Comparative development of the extensor and flexor tibiae muscles in ...

Development 101, 351-361 (1987) Printed in Great Britain © The Company of Biologists Limited 1987

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Comparative development of the extensor and flexor tibiae muscles in the legs of the locust, Locusta migratoria CAMILLA M. MYERS and ELDON E. BALL Developmental Neurobiology Croup, Research School of Biological Sciences, Australian National University, PO Box 475, Canberra City, ACT2601, Australia

Summary The embryonic development of the extensor and flexor tibiae muscles in the pro- and mesothoracic legs of the locust, Locusta migratoria, is described, and compared to the previously described development of these muscles in the metathoracic legs. The basic pattern of development of each muscle is the same in all three pairs of legs. The extensor tibiae (ETi) forms a giant syncytium, or supramuscle pioneer, which then breaks up into a series of muscle pioneers. The flexor tibiae muscle (FITi) is formed directly by sequential addition of individual muscle pioneers. Thus, there are at least two fundamentally different patterns of muscle development in the embryonic locust, as exemplified by these two muscles, and supramuscle pioneer formation is not a unique feature of the metathoracic ETi associated with its evolutionary hypertrophy. In spite of a basically similar pattern of development of homologous muscles in all three pairs of legs, there

Introduction

The embryonic development of the metathoracic extensor tibiae muscle (ETi) of the grasshopper has recently been described (Ball, Ho & Goodman, 1985; Ball & Goodman, 1985a). During its early embryonic development, this muscle passes through a syncytial stage, called a supramuscle pioneer (supraMP), which ultimately contains hundreds of nuclei. Between 50 and 60% of embryonic development this syncytium breaks up into a series of smaller syncytia, each of which corresponds to a future muscle bundle. The flexor tibiae muscle, by contrast, does not pass through a supraMP stage, but directly forms the smaller syncytia corresponding to future muscle bundles. By 60 % of embryonic development, both muscles have arrived at a similar condition and from that stage

are significant developmental differences between the metathoracic ETi and FITi and their homologues in the anterior legs. First, during development of the ETi muscles, the ETi MP forms a double row of attachment sites along both walls of the metathoracic leg, while in the anterior legs there is only a single row. Second, during development of the FITi muscles, the proximal MP, which lies at the tip of the apodeme, dies and breaks down in the metathoracic limbs. In the pro- and mesothoracic limbs it remains intact and eventually forms a large proximal muscle bundle. Third, the accessory ETi and FITi muscles, which develop in the metathoracic legs, are not formed in the anterior legs. Key words: muscle development, locust, Locusta migratoria, muscle pioneer, extensor tibiae muscle, flexor tibiae muscle.

onward both develop in a similar manner, so there is no obvious reason why their early development should be so different. One possibility is that the pattern of development of the metathoracic ETi is somehow related to the evolutionary hypertrophy of this muscle in relation to its function of powering the jump. One way of testing this hypothesis and of simultaneously establishing the degree of embryonic similarity between legs which are morphologically quite different in the adult, is to examine the development of the homologous muscle in the pro- and mesothoracic legs, which are not specialized for jumping. These legs are much smaller than the metathoracic and function quite differently, the prothoracic legs being specialized for exploration and walking, and the mesothoracic legs playing a role in postural support and walking (Burns, 1973).

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C. M. Myers and E. E. Ball

Materials and methods Embryos Qutches of eggs of Locusta migratoria were laid in cups of damp sand and incubated at 28°C. Under these conditions embryonic development takes approximately 14 days. Embryos were staged according to the criteria given by Bentley, Keshishian, Shankland & Toroian-Raymond (1979) for Schistocerca. These criteria can readily be applied to Locusta and the ages given in this paper are based on them. We have not established whether the criteria correspond to exactly the same absolute ages in the two species although they do not appear to differ dramatically.

Antibody

t proximal bundle FITi

FITi AETi FCO I A FITi

treatment

Embryos intended for staining with monoclonal antibodies were dissected in locust Ringer (NaCl, 8-7 gl" 1 ; KC1, 0 - 2 2 g r ' ; CaCl 2 .2H 2 O, 0-29gl"'; MgS0 4 .7H 2 O, 0-25 gl" 1 ; TES, l-15gl"'; pH7-0) and quickly transferred to 2 % paraformaldehyde in Millonig's phosphate buffer. Embryos older than 40 % were treated with chitinase (Sigma) to permeabilize or remove the cuticle that begins to be laid down about that time. The antibodies used in this study were 1-5 (Chang, Ho & Goodman, 1983), which stains neuronal tissue and muscle pioneers, and Mes-3 (Kotrla & Goodman, 1984), which stains a small subset of neurones in the central nervous system plus muscle pioneers. Following chitinase treatment the embryos were thoroughly washed in phosphate-buffered saline (PBS) and then placed in a solution containing the antibody, 2 % bovine serum albumen and 1 % Triton X-100 on a shaker overnight at room temperature. Monoclonal antibody binding was visualized with biotin-streptavidin-HRP (Amersham) using standard techniques. The embryos were washed, then dehydrated and cleared through a glycerin series and photographed in whole mount. Intracellular dye fills For intracellular injection of MPs, a mixture of 4 % horseradish peroxidase (HRP, Sigma) and 1 % Lucifer Yellow (LY, Aldrich) was made up in distilled water. This mixture was then centrifuged at 45 000 revs min" 1 for 5min and the supernatant was injected using either current or pressure (Picospritzer, General Valve Corp.). Injected embryos were fixed immediately in 2 % paraformaldehyde and 2-5 % glutaraldehyde in Millonig's phosphate buffer (Bate, 1976). They were then washed, treated with chitinase for a variable time depending on age, washed again and the HRP was then visualized using 3,3-diaminobenzidine and H 2 O 2 . In some preparations, the HRP was intensified with CoCl2 using the techniques of Watson & Burrows (1981). The embryos were then dehydrated and cleared through a glycerin series and viewed and photographed in whole mount.

Terminology The pro-, meso- and metathoracic legs of a locust are shown drawn to the same scale in Fig. 1A. Fig. IB shows the organization and relative sizes of the extensor and flexor tibiae muscles within the basal segments of a mesothoracic leg (the prothoracic leg is similar). The position of the

FITi

Fig. 1. Comparative morphology of the legs of a locust and the organization of their femoral muscles. (A) The legs, all drawn to the same scale. Note the hypertrophy of the metathoracic leg and especially of its femur. (B) Idealized drawing of the femoral musculature of a mesothoracic leg. (The prothoracic femur is similarly organized except that it is slightly smaller as is its femoral chordotonal organ). Note the large proximal FITi bundle which is missing in the metathoracic leg. (C) Idealized drawing of the femoral musculature of a metathoracic leg. Both the extensor and flexor tibiae are larger than they are in the other two legs, but the extensor has become disproportionately enlarged. For clarity, the retractor unguis muscle has been omitted in both B and C. Abbreviations: AETi, accessory extensor tibiae muscle; AFITi, accessory flexor tibiae muscle; c, coxa; ETi, extensor tibiae muscle; FCO, femoral chordotonal organ; f, femur; FITi, flexor tibiae muscle; meso, mesothoracic leg; meta, metathoracic leg; pro, prothoracic leg; fa, tarsus; ti, tibia; tr, trochanter. femoral chordotonal organ (FCO) is shown by the shaded area in the proximal portion of the femur. The same features of a metathoracic leg are shown in Fig. 1C. Note the hypertrophy of the extensor tibiae muscle and the distal position of the FCO. The axes referred to in this paper (Fig. 2) are those of the adult legs shown in Fig. 1. In young embryos, these designations are incorrect by 90° since the future dorsal side of the leg points anterior. During embryonic development the leg gradually rotates into the adult position. Since insects have an external rather than an internal skeleton, their muscles have a different relationship to the skeleton than do those of vertebrates. Apodemes, which are cuticular processes extending inward from the body wall (Snodgrass, 1935), are functionally analogous to vertebrate tendons in that they link muscle to the skeleton. Apodemes originate from invaginations of the ectoderm at the joint between two leg segments. The invaginated ectoderm extends proximally within the leg segment and secretes the cuticular apodeme on its external surface, which in this case faces the inner cavity of the invagination and is continuous

Femoral muscle development in locust embryos dorsal

Fig. 2. Diagram of an embryonic leg at approximately 45 % indicating terminology used in this paper. Abbreviations: f, femur; ta, tarsus; ti, tibia. with the cuticle covering the leg. The ETi and FITi muscle bundles attach proximally to the cuticulanzed ectoderm of the wall of the femur and distally to the cuticulanzed ectoderm of the apodeme. Contraction of the muscle exerts force on the apodeme, thus moving the next distal leg segment at the joint. In embryonic locusts, the apodeme surrounds a substantial cavity, which is continuous with the space outside the embryo (e.g. Figs 5A, 6C, 7A). Results Development of the metathoracic extensor and flexor tibiae muscles

The metathoracic extensor (ETi) and flexor (FITi) tibiae muscles of Locusta migratoria both develop in a manner very similar to that described in the metathoracic leg of Schistocerca gregaria (Ball et al. 1985;

Ball & Goodman, 1985a,b). The ETi muscle begins as a single mesoderm cell which attaches the ectoderm of the invaginating apodeme to the wall of the femur, and is first visible at around 35%. This cell then enlarges dramatically by fusion with surrounding mesoderm cells to form a giant, horseshoe-shaped syncytium around the apodeme, called the supramuscle pioneer (supraMP). At about 45 % of development, however, the supraMP in Locusta deviates from the developmental pattern of Schistocerca, splitting into two halves at the bridge-like connection at the proximal tip of the apodeme. The evidence for this is that HRP injected into one of the lateral arms at this and later stages remains only in that arm. At the same time, the lateral edges of the supraMP

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become scalloped, with certain regions adhering tightly to the ectoderm and neighbouring regions receding from it (Fig. 3A,D). At about 55% the halves of the supraMP begin to break up further and form a series of periodic bridges which connect the apodeme to the wall of the femur. Each bridge forms the core around which a muscle bundle will develop. In contrast, the FITi muscle does not develop from a syncytial supraMP, but rather by the sequential recruitment of individual MPs from the undifferentiated mesoderm cells surrounding the apodeme. The first FITi MPs appear as a symmetrical pair at the tip of the invaginating apodeme at around 37 %. A third MP soon appears between this pair, but this MP later dies at around 47 % (approximately 2 % later than in Schistocerca). Further growth occurs by symmetrical addition of pairs of MPs distally along either side of the lengthening apodeme. Each MP develops into a bundle of muscle fibres by a cycle of fusion with surrounding mesoderm cells followed by division and differentiation into the muscle fibres. Development of the ETi and FITi muscles of the prothoracic and mesothoracic legs, which has not previously been described, is the topic of the following sections. The pattern of development is so similar in the two legs that a single description will suffice for both. Development of the prothoracic and mesothoracic extensor tibiae muscles

The extensor tibiae muscles of the prothoracic and mesothoracic legs of the locust embryo develop from a single muscle pioneer (Fig. 4A,B), in a similar manner to that already described for the metathoracic leg (Ball et al. 1985; Ball & Goodman, 1985a). However, development of the muscles in the two more anterior legs lags behind that in the metathoracic leg by a few percent, depending upon the stage of development. Thus, the first muscle pioneer is not recognizable until around 37 %. This cell then begins gradually to enlarge until by around 42 % it has formed a slightly curved, disc-shaped multinucleated cell over the tip of the apodeme, directly beneath the dorsolateral ectoderm. At this stage, the developing femoral chordotonal organ (FCO) takes up a large proportion of the total volume of the proand mesothoracic femurs (Fig. 4A,D). The development of this large proprioceptive organ in all three legs of Melanoplus has been described by Slifer (1935). It is formed from an invagination of the dorsal ectoderm of the femur, from which the sensory cells and accessory cells of the scoloparia differentiate. As a consequence of the presence of the FCO, the ETi apodeme is not straight, as it is in the metathoracic femur, but curved around to the dorsal posterior edge

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ectoderm of wall of femur

pro & meso

scalloping

individual MPs

meta leg

ventral bridges

individual MPs

Fig. 3. Schematic diagrams summarizing the differences between the development of the pro/mesothoracic ETi muscles and the metathoradc ETi muscle during the period when the supraMP is dividing to form an array of individual MPs. (A) The ETi supraMP consists of two large syncytial arms which connect the ectoderm of the apodeme to the ectoderm of the wall of the femur. At this stage, the supraMP appears similar in all three legs except for differences in size. (B-C) Prothoradc and mesothoracic legs. (B) The scalloping along the lateral edges of the supraMP deepens, with the result that on either side of the apodeme there is a single row of cytoplasmic bridges, connected at their bases, linking the ectoderm of the walls of the femur to that surrounding the apodeme. (C) Each of these cytoplasmic bridges then separates from its neighbours and becomes an individual MP. (D-E) Metathoracic legs. (D) Each arm of the supraMP divides to form a dorsal and a ventral row of cytoplasmic bridges which are linked at their bases and connect the lateral walls of the femur to the apodeme. (E) The links between the cytoplasmic bridges break to form two rows of individual MPs on either side of the apodeme. Abbreviations: ETi, extensor tibiae; MP, muscle pioneer; pro, prothoracic; meso, mesothoracic; meta, metathoradc; supraMP, supramuscle pioneer. of the leg. The supraMP continues to expand gradually (Fig. 4C,D), presumably by fusion with other mesoderm cells (Ball & Goodman, 1985a) and at around 47-48 % it begins to extend distally over the apodeme (Fig. 4E). At the same time, the proximal edge of the supraMP, which lies between the dorsal

ectoderm and the ectodermal invagination of the FCO, begins to become scalloped (Fig. 4F). Between 50 and 52 % the supraMP begins to split into two arms, which often remain connected at the distal end. As the arms grow out they gradually move ventrally to lie on either side of the apodeme (Fig. 5A,B),

Femoral muscle development in locust embryos

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Fig. 4. Development of the prothoracic and mesothoracic ETi muscles. (A) Lateral view of HRP-filled mesothoracic ETi MP at 40 %. Note its relation to its own apodeme and the FCO. Bar: 20fan. (B) Close-up dorsolateral view of the same MP to show its multinucleate nature and how it connects to the apodeme ectoderm. Bar: 10/im. (C) Mesothoracic 1-5 stained ETi MP at approx. 46%. It now contains many more nuclei, and filopodia can be seen radiating from it. Dorsolateral view. Bar: \0fim. (D) 1-5 stained prothoracic ETi MP from the same 46% animal in lateral view, showing how it connects the apodeme ectoderm to the ectoderm of the wall of the leg. The FCO ectoderm is also clearly visible. Bar: 20fim. (E) HRP-filled prothoracic ETi MP at approx. 48%. Note numerous nuclei and filopodia radiating in all directions. Bar: 15,um. (F) By 50% the edges of the ETi MP are becoming scalloped (arrows), as shown in this dorsolateral view of an HRP-filled MP. Bar: 20fan. NB. Proximal is to the left in each micrograph. Abbreviations: apo, apodeme; ect, ectoderm; £77, extensor tibiae; FCO, femoral chordotonal organ.

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FCO ect

Fig. 5. Further development of the ETi muscle. (A) Lateral view of a prothoracic ETi MP at approx. 53 %, showing its position in relation to the FCO ectoderm. Bar: 20/im. (B) HRP-filled ETi MP at approx. 54%. By this stage the horseshoe-shaped MP is separating into two arms. In this preparation, the arms have separated proximally and are linked only by a thin connection distally (arrow) through which the HRP passed. Dorsal view. Bar: 15 fim. (C) A prothoracic ETi MP stained with 1-5 at approx. 56%. The horseshoe is now breaking up into groups of cytoplasmic bridges connecting the ectoderm of the wall of the leg with that of the apodeme. Dorsal view. Bar: 50jtm. (D) HRP fill of prothoracic ETi MP at 57% fills only a small portion of the horseshoe-shaped structure revealed by 1-5. Dorsal view. Bar: 30 pm. (E) The same preparation in lateral view reveals that the prothoracic ETi consists of a single line of cytoplasmic bridges, in contrast to that of the metathoracic leg (Ball & Goodman, 1985a). Bar: 30ftm. Proximal is to the left in each micrograph. Abbreviations: apo, apodeme; ect, ectoderm; ETi, extensor tibiae; FCO, femoral chordotonal organ. which is now relatively straight since by this stage the femur has grown considerably in length, with the FCO now restricted to the proximal end. From 53 to 55 % (Fig. 5B,C) the supraMP looks quite similar to that in the metathoracic femur between 45 and 5 0 % , and the outer edges of the MP have begun to pull away from certain regions of the ectoderm to give a scalloped appearance (Figs 3A,B, 4F). However, the situation in the pro- and mesothoracic legs differs from that in the metathoracic ETi at this stage, where the scalloping of the arms of the supraMP leads to the formation of four rows of cytoplasmic bridges (Fig. 3D,E). These connect the apodeme ectoderm to the ectoderm of the wall of the femur (two rows medially and two laterally; i.e. one dorsal row and

one ventral row on each side; Ball & Goodman, 1985a), as shown in Fig. 3E. In the pro- and mesothoracic femurs, the ETi supraMP forms only two rows of cytoplasmic connections between the apodeme and the ectoderm of the femur wall, one medially and one laterally (Figs 3B,C, 5E). By 56-57 % (Fig. 5C-E) the supraMP has begun to split up into individual MPs and intracellularly injected HRP no longer fills the entire supraMP (Fig. 5D). A second major difference between the development of the metathoracic ETi MP and those of the anterior legs is the separation of the accessory extensor tibiae (AETi) MP from the former. The A ETi first becomes apparent as an expansion at the distal end of the medial arm of the horseshoe-shaped ETi at

Femoral muscle development in locust embryos about 45 % (Ball & Goodman, 1985a). It then pinches off from the main body of the ETi and moves laterally, eventually becoming a V-shaped MP symmetrically arranged over the apodeme by about 52 %. This MP subsequently divides into two at the base of the V, with each of the resulting MPs remaining dorsal to the apodeme and eventually forming one of

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the AETi muscle bundles. AETi MPs are neither formed in the anterior legs during the period up to 60 % of embryonic development, nor are they present in the adult. With the above exception, development and differentiation of the ETi muscle bundles from the individual MPs appear to follow the same pattern as that already described for the metathoracic ETi, although this period of development was not studied in detail. Development of the prothoracic and mesothoracic flexor tibiae muscles The flexor tibiae muscles of the prothoracic and mesothoracic legs develop in a very similar manner to the metathoracic FITi and, unlike the ETi supraMPs, their morphology is unaffected by the FCOs, since they lie on the ventral surface of the femur. The FITi MPs first appear as a symmetrical pair of cells at the tip of the ingrowing apodeme at around 3 8 % . A third cell (Fig. 6A) soon appears between the first two, as was described in the metathoracic leg. By approximately 44 %, the proximal MP has moved away from the lateral MPs (Fig. 6B). The three initial MPs appear to maintain their contact with the ectoderm of the wall of the leg, moving proximally as cell division occurs at the distal end of the femur (Fig. 6B,C). Mesoderm cells are recruited and added to the lateral sets of MPs as the leg grows (Fig. 6B). By 47 %, the FITi MPs in the three legs have reached the condition shown in Fig. 7 A - C . The major difference between the legs at this age is that, in the metathoracic leg, the MPs are regularly arranged around the proximal end of the apodeme (Fig. 7C), rather than being arranged in three discrete groups as is the case in the pro- and mesothoracic legs. Another major difference between the legs becomes apparent at about 48-49%, when the central MP at the proximal tip of the apodeme in the metathoracic leg dies and breaks down (Fig. 7D), while the comparable MP in the pro- and mesothoracic legs remains intact and continues to grow.

apo

Fig. 6. Early development of the prothoracic and mesothoracic FITi muscles. (A) The mesothoracic FITi muscles at 41 % of embryonic development (1-5 stained) consists of two lateral cells symmetrically placed around the apodeme (one of these is out of the plane of focus in this preparation: dotted area) and a larger central cell at the proximal end of the apodeme. Ventral view. Bar: 10^m. (B) Prothoracic FITi at 44% of embryonic development, additional MPs are being added laterally as the first-formed MPs move proximally (1-5). Ventral view. Bar: 15 ^m. (C) Lateral view of the preparation shown in B to show the position of the MP in relation to the apodeme and the ectoderm of the wall of the leg. Bar: 25iim. Proximal is to the left in each micrograph. Abbreviations: apo, apodeme; ect, ectoderm; FITi, flexor tibiae; lat, lateral; prox, proximal.

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Fig. 7. Comparative development of the FlTi muscles after 45 %. All in ventral view. (A-C) FlTi at 47 % in the pro(A), meso- (B), and metathoracic (C) legs of the same animal (1-5). Bars: 20\im. (D) Metathoracic FlTi at 49 %. Note debris (deb) of the proximal MP. Bar: 30jum. (E) Prothoracic FlTi at 55% (1-5). The proximal MP is still clearly present. Bar: 30f*m. (F) Two separate HRP fills of the proximal and a lateral MP of the mesothoracic FlTi at 54%, demonstrating that each cytoplasmic bridge is discrete and that the proximal MP is still present, in contrast to the condition in the metathoracic leg. Bar: 20/jm. Proximal is to the left in each micrograph. Abbreviations: apo, apodeme; deb, debris; FlTi, flexor tibiae; lat, lateral; prox, proximal. Thus by 55 % the central MP is a great deal longer than the lateral MPs and connects the tip of the apodeme to the ventral ectoderm of the trbchanter close to the posterior border of the coxa (Fig. 7E). As

was found in Schistocerca (Ball & Goodman, 1985b) the 1-5 MAb staining becomes inconsistent after about 50% of development, resulting in markedly different staining intensities between neighbouring

Femoral muscle development in locust embryos

MPs (Fig. 7E). That the central MP in the pro- and mesothoracic legs does not break down at a later stage was confirmed by intracellular dye filling with HRP (Fig. 7F). As is the case during the development of the ETi muscles, a further difference between the development of the metathoracic FITi muscles and the proand mesothoracic muscles is the absence of the two accessory flexor tibiae (AFlTi) muscle bundles in the anterior two pairs of legs. In the metathoracic legs, the two distal-most lateral MPs form the AFlTi muscle bundles. This differentiation does not occur in the pro- and mesothoracic legs.

Discussion The embryonic development of the extensor (ETi) and flexor (FITi) tibiae muscles of the pro- and mesothoracic legs of the locust has been shown to be very similar to that already described for the metathoracic legs (Ball et al. 1985; Ball & Goodman, 1985a,b). As in the metathoracic legs, the pro- and mesothoracic ETi muscles develop from a large syncytium, the supramuscle pioneer (supraMP), which subsequently breaks up into a series of individual MPs each of which eventually forms a muscle bundle (Figs 3, 8). The pro- and mesothoracic FITi muscles also follow the same pattern as the metathoracic FITi. They do not form from a supraMP, but directly from an array of individual MPs, again corresponding to the future muscle bundles (Fig. 9). Thus it seems that there are at least two distinct, general patterns of muscle development in the locust, and that the metathoracic ETi is not unique in

forming a s u p r a M P . F u r t h e r m o r e , the formation of the giant syncytium or s u p r a M P is not related to the evolutionary hypertrophy of the E T i muscle in the metathoracic leg, since it also occurs in the c o m p a r a tively unspecialized ETi muscles of the pro- and mesothoracic legs. There a r e , however, some specializations of the pro- and mesothoracic legs related to their roles in walking, feeding and postural support, which are revealed during their development. T h e flexor tibiae muscles play a much greater role in these behaviours than they do in the metathoracic legs, where their major role is to flex the tibia in preparation for the jump (Heitler, 1974, 1977). In the m o r e anterior legs, the flexors are larger and m o r e powerful than the extensors. This is the reverse of the situation in the metathoracic femur (Fig. 1C), where the extensor tibiae muscle, being specialized for powering the j u m p , is far larger than the flexor tibiae muscle (Snodgrass, 1929; Burns & U s h e r w o o d , 1979). This situation is mirrored in the e m b r y o , where it was found that the syncytium of the developing E T i in the pro- and mesothoracic legs is much smaller, and grows more slowly, than that in the metathoracic legs, particularly during the earlier stages of development. This reduced size may b e related to another specialization of the fore and middle legs, the large femoral chordotonal organ ( F C O ) , which in these legs is composed of two parts, the proximal and distal scoloparia. This organ lies in the proximal p o r t i o n of the leg and plays an important role in giving proprioceptive information on the position of the tibia and tibial velocity during walking movements (Burns, 1974). In the e m b r y o , one of the most conspicuous features of the developing pro- and mesothoracic limbs is the F C O , which, especially in early e m b r y o s , occupies a large proportion of the femur. T h e presence of the F C O in these legs results in compression of the ETi between the invaginated ectoderm of the F C O and the ectoderm of the dorsal and lateral leg

500 ^m Fig. 8. Overview of development of the pro- and mesothoracic extensor tibiae muscles between 40 % and 57 % of embryonic development. All views are perpendicular to the plane of the ETi although this plane changes within the femur during development. Bar: 500 [im.

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500 urn Fig. 9. Overview of development of pro- and mesothoracic flexor tibiae muscles between 40 % and 55 % of embryonic development. Ventral views. Bar: 500 ^m.

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wall. Thus instead of forming the double horseshoe shape so characteristic of the metathoracic ETi supraMP from 42 to 45 % of development (Ball & Goodman, 1985a), the pro- and mesothoracic ETi supraMPs form a flattened 'cap' over the tip of the apodeme (Fig. 4C). Only after about 46%, when the femur begins to extend, does the ETi apodeme straighten out, and the supraMP begin to grow out along its apodeme distal to the FCO (Fig. 4E,F). Slifer (1935) described formation of the FCO apodeme by a fusion of proximal and distal ectodermal invaginations in Melanoplus. In Locusta, we have been unable to find a proximal invagination and it appears to us that the FCO apodeme is extended in the same manner as the ETi and FITi apodemes in that it becomes attached to the ectoderm proximally, early in development, and then lengthens by ectodermal cell proliferation between its proximal and distal ends. Another interesting difference between the development of ETi in the pro- and mesothoracic legs and the metathoracic legs is in the number and organization of attachment sites of the muscle bundles. As previously described for the development of the metathoracic ETi (Ball & Goodman, 1985a), the two arms of the supraMP each divide to form a dorsal and a ventral row of cytoplasmic bridges to the ectoderm of the femur wall (Fig. 3A,D,E). Each of these cytoplasmic bridges eventually forms the individual muscle bundles which make up the ETi muscle. The positions of the insertions of these bundles between sclerotized ridges in the cuticle can clearly be seen as two rows of chevrons on each side of the adult metathoracic femur (Fig. 1A). In the adult pro- and mesothoracic legs, there is only one row of muscle bundles inserted into each of the lateral and medial walls of the femur. The present study reveals that, during the development of the pro- and mesothoracic ETis, only one row of cytoplasmic bridges is formed between the giant syncytium and the lateral and medial walls of the femur (Figs 3B,C, 5E). This reinforces the finding of Ball et al. (1985) that every muscle bundle in the legs of the locust is preceded by the earlier appearance of a single muscle pioneer. Related to this finding is the lack of accessory ETi muscle bundles in the pro- and mesothoracic legs. Presumptive AETi MPs do not separate from the ETi supraMP in these legs, as they do in the metathoracic legs. This contrasts with the situation in other muscles where the absence of particular muscle bundles can be traced back to selective cell death of MPs. during development. Contraction of the AETi muscles would pull the ETi apodeme proximodorsally, although their exact functional role is unknown. Presumably, their presence in the metathoracic legs is

related to the specializations of these legs for escape and defensive behaviours. The proximal myogenic bundle of the metathoracic ETi has also been suggested to be a specialization of this limb for aiding blood flow along the leg (Usherwood, 1974; Evans & O'Shea, 1978). The pro- and mesothoracic ETi muscles do not possess a myogenic bundle (Burns & Usherwood, 1979). However, our studies have not revealed any developmental basis for this difference. As discussed above, the flexor tibiae muscle in the pro- and mesothoracic femurs differs from that in the metathoracic femur of the adult in function, structure, innervation (Theophilidis & Burns, 1983) and degree of development relative to the extensor tibiae. The present study has shown that although the basic pattern of FITi development is the same in all three legs, there are two major differences between the development of the pro- and mesothoracic FITi and that of the metathoracic FITi. First, the central proximal muscle pioneer, which during the development of the metathoracic FITi breaks down and disappears, remains intact in the pro- and mesothoracic legs, and continues to grow and eventually to differentiate. This proximal muscle pioneer forms the very large proximal, phasic bundle of the pro- and mesothoracic flexors previously described in the adult by Snodgrass (1929) and Theophilidis & Burns (1983), and shown in Fig. IB. This proximal bundle links the tip of the apodeme to the proximal border of the trochanter, and is thought to be the main bundle of phasic fibres involved in the relatively fast tibial flexions involved in walking movements, for which these legs are adapted. There is no homologous proximal bundle of the metathoracic FITi (Fig. 1C), owing to the death of its muscle pioneer (Fig. 7D). Coincidentally, in the metathoracic leg the femoral/ trochanteral joint is quite rigid, and the trochanter is vastly reduced in size such that it forms only a wedgeshaped segment between the femur and coxa (Fig. 1A,C). The second major difference is the formation of AFlTis in the metathoracic leg, but not in the other legs. The function of the AFlTis does not appear to have been studied, but their contraction would presumably pull the FITi apodeme proximodorsally. It is clear from our findings that differing patterns of adult musculature in homologous structures such as legs can arise in two ways. First, comparable muscle pioneers may form in all cases, but then selectively die, as exemplified by the proximal bundle of the metathoracic FITi. Second, a muscle pioneer, and consequently a muscle, may never form, as exemplified by the AETis and AFlTis of the anterior legs.

Femoral muscle development in locust embryos The break-up of the metathoracic proximal FITi MP, while its homologues survive, brings into focus the whole question of the role of programmed cell death in muscle development, and what factors control it. The timing and extent of innervation of the muscle bundle are two possibly significant factors, particularly since development of the metathoracic limbs precedes that of the pro- and mesothoracic limbs. Little has been published concerning the arrival time of neuronal growth cones at FITi or about early patterns of innervation there. However, one possible mechanism for obtaining selective cell death might be the timed release of a hormone which triggers the breakdown of uninnervated or uniquely innervated bundles of FITi. It is interesting to note that while the metathoracic FITi is innervated by thirteen identified neurones (nine excitatory motor neurones, two inhibitors and two DUM cells (Phillips, 1980, 1981)), the pro- and mesothoracic FITi muscles are each innervated by an additional three excitatory motor neurones (Phillips, 1981; Theophilidis & Burns, 1983). Furthermore, two of the three additional neurones exclusively innervate the proximal bundle (Theophilidis & Burns, 1983). The flexor tibiae muscles of locust legs might thus be a particularly accessible system in which to study programmed cell death during development. We thank the Goodman laboratory for the 1-5 and Mes-3 monoclonal antibodies and Drs Corey Goodman, David Reye and Paul Whitington for comments on the manuscript. References E. E. & GOODMAN, C. S. (1985a). Muscle development in the grasshopper embryo. II. Syncytial origin of the extensor tibiae muscle pioneers. Devi Biol. Ill, 399-416. BALL, E. E. & GOODMAN, C. S. (19856). Muscle development in the grasshopper embryo. III. Sequential origin of the flexor muscle pioneers. Devi Biol. Ill, 417-424. BALL, E. E., HO, R. K. & GOODMAN, C. S. (1985). Muscle development in the grasshopper embryo. I. Muscles, nerves and apodemes in the metathoracic leg. Devi Biol. 111,383-398. BATE, C. M. (1976). Pioneer neurones in an insect embryo. Nature, Lond. 260, 54-56. BALL,

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control of walking in Orthoptera. II. Motor neurone activity in normal free-walking animals. J. exp. Biol. 79, 69-98. CHANG, S., HO, R. K. & GOODMAN, C. S. (1983). Selective groups of neuronal and mesodermal cells recognized early in grasshopper embryogenesis by a monoclonal antibody. Devi Brain Res. 9, 297-304. EVANS, P. D. & O'SHEA, M. (1978). The identification of

an octopaminergic neurone and the modulation of a myogenic rhythm in the locust. J. exp. Biol. 73, 235-260. HEITLER, W. J. (1974). The locust jump. Specialisations of the metathoracic femoral-tibial joint. /. comp. Physiol. 89, 93-104. HEITLER, W. J. (1977). The locust jump. III. Structural specializations of the metathoracic tibiae. J. exp. Biol. 67, 29-36. KOTRLA, K. & GOODMAN, C. S. (1984). Transient expression of a surface antigen on a small subset of neurones during embryonic development. Nature, Lond. 311, 151-153. PHILLIPS, C. E. (1980). An arthropod muscle innervated by nine excitatory motor neurones. J. exp. Biol. 88, 249-258. PHILLIPS, C. E. (1981). Organization of motor neurons to a multiply innervated insect muscle. /. Neurobiol. 12, 269-280. SLIFER, E. H. (1935). Morphology and development of the femoral chordotonal organs of Melanoplus differentialis (Orthoptera, Acrididae). J. Morph. 38, 615-637. SNODGRASS, R. E. (1929). The thoracic mechanism of a grasshopper and its antecedents. Smithsonian Misc. Collect. 82, 1-111. SNODGRASS, R. E. (1935). Principles of Insect Morphology.

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