It is known that myogenic cells in limb buds are derived from somites. In order to examine the potential of the limb primordium (presumptive limb somatopleure) to induce myogenic cell migration, we transplanted chick presumptive limb somatopleure to the flank region of an embryo, a region that does not normally contribute myogenic cells to the limb. Somitic cell migration was examined using a vital labeling technique. When the presumptive limb somatopleure was transplanted and was in contact with the host flank somite, somitic-cell migration toward the graft was observed. The labeled somitic cells within the graft were identified as myogenic cells in two ways: first, we found that N-cadherin-expressing cells appeared in the graft. Second, after 3 further days of incubation, the somitic cells formed dorsal and ventral masses and expressed sarcomeric myosin heavy chain within the graft. Cell migration occurred only when the somite was in contact with the medial region of the presumptive limb somatopleure. When the somite was not in contact with the limb somatopleure, or when the somite was in contact with the lateral region of the limb somatopleure, migration did not occur. These observations indicate that the potential to induce myogenic cell migration is restricted to the medial region of the presumptive limb somatopleure and that tissue contact is required.

Cell migration is one of the most prominent morphogenetic movements in development. However, mechanisms that regulate initiation, pathway and target of migration are not well understood. Myogenic cells in limb buds are known to originate from somites. (Chevallier et al., 1977; Christ et al., 1977). Cells at the lateral edge of the somite at the limb level migrate into the limb bud. This phenomenon provides a simple system to investigate the mechanism of cell migration, since (1) the migrating cells are a homogeneous population with a predetermined developmental fate, (2) the migration is completed within a short period of time (from stage 15 until stage 18 at chick wing level, about 10 hours) and (3) the pathway is simple and unique: from somite to adjacent somatopleure. These characteristics are in contrast with the migration of neural crest cells. Neural crest cells are the precursors of several cell types (fibroblasts, chondrocytes, neural cells, pigment cells), their migration lasts for a long period (from stage 12 until stage 22 at wing level of chick embryo, for example, about 36 hours) and proceeds along several pathways (dorsoventral pathway and ventral pathway in the trunk region). In spite of the simplicity of the process, myogenic cell migration into the limb bud has not been well investigated. One of the reasons for this is that there had been no molecular markers for the migrating myogenic cells untill the recent finding of Pax-3 expression in these cells (Bober et al., 1994; Goulding et al., 1994; Williams and Ordahl, 1994). The migrating myogenic cells in the limb bud do not express detectable levels of myogenic factors (MyoD, myogenin, Myf-5 or MRF-4) for at least 24 hours following migration to the developing limb bud (Bober et al., 1991; Pownall and Emerson, 1992). They do not differentiate to muscle cells until stage 25 in chick.

In order to trace myogenic cell migration, we previously developed a vital labeling technique in which somitic cells are labeled by fluorescent dye in situ (Hayashi and Ozawa, 1991). This is the most simple and reliable method, especially for observing the early events of somitic cell migration, because it does not require any microsurgery, such as somite trans-plantation, which may result in artifacts. The vital labeling method enables us to design experiments to investigate the regulatory mechanisms of myogenic cell migration.

The myogenic cells of flank somites do not contribute to limb muscle cells. The cells at the lateral edge of the flank somite retain epithelial structure (Christ et al., 1983), whereas the somite cells at limb levels migrate out of the somite. This level difference in myogenic cell migration is not due to position specificity of the somite. When transplanted to the limb levels, the transplanted somites, regardless of their origin, can contribute to limb muscle cells (Chevallier, 1978). Thus, somitic cells may not possess position specificity as regards their ability to migrate into limb bud. The fact that Pax-3, which is expressed in migratory myogenic cells, is also expressed in the ventrolateral bud of the flank somites supports this notion (Bober et al., 1994; Goulding et al., 1994; Williams and Ordahl, 1994). One possible explanation for the position difference of the myogenic cell behavior is that the growing limb buds induce migration of Pax-3-positive myogenic cells.

To examine the potential of limb primordium (presumptive limb somatopleure) to induce myogenic cell migration, we transplanted it to the flank region. We demonstrate here that the medial region of the presumptive limb somatopleure exerts some influence on somitic cell migration, and that this influence requires tissue contact.

Animals

Fertilized eggs of domestic chick were incubated at 37.5°C. Embryos were staged as described by Hamburger and Hamilton (1951). The most anterior pair of somites without a clear anterior boundary was counted as somite pair number one (Beresford, 1983).

Fluorescent labeling

Fluorescent labeling of somites was performed as described by Hayashi and Ozawa (1991). One mg of DiI (1,1’,dioctadecyl-3,3,3’,3’,-tetramethylindo-carbocyanine perchlorate, Molecular Probes, OR), and 800 μg of phosphatidylcholine were sonicated together in 100 μl of Tyrode’s solution. The solution was diluted to 1 ml with Tyrode’s solution, vortexed, and briefly centrifuged. A small volume of the resulting suspension was injected into the lumen under the dermomyotome with a glass microcapillary. In some cases, 5% (saturated DiI with ethanol) in 0.3 M sucrose was injected. The results obtained using the two solutions were indistinguishable, but the latter was easier to use, because it did not contain aggregates that could clog the capillary.

Fluorescent labeling of grafts was performed as follows. DiO (3,3’-dioctadecyloxa-carbocyanine perchlorate, Molecular Probes, OR) was sonicated in a phosphatidylcholine solution as described above, centrifuged at high speed and passed through a Millipore filter (0.22 μm pore size) to remove dye aggregates completely. Tissues were immersed in this solution for 5 minutes and washed with Tyrode’s solution several times.

Transplantation experiments

Transplantation procedures are illustrated in Fig. 1. Manipulations were performed through a window in the shell. To create a lesion at the graft site in a host embryo, the somatopleure at the flank level (at the level of somites 23-25) was excised with sharpened tungsten wires (Dossel, 1958) and microscissors, and discarded. Grafts were fragments of somatopleure (parietal layer of lateral plate with ectoderm) that were excised from the wing region (at the level of somites 17-19) or flank region (at the level of somites 23-25) of donor embryos (at stage 14-16). They were labeled with DiO as described above and transplanted to the lesion of the host (at stage 14-16). The host embryos were incubated for several hours until the explant adhered to the embryo. Then somite 24 was labeled with DiI as described above (at stage 15-17). The window was sealed with adhesive tape and the egg was returned to the incubator.

Fig. 1.

Diagram illustrating the transplant procedure. Grafts were labeled with DiO and transplanted into the flank region (at the level of somites 23-25) of the host embryos. After several hours, somite 24 of the host was injected with DiI. After about 18 hours, the embryos were fixed and observed.

Fig. 1.

Diagram illustrating the transplant procedure. Grafts were labeled with DiO and transplanted into the flank region (at the level of somites 23-25) of the host embryos. After several hours, somite 24 of the host was injected with DiI. After about 18 hours, the embryos were fixed and observed.

Four series of experiments were performed (Fig. 2).

Fig. 2.

Schematic representation of the four transplantation experiments. (A) Exp. 1. Flank somatopleure (level of somites 23-25) was labeled and transplanted to the flank region of the host (level of somites 23-25). (B) Exp. 2. Wing somatopleure (level of somites 18-20) was transplanted to the flank region. (C) Exp. 3. Wing somatopleure was transplanted to the flank region in reversed mediolateral orientation. (D) Exp. 4. Wing somatopleure was transplanted to a site about 100 μm laterally from the flank somite. A fragment of the host flank somatopleure (asterisk) remained between the somite and the graft. The development of the grafts after 18-hour incubation is illustrated on the right. The medial region of the wing somatopleure is cross-hatched.

Fig. 2.

Schematic representation of the four transplantation experiments. (A) Exp. 1. Flank somatopleure (level of somites 23-25) was labeled and transplanted to the flank region of the host (level of somites 23-25). (B) Exp. 2. Wing somatopleure (level of somites 18-20) was transplanted to the flank region. (C) Exp. 3. Wing somatopleure was transplanted to the flank region in reversed mediolateral orientation. (D) Exp. 4. Wing somatopleure was transplanted to a site about 100 μm laterally from the flank somite. A fragment of the host flank somatopleure (asterisk) remained between the somite and the graft. The development of the grafts after 18-hour incubation is illustrated on the right. The medial region of the wing somatopleure is cross-hatched.

Exp. 1. For negative control, flank somatopleure (at the level of somites 23-25) was transplanted to the flank region (the corresponding site) of the host. The graft site was adjacent to the somites (Fig. 2A).

Exp. 2. Wing somatopleure (at the level of somites 17-19) was transplanted to the flank region in the original anteroposterior and mediolateral orientations. The graft site was adjacent to the host flank somites (Fig. 2B).

Exp. 3. Wing somatopleure was transplanted in reverse mediolateral orientation in the following two ways. In some experiments, wing somatopleure of the left side of the donor embryos was transplanted to the right side of the host flank region in the original anteroposterior orientation. This causes reversal in mediolateral orientation. In other cases, wing somatopleure of the right side of the donor embryo was rotated 180° around the dorsoventral axis and transplanted to the right side of the host flank region. This causes reversals in both anteroposterior and mediolateral orientations. The graft site was adjacent to the host flank somites (Fig. 2C).

Exp. 4. Wing somatopleure was transplanted in the original anteroposterior and mediolateral orientations as in the case of Exp. 2; however, in this experiment, the graft site was about 100 μm laterally from the host flank somites (Fig. 2D). This results in an intercalation of host flank somatopleural tissue between host somites and the graft (asterisk in Fig. 2D).

Observation

After incubation for about 18 hours, the embryos (at stage 19-21) were fixed in 4% paraformaldehyde, 0.25% glutaraldehyde/0.1 M phosphate buffer overnight and washed in 0.1 M phosphate buffer. After whole-mount observation with an epi-fluorescence microscope, they were soaked in 15% sucrose/phosphate buffer, embedded in 7.5% gelatin/15% sucrose and frozen. Cryosections 15 μm thick were cut and immediately observed. DiI was observed as red through rhodamine filters and as yellow through FITC filters. DiO was observed as green through FITC filters, but could not be observed with rhodamine filters (Honig and Hume, 1989). Migration was assessed on a cross section through the center of the labeled somite. The sections containing 0-4 cells migrating out of the somite were scored as -, those containing 5-9 cells as +-, those containing 10-29 cells as + and those containing more than 29 cells as ++.

Immunohistochemistry

Samples for immunohistochemistry were fixed in 2% paraformaldehyde in 0.1 M phosphate buffer. Cryosections, 7 μm thick, were incubated in 0.5% casein, 0.5% gelatin in PBS for more than 30 minutes and subsequently in primary antibody for 1 hour at room temperature. They were washed in PBS for 30 minutes and then incubated in a second antibody for 1 hour. After washing in PBS for 30 minutes, sections were observed with an epifluorescence microscope. For staining N-cadherin, monoclonal antibody NCD-2 (kindly provided by Dr Masatoshi Takeichi), biotin-conjugated anti-rat IgG and rhodamine-conjugated avidin (Vector Laboratories, CA) were used. For staining sarcomeric myosin heavy chain, monoclonal antibody MF20 (Bader et al., 1982; kindly provided by Dr Takashi Obinata) and FITC-conjugated anti-mouse IgG (Kirkegaad and Perry Lab., Inc.) were used.

Myogenic cell migration in normal embryos

In order to determine from which somites myogenic cells migrate laterally and from which somites they do not, we injected dye into various somites. Dye was injected at the stages indicated in Table 1. More than 10 hours after injection, when the embryos were at stages 19-21, we observed the distribution of labeled cells in whole-mount preparations from the dorsal side (Table 1). Lateral migration of labeled cells was detected from somites 16-21 and 27-33 (Fig. 3A) but not from somites 22-26 (Fig. 3B) even after 27 hours (at stage 21, Fig. 3C). From somites 20, 21 and 27 migration was inconsistent (Table 1). This is probably due to the individual variations among embryos or errors in counting of somites. Generally, the number of migrating cells from these somites was small. Our observations are roughly consistent with the findings of previous transplantation studies that the most posterior somite that contributes to wing muscle cells is somite 21 (Beresford, 1983), and the most anterior somite that contributes to leg muscle cells is 26 (Lance-Jones, 1988).

Table 1.
Somitic cell migration in normal embryos
graphic
graphic
Fig. 3.

Somitic cells labeled with fluorescent dye in normal embryos. (A) Whole-mount observation of the leg region at stage 20. Dye was injected into somite 30 at stage 18. Right is cranial. Lateral migration of labeled cells was clearly detected (arrow). (B) Whole-mount observation of the flank region at stage 20. Dye was injected into somite 24 at stage 16. The lateral edge of the somite was smooth (arrow) and no labeled cells were detected in the somatopleure. (C) Wholemount observation of the flank region at stage 21. Dye was injected into somite 24 at stage 16. Lateral migration of the somitic cells was not recognized (arrow). (D) Frontal section of the wing level of stage 19 embryo. Dye was injected into somite 18 at stage 15. Labeled cells were observed in the proximal region of the wing bud (arrow), but not in the distal or ventral regions (small arrows). (E) Frontal section of the flank level at stage 20. Dye was injected into somite 24 at stage 16. The lateral ventral edge of the somite forms an epithelial structure (arrow). n, neural tube; t, tail; s, somite. Bars are 200 μm.

Fig. 3.

Somitic cells labeled with fluorescent dye in normal embryos. (A) Whole-mount observation of the leg region at stage 20. Dye was injected into somite 30 at stage 18. Right is cranial. Lateral migration of labeled cells was clearly detected (arrow). (B) Whole-mount observation of the flank region at stage 20. Dye was injected into somite 24 at stage 16. The lateral edge of the somite was smooth (arrow) and no labeled cells were detected in the somatopleure. (C) Wholemount observation of the flank region at stage 21. Dye was injected into somite 24 at stage 16. Lateral migration of the somitic cells was not recognized (arrow). (D) Frontal section of the wing level of stage 19 embryo. Dye was injected into somite 18 at stage 15. Labeled cells were observed in the proximal region of the wing bud (arrow), but not in the distal or ventral regions (small arrows). (E) Frontal section of the flank level at stage 20. Dye was injected into somite 24 at stage 16. The lateral ventral edge of the somite forms an epithelial structure (arrow). n, neural tube; t, tail; s, somite. Bars are 200 μm.

Close observation of cross sections at the wing level at stage 19 revealed that the labeled cells colonize within the dorsoproximal region of the limb bud, and that they do not invade the distal and ventral regions (Fig. 3D). At the flank level, cells at the lateral edge of the somite, which did not migrate out of the somite by stage 20 (Fig. 3E), retained epithelial structure (referred to as myocoel by Christ et al., 1983). A few labeled cells were sometimes found among the flank somatopleural mesenchyme. They were located at the basal surface of coelomic epithelia, and are probably endothelial cells, as mentioned by Schramm and Solursh (1990).

Transplantation of flank somatopleure (Exp. 1)

The flank somatopleura were labeled with DiO and transplanted into the flank regions of host embryos (Fig. 2A). The host flank somite was injected with DiI. During the incubation of about 18 hours, the grafts grew larger in area but did not thicken (Fig. 4A). In all 8 cases, the host flank somite was in contact with the graft. The lateral edge of the somite formed a compact cell mass without intermingling with somatopleural cells (Table 2, Fig. 4B). In 2 cases, about 5 DiI-labeled cells were found within the grafts. It is not known whether these cells were myogenic cells.

Table 2.
Somitic cell migration in transplantation experiments
graphic
graphic
Fig. 4.

Transplantation experiments (Exp. 1). DiO-labeled flank somatopleure was transplanted to the flank region, and the flank somite was labeled with DiI. (A) After 18 hours the cross section was observed through fluorescent filters. The lateral edge of the somite (yellow) and the graft (green) were in contact (arrow). (B) The same section as in A observed through rhodamine filters. Somitic cells (red) did not migrate laterally (arrow). Bar is 200 μm.

Fig. 4.

Transplantation experiments (Exp. 1). DiO-labeled flank somatopleure was transplanted to the flank region, and the flank somite was labeled with DiI. (A) After 18 hours the cross section was observed through fluorescent filters. The lateral edge of the somite (yellow) and the graft (green) were in contact (arrow). (B) The same section as in A observed through rhodamine filters. Somitic cells (red) did not migrate laterally (arrow). Bar is 200 μm.

Transplantation of wing somatopleure (Exp. 2)

To examine the potential of wing somatopleure to induce somitic cell migration, we transplanted it to the flank region. Grafts were transplanted in the original anteroposterior and mediolateral orientations (Fig. 2B). During the incubation of about 18 hours, the graft grew well and formed an extra limb bud at the host flank region (Fig. 5A,B). In all 8 cases, DiI-labeled cells were found within the graft as dispersed single cells (Fig. 5D). This indicates that the wing somatopleure has the potential to induce somitic cell migration.

Fig. 5.

Transplantation experiments (Exp. 2). DiO-labeled wing somatopleure was transplanted to the host flank region, and the flank somite was labeled with DiI. (A) After 18 hours a whole-mount preparation was observed through fluorescence filters. The graft formed an extra limb bud in the flank region of the host embryo. Right is cranial. (B) The same preparation was observed through rhodamine filters. DiI-labeled somitic cells migrated into the graft (arrow). (C) The cross section of the flank region of the embryo in Exp. 2 was observed through fluorescent filters. The lateral edge of the somite (yellow) was in contact with the graft (green, arrow). (D) The same section observed through rhodamine filters. DiI-labeled cells were observed within the graft (arrow). Bars are 200 μm.

Fig. 5.

Transplantation experiments (Exp. 2). DiO-labeled wing somatopleure was transplanted to the host flank region, and the flank somite was labeled with DiI. (A) After 18 hours a whole-mount preparation was observed through fluorescence filters. The graft formed an extra limb bud in the flank region of the host embryo. Right is cranial. (B) The same preparation was observed through rhodamine filters. DiI-labeled somitic cells migrated into the graft (arrow). (C) The cross section of the flank region of the embryo in Exp. 2 was observed through fluorescent filters. The lateral edge of the somite (yellow) was in contact with the graft (green, arrow). (D) The same section observed through rhodamine filters. DiI-labeled cells were observed within the graft (arrow). Bars are 200 μm.

Transplantation of wing somatopleure in reverse orientation (Exp. 3)

Our observation that the myogenic cells did not colonize at the ventral region of the limb buds led us to hypothesize that this region had a character different from that of the dorsoproximal region. In order to assess the potential of the ventral region of the limb bud (which corresponds to the lateral region of the presumptive limb somatopleure) to induce somitic cell migration, wing somatopleure was transplanted in the reverse mediolateral orientation (Fig. 2C), so that the lateral region of wing somatopleure contacted the host somite. During the incubation of about 18 hours, the graft grew well and formed an extra limb bud at the flank region of the host (Fig. 6A). Although in all 8 cases the DiI-labeled somite was in contact with the graft, the lateral edge of the somite formed a compact cell mass without intermingling with somatopleural cells (Fig. 6B). In 2 cases, about 5 labeled cells were found within the grafts. It is not known whether these cells are myogenic cells. The results of experiments in which wing somatopleure was transplanted from the right side were indistinguishable from those of transplants from the left side of the donor.

Fig. 6.

Transplantation experiments (Exp. 3). DiO-labeled wing somatopleure was transplanted to the host flank region in reverse mediolateral orientation, and the flank somite was labeled with DiI. (A) After 18 hours the cross section was observed through fluorescence filters. The flank somite (yellow) and the graft (green) were in contact (arrow). (B) The same section as in A observed through rhodamine filters. Somitic-cell migration was not seen (arrow). Bar is 200 μm.

Fig. 6.

Transplantation experiments (Exp. 3). DiO-labeled wing somatopleure was transplanted to the host flank region in reverse mediolateral orientation, and the flank somite was labeled with DiI. (A) After 18 hours the cross section was observed through fluorescence filters. The flank somite (yellow) and the graft (green) were in contact (arrow). (B) The same section as in A observed through rhodamine filters. Somitic-cell migration was not seen (arrow). Bar is 200 μm.

Transplantation of wing somatopleure to a site nonadjacent to flank somites (Exp. 4)

To examine whether the induction of cell migration requires tissue contact, wing somatopleure was transplanted to a site approximately 100 μm laterally from the flank somites (Fig. 2D). During the incubation of about 18 hours, the graft attached to the free edge of the host flank somatopleure, and formed an ectopic limb bud at the lateral side of the abdominal wall (13 cases). In 9 of 13 cases, the lateral edge of the flank somite elongated into somatopleure as the normal somites do, but it did not contact the graft. The cells at the edge formed a compact cell mass without dispersing (Table 2, Fig. 7B). In the other 4 cases, the lateral edge of the somite elongated through the flank somatopleure and contacted the graft. In these cases the lateral edge of the somite was irregularly shaped at the contact site, and labeled cells were detected within the graft (Table 2, Fig. 7C,D).

Fig. 7.

Transplantation experiments (Exp. 4). DiO-labeled wing somatopleure was transplanted to a site nonadjacent to the flank somite, and the somite was labeled with DiI. (A) After 18 hours, the cross section was observed through fluorescent filters. The host somatopleural mesoderm (asterisk) prevented contact between the somite (yellow) and the graft (green). (B) The same section as in A observed through rhodamine filters. The somitic cells did not migrate (arrow). (C) In this case, the somite elongated through the host somatopleure (asterisk) and contacted the graft (green). (D) The same section as in C was observed through rhodamine filters. DiI-labeled cells were observed within the graft (arrow).

Fig. 7.

Transplantation experiments (Exp. 4). DiO-labeled wing somatopleure was transplanted to a site nonadjacent to the flank somite, and the somite was labeled with DiI. (A) After 18 hours, the cross section was observed through fluorescent filters. The host somatopleural mesoderm (asterisk) prevented contact between the somite (yellow) and the graft (green). (B) The same section as in A observed through rhodamine filters. The somitic cells did not migrate (arrow). (C) In this case, the somite elongated through the host somatopleure (asterisk) and contacted the graft (green). (D) The same section as in C was observed through rhodamine filters. DiI-labeled cells were observed within the graft (arrow).

Specimens in which the graft did not attach to the host somatopleure but to the splanchnic mesoderm were excluded.

N-cadherin expression in myogenic cells

It is known that cultured myoblasts express N-cadherin prior to fusion (Pouliot et al., 1990). The level of the expression increases as the myoblast reaches maturity, and the maximal level of N-cadherin expression prior to myoblast fusion is thought to be involved in the fusion process (Knudsen et al., 1989; Pouliot et al., 1990). We investigated N-cadherin expression during the development of the normal limb bud. At stage 18, the neural tube and the dermo-myotome expressed N-cadherin strongly, but we could not detect N-cadherin in the wing bud (data not shown). At stage 19, however, we detected N-cadherin-expressing cells in the wing bud. These cells were distributed in the dorsomedial region of the bud (Fig. 8A), showing a pattern very similar to that of the dye-labeled somite-derived cells (cf. Fig. 3D). At stage 25, N-cadherin-expressing cells were restricted to the dorsal and the ventral myogenic masses (Fig. 8B). These cells were not yet fully differentiated, but some of them expressed sarcomeric myosin heavy chain (Fig. 8C). At the flank level, there were no N-cadherin-expressing cells among the somatopleural mesoderm (Fig. 8D). These results suggest that N-cadherin-expressing cells within the limb bud are myogenic cells.

Fig. 8.

N-cadherin expression in normal embryos (A-D) and in the transplantation experiments (E,F). (A) Cross section of wing bud of normal embryo at stage 19, stained with an anti-N-cadherin antibody. N-cadherin-expressing cells were detected in the proximal region of the bud (arrow), but not in the distal and ventral regions (small arrows). (B) Wing bud at stage 25. N-cadherin-expressing cells were present in the dorsal (d) and ventral (v) regions of the bud. (C) The section adjacent to that in B was stained with an anti-sarcomeric myosin heavy chain antibody. Several cells within the dorsal and ventral regions expressing N-cadherin were stained. (D) Cross section of flank region of normal embryo at stage 20, stained with anti-N-cadherin antibody. The somitic cells expressed N-cadherin (arrow). (E) Wing somatopleure was transplanted to host flank. After 18 hours, a cross section through the graft was stained with an anti-N-cadherin antibody. We recognized several N-cadherin-expressing cells in the proximal region of the graft (arrow). (F) Wing somatopleure was transplanted to a site nonadjacent to the somite in the host flank region. After 18 hours the cross section was stained with an anti-N-cadherin antibody. N-cadherin-expressing cells were not found in the host somatopleural mesoderm (asterisk) or in the graft. Arrows indicate the contact area between the host tissue and the graft. Bars are 200 μm.

Fig. 8.

N-cadherin expression in normal embryos (A-D) and in the transplantation experiments (E,F). (A) Cross section of wing bud of normal embryo at stage 19, stained with an anti-N-cadherin antibody. N-cadherin-expressing cells were detected in the proximal region of the bud (arrow), but not in the distal and ventral regions (small arrows). (B) Wing bud at stage 25. N-cadherin-expressing cells were present in the dorsal (d) and ventral (v) regions of the bud. (C) The section adjacent to that in B was stained with an anti-sarcomeric myosin heavy chain antibody. Several cells within the dorsal and ventral regions expressing N-cadherin were stained. (D) Cross section of flank region of normal embryo at stage 20, stained with anti-N-cadherin antibody. The somitic cells expressed N-cadherin (arrow). (E) Wing somatopleure was transplanted to host flank. After 18 hours, a cross section through the graft was stained with an anti-N-cadherin antibody. We recognized several N-cadherin-expressing cells in the proximal region of the graft (arrow). (F) Wing somatopleure was transplanted to a site nonadjacent to the somite in the host flank region. After 18 hours the cross section was stained with an anti-N-cadherin antibody. N-cadherin-expressing cells were not found in the host somatopleural mesoderm (asterisk) or in the graft. Arrows indicate the contact area between the host tissue and the graft. Bars are 200 μm.

In order to confirm that the somitic cells migrating in the transplanted wing somatopleure were myogenic cells, we examined N-cadherin expression in the graft. The wing somatopleure was transplanted to the flank region and incubated for about 18 hours (as in Exp. 2). In the cross section of the graft, we found scattered N-cadherin-expressing cells (Fig. 8E). We concluded that these cells are probably not of graft origin, because the graft was excised from the embryo before initiation of myogenic cell migration (at stage 15; Solursh et al., 1987) and because N-cadherin-positive cells were not found in the graft when the somite was not in contact with the graft (as in Exp. 4, Fig. 8F).

Further development of the transplanted wing bud

In order to verify further that the cells migrating in the graft were myogenic cells, the host embryos in experiment 2 were incubated for a further 3 days (total 5.5 days), after which we examined the expression of sarcomeric myosin heavy chain within the graft. In this case, the graft was not labeled and the somite was labeled with DiO. The graft formed a well-grown extra limb bud at the flank of the host embryo (Fig. 9A). In the longitudinal section of the extra limb bud, we found that the DiO-labeled cells formed dorsal and ventral masses (Fig. 9B). These masses were recognized by anti-sarcomeric myosin heavy chain antibody (Fig. 9C). This experiment indicates that the somitic cells migrating in the graft are myogenic cells.

Fig. 9.

Further development of the transplanted wing somatopleure in the flank region and expression of sarcomeric myosin heavy chain. (A) Wing somatopleure was transplanted to the flank site, and the flank somite was labeled with DiO. After 3 days, the graft formed an extra limb bud (arrowhead) between the host wing bud and the leg bud. (B) Longitudinal section of the graft. DiO-labeled somitic cells accumulated as dorsal (d) and ventral (v) masses within the graft. (C) The same section as in B was stained with anti-sarcomeric myosin heavy chain antibody and rhodamine-conjugated second antibody. The dorsal and the ventral masses were stained. This suggests that the DiO-labeled cells differentiated to muscle cells. The bar is 200 μm.

Fig. 9.

Further development of the transplanted wing somatopleure in the flank region and expression of sarcomeric myosin heavy chain. (A) Wing somatopleure was transplanted to the flank site, and the flank somite was labeled with DiO. After 3 days, the graft formed an extra limb bud (arrowhead) between the host wing bud and the leg bud. (B) Longitudinal section of the graft. DiO-labeled somitic cells accumulated as dorsal (d) and ventral (v) masses within the graft. (C) The same section as in B was stained with anti-sarcomeric myosin heavy chain antibody and rhodamine-conjugated second antibody. The dorsal and the ventral masses were stained. This suggests that the DiO-labeled cells differentiated to muscle cells. The bar is 200 μm.

The present study demonstrates the potential of presumptive wing somatopleural mesoderm to induce myogenic cell migration from somites. It also demonstrates that the potential is restricted to the medial region of the presumptive limb somatopleure, and this influence is inhibited by intervening tissues not possessing induction potential, such as the lateral region of presumptive limb somatopleure or the flank somatopleure.

The vital labeling method (Hayashi and Ozawa, 1991) was revealed to be very useful in this study. First, because this technique does not require any surgical operations in itself, it can be combined with transplant operations. Second, the use of two different dyes allowed simultaneous identification of graft and host somitic cells.

The migrating myogenic cells within the limb bud do not express myogenic factors, although these cells are already determined as muscle precursors (Bober et al., 1991; Pownall and Emerson, 1992). It is very interesting to study the molecular differences between myogenic cells and other mesenchymal cells. The Pax-3 gene was recently revealed to be expressed in the migrating myogenic cells (Bober et al., 1994; Goulding et al., 1994; Williams and Ordahl, 1994). Its expression is transient prior to the expression of myogenic factors. In the mouse mutant, Splotch, bearing a defect in this gene, axial muscles develop normally but limb muscle formation is impaired, suggesting that this gene functions in the migration of myogenic cells rather than in the differentiation to muscle cells. To investigate other molecular differences between myogenic cells and other mesenchymal cells, we examined N-cadherin expression of myogenic cells in vivo, because it is known that cultured myoblasts express N-cadherin prior to the fusion. We found that myogenic cells in the limb bud express N-cadherin after stage 19, considerably earlier than the onset of expression of any myogenic factors. Its expression could be used to distinguish the myogenic cells from other limb mesenchymal cells. Transient expression of vimentin in migrating cells in limb buds (Hayashi et al., 1993) might be another molecular characteristic of myogenic cells in limb buds. In this study, we based our identification of the migrating somitic cells as myogenic cells on two findings. First, N-cadherin-expressing cells were found within the transplanted wing somatopleure only in cases in which the graft was in contact with host somite. Second, after 3 further days of incubation, the somitic cells formed dorsal and ventral masses and expressed sarcomeric myosin heavy chain within the graft.

Myogenesis in somites is influenced by other tissues such as neural tube and notochord (Goulding et al., 1994; Rong et al., 1992). As to the migration of myogenic cells from somites, it has been shown that when the flank somite is transplanted to the limb level, myogenic cell migration is induced (Chevallier et al., 1977). This experiment indicates that myogenic cell migration is influenced by the environment within the embryo; however, it does not define the component that exerts the influence. The present study demonstrates that it is the somatopleural component that exerts inductive influence on myogenic cell migration.

The molecular nature of the inducing mechanism is unknown. Our finding that the potential to induce myogenic cell migration is localized in the medial region of the presumptive limb somatopleure suggests localization of an induction molecule in this region. An alternative possibility, which cannot be excluded, is that a repulsive molecule, which inhibits emigration of myogenic cells from the somite, is localized in the lateral region of the presumptive limb somatopleure and in the flank somatopleure. In any case, it is likely that cells at the ventrolateral edge of the somite, regardless of whether it is located at the limb level or flank level, have the potential to migrate from the somite under suitable environmental conditions. The expression of Pax-3 in the lateral ventral bud of the flank somite as well as of the limb-level somite (Bober et al., 1994; Goulding et al., 1994; Williams and Ordahl, 1994) supports this notion.

The possibility that myogenic cells migrate in response to a chemotactic attractant produced by limb buds was proposed by several authors. Gumpel-Pinot et al. (1984) reported that myogenic cell migration within the limb bud stopped after removal of the apical ectodermal ridge (AER) of the limb, and proposed that myogenic cells were attracted by a factor derived from the AER. However, this idea is contradicted by findings of Brand-Saberi et al. (1989) who observed that in some situations, myogenic cells did migrate in the absence of AER. Venkatasubramanian and Solursh (1984) reported that when limb mesenchyme was tested in a chemotactic chamber, PDGF attracted limb bud cells, which may have been myogenic cells. However, Solursh et al. (1987) observed cell migration from the lateral edge of the somite with a scanning electron microscope, and found that the myogenic cells did not migrate until the lateral plate contacted the lateral margin of the somites. This observation is consistent with our results that the flank somitic cells were not induced to migrate unless the transplanted wing somatopleure contacted the somites. Although these results indicate that nondiffusible materials are involved, it is still possible that diffusible factors, such as PDGF or HGF, are also involved in the induction of migration. The effects of any such factors would most likely extend only over a very short distance, as in the case of paracrine systems, or in the case of growth factors fixed on the extracellular matrix.

Our observations revealed that myogenic cells colonize within a restricted area in the limb bud; they colonize in the dorsoproximal region but do not invade the ventral and distal regions of the limb bud. This suggests that the distribution of myogenic cells within limb buds is highly regulated. Specific mechanisms may regulate myogenic cell distribution, as evidenced by the fact that the distribution patterns of endothelial cells and pigment cells in the limb bud are different from that of myogenic cells. For instance, endothelial cells, but never myogenic cells, invade the distal and ventral regions of the limb bud. One possible mechanism by which myogenic cell distribution is regulated is different affinities for myogenic cells of the dorsoproximal and the ventrodistal regions. The segregation of chondrogenic and myogenic regions is thought to involve the sorting out of their precursor cells as a result of differential affinity. The exclusive interaction between chondrogenic cells and myogenic cells was proved to depend on direct cell-cell or cell-extracellular matrix contact (Schramm and Solursh, 1990; Schramm et al., 1994).

After the establishment of myogenic masses, these cells continue migrating, catching up with the out-growth of the limb bud. This migration has been observed as follows. When a myogenic region of a quail limb bud was grafted into the wing bud of a chick embryo, quail myoblasts within the graft migrated distally in the host wing bud and differentiated into muscle cells (Wachtler et al., 1981). However, when somites were grafted into a wing bud, they migrated only slightly (Chevallier and Kieny, 1982; Gumpel-Pinot et al., 1984; Brand-Saberi et al., 1989). It is likely that the mechanism of migration of myoblasts within the limb bud is not identical to that of emigration of myogenic cells from somites.

We thank Dr Takashi Obinata (Chiba University, Japan) and Dr Masatoshi Takeichi (Kyoto University, Japan) for their kind gifts of antibodies and Dr Keiko Takiguchi-Hayashi (Mitsubishi Kasei Institute of Life Sciences, Japan) for her technical advice on microsurgery.

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