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J. Embryol. exp. Morph. 78, 67-82 (1983) Printed in Great Britain © The Company of Biologists Limited 1983 The distribution of muscle fibre types in chick embryo wings transplanted to the pelvic region is normal By N. G. LAING 1 AND A. H. LAMB 1 From the Department of Pathology, University of Western Australia SUMMARY Chick embryo wing buds were transplanted to the pelvic region in place of, or in addition to, the hindlimb bud prior to innervation. The wrist muscle ulnimetacarpalis dorsalis (umd) wa$ innervated by middle-dorsal or middle-ventral motoneurons in the lumbar lateral motor column (LMC) in a rostrocaudal position which varied with the rostrocaudal position of the wing. Despite the heterotopic innervation the subsequent development of the distributions of fast and slow muscle fibres, as judged by ATPase staining, was normal in all muscles examined. The pattern of innervation in the umd, as judged by acetylcholinesterase staining also developed normally. It is probable that muscle fibre type is intrinsically, not neurogenically, determined. INTRODUCTION In the previous paper we observed that muscle fibre types differentiate early in the development of the wrist muscle ulnimetacarpalis dorsalis (umd). In this paper we set out to investigate the factors which determine muscle fibre type. The prevailing hypothesis is that the muscle fibres have their type imposed by the innervating nerve (Buller, Eccles & Eccles, 1960; Bennett & Pettigrew, 1974a). The only experimental evidence for the hypothesis is from studies of cross innervation by foreign nerves in juveniles and adults (e.g. Bennett & Pettigrew, 1974a) when the nerves have already been in contact with muscle. This contact may itself have specified the motoneurones. We therefore decided to carry out a cross innervation study in which the nerves had not previously contacted muscles. Wings were transplanted to the pelvic region of chick embryos at E3, which is before axons have grown into the limbs (Oppenheim & Heaton, 1975; Roncali, 1970). The distribution of muscle fibre types was found to be remarkably similar to that in control wings. Some of the results have previously been reported in brief (Laing & Lamb, 1982). METHODS Fertile eggs from a local hatchery were incubated in a humidified forceddraught incubator at 38°C. At stages 18-19 (Hamburger & Hamilton, 1951), 1 Authors' address: Department of Pathology, University of Western Australia, Queen Elizabeth II Medical Centre, Nedlands, 6009, Perth, Western Australia. 68 N. G. LAING AND A. H. LAMB which were found to be the best stages for operations, the embryo was exposed and the right wing bud removed by cutting with electrolytically sharpened tungsten needles as close as possible to the posterior cardinal vein. One of two experimental regimes was followed. In one, the right leg bud was removed in a similar manner to the wing bud and replaced by the wing bud. In the other, the wing bud was pushed into a slit made between the hindlimb bud and the vein to produce a supernumerary wing (Hamburger, 1939). The hole in the shell was sealed with Sellotape and the egg replaced in the incubator. At 17 days of incubation (E17) some of the embryos were re-exposed and horseradish peroxidase (HRP) injected into the umd to label the motoneuron pool. All embryos were fixed at E18 and processed for HRP histochemistry and/or ATPase and acetylcholinesterase (ACh.E) histochemistry. All the methods were described in detail in the previous paper (Laing & Lamb, 1983). RESULTS Several outcomes of the operation were observed. In many cases the wing was absent. When present it was often internalized and growing in the coelomic cavity, or it was highly abnormal. Well-formed wings were found in 71 % of surviving embryos (59% of operated embryos died prior to E17/E18). However, even in the well-formed wings there was often a complete or partial absence of musculature particularly distal to the elbow or wrist. The variations in the amount of muscle may relate to variations in the innervation of such grafts as observed by Hamburger (1939). He found grafts were best innervated when they arose dorsally close to the vertebral column. Movements of the thumb (digit 2: Sullivan, 1962) in ovo were a good indication of a well-muscled limb. Such a limb stained for ACh.E is shown in Fig. 1. Acetylcholinesterase staining The pattern of end plates in the wrist muscle ulnimetacarpalis dorsalis {umd) as displayed by ACh.E staining was the same in the displaced wings as on the contralateral control side (Fig. 2). ACh.E spots were distributed throughout the length of the slow head. The fast head usually had one or two bands of end plates. The distributions of end plates on individual muscle fibres confirmed the differences apparent in whole mounts. In five embryos 100 ACh.E-stained muscle fibres were teased from both the fast and slow heads of the umd of control and displaced wings. The majority of muscle fibres teased from the slow head of both control and operated umds had multiple end plates (76 ± 5 % and 72 ± 8 % respectively, n = 5 in both cases). The majority of fibres in the fast heads had a single end plate (99 ± 1 % and 98 ± 1 %, n = 5). The values for the operated, control and normal wings are not significantly different (P > 0-1, Mann-Whitney U-test) (see Laing & Lamb, 1983 for data on normal wings). In the slow heads Muscle fibre types in transplanted wings 69 1 Fig. 1. Whole-mount preparation of a hind limb from an E18 embryo with a supernumerary wing stained for ACh.E. Bar = 5 mm. there was a relationship between the length of a teased muscle fibre fragment and the number of end plates upon it (Fig. 3). The distributions of values for three arbitrarily chosen ranges of fragment lengths were similar in the control and operated muscles. The slow head tended to be shorter in the displaced wings than in the control wings (Table 1) and thus there were fewer long fragments in the operated sample. Such variations within the subdivisions of lengths account for the slight variations between the control and operated samples. In the control and operated fast heads the distributions of muscle fibres with single and multiple end plates were almost identical (Fig. 4). There was no sign of a 'half-way' state with approximately 50 % of the muscle fibre fragments singly and 50 % multiply innervated in either of the 'operated' umd heads. 70 N . G. LAING AND A. H. LAMB 2A Fig. 2. Comparison of the umd muscles in normal (A) and displaced (B) wings from E18 embryos stained for ACh.E. Bar = 0-5 mm. There was no significant difference (P>0-05, Kolmogorov-Smirnov twosample test) in the inter end-plate distances in the slow head of the umds of control and displaced wings (Fig. 5). ATPase staining Transverse sections cut through the forearm of E18 embryos and stained for ATPase at pH 4-3 revealed the pattern of acid-labile (pale) and acid-stable (dark) fibres within the whole forearm musculature. Most of the muscles displayed a mixture of pale and dark fibres. The distribution of the mixture was characteristic of each muscle and was retained in the displaced wings. Sections taken through the mid forearm in both control and displaced wings show how accurately the pattern is conserved (Fig. 6). The supinator is closely applied to the radius and its dorsal part has many dark fibres whereas its ventral part is composed entirely of pale fibres. The pronator sublimis has very few dark fibres whereas its neighbour the pronatorprofundus has many. The extensor metacarpi radialis has few dark fibres Muscle fibre types in transplanted wings 71 control (n = 337) operated (n = 349) "rrrrr: 3020109 10 B 40- 3 2 "3 20-1 * 10H 11 12 13 control (n = 121) operated (n = 137) 30- 1 2 3 4 5 6 9 10 11 12 13 - control (n = 42) . operated (n = 14) C 403020101 2 3 4 5 6 7 8 9 10 11 12 Number of end plates per fragment 13 Fig. 3. Comparison of the length of an E18 muscle fibre fragment and the number of end plates upon it for the slow head of the umd in displaced and control wings. A: fragment length less than 500[xm, B: 500-1000(im and C: greater than 1000jum. Table 1. Length of umd muscle heads in control and displaced wings Slow heads A) Embryo Control 1 2 3 4 5 Operated Percentage (m) (jmi) 2029 2206 2265 2324 1765 1735 1471 1706 1794 1324 Fast heads B) 86% 67% 75% 77% 75% Embryo Control 1 2 3 4 5 Operated Percentage (jum) (jum) 1029 950 857 909 971 971 900 824 771 1152 94% 95% 96% 85% 119% 72 N. G. LAING AND A. H. LAMB A 100- B 100501-1000 fan ^ 5 0 0 jum 90- 90- —control (n = 490) ... operated (n = 470) 30- 30- 20- 20- 10- control (n = 10) operated (n = 30) 10- 0 0 Number of end plates per fragment Fig. 4. Comparison of the length of a muscle fibre fragment and the number of end plates upon it for the fast heads in displaced and control wings. A: fragment length less than 500^m, (control and operated histograms were identical). B: fragment length 500-1000jum. Data from the same E18 embryos as in Fig. 3. 2220control (n = 1144) operated (n = 873) 1816•a 1 4 - 12- 642- 100 200 300 400 500 Distance between end plates 600 Fig. 5. Distribution of inter end plate distances on single muscle fibre fragments teased from the slow heads of displaced and control wings. Data from the same E18 embryos as Fig. 3. Muscle fibre types in transplanted wings 73 D emr 6A B Fig. 6. Distribution of muscle fibre types as shown by ATPase staining at pH4-3: slow fibres are dark. Transverse sections cut through the mid-forearm of E18 embryos. A: control wing, B: displaced wing, emr: extensor metacarpi radialis, FCU: flexor carpi ulnaris, pp: pronator profundus, ps: pronator sublimis, s: supinator, r: radius, u: ulna, D: dorsal, V: ventral. Bar = 0-5 mm. 74 N. G. LAING AND A. H. LAMB * * * ' • ..VV".'-'••"-'-•WRi 7A f >•"•*-• .'• ^i^'* /I— B Fig. 7. Distribution of muscle fibre types as shown by ATPase staining at pH4-3 (slow fibres are dark). Transverse sections through E18 forearms near elbow. A: control wing. B: displaced wing, b: brachialis, r. radius, u: ulna. Bar = 0-5 mm. Muscle fibre types in transplanted wings pH 9-4 pH 4-3 - v * •> Fig. 8. ATPase staining of transverse sections of the umd. A: control, B-E various operated umds showing degrees of loss of the fast head. F: fast head, S: slow head. Bar = 0-5 mm. 75 76 N. G. LAING AND A. H. LAMB Fig. 9. HRP-labelled motoneurons (arrows) in the lumbar spinal cord following injection of the slow head of the umd in displaced wings. Dark-field illumination. Bar = 0 1 mm. Muscle fibre types in transplanted wings 11 and the flexor carpi ulnaris has a pattern of dark fibres at one end with very few at the other as it twists round on itself. Sections taken nearer the elbow show the brachialis to be composed almost exclusively of dark fibres in both control and displaced wings (Fig. 7). In confirmation of the findings with ACh.E staining, the fast and slow heads of the umd are easily recognizable after ATPase staining of the displaced wings (Fig. 8). An interesting finding depicted in Fig. 8 was that the fast head was in most cases smaller than on the control side and sometimes entirely absent. This is presumably a mild manifestation of the process which sometimes results in no muscle distal to the elbow. It is perhaps significant that it is the later developing fast head (Laing & Lamb, 1983) which is affected. The loss of the fast head was not related to the type of graft. A variable number of pale fibres appeared in the predominantly dark slow head, the number often being elevated compared to the control side. Also, a variable number of dark fibres appeared in the fast head. If there were no dark fibres in the control muscle there tended to be none in the displaced muscle and if there were a few in the control muscle there were also a few in the displaced muscle. N=4 n = 34 N=4 n = 15 Fig. 10. Composite diagrams showing the position of all labelled motoneurons obtained from each of the four types of displaced wings. A: wing replacing leg, B: wing rostral to leg, C: wing dorsal to and level with leg, D: wing caudal to leg. The patterns were consistent from embryo to embryo. The position of each labelled cell was drawn by lining up the different sections using the central canal and the edges of the white and grey matter as markers. N: number of embryos, n: number of cells. 78 N . G. LAING AND A. H. LAMB A 101 8- N =4 n = 34 64200 B 10- 10 20 30 40 50 60 70 80 90 100 8- N=4 n = 15 64200 10 20 30 40 50 60 70 80 90 100 = 24 0 10 20 30 40 50 60 70 80 90 100 10 20 30 40 50 60 70 80 90 100 D 10-1 0 Fig. 11. Distribution of the labelled cells in the rostrocaudal axis. Different embryos normalized as in the previous paper (Laing & Lamb, 1983). A: wing replacing leg, B: wing rostral to leg, C: wing dorsal to and level with leg, D: wing caudal to leg. N: number of embryos, n: number of cells. Muscle fibre types in transplanted wings 79 Horseradish peroxidase labelling of motoneurons The slow head of the umd was chosen for HRP injection because of its superficial position and because the motoneuron pools to the two heads are indistinguishable in normal embryos (Laing & Lamb, 1983). The position of labelled motoneurons in the mediolateral axis of the lumbar lateral motor column (LMC) varied considerably, but tended to be middle dorsal or middle ventral (Figs 9 and 10). These positions correspond to those of the motor pools to the extensor hallucis brevis and extensor digitorum brevis (Hollyday, 1980). Such correlations in the mediolateral axis are, however, of limited value because the motoneurons surviving after grafting may not be the ones which survive normal motoneuron death, (e.g. all the normal medial motoneurons may die after grafting so that the most medial motoneurons seen would be lateral in a normal embryo). In the rostrocaudal axis, the position of the labelled cells depended to a large extent on the position of the grafted wing. With the wing replacing the leg, or lying caudal or dorsal supernumerary to the leg, the rostral end of the motoneuron pool was around 40 % of the length of the LMC from its rostral end (Fig. 11). In ten such embryos none of the 152 labelled cells were found further rostrally. This position corresponds to the position of the pools to the short extensors in the hind limb (Hollyday, 1980). On the other hand, in each of four embryos in which the wing was rostral to the leg, cells were found rostral to the 40% mark (Fig. 11). Nevertheless, the fact that they were in an intermediate position in the mediolateral axis is consistent with previous observations, that muscles derived from the dorsal muscle mass are always innervated by intermediate or lateral LMC motoneurons (Hollyday, 1981). DISCUSSION The distributions of muscle fibre types were remarkably unchanged in wings innervated by lumbar motoneurons. There are a number of possible explanations for this. Axonal guidance within the limb may be under such precise control that the appropriate types of axons are always led to correct regions of the limb, corresponding to the observed patterns of fast and slow fibres. In this way it would still be possible for fibre type to be determined by the innervating axon (neurogenically determined). However, such an explanation places extraordinary demands on the mechanism of axon guidance. This is especially true in view of the normal pattern even when the wing muscles received innervation from regions of the spinal cord which do not normally innervate homologous regions of the hind limb (e.g. umd innervated by rostral motoneurons). Neurogenic determination is also unlikely following the recent work of Butler, Cosmos & Brierley (1982) on the development of fibre types in aneural wings. They found that despite the lack of innervation, the ATPase patterns of the wing were the same as normal for similar stages of development. 80 N. G. LAING AND A. H. LAMB The probability that muscle fibres are intrinsically determined raises some interesting questions. How do the motoneurons become correctly matched with the appropriate muscle fibres? This depends on whether or not the motoneurons are themselves intrinsically determined. There is some evidence for intrinsic determination of the spatial type of motoneurons in that axons from motoneurons in certain regions of the spinal cord preferentially grow towards certain regions of the limb (Lance-Jones & Landmesser, 1981) and certain motoneurons appear to be incompatible with certain limb regions (Lamb, 1979, 1981). However, it is not known whether motoneurons are intrinsically determined for fast/slow type. If they are, then it would be easy to envisage a simple matching process whereby motoneurons can only make or retain contact with the appropriate fibre type. If they are not, it would be necessary for the motoneuron to be specified by the muscle fibres it contacts. However, at this stage of development chick embryo muscle fibres are multiply innervated (Srihari & Vrbova, 1978; Pettigrew, Lindeman & Bennett, 1979; Pockett, 1981), begging the question of whether single motoneurons contact more than one muscle fibre type. Our results are at variance in some respects with a recent study by Khaskiye et al. (1980), who transplanted lumbar spinal cord in place of brachial cord prior to limb innervation. They found that the resulting patterns of end-plate distributions, as revealed by ACh.E staining, in the anterior and posterior latissimus dorsi muscles (ALD and PLD) were altered, the main change being a shift towards distributed innervation in the normally focally innervated PLD. However, the ATPase patterns were close to normal. Thus, there was a dissociation of ATPase typing from the cholinesterase patterns. A similar dissociation is seen in muscles of embryos paralysed by neuromuscular blockade (Laing, 1982a and unpublished observations) which suggests there may be a functional reason for the discrepancy between the effects of cord and limb transplants. Nevertheless, the fact that the ATPase patterns are unchanged in either procedure adds further support to intrinsic muscle fibre type determination. If motoneurons are specified for fast/slow properties, the unchanged patterns of muscle fibre types in wings innervated by lumbar motoneurons indicate that fast/slow properties are important in matching motoneuron and muscle fibre. This means that the 'hierarchy of neuronal specificities' sometimes hypothesized to account for the formation of neuromuscular connections (e.g. Hollyday, 1981) must be extended to include fast/slow properties. The formation of the patterns of muscle fibre types may provide an explanation for the puzzling phenomenon of motoneuron death in which approximately 50 % of embryonic motoneurons die (Prestige, 1967; Hamburger, 1975; Oppenheim & Majors-Willard, 1978; Laing, 1982/?; Lance-Jones, 1982). If motoneurons and muscle fibres are both intrinsically specified, then motoneuron death might be acting to correct any imbalances between the proportions of fast and slow motoneurons innervating a muscle and the proportions of fast and slow Muscle fibre types in transplanted wings $1 muscle fibres. Studies of the proportions of axons initially invading each muscle may solve this question. Throughout these experiments we received excellent technical assistance from Jane Eccleston, Jane Diggins and Janine van Noort. The work was supported by the National Health and Medical Research Council of Australia and the Muscular Dystrophy Research Association of Western Australia. We would like to thank Philip Sheard for his comments on the manuscript. REFERENCES M. R. & PETTIGREW, A. G. (1974a). The formation of synapses in striated muscle during development. /. Physiol. 241, 515-545. BENNETT, M. R. & PETTIGREW, A. G. {191 Ab). 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