The organisational principles of locomotor networks are less well understood than those of many sensory systems, where in-growing axon terminals form a central map of peripheral characteristics. Using the neuromuscular system of the Drosophila embryo as a model and retrograde tracing and genetic methods, principles underlying the organisation of the motor system have been uncovered. Dendritic arbors of motor neurons, rather than their cell bodies, are partitioned into domains to form a myotopic map, which represents centrally the distribution of body wall muscles peripherally. While muscles are segmental, the myotopic map is parasegmental in organisation. It forms by an active process of dendritic growth independent of the presence of target muscles, proper differentiation of glial cells, or (in its initial partitioning) competitive interactions between adjacent dendritic domains. The arrangement of motor neuron dendrites into a myotopic map represents a first layer of organisation in the motor system. This is likely to be mirrored, at least in part, by endings of higher-order neurons from central pattern-generating circuits, which converge onto the motor neuron dendrites. These findings will greatly simplify the task of understanding how a locomotor system is assembled. These results suggest that the cues that organise the myotopic map may be laid down early in development as the embryo subdivides into parasegmental units (Landgraf, 2003).

The analysis began by correlating the positions of motor neuron dendrites with the distribution of their muscle targets in the periphery. Motor neurons were retrogradely labelled in a pairwise fashion and the positions of their dendritic arbors were mapped. Because of an interest in the mechanisms that underlie the assembly of the motor system, focus was placed on stages when each motor neuron first establishes a characteristic domain of arborisation within the neuropile (early stage 17, 15h after egg-laying [AEL]) (Landgraf, 2003).

Motor axons project into the muscle field via two main nerves, the intersegmental (ISN) and the segmental nerve (SN). The transverse nerve (TN) runs along the segment border and has few motor axons. Choice of nerve root is one of several features that divide the motor neurons into two principal sets, the ISN and SN. (1) The cell bodies of SN motor neurons are located in the same segment as the muscles that they innervate, whereas ISN motor neuron somata are located in the segment next anterior (with the exception of the RP2 and two neuromodulatory efferent ventral unpaired median [VUM] neurons. (2) ISN motor neurons innervate internal muscles, which span a segment from anterior to posterior, whereas SN (and the TN) motor neurons innervate external muscles. External muscles are distinct from the internal set in several respects: (1) they are generally transverse; (2) unlike internal muscles, they require wingless (wg) signalling for their specification; (3) external (but not internal) muscles and their innervating motor neurons express the cell adhesion molecule (CAM) Connectin, with the single exception of muscle ventral transverse 1 (VT1) (Landgraf, 2003 and references therein).

In addition, ISN and SN motor neurons elaborate their dendrites in distinct regions of the neuropile. Dendrites of ISN motor neurons occupy a domain extending posteriorly from the posterior part of one neuromere into the anterior part of the next. SN motor neuron dendrites occupy a domain that lies between the domains of ISN motor neuron arbors (Landgraf, 2003).

Thus, the organisation of the body wall muscles into internal and external sets is reflected centrally in patterns of motor neuron arborisations. The innervating motor neurons project their axons through different nerves and elaborate their dendritic fields in distinct regions of the neuropile. Although dendritic arbors become progressively more elaborate and extensive over developmental time, their separate domains remain clearly recognisable and appear to be maintained at least until the motor system is fully functional (18 h AEL) (Landgraf, 2003).

Having established that there is a central representation of the muscle field, the organisation of the motor neuron dendrites was analyzed in greater detail. (1) The set of external muscles and their innervating (SN) motor neurons were examined. Muscles of similar anteroposterior positions, such as the ventral acute muscle (VA3) and the segment border muscle (SBM), are innervated by motor neurons whose dendritic arbors lie in a common region of the neuropile. Conversely, motor neurons supplying the anterior (lateral transverse 1-2 [LT1-LT2]) versus the posterior (SBM) muscles have dendritic arbors that are correspondingly separated in the anteroposterior axis of the CNS (Landgraf, 2003).

To put the idea of a regular map to the test, focus was placed on an unusual external motor neuron-muscle pair. Muscle VT1 is innervated by a TN rather than an SN motor neuron. However, VT1 lies at the same place in the anteroposterior axis as the SBM, although VT1 is ventral and the SBM more dorsal. The VT1 motor neuron dendritic field is found to overlaps with that of the SBM motor neuron. For the external set, it is concluded that differences in target muscle location in the anteroposterior axis are mapped centrally as regular differences in dendritic position, but dorsoventral distinctions are not (Landgraf, 2003).

It was next asked whether there is a similarly regular representation of the internal muscles in the developing CNS. While most external muscles are transverse and have unique anteroposterior locations, the internal muscles span the width of a segment so that positional distinctions between them are solely in the dorsoventral axis. It was found that the set of internal muscles is represented centrally by three dendritic domains. Motor neurons innervating ventral internal muscles elaborate their dendritic arbors in the anterior half of the ISN dendritic domain. Motor neurons with dorsolateral internal muscle targets (lateral longitudinal [LL] 1, dorsal acute [DA] 3, dorsal oblique 3-5 [DO3-DO5]) put their arbors into the posterior part of the ISN dendritic domain. Finally, dorsal muscles are represented by a motor neuron dendritic domain that lies between those representing ventral (anterior) and dorsolateral (posterior) internal muscle groups. Thus, the internal muscles are represented in the neuropile by three domains of dendritic arborisation that reflect their different dorsoventral locations in the periphery. Once again, it is concluded that there is a regular mapping of muscle position in the neuropile: in this case, it is positions in the dorsoventral axis peripherally that are represented centrally as differences in the anteroposterior locations of dendrites (Landgraf, 2003).

To test the idea that dendritic arbor positions relate to the distribution of muscles, an atypical motor neuron-muscle pair was examined. The RP2 motor neuron is reported to innervate dorsal muscle DA2, yet its dendrites span the domains that represent both dorsal and dorsolateral internal muscles. However, on careful analysis it was found that DA2 is, in fact, specifically innervated by a U neuron whose dendrites lie in the dorsal internal domain, whereas the RP2 axon forms endings generally on all dorsolateral and dorsal muscles by 19 h AEL. These seem to correspond to the type 1s boutons found in late larvae. Thus, the RP2 neuron puts its dendrites into a region of the neuropile that does indeed represent its targets, namely the dorsolateral and dorsal internal muscles (Landgraf, 2003).

Like the muscle field itself, the map of motor neuron dendrites is metamerically repeated. However, the boundaries of these two units are out of register with one another, since the dendrites of the motor neurons innervating internal muscles lie in the next anterior neuromere. The anterior border of the dendritic map, as defined by the extent of these anterior dendrites, coincides with the anterior margin of engrailed (en) expression. Thus, while the muscles are segmental in their organisation, the domains occupied by the dendrites of their innervating motor neurons are parasegmental (Landgraf, 2003).

To test whether genes that implement the parasegmental pattern in the epidermis are also required for the formation of the parasegmental organisation of the neuromuscular system, the formation of SN and ISN dendritic fields was studied in embryos singly mutant for the following segment polarity genes: en/invected (Df(en^E)), wg (wg^CX4), naked (nkd²), patched (ptc⁹), hedgehog (hh²¹), and gooseberry (Df2R(gsb)). Every one of the six different mutants that were analysed has partially aberrant patterns of neuroblasts (NBs). Nevertheless, SN and ISN motor neurons still form and can be identified by their characteristic axonal projections into the periphery. In addition, it was found that the fundamental separation between SN and ISN dendritic domains is present despite often severe perturbations in CNS structure. For example, in gsb mutant embryos, both nerve roots are frequently fused so that the SN and ISN share a common CNS exit point. Nevertheless, SN and ISN axons as well as their dendritic fields do not intermingle but remain separate. These results suggest that the subdivision of the neuropile into the principal ISN and SN dendritic domains is a robust feature of the system, which appears to be specified early in development, since the embryo subdivides into parasegmental units (Landgraf, 2003).

It was next asked what mechanisms underlie the formation of the myotopic map. Because ISN and SN motor neurons lie at different positions in the CNS and their axons grow out into the muscle field through different nerves, it is reasonable to suppose that at least the major subdivision of dendritic arborisations into internal and external domains could be a byproduct of the locations at which the motor neurons are generated and the paths taken by their growing axons. This ‘passive mapping' explanation can be excluded by considering a single motor neuron-muscle pair, namely dorsal transverse 1 (DT1) and its innervating motor neuron. DT1 is an external muscle (by position, orientation, wg dependence, and Connectin expression), yet its motor neuron is clustered with the internal muscle innervating set and its axon (uniquely for the external muscles) grows out through the ISN. Despite its packing within the ‘internal motor neuron' set, the DT1 motor neuron makes a long posterior projection through the internal muscle domain of the myotopic map to reach the external domain, where it arborises appropriately, reflecting the orientation and external nature of its target muscle. In contrast, motor neurons derived from the same NB as DT1 innervate neighboring internal muscles DO3-DO5 and put their dendrites in a more anterior region characteristic of the dorsolateral muscles. These findings strongly suggest that the mapping of the muscle field within the CNS is an active process of growth and arborisation that partitions dendrites into subdomains of the neuropile that are appropriate to their function, rather than a passive subdivision of available space by position of origin or axon trajectory (Landgraf, 2003).

Since dendritic arbors form after motor axons have reached their targets, the muscles could be instrumental in dictating the organisation of the central map. To test this idea, the UAS/GAL4 system was used to misexpress an activated form of Notch (Kidd et al. 1998) in the developing mesoderm, suppressing the formation of muscle founder cells while leaving other tissues intact. In such muscleless embryos, the main nerve trunks, SN and ISN, still form and project into the periphery. Retrograde labellings of these nerves show that SN and ISN motor neurons form relatively normal dendritic arbors that consistently conform to the characteristic separation of SN and ISN dendrites. Thus, the neuropile is partitioned into distinct fields of dendritic arborisation independently of the muscles. It is concluded that the mapping process is likely to be an autonomous property of the motor neurons and their neighboring cells (Landgraf, 2003).

It was next asked whether motor neuron dendritic fields could be patterned by the substrates on which they grow. In the Drosophila ventral nerve cord (VNC), motor neuron dendrites form in the dorsal-most region of the neuropile, sandwiched between longitudinal glia above and the underlying scaffold of axons. Glial cells can act as substrates for supporting and guiding axonal growth. To test whether they might also be required for the growth and spatial patterning of dendritic fields, dendritic arbors were analysed in glial cells missing (gcm) mutant embryos, which are defective in glial cell differentiation. Although the structure of the nervous system is disrupted in gcm mutant embryos and the dendritic arbors are abnormal, they continue to form in their characteristic locations and the fundamental distinction between the ISN and SN motor neuron dendritic fields is maintained. Remarkably, even the long posterior dendritic projection of the DT1 motor neuron forms and reaches its target region, the SN external muscle dendritic domain. These results suggest that the patterning of the neuropile into distinct motor neuron dendritic domains is a process that appears to be intrinsic to the motor neurons and their neighboring neurons, but does not require proper glial cell differentiation (Landgraf, 2003).

One likely explanation for the division of dendrites into separate domains is that there is a process of mutual exclusion between the arborisations of neighboring cells. Such a process of dendritic ‘tiling' has so far only been documented between particular classes of sensory neurons, but could also occur in the motor system. The idea of tiling was tested by considering two groups of motor neurons whose axons have a common trajectory, but whose dendritic fields form in adjacent territories. The DO3-DO5 and DT1 motor neurons project their dendrites posteriorly, and at their most-anterior point, these dendrites meet the axons and dendrites of the anterior corner cell (aCC) and U/CQ neurons. To show whether the aCC and U/CQ axons and/or dendrites inhibit the growth of DO3-DO5 and DT1 dendrites anteriorly, these neurons (as well as RP2 and the posterior corner cell [pCC] interneuron) were selectively ablated. Using anti-Even-skipped (Eve) staining as a marker for aCC, RP2, and U/CQs (there are an additional two medially located eve-expressing interneurons, pCC and friend of pCC [fpCC], it was found that these neurons can be selectively ablated before they form dendrites (at approximately 11 h AEL): on average, by 10.5 h AEL all but 0.6 and by 12 h AEL all but 0.06 of the seven medially located eve-expressing neurons have been ablated per half-neuromere. In no instance was a concomitant anterior expansion of the DO3-DO5 and DT1 motor neuron dendrites into the regions vacated by the aCC and U/CQ dendrites observed. It is concluded that, at least in this instance, the initial dendritic territory of one set of motor neurons (DO3-DO5 and DT1) is not defined by a process of tiling, in which they are excluded by neighboring (aCC and U/CQ) dendritic arbors. However, it is possible that the elaboration of motor neuron dendritic arbors during later developmental stages may involve interactions between neighboring dendritic territories, activity-dependent processes, or both (Landgraf, 2003).

Thus, in summary, these results suggest that the mechanisms that subdivide the neuropile into distinct dendritc domains are very robust and refractory to perturbations. They further suggest that the cues that organise the map may be laid down early in development as the embryo subdivides into parasegmental units (Landgraf, 2003).

Glial cells play important roles in the developing brain during axon fasciculation, growth cone guidance, and neuron survival. In the Drosophila brain, three main classes of glia have been identified including surface, cortex, and neuropile glia. While surface glia ensheaths the brain and is involved in the formation of the blood-brain-barrier and the control of neuroblast proliferation, the range of functions for cortex and neuropile glia is less well understood. This study used the nirvana2-GAL4 driver to visualize the association of cortex and neuropile glia with axon tracts formed by different brain lineages and to selectively eliminate these glial populations via induced apoptosis. The larval central brain consists of approximately 100 lineages. Each lineage forms a cohesive axon bundle, the secondary axon tract (SAT). While entering and traversing the brain neuropile, SATs interact in a characteristic way with glial cells. Some SATs are completely invested with glial processes; others show no particular association with glia, and most fall somewhere in between these extremes. The results demonstrate that the elimination of glia results in abnormalities in SAT fasciculation and trajectory. The most prevalent phenotype is truncation or misguidance of axon tracts, or abnormal fasciculation of tracts that normally form separate pathways. Importantly, the degree of glial association with a given lineage is positively correlated with the severity of the phenotype resulting from glial ablation. Previous studies have focused on the embryonic nerve cord or adult-specific compartments to establish the role of glia. This study provides, for the first time, an analysis of glial function in the brain during axon formation and growth in larval development (Spindler, 2009).

Secondary neurons, which are born during the larval period, form SATs that have to extend over relatively long distances, finding their way amidst a complex array of (primary) axons, dendrites, and glia. The association of glia and SATs varies for different lineages. SATs either (1) remained wrapped within the neuropile or joined other tracts that were then ensheathed as a larger tract system, (2) encountered strands of glial condensations, or (3) had no association with glial sheaths. The association between individual SATs and glia was highly invariant. Thus, if SAT A joined SAT B to form a larger tract system that became wrapped by glia, the same densities of glia could be observed in other brains for the corresponding SATs (Spindler, 2009).

To address the role of glia during SAT fasciculation, growth, and guidance, cortex and neuropile glia, the two glial types in contact with growing SATs, were selectively eliminate. Expression of the pro-apoptotic proteins Hid and Rpr were effective in inducing apoptosis of most cortex and neuropile glia by the early or mid larval stage. It can be assumed that the primary axon tract formation is not disrupted, given that expression of the nrv2-GAL4 driver line does not set in prior to stage 12, primary axon tract (PAT) patterning is complete before glia invade the neuropile, and the first signs of apoptosis appear after hatching. In addition, the surface glia remain intact, providing general ensheathment around the brain, a functional blood–brain barrier, and potential signaling molecules used to control neuroblast proliferation (Spindler, 2009).

Upon the elimination of glia, frequent abnormalities are seen in the pattern of SATs. Importantly, the strength of an SAT phenotype appears to correlate with the degree of glial association of that SAT in a wild-type brain. Of all SATs analyzed, the mushroom body (associated with a complete glial sheath) exhibits the most severe defects, including complete SAT misguidance and aberrant fasciculation of neurites from adjacent SATs. In contrast, SATs that were less endowed with glia in the wild type typically had a normal projection pattern (Spindler, 2009).

This study does not differentiate between a role for glia in producing chemo-attractant signals or acting as a physical scaffold for guidance. In previous studies, ectopic expression of dominant-negative Drosophila E-cadherin in either cortex/neuropile glia or SATs themselves results in non-radial trajectories of SATs into the neuropile. This suggests a requirement for SAT-glia adhesion as secondary axons project toward the cortex-neuropile boundary. However, the direction of neuroblast division is also disrupted with DE-cadherin knock-down, thus aberrant SAT trajectories may be a secondary effect of abnormal cell body layering within the cortex. In support of a signaling role for glia, the midline guidance defect of the CP1 SAT (anteromedially, crossing the peduncle and entering the diagonal commissure) is reminiscent of the robo-slit phenotypes in the Drosophila ventral nerve cord. Whether robo/slit signaling is also used between SATs and glia in the brain is a question that warrants further investigation (Spindler, 2009).

The situation that SATs in part require glia for pathway guidance in the neuropile is different than the embryonic brain in which pioneer axons and PATs are formed in the absence of extensive glial processes. Why are SATs different? One can imagine the scenario in which the PATs generate the neuropile de novo, forcing cell body movement outwards as the central neuropile grows. By first instar, a full neuropile is established, and SATs must guide through a dense maze of neurites, glia, and trachea. It therefore appears that: (1) PATs establish the initial connectivity of the brain; (2) glia grow in around this initial scaffolding, and finally (3) SATs use both the PAT scaffolding and the glial boundaries for guidance into and around the neuropile (Spindler, 2009).

Insects have long been used to evaluate glial-neuronal interactions from embryonic to adult stages. An important focus of these studies was whether or not glia or axon tracts appear first, and in how far axonal pathfinding is disrupted if glia is ablated. In this regard, clear developmental differences have been found between distinct regions of the nervous system, as well as between insect species for homologous nervous system domains (Spindler, 2009).

In the Drosophila ventral nerve cord, two subpopulations of neuropile glia were studied in the context of axonal pathfinding: midline glia and longitudinal glia. Both types of glial cells appear around the same stage when pioneer neurons extend their axons. The proper number and positioning of midline glia is clearly required for the formation of commissural axon tracts. The loss of longitudinal glia (by ablation and in embryos mutant for the gcm gene) primarily affects the defasciculation and fasciculation events of longitudinal pioneer tracts, subsequently affecting the follower neuron trajectories. Note that gcm is required for all classes of glia, including surface glia; thus, in very late gcm mutant embryos, severe disruptions of the entire neuropile result from the fact that, with the onset of embryonic movement, the CNS lacking surface glia is literally 'shredded to pieces' (Spindler, 2009).

In the embryonic Drosophila peripheral nervous system, ablation of the peripheral glia (i.e. exit glia) via targeted overexpression of the cell death genes grim and ced-3 lead to aberrant pathfinding of both motor and sensory axons as they exited the CNS; although the motor neurons eventually overcame the absence of peripheral glia finding correct muscle targets, suggesting a limited role for the peripheral glia in the initial trajectory of motor neurons. In grasshopper, ablation of the cell-segment boundary glial guidepost cell lead to more severely aberrant axon trajectories. Ablation of glia surrounding the antennal lobe of adult Manduca generates olfactory axon de-fasciculation and misguidance. Finally, guidance phenotypes have also been observed with disruption of the Drosophila lamina glia; R1-R6 photoreceptor axons show aberrant guidance past the lamina into the medulla of the optic lobe (Spindler, 2009 and references therein).

An important aspect of the developmental role of glia added by this study is the focus on the correlation between closeness of axon tract/glia association, and axon tract abnormality in the absence of glia. In other words: the nervous system is formed by a large number of fascicles, and these fascicles vary in their degree of glial wrapping. It would be misleading when carrying out a genetic study to only focus on a single fascicle (or small subset of fascicles), and extrapolate from the phenotype observed for this fascicle onto the brain as a whole. In this analysis, SATs of several lineages, notably those that in normal brains have little glia covering them in the neuropile, show few abnormalities in glia-less brains. By contrast, other lineages were affected in the majority of cases, and typically, these lineages also were the ones whose SATs were associated more closely with glia (Spindler, 2009).

Neuropile compartments are formed by terminal branches of axons and dendrites and their synapses. For example, the antennal lobe of insects consists of the axonal terminals of sensory neurons located in the antenna, and dendritic terminals of antennal projection neurons and local interneurons located in the deuterocerebrum, aside from a relatively small number of other modulatory neurons. Sensory neurons expressing the same olfactory receptor all converge onto the dendrites of a small number of projection neurons to form an olfactory glomerulus. In many insects, notably Manduca, olfactory glomeruli are individually compartmentalized by neuropile glia, and it has been shown that glia plays a prominent role in establishing the glomeruli organization. According to the prevailing view, glomeruli are initially ordered by the specialized endings of sensory terminals into protoglomeruli; however, glial processes soon invade the space in between protoglomeruli and restrict the arborization of receptor axons. Therefore, in Manduca antennal lobes, glia is required for early maintenance of the glomerular map (Spindler, 2009 and references therein).

A recent analysis of the time course of glial development in the Drosophila antennal lobe suggested that in this species, glia plays an even lesser role, since glomeruli are only incompletely, and at a late time point, wrapped by glia. While glia were never ablated in those studies, later work found that elimination of Neuroglian from midline glia resulted in an inability of ORN axons to cross through the antennal commissural tract to the contralateral lobe. In this study, most adult brains lacking cortex and neuropile glia still form antennal lobes with glomeruli, however in some samples the glomeruli are poorly defined, and cannot be identified by their position and shape in the antennal lobe. The variability in phenotype penetrance likely stems from larvae containing the most glia surviving to eclosion; therefore, a relatively normal looking adult brain could stem from a weak glial apoptosis early in development. Whether the antennal lobe disorganization is a secondary defect due to a lack of definition normally provided by the presence of glia, a result of SATs that normally contribute to the AL misguiding or truncating early, or a maintenance defect in which axons begin inappropriately intermingling among glomeruli is not clear. Perhaps the Drosophila antennal lobe glia is required for glomeruli maintenance even after glomeruli organization is established, and future studies will hopefully address this possibility (Spindler, 2009).

In the post-embryonic midline of Drosophila, ablation of the transient interhemispheric fibrous ring (TIFR), a transient population of midline glia, by ectopic expression of the pro-death gene hid, generates defects in the adult central complex. What is unclear, however, is the cellular event that is effected to cause the defects. This study suggests that morphological abnormalities in adult compartments from glial manipulation are due to the misguidance of larval SATs to the correct neuropile compartment in the brain, affecting the formation of adult neuropile structures. This study presents evidence that glia is an important mediator of axon guidance in the Drosophila larval brain; the mechanism for glia-neuron communication during this process is an exciting area for future investigation (Spindler, 2009).