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).

GENE STRUCTURE

Exons - 4

PROTEIN STRUCTURE

Amino Acids - 610

Structural Domains

Both Drl and Doughnut (Dnt), two related Drosophila receptor tyrosine kinases, have novel, short, extracellular domains that contain distinct regions of amino acid identity. The two proteins also share unique amino acid substitutions within the catalytic domain, which, together with their extracellular domains, place them in a new receptor protein tyrosine kinase subfamily. Despite the catalytic domain substitutions, the presence of those amino acids thought to be essential for protein-tyrosine kinase function predicts that both Drl and Dnt are likely to be catalytically active (Callahan, 1995).

derailed individuals carrying a null mutation are viable, indicating that the derailed gene is not required for vital functions. The derailed gene encodes a putative receptor tyrosine kinase, homologous to the human protein receptor tyrosine kinase. These products are unique among receptor tyrosine kinases, since they possess a short extra cellular domain, and a modified intracellular catalytic domain. In particular, the subdomains directly involved in ATP binding and phosphotransfer reaction display remarkable variations. These results suggest that derailed is part of a novel signal transduction cascade involved in learning and memory (Dura, 1995).

date revised: 30 May 2008

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