Engrailed controls the organization of the ventral nerve cord through frazzled regulation

In Drosophila, the ventral nerve cord (VNC) architecture is built from neuroblasts that are specified during embryonic development, mainly by transcription factors. Engrailed, a homeodomain transcription factor known to be involved in the establishment of neuroblast identity, is also directly implicated in the regulation of axonal guidance cues. Posterior commissures (PC) are missing in engrailed mutant embryos, and axonal pathfinding defects are observed when Engrailed is ectopically expressed at early stages, prior to neuronal specification. frazzled, enabled, and trio, all of which are potential direct targets of Engrailed and are involved in axonal navigation, interact genetically with engrailed to form posterior commissures in the developing VNC. The regulation of frazzled expression in engrailed-expressing neuroblasts contributes significantly to the formation of the posterior commissures by acting on axon growth. A small genomic fragment within intron 1 of frazzled can mediate activation by Engrailed in vivo when fused to a GFP reporter. These results indicate that Engrailed's function during the segregation of the neuroblasts is crucial for regulating different actors that are later involved in axon guidance (Joly, 2007).

During embryogenesis, Engrailed is first expressed in posterior epidermal cells within each segment, and then later in NBs, GMCs, and neurons. Present at all developmental stages in a subpopulation of neural cells, Engrailed is a good candidate for a factor participating in neuronal determination. Several Engrailed target genes involved in neurogenesis have been identified, and in particular in axonal guidance, including eg, con, comm, fra, ena, and trio. This suggested an important role for engrailed in this process (Joly, 2007).

Interestingly, Trio and Ena were recently found to function as effectors of Fra signalling and to act together in the formation of commissural axons. In particular, they were shown to physically interact, suggesting a potential mechanism by which Fra might coordinate the actin cytoskeletal dynamics necessary for axonal cone growth. This study shows that en genetically interacts not only with fra, but also with ena and trio, to form the posterior commissures. En thus appears to directly regulate PC formation by acting at different levels to ensure axon growth through a complex signalling network that involves Fra (Joly, 2007).

Transheterozygous embryos with alterations in both en and in any of several potential targets present axonal defects that are very similar to those observed in homozygous en mutant embryos. Overexpressing Fra using the prd-Gal4 driver cannot rescue the axonal defects of homozygous en mutant embryos. This confirms that En plays an important role in axonal guidance by regulating various target genes, including ena, trio, commissureless (comm), and transcription factors such as eg, that have been identified as potential En targets. While En is often identified as a repressor, there is no evidence for a role for En in the repression of genes that instruct neurons to choose the AC, such as Wnt5/Drl components. This study demonstrates instead that En regulates axonal guidance and growth by activating components necessary for the establishment of neuronal posterior connectives (Joly, 2007).

Several lines of evidence are provided that fra expression is directly controlled by Engrailed. For example, genomic fragment 2C5 was found to bind En in vivo, first during embryogenesis (as assayed by ChIP) and later in larvae (as assayed by immuno-FISH. In addition, this genomic fragment is shown in this study to be able to mediate activation by En in transgenic flies. However, even though it is known to bind En in embryos, 2C5 is not able to drive GFP expression during embryogenesis, suggesting that it recapitulates only a fraction of the frazzled regulatory sequences (Joly, 2007).

Genetic data is provided arguing that fra is regulated by En during embryogenesis. en and fra interact genetically to ensure the formation of a correct scaffold within the VNC. In homozygous en mutant embryos, fra expression is affected by early stage 11, and Fra immunostaining is absent in the PCs at stage 14, correlating with a loss of posterior commissures (Joly, 2007).

This study shows that PC formation requires an early function of En that acts prior to the specification of neuronal cell fate and to axon growth. Indeed, only the ectopic expression of En at early stages leads to axonal misrouting, whereas the use of pan-neuronal drivers does not cause any axonal defects. Once neurons are specified, En is no longer able to change their fate and hence affect their axonal navigation. This confirms a role for En during NB segregation, and suggests that the neuronal expression of Engrailed is not essential for the formation of the VNC (Joly, 2007).

During NB segregation, Engrailed may participate in the specification of pioneer neurons. Indeed, it was observed that not all the axons that form PCs come from en-expressing cells. Moreover, in homozygous mutant en embryos, it was found that the pioneer marker BP102 was affected in PCs. This suggests that a cluster of En-positive neurons corresponds to the pioneers, which are normally required for normal pathfinding by later outgrowing neurons. This could explain the absence of PCs in engrailed homozygous mutant embryos. Interestingly, the use of a late eve-Gal4 driver to ectopically express En in aCC/RP2 pioneer neurons had no effect on axonal pathfinding. This confirms that the En-sensitive period occurs before the specification of the neurons, including the pioneers (Joly, 2007).

This study shows that the En/fra interaction is important for the formation of the PCs, since PCs are not formed in transheterozygous mutant en/fra embryos. This absence of PCs might result from a loss of axonal growth, which is known to involve Fra. This might also account for the PC defects that are observed in homozygous en mutant embryos (Joly, 2007).

Since the function of En in establishing the axon scaffold within the VNC is essential during NB segregation, it is suspected that the regulation of En target genes involved in axonal pathfinding might also occur at early stages. Indeed, it was possible to confirm that the axonal defects detected in en/fra transheterozygous embryos required the loss of early fra activation during NB segregation. This was shown by RNA in situ hybridization and by rescue experiments: PC axons of stage 15 enX31/fra1 embryos only develop normally when Fra expression is recovered before the specification of the neurons, but do not form properly once neurons are formed. Therefore, one possible hypothesis is that the activation of fra in NBs allows the axonal growth of the PC pioneers (Joly, 2007).

The data suggest that the fra level in NBs and neurons is crucial for axon growth. Because mutations affecting axon growth must be dominant over axonal guidance problems, it is logical that the VNCs of both en/en and en/fra present the same missing PC phenotype. Indeed, with fra being a direct target of En, it can be assumed that in the absence of the En activator, fra expression will be lower or lost. Indeed, it was noticed that en/fra embryos phenocopy fra/fra embryos: in both cases PCs are missing, and neurons express En but show defects in their positioning. Therefore, these changes in neuronal cell fate can be attributed to a change in fra expression. One open question concerns the sensitive period of Fra in this process: frazzled is activated by Engrailed during the segregation of the NBs, but Fra protein is only detectable in neurons. One possible explanation is that Fra protein is present at early stages, but is under the threshold of detection. Another explanation can also be drawn from previous work in vertebrates, where it has been shown that growth cones possess the machinery necessary for protein translation and can translate guidance molecules locally. The resulting rapid changes in protein levels were shown to be involved in axon guidance. Therefore, one hypothesis is that the fra RNA pool in NBs is rapidly translated in growth cones in order to cause changes in the cytoskeleton necessary for axon growth and their further guidance (Joly, 2007).

These results give new insights into En function during neurogenesis and show that En can alter the VNC architecture at different levels to form PCs, playing on axonal pathfinding and axon growth. Indeed, en mutant embryos present PCs that are not properly positioned or not even formed in most segments. Further, ectopic expression of En leads to abnormal axonal pathfinding. Both loss and gain of function of en could be associated with changes in the identity of the NBs (data not shown), confirming a role for En in this process (Joly, 2007).

En functions during neurogenesis act through the regulation of different target genes. One way is through the regulation of transcription factors such as eagle, but it also regulates the expression of fra, trio and ena, which are more directly involved in axon growth and which participate with En in the formation of the PCs. Indeed, monitoring eg-expressing neurons in an en/fra genetic background showed that axons projecting through PCs do not grow properly, confirming that en and fra are involved in this process (Joly, 2007).

Together, these results illustrate how En can act during NB segregation to build a wild-type VNC. Recent results in vertebrates suggest that the regulatory pathway that this study has identified between En and fra (EN1 and DCC in vertebrates) may be evolutionarily conserved. Elucidating the molecular events that allow En/Fra-positive neurons to specifically project axons through PCs but not ACs will be the next challenge to explore in order to better understand axonal guidance (Joly, 2007).

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).

Midline governs axon pathfinding by coordinating expression of two major guidance systems

Formation of the neural network requires concerted action of multiple axon guidance systems. How neurons orchestrate expression of multiple guidance genes is poorly understood. This study shows that Drosophila T-box protein Midline controls expression of genes encoding components of two major guidance systems: Frazzled, ROBO, and Slit. In midline mutant, expression of all these molecules are reduced, resulting in severe axon guidance defects, whereas misexpression of Midline induces their expression. Midline is present on the promoter regions of these genes, indicating that Midline controls transcription directly. It is proposed that Midline controls axon pathfinding through coordinating the two guidance systems (Liu, 2009).

To address how Mid activates expression of the three axon guidance genes, the binding sequence of Mid was determined using an in vitro binding site selection method. Mid-binding sequence was selected from a pool of random oligonucleotides using Mid protein affinity-purified from an embryonic extract. The consensus sequence deduced from the selected oligonucleotides was (G/A/T)NA(A/T)N(T/G)(A/G)GGTCAAG. This sequence was found in the upstream regions or an intron of slit, frazzled, and robo, and all of these sites were conserved among several Drosophila species. To determine whether Mid binds to these regions in vivo, chromatin immunoprecipitation (ChIP) was performed using anti-Mid antibody. In all three genes, Mid was present around the Mid-binding sites, but not on regions without the binding site. In contrast, a potential Mid-binding site 32-kb upstream of the commisureless gene, whose expression is not affected in mid mutants, was not occupied by Mid (Liu, 2009).

The importance of the Mid-binding sites in frazzled and slit was assessed by transgenic reporter assays. To test the role of the Mid site in frazzled, reporter genes were constructed that contain the transcription start site of frazzled and an upstream region including a wild-type Mid-binding site (fraPlacZ) or a mutated site (fraMPlacZ). Compared with the wild-type reporter gene, the reporter with a mutated binding site showed reduced expression levels (33% reduction). Thus Mid-binding site is indeed required for the proper expression of frazzled. Mutating the Mid-binding site in slit also caused a severe effect on slit expression. The lacZ expression in sliPlacZ is driven by the slit regulatory element and the endogenous promoter. While sliPlacZ with the wild-type binding site recapitulated the slit expression in the midline glia and lateral cells, base substitutions in the Mid-binding site in sliMPlacZ abolished the lacZ expression. It is possible that the Mid-binding site resides in an essential promoter element of slit, and hence, the base substitutions abolished slit transcription in all cells. However, the same results were obtained using sli4.5HHlacZ and sliM4.5HhlacZ in which the slit regulatory element is fused to a heterologous hsp70 promoter. Since mid was expressed in the lateral cells but not in midline glia, these results suggest that Mid-binding sites in slit control slit transcription via binding to multiple factors: Mid in lateral cells and unknown factor(s) in midline glia. Taken together, these results demonstrate a direct role for Mid in the regulation of frazzled and slit, and suggest that Mid governs the expression of multiple axon guidance genes through directly binding of the Mid sites in their regulatory regions (Liu, 2009).

This study has shown that Mid directly controls transcription of key components of the two major axon guidance systems: the Netrin/Frazzled system and the Slit/ROBO system. Because these two systems are considered to have opposing outputs, it is interesting that the expression of both systems are induced by the same transcription factor, Mid. Dynamic expression of Frazzled and ROBO is required for growth cones to simultaneously respond to both attractants and repellents, integrate these signals, and then respond to the relative balance of forces. These molecules also provide nonautonomous functions required for cell motility, such as mediating cell adhesion and promoting axon elongation. The coordination of axon guidance systems by Mid may thus ensure cooperative actions of multiple guidance molecules in growth cone dynamics, axonal adhesion, and elongation. The role of Mid in the transcriptional regulation of axon guidance might be a conserved function, because its orthologs of human, mouse, and zebrafish Tbx20 are also expressed in motor neurons (Liu, 2009).

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

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).

Different levels of the Tripartite motif protein, Anomalies in sensory axon patterning (Asap), regulate distinct axonal projections of Drosophila sensory neurons

The axonal projection pattern of sensory neurons typically is regulated by environmental signals, but how different sensory afferents can establish distinct projections in the same environment remains largely unknown. Drosophila class IV dendrite arborization (C4da) sensory neurons project subtype-specific axonal branches in the ventral nerve cord, and it was shown that the Tripartite motif protein, Anomalies in sensory axon patterning (Asap) is a critical determinant of the axonal projection patterns of different C4da neurons. Asap is highly expressed in C4da neurons with both ipsilateral and contralateral axonal projections, but the Asap level is low in neurons that have only ipsilateral projections. Mutations in asap cause a specific loss of contralateral projections, whereas overexpression of Asap induces ectopic contralateral projections in C4da neurons. Biochemical and genetic analysis has shown that Asap regulates Netrin signaling, at least in part by linking the Netrin receptor Frazzled to the downstream effector Pico. In the absence of Asap, the sensory afferent connectivity within the ventral nerve cord is disrupted, resulting in specific larval behavioral deficits. These results indicate that different levels of Asap determine distinct patterns of axonal projections of C4da neurons by modulating Netrin signaling and that the Asap-mediated axonal projection is critical for assembly of a functional sensory circuit (Morikawa, 2011).

The class IV dendrite arborization (C4da) sensory neurons in the Drosophila peripheral nervous system (PNS) provide an in vivo system for investigating the molecular mechanisms that regulate distinct sensory axon projections. C4da neurons comprise three subtypes positioned at distinct regions in each abdominal hemisegment: ddaC, v’ada, and vdaB. In contrast to the similar patterns of their dendrite arbors on the epidermis, the three C4da subtypes exhibit distinctive branching patterns of axonal terminals in the ventral nerve cord (VNC). Upon entering the VNC, the v’ada axons extend terminal branches along the longitudinal axis and likely synapse onto neurons on the ipsilateral side, whereas the ddaC and vdaB neurons additionally develop terminal branches projecting across the midline to form synapses on the contralateral side. These structures suggest that the three C4da subtypes project distinctive axonal terminals in response to the same environmental cues in the VNC, but the origin of the subtype-specific differences in C4da axonal arborization remains unknown (Morikawa, 2011).

To explore molecular mechanisms responsible for the specific projection pattern of distinct C4da neuron axons, a genetic screen was performed for mutants specifically defective in the axonal patterning in C4da neurons, and the protein Anomalies in sensory axon patterning (Asap) was identified as an axon-specific determinant for the subtype-specific contralateral projections in C4da neurons. Further genetic and biochemical analyses indicate that Asap modulates Netrin signaling. These findings suggest that Asap establishes distinct patterns of sensory axon projections by altering the susceptibility of each C4da subtype to the environmental cue Netrin in a dose-dependent manner and contributes to the assembly of the functional sensory circuits that involve perception through C4da neurons (Morikawa, 2011).

asap encodes a member of the Tripartite motif (TRIM) family of proteins, which are characterized by a tripartite motif composed of a Really Interesting New Gene (RING) domain, one or two B-Box domains, and a coiled-coil region. Asap additionally contains a fibronectin type III (FNIII) domain and a spla kinase and ryanodine receptor (SPRY) domain in the C terminus. Of more than 50 TRIM proteins found in mammals, Asap shows the highest homology to a subgroup that contains Trim9, Trim67, midline 1 (MID1; Trim18), and midline 2 (MID2; Trim1). Drosophila and Caenorhabditis elegans each have a single gene, asap and muscle arm development defective (madd)-2 respectively, that belongs to this subgroup (Morikawa, 2011).

Asap was identified as the critical determinant of the projection pattern of axonal terminals in C4da sensory neurons. Asap-mutant C4da axons failed to form commissural fascicles because of the specific loss of the contralateral projections in ddaC and vdaB axons, and these defects could be ameliorated substantially by expression of asap in mutant C4da neurons, indicating that Asap functions in a cell-autonomous manner to regulate the contralateral axonal projection. It was also found that axon terminal processes in v’ada neurons, as well as in ddaC and vdaB, were oriented preferentially to the midline and that this biased orientation disappeared in asap mutants. These data indicate that Asap is required for asymmetric orientation of the axonal terminal processes in all C4da neurons as well as for contralateral projections in ddaC and vdaB axons. Given the expression patterns of Asap in C4da neurons, it is proposed that high Asap levels promote contralateral projections and that low levels are sufficient for asymmetric orientation of the terminal processes toward the midline. In support of this model, overexpression of Asap in v’ada neurons, which express low levels of Asap, induced ectopic contralateral projections (Morikawa, 2011).

How could Asap control axonal patterns in C4da neurons in a dose-dependent manner? Given that Asap functions through the Netrin signaling pathway, one possibility is that Asap may modulate axonal responses to the midline attractant Netrin differentially, according to its level. More specifically, Asap levels in ddaC and vdaB neurons might be high enough to respond fully to the Netrin attraction signal to form contralateral projections, whereas low Asap levels in v’ada neurons might be insufficient to induce contralateral projections but could orient them toward the midline. Netrin signaling has been reported to regulate diverse aspects of neuronal development, such as migration, axonal initiation, outgrowth, guidance, and synaptogenesis. For instance, in C. elegans, glia-derived Netrin signal facilitates axonal extension in the RIA interneuron, whereas the same Netrin signal promotes specification of presynaptic terminals but not axonal extension in the neighboring AIY interneuron. However, it has been unclear how the same receptor and ligand elicit diverse cellular responses in distinct neurons. The current findings suggest that Asap can modulate Netrin signaling to generate diverse neurite behaviors and thus raise the possibility that different levels of Asap in neurons may contribute to distinctive cellular responses to the same guidance cue, Netrin. Interestingly, although immunolabeling revealed that Asap is expressed at different levels in different subsets of PNS neurons, it was found that most PNS neurons appear to express similar levels of Fra receptor. Given that different classes of PNS neurons likely elaborate axonal arbors with distinctive projection patterns in the same VNC environment, it is conceivable that Asap levels account for the differences in response to the Netrin signal in the PNS neurons (Morikawa, 2011).

The mechanism that regulates Asap levels in C4da neurons currently is unknown. Previous studies have shown that specification of each sensory neuron is regulated in part by the combinatorial actions of transcription factors. This study found several consensus sequences potentially recognized by specific transcription factors in the 5'UTR and the first intron of asap gene. Thus, it will be of interest to determine whether these transcription factors may control Asap levels as well as axonal projection patterns in individual C4da neurons (Morikawa, 2011).

TRIM family proteins are characterized by the conserved Tripartite domains, including the RING domain, and several TRIM proteins likely act as E3 ligases through the RING domain to target substrates for destruction via the ubiquitin-proteasome system (Meroni, 2005). Indeed, recent studies demonstrated that MADD-2, the nematode homolog of Asap, requires the RING domain for its roles in muscle arm extension and ventral guidance of HSN axons, implying that MADD-2 functions as an ubiquitin ligase in this context (Alexander, 2010; Hao, 2010; Song, 2011). By contrast, the genetic rescue experiments in C4da neurons revealed that the RING domain is dispensable and that instead the FNIII domain is essential for the Asap function in axonal projection, presumably by promoting Asap. Pico interaction. These results suggest that Asap/MADD-2 may have distinct functions in different cellular contexts (Morikawa, 2011).

Behavioral analysis suggests that the Asap-mediated axonal projections in the VNC are essential for functional circuits to process properly the sensory information that is received by C4da neurons. Trim9, a mammalian homolog of Asap, is expressed specifically in the nervous system. Given that Trim9 and Asap likely link the Netrin receptors to the downstream effectors, Asap/Trim9 might play a conserved role in formation of functional neuronal circuits by modulating Netrin signaling. Further studies of the function of Asap in C4da neurons should help elucidate how the diversity of axonal projection patterns and precise neural circuit assembly is achieved during nervous system development (Morikawa, 2011).

Netrin and frazzled regulate presynaptic gap junctions at a Drosophila giant synapse

Netrin and its receptor, Frazzled, dictate the strength of synaptic connections in the giant fiber system (GFS) of Drosophila melanogaster by regulating gap junction localization in the presynaptic terminal. In Netrin mutant animals, the synaptic coupling between a giant interneuron and the 'jump' motor neuron was weakened and dye coupling between these two neurons was severely compromised or absent. In cases in which Netrin mutants displayed apparently normal synaptic anatomy, half of the specimens exhibited physiologically defective synapses and dye coupling between the giant fiber (GF) and the motor neuron was reduced or eliminated, suggesting that gap junctions were disrupted in the Netrin mutants. When the gap junctions were examined with antibodies to Shaking-B (ShakB) Innexin, they were significantly decreased or absent in the presynaptic terminal of the mutant GF. Frazzled loss of function mutants exhibited similar defects in synaptic transmission, dye coupling, and gap junction localization. These data are the first to show that Netrin and Frazzled regulate the placement of gap junctions presynaptically at a synapse (Orr, 2014).

The results show for the first time that Netrin-Frazzled signaling is specifically responsible for localizing gap junctions presynaptically at the GF-TTMn synapse. In the absence of Netrin, the gap junctions are not assembled in the presynaptic terminal and dye coupling is weak or absent in otherwise anatomically normal synapses. Similarly, Frazzled LOF mutants disrupted gap junctions and synaptic transmission. Finally, presynaptic expression of the dominant-negative Frazzled construct that is missing the intracellular domain also disrupts gap junction assembly, dye coupling, and synaptic transmission. In Netrin LOF mutants, axonal pathfinding is normal because the GF always projects into the target region and occasionally branches ectopically in the target region. However, dendritic path finding is dependent on Netrin-Frazzled signaling. In Netrin LOF mutants, the TTMn dendrite that normally projects toward the midline is often missing, as observed in other motor neurons. Finally, Netrin-Frazzled signaling is implicated in target selection, because GFs that reach the target area often do not build synapses, as seen in other model systems (Orr, 2014).

It was hypothesized that the physiological defect seen in Netrin and frazzled mutants arises from a reduction in trans-synaptic coupling between presynaptic and postsynaptic Innexins. Similar phenotypes, long latency, and lack of dye coupling have been observed in the shakB2 mutant, which lacks gap junctions at the GF-TTMn synapse. The data suggest that when presynaptic and postsynaptic cells make contact, Netrin-Frazzled signaling is instructive for presynaptic localization of Innexins in the GF terminal to form trans-synaptic gap junctions (Orr, 2014).

Two roles were identified for Netrin-Frazzled signaling in assembly of the giant fiber system. Netrin was shown to act as a cue to direct the GF to select a target. Netrin-Frazzled signaling was also a local guidance cue for the GF and the medial dendrite of TTMn. The TTMn medial dendrite grows toward the midline glia, which were shown to be a source of Netrin. Second, it was hypothesized that Netrin bound on the postsynaptic Frazzled receptors serves as a synaptogenic cue for presynaptic Frazzled located on the GF. It is proposed that the bound Frazzled receptors directed presynaptic synaptogenesis and Innexin localization in the presynaptic terminal (Orr, 2014).

The frazzled dominant-negative construct supports the hypothesis that Netrin-Frazzled signaling is instructive in GF-TTMn synaptogenesis and function. Expression of fraC presynaptically disrupts the circuit by interrupting wild-type Netrin-Frazzled signaling. This was demonstrated through disruption of GF-TTMn synaptogenesis and the absence of gap junctions in the presynaptic terminal. However, the expression of UAS-fraC postsynaptically did not disrupt function, but did disrupt the morphology of the postsynaptic neuron. Postsynaptic expression of UAS-fraC disrupted dendritic maturation, resulting in medial dendrite pruning defects and lateral dendrite extension defects. The fraC experiments are interpreted as providing some evidence for Frazzled's cell autonomous role in building this giant synapse. More direct evidence would require rescue experiments. Unfortunately, the relevant genes are located very close to one another, making it difficult to obtain the appropriate recombination event. Future experiments will use recently acquired GAL4 drivers on the third chromosome to clarify this issue. The Frazzled RNAi experiments were uninformative, possibly because RNAi is not a strong enough disruption of frazzled to cause effects in the GFS. In brief, the cell autonomous function of Frazzled warrants further investigation (Orr, 2014).

When UAS-fraC was expressed in the embryo in the Netrin LOF background, it revealed that the disruption of commissures was Netrin dependent. An interaction experiment (NetAΔBΔ/+; A307/+; UAS-fraC/+) revealed a different mechanism by which the dominant-negative fraC obstructed synaptogenesis. In a heterozygous Netrin LOF background, the mutant version of Frazzled was expressed, further knocking down Netrin-Frazzled signaling to disrupt synaptogenesis. The results suggested that fraC was acting as a Netrin sink by binding to secreted Netrin, limiting the amount of Netrin that could bind to wild-type Frazzled receptors (Orr, 2014).

The chemical synaptic component of the GF-TTMn synapse was observed in the Net LOF mutants using antibodies against the presynaptic density protein Bruchpilot (T-bars) with anti-NC82 staining. However, the Bruchpilot labeling was not informative. No further effort was made because the cholinergic component has no effect on synaptic circuit function in the adult (Orr, 2014).

In contrast to the GF-TTMn synapse, the GF-PSI synapse is unaffected by the absence of Netrin, Frazzled, or the expression of the dominant-negative Frazzled dominant-negative. This shows that the GF-TTMn synapse specifically is dependent on Netrin-Frazzled signaling for function. This mechanism for gap junction insertion is so specific that neighboring electrical synapses that share the same presynaptic terminal (GF) use different mechanisms for gap junction localization (Orr, 2014).

Netrin is secreted from two known sources, the midline glia and the postsynaptic target TTMn. A model is presented for Netrin localization and function in which Netrin is captured on the surface of one neuron (TTMn) by Frazzled and is then presented to Frazzled receptors on another neuron (GF) to transmit signaling. During development, the TTMn extends its medial dendrite toward a source of Netrin, the midline glia. After the TTMn dendrite has grown into the synaptic area by 9% of PD, both the midline glia and TTMn are labeled with Netrin. It is hypothesized that this is important in the induction of synaptic maturation of this synapse (Orr, 2014).

Rescuing Netrin LOF mutants by expressing a secreted form of Netrin specifically in either TTMn or midline glia supports a model that Netrin is presented to the GF to promote synapse formation. The secreted Netrin rescue experiments were effective because Netrin could localize where it would normally as long as it was secreted by a nearby endogenous source. This could explain why it was possible to rescue the Netrin LOF mutants in a non-cell-autonomous fashion by expressing secreted Netrin in either midline glia or the TTMn independently. Postsynaptic expression of the Frazzled dominant-negative also supports the presentation model. When two copies of Frazzled lacking its intracellular domain were expressed on the TTMn, Netrin could bind to the mutant Frazzled, be presented to the GF, and support normal synaptic function regardless of disrupted intracellular signaling in the TTMn by the deletion of the intracellular domain (Orr, 2014).

In contrast, expressing membrane-tethered UAS-NetBCD8-TM on either the midline glia or TTMn failed to rescue function of the circuit because localization and secretion of Netrin was disrupted. When attempts were made to rescue the Netrin LOF mutants by expressing membrane-tethered NetrinB postsynaptically, the defects were enhanced and the medial dendrite did not extend to the midline in 90% of specimens. However, in the tethered NetB mutant, tethered NetrinB was expressed in both of its endogenous sources, midline glia and TTMn, and the synapse functioned normally. While being expressed under its endogenous promoter, tethered Netrin supported normal synaptogenesis. It is possible that, through the endogenous expression pattern, cells not identified in this study could contribute to the normal phenotype seen in the mutants in a nonlocal manner. However, it is hypothesized that the tethered NetrinB mutant does not behave in a predictable way. It is suggested that this protein is not as tightly membrane bound as the UAS-NetBCD8-TM protein product due to the added extracellular myc domains in the tethered mutant. The tethered mutant's additional myc domains may account for differences in phenotypes due to increased protein flexibility or possible cleavage and secretion from the cell of origin. Considering this, non-cell-autonomous expression of a secreted Netrin rescued Netrin LOF defects, whereas expression of the tethered version using the same GAL4 drivers could not rescue the defects. This is evidence for the importance of Netrin secretion in GFS synaptogenesis (Orr, 2014).



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)

Localized netrins act as positional cues to control layer-specific targeting of photoreceptor axons in Drosophila

A shared feature of many neural circuits is their organization into synaptic layers. However, the mechanisms that direct neurites to distinct layers remain poorly understood. This study identified a central role for Netrins and their receptor Frazzled in mediating layer-specific axon targeting in the Drosophila visual system. Frazzled is expressed and cell autonomously required in R8 photoreceptors for directing their axons to the medulla-neuropil layer M3. Netrin-B is specifically localized in this layer owing to axonal release by lamina neurons L3 and capture by target neuron-associated Frazzled. Ligand expression in L3 is sufficient to rescue R8 axon-targeting defects of Netrin mutants. R8 axons target normally despite replacement of diffusible Netrin-B by membrane-tethered ligands. Finally, Netrin localization is instructive because expression in ectopic layers can retarget R8 axons. It is proposed that provision of localized chemoattractants by intermediate target neurons represents a highly precise strategy to direct axons to a positionally defined layer (Timofeev, 2012).

Recent studies identified at least four molecular mechanisms that control layer-specific targeting in the nervous system by cell-cell interactions independently of neural activity. First, combinatorial expression of homophilic cell surface molecules promotes the recognition and stabilization of contacts between matching branches of pre- and postsynaptic neuron subsets. For instance, four members of the immunoglobulin superfamily of cell adhesion molecules, Sidekick 1 and 2 and Dscam and DscamL, are expressed and required in subsets of bipolar, amacrine, and retinal ganglion cells for targeting to different inner plexiform sublayers (IPLs) in the chick retina. In Drosophila, the leucine-rich repeat protein Caps may play an analogous role, as it is specifically expressed in R8 cells and target layers M1-M4 and, thus, could promote homophilic interactions to stabilize connections within correct columns and layers. Second, concise temporal transcriptional control is used to regulate the levels of ubiquitous cell surface molecules and, thus, adhesiveness of afferent and target neurons to balance branch growth and targeting. This mechanism is supported by findings in the fly visual system where the transcription factor Sequoia controls R8 and R7 axon targeting by the temporal regulation of N-Cadherin (CadN) expression levels. Third, repellent guidance cues are utilized to exclude projections from some layers, as has been shown for membrane-bound Semaphorin family members and Plexin receptors in the IPL of the mouse retina. Fourth, recent studies also implicated the graded expression of extracellular matrix-bound guidance cues such as Slit in the organization of layered connections in the zebrafish tectum. The current findings for the essential role of Netrins and Fra in visual circuit assembly provide evidence for a different strategy: a localized chemoattractant guidance cue is used to single out one layer, thus providing precise positional information required for layer-specific axon targeting of cell types expressing the receptor. Unlike in the ventral nerve cord, where the Netrin/Fra guidance system controls growth across the midline, in the visual system, it mediates target recognition by promoting axon growth into but not past the Netrin-positive layer (Timofeev, 2012).

Rescue experiments support the model that Netrins are primarily provided by the axon terminals of lamina neurons L3 in the M3 layer. During early pupal stages, Fra-positive R8 axons pause in their temporary layer at the distal medulla neuropil border. From midpupal development onward, upon release from this block, Fra-positive R8 axons are guided to the Netrin-expressing M3 layer (Timofeev, 2012).

Axons can use intermediate target cells either along their trajectory to guide them toward their target areas or within the target area to bring putative synaptic partners into close vicinity. Although R8 axons and lamina neurons L3 terminate closely adjacent to each other in the same layer, they have been described to not form synaptic connections with each other but to share common postsynaptic partners such as the transmedullary neuron Tm9. Thus, the results suggest that layer-specific targeting of R8 axons relies on the organizing role of lamina neurons L3 as intermediate targets in the M3 layer rather than direct interactions with postsynaptic partners. Consistent with this notion, axons of lamina neurons L3 timely extend between the temporary layers of R8 and R7 axons from early pupal stages onward, and targeting of their axons is independently controlled by other cell surface molecules such as CadN. Further studies will need to identify potential Fra-positive synaptic partners in the medulla and test whether this guidance receptor equally controls targeting of their dendritic branches, thus bringing pre- and postsynaptic neurites into the same layer. Additional mechanisms likely mediate cell-cell recognition and synaptic specificity, as electron microscopic analysis showed that presynaptic sites in R8 axons are not restricted to the M3 layer but distributed along the axon (Timofeev, 2012).

Netrins are diffusible guidance cues acting both at long range in a gradient and at short range when immobilized. Consistent with studies in the Drosophila embryo, it was observed in this study that NetB in the visual system acts at short range, as R8 axon targeting is normal when solely membrane-tethered NetB is available at near-endogenous levels. Secreted Netrins are converted into a short-range signal because they are locally released by lamina neurons L3 and prevented to diffuse away through a Fra-mediated capturing mechanism. Filopodial extensions could enable R8 growth cones to bridge the distance to NetB-expressing lamina neuron L3 axon terminals (Timofeev, 2012).

Although in principle Netrins could be secreted by both dendritic and axonal arbors of complex neurons, the results support the notion that axon terminals are the primary release sites to achieve layer-specific expression. This may be mediated by a cargo transport machinery along polarized microtubules similar to that used by synaptic proteins or neurotransmitters. Consistently, recent findings in C. elegans identified proteins involved in motor cargo assembly and axonal transport as essential for Netrin localization and secretion. Intermediate target neurons may thus constitute an important strategy to draw afferent axons into a layer, if guidance cues are preferentially released by axon terminals and not by dendritic branches of synaptic partner neurons. Netrin-releasing lamina neurons L3 form dendritic spines in the lamina and axon terminals in the medulla. Similarly, Netrin-positive transmedullary neuron subtypes such as Tm3 and Tm20 form dendritic branches in the medulla and extend axons into the lobula. Thus, a mechanism, whereby neurons in one brain area organize the connectivity in the next, may be used at least twice in the visual system (Timofeev, 2012).

Knockdown of fra in the target area strongly reduced NetB in the M3 layer, supporting the notion that a receptor-mediated capturing mechanism controls layer-specific Netrin accumulation. Despite the use of multiple genetic approaches, no R8 axon-targeting errors were observed when manipulating Fra levels exclusively in target . This could be attributed to the technical limitation that knockdown is incomplete owing to the activity of the ey enhancer in around 50% of medulla neurons. However, as lamina neurons L3 continue to locally release Netrins, remaining ligands may likely be sufficient to guide fully responsive R8 axons to their target layer (Timofeev, 2012).

Unlike in the fly embryonic CNS, where Netrins are captured by Fra and presented to growth cones expressing a Netrin receptor other than Fra, or in C. elegans, where Unc-6 is captured at the dendrite tips of nociceptive neurons by Unc-40 to interact with Unc-5 (Smith, 2012), genetic analyses indicate that fra is required in R8 axons. Hence, Netrins captured by Fra-positive target neurons may either be presented to Fra-expressing R8 axons in a dynamic fashion, or R cell- and target neuron-derived Fra interact with Netrins in a ternary complex in trans. This is conceivable since (1) the vertebrate counterpart Netrin-1 shows a high binding affinity for DCC; (2) DCC can bind Netrins with multiple domains (DCC, fourth and fifth fibronectin type III domains; Netrins, Laminin N-terminal (LamNT) and three Laminin-type epidermal growth factor [EGF]-like domains); and (3) at least in cis, Netrins can bind and aggregate multiple DCC ectodomain molecules. Ligand capture and presentation by receptors have also been reported for F-spondin and lipoprotein receptor-related protein (LRP) at the vertebrate floor plate. Netrins have previously been shown to promote exocytosis and recruitment of their receptor to distinct subcellular locations on cell surfaces. Moreover, in the visual system, Netrins may increasingly draw neurites of Fra-positive target neurons into layer M3, which in turn could promote further ligand accumulation. Thus, additional feedback loops may contribute to the specific enrichment of both Netrins and Fra in the M3 layer (Timofeev, 2012).

R8 axon targeting involves multiple successive steps: (1) the selection of the retinotopically correct column; (2) pausing in the temporary layer; (3) timely release from the temporary layer and extension of a filopodium; (4) bypassing of incorrect neuropil layers; (5) correct identification and targeting to the M3 layer; (6) stabilization of connections in the correct layer and column and transformation of growth cones into mature terminals; and (7) formation of the correct repertoire of synaptic contacts. Strong early defects would likely impact on subsequent steps (Timofeev, 2012).

Within this sequence of events, interactions of Golden goal (Gogo) and Flamingo (Fmi) in cis within R8 axons and in trans with Fmi-positive neuronal processes in the emerging M1, M2, and lower M3 layers have been shown to contribute to the timely release of R8 growth cones from their temporary layer and, consequently, enable correct targeting to the M3 layer (steps 3 and 6). Caps may specifically promote cell-cell recognition and stabilize interactions between R8 axons and target neuron branches within their correct column and target layer (step 6). However, an alteration of adhesiveness may not be sufficient to promote the extension of filopodia toward the correct layer, and additional attractive guidance forces are required. The Netrin/Fra guidance system is well suited to play such a role by providing the necessary positive forces directing filopodia toward deeper layers and by promoting recognition of a single layer at a given position (steps 4 and 5). This notion is supported by observations that loss of Fra or Netrins causes many R8 axons to stall at the distal medulla neuropil border and to terminate at interim positions in layers M1/M2. Furthermore, ectopic expression of membrane-tethered NetB is sufficient to retarget a significant proportion of R8 axons. Unlike Caps and Gogo/Fmi, whose ectopic expression can promote targeting of some R7 axons to the M3 layer, Fra was not sufficient to redirect R7 axons from the M6 to the M3 layer. A likely explanation is that the effects of R7-specific guidance determinants cannot be overwritten, or essential cooperating receptors or downstream components of Fra present in R8 are missing in R7 cells. Furthermore, overexpression of Fra causes many R8 axons to stall at the medulla neuropil border, suggesting that tight temporal regulation of receptor levels in R8 axons is essential for the integration of an additional potential repellent input (Timofeev, 2012).

Together, these findings in the Drosophila visual system suggest that the dynamic coordinated actions of chemotropic guidance cues and cell adhesion molecules contribute to layer-specific targeting of specific cell types. A similar molecular mechanism relying on Netrins or other localized attractive guidance cues and their receptors may be more widely used for the assembly of laminated circuits (Timofeev, 2012).

Netrin-dependent downregulation of Frazzled/DCC is required for the dissociation of the peripodial epithelium in Drosophila

Netrins are secreted chemoattractants with roles in axon guidance, cell migration and epithelial plasticity. Netrin-1 also promotes the survival of metastasized cells by inhibiting the pro-apoptotic effects of its receptor Deleted in Colorectal Carcinoma (DCC). This study reports that Netrins can also regulate epithelial dissociation during Drosophila wing eversion. During eversion, peripodial epithelial cells lose apico-basal polarity and adherens junctions, and become migratory and invasive -- a process similar to an epithelial-mesenchymal transition. Loss of netrinA inhibits the breakdown of cell-cell junctions, leading to eversion failure. In contrast, the Netrin receptor Frazzled blocks eversion when overexpressed, whereas frazzled RNAi accelerates eversion in vitro. In peripodial cells Frazzled is endocytosed, and undergoes NetA-dependent degradation, which is required for eversion. Finally, evidence is provided that Frazzled acts through the ERM-family protein Moesin to inhibit eversion. This mechanism may also help explain the role of Netrin and DCC in cancer metastasis (Manhire-Heath, 2013).

The results delineate a novel regulatory mechanism controlling wing disc eversion in which a Fra-Moe pathway required for maintenance of epithelial adhesion junctions is inhibited by Netrin-dependent degradation of the receptor. The Netrin/Fra pathway appears to act in parallel to the JNK pathway, as loss of netA did not prevent JNK activation and loss of 1NK activation did not affect Fra or NetA levels. Given the intermediate penetrance of netA-IR phenotypes, the reduction of Fra must be only one of several redundant mechanisms required for eversion. The idea that Fra may act as an epithelial maintenance factor is supported by recent findings in which loss of netrins or fra causes defects in the formation of the embryonic midgut epithelium. In vertebrates, DCC expression in epithelia has been reported for a variety of tissues such as the skin, gut, lung and bladder, and DCC has been shown to increase cell-cell adhesion in both HT-29 cells and fibroblasts. Given the punctate distribution of Fra in netA-IR discs, its ability to stabilize the ZA is presumably not through some structural or adhesive role at the ZA but rather via signalling from endosomes. Elucidating the molecular pathway linking Fra, Moe and ZA maintenance, and understanding how that pathway interacts with molecular processes acting downstream of JNK activation (such as MMP breakdown of the basement membrane) are important future goals (Manhire-Heath, 2013).

The opposing roles for NetA and Fra demonstrated here correlate with previous descriptions of Netrin-1 as an oncogene and DCC as a tumour suppressor. Netrin-1 levels are elevated in metastatic breast cancers and strongly overexpressed in human pancreatic cancer. Further, overexpression of Netrin-1 is associated with tumour formation and progression in mice, whereas in mammary metastatic tumour cell lines, metastatic progression was blocked when Netrin-1 expression was decreased. Although current research focuses largely on the role of DCC as a dependence receptor the effects of increased Netrin-1 or decreased DCC expression on cells at early stages of metastasis are unclear. The findings from this study raise the possibility that Netrins may not only promote cell migration and the subsequent survival of metastasized cells but also influence the initial loss of the epithelial state (Manhire-Heath, 2013).

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. ftzng-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 ftzng-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 ftzng-GAL4 line. Similar results were obtained with eveng-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 ftzng-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 ftzng-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/+;ftzng-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;+/+;ftzng-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).

Midline signalling systems direct the formation of a neural map by dendritic targeting in the Drosophila motor system

A fundamental strategy for organising connections in the nervous system is the formation of neural maps. Map formation has been most intensively studied in sensory systems where the central arrangement of axon terminals reflects the distribution of sensory neuron cell bodies in the periphery or the sensory modality. This straightforward link between anatomy and function has facilitated tremendous progress in identifying cellular and molecular mechanisms that underpin map development. Much less is known about the way in which networks that underlie locomotion are organised. In the Drosophila embryo, dendrites of motorneurons form a neural map, being arranged topographically in the antero-posterior axis to represent the distribution of their target muscles in the periphery. However, the way in which a dendritic myotopic map forms has not been resolved and whether postsynaptic dendrites are involved in establishing sets of connections has been relatively little explored. This study shows that motorneurons also form a myotopic map in a second neuropile axis, with respect to the ventral midline, and they achieve this by targeting their dendrites to distinct medio-lateral territories. This map is 'hard-wired'; that is, it forms in the absence of excitatory synaptic inputs or when presynaptic terminals have been displaced. The midline signalling systems Slit/Robo and Netrin/Frazzled are the main molecular mechanisms that underlie dendritic targeting with respect to the midline. Robo and Frazzled are required cell-autonomously in motorneurons and the balance of their opposite actions determines the dendritic target territory. A quantitative analysis shows that dendritic morphology emerges as guidance cue receptors determine the distribution of the available dendrites, whose total length and branching frequency are specified by other cell intrinsic programmes. These results suggest that the formation of dendritic myotopic maps in response to midline guidance cues may be a conserved strategy for organising connections in motor systems. It is further proposed that sets of connections may be specified, at least to a degree, by global patterning systems that deliver pre- and postsynaptic partner terminals to common 'meeting regions' (Mauss, 2009).

How different dendritic morphologies and territories are generated in a motor system was investigated using the neuromuscular system of the Drosophila embryo as a model. Its principal components are segmentally repeated arrays of body wall muscles (30 per abdominal half segment), each innervated by a specific motorneuron. The motorneuron dendrites are the substrate on which connections with presynaptic cholinergic interneurons form. 180 cells (on average 11.25 for each identified motorneuron and a minimum of five) were labelled, and the dendritic morphologies and territories of the motorneurons that innervate the internal muscles were charted using retrograde labelling with the lipophilic tracer dyes 'DiI'and 'DiD.' This was done in the context of independent landmarks, a set of Fasciclin 2-positive axon bundles, at 18.5 h after egg laying (AEL), when the motor system first becomes robustly functional and the geometry of motorneuron dendritic trees has become sufficiently invariant to permit quantitative comparisons (Mauss, 2009).

Three classes of motorneurons were found based on dendritic arbor morphology and territory with respect to the ventral midline: (1) motorneurons with dendrites in the lateral neuropile (between the lateral and intermediate Fasciclin 2 tracts); (2) in the lateral and intermediate neuropile (between the intermediate and medial Fasciclin 2 tracts), and (3) in the lateral, intermediate plus medial neuropile (posterior commissure) (Mauss, 2009).

Moreover, the medio-lateral positions of motorneuron dendrites correlate with the dorsal to ventral locations of their target muscles in the periphery. Motorneurons with dorsal targets (DA1, DA3, DO1-5) have their dendrites in the lateral neuropile, while those innervating ventral and lateral muscles (LL1, VL2-4, VO1-2) also have dendrites in the intermediate neuropile. Coverage of the medial neuropile is particular to motorneurons innervating the most ventral group of muscles (VO3-6). These dendritic domains are arranged in the medio-lateral axis of the neuropile in such a way that they form a neural, myotopic representation of the distribution of body wall muscles in the periphery. Only a single motorneuron deviates from this clear-cut correlation between dendritic medio-lateral position and target muscle location: MN-DA2 has dendrites not only in the lateral neuropile, like other motorneurons with dorsal targets, but also in the intermediate neuropile (Mauss, 2009).

Previously studies have shown that motorneurons in the Drosophila embryo distribute their dendrites in distinct anterior to posterior domains in the neuropile, forming a central representation of target muscle positions in the periphery. The mechanisms required for the generation of this dendritic myotopic map remain elusive. In this study, dendritic myotopic organisation was characterized in a second dimension, with respect to the ventral midline, and the main molecular mechanism that underlies the formation of this dendritic neural map were identified, namely the combinatorial action of the midline signalling systems Slit/Robo and Netrin/Frazzled (Mauss, 2009).

Neural maps are manifestations of an organisational strategy commonly used by nervous systems to order synaptic connections. The view of these maps has been largely axonocentric and focused on sensory systems, though recent studies have challenged the notion of dendrites as a 'passive' party in arranging the distribution of connections. This study has demonstrated that motorneuron dendrites generate a neural, myotopic map in a motor system and that this manifest regularity can form independently of its presynaptic partner terminals (Mauss, 2009).

An essential feature of neural maps is the spatial segregation of synaptic connections. In the Drosophila embryonic nerve cord, there is some overlap between dendritic domains in the antero-posterior neuropile axis. Overlap of dendritic territories is also evident in the medio-lateral dimension, since all motorneurons have arborisations in the lateral neuropile, though distinctions arise by virtue of dendrites in additional intermediate and medial neuropile regions. The combination of myotopic mapping in both dimensions may serve to maximise the segregation between dendrites of different motorneuron groups. For example, the dendritic domain of motorneurons with dorsal targets differs from the territory innervated by ventrally projecting motorneurons in the antero-posterior location and the medio-lateral extent. Myotopic mapping in two dimensions could also provide a degree of flexibility that could facilitate wiring up in a combinatorial fashion. For instance, muscle LL1 lies at the interface between the dorsal and ventral muscle field; its motorneuron, MN-LL1, has one part of its dendritic arbor in the lateral domain that is characteristic for dorsally projecting motorneurons, while the other part of the dendritic tree innervates the intermediate neuropile precisely where ventrally projecting motorneurons put their dendrites (Mauss, 2009).

Myotopic dendritic maps might constitute a general organisational principle in motor systems. In insects, a comparable system of organisation has now been demonstrated also for the adult motor system of Drosophila (Brierley, 2009; Baek, 2009) and a degree of topographic organisation had previously been suggested for the dendrites of motorneurons that innervate the body wall muscles in the moth Manduca sexta. In vertebrates too, there is evidence that different motor pools elaborate their dendrites in distinct regions of the spinal cord in chick, turtle, and mouse. Moreover, elegant work in the mouse has shown that differences in dendritic territories correlate with and may determine the specificity of proprioceptive afferent inputs (Mauss, 2009 and references therein).

The neural map characterised in this study is composed of three morphological classes of motorneurons with dendrites innervating either (1) the lateral or (2) the lateral and intermediate or (3) the lateral, intermediate, and medial/midline neuropile (Mauss, 2009).

The motorneuron dendrites are targeted to these medio-lateral territories by the combinatorial, cell-autonomous actions of the midline guidance cue receptors Robo and Frazzled. The formation of dendritic territories by directed, targeted growth appears to be an important mechanism that may be more widespread than previously anticipated, though the underlying mechanisms may vary. Global patterning cues have been implicated in the vertebrate cortex (Sema3A). In the zebrafish retina, live imaging has shown that retinal ganglion cells put their dendrites into specific strata of the inner plexiform layer, but the roles of guidance cues and interactions with partner (amacrine) cells have not yet been studied (Mauss, 2009).

Slit/Robo and Netrin/Frazzled mediated gating of dendritic midline crossing has been previously documented in Drosophila embryos and zebrafish. This study demonstrated that dendrites are targeted to distinct medio-lateral territories by the combinatorial, opposing actions of Robo and Frazzled and that this is the main mechanism underlying the formation of the myotopic map. Strikingly, the same signalling pathways also regulate dendritic targeting of adult motorneurons in Drosophila, suggesting this to be a conserved mechanism (Brierley, 2009). Robo gates midline crossing of dendrites and in addition, at progressively higher signalling levels, restricts dendritic targeting to intermediate and lateral territories. Frazzled, on the other hand, is required for targeting dendrites towards the midline into intermediate and medial territories. The data argue that Frazzled is expressed by representatives of all three motorneuron types. Recently, Yang (2009) has shown that expression of frazzled leads to a concomitant transcriptional up-regulation of comm, thus linking Frazzled-mediated attraction to the midline with a decrease in Robo-mediated repulsion. While this has been demonstrated for midline crossing of axons in the Drosophila embryo, this study found that, at least until 18.5 h AEL, expression of UAS-frazzled alone was not sufficient to induce midline crossing of dendrites in MN-LL1 and MN-DA3. It is conceivable that differences in expression levels and/or timing between CQ-GAL4 used in this study and egl-GAL4 used by Yang might account for the differences in axonal and dendritic responses to UAS-frazzled expression. Moreover, the widespread expression of Frazzled in motorneurons and other cells in the CNS may point to additional functions, potentially synaptogenesis, as has been shown in C. elegans (Mauss, 2009).

Strikingly, neither synaptic excitatory activity nor the presynaptic (cholinergic) partner terminals seem to be necessary for the formation of the map. The map is already evident by 15 h AEL, before motorneurons receive synaptic inputs. It also forms in the absence of acetylcholine, the main (and at that stage probably exclusive) neurotransmitter to which motorneurons respond. Moreover, motorneuron dendrites innervate their characteristic dendritic domains when the cholinergic terminals have been displaced to outside the motor neuropile. However, interactions with presynaptic partners seem to contribute to its refinement. First, it was found that dendritic mistargeting phenotypes show a greater degree of penetrance earlier (15 h AEL) than later (18.5 h AEL) in development. Secondly, when interactions with presynaptic partner terminals are reduced or absent, dendritic arbor size increases and the distinction between dendritic territories is less evident than in controls. Fine-tuning of terminal arbors and sets of connections through contact and activity-dependent mechanisms is a well-established feature of neural maps in sensory systems and the current observations suggest that this may also apply to motor systems (Mauss, 2009).

The formation of the myotopic map is the product of dendritic targeting. It is therefore intimately linked with the question of how cell type-specific dendritic morphologies are specified. For instance, changing the balance between the Robo and Frazzled guidance receptors in motorneurons is sufficient to 'convert' dendritic morphologies from one type to another. The importance of target territories for determining dendritic arbor morphology has recently been explored in a study of lobula plate tangential cells in the blowfly, where the distinguishing parameter between the dendritic trees of four functionally defined neurons were not growth or branching characteristics but the regions where neurons put their dendrites (Mauss, 2009).

Because Slit/Robo and Netrin/Frazzled signalling have been reported to affect dendritic and axonal branching as well as axonal growth, respectively, it was asked what the effect was on motorneuron dendrites of altered Robo and Frazzled levels. It was found that in the wild-type different motorneurons generate characteristically different amounts of dendritic length and numbers of branch points (MN-DA1/aCC and MN-VO2/RP1, RP2, MN-DA3 and MN-LL1). In the Drosophila embryo and larva, Slit/Robo interactions have been suggested to promote the formation of dendrites and/or branching events, similar to what has been shown for cultured vertebrate neurons. The current data on embryonic motorneurons are not compatible with this interpretation. First, when altering the levels of Robo (or Frazzled) in individual motorneurons and mistargeting their dendrites, no statistically significant changes were detected in total dendritic length or number of branch points. Instead, for MN-DA3 and MN-LL1, it was observed that dendritic arbors respond to changes in the expression levels of midline cue receptors by altering the amount of dendritic length distributed to the medial, intermediate, and lateral neuropile. Secondly, in nerve cords entirely mutant for the Slit receptor Robo an increase is seen in dendrite branching at the midline. These observations suggest that for Drosophila motorneurons Slit/Robo interactions negatively regulate the establishment and branching of dendrites and thus specify dendritic target territories by defining 'exclusion' zones in the neuropile. The quantitative data from this study suggest that dendritic morphology is the product of two intrinsic, genetically separable programmes: one that specifies the total dendritic length to be generated and the frequency of branching; the other implements the distribution of these dendrites in the target territory, presumably by locally modulating rates of extension, stabilisation, and retraction of branches in response to extrinsic signals (Mauss, 2009).

The question of how neural circuits are generated remains at the heart of developmental neurobiology. At one extreme, one could envisage that every synapse was genetically specified, the product of an exquisitely choreographed sequence of cell-cell interactions. At the other extreme, neural networks might assemble through random cell-cell interactions and feedback processes enabling functional validation. The latter view supposes that neurons inherently generate polarised processes, have a high propensity to form synapses, and arrive at a favourable activity state through homeostatic mechanisms. Current evidence suggests that, at least for most systems, circuits form by a combination of genetic specification and the capacity to self-organise (Mauss, 2009).

This study has demonstrated that the postsynaptic structures of motorneurons, the dendrites, form a neural map. It was also shown that dendrites are closely apposed to cholinergic presynaptic specialisations in their target territories, suggesting that the segregation of dendrites may be a mechanism that facilitates the formation of specific sets of connections. Strikingly, this map of postsynaptic dendrites appears to be 'hard-wired' in that it can form independently of its presynaptic partners and it is generated in response to a third party, the midline guidance cues Slit and Netrin. A comparable example is the Drosophila antennal lobe, where projection neurons form a neural map independently of their presynaptic olfactory receptor neurons, though in this sensory system the nature and source of the cue(s) remain to be determined. This study complements previous work that demonstrated the positioning of presynaptic axon terminals by midline cues, also independently of their synaptic partners. Together, these results suggest that global patterning cues set up the functional architecture of the nervous system by independently directing pre- and postsynaptic partner terminals towards common 'meeting' areas (Mauss, 2009).

Clearly, such global guidance systems deliver a relatively coarse level of specificity and there is ample evidence for the existence of codes of cell-adhesion molecules and local receptor-ligand interactions capable of conferring a high degree of synaptic specificity. Therefore, one has to ask what the contribution is of global partitioning systems in establishing patterns of connections that lead to a functional neural network. A recent study in the Xenopus tadpole spinal cord has addressed this issue. Conducting patch clamp recordings from pairs of neurons, it has been found that the actual pattern of connections in the motor circuit reveals a remarkable lack of specificity. Furthermore, the segregation of axons and dendrites into a few broad domains appears to be sufficient to generate the connections that do form and to enable the emergence of a functional network. The implication is that neurons might be intrinsically promiscuous and that targeting nerve terminals to distinct territories by global patterning cues, as has been shown in this study, is important to restrict this synaptogenic potential and thereby confer a degree of specificity that is necessary for the emergence of network function (Mauss, 2009).

Dendritic targeting in the leg neuropil of Drosophila: the role of midline signalling molecules in generating a myotopic map

Neural maps are emergent, highly ordered structures that are essential for organizing and presenting synaptic information. Within the embryonic nervous system of Drosophila motoneuron dendrites are organized topographically as a myotopic map that reflects their pattern of innervation in the muscle field. This fundamental organizational principle exists in adult Drosophila, where the dendrites of leg motoneurons also generate a myotopic map. A single postembryonic neuroblast sequentially generates different leg motoneuron subtypes, starting with those innervating proximal targets and medial neuropil regions and producing progeny that innervate distal muscle targets and lateral neuropil later in the lineage. Thus the cellular distinctions in peripheral targets and central dendritic domains, which make up the myotopic map, are linked to the birth-order of these motoneurons. Developmental analysis of dendrite growth reveals that this myotopic map is generated by targeting. The medio-lateral positioning of motoneuron dendrites in the leg neuropil is controlled by the midline signalling systems Slit-Robo and Netrin-Fra. These results reveal that dendritic targeting plays a major role in the formation of myotopic maps and suggests that the coordinate spatial control of both pre- and postsynaptic elements by global neuropilar signals may be an important mechanism for establishing the specificity of synaptic connections (Brierley, 2009).

Neural maps are emergent, highly ordered structures that are essential for organizing and presenting synaptic information. The architecture of dendrites and the role they play in establishing connectivity within maps has been somewhat overlooked. Classic cell-labelling studies in the moth Manduca sexta revealed that the dendrites of motoneurons are topographically organized to reflect their site of innervation in the bodywall. More recent work by Landgraf and colleagues has demonstrated that motoneurons in Drosophila embryos generate a detailed dendritic (myotopic) map of body wall muscles within the CNS. Alongside these data, studies on the architecture of the spinal cord also suggest that similar design principles may play a role in organizing information in vertebrate motor systems. How such dendritic maps are built is still largely unknown. This study describes the role dendritic targeting plays in constructing a myotopic map and the molecular mechanisms that control it (Brierley, 2009).

The majority of leg motoneurons in a fly are born postembryonically and most of those are derived from a single neuroblast lineage, termed lineage 15. Perhaps the most striking feature of this lineage is its birth-order-based pattern of innervation along the proximo-distal axis of the leg. Using mosaic analysis, the sequential production was observed of four neuronal subtypes during larval life, each elaborating stereotyped axonal and dendritic projections in the adult. The axon of the first-born neuron innervates a muscle in the body wall and subsequent neurons innervate more distal targets in the leg. This organization has also been reported by Baek (2009) (Brierley, 2009).

This birth-order-based peripheral pattern of lineage 15 is mirrored in the CNS, where dendrites generate a stereotyped anatomical organization. Dendrites of early-born cells span medial to lateral territories, whereas late-born cells elaborate dendrites in the lateral neuropil and cells born between these times occupy intermediate territories. The sequential production of neuronal subtypes by neural precursor cells is a common mechanism for generating a diversity of circuit components. A similar birth-order-based specification of axonal and dendritic projection patterns has previously been described for projection neurons in the fly's olfactory system (Brierley, 2009).

The data reveal the existence of a myotopic map in the adult fly and supports the proposition that dendritic maps are a common organizing principle of all motor systems. Mauss (2009) also reveal a map in the embryonic CNS of Drosophila, where the dendrites of motoneurons are organized along the medio-lateral axis of the neuropil representing dorsoventral patterns of innervation in the body wall muscles (Brierley, 2009).

How are dendritic maps built? The myotopic map seen in the leg neuropil could be generated by two distinctly different mechanisms. Neurons could elaborate their dendrites profusely across a wide field and then remove branches from inappropriate regions or, alternatively, they could target the growth of dendrites into a distinct region of neuropil throughout development. Both mechanisms can generate cell-type-specific projection patterns as seen in the vertebrate retina. To reveal which mechanism is deployed in the leg motor system of Drosophila, single-cell clones of motoneuron subtypes generated by heatshocks at 48 and 96 h AH were imaged, since their final dendritic arborizations cover clearly distinct territories within the map. The dendrites of both elaborate branches only in territories where the mature arborizations eventually reside, which strongly supports the notion that this myotopic map is generated by targeting and not large-scale branch elimination. Importantly, this developmental timeline also revealed that the motoneurons elaborate their dendrites synchronously, regardless of the birth date of the cell. This observation suggests that a 'space-filling/occupancy based' model, where later-born neurons are excluded from medial territories by competitive interactions is unlikely. Similarly, heterochronic mechanisms where different members of the lineage experience different signalling landscapes due to differences in the timing of outgrowth are not likely either. With synchronous outgrowth dendrites experience the same set of extracellular signals, suggesting that the intrinsic properties of cells, defined by their birth order, may be more important for the generation of subtype-specific projections. Such intrinsic properties could include cell-cell recognition systems such as adhesion molecules, e.g., Dscams or classical guidance receptors, that could interpret extracellular signals. In the Drosophila embryo motoneurons also use dendritic targeting to generate a myotopic map (Brierley, 2009).

It is emerging that dendrites are guided by the same molecules that control axon pathfinding. The medio-lateral organization of leg motoneuron dendrites within the leg neuropil prompted an investigation as to whether the midline signalling molecules Slit and Netrin and their respective receptors Roundabout and Frazzled could be involved in targeting growth to specific territories (Brierley, 2009).

Using mosaic analysis it was found that both the 48 and 96 h AH motoneuron subtypes require Robo to generate their appropriate shape and position within the medio-lateral axis. When Robo was removed from the 48 h AH subtype the mean centre of arbor mass was shifted toward the midline. The dendrites of 96 h AH neurons showed a shift in distribution in the absence of Robo but still failed to reach the midline, suggesting that only part of this cell's targeting is due to repulsive cues mediated by the Robo receptor. It was predicted that if Robo levels played an instructive role in dendrite targeting it would be possible to shift dendrites laterally by cell autonomously increasing Robo. This was found to be the case in both subtypes. Taken together these data suggest that differences in the level of Robo signalling may provide a mechanism by which Slit could be differentially interpreted to allow subtype-specific targeting along the medio-lateral axis (Brierley, 2009).

The Robo receptor is part of a larger family of receptors that includes Robo2 and Robo3. This family of receptors have been found to be important for targeting axons to the appropriate longitudinal pathway in the embryonic CNS. Comm plays a key role in allowing contralaterally projecting neurons to cross the midline, and its ectopic expression (CommGOF) is known to robustly knock down Robo and Robo2 and 3. Comm was cell autonomously expressed in both lineage 15 subtypes and shifts to the midline were found in both 48 and 96 h AH neurons. For the 48 h AH neurons, Robo LOF data and CommGOF data are comparable, suggesting that Robo alone plays a major role in the positioning dendrites of these cells. In contrast, in the 96 h AH subtype RoboLOF and CommGOF effects were found to be significantly different, suggesting that the 96 h AH subtype may not only use the Robo receptor but additional Robos as well. Knockdown of Slit also supports this idea, as the branches of late-born neurons were occasionally found reaching the midline, something that was never see in RoboLOF clones. Thus, one way of establishing differences in the medio-lateral position could be through a dendritic “Robo code” where early-born cells express Robo and late-born cell express multiple Robo receptors (Brierley, 2009).

With Netrin being expressed in the midline cells during the pupal-adult transition it was asked whether attractive Netrin-Fra signalling could also contribute to positioning dendrites in the leg neuropil. When Fra was removed from the 48 h AH subtype it was found that the arborization was shifted laterally, whereas removing it from the 96 h AH subtype had little effect, and neither did the removal of Netrin A and B from the midline, suggesting that Netrin-Fra signalling may not play a role in dendritic targeting in the later-born cell. It may be that Fra is expressed in early-born cells within the lineage and then down-regulated, although it cannot be excluded that Netrin-Fra signalling was masked by the repulsion from Slit-Robo signalling. These data are consistent with Fra being a major player in targeting the dendrites of the 48 h AH cell. The fact that both Fra and Robo are required for normal morphogenesis of 48 h AH neurons raises the possibility that members of lineage 15 could use a 'push-pull' mechanism for positioning their dendrites, where the blend of receptors within a cell dictates the territory within the map that they will innervate (Brierley, 2009).

How could such subtype-specific blends of receptors be established? A number of studies have revealed that spatial codes of transcription factors are important for specifying the identity of motoneuron populations. Within lineage 15 it is possible that temporal, rather than spatial, transcription factor codes are important for regulating the blend of guidance receptors. A number of molecules have been identified that control the sequential generation of cell types within neuroblast lineages. Chief amongst these are a series of transcription factors that include Hunchback, Krüppel, Pdm, Castor and Seven-up. These temporal transcription factors are transiently expressed within neuroblasts and endow daughter neurons with distinct “temporal identities”. Castor and Seven-up are known to schedule transitions in postembryonic lineages, regulating the neuronal expression of BTB-POZ transcription factors Chinmo and Broad. It is possible that the temporal transcription factors Broad and Chinmo could control the subtype-specific expression of different Robo receptors or the Netrin receptor Frazzled in leg motoneurons. There is a precedent for this in the Drosophila embryo, where motoneuron axon guidance decisions to distal (dorsal) versus proximal (ventral) targets are orchestrated by Even-Skipped, a homeobox transcription factor, which in turn controls the expression of distinct Netrin receptor combinations (Brierley, 2009).

Studies focusing on the growth of olfactory projection neuron dendrites in Drosophila reveal that they elaborate a glomerular protomap prior to the arrival of olfactory receptor neurons suggesting that target/partner-derived factors may not be necessary for establishing coarse patterning of synaptic specificity. The global nature of the signals describe in this study and their origin in a third-party tissue is a fundamentally different situation to that where target-derived factors instruct partner cells, such as presynaptic amacrine cells signalling to retinal ganglion cell dendrites in the zebrafish retina. Furthermore, although this study shows that Slit and Netrin control the positioning of dendrites across the medio-lateral axis of the CNS, it may be that other similar guidance signals are important for patterning dendrites in other axes. There is a striking conservation of the molecular mechanisms that build myotopic maps in the embryo and pupae. Understanding the similarities and differences between these myotopic maps, from an anatomical, developmental, and functional perspective, may give insight into the evolution of motor systems and neural networks in general (Brierley, 2009).

This study found that individual leg motoneurons that lacked Robo signalling appeared to have more complex dendritic arborizations. The working hypothesis, that dendrites invaded medial territories because of a failure of Slit-Robo guidance function, did not take into account the possibility that cells may generate more dendrites due to a change in a cell-intrinsic growth program. Thus the changes seen in dendrite distribution relative to the midline could formally be a result of 'spill-over' from that increase in cell size/mass. To determine whether this was the case larger cells were generated by activating the insulin pathway in single motoneurons. It was found the dendrites of these 'large cells' remained within their normal neuropil territory, supporting the idea that the removal of Robo-Slit signalling results in a disruption in guidance, not growth. These data underline the fundamental importance of midline signals in controlling the spatial coordinates that these motoneuron dendrites occupy, i.e., that a neuron twice the size/mass of a wild-type cell is still marshalled into the same volume of neuropil (Brierley, 2009).

When the image stacks were reconstructed to look at the distribution of the dendrites in the dorso-ventral axis, it was found that the apparent increase in size was in fact a redistribution of the dendrites from ventral territories into more dorsal medial domains. This was unexpected and suggests that changes in midline signalling can also impact the organization of dendrites in the dorso-ventral axis. So CommGOF 96 h AH neurons may not only encounter novel synaptic inputs by projecting into medial territories, but they may also lose inputs from the ventral domains of neuropil they have vacated. These observations suggest that motoneurons within lineage 15 have a fixed quota of dendrites and where it is distributed in space depends on cell-intrinsic blends of guidance receptors. Taken together these data support the idea that growth and guidance mechanisms are genetically separable programs. In identified embryonic motoneurons where Slit-Robo and Netrin-Fra signalling has been disrupted, quantitative analysis reveals dendrites also show no measurable difference in their total number of branch tips or length (Mauss, 2009). Moreover, recent computational studies in larger flies reveal that dendritic arborizations generated by the same branching programs can generate very different shapes depending on how their 'dendritic span' restricted within the neuropil. Previous work in both vertebrates and Drosophila has shown that a loss of Slit-Robo signalling results in a reduction in dendrite growth and complexity, but this study found no evidence to support this (Brierley, 2009).

Neural maps and synaptic laminae are universal features of nervous system design and are essential for organizing and presenting synaptic information. How the appropriate pre- and postsynaptic elements within such structures are brought together remains a major unanswered question in neurobiology. Studies in recent years have shown that neural network development involves both hardwired molecular guidance mechanisms and activity-dependent processes; the relative contribution that each makes is still unclear. Work on the spinal cord network of Xenopus embryos revealed that seven identifiable neuron subtypes can establish connections with one another and that the key predictor of connectivity was their anatomical overlap. This could be interpreted to mean that connectivity is promiscuous and that the major requirement for the generation of synaptic specificity is the proximity of axons and dendrites. This is particularly interesting in light of the current dendrite targeting data and the observation that both sensory neurons and interneurons in Drosophila use the same midline cues to position their pre-synaptic terminals in the CNS. Moreover, a recent study has shown that Semaphorins control the positioning of axons within the dorso-ventral axis. Taken together these observations suggest that during development the coordinated targeting of both pre- and postsynaptic elements into the same space using global, third-party guidance signals could provide a simple way of establishing the specificity of synaptic connections within neural networks. This idea is akin to 'meeting places' such as the traditional rendezvous underneath the four-sided clock at Waterloo railway station where two interested parties organize to meet. Understanding how morphogenetic programs contribute to the generation of synaptic specificity is likely to be key to solving the problem of neural network formation (Brierley, 2009).

Frazzled cytoplasmic P-motifs are differentially required for axon pathway formation in the Drosophila embryonic CNS

Frazzled is a Netrin-dependent chemoattractive receptor required for axon pathway formation in the developing Drosophila embryonic CNS. The cytoplasmic domain is important and contains three conserved P-motifs (P1, P2, and P3) thought to initiate intracellular signaling cascades and to crosstalk with other receptors during axon pathway formation. This study rescued homozygous frazzled embryos by pan-neurally expressing a series of mutants lacking either the cytoplasmic domain or one of the conserved P-motifs and assessed the ability of these mutants to rescue frazzled defects in commissural, longitudinal and motor axon pathways. Surprisingly, while the cytoplasmic domain is required, removal of an individual P-motif does not prevent gross formation of commissures. However, removal of P3 from Fra does prevent Eagle-expressing commissural axons from crossing the midline in the posterior commissure suggesting that some neurons have a stronger requirement for P3-dependent signaling. Indeed, axons within the longitudinal connective as well as a small subset of motor neurons within the ISNb pathway also specifically require P3 to project to their targets correctly. In these latter axon projections, deleting the P1-motif appears to de-regulate the receptor's activity, actually increasing the frequency of motor neuron projection errors and inducing ectopic midline crossing errors. Collectively, these data demonstrate the critical nature of both the P1 and the P3-motifs to Frazzled function in vivo during axon pathway formation (Dorsten, 2008).

In the absence of frazzled over-expression of Abelson tyrosine kinase disrupts commissure formation and causes axons to leave the embryonic CNS

In the Drosophila embryonic nerve cord, the formation of commissures require both the chemoattractive Netrin receptor Frazzled (Fra) and the Abelson (Abl) cytoplasmic tyrosine kinase. Abl binds to the cytoplasmic domain of Fra and loss-of-function mutations in abl enhance fra-dependent commissural defects. To further test Abl's role in attractive signaling, Abl was over-expressed in Fra mutants anticipating rescue of commissures. The Gal4-UAS system was used to pan-neurally over-express Abl in homozygous fra embryos. Surprisingly, this led to a significant decrease in both posterior and anterior commissure formation and induced some commissural and longitudinal axons to project beyond the CNS/PNS border. Re-expressing wild-type Fra, or Fra mutants with a P-motif deleted, revert both commissural and exiting phenotypes, indicating that Fra is required but not a specific P-motif. This is supported by S2 cell experiments demonstrating that Abl binds to Fra independent of any specific P-motif and that Fra continues to be phosphorylated when individual P-motifs are removed. Decreasing midline repulsion by reducing Robo signaling had no effect on the Abl phenotype and the phenotypes still occur in a Netrin mutant. Pan-neural over-expression of activated Rac or Cdc42 in a fra mutant also induced a significant loss in commissures, but axons did not exit the CNS. Taken together, these data suggest that Fra activity is required to correctly regulate Abl-dependent cytoskeletal dynamics underlying commissure formation. In the absence of Fra, increased Abl activity appears to be incorrectly utilized downstream of other guidance receptors resulting in a loss of commissures and the abnormal projections of some axons beyond the CNS/PNS border (Dorsten, 2010).

Frazzled and Abelson Tyrosine kinase activity clearly cooperate during the formation of embryonic commissures. In the absence of Fra, detection of Netrin-dependent chemoattraction is compromised and many posterior commissures fail to form. Both anterior and posterior commissures are absent if fra and abl activity is lost. This presumably reflects the ability of abl mutations to interact with a second Netrin receptor, Dscam, as well as Netrin independent receptors (e.g. Turtle) known to be important for commissure formation. Finally, as most commissures are also lost when both maternal and zygotic contributions of Abl are genetically removed, it seems Abl itself is required for commissure formation. Given these different observations, it seemed plausible that over-expressing Abl in fra null embryos would improve commissure formation. However, instead of an improvement, this study clearly documented a major decrease in both anterior and posterior commissures and the induction of a novel phenotype whereby axons normally confined to the CNS now project into the periphery (AEP defects). It is worth emphasizing that these phenotypes occur even with the over-expression of a wild-type Abl transgene that retains its autoinhibitory domain and must be activated by endogenous mechanisms. These phenotypes are completely dependent on the absence of Fra but not any specific P-motif, occur in the absence of Netrins as well, and are not alleviated if Robo-dependent midline repulsion is reduced. Interestingly, the loss of commissures, but not the Axons Exiting to Periphery (AEP) defects, is also observed when activated Rac or Cdc42 GTPases are over-expressed in a homozygous fra mutant. Taken together, it is proposed that during exploration of the midline, Fra is a key regulator of Abl activity and helps determine how the cytoskeletal machinery utilizes Abl. In the absence of Fra, axon outgrowth does not simply stall; but rather, axons follow a variety of aberrant trajectories away from the midline. This suggests that Fra normally competes with several other receptor systems to dictate how Abl functions to regulate the cytoskeletal machinery. While competitors undoubtedly include other midline guidance cues, the emergence of AEP defects suggests that Fra also competes with guidance systems not normally associated with the midline. In the absence of Fra, these other receptors appear to utilize the extra Abl to alter cytoskeletal dynamics at a variety of choice points, ultimately preventing commissure formation and directing some axons out of the CNS (Dorsten, 2010).

The Abl gain-of-function phenotype described in this study occurs only if fra is absent. That is, commissures form correctly in a heterozygous fra mutant or when partially active Fra transgenes with a single P-motif deleted are re-expressed with Abl. In S2 cell immunoprecipitation experiments, both Abl and BcrAbl bind to the cytoplasmic tail of Fra independent of any specific P-motif. While surprising given the conservation of these P-motifs and their known importance to Fra function, the lack of P-motif specificity is consistent with genetic rescue experiments. All three P-motif deletion mutants rescue commissure formation and the AEP defects elicited by over-expression of either wild-type Abl or BcrAbl in fra embryos. The ectopic midline crossovers (fuzzy commissures) observed with only BcrAbl also depend on Fra and specifically the P3-motif. However, BcrAbl is not an endogenous Drosophila protein and, the human Bcr domain may induce neomorphic phenotypes. Because BcrAbl does not preferentially bind to the P3 motif, and wild-type Abl does not elicit crossover defects, it is now suspected that the ectopic crossovers are an example of a neomorphic phenotype, a hypothesis that will be extensively addressed in future work (Dorsten, 2010).

The physical interaction between Fra and either Ablwt or BcrAbl in immunoprecipitation assays can reflect direct or indirect association between the two proteins. It is possible that the failure to observe P-motif dependence reflects the binding of Abl to multiple P-motifs or the use of scaffold proteins associated with more then one P-motif. Given that it has been demonstrated that the cytoplasmic tail of Fra fused to glutathionine-S-transferase (GST) binds to in vitro translated Abl, direct binding of Abl to Fra is clearly possible. If so, these experiments suggest that Abl may bind to Fra in the regions between P-motifs, which is, in fact, where most of the tyrosine residues within the cytoplasmic domain of Fra reside. Moreover, in S2 cells, tyrosine phosphorylation of Fra is not affected by removal of the P1 or P2 motif and may actually increase when P3 is removed. This is intriguing as removal of the P3-motif is known to significantly affect Fra signaling in vivo and the FraΔP3 transgene is the least capable of rescuing the AEP defects caused by Ablwt or BcrAbl expression. Immunoblots of Fra phosphorylation in the absence of pervanadate pretreatment also suggest the steady-state level of tyrosine phosphorylation may be relatively low, or highly dynamic. S2 cells are known to express tyrosine phosphatases that antagonize Abl activity for some substrates, and antagonistic action between Abl and several phosphatases during nerve cord development has been documented. Since tyrosine phosphorylation of vertebrate DCC is required for attractive responses, axon outgrowth and orientation of the axon, it will be important to systematically assess how Fra and Abl physically interact to regulate each other's activity during midline guidance (Dorsten, 2010).

Both of the phenotypes observed when Abl activity is elevated in a fra mutant, the loss of commissures and the exiting of CNS axons to the periphery, suggest these embryos are experiencing an excess of midline repulsion. During commissure formation, Slit dependent repulsion prevents commissural axons from crossing unless Commissureless prevents the Slit receptor Robo from accumulating on the cell surface. Before commissural axons extend towards the midline, Fra activity may help increase Comm expression so Comm levels are expected to be reduced in fra mutants leading to an increase in Robo-dependent repulsion. Since increasing Robo activity in a fra embryo is sufficient to reduce commissure formation, it was important to test whether an excess of Robo-dependent repulsion underlies the Abl over-expression phenotypes. However, introduction of one null allele of robo (Robo1) had no discernable affect on the Abl gain-of-function phenotypes, even though, in previous work, elevating Abl activity in a heterozygous robo mutant induces ectopic midline crossing errors. Given the absence of even a minimal suppression, it seems unlikely that the loss of commissures and/or AEP defects noted in mutants reflects an increase in Robo-dependent midline repulsion. While two other Robo receptors, Robo2 and Robo3, operate during midline guidance and could conceivably contribute to these Abl phenotypes, neither of these receptors have the conserved CC3 cytoplasmic domain known to be important for Abl binding to Robo1. Moreover, while certain Netrin receptors (e.g., Unc5) can also elicit a repulsive response, both commissure loss and AEP defects still occur when Abl is over-expressed in a Netrin mutant. This provides strong evidence that abnormal signaling by other Netrin receptors is not responsible for these phenotypes (Dorsten, 2010).

In terms of the AEP defects, which also point to excess repulsion, it is worth noting that axons do not leave the CNS in a commissureless mutant experiencing very high levels of Slit-dependent repulsion, nor do they appear evident in published figures of fra Dscam double mutants, or even fra Dscam abl triple mutants. While the identity of all the axons leaving the CNS has not been established, ut was confirmed that at least two subtypes of CNS axons are exiting: both FasII expressing interneurons and sema2b commissural axons. FasII axons do not leave the CNS when midline repulsion is elevated in a commissureless mutant, and while the level of Abl activity affects the trajectory of FasII interneurons, in most cases altering Abl activity leads to midline crossing errors rather than an exit from the CNS. Over-expressing Abl in a fra mutant also affects several different guidance decisions by Sema2b-expressing commissural axons. While the cues guiding these neurons are not well understood, the spectrum of defects observed both before and after they cross the midline suggest that these neurons are responding to more then just midline repulsion. Thus, if a repulsive mechanism is functioning, as initially suggested by the phenotype, the origin of the signal remains to be determined. Indeed, data using activated forms of Rac and Cdc42 suggest that the primary defect lies in the ability of growth cones to properly regulate actin dynamics underlying axon outgrowth and steering. This could involve both attractive and repulsive systems (Dorsten, 2010).

Proper axon guidance also requires concerted regulation of the cytoskeletal dynamics underlying axon outgrowth and steering. Like most guidance receptors, Fra, or its vertebrate and C. elegans homologues, is known to initiate signaling pathways affecting cytoskeletal dynamics. Abl is also a key regulator of actin dynamics in vertebrate cells and of the development of the Drosophila nervous system. Mutations in abl interact with several cytoskeletal regulators to affect axon pathway formation: kette, capulet, chicadee (Profilin), enabled and trio. Thus, in the absence of Fra-dependent regulation, does elevated Abl activity affect the cytoskeletal machinery to indirectly cause a reduction in commissures and AEP defects? This study tested this basic concept by expressing in fra mutants other key regulators of cytoskeletal dynamics known to affect midline guidance. Surprisingly, over-expression of activated Rac and Cdc42 in a fra mutant replicates the loss of commissure phenotype (but not the AEP defects) observed with Abl. The Cdc42 result is most intriguing as expression in a wild-type or heterozygous fra embryo results in fused commissures and gaps in the longitudinal connectives. Yet, upon complete removal of Fra, commissures do not form and the longitudinal connectives reform. Thus, in the absence of Fra, commissure formation, but not AEP defects, appears to be particularly sensitive to manipulation of actin-based processes. It is possible that the manipulation of Cdc42 and Rac activity in a fra mutant is affecting shared processes related to actin polymerization. For example, in vertebrate studies, Cdc42 and Abl work in parallel to regulate actin polymerization and Abl may activate Rac in response to cell adhesion. If so, the data suggest that in the absence of Fra activity these key regulators are being used by other surface receptors to regulate actin dynamics in a manner that ultimately prevents commissure formation. This is certainly consistent with the number of guidance systems that have been linked to these regulators and the scope of guidance detected defects. Minimally, the Cdc42 and Rac data continue to highlight the degree and importance of Fra-dependent regulation of cytoskeletal dynamics, especially actin-based processes, during commissure formation. Moreover, they point to a highly competitive process between multiple surface receptors and the cytoskeletal machinery where Fra is a major player. While competition between midline attractive and repulsive cues has been recognized, in the current experiments, midline repulsive activity had no affect on the Abl phenotypes. Therefore, it seems likely that Fra is competing with several other receptor systems whose presence (but not identity) has been uncovered by over-expression of Abl, Rac or Cdc42 in homozygous fra embryos. Which guidance events are being affected has not yet been determined, but a few candidates exist. In addition to fra and Dscam, loss-of-function mutations in abl interact with the cell-cell adhesion molecules neurotactin and amalgam, fasI, midline-fasciclin and turtle to reduce commissure formation and some of these are fairly ubiquitously expressed in the nerve cord. Abl has also been linked to the regulation of cell-cell adhesion molecules alone or in combination with receptor systems such as Notch (Dorsten, 2010).

In summary, these data suggest a model whereby Fra activity initiates key signaling events that dictate when and how Abl activity is utilized during commissure formation. Rac and Cdc42 are probably also involved in this process, and, together with Abl, help regulate key aspects of actin dynamics underlying commissure formation. In the absence of Fra other midline guidance systems are still functioning well enough to form most commissures, but they are clearly sensitive to perturbation of intracellular signaling pathways regulating cytoskeletal dynamics. Thus, when Fra is removed, other guidance systems appear to recruit Abl, Rac or Cdc42 activity to misdirect axon outgrowth, ultimately preventing commissure formation and, with Abl, causing some axons to exit the CNS. Thus, in a normal embryo, Fra must be sending information that allows it to compete very well against these other guidance receptors to properly regulate axon outgrowth and steering during commissure formation. While an alteration in midline guidance decisions may also account for the AEP defects, the scope of guidance errors observed in neurons leaving the CNS suggest that increasing Abl activity could also be affecting other guidance systems not directly associated with the midline. While the identity and specific role of these guidance systems awaits discovery, the sensitivity of the CNS axon scaffold to Abl over-expression will be an important tool for identifying these competing pathways (Dorsten, 2010).

Netrin-guided accessory cell morphogenesis dictates the dendrite orientation and migration of a Drosophila sensory neuron

Accessory cells, which include glia and other cell types that develop in close association with neurons, have been shown to play key roles in regulating neuron development. However, the underlying molecular and cellular mechanisms remain poorly understood. A particularly intimate association between accessory cells and neurons is found in insect chordotonal organs. This study found that the cap cell, one of two accessory cells of v'ch1, a chordotonal organ in the Drosophila embryo, strongly influences the development of its associated neuron. As it projects a long dorsally directed cellular extension, the cap cell reorients the dendrite of the v'ch1 neuron and tows its cell body dorsally. Cap cell morphogenesis is regulated by Netrin-A, which is produced by epidermal cells at the destination of the cap cell process. In Netrin-A mutant embryos, the cap cell forms an aberrant, ventrally directed process. As the cap cell maintains a close physical connection with the tip of the dendrite, the latter is dragged into an abnormal position and orientation, and the neuron fails to undergo its normal dorsal migration. Misexpression of Netrin-A in oenocytes, secretory cells that lie ventral to the cap cell, leads to aberrant cap cell morphogenesis, suggesting that Netrin-A acts as an instructive cue to direct the growth of the cap cell process. The netrin receptor Frazzled is required for normal cap cell morphogenesis, and mutant rescue experiments indicate that it acts in a cell-autonomous fashion (Mrkusich, 2010).

Many sense organs in insects are multicellular, consisting of a neuron and two or more closely associated cells, which collaborate to transduce sensory stimuli into electrical activity in the mature organ. This study has revealed that the cap cell, one of the accessory cells of the v'ch1 chordotonal organ, also plays a key role in the morphogenesis of its associated neuron (Mrkusich, 2010).

A number of lines of evidence suggest that dorsally directed extension of the cap cell both tows the v'ch1 neuron cell body from its birthplace into its final position in the dorsolateral region of the body wall and also pulls its growing dendrite into a stereotypic orientation. These include: the tight physical connection, which is maintained throughout development, between the cap cell and the tip of the dendrite in both wild-type and NetA mutant embryos; the relative timing and common direction of cap cell extension, dendrite reorientation and neuron migration observed in wild-type embryos; the tight correlation between aberrant direction of cap cell extension, failure of neuron migration and inappropriate dendrite orientation seen in NetA, fra mutants and NetA misexpression embryos; and the consistent failure of neuron migration when the cap cell fails to extend dorsally following misexpression of NetA (Mrkusich, 2010).

The variability in v'ch1 dendrite position seen at early stages of dendrite growth in wild-type embryos probably reflects a degree of imprecision in the mechanisms that specify the initial direction of dendrite outgrowth. Whether a neuron-intrinsic cue, related to the plane of division of the neuron progenitor cell, or some external cue determines the site of dendrite emergence remains to be determined. In any event, it is clear that NetA plays no role in this early phase of dendrite growth, as it is unaffected in NetA mutant embryos (Mrkusich, 2010).

The extent to which the dendrite can be relocated after it has first emerged from the v'ch1 neuron cell body in NetA and fra mutants is surprising: such a phenomenon of neurite repositioning has not previously been described. It implies a considerable flexibility in the cellular machinery for anchoring the base of the dendrite (Mrkusich, 2010).

v'ch1 and the lch5 cluster are the only sensory neurons in the body wall of the Drosophila embryo to undergo significant movements during normal development: v'ch1 migrates dorsally, whereas the lch5 cluster moves ventrally. The findings suggest that the v'ch1 neuron does not actively migrate into a more dorsal position: rather, it is passively towed by the cap cell. A different view of chordotonal organ migration has been presented. It has been suggested that the chemo-repellent Slit acts directly on thoracic chordotonal neurons via Robo2 (Leak - FlyBase) receptors, blocking their response to ventral attractants that promote a ventral migration of lch5 neurons in abdominal segments. However, the chordotonal neuron migration phenotypes observed by in this previous study could be secondary to abnormal morphogenesis of associated ligament and/or scolopale cells. Indeed, an earlier study has suggested that ligament cells pull the lch5 neurons from a dorsal to a ventral position, and time-lapse observations made of lch5 migration in the current study support that view (Mrkusich, 2010).

The dramatic morphogenetic changes that the cap cell undergoes during its dorsal extension provide a tractable model for dissecting the molecular basis for cell migration. The cap cell is large, readily visualised both in fixed and living embryos and is potentially accessible for direct surgical manipulations. It shows features of both cell migration (lamellipodial extension and nuclear translocation) and of axon growth (growth cone extension with filopodia) (Mrkusich, 2010).

The dorsally directed extension of the v'ch1 cap cell is dependent upon NetA function. In NetA mutants, the cap cell undergoes morphogenesis, but extends a process in a ventral, rather than a dorsal, direction. The pattern of cap cell process extension seen when NetA is expressed ventral to the cap cell suggests that NetA acts as an instructive guidance factor for cap cell growth. This is supported by the normal expression pattern of NetA: the final insertion point of the cap cell process is located near the middle of the patch of epidermal NetA mRNA expression (Mrkusich, 2010).

In NetA mutants the cap grows quite consistently in an anteroventral direction and inserts at a specific location in the epidermis, close to the site of insertion of the vchB cap cell. This suggests that, in the absence of its normal guidance cue NetA, the v'ch1 cap cell is responding to the same attractive cues that guide the extending vchB cap cell. The fact that the v'ch1 cap cell can reliably grow towards this alternative location via a totally different route to that used by the vchB cap cell suggests that this cue functions as a chemoattractant (Mrkusich, 2010).

In all of its previously described developmental roles, NetA appears to act redundantly to NetB. This generalisation does not hold for guidance of v'ch1 cap cell growth, as NetB appears to play no role in this process. NetB mutant embryos do not display cap cell defects, the phenotypes of NetA,B mutants are similar to NetA mutants and ectopic expression of NetB in oenocytes, unlike NetA, has no effect on v'ch1 migration or dendrite growth (Mrkusich, 2010).

In many developmental contexts, binding of Netrin to its receptor, UNC-40/DCC/Fra, directly elicits a cellular response in the cell bearing the receptor, whereas in other situations Fra acts in a non-cell-autonomous fashion (Mrkusich, 2010).

This study found that guidance of v'ch1 cap cell growth by NetA requires Fra activity: fra mutants show the same dendrite and cell migration and cap cell defects as NetA mutants. fra mutant rescue experiments suggest that Fra regulates cap cell morphogenesis via a cell-autonomous mechanism: defective cap cell phenotypes in fra mutants are almost completely rescued by driving a wild-type fra gene construct with P0163-GAL4, which drives gene expression in the whole v'ch1 sense organ. By contrast, there is no significant rescue of the mutant phenotype with the neuronal driver line elav-GAL4, which expresses only rarely in the cap cell (Mrkusich, 2010).

Netrins guide migration of distinct glial cells in the Drosophila embryo

Development of the nervous system and establishment of complex neuronal networks require the concerted activity of different signalling events and guidance cues, which include Netrins and their receptors. In Drosophila, two Netrins are expressed during embryogenesis by cells of the ventral midline and serve as attractant or repellent cues for navigating axons. It was asked whether glial cells, which are also motile, are guided by similar cues to axons, and the influence of Netrins and their receptors on glial cell migration was analyzed during embryonic development. In Netrin mutants, two distinct populations of glial cells are affected: longitudinal glia (LG) fail to migrate medially in the early stages of neurogenesis, whereas distinct embryonic peripheral glia (ePG) do not properly migrate laterally into the periphery. It is further shown that early Netrin-dependent guidance of LG requires expression of the receptor Frazzled (Fra) already in the precursor cell. At these early stages, Netrins are not yet expressed by cells of the ventral midline, and evidence is provided for a novel Netrin source within the neurogenic region that includes neuroblasts. Later in development, most ePG transiently express uncoordinated 5 (unc5) during their migratory phase. In unc5 mutants, however, two of these cells in particular exhibit defective migration and stall in, or close to, the central nervous system. Both phenotypes are reversible in cell-specific rescue experiments, indicating that Netrin-mediated signalling via Fra (in LG) or Unc5 (in ePG) is a cell-autonomous effect (von Hilchen, 2010).

Based on the present data, a dual role is postulated for Netrin-mediated signalling in glial cell migration. According to this model, early in neurogenesis, Netrins guide the LGB and its progeny from the lateral edge of the neuroectoderm towards a medial position. At these early stages, Netrins are expressed by cells of the neuroectoderm as well as by NBs, and this Netrin source most likely attracts the LGB via Fra. Ectopic expression of Netrins in the vicinity of the LGB might abolish a possible ventral-to-dorsal gradient and hence (occasionally) results in ectopic clusters. Additionally, attempts were made to express Netrins in the dorsal area of the embryo and thereby attract the LGB and its progeny in the wrong direction. Unfortunately, none of the tested drivers showed Gal4 expression at the appropriate stage and intensity. Further experiments are needed to prove this model (von Hilchen, 2010).

The LGB delaminates from the lateral neuroectoderm close to the sensory organ precursor-derived ePG11 (60%-65% dorsoventral axis, where 0% is the ventral midline). In wild type, it migrates medially while proliferating, whereas it is believed that in Netrin and fra mutants the LGB remains (and proliferates) at its place of birth and does not migrate at all in affected hemisegments. Morphogenetic movements during germ band retraction, mesoderm migration and dorsal expansion of the epidermis complicate this issue. Nevertheless, ectopic clusters mainly remain in close proximity to ePG11. Although ectopic LG have no contact to axons, the lineage develops normally with respect to cell number and marker gene (Msh, the LG-specific marker Naz, Pros) expression. This is contrary to published data on the development and differentiation of LG, which have been postulated to depend on an intimate interaction with longitudinal axons. Nevertheless, LG play an important role in the navigation and fasciculation of longitudinal axons. Accordingly, in hemisegments of Netrin and fra mutants, in which LG are mispositioned from the earliest stages, it was found that the longitudinal axon tracts are thinner, show aberrant projections and fasciculation defects. These neuronal phenotypes were reported previously, but without noticing that the LG are missing in these hemisegments. Similar neuronal phenotypes can be induced by ectopic expression of unc5 in glial cells (repo>unc5), which only affects LG and shifts them away from the midline to more lateral positions or even into the PNS. In some hemisegments with ectopic LG clusters, longitudinal tracts show a weaker phenotype. In these cases, other glial cells (from within the same hemisegment or an adjacent hemisegments) fill the gaps on top of the longitudinal axons and hence seem to compensate for the loss of LG (von Hilchen, 2010).

From these data, it is concluded that the longitudinal axon phenotypes observed in Netrin and fra mutants are, at least partially, a secondary effect of the lack of LG in corresponding neuromeres. Additionally (and somewhat confusingly), these neuronal phenotypes in fra mutants can be partially rescued by elav-Gal4 and Mz605-Gal4, but neither rescues the LG phenotype. Further experiments with other Gal4 drivers that allow a more restricted spatio-temporal expression of UAS-fra might help to resolve this issue (von Hilchen, 2010).

The second population of glial cells that is guided by Netrin-mediated signalling comprises ePG. Nine ePG migrate from the ventral nerve cord into the PNS of each abdominal hemisegment, but it is ePG6 and ePG8 in particular, both progeny of NB2-5, that show a stalling phenotype in NetABδ, NetBδ and unc58 mutants. It was shown by rescue experiments and analysis of Netrin single mutants that only NetB provided by cells of the ventral midline repels ePG6 and ePG8 via the Unc5 receptor. Although both Netrins are expressed by the ventral midline, they clearly do not share a redundant function for ePG guidance. A similar observation has been reported for unc5-expressing motoaxons, which respond differently to each Netrin. To date, the nature of these differences between the two Netrins in combination with Unc5 remains unresolved. In NetA?-NetB™, only NetB is expressed, but is tethered to the membrane of the cell. In these embryos, no ePG stalling was observed, further supporting the notion that only NetB is required for ePG migration and indicating that this signalling is at short range. But why do all ventrally derived ePG express unc5 mRNA transiently during their migratory phase? Further work will be needed to clarify why NetB-Unc5 signalling is selectively required for normal migration of ePG6 and ePG8 (von Hilchen, 2010).

It is widely accepted that embryonic glial cells use neurons or neuronal processes as the substrate for migration. It was shown previously that most migrating ePG follow certain axonal projections. Could such neuron-glia contact be sufficient for proper guidance? The questions would then be (1) how do glial cells actually identify their respective neuronal projections and (2) how is directionality of migration given? Four-dimensional analysis of ePG migration, however, has revealed that ePG6 and ePG8 do not necessarily follow axons, but may also use other glial cells as substrate. These two cells leave the CNS later than other ePG, they are the only ePG that can overtake other cells, and they may migrate on top of ePG rather than along peripheral nerves. Is this possible lack of axonal association (and perhaps adhesion) the reason why these cells need an additional guidance system? (von Hilchen, 2010).

The initial migration of the LGB and its early progeny cannot occur along axons because at these early stages axonal projections are not yet established. As discussed, in most cases the LG phenotype affects the entire LGB lineage and hence is an early guidance defect. So it might well be that early LGB guidance is dependent on Netrin-Fra signalling without any neuronal contribution. After the first division of the LGB, neuronal projections are established, Net-Fra attraction is no longer required and fra expression is switched off. The results of the fra rescue experiments show that at least the timing of fra expression is crucial: gcm-Gal4-induced fra expression can rescue the LG phenotype, whereas a slightly later expression driven by repo-Gal4 cannot. The expressivity of the LG phenotype in NetAB? mutants is only 30%. How are 70% of the LGB 'rescued'? A second, as yet unknown, signalling mechanism could guide the LGB medially, either by ventral attraction or dorsal repulsion. Similar redundancies have been reported, e.g. for border cell migration in the ovary or germ cell migration in early embryogenesis (von Hilchen, 2010).

In addition to the selectivity of the phenotypes in different populations of glial cells, as discussed above, another interesting observation comes from the rescue experiments in unc58 and fra3/fra4 mutants. Control experiments were performed to test whether pan-glial expression of either receptor affects glial cell migration. Glial expression of UAS-fra in an otherwise wild-type background does not alter the glial pattern in the CNS or PNS. Since unc5 is expressed normally in these experiments, repulsion of ePG6 and ePG8 into the periphery occurs as normal. By contrast, pan-glial expression of unc5 is able to shift LG to a more lateral position in the CNS and LG can even leave the CNS and lie in the periphery, whereas all other glial cells are properly positioned. Why do only certain glial cells react upon Netrin-mediated signalling? More precisely, what conveys the ability for LG to 'read' Netrin-mediated signalling? In addition to the receptors, cells might require downstream molecules that could be differentially expressed and hence provide competence to react to Netrins. Several such molecules have been described for both vertebrates and invertebrates. Loss-of-function mutants for Drosophila homologues of these possible downstream molecules were analyzed, but none showed phenotypes comparable to those of fra3/fra4 or unc58. Previous data demonstrate a function for the small GTPases Rac1 and Rho1 in ePG migration in Drosophila, and it was recently shown that they can act downstream of Unc5 signalling in vertebrates. A dominant-negative form of Rho1 was expressed using cas-Gal4 in an otherwise wild-type background and stalling of ePG6 and ePG8 (cas>Rho1N19) was obtained. Expression of a constitutively active form of Rho1 in ePG6 and ePG8 in an unc5 mutant background (unc58; cas>Rho1V14), however, did not restore their stalling phenotype. Although the possiblity cannot be ruled out that ectopic expression of such constructs leads to artificial phenotypes, these data indicate that Rho1 is not downstream of Unc5, but rather acts in parallel. Further experiments will be needed to unravel the signalling complex of Unc5 and Fra/Dcc, and glial cell migration in the Drosophila embryo might serve as a powerful model system for this purpose (von Hilchen, 2010).


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

date revised: 10 October 2014

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