comparative development of the extensor and flexor tibiae … · intracellular dye fills for...

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

351

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 flexortibiae muscles in the pro- and mesothoracic legs of thelocust, Locusta migratoria, is described, and com-pared to the previously described development of thesemuscles in the metathoracic legs. The basic pattern ofdevelopment of each muscle is the same in all threepairs of legs. The extensor tibiae (ETi) forms a giantsyncytium, or supramuscle pioneer, which thenbreaks up into a series of muscle pioneers. The flexortibiae muscle (FITi) is formed directly by sequentialaddition of individual muscle pioneers. Thus, thereare at least two fundamentally different patterns ofmuscle development in the embryonic locust, asexemplified by these two muscles, and supramusclepioneer formation is not a unique feature of themetathoracic ETi associated with its evolutionaryhypertrophy.

In spite of a basically similar pattern of developmentof homologous muscles in all three pairs of legs, there

are significant developmental differences between themetathoracic ETi and FITi and their homologues inthe anterior legs. First, during development of the ETimuscles, the ETi MP forms a double row of attach-ment 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, theproximal MP, which lies at the tip of the apodeme, diesand breaks down in the metathoracic limbs. In thepro- and mesothoracic limbs it remains intact andeventually forms a large proximal muscle bundle.Third, the accessory ETi and FITi muscles, whichdevelop in the metathoracic legs, are not formed in theanterior legs.

Key words: muscle development, locust, Locustamigratoria, muscle pioneer, extensor tibiae muscle, flexortibiae muscle.

Introduction

The embryonic development of the metathoracicextensor tibiae muscle (ETi) of the grasshopper hasrecently been described (Ball, Ho & Goodman, 1985;Ball & Goodman, 1985a). During its early embryonicdevelopment, this muscle passes through a syncytialstage, called a supramuscle pioneer (supraMP),which ultimately contains hundreds of nuclei. Be-tween 50 and 60% of embryonic development thissyncytium 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 passthrough a supraMP stage, but directly forms thesmaller syncytia corresponding to future musclebundles.

By 60 % of embryonic development, both muscleshave arrived at a similar condition and from that stage

onward both develop in a similar manner, so there isno obvious reason why their early developmentshould be so different. One possibility is that thepattern of development of the metathoracic ETi issomehow related to the evolutionary hypertrophy ofthis muscle in relation to its function of powering thejump. One way of testing this hypothesis and ofsimultaneously establishing the degree of embryonicsimilarity between legs which are morphologicallyquite different in the adult, is to examine the develop-ment of the homologous muscle in the pro- andmesothoracic legs, which are not specialized forjumping. These legs are much smaller than themetathoracic and function quite differently, the pro-thoracic legs being specialized for exploration andwalking, and the mesothoracic legs playing a role inpostural support and walking (Burns, 1973).

352 C. M. Myers and E. E. Ball

Materials and methods

EmbryosQutches of eggs of Locusta migratoria were laid in cups ofdamp sand and incubated at 28°C. Under these conditionsembryonic development takes approximately 14 days. Em-bryos were staged according to the criteria given byBentley, Keshishian, Shankland & Toroian-Raymond(1979) for Schistocerca. These criteria can readily be appliedto Locusta and the ages given in this paper are based onthem. We have not established whether the criteria corre-spond to exactly the same absolute ages in the two speciesalthough they do not appear to differ dramatically.

Antibody treatmentEmbryos intended for staining with monoclonal antibodieswere dissected in locust Ringer (NaCl, 8-7 gl"1; KC1,0-22 g r ' ; CaCl2.2H2O, 0-29gl"'; MgS04.7H2O, 0-25gl"1; TES, l-15gl"'; pH7-0) and quickly transferred to2 % paraformaldehyde in Millonig's phosphate buffer.Embryos older than 40 % were treated with chitinase(Sigma) to permeabilize or remove the cuticle that begins tobe laid down about that time. The antibodies used in thisstudy were 1-5 (Chang, Ho & Goodman, 1983), which stainsneuronal tissue and muscle pioneers, and Mes-3 (Kotrla &Goodman, 1984), which stains a small subset of neurones inthe central nervous system plus muscle pioneers. Followingchitinase treatment the embryos were thoroughly washed inphosphate-buffered saline (PBS) and then placed in asolution containing the antibody, 2 % bovine serum albu-men and 1 % Triton X-100 on a shaker overnight at roomtemperature. Monoclonal antibody binding was visualizedwith biotin-streptavidin-HRP (Amersham) using standardtechniques. The embryos were washed, then dehydratedand cleared through a glycerin series and photographed inwhole mount.

Intracellular dye fills

For intracellular injection of MPs, a mixture of 4%horseradish peroxidase (HRP, Sigma) and 1 % LuciferYellow (LY, Aldrich) was made up in distilled water. Thismixture was then centrifuged at 45 000 revs min"1 for 5minand the supernatant was injected using either current orpressure (Picospritzer, General Valve Corp.). Injectedembryos were fixed immediately in 2 % paraformaldehydeand 2-5 % glutaraldehyde in Millonig's phosphate buffer(Bate, 1976). They were then washed, treated with chitin-ase for a variable time depending on age, washed again andthe HRP was then visualized using 3,3-diaminobenzidineand H2O2. In some preparations, the HRP was intensifiedwith CoCl2 using the techniques of Watson & Burrows(1981). The embryos were then dehydrated and clearedthrough a glycerin series and viewed and photographed inwhole mount.

TerminologyThe pro-, meso- and metathoracic legs of a locust are showndrawn to the same scale in Fig. 1A. Fig. IB shows theorganization and relative sizes of the extensor and flexortibiae muscles within the basal segments of a mesothoracicleg (the prothoracic leg is similar). The position of the

t proximal bundle FITi FITi

AETiFCO I A FITi

FITi

Fig. 1. Comparative morphology of the legs of a locustand the organization of their femoral muscles. (A) Thelegs, all drawn to the same scale. Note the hypertrophyof the metathoracic leg and especially of its femur.(B) Idealized drawing of the femoral musculature of amesothoracic leg. (The prothoracic femur is similarlyorganized except that it is slightly smaller as is its femoralchordotonal organ). Note the large proximal FITi bundlewhich is missing in the metathoracic leg. (C) Idealizeddrawing of the femoral musculature of a metathoracicleg. Both the extensor and flexor tibiae are larger thanthey are in the other two legs, but the extensor hasbecome disproportionately enlarged. For clarity, theretractor unguis muscle has been omitted in both B andC. Abbreviations: AETi, accessory extensor tibiaemuscle; AFITi, accessory flexor tibiae muscle; c, coxa;ETi, extensor tibiae muscle; FCO, femoral chordotonalorgan; f, femur; FITi, flexor tibiae muscle; meso,mesothoracic leg; meta, metathoracic leg; pro, prothoracicleg; fa, tarsus; ti, tibia; tr, trochanter.

femoral chordotonal organ (FCO) is shown by the shadedarea in the proximal portion of the femur. The samefeatures of a metathoracic leg are shown in Fig. 1C. Notethe hypertrophy of the extensor tibiae muscle and the distalposition of the FCO.

The axes referred to in this paper (Fig. 2) are those of theadult legs shown in Fig. 1. In young embryos, thesedesignations are incorrect by 90° since the future dorsal sideof the leg points anterior. During embryonic developmentthe leg gradually rotates into the adult position.

Since insects have an external rather than an internalskeleton, their muscles have a different relationship to theskeleton than do those of vertebrates. Apodemes, whichare cuticular processes extending inward from the body wall(Snodgrass, 1935), are functionally analogous to vertebratetendons in that they link muscle to the skeleton. Apodemesoriginate from invaginations of the ectoderm at the jointbetween two leg segments. The invaginated ectodermextends proximally within the leg segment and secretes thecuticular apodeme on its external surface, which in this casefaces the inner cavity of the invagination and is continuous

Femoral muscle development in locust embryos 353

dorsal

Fig. 2. Diagram of an embryonic leg at approximately45 % indicating terminology used in this paper.Abbreviations: f, femur; ta, tarsus; ti, tibia.

with the cuticle covering the leg. The ETi and FITi musclebundles attach proximally to the cuticulanzed ectoderm ofthe wall of the femur and distally to the cuticulanzedectoderm of the apodeme. Contraction of the muscle exertsforce on the apodeme, thus moving the next distal legsegment at the joint. In embryonic locusts, the apodemesurrounds a substantial cavity, which is continuous with thespace outside the embryo (e.g. Figs 5A, 6C, 7A).

Results

Development of the metathoracic extensor and flexortibiae muscles

The metathoracic extensor (ETi) and flexor (FITi)tibiae muscles of Locusta migratoria both develop in amanner very similar to that described in the meta-thoracic leg of Schistocerca gregaria (Ball et al. 1985;Ball & Goodman, 1985a,b). The ETi muscle begins asa single mesoderm cell which attaches the ectodermof the invaginating apodeme to the wall of the femur,and is first visible at around 35%. This cell thenenlarges dramatically by fusion with surroundingmesoderm cells to form a giant, horseshoe-shapedsyncytium around the apodeme, called the supra-muscle pioneer (supraMP). At about 45 % of devel-opment, however, the supraMP in Locusta deviatesfrom the developmental pattern of Schistocerca, split-ting into two halves at the bridge-like connection atthe proximal tip of the apodeme. The evidence forthis is that HRP injected into one of the lateral armsat this and later stages remains only in that arm. Atthe same time, the lateral edges of the supraMP

become scalloped, with certain regions adheringtightly to the ectoderm and neighbouring regionsreceding from it (Fig. 3A,D). At about 55% thehalves of the supraMP begin to break up further andform a series of periodic bridges which connect theapodeme to the wall of the femur. Each bridge formsthe core around which a muscle bundle will develop.In contrast, the FITi muscle does not develop from asyncytial supraMP, but rather by the sequentialrecruitment of individual MPs from the undifferen-tiated mesoderm cells surrounding the apodeme. Thefirst FITi MPs appear as a symmetrical pair at the tipof the invaginating apodeme at around 37 %. A thirdMP soon appears between this pair, but this MP laterdies at around 47 % (approximately 2 % later than inSchistocerca). Further growth occurs by symmetricaladdition of pairs of MPs distally along either side ofthe lengthening apodeme. Each MP develops into abundle of muscle fibres by a cycle of fusion withsurrounding mesoderm cells followed by division anddifferentiation into the muscle fibres.

Development of the ETi and FITi muscles of theprothoracic and mesothoracic legs, which has notpreviously been described, is the topic of the follow-ing sections. The pattern of development is so similarin the two legs that a single description will suffice forboth.

Development of the prothoracic and mesothoracicextensor tibiae muscles

The extensor tibiae muscles of the prothoracic andmesothoracic legs of the locust embryo develop froma single muscle pioneer (Fig. 4A,B), in a similarmanner to that already described for the metathoracicleg (Ball et al. 1985; Ball & Goodman, 1985a).However, development of the muscles in the twomore anterior legs lags behind that in the meta-thoracic leg by a few percent, depending upon thestage of development. Thus, the first muscle pioneeris not recognizable until around 37 %. This cell thenbegins gradually to enlarge until by around 42 % ithas formed a slightly curved, disc-shaped multi-nucleated cell over the tip of the apodeme, directlybeneath the dorsolateral ectoderm. At this stage, thedeveloping femoral chordotonal organ (FCO) takesup a large proportion of the total volume of the pro-and mesothoracic femurs (Fig. 4A,D). The develop-ment of this large proprioceptive organ in all threelegs of Melanoplus has been described by Slifer(1935). It is formed from an invagination of the dorsalectoderm of the femur, from which the sensory cellsand accessory cells of the scoloparia differentiate. Asa consequence of the presence of the FCO, the ETiapodeme is not straight, as it is in the metathoracicfemur, but curved around to the dorsal posterior edge


ectodermof wall

of femur

pro & meso meta leg

scallopingventralbridges

individualMPs

individualMPs

Fig. 3. Schematic diagrams summarizing the differences between the development of the pro/mesothoracic ETi musclesand 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 ectodermof 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 theresult that on either side of the apodeme there is a single row of cytoplasmic bridges, connected at their bases, linkingthe ectoderm of the walls of the femur to that surrounding the apodeme. (C) Each of these cytoplasmic bridges thenseparates from its neighbours and becomes an individual MP. (D-E) Metathoracic legs. (D) Each arm of the supraMPdivides to form a dorsal and a ventral row of cytoplasmic bridges which are linked at their bases and connect the lateralwalls of the femur to the apodeme. (E) The links between the cytoplasmic bridges break to form two rows of individualMPs 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 gradu-ally (Fig. 4C,D), presumably by fusion with othermesoderm cells (Ball & Goodman, 1985a) and ataround 47-48 % it begins to extend distally over theapodeme (Fig. 4E). At the same time, the proximaledge of the supraMP, which lies between the dorsal

ectoderm and the ectodermal invagination of theFCO, begins to become scalloped (Fig. 4F). Between50 and 52 % the supraMP begins to split into twoarms, which often remain connected at the distal end.As the arms grow out they gradually move ventrallyto lie on either side of the apodeme (Fig. 5A,B),


Fig. 4. Development of the prothoracic and mesothoracic ETi muscles. (A) Lateral view of HRP-filled mesothoracicETi MP at 40 %. Note its relation to its own apodeme and the FCO. Bar: 20fan. (B) Close-up dorsolateral view of thesame MP to show its multinucleate nature and how it connects to the apodeme ectoderm. Bar: 10/im. (C) Mesothoracic1-5 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, showinghow 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 alldirections. Bar: 15,um. (F) By 50% the edges of the ETi MP are becoming scalloped (arrows), as shown in thisdorsolateral 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.


FCOect

Fig. 5. Further development of the ETi muscle. (A) Lateral view of a prothoracic ETi MP at approx. 53 %, showing itsposition in relation to the FCO ectoderm. Bar: 20/im. (B) HRP-filled ETi MP at approx. 54%. By this stage thehorseshoe-shaped MP is separating into two arms. In this preparation, the arms have separated proximally and arelinked 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 ofcytoplasmic 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 singleline of cytoplasmic bridges, in contrast to that of the metathoracic leg (Ball & Goodman, 1985a). Bar: 30ftm. Proximalis to the left in each micrograph. Abbreviations: apo, apodeme; ect, ectoderm; ETi, extensor tibiae; FCO, femoralchordotonal organ.

which is now relatively straight since by this stage thefemur has grown considerably in length, with theFCO now restricted to the proximal end. From 53 to55 % (Fig. 5B,C) the supraMP looks quite similar tothat in the metathoracic femur between 45 and 50%,and the outer edges of the MP have begun to pullaway from certain regions of the ectoderm to give ascalloped appearance (Figs 3A,B, 4F). However, thesituation in the pro- and mesothoracic legs differsfrom that in the metathoracic ETi at this stage, wherethe scalloping of the arms of the supraMP leads to theformation of four rows of cytoplasmic bridges(Fig. 3D,E). These connect the apodeme ectoderm tothe ectoderm of the wall of the femur (two rowsmedially 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 meso-thoracic femurs, the ETi supraMP forms only tworows of cytoplasmic connections between the apo-deme and the ectoderm of the femur wall, onemedially and one laterally (Figs 3B,C, 5E). By56-57 % (Fig. 5C-E) the supraMP has begun to splitup into individual MPs and intracellularly injectedHRP no longer fills the entire supraMP (Fig. 5D).

A second major difference between the develop-ment of the metathoracic ETi MP and those of theanterior legs is the separation of the accessory exten-sor tibiae (AETi) MP from the former. The A ETifirst becomes apparent as an expansion at the distalend of the medial arm of the horseshoe-shaped ETi at


about 45 % (Ball & Goodman, 1985a). It then pinchesoff from the main body of the ETi and moveslaterally, eventually becoming a V-shaped MP sym-metrically arranged over the apodeme by about 52 %.This MP subsequently divides into two at the base ofthe V, with each of the resulting MPs remainingdorsal to the apodeme and eventually forming one of

apo

the AETi muscle bundles. AETi MPs are neitherformed in the anterior legs during the period up to60 % of embryonic development, nor are they presentin the adult.

With the above exception, development and differ-entiation of the ETi muscle bundles from the indi-vidual MPs appear to follow the same pattern as thatalready described for the metathoracic ETi, althoughthis period of development was not studied in detail.

Development of the prothoracic and mesothoracicflexor tibiae muscles

The flexor tibiae muscles of the prothoracic andmesothoracic legs develop in a very similar manner tothe metathoracic FITi and, unlike the ETi supraMPs,their morphology is unaffected by the FCOs, sincethey lie on the ventral surface of the femur.

The FITi MPs first appear as a symmetrical pair ofcells at the tip of the ingrowing apodeme at around38%. A third cell (Fig. 6A) soon appears betweenthe first two, as was described in the metathoracic leg.By approximately 44 %, the proximal MP has movedaway from the lateral MPs (Fig. 6B). The three initialMPs appear to maintain their contact with the ecto-derm of the wall of the leg, moving proximally as celldivision occurs at the distal end of the femur(Fig. 6B,C). Mesoderm cells are recruited and addedto the lateral sets of MPs as the leg grows (Fig. 6B).By 47 %, the FITi MPs in the three legs have reachedthe condition shown in Fig. 7A-C. The major differ-ence between the legs at this age is that, in themetathoracic leg, the MPs are regularly arrangedaround the proximal end of the apodeme (Fig. 7C),rather than being arranged in three discrete groups asis the case in the pro- and mesothoracic legs.

Another major difference between the legs be-comes apparent at about 48-49%, when the centralMP at the proximal tip of the apodeme in themetathoracic leg dies and breaks down (Fig. 7D),while the comparable MP in the pro- and meso-thoracic legs remains intact and continues to grow.

Fig. 6. Early development of the prothoracic andmesothoracic FITi muscles. (A) The mesothoracic FITimuscles at 41 % of embryonic development (1-5 stained)consists of two lateral cells symmetrically placed aroundthe apodeme (one of these is out of the plane of focus inthis preparation: dotted area) and a larger central cell atthe proximal end of the apodeme. Ventral view. Bar:10^m. (B) Prothoracic FITi at 44% of embryonicdevelopment, additional MPs are being added laterally asthe first-formed MPs move proximally (1-5). Ventralview. Bar: 15 ^m. (C) Lateral view of the preparationshown in B to show the position of the MP in relation tothe 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, flexortibiae; lat, lateral; prox, proximal.


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 %. Notedebris (deb) of the proximal MP. Bar: 30jum. (E) Prothoracic FlTi at 55% (1-5). The proximal MP is still clearlypresent. 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 thecondition 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 longerthan the lateral MPs and connects the tip of theapodeme to the ventral ectoderm of the trbchanterclose to the posterior border of the coxa (Fig. 7E). As

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


MPs (Fig. 7E). That the central MP in the pro- andmesothoracic legs does not break down at a laterstage was confirmed by intracellular dye filling withHRP (Fig. 7F).

As is the case during the development of the ETimuscles, a further difference between the develop-ment of the metathoracic FITi muscles and the pro-and mesothoracic muscles is the absence of the twoaccessory flexor tibiae (AFlTi) muscle bundles in theanterior two pairs of legs. In the metathoracic legs,the two distal-most lateral MPs form the AFlTimuscle bundles. This differentiation does not occur inthe pro- and mesothoracic legs.

Discussion

The embryonic development of the extensor (ETi)and flexor (FITi) tibiae muscles of the pro- andmesothoracic legs of the locust has been shown to bevery similar to that already described for the meta-thoracic legs (Ball et al. 1985; Ball & Goodman,1985a,b). As in the metathoracic legs, the pro- andmesothoracic ETi muscles develop from a largesyncytium, the supramuscle pioneer (supraMP),which subsequently breaks up into a series of indi-vidual MPs each of which eventually forms a musclebundle (Figs 3, 8). The pro- and mesothoracic FITimuscles also follow the same pattern as the meta-thoracic FITi. They do not form from a supraMP, butdirectly from an array of individual MPs, againcorresponding 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

500 ̂ m

Fig. 8. Overview of development of the pro- andmesothoracic extensor tibiae muscles between 40 % and57 % of embryonic development. All views areperpendicular to the plane of the ETi although this planechanges within the femur during development. Bar:500 [im.

forming a supraMP. Fur thermore , the formation ofthe giant syncytium or supraMP is not related to theevolutionary hypertrophy of the ETi muscle in themetathoracic leg, since it also occurs in the compara-tively unspecialized ETi muscles of the pro- andmesothoracic legs.

There are , however, some specializations of thepro- and mesothoracic legs related to their roles inwalking, feeding and postural support , which arerevealed during their development . The flexor tibiaemuscles play a much greater role in these behavioursthan they do in the metathoracic legs, where theirmajor role is to flex the tibia in preparat ion for thejump (Heitler, 1974, 1977). In the more anterior legs,the flexors are larger and more powerful than theextensors. This is the reverse of the situation in themetathoracic femur (Fig. 1C), where the extensortibiae muscle, being specialized for powering thejump, is far larger than the flexor tibiae muscle(Snodgrass, 1929; Burns & Usherwood, 1979). Thissituation is mirrored in the embryo, where it wasfound that the syncytium of the developing ETi in thepro- and mesothoracic legs is much smaller, andgrows more slowly, than that in the metathoracic legs,particularly during the earlier stages of development .This reduced size may be related to another specializ-ation of the fore and middle legs, the large femoralchordotonal organ (FCO) , which in these legs iscomposed of two parts, the proximal and distalscoloparia. This organ lies in the proximal por t ion ofthe leg and plays an important role in giving proprio-ceptive information on the position of the tibia andtibial velocity during walking movements (Burns,1974). In the embryo, one of the most conspicuousfeatures of the developing pro- and mesothoraciclimbs is the F C O , which, especially in early embryos ,occupies a large proportion of the femur. The pres-ence of the F C O in these legs results in compressionof the ETi between the invaginated ectoderm of theFCO and the ectoderm of the dorsal and lateral leg

500 urn

Fig. 9. Overview of development of pro- andmesothoracic flexor tibiae muscles between 40 % and55 % of embryonic development. Ventral views. Bar:500 ̂ m.


wall. Thus instead of forming the double horseshoeshape so characteristic of the metathoracic ETisupraMP from 42 to 45 % of development (Ball &Goodman, 1985a), the pro- and mesothoracic ETisupraMPs form a flattened 'cap' over the tip of theapodeme (Fig. 4C). Only after about 46%, when thefemur begins to extend, does the ETi apodemestraighten out, and the supraMP begin to grow outalong its apodeme distal to the FCO (Fig. 4E,F).

Slifer (1935) described formation of the FCOapodeme by a fusion of proximal and distal ectoder-mal invaginations in Melanoplus. In Locusta, we havebeen unable to find a proximal invagination and itappears to us that the FCO apodeme is extended inthe same manner as the ETi and FITi apodemes inthat it becomes attached to the ectoderm proximally,early in development, and then lengthens by ectoder-mal cell proliferation between its proximal and distalends.

Another interesting difference between the devel-opment of ETi in the pro- and mesothoracic legs andthe metathoracic legs is in the number and organiz-ation of attachment sites of the muscle bundles. Aspreviously described for the development of themetathoracic ETi (Ball & Goodman, 1985a), the twoarms of the supraMP each divide to form a dorsal anda ventral row of cytoplasmic bridges to the ectodermof the femur wall (Fig. 3A,D,E). Each of thesecytoplasmic bridges eventually forms the individualmuscle bundles which make up the ETi muscle. Thepositions of the insertions of these bundles betweensclerotized ridges in the cuticle can clearly be seen astwo rows of chevrons on each side of the adultmetathoracic femur (Fig. 1A). In the adult pro- andmesothoracic legs, there is only one row of musclebundles inserted into each of the lateral and medialwalls of the femur. The present study reveals that,during the development of the pro- and mesothoracicETis, only one row of cytoplasmic bridges is formedbetween the giant syncytium and the lateral andmedial walls of the femur (Figs 3B,C, 5E). Thisreinforces the finding of Ball et al. (1985) that everymuscle bundle in the legs of the locust is preceded bythe earlier appearance of a single muscle pioneer.

Related to this finding is the lack of accessory ETimuscle bundles in the pro- and mesothoracic legs.Presumptive AETi MPs do not separate from the ETisupraMP in these legs, as they do in the metathoraciclegs. This contrasts with the situation in other muscleswhere the absence of particular muscle bundles canbe traced back to selective cell death of MPs. duringdevelopment. Contraction of the AETi muscleswould 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 escapeand defensive behaviours.

The proximal myogenic bundle of the metathoracicETi has also been suggested to be a specialization ofthis limb for aiding blood flow along the leg (Usher-wood, 1974; Evans & O'Shea, 1978). The pro- andmesothoracic ETi muscles do not possess a myogenicbundle (Burns & Usherwood, 1979). However, ourstudies have not revealed any developmental basis forthis difference.

As discussed above, the flexor tibiae muscle in thepro- and mesothoracic femurs differs from that in themetathoracic femur of the adult in function, struc-ture, innervation (Theophilidis & Burns, 1983) anddegree of development relative to the extensor tibiae.The present study has shown that although the basicpattern of FITi development is the same in all threelegs, there are two major differences between thedevelopment of the pro- and mesothoracic FITi andthat of the metathoracic FITi. First, the centralproximal muscle pioneer, which during the develop-ment of the metathoracic FITi breaks down anddisappears, remains intact in the pro- and meso-thoracic legs, and continues to grow and eventually todifferentiate. This proximal muscle pioneer forms thevery large proximal, phasic bundle of the pro- andmesothoracic flexors previously described in the adultby Snodgrass (1929) and Theophilidis & Burns(1983), and shown in Fig. IB. This proximal bundlelinks the tip of the apodeme to the proximal border ofthe trochanter, and is thought to be the main bundleof phasic fibres involved in the relatively fast tibialflexions involved in walking movements, for whichthese legs are adapted. There is no homologousproximal 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 isvastly reduced in size such that it forms only a wedge-shaped segment between the femur and coxa(Fig. 1A,C).

The second major difference is the formation ofAFlTis in the metathoracic leg, but not in the otherlegs. The function of the AFlTis does not appear tohave been studied, but their contraction would pre-sumably pull the FITi apodeme proximodorsally.

It is clear from our findings that differing patternsof adult musculature in homologous structures suchas legs can arise in two ways. First, comparablemuscle pioneers may form in all cases, but thenselectively die, as exemplified by the proximal bundleof the metathoracic FITi. Second, a muscle pioneer,and consequently a muscle, may never form, asexemplified by the AETis and AFlTis of the anteriorlegs.


The break-up of the metathoracic proximal FITiMP, while its homologues survive, brings into focusthe whole question of the role of programmed celldeath in muscle development, and what factors con-trol it. The timing and extent of innervation of themuscle bundle are two possibly significant factors,particularly since development of the metathoraciclimbs precedes that of the pro- and mesothoraciclimbs. Little has been published concerning thearrival time of neuronal growth cones at FITi or aboutearly patterns of innervation there. However, onepossible mechanism for obtaining selective cell deathmight be the timed release of a hormone whichtriggers the breakdown of uninnervated or uniquelyinnervated bundles of FITi. It is interesting to notethat while the metathoracic FITi is innervated bythirteen identified neurones (nine excitatory motorneurones, two inhibitors and two DUM cells (Phil-lips, 1980, 1981)), the pro- and mesothoracic FITimuscles are each innervated by an additional threeexcitatory motor neurones (Phillips, 1981; Theophili-dis & Burns, 1983). Furthermore, two of the threeadditional neurones exclusively innervate the proxi-mal bundle (Theophilidis & Burns, 1983). The flexortibiae muscles of locust legs might thus be a particu-larly accessible system in which to study programmedcell death during development.

We thank the Goodman laboratory for the 1-5 andMes-3 monoclonal antibodies and Drs Corey Goodman,David Reye and Paul Whitington for comments on themanuscript.

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(Accepted 22 June 1987)

comparative development of the extensor and flexor tibiae … · intracellular dye fills for...

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