InteractiveFly: GeneBrief

roundabout 2: Biological Overview | References


Gene name - roundabout 2

Synonyms - leak

Cytological map position - 22A1-22A1

Function - transmembrane receptor

Keywords - CNS, brain, heart, trachea axon guidance, dendrites

Symbol - robo2

FlyBase ID: FBgn0002543

Genetic map position - chr2L:1380086-1420449

Classification - Immunoglobulin domain, Fibronectin type III domain

Cellular location - surface transmembrane



NCBI links: Precomputed BLAST | EntrezGene
Recent literature
Evans, T.A., Santiago, C., Arbeille, E. and Bashaw, G.J. (2015). Robo2 acts in trans to inhibit Slit-Robo1 repulsion in pre-crossing commissural axons. Elife 4. PubMed ID: 26186094
Summary:
During nervous system development, commissural axons cross the midline despite the presence of repellant ligands. In Drosophila, commissural axons avoid premature responsiveness to the midline repellant Slit by expressing the endosomal sorting receptor Commissureless, which reduces surface expression of the Slit receptor Roundabout1 (Robo1). This study describes a distinct mechanism to inhibit Robo1 repulsion and promote midline crossing, in which Roundabout2 (Robo2) binds to and prevents Robo1 signaling. Unexpectedly, it was found that Robo2 is expressed in midline cells during the early stages of commissural axon guidance, and that over-expression of Robo2 can rescue robo2-dependent midline crossing defects non-cell autonomously. It was shown that the extracellular domains required for binding to Robo1 are also required for Robo2's ability to promote midline crossing, in both gain-of-function and rescue assays. These findings indicate that at least two independent mechanisms to overcome Slit-Robo1 repulsion in pre-crossing commissural axons have evolved in Drosophila.
Sasse, S. and Klambt, C. (2016). Repulsive epithelial cues direct glial migration along the nerve. Dev Cell 39(6): 696-707. PubMed ID: 27997826
Summary:
Most glial cells show pronounced migratory abilities and generally follow axonal trajectories to reach their final destination. However, the molecular cues controlling their directional migration are largely unknown. To address this, glial migration onto the developing Drosophila leg imaginal disc was developed as a model. Here, CNS-derived glial cells move along nerves containing motoaxons and sensory axons. Along their path, glial cells encounter at least three choice points where directional decisions are needed. Subsequent genetic analyses allowed uncovering mechanisms that escaped previous studies. Most strikingly, it was found that glial cells require the expression of the repulsive guidance receptors PlexinA/B and Robo2 to prevent breaking away from the nerve. Interestingly, the repulsive ligands are presented by the underlying leg imaginal disc epithelium, which appears to push glial cells toward the axon fascicle. In conclusion, nerve formation not only requires neuron-glia interaction but also depends on glial-epithelial communication.
Vaughen, J. and Igaki, T. (2016). Slit-Robo repulsive signaling extrudes tumorigenic cells from epithelia. Dev Cell 39: 683-695. PubMed ID: 27997825
Summary:
Cells dynamically interact throughout animal development to coordinate growth and deter disease. For example, cell-cell competition weeds out aberrant cells to enforce homeostasis. In Drosophila, tumorigenic cells mutant for the cell polarity gene scribble (scrib) are actively eliminated from epithelia when surrounded by wild-type cells. While scrib cell elimination depends critically on JNK signaling, JNK-dependent cell death cannot sufficiently explain scrib cell extirpation. Thus, how JNK executed cell elimination remained elusive. This study shows that repulsive Slit-Robo2-Ena signaling exerts an extrusive force downstream of JNK to eliminate scrib cells from epithelia by disrupting E-cadherin. While loss of Slit-Robo2-Ena in scrib cells potentiates scrib tumor formation within the epithelium, Robo2-Ena hyperactivation surprisingly triggers luminal scrib tumor growth following excess extrusion. This extrusive signaling is amplified by a positive feedback loop between Slit-Robo2-Ena and JNK. These observations provide a potential causal mechanism for Slit-Robo dysregulation in numerous human cancers.



BIOLOGICAL OVERVIEW

Transcription factors establish neural diversity and wiring specificity; however, how they orchestrate changes in cell morphology remains poorly understood. The Drosophila Roundabout (Robo) receptors regulate connectivity in the CNS, but how their precise expression domains are established is unknown. This study shows that the homeodomain transcription factor Hb9 acts upstream of Robo2 and Robo3 to regulate axon guidance in the Drosophila embryo. In ventrally projecting motor neurons, hb9 is required for robo2 expression, and restoring Robo2 activity in hb9 mutants rescues motor axon defects. Hb9 requires its conserved repressor domain and functions in parallel with Nkx6 to regulate robo2. Moreover, hb9 can regulate the mediolateral position of axons through robo2 and robo3, and restoring robo3 expression in hb9 mutants rescues the lateral position defects of a subset of neurons. Altogether, these data identify Robo2 and Robo3 as key effectors of Hb9 in regulating nervous system development (Santiago, 2014).

Combinations of transcription factors specify the tremendous diversity of cell types in the nervous system. Many studies have identified requirements for transcription factors in regulating specific events in circuit formation as neurons migrate, form dendritic and axonal extensions, and select their final synaptic targets. In most cases, the downstream effectors through which transcription factors control changes in neuronal morphology and connectivity remain unknown, although several functional relationships have been demonstrated (Santiago, 2014).

Conserved homeodomain transcription factors regulate motor neuron development across phyla. Studies in vertebrates and invertebrates have shown that motor neurons that project to common target areas often express common sets of transcription factors, which act instructively to direct motor axon guidance. In mouse and chick, Nkx6.1/ Nkx6.2 and MNR2/Hb9 are required for the specification of spinal cord motor neurons, and for axon pathfinding and muscle targeting in specific motor nerves. In Drosophila, Nkx6 and Hb9 are expressed in embryonic motor neurons that project to ventral or lateral body wall muscles, and although they are not individually required for specification, they are essential for the pathfinding of ventrally projecting motor axons. Axons that project to dorsal muscles express the homeodomain transcription factor Even-skipped (Eve), which regulates guidance in part through the Netrin receptor Unc5. Eve exhibits cross-repressive interactions with hb9 and nkx6, which function in parallel to repress eve and promote islet and lim3. Hb9 and Nkx6 act as repressors to regulate transcription factors in the spinal cord; however, guidance receptors that act downstream of Hb9 and Nkx6 have not been characterized. Interestingly, in both flies and vertebrates, Hb9 and Nkx6 are also expressed in a subset of interneurons, and knockdown experiments in Drosophila have suggested a role for hb9 in regulating midline crossing (Santiago, 2014).

Roundabout (Robo) receptors regulate midline crossing and lateral position within the developing CNS of invertebrates and vertebrates. Two recent studies in mice have also identified a role for Robos in regulating motor axon guidance in specific motor neuron populations (Bravo-Ambrosio, 2012; Jaworski, 2012). The three Drosophila Robo receptors have diversified in their expression patterns and functions. Robo2 is initially expressed in many ipsilateral pioneers and also contributes to Slit-mediated repulsion. Subsequently, robo2 expression is more restricted, and it is required to specify the medio-lateral position of axons. Robo3 is expressed in a subset of CNS neurons and also regulates lateral position (Santiago, 2014).

Characterization of the expression domains of the Drosophila Robos revealed an intriguing pattern, in which Robo1 is expressed on axons throughout the width of the CNS, Robo3 is found on axons in intermediate and lateral zones, and Robo2 is enriched on the most lateral axons. These patterns are transcriptional in origin, as replacing any robo gene with the coding sequence of another Robo receptor results in a protein distribution that matches the endogenous expression of the replaced gene (Spitzweck, 2010). A phenotypic analysis of these gene-swap alleles revealed the importance of transcriptional regulation for the diversification of robo gene function (Spitzweck, 2010). Robo2 and robo3's roles in regulating lateral position are largely dependent on their expression patterns, although unique structures within the Robo2 receptor are also important for its function in lateral position (Evans, 2010; Spitzweck, 2010). In the peripheral nervous system, the Atonal transcription factor regulates robo3 in chordotonal sensory neurons, directing the position of their axon terminals. In the CNS, the transcription factors lola and midline contribute to the induction of robo1. However, how the expression patterns of robo2 and robo3 are established to direct axons to specific medio-lateral zones within the CNS remains unknown (Santiago, 2014).

This study identifies a functional relationship between Hb9 and the Robo2 and Robo3 receptors in multiple contexts. Hb9 acts through Robo2 to regulate motor axon guidance and can direct the medio-lateral position of axons in the nerve cord through its effects on robo2 and robo3. Furthermore, hb9 interacts genetically with nkx6 and requires its conserved repressor domain to regulate robo2. Together, these data establish a link between transcriptional regulators and cell surface guidance receptors, providing an example of how upstream factors act through specific guidance receptors to direct circuit formation (Santiago, 2014).

This study has demonstrated a functional relationship between Hb9 and the Robo2 and Robo3 receptors in multiple contexts in the Drosophila embryo. In the RP motor neurons, hb9 is required for robo2 expression, and genetic rescue experiments indicate that robo2 acts downstream of hb9. Hb9 requires its conserved repressor domain and acts in parallel with Nkx6 to regulate robo2 and motor axon guidance. Moreover, hb9 contributes to the endogenous expression patterns of robo2 and robo3 and the lateral position of a subset of axons in the CNS, and can redirect axons laterally when overexpressed via upregulation of robo2. Finally, restoring Robo3 rescues the medial shift of MP1 axons in hb9 mutants, indicating that hb9 acts through robo3 to regulate medio-lateral position in a defined subset of neurons (Santiago, 2014).

Hb9 and nkx6 are required for the expression of robo2 in motor neurons, and rescue experiments suggest that the loss of robo2 contributes to the phenotype of hb9 mutants. However, nkx6 mutants and hb9 mutants heterozygous for nkx6 have a stronger ISNb phenotype than robo2 mutants, implying the existence of additional downstream targets. One candidate is the cell adhesion molecule FasIII, which is normally expressed in the RP motor neurons and appears reduced in nkx6 mutant embryos. Identifying the constellation of effectors that function downstream of Hb9 and Nkx6 will be key to understanding how transcription factors expressed in specific neurons work together to drive the expression of the cell surface receptors that regulate axon guidance and target selection (Santiago, 2014).

Robo2's activity in motor axon guidance appears distinct from the previously described activities of the Drosophila Robo receptors. Although Robo1 can replace Robo2's repulsive activity at the midline (Spitzweck, 2010), Robo2's function in motor axon guidance is not shared by either Robo1 or Robo3. Moreover, Robo2's antirepulsive activity at the midline and its ability to shift axons laterally when overexpressed both map to Robo2's ectodomain, whereas this study has found that Robo2's activity in motor axon guidance maps to its cytodomain (Evans, 2010; Spitzweck, 2010). The signaling outputs of Robo2's cytodomain remain unknown, as it lacks the conserved motifs within Robo1 that engage downstream signaling partners. How does Robo2 function during motor axon guidance? In mice, Robo receptors are expressed in spinal motor neurons and prevent the defasciculation of a subset of motor axons (Jaworski, 2012). Does Drosophila Robo2 regulate motor axon fasciculation? The levels of adhesion between ISNb axons and other nerves must be precisely controlled during the different stages of motor axon growth and target selection, and several regulators of adhesion are required for ISNb guidance. Furthermore, whereas Slit can be detected on ventral muscles, it is not visibly enriched in a pattern that suggests directionality in guiding motor axons, making it difficult to envision how Robo2-mediated repulsive or attractive signaling might contribute to ISNb pathfinding. Future work will determine how Robo2's cytodomain mediates motor axon guidance, whether this activity is Slit dependent, and whether Robo2 signals attraction, repulsion, or modulates adhesion in Drosophila motor axons (Santiago, 2014).

Elegant gene-swap experiments revealed the importance of transcriptional regulation in establishing the different expression patterns and functions of the Drosophila Robo receptors (Spitzweck, 2010). By analyzing a previously uncharacterized subset of axon pathways, this study has uncovered a requirement for Hb9 in regulating lateral position in the CNS. Although Hb9 can act instructively to direct lateral position when overexpressed, its endogenous expression in a subset of medially projecting neurons suggests that its ability to shift axons laterally is context dependent. A complex picture emerges in which multiple factors act in different groups of neurons to regulate robo2 and robo3. In a subset of interneurons, hb9 is endogenously required for lateral position through the upregulation of robo3 and likely robo2. In other neurons, such as those that form the outer FasII tracts, the expression patterns of robo2 and robo3 rely on additional upstream factors. What might be the significance of a regulatory network in which multiple sets of transcription factors direct lateral position in different groups of neurons? One possibility is that hb9-expressing neurons may share specific functional properties, such as the expression of particular neurotransmitters or ion channels. Alternatively, hb9 may regulate other aspects of connectivity. Indeed, Robo receptors mediate dendritic targeting in the Drosophila CNS, raising the exciting possibility that hb9 regulates both axonal and dendritic guidance through its effects on guidance receptor expression (Santiago, 2014).

What is the mechanism by which Hb9 regulates the expression of robo2, robo3, and its other downstream effectors? This study has found that Hb9 requires its conserved putative repressor domain and acts in parallel with Nkx6 to regulate robo2 and motor axon guidance. It has previously been shown that hb9 and nkx6 function in parallel to regulate several transcription factors. Hb9, nkx6 double mutants show decreased expression of islet and lim3 and upregulation of eve and the Nkx2 ortholog vnd. Are Hb9 and Nkx6 regulating robo2 or robo3 through any of their previously identified targets? Hb9 and nkx6 single mutants show no change in islet, lim3, or vnd expression, arguing that hb9 and nkx6 do not act solely through these factors to regulate robo2 or robo3. Eve expression is unaffected in nkx6 mutants, and whereas it is ectopically expressed in two neurons per hemisegment in hb9 mutants, these do not correspond to RP3 or MP1, the identifiable cells in which changes can be detected in robo2 and robo3. Therefore, the data do not support the hypothesis that Hb9 and Nkx6 regulate robo2 or robo3 primarily through their previously identified targets islet, lim3, vnd, or eve (Santiago, 2014).

Gain-of-function experiments in vertebrates suggest that Hb9 and Nkx6 act as repressors to regulate gene expression in the spinal. The finding that Hb9's Eh domain is required for motor axon pathfinding and robo2 regulation suggests that Hb9 acts as a repressor in this context as well, most likely through a previously unidentified intermediate target. In contrast, the Eh domain is not required for Hb9's ability to regulate robo3 or lateral position in hb9GAL4+ neurons that project to intermediate zones of the CNS. The finding that Hb9;delta;Eh retains significant activity in rescuing lateral position and robo3 expression indicates that Hb9 may regulate robo2 and robo3 via distinct mechanisms, perhaps involving different transcriptional cofactors or intermediate targets. In support of this hypothesis, hb9 overexpression in the ap neurons can induce robo2, but not robo3. These data raise the intriguing possibility that Hb9's ability to regulate robo2 and robo3 via different mechanisms contributed to the diversification of their expression patterns in the CNS. Determining how Hb9 and Nkx6 regulate their effectors will be key to achieving a complete understanding of how these conserved transcription factors control changes in cell morphology and axon pathfinding during development. Of note, Hb9 mutant mice exhibit defects in a subset of motor nerves, including the phrenic and intercostal nerves, which are also affected in Robo mutants. It will be of great interest to determine if despite the vast divergence in the evolution of nervous system development between invertebrates and vertebrates, Hb9 or Nkx6 has retained a role for regulating Robo receptors across species (Santiago, 2014).

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

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

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

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

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

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 (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 (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 (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 (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 (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 (Robo1R2I1+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 (Robo2R1I+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 (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 (Robo1R2I1 and Robo1R2I3) or in combination (Robo1R2I1+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 (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 (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-Robo2R1I1+2 and UAS-Robo1R2I3, but not by UAS-Robo1R2I1+2 or UAS-Robo2R1I1-3. 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 (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 (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; Robo2ΔIg1+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 (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 (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 (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., Robo2R1I1+2, Robo1R2I1+3, Robo1R2I1, and Robo1R2I3) 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 (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 (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 (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 Robo1R2I1+2 and Robo2R1I1+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 Robo2R1I1+2 receptor fragment (containing Ig3-5 of Robo2) was found to exhibit Robo2-like Slit-independent dimerization, whereas the Robo1R2I1+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 (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 (Evans, 2010).

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 (FruM) acts in foreleg GRNs to promote midline crossing by their axons, whereas midline crossing is repressed in females by female-specific Dsx (DsxF). In addition, midline crossing by these neurons might be promoted in males by male-specific Dsx (DsxM). The roundabout (robo) paralogs also regulate midline crossing by these neurons, and evidence is provided that FruM 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 FruM function. Specifically, FruMC 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 DsxM have somewhat fewer contralateral GRN projections, and (2) DsxF prevents the appearance of contralateral GRN axons in females (Mellert, 2010).

The finding that FruM regulates GRN axon midline crossing is consistent with previous findings that, in some neurons, FruM regulates axonal morphology. Regulation of axonal morphology is likely to alter synaptic connectivity, suggesting that one of the roles of FruM 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 FruM 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 FruMC in female GRNs using poxn-Gal4 is sufficient to induce midline crossing, suggesting that the sex-nonspecific GRNs are not intrinsically nonresponsive to FruM (Mellert, 2010).

With respect to the latter result, it is worth considering the contrast between females that are masculinized with fruδtra, where no contralateral GRN projections are observed, and females in which poxn-Gal4 is used to drive the expression of UAS-fruMC::AU1 (AU epitope tagged Fruitless) in females, where GRN midline crossing is observed. In the case of females masculinized by fruδtra it was shown that the absence of contralateral GRN projections was due to DsxF functioning to prevent midline crossing in a manner that was epistatic to fruM function. One attractive explanation for the difference between these two situations is based on the fact that masculinization by fruδtra occurs via FruM produced from the endogenous fruitless locus, whereas masculinization by UAS-fruMC::AU1, occurs via fruMC expressed from a UAS construct that contains none of the untranslated sequences present in endogenous fruM transcripts. Thus, it might be that the difference in midline crossing seen in these two situations is due to DsxF directly regulating fruM expression through noncoding fru sequences that are present in the endogenous fru gene, but absent in the fru cDNA expressed from UAS-fruMC::AU1. It is not likely that DsxM represses fruM transcription, fruP1.LexA was seen to be expressed in GRNs in both males and females. Thus, if fruM is downstream of dsx in these cells, DsxF probably affects the processing or translation of fruM transcripts through sequences not present in the UAS-fruMC::AU1 construct. Alternatively, differences between these two situations in expression levels or patterns of expression might result in differences in the ability of FruM versus FruMC to overcome a parallel repressive effect of DsxF (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 fruM-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 fruM regulate midline crossing? The data indicate that robo lies genetically downstream of fruM. The most straightforward mechanistic explanation is that FruM suppresses the activity of the Robo signaling pathway. Several ways that this might occur can be envisioned. First, fruM 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 fruM regulates comm, it does so subtly. It is more probable that fruM regulates the expression of either other regulators of robo signaling, robo itself, or robo effectors. Strategies are being pursued to identify candidate FruM 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 fruM, and that fruM 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 fruM-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, fruM, 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).

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).

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 cardiac cell polarity and morphogenesis

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

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

To assess the role of Slit in Drosophila heart, slit null-mutant embryos (slit2) were analyzed by labeling the heart with antibodies against Dmef2, a muscle-specific transcription factor expressed in all myocardial and other muscle cells. When the bilateral rows of myocardial progenitors have reached the dorsal midline, they fail to align properly in slit mutants compared to wild-type. A similar phenotype is observed in robo,robo2 double-mutant embryos. In contrast, only subtle alignment defects are found in robo or robo2 single mutants. Unlike robo or robo2,robo3 mutants in combination with robo or robo2 do not cause additional heart defects, and thus robo3 is unlikely involved in cardiac development. Similar alignment phenotypes were observed with nmrH15lacZ reporter, a marker for myocardial nuclei, in slit mutants. Although the dorsal migration of the myocardial progenitors does not seem to be affected, their highly regular arrangement is already perturbed before they reach the midline, as manifested in gaps and double rows. Visualization of the pericardial cells with Zfh-1 shows that their alignment with the myocardial cells is also perturbed in slit-robo mutants. At stage 16, two types of phenotypes can be distinguished: Type I consists of irregular cell arrangements, and type II, in addition, has large gaps. These two types of phenotypes are found in roughly equal proportion in slit and robo,robo2 double mutants. These defects are unlikely caused by abnormalities in cardiac lineage specification or in ectodermal epithelium formation during dorsal closure (Qian, 2005).

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

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

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

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

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

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

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

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

Conserved sequence block clustering and flanking inter-cluster flexibility delineate enhancers that regulate nerfin-1 expression during Drosophila CNS development

Nerfin-1 is a nuclear regulator of axon guidance required for a subset of early pathfinding events in the developing Drosophila CNS. Nerfin-1 belongs to a highly conserved subfamily of Zn-finger proteins with cognates identified in nematodes and man. The neural precursor gene prospero is essential for nerfin-1 expression. Unlike nerfin-1 mRNA, which is expressed in many neural precursor cells, the encoded Nerfin-1 protein is only detected in the nuclei of neuronal precursors that will divide just once and then transiently in their nascent neurons. Although nerfin-1 null embryos have no discernible alterations in neural lineage development or in neuronal or glial identities, CNS pioneering neurons require nerfin-1 function for early axon guidance decisions. Furthermore, nerfin-1 is required for the proper development of commissural and connective axon fascicles. Nerfin-1 is essential for the proper expression of robo2, wnt5, derailed, G-oα47A, Lar, and futsch<, genes whose encoded proteins participate in these early navigational events (Kuzin, 2005).

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

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

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

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

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

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

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

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

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

Because Robo is expressed rather generally in olfactory neurons, loss-of-function was studied by targeted misexpression of antagonists of signaling, rather than in clones. SG18.1-Gal4 expresses in a large fraction of olfactory neurons thus revealing most of the glomeruli as well as the antennal commissure. Ectopic expression of commissureless (comm) using SG18.1-Gal4 resulted in disorganization of glomerular patterning with a weak effect on the commissure. Comm has been shown to downregulate Robo, although its effect on Robo2 and Robo3 is less well understood. The phenotype of Comm ectopic expression suggests that Robo is necessary for determining sensory neuron position within the lobe. Abelson kinase (Abl) phosphorylates the CC0 and CC1 domains of Robo, thus antagonizing signaling. Ectopic expression of either Abl or a constitutively active Dcdc42v12 completely abolishes glomerular formation. Sensory neurons expressing Dcdc42v12 (SG18.1::Dcdc42v12) show an attraction for the midline and terminate there forming 'glomerular-like' structures at the midline. Results from loss-of-function clones predict such a phenotype for robo2 nulls. Constitutive activation of Dcdc42 is known to affect cytosketal dynamics generally, and could phenocopy a loss-of-function of all Robo receptors (Jhaveri, 2004).

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

Whether the genetic elements participating with Robo signaling in other well-studied systems also operate in the Drosophila adult olfactory system was also tested. A deficiency for the Slit region was crossed into an SG18.1 UAS-GFP UAS-robo2 recombinant. In this situation, where endogenous levels of the ligand were reduced by 50%, commissure formation, which is disrupted by the ectopic expression of Robo2, was restored and glomerular morphology also returned to normal. Targeted down-regulation of Robo signaling by misexpression of Comm or activated Dcdc42v12, respectively, also suppress the phenotype caused by elevated Robo2 (Jhaveri, 2004).

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

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

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

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

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

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).

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 ┬Ám 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 RoboDelta (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 RoboDeltaCC2+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).

Attractive and repulsive functions of Slit are mediated by different receptors in the Drosophila trachea

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).

Short-range and long-range guidance by Slit and its Robo receptors: Robo and Robo2 play distinct roles in midline guidance

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).

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).

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).

Short-range and long-range guidance by Slit and its Robo receptors: A combinatorial code of Robo receptors controls lateral position

Slit is secreted by midline glia in Drosophila and functions as a short-range repellent to control midline crossing. Although most Slit stays near the midline, some diffuses laterally, functioning as a long-range chemorepellent. A combinatorial code of Robo receptors controls lateral position in the CNS by responding to this presumptive Slit gradient. Medial axons express only Robo, intermediate axons express Robo3 and Robo, while lateral axons express Robo2, Robo3, and Robo. Removal of robo2 or robo3 causes lateral axons to extend medially; ectopic expression of Robo2 or Robo3 on medial axons drives them laterally. Precise topography of longitudinal pathways appears to be controlled by a combination of long-range guidance (the Robo code determining region) and short-range guidance (discrete local cues determining specific location within a region) (Simpson, 2000b).

Robo and Robo2 together play an early function in the control of midline crossing. robo continues to be expressed by all neurons, and Robo protein appears at high levels on axons either after they cross the midline, or from the outset if they never cross the midline. Robo2 is more dynamic in its pattern of expression. Initially, it is expressed by a wide range of neurons, including all of the early pioneer neurons whose axons do not cross the midline (e.g., pCC, MP1, dMP2, and vMP2). But during the period around late stage 13 in which these axons selectively defasciculate to form the medial pCC pathway and the intermediate MP1 pathway, the expression of Robo2 declines in many of these neurons. It is during this same period (late stage 13 to stage 14) that Robo3 begins to be expressed by a subset of neurons (Simpson, 2000b).

From stage 14 onward, as multiple longitudinal pathways form, all three Robos are expressed on some or all longitudinal axon tracts and are excluded from commissural axon tracts. Within the longitudinal tracts, their expression patterns differ dramatically. Robo is found on all longitudinal axon pathways. The second phase of Robo2 expression, and the only phase of Robo3 expression, have a common quality. Both are expressed on a subset of axons that extend in specific lateral positions of the developing CNS. Robo3 is expressed only on axons that extend in the outer two-thirds of the longitudinal pathways (the intermediate and lateral regions). A high level of Robo2 expression is restricted to axons that extend in the outer third of the longitudinal pathways (the lateral region), farthest from the midline and thus farthest from the source (Simpson, 2000b).

All three Robos show relatively tight boundaries. All three are absent from the commissures, and Robo3 and Robo2 are restricted to certain regions of the longitudinal pathways. The expression of Robo3 and Robo2 is not graded, but rather appears to form regional boundaries. While the high level of Robo2 is restricted to the lateral pathways, a lower level of Robo2 expression is detected on some of the intermediate pathways (the more lateral ones). The low level of Robo2 expression begins right in the middle of the intermediate Fas II pathway. This step-wise expression of Robo2 (from none on the medial portion of the intermediate pathways, to a low level on the lateral portion of the intermediate pathways, to a high level laterally) reveals further regional subdivisions of the longitudinal pathways (Simpson, 2000b).

The pattern of expression of Robo3 and Robo2 in individual identified neurons is consistent with their overall patterns of expression. For example, robo3 RNA is expressed in the MP1 neuron whose axon pioneers the intermediate Fas II pathway. robo3 is largely absent from pCC and other neurons whose axons pioneer the medial Fas II pathway. All of these neurons (e.g., MP1, pCC) transiently express robo2 when they are making the earlier decision not to cross the midline, but such expression declines by the time the medial and intermediate Fas II longitudinal pathways separate from one another. This is consistent with the presence of Robo2 only on lateral axons during later stages of development (Simpson, 2000b).

All three Robos are expressed on specific growth cones and filopodia. Commissural growth cones and axons are devoid of all three Robos. All ~150 longitudinal axons express Robo. The ~50 intermediate axons and the ~50 lateral axons express Robo3 (with one exception). The ~50 lateral axons express Robo2. Most lateral axons express Robo2 and Robo3, with one exception; the most lateral axon bundle of ~10 axons expresses high levels of Robo2, but is largely or completely devoid of Robo3 (Simpson, 2000b).

The mutant phenotypes of robo, robo2, and robo3 support the hypothesis that the Robos specify lateral position with respect to the midline. robo mutants show axons ectopically crossing and recrossing the midline. These axons are predominantly those of the innermost part of the longitudinal scaffold. When a robo mutant is examined with anti-Fas II (mAb 1D4), only the medial Fas II pathway crosses the midline. The intermediate and lateral Fas II pathways stay on their own side. One possible interpretation is that the intermediate and lateral expression of Robo3 and Robo2 keeps these axons from crossing the midline in a robo mutant. In a robo, robo2 double mutant, all axons go to the midline and do not leave it (and thus it looks like a slit mutant) (Simpson, 2000b).

robo2 loss-of-function mutations show occasional ectopic midline crossing, but, more prominently, they show abnormalities in lateral positioning. The most common phenotype as revealed with anti-Fas II staining is crossovers and 'braiding' between the intermediate and lateral Fas II pathways (and sometimes between the medial and intermediate Fas II pathways). Although superficially the axon scaffold looks relatively normal in a robo2 mutant (when visualized with mAb BP102, which labels all axons), the lateral positions of the longitudinal pathways are altered in the absence of Robo2 (Simpson, 2000b).

The role of Robo3 in lateral positioning was examined using RNA interference (RNAi). Injection of robo3 dsRNA causes the stage 16 embryo to have two Fas II longitudinal pathways instead of three. The intermediate pathway is missing, and the medial pathway is larger than normal. The lateral (normally Robo2 expressing) Fas II pathway appears normal. In the absence of Robo3, the medial and intermediate Fas II pathways fail to separate, and the intermediate (normally Robo3 expressing) pathway does not properly form (Simpson, 2000b).

The robo2, robo3 double mutant, generated by injecting robo3 dsRNA into a robo2 mutant, contains a large single Fas II longitudinal pathway. This single fascicle is thicker than wild-type Fas II pathways and is close to the midline in the normal location of the medial Fas II pathway. In the absence of both Robo3 and Robo2, it appears as if most (and in some cases all) of the Fas II axons selectively fasciculate into one Fas II pathway in the medial position. This suggests that Robo3 and Robo2 are required for the normal formation of the Robo3 expressing intermediate Fas II pathway and the Robo2/Robo3 expressing lateral Fas II pathway (Simpson, 2000b).

Unlike robo mutants alone, robo, robo3 embryos (robo mutants with robo3 dsRNA) exhibit ectopic crossing of the intermediate Fas II pathway as well as the medial one. This resembles the addition of the two individual phenotypes: lack of Robo3 causes the intermediate pathway to join the medial pathway, and the lack of Robo allows this fused Fas II pathway to weave back and forth across the midline. In the robo, robo3 double mutant, the outer Fas II pathway remains on its appropriate side, presumably due to the presence of Robo2 (Simpson, 2000b).

These phenotypes support the model that Robo is the most important contributor to maintaining the Fas II pathways on the appropriate side of the midline, but that Robo2 and Robo3 determine the lateral position of these and other longitudinal pathways. Robo3 specifies the intermediate region and its pathways, while Robo2 specifies the lateral region and its pathways (Simpson, 2000b).

Overexpression of Robo2 supports the model that Robo2 levels contribute to the lateral position of axons. Overexpression of UAS-robo2 in all CNS axons using the elav-GAL4 driver results in a commissureless-like phenotype (i.e., appearing like the commissureless mutant). There are a number of other genetic combinations that result in a commissureless-like phenotype. These all look the same when examined with mAb BP102 that stains all axons: the commissures are missing. But the appearance of the three Fas II pathways differs, depending upon the genetic makeup of the embryo (Simpson, 2000b).

When Robo expression is increased on all axons, by either directly driving more Robo or in a comm mutant (in which Comm no longer downregulates Robo), three distinct Fas II pathways are still detected. This is true even when the Robo Y-F 'hyperactive' receptor is transgenically expressed on all axons. Under these various conditions, there is disorganization of longitudinal pathways, but the three Fas II pathways can generally be detected (Simpson, 2000b).

But when Robo2 is ectopically expressed on all axons, the lateral position of the pathways is disrupted, and as a result, all three Fas II pathways are bundled together into a single, thick tract. Ectopic Robo2, but not ectopic Robo or loss of Comm, is sufficient to override the endogenous positional information that specifies the locations of the three Fas II pathways (Simpson, 2000b).

Overexpression of Robo3 in all CNS axons (using the elav-GAL4 driver) results in a weakly commissureless-like phenotype. As with UAS-robo and UAS-robo2, the gain-of-function commissureless phenotype of Robo3 requires two copies of the UAS-robo3 reporter to generate commissureless segments. Some of the Fas II bundles are fused, but at least two distinct fascicles are still visible at this level of overexpression (Simpson, 2000b).

Expressing Robo2 or Robo3 in subsets of neurons whose axons normally extend in medial longitudinal pathways can drive these axons to assume more lateral positions. Although both can drive medial axons further laterally, Robo2 and Robo3 are not identical: when tested on the same axons, Robo2 drives medial axons further laterally than does Robo3 (Simpson, 2000b).

For example, in each abdominal hemisegment, three neurons express the transcription factor Apterous (Ap). These neurons normally extend their axons toward the midline, and then turn anteriorly on their own side close to the midline. These axons turn anteriorly in the medial region. When viewed at the light level with confocal microscopy, the Ap axons sometimes look like they are running just at the lateral edge of the medial Fas II bundle, and sometimes there is a little space between them (suggesting another axon or two interposed. This staining pattern was used to infer that the Ap axons run in a medial axon pathway just lateral to the medial Fas II tract (Simpson, 2000b).

When these axons ectopically express Robo2 under control of Apterous-GAL4 (Ap-GAL4), they move laterally and extend anteriorly in a specific location between the intermediate and the lateral Fas II pathways. In fact, they extend in the medial-most region of the endogenous Robo2 expression zone. The Ap axons from neighboring segments appear to pick the same lateral pathway, and to fasciculate together as they extend anteriorly from segment to segment. Two different GAL4 reporters were tested. Both drive different levels of Robo2 expression as indicated by their different strengths of pan-neural gain-of-function phenotypes. Nevertheless, both drive the Ap axons to the same lateral location. There is a second argument that supports this same conclusion. It is well known that the GAL4 expression system drives different levels of expression from a UAS transgene in different cells and segments of the same embryo. Yet, from cell to cell, segment to segment, and embryo to embryo, the Ap axons (expressing what is presumed to be variable levels of Robo2) always turn anteriorly in the same lateral location between the intermediate and lateral Fas II pathways (Simpson, 2000b).

This suggests that Robo2 reading of the Slit gradient drives axons to a rough lateral position, regardless of the precise level of Robo2, after which local cues determine which specific pathway a particular axon joins. Thus, Robo2 drives the Apterous axons to the lateral third of the scaffold, and regardless of the precise level of Robo2, this is sufficient to allow their final pathway choice to be precisely and uniformly dictated by some unknown but specific local cue. All of these experiments are done at different Robo2 levels within a relatively narrow range. It is conceivable that much higher levels of Robo2, or Robo3, might drive axons further laterally, even into more lateral zones (Simpson, 2000b).

Using the same Ap-GAL4 transgene to drive overexpression of Robo3 in the Ap axons leads to a different alteration in lateral position. Whereas ectopic Robo2 drives these axons quite far laterally to a position between the intermediate and lateral Fas II pathways, the ectopic expression of Robo3 drives them to an intermediate position, just medial to the intermediate Fas II pathway. Thus, Robo3 and Robo2 can both drive the medial Ap axons to more lateral positions, but they do so to different extents, as might be expected by their normal patterns of expression. Robo2 drives axons further laterally than does Robo3, relative to the intermediate Fas II bundle (Simpson, 2000b).

Using the same Ap-GAL4 transgene to drive overexpression of either Robo or the hyperactive Robo Y-F in the Ap axons leads to no alteration in lateral position. Even with more Robo, these axons still continue to extend in their normal medial location. These axons, and for that matter all longitudinal axons, normally express Robo, and it is inferred from this result that increasing the level of Robo does not alter the choice of lateral position by typical follower growth cones. The choice of lateral position by these axons is exquisitely sensitive to the presence of Robo2 and Robo3, but apparently not to the level of Robo. The ability of Robo2 to lateralize these axons does not depend on the presence of Robo: lateralization occurs in neurons that coexpress transgenic Robo2 and a Robo dominant negative receptor, or when Robo2 is expressed ectopically in a robo null mutant background (Simpson, 2000b).

The role of the Robo code in determining lateral position, and the potential interplay of this long-range guidance system were further analyzed using the presumptive Slit gradient with other local cues. Are all medial axons driven to the same lateral positions by Robo3 and Robo2? Or, alternatively, are they driven to cell-specific lateral positions? If the latter is the case, are their other cues that help predict the specific location? To this end, other GAL4 lines were used to drive the expression of various Robo family members in other subsets of axons (Simpson, 2000b).

The 15J2-GAL4 line drives expression in the dMP2 and vMP2 neurons (and variably in a few other neurons). These two neurons normally express Fas II, and normally extend in the medial Fas II pathway. Ectopic expression of Robo2 in these neurons leads to a bimodal phenotype. The dMP2 and vMP2 axons always appear to extend in a Fas II pathway, but they now pick either the intermediate or lateral Fas II pathways. These axons are never found medially, are often found in the intermediate Fas II pathway, and occasionally are found in the lateral Fas II pathway. These are distinctly different locations from where the Ap neurons are driven by Robo2 expression. It is sometimes difficult to determine which pathway the dMP2 and vMP2 axons are in (i.e., intermediate vs. lateral) because the two pathways intertwine in these gain-of-function embryos. Comparing the two experiments, Robo2 drives the Ap neurons to a non-Fas II pathway, while it drives dMP2 and vMP2 (which normally follow the medial Fas II pathway) into either the intermediate or lateral Fas II pathway (Simpson, 2000b).

It is concluded that precise topography requires more than just the Robo code. Robo3 and Robo2 expression define specific lateral regions. Ectopic Robo3 drives axons into the intermediate region, while ectopic Robo2 drives them even further laterally. But axons respond in a cell-specific fashion. Ectopic Robo2 drives the three Ap axons to between the intermediate and lateral Fas II pathways, while it drives the dMP2 and vMP2 axons into either the intermediate or lateral Fas II pathway. The control of location appears irrespective of level, since the result is consistent in spite of the different levels of expression generated by different Robo2 reporter lines, and by the variability in expression as driven with the GAL4 system. How can this precision be explained (Simpson, 2000b)?

Many models for topographic specificity involve the notion of two opposing gradients, either both of the same sign (i.e., both either attractive or repulsive) in the opposite orientation, or both of different signs in the same orientation. Such models are very attractive to explain certain aspects of sensory maps in the brain. However, such models need not apply to all topographic projections. Thus far, no evidence has been found for a second gradient working in concert with the repulsive Slit gradient. The most parsimonious model is that precise topography in the medial-lateral axis of the Drosophila CNS requires two opposing forces: long-range repulsion and short-range attraction. Cues might exist, for example, that mark the boundary of the neuropil. But in terms of location with the neuropil, all that is required is an opposing force to the Slit gradient -- it need not be a long-range gradient itself. Discrete local cues would be sufficient. Clearly, the long-range repulsion is controlled by the Slit gradient and the Robo code. It is proposed that the opposing force is short-range attraction as controlled by discrete local cues, one of which is Fasciclin II. In this way, the Robo code specifies the lateral region, while local cues specify precise location within that region (Simpson, 2000b).

The strongest support of this model involves the specification of the three major Fas II pathways. Fas II is a homophilic cell adhesion molecule expressed on axons that fasciculate together in three major longitudinal pathways: one medial, one intermediate, and one lateral. Growth cones expressing Fas II and Robo pick the medial Fas II pathway. Growth cones expressing Fas II, Robo3, and Robo pick the intermediate Fas II pathway. Presumably, the attraction of the medial Fas II pathway is insufficient to balance the repulsion mediated by Robo3. Growth cones expressing Fas II, Robo2, Robo3, and Robo pick the lateral Fas II pathway. In this case, it is not until they contact the lateral Fas II pathway that the Fas II-mediated attraction is stronger than the Robo2-mediated repulsion. Removal of Robo3 leads to only two Fas II pathways in which the intermediate pathway is missing, and instead the medial pathway is twice as thick. Ectopic expression of Robo2 in the dMP2 and vMP2 neurons, which normally extend in the medial Fas II pathway, drives their axons into either the intermediate or lateral Fas II pathway. Specificity is determined by the combination of Fas II and the particular Robo family members (Simpson, 2000b).

It is proposed that other pathways are specified by other pathway labels. For example, two pathways -- one medial and the other lateral -- express Connectin, another homophilic cell adhesion molecule. Growth cones expressing Connectin and Robo pick the medial Connectin pathway, while growth cones expressing Connectin and Robo2 (and presumably Robo and Robo3) pick the lateral Connectin pathway. Removal of Robo2 leads to only one fused medial Connectin pathway (Simpson, 2000b).

How do Robos read and respond to the Slit gradient? How are Robo3 and Robo2 different from Robo? How do Robo3 and Robo2 specify lateral position? Why does Robo3 drive axons into the intermediate region, while Robo2 drives them into the lateral region? Robo3 and Robo2 must differ from one another in either their ectodomains (and thus have different abilities to read the Slit gradient), or in their cytoplasmic domains (and thus have different abilities to signal), or both. What are the key differences that allow them to drive axons to different lateral regions? Both of these receptors (Robo3 and Robo2) differ from Robo in some quality of their signaling, either having some additional output or missing some output found in Robo. Their cytoplasmic domains are quite different from Robo, but what differences are key for determining lateral position (Simpson, 2000b)?

It will be of interest to determine to what extent different chimeric receptors and mutated receptors can drive lateralization. Preliminary collaborative results suggests that it should be possible to separate the functions of the various ectodomains and cytoplasmic domains (Simpson, 2000b).

Selecting a longitudinal pathway: Robo receptors specify the lateral position of axons in the Drosophila CNS

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).


REFERENCES

Search PubMed for articles about Drosophila Robo2

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

Bravo-Ambrosio, A., Mastick, G., Kaprielian, Z. (2012) Motor axon exit from the mammalian spinal cord is controlled by the homeodomain protein Nkx2.9 via Robo-Slit signaling. Development 139: 1435-1446. PubMed ID: 22399681

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

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

Godenschwege, T. A., et al. (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. J. Neurosci. 22(8): 3117-3129. 11943815

Jaworski, A., Tessier-Lavigne, M. (2012) Autocrine/juxtaparacrine regulation of axon fasciculation by Slit-Robo signaling. Nat Neurosci 15: 367-369. PubMed ID: 22306607

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

Kuzin, A., Kundu, M., Ekatomatis, A., Brody, T. and Odenwald, W. F. (2009). Conserved sequence block clustering and flanking inter-cluster flexibility delineate enhancers that regulate nerfin-1 expression during Drosophila CNS development. Gene Expr. Patterns 9(2): 65-72. PubMed ID: 19056518

Mellert, D. J., Knapp, J. M., Manoli, D. S., Meissner, G. W. and Baker, B. S. (2010). Midline crossing by gustatory receptor neuron axons is regulated by fruitless, doublesex and the Roundabout receptors. Development 137(2): 323-32. PubMed Citation: 20040498

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

Rajagopalan, S., et al. (2000). Selecting a longitudinal pathway: Robo receptors specify the lateral position of axons in the Drosophila CNS. Cell 103: 1033-1045. PubMed Citation: 11163180

Santiago, C., Labrador, J. P., Bashaw, G. J. (2014) The Homeodomain Transcription Factor Hb9 Controls Axon Guidance in Drosophila through the Regulation of Robo Receptors. Cell Rep 7: 153-165. PubMed ID: 24685136

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

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

Simpson, J. H., et al. (2000b). Short-range and long-range guidance by Slit and its Robo receptors: A combinatorial code of Robo receptors controls lateral position. Cell 103: 1019-1032. PubMed Citation: 11163179

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

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

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


Biological Overview

date revised: 25 July 2014

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