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 (dockmat), phenotypes reminiscent of loss of robo function can often be seen. dockmat 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 nmrH15lacZ 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 shotgun (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).

Genetic control of cell morphogenesis during Drosophila melanogaster cardiac tube formation

Tubulogenesis is an essential component of organ development, yet the underlying cellular mechanisms are poorly understood. This study analyzed the formation of the Drosophila cardiac lumen that arises from the migration and subsequent coalescence of bilateral rows of cardioblasts. This study of cell behavior using three-dimensional and time-lapse imaging and the distribution of cell polarity markers reveals a new mechanism of tubulogenesis in which repulsion of prepatterned luminal domains with basal membrane properties and cell shape remodeling constitute the main driving forces. Furthermore, a genetic pathway is identified in which roundabout, slit, held out wings, and dystroglycan control cardiac lumen formation by establishing nonadherent luminal membranes and regulating cell shape changes. From these data a model is proposed for Drosophila cardiac lumen formation, which differs, both at a cellular and molecular level, from current models of epithelial tubulogenesis. It is suggested that this new example of tube formation may be helpful in studying vertebrate heart tube formation and primary vasculogenesis (Medioni, 2008).

The analysis provided here establishes the cellular basis of lumen formation of the Drosophila cardiac tube. The lumen of the tube is formed from the migration of two bilateral rows of polarized cardioblasts (CBs), which join at the dorsal midline. One main result of this study is the characterization of two types of cell membrane domains directly involved in lumen formation, the luminal domains (L domains) and adherent domains (J domains). Adherens junctions that are responsible for sealing the tube originate from the J domain, whereas the membrane walls of the lumen originate from the L domain (Medioni, 2008).

Remarkably, the L domain displays characteristics of basal membranes, revealed by expression of molecular markers normally associated with a basal membrane. Furthermore, specification of the L and J domains takes place very early in the tubulogenesis process, significantly before coalescence of the bilateral rows of CBs at the dorsal midline. Finally, during CB migration, membrane domains undergo remodeling, concomitant with profound cell shape changes. These two cellular processes appear to be closely connected and are probably regulated by the cellular environment of the CBs composed by the overlying dorsal ectoderm and the amnioserosa cells. These interactions will be investigated in a future work (Medioni, 2008).

The mechanism of Drosophila cardiac lumen formation reported in this study is thus notably different from the previously described mechanisms of epithelial tubulogenesis. In epithelial tubulogenesis, after receiving a polarization signal that sets apicobasal polarity, the cells or group of cells establish a basal surface and generate vesicles carrying apical membrane proteins. The vesicles are targeted to the prospective apical region, where they fuse with the existing membrane or with each other to form a lumen. Finally, continued vesicle fusion and apical secretion expand the lumen (Medioni, 2008).

In contrast, constriction of the leading edge domain during cardioblast (CB) migration, precise control of cell shape changes, and delimitation of specific membrane domains appear to be the driving forces of Drosophila cardiac lumen formation. Cells forming the dorsal vessel have the features of migrating cells. In contrast to epithelial tubulogenesis, which involves apical membrane domains, the apex of polarized CBs constricts, forms adherens junctions, and consequently does not constitute the L domain. Instead, the luminal membrane domain possesses basal membrane characteristics, as is also the case in endothelial cells. Moreover, the size of the cardiac lumen is determined by the isotropic growth of CBs, and not, as in other models, by anisotropic extension of the L domain involving apical membrane vesicles.

Finally, the genetic control of the process involves gene products of slit, robo, how, and dg, which are not known regulators of lumen formation in epithelial tubes (Medioni, 2008).

This study leads to the identification of a genetic pathway, including slit, robo, how, and dg, controlling membrane domain specification and dynamics during cardiac lumen formation. Within this pathway, Slit appears to play a central role and a previously unrecognized function in cell morphogenesis (Medioni, 2008).

Several studies have shown that Slit-Robo function is essential for cardiac tube formation by controlling the proper migration, cohesion, and alignment of the two rows of CBs. The results reported in this study show that Slit is also involved in the correct specification of the L domain and its distinct features with respect to the adjacent J domains. Activation of Slit-Robo signaling determines the respective size of these two types of domains (Medioni, 2008).

The data suggest that activation of this pathway inhibits the formation of adherens junctions. This possibility is supported by recent findings in chick retina cells, where activation of the Slit-Robo pathway leads to the inactivation of β-catenin (Arm in Drosophila), resulting in the dissociation of N-cadherin from the junctional complex and preventing the formation of adherens junctions. Consistent with these observations, DE-Cad (Shg) is expressed in the J domains of CBs and is required for cardiac tube morphogenesis. Moreover, slit and shg show genetic interaction in cardiac tube morphogenesis. In the absence of slit function, the size of the L domain is strongly reduced, suggesting that Slit-Robo signaling prevents the formation of Arm/DE-Cad-mediated adherens junctions in the L domain (Medioni, 2008). How encodes an RNA-binding protein involved in mRNA metabolism, and given its exclusive nuclear localization at this stage of development, How may regulate slit splicing. In the absence of the How protein, the gene splicing could be affected, producing a Slit protein unable to correctly localize at the L domain. This hypothesis is consistent with the fact that expression of wild-type Slit in CBs can suppress the effect of how18 mutation on Slit localization and lumen formation. How has also recently been shown to regulate the splicing of neuronal membrane proteins such as neurexin. Moreover, How is expressed in the midline glia with Slit and Dg, suggesting that interaction among these three genes is part of a general mechanism by which junctions and lumen formation are controlled (Medioni, 2008).

A model is preposed for the genetic control of lumen formation in the cardiac tube. According to this model, How could directly regulate Slit by controlling its splicing and targeting the luminal compartment. Consequently, Slit binds to Robo activating the signaling pathway, which in turn inhibits Arm/DE-Cad-mediated adherens junction formation in the luminal compartment, leading then to the specification of distinct J and L domains. Parallel to this, activation of Slit-Robo signaling modulates the actin cytoskeleton and triggers CB cell shape remodeling required for lumen formation and growth. As How is able to act on many targets, it could also directly control the actin cytoskeleton by targeting an actin-binding molecule. Concerning Dg, it was shown that dg and slit genetically interact; however, overexpression of Slit does not rescue the lumen phenotype observed in dg mutants, contrasting with how mutations. Thus, it is proposes that Dg could regulate Slit localization at the L domain by its function in the specification and differentiation of the L domain, and therefore acts parallel to slit for lumen formation, behaving, for example, as a coreceptor of Robo. In addition, Dg could control actin cytoskeleton dynamics via its interaction with Dystrophin (Medioni, 2008).

The data clearly show that cardiac tube formation in Drosophila differs substantially from all other described mechanisms of tubulogenesis. Is this mechanism of tubulogenesis unique or is it shared with other organs and/or other organisms? Primary vasculogenesis in vertebrates leads to the formation of large median vessels, the dorsal aorta and the cardinal vein. These vessels arise from migrating mesenchymal cells of the lateral mesoderm, termed angioblasts, that are organized in bilateral groups of cells. Angioblasts migrate toward the midline as a cohort of cells, coalesce, and form a lumen. At this stage, as in flies, cells around the lumen show a crescentlike shape and an extracellular matrix is deposited at the internal face of luminal membranes. Similar cellular events are also observed during the formation of the primitive cardiac tube in vertebrates, suggesting that a common mechanism of tubulogenesis might exist for all tubes that arise from the coalescence of migrating bilateral mesenchymal cells (Medioni, 2008).

The Drosophila cardiac tube, or dorsal vessel, shares many similarities with the cardiovascular system of vertebrates. A significant fraction of genes expressed in the Drosophila cardiac tube are also annotated to be expressed in vertebrate blood vessels, suggesting that vasculogenesis and dorsal vessel morphogenesis might share common genetic regulators (Medioni, 2008).

Finally, components of the genetic pathway controlling cardiac lumen formation that are described in this study have potentially similar functions in vertebrates. It has been shown that numerous proteins involved in axon guidance are expressed in vertebrate blood vessels. In particular, the Slit-Robo signaling pathway has been involved in promoting tumor vascularization, hSlit2 being expressed in tumor cells and hRobo1 in endothelial cells. Moreover, mSlit3 has been implicated in mammalian cardiogenesis, and Quaking, the mouse homologue of How, is required for vasculogenesis and expressed in the developing heart (Medioni, 2008).

In conclusion, analysis of CB morphogenesis during development of the Drosophila cardiovascular system provides evidence for a new model of biological tube formation. It is proposed that this mechanism might also be used for the formation of the large median vessels and primitive heart tube in vertebrates (Medioni, 2008).

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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 Dcdc42v12 completely abolishes glomerular formation. Sensory neurons expressing Dcdc42v12 (SG18.1::Dcdc42v12) 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 Dcdc42v12, 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).

Talin is required to position and expand the luminal domain of the Drosophila heart tube

Fluid- and gas-transporting tubular organs are critical to metazoan development and homeostasis. Tubulogenesis involves cell polarization and morphogenesis to specify the luminal, adhesive, and basal cell domains and to establish an open lumen. This study explores a requirement for Talin, a cytoplasmic integrin adaptor, during Drosophila embryonic heart tube development. Talin marked the presumptive luminal domain and was required to orient and develop an open luminal space within the heart. Genetic analysis demonstrated that loss of zygotic or maternal-and-zygotic Talin disrupted heart cell migratory dynamics, morphogenesis, and polarity. Talin is essential for subsequent polarization of luminal determinants Slit, Robo, and Dystroglycan as well as stabilization of extracellular and intracellular integrin adhesion factors. In the absence of Talin function, mini-lumens enriched in luminal factors form in ectopic locations. Rescue experiments performed with mutant Talin transgenes suggested actin-binding was required for normal lumen formation, but not for initial heart cell polarization. The study proposes that Talin provides instructive cues to position the luminal domain and coordinate the actin cytoskeleton during Drosophila heart lumen development (Vanderploeg, 2015).

These experiments establish an essential function for the integrin adapter Talin in the assembly of the Drosophila embryonic heart. During the cardioblast (CB) migratory phase preceding tubulogenesis, Talin localizes along the CB apical surface, immediately ventral to the leading edge which extends towards the dorsal midline. As this Talin rich domain persists throughout embryonic heart assembly, eventually surrounding the lumen of the open cardiac tube, this surface is termed the pre-luminal domain. Talin is essential for the dynamic cell morphology and the leading edge features that characterise collective cardial cell migration. Furthermore, following migration, Talin is required to enclose a continuous lumen between the bilateral CB rows (Vanderploeg, 2015).

Analysis of late stage hearts in rhea zygotic mutants reveals that Talin is essential to correctly orient the CB polarity such that a continuous lumen is enclosed along the midline. In wildtype, many membrane receptors including Robo, Dg, Unc5, and Syndecan accumulate along the luminal domain. E-cadherin, Dlg, and other cell-cell adhesion factors are restricted to cell contact points immediately dorsal and ventral to the lumen and to the lateral cell domains between ipsilateral CBs. As evidenced by Robo and Dg immunolabeling experiments, the midline luminal domain is absent or, at best, is discontinuous along the midline in rhea mutant embryos. However, the Robo and Dg enriched luminal domains are not completely absent in null rhea homozygotes, but are found ectopically along lateral membranes between ipsilateral CBs. Robo's ligand, Slit, is also detected within these ectopic lumina. Similar ipsilateral Slit and Robo accumulations were observed in embryos mutant for the integrin subunit genes scab (αPS3) or mys (βPS1). Thus, the expanded Dlg-rich adhesive contact observed in rhea null embryonic hearts is consistent with a model in which integrins and Talin instruct the localization of Slit and Robo. These cues are essential to orient the lumen and to restrict the adhesive regions. In the absence of Talin, other components of the luminal structure, including Dg and the Slit-Robo complex, can self-assemble and create non-adherent luminal domains. However, proper midline positioning of the lumen requires Talin function (Vanderploeg, 2015).

Using an array of Talin transgenes previously shown to modify integrin adhesion strength and actin recruitment, this study assessed and compared the importance of these Talin-dependent processes. Binding of Talin's integrin binding site 1 (IBS1) to a membrane proximal NPxY motif on the β-integrin tail induces conformational changes within the integrin dimer, activating it and increasing the affinity for ECM ligands. Integrin activation is likely required prior to Talin IBS2 binding, an interaction which promotes a strong and stable integrin-cytoplasmic adhesome linkage. The current data indicates that either of Talin's two integrin binding sites are sufficient to promote CB morphogenesis and heart tube assembly. The ability of the heart to form in the presence of only IBS1 or IBS2 suggests that strong, long-lasting integrin-mediated adhesions are unnecessary. This idea is reinforced by the late accumulation of CAP, a protein recruited to more mature muscle adhesions. It is likely that transient adhesions are sufficient for lumenogenesis. It remains possible that an essential role for either IBS1 or IBS2 is masked by the perdurant maternal Talin in zygotic mutants. However, the functional redundancy of these domains is consistent with in vitro and in vivo studies suggesting that a subset of Talin functions can be fulfilled by either IBS domain (Vanderploeg, 2015).

Talin links integrins to the actin cytoskeleton both directly through an actin binding domain, or indirectly through recruitment of actin regulators such as Vinculin. Bond force studies of the C-terminal ABD suggest that although the ABD-actin linkage is direct, it is a weak bond which likely relies on additional direct or indirect Talin-actin linkages to form a strong and stable connection. Supporting this, TalinABD is essential for morphogenetic processes which rely on transient and dynamic integrin-actin linkages, but it is at least partially dispensable for longer-lasting adhesions which are likely stabilized by indirect Talin-actin interactions through Vinculin. The current studies demonstrate that Drosophila heart development is sensitive to disruptions in Talin's C-terminal ABD, which implicates cytoskeletal reorganization as a key process downstream of integrins during tubulogenesis. Supporting this, expression of constitutively active Diaphanous or dDAAM, formin proteins which promote actin polymerization, induced ectopic lumina similar to those that have been characterized in rhea mutants. These data are consistent with Talin promoting CB morphogenesis and lumen formation through direct, but dynamic actin linkages and suggest that formins may act downstream of Talin in apicalizing lumen formation (Vanderploeg, 2015).

To date, most studies on the Drosophila embryonic heart have focused on cell surface factors including receptors and their respective ligands; few studies have moved into the cell to establish the downstream signaling pathways involved. Insights into in vitro models suggest that polarity pathways and vesicle trafficking will be informative areas of study. For example, in the MDCK cyst model, the small GTPases Rab8a and Rab11a coordinate with the exocyst complex to deliver luminal factors to the pre-luminal initiation site. It remains to be determined whether similar exocytosis or secretion mechanisms are required for Drosophila heart lumen initiation or expansion. Furthermore, although it is unclear which classical apical polarity proteins are conserved in the Drosophila heart, epithelial and endothelial models suggest that the Cdc42-Par6-aPKC complex is a conserved master regulator of tube formation in both vertebrates and flies. Indeed, Drosophila heart tubulogenesis fails in embryos with heart specific inhibition of Cdc42 and expression of activated Cdc42 results in lateral lumina reminiscent of those characterized in rhea homozygotes. A mechanism is envisioned of heart tubulogenesis in which Talin provides instructive cues to the vesicle trafficking and polarity networks that target luminal factors and inhibit the assembly of cell-cell adhesion structures within the pre-luminal domain (Vanderploeg, 2015).

Slit/Robo signaling regulates cell fate decisions in the intestinal stem cell lineage of Drosophila

In order to maintain tissue homeostasis, cell fate decisions within stem cell lineages have to respond to the needs of the tissue. This coordination of lineage choices with regenerative demand remains poorly characterized. This study identified a signal from enteroendocrine cells (EEs) that controls lineage specification in the Drosophila intestine. EEs secrete Slit, a ligand for the Robo2 receptor in intestinal stem cells (ISCs) that limits ISC commitment to the endocrine lineage, establishing negative feedback control of EE regeneration. Furthermore, this lineage decision was shown to be made within ISCs and requires induction of the transcription factor Prospero in ISCs. This work identifies a function for the conserved Slit/Robo pathway in the regulation of adult stem cells, establishing negative feedback control of ISC lineage specification as a critical strategy to preserve tissue homeostasis. The results further amend the current understanding of cell fate commitment within the Drosophila ISC lineage (Biteau, 2014).

The data support a model in which Slit/Robo2 controls cell fate decisions in the ISC lineage by regulating the specification of ISCs into Prospero-expressing EE precursors before or during mitosis. Interestingly, manipulating the activity of Robo2 in ISCs did not affect the phenotype generated by expression of NotchRNAi (in which the formation of EC-committed EBs is specifically inhibited). In addition, no evidence was found that loss of Robo2 affects Delta expression in ISCs. Finally, the activation of the Notch pathway is sufficient to promote differentiation independently of Robo2 signaling. This supports the idea that Robo2 acts upstream and independently of the activation of the Notch signaling pathway, regulating lineage commitment in ISCs, whereas Notch specifically controls differentiation of daughter cells into the EC fate. In this model, the absence of Notch signaling results in default commitment of ISC daughter cells into an EE fate, and lineage commitment thus becomes independent of Robo2/Slit signaling, because EC differentiation is impaired (Biteau, 2014).

It is interesting to note that the intensity of the Slit signal is integrated by ISCs to generate an all-or-nothing response: above a defined Slit threshold, Prospero is expressed by around 6% of mitotic ISCs, whereas below this level, 15%-20% of ISCs express Prospero, and no intermediate expression of Prospero can be detected. Further studies will be required to characterize the signaling cascade that controls Prospero expression downstream of the Robo2 receptor in ISCs (Biteau, 2014).

Robo4 has recently been identified as a regulator of hematopoietic stem cell homing in mice. In addition, proteins of the Slit and Robo families have been suggested to act as tumor suppressors and be directly involved in the tumorigenesis process. This study identifies a mechanism by which differentiated cells engage this pathway to directly regulate stem cell function and lineage commitment. A role for Slit/Robo signaling in the control of fate decisions in mammalian normal or cancer stem cell lineages has not yet been tested. However, based on the conservation of mechanisms that control Drosophila ISC self-renewal and differentiation, it can be anticipated that this feedback control of stem cell fate decisions through Slit/Robo signaling also controls adult tissue homeostasis in higher organisms (Biteau, 2014).

Coordinate regulation of stem cell competition by Slit-Robo and JAK-STAT signaling in the Drosophila testis

Stem cells in tissues reside in and receive signals from local microenvironments called niches. Understanding how multiple signals within niches integrate to control stem cell function is challenging. The Drosophila testis stem cell niche consists of somatic hub cells that maintain both germline stem cells and somatic cyst stem cells (CySCs). This study shows a role for the axon guidance pathway Slit-Roundabout (Robo) in the testis niche. The ligand Slit is expressed specifically in hub cells while its receptor, Roundabout 2 (Robo2), is required in CySCs in order for them to compete for occupancy in the niche. CySCs also require the Slit-Robo effector Abelson tyrosine kinase (Abl) to prevent over-adhesion of CySCs to the niche, and CySCs mutant for Abl outcompete wild type CySCs for niche occupancy. Both Robo2 and Abl phenotypes can be rescued through modulation of adherens junction components, suggesting that the two work together to balance CySC adhesion levels. Interestingly, expression of Robo2 requires JAK-STAT signaling, an important maintenance pathway for both germline and cyst stem cells in the testis. This work indicates that Slit-Robo signaling affects stem cell function downstream of the JAK-STAT pathway by controlling the ability of stem cells to compete for occupancy in their niche (Stine, 2014: PubMed).

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 (sliGA178, sliGA945) 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 AblGOF 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 can be visualized at stage 17 with anti-Fasciclin II (FasII, Mab1D4). In late-stage wild-type embryos (stage 17), FasII is excluded from the midline. However, in orbit/MAST mutants, ectopic midline crossing was detected, primarily by the midline-proximal MP1 axon pathway. This phenotype is qualitatively identical to that seen in Abl zygotic mutants (note that loss of maternal and zygotic Abl generates catastrophic axonal defects, underlining Abl's central role in axonal development). Since the exclusion of FasII from axon commissures reflects a redistribution of protein that could be dependent on Orbit/MAST, it was important to confirm the guidance defects with an alternative marker. Using a Tau-LacZ fusion protein under control of an Apterous promotor expressed in two medial ipsilateral axons that never cross the midline, frequent ectopic crossing of these axons was found in orbit/MAST mutants (Lee, 2004).

The late-stage axon pathway defects in orbit/MAST mutants suggest a failure in the repellent effects of Slit on growth cone orientation. To be certain that the orbit/MAST phenotype reflects a loss of growth cone orientation and not simply a change in patterns of axon fasciculation, axon trajectories of pioneer neurons was inspected before other axons were available to serve as a substrate for fasciculation. At late stage 12, the posterior corner cell (pCC) helps to pioneer the MP1 pathway proximal to the midline; pCC neurites extend anteriorly and slightly away from the midline in wild-type. In orbit/MAST homozygotes, the pCC often orients toward the midline, sometimes crossing to meet its contralateral homolog. This shows that Orbit/MAST is required for accurate directional specificity of axon growth (Lee, 2004).

In addition to controlling midline crossing of axons, Slit repulsion determines the lateral position of longitudinal axon fascicles within the CNS neuropil. A marker for a mediolateral axon fascicle (Sema2b-Tau-myc) was used to examine this later function of Slit and its Robo receptors. In wild-type, Sema2b-positive axons cross the midline, turn, and extend along a straight longitudinal trajectory. In orbit/MAST mutants, a few Sema2b-positive axons meandered toward the midline from lateral positions. However, measurement of the lateral separation of these axon tracts reveals a significant inward shift in orbit/MAST mutants. Together, these genetic data demonstrate that Orbit/MAST performs a cell-autonomous postmitotic function during growth cone navigation (Lee, 2004).

The interaction between Orbit/MAST and Abl in the retina predicted that these proteins might cooperate to mediate axon guidance choices. However, since Abl plays both positive and negative roles in Slit signaling, it was important to test the polarity of genetic interactions in the context of embryonic development. Abl and Orbit/MAST levels were elevated, alone or in combination in postmitotic neurons. A mild synergy between the two genes during midline guidance was found that is consistent with cooperation. Interestingly, overexpression of Orbit/MAST alone induces a low but significant number of guidance errors at the midline. Stronger interactions were observed through LOF analysis. Double homozygous LOF mutants showed substantially increased ectopic midline crossing compared to single mutant controls, reminiscent of mutations in robo itself. Due to large maternal contributions of Abl and Orbit/MAST, even amorphic alleles are not zygotic null. Thus, it is not possible to use the double LOF mutant to conclude that both proteins act in a common pathway; however, the observed synergy does show that Abl and Orbit/MAST cooperate during midline axon guidance (Lee, 2004).

Since Abl is also required for motor axon pathfinding in the periphery, intersegmental nerve b (ISNb) morphology was compared in double and single mutants. Overexpression of Abl generates an ISNb bypass phenotype where this group of axons fail to enter their target domain. Coexpression of Abl and Orbit/MAST does enhance the expressivity of phenotype slightly, but the effect is subtle. Once having entered the ventral target domain, wild-type ISNb axons innervate the clefts between muscles 6, 7, 12, and 13. In Abl LOF mutants, ISNb stops short of its final targets, often terminating at muscle 13. A similar ISNb growth cone arrest phenotype is observed at very low penetrance in orbit/MAST LOF alleles. However, comparison of these phenotypes to orbit,Abl recombinant homozygotes revealed a strong enhancement of ISNb arrest in double LOF mutants, increasing the frequency of defects and shifting arrest to a more proximal position at the muscle 6/7 cleft. Thus, Abl and Orbit/MAST cooperate during axon guidance decisions in multiple contexts (Lee, 2004).

Analysis of CNS axons suggested that Orbit/MAST is an effector in the Slit/Robo repellent pathway. To test the hypothesis, the same genetic assay was used that was used to identify Slit as the ligand for the Robo receptor family. While heterozygotes lacking one copy of Slit or its receptors show very few guidance errors at the midline choice point, transheterozygotes that also remove one copy of a second gene in the pathway often reveal strong, synergistic phenotypes. Indeed, while orbit/MAST heterozygotes show no significant midline defects, very strong synergy is observed with mutations in slit (roughly 10-fold). As a control for the specificity of the interaction, embryos were examined lacking different alleles of orbit/MAST and an allele of capulet (capt), an actin binding protein that shows strong interactions with both slit and Abl (Wills, 2002). No synergy was observed between capt and orbit/MAST. The same transheterozygote analysis was performed with single mutations in the repellent receptors; orbit/MAST was found to enhance robo. Additional crosses revealed that orbit/MAST interacts with robo and robo2 but not with robo3, consistent with the specialization of Robo and Robo2 for midline crossing. To be certain that Orbit/MAST is not required simply for the expression or delivery of Slit and/or Robo protein, staining in wild-type and orbit/MAST embryos was compared, but no obvious differences were seen (Lee, 2004).

While all the data supported the model that Orbit/MAST is necessary for Abl function during axon guidance, a more rigorous test was desired. If Orbit/MAST acts as an effector of Abl, orbit/MAST mutations would be expected to be epistatic to an Abl GOF phenotype. The fact that Abl acts in both positive and negative capacities during midline guidance complicates the interpretation of such an experiment within the CNS; however, Abl plays a less complex role for ISNb motor axons. When overexpressed under a strong postmitotic neural GAL4 source, Abl generates an ISNb bypass phenotype; neuronal expression of GAL4 alone has no effect. However, when Abl is overexpressed in an orbit/MAST homozygous background, the frequency of ISNb bypass drops approximately 2-fold. This indicates that Orbit/MAST acts genetically downstream of Abl in embryonic growth cones (Lee, 2004).

Compartmentalization of visual centers in the Drosophila brain requires Slit and Robo proteins

Brain morphogenesis depends on the maintenance of boundaries between populations of non-intermingling cells. Molecular markers have been used to characterize a boundary within the optic lobe of the Drosophila brain; Slit and the Robo family of receptors, well-known regulators of axon guidance and neuronal migration, were found to inhibit the mixing of adjacent cell populations in the developing optic lobe. The data suggest that Slit is needed in the lamina to prevent inappropriate invasion of Robo-expressing neurons from the lobula cortex. Slit protein surrounds lamina glia, while the distal cell neurons in the lobula cortex express all three Drosophila Robos. The function of these proteins in the visual system was examined by isolating a novel allele of slit that preferentially disrupts visual system expression of Slit and by creating transgenic RNA interference flies to inhibit the function of each Drosophila Robo in a tissue-specific fashion. Loss of Slit or simultaneous knockdown of Robo, Robo2 and Robo3 causes distal cell neurons to invade the lamina, resulting in cell mixing across the lamina/lobula cortex boundary. This boundary disruption appears to lead to alterations in patterns of axon navigation in the visual system. It is proposed that Slit and Robo-family proteins act to maintain the distinct cellular composition of the lamina and the lobula cortex (Tayler, 2004).

The optic lobes are comprised of four processing centers derived from two distinct populations of precursor cells. In several regions of the optic lobe, cells derived from these different sets of progenitors lie immediately adjacent to one another but do not intermingle. This type of organization is found at the interface of the lamina and the lobula cortex, which are derived from the outer and inner optic anlagen, respectively. Distal cell neurons form the anterior edge of the lobula cortex and are located immediately adjacent to the posterior face of the lamina. Distal cell neurons are closely appositioned to glia at the posterior edge of the developing lamina. This study examines the mechanisms that prevent the distal cell neurons of the lobula cortex from intermingling with the lamina glia (Tayler, 2004).

A novel role has been identified for Slit and the Robo receptors as key factors that prevent mixing between adjacent groups of cells in the fly brain. The secreted protein Slit surrounds the lamina glia on one side of the boundary while Robo family proteins (receptors for Slit) are expressed by the distal cell neurons on the other side of the boundary. Loss of Slit expression or tissue-specific inhibition of Robo family expression in distal cell neurons causes the intermingling of lamina glia and distal cell neurons. It is proposed that Slit protein in the lamina keeps Robo-expressing neurons within the normal confines of the lobula cortex, establishing the sharp boundary between these two regions. Given the conservation of Slit and Robo signaling in axon guidance throughout evolution, Slit and Robo family members may also regulate boundary formation in the brains of other animals. Interestingly, humans with mutations in ROBO3 exhibit defects in hindbrain morphology, although the underlying developmental defect in humans is not known (Tayler, 2004).

RNAi knockdown of Robo family protein expression in the optic lobe using the Sca-Gal4 driver causes robust defects in distal cell neuron positioning. In addition to driving gene expression in the inner proliferation center neuroblasts and distal cell neurons, Sca-Gal4 also drives expression in R8 photoreceptor axons and neuroblasts of the outer proliferation center and neurons of the medulla cortex. Inhibition of Robo family expression only in the photoreceptors caused no detectable defects. In addition, knockdown of all three Robo family proteins in the medulla cortex using apterous-Gal4 had no effect on distal cell neuron behavior, and no defects in medulla neuron movement or axon targeting were identified in either slit mutants or Robo family knockdowns. Taken together with Robo family protein expression data, the Robo family knockdown analysis strongly supports a requirement for Robo family receptors in distal cell neurons in preventing them from invading the lamina neuropil (Tayler, 2004).

In the Drosophila visual system, Slit protein is present in a continuous zone from the base of the lamina into the underlying medulla neuropil. Although Slit mRNA is detected within the optic lobe, and Slit:lacZ expression is detected in medulla glia at the base of the lamina and in medulla cortex neurons, the optic lobe does not appear highly sensitive to the precise source or concentration of Slit. Attempts to use mosaic analysis to further define the cells in which slit function was required were unsuccessful, since no phenotypes were observed, despite the generation of large marked patches of slit2 mutant tissue in the visual system and the use of the Minute technique to maximize mutant clone size. It is suspected that the diffusibility of Slit protein combined with the large number of Slit-expressing cells in the optic lobe permitted the remaining heterozygous and wild-type cells in the mosaic animals to provide sufficient Slit to support proper optic lobe development. In addition, expression of Slit in photoreceptors under the control of GMR-Gal4 rescued the photoreceptor projection phenotype of slit mutants as effectively as more general expression of Slit in the optic lobe using Omb-Gal4. Thus, delivery of Slit to these neuropil regions may be sufficient to restore the boundary between the lobula cortex and the lamina (Tayler, 2004).

The effects of overexpression and ectopic expression of Slit and Robo proteins were examined in the optic lobe. Overexpression of Slit in the optic lobe using GMR-Gal4, Sca-Gal4, Omb-Gal4 or the more ubiquitously expressed Tubulin-Gal4 did not generate detectable phenotypes in the optic lobe. The failure to generate strong overexpression phenotypes could reflect the increased Slit expression within the lamina that accompanied overexpression in other regions using these Gal4 drivers. However, overexpression of Robo2 under the control of Sca-Gal4 dramatically distorted the shape of the lobula cortex, causing the distal cell neurons to move around the ventral and dorsal edges of the lamina. Since distal cell neurons normally encounter Slit protein at the posterior face of the lamina, this redistribution could reflect repulsion from regions of Slit expression. Overexpression of Robo or Robo3 caused no detectable defects (Tayler, 2004).

Slit coordinates cardiac morphogenesis in Drosophila

Slit is a secreted guidance cue that conveys repellent or attractive signals from target and guidepost cells. In Drosophila, responsive cells express one or more of three Robo receptors. The cardial cells of the developing heart express both Slit and Robo2. This is the first report of coincident expression of a Robo and its ligand. In slit mutants, cardial cell alignment, polarization and uniform migration are disrupted. The heart phenotype of robo2 mutants is similar, with fewer migration defects. In the guidance of neuronal growth cones in Drosophila, there is a phenotypic interaction between slit and robo heterozygotes, and also with genes required for Robo signaling. In contrast, in the heart, slit has little or no phenotypic interaction with Robo-related genes, including Robo2, Nck2, and Disabled. However, there is a strong phenotypic interaction with Integrin genes and their ligands, including Laminin and Collagen, and intracellular messengers, including Talin and ILK. This indicates that Slit participates in adhesion or adhesion signaling during heart development (MacMullin, 2006).

This study shows that slit is required for the ordered migration of cardial cells to meet their contralateral partners at the dorsal midline. This function is independent of dorsal closure. The cells that secrete Slit also express the Slit receptor, Robo2. robo2 has a mild heart phenotype; Drosophila doubly mutant for robo1 and robo2 have a more severe phenotype. When tested genetically, it was found that the functional interaction between Robo2 and Slit is weak. In contrast, reduced levels of function of the alphaPS3/alphaPS1 Integrin, its ligands, or cytoplasmic linkers all acted to enhance the phenotype of slit (MacMullin, 2006).

Defects in cardial cell morphogenesis were seen at all stages of heart development in slit mutants, suggesting a continuous requirement for Slit. Errors included gaps in the ribbon of migrating cells, delayed cell migration, gaps, clumping or blisters in the midline alignment of cardial cells, and the lack of a heart lumen. All of these defects reflect changes in cell interaction with the ECM, or cell polarization, and do not clearly define a role for Slit in attractive or repellent guidance signaling (MacMullin, 2006).

In ectodermal and mesodermal guidance, Robo2 participates in both attractive and repellent signaling. The developing heart is the only tissue known where Slit and a Robo receptor are expressed in the same cell, and this complicates a model of cardial cell migration by attractive guidance. Similarly, ephrins and EphA receptors are co-expressed in retinal ganglion cells and in spinal motoneurons. It has not been resolved whether co-expression of ephrin and EphA silences receptors by desensitization, or if subcellular segregation of ligand and receptor prevents autocrine signaling (MacMullin, 2006).

Slit is concentrated at the apical surface, which reflects the apical location of its receptor. There is no source of Slit available to the basal surface of heart, so it is unlikely that Robo signals from the basal domain. Slit function as an attractive or repellent guidance ligand in the nervous system is reflected by a strong genetic interaction between slit and robo, and between slit and genes for molecules required for Robo signaling, such as Nck (dock), Disabled (dab), and Abelson (abl). Not similar genetic interaction was observed in heart assembly, suggesting the functional relationship between Slit and Robo2 involves other signals, such as adhesion signals (MacMullin, 2006).

Robo2, which has 5 Ig and 3 fibronectin-like domains, is a member of the Ig Cell Adhesion Molecule (CAM) superfamily. Heterophilic and homophilic adhesion between Robos has been demonstrated. It is possible that Robo2 homophilic interactions, and Robo2-Slit binding act to adhere, align, and polarize migrating cardial cells. Heart morphogenesis was partially restored in slit mutants that express a slit transgene unable to bind Robo. Direct Slit-Robo interaction may not be essential to slit function in the heart (MacMullin, 2006).

At the genetic level, it has been established that Integrins and Integrin ligands interact with Slit in axon guidance in the nervous system. In this context, decreased levels of Integrin function made axons more sensitive to changes in guidance signaling. It is proposed that Integrin signaling or adhesion act to raise the threshold of growth cones to respond to guidance signals (MacMullin, 2006).

In the Drosophila heart, Slit function may include adhesion-related functions. This would account for the similarity of heart assembly phenotypes between slit alphaPS3/ alphaPS1 Integrin and Integrin ligands (Laminin and Collagen IV). Simultaneous reduction of Slit and Integrin function compromised heart assembly. This may involve adhesive signals from Integrins, given the genetic interaction between slit and two downstream Integrin linkers, talin (rhea) and ILK. Two models emerge from these data. The first is that Slit and Integrins function in parallel pathways, both of which converge upon adhesion dependent regulation of cell migration and morphogenesis. The second is that Slit and Integrin function in a common pathway-perhaps as ligand and receptor (MacMullin, 2006).

Development of the lumen of the heart requires the ECM protein Pericardin, and cell surface receptors Toll, Faint sausage and DE-cadherin. Possible outside-in signaling by these molecules remains to be explored (MacMullin, 2006).

There is strong evidence that other guidance molecules act in a parallel path to affect Integrin function. Sema3A expressed by vascular endothelia during angiogenesis acts to reduce Integrin function at focal adhesions, perhaps to facilitate branching angiogenesis. In contrast, activation of ephrin A2 or A5 results in increased adhesive function of Integrins. In both cases, second messenger signaling is implicated to regulate Integrin function. The pattern of gene interactions with slit in the nervous system are consistent with a role for second messengers downstream of guidance cue receptors, including Nck2 (dock), Abelson, and Myosin Light Chain Kinase. In contrast, a survey of modifiers of slit in the heart has revealed only Integrin linked proteins, implicating integrin dependent Slit function (MacMullin, 2006).

The structures of Slit and Netrin share similarities with Laminins, raising the possibility that these guidance molecules may link cells to the ECM. Like Laminin, Slit contains multiple EGF-like repeats, and a globular 'G' (also known as ALPS) domain. Neither of these domains are linked to Robo signaling. The EGF domains of Laminin are functionally associated with linking to other ECM proteins like Nidogen. The G domain is involved in association with Integrins and alpha-Dystroglycan. Slit contains domains that suggest association with the ECM, and this association may play a role in the formation of a Slit gradient. However, Slit localization determined by light or electron microscopy finds the protein on cell or axon surfaces, unlike the distribution of basement membrane proteins like Laminin or Perlecan. Cell surface labeling for Slit has only been reported on Slit or Robo expressing cells. It is suggested that Slit is localized on cardial cells by association with Robo, and possibly also alphaPS3/alphaPS1 Integrin. This positions Slit to participate in linking the cell to the ECM as well as to trigger intracellular signals through both Integrins and Robos. Slit likely associates with the ECM. Biochemical studies have implicated Laminin and Glypican as vertebrate Slit ligands. It is suggested that Slit facilitates cardial cell adhesion, migration, polarization, and lumen formation by physical interaction with Robo2, integrin receptors and ECM ligands (MacMullin, 2006).

In contrast to other guidance receptor systems, motifs associated with structural proteins of the ECM prevail in the structure of Slit and Netrin and their receptors Robo and DCC/Neogenin. The receptor-ligand binding domains have been identified, and the functions of other conserved domains remain undefined. Experiments aimed at uncovering the functions of the EGF and G domains of Slit and Netrin will clarify the multifunctional nature of these proteins (MacMullin, 2006).

Lateral positioning at the dorsal midline: Slit and Roundabout receptors guide Drosophila heart cell migration

Heart morphogenesis requires the coordinated regulation of cell movements and cell–cell interactions between distinct populations of cardiac precursor cells. Little is known about the mechanisms that organize cardiac cells into this complex structure. In this study, the role of Slit, an extracellular matrix protein and its transmembrane receptors Roundabout (Robo) and Roundabout2 (Robo2) were analyzed during morphogenesis of the Drosophila heart tube, a process analogous to early heart formation in vertebrates. During heart assembly, two types of progenitor cells align into rows and coordinately migrate to the dorsal midline of the embryo, where they merge to assemble a linear heart tube. Cardiac-specific expression of Slit is required to maintain adhesion between cells within each row during dorsal migration. Moreover, differential Robo expression determines the relative distance each row is positioned from the dorsal midline. The innermost CBs express only Robo, whereas the flanking pericardial cells express both receptors. Removal of robo2 causes pericardial cells to shift toward the midline, whereas ectopic robo2 in CBs drives them laterally, resulting in an unfused heart tube. A model is proposed in which Slit has a dual role during assembly of the linear heart tube, functioning to regulate both cell positioning and adhesive interactions between migrating cardiac precursor cells (Santiago-Martínez, 2006).

Morphogenesis of the heart is a complex process requiring the coordinated regulation of cell positioning and adhesive interactions between distinct populations of migrating precursor cells. In this study, the results are consistent with the model that Slit and Robos are required for both of these functions. The differential expression of Robo and Robo2 is important for maintaining the relative positioning of the two distinct populations of cells during this dorsal migration. The inner rows of CBs express the single Robo receptor, whereas the PCs, which are positioned more laterally, express both receptors. Removal of robo2 may cause individual PCs to shift toward the midline, whereas ectopic expression of Robo2 in CBs drives the rows of cells laterally, resulting in an unfused heart tube (Santiago-Martínez, 2006).

Furthermore, loss of slit, or both robo and robo2, causes defects in cell adhesion, resulting in gaps in the rows of CBs and PCs. Often the gaps in the rows of CBs correspond with the gaps in the PC rows, suggesting that these two cell types must also be adhered to each other. The phenotypes observed may also be due to a loss of adhesion between these cardiac cells and the overlying dorsal ectoderm. Indeed, evidence has been provided that the dorsal ectoderm coordinately migrates with the heart cells during dorsal closure. Although no significant defects were observed in dorsal closure in slit mutants, it is possible that Slit may also be playing a role in adhesion between the overlying dorsal epithelium and the cardiac cells. How is cell adhesion between adjoining groups of cells regulated? It is likely that Slit or Robo receptors have parallel or cooperative roles with cell adhesion systems during heart formation. In vitro, activation of Robo by Slit interferes with N-cadherin-mediated adhesion. Future studies will reveal whether Slit and Robos cooperate with homophilic cell adhesion molecules in the developing heart (Santiago-Martínez, 2006).

Two papers have been recently published that also implicate Slit in Drosophila heart patterning. Both of these studies support the findings that Slit plays an important role in regulating cell adhesion between migrating groups of CBs during heart tube assembly. However, these papers differ somewhat, both from each other and from the current study, in their assessment of the role of the two receptors for Slit, Robo and Robo2, during this process. One issue on which these studies disagree is in the expression patterns of Robo and Robo2 in the cells of the heart. In this study, Robo and Robo2 were found to be differentially expressed in the heart. Specifically, the current analysis of the expression of both receptors reveals that at the dorsal midline, the inner rows of CBs express Robo, whereas the flanking rows of PCs express both receptors. Interestingly, this expression pattern is similar to what is observed for Robo and Robo2 in the ventral midline of the CNS, and it is believed this similarity also reflects a comparable function for these receptors at both the ventral and dorsal midlines. These findings, which were confirmed at both the protein and mRNA levels, were not observed in the two other studies. For example, the authors of one study failed to report the coexpression of Robo with Robo2 that was observed in PCs. The weaker expression of Robo in PCs as compared with its expression in CBs may account for the fact that this expression pattern was not reported. In one of the studies, the robo2 (and not Robo, as this study reports) was found to be coexpressed with Slit in CBs. Surprisingly, the expression of robo was not examined in this study. That these results were based solely on in situ hybridization and were not supported by analysis of protein expression may account for the significant differences between the findings. Another difference between these studies lies in the current gain-of-function experiments with Robo2. Specifically, this analysis revealed an important role for Robo2 in specifying the distance a migrating PC maintains from the midline. This phenotype is similar to what is observed at the ventral midline for CNS axons ectopically expressing Robo2 and provides strong support for the positioning model presented in this study (Santiago-Martínez, 2006).

Together, these differences in have led to the proposal of an alternative model for Slit and Robo receptors in heart cell positioning at the dorsal midline, whereby the combinatorial expression of Robo receptors controls the relative position of individual rows of migrating cells from the dorsal midline during heart tube assembly. Why do cells that express both Robo and Robo2 receptors stay farther away from the dorsal midline than cells that express only Robo? The results are similar to what is observed at the ventral midline of the Drosophila CNS, where Slit, secreted from the midline glial cells, functions as a repellent to specify the lateral positioning of axons according to the specific combination of Robo receptors that these axons express. During development of the CNS, medial axons expressing the Robo receptor are positioned closer to the ventral midline than lateral axons expressing both Robo and Robo2. From loss- and gain-of-function genetic experiments presented in this study, a similar model is proposed for heart cell positioning at the dorsal midline during heart tube formation. Rows of CBs that express the single Robo receptor migrate closer to the dorsal midline than PCs that express both Robo and Robo2. These development events, although seemingly diverse, share a key similarity. In both cases, bilateral populations of migrating cells are organizing themselves relative to a midline. However, there is also a notable difference between these two circumstances. In the CNS, Slit secreted from the ventral midline glial cells prevents migrating Robo-expressing axonal growth cones from crossing into ligand-expressing territory. At the dorsal midline, Slit is secreted by the innermost CB cells, which are also cells that respond to Slit. This represents a novel intrinsic function for Slit-Robo signaling. Cells expressing Slit are organizing themselves and neighboring cells by virtue of which Robo receptor they express. Further study will reveal the precise nature of Slit’s role in this process and will have important implications for understanding mechanisms of organ self assembly (Santiago-Martínez, 2006).

A major weakness of the current positioning model is in the current analysis of the weak robo or robo2 loss-of-function phenotypes. For example, the model would predict that removal of robo2 from PCs would cause these cells to shift to a position closer to the dorsal midline. Although mispositioned PCs in robo2 mutants was occasionally detected, the phenotypes observed are not very penetrant or striking. Likewise, robo also has a very mild cardiac phenotype. Although these observations may reflect a flaw in the model, the lack of strong phenotypes for robo or robo2 single mutants could also be explained by the additional roles these molecules play in cell–cell adhesion. By this reasoning, the loss of a single receptor may not be enough to disrupt the adhesion between adjacent cells. The same findings were observed in gain-of-function experiments with Robo2. Overexpression of Robo2 in CBs in a robo mutant background results in cardiac cell mispositioning, but the adhesion between the rows of cell is maintained. However, ectopic Robo2 in a robo,robo2 double mutant background revealed strong defects in both processes (Santiago-Martínez, 2006).

Genetic modifier screens reveal new components that interact with the Drosophila dystroglycan-dystrophin complex

The Dystroglycan-Dystrophin (Dg-Dys) complex has a capacity to transmit information from the extracellular matrix to the cytoskeleton inside the cell. It is proposed that this interaction is under tight regulation; however the signaling/regulatory components of Dg-Dys complex remain elusive. Understanding the regulation of the complex is critical since defects in this complex cause muscular dystrophy in humans. To reveal new regulators of the Dg-Dys complex, genetic interaction screens to identify modifiers of Dg and Dys mutants in Drosophila wing veins. These mutant screens revealed that the Dg-Dys complex interacts with genes involved in muscle function and components of Notch, TGF-β and EGFR signaling pathways. In addition, components of pathways that are required for cellular and/or axonal migration through cytoskeletal regulation, such as Semaphorin-Plexin, Frazzled-Netrin and Slit-Robo pathways show interactions with Dys and/or Dg. These data suggest that the Dg-Dys complex and the other pathways regulating extracellular information transfer to the cytoskeletal dynamics are more intercalated than previously thought (Kucherenko, 2008).

There are only a few examples of signaling pathways that have been shown to transmit information from outside the cell that results in cytoskeletal rearrangements inside the cell. Slit-Robo, Netrin-Frazzled and Semaphorin-Plexin pathways are examples of such activity. Dg-Dys complex appears also regulate the cytoskeleton based on extracellular information. Interestingly, the interaction screens described in this paper show that these aforementioned pathways are much more intricately connected than previously thought. The Robo and Netrin Receptor (DCC) pathways have previously been shown to interact, now this study reports that Dg-Dys complex interacts with these pathways as well (Kucherenko, 2008).

The interactions seen in wing development involving the Drosophila DGC and the genes that affect neuronal guidance (sli, robo, fra, sema-2a, sema-1a, Sdc) might be explained by their possible role in hemocyte (insect blood cell) migration. Analysis done in Drosophila shows that known axon guidance genes (sli, robo) are also implicated in hemocyte migration during development of the central nervous system. Similar findings have been reported in mammals, where blood vessel migration is linked to the same molecular processes as axon guidance. Both sli and robo have been implicated in the vascularization system in vertebrates. A recent study demonstrated that proper hemocyte localization is dependent upon Dys and Dg function in pupa wings. Mutations in these genes result in hemocyte migration defects during development of the posterior crossvein. Hence, it is speculated that the neuronal guidance genes that were found may interact with the DGC in wing veins by having a role in the migration process (Kucherenko, 2008).

Similar to sli and robo, the Dys and Dg mutants also affect photoreceptor axon pathfinding in Drosophila larvae. It is therefore possible that this group of modifiers will interact with the DGC in axon pathfinding and other processes. Supportive of that notion is the fact that mammalian Syndecan-3 and Syndecan-4 are essential for skeletal muscle development and regeneration. In addition slit-Dg interaction has previously been observed in cardiac cell alignment. Sequence analysis of slit reveals that it possesses a laminin G-like domain at its C-terminus. Dystroglycan's extracellular domain has laminin G domain binding sites and has been shown to bind 2 of the five laminin G domains in laminin. It is therefore possible that slit, through its laminin G-like domain, binds to Dystroglycan and that Dystroglycan is a slit receptor. It will be informative to reveal the mechanisms and nature of these interactions (Kucherenko, 2008).

Interdependence of macrophage migration and ventral nerve cord development in Drosophila embryos

During embryonic development, Drosophila macrophages (haemocytes) undergo a series of stereotypical migrations to disperse throughout the embryo. One major migratory route is along the ventral nerve cord (VNC), where haemocytes are required for the correct development of this tissue. A reciprocal relationship exists between haemocytes and the VNC; defects in nerve cord development prevent haemocyte migration along this structure. Using live imaging, it was demonstrated that the axonal guidance cue Slit and its receptor Robo are both required for haemocyte migration, but signalling is not autonomously required in haemocytes. The failure of haemocyte migration along the VNC in slit mutants is not due to a lack of chemotactic signals within this structure, but rather to a failure in its detachment from the overlying epithelium, creating a physical barrier to haemocyte migration. This block of haemocyte migration in turn disrupts the formation of the dorsoventral channels within the VNC, further highlighting the importance of haemocyte migration for correct neural development. This study illustrates the important role played by the three-dimensional environment in directing cell migration in vivo and reveals an intriguing interplay between the developing nervous system and the blood cells within the fly, demonstrating that their development is both closely coupled and interdependent (Evans, 2010).

Haemocyte migration is dependent upon the correct development of the VNC. Haemocytes fail to migrate along the VNC midline in slit and robo1,2 mutants, which have related VNC defects due to axonal pathfinding defects and glial mispositioning. However, Robo signalling is not required in haemocytes, nor are haemocytes attracted or repelled by Slit. Instead, VNC defects in these mutants result in a physical barrier to haemocyte progression down the ventral midline. Previously, it was assumed that haemocytes fail to migrate down the midline in sim mutants because of a loss of Pvf ligand expression; however, slit mutants exhibit a similar, although less severe, phenotype to sim mutants, despite maintaining Pvf2 and Pvf3 expression. Therefore, it seems that the failure of the VNC and epidermis to separate and consequently provide a suitable migratory substrate is also a crucial factor in the regulation of haemocyte migration. The failure in separation might be due to both axonal pathfinding defects and glial mispositioning, resulting in a failure of midline cells to relinquish contact with the epidermis; apoptosis of midline cells has previously been shown to contribute to separation. Lastly, it was shown that in the absence of haemocyte migration along the VNC, the dorsoventral channels in this structure fail to form correctly, underlining the importance of haemocyte migration for correct morphogenesis of the VNC (Evans, 2010).

The migration of haemocytes along the VNC is a key process in VNC morphogenesis. Conversely, perturbing VNC development blocks haemocyte migration along the midline and prevents haemocytes from reaching mid-trunk neuromeres and other potential sites of function; hence, haemocyte migration and VNC development are interdependent processes. This interdependence is particularly fascinating given that the key growth factors in haemocyte migration (Pvf2 and Pvf3) also regulate glial cell migrations within the VNC. Reiterative use of growth factors, such as the Pvfs, reduces the number of genes required to regulate the multitude of processes crucial for development. Furthermore, using the same genes to regulate more than one process enables temporal and spatial coupling of such processes. In this example, sim coordinates haemocyte migration and epidermis-VNC separation through the expression of genes such as slit and Pvf2 (Evans, 2010).

Most VNC defects caused by failures in haemocyte migration are visible at later stages of development, but this study detects an early (stage 13/14) structural difference in the VNC, with a reduction in the diameter of the dorsoventral channels when haemocytes are prevented from leaving the head. It remains to be seen whether this phenotype is purely due to the lack of haemocytes sitting in these channels or whether haemocyte-derived matrix is required for their normal formation. The precise function of these channels is unknown, but they are lined by a specific subset of glia and so presumably fulfil an important role. One potential purpose might be to facilitate haemocyte migration, enabling cues produced dorsally to diffuse through to the ventral surface (Evans, 2010).

Several papers have focused on the role of the Pvfs in haemocyte migration along the VNC midline. This study demonstrates that expression of Pvfs alone is not sufficient for this process (or even required for migration along the dorsal side of the VNC) and that the integrity of the VNC is also fundamental. The loss of other chemoattractants in slit and robo1,2 mutants cannot be excluded, but the failure of the VNC to separate from the epidermis seems a more likely explanation: first, unlike sim mutants, slit mutants maintain expression of many midline genes; second, the sim and slit haemocyte phenotypes are very similar, suggesting that those genes that are lost play minor roles, if any, in the regulation of this process (Evans, 2010).

The current understanding of haemocyte migration along the VNC is as follows: haemocytes require activation of Pvr by Pvfs to direct migration into the germ band and along the VNC and for their survival. Concurrently, the VNC must separate from the epidermis to provide space for them to move down the midline, where they can secrete matrix and remove dying glial and neuronal cells. This process is aberrant in slit mutants and haemocytes can only reach mid-trunk neuromeres by moving to the lateral edges of the VNC to bypass regions where the epidermis and VNC have failed to separate. Cell-cell repulsion between haemocytes exiting the head and germ band might also help drive haemocytes onto the attractive 'track' of Pvfs and matrix (derived from glia and preceding haemocytes), enabling them to spread along the developing VNC, and might also contribute to lateral migration. Other partially redundant cues might also aid haemocyte dispersal (Evans, 2010).

The regulation of cell migration by the presence or absence of physical barriers has been shown in other systems. For example, dendritic cells migrate into lymphatic vessels through 'preformed portals' in the basement membrane. Conversely, the three-dimensional environment can physically constrain cells, inhibiting their dispersal, such that its remodelling by distinct leading cells (e.g. fibroblasts) is required before invasive migration can begin. Therefore, it seems likely that the availability or constriction of such routes is an important means to regulate cell migration (Evans, 2010).

The reciprocal nature of VNC development and haemocyte migration shows the importance of coordinating developmental events. Haemocytes are programmed to track along the VNC by following Pvf signals and to migrate into the gap between the separating VNC and epidermis at the exact time when they are needed to clear apoptotic cells. Vertebrate endothelial cells and hematopoietic lineages are thought to be derived from common hemangioblast precursors; like haemocytes, vertebrate endothelial cells respond to VEGF ligands and, additionally, to a variety of neuronal guidance cues, enabling the vasculature to closely follow the pattern of nerves. Although Drosophila possess an open circulatory system, as opposed to the closed vasculature of vertebrates, it is intriguing that haemocytes use proteins that are related to those employed in vertebrate haematopoiesis (Serpent and Lozenge are members of the GATA and RUNX transcription factor families, respectively) and to those employed in the generation of a vascular network (Pvr and the Pvfs are related to VEGFRs and VEGFs, respectively), particularly as haemocyte routes, similar to those of vertebrate endothelial cells, closely follow the routes of neuronal tracts. Therefore, because the control of haemocyte migration appears to have evolved in an analogous way to endothelial guidance in vertebrates, haemocytes might represent a useful model with which to study the evolution of neurovascularisation and could represent an early step in the evolution of a vascular network (Evans, 2010).

A genetic screen for mutations affecting gonad formation in Drosophila reveals a role for the slit/robo pathway

Organogenesis is a complex process requiring multiple cell types to associate with one another through correct cell contacts and in the correct location to achieve proper organ morphology and function. To better understand the mechanisms underlying gonad formation, a mutagenesis screen was performed in Drosophila and twenty-four genes were identified that were required for gonadogenesis. These genes affect all different aspects of gonad formation and provide a framework for understanding the molecular mechanisms that control these processes. Gonad formation is found to be regulated by multiple, independent pathways; some of these regulate the key cell adhesion molecule DE-cadherin, while others act through distinct mechanisms. In addition, it was discovered that the Slit/Roundabout pathway, best known for its role in regulating axonal guidance, is essential for proper gonad formation. These findings shed light on the complexities of gonadogenesis and the genetic regulation required for proper organ formation (Weyers, 2011).

Although all three Robos function in gonad formation, their phenotypes and localization patterns suggest that each one has a slightly different role in the process. Robo2 is the only Robo expressed at detectable levels as somatic gonadal precursor (SGP) cluster fusion occurs, as well as the only robo gene that causes a substantial cluster fusion defect when mutated. Therefore, Robo2 appears to be the principal Robo protein mediating cluster fusion. Once the SGP clusters have merged, all three Robos contribute to gonad compaction and ensheathment, as all exhibit defects in these processes when mutant. Though their mutant phenotypes are similar, Robo and Robo2 have slightly different localizations, with Robo more prominent between SGPs and germ cells (GCs), and Robo2 between SGPs as well as between SGPs and GCs. This suggests potentially distinct functions, and would be consistent with other reports that the Robos perform separate functions. In contrast, slit mutants have stronger compaction and SGP cluster fusion defects than mutants for any one robo, suggesting Slit is the required ligand for Robo function, and that there is also some functional redundancy between the different Robo receptors during these aspects of gonad formation (Weyers, 2011).

Though slit mutants demonstrate a gonad phenotype, no zygotic Slit expression was detected within or immediately surrounding the embryonic gonad. Therefore, Slit could be acting upon the gonad from a distance. Potential sources of Slit include the ectoderm at muscle attachment sites and the walls of the gut, both of which are adjacent to the mesoderm where the gonad is located. These locations suggest that Slit repels migrating SGPs away from these surrounding tissues, preventing SGPs from exploring too far medially or laterally, and in effect guiding SGP clusters together and helping them to condense together during compaction. In an effort to explore this model further, Slit was expressed in various tissues surrounding the gonad, however, these experiments gave ambiguous results which neither refuted nor supported the model of Slit as a guidance factor in gonadogenesis. Rather, these results suggested that the amount of Slit present was more important than the location. Thus, it is also possible that Slit provides a permissive signal to gonadal cells, rather than a directionally instructive one (Weyers, 2011).

The Robos may also contribute to gonad formation through adhesive mechanisms; Robo and Robo2 exhibit both homophilic and heterophilic adhesion properties. Studies with shg (DE-cad) or foi mutants indicate that a loss of adhesion in the gonad can account for incomplete compaction and ensheathment. Therefore, the defects observed in mutants for the robo genes may be due to a disruption in Robo-mediated adhesion within the gonad. Robo adhesion can occur independently of Slit and would therefore account for the SLIT independent nature of ensheathment (Weyers, 2011).

The Slit/Robo pathway may also influence cadherin-mediated adhesion in the gonad. While some studies have demonstrated a negative relationship between cadherin-based adhesion and Slit/Robo signaling, Slit/Robo have also been observed to upregulate cadherins. While no significant change was observed in DE-CAD expression in Slit/Robo pathway mutants, it remains possible that these two important mechanisms for regulating gonad formation are cooperating in this process (Weyers, 2011).

Drosophila syndecan regulates tracheal cell migration by stabilizing Robo levels

This study identified a new role for Syndecan (Sdc), the only transmembrane heparan sulphate proteoglycan in Drosophila, in tracheal development. Sdc is required cell autonomously for efficient directed migration and fusion of dorsal branch cells, but not for dorsal branch formation per se. The cytoplasmic domain of Sdc is dispensable, indicating that Sdc does not transduce a signal by itself. Although the branch-specific phenotype of sdc mutants resembles those seen in the absence of Slit/Robo2 signalling, genetic interaction experiments indicate that Sdc also helps to suppress Slit/Robo2 signalling. It is concluded that Sdc cell autonomously regulates Slit/Robo2 signalling in tracheal cells to guarantee ordered directional migration and branch fusion (Schulz, 2011; Full text of article).

A non-signaling role of Robo2 in tendons is essential for Slit processing and muscle patterning

Coordinated locomotion of an organism relies on the development of proper musculoskeletal connections. In Drosophila, the Slit-Robo signaling pathway guides muscles to tendons. This study shows that the Slit receptor Roundabout 2 (Robo2) plays a non-cell-autonomous role in directing muscles to their corresponding tendons. Robo2 is expressed by tendons, and its non signaling activity in these cells promotes Slit cleavage producing a cleaved Slit-N-terminal guiding signal, which provides short-range signaling into muscles. Consistently, robo2 mutant embryos exhibited a muscle phenotype similar to that of slit, which could not be rescued by a muscle-specific Robo2 expression but rather by an ectodermally derived Robo2. Alternatively this muscle phenotype could be induced by tendon-specific robo2 RNAi. It was further shown that membrane immobilization of Slit, or its N-terminal cleaved form on tendons bypasses the functional requirement for Robo2 in tendons, verifying that the major role of Robo2 is to promote the association of Slit with the tendon cell membrane. Cleaved Slit (Slit-N) tends to oligomerize whereas full-length uncleavable Slit does not. It is therefore proposed that Slit-N oligomers produced at the tendon membrane by Robo2 signal to the approaching muscle by combined Robo;Robo3 activity. These findings establish a Robo2-mediated mechanism, independent of signaling essential to limiting Slit distribution, which might be relevant to the regulation of Slit-mediated short-range signaling in additional systems (Ordan, 2015).

To determine the individual contribution of Robo family receptors to the embryonic muscle pattern, the orientation of the three lateral transverse muscles (LT1-3) and of the dorsal acute muscle 3 (DA3) was analyzed in Drosophila embryos at stage 16 lacking distinct Robo receptors, and it was compared to that of slit. In slit mutant embryos the LT muscles are closer to the posterior segment border and are not spaced correctly, and the DA3 muscle does not extend its leading edge at orthogonal fashion. The orientation of the LT and the DA3 muscles in robo or in robo3 mutants, exhibited almost normal positioning relative to their wild-type (WT) counterparts. Quantification of the ratio between the distance of LT3 muscle and the posterior segment border, divided to overall segmental width (DLT3/Ds) revealed no significant difference between robo and WT, or robo3 and WT. However, in the double robo;robo3 mutant embryos the orientation of these muscles resembled that of slit. robo2 mutants exhibited a phenotype that was comparable to that of slit mutants; the LT muscles were not equally spaced and were closer to the posterior segmental border, a phenotype observed in 85% of the segments. Quantification of DLT3/Ds revealed a smaller value in robo2 mutant. In addition, in 33% of the robo2 mutant segments, the DA3 muscle lost its diagonal orientation and often migrated in a straight ventral-dorsal orientation (Ordan, 2015).

To reveal the dynamic nature of the process of muscle elongation in robo2 mutants, individual muscles were followed in live robo2 mutant and control embryos using specific GFP-marked LT, or DA3 muscles in stage 13-16 throughout muscle elongation. This analysis showed that the polarity of the mutant muscles was similar to that of WT control muscles; however, the LT muscles tended to elongate closer to the segment border and were not evenly spaced. The DA3 muscle often did not turn towards the posterior segment, and both muscles reached their target tendons more slowly. These results suggested differential contributions of the Robo receptors to the directed elongation of LT and DA3 muscles (Ordan, 2015).

To reveal the relative distribution and expression levels of the Robo receptors in muscles, advantage was taken of flies carrying HA tag that was knocked-in within the genomic region of either of the Robo receptors, and embryos were labeled with anti-HA. Robo-HA accumulated at the muscle-tendon junction, and was observed at low levels on the muscle surfaces. Robo2 levels were prominent along ectodermal stripes, but not on muscles, and Robo3 labeling was indistinguishable from background. Double labeling with anti-Robo2 and Stripe or with CD8-GFP driven by the stripe-GAL4 driver verified the ectodermal expression of Robo2 at the surfaces of the tendon cells. The relatively high ectodermal expression of Robo2-HA and its lack of staining in the affected LT and DA3 muscles were consistent with a muscle non-autonomous function of Robo2 in mediating LT and DA3 muscle-patterning (Ordan, 2015).

To address whether undetectable Robo2 protein functioning in the muscles, the ability of Robo2 to rescue the robo2 mutant muscle phenotype was tested when expressed in muscles. Driving Robo2 expression in muscles of robo2 mutant embryos using the mef2-GAL4 driver did not rescue the muscle phenotype, but rather worsen the muscle pattern in all embryos tested compared to wild type embryos overexpressing Robo2. In contrast, knockdown of Robo2 in tendons of robo2 -/+ heterozygous embryos (using sr-GAL4>robo2 RNAi) led to a phenotype similar to that of robo2 mutant; the LT muscles were mis-patterned in 84% of the segments, and the DA3 muscle was aberrant in 31% of the segments. Importantly, partial rescue of the LT muscle pattern was achieved by expressing Robo2 in the ectoderm using the 69-GAL4 driver. Moreover, embryos in which Robo has been inserted into the robo2 locus, rescued the robo2 muscle phenotype, supporting the critical contribution of Robo2 unique ectodermal distribution rather than its signaling activity. In summary, the ectodermal expression of Robo2, its knockdown in tendons, and the ectodermal rescue imply that Robo2 functions in the ectoderm to induce the muscle pattern (Ordan, 2015).

Next, the requirement was addressed for Slit in the ectodermal activity of Robo2, associated with guiding the LT and DA3 muscles. Strong genetic interaction was found between robo2 and slit in inducing the LT and DA3 muscle pattern. Trans-heterozygous embryos for both robo2 and slit exhibited an aberrant pattern of the LT muscles in 82% of the segments relative to 0 or 10% in robo2 /+ or slit/+ embryos, respectively. In the DA3 muscle, 10% of the segments of the trans-heterozygous slit/robo2 embryos exhibited aberrant muscle orientation, relative to 0% in either of the single heterozygous mutants. These results imply that Robo2 activity is mediated through its ligand Slit (Ordan, 2015).

Next, whether Robo2 affects Slit cleavage, which is critical to short-range Slit signaling, was tested. To this end, Robo2 was overexpressed in the entire embryonic ectoderm using the 69B-GAL4 driver and followed endogenous Slit cleavage by western blotting using anti-Slit antibody, reactive with the C-terminal of Slit; this antibody recognizes both full-length Slit (~170kDa) and the C-terminal cleaved polypeptide, Slit-C, (~80kDa). Strikingly, overexpression of Robo2 in the ectoderm led to an average 9.7-fold increase in the ratio of Slit-C relative to Slit-FL levels, implicating Robo2 in promoting Slit cleavage in the ectoderm. In contrast, overexpression of Robo2 in the muscles (using the mef2-GAL4 driver) did not have this effect. Remarkably, similar enhanced Slit cleavage was observed following overexpression of truncated Robo2 lacking the cytoplasmic domain (Evans and Bashaw, 2010). Notably, overexpression of Robo in the ectoderm similarly enhanced Slit cleavage (data not shown). These results implicated a non-signaling function for Robo2 in promoting Slit cleavage (Ordan, 2015).

It was reasoned that covalently bound Slit oligomers, shown to form by previous crystallographic studies, would enable Slit to bind both juxtaposing Robo receptors, on the muscle side (Robo and/or Robo3) and on the tendon side (Robo2). Therefore Slit oligomerization was tested for in embryos. Overexpression of either GFP-tagged cleaved Slit-N (Slit-N-GFP), or uncleavable full length Slit-UC-myc, was induced in the embryonic ectoderm with 69B-GAL4 driver. The embryo extracts were then boiled under reducing (with β- mercaptoethanol, BME) or non-reducing conditions (without BME), separated on SDS-PAGE, and further reacted by western, either with anti-GFP, or anti-Myc. Under non-reducing conditions, Slit-N-GFP exhibited slower electrophoretic mobility relative to its mobility in reduced conditions (expected to be 124kDa), supporting the formation of Slit-N-GFP oligomers by disulfide bonds. In contrast, uncleavable Slit (Slit-UC-myc) did not exhibit differential electrophoretic mobility in natured versus denatured conditions. This result is consistent with the unique ability of Slit-N to oligomerize (Ordan, 2015).

Taken together, these results suggest that Robo2 in tendons enhances Slit cleavage, producing Slit-N oligomers at the tendon-cell membrane that are potentially capable of binding both Robo2 in cis, and Robo/Robo3 in trans, leading to spatially restricted Slit signaling to the elongating muscle. Knocked-in membrane-bound full length Slit bypasses the requirement for Robo2. It was reasoned that if the primary function of Robo2 is to promote localized Slit-N oligomers, the knocked-in membrane-bound uncleavable Slit, (Slit- uncleavable(UC)-CD8), would rescue the phenotype of robo2. Knocked-in Slit-UC-CD8 was recombined with robo2 and the phenotype of the DA3 and LT muscles was analyzed in homozygous embryos. Whereas in robo2 mutants, the orientation of the LT muscles was defective in 85% of the segments, the addition of knocked-in Slit-UC-CD8 showed only 37% defective segments. Likewise the DLT3/Ds ratio was rescued in the robo2;Slit-UC-CD8 embryos. Given that Slit-UC-CD8 by itself produced a moderate muscle phenotype, it is concluded that Slit-UC- CD8 is capable of rescuing the robo2 phenotype of the LT muscles. For the DA3 muscle, whereas 32% of the segments showed a phenotype in robo2 mutants, robo2 mutant embryos recombined with Slit-UC-CD8 showed a muscle phenotype in only 13% of the segments, and Slit-UC-CD8 mutants showed only 5% defective segments (Ordan, 2015).

Moreover, consistent with a major function of Robo2 in promoting Slit cleavage, overexpression of the active cleaved Slit-N-GFP in tendon cells rescued the robo2 mutant LT muscle phenotype in 73% of the segments, whereas expression of Slit-UC-myc alone did not. These results are consistent with a major function for Robo2 in promoting Slit-N accumulation at the tendon cell- membrane, presumably by retaining Slit-FL and enhancing its availability proteolytic cleavage. Slit-N oligomers potentially bind to both Robo2 at the tendon side as well as to Robo and/or Robo3 at the muscle side to promote unidirectional short-range Slit-repulsive signal in muscles. In this context Robo2 cytoplasmic signaling domain is dispensable, consistent with Robo2 function in chordotonal organs and with Robo2 non autonomous function in the CNS (Ordan, 2015).

In conclusion, the findings of this study demonstrate a novel, non-signaling role for Robo2 in tendons in regulating the local distribution of active cleaved Slit oligomers at the tendon cell-membrane. This signal is critical in promoting Slit short-range signaling in muscles essential for directional muscle elongation. Such mechanism might be significant in other setups where Slit promotes signaling between neighboring cells (Ordan, 2015).


REFERENCES

Andrews, W., et al. (2006). Robo1 regulates the development of major axon tracts and interneuron migration in the forebrain. Development 133(11): 2243-52. 16690755

Baek, M. and Mann, R. (2009). Lineage and birth date specify motor neuron targeting and dendritic architecture in adult Drosophila. J. Neurosci. 29: 6904-6916. PubMed Citation: 19474317

Bagri, A., et al. (2002). Slit proteins prevent midline crossing and determine the dorsoventral position of major axonal pathways in the mammalian forebrain Neuron 33: 233-248. 11804571

Barresi, M. J. F., et al. (2005). Hedgehog regulated Slit expression determines commissure and glial cell position in the zebrafish forebrain. Development 132: 3643-3656. 16033800

Battye, R., Stevens, A. and Jacobs, J. R. (1999). Axon repulsion from the midline of the Drosophila CNS requires slit function Development 126: 2475-2481. PubMed Citation: 10226006

Bashaw, G. J. and Goodman, C. S. (1999). Chimeric axon guidance receptors: The cytoplasmic domains of Slit and Netrin receptors specify attraction versus repulsion. Cell 97: 917-926. PubMed Citation: 10399919

Battye, R., et al. (2001). Repellent signaling by Slit requires the leucine-rich repeats. J. Neurosci. 21(12): 4290-4298. 11404414

Biteau, B. and Jasper, H. (2014). Slit/Robo signaling regulates cell fate decisions in the intestinal stem cell lineage of Drosophila. Cell Rep 7(6): 1867-75. PubMed ID: 24931602

Brenman, J. E., Gao, F.-B., Jan, L. Y. and Jan, Y. N. (2001). Sequoia, a Tramtrack-related zinc finger protein, functions as a pan-neural regulator for dendrite and axon morphogenesis in Drosophila. Dev. Cell 1: 667-677. 11709187

Brierley, D. J., Blanc, E., Reddy, O. V., VijayRaghavan, K. and Williams, D. W. (2009). Dendritic targeting in the leg neuropil of Drosophila: the role of midline signalling molecules in generating a myotopic map. PLoS Biol. 7(9): e1000199. PubMed Citation: 19771147

Brose, K., et al. (1999). Slit proteins bind Robo receptors and have an evolutionarily conserved role in repulsive axon guidance. Cell 96(6): 795-806. PubMed Citation: 10102268

Bülow, H. E., et al. (2008). Extracellular sugar modifications provide instructive and cell-specific information for axon-guidance choices. Curr. Biol. 18(24): 1978-85. PubMed Citation: 19062279

Campbell, D. S., et al. (2007). Slit1a inhibits retinal ganglion cell arborization and synaptogenesis via Robo2-dependent and -independent pathways. Neuron 55(2): 231-45. PubMed citation: 17640525

Campbell, D. S. and Okamoto, H. (2013). Local caspase activation interacts with Slit-Robo signaling to restrict axonal arborization. J Cell Biol 203: 657-672. PubMed ID: 24385488

Causeret, F., et al. (2002). Slit antagonizes Netrin-1 attractive effects during the migration of inferior olivary neurons. Dev. Biol. 246: 429-440. 12051827

Chang, C., et al. (2006). MIG-10/lamellipodin and AGE-1/PI3K promote axon guidance and outgrowth in response to slit and netrin. Curr. Biol. 16(9): 854-62. 16618541

Chen, E. H., Pryce, B. A., Tzeng, J. A., Gonzales, G. A. and Olson, E. N. (2003). Control of myoblast fusion by a guanine nucleotide exchange factor, loner, and its effector ARF6. Cell 114: 751-762. 14505574

Crowner, D., Madden, K., Goeke, S. and Giniger, E. (2002). Lola regulates midline crossing of CNS axons in Drosophila. Development 129: 1317-1325. 11880341

Dascenco, D., Erfurth, M. L., Izadifar, A., Song, M., Sachse, S., Bortnick, R., Urwyler, O., Petrovic, M., Ayaz, D., He, H., Kise, Y., Thomas, F., Kidd, T. and Schmucker, D. (2015). Slit and Receptor tyrosine phosphatase 69D confer spatial specificity to axon branching via Dscam1. Cell 162: 1140-1154. PubMed ID: 26317474

Englund, C., et al. (2002). Attractive and repulsive functions of Slit are mediated by different receptors in the Drosophila trachea. Development 129: 4941-4951. 12397103

Erskine, L., et al. (2000). Retinal ganglion cell axon guidance in the mouse optic chiasm: Expression and function of robos and slits. J. Neurosci. 20(13): 4975-4982. 10864955

Evans, I. R., Hu, N., Skaer, H. and Wood, W. (2010). Interdependence of macrophage migration and ventral nerve cord development in Drosophila embryos. Development 137(10): 1625-33. PubMed Citation: 20392742

Evans, T. A., Bashaw, G. J. (2010) Functional diversity of Robo receptor immunoglobulin domains promotes distinct axon guidance decisions. Curr Biol 20: 567-572. PubMed ID: 20206526

Fabre C. C. G., Casal J. and Lawrence P. A. (2008). The abdomen of Drosophila: does planar cell polarity orient the neurons of mechanosensory bristles? Neural Dev. 3: 12. PubMed Citation: 18668998

Fabre, C. C., Casal, J. and Lawrence, P. A. (2010). Mechanosensilla in the adult abdomen of Drosophila: engrailed and slit help to corral the peripheral sensory axons into segmental bundles. Development 137(17): 2885-94. PubMed Citation: 20667917

Fan, X., Labrador, J. P., Hing, H. and Bashaw, G. J. (2003). Slit stimulation recruits Dock and Pak to the Roundabout receptor and increases Rac activity to regulate axon repulsion at the CNS midline. Neuron 40: 113-127. 14527437

Farmer, W. T., et al. (2008). Pioneer longitudinal axons navigate using floor plate and Slit/Robo signals. Development 135(22): 3643-53. PubMed Citation: 18842816

Fish, J. E., et al. (2011). A Slit/miR-218/Robo regulatory loop is required during heart tube formation in zebrafish. Development 138(7): 1409-19. PubMed Citation: 21385766

Furrer, M. P., Kim, S., Wolf, B. and Chiba, A. (2003). Robo and Frazzled/DCC mediate dendritic guidance at the CNS midline. Nat. Neurosci. 6: 223-230. PubMed citation: 12592406

Furrer, M. P., Vasenkova, I., Kamiyama, D., Rosado, Y. and Chiba, A. (2007). Slit and Robo control the development of dendrites in Drosophila CNS. Development 134(21): 3795-804. PubMed citation: 17933790

Ghavi-Helm, Y., Klein, F. A., Pakozdi, T., Ciglar, L., Noordermeer, D., Huber, W., Furlong, E. E. (2014) Enhancer loops appear stable during development and are associated with paused polymerase. Nature 512(7512): 96-100. PubMed ID: 25043061

Gibson, D. A., Tymanskyj, S., Yuan, R. C., Leung, H. C., Lefebvre, J. L., Sanes, J. R., Chedotal, A. and Ma, L. (2014). Dendrite self-avoidance requires cell-autonomous slit/robo signaling in cerebellar purkinje cells. Neuron 81: 1040-1056. PubMed ID: 24607227

Grieshammer, U., et al. (2004). SLIT2-mediated ROBO2 signaling restricts kidney induction to a single site. Dev. Cell 6: 709-717. 15130495

Guan, C. B., Xu, H. T., Jin, M., Yuan, X. B. and Poo, M. M. (2007). Long-range Ca2+ signaling from growth cone to soma mediates reversal of neuronal migration induced by slit-2. Cell 129(2): 385-95. PubMed citation: 17448996

Hao, J. C., et al. (2001). C. elegans Slit acts in midline, dorsal-ventral, and anterior-posterior guidance via the SAX-3/Robo receptor. Neuron 32: 25-38. 11604136

Hammond, R., et al. (2005). Slit-mediated repulsion is a key regulator of motor axon pathfinding in the hindbrain. Development 132(20): 4483-95. 16162649

Hocking, J. C., Hehr, C. L., Bertolesi, G. E., Wu, J. Y. and McFarlane, S. (2010). Distinct roles for Robo2 in the regulation of axon and dendrite growth by retinal ganglion cells. Mech. Dev. 127(1-2): 36-48. PubMed Citation: 19961927

Holmes, G. P., et al. (1998). Distinct but overlapping expression patterns of two vertebrate slit homologs implies functional roles in CNS development and organogenesis. Mech. Dev. 79(1-2): 57-72. PubMed Citation: 10349621

Itoh, A., et al. (1998). Cloning and expressions of three mammalian homologues of Drosophila slit suggest possible roles for Slit in the formation and maintenance of the nervous system. Brain Res. Mol. Brain Res. 62(2): 175-86. PubMed Citation: 9813312

Jacobs, J. R. (1993). Perturbed glial scaffold formation precedes axon tract malformation in Drosophila mutants. J Neurobiol 24 (5): 611-626. PubMed Citation: 8326301

Jaumouille, E., Machado Almeida, P., Stahli, P., Koch, R. and Nagoshi, E. (2015). Transcriptional regulation via nuclear receptor crosstalk required for the Drosophila circadian clock. Curr Biol 25: 1502-1508. PubMed ID: 26004759

Jhaveri, D., Saharan, S., Sen, A. and Rodrigues, V. (2004). Positioning sensory terminals in the olfactory lobe of Drosophila by Robo signaling. Development 131: 1903-1912. 15056612

Jin, Z., et al. (2003). Irx4-mediated regulation of Slit1 expression contributes to the definition of early axonal paths inside the retina. Development 130: 1037-1048. 12571096

Johnson, K. G., et al. (2004). Axonal heparan sulfate proteoglycans regulate the distribution and efficiency of the repellent Slit during midline axon guidance. Curr. Biol. 14: 499-504. 15043815

Keleman, K. and Dickson, B. J. (2001). Short- and long-range repulsion by the Drosophila Unc5 Netrin receptor. Neuron 32: 605-617. 11719202

Kidd, T., Bland, K. S. and Goodman, C. S. (1999). Slit is the midline repellent for the robo receptor in Drosophila. Cell 96(6): 785-94. PubMed Citation: 10102267

Kim, Y.-S., et al. (2002). Constitutively active myosin light chain kinase alters axon guidance decisions in Drosophila embryos. Dev. Bio. 249: 367-381. 12221012

Knöll, B., et al. (2003). On the topographic targeting of basal vomeronasal axons through Slit-mediated chemorepulsion. Development 130: 5073-5082. 12954717

Kucherenko, M. M., et al. (2008). Genetic modifier screens reveal new components that interact with the Drosophila dystroglycan-dystrophin complex. PLoS ONE 3(6): e2418. PubMed Citation: 18545683

Landgraf, M., Jeffrey, V., Fujioka, M., Jaynes, J. B. and Bate, M. (2003). Embryonic origins of a motor system: motor dendrites form a myotopic map in Drosophila. PLoS Biol. 1: E41. PubMed Citation: 14624243

Lavado, A., Ware, M., Pare, J. and Cao, X. (2014). The tumor suppressor Nf2 regulates corpus callosum development by inhibiting the transcriptional coactivator Yap. Development 141: 4182-4193. PubMed ID: 25336744

Lee, H., et al. (2004). The microtubule plus end tracking protein Orbit/MAST/CLASP acts downstream of the tyrosine kinase Abl in mediating axon guidance. Neuron 42: 913-926. 15207236

Lewis, J.O., and Crews, S.T. (1994). Genetic analysis of the Drosophila single-minded gene reveals a central nervous system influence on muscle development. Mech. Dev. 48: 81-91. PubMed Citation: 7873405

Li, H. S., et al. (1999). Vertebrate slit, a secreted ligand for the transmembrane protein roundabout, is a repellent for olfactory bulb axons. Cell 96(6): 807-18. PubMed Citation: 10102269

Liang, Y., et al. (1999). Mammalian homologues of the Drosophila Slit protein are ligands of the heparan sulfate proteoglycan Glypican-1 in brain. J. Biol. Chem. 274: 17885-17892. PubMed Citation: 10364234

Liu, Q. X., et al. (2009). Midline governs axon pathfinding by coordinating expression of two major guidance systems. Genes Dev. 23(10): 1165-70. PubMed Citation: 19451216

Long, H., et al. (2004). Conserved roles for Slit and Robo proteins in midline commissural axon guidance. Neuron 42: 213-223. 15091338

Ma, Y., et al. (2000). Functional interactions between Drosophila bHLH/PAS, Sox, and POU transcription factors regulate CNS midline expression of the slit gene. J. Neurosci. 20(12): 4596-4605. 10844029

MacMullin, A. and Jacobs, J. R. (2006). Slit coordinates cardiac morphogenesis in Drosophila. Dev. Biol. 293(1): 154-64. 16516189

Marín, O., et al (2003). Directional guidance of interneuron migration to the cerebral cortex relies on subcortical Slit1/2-independent repulsion and cortical attraction. Development 130: 1889-1901. 12642493

Mauss, A., Tripodi, M., Evers, J. F. and Landgraf, M. (2009). Midline signalling systems direct the formation of a neural map by dendritic targeting in the Drosophila motor system. PLoS Biol. 7(9): e1000200. PubMed Citation: 1977114

Medioni, C., et al. (2008). Genetic control of cell morphogenesis during Drosophila melanogaster cardiac tube formation. J. Cell Biol. 182(2): 249-61. PubMed Citation: 18663140

Mehta, B. and Bhat, K. M. (2001). Slit signaling promotes the terminal asymmetric division of neural precursor cells in the Drosophila CNS. Development 128: 3161-3168. 11688564

Miyashita, T., et al. (2004). PlexinA4 is necessary as a downstream target of Islet2 to mediate Slit signaling for promotion of sensory axon branching. Development 131: 3705-3715. 15229183

Nambu, J.R., Franks, R.G., Hu, S. and Crews, S.T. (1990). The single-minded gene of Drosophila is required for the expression of genes important for the development of CNS midline cells. Cell 63: 63-75

Nguyen Ba-Charvet, K. T., et al. (1999). Slit2-Mediated chemorepulsion and collapse of developing forebrain axons. Neuron 22(3): 463-73

Nusslein-Volhard, C., Wieschaus, E. and Kluding, H. (1984). Mutations affecting the pattern of the larval cuticle in Drosophila melanogaster. I. Zygotic loci on the second chromosome. Roux's Arch. Dev. Biol. 193: 267-282

Oda, H., et al. (1994). A Drosophila homolog of cadherin associated with armadillo and essential for embryonic cell-cell adhesion. Dev. Biol. 165: 716-26

Önel, S., Bolke, L. and Klämbt, C. (2004). The Drosophila ARF6-GEF Schizo controls commissure formation by regulating Slit. Development 131: 2587-2594. 15148300

Ordan, E. and Volk, T. (2015). A non-signaling role of Robo2 in tendons is essential for Slit processing and muscle patterning. Development 142: 3512-3518. PubMed ID: 26400093

Ozdinler, P. H. and Erzurumlu, R. S. (2002). Slit2, a branching-arborization factor for sensory axons in the Mammalian CNS. J. Neurosci. 22: 4540-4549. PubMed citation: 12040061

Patel, K., et al. (2001). Slit proteins are not dominant chemorepellents for olfactory tract and spinal motor axons. Development 128: 5031-5037. 11748139

Patthy, L. (1992). A family of laminin-related proteins controlling ectodermal differentiation in Drosophila. FEBS Lett. 298: 182-4

Piper, M., et al. (2006). Signaling mechanisms underlying Slit2-induced collapse of Xenopus retinal growth cones. Neuron 49(2): 215-28. 16423696

Plump. A. S., et al. (2002). Slit1 and Slit2 cooperate to prevent premature midline crossing of retinal axons in the mouse visual system. Neuron 33: 219-232. 11804570

Qian, L., Liu, J. and Bodmer, R. (2005). Slit and Robo control cardiac cell polarity and morphogenesis. Curr. Biol. 15(24): 2271-8. 16360689

Quinn, C. C., et al. (2006). UNC-6/netrin and SLT-1/slit guidance cues orient axon outgrowth mediated by MIG-10/RIAM/lamellipodin. Curr. Biol. 16(9): 845-53. 16563765

Rothberg, J.M., et al. (1988). An EGF-homologous locus of D. melanogaster involved in development of the embryonic central nervous system. Cell 55: 1047-59

Rothberg, J.M., Jacobs, J.R., Goodman, C.S. and Artavanis-Tsakonas, S. (1990). slit: an extracellular protein necessary for development of midline glia and commissural axon pathways contains both EGF and LRR domains. Genes & Dev. 4: 2169-87

Rothberg, J.M. and Artavanis-Tsakonas, S. (1992). Modularity of the slit protein. Characterization of a conserved carboxy-terminal sequence in secreted proteins and a motif implicated in extracellular protein interactions. J. Mol. Biol. 227: 367-70

Sabatier, C., et al. (2004). The divergent Robo family protein rig-1/Robo3 is a negative regulator of slit responsiveness required for midline crossing by commissural axons. Cell 117(2): 157-69. 15084255

Santiago-Martínez, E., Soplop, N. H. and Kramer, S. G. (2006). Lateral positioning at the dorsal midline: Slit and Roundabout receptors guide Drosophila heart cell migration. Proc. Natl. Acad. Sci. 103: 12441-12446. Medline abstract: 16888037

Schimmelpfeng, K., Gögel, S. and Klämbt, C. (2001). The function of leak and kuzbanian during growth cone and cell migration. Mech. Dev. 106: 25-36. 11472832

Schulz, J. G., et al. (2011). Drosophila syndecan regulates tracheal cell migration by stabilizing Robo levels. EMBO Rep. 12(10): 1039-46. PubMed Citation: 21836636

Shiau, C. E. and Bronner-Fraser, M. (2009). N-cadherin acts in concert with Slit1-Robo2 signaling in regulating aggregation of placode-derived cranial sensory neurons. Development 136(24): 4155-64. PubMed Citation: 19934013

Simpson, J. H., et al. (2000). Short-range and long-range guidance by Slit and its Robo receptors: Robo and Robo2 play distinct roles in midline guidance. Neuron 28: 753-766. PubMed Citation: 11163264

Sonnenfeld, M.J. and Jacobs, J.R. (1994). Mesectodermal cell fate analysis in Drosophila midline mutants. Mech. Dev. 46: 3-13

Sonnenfeld, M. J. and Jacobs, J. R. (1995). Apoptosis of the midline glia during Drosophila embryogenesis: a correlation with axon contact. Development 121: 569-578

Spitzweck, B., Brankatschk, M., Dickson, B. J. (2010) Distinct protein domains and expression patterns confer divergent axon guidance functions for Drosophila Robo receptors. Cell 140: 409-420. PubMed ID: 20144763

Steigemann, P., Molitor, A., Fellert, S., Jackle, H. and Vorbruggen, G. (2004). Heparan sulfate proteoglycan syndecan promotes axonal and myotube guidance by slit/robo signaling. Curr. Biol. 14(3): 225-30. 14761655

Stine, R. R., Greenspan, L. J., Ramachandran, K. V. and Matunis, E. L. (2014). Coordinate regulation of stem cell competition by Slit-Robo and JAK-STAT signaling in the Drosophila testis. PLoS Genet 10: e1004713. PubMed ID: 25375180

Stevens, A. and Jacobs, J. R. (2002). Integrins regulate responsiveness to Slit repellent signals. J. Neurosci. 22(11): 4448-4455. 12040052

Taguchi, A., et al. (1996). Molecular cloning of novel leucine-rich repeat proteins and their expression in the developing mouse nervous system. Brain Res. Mol. Brain Res. 35(1-2): 31-40

Tayler, T. D., Robichaux, M. B. and Garrity, P. A. (2004). Compartmentalization of visual centers in the Drosophila brain requires Slit and Robo proteins. Development 131(23): 5935-45. 15525663

Vanderploeg, J. and Jacobs, R. (2015). Talin is required to position and expand the luminal domain of the Drosophila heart tube. Dev Biol [Epub ahead of print]. PubMed ID: 25958089

Wang, K. H., et al. (1999). Biochemical purification of a mammalian Slit protein as a positive regulator of sensory axon elongation and branching. Cell 96(6): 771-84. PubMed Citation: 10102266

Ward, M. E., Jiang, H. and Rao, Y. (2005). Regulated formation and selection of neuronal processes underlie directional guidance of neuronal migration. Mol. Cell. Neurosci. 30: 378-387. PubMed citation: 16154761

Weyers, J. J., et al. (2011). A genetic screen for mutations affecting gonad formation in Drosophila reveals a role for the slit/robo pathway. Dev. Biol. 353(2): 217-28. PubMed Citation: 21377458

Wharton, K.A. and Crews, S.T. (1993). CNS midline enhancers of the Drosophila slit and Toll genes. Mech. Dev. 40: 141-54. PubMed Citation: 8494768

Wharton, K.A., et al. (1994). Control of CNS midline transcription by asymmetric E-box like elements: similarity to Xenobiotic responsive regulation. Development 120: 3563-69. PubMed Citation: 7821222

Whitford, K. L. et al. (2002). Regulation of cortical dendrite development by slit-robo interactions. Neuron 33: 47-61. 11779479

Wills, Z., et al. (2002). A Drosophila homolog of cyclase-associated proteins collaborates with the Abl tyrosine kinase to control midline axon pathfinding. Neuron 36: 611-622. 12441051

Wong, K., et al. (2001). Signal transduction in neuronal migration: Roles of GTPase activating proteins and the small GTPase Cdc42 in the Slit-Robo pathway. Cell 107: 209-221. 11672528

Wu, W., et al. (1999). Directional guidance of neuronal migration in the olfactory system by the protein Slit. Nature 400(6742): 331-6. PubMed ID: 10432110

Xu, Y. and Quinn, C. C. (2012). MIG-10 functions with ABI-1 to mediate the UNC-6 and SLT-1 axon guidance signaling pathways. PLoS Genet 8: e1003054. PubMed ID: 23209429

Yang, L. and Bashaw, G. J. (2006). Son of sevenless directly links the Robo receptor to rac activation to control axon repulsion at the midline. Neuron 52(4): 595-607. Medline abstract: 17114045

Yang, L., Garbe, D. S. and Bashaw, G. J. (2009). A frazzled/DCC-dependent transcriptional switch regulates midline axon guidance. Science 324: 944-947. PubMed Citation: 19325078

Yeo, S.-Y., et al. (2001). Overexpression of a Slit homologue impairs convergent extension of the mesoderm and causes cyclopia in embryonic zebrafish. Dev. Bio. 230: 1-17. 11161558

Yeo, S.-Y., et al. (2004). Involvement of Islet-2 in the Slit signaling for axonal branching and defasciculation of the sensory neurons in embryonic zebrafish. Mech. Dev. 121: 315-324. 15110042

Yuan, W., et al. (1999). The mouse SLIT family: secreted ligands for ROBO expressed in patterns that suggest a role in morphogenesis and axon guidance. Dev. Biol. 212(2): 290-306

Zou, Y., et al. (2000). Squeezing axons out of the gray matter: A role for slit and semaphorin proteins from midline and ventral spinal cord. Cell 102: 363-375. PubMed ID: 10975526


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

date revised: 5 November 2015

Home page: The Interactive Fly © 1997 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.