frazzled

Regulation

Transcriptional Regulation

The wide-ranging defects in dendrites and axons indicate that sequoia functions to regulate axonal and dendritic morphogenesis in most neurons. Alternatively, it is conceivable that sequoia regulates the expression of genes generally required for neuronal differentiation. To gain mechanistic insight into sequoia function, the transcript profiles in wild-type and sequoia mutant embryos were compared based on microarray analyses of over 3,000 genes or ESTs, corresponding to about 25% of the Drosophila genome. The vast majority of these genes show comparable expression levels, including genes for cytoskeletal elements, genes that specify neuronal cell fates, and genes generally required for neurite outgrowth such as cdc42. Interestingly, a small fraction of the genes/ESTs analyzed showed clearly distinct expression ratios in sequoia mutants. Of these, 93 (3.1%) different transcripts were reduced by at least one-third of the wild-type level, and 34 (1.1%) different transcripts were increased by at least 75% of the wild-type level. A number of genes that appear to be regulated by sequoia, directly or indirectly, correspond to genes implicated in the control of axon morphogenesis rather than neuronal fate. These include known genes such as connectin, frazzled, roundabout 2, and longitudinals lacking, in addition to novel molecules with homology to axon guidance molecules including slit/kekkon-1 and neuropilin-2. It is noteworthy that two of the genes showing increased transcript ratios, roundabout 2 and CG1435, a novel calcium binding protein, were both also identified in a gain-of-function screen affecting motor axon guidance and synaptogenesis. In addition to genes that have clearly been implicated in axon development based on previous studies or sequence similarity, microarray data reveal that other genes potentially regulated by sequoia include peptidases, lipases, and transporters, as well as novel zinc finger proteins. It should be noted that transcripts that are broadly expressed and increased or decreased in sequoia mutants may actually be altered to a greater extent within neurons, because sequoia likely functions cell autonomously and is only expressed in the nervous system (Brenman, 2001).

Chromatin immunoprecipitation after UV crosslinking of DNA/protein interactions was used to construct a library enriched in genomic sequences that bind to the Engrailed transcription factor in Drosophila embryos. Sequencing of the clones led to the identification of 203 Engrailed-binding fragments localized in intergenic or intronic regions. Genes lying near these fragments, which are considered as potential Engrailed target genes, are involved in different developmental pathways, such as anteroposterior patterning, muscle development, tracheal pathfinding or axon guidance. This approach was validated by in vitro and in vivo tests performed on a subset of Engrailed potential targets involved in these various pathways. Strong evidence is presented showing that an immunoprecipitated genomic DNA fragment corresponds to a promoter region involved in the direct regulation of frizzled2 expression by engrailed in vivo (Solano, 2003).

the expression of 14 genes was studied that are localized close to the genomic DNA fragments isolated in the library and tested previously for their Engrailed-specific binding ability. The results are shown for four genes (frizzled2, hibris, branchless, frazzled) that are representative of the different pathways where engrailed seems to be involved. frizzled 2 expression is activated in the presence of (VP16-En) and repressed in the presence of En. This suggests that engrailed might act as a repressor on fz2 expression. hibris is expressed along the wing margin and in the presumptive region of wing vein L3 and L4 in wild type. This expression is slightly activated in the presence of (VP16-En), but strongly repressed when En is overexpressed, suggesting that hbs expression is regulated by engrailed in vivo. branchless is essentially expressed in a dorsal/posterior territory surrounding the wing pouch in wild type. In the presence of (VP16-En), several additional patches of bnl expression are detected within the wing pouch, whereas no activation of bnl is observed after wild type En overexpression. As expected, because MS1096 drives Gal4 expression only in the wing pouch, endogenous bnl expression outside the wing pouch is not affected, showing the specificity of the experiment. Finally, frazzled is slightly expressed in wild-type wing disc. This expression is activated when (VP16-En) is overexpressed, and repressed upon En overexpression (Solano, 2003).

Protein Interactions

Netrin is a secreted protein that can act as a chemotropic axon guidance cue. Two classes of Netrin receptor, DCC and UNC-5, are required for axon guidance and are thought to mediate Netrin signals in growth cones through their cytoplasmic domains. However, in the guidance of Drosophila photoreceptor axons, the DCC ortholog Frazzled is required not in the photoreceptor neurons but instead in their targets, indicating that Frazzled also has a non-cell-autonomous function. This study shows that Frazzled can capture Netrin and 'present' it for recognition by other receptors. Moreover, Frazzled itself is actively localized within the axon through its cytoplasmic domain, and thereby rearranges Netrin protein into a spatial pattern completely different from the pattern of Netrin gene expression. Frazzled-dependent guidance of one pioneer neuron in the central nervous system can be accounted for solely on the basis of this ability of Frazzled to control Netrin distribution, and not by Frazzled signaling. A model of patterning mechanism is proposed in which a receptor rearranges secreted ligand molecules, thereby creating positional information for other receptors (Hiramoto, 2000).

In vitro chemotropic responses of growth cones to Netrin indicate that graded distribution of Netrin may be important for guiding axons in vivo. A Netrin gradient could be produced by constant secretion followed by diffusion and degradation. However, in the ventral nerve cord of the Drosophila embryo the distribution of Netrin protein cannot be explained by such a mechanism. Drosophila Netrin is encoded by two genes, Netrin-A and Netrin-B. Although Netrin messenger RNA is abundant in the midline and the ventral region of the nerve cord, Netrin-A and Netrin-B proteins localize in the dorsolateral region, where no Netrin mRNA is detected. Even when Netrin-B transcription is artificially restricted to midline cells, Netrin-B still accumulates in the dorsolateral region as in wild-type embryos, rather than forming a gradient centered at the midline. This suggests that Netrin is either transported to the dorsolateral region or is selectively captured there after secretion (Hiramoto, 2000).

Frazzled is a good candidate for a molecule that relocalizes Netrin. Its accumulation is most evident on axon stalks of the commissural region, and its ortholog, DCC, is known to bind Netrin. Moreover, the dorsolateral Netrin-positive region precisely matches Frazzled distribution. In the absence of Frazzled, Netrin does not accumulate dorsolaterally and Netrin-B is observed only on cell bodies that express Netrin-B mRNA. Moreover, when Frazzled is misexpressed in ventral unpaired median (VUM) cells, ectopic Netrin-B protein is found on their surface even though these cells do not express Netrin-B. These data indicate that ectopic Frazzled can capture Netrin synthesized elsewhere, and suggest that Frazzled localizes Netrin in the dorsolateral region of ventral nerve cord. As expected, Frazzled distribution is unaltered in Netrin-A, Netrin-B double-mutant embryos (Hiramoto, 2000).

Frazzled itself is not found uniformly throughout the membrane, but is concentrated in specific regions of the axon, indicating that its distribution may also be regulated. Localized distribution within the neuron has been observed for Roundabout (Robo), a transmembrane receptor for another guidance molecule, Slit, and the localization signal of Robo has been mapped to its cytoplasmic or transmembrane domain. Similarly, Frazzled lacking its cytoplasmic domain (Fra-deltaC) is distributed throughout the cell membrane. Furthermore, Robo-Fra, a chimaera with the extracellular and transmembrane domain of Robo and the cytoplasmic domain of Frazzled, is distributed in the same way as full-length Frazzled. This shows that the cytoplasmic domain of Frazzled is necessary and sufficient for proper localization. Fra-Robo, a chimaera with the extracellular and transmembrane domain of Fra and the cytoplasmic domain of Robo, was also expressed in frazzled minus animals. In such embryos, the Fra-Robo fusion protein fails to distribute in the wild-type Frazzled pattern, and Netrin-B is mislocalized to many of the sites of Fra-Robo accumulation. These data show that Frazzled captures Netrin with its extracellular domain, whereas Frazzled distribution is controlled by a localization signal in the cytoplasmic domain (Hiramoto, 2000).

An investigation was carried out to see how axons are guided by the Netrin that is captured by Frazzled. Focused was placed on an identified pioneer neuron, dMP2, that requires Netrin-A/Netrin-B and frazzled function. dMP2 axons extend laterally and then turn posteriorly to form the initial longitudinal axon pathway. Precisely at the turning point, the medial edge of dorsolateral Netrin accumulation abuts the dMP2 pathway. dMP2 axons make pathfinding errors in both Netrin-A, Netrin-B double mutants and frazzled mutants, and such defects are often accompanied by severe disorganization of longitudinal tracts. These data may indicate that dMP2 axon guidance by Frazzled and Netrin is essential for the formation of the longitudinal axon pathway (Hiramoto, 2000).

To investigate how Frazzled functions in the guidance of dMP2, a test was performed to see whether frazzled is required in the dMP2 neuron itself. Contradictory to the idea that Frazzled is a Netrin sensor in dMP2 growth cones, Frazzled protein is not detected in dMP2. Moreover, expressing Frazzled in dMP2 in a frazzled minus background does not rescue the defects in dMP2 axon guidance. In contrast, when Frazzled is expressed in many central neurons in the frazzled minus background, the defects of dMP2 axon guidance are rescued, even though Frazzled is not expressed in dMP2. These data indicate that, for this guidance decision, Frazzled acts as a pathway marker and not as a sensor in growth cones (Hiramoto, 2000).

The ability of Frazzled to capture Netrin raises the possibility that Frazzled guides dMP2 by capturing and presenting Netrin to dMP2. To test this, Fra-deltaC was expresssed in a frazzled-mutant background to create an ectopic Netrin-B positive region near the axon pathway of dMP2 without changing the pattern of Netrin transcription. In such embryos, dMP2 growth cones spread abnormally over this surface of artificial Netrin accumulation. Also, Netrin-B was directly misexpressed in cell bodies located near the dMP2 axon pathway. Again, dMP2 growth cones respond to the ectopic Netrin-B-positive region. This strongly suggests that the response of dMP2 to the ectopic Frazzled extracellular domain is due to a response to the Netrin bound to the domain (Hiramoto, 2000).

An implication of these data is that dMP2 uses a Netrin receptor other than Frazzled to respond to Netrin. Redirection of dMP2 growth cones to ectopic Netrin indicates that Netrin is perceived as an attractive cue to dMP2. As the Drosophila genome does not contain any other genes with significant homology to DCC, it is expected that the Netrin receptor expressed in dMP2 is structurally different from the DCC class of Netrin receptors (Hiramoto, 2000).

These data indicate that Frazzled captures and rearranges Netrin, and presents it to other growth cones. The capture/relocation mechanism can create a precise Netrin distribution even in regions that are quite distant from the source of Netrin protein. Just as Frazzled presents Netrin to the dMP2 axon at its lateral turning point, the vertebrate Frazzled ortholog DCC also captures Netrins, and is localized to the point where the commissural axons turn longitudinally. Presentation of Netrin may thus be a general feature of DCC proteins. How Netrin reaches its final location is not yet clear. As Netrin-B does not localize to all Fra-Robo positive regions even when they are close to a source of Netrin-B , relocation of Netrin is likely to involve transport along axons rather than diffusion alone. Perhaps active relocalization of receptors such as Frazzled or Robo may be used to transport ligands to the final target area, where they are interpreted by other receptors. In addition to neuronal axons, extended cellular processes, such as the cytonemes of Drosophila imaginal discs and vertebrate limb buds, have been implicated in other patterning systems. It will be interesting to see whether such systems also use capture/relocation mechanisms to generate precise spatial patterns away from the source of the diffusible morphogen (Hiramoto, 2000).

The Abelson tyrosine kinase, the Trio GEF and Enabled interact with the Netrin receptor Frazzled in Drosophila

The attractive Netrin receptor Frazzled (Fra), and the signaling molecules Abelson tyrosine kinase (Abl), the guanine nucleotide-exchange factor Trio, and the Abl substrate Enabled (Ena), all regulate axon pathfinding at the Drosophila embryonic CNS midline. Genetic and/or physical interactions between Fra and these effector molecules suggest that they act in concert to guide axons across the midline. Mutations in Abl and trio dominantly enhance fra and Netrin mutant CNS phenotypes, and fra;Abl and fra;trio double mutants display a dramatic loss of axons in a majority of commissures. Conversely, heterozygosity for ena reduces the severity of the CNS phenotype in fra, Netrin and trio,Abl mutants. Consistent with an in vivo role for these molecules as effectors of Fra signaling, heterozygosity for Abl, trio or ena reduces the number of axons that inappropriately cross the midline in embryos expressing the chimeric Robo-Fra receptor. Fra interacts physically with Abl and Trio in GST-pulldown assays and in co-immunoprecipitation experiments. In addition, tyrosine phosphorylation of Trio and Fra is elevated in S2 cells when Abl levels are increased. Together, these data suggest that Abl, Trio, Ena and Fra are integrated into a complex signaling network that regulates axon guidance at the CNS midline (Forsthoefel, 2005).

The interactions of Abl with Fra are intriguing, since they suggest that in Drosophila, as in other organisms, this evolutionarily conserved guidance receptor is regulated by tyrosine phosphorylation, and also that Fra may regulate Abl substrates. Other studies have demonstrated Netrin-dependent tyrosine phosphorylation of DCC, Netrin/DCC-dependent activation of the tyrosine kinases FAK, Src and Fyn, and the requirement of DCC tyrosine phosphorylation for Netrin-dependent Rac1 activation and growth cone turning. Interestingly, the tyrosine residue in DCC identified as the principal target of Fyn/Src kinases is not conserved in Drosophila Fra or C. elegans UNC-40, suggesting that the precise mechanisms by which Fra/DCC/UNC-40 signaling is regulated by tyrosine kinases may differ between organisms. Tyrosine phosphorylation of UNC-40 has also been observed, and although the kinase(s) responsible has not been identified, genetic interactions suggest that UNC-40 signaling is regulated by the RPTP CLR-1, supporting the idea that regulation of tyrosine phosphorylation is a consequence of UNC-6/Netrin signaling in C. elegans as well. In this study, more robust tyrosine phosphorylation of Fra was observed in cells with pervanadate stimulation than with Abl overexpression alone, raising the possibility that additional kinase(s) may function during Fra signaling. Further investigation will be needed to address this issue and to determine how Abl-mediated phosphorylation of Fra modulates commissural growth cone guidance (Forsthoefel, 2005).

Abl is thought to control actin dynamics in part through its ability to regulate other proteins through tyrosine phosphorylation. Thus, in addition to potential regulation of Fra, Fra may recruit Abl to regulate other Abl substrates. Abl interacts genetically with trio, and in this study, Trio was found to physically interact with Abl in vitro, and Trio tyrosine phosphorylation increases dramatically with co-expression of Abl. Phosphorylation of Trio may affect its activity, as observed for other GEFs. For example, Abl regulates phosphorylation and Rac-GEF activity of Sos1, and Lck, Fyn, Hck and Syk kinases tyrosine phosphorylate Vav GEF and stimulate its activity (Forsthoefel, 2005).

Trio physically interacts with Fra in vitro and in S2 cells, suggesting that Fra can recruit Trio directly. In addition, heterozygosity for trio dominantly modifies the Robo-Fra chimeric receptor phenotype, consistent with a positive role for Trio as a downstream effector of Fra signaling in vivo. As a Rac/Rho GEF, Trio may link Netrin-Fra signaling to the regulation of Rho-family GTPases in commissural axons. Rho-family GTPases have been rigorously studied with regard to their role in the regulation of cytoskeletal dynamics and axon guidance, outgrowth and branching. Although positive roles for GTPases in commissure formation in the Drosophila embryo have not been directly demonstrated, trio and GEF64C, a Rho GEF, interact genetically with fra leading to the dramatic disruption of commissures. Additionally, expression of constitutively active or dominantly negative isoforms of both Rac and Rho, as well as constitutively active Cdc42, causes axons to cross the CNS midline inappropriately. Recent studies have implicated Cdc42 and Rac1/CED-10 as effectors of DCC and UNC-40 signaling, but reaching an understanding of the biochemical mechanisms by which GTPases are regulated has been elusive. Future experiments must determine whether Netrin-Fra signaling modulates the GEF activity of Trio, and how this occurs (Forsthoefel, 2005).

Reducing the genetic dose of ena causes either more or fewer axons to cross the CNS midline, depending on the genetic background, suggesting that the role of Ena in the growth cone is complex. Heterozygosity for ena in embryos expressing the Robo-Fra chimeric receptor reduces the number of axon bundles that inappropriately cross the CNS midline, consistent with a role for Ena as a positive effector of Fra signaling. Ena/UNC-34 has been identified genetically as an effector of DCC/UNC-40 in C. elegans. In cultured mouse neurons, Ena/VASP proteins are required for Netrin-DCC-dependent filopodia formation, and Mena is phosphorylated at a PKA regulatory site in response to Netrin stimulation. In migrating fibroblasts, increasing Ena/VASP proteins at the leading edge leads to unstable lamellae and decreased motility; by contrast, increasing Ena/VASP levels at the leading edge in growth cones causes filopodia formation, possibly due to differences in the distribution of actin bundling or branching proteins. Although the role of Ena in actin reorganization in Drosophila has not been rigorously studied, Ena localizes to filopodia tips in cultured Drosophila cells, suggesting that the role of Ena in filopodia formation may be conserved (Forsthoefel, 2005).

No direct biochemical interaction was observed between Fra and Ena. However, Abl binds and phosphorylates Ena, and heterozygosity for both Abl and ena further suppresses the Robo-Fra phenotype, suggesting that Fra may recruit Abl to regulate filopodial extension through Ena. Alternatively, Fra may regulate Ena through other molecule(s), and the synergistic suppression of the Robo-Fra phenotype by Abl and ena is a result of the compromise of parallel pathway(s) regulated by Fra. It is important to note that the functional consequences of biochemical interactions between Abl and Ena are not yet understood. Therefore it will be of particular interest to determine whether Ena is tyrosine phosphorylated in response to Netrin-Fra signaling, and if Ena phosphorylation regulates its activity during filopodial extension (Forsthoefel, 2005).

In addition to suppressing the Robo-Fra chimeric receptor phenotype, mutations in ena also suppress the loss-of-commissure phenotype in fra, Netrin, trio and Abl mutant combinations. In Drosophila (as well as in C. elegans), Ena interacts genetically and biochemically with the repulsive receptor Robo, indicating that Ena may restrict axon crossing at the midline. Thus, the fact that mutations in ena dominantly suppress fra, Netrin, trio and Abl CNS phenotypes could simply reflect the compromise of a parallel, opposing signaling pathway. Consistent with this idea, some axons that cross the midline in ena heterozygous, trio,Abl homozygous embryos are Fas2 positive, indicating a partial reduction in repulsive signaling. However, ena also dominantly suppresses fra and Netrin commissural pathfinding defects, without causing longitudinal Fas2-positive axons to cross the midline. Reductions in Robo signaling therefore may not fully explain the ability of ena to suppress defects in fra, Netrin, Abl and trio mutants (Forsthoefel, 2005).

Based on the fact that mutations in ena suppress a number of Abl mutant phenotypes, it has been proposed that Abl antagonizes Ena function. In Abl mutant embryos, Ena and actin mislocalize during dorsal closure and cellularization, and apical microvilli are abnormally elongated, indicating that Abl regulates the localization of Ena. In migrating fibroblasts, increasing Ena/VASP levels at the leading edge results in long, unbranched actin filaments, unstable lamellae, and decreased motility due to increased antagonism of capping protein. Interestingly, mutations in the gene encoding Drosophila capping protein ß enhance CNS axon pathfinding defects in Abl mutants, including commissure formation. Therefore, if Fra and/or Abl regulate Ena localization in commissural axons, then in fra, Netrin or Abl mutants, Ena may be mislocalized in the growth cone, leading to inappropriate inhibition of capping protein and excessive F-actin filament elongation. Additionally, reducing regulation of Ena by Fra or Abl may also allow greater Ena regulation by Slit-Robo signaling. In either case, reducing the gene dose of ena in fra, Netrin and trio,Abl mutant embryos would partially relieve these effects, allowing axons to respond more efficiently to other cues and cross the midline, as was observed. Consistent with this idea, it was found that either increasing or decreasing Ena/VASP proteins at the leading edge impairs the elaboration of growth cone filopodia in response to Netrin-DCC signaling, suggesting that Ena/VASP levels must be tightly regulated in order for the growth cone to respond optimally to extracellular signals (Forsthoefel, 2005).

The role of Abl in the growth cone is also likely to be complex. The observations implicate Abl as an effector of attractive Fra signaling. In addition, tyrosine phosphorylation of Robo by Abl is thought to negatively regulate repulsive signaling by Robo. Paradoxically though, loss-of-function mutations in Abl, robo and slit interact genetically, resulting in inappropriate axon crossing at the midline, and indicating that Abl may also promote repulsion in longitudinally migrating growth cones. Obviously, much remains to be understood about the molecular basis for genetic interactions of Abl, particularly how Abl and its various substrates cooperate with different growth cone receptors to yield specific cytoskeletal outputs (Forsthoefel, 2005).

In summary, genetic and biochemical interactions indicate that Abl, Trio and Ena are integrated into a complex signaling network with Fra and the Netrins during commissure formation. These observations identify another receptor that acts through these effectors, and provide a framework for further investigation of signaling by this key, evolutionarily conserved guidance receptor (Forsthoefel, 2005).

DEVELOPMENTAL BIOLOGY

Embryonic

See the embryonic expression pattern of fra at the Berkeley Drosophila Genome Project Patterns of Gene Expression Site.

Frazzled is expressed on developing axons and epithelia in the embryo. Frazzled is expressed at high levels on commissural and longitudinal axons in the developing CNS and is detected at stage 13 on the earliest commissural axons. Frazzled is expressed at lower levels on peripheral motor axons that extend outward on the intersegemental and segmental nerves, on the surface of midgut epithelial cells beginning at stage 12, and on epidermis. Frazzled does not appear to be expressed on tissues that are thought to express ligands required for motor and CNS axon pathfinding, such as muscle, glia, or midline cells (Kolodziej, 1996)

Effects of Mutation or Deletion

In fra mutants partially penetrant defects are observed in the earliest stages of the development of commissures. Commissures are sometimes thin or absent, the posterior commissure being more severly affected than the anterior. Commissures that appear to be relatively normal in thickness are often less well organized than normal (Kolodziej, 1996).

Most of the neurons of the ventral nerve cord send out long projecting axons that cross the midline. In the Drosophila CNS, cells of the midline give rise to neuronal and glial lineages with different functions during the establishment of the commissural pattern. The development of midline cells is fairly well understood. In the developing ventral neural cord, 7-8 midline progenitor cells per abdominal segment generate about 26 glial and neuronal cells, i.e. 3-4 midline glial cells, 2 MP1 neurons, 6 VUM neurons, 2 UMI neurons, as well as the median neuroblast and its support cells. The VUM neurons comprise motoneurons as well as interneurons, which project through the anterior and posterior commissures. Genetic studies indicate that the VUM neurons are involved in the initial attraction of commissural growth cones. The MP1 neurons are ipsilateral projecting interneurons, which participate in the formation of specific longitudinal axon pathways. The median neuroblast divides during larval and pupal stages. Contrary to what occurs in the grasshopper CNS, the Drosophila median neuroblast does not generate midline glial cells. In Drosophila, the midline glial cells develop from a set of 2-3 progenitors located in the anterior part of each segment. A function of the midline glial cells during the maturation of the segmental commissures has been found, such that two midline glial cells migrate along cell processes of the VUM-midline neurons to separate anterior and posterior axon commissures. If this migration is blocked, a typical fused commissure phenotype develops. Toward the end of embryogenesis, midline glial cells are required for the formation of individual fascicles within the commissures (Hummel, 1999 and references).

Independent of whether Netrin acts by a repulsive or attractive mechanism, evidence is provided that beside the Netrin/Frazzled (DCC) signaling system an additional attractive system(s) is operating in the developing embryonic nervous system of Drosophila. Attractive cues appear to be provided by the midline neurons. The genes schizo and weniger are likely to encode either additional components of the Netrin signaling system or define a second attractive guidance system. In order to obtain further insights in the function of these genes, several double mutant combinations were generated. If schizo or weniger act downstream in the netrin-frazzled pathway, no enhancement of the commissural phenotype would be expected, as compared to the frazzled deficiency phenotype. In embryos homozygous for a hypomorphic frazzled allele or mutant for schizo, only some commissural connections are missing. weniger mutant embryos have a penetrant CNS phenotype and all neuromeres are affected. However, embryos double mutant for frazzled and schizo lack most commissural axons. Similar synergistic effects are seen in frazzled/weniger or in schizo/weniger double mutant embryos. These double mutant analyses also indicate that axons crossing the midline in fra and netrin mutant embryos do not do so because of a loss of a repulsive Netrin signal. In the light of the synergistic effect seen in the frazzled/schizo double mutant it is suggested that, beside Netrin and its receptor, other proteins are required to guide commissural growth cones toward the midline. Furthermore, in the absence of two of the attractive signaling components, the existence is revealed of repulsive functions of the CNS midline. In the double mutant, the repulsive function predominates and directs axons out of the CNS (Hummel, 1999).

What is the function of midline neurons in commissure formation? Attractive and repulsive signal originating from the midline are required for normal commissure development. The Drosophila midline comprises glial and neuronal cell lineages. These data indicate that these two cell types exert distinct functions during commissure formation. The first commissural growth cones invariably steer toward the anterior-most VUM neurons where these growth cones cross the midline to form the posterior commissure. This indicates that initially the midline neurons attract the commissural growth cones. The netrin genes that encode an attractive signal for commissural growth cones are expressed in midline neurons and glial cells during initial commissure formation. However, the number of commissural fibers is normal in mutations affecting the development of the midline glia. Similarly, ablation experiments using the directed expression of reaper and grim in the midline glial cells result in a fused commissure phenotype and do not lead to a reduction in the number of commissural axons crossing the midline. Thus, it is proposed that the midline glial cells do not play an essential role in attracting the commissural growth cones. The glial derived Netrin signal could be required to counteract repulsive signals. Additional support for the assumption that the midline neurons attract commissural growth cones is provided by the orthodenticle mutant phenotype. Here some midline neurons as well as one of the two segmental commissures is missing. Similarly, expression of dominant negative Jun in all midline cells results in a loss of midline neurons and a concomitant loss of all commissures. Furthermore, in patched mutant embryos the midline glial cells are almost absent and appear to be transformed into midline neurons. Attraction of commissural growth cones is normal in these embryos, however commissural axons stall at the midline. This suggests that the midline glial cells do not participate in attracting commissural growth cones but provide locally acting, contact dependent cues helping growth cones across the midline. Similarly, in the vertebrate neural tube, changes in growth cone morphology have suggested that commissural axons are guided by a contact dependent mechanism across the floor plate (Hummel, 1999 and references).

The following model is proposed for commissure formation. The initial growth of commissural growth cones towards the midline in stage 12 embryos is guided by an attractive signal expressed by the midline neurons. Presumably, this attraction is mediated by early Netrin expression in the midline neurons or alternatively by the action of a Schizo/Weniger attractive system. At this early developmental stage the midline glial cells are elongated in shape, contacting the epidermis with their basal side and are assumed to send out cellular processes contacting the VUM-midline neurons at the dorsal side of the nervous system. The midline glial cells express a repulsive signal that is conveyed to lateral axons via the Robo receptor and/or the karussell gene product. This repulsive function restricts the first axons to cross the midline just anterior of the VUM neurons. The midline glial cells also express a contact dependent permissive guidance cue helping the axons to cross the midline. Subsequently, neuron-glia interaction at the midline results in the migration of the midline glial cells along processes of the VUM neurons (Hummel, 1999).

Frazzled (Fra) is the DCC-like Netrin receptor in Drosophila that mediates attraction; Roundabout (Robo) is a Slit receptor that mediates repulsion. Both ligands, Netrin and Slit, are expressed at the midline; both receptors have related structures and are often expressed by the same neurons. To determine if attraction versus repulsion is a modular function encoded in the cytoplasmic domain of these receptors, chimeras were created carrying the ectodomain of one receptor and the cytoplasmic domain of the other and their function in transgenic Drosophila was tested. Fra-Robo (Fra's ectodomain and Robo's cytoplasmic domain) functions as a repulsive Netrin receptor; neurons expressing Fra-Robo avoid the Netrin-expressing midline and muscles. Robo-Fra (Robo's ectodomain and Fra's cytoplasmic domain) is an attractive Slit receptor; neurons and muscle precursors expressing Robo-Fra are attracted to the Slit-expressing midline (Bashaw, 1999).

In Drosophila, the same midline cells normally secrete both Netrins and Slit. Growth cones can simultaneously respond to both ligands in a cell-specific fashion. Some growth cones express high levels of Fra and low levels of Robo, and they extend toward and across the midline. Other growth cones appear to express high levels of both receptors, and they can extend toward the midline, but they do not cross it. Growth cones can dramatically change their levels of Robo expression; once they cross the midline, growth cones increase their level of Robo, a change that prevents them from crossing the midline again. Such complex and dynamic behavior requires growth cones to be able to simultaneously respond to both attractants and repellents and to integrate these signals and respond to the relative balance of forces. Introducing a chimeric receptor into this finely tuned system leads to dramatic phenotypes. Adding a receptor that responds to Netrin as a repellent leads to a comm-like phenotype in which too few axons cross the midline. Adding a receptor that responds to Slit as an attractant leads to the opposite robo- or slit-like phenotypes, in which too many axons cross the midline or remain at the midline, respectively. These phenotypes are dose dependent, suggesting that by adding more chimeric receptor, the relative balance can be tipped and in tis way the growth cone's response is selectively controlled. This striking dosage sensitivity raises the possibility of using these phenotypes as the basis for genetic suppressor screens to identify signaling components that function downstream of attractive and repulsive guidance receptors (Bashaw, 1999).

Another finding of this study is that the signal transduction machinery for attraction and repulsion downstream of these receptors appears to be present in all neurons, and probably in all migrating muscle precursors as well. All neurons expressing either Fra-Robo or Robo-Fra appear to behave the same, regardless of their environment: if they express Fra-Robo, they stay away from the midline; if they express Robo-Fra, they extend toward the midline. No other factor appears to intrinsically commit one growth cone or another to only one kind of response. The same is true for migrating muscle precursors. Normally, many of them express Robo and migrate away from the Slit-expressing midline. However, given the opportunity (by transgenic expression of Robo-Fra), they clearly contain the full machinery for the opposite response. In all these transgenic experiments, the growth cone or muscle response always correlated with the level of receptor (Brashaw, 1999 and references).

The finding that the cytoplasmic sequence determines the response of a guidance receptor raises a number of interesting questions. Attraction might lead to a local change favoring actin polymerization over depolymerization, while repulsion might lead to the opposite change. But is guidance that simple? The cytoplasmic sequences of five different families of repulsive guidance receptors are now known: UNC-5s, Eph receptors, Neuropilins, Plexins, and Robos. Interestingly, they appear to share little if any sequence similarity to one another in their cytoplasmic domains. It is possible, of course, that they bind different adapter proteins that converge on the same repulsive motility machinery. But it is equally likely that not all repulsion is the same and that different classes of repulsive receptors mediate different types of responses in the growth cone. It could be that what is lumped together under the term 'repulsion' actually represents several molecularly distinct mechanisms that negatively influence local growth cone behavior. Just what these different cytoplasmic domains do, and how many different types of repulsion exist, awaits future investigation (Bashaw, 1999 and references).

Several recent experiments point to the modular design of axon guidance receptors, in which the extracellular domain determines the ligand specificity while the cytoplasmic domain dictates the response of the growth cone. In particular, It has demonstrated that a DCC-UNC5H2 chimeric receptor consisting of the extracellular domain of DCC and the cytoplasmic domain of UNC5H2 is as effective as wild-type UNC5H2 in repelling Xenopus spinal axons away from a Netrin source in vitro. This finding was tested in vivo. In addition, attempts were made to extend this result by testing the prediction that a reciprocal UNC5-DCC chimera should mediate attraction to Netrin (Keleman, 2001).

UAS transgenes were prepared encoding chimeric Fra-Unc5 and Unc5-Fra receptors, in which the cytoplasmic domains of the two Netrin receptors had been swapped immediately proximal to their transmembrane domains. To test the prediction that the cytoplasmic domain of Unc5 specifies repulsion, the CNS of embryos in which one or another of these chimeras was expressed using the elav-GAL4 driver was examined. As expected, pan-neural expression of the Fra-Unc5 chimera results in a commissureless phenotype just as strong as that observed with the full-length Unc5 receptor. Ectopic expression of Unc5-Fra has no obvious effect, as previously found to be the case also for full-length Fra (Keleman, 2001).

Does the Unc5-Fra chimera act as an attractive Netrin receptor? If so, pan-neural expression of this receptor, like that of Fra itself, should at least partially rescue the frazzled mutant phenotype. This is indeed the case. Each of two UAS-Unc5-fra transgene insertions tested almost completely rescue the frazzled null mutant. UAS-Unc5-fra rescues both the commissural and longitudinal axon defects of frazzled mutants just as efficiently as does UAS-fra. It is therefore concluded that Unc5-Fra is an attractive Netrin receptor, formally completing the demonstration that Netrin receptors are modular: the growth cone response (attraction or repulsion) is determined by the cytoplasmic domain (DCC or UNC5, respectively), irrespective of the Netrin binding extracellular domain to which it is attached (Keleman, 2001).

Experiments using the elav-GAL4 driver show that Unc5 is a potent mediator of Netrin repulsion at short range, preventing commissural axons from crossing the midline. In a final set of experiments, these observations were extended by asking how an ipsilateral interneuron -- one that does not normally cross the midline -- would respond to ectopic expression of Unc5. For this, the Ap-GAL4 driver was used. This line expresses GAL4 in three neurons (termed the Ap neurons) in each hemisegment. Their cell bodies are positioned laterally within the nerve cord, several cell diameters from the midline. One is located dorsally, the other two ventrally. All three are intersegmental interneurons. Their axons first grow toward the midline, but they do not cross it, instead turning anteriorly to continue along the medial edge of the ipsilateral longitudinal tract. In the experiments reported here, focus was placed on the behavior of the dorsal Ap neuron (Keleman, 2001).

Expression of Unc5 in this neuron has remarkable consequences. Rather than growing toward the midline, its axon now grows laterally away from the midline to exit the CNS and continue on a motor trajectory into the periphery. This phenotype is highly penetrant: 91% of dorsal Ap axons examined exited the CNS in these embryos. Thus, Unc5 can repel axons away from the midline at long range, forcing them 180° off course. All of the mutant Unc5 proteins tested in the midline crossing assay were also found to be defective in this assay (Keleman, 2001).

This long-range repulsion by Unc5 requires Netrin function, as expected. However, unlike the short-range repulsion of commissural axons at the midline, long-range repulsion of Ap axons is partially dependent on frazzled function. In frazzled mutant embryos, only 59% of Ap axons exited the CNS upon ectopic Unc5 expression. To determine whether this reflects an autonomous requirement for frazzled, its function was restored specifically in the Ap neurons by introducing a UAS-fra transgene into these embryos. The percentage of Ap axons exiting the CNS rose to 97%, demonstrating that potent long-range repulsion of Ap axons requires expression of both Unc5 and Fra in the Ap neurons themselves (Keleman, 2001).

A majority of neurons that form the ventral nerve cord send out long axons that cross the midline through anterior or posterior commissures. A smaller fraction extend longitudinally and never cross the midline. The decision to cross the midline is governed by a balance of attractive and repulsive signals. This study has explored the role of a G-protein, Galphaq (G protein alpha49B), in altering this balance in Drosophila. Dgq was originally identified from a head cDNA library as a homolog of mammalian Galphaq. Initial functional characterization had suggested that it was a visual-specific G-protein essential for Drosophila visual transduction. A splice variant of Galphaq, dgqalpha3, is expressed in early axonal growth cones, which go to form the commissures in the Drosophila embryonic CNS. Misexpression of a gain-of-function transgene of dgqalpha3 (AcGq3) leads to ectopic midline crossing. Analysis of the AcGq3 phenotype in roundabout and frazzled mutants shows that AcGq3 function is antagonistic to Robo signaling and requires Frazzled to promote ectopic midline crossing. These results show that a heterotrimeric G-protein can affect the balance of attractive versus repulsive cues in the growth cone and that it can function as a component of signaling pathways that regulate axonal pathfinding (Ratnaparkhi, 2002).

cDNA clones corresponding to the dgq gene were isolated in library screens using a fragment from the eye-specific splice variant dgqalpha1. Libraries derived from either embryo or appendage RNAs were screened and dgq-positive cDNA clones were analyzed by restriction digests and PCR. Three classes of cDNA clones were obtained. In the region of the open-reading frame, one of these classes corresponds to a splice variant transcript of the dgq gene, dgqalpha3, known to be expressed in several adult tissues. This class was isolated repeatedly from the embryo cDNA library, as judged by extensive PCR analysis. dgqalpha3-specific transcripts are present in poly(A⁺) RNA extracted from heads, appendages, male and female bodies, and embryos. Another class of cDNA clones was found only in the appendage library and appeared identical to the adult visual Galphaq splice form (dgqalpha1) (Ratnaparkhi, 2002).

The presence of the Dgqalpha3 protein in Drosophila embryos was examined by Western blot analysis of embryo extracts. The antiserum used recognizes the C-terminal end of the mammalian Gq protein. In Drosophila Gq this C-terminal sequence is conserved only in the Dgqalpha3 form. The results obtained indicate that a 39 kDa band, corresponding to the predicted size of the Dgqalpha3 protein, is present in embryos throughout development from as early as 0-8 hr (Ratnaparkhi, 2002).

Presence of dgqalpha3 RNA and protein in embryos suggests an involvement of the dgq gene in Drosophila development. The expression pattern of dgqalpha3 during embryonic development was examined by in situ hybridization with a dgqalpha3 splice variant-specific probe. Although dgqalpha3 RNA is present in earlier stages, tissue-specific expression of dgqalpha3 is first seen in the brain and ventral nerve cord at stage 13. This expression persists until late in development, where in addition, strong expression is seen in an anterior sense organ. This organ corresponds in position to the Bolwig's organ or the larval eye (Ratnaparkhi, 2002).

Expression of Dgqalpha3 during development of the embryonic nervous system was further confirmed by immunohistochemical staining of wild-type embryos with the Gq antiserum. The first indication of Dgqalpha3 expression in the CNS is at early stage 12. This is also the stage at which the pioneer neurons begin formation of axon pathways that give rise to the typical ladder-like appearance of the embryonic CNS, consisting of longitudinal tracts and anterior and posterior commissures that can be visualized with the axonal marker mAb BP102. A similar pattern of expression of anti-Gq and the axonal marker mAb BP102 at early stage 12 suggests that Dgqalpha3 is expressed in the pioneer growth cones that give rise to the commissures. At later stages of development Dgqalpha3 protein expression increases in the axonal tracts of the CNS. In addition, Dgqalpha3 expression was visible in the midgut epithelium at stages 12 (Ratnaparkhi, 2002).

Axonal guidance in the Drosophila CNS requires the interpretation of both attractive and repulsive cues, generated by cells that lie in the midline. The expression pattern of Dgqalpha3 protein suggested that it might be required in early growth cones for the interpretation of these cues. To address this possibility, it was essential to alter Galphaq signaling in a tissue and cell-specific manner. Therefore, transgenic strains were created with a dominant active form of Dgqalpha3, in which a glutamine residue at position 203 was mutated to a leucine. The mutation was made based on previous studies on dominant active forms of Galphaq from mammalian cells and Drosophila. As controls, transgenic lines carrying the wild-type form of Dgqalpha3 were created. Both activated dgqalpha3 (UAS-AcGq3) and dgqalpha3 (UAS-Gq3) cDNAs were placed under the control of the GAL4-inducible UAS promoter that would allow tissue and cell-specific expression. Initially, the C155-GAL4 line, which expresses in all postmitotic neurons, was used in order to study the effect of UAS-AcGq3 expression on axonal development. When stained with mAb BP102, the CNS of C155-GAL4;UAS-Gq3 embryos looked normal. In embryos expressing AcGq3, the pattern of the CNS appeared mildly deranged in that the commissures were thicker, and the neuropil region was broader than usual. More significant differences between the two genotypes were obvious when a monoclonal antibody against Fasciclin II (mAb 1D4) was used. At stage 13, anti-Fasciclin II (anti-Fas II) marks the pioneer axons that go to form the first longitudinal axon pathway, which by stage 16, defasciculates to form three distinct fascicles. These axons project ipsilaterally and do not cross the midline. In embryos of the genotype C155-GAL4;UAS-Gq3, this projection pattern was identical to wild-type embryos, indicating that overexpression of Dgqalpha3 has no effect on Fas II-expressing axons. However, in embryos expressing AcGq3, Fas II-positive axons appeared abnormal in all the embryos examined with variations in the extent of abnormality. One obvious phenotype observed was that of 'stalling' of Fas II-positive axons, which could be seen clearly at late stage 13. At this stage, minute outgrowths from the cell bodies and axonal tracts were also visible. From stage 15 onward, Fasciclin II-expressing axons could be seen crossing the midline. Occasionally a whirling phenotype similar to that observed in robo mutant alleles was seen (Ratnaparkhi, 2002).

From these experiments the fate of the axons that cross the midline was unclear. For this purpose a strain with the Apterous tau-ßgalactosidase (Ap-taußgal) construct was created in which single axons could be observed. Ap-taußgal marks specific Apterous-expressing neurons in each hemisegment of the embryo. Normally these axons project anteriorly on the ipsilateral side to form a distinct Apterous fascicle. In embryos of the genotype C155; UAS-AcGq3, axons from Apterous-expressing neurons no longer remain on the ipsilateral side but are now able to cross the midline. However, unlike axons that crossover in robo mutant embryos, these appear to stall after reaching and crossing the midline (Ratnaparkhi, 2002).

The phenotypes observed in embryos expressing AcGq3 suggest that Gq signaling can drive formation of the commissures and longitudinal tracts. This idea is supported by the phenotype observed in embryos homozygous for Df(2R)vg-C (which uncovers dgq). In these embryos the commissures appear thinner, and there are extensive breaks in the longitudinal tracts. These phenotypes are considerably stronger than those observed for frazzled mutants, which is also uncovered by the same deficiency, indicating that the effect of removing both Dgq and Frazzled is additive. However, these defects could be either caused by erroneous signaling within neurons so that they misinterpret existing cues, or by a non-autonomous mechanism that affects midline guidance cues. The latter would result in misplaced neurons or glia or neurons with changed identity. In Df(2R) vg-C embryos, the pattern of neurons expressing the Even-skipped (Eve) protein appear normal, indicating that the defects seen occur after neuronal patterning is complete (Ratnaparkhi, 2002).

To confirm that the phenotype seen by expression of AcGq3 in the CNS is caused by altered signaling within neurons expressing AcGq3, more restrictive GAL4 drivers were used to express UAS-AcGq3 in specific subsets of neurons of the embryonic CNS. ftz_ng-GAL4 expresses in a small subset of neurons that include mostly motor neurons and some interneurons like vMP2, pCC, dMP2, and MP1. These interneurons pioneer the longitudinal axon tracts, which stain positive for Fasciclin II. In addition, these axons never cross the midline. On expressing UAS-AcGq3 with ftz_ng-GAL4, midline crossing by Fasciclin II-positive axons could be observed. At stage 13, the pCC axon, which normally projects anteriorly on the ipsilateral side, could be seen turning toward the midline. At stage 16, aberrant midline crossing by the medial fascicle could be observed. The number of midline crossovers at this stage is less as compared with C155-GAL4, presumably because of the restricted and comparatively weak expression of the ftz_ng-GAL4 line. Similar results were obtained with eve_ng-GAL4, which expresses in aCC, pCC, and RP2 neurons. The pCC axon can be seen crossing the midline, whereas the aCC and RP2 projections look normal on expression of AcGq3. Axons from Apterous-expressing dorsal cells (dc) can also change their trajectory on expression of AcGq3. Instead of projecting toward the anterior and in an ipsilateral direction as is normal, a fraction of the axons can be seen drifting across the midline. The autonomy of AcGq3 function is further supported by the observation that neurons and glia are patterned normally in C155-GAL4/UAS-AcGq3 embryos, as judged by staining with anti-Eve and anti-Repo antibodies. Taken together these data demonstrate that specific activation of Dgqalpha3 in ipsilaterally projecting neurons causes changes in their axonal trajectories so that they are now able to project across the midline (Ratnaparkhi, 2002).

To understand how Dgqalpha3 acts to change axonal paths, possible interactions with genes known to affect midline guidance were sought. Axons that cross the midline and project along the contralateral longitudinal tract normally need to downregulate expression of Robo, which acts as a receptor for the midline repellant Slit. It is known that Robo downregulation requires Commissureless, but the precise mechanism is not understood. A possible mechanism by which AcGq3 could promote midline crossing was by downregulating Robo. To test this hypothesis, Robo expression was examined in ftz_ng-GAL4;UAS-AcGq3 embryos. Interestingly, Robo is not downregulated visibly in axons that ectopically cross the midline under the influence of AcGq3. The extent of Robo staining seen on these axons that aberrantly cross the midline is comparable with that seen on the longitudinal tracts. Thus, constitutive activation of Dgqalpha3 results in aberrant midline crossing of axons by a mechanism that is independent of Robo downregulation (Ratnaparkhi, 2002).

Another mechanism by which AcGq3 could induce midline crossing is through inhibition of the repulsive signal mediated by Robo. If this were so, then reducing levels of Robo by genetic means should enhance the phenotype of AcGq3. To test this, AcGq3 was expressed using ftz_ng-GAL4 in embryos carrying a single copy of the robo¹ mutant allele. robo¹ is a recessive mutation. However, embryos with one copy of this mutation show midline crossing at a frequency of ~10%. When UAS-AcGq3;robo¹/+;ftz_ng-GAL4 embryos were stained with mAb 1D4, a significant increase in the number of midline crossovers was observed as compared with embryos of the genotype UAS-AcGq3;+/+;ftz_ng-GAL4. This suggests that activation of Dgqalpha3 antagonizes the repulsive output through Robo resulting in excessive midline crossing. The antagonism could be mediated either through phosphorylation of Robo or signaling components that function downstream and/or in parallel with Robo (Ratnaparkhi, 2002).

Phosphorylation of a single tyrosine residue on Robo by Abelson (Abl) tyrosine kinase inhibits Robo repulsive signaling and is needed for normal midline crossing to take place. Expression of a mutant form of Robo in which this tyrosine residue (Y1040) has been replaced with a phenylalanine (in a transgenic strain referred to as UAS-roboY-F), leads to constitutive Robo signaling such that no axons cross the midline, resulting in a complete absence of commissure formation. If AcGq3 acts upstream of Robo, it was predicted that ectopic midline-crossovers, induced by expression of AcGq3, would be reduced in the presence of Robo Y-F. In fact, in embryos expressing both AcGq3 and Robo Y-F, no ectopic crossovers are seen, indicating that AcGq3 could inhibit Robo signaling by promoting Robo phosphorylation. This finding is also supportive of the fact that AcGq3 exerts its effect independent of Commissureless-mediated Robo downregulation. It is possible however, that AcGq3 acts through a parallel pathway that is no longer effective in the presence of Robo Y-F (Ratnaparkhi, 2002).

Both the spatiotemporal pattern of expression and functional analysis of dgq indicate that Gq activation in vivo promotes midline crossing. Axons that cross the midline need to down-modulate their repulsive signaling pathway(s) as well as respond positively to attractive cues. Therefore, whether changes in the levels of 'attractive' signaling such as the Netrin-Frazzled pathway affect the phenotype of AcGq3 was examined. Interestingly, AcGq3 phenotype shows a dosage-dependent interaction with Fra. Removal of a single copy of the Fra gene leads to a threefold reduction in the number of midline crossovers induced by AcGq3. A further reduction was observed on removal of both copies of the Fra gene as seen in embryos of the genotype C155-GAL4/UAS-AcGq3;fra³/fra⁴. Signaling through AcGq3 is thus sensitive to levels of Frazzled in the CNS (Ratnaparkhi, 2002).

To examine the effect, if any, of AcGq3 on the frazzled mutant phenotype, embryos of the genotype C155-GAL4/UAS-AcGq3;fra³/fra⁴ were examined with anti-connectin antibody and BP102. Anti-connectin labels a distinct axon fascicle in the longitudinal connectives, axon projections of SP1 and RP1 neurons that project through the anterior commissure, and a subset of axons that project through the posterior commissure to their contralateral targets. In embryos of the genotype C155-GAL4/+; fra³/fra⁴, breaks were observed in connectin-positive commissural axons and longitudinal tracts. Embryos of the genotype C155-GAL4/UAS-AcGq3;fra³/fra⁴ also show similar breaks, indicating that AcGq3 does not have an effect on the frazzled mutant phenotype. Similar results were obtained by staining with BP102 (Ratnaparkhi, 2002).

The induction of ectopic midline crossing by AcGq3 suggests that Dgqalpha3 function might be required during commissural growth. What activates Dgqalpha3 in vivo? In Drosophila, the only pathway so far known to mediate attraction toward the midline, is the Netrin-Frazzled signaling pathway. However, null mutants for netrins and frazzled continue to show formation of commissures, albeit thin and poorly organized. The failure to show a complete absence of commissures suggests that an alternate signaling pathway or pathways exists at the midline, one that promotes commissural growth. The presence of a second attractive signaling pathway operating at the midline has also been suggested based on analysis of mutants involved in formation of commissures. Dgqalpha3 might act as a component of this alternate pathway to promote commissural growth (Ratnaparkhi, 2002).

Signaling mechanisms involved in DCC/Frazzled-mediated attraction are poorly understood in vertebrates as well as invertebrates. In vitro studies using pharmacology in vertebrate systems have shown that guidance mediated by Netrin-1 is dependent on cAMP levels in the growth cone. Increase in cAMP levels results in attraction, whereas low levels of the cyclic nucleotide causes repulsion. In Xenopus cultured neurons, Netrin-1-induced turning response has also been shown to depend on Ca²⁺ influx through the plasma membrane and Ca²⁺-induced Ca²⁺ release through intracellular stores. The involvement of second messengers such as Ca²⁺ and cAMP suggests that G-protein-coupled signaling pathways might be involved. Heterotrimeric G-proteins are also thought to play a role in neuronal migration and growth cone collapse (Ratnaparkhi, 2002).

The Adenosine A2b receptor has been implicated in Netrin-1 signaling. However, it has been shown that DCC can bind Netrin-1 and signal attraction independent of the Adenosine A2b receptor. DCC undergoes a ligand-dependent dimerization essential for its signaling that remains unaffected even in the presence of antagonists to adenosine receptors, thus providing evidence that DCC alone is central to Netrin-1 signaling. As compared with vertebrates, the mechanism of Netrin signaling in Drosophila is still obscure. Given the evolutionarily conserved nature of both, the ligand and the receptor, similar downstream signaling elements are very likely involved in mediating attraction. It is possible that a seven transmembrane domain receptor activates Dgqalpha3 signaling in response to novel attractive cues or Netrins leading to increase in Ca²⁺ levels and thus promoting attraction (Ratnaparkhi, 2002).

The results from the genetic analysis of AcGq3 and frazzled suggest that Frazzled function is essential for AcGq3-mediated ectopic midline crossing. In addition, they also indicate that Dgqalpha3 does not function downstream of frazzled signaling. A simple explanation for these observations could be that activity of Dgqalpha3 and Frazzled are both essential to promote midline crossing. The effects of the two signaling pathways are additive; activation of Frazzled and Dgqalpha3 are both necessary to elicit attraction. Removal of one or both copies of frazzled in the presence of AcGq3 simply reduces the sum total of attraction sensed by the growth cone, thus inhibiting aberrant midline crossing of ipsilateral axons (Ratnaparkhi, 2002).

The antagonism between AcGq3 and Robo suggests that AcGq3 operates by modulating repulsion from the midline during commissural growth. It has been demonstrated that Robo signaling is negatively modulated by tyrosine phosphorylation by Abelson kinase. AcGq3 could inhibit Robo signaling by a similar mechanism of phosphorylating Robo. It could perhaps do this by activating a kinase cascade involving a nonreceptor tyrosine kinase such as Bruton's tyrosine kinase (BTK or Tec kinase) which, in mammalian cells, has been shown to be a direct effector of Gq signaling. The results are equally consistent with the possibility that AcGq3 and Robo act through parallel pathways, such that AcGq3 induced midline crossing requires downregulation of Robo signaling (Ratnaparkhi, 2002).

Based on the results obtained from genetic analysis of AcGq3 with frazzled and robo, the following models can be proposed to explain the function of Dgqalpha3. In the first, Dgqalpha3 can be thought of as being a component of the attractive signaling pathway alone. Expression of the activated form of the protein functions to override the repulsive cues at the midline and promote ectopic midline crossing. In such a scenario, one would argue that the synergism observed between AcGq3 and robo¹ is a consequence of the combined effect of reduced Robo signaling and excess attractive signaling induced by AcGq3 leading to an increase in the number of midline crossovers. In the presence of UAS-RoboY-F, repulsive signaling increases to a level that cannot be overriden by AcGq3-attractive signaling. A second possibility is that Dgqalpha3 is a component of an attractive signaling pathway, which functions to potentiate Frazzled signaling by negatively modulating the repulsion mediated by Robo signaling. This could be through phosphorylation of Robo. A recent study using spinal axons from stage 22 Xenopus embryos has shown that the repulsive ligand Slit can 'silence' the Netrin-mediated attraction through a direct physical interaction between the cytoplasmic domains of Robo and Frazzled. This ligand-dependent silencing effect serves to promote repulsion of growth cones from the midline during the development of commissures. Dgqalpha3 might function conversely at the level of downstream effector molecules to inhibit repulsion in response to attractive cues to promote midline crossing (Ratnaparkhi, 2002).

In summary, these results predict the involvement of a Gq-mediated signaling pathway in regulating midline crossing in Drosophila. In addition, they also support the notion that balance between attraction and repulsion is a crucial factor that determines the final response of a growth cone to different cues. Inhibition of dgq function specifically in the growth cones should prove useful in dissecting out other components of this pathway which regulates midline crossing (Ratnaparkhi, 2002).

pCC/MP2 neurons pioneer the longitudinal connectives by extending axons adjacent to the midline without crossing it. These axons are drawn toward the midline by chemoattractive Netrins, which are detected by their receptor Frazzled (Fra). However, these axons are prevented from crossing by Slit, an extracellular matrix ligand expressed by glial cells and recognized by Roundabout (Robo), a receptor on the axons of most neurons. Conventional myosin II activity provides the motile force for axon outgrowth, but to achieve directional movement during axon pathway formation, myosin activity should be regulated by the attractive and repulsive guidance cues that guide an axon to its target. Evidence for this regulation is obtained by using a constitutively active Myosin Light Chain Kinase (ctMLCK) to selectively elevate myosin II activity in Drosophila CNS neurons (Kim, 2002).

Expression of ctMLCK pan-neurally or in primarily pCC/MP2 neurons causes these axons to cross the midline incorrectly. This occurs without altering cell fates and is sensitive to mutations in the regulatory light chains. These results confirm the importance of regulating myosin II activity during axon pathway formation. Mutations in the midline repulsive ligand Slit, or its receptor Roundabout, enhance the number of ctMLCK-induced crossovers, but ctMLCK expression also partially rescues commissure formation in commissureless mutants, where repulsive signals remain high. Overexpression of Frazzled, the receptor for midline attractive Netrins, enhances ctMLCK-dependent crossovers, but crossovers are suppressed when Frazzled activity is reduced by using loss-of-function mutations. These results confirm that proper pathway formation requires careful regulation of MLCK and/or myosin II activity and suggest that regulation occurs in direct response to attractive and repulsive cues (Kim, 2002).

The general importance of regulating myosin II activity during axon guidance decisions is confirmed by observation that pan-neural expression of ctMLCK, but not wtMLCK, in Drosophila embryos causes axons within the pCC/MP2 pathway to project across the midline incorrectly. In crossing the midline, axons in the pCC/MP2 pathway either over-respond to midline attractive cues leading them across the midline or fail to respond to repulsive signals preventing them from crossing. Indeed, it is likely that both processes are operating. Axons within the pCC/MP2 pathway move toward the midline as Fra receptors detect chemoattractive Netrins. However, they are prevented from crossing by the repulsive ligand Slit, detected by Robo, the cell surface receptor present on most growth cones. Expression of ctMLCK does not alter the onset of axon extension nor the initial pioneering events of pCC/MP2 neurons, but is sufficient to allow these axons to overcome the repellent Slit barrier and cross the midline. If midline repulsive signals are reduced by using heterozygous mutations of either slit or robo, ctMLCK expression induces many more pCC/MP2 axons to cross the midline, and decreasing myosin II activity using sqh mutations that lower the activity of the regulatory light chains suppresses some of the crossovers observed in heterozygous robo mutants. Thus, it seems that myosin II activity must be maintained below a certain threshold in order for Robo to prevent axons from crossing the midline. When myosin II activity exceeds that threshold, as in embryos expressing ctMLCK, the growth cone is unable to respond appropriately to activation of Robo (Kim, 2002).

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date revised: 15 October 2005 

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