slit

DEVELOPMENTAL BIOLOGY

Embryonic

Slit protein is found localized to the midline neuroepithelium after germ band extention [Images]. Throughout germband shortening, Slit protein becomes confined to specific medial ectodermal cells identified as glial cells (Rothberg 1988, 1990). Slit is secreted by glial cells and distributed along axon tracts. Expression is also seen in cardioblast precursors of the dorsal aorta, hindgut, midgut and brain (Wharton, 1993).

Slit stimulation recruits Dock and Pak to the Roundabout receptor and increases Rac activity to regulate axon repulsion at the CNS midline

Drosophila Roundabout is the founding member of a conserved family of repulsive axon guidance receptors that respond to secreted Slit proteins. Evidence is presented that the SH3-SH2 adaptor protein Dreadlocks (Dock), the p21-activated serine-threonine kinase (Pak), and the Rac1/Rac2/Mtl small GTPases can function during Robo repulsion. Loss-of-function and genetic interaction experiments suggest that limiting the function of Dock, Pak, or Rac partially disrupts Robo repulsion. In addition, Dock can directly bind to Robo's cytoplasmic domain, and the association of Dock and Robo is enhanced by stimulation with Slit. Furthermore, Slit stimulation can recruit a complex of Dock and Pak to the Robo receptor and trigger an increase in Rac1 activity. These results provide a direct physical link between the Robo receptor and an important cytoskeletal regulatory protein complex and suggest that Rac can function in both attractive and repulsive axon guidance (Fan, 2003).

Strong defects in embryonic axon guidance are observed only when both the maternal and zygotic components of dock function are removed. In these maternal minus dock mutants (dock^mat), phenotypes reminiscent of loss of robo function can often be seen. dock^mat embryos examined with an antibody that labels all axons frequently show thickening of commissural axon bundles and a commensurate reduction in the thickness of longitudinal axon bundles. Staining these embryos with an antibody that selectively labels noncrossing axons (anti-fasII) reveals a significant degree of ectopic midline crossing. These phenotypes are similar to, but considerably less severe than, those observed in robo mutants. The similarity in mutant phenotypes that is observed provides genetic support for the idea that dock could contribute to Robo repulsion (Fan, 2003).

If dock and robo function together during midline guidance, they should be coexpressed in embryonic axons. This is indeed the case. Double labeling of embryos with antibodies raised against Dock and Robo reveals substantial coexpression of the two proteins. Both Dock and Robo show enriched expression on CNS axons beginning as early as stage 12, corresponding to the time of initial axon outgrowth. At these early stages of axon growth, Dock is detected in the pCC axon, a cell known to express Robo, as revealed by double labeling with FasII. Interestingly, while Robo shows a regionally restricted expression pattern with high levels of expression on longitudinal portions of axons and low levels in commissural axons, Dock is expressed equivalently in both commissural and longitudinal axon segments. This observation raises the possibility that Dock could have additional roles in the guidance of commissural axons not shared by Robo. These observations show that Dock and Robo are both present at the right time and place to function together during midline repulsion (Fan, 2003).

Biochemical data suggests that the interaction between Dock and Robo is an SH3-dependent interaction and that the first two SH3 domains of Dock are most important for mediating Robo binding. Based on the observations that a three-protein interaction can be detected between Robo, Dock, and Pak and that Pak has been shown to interact with the SH3-2 domain of Dock, it is believed that the SH3-1 domain is the most important for Robo and Dock binding. Furthermore, Slit stimulation enhances Dock's ability to bind to Robo, suggesting a ligand-regulated SH3 domain interaction. This represents a different kind of adaptor interaction to many that have been observed previously, where Nck appears to interact with a number of tyrosine-kinase receptors through an SH2 domain/phosphotyrosine interaction. In the latter case, how ligand binding to the receptor regulates the Nck SH2 domain interaction is quite well understood. The observation that the Robo receptor shows a ligand-regulated SH3 domain interaction with Dock/Nck suggests that somehow ligand binding results in an increased availability of the SH3 binding sites in the receptor (Fan, 2003).

The implication of Rac in Robo repulsion (dominant negative Rac1 shows a strong enhancement of slit;robo/+ defects) was unexpected in view of the well-established role of Rac as a positive regulator of axon outgrowth. On the surface, this finding appears quite contradictory to the function of Rac to promote actin polymerization at the leading edge of motile cells and axons. One possible explanation of this finding is that perhaps Rac can have different or even opposite effects on the actin cytoskeleton, depending on the molecular context in which it is activated and its overall level of activity. For example, depending on the coordinate local function of other small GTPases and actin regulatory proteins, the consequences of Rac function could be different. It is interesting to note that in addition to a role for Rac, genetic analysis and previously published data also support an important role for Rho in midline repulsion. Furthermore, in addition to strongly stimulating Rac activity, Slit has been shown have a modest stimulatory effect on Rho activity. The implication of both Rac and Rho in mediating repulsive responses has also been suggested to explain the output of the Plexin receptor. It will be interesting in the future to determine the interrelationship between Rac and Rho outputs in the context of Robo repulsion as well as in signaling downstream of other attractive and repulsive axon guidance receptors (Fan, 2003).

As an alternative to the context- and level-dependent explanation of the role of Rac in Robo repulsion, the observed axon steering defects in embryos where both Rac and Slit function are reduced, or in embryos deficient for multiple rac genes, could be explained as a secondary consequence of defects in the rate of axon extension. In this scenario, Rac's role in repulsive axon guidance would be intimately coupled with its role in axon outgrowth. That is to say, that appropriate steering decisions go hand and hand with the appropriate regulation of the rate of axon outgrowth (e.g., you are more likely to miss your exit if you are driving too fast). In this regard, it is important to emphasize that even repulsive cues can have stimulatory effects on axon extension. For example, in addition to repelling Xenopus spinal neurons, Slit also has a stimulatory effect on the rate of axon extension (Fan, 2003 and references therein).

Biochemical data support the idea that Slit stimulation of Robo can regulate the recruitment of Dock and Pak to the Robo receptor and also trigger an increase in Rac activity. Both of these events are dependent on the CC2 and CC3 sequences in Robo's cytoplasmic domain. Thus, the observations are consistent with either a Dock-dependent or a Dock-independent recruitment of Rac to Robo. Based on the known physical interactions between Dock and Pak and between Pak and Rac, it is likely that the recruitment of Rac is dependent on Dock. Alternatively, another protein interacting through CC2 and/or CC3 could function to recruit Rac in a Dock-independent fashion (Fan, 2003).

Regardless of whether the recruitment of Rac to Robo is dependent on Dock and Pak or is an independent event, the data cannot explain how Slit stimulation of Robo results in increased Rac activity. Two obvious types of molecules that are missing from the model and the protein complex are the upstream regulators of Rac, the GEF and GAP proteins. Intriguingly, in the course of a genome-wide analysis of all RhoGEFs and RhoGAPs in Drosophila, one Rac-specific GAP has been identified that when overexpressed results in phenotypes reminiscent of robo loss of function (H. Hu et al., submitted, reported in Fan, 2003). There are a number of candidate GEFs that could explain how Rac activity is upregulated by Slit activation of Robo, most notably Sos, rtGEF (pix), and Trio. It will be interesting to determine which if any of these molecules could play such a role in Robo signaling (Fan, 2003).

Son of sevenless directly links the Robo receptor to rac activation to control axon repulsion at the midline

Son of sevenless (Sos) is a dual specificity guanine nucleotide exchange factor (GEF) that regulates both Ras and Rho family GTPases and thus is uniquely poised to integrate signals that affect both gene expression and cytoskeletal reorganization. Sos is recruited to the plasma membrane, where it forms a ternary complex with the Roundabout receptor and the SH3-SH2 adaptor protein Dreadlocks (Dock) to regulate Rac-dependent cytoskeletal rearrangement in response to the Slit ligand. Intriguingly, the Ras and Rac-GEF activities of Sos can be uncoupled during Robo-mediated axon repulsion; Sos axon guidance function depends on its Rac-GEF activity, but not its Ras-GEF activity. These results provide in vivo evidence that the Ras and RhoGEF domains of Sos are separable signaling modules and support a model in which Robo recruits Sos to the membrane via Dock to activate Rac during midline repulsion (Yang, 2006).

Sos was identified in Drosophila as a GEF for Ras in the sevenless signaling pathway during the development of the Drosophila compound eye, where it activates the Ras signaling cascade to determine R7 photoreceptor specification. Studies in mammalian cell culture demonstrated that Sos functions as a GEF for both Ras and Rac in the growth factor-induced receptor tyrosine kinase (RTK) signaling cascade. Upon RTK activation, the SH3/SH2 adaptor protein Grb2/Drk recruits Sos to autophosphorylated receptors at the plasma membrane, where Sos activates membrane-bound Ras. In a later event downstream of RTK activation, Sos is thought to be targeted to submembrane actin filaments by interaction with another SH3 adaptor, E3b1(Abi-1), where Sos activates Rac . Whether the activation of Rac by Sos is strictly dependent on prior activation of Ras remains controversial, nor is it clear how Sos coordinates the activity of its two GEF domains in vivo (Yang, 2006 and references therein).

Evidence is provided that Sos functions as a Rac-specific GEF during Drosophila midline guidance. Sos is enriched in developing axons, and sos exhibits dosage-sensitive genetic interactions with slit and robo. Strikingly, genetic rescue experiments show that the Dbl homology (DH) RhoGEF domain of Sos, but not its RasGEF domain, is required for its midline guidance function. Biochemical experiments show that Sos physically associates with the Robo receptor through Dock in both mammalian cells and Drosophila embryos. Furthermore, Slit stimulation of cultured cells results in the rapid recruitment of Sos to membrane Robo receptors. These results provide a molecular link between the Robo receptor and Rac activation, reveal an independent in vivo axon guidance function of the DH RhoGEF domain of Sos, and support the model that Slit stimulation recruits Sos to the membrane Robo receptor via Dock to activate Rac-dependent cytoskeletal changes within the growth cone during axon repulsion (Yang, 2006).

These data support the idea that Sos provides a direct molecular link between the Robo receptor and the activation of Rac during Drosophila midline guidance. Genetic interactions between sos, robo, dock, crGAP/vilse, and the Rho family of small GTPases strongly suggest that Sos functions in vivo to regulate Rac activity during Robo signaling. Genetic rescue experiments indicate that sos is required specifically in neurons to mediate its axon guidance function. Furthermore, genetic data establish that, in the context of midline axon guidance, the Ras-GEF and Rac-GEF activities of Sos can be functionally uncoupled. Biochemical experiments in cultured cells and Drosophila embryos show that Sos is recruited into a multiprotein complex consisting of the Robo receptor, the SH3-SH2 adaptor protein Dock, and Sos, in which Dock bridges the physical association between Robo and Sos. Finally, experiments in cultured cells support the idea that Slit activation of Robo can recruit Sos to the submembrane actin cytoskeleton to regulate cell morphology. Together, these results suggest a model in which Slit stimulation recruits Sos to the Robo receptor via Dock to regulate Rac-dependent cytoskeletal changes within the growth cone during axon repulsion (Yang, 2006).

Based on previous work implicating rac in Robo repulsion, as well as in vitro studies demonstrating that Sos exhibits GEF activity for Rac, but not Rho or Cdc42, Rac seemed the most likely Sos substrate. However, rho has also been implicated in mediating Robo repulsion, and genetic interactions between sos and dominant-negative Rho have been interpreted to suggest that Sos could act as a GEF for Rho. This question was investigated further, and two types of genetic evidence have been presented that suggest that indeed Rac is the favored substrate of Sos. First, ectopic expression experiments in the eye reveal interactions exclusively between sos and rac. Second, genetic interaction experiments using loss of function mutations in rac and rho (rather than the more problematic dominant-negative forms of the GTPases) reveal strong dose-dependent interactions between sos and rac, but not sos and rho during midline axon guidance. Together, these observations argue in favor of Rac as the primary in vivo Sos substrate. Nevertheless, the possibilities that Sos also contributes to Rho activation and that the combined activation of Rac and Rho is instrumental in mediating the Robo response cannot be excluded (Yang, 2006).

Previous studies have demonstrated that Slit stimulation of the Robo receptor leads to a rapid increase in Rac activity in cultured cells. However, the mechanism by which Rac is activated downstream of Robo was not clear. This study provides direct genetic and biochemical evidence that Sos is coupled to the Robo receptor through the Dock/Nck SH3-SH2 adaptor, where it can regulate local Rac activation. Studies in cultured mammalian cells have highlighted the importance of distinct Sos/adaptor protein complexes in controlling the subcellular localization and substrate specificity of Sos. In the context of Rac activation, the E3b1 (Abi-1) adaptor has been shown to play a critical and rate-limiting role in Sos-dependent Rac activation and subsequent formation of membrane ruffles (Innocenti, 2002). Could Sos regulation of Rac activity during Robo repulsion be similarly limited by the availability of specific adaptor proteins? It is interesting to note in this context that overexpression of dock does not lead to ectopic axon repulsion, suggesting that Dock may not be limiting for Robo signaling. However, although dock mutants do have phenotypes indicative of reduced Robo repulsion, their phenotype is considerably milder than that seen in robo mutants, raising the possibility that there may be additional links between Robo and Sos (Yang, 2006).

A number of studies in cultured mammalian cells have suggested that Rac activation induced by activated growth factor receptors requires the prior activation of Ras. For example, PDGF-induced membrane ruffling can be promoted or inhibited by expression of constitutively active or dominant-negative Ras, respectively. However, other studies have suggested that in Swiss 3T3 cell lines RTK activation of Rac is Ras independent. In addition, the observation that Ras activation and Rac activation display very different kinetics, with Rac activation persisting long after Ras activity has returned to basal levels, has been used to argue against an obligate role for Ras in Rac activation. In this study, using a genetic rescue approach, whether the ability of Sos to activate Rac during axon guidance in an intact organism requires its Ras-GEF function was directly tested. Genetic data indicate that the RasGEF domain of Sos is dispensable for axon guidance, while the DH RhoGEF domain is strictly required. This observation argues strongly in favor of the model that in vivo Sos activation of Rac does not strictly require Sos activation of Ras (Yang, 2006).

It is clear that subcellular localization plays a major role in regulating Sos activity and that different protein complexes containing Sos exist in different locations in the cell. This study has shown that activation of the Robo receptor by Slit triggers the recruitment of Sos to Robo receptors at the plasma membrane. Biochemical data argue that the adaptor Dock/Nck is instrumental in bridging this interaction, and given the diverse interactions between Dock/Nck and guidance receptors, it seems likely that Dock/Nck could fulfill this role in many guidance receptor contexts. This bridging function of Dock/Nck and guidance receptors is analogous to the role of Grb2 for growth factor receptors only insomuch as it brings signaling molecules to the receptor—the mechanism of interaction is distinct, since it is mediated through SH3 domain contacts rather than SH2/phosphotyrosine interactions. These observations suggest that there may be an additional pool of Sos that can function in a distinct adaptor protein/guidance receptor complex to regulate cell morphology in response to extracellular guidance cues (Yang, 2006).

Is regulating subcellular localization the only mechanism by which Sos activity is controlled? This seems unlikely. Indeed, a recent study has implicated tyrosine phosphorylation of Sos by Abl as an additional mechanism to activate the Rac-specific GEF activity of Sos in vertebrate cell culture models. This raises the intriguing possibility that Abl may fulfill a similar role for Robo signaling. This is a particularly appealing idea given the well-documented genetic and physical interactions between Robo and Abl. Indeed, sos and abl exhibit dose-dependent genetic interactions during midline axon guidance. A clear genetic test of whether Abl activates the Rac-GEF activity of Sos downstream of Robo may be complicated by the fact that Abl appears to play a dual role in Robo repulsion: both increasing and decreasing abl function lead to disruptions in Robo function. Nevertheless, it should be possible in the future to generate mutant versions of Sos that are refractory to Abl activation and to test whether these alterations disrupt the Sos guidance function. It will also be of great interest to determine whether the redistribution of Sos can also be observed in response to guidance receptor signaling in navigating growth cones, and if so, then what changes in actin dynamics and growth cone behavior are elicited (Yang, 2006).

Slit and Robo control cardiac cell polarity and morphogenesis

Basic aspects of heart morphogenesis involving migration, cell polarization, tissue alignment, and lumen formation may be conserved between Drosophila and humans, but little is known about the mechanisms that orchestrate the assembly of the heart tube in either organism. The extracellular-matrix molecule Slit and its Robo-family receptors are conserved regulators of axonal guidance. This study reports a novel role for the Drosophila slit, robo, and robo2 genes in heart morphogenesis. Slit and Robo proteins specifically accumulate at the dorsal midline between the bilateral myocardial progenitors forming a linear tube. Manipulation of Slit localization or its overexpression causes disruption in heart tube alignment and assembly, and slit-deficient hearts show disruptions in cell-polarity marker localization within the myocardium. Similar phenotypes are observed when Robo and Robo2 are manipulated. Rescue experiments suggest that Slit is secreted from the myocardial progenitors and that Robo and Robo2 act in myocardial and pericardial cells, respectively. Genetic interactions suggest a cardiac morphogenesis network involving Slit/Robo, cell-polarity proteins, and other membrane-associated proteins. It is concluded that Slit and Robo proteins contribute significantly to Drosophila heart morphogenesis by guiding heart cell alignment and adhesion and/or by inhibiting cell mixing between the bilateral compartments of heart cell progenitors and ensuring proper polarity of the myocardial epithelium (Qian, 2005).

Early embryonic events of heart formation are remarkably similar between Drosophila and vertebrates, in that two bilaterally symmetrical strips of precardiac mesoderm fuse as a linear tube at the ventral or dorsal midline in both systems. Although there is much interest in understanding the basis of heart-tube assembly, little is known about the underlying molecular-genetic mechanisms that orchestrate this and other morphogenetic processes. Drosophila Slit, an EGF- and LRR-containing secreted protein, is expressed in the heart, and thus may participate in heart morphogenesis. Slit functions as a repulsive ligand for the Roundabout (Robo) family of receptors in the CNS and acts both attractively and repulsively in trachea and somatic muscles. In vertebrates, there are three slit and three robo genes. Among them, Slit3 is expressed prominently in the developing atrial walls of the murine heart. A Slit3 gene-trap mouse exhibits abnormal heart formation, including an apparent enlargement of the right ventricle. Whether or not this heart defect is secondary to other embryonic defects is not known, nor is the genetic or cellular mechanism underlying this phenotype. It is also not known which of the Robo receptors and other Slit proteins play a role in heart development (Qian, 2005).

To assess the role of Slit in Drosophila heart, slit null-mutant embryos (slit²) were analyzed by labeling the heart with antibodies against Dmef2, a muscle-specific transcription factor expressed in all myocardial and other muscle cells. When the bilateral rows of myocardial progenitors have reached the dorsal midline, they fail to align properly in slit mutants compared to wild-type. A similar phenotype is observed in robo,robo2 double-mutant embryos. In contrast, only subtle alignment defects are found in robo or robo2 single mutants. Unlike robo or robo2,robo3 mutants in combination with robo or robo2 do not cause additional heart defects, and thus robo3 is unlikely involved in cardiac development. Similar alignment phenotypes were observed with nmr^H15lacZ reporter, a marker for myocardial nuclei, in slit mutants. Although the dorsal migration of the myocardial progenitors does not seem to be affected, their highly regular arrangement is already perturbed before they reach the midline, as manifested in gaps and double rows. Visualization of the pericardial cells with Zfh-1 shows that their alignment with the myocardial cells is also perturbed in slit-robo mutants. At stage 16, two types of phenotypes can be distinguished: Type I consists of irregular cell arrangements, and type II, in addition, has large gaps. These two types of phenotypes are found in roughly equal proportion in slit and robo,robo2 double mutants. These defects are unlikely caused by abnormalities in cardiac lineage specification or in ectodermal epithelium formation during dorsal closure (Qian, 2005).

Given the cardiac abnormalities of slit and robo mutants, the expression pattern of slit and its receptors in the developing heart were examined. Slit protein is first detected in the heart at stage 14, uniformly distributed within the myocardial cytoplasm. As the bilateral rows of cardiac progenitors align at the dorsal midline, Slit accumulates at the contact sites between them. Like Slit, Robo initially displays a similarly uniform cortical localization within myocardial cells. Once they reach the midline, Robo enriches strongly at the dorsal (apical) surface of the cell. In contrast, Robo2 is present in pericardial cells located ventrally to the myocardial cells. Unlike Slit and Robo, Robo2 does not accumulate at the midline but remains in pericardial cells. In robo mutants, however, robo2 is ectopically expressed in myocardial cells and enriches at the dorsal midline, similar to Robo in wild-type embryos. Thus, robo2 apparently compensates for a myocardial loss-of-robo function, and this compensation is consistent with their redundant requirement in cardiac morphogenesis (Qian, 2005).

Although Slit and Robo are indeed expressed in the heart, indirect effects cannot be ruled out because they function in multiple tissues. To address whether slit/robo acts autonomously within the heart, tissue- and cell-type-specific rescue experiments were performed. slit and robo expression within myocardial cells is sufficient to rescue the slit and robo,robo2 phenotype, respectively, in promoting normal heart morphogenesis (Qian, 2005).

Because slit and robo are expressed at the cardiac midline and are required for heart cell alignment, it was asked if local mislocalization of these proteins also causes cardiac morphogenesis defects. Myocardial-specific (tinCΔ4-driver) or pan-mesodermal (twi24B-driver) overexpression of slit does not produce significant cardiac alignment defects or only with low penetrance, suggesting that augmenting Slit levels in myocardial cells hardly perturbs cardiac cell alignment. Mesodermal robo overexpression, however, results in frequent alignment defects, as does ectopic expression of slit in pericardial cells. Interestingly, in those embryos that exhibit virtually normal cardiac alignment, Slit accumulates continuously at the cardiac midline. In contrast, the embryos with significant abnormalities also mispattern Slit. Precise midline accumulation of Slit thus seems to be critical to correctly align and assemble the heart tube (Qian, 2005).

Because similar cardiac misalignment defects occur in robo,robo2 as in slit mutants, it was asked whether Slit accumulation is affected in robo,robo2 embryos. Indeed, without robo and robo2, Slit no longer concentrates evenly at the contact point between the myocardial cells. Thus, loss of Robo receptors compromises Slit accumulation at the dorsal midline. When robo2 is misexpressed in myocardial cells by using tinCΔ4-Gal4, a premature midline accumulation of Slit is observed, and upon contact of the bilateral cardiac rows, Slit no longer concentrates at the cardiac midline. It may be also that misexpressed Robo2 receptors trap Slit in the cytoplasm and prevent its proper secretion. When Robo or Robo2 is expressed throughout the mesoderm, the Slit pattern is also severely disrupted, and the heart tube is frequently misaligned. Because pan-mesodermal expression of slit is of little consequence, it may be that the localization of Robo is crucial for Slit accumulation at the midline. However, slit mutants do not exhibit correct Robo patterning either, thus implying that slit is necessary but not sufficient (or instructive) for Robo localization (Qian, 2005).

Previous reports suggest a role of cardiac cell-polarity acquisition in heart morphogenesis. Failure to correctly polarize the cardiac epithelium may result in misalignments that are independent of the earlier specification and differentiation events. To study the polarity of the cardiac epithelium in slit mutants, Dlg was examined. Dlg localizes to the baso-lateral sides of myocardial epithelium before contact of the bilateral rows is established, and to the apical-lateral sides after contact. Unlike in the dorsal ectoderm, cardiac Dlg localization is severely compromised in slit mutants as the bilateral heart primordia come in contact. Because a polarity phenotype is manifest only upon heart-tube assembly, slit does not appear to be required for guiding the cardiac epithelium to the dorsal midline or for initiating its polarity before contact, but rather for correctly switching its polarity from basal-lateral to apical-lateral. Examination of myocardial polarity of slit mutants with two other basal-lateral to apical-lateral makers, α-Spectrin and Armadillo, shows defects similar to those observed with Dlg. In addition, the transmembrane protein Toll, which is present on the apical-lateral surface of myocardial cells during, but not before, the cardiac alignment process, was examined. As with Dlg, α-Spectrin, and Armadillo, Toll protein is no longer restricted to the apical-lateral sides of the myocardial cells in slit mutants. Toll mislocalization can be rescued by expressing a slit transgene in the hearts of slit mutants. The disruption in apical-lateral patterning of all cell-polarity makers examined suggests an important function of slit in polarity acquisition and maintenance. Consistent with this conclusion is the accumulation of Slit and Robo at the dorsal myocardial midline, which potentially mediates the switch in myocardial cell polarity as a prerequisite for heart-tube formation (Qian, 2005).

In contrast to the apical-lateral localization of the previous markers, Dystroglycan (Dg) is heavily enriched at both apical and basal sides of myocardial membrane, but is excluded laterally. Interestingly, in slit mutant hearts, polarized Dg localization does not seem to be significantly altered despite the severe cardiac morphogenetic defects. This is in contrast to Tbx20 neuromancer (nmr) mutants, in which myocardial polarity is also disrupted, including Dg localization (Qian, 2005).

It was anticipated that there are numerous molecules involved in generating or maintaining cardiac cell polarity in conjunction with slit/robo during heart morphogenesis, but mutants of some key factors may be early lethal or have pleiotropic effects. Thus, genetic interactions between cell-polarity genes and slit were examined in relation to cardiac morphogenesis. For this purpose, various transheterozygous combinations between were made between slit and polarity genes that are expressed in the heart, including dg, dlg, and shortgun (shg), encoding E-cadherin, and mutants previously shown to have cardiac defects. As single heterozygotes, they do not have detectable heart abnormalities, but removal of one copy of slit and dg, shg, or dlg results in defective cardiac morphogenesis. In contrast, crumbs(crb) does not interact with slit in the heart, which is consistent with the lack of (polarized) Crb localization in the cardiac epithelium. Taken together, these observations suggest that slit and cell-polarity genes cooperate in aligning the myocardium. Slit/Robo localization is also perturbed in nmr mutants, suggesting that Tbx20-mediated transcriptional activities also influence Slit/Robo localization in the heart (Qian, 2005).

Slit is well known as a repellent signal that emanates from the CNS midline and patterns axon tracks, muscles, and tracheal branches. Slit can also act as an attractant, but in all cases seems to be secreted from another cell type from its receptors. In contrast, during Drosophila heart morphogenesis, both Slit and Robo originate from the same cells, i.e., from the cardiomyocytes as they align at the dorsal midline. During this apparently autocrine process, Slit ligands and Robo receptors relocalize from the myocardial circumference to accumulate between the bilateral cell rows, mediating aligned adhesion between these rows. It is presently unknown how Slit and Robo relocalize to the apical side of the heart, but this process is likely to require the function of cell-polarity genes, such as dlg and others, that genetically interact with slit and are repolarized themselves. It may also be that a Slit molecule can bind Robo receptors on both sides of the midline, perhaps in a cooperative manner, which would then lead to a progressive accumulation of both receptors and ligands at the midline and thus to a precise alignment of the bilateral rows. This is reminiscent of the attractive Robo-Slit interaction during muscle patterning: Robo is made in the muscles of adjacent segments and accumulates at the Slit-secreting muscle-attachment sites between the segments. Regardless of the difference in cellular origin, Slit may bind Robo receptors across the segment boundary, just Slit may interact with Robo proteins across the midline between the myocardial rows. Such a Robo-Slit-mediated adhesion process is also consistent with the observed myocardial-epithelium repolarization, which would bring the bilateral rows of cells in close proximity. In slit mutants, morphogenetic defects not only include failed alignments but also double alignments and intercalation. Thus, mutant cardiomyocytes often reach the midline and get in close proximity with the contralateral side but then seem to intermix. Therefore, it is proposed that Robo-Slit act as heterophilic cell-adhesion molecules mediating coordinated stereotyped alignment as well as inhibiting cell mixing. In conclusion, it is proposed that Slit/Robo proteins act in concert with cell-polarity genes in guiding and maintaining myocardial (and pericardial) cell alignment, which is likely a prerequisite for later morphogenetic events, such as formation of a continuous cardiac lumen precisely at the position of Slit localization (Qian, 2005).

Pupal

Positioning sensory terminals in the olfactory lobe of Drosophila by Robo signaling

Olfactory receptor neurons and the interneurons of the olfactory lobe are organized in distinct units called glomeruli. Expression patterns and genetic analysis has been used to demonstrate that a combinatorial code of Roundabout (Robo) receptors act to position sensory terminals within the olfactory lobe. Groups of sensory neurons possess distinct blends of Robo and Robo3 and disruption of levels by loss-of-function or ectopic expression results in aberrant targeting. In wild type, most of the neurons send collateral branches to the contralateral lobe. The data suggest that guidance of axons across brain hemispheres is mediated by Slit-dependent Robo2 signaling. The location of sensory arbors at distinct positions within the lobe allows short-range interactions with projection neurons leading to formation of the glomeruli (Jhaveri, 2004).

The Drosophila olfactory lobe is composed of about 50 glomeruli located at fixed positions within the mediolateral, anteroposterior and dorsoventral axis. Sensory neurons expressing a given candidate odorant receptor target to the same glomeruli and also send projections to the contralateral lobe. Adult olfactory neurons differentiate within the first one-third of pupal life, radiate over the lobe anlage and transit across the midline. Sensory neurons invade the lobe during the next one-third of pupation and form distinct glomeruli (Jhaveri, 2004).

Antibodies against the three Robo receptors were used to examine their localization in olfactory neurons during pupal life. The patterns of Robo, Robo2 (Leak -- FlyBase) and Robo3 are rather dynamic and appear markedly different when examined early during lobe development, when compared with later after glomeruli are formed. During the first ~20 hours after puparium formation (APF), when the olfactory neurons are on the surface but have not yet invaded the lobe, Robo is expressed uniformly on all afferent axons. Robo2 is present at low levels in all neurons but is enriched in regions lateral to the commissure. A careful examination of confocal sections through a number of pupal lobes stained with anti-Robo2 suggests that immunoreactivity is lower as axons transit the midline than just prior to/after crossover. Expression of Robo2 declines in older pupae and is no longer detectable by ~40 hours APF. Axons that express high Robo3, lie at more medial positions in the outer nerve layer. The analysis of patterns of expression indicates that populations of neurons possess unique combinations of Robo, Robo2 and Robo3 that change during development (Jhaveri, 2004).

robo3 expression in the embryonic peripheral nervous system has been shown to be regulated by the proneural gene atonal (ato). In the adult olfactory system, ato specifies a subset of neurons that are the first to develop and appear to guide the rest of the axons into the lobe. In ato1/Df(3R)p13 animals, these 'pioneers' fail to form and the rest of the neurons stall at the entry to the olfactory lobe. A subset of the Ato-independent neurons express Robo3. Furthermore, only a subset of the Ato-dependent neurons visualized by Ato::GFP express Robo3. As expected, these occupy medial positions in the outer nerve layer. These data together suggest that Robo3 is not expressed in genetically defined subset of neurons in the pupal olfactory system (Jhaveri, 2004).

Sensory neurons begin to invade the lobe from about 25 hours APF and the first signs of glomerular organization become apparent by around 36 hours APF. Glomerular formation occurs sequentially and by 60 hours APF most of the glomeruli have formed. The entry of glial cell processes and concomitant increase in lobe volume, results in some re-organization of glomerular position and the adult pattern can only be discerned by about 80 hours APF. Robo and Robo3 are enriched in subsets of sensory neurons as they terminate within the lobe. Robo is detected in most axons, although at differing levels, while Robo3 is strongly enriched in terminals within a smaller number of glomeruli. A comparison of stained 60 hour APF lobes with the adult glomerular map suggests that Robo3-expressing neurons tend to preferentially target more dorsomedial locations. An estimation of Robo and Robo3 immunoreactivity in identified glomeruli supports the idea of a combinatorial code in determining sensory neuron position (Jhaveri, 2004).

Brains at different pupal ages were stained with antibodies against the secreted ligand Slit. A sheet of cells in the midline of the sub-esophageal ganglion expresses high levels of Slit. Immunoreactivity declines in later pupae (after 60 hours APF) and is absent in the adult. The midline cells do not express the glial marker Reverse Polarity (Repo). Other regions in the midbrain closely associated with groups of Repo-positive glial cells were also labeled by anti-Slit. The diffuse nature of the staining makes it difficult to ascertain whether the glia are the source of secreted Slit in the midbrain. At 20 hours APF, the boundaries of the olfactory lobes are clearly demarcated by the presence of surrounding glial cells. Slit levels within the lobe neuropil is significantly higher than that of the background. Expression can be detected from 14 hour APF and begins to decline by 60 hours APF (Jhaveri, 2004).

The MARCM method combined with ey-FLP generates large patches of homozygous tissue in the eye-antennal disc. Since flip-out occurs early, phenotypes generated in mature neurons result from a lack of gene function from the beginning of differentiation. Clones of robo2¹ and robo3¹ were generated and targeting of a small number of sensory neurons marked by the Or22a-Gal4 transgene was examined. Sensory neurons expressing Or22a normally project to glomerulus designated DM2 and cross-over to the contralateral lobe in the inter-antennal commissure (Jhaveri, 2004).

Neurons lacking Robo2 function (robo2¹ clones) fail to cross over to the contralateral lobe and terminate at the midline forming small 'glomerular-like' structures. The terminals show immunoreactivity against the synaptic marker nc82. Targeting to DM2 occurs normally although in many (13 out of 16) cases the glomeruli appear less intensely innervated by GFP-expressing neurons. Loss of Robo3 function (robo3¹ clones), however, affected targeting of axons rather dramatically. In all cases, some mutant neurons did project correctly to DM2 although a subset of axons strayed to ectopic sites. Commissure formation was unaffected. The erroneously placed terminals formed 'glomerular-like' organizations as revealed by staining with mAbnc82, but these did not correspond in shape or position to those previously identified. A large irregular shaped 'glomerulus' located ventrally in the posterior region of the lobe was most frequently observed. In about half the preparations, an additional site was observed in a dorsolateral location. Such ectopic targets were never found in control animals carrying the or22a-Gal4 (14.6) transgene (Jhaveri, 2004).

Because Robo is expressed rather generally in olfactory neurons, loss-of-function was studied by targeted misexpression of antagonists of signaling, rather than in clones. SG18.1-Gal4 expresses in a large fraction of olfactory neurons thus revealing most of the glomeruli as well as the antennal commissure. Ectopic expression of commissureless (comm) using SG18.1-Gal4 resulted in disorganization of glomerular patterning with a weak effect on the commissure. Comm has been shown to downregulate Robo, although its effect on Robo2 and Robo3 is less well understood. The phenotype of Comm ectopic expression suggests that Robo is necessary for determining sensory neuron position within the lobe. Abelson kinase (Abl) phosphorylates the CC0 and CC1 domains of Robo, thus antagonizing signaling. Ectopic expression of either Abl or a constitutively active Dcdc42^v12 completely abolishes glomerular formation. Sensory neurons expressing Dcdc42^v12 (SG18.1::Dcdc42^v12) show an attraction for the midline and terminate there forming 'glomerular-like' structures at the midline. Results from loss-of-function clones predict such a phenotype for robo2 nulls. Constitutive activation of Dcdc42 is known to affect cytosketal dynamics generally, and could phenocopy a loss-of-function of all Robo receptors (Jhaveri, 2004).

Ectopic expression demonstrates that levels and location of Robo receptor expression are important for three-dimensional patterning of sensory terminals. Robo was ectopically in sensory neurons to test whether the domains and levels of receptors are instructive in positioning of sensory terminals within the lobe. SG18.1::GFP was used to drive Robo in olfactory neurons; the positions and morphology of glomeruli could be visualized by GFP. Robo is expressed endogenously in all olfactory neurons and the small increase in level caused by driving a single copy of the UAS-robo transgene did not significantly alter lobe morphology. Higher levels achieved by driving three copies of the transgene abrogated glomerular formation. Changing the nature of the Robo code by misexpressing Robo3, however, resulted in a dorsomedial shift of projections. The commissure forms normally when either Robo or Robo3 are misexpressed. Ectopic expression of Robo2, however, completely abolishes commissure formation with a less severe effect on glomerular morphology (Jhaveri, 2004).

Whether the genetic elements participating with Robo signaling in other well-studied systems also operate in the Drosophila adult olfactory system was also tested. A deficiency for the Slit region was crossed into an SG18.1 UAS-GFP UAS-robo2 recombinant. In this situation, where endogenous levels of the ligand were reduced by 50%, commissure formation, which is disrupted by the ectopic expression of Robo2, was restored and glomerular morphology also returned to normal. Targeted down-regulation of Robo signaling by misexpression of Comm or activated Dcdc42^v12, respectively, also suppress the phenotype caused by elevated Robo2 (Jhaveri, 2004).

These data argue that sensory neuron positioning within the lobe is determined by signaling through the Robo receptors. Reduction of Slit levels suppress the effect of receptor overexpression, demonstrating that the phenotypes are mediated through endogenous ligand. In this case, alteration of the geometry of the Slit gradient by misexpression would be expected to alter terminal positioning of sensory neurons. High Slit expression was driven in glial cells around and within the lobe using loco-Gal4. Staining of the adult lobes in these animals with an antibody against the synaptic marker mAbnc82 revealed the presence of ectopic glomeruli outside the normal lobe circumference. Increasing Slit levels further using multiple copies of the transgene led to more severe effects (Jhaveri, 2004).

The model proposes that olfactory neurons traveling in the outer nerve layer possess a different combination of Robo receptors that respond to Slit by branching into the lobe and arborizing at specific positions. In order to understand this positional code, a Gal4 line was selected that would allow expression in a set of neurons projecting to identified glomeruli to be driven from early during development. lz-Gal4;UAS-GFP labels two glomeruli -- DM6 and DL3 -- during development and in the adult brain, thus providing a means to examine the location of selective sensory neurons when the combinations of Robo are altered. A change in the levels of any of the three Robo receptors, caused by misexpression using the lz-Gal4 driver, altered the positions of these identified terminals. The phenotypes showed variable expressivity; however, it was possible to categorize preferred positions for the terminals in each treatment (Jhaveri, 2004).

Elevated Robo levels shift DL3/DM6 neurons to more central locations. Robo3 misexpression shifted the positions of the arbors most frequently to a mediodorsal axis. Large irregular-shaped glomeruli were frequently observed. The changes in neuronal positions observed by Robo2 misexpression were somewhat surprising given the hypothesis that Robo2 is involved largely in commissure formation. It is suggested that high levels of Robo2 induced by lz-Gal4 could interfere with the function of endogenous receptors. Robo2 misexpression most frequently produced cases where projections were seen terminating within a single lobe (Jhaveri, 2004).

The ectopic 'glomeruli' produced by alterations in the Robo code showed a normal organization of cellular elements. In the wild type, terminal branches of sensory neurons remain at the periphery of each glomerulus. Dendritic arbors of the lobe interneurons, filled the entire glomerulus as seen by GFP driven by GH146-Gal4 or the synapse specific marker mAbnc82. Glomeruli produced by misexpression of any of the Robo receptors also showed a similar organization as evidenced by mAbnc82 staining (Jhaveri, 2004).

This expression and genetic data suggests a model for axon guidance in the olfactory lobe. Neurons arriving at the olfactory lobe in the antennal nerve express Robo, and those expressing high levels of Robo3 additionally decussate onto the medial side of the outer nerve layer. The position of an axon in the nerve layer is influenced by Slit levels, although the identity of the cells that contribute Slit still needs to be elucidated. Several regions of Slit expression have been detected in the brain, although the cells at the midline express highest levels. Robo2, which is expressed at very low levels in all sensory neurons, is elevated after the axons cross the midline thereby preventing re-crossing. Later during pupation, sensory axons branch into the lobe and terminate at distinctive positions regulated by their unique Robo code in response to Slit levels. This allows short-range interactions with the dendritic arbors of projection neurons leading to formation of glomeruli (Jhaveri, 2004).

Slit and Robo control the development of dendrites in Drosophila CNS

The molecular mechanisms that generate dendrites in the CNS are poorly understood. The diffusible signal molecule Slit and the neuronally expressed receptor Robo mediate growth cone collapse in vivo. However, in cultured neurons, these molecules promote dendritic development. This study examined the aCC motoneuron, one of the first CNS neurons to generate dendrites in Drosophila. Slit displays a dynamic concentration topography that prefigures aCC dendrogenesis. Genetic deletion of Slit leads to complete loss of aCC dendrites. Robo is cell-autonomously required in aCC motoneurons to develop dendrites. These results demonstrate that Slit and Robo control the development of dendrites in the embryonic CNS (Furrer, 2007).

Previous studies have suggested that Slit and Robo promote collateral neurite formation in cultured neurons. The goal of this study was to examine the role of Slit and Robo in the context of in vivo dendrogenesis in the CNS. Dendrogenesis is a late-stage event in the differentiation of neurons. Thus, to uncover the specific role of molecules responsible for dendrogenesis, one must not only demonstrate their loss-of-function phenotype but also isolate their cell-autonomous operation. Furthermore, it is also necessary to uncouple the earlier contribution of the molecules, to either neurogenesis or axogenesis, from their direct contribution during dendrogenesis. Focus was placed on the aCC motoneuron, one of the first CNS neurons to generate dendrites in Drosophila embryos and also one that can be genetically manipulated and visualized at the single-cell level. The results support the conclusion that in neurons Slit, signaling through Robo, is responsible for controlling the timing, positioning, and size of dendrites in the embryonic CNS. They also offer insights into the complexity that surrounds the development of dendrites in vivo (Furrer, 2007).

In robo/robo embryos, the aCC produces small dendrites this residual dendrogenesis reflects partial functional redundancy among Robo family receptors. RNAi against the robo gene in the aCC also results in small dendrites. Conversely, cell-specific resupply of wild-type Robo in the aCC reinstates its ability to grow dendrites. These results, together with the fact that the aCC neurons in robo/robo embryos have no other defects prior to the onset of dendrogenesis, support the specific role of Robo in dendritic development (Furrer, 2007).

The dendrogenic role of Robo was first demonstrated by Whitford (2002). The key experiment was inhibition of neurite branching in cultured neurons through overexpression of the cytoplasmically truncated Robo. Similar attempts to use a Drosophila version of cytoplasmically truncated Robo have failed to induce any extra or abnormal dendrogenesis in vivo. Instead, it was shown that both genetic deletion and RNAi against the robo gene cause dendrogenesis defects in uniquely identified CNS neurons. The difference in effectiveness of dominant-negative proteins between the mammalian and Drosophila neurons might simply reflect whether or not Robo is a rate-limiting factor in a given neuron (Furrer, 2007).

Robo is expressed throughout neuronal development, not just during the period of axon guidance analyzed by the majority of in vivo studies to date. Single-cell analyses in the embryonic Drosophila CNS have shown a role for Robo in directing growth cones away from the Slit-secreting midline. Without Robo, the axons of RP2 motoneurons are misguided medially. Later, the same Robo-lacking RP2 neurons also misguide their dendritic growth cones towards the midline (Furrer, 2003). In comparison to RP2, aCC motoneurons do not normally rely on Robo to properly orient axonal and dendritic growth cones. However, when Robo is overexpressed in the aCC, its dendritic growth cone can be made to avoid the midline. In all these cases, it would appear that Robo causes growth cone collapse upon detecting Slit at the midline. By contrast, this study supports a role for Robo in promoting the formation of collateral dendritic processes. aCC motoneurons cell-autonomously require Robo during dendrogenesis. Clearly, the same receptor has distinct roles, either collapsing growth cones or promoting collateral dendrogenesis, i.e. two seemingly opposite types of cellular responses, sometimes even within a single neuron. Although the underlying mechanism is not yet known, it is intriguing that migrating myoblasts also exhibit a developmentally regulated response switch of Slit-Robo signaling from repulsion to attraction in Drosophila embryos (Furrer, 2007).

The Slit concentration topography of the embryonic CNS exhibits a dynamic four-dimensionality. Previously, it was postulated that there is a descending gradient of Slit from its source. Indeed, both in culture media and within imaginal discs, diffusible signaling molecules set up gradients that descend from their source. The actual Slit topography in the embryonic CNS is much more complex. Unlike culture media, the embryonic CNS redistributes molecules such as Netrin and Slit from their original source. Already by hour 14, the time when the first dendrites begin to form, a prominent secondary accumulation of Slit is present locally 10-20 µm away from the midline source of Slit. The local concentration is approximately 43% of that at the midline, and the amount of Slit that is found beyond 10 µm from the midline, the local minimum, is 56% of the total extracellular Slit in the whole CNS (Furrer, 2007).

How does Slit reach the neuropil in such abundance? Slit could accumulate there either through diffusion or direct filopodia-mediated delivery. Once there, Syndecan plays a role in capturing the extracellular Slit. The current study suggests that the presence of Robo at the neuropil also contributes to Slit capture on cell surface. In addition to Syndecan and Robo, at least two more Slit receptors, Robo2 and Robo3, are known in Drosophila. When bound to such receptors on the surface of migrating axons, Slit could be transported along commissural and longitudinal fascicles. Individual Robo receptors are expressed in overlapping but distinct sets of neurons. Plenty of molecular heterogeneity and cellular dynamics exists within the developing nervous system that could contribute to an extensive redistribution of Slit within the CNS (Furrer, 2007).

It is proposed that neural development utilizes the complex Slit topography to control dendrogenesis. First, the position of the aCC collateral dendrogenesis coincides with the local Still accumulation. Except for the slit/slit embryos where there is no Slit present, all other genetic backgrounds examined in this study have aCC dendrites developing where Slit accumulates locally. Second, there is a positive correlation between the size of aCC dendrites and the amount of Slit present. A notable exception is the robo/robo embryos, in which the size of aCC dendrites is attenuated due to the loss of Robo, a Slit receptor, in the neuron. In Drosophila embryos, evidence for Slit proteolysis has been presented. Because the Slit antibody recognizes the carboxyl terminus region of the protein, it does not distinguish between full-length Slit, which is capable of activating Robo, and the carboxyl-terminus fragment of the proteolytic product, which is not. This study assessed the developmental control over Slit proteolysis. The quantification shows that, at hour 14, the proteolysis affects only about 8% of the total volume of Slit. Independent data also suggest that Slit at the neuropil and beyond is indeed in the form that is capable of stimulating Robo. In this study, it is assumed that a majority of Slit protein detected by the antibody in the neuropil is in the full-length form, and the positive correlation between the Slit profile and aCC dendrogenesis is taken to suggest that Slit acts in an instructive role, setting the size of dendrites. Also, the time at which Slit begins to accumulate at the emerging neuropil immediately precedes the initiation of collateral dendrogenesis in the aCC. This indicates that Slit accumulation is not simply a consequence of dendritic development. Instead, the tight spatiotemporal correlation between Slit topography and aCC dendrogenesis supports a model in which Slit plays a crucial role (Furrer, 2007).

The slit/slit phenotype during the period of dendrogenesis is dramatic. Visualization with the anti-HRP antibody and retrograde DiI labeling in late-stage slit/slit embryos reveals that many motoneurons extend out axons in the CNS without Slit. Yet, they fail to initiate dendrites. Thus, the phenotype that motoneurons such as aCCs and RP2s exhibit is unique. However, there is a problem in attributing a direct cause of the dendrite-less motoneurons to the absence of Slit. This is because slit/slit embryos form very few axon fascicles, resulting in a virtually neuropil-less CNS. Therefore, the dendrogenesis defects observed in slit/slit embryos could be accounted for by any of the following three scenarios: (1) the neuropil, not Slit, induces dendrogenesis, (2) Slit alone is required, or (3) both the neuropil and Slit are required. Of these, the third scenario is the most likely (Furrer, 2007).

Hints about the additional factors that impact dendrogenesis are available not only where neurons develop dendrites, but also where they do not. Except for slit/slit, all other genotypes examined in this study develop a neuropil in the CNS. In all cases, the aCC forms collateral dendrites at the neuropil, but not anywhere else. However, no neuron that extends its axon or dendrite across the midline develops dendritic branches at the midline despite the fact that the midline is the sole source of Slit in the CNS. Furthermore, it was found that, unlike dissociated neurons in culture, ectopic Slit presented outside of the CNS, at muscle-12, does not induce collateral dendrogenesis in aCC motoneurons. What are the factors that spatially restrict the dendrogenic function of Slit-Robo signaling to the neuropil? It is possible that such factors are present at the neuropil itself, serving a permissive role. However, it is also conceivable that the active suppression of dendrogenesis occurs outside the neuropil, including at the CNS midline and outside the CNS. In addition to such extrinsic factors, each neuron could display intrinsic molecular biases towards a certain portion of its axon. If this were true, then one might anticipate finding mutations that cause reduced dendrites at the neuropil, as well as mutations that cause ectopic collateral dendrogenesis outside the neuropil. Recently, several mutants have been found that fit both of these categories. Characterization of these mutations will not only help identify additional factors that impact dendrogenesis, but also offer insights into the general question of how spatiotemporal precision in dendrogenesis is regulated within the CNS (Furrer, 2007).

In the developing Drosophila CNS, the initial Slit topography before hour 14 is relatively simple, with a single peak at the midline. There, Slit-Robo signaling repels axonal growth cones from the midline and coordinates positioning of longitudinal fascicles. As long as the midline peak persists, it continues to repel dendritic growth cones. However, at the emerging neuropil, the concentration of extracellular Slit also rises steadily, creating a second Slit-enriched region within the developing CNS. Here, Slit-Robo signaling has an additional role as a promoter for dendrogenesis. Thus, the same Slit-Robo signaling that repels growth cones from the midline, also produces dendrites at the neuropil, thereby sculpting the neural architecture at multiple stages. How various molecules that are known to impact dendritic morphology may be linked to Slit-Robo signaling remains an open question. Future study is needed to address how Slit and its receptor Robo collaborate with diverse signaling partners at multiple steps of neural development, to serve as the 'architects' of the developing CNS (Furrer, 2007).

Effects of Mutation or Deletion

slit mutants exhibit disruptions in midline neuronal precursors. Some cells are absent and others die in abnormal positions along the ventral surface of the nerve cord (Rothberg, 1990). This results in a collapse and fusion in longitudinal connectives in the midline similar to what is seen in single-minded mutants (Jacobs, 1993 and Sonnenfeld, 1994).

Within cell lineages, cell death was examined in the midline of Drosophila embryos. Approximately 50% of cells within the anterior, middle and posterior midline glial (MGA, MGM and MGP) lineages die by apoptosis after separation of the commissural axon tracts. Glial apoptosis is blocked in embryos deficient for reaper, where greater than wild-type numbers of midline glia (MG) are present after stage 12. Quantitative studies reveal that MG death followed a consistent temporal pattern during embryogenesis. Apoptotic MG are expelled from the central nervous system and are subsequently engulfed by phagocytic haemocytes. MGA and MGM survival is apparently dependent upon proper axonal contact. In embryos mutant for the slit gene, MGA and MGM maintain contact with longitudinally and contralaterally projecting axons, and MG survival is comparable to that in wild-type embryos (Sonnenfeld, 1995).

Guidance of axons toward or away from the midline of the central nervous system during Drosophila embryogenesis reflects a balance of attractive and repulsive cues originating from the midline. Slit, a protein secreted by the midline glial cells provides a repulsive cue for the growth cones of axons and muscle cells. Embryos lacking slit function show a medial collapse of lateral axon tracts and ectopic midline crossing of ventral muscles. Transgene expression of slit in the midline restores axon patterning. Ectopic expression of slit inhibits formation of axon tracts at locations of high Slit production and misdirects axon tracts towards the midline. slit interacts genetically with roundabout, which encodes a putative receptor for growth cone repulsion (Battye, 1999).

The function of leak and kuzbanian during growth cone and cell migration. kuzbanian genetically interacts with slit

Axonal growth cones require an evolutionary conserved repulsive guidance system to ensure proper crossing of the CNS midline. In Drosophila, the Slit protein is a repulsive signal secreted by the midline glial cells. It binds to the Roundabout receptors, which are expressed on CNS axons in the longitudinal tracts but not in the commissural tracts. An analysis of the genes leak (referred to in much of the literature as roundabout 2) and kuzbanian is presented: both genes are involved in the repulsive guidance system operating at the CNS midline. Mutations in leak, were first recovered by Nusslein-Volhard (1984) based on defects in the larval cuticle. Analysis of the head phenotype suggests that slit may act as an attractive guidance cue while directing the movements of the dorsal ectodermal cell sheath. kuzbanian regulates midline crossing of CNS axons. It encodes a metalloprotease of the ADAM family and genetically interacts with slit. Expression of a dominant negative Kuzbanian protein in the CNS midline cells results in an abnormal midline crossing of axons and prevents the clearance of the Roundabout receptor from commissural axons. These analyses support a model in which Kuzbanian mediates the proteolytic activation of the Slit/Roundabout receptor complex (Schimmelpfeng, 2001).

Complementation analysis reveals that leak is indeed allelic to robo2, indicating that leak encodes robo2. Mutations in the slit gene, as with leak, were initially identified based on their common head phenotype (Nusslein-Volhard, 1984). The head defects of leak/robo2 are indicative for abnormal head involution. In wild type embryos this process starts during stage 11/12 when the brain neuroblasts invaginate into the interior of the embryo. Subsequently, a complex set of morphogenetic movements results in a closure of the gap dorsal to the forming brain lobes. The posterior dorsal ectoderm continues to extend anteriorly and together with the adverse movement of the dorsal clypeolabrum leads to the formation of the dorsal pouch. At the end of embryogenesis the dorsal ectoderm has enclosed the anterior tip of the embryo. slit and leak are expressed in different ectodermal domains of the head. Digoxygenin labeled anti-sense slit RNA was used to probe slit expression during head development. slit expression can be observed in the ectoderm just ventral to the invaginating foregut as soon as the CNS midline expression of slit can be detected. In addition, slit expression is detected in the clypeolabrum. In stage 12 embryos three expression domains can be recognized. By stage 13 the middle domain resolves into two distinct domains. Domain 'd' has broad lateral extension. In stage 16 embryos, domains 'a-c' are found inside the embryo. Also, ectodermal slit expression in domain 'd' is found dorsal to the pharynx musculature in the dorsal pouch (Schimmelpfeng, 2001).

Leak protein expression was monitored using a polyclonal antiserum. In wild type stage 12 embryos high levels of Leak protein are found ventrally to the foregut adjacent to but in parts overlapping with the slit-expressing domain. This expression mode is different from the ventral nerve cord where Leak protein is excluded from the midline. However, during stage 13/14 Leak expression is lost in the ectodermal midline cells in the head. During head involution, the dorsal ectoderm slides over the forming brain lobes and over the developing foregut region. High levels of Leak expression are found in the leading edge cells of the migrating dorsal ectoderm. In stage 17 embryos Leak expression prefigures the forming mouth hooks (Schimmelpfeng, 2001).

Within the ventral nerve cord of slit mutant embryos, the ventral expression domain of Leak shifts toward the midline. Interestingly, Leak expression in the anterior ventral ectoderm appears to remain unchanged. In the dorsal expression domain, leak expressing ectodermal cells do not migrate as fast toward the anterior as they do in the wild type. In fact, in slit mutant embryos the dorsal ectoderm fails to migrate toward the anterior tip of the embryo. In addition, Leak expressing cells do not properly prefigure the mouth hooks. The medial connection is not formed but rather one can observe that the Leak expressing cells fan out laterally (Schimmelpfeng, 2001).

In summary, Leak appears to act as a Slit receptor during head involution. During this process it controls morphogenetic movements rather than directed growth of axons or filopodia. The observed migration defects in slit mutants may indicate that Slit/Leak interaction does not necessarily lead to a repulsive signaling mechanism (Schimmelpfeng, 2001).

In addition to its role during axon guidance, leak also functions during cell migration. About 20 years ago, mutations in leak were identified based on their characteristic head defects that suggested a function during head involution, a process that requires morphogenetic cell movements. The role of Roundabout receptors in cell migration is not surprising since mutations in the Roundabout ligand Slit result in defects in the migration of mesodermal cells. slit mutants also exhibit similar defects in head involution as seen in leak mutant embryos (Nusslein-Volhard, 1984). Similarly, slit function in vertebrates has been associated with cell migration. The phenotypic analysis of the slit/leak mutant phenotypes may, however, indicate that Slit does not act as a chemorepellent during head involution. slit is expressed in the dorsal pouch along which leak expressing cells migrate during head involution. In slit mutants, migration of this cell sheath is reduced. A simple explanation of this phenotype could be that the slit expressing dorsal pouch cells are attractive for the leak expressing ectodermal cells -- alternatively one would have to postulate a more caudally located slit source driving the ectodermal cells toward the anterior of the embryo by a repulsive mechanism. However, no such slit expressing cells were detected, suggesting that Slit, as Netrins, can act in both ways: as repulsive and as attractive guidance cues (Schimmelpfeng, 2001).

Another complementation group consisting of five alleles that leads to abnormal commissure formation maps to the genomic interval 34C/D. Subsequent complementation analyses indicates that these alleles are EMS-induced alleles of kuzbanian. In kuzbanian mutant embryos, many Fasciclin II positive axons appear to cross the CNS midline. In fact, it appears as if many, if not all, commissural axons abnormally express the Fasciclin II antigen. As in roundabout or leak, the longitudinal axon tracts of kuzbanian mutant embryos are reduced in size but they are generally found in more lateral positions (Schimmelpfeng, 2001).

The kuzbanian phenotype suggests that kuzbanian might be involved in the repulsive signaling system operating at the CNS midline. Other work has also suggested that Kuzbanian participates in the processing of the secreted Slit protein (Brose, 1999). A possible genetic interaction of the two mutations was tested by first analyzing the CNS phenotype of slit kuzbanian/++ embryos. When heterozygous, both mutations do not lead to a detectable embryonic CNS phenotype. In contrast to this, in 12% of the slit/kuzbanian double heterozygote embryos (or in 6% of the neuromeres out of 650 neuromeres counted), a roundabout-like mutant CNS phenotype was found. In embryos lacking zygotic kuzbanian function, in addition to being heterozygous for slit, a kuzbanian-like CNS phenotype emerged. The position of the longitudinal connectives, however, is shifted toward the CNS midline. Removal of one copy of kuzbanian in a slit mutant background does not enhance the slit phenotype (Schimmelpfeng, 2001).

This could be interpreted such that the Kuzbanian protease is required to activate the Slit protein, which serves as a ligand of the Roundabout receptors. Different allelic combinations of roundabout and kuzbanian were analyzed. roundabout;kuzbanian double heterozygote embryos appear wild type. When one copy of kuzbanian is removed in a roundabout mutant background, a roundabout CNS phenotype developes. The phenotype revealed by Fasciclin II staining may be slightly more extreme compared to the roundabout phenotype, since the lateral Fasciclin II positive fascicles are also affected. The overall axon pattern of embryos homozygous for roundabout and kuzbanian appears to be a slightly more severe phenotype compared to the roundabout phenotype, since axons running along the CNS midline were frequently detected. This is also evident following a Fasciclin II staining (Schimmelpfeng, 2001).

slit and kuzbanian also genetically interact. Since slit is required for head formation as well, the cuticle phenotype of kuzbanian mutant larvae was examined. Removal of the zygotic expression did not lead to a head defect as observed for slit or leak mutant larvae. It is presumed that maternal kuzbanian function may mask a head phenotype. Removal of maternal and zygotic kuzbanian function leads to a neurogenic phenotype with no recognizable head structures (Schimmelpfeng, 2001).

In kuzbanian mutants Leak expression is unaffected. To address the question whether Kuzbanian affects the expression of Roundabout an anti-Robo antibody was used. In wild type embryos, the Roundabout protein is always found on the longitudinal connectives. No Roundabout protein is expressed on commissural tracts. In kuzbanian mutant embryos, however, Roundabout can be detected on commissural axons crossing the CNS midline. Thus, Kuzbanian may function to clear Roundabout/Slit receptor-ligand complex from axons crossing the CNS midline (Schimmelpfeng, 2001).

During development, kuzbanian is expressed ubiquitously and most, if not all, CNS neurons appear to express the gene. Expression of a dominant negative Kuzbanian protein in all CNS neurons using the elav promoter leads to a kuzbanian-like CNS axon phenotype. To better analyze which cells in the developing CNS have to express the Kuzbanian protein in order to prevent the inappropriate crossing of the CNS midline by navigating axons, the GAL4 system was used. Using a single-minded GAL4 driver strain, a dominant negative Kuzbanian protein was expressed in all CNS midline cells. In 18% of the embryos no gross CNS defects were observed; in the remaining embryos, inappropriate midline crossing of Fasciclin II positive axons was found. 40% of these embryos displayed an almost roundabout-like CNS axon phenotype. Additionally, the formation of the lateral Fasciclin II positive axon tracts was affected, pointing toward a non-cell-autonomous function of the dominant negative Kuzbanian protein when expressed in the CNS midline. This is also suggested by the observation that in embryos expressing the dominant negative Kuzbanian protein, muscle fibers traverse the CNS dorsally as observed in slit or leak mutant embryos. It was next of interest to find out whether the expression of dominant negative Kuzbanian in the CNS midline cells also affects the clearance of the Roundabout protein from commissural axons. As observed in kuzbanian mutant embryos, Roundabout is found on commissural axon tracts, suggesting that Kuzbanian participates in the down-regulation of Roundabout expression on commissural axons. This also shows that axons can cross the midline despite the expression of Roundabout (Schimmelpfeng, 2001).

This phenotypic analysis has indicated that in kuzbanian mutant embryos, longitudinally projecting axons expressing Fasciclin II inappropriately cross the midline. These commissural axons also ectopically express the Roundabout receptor, which they never do in wild type embryos. Together with the data obtained from analyzing kuzbanian function during Delta-Notch signaling, this may be interpreted either as a requirement of kuzbanian in the CNS midline to generate the fully active Slit protein or it may indicate that Kuzbanian activates one or more Roundabout receptors in the lateral CNS (Schimmelpfeng, 2001).

Therefore, to further elucidate the function of kuzbanian, a dominant negative version was expressed in all CNS midline cells. Interestingly, this results in a mutant CNS phenotype combining phenotypic traits of roundabout and leak: axons and muscle fibers cross the CNS midline. This may indicate a function of Kuzbanian during Slit processing, possibly influencing the formation of the Slit gradient in the developing Drosophila nervous system. Alternatively, it may be interpreted in such a way that the cleavage of the Roundabout/Slit complex is required to facilitate growth cone retraction at the midline. This latter possibility is reminiscent of recent findings regarding the function of Ephrin signaling. The membrane-anchored Ephrin ligands constitute a large family of axon guidance molecules, which upon binding to Eph-receptor tyrosine kinases frequently mediate repulsive signals. In order to form the receptor-ligand complex, which triggers signaling to the cellular cytoskeleton, the two cells involved must adhere and subsequently cannot pull their plasma membranes apart. Only after the membrane anchored Ephrin ligand is cleaved by the Kuzbanian metalloprotease is the retraction process initiated (Schimmelpfeng, 2001).

In wild type embryos, Commissureless is expressed in the CNS midline cells and down-regulates the expression of Roundabout on commissural axons. It has been suggested that this down-regulation is required in order to allow crossing of contralateral projecting axons. In roundabout mutant embryos, axons frequently cross or even recross the CNS midline since the midline repellent Slit cannot be perceived. Conversely, high levels of roundabout expression result in a commissureless mutant phenotype. In kuzbanian mutant embryos, axons are able to cross the midline despite the expression of Roundabout on commissural axons. The local expression of a membrane-bound dominant negative Kuzbanian protein in the CNS midline mimics this phenotype. Thus, kuzbanian functions at the CNS midline in the clearance of the Roundabout receptor from commissural axons. This process may involve calmodulin and Sos. In the slit;kuzbanian double mutant, too, axons cross the midline and Roundabout protein is found on the surface of commissural axons. Furthermore, in kuzbanian mutants, the function of roundabout appears to be reduced since axons cross the midline, indicating that only the activated Slit/Roundabout complex can induce its clearance from commissural axons (Schimmelpfeng, 2001).

Slit signaling promotes the terminal asymmetric division of neural precursor cells in the Drosophila CNS

The bipotential ganglion mother cells, or GMCs, in the Drosophila CNS asymmetrically divide to generate two distinct post-mitotic neurons. The midline repellent Slit (Sli), via its receptor Roundabout (Robo), promotes the terminal asymmetric division of GMCs. In GMC-1 of the RP2/sib lineage, Slit promotes asymmetric division by down regulating two POU proteins, Nubbin and Mitimere. The down regulation of these proteins allows the asymmetric localization of Inscuteable, leading to the asymmetric division of GMC-1. Consistent with this, over-expression of these POU genes in a late GMC-1 causes mis-localization of Insc and symmetric division of GMC-1 to generate two RP2s. Similarly, increasing the dosage of the two POU genes in sli mutant background enhances the penetrance of the RP2 lineage defects whereas reducing the dosage of the two genes reduces the penetrance of the phenotype. These results tie a cell-non-autonomous signaling pathway to the asymmetric division of precursor cells during neurogenesis (Mehta, 2001).

To determine the requirement for Sli signaling in precursor cell division, focus was placed on two GMC lineages in the ventral nerve cord of the Drosophila embryo: the GMC-1-->RP/sib lineage generated by NB4-2 and the GMC1-1a-->aCC/pCC lineage, generated by NB1-1. The GMC-1 asymmetrically divides at ~7.5-7.45 hours of development to generate the RP2 motoneuron and its sibling (sib) cell whereas GMC1-1a asymmetrically divides at approx. 7 hours of development to generate aCC and pCC neurons. All these cells can be reliably identified in several ways. Initially, embryos mutant for sli were examined with anti-Even-skipped (Eve) antibody. Eve is first expressed in GMC-1 of the RP2/sib lineage; it is also expressed in a newly formed RP2 and sib. The sib eventually loses Eve expression whereas RP2 maintains Eve. Eve staining of sli mutant embryos reveals that the GMC-1 in sli mutants frequently divides symmetrically to generate two RP2s instead of an RP2 and a sib. Thus, while ~7-hour old sli embryos had only one GMC-1 as in wild type, in 10% of the hemisegments, ~8.5-hour old mutant embryos had two cells of equal sizes, both expressing Eve. This is in contrast to the wild type where a larger RP2 and a smaller sib are faithfully observed by 7.5-8 hours of age). It must be pointed out that there is no de novo synthesis of Eve in sib; thus the entire stock of Eve protein in a sib is inherited from GMC-1 and thus, the immunoreactivity for Eve in sib is an ancestry dependent/indicator. Similarly, in wild type the difference in the size of the nuclei between RP2 and sib is generated prior to cytokinesis, and thus inherent to the lineage. When ~10-hour old sli embryos were examined with Eve, 7% of the hemisegments had two RP2s. In 13-hour old sli embryos, two RP2s of equal sizes were observed in 11% of the hemisegments. Moreover, symmetric division of GMC-1 to generate two RP2s was also observed in single-minded (sim) embryos in 9% of the hemisegments. Sim is a transcription factor and is the upstream activator of sli. RP2 and aCC neurons are occasionally misplaced within a hemisegment in the CNS of 14-hour or older sli mutant embryos, however, in embryos that are less than 10 hours old the CNS is not severely affected and these cells do not cross the midline or segmental boundary. Thus, the RP2 or aCC duplications observed here are not due to migration of RP2s across the midline or segmental boundaries (Mehta, 2001).

More direct evidence was sought for the symmetric mitosis of GMC-1. If the GMC-1 in a sli mutant embryo divides symmetrically to generate two RP2s, the cytokinesis and nuclear division of GMC-1 must also be symmetrical as opposed to the non-symmetrical nuclear and cytokinesis of GMC-1 in wild type. Thus, it must be possible to observe symmetrical versus non-symmetrical division by examining GMC-1s that are undergoing cytokinesis with cell cortex markers in combination with nuclear markers. Therefore, sli mutant embryos were stained with the cell cortex marker spectrin and the lineage specific nuclear marker Eve. The asymmetric cytokinesis of GMC-1 (and the unequal nuclear sizes of daughter nuclei) to generate two unequal cells in wild type can be faithfully observed using these markers. However, in sli mutant embryos the GMC-1 undergoes a symmetric cytokinesis with two nuclei of equal sizes to generate two equal sized cells. These results indicate that GMC-1 in sli or sim mutants undergoes a symmetrical division to generate two RP2s (Mehta, 2001).

The generation of an RP2 at the expense of the sib was further confirmed using additional cell-type specific markers. Initially, mutant embryos were double stained for Eve and Zfh-1. In wild type, Zfh-1 is never expressed in GMC-1, GMC1-1a, sib, pCC, or newly formed RP2 and aCC. Zfh-1 begins to be expressed in RP2 and aCC at ~9 hours of development (at 22°C) and continues to be expressed thereafter. In ~10-hour old sli embryos, both the progeny of GMC-1 of the RP2/sib lineage co-express high levels of Eve and Zfh-1 and they continue to co-express high levels of Eve and Zfh-1 in ~13-hour or older mutant embryos. In the aCC/pCC lineage, both the progeny of GMC1-1a co-express Eve and Zfh-1 in 8% of the hemisegments, indicating that the two daughter cells have adopted an aCC identity in these hemisegments. Consistent with the possibility that the duplicated Eve- and Zfh-1-positive cells are RP2 and aCC neurons in sli embryos, they express 22C10 and have an axon trajectory of an RP2 and aCC, respectively (Mehta, 2001).

The symmetric division of GMCs in sli mutants is similar to that observed in insc, Notch or rapsynoid (raps; also known as pins) mutants and opposite that of nb. Previous results show that the cytoplasmic adaptor protein Insc is required for the asymmetric division of GMC-1 into RP2 and sib. During GMC-1 division, Insc protein localizes to the apical side and Nb to the basal side. The Nb-negative daughter cell becomes specified as sib by Notch signaling whereas the cell that inherits Nb becomes an RP2 owing to the blocking of Notch signaling by Nb. Thus, in insc mutants, both cells inherit Nb and are specified as RP2 while in nb mutants both progeny becomes sib. Given the similarity of sli, sim and insc mutant phenotypes, the relationship between Sli and Insc was examined. First, in sli mutants the localization of Insc in GMC-1, when examined, is not asymmetric. About 7% of the hemisegments show this phenotype. A similar non-localization of Insc was also observed in GMC1-1a of the aCC/pCC lineage. In raps mutant embryos, Insc is also not localized and as in sli the GMC-1 divides symmetrically to generate two RP2s. Thus, failure to localize Insc in these GMCs in sli mutants is responsible for their symmetric mitosis. In insc;nb double mutants both the daughters of GMC-1 are specified as sib by Notch signaling. In sli;nb (or sim;nb) double mutant embryos also, both the progeny of GMC-1 adopt a sib fate. Thus, Sli is required upstream of Nb during the asymmetric division of GMC-1. Since the GMC-1 symmetrically divides to yield two RP2s in Notch;nb double mutants, and two sibs in sli;nb double mutants, Sli is also upstream of Notch signaling during the asymmetric division of GMC-1. These results also indicate that when the GMC-1 in sli mutants symmetrically divides, both daughters inherit Nb (Mehta, 2001).

Since previous results tie the two POU genes, miti and nub, to the normal elaboration of the GMC-1->RP2/sib lineage, the expression of these genes was examined in sli mutant embryos. In wild type, the levels of Nub (or Miti), which are normally high in a newly formed GMC-1, are down regulated prior to the asymmetric division of GMC-1. In sli mutants the expression of Nub (or Miti) in a newly formed GMC-1 is comparable to that of wild type, but, in a late GMC-1 the level remains high compared to wild type. A brief ectopic expression of these POU genes from the hsp70 promoter prior to GMC-1 division induces GMC-1 to divide symmetrically to generate two GMC-1s; each then divides asymmetrically to generate an RP2 and a sib. If the symmetric division of GMC-1 in these mutants has anything to do with the lack of down regulation of Nub and Miti in GMC-1, ectopic expression of miti or nub should also induce GMC-1 to divide symmetrically to generate two RP2 neurons. Indeed, a brief over-expression of miti (or nub) in a late GMC-1 causes this GMC to divide symmetrically into two RP2 neurons in 27% of the hemisegments (Mehta, 2001).

The loss-of-function effects of sli on the distribution of Insc in GMC-1 (and thus the symmetrical division of GMC-1) could be due to this lack of down regulation of Miti and Nub in GMC-1. To test this possibility, the miti transgene was ectopically expressed from the hsp70 promoter. A 25-minute induction of miti was sufficient to alter the localization of Insc and the distribution of Insc in these embryos resembled the distribution of Insc in sli embryos (Mehta, 2001).

The penetrance of the symmetrical division phenotype in sim mutant is sensitive to the dosage of nub and miti genes. The penetrance of the symmetric division of GMC-1 phenotype in sli and sim mutants is ~10%, indicating a partial genetic redundancy for this pathway. Since the loss of asymmetric division of GMC-1 in sli or sim appears to be due to a failure in the down regulation of Nub and Miti, it was reasoned that the penetrance of the phenotype might be enhanced by increasing the copy numbers of these POU genes in sli or sim background. Using a duplication for nub and miti embryos were examined for the GMC-1 division phenotype. The penetrance of the phenotype in these embryos was enhanced to 42%. Similarly, halving the copy numbers of the two POU genes in sim background suppresses the phenotype to 1.4% (Mehta, 2001).

The above results indicate that the symmetrical division of GMC-1 in sli mutants is due to the up regulation of the two POU genes and that these two POU genes are the targets of Sli signaling in GMC-1; however, the partial penetrance of these phenotypes in sli mutants indicate that additional pathways also mediate this very same process and regulate the levels of the two POU proteins in GMC-1. Since the penetrance in insc mutants is also partial, additional pathways must exist to mediate the asymmetric division of GMC-1 to partially complement the loss of the Insc/Sli pathway (Mehta, 2001).

How is the Sli signal transmitted from outside to inside? Previous results show that one of the receptors for Sli is the transmembrane protein encoded by the robo locus. To determine if the effect of Sli signaling on GMC-1 is mediated via Robo, the expression of Robo in the GMC-1->RP2/sib and GMC-1-1a->aCC/pCC lineages was examined. Double staining of wild-type embryos with anti-Eve and anti-Robo shows that both these GMCs express Robo. Consistent with this, in robo null mutants, GMC-1 and GMC1-1a were found to divide symmetrically to generate two RP2s and two aCCs at the expense of sib and pCC. Although the penetrance of the RP2 lineage phenotype was low in robo mutants, the facts that Robo is expressed in GMC-1 and that the phenotype was observed only in robo null mutants argue that Robo at least partially transmits the Sli signal and promotes the asymmetric division of GMC-1 into RP2 and sib. Since three additional robo genes, robo2, robo3 and robo4 exist in Drosophila, the weak penetrance is likely to be due to genetic redundancy between these robo genes (Mehta, 2001).

The following picture emerges from this study. The Sli-Robo signaling down regulates the levels of Nub and Miti in late GMC-1, allowing the asymmetric localization of Insc and the asymmetric division of GMC-1. The possibility is entertained that loss of sibling cells in sli mutants would mean that some projections will be duplicated, while others are eliminated. Depending upon the extent, this might have an overall bearing on the pathfinding defects in sli mutants. Since Sli signaling is conserved in vertebrates, it is possible that this signaling may regulate generation of asymmetry during vertebrate neurogenesis as well (Mehta, 2001).

Repellent signaling by Slit requires the leucine-rich repeats

Slit is a repellent axon guidance cue produced by the midline glia in Drosophila that is required to regulate the formation of contralateral projections and the lateral position of longitudinal tracts. Four sequence motifs comprise the structure of Slit: a leucine-rich repeat (LRR), epidermal growth factor-like (EGF) repeats, a laminin-like globular (G)-domain, and a cysteine domain. The LRR is required for repellent signaling and in vitro binding to Robo. Repellent signaling by slit is reduced by point mutations that encode single amino acid changes in the LRR domain. By contrast to the EGF or G-domains, the LRR domain is required in transgenes to affect axon guidance. The midline repellent receptor, Robo, binds Slit proteins with internal deletions that also retain repellent activity. However, Robo does not bind Slit protein missing the LRR. Taken together, these data demonstrate that Robo binding and repellent signaling by Slit require the LRR region (Battye, 2001).

The 13 alleles of slit examined in this study cause a number of midline guidance errors in axons and ventral muscles. Muscle phenotypes range from midline crossovers of all ventral oblique muscles to midline crossing of a few muscle cells per embryo. Axon phenotypes vary from complete midline fusion of all CNS axons to midline crossings of only the most medial axons. Lateral axon tracts are relatively unaffected in mild EMS alleles and P-element insertion alleles of slit. All three P-element insertions have been mapped 10-100 bp upstream of the transcription initiation site and apparently reduce the level of Slit protein produced. It is possible that medial axons, being closer to the midline source of secreted Slit, have a higher threshold to respond to Slit and are thus more sensitive to reduced protein levels in mild alleles. Guidance and lateral positioning of the more lateral axon tracts are regulated by the Robo2 and Robo3 receptors, which may respond at a lower threshold to Slit (Battye, 2001).

Three mutations that result in single amino acid changes map to the LRR domain. Point mutations in the ß-sheet of LRR 1 and 2 (sli^GA178, sli^GA945) generate a severe phenotype. The sensitivity of slit phenotype to these conservative coding changes suggests a critical requirement of the LRR domain in repellent signaling. All other sequence changes result in truncated proteins with variable portions of the EGF domain preserved. Truncated proteins lacked the epitope recognized by the antibody used; therefore, it was not possible to assess their stability or distribution (Battye, 2001).

Drosophila Slit is likely proteolytically cleaved at the beginning of the sixth EGF repeat. Slit synthesized in sli⁵⁵⁰ mutants has an incomplete sixth EGF repeat and an altered C-terminal sequence. If the altered C-terminal sequence does not destabilize the protein, it would be anticipated that sli⁵⁵⁰ could act as a hypermorph, signaling in a manner comparable to the N-terminal portion of endogenously cleaved Slit. slit⁵⁵⁰ had the most variable penetrance of all the alleles examined. Nevertheless, the axon guidance phenotype, viability, and robo interaction suggest that sli⁵⁵⁰ is a hypomorph (Battye, 2001).

Although the laminin-like globular domain and the cysteine domain of slit represent one-fourth of the coding region, no sequence changes map to this region. It is possible that many point mutations in this region are not lethal and would not have been isolated by mutagenesis (Battye, 2001).

To learn more about the structural requirements for repellent signaling by Slit, attempts were made to rescue slit mutants with midline expression of slit transgenes lacking internal sequences. A slit transgene lacking the LRR fails to restore midline guidance and fails to generate effects after ectopic expression. Furthermore, in vitro translated Slit, which lacks a full LRR, does not bind to Robo, the repellent receptor. Point mutations encoding single amino acid changes in the LRR also greatly reduced repellent signaling. These data indicate that the LRR of Slit is required for receptor binding and repellent signaling (Battye, 2001).

Slit is the first protein for which receptor binding and signaling have demonstrated a requirement for the LRR. The LRR defines a superfamily of proteoglycans of the ECM having tandem repeats of xxI/V/LxxxxF/P/LxxL/PxxLxxL/IxLxxNxI/L, where x is any amino acid. The best-characterized members are decorin and biglycan. A Drosophila cell surface receptor (Toll), a GPI-linked proteoglycan (Connectin), and transmembrane Chaoptin also contain LRR. LRR-containing proteins of the ECM are implicated in binding of collagen [fibromodulin, decorin, lumican, and biglycan, laminin (biglycan), and fibronectin [decorin and biglycan]. Biglycan, decorin, and fibromodulin bind TGF-ß and may act as a tumor suppresser (Battye, 2001).

Fly LRR-containing proteins are involved in cell adhesion. Connectin is required for homophilic adhesion during motoneuron pathfinding and target recognition in Drosophila. Toll promotes homophilic adhesion but also appears to inhibit formation of neuromuscular junctions on Toll-expressing muscle. Chaoptin is required for fasciculation of photoreceptor axons. Repellent signaling by Toll and Connectin has been suggested and later discounted. The possibility of common functions of LRRs deserves reexamination (Battye, 2001).

It has been suggested that full-length Slit associates with the cell surface and that proteolytic cleavage at the start of the sixth EGF repeat generates two fragments. Three independent lines of evidence considered here identify a requirement for the LRR, in the N-terminal proteolytic fragment, for Robo binding and repellent signaling. Deletion of the EGF or G-domain does not reduce repellent signaling. These transgenes are more potent than full-length Slit in restoring midline axon guidance in slit mutants. This may be because these truncated proteins are more stable or are expressed at higher levels. Epitope tag labeling of the transgene products verified the presence of all transgene products but could not resolve relative levels of expression. An alternative interpretation is that the EGF or G-domain may have a regulatory influence on repellent signaling by the LRR, perhaps in establishing the gradient of repellent signal by retention on the MG cell surface. Functions of the EGF and G-domains deserve further investigation (Battye, 2001).

Robo binding does not require an intact EGF, G-, or cysteine domain. Internal deletion of the EGF domains also removes the putative cleavage site in EGF repeat 6; nevertheless, this protein still signals as a repellent. Therefore, it is likely that uncleaved Slit has repellent signaling function (Battye, 2001).

Protein interactions of the EGF, G-, and cysteine domains await analysis. They may play a role in binding laminin and Netrin, which bind vetebrate Slit. The EGF repeats of Slit are very similar to the non-calcium-binding repeats of Notch and Delta. Notch, Delta, Slit, laminin, and sea urchin fibropellin share a PGYTG motif within the EGF region. The EGF domains are implicated in specific protein recognition events, for instance, the recognition of Notch by Delta. The laminin EGF domain can promote neurite extension and modulate attractive signaling by netrin (Battye, 2001).

The G-domain of laminin (also termed the ALPS motif) is also found in Slit, neurexin, agrin, and perlecan, juxtaposed with EGF repeats. All members of this family participate in morphogenetic activities in the ECM. Laminin, neurexin, and agrin are also implicated in cell signaling during cell differentiation. Laminin binding to syndecan and integrin requires the G-domain. The G-domains of agrin promote postsynaptic differentiation of the neuromuscular synapse. Removal of the adjacent EGF repeats enhances this activity. This study did not reveal an essential role for the G-domain in repellent signaling by Slit. The role of this domain in Slit may be revealed when its binding partner is identified (Battye, 2001).

The C terminus of vertebrate Slit contains a cysteine knot, also found in growth factors that dimerize. The C terminal of Drosophila Slit, also cysteine rich, does not have appropriately spaced cysteines to form looped intermolecular disulfate bonds. Internal deletion constructs of Slit that retain repellent effects do not remove the cysteine domain; therefore, a contribution from this domain in Slit signaling cannot be excluded. In contrast to the other domains of the Slit protein, only the LRR domain is required in slit transgenes to restore midline guidance. Furthermore, translated Slit that lacks a full LRR does not bind to Robo, the repellent receptor. These data and the mutant sequence data indicate that LRR of Slit is required for, although not necessarily sufficient for, receptor binding and repellent signaling. This is the first demonstration that the LRR motif can function as a ligand for signal transduction (Battye, 2001).

Short- and long-range repulsion by the Drosophila Unc5 Netrin receptor

Given that Slit can bind directly to Netrin, and can also act via Robo receptors to silence Netrin attraction, might midline repulsion by unc-5 depend in any way on repulsion mediated by Slit and its Robo receptors? To test this, embryos were generated carrying both the elav-GAL4 and UAS-Unc5 transgenes, and that also were homozygous for one or more of the null alleles slit², robo¹, and robo2⁴ (Keleman, 2001).

The commissureless phenotype of pan-neural Unc5 embryos is essentially unaltered in the robo and robo2 single mutant backgrounds. The phenotype is more difficult to interpret when either slit or both robo and robo2 function is eliminated. The CNS phenotype observed in these embryos is intermediate between that of pan-neural Unc5 embryos and either slit or robo robo2 embryos. In some segments, axons are entirely collapsed at the midline as in slit or robo robo2 mutants, but in other segments axons are separated into two bundles, one on each side of the midline. This argues against a direct role for Slit in Unc5-mediated repulsion, since clearly Unc5 misexpression does have an effect in the absence of Slit. It does, however, suggest an indirect role. For example, repulsion by Netrin and Unc5 may only be effective in keeping axons away from the midline when it is added on top of the repulsive signal transduced via Slit and its Robo receptors. Another, not exclusive, possibility is that this intermediate phenotype is due to the ventral displacement of midline cells that occurs in both slit and robo robo2 mutants (Keleman, 2001).

Integrins regulate responsiveness to Slit repellent signals

Integrins are concentrated within growth cones, but their contribution to axon extension and pathfinding is unclear. Genetic lesion of individual integrins does not stop growth cone extension or motility, but does increase axon defasciculation and axon tract displacement. In this study, a dosage-dependent phenotypic interaction is documented between genes for the integrins, their ligands, and the midline growth cone repellent, Slit, but not for the midline attractant, Netrin. Longitudinal tract axons in Drosophila embryos doubly heterozygous for slit and an integrin gene, encoding alphaPS1, alphaPS2, alphaPS3, or ßPS1, take ectopic trajectories across the midline of the CNS. Drosophila doubly heterozygous for slit and the genes encoding the integrin ligands Laminin A and Tiggrin reveal similar errors in midline axon guidance. It is proposed that the strength of adhesive signaling from integrins influences the threshold of response by growth cones to repellent axon guidance cues (Stevens, 2002).

Axon fasciculation and guidance were studied in the CNS of embryos mutant for the ß integrin gene myospheroid (mys) and three alpha integrins, alphaPS1 (mew), alphaPS2 (if), and alphaPS3/4 (scb). It is not known whether the scb locus affects alphaPS3 or alphaPS4 or both, because the alphaPS4 locus is separated from alphaPS3 by 259 bp. Variation in penetrance of mutant phenotype was observed with all integrin alleles. The three longitudinal fascicles are intact in loss-of-function mutations in mys, which encodes the only ß integrin expressed in the CNS; however, midline fusions of the most medial tract are seen in some segments. Axon tract structure is least disrupted in mew mutant embryos. No midline guidance errors are seen; however, the longitudinal fascicles appeared to be thinner, with occasional defasciculation. if mutant embryos are similar in phenotype to mew, but also reveal a low frequency of midline crossover errors. Midline fusions as well as transient merging of lateral fascicles are seen in scb mutant embryos (Stevens, 2002).

Axon tract fasciculation appears normal in embryos heterozygous for mutations in one integrin gene and also in embryos heterozygous for mutations in two integrin genes. However, all four integrin mutations reveal a semidominant phenotype when doubly heterozygous with slit. In all instances, the frequency of midline guidance errors is increased over the levels seen in homozygous integrin mutants. Apart from midline axon crossings in one-third of the segments, the LT appeared normal in mys/+;sli/+ embryos. Less than 10% of segments revealed midline guidance errors in mew/+;sli/+ embryos. In contrast, 40% and 58% of segments had midline guidance errors in if/+;sli/+ and scb/sli embryos. Only in scb/sli double heterozygotes are the middle and most lateral axon tracts affected, indicating a strong phenotypic interaction between scab and slit (Stevens, 2002).

Midline guidance phenotypes are observed in integrin homozygotes that are also haplosufficient for slit. In particular, the frequency of midline crossing is much higher in mew/Y;sli/+ and if/Y;sli/+ mutants and also involves more lateral axon tracts (Stevens, 2002).

A scb,sli recombinant could not be isolated, in order to assess the scb,sli/scb phenotype. These genes would be expected to recombine in 1 of 25 chromosomes; however, no recombinants were isolated in 200 chromosomes screened. It was possible to investigate the interaction of these genes by using overlapping deficiencies. Midline guidance was assessed in scb and sli heterozygotes, in trans to a deficiency that uncovers either scb or sli or both genes. Consistent with the characterization of the sli and scb phenotypes, sli in trans to a deficiency uncovering sli has complete midline fusion of all axon tracts, and scb in trans to a deficiency uncovering only scb has an integrin mutant phenotype. The semidominant interaction of sli and scb is also confirmed when scb is trans to a deficiency uncovering only slit or when sli is trans to a deficiency uncovering only scb. A synthetic scb homozygote and sli heterozygote phenotype was generated in embryos with a scb mutant allele in trans to a deficiency uncovering both scb and sli. This phenotype is qualitatively similar to the scb/sli phenotype, revealing frequent midline crossing or fusion of the two most medial but not the most lateral axon fascicles (Stevens, 2002).

The midline axon phenotype of integrin mutants is part of a more complex phenotype involving defasciculation, irregular fascicle position, and 'wavy' axon trajectories. This phenotype emerges even when slit function is normal and may reflect axon guidance functions of known integrin ligands in the nervous system. Two integrin ligands have been identified within the LT: Laminin, and Tiggrin. The Fas II phenotypes of embryos mutant for these ECM proteins were characterized to clarify their possible contribution to axon guidance (Stevens, 2002).

Tiggrin is a secreted glycoprotein that contains an RGD motif and is considered to be a ligand of the PS2 integrin. Embryos homozygous for a loss of function allele of Tiggrin have a subtle Fas II phenotype reminiscent of integrin mutants. CNS axon tracts are wavy, and no midline axon guidance errors are seen. Labeling of the most lateral axon tract is interrupted between segments. Like the integrin genes, tig also has a semidominant interaction with slit. Fas II labeling of fascicles between segments is reduced. Midline guidance errors are seen in one in three segments (Stevens, 2002).

Drosophila Laminin is a trimer of three proteins: Laminin A, B1, and B2. Laminin is known to be a ligand of PS1 integrin and possibly other integrins as well. Mutants have not been isolated for the B1 and B2 chains; however, a loss of function allele for lanA encoding the A chain has been characterized. The Fas II phenotype of the lanA mutant is nearly wild type, revealing midline guidance errors in 4% of segments. When doubly heterozygous with sli, in sli/+;lanA/+ embryos, the frequency of midline crossovers is >30% (Stevens, 2002).

Does a change in lanA function also affect integrin function in CNS axon tract formation? lanA interaction with scb was examined because Laminin is not known to be a ligand of alphaPS3/4 (encoded by scb), and scb has a strong semidominant interaction with slit. Both lanA and scb reveal midline guidance errors when homozygous. However, in the scb/+;lanA/+ double heterozygote, midline guidance errors are not seen. Nevertheless, this genotype shares aspects of the integrin CNS phenotype: defasciculation and interruptions in Fas II labeling of the most lateral fascicle. This suggests function of both genes in a common or parallel pathway. If the interaction of scb and lanA is independent of the interaction of either gene with sli, then the phenotype of the triple heterozygote scb/sli;lanA/+ would reflect the addition of the scb/sli, scb/+;lanA/+, and sli/+;lanA/+ phenotypes. The degree of defasciculation and midline guidance errors in all axon tracts of the triple heterozygote appears to be additive. However, a narrowing of the CNS and the medial displacement of all axon tracts are also seen in the triple heterozygote. This phenotype is typical of mutants in genes required for midline guidance and is not a component of the integrin mutant phenotype. The synergistic interaction of these three genes suggests dosage-dependent function for each gene in common or parallel pathways (Stevens, 2002).

Given that genes that function in cell to ECM adhesion interact with axon repellent signals, it was of interest to see whether adhesion gene phenotypes interact similarly with midline attractant signals. Whether embryos homozygous or heterozygous for a deficiency that uncovers both Netrin genes, netA and netB, affect midline guidance was examined in a gene interaction assay. Embryos that lack netrin function have few commissural axons, and most commissures are missing. Embryos with one copy of each netrin gene (NP5/+) have normal commissures; however, the LTs, visualized with BP102, are thinner between segments and thicker within segments. Embryos that are heterozygous for mutations in both an integrin gene and the netrins show no enhancement or suppression of this phenotype (Stevens, 2002).

These data suggest that there may be a dosage-sensitive function of netrins in the organization of the LT. The morphology of Fasciclin II axon bundles was examined more closely. Embryos homozygous or heterozygous for the netrin deficiency reveal irregularity and interruptions in longitudinal Fasciclin II bundles. The heterozygote netrin phenotype was not enhanced in embryos also heterozygous for slit, robo, or scab function. In contrast, the frequency of midline guidance errors was increased in slit/+ or robo/+ but not scb/+ embryos when netrin function was also reduced or removed. These data indicate that repellent signaling is significantly more dosage sensitive than attraction in midline phenotypes (Stevens, 2002).

Thus, a reduced level of expression of the genes for four integrins (alphaPS1, alphaPS2, alphaPS3/4, and ßPS1) or two integrin ligands (Tiggrin and Laminin) increases the probability that CNS axons make pathfinding errors when slit expression is reduced. Expression of the integrins Tiggrin and Laminin A has been demonstrated in the CNS. Integrin expression is not localized and may be expressed in both glia and neurons. Overexpression of alphaPS3 or Laminin A in motoneurons affects axon guidance. Loss of function of the integrins disrupts axon fasciculation and longitudinal axon fascicle placement in the embryonic nerve cord but does not clearly affect axon guidance. These observations have been extended in this study, with different alleles of the integrins, demonstrating a similar function for alphaPS3/4, and revealing axon fascicle phenotypes for loss of function of integrin ligands Tiggrin and Laminin A. The mutant phenotypes share common elements: mild phenotypes show wavy axon tracts and reduced Fas II labeling between segments, whereas severe phenotypes include defasciculation and fascicle displacement, including midline axon guidance errors. The integrins have different extracellular ligands. Therefore, the integrins contribute similarly to axon tract integrity, independent of the ligand that they bind (Stevens, 2002).

Integrin phenotypes in the CNS do not demonstrate a direct role for integrins in growth cone guidance. In contrast, perturbation of midline growth cone repellent signals results in a medial narrowing of the CNS and ectopic midline crossing of longitudinally projecting axons, rather than defasciculation and displacement of axon tracts. One feature of integrin and tiggrin phenotypes shared with robo and dock mutant phenotypes is a thinning or loss of Fas II labeling in the most lateral axon fascicle. This fascicle expresses Fas II late in embryogenesis. This phenotype may reflect impaired or delayed development of independent fascicles in the nerve cord, as implicated by studies of robo function (Stevens, 2002).

Axon guidance cues such as Netrin or Slit are secreted proteins that associate with the ECM. Vertebrate Slit, for instance, binds to Laminin, Netrin, and Glypican. These cues also act at a distance from the cells that synthesize them. Whether or not Slit forms a detectable gradient in the ECM, the amount of protein alters the potency of repellent signaling. A robo-like phenotype is seen in hypomorphs of slit that produce less protein, and overproduction of slit reduces the number of commissural axons; therefore, Slit signaling is dosage sensitive. If reduced expression of another gene enhances the midline guidance phenotype of slit, then the normal function of that gene contributes functionally to inhibit axons from crossing the midline. This may reflect function in the production or transduction of the repellent signal or another function in the growth cone that reduces the probability of a growth cone approaching the midline (Stevens, 2002).

The semidominant interaction of all integrins, Tiggrin, and Laminin A with slit is more prevalent than might be expected if the integrins play a specialized role in Slit signaling. scab also has a dramatic semidominant interaction with dock (Nck), which functions in diverse axon guidance events. scb/dock double heterozygotes have disrupted longitudinal, commissural, and peripheral axon tracts. A similar genetic test suggests that ßPS integrin modulates RhoA activity and axon stability in the mushroom body. These diverse phenotypes reflect an adhesive function of the integrins that reduces the responsiveness of growth cones to guidance signals. Independent evidence suggests that this occurs in the growth cone, but a role for integrin in the glia that emit guidance signals cannot be discounted (Stevens, 2002).

Mutations in the netrinA and netrinB genes do not reveal semidominant interactions with genes for integrin function. Therefore Netrin signaling is not dosage sensitive in this genetic assay. Although Netrin might form a gradient in vivo, these data suggest that Slit may more effectively communicate positional information than does Netrin. Double mutants of netrin and slit have a slit phenotype, indicating that Netrin signaling acts genetically upstream of repulsion and also that attraction to the midline persists in the absence of Netrin. These data suggest that Netrin is not the sole midline attractant in Drosophila: more axons approach the midline in a netrin, slit double heterozygote than would be the case if only slit function is reduced. Therefore Slit and Netrin do not generate independent, additive guidance signals. Netrin can bind to Slit. Furthermore, the Slit receptor may silence attractant signaling by the Netrin receptor. Copresentation of Slit and Netrin to the receptors on the growth cone may enhance the repellent signal. Attraction to the midline requires silencing of Slit signaling (Stevens, 2002).

Integrins are concentrated in the growth cones of Drosophila axons, and their ligands are uniformly distributed over pathways of axon extension. Integrins facilitate the growth of axons by providing a link between the ECM and the cytoskeleton of the growth cone. Defasciculation and guidance errors seen in integrin mutants reflect decreased adhesion to the ECM and a lower threshold to errors in guidance. Axon extension is not impaired in Drosophila integrin mutants, although it is possible that a maternal contribution of integrin may mask this requirement (Stevens, 2002).

Part of the adhesive function of integrins is to activate intracellular signals that alter motility and axon outgrowth. During ligand binding, ß integrins may activate Focal Adhesion Kinase and Rho, which stabilize actin structures, permit actin filament growth, and facilitate the formation of focal adhesions. Intracellular signals can also modify adhesiveness by altering the affinity of integrins for their ligands. These signals can combine to cluster integrins and strengthen their attachment to the ECM. This increased adhesiveness can act in opposition to factors that decrease adhesion and axon extension, such as myelin or aggrecan. Furthermore, integrin signaling can influence other adhesion systems active in the growth cone (Stevens, 2002).

Similarly, integrin function alters the sensitivity of growth cones to repellent signaling by Slit. When slit expression and integrin function are both reduced, growth cones are more likely to respond to attractive guidance from the midline. Signals from axon guidance receptors promote growth cone reorientation and remodeling of the growth cone cytoskeleton. Integrin adhesion promotes local stabilization of cytoskeletal links to the ECM, which antagonizes growth cone reorientation. The threshold of response of growth cones to axon guidance signals is therefore regulated by the ability of guidance signals to reduce the stability of ECM to cytoskeletal linkages. This threshold may be reached at axon guidance choice points, including the segment boundary and the commissures, where clustering of errors in axon guidance occur. The efficacy of guidance signals may be reduced between choice points by local increases in ECM affinity (Stevens, 2002).

Integrin-ligand affinity and integrin-cytoskeletal linkages are logical targets of axon guidance signals. Further studies of the targets of integrin and guidance signals should reveal how growth cones integrate information from the ECM (Stevens, 2002).

Constitutively active myosin light chain kinase, acting downstream of Slit signaling, alters axon guidance decisions in Drosophila embryos

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).

A Drosophila homolog of cyclase-associated proteins functioning downstream of Slit, collaborates with the Abl tyrosine kinase to control midline axon pathfinding

Drosophila capulet (capt), a homolog of the adenylyl cyclase-associated protein that binds and regulates actin in yeast, associates with Abl in Drosophila cells, suggesting a functional relationship in vivo. A robust and specific genetic interaction is found between between capt and Abl at the midline choice point where the growth cone repellent Slit functions to restrict axon crossing. Genetic interactions between capt and slit support a model where Capt and Abl collaborate as part of the repellent response. Further support for this model is provided by genetic interactions that both capt and Abl display with multiple members of the Roundabout receptor family. These studies identify Capulet as part of an emerging pathway linking guidance signals to regulation of cytoskeletal dynamics and suggest that the Abl pathway mediates signals downstream of multiple Roundabout receptors (Wills, 2002).

Since analysis of Abl loss-of-function would predict cooperation between Abl and other genes in the repellent pathway, genetic interactions in embryos transheterozygous for Abl and either slit or combinations of mutations in different roundabout genes (ie. slit/+;Abl/+ or robo,robo2/+,+;Abl/+) were assayed. Surprisingly, these embryos displayed striking midline phenotypes far stronger than control genotypes. For example, slit²/+;Abl²/+ transheterozygotes show a 24-fold enhancement of the slit²/+ phenotype. This experiment strongly supports the model that Abl acts positively in the Slit pathway, consistent with the phenotypes of Abl homozygotes and of all the capulet genetic interactions observed (Wills, 2002).

The microtubule plus end tracking protein Orbit/MAST/CLASP acts downstream of the tyrosine kinase Abl in mediating axon guidance

Axon guidance requires coordinated remodeling of actin and microtubule polymers. Using a genetic screen, the microtubule-associated protein Orbit/MAST (proper FlyBase designation Chromosome bows) has been identified as a partner of the Abelson (Abl) tyrosine kinase. Identical axon guidance phenotypes are found in orbit/MAST and Abl mutants at the midline, where the repellent Slit restricts axon crossing. Genetic interaction and epistasis assays indicate that Orbit/MAST mediates the action of Slit and its receptors, acting downstream of Abl. Orbit/MAST protein localizes to Drosophila growth cones. Higher-resolution imaging of the Orbit/MAST ortholog CLASP in Xenopus growth cones suggests that this family of microtubule plus end tracking proteins identifies a subset of microtubules that probe the actin-rich peripheral growth cone domain, where guidance signals exert their initial influence on cytoskeletal organization. These and other data suggest a model where Abl acts as a central signaling node to coordinate actin and microtubule dynamics downstream of guidance receptors (Lee, 2004).

Orbit/MAST was identified as a candidate partner of Abl in a post-embyonic screem. In a retinal screen, overexpression of orbit/MAST enhanced the Abl^GOF phenotype, suggesting that these two proteins cooperate in vivo. However, validation of the screen required analysis of mutations in orbit/MAST (Lee, 2004).

Orbit/MAST was initially identified as a maternal effect lethal locus with defects in mitotic spindle and chromosome morphology; however, zygotic mutants display no defects in cell division, presumably due to maternal stores of the protein required for oogenesis. Independent LOF alleles were examined for zygotic phenotypes. Axon fascicles that are restricted to either side of the central nervous system (CNS) midline by Slit signaling