of the embryonic VNC and of the larval brain, even though drl is expressed in neural cells in the embryo and in glial cells in the larval brain (Hitier, 2000).

The drl product is required for shaping the TIFR, not for cell survival The expression of drl is required to build up a correct TIFR (Simon, 1998). However, since the TIFR could be observed only in the hypomorphic lindrlP mutant it has not been clear if drl expression is required for the formation and survival of the four IJ cells or for the TIFR fasciculation per se. A staining experiment performed on lio2;PX422/+ third instar larvae reveals that the four interhemispheric nuclei are normally present in an amorphic drl background. At the pupal stage, using the 442-gal4 construct and the axon targeted tau-based reporter it has been shown that the TIFR is drastically disorganized in the amorphic context. drl is thus required for glial fibrous fasciculation and/or pathway selection in the interhemispheric junction, as is observed for neurons at the embryonic midline, but not for cell survival (Hitier, 2000).

In order to characterize the TIFR function in adult brain development the brain of 442-gal4/+; UAS-hid/+ flies, in which the TIFR was destroyed by hid-induced apoptosis, was examined. Killing specifically the TIFR cells leads to a late partial pupal lethality. Interestingly, the adult 442-gal4/+;UAS-hid/+ shows a strong drl-like phenotype, i.e. protocerebral bridge disorganization, fibers crossing the midline above the fan-shaped body, flat fan-shaped body and fused MB lobes. This result confirms that the lack of drl expression in the TIFR is responsible for most of the adult phenotype displayed by the drl mutant and that the TIFR is a key cellular structure for the midline choice in the central brain. However, unlike the drl amorphic mutant, the alpha lobes are still present in 442-gal4/+;UAS-hid/+ individuals. In drl mutants the absence of a lobe phenotype could thus be due to the lack of drl expression in the MB itself (Hitier, 2000).

The TIFR destruction leads to a new phenotype compared to the drl mutant: the protocerebral bridge was found to be severely disorganized or absent, and the ellipsoid body ventrally opened and flattened. The fact that the amorphic drl mutant does not exhibit such a drastic phenotype confirms the observation that drl is not required for the existence of the four interhemispheric cells. Interestingly, this additional phenotype partially matches that of the nob mutant phenotype in a Canton-S background. It is hypothesized that the nob gene might play a role in the TIFR and interact with the drl gene (Hitier, 2000).

To assess the drl-nob interaction, drl expression in the nob mutant was followed in third instar larvae brains. In nob mutants drl expression is not detectable in the anterior fibers crossing the interhemispheric junction. In the optic lobes drl expression does not seem to be affected by the nob mutation and the drl product is still detectable in the posterior fibers of the interhemispheric junction. In nob/Y; lio1/+ larvae the beta-galactosidase expression driven by the lio1 PlacZ insertion specifically disappears in the four interhemispheric nuclei corresponding to the TIFR. This effect is nob-specific since drl larval interhemispheric expression is normal in several other central brain mutants, including central body defect (cbd), central complex broad (ccb), central complex deranged (ccd), central brain deranged (ceb), central complex (cex), ellipsoid body open (ebo) and mushroom body deranged (mbd) (Hitier, 2000).

In nob/Y; PX425/+ and nob/Y; PX432/+ larvae the interhemispheric expression is specifically absent, as is the pupal TIFR expression. Altogether these results imply either that the nob gene controls drl expression in the TIFR or that the nob gene is required for the TIFR existence. The gal1916 enhancer-trap line inserted in the brain washing gene was initially proposed to stain the TIFR. Recent confocal microscopy study has indicated that the gal1916 line actually stains a neighboring structure. Thus, the drl expression remains the only marker available for the TIFR, so it is impossible to determine whether nob is required for drl expression in the TIFR or for the TIFR existence. However, the fact that nob flies do not exhibit the complete phenotype of 442-gal4/+;UAS-hid/+ flies, in which interhemispheric cells are missing argues for transcriptional regulation of the drl gene by nob. Such transcriptional regulation could nevertheless be indirect and cloning of the nob gene should help address this issue (Hitier, 2000).

Derailed controls key guidance events in the developing nervous system and mesoderm. Like other members of the 'related to tyrosine kinases' (RYK) subfamily of RTKs, Drl has several highly unusual amino acid substitutions within the catalytic domain. All RYK subfamily members contain the 11 subdomains that are hallmarks of the broad family of protein kinases. Furthermore, each member has the invariant lysine of subdomain II that is essential for the phosphotransfer reaction of active kinases. However, all subfamily members share several unusual amino acid substitutions in regions of the catalytic domain that are normally highly conserved in other RTKs. The most notable of these are substitution of the first glycine within the subdomain I nucleotide binding motif [(Q/M/K)XGXXG for GXGXXG] and substitutions within the canonical activation loop motif of subdomain VII [D(S/N)(A/S) for DFG]. The unusual catalytic domain of Drl raises the possibility that members of this subfamily are catalytically inactive. To test the role of Drl kinase activity in vivo, the invariant lysine required for catalytic activity of known kinases was mutated and the ability of this mutant to function in two assays was examined: a dominant gain-of-function axon switch assay in the nervous system and phenotypic rescue of muscle attachment in drl mutants. This predicted kinase-deficient Drl mutant is capable of functioning in both assays. These results indicate that Drl does not require kinase activity in vivo and suggest that members of the RYK subfamily of RTKs transduce signals unconventionally. Transduction of Drl signals might involve the formation of heterodimers with another active RTK, in which case its signaling could be dependent on transphosphorylation of its cytoplasmic domain by this partner. A precedent for this scenario is the formation of heterodimers between the catalytically inactive ErbB3 RTK and other active members of the ErbB subfamily. An alternative mode for Drl action could involve the recruitment of signaling components independent of its phosphorylation state. For example, ligand binding might induce a conformational change of the Drl cytoplasmic domain that would subsequently allow binding of the appropriate signaling components. In either event, these in vivo results indicate that Drl and possibly the entire RYK subfamily of atypical RTKs transduce signals in an unconventional manner (Yoshikawa, 2001).

castor encodes a zinc finger protein expressed in a subset of Drosophila embryonic neuroglioblasts where it controls neuronal differentiation. cas is expressed at larval and pupal stages in brain cell clusters where it participates in the elaboration of the adult structures. In particular using the MARCM system (mosaic analysis with a repressible cell marker), it has been shown that cas is required postembryonically for correct axon pathfinding of the central complex (CX) and mushroom body (MB) neurons. The derailed gene, alternatively termed linotte (lio) in this study, encodes a transmembrane protein expressed at larval/pupal stage in a glial structure, the TIFR, and interacts with the no-bridge (nob) gene. cas interacts genetically with derailed and nob. These interactions do not involve direct transcription regulation but probably cellular communication processes (Hitier, 2001).

Derailed/Linotte is expressed at the embryonic stage in neurons of the VNC and of the procephalic region, and in the late third instar larvae in a glial transient interhemispheric fibrous ring (TIFR) that persists at the early pupal stages and disappears before adulthood. drl null mutants are viable and drl has been implicated in axon pathway selection in the embryonic VNC, and in adult brain development at metamorphosis. The no-bridge mutant, which exhibits adult brain defects, interacts with drl via the TIFR. Using a genetic screen designed to isolate mutations interacting with drl from a collection of Gal4 lines, a new hypomorphic cas allele (cas3921) has been identified. Cas protein is expressed in larval and pupal brain in cell clusters. Analysis of mutant clones generated with the MARCM method demonstrates that cas expression is required during larval life to control axonal outgrowth in CX and MB neurons. Although single mutants show only weak brain defects, double mutants lio;cas3921 and liodrlP;cas3921 exhibited strong defects in MB and CX indicating that drl and cas interact to build up the adult brain. cas expressing cells are disorganized in the third instar larva brain of drl mutants, whereas no defect is detected in drl embryos. Moreover, nob also interacts genetically with cas. Altogether these data indicate that cas is involved in postembryonic brain development where it interacts with drl and nob, these interactions probably involve cell/cell communication (Hitier, 2001).

Anti-Cas antibodies have revealed that the Cas protein is present in the CNS during larval and pupal stages confirming the cas3921 enhancer trap expression pattern. In larva, cas was found expressed in disseminated cells on the ventral side of the VNC. In the dorsal part of the larval brain, cas is found expressed in five linearly organized cells clusters on both sides of the interhemispheric junction. Twenty-four hours after pupariation, expression progressively disappears and no clear signal is detected in the adult brain. The cas3921 line led to adult expression in a subset of ellipsoid body and fan-shaped body fibers, and in the pars intercerebralis. Since the enhancer trap expression of cas3921 is very similar to that of Cas expression during embryonic, larval and pupal stages, it is speculated that the adult CX expression displayed by cas3921 actually reflects cas expression. The stability of Gal4 and ß-gal protein might allow detection in the adult where Cas might be present at weak level. Alternatively the adult Cas product might not be recognized by the antibody because the protein is modified. Cas expression appears normal in the cas3921 mutant, confirming that cas3921 is a weak hypomorph allele (Hitier, 2001).

Since viable cas mutants are hypomorphic, to fully assess the postembryonic role played by cas in adult brain development the MARCM method was used in combination with a cas null mutation and the UAS-cd8-GFP reporter. Clones were analyzed in paraffin section with anti-GFP antibody rather than with confocal microscopy in whole amount preparations to allow for the detection of non-autonomous effect of the mutant clones on adjacent brain structures. Mutant cas clones induced during larval stages lead to EB and MB defects, indicating that the postembryonic cas expression is required for CX and MB development. No obvious defects are observed with cas- clones in the lobula, the medulla and the antennal lobes, all regions, where no cas expression is detected. Multi-cellular clones were observed in the CX and in the MBs, indicating that the cas null mutation is not lethal for neuroblasts or ganglion mother cells. Nevertheless, large cas- clones in the central brain lead to the death of the individual. This probably prevents the observation of wider brain defects. In particular this could explain why the cas clone experiment did not lead to the defect observed in the brain of cas3921/cas290 individuals. When only a small subset of EB neurons are mutant for cas, neither cas- fibers nor the EB complete structure exhibit any obvious defects. However when larger cas- clones occur in neurons presumably identified as large field R2 or R4 neurons, the EB exhibits a ventral cleft. Interestingly the defect also affects EB neurons that are not mutant for cas, showing that cas is locally non-autonomous. In the MBs non-autonomous defects are also observed: when a subset of ß or ß' neurons lack the Cas product, MBs exhibit a severe fusion of ß and/or ß' lobes. The fusion comprises cas- fibers but also cas+ fibers. Moreover, although cas clones occurring in gamma neurons do not lead to any obvious intrinsic defects, they nevertheless induce cas+ ß lobes to fuse. Since gamma lobes differentiate before ß lobes, one can hypothesize that cas controls the expression of a gamma lobe signal that guides ß fibers during pupal differentiation. Alternatively, since a Elav-gal4 driver was used to detect cas clones, only the neuronal component of clones was observed (Hitier, 2001).

The possibility cannot be excluded that glial cas- cells are responsible for cas+ fibers misrooting. This idea is supported by the fact that during embryogenesis Cas is expressed in midline glial precursor cells (Hitier, 2001 and references therein).

cas is shown to interact genetically with drl to build up the adult brain and drl is required for the correct organization of cas cell clusters. Neither drl nor cas control the expression of the other gene, and neither mutation affects the correct development of the cells expressing the second gene during embryogenesis. However, subtle defects might have escaped analysis. At the third instar larvae the situation is different. The five clusters of Cas positive cells linearly organize in a wild-type context but appear disorganized in derailed mutants. Cluster positioning is disturbed and some clusters appeared 'fused' together. Analysis of cas and drl expression in the double mutant third instar larvae CNS suggests that drl and cas are not expressed in the same cells. In particular cas expressing fibers in the central brain did not include the TIFR where drl is expressed. These results suggest that the drl/cas post embryonic interaction does not involve direct transcriptional regulation but rather cellular interactions, between cas expressing clusters and interhemispheric glial cells expressing Drl (Hitier, 2001).

The Drosophila Wnt5 protein, acting through Derailed, 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. 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. Wnt5 protein is predominantly present on posterior commissural axons and at a low level on the anterior commissure and longitudinal projections. Transcriptional repression of Wnt5 in AC neurons by the Wnt5 receptor, Derailed, contributes to this largely posterior commissural localization of Wnt5 protein (Fradkin, 2004).

The correct wiring of a nervous system requires that a large number of neurons stereotypically extend their axonal processes to make synaptic contacts with their muscle and neuronal targets. The leading portion of the axon, the growth cone, is faced with a bewildering number of routing decisions as it travels, frequently many hundreds of cell body diameters, to its target (Fradkin, 2004).

Path finding relies on the growth cone receiving and interpreting guidance cues presented to it at intermediate points in its journey. While the growth cone likely integrates multiple signals at many points, the initial extension of axons has received the most scrutiny; several of the important attractive and repulsive guidance cues and their neuronal receptors have been identified (Fradkin, 2004).

In Drosophila, the majority of the neurons of the embryonic ventral cord are born near the ventral midline, a morphological and functional line of symmetry whose vertebrate equivalent is the floorplate. Axon tracts in the mature embryo form a characteristic “ladder-like” structure reflecting the presence of two longitudinal tracts that extend in the anterior–posterior axis and two commissural tracts, the anterior (AC) and posterior commissures (PC) that bridge the longitudinal pathways in every segment (Fradkin, 2004).

The first choice for many newly extending axons is whether to cross the ventral midline. The axons of certain neurons, the ipsilaterals, do not cross the midline, but instead project with other longitudinal axons toward the anterior or posterior. Other axonal pathways, the contralaterals, cross the midline, extend along the longitudinal tracts and do not recross. The decision to cross or not and the prevention of repeated midline crossing are regulated by interactions between the extending axons and the midline glia cells (MG), specialized cells that emanate repulsive and attractive signals and underlie both the AC and PC at the ventral midline. The Netrin and Slit proteins are among the best characterized of the midline-derived cues. Netrins, signaling through axonal Frazzled receptors, primarily act as attractants, although repulsive Netrin-dependent signaling has also been reported. Slit protein signaling through the axonal Robo family of receptor proteins repels axons away from the midline, thus preventing them from recrossing. In addition to their midline roles, evidence has been provided that the expression domains of the three Robo proteins also delimit lateral domains within the longitudinal axon tracts (Fradkin, 2004).

Initial outgrowth and path finding of pioneering axons is, at least in part, dependent on both their interactions with the glial cell scaffold and their responsiveness to midline-derived cues. Subsequently, “follower” axons fasciculate with the pioneers to form multiaxon fascicles. Regulation of the relative balance between fasciculation and defasciculation through regulation of cell adhesion molecule activities at specific points allows individual axons to branch off to follow separate trajectories. Several cell adhesion proteins have been shown to act in the fasciculation of embryonic Drosophila axons. Embryos bearing mutations in the Drosophila NCAM ortholog, fasciculin II (fasII), display inappropriately defasciculated axons, whereas axons that overexpress fasII become hyperfasciculated. The Connectin (Conn) protein effects homophilic interactions between motoneurons. The beaten path (beat-Ia) gene encodes an Ig domain-containing protein secreted from axonal growth cones. In beat-Ia mutants, axons become hyperfasciculated in a manner suppressible by fasII and conn mutations, suggesting that Beat-Ia acts as a secreted anti-adhesive factor. There are 13 other Drosophila beat genes; interestingly, those four whose full ORF sequences have been determined encode transmembrane or GPI-linked proteins. Genetic interactions between beat-Ia and beat-Ic (encoding a transmembrane Beat protein) indicate their complementary functions, with beat-Ic and beat-Ia acting to increase and decrease adhesiveness, respectively. The Beat receptor(s) have not yet been identified (Fradkin, 2004).

Most of the mechanisms regulating guidance across the midline that have been uncovered thus far operate in both the AC and PC. Little is known about the mechanisms that underlie the choice of axons to go through either the AC or the PC. One gene implicated in this process is derailed (drl), a member of RYK subfamily of receptor tyrosine kinases. Recent studies have demonstrated that interactions between the Drl and Wnt5 proteins play an important role in preventing AC axons from inappropriately crossing in the PC (Yoshikawa, 2003). AC axons are less tightly fasciculated in the drl mutant, suggesting that Drl may act to regulate interneuronal adhesion (Fradkin, 2004).

Wnt5 is a member of the Wnt gene family, a large group of evolutionarily conserved genes encoding secreted glycoproteins that play roles at many developmental stages in a variety of tissues. Among other roles in the nervous system, Wnt proteins act in cell fate determination, synapse formation and maintenance and as mitogens (Fradkin, 2004).

Evidence has been provided that the Drosophila Wnt5 protein is found on the embryonic CNS axon tracts. The Wnt5 gene encodes a highly unusual Wnt protein that bears a long amino terminal extension to the Wnt-homologous domain that contains no known conserved domains. This report presents a detailed analysis of the Wnt5 mRNA and protein expression domains and demonstrates a crucial role for Wnt5 in formation of the major axon tracts during embryonic CNS development through examination of embryos lacking Wnt5. Wnt5 protein is required for the separation or defasciculation of early axonal projections that subsequently form the mature commissural and longitudinal connectives. Evidence is provided that the porcupine (porc) gene is a member of theWnt5 signaling pathway and that the Wnt5 gene is itself one of the downstream targets (Fradkin, 2004).

Wnt5 mRNA is most highly expressed by a large number of neurons predominantly in lateral and midline clusters underlying the PC, but is also found in a few cell bodies more closely associated with the AC. Wnt5 is not apparently expressed by the midline or lateral glia cells. While Wnt5 protein is detected on both AC and PC axons, as well as the longitudinal axon tracts, Wnt5 protein is expressed at higher levels on the PC as compared with the AC from the earliest stages when axons begin to extend and throughout embryonic CNS development (Fradkin, 2004).

Wnt5 null alleles were generated to determine the role of Wnt5 in CNS development. Early BP102+ commissural axons that pioneer first the PC, and subsequently the AC, are unaffected in the Wnt5 mutant. In the wild-type embryo, the BP102+ AC and PC pioneers and their early followers are closely associated near the midline, but subsequently separate at stage 13, to begin the formation of the two distinct commissures. This separation does not take place in the majority of Wnt5 mutant segments (Fradkin, 2004).

Visualization of the Sema2b+ axons, followers that cross in the wild-type AC at early stage 15, reveals that they largely fail to do so in the Wnt5 mutant and fasciculate inappropriately with ipsilateral sibling longitudinal projections or cross the midline in the region between the AC and PC. The PC axon trajectories were visualized by panneural staining and in the Eg+ PC-crossing lineage and no major alterations were seen in the Wnt5 mutant (Fradkin, 2004).

In addition to the commissural defects in Wnt5 mutant embryos, alterations to the longitudinal pathway projections were observed. Wnt5 mutant embryos display differential phenotypes with respect to the FasII+ longitudinal pathways: the medial or innermost pathway is largely unaffected; however, breaks are found in the intermediate pathway and the lateral pathways. Supporting these observations, the Ap+ projections, which in wild type follow the ipsilateral medial pathway, were unaffected by the absence of Wnt5. Visualization of the intermediate and lateral pathways with anti-Robo3 and anti-Robo2, respectively, further indicated that disruption became more severe in the pathways more lateral to the ventral midline (Fradkin, 2004).

Analyses of the longitudinal pioneer neurons indicate that, when specific axons have to selectively defasciculate to pioneer new pathways, particularly the intermediate and lateral pathways, they do not do so in the absence of Wnt5. Examination of the Robo2+ neurons indicates that after a limited period of extension, they stop and fail to form the continuous fascicle seen in the wild-type embryo. The Wnt5 longitudinal phenotypes are unlikely to simply reflect the failure of the axons that normally transit the AC to cross based on the following observations: (1) early defasiculation defects in the longitudinal pioneering projections at times where the commissures themselves are being pioneered, (2) discontinuities in ipsilaterally projecting pathways that do not cross the midline, and (3) apparently normal Fas2+ longitudinal pathways in the comm null mutant, where few if any axons cross in either commissure (Fradkin, 2004).

The VUM neurons, which normally aid in separating the commissures by sending their projections in between the PC and AC BP102+ pioneers, project abnormally in the Wnt5 mutant. Migration of the VUM cell bodies and the MG, which are also involved in establishing the physical separation between the AC and PC, do, however, occur normally in the Wnt5 mutant. Because the failure to establish the AC in the Wnt5 mutant occurs with a similar frequency (67%) to that observed for the abnormal VUM projections (70%), the VUM abnormalities likely contribute to the later failures of the follower axons to form the AC. The VUMs express Wnt5 mRNA at the time of commissure separationand may therefore represent the chief source of Wnt5 protein mediating separation of the AC and PC (Fradkin, 2004).

How does Wnt5 mediate the formation of the AC and the lateral longitudinal pathways? The data, including the observation that Wnt5 protein is most highly expressed on the PC, support the previously proposed role for PC-expressed Wnt5 as a repellent for Drl+ axons (Yoshikawa, 2003), but reveal the likely nature of Wnt5-mediated repulsion required to effect the formation of the AC and more lateral longitudinal pathways. It is suggested that the major role of Wnt5 is to mediate the selective defasciculation of the early commissural and longitudinal pathways necessary for them to separate into distinct commissures or pioneer new pathways, respectively. As defasciculation may be viewed as local repulsion or decreased attraction between axons, this interpretation of the early commissural defects in the Wnt5 mutant is therefore not at odds with Wnt5 acting as a PC-derived repellent. However, Wnt5 appears to act by facilitating the defasciculation of Drl+ axons from their siblings necessary for formation of the AC. The role of drl in the Wnt5 mutant longitudinal pathway defasciculation defects is presently unclear. Interestingly, studies of the drl mutant phenotype described the inappropriate defasiculation of axons lacking drl. Similar drl null phenotypes were observed as previously reported; however, the drl CNS phenotypes are consistently less severe than those seen in the Wnt5 null, suggesting that other genes may also interact with Wnt5 to effect selective fasciculation (Fradkin, 2004).

Tescue experiments demonstrated that the primary requirement for Wnt5 expression during embryonic CNS development is in neurons. Panneural Wnt5 expression rescued both the commissural and longitudinal defects. Strikingly, high levels of Wnt5 secreted throughout the CNS-driven by the Repo-Gal4 lateral glial cell driver in the Wnt5 mutant background fail to rescue, suggesting that neurons may process Wnt5 differently than the lateral glia. In support of this possibility, evidence has been presented that Wnt5 protein secreted from tissue culture cells and produced in embryos, respectively, is proteolytically processed (Fradkin, 2004).

Wnt5 overexpression at the AC midline (Sim-Gal4 driver), but not the PC midline (Btl-Gal4 driver) results in a failure to form the AC without noticeable effects on the PC. The noncrossing axons appear tightly associated in the longitudinals, suggesting that Wnt5 protein levels may be important in the regulation of axonal adhesion, with either too little or too much Wnt5 resulting in overly tight fasciculation. The Sim-Gal4-driven Wnt5 overexpression phenotype has also been interpreted to indicate that ectopic Wnt5 repulses all Drl+ AC axons (Yoshikawa, 2003). However, given the current absence of data showing that Wnt5 can directly collapse Drl+ growth cones, it is equally possible that the overly tight fasciculation of those axons precludes their midline crossing (Fradkin, 2004).

This study has shown that the differing levels of Wnt5 protein on the PC vs. AC are maintained, at least in part, from repression of Wnt5 transcription in AC neurons by Drl. Furthermore, apparently normal CNS architecture is observed when Wnt5 is pan-neurally expressed in a wild-type background (in Elav-GAL4 X UAS-Wnt5 embryos) resulting in high levels of Wnt5 protein on both commissures. The question arises as to why such regulation of Wnt5 in the AC exists? One possibility is that, although high levels of Wnt5 protein on both commissures result in rescue of the Wnt5 mutant phenotype and a lack of an observable phenotype in the wild-type background, respectively, the active species of Wnt5 protein may be asymmetrically generated with respect to the commissure of origin, reflecting PC- vs. AC-specific Wnt5 protein processing. Alternatively, the Drl-mediated repression of AC Wnt5 expression may reflect a requirement for low levels of Wnt5 in AC axons while precluding the higher levels that effect defasciculation, likely via the Drl receptor (Fradkin, 2004).

This study found that porc gene expression is required for the MG Wnt5 overexpression phenotype. Since porc aids in Wnt5 protein secretion (Tanaka, 2002), these data indicate porc-mediated Wnt5 secretion is required for Wnt5 signaling in this overexpression assay. porc function appears to be limiting for Wnt5 secretion in this assay as reduction of porc gene dosage by half in the porc/+ females is sufficient to suppress the Wnt5 midline overexpression phenotype. Is porc required for wild-type Wnt5-mediated signaling? The BP102+ axon tracts are disorganized in porc germline clone mutants lacking both maternal and zygotic porc (Tanaka, 2002); however, interpretation of this phenotype is complicated by the requirement for porc in wg signaling, which in turn plays roles in segmentation and neuroblast specification. In porc zygotic mutants, the BP102+ axon scaffold is only slightly affected; however, the maternally contributed porc mRNA may mask a requirement for porc in Wnt5 signaling in the zygotic porc mutant embryo. The demonstration of a requirement for porc in establishing the Wnt5 midline overexpression phenotype suggests that this assay will be a useful tool in uncovering novel members of the Wnt5 signaling pathway and its targets, which likely include cell surface adhesion proteins facilitating selective fasciculation and modulators of their activities (Fradkin, 2004).

The RYK subfamily of receptor tyrosine kinases is characterised by unusual, but highly conserved, amino acid substitutions in the kinase domain. The derailed gene encodes a Drosophila RYK subfamily member involved in embryonic and adult central nervous system development. Previous studies have shown that the kinase activity of this receptor is not required in vivo for its embryonic function. In this study, the role was investigated of the cytoplasmic domain and the kinase activity of the Derailed receptor tyrosine kinase in adult brain development. The results indicate that these domains are not essential for adult brain development but they are required for the proper regulation of the activity of this receptor. This sheds light on a regulatory role for the kinase activity of a RYK subfamily member (Taillebourg, 2005).

Respective roles of the DRL receptor and its ligand WNT5 in Drosophila mushroom body development

In recent decades, Drosophila mushroom bodies (MBs) have become a powerful model for elucidating the molecular mechanisms underlying brain development and function. Derailed receptor tyrosine kinase as an essential component of adult MB development. Using MARCM clones, a non-cell-autonomous requirement has been demonstrated for the Drl receptor in MB development. This result is in accordance with the pattern of Drl expression, which occurs throughout development close to, but not inside, MB cells. While Drl expression can be detected within both interhemispheric glial and commissural neuronal cells, rescue of the drl MB defects appears to involve the latter cellular type. The WNT5 protein has been shown to act as a repulsive ligand for the Drl receptor in the embryonic central nervous system. This study shows that WNT5 is required intrinsically within MB neurons for proper MB axonal growth and probably interacts with the extrinsic Drl receptor in order to stop axonal growth. It is therefore proposed that the neuronal requirement for both proteins defines an interacting network acting during MB development (Grillenzoni, 2007).

This study has shown that a drl LOF mutation affects MB development as early as at the newly hatched first instar larval stage. It is at (or just before) this stage that the axons of the first MB intrinsic neurons to be born form the median and vertical lobes. It can be hypothesized that the MB defects displayed by drl LOF adult flies are at least partially due to aberrant early MB development. The Drl protein is not expressed within the MB intrinsic neurons at any developmental stage analyzed. This result is strengthened by clonal analysis experiments, which showed that the early removal of the wild-type drl gene in a subset of the three classes of MB intrinsic neurons does not alter their axonal morphology. The clonal analysis results demonstrate sensu stricto a non-cell-autonomous requirement for the drl gene in the MB intrinsic neurons; it does not, however, completely exclude the possibility that drl mutant clones develop properly due to the expression of the Drl protein in MB intrinsic neurons outside the clones. This would imply that the Drl expression level in the MBs is below the level required by the detection method used in this study. However, restoring the expression of the drl gene solely in a subpopulation of MB intrinsic neurons with the GAL4-247 line, or even in most if not all MB intrinsic neurons with GAL4-OK107, is insufficient to rescue the MB defects induced by the drl LOF mutation. The partial rescue obtained previously with the GAL4-c739 line is likely to be due to some transient expression outside the MBs during development. The fact that the mutant phenotype cannot be rescued by two other GAL4 lines that are expressed either more specifically (GAL-247) or in more MB neurons (GAL-OK107) is ruling out a role of the MB expression of GAL-c739 in the weak rescuing effect. Based on the overall results obtained, the hypothesis is favored that drl gene function is required extrinsically by MBs for their proper development. Finally, the MARCM technique allowed visualization of the morphology of single-side median MB axons in drl LOF individuals. This analysis revealed that the mutant phenotype is not simply a fusion of the median contralateral lobes at the midline, but rather a real crossing of the axons, which then intermingle with their contralateral equivalents (Grillenzoni, 2007).

The function of the Drl protein is required extrinsically by the MBs for their proper development. The data show that the protein is expressed from the onset of brain commissural formation in a subset of neurons crossing the midline. This pattern is a remnant of Drl expression in the embryonic CNS, although at later stages the Drl-expressing brain commissural axons divide into two tracts. It is important to emphasize that the embryonic brain commissure is not identical to those of the ventral CNS, and that the molecular factors involved in their development, although often conserved, do not necessarily play the same role. Knowing that the Drl receptor is necessary cell-autonomously in the CNS to allow the correct midline crossing of a subset of anterior commissural axons, this study analyzed whether similar defects could be observed in the embryonic brain. Such defects could be the primary cause of the MB observed phenotype. This is not the case, since no embryonic brain commissural tract abnormalities were detected using different axonal markers. It has been previously suggested that Drl expression in interhemispheric glial cells during late third instar larval and pupal stages is necessary for MB axonal development. Although Drl expression could be detected in interhemispheric glial cells of third instar brains, it was not possible to rescue the MB phenotype by specific interhemispheric glial cell expression. Moreover, no glial cells expressed the Drl protein at earlier developmental stages, even though MB defects were already present in drl LOF individuals. In addition, the observed Drl expression in commissural neurons and the positive rescue results using a pan-neuronal driver lead to a postulate that Drl is required in neuronal cells extrinsic to the MBs for the correct axonal development of the latter. In conclusion, this study suggests that in the Drosophila brain, Drl expression in a subpopulation of commissural neurons is necessary not for their own axonal development but rather for the guidance of MB intrinsic neurons that do not express the Drl protein (Grillenzoni, 2007).

This neuronal hypothesis is particularly attractive when the Wnt5 results are taken into account. Wnt5 mutants were tested because Wnt5 was described as being a ligand for the Drl receptor in the ventral CNS. Clear MB phenotypes were found in Wnt5 mutant brains. The Wnt5 MB mutant phenotype is most consistent with Wnt5 being required for neurite outgrowth. It is striking that these mutant phenotypes resemble those described for drl+ overexpression. It is proposed that this GOF phenotype is due to drl+ expression within or close to MB cells, where the ectopic Drl protein can bind to the Wnt5 protein and prevent its function. Therefore, a general model is proposed for the role of the Wnt5-drl pair in building normal MBs: Wnt5 is expressed and required within MB cells in order to insure proper axonal growth. One can propose that during this process the secreted Wnt5 activates an MB intrinsic receptor, which seems not to be of the fz type, in order to activate axonal growth. When Wnt5 is absent, e.g. in a Wnt5 mutant MB, then the axons fail to grow properly. In the normal situation, these MB intrinsic axons will stop growing at the midline when they reach extrinsic axons expressing Drl, because Wnt5 is trapped by the Drl receptor. In drl mutant individuals, however, the MB axons will continue to grow, because Wnt5 is not trapped by the Drl receptor. Although the biochemical relationship between the ligand and receptor is conserved from the embryonic ventral CNS to the adult brain, it should be stressed that MB development involves neurons that express Wnt5 and not Drl, which is exactly opposite to the case in the embryo, where the mutant phenotype involves neurons that express the drl gene and not Wnt5. This is why drl and Wnt5 mutants have the same phenotype in the embryonic ventral CNS but have opposite phenotypes in adult MBs (Grillenzoni, 2007).

The genetic control of brain development requires both intrinsic and extrinsic clues. The perfect crosstalk between both types of molecular information, coming from neurons of different types of brain substructures, ultimately ensures the development of a harmonious and functional brain. It is central for neurobiology to decipher these interacting and developing neuronal networks at the cellular and molecular levels. This study has describe a clear case in which drl, a receptor tyrosine kinase, is required within the brain for the normal development of MBs, although it is neither expressed nor required intrinsically within the MB neurons. Further, it is proposed that the Wnt5 signaling molecule is the intrinsic MB axon target that needs to interact with the extrinsic Drl receptor in order to construct proper MBs within the brain (Grillenzoni, 2007).

Differentially expressed Drl and Drl-2 play opposing roles in Wnt5 signaling during Drosophila olfactory system development

In Drosophila, odor information received by olfactory receptor neurons (ORNs) is processed by glomeruli, which are organized in a stereotypic manner in the antennal lobe (AL). This glomerular organization is regulated by Wnt5 signaling. In the embryonic CNS, Wnt5 signaling is transduced by the Drl receptor, a member of the Ryk family. During development of the olfactory system, however, it is antagonized by Drl. This study identified Drl-2 as a receptor mediating Wnt5 signaling. Drl is found in the neurites of brain cells in the AL and specific glia, whereas Drl-2 is predominantly found in subsets of growing ORN axons. A drl-2 mutation produces only mild deficits in glomerular patterning, but when it is combined with a drl mutation, the phenotype is exacerbated and more closely resembles the Wnt5 phenotype. Wnt5 overexpression in ORNs induces aberrant glomeruli positioning. This phenotype is ameliorated in the drl-2 mutant background, indicating that Drl-2 mediates Wnt5 signaling. In contrast, forced expression of Drl-2 in the glia of drl mutants rescues the glomerular phenotype caused by the loss of antagonistic Drl function. Therefore, Drl-2 can also antagonize Wnt5 signaling. Additionally, genetic data suggest that Drl localized to developing glomeruli mediates Wnt5 signaling. Thus, these two members of the Ryk family are capable of carrying out a similar molecular function, but they can play opposing roles in Wnt5 signaling, depending on the type of cells in which they are expressed. These molecules work cooperatively to establish the olfactory circuitry in Drosophila (Santschi, 2009).

Several lines of evidence support a role for Drl-2 in Wnt5 signaling. First, in drl-2 mutants, two glomeruli shifted to abnormal positions as observed in Wnt5 mutants. Second, when drl-2 was combined with drl, the phenotype more closely resembled that of the Wnt5 mutant. Third, the abnormal commissural distribution of the presynaptic protein Bruchpilot, which was induced by overexpression of Wnt5 in ORNs, was restored in drl-2 mutants. Moreover, the ectopic projection pattern of ORNs, which was caused by Wnt5 overexpression in drl mutants, was mostly ameliorated by the absence of drl-2. Thus, drl-2 is epistatic to both Wnt5 and drl, and Drl-2 mediates Wnt5 signaling. This Drl-2 activity is antagonized by Drl during development of the olfactory system. However, the glomerular defects of drl drl-2 mutants are milder than those of Wnt5 mutants. Additional Wnt5 receptors that have yet to be identified may contribute to these phenotype differences (Santschi, 2009).

Antibody staining revealed that the expression patterns of Drl and Drl-2 differ in the developing olfactory system. Drl is expressed by glia and brain cells extending neurites to the AL but is not detected on ORN axons. In contrast, Drl-2 is predominantly detected on ORN axons as well as in a region adjacent to the exit site of ORN axons on the dorsal side of the AL. Thus, the expression patterns of Drl and Drl-2 are complex and mostly nonoverlapping, suggesting that multiple processes use Wnt5 signaling during olfactory system development. To reveal whether Drl-2 functions in ORNs, clonal analyses was performed in which drl-2 clones were generated in ORNs. Rescue experiments were also conducted by expressing Drl-2 in the ORNs of drl-2 mutants with pebbled-Gal4. However, the mild drl-2 phenotype did not allow a clearly determination of the site(s) where Drl-2 functions (Santschi, 2009).

The genetic analysis suggests that Drl and Drl-2 can both play opposing roles in Wnt5 signaling during olfactory system development. The DA1 glomerulus in drl mutant lNB clones shifts in a pattern similar to that of Wnt5 mutants, suggesting that Drl may transduce Wnt5 signaling in the developing AL. In addition to this possible transducing activity of Drl, the antagonistic action of Drl and the transducing activity of Drl-2 can explain other mutant glomerular phenotypes observed in this study. In drl mutant brains, in which both transducing and antagonistic activities of Drl are lost, an excess amount of Wnt5 ligand signals through Drl-2 and causes several glomeruli to shift in the direction opposite to that in Wnt5 mutants. However, in drl drl-2 mutants, in which transducing activities of both Drl and Drl-2 are lost, the glomerular defects resemble those of Wnt5 mutants. Thus, the opposing actions of Drl may both be essential for olfactory system development. In addition, it was demonstrated that Drl-2 can antagonize Wnt5 signaling when ectopically expressed in glia. Therefore, Drl and Drl-2 can each potentially mediate or antagonize Wnt5 signaling, depending on the cells in which they are expressed (Santschi, 2009).

Wnt–Ryk signaling mediates both repulsion of developing axons and induction of neurite growth in Drosophila and vertebrates. In the Drosophila olfactory system, Drl localized to the neurites of AL neurons may mediate both of these activities, because drl NB clones exhibit two distinct phenotypes. Neurites projecting to VA1 were stunted in drl vNB clones, suggesting that Drl may mediate neurite growth. In drl lNB clones, the position of DA1 was shifted ventrally, which may reflect the loss of neurite repulsion triggered by Wnt5 originating from growing ORN axons. Drl-2 on ORN axons can also mediate either of these two neurite activities to control ORN projections to the AL. Thus, proper AL development may be regulated by Wnt–Ryk signaling that mediates both the repulsion and outgrowth of neurites innervating the AL. The development of the Drosophila olfactory system appears to be controlled by a complex network of Wnt5 signaling among ORNs, interneurons, and glia (Santschi, 2009).

In summary, although each of the drl and drl-2 genes has acquired a specific expression pattern during evolution, both products can either mediate or antagonize Wnt5 signaling in a cell-type specific manner. In this manner, Drl and Drl-2 may regulate either the repulsion or outgrowth of neurites, perhaps in accordance with additional mechanisms. Additional studies will reveal the details of the complex Wnt5 signaling that control the formation of an accurate glomerular map of the olfactory circuitry (Santschi, 2009).

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). This study reports 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. 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. It is proposed 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 (Wu, 2014).


EVOLUTIONARY HOMOLOGS

Doughnut is a second Drosophila RYK receptor tyrosine kinase

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).

C. elegans LIN-18 is a Ryk ortholog and functions in parallel to LIN-17/Frizzled in Wnt signaling

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).

Opposing Wnt pathways orient cell polarity during organogenesis

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).

The conserved transmembrane RING finger protein PLR-1 downregulates Wnt signaling by reducing Frizzled, Ror and Ryk cell-surface levels in C. elegans

Wnts control a wide range of essential developmental processes, including cell fate specification, axon guidance and anteroposterior neuronal polarization. This study identified a conserved transmembrane RING finger protein, PLR-1, that governs the response to Wnts by lowering cell-surface levels of the Frizzled family of Wnt receptors in Caenorhabditis elegans. Loss of PLR-1 activity in the neuron AVG causes its anteroposterior polarity to be symmetric or reversed because signaling by the Wnts CWN-1 and CWN-2 are inappropriately activated, whereas ectopic PLR-1 expression blocks Wnt signaling and target gene expression. Frizzleds are enriched at the cell surface; however, when PLR-1 and Frizzled are co-expressed, Frizzled is not detected at the surface but instead is colocalized with PLR-1 in endosomes. The Frizzled cysteine-rich domain (CRD) and invariant second intracellular loop lysine are crucial for PLR-1 downregulation. The PLR-1 RING finger and protease-associated (PA) domain are essential for activity. In a Frizzled-dependent manner, PLR-1 reduces surface levels of the Wnt receptors CAM-1/Ror (see Drosophila Ror) and LIN-18/Ryk. PLR-1 is a homolog of the mammalian transmembrane E3 ubiquitin ligases RNF43 and ZNRF3, which control Frizzled surface levels in an R-spondin-sensitive manner. It is proposed that PLR-1 downregulates Wnt receptor surface levels via lysine ubiquitylation of Frizzled to coordinate spatial and temporal responses to Wnts during neuronal development (Moffat, 2014).

Mammalian RTK family members

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).

Mammalian Ryk is a Wnt coreceptor required for stimulation of neurite outgrowth

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).

Cleavage of the Wnt receptor Ryk regulates neuronal differentiation during cortical neurogenesis

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).

Ryk and axon guidance

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).


REFERENCES

Search PubMed for articles about Drosophila Derailed

Finley, K. D., et al. (1997). dissatisfaction, a gene involved in sex-specific behavior and neural development of Drosophila melanogaster. Proc. Natl. Acad. Sci. 94: 913-918.

Finley, K. D., et al. (1998). dissatisfaction encodes a Tailless-like nuclear receptor expressed in a subset of CNS neurons controlling Drosophila sexual behavior. Neuron 21(6): 1363-74.

Pitman, J. L., et al. (2002). DSF nuclear receptor acts as a repressor in culture and in vivo. Dev. Biol. 245: 315-328. 11977984

Yamamoto, D., Fujitani, K., Usui, K., Ito, H. and Nakano, Y. (1998). From behavior to development: genes for sexual behavior define the neuronal sexual switch in Drosophila. Mech. Dev. 73(2): 135-146.


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date revised: 5 February 2015

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