roundabout

DEVELOPMENTAL BIOLOGY

Embryonic

The robo expression pattern was examined in the embryonic CNS. The in situ hybridization pattern of ROBO mRNA in Drosophila shows it to have elevated and widespread expression in the CNS. Multiple monoclonal and serum antibodies were raised against portions of Robo protein and the same staining pattern was observed with all of them. Robo is first seen in the embryo weakly expressed in lateral stripes during germband extension [Images]. At the onset of germband retraction, Robo expression is observed in the neuroectoderm. By the end of stage 12, as the growth cones first extend, Robo is seen on growth cones which project ipsilaterally, including pCC, aCC, MP1, dMP2, and vMP2. Strikingly, little or no Robo expression is observed on commissural growth cones as they extend toward and across the midline. However, as these growth cones turn to project longitudinally, their level of Robo expression dramatically increases. Robo is expressed at high levels on all longitudinally projecting growth cones and axons. In contrast, Robo is expressed at nearly undetectable levels on commissural axons. This is striking since 90% of axons in the longitudinal tracts also have axon segments crossing in one of the commissures. Thus, Robo expression is regionally restricted. Robo expression is also seen at a low level throughout the epidermis and at a higher level at muscle attachment sites. In stage 16-17 embryos, faint Robo staining can be seen in the commissures but at levels much lower than observed in the longitudinal tracts. For those axons that never cross the midline, Robo is expressed on their growth cones from the outset; for the majority of axons that do cross the midline, Robo is expressed at high levels on their growth cones only after they cross the midline (Kidd, 1998a).

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

The glial cells present repulsive signals to the Roundabout receptor in addition to a permissive contact-dependent signal helping commissural growth cones across the midline. A novel repulsive component is encoded by the karussell gene. In stage 12 karussell mutant embryos, several short FasII-positive cell processes project toward the CNS midline. In older embryos, FasII expression is found on axons crossing the midline in more than 50% of the neuromeres. The distribution of the 22C10 antigen (see Futsch) in karussell embryos uncovers only a few abnormalities. Thus a small subset of normally ipsilateral axons project contralateral in karussell mutants. The majority of axons found in the circles around the RP1 neurons are likely to be commissural axons that cross the midline more than once. This suggests that karussell encodes a novel component of the repulsive signaling pathway. karussell is shown to act in parallel to commissureless and roundabout gene functions, or it may act downstream in a common regulatory hierarchy (Hummel, 1999).

To determine which CNS midline cells present the repulsive signal recognized by Robo, double mutant embryos were analyzed. In commissureless mutant embryos no commissures are formed. The gene pointed is specifically expressed in the midline glial cells and controls differentiation of this cell type. In commissureless pointed mutant embryos few commissures are formed. Similarly in commissureless/slit mutant embryos commissures do form. Since pointed as well as slit affect differentiation of the midline glial cells it is suggested that these cells present the repulsive ligand to Robo. In the absence of differentiated midline glial cells no repulsive ligand can be present and growth cones can cross the midline. This finding implies that disruption in glial differentiation at the midline should lead to a robo-like phenotype as well (Hummel, 1999).

What is the function of midline glia in commissure formation? Genetic data suggest that, in addition to being a permissive substrate for commissural growth, the midline glial cells present the repulsive signal to axons that should not cross the midline. Indeed, many examples of FasciclinII-positive axons crossing the midline are found in mutants in which the development of the midline glial cells is affected. The same defect can be observed when differentiation of the midline glia is impaired by directed overexpression of argos, which is a negative regulator of the EGF-receptor pathway. This finding was not unexpected since the Commissureless protein, which regulates Robo expression, is found in high levels in these cells. It is proposed that one important function of the midline glial cells is to act as a control post dictating who can and cannot cross. They prevent commissural axons from crossing the midline more than once and ensure that ipsilateral projecting axons never cross the midline. These two processes might be regulated by different processes (karussell/roundabout) (Hummel, 1999).

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

In situ hybridization and immunocytochemistry studies show that all three robos are expressed in the embryonic CNS during the period of axon outgrowth. robo expression begins first at embryonic stage 10. robo2 expression is first visible at stage 11 and becomes restricted to a smaller subset of neurons later in development (by stage 15). robo3 expression does not begin until late stage 13 and is limited to fewer neurons. Comparing the cells that express robo, robo2, and robo3 gives clues about the potential roles the three different Robo receptors might play during axon guidance in terms of two different events. They function both during the early establishment of midline crossing decisions and later during the establishment of lateral position (i.e., the location and choice of specific longitudinal axon pathways in the medial-lateral axis). robo2 RNA can be detected in the aCC and pCC neurons at early stage 13. The expression level of robo2 in these cells increases throughout stage 13. robo2 is transiently expressed in a variety of other pioneer neurons in the CNS, including MP1, dMP2, and vMP2. All of these growth cones normally project ipsilaterally without crossing the midline. The four axons from pCC, vMP2, MP1, and dMP2 initially selectively fasciculate as they extend in a pairwise fashion and transiently display a high affinity for one another; they all express high levels of Fas II. However, they subsequently selectively defasciculate during the time that pCC and vMP2 pioneer the medial Fas II pathway, while MP1 ultimately pioneers the intermediate Fas II pathway. The defasciculation of these axons and their separation to form these two distinct longitudinal pathways occurs when robo2 expression in all of these neurons declines; this is the same period when robo3 appears in a subset of these neurons (Simpson, 2000a).

robo3 is expressed later than robo2 and in a highly restricted subset of CNS neurons. robo3 is not expressed at early or midstage 13 but, by late stage 13, begins to be expressed in MP1 (which pioneers the intermediate Fas II pathway) and aCC (which is a motoneuron that exits the CNS and extends into the periphery). robo3 expression increases throughout stage 14 in both MP1 and aCC. robo3 mRNA is not detected in pCC, vMP2, or dMP2 (Simpson, 2000a).

The pCC, vMP2, MP1, and dMP2 growth cones pioneer the first two longitudinal axon pathways. All four growth cones initially extend right next to the midline but normally do not cross it. In a robo mutant, all four growth cones cross and recross the midline. In a slit mutant, all four growth cones enter the midline and do not leave it. From the beginning of axon outgrowth, robo is expressed in all four neurons. Similarly, robo2 is transiently expressed in all four neurons by early stage 13. However, it is not until late stage 13 that robo3 is expressed at low levels in two of these four neurons. Thus, robo and robo2 are expressed early enough in these ipsilaterally projecting pioneer neurons to prevent them from entering or crossing the midline, whereas robo3 is not. As robo3 expression begins, robo2 expression becomes more restricted. As development proceeds, both robo2 and robo3 expression becomes restricted to a pattern that specifies the lateral position of axons (Simpson, 2000a).

Antibody staining using monoclonal and polyclonal antisera raised (in mouse) against the three different Robos supports the mRNA expression data. Robo and Robo2 proteins appear earlier than Robo3 and, in general, appear to be expressed on many if not all of the early ipsilaterally projecting axons. Later in development, as Robo3 protein appears, the patterns of expression resolve into a restricted pattern for Robo2 and Robo3. Robo, Robo2, and Robo3 are found on the longitudinal tracts of the CNS scaffold but not in the commissural segments of contralaterally projecting axons. All three Robos are expressed on growth cones as revealed by immunoelectron microscopic analysis. Robo is present across the entire medial-lateral span of the longitudinal pathways, while Robo3 is expressed on axons in the lateral two thirds, and Robo2 is further restricted to the lateral third only of the longitudinal axon pathways. Immunocytochemistry also shows that the Robo2 protein is found in the heart, the early trachea, and the lateral body wall muscles, where it subsequently resolves to the muscle attachment sites (Simpson, 2000a).

Slit and Robo control cardiac cell polarity and morphogenesis

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

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

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

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

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

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

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

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

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

It was anticipated that there are numerous molecules involved in generating or maintaining cardiac cell polarity in conjunction with slit/robo during heart morphogenesis, but mutants of some key factors may be early lethal or have pleiotropic effects. Thus, genetic interactions between cell-polarity genes and slit were examined in relation to cardiac morphogenesis. For this purpose, various transheterozygous combinations between were made between slit and polarity genes that are expressed in the heart, including dg, dlg, and 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).

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).

Pupal

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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).

Effects of mutation and ectopic expression

On each side of the midline of the Drosophila CNS, axons are organized into a series of parallel pathways. The midline repellent Slit, previously identified as a short-range signal that regulates midline crossing, also functions at long range to pattern these longitudinal pathways. In this long-range function, Slit signals through the receptors Robo2 and Robo3. Axons expressing neither, one, or both of these receptors project in one of three discrete lateral zones, each successively further from the midline. Loss of robo2 or robo3 function repositions axons closer to the midline, while gain of robo2 or robo3 function shifts axons further from the midline. Local cues further refine the lateral position. Together, these long- and short-range guidance cues allow growth cones to select with precision a specific longitudinal pathway (Rajagopalan, 2000).

Forced expression of Robo2 or Robo3 repositions axons further from the midline. Increased expression of Robo does not. Clearly, the repulsive signal provided by Robo2 and Robo3 is qualitatively different from the Robo signal. What is the basis for this difference? One interesting possibility is that only Robo2 and Robo3 detect the long-range graded Slit signal, while Robo responds only to the short-range signal that regulates midline crossing. In vivo, Slit exists in a least three isoforms: a full-length 190 kDa glycoprotein, and 140 kDa N-terminal and 55 kDa C-terminal fragments produced by proteolytic cleavage of the full-length protein. At present, it is not known in which of these isoforms the various activities of Slit reside. Once this issue has been resolved, it will be interesting to test this idea by comparing the affinities of each of the Robo receptors for the different Slit isoforms (Rajagopalan, 2000).

Another possibility is that Robo2 and Robo3 transduce a qualitatively different signal from Robo by activating a different set of signal transduction pathways inside the growth cone. This is an appealing idea, since it is in their cytoplasmic domains that Robo2 and Robo3 differ most from Robo. Both Robo2 and Robo3 lack cytoplasmic motifs that are found in all other known Robo family receptors in various species, and are required in Robo for it to regulate midline crossing. In Robo signaling, these motifs are thought to mediate interactions with Ena and Abl, though it is evident that Robo must signal through other pathways as well. Receptor tyrosine phosphatases and the calmodulin and Sos-Ras pathways have also been implicated in Robo signaling, though their roles are even less clear. Too little is known about Robo signal transduction at this point to predict how the pathways activated by Robo2 and Robo3 might differ (Rajagopalan, 2000).

What forces counter the Slit gradient in the longitudinal pathways to prevent axons from simply continuing down the gradient and out to the periphery? One possibility would be a second gradient. It could be a repulsive countergradient or a parallel attractive gradient. In the vertebrate spinal cord, Slit and Semaphorin chemorepellents are expressed on both sides of the longitudinal pathways, 'squeezing' axons into a narrow corridor between the two repulsive centers. In Drosophila, there is little to suggest that such squeezing occurs. No known chemorepellent is expressed at the lateral edges of the CNS. If not by a repulsive countergradient, then might Slit instead be balanced by the parallel gradient of an attractant secreted from the midline? Netrins would be an obvious candidate for this attractant. However, the current model for guidance at the midline proposes that commissural axons lose sensitivity to Netrins and any other midline attractants as they cross. This remains to be tested in Drosophila, but if it is true, as seems likely, then the fact that most longitudinal axons have first crossed the midline would argue against the idea that Slit is balanced by a graded midline attractant (Rajagopalan, 2000).

A second graded signal to balance the Slit gradient therefore seems unlikely. In contrast, there is strong evidence that repulsion by Slit is balanced by local interactions within the longitudinal tract. This is revealed by the behavior of the Ap axons when they are forced to misexpress Robo2 or Robo3. As a result, they move down the Slit gradient, but not uniformly, and not out of the CNS. Instead, they appear to latch on to one of two alternative lateral pathways. This strongly suggests that local cues within the longitudinal tract provide a short-range attractive force that can overcome the long-range repulsive influence of Slit (Rajagopalan, 2000).

There is also other evidence to support the notion that local cues counter the Slit signal: this comes from the initial experiments that led to the formulation of the labeled pathways hypothesis itself. The idea of specific pathway labels was inspired largely by the behavior of a single neuron, called the G neuron, in the grasshopper embryo. This neuron extends an axon that grows across the midline and contralateral longitudinal tract until its growth cone meets a lateral fascicle known as the A/P fascicle. It then turns anteriorly along this pathway, fasciculating tightly with the P axons. What does the G growth cone do when the P axons are ablated? It continues further laterally! This behavior, a mystery when it was first observed in the 1980s, can now be readily understood as the continued extension of the G growth cone down the Slit gradient. At the same time, it provides further evidence that long-range repulsion from the midline is balanced by short-range cues provided by single fascicles within the longitudinal tract (Rajagopalan, 2000).

It is proposed that lateral pathway choices are specified by two interdependent mechanisms: a Robo code and a fasciculation code. The Robo code specifies the broad zone within which a growth cone should select a pathway, while the final choice of a pathway within that zone is specified by its fasciculation code. The two systems therefore act as the coarse and fine tuning for lateral pathway selection. With such a Robo code in place, it is necessary only to differentially label the pathways within a given zone. For this a relatively small number of surface molecules should suffice (Rajagopalan, 2000).

Two groups of axons, the Sema2b and the Ap axons provide an instructive example to illustrate how this system might work. Sema2b axons occupy a lateral position in the nerve cord and extend axons across the midline in the anterior commissure. The cell bodies of AP axons are located laterally, and these axons grow initially toward the midline before turning, without crossing, to continue anteriorly near the medial edge of the ipsilateral longitudinal tract. The Sema2b neurons have the Robo code of Robo+Robo3 and an unknown fasciculation code, and project their axons along a fascicle near the middle of the longitudinal tract. The Sema2b growth cones approach their target fascicle from the medial side, having crossed the midline and so, most likely, having lost their senstivity to the long-range attractive cues it provides. Within the medial (Robo-only) zone, they encounter a fascicle that expresses the appropriate fasciculation code. They do not select this pathway, however, because the long-range repulsive influence of Slit at this point is stronger than the short-range attractive forces provided by these fasciculation cues. Instead, they continue to migrate down the Slit gradient into the next zone, the intermediate Robo+Robo3 zone. Here they encounter another fascicle with the same fasciculation code and now, since the Slit signal has become weaker, short-range attraction exceeds long-range repulsion and they turn to follow this pathway. When robo3 function is removed, the Sema2b growth cones no longer detect the long-range repulsive Slit signal, and so they select instead the first attractive pathway they encounter (Rajagopalan, 2000).

The Ap neurons have a Robo code of Robo-only, and, as for the Sema2b neurons, their fasciculation code too is unknown. Their growth cones make a lateral approach toward their medial target fascicle. As ipsilateral axons that project toward but not across the midline, they respond to both its long-range attractive signals (most likely the Netrins) and its short-range repulsive cue (Slit). They also respond to short-range attractive cues (pathway labels), and, when forced to express Robo2 or Robo3 will also respond to long-range repulsion from the midline (Slit again). Initially, long-range attraction is the predominant force, and the Ap growth cones migrate toward the midline. En route to their medial target fascicle they encounter two alternative pathways that express the appropriate fasciculation cues. However, the short-range attraction these pathways offer is insufficient to overcome the pull of the midline. It is not until the Ap growth cones are closer to the midline, and begin to sense Slit as a short-range repellent (acting through Robo), that the midline loses its appeal and the Ap growth cones turn to follow instead the short-range attractive cues of their target fascicle. If the Ap axons are forced to express either Robo2 or Robo3, they can also sense Slit as a long-range repellent. The midline no longer beckons, and so the Ap growth cones are far more likely to take one of the alternative pathways they encounter out in the lateral or intermediate zones. Most often they choose the one in the intermediate Robo+Robo3 zone (Rajagopalan, 2000).

A delicate interplay between long-range graded cues and short-range pathway labels thus underlies the exquisite precision of lateral pathway selection in the Drosophila CNS. It would not be surprising to find similar mechanisms at work in the many other regions of invertebrate and vertebrate nervous systems in which axons are patterned into a series of parallel pathways (Rajagopalan, 2000).

Roundabout (Robo) in Drosophila is a repulsive axon guidance receptor that binds to Slit, a repellent secreted by midline glia. In robo mutants, growth cones cross and recross the midline, while, in slit mutants, growth cones enter the midline but fail to leave it. This difference suggests that Slit must have more than one receptor controlling midline guidance. In the absence of Robo, some other Slit receptor ensures that growth cones do not stay at the midline, even though they cross and recross it. The Drosophila genome is shown to encode three Robo receptors and Robo and Robo2 have distinct functions, which together control repulsive axon guidance at the midline. The robo,robo2 double mutant is largely identical to slit (Simpson, 2000a).

Mutations in robo2 were generated to determine if Robo2 has an essential function -- whether it plays a role in midline guidance, and, in particular, whether its presence drives axons to leave the midline in robo mutants. When examined with mAb BP102 against all CNS axons, the robo2 mutant looks slightly abnormal but much closer to wild-type than does the robo mutant. In the robo2 mutant, some axons ectopically cross the midline. This ectopic crossing phenotype is much weaker and less penetrant than in the robo mutant. In the robo2 mutant there is disorganization of the longitudnal tracts. At stage 16, Fas II is normally expressed on four major longitudinal axon pathways, of which three are clearly visible in a single optical focal plane and are diagnostic for lateral positioning. One of the Fas II pathways (the pCC pathway) is medial, another is intermediate (the MP1 pathway), and a third is lateral (this one is the last to form). A fourth Fas II pathway is more ventral directly below the medial Fas II pathway (Simpson, 2000a).

The disorganization of the Fas II pathways appears as 'braiding,' since, instead of maintaining their parallel alignment (i.e., medial, intermediate, and lateral), the three diagnostic Fas II bundles on each side of the CNS now cross over and intermittently join with each other on their own side. Segments that show misrouting of axons between bundles on the same side of the midline are more common than those that show axons crossing the midline. The frequency of aberrations is higher in the excision/deficiency embryos as compared to the excision/excision embryos, but this may be due to the fact that the deficiency removes a number of genes in addition to robo2 -- notably robo3. Heterozygosity for one robo can enhance the null phenotype of another; robo2 dominantly enhances a robo mutation. Thus, it is plausible that the increase in robo2 defects in the excision/deficiency combination is due to heterozygosity for robo3 rather than to any additional reduction in Robo2. The robo2 phenotype can also be visualized using anti-Connectin mAb. Connectin is a cell adhesion molecule that is expressed in the CNS by a subset of axons that fasciculate in two longitudinal axon pathways, one medial and the other intermediate to lateral. Some of these axons cross in the anterior commissure, where they also express Connectin. In robo2 mutants, the two Connectin pathways are often fused together into a single group of axons. The Fas II and Connectin staining patterns suggest that the loss of function of robo2 affects the ability of these axons to locate their correct lateral position and to form their correct pattern of longitudinal axon pathways. robo mutants, however, still show two Connectin pathways, but axons in the medial of the two Connectin pathways appear to ectopically cross the midline (just as the medial Fas II axons abnormally cross the midline) (Simpson, 2000a).

The ectopic crossing of axons in robo2 mutants indicates that Robo2 does indeed contribute to midline guidance as well as to lateral position. To determine if Robo2 supplies the repulsive force that drives axons to leave the midline in robo mutants, robo,robo2 double mutants were generated by recombination. The robo, robo2 double mutants were examined with mAbs 1D4 and BP102 and found to be phenotypically identical to slit. All axons are initially attracted to the midline (presumably guided in part by Netrins). But once these axons enter the midline, they are unable to leave. In a robo mutant alone, the axons leave the midline but recross it. In the double mutant, they never leave the midline, just as in a slit mutant. Thus, Robo and Robo2 together can account for all of the function of Slit in midline guidance. In the absence of Robo, it is the small amount of Robo2 on the growth cones that drives them to leave the midline, even though they can cross and recross the midline (Simpson, 2000a).

The relative contribution of Robo and Robo2 to prevention of crossing can be clarified by examining their ability to dominantly enhance each other (i.e., the phenotype generated by removing 100% of one protein is enhanced by removing 50% of the other protein). Heterozygosity for robo in a robo2 null background (robo^+/- robo2^-/-) increases the midline disruption. These embryos show a dramatic increase in ectopic midline crossing as compared to robo2 mutants alone, and the crossing involves all three of the Fas II longitudinal pathways (not just the medial Fas II pathway, as seen in robo mutants alone). Thus, one copy of robo (presumably producing 50% of protein) is not sufficient to prevent crossing, but it is sufficient to prevent axons from lingering at the midline in the absence of robo2 (Simpson, 2000a).

Heterozygosity for robo2 in a robo null background (robo^-/-robo2^+/-) leads to a different enhancement in the midline phenotype. Just as in a robo mutant, so too in a robo^-/-robo2^+/- mutant; it is only the axons in the medial Fas II pathway that ectopically enter and cross the midline. However, this subset of axons usually does not leave the midline, and, instead, the two medial Fas II pathways fuse and run along the midline. (In a slit mutant -- or robo,robo2 double homozygous mutant -- all three Fas II pathways are fused along the midline.) Thus, whereas one copy of robo (in the absence of robo2) is sufficient to prevent axons from staying at the midline, one copy of robo2 (in the absence of robo) is not (Simpson, 2000a).

Robo and Robo2 also cooperate in other developmental processes. Slit, Robo, and Robo2 function during mesoderm migration. After gastrulation in Drosophila, many myoblasts migrate laterally away from the ventral midline. In slit mutant embryos, some mesoderm cells do not migrate away from the midline and, instead, form muscles abnormally near the midline that often stretch across the midline. A weak version of this phenotype is observed in the robo mutant, suggesting that it alone cannot control mesoderm migration away from Slit. A similarly weak phenotype is observed in the robo2 mutant. However, a strong phenotype is observed in the robo,robo2 double mutant. This phenotype is very similar to the slit phenotype; many mesodermal cells do not migrate away from the midline, and, instead, some developing muscles are found ectopically crossing the midline. Thus, Robo and Robo2 appear to cooperate in controlling mesoderm migrations away from the midline. Robo and Robo2 also appear to cooperate in governing proper cell migrations and alignment of cardioblasts in the embryonic heart and in the further development of muscle, including the identification of proper insertion sites (Simpson, 2000a).

Overexpression of robo2 demonstrates that Robo2 can act as a repulsive axon guidance receptor. Moreover, it reveals an important difference between Robo and Robo2. The UAS-GAL4 system was used to drive robo2 expression in all neurons in the embryonic CNS. An expression series of increasing levels of Robo2 was generated. A characteristic phenotypic series was observed based on increasing levels of Robo2 that is different from what is seen with Robo. At the high end of expression levels, both genes generate a commissureless-like phenotype in which no axons cross the midline. However, increasing Robo expression leads to a simple phenotypic series of increasing severity of the commissureless phenotype. Interestingly, something quite different is observed with Robo2. A low level of Robo2 overexpression results in inappropriate midline crossing reminiscent of a partial robo loss-of-function phenotype and, with increasing levels of Robo2, of a complete loss of function of robo. As levels of Robo2 continue to increase, the response becomes biphasic. The proclivity to cross the midline (and thus mimic the robo loss of function) is replaced at higher levels of Robo2 by an increasing tendency to avoid the midline (and thus mimic the robo gain of function). This biphasic phenotypic series with increasing levels of Robo2 is different from what is observed with Robo and suggests two opposing functions with different thresholds. In one case, moderate levels of Robo2 appear to be able to interfere with midline repulsion. One interpretation is that Robo2 disrupts Robo signaling, either by competing for Slit binding or by decreasing Robo's output strength. Robo2 is found to be capable of heterodimerizing with Robo (as well as both receptors being capable of homodimerizing). If the heterodimer has a weaker repulsive output than a Robo homodimer, then this could explain the decrease in midline repulsion at low increased levels of Robo2 (Simpson, 2000a).

However, Robo2 does not just interfere with midline repulsion; it can also mediate it. Higher levels of ectopic Robo2 lead to the opposite phenotype in which axons fail to cross the midline. Evidently, Robo2 does have a repulsive output, just not as strong as that of Robo. Sufficient levels of Robo2 are capable of generating a complete commissureless phenotype. Thus, at low levels, Robo2 decreases the strength of Robo signaling and permits inappropriate midline crossing, while, at higher levels, Robo2 is capable of mediating sufficient repulsive signaling to prevent midline crossing entirely (Simpson, 2000a).

The commissureless phenotype observed at the higher levels of Robo2 overexpression can be partially genetically suppressed by heterozygosity (i.e., removing one copy) of robo, slit, or enabled. Although the number of commissures that form in these backgrounds is increased, the phenotype is more complex than simple suppression because in many cases the crossovers that now occur are inappropriate. Adding a robo dominant-negative transgene (truncated just after the transmembrane domain) changes the phenotype at all levels of Robo2. The Robo dominant negative (roboDN) increases the ectopic crossing seen at low levels of Robo2 overexpression, and it causes ectopic crossing at higher levels of Robo2 overexpression as well. It is unclear whether this is suppression by interference with Robo2 repulsion directly or, alternatively, whether it results from cumulative loss of repulsion by reducing the efficacy of the Robo pathway. However, increasing levels of RoboDN in a wild-type background only look like a robo loss of function, no matter how much RoboDN is added, and not like a robo,robo2 double mutant or slit mutant. This suggests that the RoboDN affects Robo output and not Robo2 output, making the second alternative above seem more likely (Simpson, 2000a).

Ectopic expression of low levels of Robo2 by all neurons causes ectopic crossing of axons reminiscent of a robo mutant. A possible explanation is that small amounts of Robo2 can interfere with repulsion by Robo. Perhaps Robo2, which lacks some of the conserved motifs found in the Robo cytoplasmic domain, has a less robust repulsive output than Robo. Extra Robo2 could interfere with Robo by dimerizing with it and creating a weaker receptor. Alternatively, Robo2 might interfere by competing for Slit binding or by sequestering downstream signaling components needed by Robo. In vitro analysis shows that the cytoplasmic domains of Robo2 and Robo can bind to one another (and homodimerize), suggesting that the interference might be direct (Simpson, 2000a).

The in vitro translated cytoplasmic domains of Robo and Robo2 can bind to GST-fusion proteins containing the cytoplasmic domain of Robo or Robo2. The homodimeric interactions are favored over the heterodimer by ~4-fold. The binding of Robo to Robo2 and of Robo to itself is not altered in GST-Robo fusion proteins individually lacking conserved motif CC1, 2, or 3, nor in one lacking the 67 amino acids closest to the transmembrane domain. Further experiments to determine which cytoplasmic domains are sufficient and necessary for in vitro Robo and Robo2 dimerization are in progress.

Although Robo and Robo2 can interact in vitro, it is not known if they heterodimerize in vivo. They are coexpressed in certain cells and thus have the opportunity to function cooperatively, but they can clearly function independently, presumably as homodimers. Robo can maintain a relatively normal CNS scaffold in the absence of Robo2. Robo2 can prevent the medial and lateral pathways from crossing the midline and all axons from lingering at the midline, in the absence of Robo. Although heterodimers have not yet been detected in vivo due to problems with coimmunoprecipitation sensitivity in whole-embryo preparations, the genetic results described above (i.e., the biphasic phenotypic series with increasing levels of Robo2) are consistent with this possibility (Simpson, 2000a).

Commissureless protein can downregulate Robo2 as well as Robo. comm overexpression in midline glia and early neurons using Scabrous-GAL4 can reduce the level of Robo2 protein in CNS axons just as it reduces the levels of Robo. In comm gain-of-function embryos, the phenotype is robo like, but there is more disorganization of the outer (i.e., intermediate and lateral) pathways, presumably because Comm is downregulating Robo2 as well as Robo. In comm null mutants, Robo2 is still localized to the lateral pathways of the CNS scaffold (and Robo3 to the intermediate and lateral pathways), indicating that Comm is not required for the lateral restriction of Robo2 and Robo3. This restriction of Robo2 and Robo3 to specific subsets of neurons appears to be largely transcriptional as revealed by in situ hybridization (Simpson, 2000a).

In contrast, the dramatic increase of Robo protein levels as growth cones cross the midline is, at least in part, regulated by Comm. The distinction is as follows: which neurons express any particular Robo family member (or combination of Robos) appears to be largely transcriptionally controlled, whereas when a given neuron displays on its axons any particular Robo family member (after the onset of transcription) appears to be controlled by other mechanisms, including Comm. Moreover, where a neuron expresses any particular Robo family member (i.e., the commissural versus longitudinal axon segment) also appears to be controlled by other mechanisms (Simpson, 2000a).

The comm gain of function shows that Comm can downregulate both Robo and Robo2. But does it normally regulate more than just Robo? In the original midline mutant screen paper, the robo;comm double mutant was described as looking just like robo when stained with mAb BP102 (which labels all CNS axons). If the double mutant was indeed indistinguishable from robo alone, then this would suggest that Comm normally only regulates Robo. But this is not the case; distinct differences are observed when the double (robo;comm) mutant is compared with robo alone, using mAb 1D4 to stain the three major Fas II pathways. In a robo mutant, the axons in the medial Fas II pathway cross and recross the midline, while the axons in the intermediate and lateral Fas II pathways do not cross the midline. In contrast, in a robo;comm double mutant, the intermediate Fas II pathway is also perturbed and can be seen crossing the midline. At the very least, this result shows that, in the absence of Robo, Comm still has some additional function that is revealed by removing them both together. Since this additional function affects midline guidance, it is speculated that this additional function involves its regulation of Robo2 and/or Robo3. There are several alternative ways in which one might interpret the additional phenotypes seen in the robo;comm double mutant. Distinguishing between these models requires having probes for the different subsets of Fas II axons (medial versus intermediate versus lateral); such probes are not yet available, although work is underway to generate these tools (Simpson, 2000a).

Can Comm also downregulate Robo3? It is very difficult to do the same experiment as with Robo and Robo2. Both Robo and Robo2 proteins are expressed early in both the CNS and surrounding tissues. Comm can be overexpressed early only in the CNS, and differential reduction of Robo or Robo2 protein in the CNS compared to the surrounding tissue can be assessed. However, Robo3 is neither expressed early enough nor in tissues outside the nervous system for a similar comparison. The fact that the robo,robo2;comm triple mutant looks like the robo,robo2 double mutant (in which no axons leave the midline) suggests that if loss of Comm increases the level of Robo3, it does not do so sufficiently to allow any axon to escape the midline. But Robo3 may simply be too weak on its own, even when released from putative Comm downregulation, to repel axons away from the midline. All of these results and interpretations are further complicated by the existence in the Drosophila genome of a gene encoding a second Comm-like protein. Both Comms are all capable when overexpressed of downregulating Robo and Robo2. How they function to regulate the different Robos is under investigation (Simpson, 2000a).

Comm is expressed on midline cells; Robo is expressed in a dynamic fashion on growth cones and appears to function as an axon guidance receptor. robo function is dosage-sensitive. commissureless and roundabout lead to complementary mutant phenotypes in which either too few or too many axons cross the midline. The robo;comm double-mutant phenotype is identical to robo alone, suggesting that in the absence of robo, comm is no longer required. Overexpression of comm is also dosage-sensitive and leads to a phenotype identical to robo loss-of-function. Comm controls Robo expression; increasing Comm leads to a reduction of Robo protein. The levels of Comm and Robo appear to be tightly regulated to assure that only certain growth cones cross the midline and that those growth cones that do cross never do so again (Kidd, 1998b).

A large-scale screen has been carried out for mutations that affect the development of CNS axon pathways in the Drosophila embryo. Embryos were screened from over 13,500 balanced lines and over 250 mutant lines were saved whose phenotypes suggest possible defects in growth cone guidance. Two new genes, commissureless (comm) and roundabout (robo) are described in this paper. Mutations in comm lead to an absence of nearly all CNS axon commissures, such that growth cones that normally project across the midline extend instead only on their own side. Mutations in robo lead to the opposite misrouting, such that some growth cones that normally extend only on their own side, now project across the midline. The phenotypes of these two genes suggest that they may encode components of attractive and repulsive signaling systems at the midline that either guide growth cones across the midline or keep them on their own side (Seeger, 1993).

In robo mutants, axons that normally project ipsilaterally can cross and recross the midline. Growth cones expressing Robo are believed to be repelled from the midline by the interaction of Robo and its ligand Slit, an extracellular protein expressed by the midline glia. To help understand the cellular basis for the midline repulsion mediated by Robo, time-lapse observations were taken to compare the growth cone behavior of the ipsilaterally projecting motorneuron RP2 in robo and wild-type embyros. In wild-type embryos, filopodia can project across the midline but are quickly retracted. In robo mutants, medial filopodia can remain extended for longer periods and can develop into contralateral branches. In many cases RP2 produces both ipsilateral and contralateral branches, both of which can extend into the periphery. The growth cone also exhibits longer filopodia and more extensive branching both at the midline and throughout the neuropile. Cell injections in fixed stage 13 embryos has confirmed and quantified these results for both RP2 and the interneuron pCC. The results suggest that Robo both repels growth cones at the midline and inhibits branching throughout the neuropile by promoting filopodial retraction (Murray, 1999).

The original model for Robo proposed the existence of a midline repellent ligand for which Robo was the receptor. The results presented here show that Robo's effects are not confined to the midline. In addition to preventing axons from crossing the midline, Robo also appears to inhibit filopodial extension and subsequent branch formation throughout the neuropile. Recently, in vitro binding studies and genetic analysis have identified Robo's ligand as the extracellular matrix protein Slit. As expected, in the CNS slit is only transcribed by the midline glia. The location of the Slit protein, however, is less clear. Antibodies raised against a C-terminal fragment of the protein show that Slit is found on the surface of axons throughout the neuropile. Recently it has emerged that Slit is proteolytically cleaved into two fragments, one of which, the C-terminal fragment, appears to be more readily diffusible (Brose, 1999). Thus, different antibodies may recognize different protein fragments with different expression patterns. Furthermore, evidence exists that Slit can function as a diffusible chemorepellent both in vitro (Brose, 1999) and in the Drosophila embryo (Kidd, 1999). These studies, together with the current results, suggest that Slit, or a fragment thereof, may interact with Robo in regions of the neuropile away from the midline. These results reinforce the concept that accurate growth cone guidance depends on a delicate balance of multiple attractive and repulsive cues. When a factor such as Robo is removed, the highly stereotypic trajectories of identified neurons are replaced with more variable results. Thus in the case of both RP2 and pCC, ipsilateral axons do not simply cross the midline but exhibit various trajectories and may even bifurcate into multiple axons (Murray, 1999).

The establishment of axon trajectories is ultimately determined by the integration of intracellular signaling pathways. Here, a genetic approach in Drosophila has demonstrated that both Calmodulin and Son of sevenless signaling pathways are used to regulate which axons cross the midline. A loss in either signaling pathway leads to abnormal projection of axons across the midline and these increase with roundabout or slit mutations. When both Calmodulin and Son of sevenless are disrupted, the midline crossing of axons mimics that seen in roundabout mutants, although Roundabout remains expressed on crossing axons. Calmodulin and Son of sevenless also regulate axon crossing in a commissureless mutant. These data suggest that Calmodulin and Son of sevenless signaling pathways function to interpret midline repulsive cues that prevent axons crossing the midline (Fritz, 2000).

A novel CaM inhibitor, called kinesin-antagonist (KA), has been expressed using the neurogenic enhancer element of the fushi tarazu gene (ftzng) in a subset of CNS neurons that normally do not cross the midline. KA expression decreases endogenous CaM activation of target proteins in the growth cone and this leads to specific axon guidance defects including stalls at selected choice points, failure to fasciculate properly and abnormal crossing of the midline. robo and slit mutations and KA interact synergistically to increase the number of axon bundles abnormally crossing the midline. KA also induces axon bundles to cross the midline in the absence of Comm protein. Sos-dependent crossovers are enhanced by KA or by slit mutation. KA and Sos also interact to increase the number of axon bundles crossing in a comm mutant. Thus, the data demonstrate that both CaM and Sos signaling pathways are required to prevent certain axons crossing the midline (Fritz, 2000).

Whether CaM and Sos-mediated signaling is working directly downstream of Robo or in closely associated, but parallel signaling pathways to prevent axons from crossing is difficult to ascertain from this genetic data alone. If these signaling pathways lie downstream of Robo, the data suggest that both CaM and Sos are activated upon Slit binding to Robo, and result in growth cone repulsion. Interestingly, increased levels of calcium have been implicated in growth cone retraction and growth cone collapse, two ways in which a growth cone may respond to a repulsive agent. In addition, retrograde actin flow, which leads to filopodial retraction, is stimulated by CaM activation of myosin light chain kinase. Two other CaM target proteins, cAMP adenylyl cyclase and phosphodiesterase, regulate cAMP cellular concentrations thus altering neuronal response to Netrin 1 and other guidance cues. Activation of a Sos signaling pathway can affect cytoskeletal dynamics by activating various GTPases known to regulate growth cone behavior and axon guidance. Moreover, the cytoplasmic tail of Robo, known to be essential for signaling function, has a tyrosine residue that could recruit Sos via Drk or dreadlocks (dock), another SH2-SH3 adapter protein that affects axon guidance. Alternatively, Robo may bind Enabled, a known substrate for Abelson tyrosine kinase (Abl), which has been implicated in commissure formation. If Sos binds to phosphotyrosine residues on Ena (also via an adapter protein) it could be indirectly recruited to Robo (Fritz, 2000 and references therein).

Another possibility is that a disruption in both the CaM and Sos signaling pathways indirectly causes abnormal crossovers. CaM has been identified as a player downstream of several guidance molecules. Indeed, the gaps in the longitudinal connectives observed with increasing copies of KA in a comm mutant or in KA robo mutants, which are not seen in robo mutants alone, suggest CaM may function downstream of other guidance cue receptors to allow extension through the connective. Once these signals are attenuated by expression of KA, axons may inadvertently cross the midline. However, if CaM only functions in cell adhesive mechanisms within the connectives, it is difficult to explain why axons cross the midline in comm mutants when no other axons cross and the presence of Slit is still being read by Robo (Fritz, 2000 and references therein).

Since CaM and Sos appear to interpret a midline repulsive cue, the existence of an additional midline repulsion system working in parallel to Robo represents an interesting possibility. In robo mutants, axons cross the midline but then move to the longitudinal connective, instead of collapsing at the midline as observed in slit mutants. It has been suggested that this occurs because the continued presence of Slit at the midline is detected by a second receptor system, and candidate genes include a second robo gene or karussel. As the data shows, heterozygous slit mutations interact very strongly with single copies of KA, Sos or KA Sos together, to force axons across the midline. The interaction between Sos and slit mutations, especially when compared to the lack of Sos and robo interaction, is particularly striking. It seems that if the activity of both repulsion systems is decreased due to the reduction of a common ligand (Slit), a disruption in CaM and/or Sos signaling dramatically increases midline crossing errors. Most of these results, including the synergistic effects of KA and Sos, robo and slit mutations, the robo-like phenotype of KA Sos mutants, and the enhancement of crossovers in comm mutants can be explained by a parallel decrease in both midline repulsive systems upon disruption of the CaM and Sos signaling pathways. Thus while the mechanisms by which CaM and Sos contribute to an axon guidance decision at the midline remain unclear, the data clearly indicate that CaM and Sos signaling pathways are critical to the transduction of repulsive information at the midline (Fritz, 2000 and references therein).

Axons in the bilateral CNS of Drosophila decide whether or not to cross the midline before following their specific subsequent pathways. In commissureless mutants, the RP3 and V motoneuron axons often fail to cross the midline but subsequently follow the mirror-image pathways and innervate corresponding muscle targets on the ipsilateral side. Conversely, in roundabout mutants, the RP2 and aCC motoneuron axons sometimes cross the midline abnormally but their subsequent pathways and synaptic targeting are the perfect mirror images of those seen in wild type. Furthermore, within a single segment of these mutants, bilateral pairs of motoneuron axons can make their midline decisions independent of one another. Thus, neither the particular molecular experience of the growth cones nor the decision at the midline caused by these mutations affects growth cone ability to respond normally to subsequently presented cues (Wolf, 2000).

The logic motivating these experiments is that growth cones will retain their ability to respond normally to all subsequent cues if they rely on an experience-independent preprogramming (Preprogram model). But, if their normal mode of operation is one of continual reprogramming, and the final axon pathways are a result of a specific sequence of interactive reprogramming, missing the normal cues at the midline should lead to a subsequent deviation from the normal pathways at one point or another (Reprogram model). It was reasoned that by following the axon pathways of individual neurons affected by midline mutations, these scenarios could be examined experimentally. The first set of experiments with comm mutants provided cases in which growth cones were prevented from crossing the midline. However, due to the bilateral symmetry of the molecular and cellular organizations across the midline, the RP3 and V motoneuron growth cones, when they failed to cross the midline, still found themselves surrounded by the same microenvironment that they would normally have experienced after crossing the midline. The net effect is essentially equivalent to a cellular transplant across the midline. Without experiencing the Comm protein on the midline glia, and despite the abnormal decision to not cross the midline, these growth cones are nevertheless perfectly capable of responding normally to all subsequent cues, allowing them to follow the mirror-image peripheral pathways all the way to their respective target muscles and to initiate synaptogenesis there (Wolf, 2000).

The second set of experiments with robo mutants provided complementary cases in which growth cones were made to abnormally cross the midline, presumably due to either full or partial loss of the ability of the growth cones to respond to midline repulsion signals. The Robo protein is a widely expressed neuronal growth cone receptor, and its deletion offers a means to disrupt the midline decisions of growth cones independent of comm mutations. In all cases, when they cross the midline abnormally, the RP2 and aCC motoneuron growth cones retain normal responsiveness to all subsequent cues, selecting the mirror-image pathways and muscles on the other side of the midline (Wolf, 2000).

These results clearly demonstrate that neither disrupted midline decisions nor a lack of midline signaling molecules affect the ability of the motoneuron growth cones to respond normally to cues encountered beyond the midline. It is concluded that, under the situations examined, the growth cones rely on an experience-independent preprogramming for their navigation through complex in vivo environments (Wolf, 2000).

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).

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).

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).

Receptor tyrosine phosphatases regulate axon guidance across the midline of the Drosophila embryo, interacting with the Robo system

Neural receptor-linked protein tyrosine phosphatases (RPTPs) are required for guidance of motoneuron and photoreceptor growth cones in Drosophila. These phosphatases have not been implicated in growth cone responses to specific guidance cues, however, so it is unknown which aspects of axonal pathfinding are controlled by their activities. Three RPTPs, known as DLAR, DPTP69D, and DPTP99A, have been genetically characterized thus far. The isolation of mutations in the fourth neural RPTP, DPTP10D, is reported. The analysis of double mutant phenotypes shows that DPTP10D and DPTP69D are necessary for repulsion of growth cones from the midline of the embryonic central nervous system. Repulsion is thought to be triggered by binding of the secreted protein Slit, which is expressed by midline glia, to Roundabout (Robo) receptors on growth cones. Robo repulsion is downregulated by the Commissureless (Comm) protein, allowing axons to cross the midline. The Rptp mutations genetically interact with robo, slit and comm. The nature of these interactions suggests that DPTP10D and DPTP69D are positive regulators of Slit/Roundabout repulsive signaling. Elimination of all four neural RPTPs converts most noncrossing longitudinal pathways into commissures that cross the midline, indicating that tyrosine phosphorylation controls the manner in which growth cones respond to midline signals (Sun, 2000).

The fact that longitudinal axons can be changed into commissural axons by elimination of RPTP activity suggests that tyrosine phosphorylation controls the manner in which growth cones respond to midline repulsive signals. This is consistent with the observation that pharmacological inhibition of tyrosine kinase activity in grasshopper embryos causes a robo-like phenotype in which the longitudinal axon of the pCC neuron crosses the midline and circles back to the ipsilateral side. Further evidence that the effects of the inhibitor may actually be due to blockage of Robo signaling is provided by the recent observation that the Drosophila pCC axon in robo embryos has a unique branched morphology that is identical in appearance to that of the grasshopper pCC in inhibitor-treated embryos (Sun, 2000 and references therein).

The repulsive response to midline signals is encoded within the Robo cytoplasmic domain. The cytoplasmic domains of fly, nematode and mammalian Robo family proteins (Robos) contain conserved tyrosine-containing PYATT sequence motifs, suggesting that these domains could be direct targets for tyrosine kinases. Phosphorylated tyrosine motifs usually function by binding to SH2 and PTB-domain adapter proteins that mediate downstream signaling events. Robo also contains two proline-rich sequences that could interact with SH3-domain adapters. Robo2 has the tyrosine-containing motif, but lacks the proline-rich sequences (Sun, 2000).

How are Robo signaling pathways regulated by RPTPs? There is no evidence at present that the RPTPs directly alter signaling by the Robo protein. It is possible that the RPTPs and Robo feed into separate pathways that only intersect after several signaling steps. There is, however, a known mechanism for RPTP-mediated positive regulation of tyrosine kinase pathways that suggests how DPTP10D and DPTP69D could facilitate Robo signaling. During T cell receptor (TCR) signal transduction, the RPTP CD45 removes an inhibitory C-terminal phosphate group from the Src-family tyrosine kinase Lck, thereby activating it and allowing it to phosphorylate the z chain of the TCR. The phosphorylated z chain in turn binds to an SH2-domain containing tyrosine kinase (ZAP-70), which mediates downstream signaling events. CD45 is required for TCR signaling because in its absence Lck is not activated and thus cannot efficiently phosphorylate the z chain. (Interestingly, CD45 may also be involved in the termination of the TCR signaling response, since it can dephosphorylate the z chain and prevent it from binding to ZAP-70) Another mammalian receptor phosphatase, RPTPa, also dephosphorylates and activates Src-family kinases. Fibroblasts derived from RPTPa knockout mice have reduced Src and Fyn activities, suggesting that RPTPa is an in vivo regulator of Src family kinase function (Sun, 2000 and references therein).

By analogy to these pathways, DPTP10D and DPTP69D might regulate growth cone repulsion by activating Src-family tyrosine kinase(s) that phosphorylate Robos. This could explain the genetic data, since the loss of RPTP function would be expected to cause a decrease in the extent of Robo phosphorylation. One might also propose that positive regulation of repulsion by the RPTPs occurs through direct dephosphorylation of Robos, and that dephosphorylated Robos are more active in signaling. This would be unusual, however, since normally it is the phosphorylated form of a signaling motif that binds to downstream adapters. A variant of the direct interaction model proposes that Robos become phosphorylated on tyrosines after engagement of Slit, and that DPTP10D or DPTP69D are recruited into a Robo/Slit signaling complex by their interactions with the phosphotyrosine motifs. RPTPs might remain bound to these sites for a significant time period, because they often hydrolyze phosphate-tyrosine bonds quite slowly. The RPTPs could then function as adapters themselves, binding to downstream signaling proteins and recruiting them into Robo/Slit receptor complexes. Determining which, if any, of these models is correct will require biochemical or genetic identification of in vivo substrates for RPTPs (Sun, 2000).

Roundabout signalling, cell contact and trophic support confine longitudinal glia and axons in the Drosophila CNS

The mechanisms that generate and maintain the longitudinal axon pathways of the Drosophila CNS are largely unknown. The longitudinal pathways are formed by ipsilateral pioneer axons and the longitudinal glia. The longitudinal glia dictate these axonal trajectories and provide trophic support to later-projecting follower neurons. Follower interneuron axons cross the midline once and join these pathways to form the longitudinal connectives. Once on the contralateral side, longitudinal axons are repelled from recrossing the midline by the midline repulsive signal Slit and its axonal receptor Roundabout. Longitudinal glia also transiently express roundabout, which halts their ventral migration short of the midline. Once in contact with axons, glia cease to express roundabout and become dependent on neurons for their survival. Trophic support and cell-cell contact restrict glial movement and axonal trajectories. The significance of this relationship is revealed when neuron-glia interactions are disrupted by neuronal ablation or mutation in the glial cells missing gene, which eliminates glia, when axons and glia cross the midline despite continued midline repellent signaling (Kinrade, 2001).

The longitudinal pathways are pioneered by four neurons per hemisegment: pCC, MP1, dMP2 and vMP2 -- their axons never cross the midline. These ipsilateral pioneer axons form a scaffold for the later selective fasciculation of follower axons. During the formation of the first longitudinal fascicle, pioneer growth cones also express the Robo receptor, which prevents them from crossing the midline. During their pathfinding, the pioneer axons interact with a class of glial cells, the interface glia, which at the end of embryogenesis overlie the longitudinal axons. The longitudinal glia are the interface glia derived from the segmentally repeated lateral glioblasts, located at the edge of the neuroectoderm. Longitudinal glia, like the midline glia, are reminiscent of vertebrate oligodendrocytes since they originate from highly migratory and proliferative precursors and enwrap CNS axons. The longitudinal glioblasts divide and migrate ventrally until they contact the cell bodies of the pioneer neurons, where they halt at a certain distance from the midline. The first longitudinal fascicle is formed as the descending axons of dMP2 and MP1 meet the ascending axons of pCC and vMP2. Longitudinal glia migrate anteroposteriorly slightly ahead, but in close contact with, the extending pioneer growth cones and stall at choice points relevant for axon guidance and fasciculation. Following the formation of the first longitudinal fascicle, glia continue migrating, occupying choice points to instruct axonal defasciculation and refasciculation. The final pattern of pioneer axon trajectories is dictated by glia (Kinrade, 2001 and references therein).

Interface glia normally overlie the longitudinal connectives of the CNS and are not found over the midline. Mutations in robo cause interface glia to migrate over the midline. However, not all glia migrate over the midline: some remain along the longitudinal tracts, whose position is nevertheless closer to the midline than in normal embryos. This differential effect of robo mutations on glia correlates with similar differences in the axonal phenotype. For instance, only the pCC/MP2 (central) fasII fascicle, but not the outer two fascicles, is affected in robo mutants. The longitudinal glia are responsible for the formation of the three fasII fascicles. Hence, these data suggest that either there is a differential requirement for robo among the longitudinal glia, or that other members of the robo family may also play a role in these glia or that further factors, other than robo function, may determine glial positions along the longitudinal fascicles (Kinrade, 2001).

Since in robo mutants longitudinal glia migrate over the midline, it was of interest to see if robo may be expressed in glia as well as in axons. If so, robo would keep glia away from the midline in normal embryos and would render them insensitive to midline repulsion in robo mutants. Prior to axonogenesis, robo is expressed in two broad longitudinal bands at either side of the sli-expressing midline. Sli protein is also found within these bands at stages 12.2 and 12.1. By stage 13 robo expression resolves into segmental clusters, preceding the overt expression in axons (Kinrade, 2001).

robo is also expressed in one transverse stripe per hemisegment at stage 11. These lateral stripes include the tracheal pits. The longitudinal glioblasts originate just ventrally of the tracheal pits, and they divide as they migrate medially within these lateral bands of robo expression. Glia stop migrating medially as they enter the longitudinal bands of robo expression. Within these robo expression domains glia express robo themselves. Glia also express the longitudinal glia cell membrane marker Heartless. Thus longitudinal glia express robo and respond to midline-derived repulsion within a narrow time window. Expression of robo in normal embryos disappears from glia from stage 13, as glia occupy more dorsal positions over the longitudinal tracts. By stage 14, Robo is clearly present only in axons. Since glia maintain lateral positions in the longitudinal pathways throughout embryogenesis despite ceasing to express robo, further mechanisms must restrict glial movement (Kinrade, 2001).

Trophic support between neurons and glia is a means of restricting glial movement and axonal trajectories. There are reciprocal (although asymmetric) trophic interactions between neurons and glia during pathfinding. Pioneer neurons maintain the survival of longitudinal glia, and glia maintain the survival of follower neurons. When axon-glia interactions are intact, axonal CNS patterning is virtually normal in the absence of cell death, although subtle axonal defects are found, such as longitudinal growth cones projecting toward the midline. However, when neuron-glia interactions are perturbed in rpr mutant embryos, which are unable to undergo programmed cell death, the misrouting of axons across the midline is dramatically enhanced, compared either with rpr mutants alone or with perturbing neuron-glia interactions in embryos in which apoptosis can occur normally. In embryos double mutant for gcm and rpr or upon neuronal ablation in a rpr mutant background, axonal extension along the longitudinal pathways is severely affected, as visualized with fasII. Longitudinal connectives are virtually missing and fasII-positive axons cross the midline in every segment. In the case of ablated rpr mutant embryos the phenotype often resembles (albeit more severely) the robo mutant phenotype. These midline crossing axons express robo. In embryos double mutant for gcm and rpr or upon neuronal ablation in a rpr mutant background there is no robo expression along the longitudinal connectives, but instead all axons expressing robo cross the midline. These observations imply that attraction towards the midline is the default pathway taken by axons and glia despite midline repulsion in the absence of normal axon-glia interactions in the longitudinal pathways. They also mean that the pressure for survival normally keeps cells in contact with their normal neighbors, in this case in lateral positions. Taken together, these data show that in normal embryos control of cell survival and cell-cell contact are means of confining glia and axons laterally (Kinrade, 2001).

Cells in animals are programmed to die unless they receive input from their neighbors. In the nervous system, target cells provide trophic factors to extending axons, thus ensuring correct innervation. In the Drosophila CNS, trophic support between neurons and glia plays an instructive role during the formation of longitudinal pathways. Glia numbers are depleted upon neuronal ablation, and they can be rescued by blocking programmed cell death. Longitudinal glia normally undergo apoptosis at the time they first come into axonal contact. Pioneer neurons do not require longitudinal glia for survival, but they require glia for pathfinding. Thus by regulating glia survival, the pioneer neurons anchor longitudinal glia to their axons to enable their pathfinding. Subsequently, longitudinal glia maintain the survival of follower neurons, thus aiding the maintenance of the axonal fascicles in lateral positions. Altogether these data show that survival pressure is instructive in determining the positions of glia and axons during pathfinding. Evidence is provided in support of this notion. (1) In embryos lacking programmed cell death, some axons project across the midline. (2) When neuron-glia interactions are disturbed in embryos lacking programmed cell death, axons and glia dramatically cross over the midline. These are more severe misroutings than if only neuron-glia interactions are disturbed. This reveals the roles of axon-glia interactions in keeping both axons and glia laterally, and it also shows that combining lack of programmed cell death with other genotypes does not lead to additive but synergistic phenotypes. This means that cells respond differently if their survival needs are removed. Interestingly, blocking programmed cell death has been used as a means of unravelling functions of neurotrophins other than in survival. These observations, however, imply that preventing cell death does not recreate a normal although death-free environment, but instead generates a novel one in which cells are subject to different kinds of pressures. In the normal Drosophila embryo, the pressure for continued maintenance of cell contact functions to keeps axons and glia away from the default midline pathway, and along lateral positions (Kinrade, 2001).

Altered levels of Gq activity modulate axonal pathfinding in Drosophila

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Ectopic expression in the giant fiber system of Drosophila reveals distinct roles for Roundabout (Robo), Robo2, and Robo3 in dendritic guidance and synaptic connectivity

The Drosophila giant fiber (GF) system is responsible for a jump-and-flight response to visual stimuli. This system has the advantage that a single pair of descending giant fibers contacts a pair of large target motor neurons [tergotrochanteral motor neuron (TTMn)], and the resulting central synapses can be easily studied anatomically and electrophysiologically in adults. The cell bodies of the GF are located in the brain, and they send dendritic processes into the visual and antennal centers. Each GF extends a single unbranched axon ipsilaterally from the brain to the second thoracic neuromere, where it extends laterally along the dendrites of its target, the TTMn, forming a mixed electrical and chemical synapse. In the adult giant fiber system, ectopic Robo expression can regulate the growth and guidance of specific motor neuron dendrites, whereas Robo2 and Robo3 have no effect. Ectopic expression of Robo in the TTMn results in stunted dendrites with a penetrance of 100%. The medial dendrites do not reach the midline and appear stalled and distorted 20-30 µm lateral to the midline. The lateral dendrite is often absent or abnormal in these animals as well. Consistent with the distorted anatomy of the medial dendrite, the physiology of the GF->TTMn synapse is weakened but is seldom completely disconnected. Typically the latency is increased to ~2 msec, and the following frequency is lower in every case. The weak connection suggests that despite the misguidance of the TTMn dendrites, the GF is still able to locate and synapse on its normal target, although the resulting synapse is weaker than usual. The effect of Robo on dendritic guidance can be suppressed by Commissureless coexpression. Although a role for all three Robo receptors in giant fiber axon guidance has been confirmed, the strong axon guidance alterations caused by overexpression of Robo2 or Robo3 have no effect on synaptic connectivity. In contrast, Robo overexpression in the giant fiber seems to directly interfere with synaptic function. It is concluded that axon guidance, dendritic guidance, and synaptogenesis are separable processes and that the different Robo family members affect them distinctly (Godenschwege, 2002).

The GF and the TTMn are thought to be born during the embryonic wave of neurogenesis. The GF initiates axonogenesis in the late third instar and has reached the thorax by the beginning of pupation. The GFs make their first contact with the TTMn at ~17% of pupal development. After reaching the thorax, the GF extends laterally along the TTMn and initiates synaptogenesis during the period from 25% to 50% of pupal development. During the remainder of pupal development, the GF continues to grow laterally; the presynaptic and postsynaptic processes grow in diameter as the synapse matures; and gap junctions and chemical synaptic components are put in place. Antibodies were used to determine Slit and Robo expression patterns during various pupal and adult stages during and after GF guidance. Specific Slit labeling occurs in the midline of the s neuromere and in all of the thoracic and abdominal neuromeres, presumably on the midline glia. Expression of Slit is strongest in early pupas (0%-50% of pupal development) and is not detectable in late pupas (after 75%) or in adults. Antibodies to Robo strongly label the CNS in a complementary manner; the entire neuropil is labeled with the exception of the midline at all pupal stages and is not detected in adult flies. No specific staining using Robo2 and Robo3 antibodies could be seen in the CNS in pupae or adults, suggesting that Robo2 and Robo3 are expressed weakly or not at all at these stages. However, it should be noted that the antibody to Robo2 is very buffer-sensitive and may not work well in the conditions needed to fix pupal tissues (Godenschwege, 2002).

A dramatic difference between Robo and Robo2 or Robo3 was revealed when each was expressed in the jump motor neuron (TTMn). Robo has a very powerful effect on the TTMn dendrites, repelling them from the midline, whereas Robo2 and Robo3 has no influence whatsoever on the dendritic projection. There is complementary evidence from loss-of-function experiments in the embryonic nervous system that Robo has a function in determining the dendritic projection of some motor neurons. In wild-type specimens, the dendrites of the raw prawn 2 (RP2) neuron do not cross the midline, but in the robo loss-of-function mutant, the dendrites do cross the midline. The results demonstrate that Robo is involved in the regulation of dendritic projection in this embryonic motor neuron in addition to its well known function in axons. In the case of the adult GF system, loss-of-function mutants cannot be easily examined, because the animals do not survive. Attempts were made to reveal an endogenous role by expressing Comm and Robo^Delta (lacking the Robo intracellular domain), which worked in the axons; however, no evidence was found for an endogenous role of the Robo receptor in the TTMn. How could these results, suggesting an endogenous role in embryos, and these findings in the adult GF system be integrated? The combined results suggest a model by which neurons could establish their various bilateral and unilateral symmetries. Neurons such as the embryonic RP2 may express Robo to prevent dendrites and axons from approaching or crossing the midline, whereas others may express Robo2 or Robo3, allowing their dendrites to approach or cross the midline but preventing their axons from approaching or crossing the midline. In this relatively simple manner, the laterality of many neurons in the CNS could be regulated with only a few genes. This would also explain the inability to find an endogenous role for Robo in the TTMn, because Robo in the TTMn would prevent the dendrite from approaching the midline and thereby disrupt connections with the GF (Godenschwege, 2002).

A number of possible explanations were considered for the functional differences among Robo, Robo2, and Robo3 in dendritic guidance. It is not attributable to differential receptor targeting within the neurons, because no difference in the relative distribution between Robo-myc and Robo2-myc was found. In addition, the functional difference cannot be explained by an obvious difference in their cytoplasmic domains; the cytoplasmic CC2 and CC3 motifs are present in Robo but not in Robo2 or Robo3, but their removal in the Robo receptor has no affect on dendritic guidance, suggesting that other motifs in the Robo receptors are responsible for the functional difference. Robo2 and Robo3 may be regulated separately from the regulation of Robo by Comm, and two other comm-like genes have been identified in Drosophila. If these comm-like genes downregulate Robo2 and Robo3 but not Robo and are endogenously expressed in the TTMn, the difference between the Robo receptors in their ability to affect the TTMn dendritic guidance could easily be explained. This idea that Robo and Robo2 may be processed differentially is supported by examining the myc-tagged constructs. There seems to be preferential removal of Robo2-myc in the TTMn but not in the GF. When the dosage of the gene was increased, the amount of Robo2-myc protein, as indicated by antibody staining of the TTMn axon and dendrites, did not correlate with gene dosage. Additionally, the staining of unidentified neurons outside the giant fiber system is dramatically different for Robo-myc and Robo2-myc. Finally, the lack of Robo2-myc staining in GF dendrites suggests that Robo2 may be degraded or removed preferentially from the surface of dendrites but not axons, whereas Robo is not. In summary, the distinct functions of the Robo receptors may be attributable in part to differential regulation of these proteins at the cell surface (Godenschwege, 2002).

Although Robo apparently does not function normally in the TTMn, it was possible to rescue the Robo-induced misguidance of the TTMn dendrite by Comm coexpression. This demonstrates that the ectopic Robo-Comm machinery can function in dendrites and supports the idea that Robo-Comm interaction may be used to guide dendrites in a manner similar to that seen for axons (Godenschwege, 2002).

The results reveal two relatively independent roles for the Robo receptor during synaptogenesis: (1) an indirect regulation of synapse formation by the influence of Robo receptors on anatomical overlap of the axons and dendrites of the two cells, and (2) a direct disruptive effect by weakening the synapse (Godenschwege, 2002).

There is a powerful effect of the Robos on synaptic connectivity through their regulation of presynaptic and postsynaptic anatomy. When Robo is expressed exclusively postsynaptically, the synapse is weakened in all specimens. This is correlated with the fact that the TTMn dendrites are always pushed laterally, and the GF connections never appear anatomically normal. However, simultaneous presynaptic and postsynaptic expression can improve the connection so that 22% of these flies had normal connections. Presumably by pushing the TTMn dendrite and the GF axon laterally, the chances for overlap and strengthening the connection are improved. By regulating the overlap of the axonal and dendritic processes, the Robos control whether the cells are within synaptic grasp of one another, and this provides the outlines of the circuit diagram that will emerge. This may be considered an indirect, although critical, role of the Robo receptors on synaptogenesis (Godenschwege, 2002).

In addition, Robo appears to have a direct disruptive effect on the GF->TMn synapse. A bendless-like phenotype (referring to the synaptic structure) was revealed when Robo (but not Robo2 or Robo3) was expressed in the GF. When Robo was expressed in the GF but not in the TTMn, approximately one-third of the specimens exhibited a weakened GF->TMn synapse, and half of these were anatomically ben-like. However, no ben-like phenotype was found when Robo^{DeltaCC2+DeltaCC3} (lacking two of the intracellular domains of Robo) was expressed in the GF, and the synaptic connectivity of the GF->TMn synapse was dramatically improved. Furthermore, it was possible to show that in particular the CC2 motif is essential for the induction of the ben-like phenotype. The CC2 and CC3 motifs have been shown to bind to Enable and Abelson, respectively, and to play opposing roles downstream of the Robo receptor. Consistent with these findings, a robo construct lacking the CC3 motif enhances the occurrence of the ben-like phenotype. The CC2 motif-dependent induction of the ben-like phenotype and the weakening of the GF->TMn synapse cannot be simply explained by an altered lateral position of the GF axon because of Robo-induced repulsion from the midline. Robo lacking the CC2 and CC3 motifs is still capable of deflecting the GF from the midline. More strikingly, Robo2 and Robo3 are capable of displacing the GF axon even farther from the midline, but the GF->TMn synapse in the ectopic location is physiologically completely normal. These results suggest that the presynaptic Robo-induced ben-like phenotype may not be attributable to a pathfinding error but possibly to an interference with target recognition or synaptogenesis. Interestingly, vesl, a member of the vasodilator stimulated phosphoprotein/Ena family in vertebrates, is suggested to play a role in synaptogenesis and synaptic plasticity. This implies that interfering with endogenous Drosophila Enabled and Abelson signaling by Robo overexpression may have a disruptive effect on synaptogenesis or synapse maturation of the giant fiber (Godenschwege, 2002).

Simultaneous presynaptic and postsynaptic expression enhances the penetrance of the ben-like phenotype and the disconnection phenotype, synergistically demonstrating the involvement of the postsynaptic cell in the expression of this phenotype. These findings suggest that the presynaptic and postsynaptic partners have found one another, and pathfinding is complete before the emergence of this severe synaptic defect. Furthermore, because simultaneous presynaptic and postsynaptic overexpression is supposed to compensate for the pathfinding errors, because both GF and its TTMn target are shifted laterally, the increase in the number of totally disconnected neurons is likely to be attributable to a synaptic effect rather than the secondary consequence of a guidance defect (Godenschwege, 2002).

These results are interpreted to mean that Robo expression on either side of the synapse interferes with synapse formation, but the presence of Robo on both sides synergistically enhances the disruptive effect of Robo on synapse maturation. These results suggest that possibly the Robo receptor needs to be removed from both growth cones and dendrites for synaptogenesis to proceed normally (Godenschwege, 2002).

Constitutively active myosin light chain kinase 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 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).

To test the model that capt acts in the repellent pathway, the system of receptors was examined. However, examination of single gene mutations might not be sufficient. This is because the response to Slit is mediated by multiple receptors: Robo, Robo2, and Robo3. Indeed, capt transheterozygotes lacking single alleles in robo, robo2, or robo3 alone show little if any midline phenotype. Yet, when capt alleles are combined with double mutations lacking one copy of robo and robo2 simultaneously, a phenotype almost 2-fold greater than that seen in the robo,robo2 heterozygous embryos is observed. Interestingly, capt/+ does not enhance the phenotype of robo,robo3 heterozygotes, which is already quite strong (Wills, 2002).

As capt activity is further reduced, the interaction with robo2 gets stronger; mutants lacking two copies of capt and one copy each of robo2 and robo3 display penetrant midline phenotypes. Since these allelic combinations are the most severe, they were used for more detailed phenotypic analysis. For example, since the repulsion of growth cones at the midline is dependent on the presence of the midline glia, which secrete the Slit repellent, the midline glia in these mutants were examined with anti-Wrapper antibody, which specifically stains the surface of these glial cells. Midline glia are present in capt-robo2,robo3 mutants, even where axons inappropriately crossed the midline. The first axons in the MP1 fascicle were examined just as they pioneer the ipsilateral pathway early in CNS development. At stage 12, the posterior corner cell (pCC) extends its axon along an anterior trajectory parallel to the midline in order to pioneer the most medial Fasciclin II-positive (MP1) pathway. In capt-robo2,robo3 mutants, pCC axons were sometimes found that had turned toward and crossed the midline at this early stage. This phenotype is similar to that seen in robo alleles (Wills, 2002).

As predicted from loss-of-function analysis, while expression of wild-type Robo alone has little, if any, effect on retinal patterning, the combination of Abl and Robo causes a striking increase in the severity of the Abl gain-of-function eye phenotype. Thus, Robo serves as an enhancer of Abl activity in this kinase-dependent assay. This is also true of Robo2 and of Robo3. These data support the hypothesis that all Robo receptors can engage the Abl signaling pathway. So, is this in vivo interaction dependent on the Robo domains previously shown to recruit Abl and Ena proteins? Interestingly, neither deletion of CC2 nor deletion of CC3 was found to attenuate the Abl-Robo interaction. A UAS-robo transgene lacking the motif CC1 did show a reduction in eye phenotype when combined with UAS-Abl, but the difference was slight (Wills, 2002).

To confirm that Abl can interact with Robo in a CC3 domain-independent fashion during axon guidance, embryos that overexpress Abl and either wild-type Robo(+) or mutant Robo(DeltaCC3) were examined in postmitotic neurons. Abl gain-of-function alone generates two axon guidance phenotypes: (1) ISNb motor axon bypass of ventral target muscles and (2) ectopic midline crossing. Interestingly, coexpression of Abl and either Robo(+) or Robo(DeltaCC3) dramatically enhances the ISNb axon phenotype; however, there was no effect on midline crossing in any of these genotypes. Thus, in vivo, Abl is capable of a functional interaction with all three Robo receptors via some novel mechanism. However, the midline guidance system is specifically refractory to a simultaneous increase in Abl and Robo activities, perhaps due to the dual role of Abl in this context (Wills, 2002).

A novel Dbl family RhoGEF promotes Rho-dependent axon attraction to the central nervous system midline in Drosophila and overcomes Robo repulsion

The key role of the Rho family GTPases Rac, Rho, and CDC42 in regulating the actin cytoskeleton is well established. Increasing evidence suggests that the Rho GTPases and their upstream positive regulators, guanine nucleotide exchange factors (GEFs), also play important roles in the control of growth cone guidance in the developing nervous system. The identification and molecular characterization of a novel Dbl family Rho GEF, GEF64C, is presented that promotes axon attraction to the central nervous system midline in the embryonic Drosophila nervous system. In sensitized genetic backgrounds, loss of GEF64C function causes a phenotype where too few axons cross the midline. In contrast, ectopic expression of GEF64C throughout the nervous system results in a phenotype in which far too many axons cross the midline, a phenotype reminiscent of loss of function mutations in the Roundabout (Robo) repulsive guidance receptor. Genetic analysis indicates that GEF64C expression can in fact overcome Robo repulsion. Surprisingly, evidence from genetic, biochemical, and cell culture experiments suggests that the promotion of axon attraction by GEF64C is dependent on the activation of Rho, but not Rac or Cdc42 (Bashaw, 2001).

The GEF64C overexpression phenotype is qualitatively similar to the phenotype of mutations in the Robo receptor, raising the possibility that GEF64C promotes attraction to the midline by interfering with Robo repulsion. Several observations argue against this idea. (1) There are significant differences between the GEF64C gain of function and robo loss of function phenotypes: robo mutations have more profound effects on the growth cones that pioneer the ipsilaterally projecting FasII-positive posterior corner cell (pCC) pathway than does GEF64C overexpression. (2) Overexpression of GEF64C does not appear to affect Robo protein expression or localization. (3) In terms of genetic predictions based on the function of commissureless (comm), Comm downregulates Robo receptors on commissural axons. In comm mutants no axons cross the midline; in robo;comm double mutants, the phenotype is like robo. Thus, if GEF64C overexpression were blocking robo function, the GEF64C gain of function should be at least partially epistatic to mutations in comm -- this is not the case. For these reasons, it is believed that GEF64C overexpression exerts its effects through stimulation of an attractive signaling pathway, rather than through inhibition of Robo repulsion (Bashaw, 2001).

The GEF64C gain of function phenotype suggests that by increasing the expression of an attractive signaling molecule, it is possible to overcome the normal repulsive signals that are present at the midline. To determine whether GEF64C expression would also allow axons to cross the midline in genetic backgrounds where axons are biased toward being repelled, GEF64C was coexpressed with a hyperactive mutant form of the Robo receptor: RoboY-F. Pan-neural expression of UASroboY-F results in a commissureless phenotype, in which no axons cross the midline. If in this roboY-F background GEF64C expression is simultaneously driven, many commissural axons are now able to cross the midline, and some segments appear to be nearly wild-type. Thus, even in this artificially repulsive background, GEF64C can allow significant axon growth to and across the midline, raising the exciting possibility that finding ways to stimulate the activity of functionally homologous mammalian GEFs could promote regrowth of injured axons in the adult CNS (Bashaw, 2001).

Regulation of rho family GTPases is required downstream of Robo to prevent axons from crossing the midline

Rho family GTPases are ideal candidates to regulate aspects of cytoskeletal dynamics downstream of axon guidance receptors. To examine the in vivo role of Rho GTPases in midline guidance, dominant negative (dn) and constitutively active (ct) forms of Rho, Drac1, and Dcdc42 are expressed in the Drosophila CNS. When expressed alone, only ctDrac and ctDcdc42 cause axons in the pCC/MP2 pathway to cross the midline inappropriately. Heterozygous loss of Roundabout enhances the ctDrac phenotype and causes errors in embryos expressing dnRho or ctRho. Homozygous loss of Son-of-Sevenless (Sos) also enhances the ctDrac phenotype and causes errors in embryos expressing either dnRho or dnDrac. CtRho suppresses the midline crossing errors caused by loss of Sos. CtDrac and ctDcdc42 phenotypes are suppressed by heterozygous loss of Profilin, but strongly enhanced by coexpression of constitutively active myosin light chain kinase (ctMLCK), which increases myosin II activity. Expression of ctMLCK also causes errors in embryos expressing either dnRho or ctRho. These data confirm that Rho family GTPases are required for regulation of actin polymerization and/or myosin activity and that this is critical for the response of growth cones to midline repulsive signals. Midline repulsion appears to require down-regulation of Drac1 and Dcdc42 and activation of Rho (Fritz, 2002).

Thus, when expressed alone, only ctDrac and ctDcdc42 cause midline crossing errors. However, the mutant GTPases interact genetically with mutations in robo, Sos, and chic and with overexpression of ctMLCK. The interactions are surprisingly specific. Midline crossing errors caused by expression of ctDrac or ctDcdc42 are suppressed by heterozygous loss of Profilin and enhanced by expression of ctMLCK. These results indicate that Drac1 and Dcdc42 encourage axons to cross the midline by regulating actin polymerization and/or myosin activity. CtRho and dnRho interact strongly with expression of ctMLCK or heterozygous loss of Robo, which suggests that regulation of myosin activity by Rho is crucial for midline repulsion. This work demonstrates that Rho, Drac1, and Dcdc42 are involved in dictating which axon may cross the midline, presumably by aiding in the transduction of attractive and/or repulsive cues operating at the midline. By using mutations in signaling molecules known to prevent pCC/MP2 axons from crossing the midline, this analysis concentrates on how Rho, Drac1, and Dcdc42 may regulate cytoskeletal dynamics in response to midline repulsive cues (Fritz, 2002).

Rho family GTPases activate a number of effectors that may affect axon outgrowth by regulating adhesion, myosin force generation, and/or actin polymerization. The ctDrac- and ctDcdc42-induced midline crossing errors are suppressed by heterozygous loss of Profilin, an actin-binding protein, which stimulates actin polymerization. Since reducing actin polymerization partially rescues the ctDrac and ctDcdc42 phenotypes as well as errors caused by heterozygous loss of Robo, it is likely that the midline crossing errors are caused by excessive actin polymerization. Increased actin polymerization may produce more filopodia to explore the midline, which leads to midline crossing. There are several pathways through which Drac1 and Dcdc42 might affect actin polymerization. The Cdc42/ Rac effector p21-activated kinase (PAK) activates LIM kinase to phosphorylate cofilin, an actin-depolymerizing factor required for neurite outgrowth. Cdc42 also activates actin polymerization through WASP, which stimulates polymerization by binding to the Arp2/3 complex. The activation of WASP by Cdc42 is enhanced by Profilin, which may explain why the suppression of the ctDcdc42 phenotype is stronger than that of the ctDrac-induced errors. However, actin polymerization may not be the only process regulated by Rho family GTPases to increase outgrowth (Fritz, 2002).

The data suggest that Drac1 and Dcdc42 activation must be prevented or reduced for axons to respond to repulsive signals at the midline. The midline crossing errors seen in Drac1 mutants are strongly enhanced by a partial loss of Robo, which suggests that midline repulsion requires a down-regulation of Drac1 activity. Down-regulation of Rac activity occurs in response to other repulsive signals, such as Ephrin and Semaphorin. A mechanism for this is suggested by experiments showing that Plexin-B, the receptor for Semaphorin, binds specifically to activated Rac, most likely to prevent it from activating effectors. Experiments in cell culture systems have confirmed that Robo-mediated signaling involves down-regulation of Cdc42. Activation of Robo by Slit recruits srGAP1 to the CC3 domain of Robo’s cytoplasmic tail, where it interacts with and inactivates Cdc42. Although srGAP1 does not affect the activity of Rac, srGAP2 and srGAP3 also bind to Robo, and one of these may regulate Rac activity. Down-regulation of Cdc42 and Rac by Robo-dependent repulsive signals is consistent with recent experiments showing that activation of DCC by chemoattractive Netrins stimulates neurite outgrowth and results in activation of Cdc42 and Rac1. Together, these data and the literature led to the hypothesis that Robo prevents axons from crossing the midline by decreasing Drac and Dcdc42 activity so that actin polymerization and myosin force generation are reduced (Fritz, 2002).

Down-regulation of Dcdc42 and Drac1 by Robo may also repel axons by preventing coupling of the actin cytoskeleton to the substrate. Rac is required for localization of E-Cadherin to cell-cell contacts and recruiting actin to Cadherin binding sites. Cdc42 and Rac promote Cadherin-mediated adhesion by preventing IQGAP, a CaM-binding Ras GAP, from interfering with the interaction of ß-catenin with alpha-catenin. Integrin-mediated adhesion also involves signaling through Rho family GTPases. By reducing actin and myosin dynamics and decoupling the cytoskeleton from the substrate, downregulation of Drac and Dcdc42 by repulsive guidance receptors would prevent axons from extending across the midline (Fritz, 2002).

The role of Rho in midline repulsion is more difficult to determine since both dnRho and ctRho enhance the midline crossing phenotype of heterozygous robo mutants. This is consistent with the data in which both dnRho and ctRho enhance the ctMLCK phenotype. Similar complexities are seen in the literature; expression of a Rho GEF, which is expected to increase Rho activity, leads to increased attraction to the midline, even though activation of Rho usually leads to growth cone collapse or retraction. The complexity of the Rho interactions is understandable when the dual role of myosin activity during axon guidance is considered. The most documented connection between myosin activity and Rho is through the effector Rho Kinase (RhoK). RhoK phosphorylates MLC and also inactivates myosin phosphatase by phosphorylating its myosin binding subunit, leading to increased phosphorylation of MLC and therefore increased myosin activity. Myosin activation is needed both for the retrograde flow of actin that retracts filopodia and for the force that propels the growth cone forward. Repulsive guidance signals are expected to increase retrograde flow while preventing forward movement (Fritz, 2002).

Expression of dnRho may specifically interfere with retraction of filopodia in response to repulsive cues, leading to increased midline crossing errors. A global increase in myosin activity caused by expression of either ctRho or ctMLCK, or even a Rho GEF, may cause axon guidance errors by increasing the forward movement of the growth cone. Midline attractive activity (e.g., Netrins) probably also influences how much myosin activity is available to move a growth cone over the midline. The literature and these experiments are most consistent with a model in which Rho is activated by repulsive guidance signals. Activation of ephrinA5 receptors causes an increase in Rho activity resulting in a growth cone collapse. Plexin B, the receptor for repulsive semaphorins, binds to and seems to activate Rho. Activation of Robo by Slit recruits srGAP1, which appears to prevent it from binding to and inactivating Rho. The genetic interactions seen between Sos^e49 mutations and expression of ctRho or dnRho are consistent with Sos acting as a GEF for Rho in pCC/MP2 neurons. DnRho strongly enhances the midline crossing errors caused by loss of Sos, while ctRho almost completely suppresses them. Since Sos-dependent signaling pathways are required for response to midline repulsive cues, this is further evidence that Rho is activated downstream of repulsive guidance signals, although a role downstream of selected attractants cannot be ruled out (Fritz, 2002).

Clearly, regulation of Rho family GTPase activity is necessary to prevent axons from crossing the midline inappropriately. Midline repulsive signaling involves regulation of all three GTPases; Drac1 and Dcdc42 are likely downregulated, while Rho seems to be activated downstream of repulsive signals. The Rho family GTPases influence actin polymerization and/or myosin force generation to regulate the processes of growth cone motility that are required for proper response to axon guidance signals (Fritz, 2002).

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

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

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

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

Robo and Slit and tracheal migration

Oxygen delivery in many animals is enabled by the formation of unicellular capillary tubes that penetrate target tissues to facilitate gas exchange. The tortuous outgrowth of tracheal unicellular branches towards their target tissues is controlled by complex local interactions with target cells. Slit, a phylogenetically conserved axonal guidance signal, is expressed in several tracheal targets and is required both for attraction and repulsion of tracheal branches. Robo and Robo2 are expressed in different branches, and are both necessary for the correct orientation of branch outgrowth. At the CNS midline, Slit functions as a repellent for tracheal branches and this function is mediated primarily by Robo. Robo2 is necessary for the tracheal response to the attractive Slit signal and its function is antagonized by Robo. It is proposed that the attractive and repulsive tracheal responses to Slit are mediated by different combinations of Robo and Robo2 receptors on the cell surface (Englund, 2002).

The importance of glial substrata in guiding the GB1 inside the CNS was investigated. By genetic ablation experiments, it has been shown that different glial cells provide distinct positional cues to the trachea. Longitudinal glia are first required for GB1 migration towards the midline, whereas midline and channel glia are necessary for inhibiting it from crossing the midline and to make it migrate dorsally through the neuropil. Slit signaling plays a major role in the migration of the GB1 cell. Slit is produced by midline cells and prevents GBs from crossing the midline of the VNC. Slit is also required as an attractant for the outgrowth of the primary, dorsal and visceral branches. The Slit receptors Roundabout (Robo) and Roundabout 2 (Robo2) are both required in the trachea independently of their function in axonal migration. The analysis of the tracheal robo and robo2 mutant phenotypes suggests that they may mediate different responses to the Slit signal. These results provide a first insight into the signaling mechanisms that guide the GB in the CNS, and identify an in vivo system for the study of the bi-functional role of Slit in epithelial cell guidance at the level of single cells (Englund, 2002).

A major determinant of axonal pathways inside the CNS is the repellent signal Slit. Midline cells express Slit, a large extracellular matrix protein that functions both as a short- and long-range repellent, controlling axon crossing at the midline and mesodermal cell migration away from the midline. In axon guidance, the Slit repulsive signal is mediated by the Roundabout (Robo) receptors. Different axons express different combinations of the three receptors, which determine the distance of their projections from the midline along the longitudinal fascicles. The midline crossing phenotypes of GBs in embryos expressing Ricin A in the midline glia (thus ablating these cells) suggests that Slit signaling may also guide GB1 in its turn away from the midline. Embryos expressing GFP under the control of the pan-tracheal btl-GAL4 driver, which drives expression of GAL4 in all tracheal cells from stage11, were double stained with antibodies against GFP and Slit or its receptors, and their expression was analyzed by confocal microscopy. The GB1 cell comes close to the midline source of Slit at early stage 16 but it then turns dorsally and posteriorly at the midline. Slit is also expressed in several other tissues close to the migrating tracheal branches. At early stage 14 in the dorsal side of the embryo, two rows of migrating mesodermal cells that will form the larval heart express Slit. These cardioblasts are in close proximity to the two leading cells of the tracheal dorsal branches (DBs), which also migrate towards the dorsal midline and give rise to the dorsal anastomosis (DB2) and the dorsal terminal branch (DB1). Slit expression is also detected from stage 13 on the surface of the midgut, at the sites of contact of the growing tracheal visceral branches (VBs). Finally, Slit is detected in lateral stripes of epidermal cells adjacent to the growing dorsal trunk (DT) and dorsal branches from stage 13. Are the Slit receptors expressed at this time in the trachea? Robo staining can be detected in all tracheal cells as they invaginate from the epidermis already at stage 11. Its tracheal expression is decreased by stage 13, when it is only weakly expressed in the dorsal trunk. No convincing expression of Robo was detected in the trachea after stage 14, even when serial optical sections of the GB1 cell were analyzed along its path in the CNS. Robo2 is also expressed in all tracheal cells from stage 11 and it then becomes restricted to the dorsal trunk and dorsal and visceral branches by stage 13. In contrast to Robo, which becomes undetectable in the trachea by stage 14, Robo2 expression is stronger and is maintained as late as at stage 16 in the DB1 and DB2 cells at the dorsal midline. Robo3 expression could not be detected in the trachea. The expression of Slit in tissues surrounding the developing trachea and the dynamic expression of its two receptors in different tracheal branches suggests a role for Slit signaling in tracheal branch outgrowth towards their target tissues (Englund, 2002).

The morphology of GB1 allows the separation of its tour in the CNS in two parts. In the first part, starting at the entry point into the CNS, GB1 extends broad filopodial projections and moves its cell body and nucleus ~20 µm towards the ventral longitudinal glia. In the second part, the position of the nucleus remains fixed and the tracheal cell sends a 30 µm long extension that navigates first towards the midline and then turns dorsally through a channel towards the dorsal longitudinal glia. GB1 contacts different groups of glial cells during its migration through the ventral nerve cord. The results from genetic ablation of different glial landmarks provide evidence for an instructive role of these substrates in steering GB1 migration and extension. In particular, the GB1 midline crossing phenotype observed after the ablation of midline glia argues for a repulsive signaling mechanism that redirects the cell from its route towards the midline (Englund, 2002).

The elegant analysis of axonal guidance at the midline of the fly CNS establishes the Slit repellent signal as a major determinant of axonal pathways. A gradient of Slit emanating from the midline prevents axons from crossing the midline through the activation of Robo receptors but it also functions as a long range repellent to position axons in distinct lateral fascicles. This later function is mediated by the expression of different combinations of Robo, Robo2 and Robo3 on axons that take distinct positions along the longitudinal tracts (Englund, 2002).

In CNS and muscle development Slit function is mediated by the Robo receptors. robo and robo2 are expressed in the trachea; the tracheal phenotypes of robo; robo2 double mutant embryos are very similar to the phenotypes of slit mutants, indicating that the tracheal responses to Slit are mediated by Robo and Robo2. Robo and Robo2 receptors can form homo- and hetero-dimers in vitro and the differences in their expression patterns suggests that they might mediate different responses to Slit. Indeed, the comparison of the phenotypes between the mutants for either of the two receptor genes reveals some intriguing differences. In robo embryos, the GBs erroneously cross the midline, suggesting that slit signaling via robo mediates repulsion away from the midline. In contrast, in robo2 mutants GBs fail to enter the CNS, suggesting that Robo2 may mediate an attractive response to Slit. In addition, the stalls in the migration of the dorsal branches detected in slit embryos were only found in robo2 mutants; no stalling phenotypes were detected in the tracheal branches that did not target the CNS in robo mutants. There is also a difference between the phenotypes generated by overexpression of robo and robo2. Overexpression of Robo in GB1 causes most of the branches to turn away from the midline prematurely. This phenotype is much weaker in embryos overexpressing Robo2, indicating that Robo is a more potent repulsive receptor in the GB. In addition, tracheal overexpression of Robo2 cannot rescue the robo mutant GB phenotype, even though this is possible via the tracheal expression of Robo. This result further indicates that Robo and Robo2 are not identical in their output and they cannot simply substitute for one another (Englund, 2002).

To further investigate whether different receptor complexes may mediate different responses to Slit, advantage was taken of the phenotypes caused by overexpression of Slit in the gut. In wild-type embryos, ectopic Slit can attract new visceral branches to its site of expression. This attractive function of Slit requires Robo2, as evidenced by the observation that overexpression of Slit with the same driver does not induce branch outgrowth in robo2 mutants. Robo alone cannot mediate the attractive response to Slit in the visceral branches -- instead it appears to function as an antagonist of the attractive signal mediated by Slit and Robo 2 in the visceral branches, because the number of new branches induced by Slit in robo mutants is three times higher than the number of branches induced under the same conditions in wild-type embryos (Englund, 2002).

Taken together these results suggest that there are qualitative differences between the cellular responses to Robo and Robo2 activation and that each receptor plays a unique role in the control of tracheal cell migration (Englund, 2002).

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).

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).

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).

Attention-like processes in Drosophila require short-term memory genes

Although there is much behavioral evidence for complex brain functions in insects, it is not known whether insects have selective attention. In humans, selective attention is a dynamic process restricting perception to a succession of salient stimuli, while less relevant competing stimuli are suppressed. Local field potential recordings in the brains of flies responding to visual novelty revealed attention-like processes with stereotypical temporal properties. These processes were modulated by genes involved in short-term memory formation, namely dunce and rutabaga. Attention defects in these mutants were associated with distinct optomotor effects in behavioral assays (van Swinderen, 2007).

Studies of visual discrimination in flies have revealed sophisticated perceptual effects that are relevant to selective attention, such as associative learning, context generalization, cross-modal binding, and position invariance. Visual choice behavior in Drosophila is correlated with local field potential (LFP) activity in the brain, centered around 20 to 30 Hz (van Swinderen, 2003). This activity is transiently increased in amplitude by classical conditioning, is suppressed during sleep or light anesthesia (van Swinderen, 2003), and is modulated by dopamine. Electrophysiological and behavioral measures of visual attention in flies were developed to test whether these short-term processes depend on the effect of genes involved in memory formation and plasticity (van Swinderen, 2007).

LFP responses to two distinct visual objects (a cross or a box, 180° apart, each moving around the fly once every 3 s) were investigated. When the objects were presented individually to wild-type flies, they evoked brain responses that were maximal when the single object swept directly in front of the flies. In contrast, dunce mutants (dnc¹), which are defective in short-term memory, displayed attenuated and delayed brain responses to each visual object, as compared to wild-type flies (van Swinderen, 2007).

To test for visual selection between these objects, they were presented together after having increased the salience for one object specifically in a recurrent-novelty paradigm. To measure visual selection, the 20- to 30-Hz brain response (mapped onto the 3-s sequence) was averaged for 10 s (about three rotations) after each transition to novelty, and this was compared to the response for the 10 s before novelty transitions. When wild-type flies were trained with two identical boxes for 100 s before one of the boxes changed to a cross, the response mapped selectively to the sectors of the rotation sequence associated with the (novel) cross, and the response for the competing box was significantly suppressed. Converse experiments attaching novelty salience to the alternate image (the box) after 100 s of cross training mapped 20- to 30-Hz responses to the novel box, showing that novelty selection was plastic. Novelty selection was also found to be position-invariant in a subset of trials, suggesting a cognitive effect rather than habituation (van Swinderen, 2007).

By decreasing the time between transitions in otherwise identical experiments, this paradigm provided a way to estimate the minimum exposure required for selection of recurrent novelty. When the training time was decreased to 50 s (~16 rotations), significant selection of the novel object and corresponding suppression of the competing object were still seen in wild-type flies. However, when the training time was further decreased to 25 s (about eight rotations), these novelty effects were lost (van Swinderen, 2007).

To control for the effect of change alone without novelty, transitions from a cross and a box back to two boxes were tested. In this case, an object changed to one that was already present during training. Such changes did not produce any selective 20- to 30-Hz responses for any training time in wild-type flies. The response is therefore unlikely to emanate from a startle reflex or an electrical artifact (van Swinderen, 2007).

Salience is a transient phenomenon. To investigate the extinction of novelty, the temporal sequence of selective brain responses were analyzed for successive rotations of a novel panorama after a transition. In wild-type flies, 20- to 30-Hz activity was strongly selective for the novel object (the cross) for 9 s (three successive panorama rotations) on average, and this was matched at a lower level for 10- to 20-Hz activity. Responses to training for the alternate object (making the box novel) revealed similar temporal dynamics: Wild-type flies in the 100-s paradigm stereotypically 'attended' to novelty for about 9 s or three sweeps of the panorama (van Swinderen, 2007).

The robust 100-s training effect was used for subsequent experiments in short-term memory mutants. There, dnc¹ flies failed to show any selective 20- to 30-Hz response to the novel visual stimulus after a transition. Instead, they revealed some selective responsiveness in the 10- to 20-Hz range. Further analysis of dnc¹ flies showed that brain responses in this mutant were greatest in the lower-frequency (10 to 20 Hz) bracket, as compared to greater responses at 20 to 30 Hz in the wild type (van Swinderen, 2007).

Mutations in rutabaga (rut) affect the same signaling network as mutations in dnc¹ [by producing opposite effects on adenosine 3', 5'-monophosphate (cAMP) levels], and flies display similar behavioral phenotypes in olfactory memory assays. Electrophysiology uncovered differences between rut and dnc mutants. Unlike dnc¹, rut²⁰⁸⁰ showed some responsiveness in the 20- to 30-Hz range, but without the sustained 9-s selection characteristic of wild-type flies. Similar to dnc¹, rut²⁰⁸⁰ responded strongly in the 10- to 20-Hz range (van Swinderen, 2007).

To investigate visual behavior in these strains, an optomotor paradigm was used that provides an efficient alternative to flight paradigms, because many mutant Drosophilae do not fly well [notably, dnc¹. The defective responsiveness to visual novelty seen in dnc¹ brain recordings (described above) may have predicted poor behavioral responsiveness to visual stimuli, but the opposite was the case: dnc¹ flies displayed the strongest optomotor response of ~100 different strains tested. The optomotor performance was analyzed of seven olfactory learning and memory mutants; these spanned a broad range of optomotor phenotypes. Like dnc¹, rut mutants also showed unusually strong optomotor responsiveness (van Swinderen, 2007).

To better describe optomotor performance, individual choices were filmed and quantified as flies progressed through a maze. Wild-type flies showed a preference for turning into the direction of perceived motion (a positive optomotor response) throughout most successive choice points . Another characteristic of wild-type optomotor behavior is some decreased responsiveness at choice points in the middle of the maze. In contrast, dnc¹ flies proceeded through the first two choice points without displaying any optomotor response but then responded strongly at the remaining six choice points in the maze (van Swinderen, 2007).

The delayed optomotor response in dnc¹ flies reveals a defect in processing a novel visual stimulus (a moving grating) as flies enter the maze. Attention-like behavior in dnc¹ was addressed more directly by adding a competing visual object to the optomotor paradigm. In wild-type flies, a static bar placed to one side of the transparent maze abolishes responsiveness to the moving grating, presumably by acting as a visual distractor. The effect of competing visual stimuli on optomotor responsiveness has also been previously observed in tethered flight experiments, where it has been described as evidence of limited perceptual resources (i.e., attention) partitioned among visual stimuli. In the walking analog of this paradigm, it was found that dnc¹ animals were not distracted by the competing visual stimulus (unlike wild-type flies), even though dnc¹ flies clearly perceived the distractor alone. The rut²⁰⁸⁰ brain-response defects were also matched by behavioral anomalies: The rut mutant was unresponsive to the distractor and responded more strongly than did the wild type, throughout the maze, to the grating presented alone without an initial delay. Subtle differences between rut and dnc mutants [also observed in habituation and socialization) suggest that common performance defects in these memory mutants may conceal differences at the level of short-term behavioral and brain processes (van Swinderen, 2007).

Finally, whether the conditionally expressed dnc gene product (cAMP phosphodiesterase) could modulate the corresponding electrophysiological and behavioral phenotypes described here, was investigated by expressing wild-type Dunce protein in a dnc¹ mutant background by using RU486-induced gene activation of a functional dnc transgene. When wild-type Dunce protein was expressed throughout the brain (via ElavGAL4 GeneSwitch) in adult mutant animals (by feeding adult flies RU486 for 24 hours), optomotor responsiveness remained high and brain responsiveness to novelty remained correspondingly insignificant, resembling dnc¹ flies. When the same construct was activated throughout development (by growing transgenic flies on RU486-laced food), optomotor responsiveness decreased to wild-type levels, and brain responsiveness to novelty was correspondingly increased to wild-type levels. A temporal examination of 20- to 30-Hz responses in the brain revealed that extinction dynamics were rescued as well, with the strong selective response persisting for at least 9 s in RU486-grown flies. The constitutive requirement of Dunce suggests that short-term plasticity for visual responsiveness in Drosophila adults is dependent on cAMP effects in the brain during its growth and development (van Swinderen, 2007).

Abelson tyrosine kinase and Calmodulin interact synergistically to transduce midline guidance cues in the Drosophila embryonic CNS: Interactions in robo signaling

Calmodulin and Abelson tyrosine kinase are key signaling molecules transducing guidance cues at the Drosophila embryonic midline. A reduction in the signaling strength of either pathway alone induces ectopic midline crossing errors in a few segments. When Calmodulin and Abelson signaling levels are simultaneously reduced, the frequency of ectopic crossovers is synergistically enhanced as all segments exhibit crossing errors. But as the level of signaling is further reduced, commissures begin to fuse and large gaps form in the longitudinal connectives. Quantitative analysis suggests that the level of Abelson activity is particularly important. Like Calmodulin, Abelson interacts with son-of-sevenless to increase ectopic crossovers suggesting all three contribute to midline repulsive signaling. Axons cross the midline in almost every segment if Frazzled is co-overexpressed with the Calmodulin inhibitor, but the crossovers induced by the Calmodulin inhibitor itself do not require endogenous Frazzled. Thus, Calmodulin and Abelson tyrosine kinase are key signaling molecules working synergistically to transduce both midline attractive and repulsive cues. While they may function downstream of specific receptors, the emergence of commissural and longitudinal connective defects point to a novel convergence of Calmodulin and Abelson signaling during the regulation of actin and myosin dynamics underlying a guidance decision (Hsouna, 2008).

The developmental defects observed in the formation of the CNS axon scaffold clearly point to individual and co-operative roles for CaM- and Abl-dependent signaling pathways during axon guidance at the midline. Moreover, the range of defects suggest that both CaM and Abl have multiple roles during the transduction of midline attractive and repulsive cues, and probably converge to regulate key aspects of the cytoskeletal dynamics underlying axon outgrowth and steering (Hsouna, 2008).

When the signaling strength of either pathway is individually decreased, the predominant phenotype is ectopic midline crossing errors of pCC/MP2 axons. Ectopic midline crossing errors is also the primary defect in embryos experiencing a mild, but simultaneous, reduction of both CaM and Abl signaling. CaM and Abl appear to work together in the same neurons to transduce guidance cues because ectopic crossovers are replicated if a kinase inactive Abl transgene (ftz_ng-Abl^KN) is co-expressed with the CaM inhibitor in the same neurons. It also appears that the level of Abl activity is particularly important. When both copies of the endogenous abl gene are mutated, expression of even one copy of the CaM inhibitor is sufficient to induce major defects in the axon scaffold. The converse, one copy of abl⁴ in homozygous iCaM^KA (inhibitor of CaM-KA were KA stands for a novel competitive inhibitor called Kinesin-antagonis) embryos, does not significantly affect the frequency of ectopic crossovers. Together, these data are consistent with the earlier hypothesis that both signaling pathways function to transduce midline repulsive cues (Hsouna, 2008).

Robo, the receptor for the midline repellent Slit, is expressed in pCC/MP2 neurons from the onset of axonogenesis and its activity is required to prevent them from crossing the midline. Loss-of-function robo mutations enhance the frequency of ectopic crossovers induced by either iCaM^KA expression or abl mutations. Thus, the observation that a simultaneous decrease in CaM and Abl synergistically increases ectopic crossovers was anticipated. Mechanistically, Abl is known to bind to, and phosphorylate, Robo to potentially inhibit its activity. In addition, Enabled, a known substrate for Abl, binds to Robo and is required to signal midline repulsive activity. While little is known about how CaM contributes to Robo signaling, the meandering crossovers observed in robo mutants are replicated when iCaM^KA is combined with loss-of-function mutations in sos and Sos is now known to bind to Robo. A direct role for Sos in Robo signaling is also supported by the observation that ectopic midline crossovers occur in sos abl double mutants (Hsouna, 2008).

However, it is unlikely that CaM and Abl are operating solely downstream of Robo during midline guidance. When the level of CaM and Abl activity is substantially reduced in embryos using multiple copies of iCaM^KA and abl alleles, the frequency of crossovers increase but, in addition, commissures begin to fuse and gaps in the longitudinal connectives form. These latter defects are difficult to explain solely on the basis of a disruption in Robo signaling, since they are not generally evident in robo null embryos. Thus, the efficacy of other guidance mechanisms functioning at the midline must also be affected in the CaM and Abl mutants. One obvious candidate is Netrin-dependent midline attraction (Hsouna, 2008).

Netrin is a major midline attractant required for commissure formation in the Drosophila CNS, and Frazzled is the Netrin receptor guiding many commissure axons across the midline. In the absence of Fra, most posterior commissures do not form correctly. However, it is suspected that an alteration in Fra activity is not responsible for the fused commissures and longitudinal gaps observed in iCaM^KA and abl mutants. First, in abl and fra double mutants, a loss of Abl activity in fra mutants exacerbates commissure loss, and second, expression of iCaM^KA still induces crossovers in the absence of Fra. Most vertebrate literature also predicts that CaM-dependent enzyme activity increase during Netrin-dependent attraction, not decrease, as occurs with iCaM^KA expression (Hsouna, 2008).

There is, however, growing evidence that at least one other Netrin-dependent receptor is functioning during the midline guidance of pCC/MP2 neurons. While pioneering the longitudinal connective, pCC/MP2 neurons follow an axon trajectory delineated by Netrin localized along commissure axons by Fra. To project past these Netrin-rich commissures, midline attraction must be briefly inhibited by Robo activity. If this inhibitory signal fails, pCC/MP2 axons cross the midline using the newly emerging commissures and leave gaps in the longitudinal connective. This is, in fact, similar to the defects observed in iCaM^KA and abl mutants. Importantly, while they are responding to Netrin, most pCC/MP2 neurons do not appear to express Fra. Thus, in addition to inhibiting Robo-dependent midline repulsion, a combined loss of CaM and Abl activity may be preventing Robo from blocking this Netrin-dependent attraction at the segmental boundary. Testing this hypothesis awaits characterization of the Netrin-dependent receptor that is functioning at the segmental boundary. The absence of Fra expression in pCC/MP2 neurons may also explain 1) why iCaM^KA and abl induce crossovers of pCC/MP2 neurons even though fra and abl interact to reduce commissure formation, and 2) why iCaM^KA induces pCC/MP2 axons to cross the midline even in the absence of Fra (Hsouna, 2008).

Interestingly, this Netrin-dependent, but Fra-independent attraction near the segmental boundary is known to be sensitive to IP₃ levels, which are presumably leading to an increase in intracellular calcium. Thus, the ability of these neurons to remain on the correct side of the midline is quite sensitive to the level of calcium, and now CaM, signaling. Calcium and/or CaM may function downstream of specific receptors or more generally as second messengers governing basic cell processes, such as motility. Certainly, inhibiting even a small amount of CaM activity (using one copy of iCaM^KA) sensitizes these neurons to over-expression of Fra. Moreover, the frequency of crossovers (57%) observed in this experiment is approximately the same as observed when wild type Fra is over-expressed in a heterozygous robo mutant (55%). This implies that expression of a single copy of iCaM^KA is reducing Robo activity by half, a conclusion difficult to reconcile with previous data. Therefore, it is suspected that expression of iCaM^KA is altering the spatial and temporal regulation of calcium-dependent activity underlying growth cone movement and steering. In the case of pCC/MP2 neurons, this appears to preferentially result in ectopic midline crossovers, a defect which is further enhanced when the levels of receptor for midline attraction (Fra) or repulsion (Robo) are genetically manipulated (Hsouna, 2008).

An alteration in key calcium-dependent regulatory events would also be further exacerbated by a simultaneous loss in Abl activity, especially since Abl is a key regulator of the actin and myosin dynamics underlying growth cone advance. While not previously appreciated, there is some evidence linking CaM and Abl activity during axon guidance in the embryonic axon scaffold. For example, heterozygous abl mutations suppress the ectopic midline crossing errors induced by expression of an activated Myosin Light Chain Kinase (a CaM-dependent enzyme) even when Fra is co-expressed. In addition, actin dynamics are likely to be important since both iCaM^KA and abl mutations interact with Profilin loss-of-function mutations to alter axon path finding. This study specifically demonstrates a strong, synergistic interaction between CaM- and Abl-dependent signaling during in vivo development of the embryonic CNS. Clearly, it will be important to identify where these key signaling pathways converge to regulate actin and myosin dynamics and how these regulatory events contribute to axon guidance decisions at the midline (Hsouna, 2008).

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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).

Midline crossing by gustatory receptor neuron axons is regulated by fruitless, doublesex and the Roundabout receptors

Although nervous system sexual dimorphisms are known in many species, relatively little is understood about the molecular mechanisms generating these dimorphisms. Recent findings in Drosophila provide the tools for dissecting how neurogenesis and neuronal differentiation are modulated by the Drosophila sex-determination regulatory genes to produce nervous system sexual dimorphisms. This paper reports studies aimed at illuminating the basis of the sexual dimorphic axonal projection patterns of foreleg gustatory receptor neurons (GRNs): only in males do GRN axons project across the midline of the ventral nerve cord. The sex determination genes fruitless (fru) and doublesex (dsx) both contribute to establishing this sexual dimorphism. Male-specific Fru (Fru^M) acts in foreleg GRNs to promote midline crossing by their axons, whereas midline crossing is repressed in females by female-specific Dsx (Dsx^F). In addition, midline crossing by these neurons might be promoted in males by male-specific Dsx (Dsx^M). The roundabout (robo) paralogs also regulate midline crossing by these neurons, and evidence is provided that Fru^M exerts its effect on midline crossing by directly or indirectly regulating Robo signaling (Mellert, 2010).

This study shows that the male-specific presence of contralateral GRN projections is primarily due to Fru^M function. Specifically, Fru^MC acts in foreleg GRNs to promote the crossing of the VNC midline by their axons. A role for dsx was identified in this dimorphism since (1) males that lack Dsx^M have somewhat fewer contralateral GRN projections, and (2) Dsx^F prevents the appearance of contralateral GRN axons in females (Mellert, 2010).

The finding that Fru^M regulates GRN axon midline crossing is consistent with previous findings that, in some neurons, Fru^M regulates axonal morphology. Regulation of axonal morphology is likely to alter synaptic connectivity, suggesting that one of the roles of Fru^M is to support the formation of male-specific connections, and possibly prevent the formation of female-specific connections, between neurons that are present in both sexes. Determining how such changes alter information processing will contribute to understanding how the potential for male courtship behavior is established (Mellert, 2010).

It is also notable that dsx plays a role in regulating sexually dimorphic midline crossing, given that it also specifies the sexual dimorphism in gustatory sensilla number in the foreleg. It might be that dsx regulates gustatory sensilla development independently of its regulation of GRN axon morphology. That dsx can independently specify multiple sexual dimorphisms within particular cell lineages has been previously shown for the foreleg bristles that comprise the sex comb teeth of the male foreleg and their homologous bristles in the female. There, dsx was shown to function at one time to determine the sex-specific number of bristles that are formed and at another time to determine their sex-specific morphology. In support of a similar sequential role in the developing GRNs, dsx is expressed in the gustatory sense organ precursor cells and continues to be expressed in the terminally differentiated GRNs (Mellert, 2010).

It is also possible that the effect of dsx on the presence of contralateral GRN projections is indirect. The two pools of gustatory sensilla, those that are male-specific and those that are homologous between males and females, might differ in their competence for midline crossing (i.e. only the male-specific GRNs will cross the midline when Fru^M is expressed). This is thought not to be the case for two reasons. First, dsx is expressed in the GRNs throughout their development, consistent with a role in regulating axon guidance. Second, the expression of Fru^MC in female GRNs using poxn-Gal4 is sufficient to induce midline crossing, suggesting that the sex-nonspecific GRNs are not intrinsically nonresponsive to Fru^M (Mellert, 2010).

With respect to the latter result, it is worth considering the contrast between females that are masculinized with fru^δtra, where no contralateral GRN projections are observed, and females in which poxn-Gal4 is used to drive the expression of UAS-fru^MC::AU1 (AU epitope tagged Fruitless) in females, where GRN midline crossing is observed. In the case of females masculinized by fru^δtra it was shown that the absence of contralateral GRN projections was due to Dsx^F functioning to prevent midline crossing in a manner that was epistatic to fru^M function. One attractive explanation for the difference between these two situations is based on the fact that masculinization by fru^δtra occurs via Fru^M produced from the endogenous fruitless locus, whereas masculinization by UAS-fru^MC::AU1, occurs via fru^MC expressed from a UAS construct that contains none of the untranslated sequences present in endogenous fru^M transcripts. Thus, it might be that the difference in midline crossing seen in these two situations is due to Dsx^F directly regulating fru^M expression through noncoding fru sequences that are present in the endogenous fru gene, but absent in the fru cDNA expressed from UAS-fru^MC::AU1. It is not likely that Dsx^M represses fru^M transcription, fru^P1.LexA was seen to be expressed in GRNs in both males and females. Thus, if fru^M is downstream of dsx in these cells, Dsx^F probably affects the processing or translation of fru^M transcripts through sequences not present in the UAS-fru^MC::AU1 construct. Alternatively, differences between these two situations in expression levels or patterns of expression might result in differences in the ability of Fru^M versus Fru^MC to overcome a parallel repressive effect of Dsx^F (Mellert, 2010).

robo, robo2 and robo3 are involved in GRN axon guidance. Of these three genes, robo appears to be most important in regulating GRN midline crossing because only reductions in levels of robo transcript result in midline crossing in females or fru^M-null males. Reducing levels of robo2 and robo3 transcripts in addition to robo enhances the robo phenotype but individual reductions of robo2 or robo3 function have the opposite effect, a reduction in midline crossing, suggesting that these receptors function to promote crossing in the presence of wild-type levels of robo expression (Mellert, 2010).

It is not surprising that robo differs in function from robo2 and robo3 with respect to foreleg GRN development. robo2 and robo3 are more similar in sequence to each other than to robo, and robo contains two cytoplasmic motifs not found in its paralogs. Furthermore, functional differences have been recognized since the original reports of robo2 and robo3. Finally, robo2 might promote midline crossing if pan-neuronally overexpressed at low levels and yet repress midline crossing when overexpressed at high levels. This 'switch' in function might explain why reduced midline crossing is seen under conditions of both robo2 overexpression and reduction (Mellert, 2010).

Given that the Robo receptors play such an important role in GRN development, how might fru^M regulate midline crossing? The data indicate that robo lies genetically downstream of fru^M. The most straightforward mechanistic explanation is that Fru^M suppresses the activity of the Robo signaling pathway. Several ways that this might occur can be envisioned. First, fru^M might regulate commissureless, which itself participates in the midline crossing decision by regulating the subcellular localization of Robo. No sexual dimorphism could be detected in the subcellular localization of a Robo::GFP fusion protein in GRNs in either the axons or cell body (UAS-robo::GFP), so if fru^M regulates comm, it does so subtly. It is more probable that fru^M regulates the expression of either other regulators of robo signaling, robo itself, or robo effectors. Strategies are being pursued to identify candidate Fru^M targets that might be involved in regulating midline crossing (Mellert, 2010).

How does midline crossing by GRN axons affect gustatory perception? Given that male-typical GRN morphology requires fru^M, and that fru^M has a major regulatory role for social behavior, one hypothesis is that the contralateral GRN projections in males play a role in mediating the processing of contact cues during male courtship and/or aggression. Previous reports have shown that fru^M-masculinized females, which do not have contralateral GRN projections, readily perform tapping and proceed to subsequent steps in the male courtship ritual, and behave like males with respect to aggressive behaviors. Thus, contralateral GRN projections are not necessary for the initiation and execution of these male-specific behaviors. Nevertheless, midline crossing might still be important for mediating socially relevant gustatory information. For instance, amputation experiments suggest that the detection of contact stimuli is important for courtship initiation under conditions when the male cannot otherwise see or smell the female (Mellert, 2010).

It is possible that midline crossing by GRN axons facilitates the comparison of chemical contact cues between the two forelegs. Such a comparison might help the male to determine the orientation of another fly, which would be a useful adaptation for performing social behaviors in conditions of sensory deprivation, such as in the dark. Alternatively, midline crossing might simply be a mechanism to form additional neuronal connections that integrate gustatory information into circuits underlying male-specific behaviors. Armed with the results of the present study, fru^M, dsx, and the robo genes can be used as handles for developing tools and strategies to specifically manipulate midline crossing in the foreleg GRNs, with the goal of understanding its importance with regard to male behavior (Mellert, 2010).

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 (I. R. 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 (I. R. 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 (I. R. 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 (I. R. 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 (I. R. 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 (I. R. 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 (I. R. 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 (I. R. Evans, 2010).

Distinct protein domains and expression patterns confer divergent axon guidance functions for Drosophila Robo receptors

The orthogonal array of axon pathways in the Drosophila CNS is constructed in part under the control of three Robo family axon guidance receptors: Robo1, Robo2 and Robo3. Each of these receptors is responsible for a distinct set of guidance decisions. To determine the molecular basis for these functional specializations, homologous recombination was used to create a series of 9 'robo swap' alleles: expressing each of the three Robo receptors from each of the three robo loci. The lateral positioning of longitudinal axon pathways was shown to rely primarily on differences in gene regulation, not distinct combinations of Robo proteins as previously thought. In contrast, specific features of the Robo1 and Robo2 proteins contribute to their distinct functions in commissure formation. These specializations allow Robo1 to prevent crossing and Robo2 to promote crossing. These data demonstrate how diversification of expression and structure within a single family of guidance receptors can shape complex patterns of neuronal wiring (Spitzweck, 2010).

The midline guidance cue Slit is thought to act through each of three different Robo family receptors to help form the orthogonal axonal pathways of the Drosophila ventral nerve cord. Each of the three Robos has a distinct role in forming these projections. Robo1 is primarily required to prevent longitudinal axons from crossing the midline. Robo2 has a minor role in preventing longitudinal axons from crossing, and, as this study has shown, also facilitates the crossing of commissural axons. Finally, Robo3 may also help prevent some longitudinal axons from crossing, but its major function is to direct the formation of the intermediate longitudinal pathways (Spitzweck, 2010).

The goal of this study was to assess whether these functional specializations reflect structural differences in the Robo proteins themselves or differences in robo gene regulation. To this end, gene targeting was used to replace the coding region of each robo gene with that of each other robo, creating a series of robo swap alleles. It was found that commissure formation relies on the unique structural features of both Robo1 (to prevent crossing) and Robo2 (to promote crossing). In contrast, lateral positioning of longitudinal axons does not rely on structural differences between the Robo proteins, but rather differences in robo gene expression (Spitzweck, 2010).

In the longitudinal pathways, axons are organized into discrete and stereotyped fascicles. In part, this requires selective fasciculation mediated by contact-dependent attractive or repulsive surface proteins that 'label' specific axon fascicles. This includes the FasII protein which was exploited in this study as a marker. In addition to these pathway labels, the lateral pathways are also segregated into three broad zones according to the distinct combination of Robo receptors they express. Loss- and gain-of-function genetic experiments have shown that these Robo proteins are instructive in lateral pathway selection and, hence, define a 'Robo code' (Spitzweck, 2010).

A popular model for lateral pathway selection posits that the three Robo proteins have distinct signaling properties, and that they position axons on a lateral gradient of their common ligand Slit. In this model, the Robo proteins are assumed to differ in either their affinity for Slit, the strength of their 'repulsive output,' or both. However, direct evidence for a role of Slit in lateral pathway is still lacking, and alternative models have to be considered. One such possibility is that the Robo proteins might act instead as homophilic adhesion molecules. In such a model, the Robo proteins might operate in a manner similar to other pathway labels such as FasII, but over broader zones. Regardless of whether they invoke a role for Slit, homophilic adhesion, or some other unidentified ligand, all models presented to date have assumed that there must be critical structural differences in the Robo proteins. These structural differences would form the basis of a combinatorial Robo code for lateral pathway selection (Spitzweck, 2010).

The current data demonstrate that this cannot be the case. Lateral positioning does not rely on structural differences between the Robo proteins. This is particularly clear for the distinction between the medial and the intermediate zones, which relies entirely on the selective expression of Robo3 on intermediate axons. This study found, however, that lateral positioning of these axons works surprisingly well even when Robo3 protein is replaced by either Robo1 or Robo2. Although some minor disruption in specific pathways cannot be excluded, the overall structure of the longitudinal pathways appears normal in these embryos. Notably, this includes the formation of the intermediate FasII pathway and the projections of the Sema2b axons, both of which were diagnostic for Robo3's role in lateral positioning. Thus, at least for the medial and intermediate axons, the only relevant differences between the Robos are in their patterns of gene expression. The 'Robo code' is not a protein code; it is a gene-expression code (Spitzweck, 2010).

At first glance, this result is difficult to reconcile with the previously published gain-of-function experiments. In these experiments, the various Robo proteins were expressed from GAL4/UAS transgenes in specific neurons (the Ap neurons). These Ap neurons normally express only Robo1 and hence project ipsilaterally in the medial zone. In both reports, expression of Robo3 shifted these axons into the intermediate zone, as expected, but expression of Robo1 did not. Why might Robo1 be able to replace the endogenous Robo3 in the swap experiments, but not the transgenic Robo3 in these gain-of-function studies? A trivial but unsatisfying explanation is that this was merely an artifact of the GAL4/UAS system. Prior to the advent of site-specific transgenesis, it was notoriously difficult to control for the varying expression levels from different transgene insertions, which rarely match endogenous levels. More interesting possibilities are that the discrepancy may reflect differences resulting from assaying the behavior of neurons that normally express Robo3 versus those that don't, or perhaps a 'community effect' that results from manipulating an entire cohort of neurons, not just a single neuron. In this regard it is also important to note that the Ap axons are likely to be follower axons for their specific pathway, not pioneers. Whatever the reason for this discrepancy, the substitution of the robo1 coding region into the robo3 locus is presumably the more physiologically relevant assay (Spitzweck, 2010).

How might differences in robo gene expression explain lateral positioning? One possibility is that it is only the total Robo levels that are important, with higher levels sending axons further laterally on the presumptive Slit gradient. This model fits with the results of 'supershifting' experiments, in which additional copies of the Robo3 transgene displaced the Ap axons even further from the midline. It is also supported by mathematical modeling of the Robo code. This model still invokes a role for the Slit gradient, for which there is admittedly no direct evidence. Alternatively, lateral pathway selection might rely on critical differences in the precise spatial and temporal pattern of expression, rather than differences in total Robo levels (Spitzweck, 2010).

It has long been appreciated that Robo1 is the primary receptor through which Slit repels longitudinal axons to prevent them from crossing the midline. Midline crossing errors occur in every segment of robo1 mutants, but are relatively rare in both robo2 and robo3 mutants. This study has shown that this unique function of Robo1 relies on differences in both gene regulation and protein structure. Specifically, Robo1 cannot exert its midline repulsion function when expressed in the pattern of robo2 or robo3, nor can Robo2 or Robo3 prevent midline crossing when expressed in the manner of robo1 (Spitzweck, 2010).

By examining a series of chimeric receptors consisting of distinct parts of Robo1 and Robo3, this critical and unique function of Robo1 in midline repulsion was mapped to a region of the cytoplasmic domain containing the CC1 and CC2 motifs. This conclusion is broadly consistent with previous studies that have examined Robo1 deletion mutants lacking specific CC motifs, in this case in a pan-neuronal transgenic rescue assay. Although there are subtle differences that may reflect the use of chimeric receptors versus single domain deletions, and the consequences of expressing them under the control of endogenous versus heterologous gene regulatory elements, the two studies together strongly suggest that the proline-rich CC2 motif is the critical structural determinant of Robo1's unique capability of preventing midline crossing. This domain is thought to serve as a docking site for a number of factors that contribute to Slit-dependent repulsion through Robo1, including Enabled, the Rac GTPase activating protein Vilse/CrGAP, and the SH2-SH3 adaptor Dock, the latter recruiting in turn the Rac guanine nucleotide exchange factor Sos and p21 activated kinase. CC2 is also the most broadly conserved of the cytoplasmic domains in Robo1, with the insect Robo2 and Robo3 proteins being the only known Robo receptors that lack CC2. The lack of CC2 in Robo2 and Robo3 cautions against the inference that the distinct guidance functions of these two receptors are necessarily mediated by repulsive signaling in response to activation by Slit (Spitzweck, 2010).

Indeed, this study has presented evidence that Robo2 can even act in opposition to Robo1 to promote crossing. It is assumed that Robo2 normally exerts this positive function autonomously in commissural neurons, acting in parallel to Netrin-Frazzled signaling to allow midline crossing. Two models are envisioned to account for the positive role of Robo2 in midline crossing. In one scenario, Robo2 transduces an attractive signal that promotes crossing, possibly in response to its midline ligand Slit. Such a model has previously been proposed for Robo2 in the guidance of ganglionic tracheal branches. Alternatively, Robo2 might promote crossing by antagonizing the repulsive function of Robo1, thus mediating an 'anti-repulsion' rather than an 'attraction' signal. Formally, this model is analogous to the role of Comm in Drosophila, and of Robo3/Rig-1 in vertebrates. Preliminary data are more consistent with this latter scenario (Spitzweck, 2010).

Three factors are now known that promote midline crossing: Comm, Netrin-Frazzled, and Robo2. Of these, only Comm appears to be instructive. Comm is expressed in commissural but not ipsilateral neurons, and is both necessary and sufficient for crossing. In contrast, both Frazzled and Robo2 are permissive: they are expressed in both commissural and ipsilateral neurons, and are required but not sufficient for crossing. They are also partially redundant and independent, as crossing is severely disrupted only when both are eliminated. A conceptual model for midline crossing proposes a bistable switch created by the mutual inhibition between high Robo1 levels and midline crossing: high Robo1 levels prevent crossing due to repulsive signaling, whereas crossing the midline leads to clearance of Robo1 protein from the midline axon segment. In such a model, the permissive factors (Frazzled and Robo2) may act to ensure the appropriate balance between midline attraction and midline repulsion, bringing this feedback loop into the dynamic range at which the instructive factor (Comm) can operate. In principle, any one of the three factors--Comm, Robo2, or Frazzled--could have taken on the instructive role. Comm has evidently done so in Drosophila. To the extent that a similar feedback loop operates in mice, the instructive role may have fallen in this species to the Robo2 analog, Robo3 (Spitzweck, 2010).

Functional diversity of Robo receptor immunoglobulin domains promotes distinct axon guidance decisions

Recognition molecules of the immunoglobulin (Ig) superfamily control axon guidance in the developing nervous system. Ig-like domains are among the most widely represented protein domains in the human genome, and the number of Ig superfamily proteins is strongly correlated with cellular complexity. In Drosophila, three Roundabout (Robo) Ig superfamily receptors respond to their common Slit ligand to regulate axon guidance at the midline: Robo and Robo2 mediate midline repulsion, Robo2 and Robo3 control longitudinal pathway selection, and Robo2 can promote midline crossing. How these closely related receptors mediate distinct guidance functions is not understood. This study reports that the differential functions of Robo2 and Robo3 are specified by their ectodomains and do not reflect differences in cytoplasmic signaling. Functional modularity of Robo2's ectodomain facilitates multiple guidance decisions: Ig1 and Ig3 of Robo2 confer lateral positioning activity, whereas Ig2 confers promidline crossing activity. Robo2's distinct functions are not dependent on greater Slit affinity but are instead due in part to differences in multimerization and receptor-ligand stoichiometry conferred by Robo2's Ig domains. Together, these findings suggest that diverse responses to the Slit guidance cue are imparted by intrinsic structural differences encoded in the extracellular Ig domains of the Robo receptors (T. A. Evans, 2010).

In the Drosophila embryonic central nervous system (CNS), Robo receptors are expressed in overlapping domains that divide the longitudinal axon connectives into three broad zones: axons occupying the medial zone express Robo, axons in the intermediate zone express Robo and Robo3, and axons in the most lateral zone express Robo, Robo2, and Robo3. Loss of robo2 shifts lateral axons to intermediate positions, whereas loss of robo3 shifts intermediate axons to medial positions. Conversely, ectopic expression of Robo2 or Robo3 in medial axons forces them to select more lateral pathways, whereas increased levels of Robo do not. The 'Robo code' model posits that a combinatorial code of Robo receptor expression determines the lateral position of CNS axons. To test whether a combinatorial code is necessary, the ability was tested of Robo2 and Robo3 to shift apterous axons in embryos deficient for various combinations of robo genes; removing endogenous robo or robo3 was found not to affect Robo2's ability to shift apterous axons laterally. Indeed, UAS-Robo2 was sufficient to direct the apterous axons to the lateral edge of the connectives even in robo3, robo double mutant embryos. Similarly, removal of robo2 or robo had little or no effect on the ability of UAS-Robo3 to redirect the apterous axons to more lateral pathways. Thus, it is the individual expression of Robo2 and Robo3 that dictates lateral positions of CNS axons, not a combinatorial Robo code (T. A. Evans, 2010).

Robo2 and Robo3 dictate the lateral position of axons in the Drosophila CNS, a role that is not shared by Robo. What is the basis for this differential activity? All three receptors have similar ectodomains with five immunoglobulin (Ig) domains and three fibronectin (Fn) III repeats, whereas their cytoplasmic domains are more divergent. In particular, Robo2 and Robo3 both lack two conserved motifs (CC2 and CC3) that mediate interactions with several downstream effectors and are required for Robo's midline repulsive function, leading to the speculation that distinct Robo functions are directed by their cytoplasmic domains. To determine whether the functional difference between Robo2-Robo3 and Robo is due to a qualitative difference in cytoplasmic signaling, a set of chimeric receptors was assayed for their ability to induce lateral shifting in the medial apterous axons (T. A. Evans, 2010).

First, the cytoplasmic domain of Robo was replaced with that of Robo2 or Robo3 (Robo1:2 and Robo1:3). Neither of these receptor variants was able to reposition the apterous axons. In contrast, when the cytoplasmic domains of Robo2 or Robo3 were replaced by that of Robo, the resulting chimeric receptors (Robo2:1 and Robo3:1) exhibited lateral positioning activity similar to full-length Robo2 and Robo3. These results reveal that the lateral positioning activities of Robo2 and Robo3 are specified by their ectodomains. Importantly, the cytoplasmic domains of Robo2 and Robo3 are not dispensable for lateral positioning activity, because receptors without any cytodomains are unable to redirect the apterous axons laterally. Because Robo cytoplasmic domains are functionally interchangeable for longitudinal pathway selection, any required intracellular events must be mediated by cytoplasmic sequences that are common to Robo, Robo2, and Robo3 (T. A. Evans, 2010).

To dissect the structural basis underlying the differential activities of Robo receptor extracellular domains, the relative contributions of Robo2's Ig and Fn domains were examined by generating a more restricted set of domain swaps between Robo and Robo2. Exchanging all five Ig domains between Robo and Robo2 completely swapped their lateral positioning activities. These results reveal that Robo2's ability to position axons is specified entirely by its Ig domains. However, the Fn repeats are not completely dispensable for lateral positioning activity because Robo2 variants lacking these elements displayed reduced activity. Thus, when combined with Robo2's five Ig domains, the Fn repeats and cytoplasmic domain of Robo can act permissively to facilitate lateral pathway choice (T. A. Evans, 2010).

The five Ig domains of Robo2 are necessary and sufficient to functionally distinguish it from Robo in the context of longitudinal pathway choice. To subdivide the ectodomains of Robo and Robo2 further, the presumptive Slit-binding region (Ig1) was targeted. Initially Ig1 and Ig2 were swapped together, because some evidence suggested that Ig2 could contribute to Slit binding of human Robo receptors. Robo variants possessing the first and second Ig domains of Robo2 (Robo1^R2I1+2) displayed activity comparable to full-length Robo2. However, the converse swap revealed that Robo2 still retained its activity even when its Ig1+2 was replaced with those of Robo (Robo2^R1I+2). These results reveal a bipartite contribution to Robo2's lateral positioning activity from (at least) two genetically separable elements located within Ig1+2 and Ig3-5, respectively (T. A. Evans, 2010).

Next whether Ig1 and Ig3 together could be responsible for dictating the lateral positioning activity of Robo2 was tested. Replacing Ig1 or Ig3 of Robo with those of Robo2, alone (Robo^1R2I1 and Robo^1R2I3) or in combination (Robo^1R2I1+3), was sufficient to confer Robo2-equivalent activity to Robo. Importantly, replacing Ig1-3 of Robo2 with the corresponding domains of Robo eliminated its lateral positioning activity, demonstrating that the Ig1-3 region is both necessary and sufficient to functionally distinguish Robo1 and Robo2 in the context of longitudinal pathway choice (T. A. Evans, 2010).

Ig1 and Ig3 of Robo2 can independently specify its ability to redirect medial axons to more lateral pathways. Further, the lateral positioning activities of chimeric receptors containing Ig1 or Ig3 of Robo2 were indistinguishable in the apterous neuron assay. To determine whether these receptors could also influence longitudinal pathway choice in a broader context, the effects were assayed of pan-neuronal misexpression of selected chimeric receptors on lateral positioning of FasII-positive axon pathways (T. A. Evans, 2010).

In wild-type embryos or elavGAL4;UAS-Robo embryos, three major FasII-positive tracts were detectable on either side of the midline. Pan-neuronal misexpression of Robo2, in contrast, disrupted longitudinal pathway formation such that the intermediate FasII pathway was absent in nearly all segments. Notably, this effect appeared to depend solely on Ig3 of Robo2, because it was recapitulated by UAS-Robo2^R1I1+2 and UAS-Robo^1R2I3, but not by UAS-Robo1^R2I1+2 or UAS-Robo2^R1I1-3. These observations draw a functional distinction between the activities of Ig1 and Ig3 of Robo2 and suggest that these two domains regulate longitudinal pathway choice via distinct mechanisms (T. A. Evans, 2010).

Because the Slit-binding Ig1 contributes to Robo2's lateral positioning activity, it is possible that Robo2 regulates longitudinal pathway selection in response to Slit. If so, then removing slit or disrupting its interaction with Robo2 should reduce or eliminate Robo2's lateral positioning activity. Therefore, the effects of Robo2 misexpression in apterous axons were examined in a slit mutant background. In the absence of Slit, the entire axon scaffold collapsed at the midline, and even high levels of ectopic Robo2 could not force the apterous axons laterally. This may indicate a direct requirement for Slit or instead reflect the inability of Robo2-expressing apterous axons to move outside of the collapsed axon scaffold (T. A. Evans, 2010).

Whether Robo2 could reposition axons without its Slit-binding region was examined next. To ensure complete disruption of Slit binding, both the first and second Ig domains were deleted from Robo2; Robo2^ΔIg1+2 was completely unable to reposition the apterous axons. Deleting these two domains did not interfere with expression or localization of Robo2 . Together, these results provide evidence that Robo2-directed lateral positioning is dependent on interactions with Slit; however, it is noteed that in addition to disrupting Slit binding, deletion of Ig1 and Ig2 would also disrupt other potentially important functions of these domains. Genetic analysis of the role of robo3 in the regulation of lateral chordotonal axon arborization within the CNS also supports Slit-dependent control of lateral position by Robo receptors (T. A. Evans, 2010).

Interestingly, pan-neuronal misexpression of Robo2 results in phenotypes that are inconsistent with a strictly repulsive function for Robo2. At the highest levels of overexpression, Robo2 prevents all midline crossing. However, moderate levels of Robo2 overexpression lead to ectopic midline crossing, suggesting that in some contexts Robo2 can promote midline crossing. Perhaps Robo2, like the divergent Robo receptor Rig-1/Robo3 in vertebrates, can antagonize Slit-Robo repulsion (T. A. Evans, 2010).

The panel of chimeric receptors was used to map this activity of Robo2. All of the receptor variants that contain Ig2 of Robo2 promoted midline crossing when misexpressed with elavGAL4, whereas those that contain regions of Robo2 apart from Ig2 did not. Thus, the promidline crossing activity of Robo2 is conferred by Ig2. Interestingly, rather than being excluded from the crossing portions of axons like all other Robo receptor variants, Robo2 proteins that promoted midline crossing were expressed strongly on crossing axons. This localization to crossing axons was not shared by any of the Robo3 or Robo3-Robo1 receptors (T. A. Evans, 2010).

Although the mechanism of Robo2's procrossing function cannot be addressed at this time, the fact that it is dependent on Ig2 alone suggests that it is probably not due to Robo2 binding Slit and sequestering Slit away from endogenous Robo. It is also noted that this crossing activity does not correlate with lateral positioning activity, because some variants with strong lateral positioning activity (e.g., Robo2R^1I1+2, Robo^1R2I1+3, Robo^1R2I1, and Robo^1R2I3) do not promote ectopic midline crossing. It will be interesting to determine whether Robo2 in Drosophila promotes midline crossing through inhibition of Robo or, alternatively, whether it mediates midline attraction in certain contexts. If, like Rig-1/Robo3, Robo2 acts as an antirepellent, it is likely to achieve this function through a distinct mechanism because Rig-1/Robo3's antirepellent function is specified by its cytoplasmic domain (T. A. Evans, 2010).

Because Robo2's Ig domains control lateral positioning, one possibility is that Robo2 may have a higher affinity for Slit, encouraging Robo2-expressing axons to seek out positions farther down the Slit gradient. To test this possibility, the Ig domain-containing portions of the Robo and Robo2 ectodomains were purified, and their affinities for the Robo-binding domain of Slit (Slit D2) were compared with surface plasmon resonance (SPR). It was found that Robo2 does not exhibit a higher Slit affinity than Robo; instead, the Ig1-5 region of Robo binds Slit D2 around 4-fold as strongly as the equivalent region of Robo2. Thus, the functional distinction between Robo and Robo2 for longitudinal pathway choice is not increased Slit affinity of Robo2. Furthermore, these observations suggest that the promidline crossing activity of Robo2 does not result from greater Slit affinity (T. A. Evans, 2010).

Apart from modest affinity differences, a second distinction was observed between the Slit binding profiles of Robo and Robo2. When tested against a constant amount of immobilized Slit, the maximum equilibrium binding response for Robo was approximately half of that for Robo2. Thus, at equilibrium, the same amount of Slit can bind twice as much Robo2 as Robo, suggesting a difference in receptor-ligand stoichiometry. Size-exclusion chromatography (SEC) confirmed that the Ig1-5 fragment of Robo is almost exclusively monomeric in solution, whereas Robo2 Ig1-5 appears almost exclusively as a dimer. These experiments were performed in the absence of Slit, indicating that the observed multimerization of Robo2 is at least partially ligand independent. However, the differences in maximum Slit binding response in the SPR experiments indicate that the multimerization states of Robo and Robo2 remain distinct even upon Slit binding (T. A. Evans, 2010).

To determine which region(s) of Robo2 are responsible for dimerization and whether the observed differences in receptor multimerization correlate with the two distinct lateral positioning activities observed in vivo, equivalent Ig1-5 fragments derived from the chimeric receptors Robo1^R2I1+2 and Robo2R^1I1+2 were examined via SEC. These reciprocal chimeric receptors contained distinct portions of Robo2 and exhibited distinct large-scale effects on FasII tract formation. The Robo2R^1I1+2 receptor fragment (containing Ig3-5 of Robo2) was found to exhibit Robo2-like Slit-independent dimerization, whereas the Robo1^R2I1+2 fragment (containing Ig1+2 of Robo2) did not. Thus, ectodomain-dependent dimerization of Robo2 correlates with its ability to influence large-scale longitudinal pathway choice by FasII-positive axons and may account for Ig3's contribution to the lateral positioning activity of Robo2 (T. A. Evans, 2010).

How do closely related axon guidance receptors, responding to a common ligand, generate diverse and, in some cases, opposing guidance outcomes? This study has shown that the differential roles of the Robo receptors in directing longitudinal pathway choice are determined by structural differences between receptor ectodomains. In addition, evidence is provided that a second function of Robo2 to promote midline crossing also depends on structural features of its ectodomain. It is concluded that the diversification of Robo receptor axon guidance activities is facilitated by the functional modularity of individual receptor ectodomains. Although the importance of guidance receptor cytoplasmic domains in controlling guidance decisions has been known for a decade, the results reveal that Robo receptor Ig domains play an important part in the functional diversification of this ancient and evolutionarily conserved guidance receptor family (T. A. 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).

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