melanogaster The development of indirect flight muscle innervation in Drosophila Joyce Fernandes 1 and K. VijayRaghavan 1,2, * SUMMARY

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1 Development 118, (1993) Printed in Great Britain The Company of Biologists Limited The development of indirect flight muscle innervation in Drosophila melanogaster Joyce Fernandes 1 and K. VijayRaghavan 1,2, * 1 Molecular Biology Unit, Tata Institute of Fundamental Research, Homi Bhabha Road, Colaba, Bombay , India 2 National Centre for Biological Sciences, TIFR, PO Box 1234, Indian Institute of Science Campus, Bangalore , India *Author for correspondence SUMMARY We have examined the development of innervation to the indirect flight muscles of Drosophila. During metamorphosis, the larval intersegmental nerve of the mesothorax is remodelled to innervate the dorsal longitudinal muscles and two of the dorsoventral muscles. Another modified larval nerve innervates the remaining dorsoventral muscle. The dorsal longitudinal muscles develop using modified larval muscles as templates while dorsoventral muscles develop without the use of such templates. The development of innervation to the two groups of indirect flight muscles differs in spatial and temporal patterns, which may reflect the different ways in which these muscles develop. The identification of myoblasts associated with thoracic nerves during larval life and the association of migrating myoblasts with nerves during metamorphosis indicate the existence of nerve-muscle interactions during indirect flight muscle development. In addition, the developing pattern of axonal branching suggests a role for the target muscles in respecifying neuromuscular junctions during metamorphosis. Key words: Drosophila, nerve, muscle, innervation INTRODUCTION Some of the most dramatic postembryonic changes in the nervous system take place during the metamorphic phase of holometabolous insects. Metamorphosis is a period of extensive reorganization and involves a restructuring of the larval system to accommodate elements required for the functioning of the adult organism. Studies on the reorganization of the nervous system in the moth Manduca sexta have shown that three major processes are involved: programmed cell death, neurogenesis and respecification (see Truman, 1990 for a review). Cell death removes elements that are not required for adult development. Neurogenesis results in the formation of interneurons, imaginal discderived sensory neurons and some adult-specific motorneurons. At the end of larval life, some motorneurons that lose their larval targets die, others continue to innervate persistent larval muscles, while yet others are respecified to innervate new targets (Levine and Truman, 1985; Thorn and Truman, 1989). In Drosophila, restructuring of the larval abdominal innervation is closely associated with development of the adult musculature. The metamorphosing segmental nerve trunks serve as pathways to guide myoblasts to their final destination (Currie and Bate, 1991). The involvement of the nervous system in muscle development has also been demonstrated in the case of the male-specific muscle in the 5th abdominal segment of the adult, the identity of which is controlled, at least in part, by the nerve innervating it (Lawrence and Johnston, 1986). These results show that the nervous system plays an important role in the development of musculature. The indirect flight muscles (IFMs) of the Drosophila thorax are a promising system to address questions relating to the genetic basis of nerve and muscle interactions during development. Mutants have been isolated in screens for flightlessness and several genes affecting flight musculature have already been studied (Deak, 1977; Koana and Hotta, 1978; Homyk and Shepherd, 1977; Mogami and Hotta, 1981; Deak et al., 1982; Fleming et al, 1983; Costello and Wyman, 1986). The IFMs are made up of two groups of muscles, the dorsal longitudinal muscles (DLMs) and the dorsoventral muscles (DVMs). At the onset of metamorphosis, a wave of histolysis destroys the thoracic larval body wall muscles except for certain larval muscles in the second mesothoracic segment. The muscles that escape histolysis then undergo transformations to give rise to the six DLMs. In contrast, the DVMs develop at the same time by the de novo fusion of myoblasts (Fernandes et al., 1991). The development of innervation to the IFMs can thus be examined in contrasting situations - the DLMs, where a form of target already exists, and the DVMs, where the target develops de novo. In addition, the anatomy of the adult thoracic muscles and aspects of adult IFM develop-

2 216 J. Fernandes and K. VijayRaghavan ment and function have features that may require novel mechanisms and interactions to operate (Fernandes et al., 1991). The DLM motorneurons are modified larval neurons (Hummon and Costello, 1987) and fall into the class of neurons that are respecified during metamorphosis. Using neuronal and myoblast markers, we have followed early events in the modification of larval mesothoracic nerves to innervate the developing DLM and DVM fibres. Little is known about the development of innervation to the latter group of muscles. Besides addressing the question of respecification, our studies have allowed us to investigate nerve-muscle interactions that take place during metamorphosis. Our results show that thoracic nerves harbor myoblasts during larval life and could guide IFM progenitor myoblasts during metamorphosis to their target sites. Further, the nerves themselves grow their processes and arborize in a manner that strongly suggests that their developing muscle targets provide cues for this process, which leads to the final innervation pattern seen in the adult. We discuss the mechanisms that could operate in the thoracic motor system during metamorphosis with specific reference to those that are used in nerve-myoblast and nerve-muscle interactions to specify the pattern of flight muscles and their innervation seen in the adult. MATERIALS AND METHODS Collection, timing and dissection of pupae Canton-S (CS) was used as the wild-type strain for this study. Flies were raised on standard Drosophila culture medium at 25 C. White prepupae were collected from bottles and placed on moist tissue paper in a Petri dish. The white prepupal stage, which lasts for about 1 hour at 25 C, was taken as the initial time point (0 hours APF, after puparium formation). Pupae thus collected were aged and dissected. Pupal dissections were performed as described in Fernandes et al. (1991). Larval dissections were performed as for the pupae, except that Ca 2+ -free Ringer s was used for the dissections. Antibody staining Antibodies to the twist gene product (Thisse et al., 1988), raised in rabbit, were a generous gift from Dr Fabienne Perrin-Schmitt (CNRS, Strasbourg, France). The antibody was used at a dilution of 1:500. Pupal tissue, fixed for hour, was washed thoroughly with PBT (0.3% Triton X-100 in phosphate-buffered saline (PBS: 200 g/l NaCl, 5.0 g/l KCl, 5.0 g/l KH 2PO 4, 27.8 g/l Na 2HPO 4.2H 2O), blocked with 0.5% bovine serum albumin (BSA) and incubated in the diluted TWIST antibody for 36 hours at 4 C. The tissue was then washed in PBT for 1 hour and incubated for 1 hour in 0.2% goat serum. The preparations were then bathed in biotinylated second antibody (raised in goat against rabbit immunoglobulin) for 1 hour, washed in PBT and the goat antibody coupled to an avidin-horseradish peroxidase (HRP) complex using the ABC Vectastain kit (Vector laboratories, USA) as described by the manufacturer. The HRP reaction was developed using 0.05% di-amino benzidene (DAB), and 0.015% H 2O 2. The reaction was stopped using PBT, the preparations dehydrated in alcohol, cleared in xylene and mounted in DPX (Sisco Research Labs, Bombay). The nervous system was revealed using antibodies to HRP and also with the monoclonal antibody mab 22C10 (Fujita et al., 1982; see below). After fixing and washing as described above, tissue preparations were incubated overnight at 4 C in anti-hrp antibody raised in goat (Cappel) at a dilution of 1:250. The tissue was then washed with PBT, incubated for 2 hours in 1:250 dilution of HRP coupled anti-goat immunoglobulin (Jackson Labs, USA) antibody raised in donkey, washed for 1 hour in PBT, treated with DAB, dehydrated and mounted as described above. The monoclonal antibody, mab 22C10 was provided by Dr Seymour Benzer (Caltech, USA) and was used at a dilution of 1:50. Biotinylated anti-mouse immunoglobulin from Vector Laboratories was used as secondary antibody and the reaction to reveal the antigen-antibody complex was as described above. For experiments involving labelling with two antibodies, the TWIST antibody was first applied for 36 hours, washed for 1 hour in PBT, followed by the other primary antibody (anti-hrp or mab 22C10). The TWIST antibody staining was developed first and the preparations were then processed to reveal the profile of the other primary antibody. Microscopy was done using a Zeiss compound microscope equipped with Nomarski optics. RESULTS The dorsal musculature in the mesothorax and its innervation The larval body wall musculature is a segmentally repeated arrangement of about 30 muscles per hemi-segment (Hooper, 1986). Each segment has a characteristic pattern of muscle fibre organization, with specific variations in the thoracic and certain abdominal segments (Hertweck, 1931; Crossley, 1978; Campos-Ortega and Hartenstein, 1985; Hooper, 1986; Bate, 1990). The musculature is classified as external or internal (Crossley, 1978) with respect to its proximity to the body wall. Closest to the dorsal midline is the dorsal triplet muscle, which is found in the mesothorax (T2) as well as the metathorax (T3). It belongs to the internal set of body wall muscles and is designated muscle 30 (Hooper, 1986). The other dorsal internal oblique muscles are 1, 2, 3 (Fig. 1A) and 4. Muscles 9, 10, 19 and 20 are the dorsal external oblique muscles. Of these, muscles 9, 10 and 19 are modified to serve as templates for the adult DLMs (Fig. 1B). We have earlier referred to 9, 10 and 19 as larval oblique muscles (LOMs) 1, 2 and 3 respectively (Fernandes et al., 1991). Antibodies to HRP (Jan and Jan, 1982) were used to reveal the innervation pattern of thoracic body wall muscles in T2. The segmental nerves in a larva are made up of two fascicles which are also referred to as the intersegmental nerve (ISN, the anterior fascicle) innervating dorsal targets and the segmental nerve (SN, the posterior fascicle) which innervates lateral and ventral targets (Goodman et al., 1984). In every segment, a sensory branch joins the ISN in the lateral region and serves as a useful marker through metamorphosis. (This nerve has been marked with an asterisk in Figs 1, 2, 3.) The ISN, as it traverses the dorsal region of a segment, gives off an upper branch that innervates the internal muscles and a lower branch that innervates the external muscles of the body wall. A similar pattern is seen for the abdominal muscle innervation (Johansen et al., 1989a). Thus, close to the dorsal midline, an upper branch innervates muscle 1, while a lower one innervates muscle 9. The ISN extends below muscle 1 to innervate the dorsal triplet (Fig. 1).

3 Drosophila flight muscle innervation 217 A B Fig. 1. Schematic representation of innervation to the dorsal muscles in the second (T2) thoracic segments of a third instar larva. (A) This figure is based on the observation of nerves labeled with anti-hrp antibodies and shows the intersegmental nerve innervating the dorsal musculature, comprising internal muscles 30, 1, 2, 3 and external muscles 9, 10, 19, 20. We have designated the muscle medial to 2 as 3. In its pattern of synaptic endings, muscle 3 of T2 is similar to muscle 4 of T3. The internal musculature is identical in T2 and T3 (9, 10, 19 and 20 ), and is innervated similarly. However, the muscles 9, 10 and 19 in T2 escape histolysis at the onset of metamorphosis (B) to form the DLM templates. * indicates the sensory branch of the ISN in the dorsal region. The following sections will describe how the T2 larval innervation to both the external and internal muscles undergo restructuring during metamorphosis to innervate the developing indirect flight muscles. Development of innervation to the DLM At 6 hours APF, shortly after the onset of puparium formation, most larval thoracic muscles with the exception of muscles 9, 10 and 19 have histolyzed (Fernandes et al., 1991). While the ISN maintains contact with the persistent larval muscles, there is an obvious regression of synaptic contacts (boutons) at the neuromuscular junctions (Fig. 2). Remnants of innervation to the dorsal triplet can also be seen at this stage (Fig. 2B, arrow). On the basis of bouton size, two kinds of motor endings have been identified on larval muscles (Johansen et al., 1989a). Type I processes have boutons of larger diameter than the type II processes and have a more localized distribution than the latter. At 6 hours APF, both type I and type II motor endings are seen on the persistent larval muscles (Fig. 2). At 8 hours APF (Fig. 2B), type I boutons can no longer be seen on muscles 10 and 19, but they are still present on muscle 9. This is consistent with the delay in the transformations of this, the most dorsal larval pretemplate (Fernandes et al., 1991). By 10 hours APF, a number of processes, probably neurite outgrowths, appear to emanate from the ISN at points where it contacts the muscle fibres (Fig. 2E,F). At this stage, type Fig. 2. Changes in the profile of synaptic endings on the larval muscles 9, 10 and 19 (6-10 hours APF). Dissected whole mounts of developing pupae were labelled with anti-hrp. The right hemisegment of T2 is shown in each case. Anterior is to the top. The dorsal midline (dml) is toward the right end of each frame. B,D and F are camera-lucida representations of A,C and E, respectively. * indicates the sensory branch of the ISN. (A,B) 6 hours APF. Innervation to muscles 9, 10 and 19 is in the process of regression. Remnants of the ISN branch to the dorsal triplet is marked with an arrow. Both type-i(open arrowhead) and type-ii endings (closed arrowhead) are seen on muscle 9. (C,D) 8 hours APF. Regression of larval innervation continues. (E,F) 10 hours APF. Neurite outgrowths (arrows) are seen at the larval neuromuscular junctions. The remnant of the ISN branch to the dorsal triplet is no longer seen. Bar, 50 µm.

4 218 J. Fernandes and K. VijayRaghavan I boutons are not seen. The type II endings continue to be seen until 12 hours APF and may be involved in respecification dependent on changes taking place on the target. Between 10 and 12 hours APF, the developing innervation changes very rapidly and this is probably the consequence of the onset of adult innervation developing even as regression of the larval innervation is taking place. Significant changes are seen at 12 hours APF in both the muscle and the nerve (Fig. 3A). The muscles have transformed to give rise to pretemplates which will later split by longitudinal cleavage (Fernandes et al., 1991). In place of the numerous neurite outgrowths, two branches from the ISN are seen over each pretemplate, in opposite directions. However, the pretemplate derived from muscle 10 has three branches (Fig. 3A). Since we know that finally there will be only two branches, one on each of the muscles that are derived from this pretemplate (Hummon and Costello, 1988), it is likely that retraction of improper contacts can take place even at or after 12 hours APF. Branches to the ventral most pretemplate and to the developing DVM III are not as distinct (Fig. 3A,B). The branches that can be seen have numerous fine processes. In addition, there are processes along the nerve trunk in the region of the pretemplates, which continue to be seen at 14 hours APF (Fig. Fig. 3. Modification of the ISN: establishment of innervation to the developing DLMs (12-18 hours APF) as revealed by antibodies to HRP. Anterior is to the top and dorsal midline is to the right. * indicates the sensory nerve and arrow indicates the ISN branch to DVM III. Bar, 30 µm. (A) 12 hours APF. The neurite outgrowths seen at the larval NMJs at 10 hours APF (Fig. 2E,F) have become modified into two branches of the ISN with numerous fine processes, which appear to grow towards each pretemplate. In this preparation, the second pretemplate is contacted by two posteriorly directed branches of the ISN, one of which will presumably retract later. (B) 14 hours APF. The pretemplates at this stage are splitting. The ISN branches to each pretemplate have extended along the muscles. (C) 16 hours APF. The branches of the ISN extended further and they now possess short processes that contact the developing DLM templates. (D) 18 hours APF. Branches of the ISN to the two ventral pretemplates are made up of two axons each (arrowheads).

5 Drosophila flight muscle innervation 219 3B). At this stage, the branches extend over the splitting pretemplates. At 16 hours APF, each branch to the splitting pretemplates derived from muscles 10 and 19 are made up of two components, with processes radiating out to contact the developing DLM templates (Fig. 3C). The two components are most likely the axons from two different neurons. They begin separating out by 18 hours APF (Fig. 3D) and, by 20 hours APF, each of them innervates one DLM template (Fig. 4A). It is interesting to note that the ISN branches sent out to the most dorsal larval pretemplate do not show the two components as seen for the other two pretemplates (Fig. 3C,D). This correlates with the innervation of the dorsalmost two DLM fibres by a single contralateral neuron, MN 5 (Ikeda and Koenig, 1988). Thus, events in the restructuring of larval innervation to give rise to the adult DLM innervation pattern closely parallel the transformations of the larval muscles to form the DLM templates. The possible causal relationship between nerve and muscle development in this process are discussed later. The pattern of innervation seen at 20 hours APF is very similar to that reported for the adult (Fig. 4 and Hummon and Costello, 1988). The monoclonal antibody mab 22C10 was also used to examine the development of innervation to the IFMs. This antibody stains a subset of the aspects of innervation revealed by anti-hrp staining. Neuromuscular junctions and outgrowths of the metamorphosing larval nerves can be seen with anti-hrp but not with mab 22C10. However, mab 22C10-labelled preparations allow the muscle to be seen clearly, which is an advantage. Thus, the gross pattern of changes in innervation can be followed in parallel with changes in the developing musculature. mab 22C10- stained 16, 20 and 24 hours APF preparations are shown in Fig. 5. The two components of each branch of the ISN are not obvious at hours APF, but are readily seen at 24 hours APF, by which time the DLMs have grown in volume. Development of DVM innervation In addition to developing without the use of larval templates, certain other features of the DVMs are relevant. The DVMs exist in three distinct bundles (Miller, 1950; Crossley, 1978). DVM I and DVM II develop in a lateral position with respect to the DLM fibres and are readily seen in dissected whole mounts of developing pupae (Fernandes et al., 1991). DVM III is the shortest of the three DVM bundles and is not easily seen in dissected preparations because A Fig. 4. Innervation pattern to the DLMs at 20 hours APF resembles that in the adult. (A) Camera lucida of a 20 hours preparation stained with anti-hrp. Bar, 100 µm. (B) Schematic representation of the adult innervation to the DLM, modified from Hummon and Costello (1988). B of its location below the developing DLMs. The jump muscle (the tergal depressor of the trochanter, TDT) is positioned in between DVM I and DVM II (Fig. 7C). As described earlier, innervation to the persistent larval muscles regresses by 8 hours APF. At this time, a comparable phenomenon is not observed for the DVMs (Fig. 6A), presumably because they do not use larval templates (Fernandes et al., 1991). The wave of histolysis that occurs at the onset of metamorphosis would have destroyed larval muscles in the region where the DVMs will develop and innervation to the histolysing muscles may have regressed simultaneously. The phenomenon could be similar to the manner in which larval innervation is withdrawn during adult abdominal development in Drosophila (Currie and Bate, 1991) and Manduca (Levine and Truman, 1985). At 10 hours APF, although there are no obvious signs of the DVMs, the ISN and the SN show short branches in the region, which are likely to be the beginnings of an innervation pattern to DVMs I and II (Fig. 7B). However, myoblasts are present in the region and begin to segregate into distinct clusters (Fig. 8A). At 12 hours APF, the developing DVMs are visible in preparations stained with mab 22C10 (Fig. 8B). Branches of the ISN and SN, which will innervate the TDT and DVMs I and II, are found associated with clusters of myoblasts as seen in preparations double labeled with anti-hrp and anti-twist antibodies (Fig. 7A,B). A posteriorly directed outgrowth from the metamorphosing ISN is seen growing towards the region where DVM III has begun to develop (Fig. 3A, arrow). Thus, at 12 hours APF, a certain amount of myoblast fusion has taken place, initiating DVM development, while the ISN and SN send out several processes to the developing muscles. We have previously been able to detect these muscles at 14 hours APF, using the Actin88F-lacZ transformant line (Fernandes et al., 1991) by which time segregation of myoblasts between the DVMs I and II is almost complete (Fig. 8B). At 18 hours APF, two mesothoracic nerves can be seen exiting the ventral ganglion (Fig. 7C,D). The anterior nerve, which was initially the larval ISN, gives off a branch to the TDT, then continues anteriorly towards the dorsal region and innervates DVM I. At a point just before the ISN branches innervate the developing DLMs, it gives off a posterior branch to innervate DVM III (Fig. 7C,D). The second mesothoracic nerve that leaves the ventral ganglion is the larval SN and has at least three distinct branches, the anteriormost innervating DVM II and a direct muscle (Fig. 7C,D). The pattern of innervation at this stage (18 hours APF) is likely to be similar to the adult as it does not change much during the next 6 hours, by which time the DLM innervation pattern resembles that of the adult. Association of myoblasts with nerves In close association with abdominal segmental nerves in the third instar larva, three discrete groups of myoblasts are found - the dorsal, lateral and ventral clusters (Bate et al., 1991). The progeny of these clusters give rise to the dorsal, lateral and ventral adult abdominal musculature, respectively (Currie and Bate, 1991). In contrast, myoblasts that contribute to the development of the thoracic and head musculature are associated with imaginal disc epithelia (Madhavan and Schneiderman, 1977; Poodry and Schneiderman,

6 220 J. Fernandes and K. VijayRaghavan 1970; Lawrence, 1982; VijayRaghavan and Pinto, 1984; Bate et al., 1991; Fernandes et al., 1991) and are referred to as adepithelial cells (Poodry and Schneiderman, 1970). Antibodies to the twist gene product label the myoblast clusters of the abdominal segments as well as the adepithelial cells associated with imaginal discs (Bate et al., 1991) and have been used to follow the development of muscles that they give rise to (Currie and Bate, 1991; Fernandes et al., 1991). In the thoracic segments of the third instar larva, we find twist-expressing cells associated with segmental nerves in a manner similar to the abdominal segments. The dorsal cluster of twist-expressing cells in T2 and T3 are shown in Fig. 9A. Another kind of association of myoblasts with nerves is seen later during the development of the IFMs. At 6 hours APF, myoblasts are released into the thorax as the discs evert (Fernandes et al., 1991). At Fig. 5. mab 22C10-staining profile of innervation to the developing DLMs. The muscles are seen more clearly than in the anti-hrp-labeled preparations (compare with Fig. 3). Anterior is to the bottom and dorsal midline is to the right. * indicates the sensory nerve and arrow indicates DVM III. Bar, 180 µm. (A) 16 hours APF and (B) 20 hours APF are preparations that have been double labelled with anti-twist and mab 22C10. The two components of the ISN branches as seen in preparations stained with anti-hrp (Fig. 3C, E) are not visible here. (C) 24 hours APF labelled with mab 22C10 alone. The two components of each ISN branch have separated to innervate the growing DLM fibres. Compare with Fig. 3E.

7 Drosophila flight muscle innervation 221 A B Fig. 6. Restructuring of the ISN and the SN in the region of the DVMs. There are no signs of the developing DVMs at these stages. However, larval nerves show signs of being restructured. Anterior is to the top and dorsal midline to the right. * indicates sensory nerve branch. Bar, 60 µm. (A) 8 hours APF. The anterior branch of the T2 nerve is the ISN, while the posterior one is the SN. The lateral and ventral region of the mesothorax are devoid of persistent larval muscles unlike in the dorsal region, where persistent larval muscles are present. (B) 10 hours APF. The two ISN branches that are modified to innervate the IFMs are evident at this stage. One of them proceeds dorsally to innervate the persistent larval muscles, while the other is remodelled to later innervate DVM I and the TDT. The latter branch, at this stage, shows a number of processes in the region where DVM I and the TDT will develop. type II endings seen at both stages. this time and until a few hours later (12 hours APF), myoblasts are distributed in the vicinity of the ISN and SN (Fig. 8A,B). In T3, where fewer twist-expressing cells are present, they can be seen adhering to the segmental nerve in the dorsal region (unpublished observations). In the region where the DVMs will develop, the cloud of myoblasts begins to segregate by 10 hours APF (Fig. 8A) and, by 14 hours APF, the segregation is almost complete (Fig. 7C). At this time, myoblasts expressing twist can be seen adhering to the metamorphosing ISN nerve trunk between DVM I and the DLMs. This association can be seen even at 20 hours APF by which time the six DLM templates have formed (Fernandes et al., 1991). DISCUSSION We have described the restructuring of larval mesothoracic nerves during metamorphosis to innervate the developing indirect flight muscles during the first 24 hours of metamorphosis. The events that take place during this time can be broadly divided into three major categories - regression of larval synaptic contacts, a transition phase when the environment is probably sampled by neurite outgrowths and, finally, outgrowth and elongation of the adult branches. As the larval innervation is restructured, muscle development proceeds in parallel, showing many features that suggest nerve-muscle interactions during IFM development. These features are the association of myoblasts with thoracic nerves during larval life, the close proximity of migrating myoblasts to nerves during metamorphosis and the development of nerve branching which suggests that the target muscles may influence nerve pattern. Innervation to the DLMs and the DVMs (the two groups of indirect flight muscles) develop differently and this may be a consequence of the different modes of development of these muscles. Fig. 10 compares the development of innervation to the DLMs and the DVMs, and summarizes the events involved. Larval nerves and their adult counterparts The adult targets of the modified ISN and SN include muscles from the two lineage sets identified in the adult mesothorax (Lawrence, 1982): the DLMs and DVMs belong to the dorsal lineage set (with their progenitor myoblasts associated with the wing disc) while the TDT and the leg muscles belong to the ventral lineage set (with their progenitor myoblasts associated with the mesothoracic leg disc). Thus, the ISN, which innervates solely dorsal targets in the embryo is modified to include a ventral target, the TDT, in the adult. Similarly, the SN, which innervates ventral and lateral targets in the embryo, is modified to include dorsal targets: DVM II and at least one direct muscle. It follows that adult muscles belonging to the same lineage set are not innervated by nerves that are similarly segregated. In the adult, the DLMs are innervated by motorneurons that send their axons through the posterior dorsal mesothoracic nerve (PDMN). This nerve also innervates the TDT or the jump muscle (Ikeda and Koenig, 1985). We have seen that innervation to the DLMs arises by the restructuring of the larval ISN. We have also seen that the reorganized ISN innervates DVM I and III. It follows therefore that the ISN gives rise to the PDMN. Identity of the IFM motorneurons The six DLMs of the Drosophila thorax are innervated by five motorneurons: the dorsalmost two DLMs are innervated by a single motorneuron (MN5) and the remaining DLMs are each innervated by one motorneuron (Cogshall, 1978; Ikeda and Koenig, 1985). It is possible that these motorneurons are among the ones that innervate the dorsal musculature of the larval mesothorax. While innervation to the DVMs could develop from the respecification of motorneurons that innervate larval muscles, another possibility is that these muscles are innervated by cells that have no larval targets and which differentiate into neurons and send out axons only during metamorphosis. Some leg motorneurons belong to such a class (Truman, 1990). In the lateral region of the mesothorax, where the DVMs develop, nerve endings are seen after larval muscles have degenerated. Hence, it is likely that the DVMS are innervated by persistent larval neurons. DVM III is the most dorsal of the DVMs. Since persistent nerve endings, similar to those seen

8 222 J. Fernandes and K. VijayRaghavan for DVMS I and II, are not observed in the region where DVM III (the most dorsal of the DVMs) develops, it is possible that DVM III is innervated by motorneurons that do not have larval target. Motorneurons that innervate the abdominal musculature have recently been described (Sink and Whitington, 1990). At least five motorneurons innervate the dorsal musculature, and these include RP2, acc, U and two of the VUMs. It is possible that a similar set of motorneurons innervates the dorsal musculature of the larval mesothorax and are respecified to innervate adult targets that develop in the region during metamorphosis. This is in accordance with observations in Manduca that adult targets of persistent larval motorneurons usually develop in the same general region where the larval target was located (Levine and Truman, 1985). Neurons that innervate muscle 9 in the abdominal segments have not been described. In T2, this muscle is the persistent larval muscle that gives rise to the two dorsalmost DLM fibres (Fernandes et al., 1991), which are innervated by the contralateral motorneuron MN 5 (Ikeda and Koenig, 1985). Assuming that the dorsal triplet is innervated by a different motorneuron, the dorsal musculature of the larval mesothorax may be innervated by at least seven motorneurons, some of which may be respecified to innervate the developing IFMs. Reorganization of larval nerves in Drosophila has been observed in the development of the adult mushroom bodies (Technau and Heisenberg, 1982) and also in the develop- Fig. 7. Establishment of innervation to the DVMs. C and D are camera-lucida drawings of A and B, respectively. Anterior is to the top and dorsal midline is to the right. Solid arrow indicates the ISN proceeding dorsally to innervate the persistent larval muscles. Open arrow indicates the SN branch which is later restructured to innervate the future DVM II. Closed arrowhead indicates the ISN branch that will innervate the TDT. Open arrowhead indicates ISN branch that will innervate DVM I. Small arrow indicates ISN branch that will innervate DVM I. (A,B) 12 hours APF. Double labelling with anti-twist and anti-hrp. The low signal for the TWIST antibody allows clear observation of the developing innervation. The ISN and the SN are in the process of being restructured. The ISN has two branches in the lateral region; an anterior branch, which contacts the developing DVM I, and a posterior branch, which contacts the developing jump muscle. The SN branch, which contacts the developing DVM II (open arrow), and the ISN branches have numerous small processes with myoblasts adhering to them. Bar, 40 µm. (C, D) 18 hours APF. Restructuring of the ISN and SN is complete. The nerve branches have extended and run along most of the muscle length. Innervation to DVM III is indicated by a small arrow. The jump muscle (TDT) and the direct muscle below DVM II are stippled. Bar, 50 µm (C) and 70 µm (D).

9 Drosophila flight muscle innervation 223 ment of adult innervation in the abdomen (Currie and Bate, 1991). Nerve regression at the persistent larval muscles in the thorax must be restricted to the neuromuscular junctions, since the nerve trunks of the ISN or the SN are never devoid of tracts. This is different from the reorganization in mushroom bodies, where entire axon tracts are eliminated and new axon outgrowth takes place from the persisting neurons (Technau and Heisenberg, 1982). Thus, while dye-filling experiments are required to identify the larval identity of axons that innervate adult muscles, our experiments suggest that most IFM neurons probably innervate larval targets in the region where adult muscles develop. segmental nerves. The dorsal cluster of twist-expressing cells adheres to the ISN from the region of the dorsal-triplet muscle (Fig. 1) up to the region where the DVMs will Role of nerves in harboring founder cells for the IFM pattern Apart from the myoblasts being present in imaginal discs in the thoracic regions, we have also found them along the Fig. 8. Distribution of TWIST-expressing myoblasts in the mesothorax during development of the IFMs. Dorsal midline is to the right and anterior is to the top. DVMs I and II are marked I and II, respectively. (A) 12 hours APF. This is the earliest stage that the DVMs are seen. The camera-lucida drawing is from a mab 22C10-stained preparation. The antibody at early stages marks myoblasts and continues to indicate muscle structures at least until 24 hours APF (see Fig. 5). The distribution of myoblasts is seen in relation to the ISN and SN. Bar, 80 µm. (B) 14 hours APF. Segregation of myoblasts around the developing DLM and DVM units is almost complete. At this stage, some myoblasts can be seen adhering to the ISN (arrow), which is in the process of being restructured. Bar, 60 µm. Fig. 9. Association of twist-expressing cells with segmental nerves in the third instar larva. Bar, 60 µm. (A) Photomontage of the larval body wall musculature in T2 (top half) and T3 (bottom half) stained with antibodies to the twist gene product, showing the dorsal cluster of twist-expressing cells associated with the segmental nerves. The left hemi-segment is shown. * indicates sensory nerve, arrows point to the intersegmental muscles and the arrowhead indicates the dorsal triplet. (B) An abdominal segment showing the dorsal and lateral clusters of twist-expressing cells associated with nerves.

10 224 J. Fernandes and K. VijayRaghavan develop (Fig. 7A). One possibility is that they could be pioneer (Ho et al., 1983) or founder (Bate, 1990) cells for the formation of specific thoracic muscle groups. Since the larval muscles span the region in which the DLMs will eventually develop, we had suggested earlier (Fernandes et al., 1991) that the persistent larval muscles could play a role similar to muscle pioneers in the grasshopper embryo (Ho et al., 1983). It is possible that both the persistent muscles and the twist-expressing cells associated with nerves in the region may cooperate in organizing myoblasts released from the everting wing disc, to form the thoracic musculature. The merit in this hypothesis for the role of nerve-associated myoblasts is that it reconciles the strict lineage sets observed in thoracic muscle development (Lawrence, 1982) with the apparently contradictory ability of wing-disc-associated myoblasts to contribute to diverse muscle groups (Lawrence and Brower, 1982). Could nerves direct the transformations of persistent larval muscles? In Lepidopteran larvae, nerves have been implicated in a trophic role, important only in the early stages of development (Nuesch, 1985). When nerves are severed at the onset of metamorphosis, nuclear divisions of myoblasts are reduced in number, leading to the formation of a thinner fibre. If denervation is carried out later, the adult muscle seems to be better formed (Neusch, 1985). It is unclear whether nerves play a similar role in the development of the thoracic musculature in Drosophila. However, our studies do show that growing nerves contact myoblasts. This 6h - 10h APF Events in the development of IFM innervation DLMs Synapses at larval neuromuscular junctions have regressed alomost completely at 6h Remnants of larval innervation seen at 8h Beginnings of adult outgrowth seen at 10h DVMs LARVAL INNERVATION REGRESSES AND ADULT NEURITE GROWTH BEGINS Two branches from the SN seen at 10h. One of them will innervate DVM II Two branches from the ISN visible at 10h. These will later innervate DVM I and the TDT. 10h - 14h APF EXTENSIVE BRANCHING APFOF AXONS OVER DEVELOPING MUSCLES Two ISN branches grow towards each pre-template. Numerous processes are seen along these branches (12h). A branch of the ISN that innervates DVM I and the TDT shows numerous proceses. Close to the pre-templates, an ISN branch emanates towards DVM III. The SN that innervates DVM II also shows extensive branching over the developing muscles. 14h - 18h APF THE PATTERN OF NEURITE APF GROWTH IS REFINED AND THE BASIC ELEMENTS ARE LAID DOWN Each ISN branch to the splitting pre- templates has two components (16h). The pre-templates are contacted by axonal processes. The ISN branch that innervates DLM I and the branch that innervates DVM III extend processes over their targets (16h). A similar extension of neurites emanating from the SN branch innervating DVM II (16h). 18h - 24h APF THE ADULT PATTERN OF FLIGHT MUSCLE INNERVATION IS ESTABLISHED The ISN has been modified to innervate the 6 DLM templates. Innervation to DVM I, III and the TDT develop from restructuring of the ISN. DVM II is innervated by a branch of the SN. Fig. 10. Summary of events in the development of the indirect flight muscles and their innervation (0-24 hours APF). The dots that surround the developing nerves in the schematic shown on the left of the figure represent the migrating myoblasts.

11 Drosophila flight muscle innervation 225 may be an instance where nerve-muscle interactions are necessary for subsequent events. We had earlier suggested (Fernandes et al., 1991) that splitting of the pretemplates may be directed by the developing adult nerve outgrowths. Other possible mechanisms are that the transformations of the larval muscles are a passive consequence of the growing epidermis or a cell-autonomous consequence of the onset of metamorphosis. Our studies on mutations that transform T3 nerves to T2 (J. F., S. Celniker, E. B. Lewis and K. V. R., unpublished data) suggest that changing the segmental identity of the nerve is not sufficient to generate larval templates in T3. Recently, mutations of a steroid-hormone-responsive gene have been shown to affect the DVMs significantly more than the DLMs (Restifo and White, 1992). Thus, while hormonal influences may be important in establishing the different developmental paths of these very similar muscles, it is not understood whether such regulation uses the nervous system or not. Directing myoblasts to their destination Precursors of the adult abdominal musculature are present in the third instar larva as discreet clusters of twist-expressing cells and are associated with segmental nerves. During metamorphosis, the progeny of these myoblasts use the reorganizing nerves as pathways to reach their final destination. In contrast, the bulk of the myoblasts that contribute to the indirect flight muscles are found associated with wing imaginal discs and are released into the thorax as the discs evaginate (Lawrence, 1982; Fernandes et al., 1991; Bate et al., 1991; Currie and Bate, 1991). These myoblasts are far greater in number than the abdominal muscle myoblasts and it is therefore quite unlikely that all of this population could adhere closely to T2 nerves and use them as pathways to reach their final destination in the same way as abdominal nerves are used as guides (Currie and Bate, 1991). However, myoblasts are found distributed in a characteristic pattern in the vicinity of the T2 nerves and this could be explained if myoblasts as well as nerves follow common epidermal or extracellular matrix cues. Another possibility is that the nerves, or associated glia, set up a molecular gradient, which could result in the distribution of myoblasts in close proximity of the nerves. In any event, the organization of the myoblast population over developing nerves is striking (shown as dots in Fig. 10) and could be important in preventing the mixing of groups of myoblasts that contribute to different muscles. Target roles in the establishment of IFM innervation The establishment of innervation to the IFMs and the development of the muscles are closely associated processes (see Fig. 10). Transformation of the persistent muscles begins at a time when synaptic regression is underway (8-10 hours APF). The beginnings of adult innervation are seen at 10 hours APF, in the form of neurite outgrowths that emanate from the ISN trunk. Soon afterwards, when the larval muscles are transformed into elongated pretemplates, the neurite outgrowths are replaced by well-defined branches that appear to grow toward the pretemplates. A number of minute processes radiate out from these branches as well as from the ISN trunk. The processes are present even at 14 hours APF and it is likely that they contact myoblasts in the surround. The ISN branches near the pretemplates subsequently extend and it takes until 20 hours APF (by which time six DLM templates have formed) for the innervation pattern to resemble that of the adult. The neurite outgrowths at 10 hours APF may well be the exploratory processes of motorneuron growth cones, while the 12 hours stage could be the outcome of the exploration and withdrawal of improper contacts, as seen during the development of body wall innervation in the embryo (Halpern et al., 1991). Is the close association of developing nerves and muscles a coincidence, or does it indicate a functional interaction during development? Studies in Manduca have shown that degeneration of muscle and changes in the structure of neurons, which take place during metamorphosis, are under the control of ecdysteroids. These responses, however, are thought to be independent of nerve-muscle interactions. In other words, nerve regression during the onset of metamorphosis is probably a cell-autonomous response to hormones and not target dependent (Weeks and Truman, 1985). It has been suggested that the subsequent respecification of motorneurons could be influenced by the developing adult targets (Levine and Truman, 1985), and this may well be the case with the developing IFM innervation. One kind of nerve-muscle interaction has been demonstrated in the developing Drosophila embryo, in which, prior to the establishment of contact with the muscle fibre, both the approaching growth cone and the muscle fibre transiently express Fasciclin III (Halpern et al., 1991). The growth cones send out exploratory processes to many other muscles in the vicinity and, once the right choice is made, synapse formation follows. We do not know if similar mechanisms operate in the establishment of innervation to the IFMs. In the embryo, innervation develops to muscle fibres that have already differentiated (Johansen et al., 1989b). In contrast, innervation to the indirect flight muscles seems to develop in parallel with the musculature with the branching of and changes in the neurite pattern strongly suggesting that the developing target provides cues that generate the final pattern. DVMs versus DLMs The DVMs are first seen at 12 hours APF in mab 22C10- stained preparations. The beginnings of an innervation pattern to these muscles, however, is seen at 10 hours APF, just as for the DLM. At 12 hours APF, no additional branches are formed-they only extend along the developing muscle fibres. At 18 hours APF, a final pattern is evident and must have been established a little earlier. Thus, innervation to the DVMs is established before that to the DLM fibres (20 hours APF). Unlike the DLMs, the number of fibres in the forming DVMs is the same as that found in the adult, the muscles growing in size as development proceeds. We suggest that the delay in the development of the final pattern of DLM innervation could be due to differences in the mechanism of development between the DLMs and the DVMs. One possibility is that the positioning of molecular cues, required for recognition of sites on the muscle membrane for synapse formation, are laid down later in the case of the DLMs where the remnant larval

12 226 J. Fernandes and K. VijayRaghavan membranes and associated components may need to be removed. Conclusions Our studies on the development of innervation to the indirect flight muscles have shown that the development of innervation and musculature take place in close association and we have reason to believe that nerve-muscle interactions are likely to play a significant role. We have also shown that the DLMs and DVMs differ in the timing of establishment of an adult-like innervation pattern and in their different modes of formation. The availability of mutation combinations that affect the DLMs (Fleming et al., 1983; Costello and Wyman, 1986) or the DVMs (Restifo and White, 1992) allow the detailed analysis of the mechanisms through which myoblast, nerve, muscle and epidermal cues act to form the IFMs. We are grateful to Dr Fabienne Perrin-Schmitt for a generous supply of the TWIST antibody. 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