Anat Embryol (1996) 194:327-339 �9 Springer-Verlag [996

Q i x i a Z h i �9 R u i j i n H u a n g �9 B o d o C h r i s t B e a t e B r a n d - S a b e r i

Participation of individual brachial somites in skeletal muscles of the avian distal wing

Accepted: 29 April [996

A b s t r a c t In this paper we investigate the somitic origin of the individual muscles of the forearm and hand using quail-chick chimeras. Our results show that only somites 16-21 give rise to wing muscle, but they take part in muscle formation to different extents. Somite 21 does not always participate in the formation of muscle of the forearm and hand. The most cranial somite (16) takes part in the radial muscles and the most caudal somites (20, 21) in the ulnar muscles, reflecting their position with respect to the limb bud. The centrally located so- mites (17, 18, 19) are involved in all (18) or most (17, 19) muscle primordia. This pattern of distribution is clearest in the forearm, whereas the participation of so- mites in particular muscle groups is not so distinct in the hand. Hand muscles are mainly made up of cells from somites 18-20. All brachial somites participate in dorsal (extensor) as well as ventral (flexor) muscles of the fore- arm and hand. Each somite takes part in more than three muscle primordia in a reproducible fashion, and every muscle primordium is derived from at least three so- mites. Especially the M. ulnimetacarpalis ventralis takes origin from all somites involved in limb muscle forma- tion (16-21), Apart from muscle cells, endothelial cells also and a few fibroblasts of quail origin are found in the limb bud after somite grafting.

K e y w o r d s S o m i t e �9 M y o g e n e s i s �9 Cell migration - Chick embryo

Introduction

The skeletal muscle of the avian wing and leg buds is de- rived from the ventrolateral parts of the dermomyotomes at limb bud level (Christ et al. 1974, 1977; Chevallier et al. 1977). According to Jacob et al. (1978), somites 14-21 contribute to the wing muscle. The myogenic pre-

Q. Zhi - R. Huang - B. Christ. B. Brand-Saberi (~) Anatomisches Institut der Albert-Ludwigs-Universitiit Freiburg, Albertstrasse 17, D-79104 Freiburg, Germany Tel.: 07 6l/2 03-50 86; Fax: 07 61/2 03-50 91

cursor cells of the limbs undergo epithelio-mesenchymal transformation and migrate as individual cells, apparent- ly committed to the myogenic lineage, into the limb bud mesoderm, which is a derivative of the lateral plate. The elongating limb buds are invaded by myoblasts in a prox- imo-distal direction (Brand et al. 1985; Brand-Saberi and Christ I992). During their migration, myogenic cells are undifferentiated. They can, however, be characterized by a number of genes expressed before and during this pro- cess, such as Pax-3 (Goulding et al. 1994; Williams and Ordahl 1994), FoIlistatin (Amthor et al. 1996) and by cell adhesion molecules (Brand-Saberi et al. 1996). The experimental proof of the somitic origin of limb bud muscle was made possible using the biological marker technique involving chimeras between chick and Japa- nese quail embryos (Christ et al. 1974, 1977; Chevallier et al. 1977). This method was later applied to trace the fate of single somites regarding their contribution to in- dividual muscles of the limbs. In this way, Beresford (1983) found that somites 16-21 are the source of the muscles of the avian upper arm. The somitic origin of the individual muscles of the distal wing is as yet un- known. However, exact knowledge of the origin of these muscles is the prerequisite for detailed investigations concerning the control of migration and differentiation of the avian wing musculature, Especially, the possible sources of fast and slow fibre type precursors can only be determined on the basis of single somite grafts and the knowledge of their derivatives.

In this study, we undertook interspecific homotopic transplantations of individual somites at wing bud level (Le Douarin 1969). The distribution of quail ceils in the embryo, especially in the muscle primordia of the wing zeugopod and autopod (referred to here as forearm and hand), was subsequently analyzed using serial sections incubated with a quail-specific monoclonal antibody and the Feulgen-reaction (Feulgen and Rossenbeck 1924). Our results show that somites 16-21 also give rise to the muscles of the forearm and hand. Somites 18-20 give rise to most of the muscles of the hand. Somites located midway in the brachial area contribute to most of the

Material and methods

forearm muscles. All somites of the wing level contrib- ute to dorsal (extensor) and ventral (flexor) muscles. The most cranial somites participate mainly in the radial forearm muscles, whereas the most caudal somites of the brachial area participate in the ulnar forearm muscles. In the hand, cells of the muscle primordia are more inter- mingled.

Embryos

Analysis

Eggs of the White Leghorn chick (Gallus domesticus) and the Jap- anese quail (Coturnix coturnixjaponica) were incubated at 37.8~ and 80% humidity. The embryos were staged according to Ham- burger and Hamilton (1951).

bation with the first antibody, sections were rehydrated and treated with 0.3% HaO 2 in methanol to block endogenous peroxidase ac- tivity. Unspecific binding was blocked using goat's serum (1:10 in PBS). Sections were incubated with the first antibody for 90 min at room temperature. The antibody was visualized by a second an- tibody coupled with alkaline phosphatase (dilution 1:400). Quail cells were identified as muscle cells in Feulgen-stained sections by morphological criteria and in double-labelled sections by anti-des- min (dilution 1:400) and anti-quail (dilution 1:1500) staining. The desmin antibody was detected by a second antibody coupled with peroxidase (dilution 1:300).

Sections were examined using an Axioskop routine transillu- ruination microscope and photographs were taken using Agfaortho and Ektachrome films.

Grafting procedure (Fig. 1)

Single quail somites 13-23 from the stages 13-14 HH were graft- ed homotopically to the right side of the chick host of the same stage. Correct orientation of the somite was ensured using nile blue-sulfate labelling and morphological characteristics. The graft- ed somite was always the second or third somite from caudal. The somites were counted prior to grafting. At the stages used, the first somite was completely disintegrated or covered by other structures and the second somite rudimentary. The first complete somite that was observed was somite 3.

(1

The embryos were fixed according to the method of Serra (1946) 8 days after grafting. The individual muscles of the forearm and hand were identified in paraffin sections of the wing according to Sullivan (1962) and Shellswell and Wolpert (1977). The distribu- tion of quail cells was assessed by means of the Feulgen-reaction (Feulgen and Rossenbeck 1924) and by the use of the anti-quail antibody at dilution 1:1500 (B. Carlson, Developmental Studies Hybridoma Bank) on parallel sections. Embryos destined for im- munohistochemistry were fixed in Serra's fluid (Serra 1946), de- hydrated, embedded in paraffin wax and sectioned. Prior to incu-

328

Fig. 1 Schematic illustration of the grafting procedure. A single somite of a quail embryo was grafted orthotopically into a chick embryo (Q quail donor, C chick host)

Fig. 2 a Chick embryo, 3 days after grafting of somite 19 in transverse section. Quail cells are identified by anti-quail antibody and alkaline phosphatase reaction. The cells of the grafted somite (dark spots) are found in the derivatives of the sclerotome and der- momyotome, meninges, blood vessels and dermis of the back ex- tending on to the limb base where they form a sharp border (ar- row; NT neural tube, NC notochord), x40. b Section through the same embryo. The wing bud is hit more caudally. Quail cells are located in the dorsal and ventral myogenic zone (arrows); spn spi- nal nerve, x 100

329

Results

Our results are based on 127 microsurgical operations, 64 of which (=50%) were subsequently evaluated. Of the evaluated specimens, 49 had been reincubated for up to 10 days and 15 for a shorter time in order to trace the distribution of somitic cells during early development. Fifty-two (=41%) of the operated embryos died before the end of the reincubation period; 11 embryos showed malformations or were damaged during histological pro- cessing so that they were not evaluated.

All evaluated specimens used for histological exami- nation showed no traces of abnormal development. A normal differentiation of skeleton and muscles had taken place throughout all wing levels. In sections through the bodies, no other defects were observable.

The distribution of cells from one brachial somite during early development

After 2 to 3 days of reincubation following somitic grafting, quail cells were found in all somitic deriva- tives, i.e. the sclerotome, myotome and dermis of the back. Quail cells were also found in the meninges, the mesonephros and in the endothelial lining of blood ves- sels (Fig. 2a). In the wing bud, quail cells were present in the dorsal and ventral myogenic zones (Fig. 2b). In

Fig. 3 a Frontal section through an operated embryo, two days af- ter grafting of somite 20. Quail cells make up one segment includ- ing dermis (D), myotome (/t4), sclerotome (S) and endothelial cells (arrow). xl00. b Frontal section at wing level, two days after grafting of somite 19. Myogenic cells from somite 19 are found in a central stripe of the limb bud (arrow) and in the adjacent part of the mesonephros (arrowhead); clan cranial, caud caudal, x 100

frontal section (Fig. 3a), quail cells populate a segment from one intersegmental blood vessel to the next, coin- ciding with the borders of one myotome. In frontal sec- tions cut more ventrally (Fig. 3b), quail cells can be seen to populate a corresponding area of the limb bud mesenchyme, giving the impression that myogenic cells are distributed along the lateral projections of segment boundaries into the limb bud, reminiscent of a neurolog- ical dermatome. Quail cells were always restricted to the operated side.

Participation of individual somites in muscles of forearm and hand

In order to identify the brachial muscle primordia in 9.5 to 10 day-old embryos, serial sections of 8 ~tm thickness were prepared from wings of this stage and compared to schematic diagrams and descriptions in the literature (Sullivan 1962; Shellswell and Wolpert 1977; Figs. 4, 5). Figure 6 shows transverse sections through the metacar-

330

Figs. 4-6 Schematic drawing of the dorsal muscles (Fig. 4) and the ventral muscles (Fig. 5) of the right forearm and hand of a 10- day chick embryo. Sectioning planes are indicated by broken lines. Letters (A, B, C) of sectioning planes in Figs. 4-5 correspond to sectioning level A (Fig. 6a), B (Fig. 6b) and C (Fig. 6c). x40. Lev- el A Section through metacarpals. Level B Section through mid ra- dius/ulna. Level C Section through proximal radius/ulna. The numbers refer to the list of muscles provided (U ulna, R radius, II, III, IV metacarpals II, III, IV)

Fig, 7 a Transverse section through the wing of a 10-day chick embryo after transplantation of somite 20 at level B. x40. b Detail of frame in a. Double-labelling anti-quail/anti-desmin. Labelled quail cells are indicated in the muscles of ulnar side, M. ulnimet- acarpalis ventralis (8), M. flexor carpi ulnaris (11), M. anconaeus (18) and M. extensor metacarpi ulnaris (19). xl00. c Higher mag- nification (see frame in b). Quail nuclei are dark and cytoplasm is stained for desmin in brown, x 1000

Fig. 8 Transverse section through the wing at level A (a), level B (b), and level C (c) of a 10-day embryo after transplantation of somite 18; reaction of anti-quail-antibody and alkaline phospha- tase. Quail cells are found in all muscle primordia, x40

331

+

332

Fig. 9 Frontal section through ribs and wing of a 10-day em- bryo after transplantation of somite 20. Quail cells (dark spots) are found in intercostal muscle (arrow) as well as in wing muscles (tri triceps, s scapula, R fibs, shp scapulohumeralis posterior). x40

pals (a), mid (b) and proximal (c) radius/ulna in the wing. The numbers of the muscles correspond to the list of muscles given below. When individual muscles are discussed, these numbers are added in brackets. These numbers should not be confused with somite numbers that appear in brackets as bold numerals. List of muscles:

1. M. flexor indicis and abductor indicis 2. M. abductor medius 3. M. interosseus palmaris 4. M. flexor digiti IV et M. ulnimetacarpalis dorsalis 5. M. extensor indicis brevis et M. abductor indicis 6. M. extensor medius brevis 7. M. interosseus dorsalis 8. M. ulnimetacarpalis ventralis 9. M. flexor digitorum profundus

10. M. flexor digitorum superficialis 11. M. flexor carpi ulnaris 12. M. pronator profundus 13. M. pronator superficialis 14. M. extensor medius longus 15. M. extensor indicis longus 16. M. extensor metacarpi radialis 17. M. extensor digitorum communis 18. M. anconaeus 19. M. extensor metacarpi ulnaris

Wings of operated embryos were cut in the same way and the distribution of quail nuclei was assessed using anti-quail antibodies. Quail cells were identified as mus- cle cells in double-labelled sections by anti-desmin and anti-quail antibodies (Fig. 7).

Our results showed that somites 13-15 and 22-23 did not participate in the formation of striated muscles of the forearm and hand; these muscles were only derived from somites 16-21. Somite 21 participated only sometimes in the muscle formation of the forearm and hand, but al- ways in intercostal muscle formation. Somite 20 partici- pated in wing muscles regularly and also contributed to intercostal muscles (Fig. 9). Apart from muscle cells, so-

mites 16-21 gave rise to endothelial cells and a few un- identified mesenchyme cells in the wing.

Somite 16

Five operated embryos were evaluated. Fig. 10 shows the contribution of somite 16 to the muscles of the forearm and hand in different sections in two different embryos. Grafted somitic quail cells were identified in 4 of 5 cases in M. extensor indicis brevis et M. abductor indicis (5) where they participated in myotube formation. In one specimen, quail cells were also found in M. interosseus palmaris (3), M. flexor digiti IV et M. ulnimetacarpalis dorsalis (4), M. extensor medius brevis (6) and M. inter- osseus dorsalis (7). No muscle cells of quail origin were identified in M. abductor medius (2) and M. flexor indi- cis and abductor indicis (1).

In the forearm, M. ulnimetacarpalis ventralis (8) was found to contain quail cells in all 5 specimens. In 3 and 4 cases respectively, quail cells were also found in M. ex- tensor metacarpi radialis (16) and M. extensor indicis longus (15). Quail myotubes were identified in 2 cases in M. pronator superficialis (13) and in only one case in M. flexor digitorum profundus (9). The other muscles of the forearm were free from quail cells.

In summary, myogenic cells from somite 16 mainly contributed to the formation of M. extensor indicis brevis et M. abductor indicis (5) in the hand and to M. ulnimet- acarpalis ventralis (8), M. extensor metacarpi radialis (16) and M. extensor indicis longus (15) in the forearm. M. extensor metacarpi radialis (16) and M. extensor indicis longus (15) are located at the radial side of the forearm.

Somite 17

Six operated embryos were evaluated. Fig. 11 shows one of the specimens in 3 sections. Only in one of 6 cases, quail cells were found in all muscles of the hand as well as

333

Fig. 10 Transverse section through the wing of a 10-day embryo after transplantation of somite 16 at level A (a), level B (b) and level C (e) (different specimen). Anti-quail-antibody reaction. Quail cells (dark spots') are found only in M. extensor indicis bre- vis et M. abductor indicis (5) in level A (a). In level B (b) and C (e) quail cells participate in the muscles of radial side, M. ulnimet- acarpalis ventralis (8), M. pronator superficialis (13), M. extensor indicis longus (15) and M. extensor metacarpi radialis (16). x40

Fig. 11 Transverse section through the wing of a 10-day embryo after transplantation of somite 17 at level A (a), level B (b) and level C (e). Quail cells (dark spots) are found in M. flexor digiti IV et M. ulnimetacarpalis dorsalis (4), M. extensor medius brevis (6) and M. interosseus dorsalis (7) in hand level A (a). In forearm level B (b), level C (c) apart from M. flexor digitorum superficialis (10), M. anconaeus (18) and M. extensor metacarpi ulnaris (19) quhil cells participate in all other muscles, x40. d Detail of frame in c. x l00

334

Fig. 12 Transverse section through the wing of a 10-day embryo after transplantation of somite 19, level A (a), B (b) and C (c). Quail cells (dark spots) participate in all muscle primordia in this embryo. [In section a quail cells are not seen in M. extensor indi- cis brevis et M. abductor indicis (5)]. x40

of the forearm. In the hand, quail cells were found at least in 3 of 5 cases in M. extensor indicis brevis et M. abductor indicis (5), M. extensor medius brevis (6) and M. inter- osseus dorsalis (7). In 2 cases quail cells were found in M. flexor digiti IV et M. ulnimetacarpalis dorsalis (4). In M. abductor medius (2) and M. interosseus palmaris (3), grafted cells were identified in the myotubes only in one case. Quail cells were never observed in M. flexor indicis and abductor indicis (1) except in the first case.

In the forearm, quail cells were found in all 5 cases in M. extensor indicis longus (15), M. extensor metacarpi ra- dialis (16), M. pronator profundus (12) and M. pronator su- perficialis (13). In 3-4 cases, grafted cells participated in M. extensor medius longus (14), M. ulnimetacarpalis ven- tralis (8) and M. flexor digitorum profundus (9). In M. flex- or carpi ulnaris (11), quail cells were found in two speci- mens. Quail cells were never found in muscle primordia of

Fig. 13 Transverse section through the wing of a 10-day embryo after transplantation of somite 21, level A (a), B (b) and C (c). Quail cells (dark spots) participate in all muscles of the hand in this embryo (a) - in this particular section quail cells are not seen in M. flexor indicis and M. abductor indicis (1) and M. extensor indicis brevis et M. abductor indicis (5). At forearm levels B (b) and C (c) quail cells are found only in the muscles of the ulnar side: M. ulni- metacarpalis ventralis (8), M. flexor digitorum profundus (9), M. flexor digitorum superficialis (10), M. flexor carpi ulnaris (11), M. anconaeus (18) and M. extensor metacarpi ulnaris (19). x40

the ulnar side such as M. flexor digitorum superficialis (10), M. anconaeus (18) and M. extensor metacarpi ulnaris (19).

Somite 18

The contribution of somite 18 to the muscle of the fore- arm and hand was studied in 8 embryos. Sections o f one

Table 1 Contribution of somites 16-21 to individual muscles of the forearm and hand. Each number in brackets corresponds to the number of the muscle in the figures (++ The muscle received the grafting somite cells in all evaluated chimaeras; + the muscle received the grafting somite cells in at least three evaluated chimaeras)

Somites

335

Muscle 16 17 18 19 20 21

+ + + +

+ + + + +

+ + +

+ + +

+ + + + +

+ + + + +

+

+ + + + + + + +

+ + + + +

+ + +

+ + + + + + +

+ + + +

+ + + +

+ + + + +

+ + + + + +

+ + + + + + +

+ + + + +

+ + + + + +

+ + + + +

+ + + + + +

Dorsal group of the forearm M. extensor medius longus (14) M. extensor indicis longus (15) + M. extensor metacarpi radialis (16) + M. extensor digitorum communis (17) M. anconaeus (18) M. extensor metacarpi ulnaris (19)

Ventral group of the forearm M. ulnimetacarpalis ventralis (8) ++ M. flexor digitorum profundus (9) M. flexor digitorum superficialis (10) M. flexor carpi ulnaris (11) M. pronator profundus (12) M. pronator superficialis (13)

Dorsal group of the hand M. extensor indicis brevis/M.abductor indicis + M. extensor medius brevis (6) M. interosseus dorsalis (7)

Ventral group of the hand M. flexor indicis/M, abductor indicis (1) M. abductor medius (2) M. interosseus palmaris (3) M.flexor digiti IV/M.ulnimetacarpalis dorsalis +

embryo are shown in Fig. 8. Quail cells were found to participate in all muscles of the forearm and hand at least in 5 of 8 cases. In M. interosseus dorsalis (7) of the hand and in M. ulnimetacarpalis ventralis (8) and M. extensor indicis longus (15) of the forearm, quail cells participat- ed in all specimens. We conclude from these results that somite 18 takes part in all muscles of the hand and fore- arm.

Somite 19

Six operated embryos were evaluated. Fig. 12 shows sec- tions of a representative case. Grafted somitic cells were found to participate in all muscles except M. extensor in- dicis brevis et M. abductor indicis (5) in all specimens. In M. extensor indicis brevis et M. abductor indicis (5), quail cells were found in 4 of 6 cases.

In the forearm, quail cells participated in all cases in the formation of M. ulnimetacarpalis ventralis (8), M. flexor digitorum profundus (9), M. anconaeus (18) and M. extensor metacarpi ulnaris (19). In 4-5 specimens, quail cells were also found in M. flexor digitorum super- ficialis (10), M. extensor medius longus (14), M. extensor indicis longus (15) and M. extensor digitorum communis (17). In 3 cases, they participated also in M. pronator pro- fundus (12) and M. pronator superficialis (13). Only in 2 cases did quail cells participate in M. extensor metacarpi radialis (16), which belongs to the radial group.

In summary, we found that the cells derived from somite 19 contributed to all muscles of the hand and forearm, in some cases except in M. extensor metacarpi radialis (16).

Somite 20

The distribution of myogenic cells from somite 20 was studied in 8 embryos. In the hand, all muscles except M. extensor medius brevis (6) contained quail cells in at least 7 cases. M. extensor medius brevis (6) contained grafted cells in 50% of the specimens.

In the forearm, quail cells were found in M. anconaeus (18), M. extensor metacarpi ulnaris (19), M. ulnimetacar- palis ventralis (8) and M. flexor carpi ulnaris (11) in all cases examined. In 3 4 cases, grafted cells were found ad- ditionally in M. flexor digitorum profundus (9), M. flexor digitorum superficialis (10) and M. extensor medius lon- gus (14). In M. pronator profundus (12), M. pronator su- perficialis (13), M. extensor indicis longus (15) and M. ex- tensor digitorum communis (17), quail cells participated in muscle formation only in up to 2 cases. M. extensor metacarpi radialis (16) was always devoid of quail cells. Figure 7 shows a section through one of the specimens.

Summing up, cells from somite 20 participated in all muscles of the hand. In the forearm, they took part main- ly in the formation of ulnar muscle primordia.

Somite 21

The distribution of cells from somite 21 was followed on the basis of 8 operated embryos. Figure 13 shows the sections of one of the embryos.

In these embryos, two different categories of results were observed. In one group (5 of 8 cases), no muscle cells from the grafted somites were found in the muscle primordia of forearm and hand. Only a few quail cells

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So 16

So 20

were seen as endothelial cells and unidentified mesen- chyme cells in the distal part o.f the wing. In the other group (3 of 8 cases), grafted cells participated in the for- mation of muscle in hand and forearm. They were locat- ed in all muscles of the hand in at least 2 of 3 cases. In the forearm, M. ulnimetacarpalis ventralis (8), M. flexor digitorum superficialis (10), M. flexor carpi ulnaris (11) and M. anconaeus (18) contained quail cells in all 3 cases. In one specimen, quail cells were additionally found in M. flexor digitorum profundus (9) and M. ex- tensor metacarpi ulnaris (19). This suggests that myo- genic cells from somite 21 have either the option not to participate at all in the formation of limb muscles, or to contribute mainly to the muscles of the hand and the ul- nar muscle group of the forearm.

To summarize our findings, Table 1 shows the contri- bution of the individual somites to the muscles of the forearm and hand, and the schematic diagram Fig. 14 shows the distribution of the myogenic cells in the mus- cle primordia of the forearm and hand.

Discussion

In this study, we have demonstrated the distribution of myogenic cells in the muscle primordia of the forearm and hand after single somite grafts at the brachial level. We employed the interspecific grafting technique using chick-quail chimeras (Le Douarin 1969).

By means of immunohistochemical detection of quail cells by a monoclonal antibody in serial sections, we found that the muscles of the forearm and hand are de- rived from somites 16-21. It became obvious that each muscle is made up of material originating from at least 3 somites. Especially, the M. ulnimetacarpalis ventralis re- ceives contributions from all somites (16-21).

Our results suggest that myogenic cells from individ- ual somites are not distributed randomly in the limb bud such as would result from a participation in the forma- tion of muscle primordia by chance. However, every somite appears to contribute to particular muscles (at least three muscle primordia). This is in line with the findings of Beresford (1983) for the upper arm.

The distribution of myogenic cells in the forearm and hand seems to be subjected to a set of rules: cranially lo- cated somites (16) give rise to muscles at the radial side of the forearm, whereas caudally located somites (2t), 21) contribute to muscles at the ulnar side. Centrally located somites (17, 18, 19) are involved in most (17, 19) or all (18) muscles of the forearm. This rule applies only for the forearm. In the hand, muscle primordia are mainly made

Fig. 14 Synopsis of the distribution of muscle cells from somites 16-21 in the individual muscles of the forearm and hand. Black dots represent quail cells. In this figure, only results that are based on at least three independent experiments were counted as posi- tive. Regarding somite 21, myogenic material participates only sometimes in wing muscle formation (in 3 of 8 cases)

337

h ~

1 6

[ 1 7 �84

[_18 [19 L 2 0 ....

L21

I

Fig. 15 Schematic drawing of the hypothetical distribution of myogenic cells from somites 16-21 in the wing bud. Extensive mixing occurs, especially in the hand. In the forearm, somites 16 and 17 mainly contribute to radial muscle primordia, whereas the caudal somites contribute mainly to the ulnar muscle primordia

up of myogenic cells from somites 18-20, i.e. myogenic cells from different somites mix in the periphery (Fig. 15). Moreover, all somites contribute to both dorsal (extensor) as well as ventral (flexor) muscles. It is possible that the distribution of myogenic cells from different somites mir- rors the formation of the brachial plexus from different spinal nerves. The innervation of individual muscles does not depend on the segmental origin of the muscles (Stir- ling and Summerbell 1985; Keynes et al. 1987). It there- fore seems that the distribution of both myogenic cells (Ja- cob et al. 1983) and axons in the limb bud are controlled by the stationary limb bud mesenchyme.

The process of invasion of the limb bud mesoderm by somite-derived cells has been shown to involve active cell migration (Wachtler et al. 1982; Ede et al. 1984; Gumpel-Pinot et al. 1984; Brand-Saberi et al. 1989; Krenn et al. 1991). This migration is mediated by cell- matrix (Krenn et al. 1991; Brand-Saberi et al. 1993) and cell-cell contacts (Brand-Saberi et al. 1995). As far as the extracellular matrix is concerned, myogenic cell mi- gration has been found to depend on the availability of fibronectin, since it is inhibited by injection of antibodies against the cell-attachment site of fibronectin (Brand-Sa- beri et al. 1993). Directionality is inferred from the dis- tribution of another matrix component, hyaluronan (Krenn et al. 1991). Hyaluronan is distributed with high levels at the distal end of the limb bud and low levels in the proximal areas (Kosher et al. 1981). Moreover, the amount of hyaluronan decreases with development (Krenn et al. 1991). Exogenously added hyaluronan leads to a re-initiation of myoblast migration under ex- perimental conditions that do not normally support mi- gration (Brand-Saberi and Krenn 1991; Krenn et al. 1991). On the other hand, transmembrane cell adhesion molecules (N-cadherin) have been found to be expressed by migrating myoblasts (Hayashi and Ozawa 1995) and in the stationary lateral plate-derived mesenchyme of the limb bud (Brand-Saberi et al. 1995, 1996).

By the distribution of matrix and cell-adhesion mole- cules in the limb bud mesenchyme, a primitive prepat-

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tern for the distribution of myogenic cells is laid down. N-cadherin, for instance, is found in the dorsal and ven- tral myogenic zone, but not in the chondrogenic zone during the stages of cell migration. Since N-cadherin has been shown to be involved in myoblast migration (Brand-Saberi et al. 1996), its distribution appears to re- sult in the homing of myogenic cells to the myogenic zones. In the same way, individual muscle primordia could later be partitioned off due to a refined pattern of N-cadherin distribution or that of other cell adhesion molecules, although this has not been studied.

From the present work, a second aspect of myoblast distribution has to be taken into consideration. This is the fact that the initial position of limb-bud areas (radial, cen- tral, ulnar) with respect to particular brachial somites has a bearing on the distribution of myogenic cells from these somites within the limb bud mesenchyme. This means that initially myoblasts appear to emigrate from the so- mites strictly laterally along projections of the original segmental borders into the lateral plate mesoderm. By patterning influences from the limb bud mesoderm, mix- ing of myoblasts from different somites takes place at least in the central part of the forearm: divergence of mi- grating cells in the broadening distal portion of the limb bud could be the reason for the extensive mixing of myo- blasts from different somites observed at autopod level. As long as the cranial and caudal borders of the limb bud run parallel as imaginary extensions of the axial segmen- tation, only a little mixing occurs. Another reason for dis- tal intermingling could be the high levels of hyaluronan in the distal part of the limb bud. Hyaluronan forms a pericellular coat that widens the intercellular spaces and may thus hinder the formation of stable intercellular con- tacts. This could result in a greater mobility along the an- terior-posterior axis of the limb bud.

A more detailed study of the three-dimensional pre- pattern of the limb bud mesoderm at the molecular level will be needed to give insights into the mechanisms that lead to the definite positioning of myogenic cells in the muscle primordia of the limbs. However, our results form the basis for further studies, e.g. concerning the dif- ferentiation of myoblasts from particular somites into fast and slow fibres. To answer such questions, it is nec- essary to know the exact origin and allocation of myo- blasts within the limb bud.

Acknowledgements The authors wish to thank Lidia Koschny, Monika SchtRtoff and Ellen Gimbel for excellent technical assis- tance, Christa Micucci for the photographic work and Eric Jens Burlefinger for the drawings. The manuscript was typed by Ulrike Uhl and Heike Bowe. Our studies were supported by the Deutsche Forschungsgemeinschaft Ch 44/12-2 and Br 957/2-1.

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