Netrin-A and Netrin-B



The first expression of the two genes is observed at the cellular blastoderm stage and is restricted to the presumptive mesoderm. This expression persists through gastrulation and then fades. In the visceral mesoderm, NetA continues to be expressed very strongly, and NetB weakly. Expression of NetB also remains in small patches within the somatic mesoderm layer. From stage 12 onward patches of ectodermal cells that are likely primordia for the tracheal system express NetA. Cells in the dorsal vessel and of the stomatogastric nervous system express NETB. During stage 14, accumulation of NETB is apparent in imaginal disc primordia, including cells that will give rise to the eye-antennal, labial, wing, haltere, and genital discs. Muscles from both the dorsal and ventral muscle groups express NetB. NetA is expressed by dorsal muscles 1 and 2 and is also expressed in a dorsolateral stripe in the epidermis (Harris, 1996, Mitchell, 1996).

Both NetA and NetB are strongly expressed by midline cells during the initial period of commissure formation and axonogenesis in the ventral nerve cord. NetA is initially expressed at stages 12 and 13 by the two anterior pair of midline glia (MGA and MGM) and by the VUM neurons. The expression in the VUM cluster subsequently fades, while the anterior and middle pairs of midline glia continue to express NetA strongly throughout embryogenesis. In addition, a large pair of cells located posterior to the posterior commissure also stains strongly with NetA at this stage. These cells may be associated with the median neuroblast. In contrast with the wide expression of NETA at the midline, NetB is expressed in a more restricted pattern. The midline glia express NETB very strongly, but there is no evidence of strong expression in either the VUM cluster or the MNB cluster. NetB is expressed by many more neurons than NetA. In the peripheral nervous system motor axons over the dorsal and ventral muscle groups stain for NETB protein (Mitchell, 1996 and Harris, 1996).

In the ventral nerve cord of Drosophila most axons are organized in a simple, ladder-like pattern. Two segmental commissures connect the hemisegments along the mediolateral axis and two longitudinal connectives connect individual neuromeres along the anterior-posterior axis. Cells located at the midline of the developing CNS first guide commissural growth cones toward and across the midline. The first growth cones navigate toward the anterior most ventral unpaired median (VUM) cell and thus pioneer the prospective posterior commissure. Only when the posterior commissure is established, the anterior commissure forms. In later stages, midline glial cells, migrating toward the posterior, are required to separate anterior and posterior commissures into distinct axon bundles. The VUM neurons reside ventral to the posterior commissure and project in a characteristic axon-bundle to the anterior commissure. Migration of two midline glial cells occurs along these cell processes. To unravel the genes underlying the formation of axon pattern in the embryonic ventral nerve cord, a saturating ethylmethane sulfonate mutagenesis was conducted, screening for mutations that disrupt this process. Subsequent genetic and phenotypic analyses support a sequential model of axon pattern formation in the embryonic ventral nerve cord. Specification of midline cell lineages is brought about by the action of segment polarity genes. Five genes are necessary for the establishment of the commissures. Two gene functions are required for the initial formation of commissural tracts, in addition to the function of commissureless, the netrin genes, and the netrin receptor encoded by the frazzled gene. Over 20 genes appear to be required for correct development of the midline glial cells which are necessary for the formation of distinct segmental commissures (Hummel, 1999a).

Subsequent analysis has defined four sequential steps involved in commissure development. Initially, single minded, jaywalker, Egf receptor and slit are involved in the first step in midline formation: the formation of the anlage of the CNS midline. Next the segment polarity genes hedgehog, engrailed, patched and wingless are involved in the specification of midline cell number. It is possible that midline and ectodermal pattern formations occur at the same time. In addition to the segment polarity genes other signaling mechanisms appear important. Notch, for example, is required to specify the different midline lineages. The third step in commissure formation consists of the formation of commissures. Once the midline cells have been specified, they guide commissural growth cones toward and across the midline. Here, the Netrins, frazzled, commissureless, weniger, schizo, roundabout and karussel play an essential role. The fourth step in commissure development involves the separation of the commissures. Contrary to midline specification and initial commissure formation, this process occurs relatively late during embryogenesis and thus a maternal contribution is not likely to rescue a mutant phenotype. In addition, the separation of commissures requires not only the differentiation of the midline glial cells but also the differentiation of the midline neurons as well as interactions of these two cell types for normal migration to occur. This might explain the large number of genes identified (Hummel, 1999a).

The analysis of mutations reveals two major phenotypic classes: the pointed and the tramtrack groups. pointed and tramtrack mediate different aspects of glial development. In pointed mutants no glial differentiation occurs, whereas ectopic pointed expression results in ectopic glial differentiation. tramtrack, in contrast, does not interfere with actual glial cell differentiation but appears to be required for the repression of neuronal differentiation in these cells. The pointed group consists of pointed itself, rhomboid, kastchen, klotzchen, kette, schmalspur, mochte gern, spitz, Star, cabrio and kubel. Mutations in eight other genes lead to an axon phenotype initially described for tramtrack. In tramtrack-type mutation (tramtrack, shroud, disembodied, spook, shade, shadow, phantom, and rippchen) commissures appear fused, but in contrast to pointed group mutations, connectives are not affected (Hummell, 1999a).

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

Independent of whether Netrin acts by a repulsive or attractive mechanism, evidence is provided that beside the Netrin/Frazzled (DCC) signaling system an additional attractive system(s) is operating in the developing embryonic nervous system of Drosophila. Attractive cues appear to be provided by the midline neurons. The genes schizo and weniger are likely to encode either additional components of the Netrin signaling system or define a second attractive guidance system. In order to obtain further insights in the function of these genes, several double mutant combinations were generated. If schizo or weniger act downstream in the netrin-frazzled pathway, no enhancement of the commissural phenotype would be expected, as compared to the frazzled deficiency phenotype. In embryos homozygous for a hypomorphic frazzled allele or mutant for schizo, only some commissural connections are missing. weniger mutant embryos have a penetrant CNS phenotype and all neuromeres are affected. However, embryos double mutant for frazzled and schizo lack most commissural axons. Similar synergistic effects are seen in frazzled/weniger or in schizo/weniger double mutant embryos. These double mutant analyses also indicate that axons crossing the midline in fra and netrin mutant embryos do not do so because of a loss of a repulsive Netrin signal. In the light of the synergistic effect seen in the frazzled/schizo double mutant it is suggested that, beside Netrin and its receptor, other proteins are required to guide commissural growth cones toward the midline. Furthermore, in the absence of two of the attractive signaling components, the existence is revealed of repulsive functions of the CNS midline. In the double mutant, the repulsive function predominates and directs axons out of the CNS (Hummel, 1999b).

What is the function of midline neurons in commissure formation? Attractive and repulsive signal originating from the midline are required for normal commissure development. The Drosophila midline comprises glial and neuronal cell lineages. These data indicate that these two cell types exert distinct functions during commissure formation. The first commissural growth cones invariably steer toward the anterior-most VUM neurons where these growth cones cross the midline to form the posterior commissure. This indicates that initially the midline neurons attract the commissural growth cones. The netrin genes that encode an attractive signal for commissural growth cones are expressed in midline neurons and glial cells during initial commissure formation. However, the number of commissural fibers is normal in mutations affecting the development of the midline glia. Similarly, ablation experiments using the directed expression of reaper and grim in the midline glial cells result in a fused commissure phenotype and do not lead to a reduction in the number of commissural axons crossing the midline. Thus, it is proposed that the midline glial cells do not play an essential role in attracting the commissural growth cones. The glial derived Netrin signal could be required to counteract repulsive signals. Additional support for the assumption that the midline neurons attract commissural growth cones is provided by the orthodenticle mutant phenotype. Here some midline neurons as well as one of the two segmental commissures is missing. Similarly, expression of dominant negative Jun in all midline cells results in a loss of midline neurons and a concomitant loss of all commissures. Furthermore, in patched mutant embryos the midline glial cells are almost absent and appear to be transformed into midline neurons. Attraction of commissural growth cones is normal in these embryos, however commissural axons stall at the midline. This suggests that the midline glial cells do not participate in attracting commissural growth cones but provide locally acting, contact dependent cues helping growth cones across the midline. Similarly, in the vertebrate neural tube, changes in growth cone morphology have suggested that commissural axons are guided by a contact dependent mechanism across the floor plate (Hummel, 1999b and references).

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

The Robo receptor is part of a larger family of receptors that includes robo2 and Robo3. This family of receptors have been found to be important for targeting axons to the appropriate longitudinal pathway in the embryonic CNS. Comm plays a key role in allowing contralaterally projecting neurons to cross the midline, and its ectopic expression (CommGOF) is known to robustly knock down Robo and Robo2 and 3. Comm was cell autonomously expressed in both lineage 15 subtypes and shifts to the midline were found in both 48 and 96 h AH neurons. For the 48 h AH neurons, Robo LOF data and CommGOF data are comparable, suggesting that Robo alone plays a major role in the positioning dendrites of these cells. In contrast, in the 96 h AH subtype RoboLOF and CommGOF effects were found to be significantly different, suggesting that the 96 h AH subtype may not only use the Robo receptor but additional Robos as well. Knockdown of Slit also supports this idea, as the branches of late-born neurons were occasionally found reaching the midline, something that was never see in RoboLOF clones. Thus, one way of establishing differences in the medio-lateral position could be through a dendritic “Robo code” where early-born cells express Robo and late-born cell express multiple Robo receptors (Brierley, 2009).

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

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

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

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

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

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

Pupal stage

Retinal axons in Drosophila make precise topographic connections with their target cells in the optic lobe. The role of the Netrins and their receptor Frazzled have been investigated in the establishment of retinal projections. The Netrins, although expressed in the target, are not required for retinal projections. Surprisingly, Frazzled, found on both retinal fibers and target cells, is required in the target for attracting retinal fibers, while playing at best a redundant role in the retinal fibers themselves; this finding demonstrates that target attraction is necessary for topographic map formation. Frazzled is not required for the differentiation of cells in the target. These data suggest that Frazzled does not function as a Netrin receptor in attracting retinal fibers to the target; nor does it seem to act as a homotypic cell adhesion molecule. The possibility is favored that Frazzled in the target interacts with a component on the surface of retinal fibers, possibly another Netrin receptor (Gong, 1999).

net A and net B are expressed in identical patterns: both transcripts are expressed in lamina precursors, which in wild type form an arc-shaped ribbon of cells. Thus, the Netrins are expressed in a pattern that would allow them to act as signals for incoming fibers. Fra protein, in contrast, is strongly expressed in photoreceptor axons, suggesting that retinal fibers have the ability to sense Netrin in the target. Interestingly, Fra is also expressed in the target structure, the lamina. fra transcripts are found in an arc-shaped band of cells similar to net transcripts, but double RNA in situ hybridizations reveal that fra and net transcripts do not colocalize to the same cells. Instead, fra transcripts are expressed in more mature lamina precursor cells located posteriorly adjacent to the net-expressing lamina precursor cells. While the transcript is only expressed very transiently, Fra protein expression persists and is thus present throughout the differentiated lamina and in all lamina cells (Gong, 1999).

What is the molecular function of Fra in the target cells? The fact that removal of both Netrins does not affect the retinal projection makes it unlikely that Fra functions as a Netrin receptor in the lamina target. Further, the fact that removal of Fra from the retinal fibers does not affect their projection, makes it unlikely that Fra functions as a homotypic cell adhesion molecule, directly effecting the attractive interaction between retinal fibers and their target cells. Given these findings, a third possibility is favored: Fra in the target cells may interact in a heterotypic fashion with an unidentified component on the surface of retinal fibers. It is possible that this component is another Netrin receptor. This idea is supported by the finding that Netrin misexpression in retinal fibers results in projection defects that phenotypically mimic the removal of Fra from the target, suggesting the presence in retinal fibers of another Netrin receptor in addition to Fra. The existence of additional Netrin receptors in the fly is expected. Apart from an UNC-5 type receptor, which has been found in both worms and vertebrates, a second DCC/UNC-40 homolog may also exist in the fly, based on genetic evidence that UNC40 function is partially redundant in the worm: molecular null alleles of unc40 display a less severe phenotype than some truncation alleles, suggesting that the truncated proteins interfere with a second pathway (Gong, 1999).

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Sequential axon-derived signals couple target survival and layer specificity in the Drosophila visual system

Neural circuit formation relies on interactions between axons and cells within the target field. While it is well established that target-derived signals act on axons to regulate circuit assembly, the extent to which axon-derived signals control circuit formation is not known. In the Drosophila visual system, anterograde signals numerically match R1-R6 photoreceptors with their targets by controlling target proliferation and neuronal differentiation. This study demonstrates that additional axon-derived signals selectively couple target survival with layer specificity. Jelly belly (Jeb) produced by R1-R6 axons was shown to interact with its receptor, Anaplastic lymphoma kinase (Alk), on budding dendrites to control survival of L3 neurons, one of three postsynaptic targets. L3 axons then produce Netrin, which regulates the layer-specific targeting of another neuron within the same circuit. It is proposed that a cascade of axon-derived signals, regulating diverse cellular processes, provides a strategy for coordinating circuit assembly across different regions of the nervous system (Pecot, 2014).

This study demonstrates that Jeb/Alk signaling regulates the survival of L3 neurons, one of several postsynaptic targets of R1-R6 neurons. Jeb is expressed in R1-R6 growth cones and acts at short range, prior to synapse formation, through the Alk receptor tyrosine kinase localized on budding L3 dendrites within the lamina neuropil. Jeb/Alk signaling is highly selective, as the survival of other R1-R6 postsynaptic targets (i.e., L1 and L2) is not affected when signaling is disrupted. This study also showed that, at a later stage of development, L3 growth cones produce Netrin within the medulla, which is required for the targeting of R8 growth cones to the M3 layer. It is speculated that a cascade of growth-cone-derived signals acting across different brain regions provides a general strategy for the assembly of neural circuits (Pecot, 2014).

In many regions of the developing nervous system, neurons are produced in excess, and significant cell death occurs after axons innervate their targets. In vertebrates, it is well established that target-derived neurotrophins, such as nerve growth factor, regulate neuronal numbers. These factors are produced by target neurons in limiting amounts and locally promote survival in a retrograde manner through receptors localized on axon terminals, providing a mechanism for matching the number of axons to targets. In recent years, diverse classes of molecules have been shown to control neuronal survival during development. Anterograde sources of trophic factors may also regulate survival, as denervation has been shown to induce excessive target neuron cell death. Indeed, several signals, including BDNF, are transported, in some contexts, in an anterograde manner within axons. In addition, the overexpression of BDNF in afferents can rescue cell death within the target field, and the disruption of BDNF through function blocking antibodies has been reported to decrease the number of target neurons within the rat superior colliculus. As BDNF may be produced by both axons and cells within the superior colliculus, it remains unclear whether endogenous axon-derived BDNF, and thus anterograde signaling, is required to regulate neuron survival (Pecot, 2014).

Although a role for target-derived retrograde trophic factors in vertebrate neural development was established many decades ago, trophic factors have only recently been shown to regulate neuronal development in Drosophila. Three Drosophila proteins, Neurotrophin 1, Neurotrophin 2, and Spatzle, are distantly related to vertebrate neurotrophins, and it has been shown that, like their vertebrate counterparts, they function as target-derived retrograde survival signals. Unlike their vertebrate homologs, however, which act through receptor tyrosine kinases, fly neurotrophins promote cell survival through Toll-like receptors (Pecot, 2014).

Although Jeb bears no significant homology to fly or vertebrate neurotrophins, Jeb acts through a receptor tyrosine kinase, Alk, which is distantly related to vertebrate neurotrophin receptors or Trks. Alk was originally identified as part of a fusion protein associated with large cell anaplastic lymphoma. Its role in mammals remains poorly understood. Drosophila Alk was initially found to regulate visceral mesoderm development through interaction with Jeb, and subsequently, Jeb/Alk signaling has been shown to regulate diverse cellular processes. Recent studies in vertebrates and Drosophila demonstrated that disrupting Alk function causes a decrease in the number of neurons. While in the vertebrate studies Alk's mechanism of action was not established, in Drosophila, Alk was shown to antagonize pathways that restrict neurogenesis under conditions of nutrient deprivation. Whether Jeb and Alk regulate neuronal survival in contexts outside of L3 development is not known, although Alk is widely expressed in the developing visual system, and Jeb is expressed by several populations of neurons, in addition to photoreceptors (Pecot, 2014).

The cellular specificity of the Jeb/Alk requirement is particularly surprising. Indeed, at all R1-R6 synapses containing L3 postsynaptic elements, L1 and L2 neurons each contribute a single postsynaptic element juxtaposing the same presynaptic site on R cell axons. In the absence of Jeb/Alk signaling, however, only L3 neurons die. The mechanisms that underlie this selectivity are not known. Alk is broadly expressed in the lamina, suggesting specificity may be controlled at the level of downstream signaling or that other trophic signals act redundantly with Jeb to control L1 and L2 survival. Collectively, the findings reported in this study demonstrate that anterograde Jeb/Alk signaling acts selectively to control L3 survival, providing direct evidence that anterograde signaling regulates target neuron survival in vivo (Pecot, 2014).

Several lines of evidence indicate that signaling between Jeb, expressed by R1-R6 growth cones, and Alk, localized to budding L3 dendrites, controls L3 survival between 20-40 hr APF. First, Alk mutant L3 neurons, or wild-type L3 neurons innervated by jeb mutant R1-R6 axons, die between 20-40 hr APF. Second, R cell populations containing only R1-R6 neurons are sufficient for L3 survival. Third, Alk and Jeb are expressed in a complementary fashion at the appropriate time on budding L3 dendrites and R1-R6 growth cones, respectively. And finally, L3 degeneration begins within budding L3 dendrites juxtaposed to R1-R6 growth cones. The temporal requirement for Alk/Jeb signaling corresponds to a critical and fascinating phase of lamina circuit assembly (Pecot, 2014).

R1-R6 growth cones form connections with lamina neurons in three discrete steps. First, R1-R6 growth cones from the same ommatidium associate with a single cartridge of differentiating lamina neurons. Second, through a highly stereotyped reassortment process occurring between 24-38 hr APF, these six growth cones diverge from one another and project locally to six different developing cartridges. As a consequence of this rearrangement, the R1-R6 cells that 'see' the same point in space form connections with L1, L2, and L3 neurons within the same cartridge. And third, R1-R6 then commence synapse formation at 45 hr APF, and this process continues until eclosion (~96 hr). Thus, L3 death in jeb and Alk mutants occurs prior to synapse formation, during the process of R1-R6 growth cone rearrangement. The suppression of L3 death by expression of the caspase inhibitor p35 argues that during normal development Jeb/Alk signaling acts to inhibit caspase activity. Which caspases contribute to L3 death, and whether caspases antagonize other cellular processes necessary for wiring, is not known. Regardless of how Jeb/Alk signaling functions at the molecular level, it acts to ensure that visual input from R1-R6 neurons is transmitted to the L3 pathway (Pecot, 2014).

These findings and the work of others suggest a logic underlying neural circuit assembly within the Drosophila visual system. The retina, lamina, and medulla are distinct yet interconnected regions comprising columnar modules (i.e., ommatidia, cartridges, and columns, respectively) that are matched topographically between each region. Within each module, intrinsic mechanisms and intercellular interactions control cell fate determination. For instance, R8 neurons provide a discrete locally acting signal to induce R7 development in the developing retina, while in the medulla, Notch/Delta interactions between daughter cells generated from the same ganglion mother cell promote acquisition of distinct cell fates. Superimposed upon these interactions are axon-derived signals that coordinate development between matched modules from different regions. Together, these mechanisms organize the assembly of columnar units in multiple regions (i.e., super columns), each processing visual information captured from a discrete region of the visual field. Indeed, the modular assembly of these super columns spanning different regions of the visual system reflects the function of these circuits in the parallel processing of visual information (Pecot, 2014).

R cell growth cones produce signals that regulate diverse cellular processes in the developing lamina. Hedgehog drives lamina neuronal precursors through their final division; cell adhesion proteins promote the association of columns of lamina neurons with R cell axon fascicles; EGF induces lamina neuron differentiation; a yet-to-be-identified signal regulates the development of lamina glia; and Jeb selectively regulates L3 survival. Thus, axon-derived signals act at multiple levels and in a cell-type-specific manner to regulate target development (Pecot, 2014).

Axon-derived signals also coordinate circuit assembly across topographically matched modules. Within medulla columns, L3 growth cones produce Netrin in the M3 layer, which controls the targeting of R8 growth cones to M3. Importantly, Netrin production by L3 occurs after Jeb, released from R1-R6 cells in topographically corresponding lamina cartridges, promotes L3 survival. Thus, Netrin indirectly relies upon prior Jeb signaling. As the L3 and R8 axon terminals within each medulla column transmit information captured from the same point in space to the same layer (M3) and share several postsynaptic targets, the developmental mechanisms giving rise to this circuit may reflect functional relationships between these neurons. Thus, signals produced by axons coordinate assembly of circuits between different brain regions (Pecot, 2014).

It is envisioned that intercellular signaling cascades, analogous to what are described in this study, organize other circuit modules in the fly visual system [e.g., ON (L1) and OFF (L2) circuits] comprising different cell types. As many regions of the vertebrate nervous system, including the neocortex, spinal cord, and retina, are also arranged in a hierarchically repetitive fashion, this raises the intriguing possibility that similar strategies may coordinate the development of these structures (Pecot, 2014).

Effects of Mutation or Deletion

Deletion of both NetA and NetB gives rise to thinner and sometimes absent commissures and occasional breaks in the longitudinal tracts. The posterior commissure is more severly affected that the anterior commissure. The commissural phenotype is rescued by either NetA or NetB in transgenic flies. Overexpression of NetB throughout the CNS results in thinner commisures than normal, similar to the phenotype observed in deficiency embryos. This suggests that commissural axons are either indifferent to midline Netrin owing to uniform levels throughout the CNS, or are instead now attracted to axons or cell bodies lateral to the midline. Ectopic expression of NetB in all muscles causes the intersegmental nerve to wander, branch, and sometimes stall short of its dorsal targets (Harris, 1996, Mitchell, 1996).

To assess the role of repulsion by Netrins and Unc-5 in shaping motor axon pathways, the development of these trajectories was examined in Df(1)NP5 embryos, in which both the NetA and NetB genes are deleted. For this, the general motor axon marker MAb 1D4 and anti-Unc5 were used. No abnormalities were detected in the SNa and SNc projections in these embryos. The lateral migration of peripheral and exit glia, visualized with anti-Repo antibodies, also appears normal in Netrin-deficient embryos. Double-stranded Unc5 RNA was injected into wild-type embryos in an attempt to specifically disrupt Unc5 function by RNA-mediated interference (RNAi). Although this resulted in a strong reduction in Unc5 staining, MAb 1D4 did not reveal any misrouting of SN motor axons in these embryos. Thus, while the expression data suggest a role for Unc5 in repelling SN motor axons out of the CNS and away from Netrin-expressing muscles, the genetic data indicate that repulsion by Netrins is likely to be just one of multiple guidance forces that control these projections (Keleman, 2001).

However, SNa motor axons can be repelled by Netrins. If NetB is ectopically expressed on all muscles using a 24B-GAL4 driver and a UAS-NetB transgene, SNa axons often stall at the edge of the CNS or fasciculate with the ISN (Mitchell, 1996). Does this gain-of-function phenotype depend on Unc5 function? To test this, Unc5 double-stranded RNA was injected into 24B-GAL4/UAS-NetB embryos. In control embryos that were either uninjected or injected with buffer alone, SNa motor axons fail to enter their lateral muscle target region in 54% or 57% of hemisegments, respectively. In contrast, this phenotype is seen in only 14% of hemisegments in Unc5 RNAi embryos. These data establish that SNa motor axons do indeed sense Netrin as a repulsive signal acting through the Unc5 receptor (Keleman, 2001).

The Drosophila ARF6-GEF Schizo controls commissure formation by regulating Slit: Genetic interaction with the attractive cue Netrin

The CNS of bilateral symmetric organisms is characterized by intensive contralateral axonal connections. Genetic screens in Drosophila have identified only a few genes required for guiding commissural growth cones toward and across the midline. Two evolutionarily conserved signaling molecules, Netrin and Slit, are expressed in the CNS midline cells. Netrin acts primarily as an attractive signaling cue, whereas Slit mediates repulsive functions. A detailed analysis is provided of the Drosophila gene schizo, which is required for commissure formation. schizo leads to a commissural phenotype reminiscent of netrin mutant embryos. Double-mutant analyses indicate that Netrin and Schizo act independently. The schizo mutant phenotype can be suppressed by either expressing netrin in the CNS midline cells or by a reduction of the slit gene dose, indicating that the balance of attractive and repulsive signaling is impaired in schizo mutants. Overexpression of the schizo RNA in the CNS midline using the GAL4/UAS system leads to a slit phenocopy, suggesting that schizo primarily antagonizes Slit signaling. This is further supported by cell type-specific rescue experiments. The schizo gene generates at least two proteins containing a conserved Sec7 and a pleckstrin homology domain (PH) characteristic for guanine nucleotide exchange factors (GEF) acting on ARF GTPases, which are known to regulate endocytosis, In support of the notion that schizo regulates Slit expression via endocytosis, it was found that blocking endocytosis leads to a schizo-like phenotype. It is thus proposed that the balance of the two signaling cues Netrin and Slit can be regulated, controlling membrane dynamics (Önel, 2004).

Only four zygotically active genes were found in a screen for mutations affecting commissure formation (frazzled, weniger, schizo and the netrin gene complex). Two EMS-induced schizo mutants (schizoC1-28 and schizoU112) were initially identified. Subsequently two P-element induced schizo alleles (schizol(3)3 and schizoP244) were identified. All these alleles led to a reduction in the number of commissural fibers crossing the CNS midline. Interestingly, the anterior commissures were affected more prominently. Not all neuromeres were equally affected and the strongest defects were generally observed in abdominal segments A1-A4. All CNS midline cells formed in normal number in the absence of schizo function. However, as generally observed in mutants affecting formation of commissures, the midline glial cells migrated out laterally along the few remaining commissural fibers. In addition to the commissural phenotype, defects in the longitudinal connectives were noted (Önel, 2004).

The most prominent function of schizo is its role in commissure development. Two major signaling cascades are known to control axonal growth across the midline. They are initiated by the signaling molecules Netrin and Slit, which are both secreted by the CNS midline glial cells in the Drosophila embryo. First genetic interaction studies of schizo and frazzled or schizo and netrin function demonstrate a much stronger commissural phenotype in double mutants than embryos mutant only for schizo, frazzled or netrin. The commissural phenotypes of the double-mutant embryos suggest that schizo is not acting within the Netrin signaling pathway but may be required for a Netrin-independent attractive pathway. Alternatively, schizo may be necessary for suppressing the perception or the generation of a repulsive signal normally generated by the CNS midline cells (Önel, 2004).

The main axonal repulsive signal is encoded by slit. Slit is an LLR protein secreted by the CNS midline glial cells. schizo function appears to be required to downregulate repulsive signaling, either by affecting the generation of active Slit protein or by preventing signaling in the commissural growth cones; the mutant schizo phenotype could be explained by an upregulated Slit signaling. Thus, one might expect that the schizo commissural phenotype could be suppressed by a concomitant reduction in the dose of slit function. slit-/+; schizo–/– as well as robo-/+; schizo–/– embryos were generated, and in both cases a suppression of the schizo CNS phenotype was observed. Thus schizo might be required to negatively regulate Slit signaling (Önel, 2004).

If schizo is indeed a negative regulator of slit function, an increase of schizo gene dose should result in a decrease of active Slit signaling. One might thus be able to enhance the mutant slit phenotype by using a schizo gene duplication. Following mapping of schizo to the base of the left arm of chromosome 3 a chromosomal translocation of the corresponding part of the third chromosome to the Y chromosome (Tp(3;Y)A81) was used to generate embryos with three copies of schizo. In an otherwise wild-type background, this triplication of the schizo region did not result in an abnormal CNS phenotype. However, when the schizo translocation was placed in a heterozygous slit–/+ background, a slit-like phenotype was observed that was never detected in heterozygous slit embryos (Önel, 2004).

schizo maps to chromosome region 78A/B between the genes poils aux pattes and knockout. To identify the schizo gene in this chromosomal interval P-element-induced schizo alleles were used. The chromosomal insertions of the P-elements in l(3)3 and P224 were determined by inverse PCR and Southern analyses and the results suggested that schizo corresponds to CG32434. The lethality associated with the P-element-induced l(3)3 schizo mutation could be reverted by precise excision of the P-element. Mutant l(3)3 embryos displayed a schizo phenotype with reduced commissures and defective fasciculation in the longitudinal connectives. Subsequent sequencing of cDNA clones LP01489, RE44556 and GH10594 isolated by the BDGP showed that the schizo locus encompasses 41 kb of genomic DNA. At least two different promoters direct the expression of two isoforms of 1325 amino acids (SchizoP1) and 1313 amino acids (SchizoP2) in length. Verification of the cloning of schizo was obtained by genetic rescue experiments. These deduced schizo proteins correspond to the Iso1 and Iso2 variants of the loner gene, which was recently identified in a screen for mutations affecting mesoderm development. By contrast Chen (2003) GH10594 was found to be entirely contained within the LP01489 sequence and no evidence was found for a third schizo protein isoform (Önel, 2004).

The deduced Schizo proteins share three conserved sequence modules. In the N-terminal region there is a so-called IQ domain, which is predicted to interact with calmodulin. Within the C-terminal third of the protein a Sec7 domain is directly adjacent to a PH-domain. Proteins characterized by such a domain signature are generally acting as guanine nucleotide exchange factors (GEFs). The Anopheles homolog is about 90% identical. The closest human homologs are EFA6, being 32% identical to Schizo, lacking the IQ domain, and ARF-GEP100 showing a 40% identity to Schizo. Both human proteins were shown to act as ADP ribosylation factor 6 (ARF6)-GEFs suggesting that schizo might have a similar function (Önel, 2004).

The molecular identification of schizo allowed for the determination of the expression pattern throughout development. schizo expression is already detected in the unfertilized egg, indicating a prominent maternal contribution. schizo expression stays almost uniform until the end of stage 10. Within the developing nervous system, expression can be noted in the CNS midline cells. In addition, schizo expression can be detected in the epidermis and the visceral mesoderm (Önel, 2004).

The commissural schizo phenotype does not allow the deduction of cell type in which Schizo normally acts. To test the cell-type requirement the GAL4 system was used and UAS-schizoP1 and UAS-schizoP2 transgenic flies were established. Expression of the different schizo proteins was directed in the CNS midline cells of mutant schizo embryos using the sim-GAL4 or sli-GAL4 driver strains. In both cases expression could rescue the schizo mutant CNS phenotype indicating that Schizo acts in the midline glial cells, which express both Slit and Netrin (Önel, 2004).

Genetic data indicate that schizo impairs Slit signaling in the CNS. This was further supported by overexpression of schizo. Whereas expression of schizo (P1 or P2) in all CNS midline cells of wild-type embryos did not evoke an abnormal phenotype, the same expression of schizo in heterozygous slit mutant embryos was able to induce a mild slit phenocopy. These results were similar to the ones obtained using chromosomal translocations, supporting the notion that Schizo acts in the CNS midline by regulating the level of Slit expression (Önel, 2004).

To reduce Slit activity, schizo might suppress exocytosis of Slit-containing vesicles, or it might promote endocytosis of Slit-containing vesicles from the membrane. Work from vertebrate tissue culture models has shown that Arf-GEFs such as Schizo can activate endocytosis. To test whether endocytosis might be relevant for commissure formation a dominant negative Shibire protein was expressed; this efficiently blocks endocytosis specifically in the CNS midline cells using the sim-GAL driver. In about 60% of such embryos a schizo phenocopy was observed. When higher levels of the dominant negative Shibire protein were expressed using the rho-GAL driver all embryos developed a schizo phenocopy, suggesting that endocytosis participates in the regulation of Slit function in the CNS midline cells. To further support the notion that Schizo induces endocytosis of Slit, the negative Shibire protein was expressed in a heterozygous slit mutant background. This indeed led to clear suppression of the Shibire-induced phenotype. Schizo and its vertebrate homologs exert at least part of the function through the small GTPase Arf6. Arf6 mRNA is supplied maternally and is expressed ubiquitously during embryonic development. To determine whether Schizo acts via Arf6 to control endocytosis of Slit by the midline glial cells, a dominant negative Arf6 construct (Chen, 2003) was expressed. Following expression in the midline cells using the sim-GAL driver no mutant phenotype was observed. Following expression of higher levels of Arf6DN using the rho-GAL driver, about 15% of the embryos developed a schizo phenocopy, supporting the notion that Schizo acts via Arf6-regulated endocytosis to control the level of Slit expression on the midline glial cells (Önel, 2004).

Thus genetic and molecular data support a model in which schizo negatively regulates the expression of Slit in the CNS midline cells. This study shows that schizo acts in a rather similar way to commissureless; however, rather than affecting the Roundabout receptor, schizo appears to act on the expression of the Slit ligand. First it was found that the triplication of the schizo gene interfers with slit function and that reduction of slit expression in schizo mutant embryos rescues the schizo mutant phenotype. Finally, expression of a schizo transgene in the Slit-expressing CNS midline cells (1) was able to rescue the schizo mutant phenotype and (2) could induce a slit phenocopy when expressed in wild-type embryos. The deduced nature of the Schizo protein suggests that it affects Slit expression by post-transcriptional mechanisms (Önel, 2004).

Guanine-nucleotide exchange factors (GEFs) help to convert the inactive GDP-bound form of small GTPases into a GTP-bound active form. Schizo is a new Sec7 domain containing GEF, which shows 40% homology to human Arf-GEP100. Arf-GEP100 localizes to endosomal membranes and promotes GDP/GTP exchange on ARF6. The small GTPase ARF6 is a plasma membrane-localized protein and functions in the regulation of membrane ruffling, cell motility, aspects of endocytosis and exocytosis, membrane recycling, reorganization of the cortical actin cytoskeleton and activation of phospholipase D. In Drosophila, Arf6 is remarkably well conserved, being more than 96% identical to the human counterpart (Önel, 2004).

One aspect that might hint at how Schizo regulates Slit expression is the role of ARF6 in endocytosis and exocytosis. The function of ARF6 in endocytosis is twofold. It either regulates clathrin-mediated endocytosis at the apical surface of polarized epithelial cells or it is able to regulate non-clathrin-mediated endocytosis and the recycling pathway in non-polarized cells. ARF6 has also been postulated to play a role in Ca2+-activated dense core vesicle (DCV) exocytosis by regulating phosphatidylinositol(4,5) biphosphate (PIP2). Overexpression of a UAS-ARF6 construct in midline glia cells does not result in a schizo-like phenotype, whereas expression of a dominant negative form of Arf6 results in a phenocopy of several phenotypes associated with the schizo mutant (Chen, 2003). This suggests that Arf6 might also be involved in the regulation of Slit expression (Önel, 2004).

In-vivo Slit and Netrin are both expressed by the same CNS midline cells and their expression needs to be in an intricate balance. The importance of this balance and not the individual expression levels is highlighted by the fact that it is possible to rescue the schizo mutant phenotype by both increased Netrin expression or reduced Slit expression. Within the midline glia, however, Schizo appears to primarily affect Slit expression either by inducing its endocytosis and subsequent degradation or by blocking exocytosis and thus release of Slit (Önel, 2004).

The latter case would suggest that Slit and Netrin are brought to the membrane of the midline glial cells in distinct vesicle populations, whereas the former case would require a specific membrane receptor for the Slit protein expressed by the CNS midline glia. Given the fact that the secreted Slit protein is found at very high levels at the midline glial cell membrane, this appears probable. Moreover, expression of a dominant negative Shibire protein in the midline glia leads to a schizo phenocopy. shibire encodes the Drosophila dynamin and is required for endocytosis and a block of shibire function leads to a block of endocytosis, which might result in higher levels of Slit expression. Thus, regulation of membrane dynamics appears crucial in controlling the function of the signaling molecule Slit (Önel, 2004).

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

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

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

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

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

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

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

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

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

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

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

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

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

Netrins guide migration of distinct glial cells in the Drosophila embryo

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

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

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

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

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

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

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

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

The transmembrane receptor Uncoordinated5 (Unc5) is essential for heart lumen formation in Drosophila melanogaster

Transport of liquids or gases in biological tubes is fundamental for many physiological processes. Knowledge on how tubular organs are formed during organogenesis and tissue remodeling has increased dramatically during the last decade. Studies on different animal systems have helped to unravel some of the molecular mechanisms underlying tubulogenesis. Tube architecture varies dramatically in different organs and different species, ranging from tubes formed by several cells constituting the cross section, tubes formed by single cells wrapping an internal luminal space or tubes that are formed within a cell. Some tubes display branching whereas others remain linear without intersections. The modes of shaping, growing and pre-patterning a tube are also different and it is still not known whether these diverse architectures and modes of differentiation are realized by sharing common signaling pathways or regulatory networks. However, several recent investigations provide evidence for the attractive hypothesis that the Drosophila cardiogenesis and heart tube formation shares many similarities with primary angiogenesis in vertebrates. Additionally, another important step to unravel the complex system of lumen formation has been the outcome of recent studies that junctional proteins, matrix components as well as proteins acting as attractant and repellent cues play a role in the formation of the Drosophila heart lumen. This study show the requirement for the repulsively active Unc5 transmembrane receptor to facilitate tubulogenesis in the dorsal vessel of Drosophila. Unc5 is localized in the luminal membrane compartment of cardiomyocytes and animals lacking Unc5 fail to form a heart lumen. These findings support the idea that Unc5 is crucial for lumen formation and thereby represents a repulsive cue acting during Drosophila heart tube formation (Albrecht, 2011).

The cross section of the Drosophila heart tube is constituted by opposing cardiomyocytes that cover the periphery of an inner luminal space. At the dorsal and ventral side of two facing cardiomyocytes, adherens junctions seal the lumen. Work of several laboratories recently raised evidence that the secreted extracellular matrix component Slit and its receptor Robo1/2, are specifically co-expressed at the luminal side of cardiomyocytes and provide essential cues for the process of heart lumen formation (Albrecht, 2011).

This study demonstrates that the Drosophila single-pass transmembrane receptor Unc5, together with its ligand Netrin, ensures heart lumen formation. Unc5 represents the single Drosophila homologue of a conserved receptor family, exhibiting an extracellular domain consisting of two Ig domains (both of which are essential for Netrin binding as shown for the C. elegans and the human Unc5 orthologue) and two thrombospondin type I (TSP) repeats. The Ig domains of Unc5 share homology with the first two Ig domains of Robo, which are critical for binding its ligand Slit. The binding properties of the Unc5 Ig domains are specific, and Netrin binding therefore is unique to Unc5. Conserved motifs found within the intracellular domain of Unc5 are a ZU5 motif, a DB motif and a carboxy-terminal death domain. The three vertebrate Unc5 orthologs and the single known C. elegans orthologue exhibit a similar organization (Albrecht, 2011).

Expression of Unc5 and NetrinB in the heart has been described but not connected to cardiogenesis. Therefore an analysis was performed of in which cells that constitute the Drosophila heart, Unc5 and NetrinB are expressed. Transcripts of unc5, Unc5 protein and NetrinB protein were found in all cardiomyocytes forming the cardiac tube. Because no netrinB mRNA can be detected in heart cells, the hypothesis arises that NetrinB is synthesized by non-cardiac cells and transported actively or passively to its target site as a soluble protein. Since all other proteins, known to be involved in heart lumen formation (for example Slit, Robo, Dg, Arm, DE-Cadherin), are expressed as autocrine factors by cardiac cells themselves, the Unc5/NetB system likely represents a new mode of tube size regulation during Drosophila cardiogenesis. Both proteins accumulate in a polar fashion at the luminal surface of the cardiomyocytes. From stage 15 onwards, when the two bilateral cardiac primordia migrate towards the dorsal midline, Unc5 and NetrinB appear to be exclusively enriched at the prospective luminal and the abluminal side. Transverse Z views of cardiac tubes, stained for Unc5 and specific polarity markers revealed a highly enriched localization of Unc5 in the luminal compartment of cardiomyocyte membranes. Unc5 is excluded from the junctional domains that are responsible for sealing the lumen of the heart. The same distribution is seen for NetrinB. Unc5 and NetrinB are clearly co-localized in the same cells and in the same membrane compartment. This observation argues for a cell autonomous function of the proteins Unc5 and NetrinB (Albrecht, 2011).

The most prominent cardiac defect seen in homozygous unc5 or netrin mutant animals is the lack of a heart lumen. Additionally, frequently defects were found in the alignment of cardiomyocytes along the anterior–posterior axis. In contrast to mutants of the Slit/Robo pathway, defects were never observed that can be clearly attributed to early adhesion functions or a potential role of Unc5 for specifying the polarity of the cells. Therefore the defects seen in unc5 mutants are interpreted as being specific for a late role in tubulogenesis rather than for a more general role in cell adhesion. This assumption is supported by the presence of a dorsal adherens zone between cardiomyocytes that demonstrates that a loss of Unc5 neither prevents the migration of cardioblasts from the cardiac primordium towards the midline nor the initial recognition and adhesion of cardioblasts at the dorsal midline. The cell shape of the cardiomyoblasts in unc5 mutants and in netrin deficient embryos however point to an affected remodeling process of the cytoskeleton which normally occurs during the process of lumen formation. But since downstream factors of the Netrin/Unc5 signaling cascade in Drosophila are yet not known, this issue has to be further analyzed in the future. Data focusing on Slit and Robo suggests that components regulating actin dynamics (like Enabled) may be effectors of this system during lumen formation. Work on potential interactors of the Unc5/Netrin pathway in C. elegans indicates that the Enabled homolog in worms, Unc-34, can function as a suppressor of ectopic Unc-5 during pioneer axon guidance. These observations recommend a closer inspection on the actin filament formation and actin distribution in unc5 mutants as well as an analysis of altered heart lumen formation in mutants of different cytoskeletal components or their regulators (Albrecht, 2011).

The overall polarity of the cardiomyocytes is not affected by the loss of Unc5. Even the highly specific sub-compartmental organization of the contact sides of the contralateral cardiomyocytes is correctly determined in unc5 mutants. Staining of unc5 mutant animals for junctional and luminal markers revealed that neither the localization of polarity markers nor the specification of the prospective J- and the L-domain is changed by the loss of Unc5 function (Albrecht, 2011).

These findings suggest that Unc5 is specifically required for lumen formation and the necessary processes of cell shape change rather than for general adhesion properties of the cardiomyocytes (Albrecht, 2011).

Lumen formation by cell assembly, as in the Drosophila heart, is mechanistically different from tubulogenesis in other tubular organs in which the luminal space is generated by budding, cell hollowing or wrapping. Slit and Robo1/2 are considered as being key components that define a luminal membrane compartment in cardiomyocytes and restrict the domains of junctional proteins that mediate the sealing of the heart tube. The findings indicate that the Unc5/Netrin receptor–ligand system acts at the matrix compartment of cardiomyocytes and promotes lumen formation (Albrecht, 2011).

It is proposed that Unc5/Netrin act independent of Slit and Robo on the establishment of a luminal space between the cardiomyocytes because Slit and Robo distribution is wild typic in unc5 mutant embryos. It is known from the literature that a loss of components of the Slit/Robo pathway, for example Robo itself, leads to an altered localization of Slit in the cardiomyocytes. In contrast, the loss of Unc5/Netrin function does not affect the localization of any tested protein involved in lumen formation (Albrecht, 2011).

These findings provide an independent proof for the lumen formation model. This model postulates that cell shape changes and repulsive activities at the prospective luminal membrane area, resulting in bending of the ventral side of cardiomyocytes are the driving forces for lumen formation. The extended dorsal J-domain in cardiomyocytes of unc5 mutant embryos, as well as the presence of adjacent luminal membrane compartments, exhibiting the characteristic matrix proteins of the cardiac luminal wall, argues strongly for this model. An alternative mechanism would be that cardiomyocytes first align throughout their entire luminal side and afterwards luminal space appears. This model is neither supported by the previous observations nor by findings presented in this. In summary, the data suggests the existence of a second repulsively active system present at the L-domain of cardiac cells expanding the list of key components known to be crucial for heart tubulogenesis (Albrecht, 2011).

Control of male and female fertility by the Netrin axon guidance genes

The netrin axon guidance genes have been implicated in fertility in C. elegans and in vertebrates. This study shows that adult Drosophila lacking both netrin genes, NetA and NetB, have fertility defects in both sexes together with an inability to fly and reduced viability. NetAB females produce fertilized eggs at a much lower rate than wild type. Oocyte development and ovarian innervation are unaffected in NetAB females, and the reproductive tract appears normal. A small gene, hog, that resides in an intron of NetB does not contribute to the NetAB phenotype. Restoring endogenous NetB expression rescues egg-laying, but additional genetic manipulations, such as restoration of netrin midline expression and inhibition of cell death have no effect on fertility. NetAB males induce reduced egg-laying in wild type females and display mirror movements of their wings during courtship. Measurement of courtship parameters revealed no difference compared to wild type males. Transgenic manipulations failed to rescue male fertility and mirror movements. Additional genetic manipulations, such as removal of the enabled gene, a known suppressor of the NetAB embryonic CNS phenotype, did not improve the behavioral defects. The ability to fly was rescued by inhibition of neuronal cell death and pan-neural NetA expression. Based on these results it is hypothesized that the adult fertility defects of NetAB mutants are due to ovulation defects in females and a failure to properly transfer sperm proteins in males, and are likely to involve multiple neural circuits (Newquist, 2013b).

This work characterizes the adult phenotypes of mutants lacking both the Drosophila Netrin genes. Previous work has identified locomotor and negative geotaxis defects in NetAB mutants that can be rescued in part by inhibition of apoptotic signaling or pan-neural expression of NetB (Newquist, 2013a). In this study focuses on the fertility defects of NetAB mutants and also describes viability and flight phenotypes. The results indicate that the Netrins are highly pleiotropic genes affecting multiple systems as have been observed in other species (Newquist, 2013b).

Flying requires advanced coordination between the two sides of the body, so it is not surprising that it is affected in NetAB mutants. The defects observed could be due to failure to innervate flight muscles or failures in interneuron connectivity in the CNS. Rescue of flight by pan-neural expression of the caspase inhibitor p35 indicates a neural origin for the phenotype. Flight was also rescued by pan-neural expression of NetA, which was surprising as previous work demonstrated that pan-neural expression of NetA increases axon guidance errors in the embryonic CNS (Newquist, 2013a). Nevertheless, pan-neural NetA expression can rescue the locomotor defects of NetAB flies suggesting that NetA expression may have a context dependent beneficial effect in a brain region controlling both behaviors such as the central complex (Newquist, 2013b).

NetAB males also display hyper-coordination of wing movements during the singing phase of courtship behavior. This behavior is strikingly similar to the mirror movements observed in human and mouse DCC Netrin receptor mutants. This phenotype was resistant to rescue by all the manipulations attempted, including restoration of one copy of endogenous NetB and midline NetA expression, suggesting that the behavior relies on the localized expression of NetA at a non-midline location. Alternatively, the behavior may rely on both NetA and NetB in independent roles (Newquist, 2013b).

NetAB females lay fewer eggs than wild type. The origin of this phenotype does not appear to be in oocyte or ovarian development. Ovulation is regulated at multiple levels including octopaminergic neurons innervating the reproductive tract, secretory cells in the spermathecae and parovaria glands as well as cell populations in the CNS. Disruption to any one or more of these systems could be sufficient to produce the reduced egg-laying seen in NetAB mutants. Drosophila females display a post-mating switch in which increased egg-laying and decreased mating receptivity is triggered by proteins in the male semen. The neuronal circuits stimulated by these proteins could also be disrupted in NetAB mutants. Transgenic manipulations focused on the classical roles for Netrins at the CNS midline as well as more recent roles as neuronal survival factors. None of these manipulations restored egg-laying, so it cannot be discerned whether there is a local disruption of signaling in the reproductive tract or a broader disruption of multiple cell populations (including non-neural tissues). Females have occasionally been observed in which an egg has been released from each ovary simultaneously, and these eggs appear jammed at the confluence of the common oviduct. The hypothesis is therefore favored that ovulation is disrupted by the NetAB mutations (Newquist, 2013b).

NetAB males induce reduced egg-laying in the females they mate with. Viable offspring are produced demonstrating that mating is successful, but the normal post-mating increase in egg-laying may be diminished or absent. This suggests that transfer of sperm proteins or seminal fluid (SSFT) may be disrupted. SSFT requires a male specific neural circuit, and the transfer of sperm can be genetically separated from seminal protein/fluid transfer by feminizing a small subset of cholinergic neurons (Acebes, 2004). Netrin mutations may be affecting one or more of these neural subsets with a deleterious effect on SSFT (Newquist, 2013b).

This discussion of the phenotypes observed has focused on the classical roles of Netrins in cell migration, neural connectivity and also survival. It is quite possible that the Netrins could be modulating neuronal activity in the adult animal, as the Netrin receptor DCC has been shown to modulate synaptic strength in mice. Also, altered activity in ovarian nerves is associated with infertility in humans (Newquist, 2013b).

To address the function of the Netrins in these fertility defects, there are many possible future directions. Promoter fragments fused to GAL4 for both NetA and NetB are now available and testing the ability of NetA or NetB driven by these lines to rescue the defects in NetAB flies would likely allow the site of Netrin action to be identified. The anatomy of surrounding nerves and tissues could then be examined. Localization of the Netrin proteins in pupal and adult tissues using antibody reagents would also assist in analyzing Netrin function. Functional assays of oviduct contraction would allow ovulation to be examined directly and also the role of sperm proteins to be tested. Finally, the fra Netrin receptor can produce homozygous adults that could be tested for similar defects (Newquist, 2013b).

The Drosophila Netrin genes are required in both male and female flies for optimum fertility. NetAB mutants also display decreased viability, a failure to fly, and males exhibit hyper-coordination of wings during courtship. All behaviors are rescued by restoring one copy of endogenous NetB. Flight is surprisingly restored by pan-neuronal expression of NetA, but not NetB. The control of egg-laying may reside in multiple tissues or there is a very specific requirement that is not addressed by the drivers used in this study. Future work identifying the precise positional and temporal requirements for the Netrins will provide insight into how the Netrins function in egg-laying and other adult phenotypes (Newquist, 2013b).


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Netrin-A and Netrin-B: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation

date revised: 15 February 2015

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