Motoneurons are key points of convergence within motor networks, acting as the 'output channels' that directly control sets of muscles to maintain posture and generate movement. This study used genetic mosaic techniques to reveal the origins and architecture of the leg motoneurons of Drosophila. A small number of leg motoneurons are born in the embryo but most are generated during larval life. These postembryonic leg motoneurons are produced by five neuroblasts per hemineuromere, and each lineage generates stereotyped lineage-specific projection patterns. Two of these postembryonic neuroblasts generate solely motoneurons that are the bulk of the leg motoneurons. Within the largest lineage, lineage 15, distinct birth-order differences are seen in projection patterns. A comparison of the central projections of leg motoneurons and the muscles they innervate reveals a stereotyped architecture and the existence of a myotopic map. Timeline analysis of axonal outgrowth reveals that leg motoneurons reach their sites of terminal arborization in the leg at the time when their dendrites are elaborating their subtype-specific shapes. These findings provide a comprehensive description of the origin, development, and architecture of leg motoneurons that will aid future studies exploring the link between the assembly and organization of connectivity within the leg motor system of Drosophila (Brierley, 2012).

In insects that undergo a complete metamorphosis, like Drosophila, the ventral nerve cord is produced by two distinct phases of neurogenesis. The first wave occurs during embryonic development and produces the components required for the control of larval behavior. Some of the neurons generated at this time remodel and play a role in adult circuits. The bulk of neurons found in the adult fly are produced during the second, more prolonged neurogenic phase during larval and early pupal life. Within this process, this study determined whether the leg motoneurons are produced during the embryonic or postembryonic phases of neurogenesis (Brierley, 2012).

This work identified two distinct types of motoneuron clones in the third instar larva VNC, generated by embryonic heatshocks. One type has complex, highly branched dendrites with axons that exit the nerve cord and terminate on body wall muscles. This type of neuron in insects is uniquely identifiable and can have one of two different fates during metamorphosis; some remodel and take up a new adult-specific role, whereas others undergo programmed cell death. It was not possible to determine the identity, number, or fate of specific embryonic neurons using their larval morphology alone. However, it is known that in the beetle Tenebrio molitor and the moth Manduca sexta the larval leg motoneurons remodel to become adult leg motoneurons. The second type of motoneuron clones had a simple morphology in the third instar CNS reminiscent of single-cell postembryonic clones born during larval life. This second type of neuron is similar to the flight motoneuron MN5 and the persistent Broad positive neurons seen in the embryonic CNS. These neurons remain in an immature state throughout larval life, before completing their development during metamorphosis (Brierley, 2012).

To quantify how many types of leg motoneuron are born during the embryonic wave of neurogenesis, MARCM clones were induced in the embryo, and then single-cell motoneuron clones were identified in the adult ventral nervous system with axons in the leg. These data suggest that at least seven different leg motoneuron types are born in the embryo. Using this approach their origins or how many of each type there are could not be determined. These seven types could represent progeny from seven different neuroblasts. A previous study, Baek (2009), predicted that 13 lineages generate leg motoneurons in the embryo, but their data, like the current, cannot definitively answer this question. Although it is believed that these different neurons are bona fide types, it is possible that a single motoneuron could generate two very different terminal arborizations. This seems unlikely, as all the data points toward a high degree of morphological stereotypy in embryonic motoneurons. In future the availability of many more cell type-specific markers should enable identification each of these early born neurons (Brierley, 2012).

The data reveal that neurons born during larval life make the most significant contribution to the pool of leg motoneurons. These leg motoneurons are generated by five postembryonic Nbs. Of these, two lineages generate exclusively motoneurons: lineage 15, which contains on average 28 motoneurons, and lineage 24 which contains six motoneurons. This confirms the observations of Baek (2009), who also found these lineages. Three postembryonic lineages were found that contain one or two motoneurons along with a large number of interneurons (lineages 20, 21, and 22), whereas Baek (2009) only reported one. The motoneurons within these lineages are born soon after the onset of postembryonic neurogenesis, with the first ganglion mother cell (GMC) generating two siblings, a motoneuron and a local interneuron. Following this, every time a GMC divides the motoneuron sibling undergoes apoptosis, whereas the interneuron survives. Such hemilineage-based programs of cell death play a significant role in determining the type and number of network components in the thoracic nervous system of Drosophila (Brierley, 2012).

Most knowledge of the origins of Drosophila motoneurons comes from studies in the embryo. Clonal analysis in the embryo revealed that 17 of the 31 Nbs generate motoneurons and all are born early within these lineages, with most Nbs contributing one or two motoneurons, and at most six. If every embryonic born neuron is derived from a different Nb then the maximum number of lineages generating leg motoneurons would be 12, compared with six if all are derived from a single Nb. Baek (2009) concluded that 13 lineages contribute leg motoneurons (Brierley, 2012).

The general organization of Drosophila leg motoneurons within the CNS shows great similarity with that of the grasshopper Schistocerca americana, with the neurons being clustered into groups. Each of these eight groups are presumably derived from their own single Nb, with the primary neurites inserting into characteristic position in the neuropil (Brierley, 2012).

The success of holometabolous insects as a group is due largely to their ability to produce radically different body plans at larval and adult stages that allow them to exploit very different ecological niches. Some of the most striking adaptations within the Holometabola are seen in the articulated appendages, particularly the legs. How the developmental programs that control leg motoneuron connectivity have been modified is likely to be very interesting and may provide insights into evolution of neural networks. This census of Drosophila motoneurons is likely to help with comparative studies on leg motoneurons from other insect species (Brierley, 2012).

Regardless of the exact number of lineages that generate leg motoneurons, it is striking that just two postembryonic lineages contribute the bulk of the leg motoneurons (34 of the 47) and this raises the question of whether there is something fundamentally different about leg motoneuron specification compared with what was already know from studies in the embryo (Brierley, 2012).

One of the most notable differences between the larval and adult musculoskeletal system is that the muscles of adults are multifiber and often innervated by a number of isomorphic neurons. The dendrites of most leg motoneurons are located ipsilaterally and elaborate into the same neuromere in which they are born, unlike in the embryo where motoneurons can have extensive contralateral dendrites and are parasegmental in their nature. This difference in organization, i.e., segmental vs. parasegmental dendrites, could be an adaptation for larval locomotion, where the control of the next adjacent segment is critical (Brierley, 2012).

Although there is general agreement between many of the current findings and those of Baek (2009), there are some differences in detail, which may have important implications. Unlike Baek (2009), this study found that the largest leg motoneuron lineage, lineage 15, also innervates muscles in the body wall as well as intrinsic muscles in the femur and the tibia. Lineage 15 therefore has the most extensive coverage along the proximodistal axis of the leg and does not have a distal bias, as previously suggested (Baek, 2009). The extrinsic muscles in the body wall are extremely important, as they control the bodywall/coxal joint, which is in effect a universal joint allowing the leg a near 360
rotation. Although this study has presented a more complete picture of these motoneurons, more work is needed to identify the origins of the other motoneurons that innervate this complex group of muscles. Motoneurons within lineage 24 innervate muscles in the coxa, trochanter, and femur and control the movement of the femur and tibia. In this study no neuron from this lineage was seen innervating the tibia reductor muscle group, or indeed any other glutamatergic leg motoneurons terminating on two distinct muscle targets, as suggested by Baek (2009). The three lineages, lineages 20, 21, and 22, all innervate muscle groups in the coxa (Brierley, 2012).

What this lineage analysis highlighted is that within the CNS the dendrites of each of the postembryonic lineages each occupy distinct territories along the mediolateral, anteroposterior, and dorsoventral axes. This is important, as it is known from studies on the leg sensory system that the central afferent projections of different classes of sensory neuron occupy distinctive volumes within the dorsoventral axis depending on their modality (Brierley, 2012).

Lineage 15 has the most medially projecting dendrites located in both the anterior and posterior regions of the neuropil; the dendrites of motoneurons from lineage 24 motoneurons take up more lateral territories, whereas the central projections from lineages 20, 21, and 22 occupy the most lateral neuropil domains and span the anteroposterior axis. These lineage-specific patterns are reproducible, with no obvious variation in the muscles innervated or with a significant difference in the size or morphology of the axonal arborizations. This observation emphasizes that decoding lineage-specific programs of morphogenesis is likely to hold the key for understanding the development and organization of motoneurons within the leg network (Brierley, 2012).

To explore these motor lineages in more detail, individual neurons were visualized using the MARCM technique to determine how motoneuron birth date is correlated with aspects of morphology. As well as birth-dating, the single-cell clones allowed a close look at the relationship between muscle innervation and dendrite shape (Brierley, 2012).

In lineage 15, the sequential production was found of at least five distinct motoneuron subtypes during larval life. The first-born neuron innervates a muscle in the bodywall, the next subtype targets a muscle in the proximal femur, with the following subtype targeting a muscle in the proximal tibia. The next subtype innervates targets in the distal femur and then the distal tibia. Thus, there is no simple proximal to distal filling up of the leg, based on the birth-date of neurons; instead, neurons that innervate the most proximal target of a leg segment are born first. The central projections of these motoneuron subtypes were also very stereotyped, with the dendrites of early born cells spanning medial to lateral territories and late-born cells elaborating their dendrites in the lateral and ventral neuropil. Lineage 24 also shows a stereotyped birth-order based pattern of innervation along the proximodistal axis of the leg. It was found that lineage 24 generates three subtypes during larval life with both early and late-born neurons innervating the same muscle group located in the coxa and having dendrites that target lateral regions within the CNS. The second and third subtypes target the trochanter and the femur, respectively (Brierley, 2012).

It is interesting to speculate how a lineage like 15 may have evolved from an ancestral condition. The first motoneuron subtype innervates a body wall muscle and the next the long tendon muscle located in the femur. The long tendon muscle, also called the unguis retractor, attaches to the apodeme that controls the most distal element in the leg, the pretarsus. Could it be that these early born neuron subtypes are the most 'ancient' within the lineage, while the sequential addition of the later subtypes occurred as new leg segments were introduced? It would be intriguing to look at the homologous neurons in different outgroups (Brierley, 2012).

The long tendon muscle motoneurons are also unique among the glutamatergic leg motoneurons, as they are the only ones that elaborate dendrites in the contralateral hemineuromere. It is worthy of note that there are more long tendon muscle group (ltm) motoneurons than any other leg motoneuron. This is probably due to the need for the precise control of the pretarsal claw, which is fundamental to all locomotory and nonlocomotory behavior involving the leg. The difference in the birth-order of neuron types between the different lineages is also striking. Rigid birth-order-based rules that control the targeting of terminal processes have been described for other types of secondary neurons, including the antennal lobe projection neurons found in the fly's olfactory system. The sequential production of different neuron subtypes at distinct times during development is a common mechanism for generating the diversity of circuit components in many taxa, including vertebrates. In flies, there is strong evidence that individual Nbs express a sequence of progenitor transcription factors, such as Hunchback, Kruppel, Pdm, and Castor, which in turn regulate the postmitotic transcription factors to specify a distinct identity. The differences observed between neuronal birth-date and the dendritic and axonal arborizations in lineages 15 and 24 could be due to similar transient and sequential expression of temporally controlled transcription factors, like those observed in embryonic lineages or by other transcription factors such as Chinmo and Broad, which are deployed within postembryonic neuron subtypes. Although most studies in insects emphasize stereotyped lineage-specific specification a recent report describes how local interneuron populations within the Drosophila antennal lobe can have great morphological variability. It may be that particular neuronal classes, such as those that transfer information between one part of the nervous system and another, are more developmentally hard-wired than elements that perform mainly local processing (Brierley, 2012).

The data show that soma location is not an important descriptor of identity, but rather the location of their dendritic terminals. Taken as a whole, this work reveals that, although there is great stereotypy, there is no simple organizing principle that translates birthdate into projection pattern, i.e., that early born neurons innervating proximal leg segments and late born neurons targeting distal ones. Solving lineage-based codes within this system is likely to hold the key to understanding fundamental rules about how networks are assembled (Brierley, 2012).

Understanding how ordered patterns of synaptic connectivity are established between motoneurons and the rest of the motor network is a fundamental question in neurobiology. Landgraf (2003) revealed that the dendrites of motoneurons in the Drosophila embryo are organized to reflect the innervation of muscles in the periphery. They forwarded the idea that different territories within such a 'myotopic map' reflect patterns of connectivity with premotor elements and that such maps could be a general organizational principle of all motor systems. This study was directed to establishing whether leg motoneurons generate the same kind of myotopic map and thus explore the generality of this compelling idea (Brierley, 2012).

It was found that the dendrites of leg motoneurons occupy a large volume of the leg neuropil and showed a considerable degree of overlap, even though each occupies a slightly different volume. This is in marked contrast to the myotopic map seen in the embryo, where dendrites appear to generate exclusive, nonoverlapping territories: i.e., the dendrites of motoneurons that innervate internal muscles segregate into a different neuropilar domain from those innervating external muscles, alongside which the motoneurons of the internal muscles organize themselves into a map representing the dorsoventral axis of the body wall. This difference in organization may be due to differences in the skeleton and the biomechanics of the two systems. The adult leg is a complex multijointed appendage with many degrees of freedom, where interjoint coordination between and within legs is of paramount importance. In contrast, the fly larva locomotes using simple peristaltic waves of the abdominal wall and head turns. The organization of Drosophila leg motoneuron dendrites mirrors that of vertebrate somatic motoneurons in the spinal cord, where each motoneuron type has a dendritic arborization that covers a distinctive territory but at the same time has considerable overlap with the dendrites of other types (Brierley, 2012).

To step back from this, a systematic and unbiased analysis was performed of leg motoneuron dendrite position where the prothoracic neuropil was divided into sectors and the location of the arborizations of 13 different types was measured using single-cell MARCM clones. This approach allowed determination of the volume that each of the different motoneuron types samples within the neuropil and how the different types relate to each other. The dendrogram generated shows how closely related the different subtypes are. Any branch can be reversed around a node point, and the relatedness of different arborizations can be inferred using this. Using five neurons for each type helped provide a robust measure of the similarities/differences between the different motoneurons. It was found the 13 motoneuron types clustered into nine sets. Motoneurons of the same subtype tended to group together in most examples. Some motoneurons that innervate functionally related muscles also clustered together, e.g., the ltm1 and ltm2 muscles located in the femur and the tibia. It was also found that motoneurons that innervate the tibia levator and the tibia reductor muscle also formed a group: these are synergists (Brierley, 2012).

As a counterpoint to this, a number of motoneurons were seen that innervate antagonistic muscles cluster together, e.g., the trochanter levator and trochanter depressor muscles both located in the coxa being one set, and the tarsal levator and tarsal depressor muscles that control the tarsal segments being another. Baek found four sets that clustered together;they found the motoneurons that innervate the long tendon muscles in both the femur and tibia grouped together, as did antagonistic motoneurons that innervate the coxal segment. They also saw two different types of trochanter neurons cluster, which this study did not have data for. Baek suggested that different reductors clustered together but the neuron they proposed to innervate the tibia reductor does not. This study took the clustering data and remapped this onto the neuropil. It shows clearly the large overlap of the dendrites of many of the different motoneurons. What does this overlap mean functionally? First, it is important to emphasize that just because neurites occupy the same space, judged by light microscopy, it does not mean they make connections with each other or, as in this case, receive the same types of input. In the brachial spinal cord of the bullfrog Rana catesbeiana sensory axons from the triceps brachii muscle make connections with triceps motoneurons but do not innervate subscapularis and pectoralis motoneurons, which are in very close proximity. The monosynaptic connections between triceps brachii motoneurons and sensory neurons appears relatively late in development, after the dendrites have grown into a territory that contains an extant presynaptic terminal field (Brierley, 2012).

The specific connection occurs then as soon as the motoneuron arrives. Importantly, it says that if the terminals are not within a territory they cannot make connections with inputs there. The occurrence of pre- and postsynaptic elements in space is thus necessary but not sufficient for connectivity. Other examples in the vertebrate spinal cord show that there are considerable similarities in the morphology of somatic motoneuron dendrites within large parts of their arborization, but that key differences in specific regions can occur. A good example of this is seen in the dorsal dendrites in the lumbar motoneurons of the turtle Pseudemys scripta elegans, where such specialized differences in dendrite morphology might reflect a difference in synaptic input or the processing of input. The finding that the dendrites of Drosophila motoneurons that innervate antagonistic muscle pairs are similar is interesting (Brierley, 2012).

Are such dendritic organizations important for interpreting information from similar inputs? Future work exploring connectivity using physiological approaches should allow us to address whether this is important for function or for determining patterns of connectivity during development. Although there is no simple 'easy to read' map, what the data shows is that there are robust topological relationships between these dendritic arborizations (Brierley, 2012).

As described above, there exists a diversity of dendritic and axonal projection patterns within neural maps. A key question raised by this is how do neurons within such maps ensure that both the axonal and dendritic terminals execute appropriate programs of morphogenesis. We now know that dendrites, like axons, use conserved molecular cues and various transmembrane receptors to attain their distinct organizations. One possible mechanism for generating a diversity of dendrite shapes could be retrograde signaling from the target muscle. It was of interest to look at the relative timing of axon and dendrite outgrowth in this system to see if this could be possible. The data from lineage 15 reveals that two subtypes, which innervate different long tendon muscle sets, have nearly identical dendritic trees, but their axons target muscles in different segments of the leg. The timeline data shows that motoneuron axon outgrowth in the proximal leg occurs at the same time as dendritic elaboration in the CNS. This opens the possibility that retrograde signals may play a role in the development of neurons that innervate muscle targets in the leg (Brierley, 2012).

How modular are these programs? These events must be controlled at some level by transcription factor codes regulating blends of guidance receptors. Cell intrinsic temporal transcription factors can control a combinatorial code of postmitotic transcription factors. Feedback from the muscle field could also provide patterning information as seen in the vertebrate spinal cord, where motoneuron dendrite arborizations are controlled in part by the transcription factor Pea3, which is induced by retrograde signaling from target muscles. The respective timing of outgrowth of Drosophila leg motoneuron axons and dendrites opens this up as a possibility. Previously, laser ablation studies revealed that the dendrites of Drosophila flight motoneurons change their growth following the removal of their target muscle. It will be interesting to test this hypothesis experimentally by removing the muscle targets in the leg and quantifying the dendritic arborizations of known motoneurons (Brierley, 2012).

This study has explored the origins and architecture of the leg motoneurons of Drosophila using genetic mosaic techniques.A small number of leg motoneurons are born in the embryo, but the majority are generated during larval life. These postembryonic leg motoneurons are produced by five Nbs, where the progeny of each lineage generates stereotyped, lineage-specific projection patterns. The dendrites of Drosophila leg motoneurons show similarities with spinal cord motoneurons where different types have a considerable degree of overlap but each has unique regions that it targets. These data reveal that even though there is no simple 'easy-to-read' leg myotopic map, the central projections of leg motoneurons and muscles they innervate manifest robust topological relationships. Understanding the functional relationships within this map and the molecular mechanisms that control its development will provide insights into the way ordered patterns of connectivity are established within neural networks (Brierley, 2012).