SF/HGF Is Involved in Early Limb Muscle Development 4887

Development 126, 4885-4893 (1999) 4885 Printed in Great Britain © The Company of Biologists Limited 1999 DEV2435

SF/HGF is a mediator between limb patterning and muscle development

Martin Scaal1, Alexander Bonafede1, Verena Dathe1, Martin Sachs2, Gordon Cann3, Bodo Christ1 and Beate Brand-Saberi1,* 1Institute of Anatomy, University of Freiburg, Albertstrasse 17, D-79104 Freiburg, Germany 2Department of Medical Genetics, Max-Delbrueck-Centre of Molecular Medicine, Robert-Roessle-Strasse 10, D-13122 Berlin, Germany 3Division of Oncology, Stanford University School of Medicine, Stanford CA, USA *Author for correspondence (e-mail: [email protected])

Accepted 24 August; published on WWW 6 October 1999

SUMMARY

Scatter factor/hepatocyte growth factor (SF/HGF) is posterior limb bud mesenchyme. We could identify BMP- known to be involved in the detachment of myogenic 2 as a potential inhibitor of SF/HGF expression in the precursor cells from the lateral dermomyotomes and their posterior limb bud mesenchyme. We further demonstrate subsequent migration into the newly formed limb buds. As that ZPA excision results in a shift of Pax-3-positive cells yet, however, nothing has been known about the role of the towards the posterior limb bud mesenchyme, indicating a persistent expression of SF/HGF in the limb bud role of the ZPA in positioning of the premuscle masses. mesenchyme during later stages of limb bud development. Moreover, we present evidence that, in the limb bud To test for a potential role of SF/HGF in early limb muscle mesenchyme, SF/HGF increases the motility of myogenic patterning, we examined the regulation of SF/HGF precursor cells and has a role in maintaining their expression in the limb bud as well as the inﬂuence of undifferentiated state during migration. We present a SF/HGF on direction control of myogenic precursor cells model for a crucial role of SF/HGF during migration and in limb bud mesenchyme. We demonstrate that SF/HGF early patterning of muscle precursor cells in the vertebrate expression is controlled by signals involved in limb bud limb. patterning. In the absence of an apical ectodermal ridge (AER), no expression of SF/HGF in the limb bud is observed. However, FGF-2 application can rescue SF/HGF Key words: Scatter factor/hepatocyte growth factor (SF/HGF), c-met, expression. Excision of the zone of polarizing activity (ZPA) Fibroblast growth factor (FGF), BMP-2, Pax-3, Myogenic cell results in ectopic and enhanced SF/HGF expression in the migration, Limb, Muscle, Chick

INTRODUCTION actively from the base of the limb towards its tip in a directed fashion (Wachtler et al., 1982; Brand-Saberi and Christ, 1992). In the vertebrate limb, signalling centres regulating pattern Therefore, some investigators favoured the AER as a candidate formation in all three dimensions are well established. The for direction control in myogenic cell migration (Gumpel-Pinot apical ectodermal ridge (AER) is known to be crucial for et al., 1984). However, other authors found evidence that there proximodistal limb bud outgrowth and skeletal patterning is no immediate signalling influence from the AER, but rather (Saunders, 1948). AER signalling has been demonstrated to be that properties of the stationary mesenchyme direct the mainly mediated by various fibroblast growth factors (FGFs) migrating cells towards the tip of the limb bud (Brand-Saberi expressed in the ridge (reviewed in Martin, 1998). The zone of et al., 1989). Bladt et al. (1995) found that, in mice lacking the polarizing activity (ZPA), which is located in the posterior limb c-met receptor or its ligand, scatter factor/hepatocyte growth bud mesenchyme, has been shown to regulate the development factor (SF/HGF), no muscle precursor cells enter the limb, and of anteroposterior pattern in the limb (Saunders and Gasseling, the musculature of limbs, distal tongue and diaphragma is 1968). ZPA activity has later been shown to be mediated by absent. The protooncogene c-met (Cooper et al., 1984), which Sonic Hedgehog (SHH) signalling (Riddle et al., 1993). The encodes a transmembrane tyrosine kinase, is expressed in dorsoventral axis in the limb is thought to be established by epithelia that de-epithelialize after binding of the ligand, signals from the ectoderm, for example, Wnt7a (Parr and SF/HGF (Stoker et al., 1987), a secreted 90 kDa glycoprotein McMahon, 1995). heterodimer structurally related to plasminogen (Nakamura et It has been shown that the precursor cells of skeletal limb al., 1989). Moreover, c-met has been shown to be expressed in muscles originate from the ventrolateral edges of the migrating myogenic precursor cells invading the lateral plate dermomyotomes (Christ et al., 1974, 1977; Chevallier et al., and the limb mesenchyme (Bladt et al., 1995; Yang et al., 1977). During limb bud outgrowth up to stage 25, they migrate 1996). In the chick, SF/HGF is expressed in the limb fields of 4886 M. Scaal and others the lateral plate adjacent to the lateral dermomyotomes from drawn glass pipette. Control injections were carried out with Locke which myogenic precursor cells detach, and also later in the saline solution or 100 µg/ml BSA in PBS. Embryos were reincubated developing limb bud mesenchyme (Myokai et al., 1995; Thery for 4 to 6 days, killed and treated for immunohistochemistry as et al., 1995). described below. Migration of myogenic quail cells was diagnosed when a substantial number of quail nuclei was detected in myotubes In this study, we demonstrate for the first time that SF/HGF µ expression in the chick limb is controlled by signals involved at a distance of more than 300 m to the transplant, without being continuous with stationary graft tissue. Cells were considered not to in limb bud patterning. In the absence of a functional AER, no have migrated when they were continuous with the graft or less than expression of SF/HGF in the limb bud is observed. Excision 300 µm away from stationary graft tissue. of the ZPA results in ectopic and enhanced SF/HGF expression Embryos destined for MyoD expression analysis were operated at in the posterior limb bud mesenchyme. We further demonstrate stages 20 or 21. SF/HGF was injected into the limb bud mesenchyme that this latter result is paralleled by a shift of Pax-3-expressing at a concentration of 100 µg/ml, PBS was injected as control. cells towards the posterior limb bud mesenchyme, indicating Specimens were reincubated for 24 hours, fixed and processed for in a role of the ZPA in positioning of the premuscle masses. situ hybridization as described below. Moreover, we present evidence that, in the limb bud In situ hybridization mesenchyme, SF/HGF increases the motility of myogenic precursor cells and has a role in maintaining their Embryos were rehydrated in a graded methanol series and processed as described by Nieto et al. (1996). Visualization of the hybridization undifferentiated state during migration. We present a model for product was achieved by use of the digoxigenin RNA labelling a crucial role of SF/HGF during migration and early patterning and detection kit by Boehringer (Germany) according to the of muscle precursor cells in the vertebrate limb. recommendations of the supplier. The following probes were used in this study: avian SF/HGF (kindly provided by Dr Claudio Stern, see Thery et al., 1995), a 2.3 kb insert cloned into pBluescriptSK−, quail MATERIALS AND METHODS Pax-3 (kindly provided by Dr Christophe Marcelle and Dr Michael Stark, San Diego CA), a 1543 bp insert cloned into pBluescriptSK+, Embryos and chick MyoD (kindly provided by Dr Bruce M. Paterson, Bethesda Fertilized eggs of Gallus gallus (White Leghorn) and Coturnix c. MYL), a 1518 bp insert cloned into pBluescriptKS+. japonica were obtained from a local breeder and incubated at 38¡C and 80% relative humidity for the time required. The stages of the Immunohistochemistry embryos were determined according to Hamburger and Hamilton Specimens destined for anti-quail labelling in sections were processed (1951). Limbless embryos were obtained from Drs Ursula Abbott and as described (Zhi et al., 1996). Jacqueline Pisenti, Davis CA. Microsurgery RESULTS Eggs were windowed and the vitelline membrane and the amnion slit open in the area of operation. The AER was excised at stages 18-22 Normal expression of SF/HGF in the chick embryo using ophthalmologic scissors. For ZPA removal, the posterior third Although in earlier studies (Myokai et al., 1995; Thery et al., of stage 19-22 limb buds was excised also by using ophthalmologic 1995) the expression pattern of SF/HGF in the chick embryo scissors. has been described in some detail, we reexamined SF/HGF For application of FGF-2, BMP-2 and BMP-4, heparin-coated acrylic beads of approximately 80 µm in diameter (Sigma, Germany) expression in the limb bud by whole-mount in situ were rinsed in PBS and individually transferred into protein solution hybridization from stage 18 to 26 to elucidate the possible role (FGF-2: 50 µg/ml; BMP-2: 100 µg/ml; BMP-4: 2 or 20 µg/ml). of SF/HGF in the migration control of myogenic precursor Factors were diluted in PBS+0.1% BSA and applied on stage 19-21 cells (Fig. 1). embryos. FGF-2 was obtained from Pepro Tech, Rocky Hill NJ, BMP- At stage 18, SF/HGF is expressed throughout the limb bud 2 and BMP-4 were obtained from Genetics Institute, Cambridge MA. mesenchyme. At stage 19, expression is slightly decreased in Recombinant SF/HGF was produced in Sf9 insect using the the posteriormost limb bud mesenchyme (Fig. 1A). The anterior baculovirus expression system followed by a one-step purification on bias of SF/HGF expression becomes more conspicuous at heparin sepharose (Weidner et al., 1993). Beads were soaked in factor stages 20-22 (Fig. 1B,C). At stage 22, expression decreases at 4¡C for at least 1 hour prior to implantation. To implant a bead into further in the proximal limb mesenchyme. At stage 23, SF/HGF a limb bud, a small slit was cut through the ectoderm into the dorsal mesenchyme using tungsten needles and the bead was pushed through transcripts are restricted to the anterodistal limb mesenchyme the slit into the mesoderm. N-SHH was prepared as described (Cann and a narrow band of fainter expression extending from the et al., 1999) and applied using Ni-NTA-Agarose beads (Qiagen), proximal to the distal margin of the limb in the central limb which were washed in Tris-NaCl buffer (pH 8.5), individually mesenchyme (Fig. 1D). At stage 24, expression could only be transferred into factor (0.65 mg/ml), and soaked in factor at 4¡C for detected in the anterodistal tip of the limb (Fig. 1E), where it at least 1 hour prior to implantation. Implantation procedure was persists up to stage 25 at low level. From stage 26 on expression carried out as described above. was no longer detected. The operated eggs were sealed with medical tape and reincubated for 18-24 hours. Embryos were inspected, killed and fixed in 4% SF/HGF stimulates directed myogenic cell migration paraformaldehyde overnight at 4¡C, dehydrated in a graded methanol Myogenic precursor cells in the limb bud mesenchyme are series and stored at −20¡C. For quail-chick transplantations, limb bud mesenchyme was taken known to migrate in proximodistal direction towards the tip of from quail donors at stages 19-22 and implanted into limb buds of the limb bud (Wachtler et al., 1982). SF/HGF is expressed in stage 21-24 chicken hosts, the host being always older than the donor. the limb bud mesenchyme up to stage 25, and is known to have SF/HGF was injected into the host limb mesenchyme proximal and mitogenic, motogenic and morphogenic properties in vitro and distal to the implant at a concentration of 100 µg/ml using a hand- in vivo, thereby acting in a paracrine fashion (reviewed in SF/HGF is involved in early limb muscle development 4887

Fig. 1. Normal expression of SF/HGF in the chick embryo. (A) Stage 19 chick limb bud: SF/HGF expression throughout the limb bud mesenchyme. At this stage, a slight anterior bias of the expression domain becomes detectable. (B) Stage 20: marked decrease of expression in the posterior limb bud mesenchyme (arrow). (C) Stage 22: SF/HGF expression is restricted to the anterior half of the limb bud mesenchyme. (D) Stage 23: SF/HGF transcripts in the limb bud mesenchyme are restricted to the anterodistal region and a proximodistal band of expression (short arrow). Note the metameric expression in the somites (long arrow). (E) Stage 24: SF/HGF expression is restricted to the anterodistal tip of the limb bud mesenchyme.

Matsumoto and Nakamura, 1996). To test for a possible n=12), i.e. those counted as not migrated were either in direct influence of SF/HGF on the migratory behaviour of myogenic contact with stationary graft tissue (cartilage, fibroblasts) or cells in the limb bud, we transplanted blocks of stage 19-22 emigrated less than 300 µm from this tissue. In similar quail wing bud mesenchyme containing myogenic cells into transplantation experiments without application of factors, it chick host wing buds being at least one stage more advanced. was shown that donor cells never migrate into older host tissue Proximally and distally to the transplant, we also injected (Brand-Saberi et al., 1989). Here, we could confirm this as in SF/HGF into the adjacent host mesenchyme and determined all controls without injection no migration was observed the location of quail cells in the host mesenchyme after 4 to 6 (100% not migrated, n=4). At fixation, in SF/HGF-injected days of reincubation (Fig. 2). In 86% (n=21) of the cases embryos as in controls, the transplant was situated in most examined, quail nuclei were found in myotubes of host cases in the cubital or distal humeral region of the wing. After muscles at considerable distance from the transplant, from 300 SF/HGF application quail cells were detected distal to the µm up to more than 2 mm (Fig. 2B,C). In controls after transplant in the muscles of the zeugopod and autopod (Fig. injection of BSA or Locke solution, quail cells were largely 2A), as well as in the proximal stylopod (Fig. 2D) and even in confined to the area of transplantation (75% not migrated, the pectoral muscle of the trunk (not shown).

Fig. 2. The effect of SF/HGF injection on myogenic cell migration in chick limbs. Pieces of quail wing bud mesenchyme containing myogenic cells had been transplanted into chick wing buds, and cell emigration was monitored in sections. Quail nuclei were visualized by anti-quail staining. Plane of section of A and D as shown in schematic drawing. Most grafts were found in the elbow region after reincubation (asterisk). (A) Longitudinal section through the zeugopodial and autopodial region of an 8-day chick wing, distal to the left. After SF/HGF injection distal to a transplant in the elbow region containing quail wing bud mesenchyme (arrowhead), quail nuclei can be detected in distal muscles (frame and arrow), demonstrating that myogenic cells have migrated distally. (B) Magniﬁcation of frame in A. Quail nuclei are located in the myotubes of muscle distal to the transplant (arrows). (C) Magniﬁcation of frame in D. Quail nuclei are located in myotubes of muscle proximal to the transplant (arrows). (D) Longitudinal, slightly oblique section through the stylopod of an 8- day chick wing, distal to the left. After SF/HGF injection proximal to a transplant in the distal humeral region containing quail wing bud mesenchyme (arrowhead), quail nuclei can be detected in proximal muscle (frame), demonstrating that myogenic cells have migrated proximally. Dig.3, third digit; ul, ulna; hu, humerus. 4888 M. Scaal and others

These data suggest that after SF/HGF application, myogenic Fig. 3. MyoD expression in a cells in the limb bud are motilized and migrate in a retrograd stage 24 chick embryo after or prolonged fashion, depending on the presence of SF/HGF SF/HGF injection into the in the surrounding stationary mesenchyme. However, it should wing bud. Anterior to the be noted that, after reincubation beyond stage 25, which is top. (A) In the uninjected necessary for myotubes to form, the long-range translocation wing bud, MyoD is expressed in the of myogenic quail cells in our experiments does not result from proximomedial region of the active migration but from interstitial growth in the developing limb premuscle masses wing. Nevertheless, our results indicate that the localized (arrow). (B) After SF/HGF presence of SF/HGF in the stationary mesenchyme of the limb injection into the right limb bud provides guiding cues to the migratory muscle precursor bud of the same embryo at stage 21, MyoD is downregulated in the cells in a paracrine fashion. limb premuscle masses. SF/HGF keeps myogenic precursor cells in an undifferentiated state excision (Fig. 4A-E). At all stages examined, SF/HGF In vitro studies showed that, in fibroblasts, motile and invasive expression in AER excised limb buds was severely reduced or cell behaviour can be induced by SF/HGF (Giordano et al., absent (n=14; Fig. 4B). To verify these results in a different 1993; Dugina et al., 1995). To test if SF/HGF is involved in approach, we looked at SF/HGF expression in the chick the maintenance of the undifferentiated, motile state of mutant, limbless (Prahlad et al., 1979). limbless embryos stop myogenic precursor cells in the chick limb, we monitored the limb bud outgrowth shortly after its initiation because the expression of a marker gene for differentiating muscle cells, ectoderm is not competent to form an AER in response to MyoD (Davis et al., 1987; Weintraub et al., 1989), after inducing signals from the mesenchyme (Fallon et al., 1983; application of SF/HGF to the limb bud. We injected SF/HGF Carrington and Fallon, 1988). Limbless mutants are therefore into the dorsal mesenchyme of stage 20 or 21 limb buds, where an apt system to investigate processes in the absence of a the dorsal premuscle mass is about to form. After 24 hours functional AER, without invasive manipulations (Noramly et reincubation, MyoD expression in the injected limb buds was al., 1996). As expected, we could not find SF/HGF expression severely reduced when compared to the untreated, contralateral in the rudimentary limb buds of limbless embryos from stage limb bud (94%, n=16) (Fig. 3). This indicates that, in the 20 onwards (n=12; Fig. 4C). However, weak SF/HGF presence of SF/HGF, myogenic cell differentiation is impeded. expression was detected in early stages up to stage 18/19, Thus, we conclude that SF/HGF is able to prevent premature before the newly formed AER starts to promote limb bud differentiation of myogenic cells in the limb bud, which is a outgrowth (not shown). Thus, the AER is necessary for prerequisite for continuing migration. maintenance of SF/HGF expression in the limb bud mesenchyme, but not for early SF/HGF expression in the limb The apical ectodermal ridge maintains SF/HGF field. It has been shown recently that FGF can ectopically expression in the limb bud via FGF signalling induce SF/HGF expression in the lateral plate mesoderm To reexamine the question whether the AER has a role in (Heymann et al., 1996). As the AER is the major source of myogenic cell migration in the developing limb, we tested for FGF in the growing limb bud (reviewed in Martin, 1998), we a relationship between AER signalling and SF/HGF expression speculated that the AER can induce SF/HGF expression in the by monitoring SF/HGF expression after microsurgical AER limb bud mesenchyme via FGF signalling. To test this

Fig. 4. SF/HGF expression in stage 23 chick forelimb buds in the absence of an apical ectodermal ridge. Analysis by RNA whole- mount in situ hybridization. (A-G) Anterior to the top, dorsal to the left. (H) Dorsal view, anterior to the top. (A) Normal expression in a stage 23 control embryo. (B) Forelimb bud after AER excision. The limb has an aberrant morphology and is devoid of SF/HGF expression. (C) Limb rudiment of a limbless embryo. No SF/HGF expression is detectable. (D) Forelimb bud after AER excision and implantation of a bead soaked in FGF- 2. SF/HGF is expressed in the vicinity of the bead. (E) Forelimb bud after AER excision and implantation of a PBS bead as a negative control. No SF/HGF expression is detectable. (F) Pax-3 expression in a limbless mutant embryo. As in the wild-type embryo, Pax-3-positive cells have detached from the lateral lips of the dermomyotomes at limb level. However, in the hindlimb bud Pax-3 expression is only weak (arrow), and in the forelimb rudiment it is absent (arrowhead), indicating that migrating myogenic cells have been eliminated. (G) Pax-3 expression in a wild-type control embryo. Pax-3-positive myogenic cells have detached from the dermomyotomes at limb level and have invaded the limb mesenchyme. (H) Pax-3 expression after AER removal in the right forelimb bud. The operated limb bud shows Pax-3 expression, but in a slightly aberrant pattern. SF/HGF is involved in early limb muscle development 4889 hypothesis, we implanted FGF-2-soaked beads into the distal mesenchyme of limb buds after AER excision and tested the embryos for SF/HGF expression. We observed a clear rescue of SF/HGF expression in the vicinity of the FGF beads (n=10; Fig. 4D), but not of control beads soaked in PBS (n=4; Fig. 4E). Thus, we conclude that, in the chick limb bud, the AER induces SF/HGF expression via FGF signalling. Limb rudiments of limbless mutant embryos are devoid of Pax-3-expressing cells, even though Pax- 3-positive cells detach from the somites We next examined the influence of the AER on the distribution of myogenic cells in the limb bud. Therefore, we hybridized specimens with a probe specific for Pax-3, which is a marker for undifferentiated muscle precursors in the limb (Bober et al., 1994; Goulding et al., 1994). After AER removal, Pax-3 was still detectable in the limb mesenchyme, though in a slightly aberrant pattern (n=3; Fig. 4H). This is not contradictory to the above results, as myogenic cells may well have entered the limb bud up to the time of operation, which was in this series Fig. 5. Influence of the ZPA on SF/HGF expression in stage 22/23 stage 19 or stage 21. Moreover, the short reincubation time of chick limb buds. Dorsal view, anterior to the top. The right forelimb buds have been manipulated, the contralateral sides serve as controls. 24 hours might allow for some remaining SF/HGF activity in (A) SF/HGF expression after ZPA removal. Expression is intensified the mesenchyme in the absence of an AER. In the limbless and extends ectopically into the posterior mesenchyme (arrow). mutants, however, the rudimentary forelimb buds were devoid (B) Pax-3 expression after ZPA removal. The expression domain is of any Pax-3 expression (stages 20-24) while, in the hindlimb smaller and localized more posteriorly. Note the correspondence to buds, weak Pax-3 expression was in some cases still detectable the ectopic SF/HGF domain in A. (C) SF/HGF expression after SHH (n=9; Fig. 4F). This might be due to the delayed onset of AER bead implantation. SF/HGF is upregulated not in the immediate degeneration in hindlimb buds (Noramly et al., 1996). vicinity of the bead (asterisk), but in the marginal mesenchyme However, in the somites adjacent to the limb buds, Pax-3 opposite to the bead (arrowhead). (D) SF/HGF expression after expression in the lateral tips of the dermomyotomes had BMP-2 bead implantation. SF/HGF is downregulated in the vicinity vanished, similar to the wild-type situation (Fig. 4G). In wild- of the bead (arrow). (E) SF/HGF expression after BMP-4 bead implantation. The operated limb is smaller with aberrant type embryos, these Pax-3-expressing cells detach from the morphology, but SF/HGF expression is not affected in the vicinity of dermomyotomes and invade the limb buds. As, in limbless the bead (arrowhead). embryos, no Pax-3-positive cells were seen in the forelimb buds, it is likely that these cells had been eliminated by apoptosis after having detached from the somites. Apoptosis is precursor cells in the limb, we hybridized ZPA-excised known to stop further limb outgrowth in mutant limb buds from embryos with a probe specific for Pax-3. Due to the loss of stage 19 onwards (Fallon et al., 1983). Taken together, we tissue by ZPA removal, the Pax-3 expression domain in observe persistent Pax-3 expression in AER-excised limb buds operated limbs was generally smaller than in control limbs. even though SF/HGF expression is absent whereas, in limbless Interestingly, however, we observed a shift of the Pax-3 embryos, from stage 20 onwards, neither Pax-3 nor SF/HGF expression domain, which represents the premuscle masses, transcripts can be detected in the limb rudiments. towards the posterior margin of the operated limbs (n=9; Fig. 5B). The anterior portion, which was devoid of Pax-3-positive ZPA removal leads to ectopic SF/HGF expression cells, was larger in ZPA-excised embryos. Taken together, after and correspondingly shifted distribution of ZPA removal, we observe ectopic SF/HGF expression in the myogenic cells in the limb bud posterior limb mesenchyme as well as a posterior shift of To investigate a potential role of the ZPA in myogenic cell muscle precursor cells. From this we conclude that the migration and muscle pattern formation, we analyzed SF/HGF SF/HGF expression domain is regulated by signals from the expression after microsurgical ZPA removal by in situ ZPA and is involved in directing myogenic cell migration in hybridization using a probe specific for SF/HGF (Fig. 5). At the chick limb bud, leading to the establishment of the all stages examined, after ZPA removal, we observed SF/HGF positions of the dorsal and ventral premuscle masses. expression not only in the anterodistal domain corresponding to the normal expression pattern, but also in an additional BMP-2, but not SHH, can inhibit SF/HGF expression domain along the posterior margin of the operated limb (n=30; in the limb bud Fig. 5A), which clearly exceeded the central band of fainter The finding that, in the absence of the ZPA, SF/HGF is expression. Moreover, in situ hybridizations gave an overall expressed in the posterior limb bud mesenchyme, in contrast more intense signal in the operated limb compared to the to its anterodistally restricted expression in the normal limb, contralateral side, indicating that ZPA removal allows for suggests that, in the normal limb, the ZPA represses SF/HGF enhanced SF/HGF expression in the limb bud mesenchyme. To expression in the posterior mesenchyme. Therefore, we tested test whether the altered SF/HGF expression pattern after ZPA proteins known to be synthesized by cells of the ZPA for a removal is paralleled by an altered distribution of myogenic potential inhibitory influence on SF/HGF expression. As SHH 4890 M. Scaal and others is considered to be the polarizing signal in vivo (Riddle et al., spreading, subsequent disruption of cell-cell contacts and cell 1993), we were interested in whether SHH can repress crawling (Matsumoto et al., 1994). SF/HGF expression in the limb bud. To test this, we implanted Recent work suggests an important role of SF/HGF in the beads soaked in SHH into the anterior limb bud mesenchyme recruitment and emigration of myogenic precursor cells from and tested the embryos for SF/HGF expression after 24 hours the lateral dermomyotomes into the limb buds (Bladt et al., of reincubation. Surprisingly, we did not find downregulation 1995; Brand-Saberi et al., 1996; Dietrich et al., 1999). of SF/HGF transcription in the vicinity of the beads but, on the Embryological experiments indicate that these directed cell contrary, upregulation of SF/HGF transcription in the anterior movements require signals from the proximal limb bud marginal mesenchyme of the operated limb, some distance mesenchyme (Christ et al., 1978). from the bead (n=14; Fig. 5C). The activity of the protein was Several lines of evidence indicate that SF/HGF is this evident from the occurrence of ectopic outgrowth in the postulated inducing signal. In the mouse, c-met is strongly anterior limb bud mesenchyme and from in vitro control expressed in the ventrolateral edges of the dermomyotomes, experiments (not shown). This leads us to assume that SHH is and also in cells that migrate from the somites to the limbs. not a negative regulator of SF/HGF in the posterior limb Concomitantly, around the time of emigration of myogenic mesenchyme. In a second approach, we repeated the above precursor cells, SF/HGF is expressed in the medial limb field experiment using beads soaked in BMP-2, which is also mesenchyme directly abutting the lateral dermomyotomes expressed in the ZPA (Francis et al., 1994). Here, we observed (Bladt et al., 1995). Similar expression data are known from downregulation of SF/HGF expression in the vicinity of the the chick (Thery et al., 1995; Myokai et al., 1995, own data). bead, indicating that BMP-2 can inhibit SF/HGF expression Interestingly, in SF/HGF homozygous mutant mice (Schmidt (n=6; Fig. 5D). In contrast, BMP-4-soaked beads were et al., 1995), as well as in c-met homozygous mutants (Bladt incapable of repressing SF/HGF expression in the limb bud, et al., 1995), the lateral dermomyotomes fail to de-epithelialize but resulted in an overall decrease of limb bud size probably and no emigration of myogenic cells occurs, leaving the limb due to apoptosis (n=5; Fig. 5E). Therefore, BMP-2 is a good buds devoid of skeletal muscle (Dietrich et al., 1999). In candidate for the putative repressor of SF/HGF expression in previous experiments, we could demonstrate that injection of the posterior limb bud mesenchyme. SF/HGF protein into the somatopleure of chick embryos at interlimb level induces detachment and emigration of

DISCUSSION

In this study, we analyzed the influence of SF/HGF on the behaviour of myogenic precursor cells in the chick limb bud and monitored the effect of signals from the main signalling centres in the limb, AER and ZPA, on SF/HGF expression. We found that SF/HGF keeps the migrating myogenic precursor cells in an undifferentiated state and increases their motility. Moreover, our data indicate that SF/HGF has an influence on the direction of myogenic cell migration within the limb bud mesenchyme. We present evidence that SF/HGF expression in the limb bud depends on FGF signalling from the AER, and that inhibitory signals from the ZPA restrict SF/HGF expression to the anterodistal mesenchyme of growing chick limb buds (Fig. 6). SF/HGF keeps myogenic precursor cells in a motile and undifferentiated state, thereby indirectly guiding them to distal positions SF/HGF has long been known to de-epithelialize and motilize epithelial cells in vitro (Stoker et al., 1987) and, thereby, to be involved in invasive carcinoma growth (Weidner et al., 1990). Nevertheless, SF/HGF is also involved in various Fig. 6. Schematic representation of a chick limb bud (dorsal view, developmental processes implying epitheliomesenchymal anterior to the top) and the mechanisms involved in migration control transitions, and has trophic functions for regenerating cells in of myogenic precursor cells according to the model presented here. liver, kidney and lung (reviewed in Matsumoto and FGFs from the AER maintain SF/HGF expression in the distal limb Nakamura, 1996). SF/HGF is expressed in mesenchymal mesenchyme, which in turn allows for myogenic cell migration cells and is secreted into the extracellular matrix, where it within the SF/HGF expression domain. SF/HGF could promote functions in a paracrine fashion to activate the c-met migration by inhibiting or delaying MyoD expression, which is a marker for differentiating myoblasts and not compatible with transmembrane tyrosine kinase, which is expressed in migration. BMP-2 expressed in the posterior limb bud mesenchyme epithelial cells (reviewed in Birchmeier and Birchmeier, restricts SF/HGF expression to the anterior limb bud mesenchyme. 1993). In vitro studies showed that SF/HGF-mediated The limbless gene product is an indirect prerequisite for FGF- phosphorylation of c-met and focal adhesion kinase triggers mediated SF/HGF expression because it is necessary for the the dynamic formation of focal adhesion points, cell maintenance of a functional AER. SF/HGF is involved in early limb muscle development 4891 myogenic cells from the dermomyotomes (Brand-Saberi et al., the myogenic cells migrating in its wake towards their distally 1996). located destinations. Thus, the de-epithelializing and motogenic properties of SF/HGF in myogenic cell migration are well established. Yet, SF/HGF expression in the limb bud mesenchyme is it has been unclear what might be the function of the persisting regulated by signals from the AER and the ZPA expression of SF/HGF in the limb bud mesenchyme during In the limb bud mesenchyme, SF/HGF is expressed in a later stages of limb bud outgrowth up to stage 25. spatially restricted and temporally dynamic pattern. Therefore In this study, we tested for a possible role of SF/HGF in it is likely that SF/HGF expression in the limb bud is controlled directing myogenic cells during their later migration in the by a precise regulatory mechanism, coordinating the mesenchyme of the outgrowing limb bud. We showed that expression pattern in time and space. So far, however, the SF/HGF application can strongly increase myogenic cell postulated regulatory mechanism is essentially unknown. migration and that ectopic SF/HGF is able to reinitiate and Considering that SF/HGF expression in the limb bud is, at reverse migration in the limb bud. We conclude that SF/HGF all stages examined, restricted to the mesenchyme subjacent to mobilizes myogenic cells also in normal development, and is the AER, we monitored SF/HGF expression after AER involved in directing them towards their target tissue. removal. We observed that, in the absence of the AER, However, Heymann et al. (1996) did not observe a SF/HGF transcripts cannot be detected in the operated limb. preferential migration of myogenic cells towards beads soaked Consistent with this, in limbless mutant chick embryos, which in SF/HGF and implanted into the flank of chick embryos. lack a functional AER (Prahlad et al., 1979; Fallon et al., From this, they excluded a chemotactic influence of SF/HGF 1983), the limb rudiments are also devoid of SF/HGF on myogenic cells, which was suggested by Takayama et al. expression, except in early stages before the ridge has formed. (1996) who found muscle cells in the central nervous system These results demonstrate that SF/HGF expression in the limb of transgenic mice after forced expression of SF/HGF in the bud mesenchyme requires signals from the AER. We show that neural tube. Our results do not imply a direct guiding property FGF-2 bead application to AER excised limb buds rescues of a SF/HGF source, but rather suggest that SF/HGF secreted SF/HGF expression in the vicinity of the bead. This is in line by the stationary mesenchymal cells allows for the with the findings of Heymann et al. (1996), who observed maintenance of motility in myogenic cells. We postulate that ectopic SF/HGF expression in the lateral plate mesoderm after the dynamic expression domain of SF/HGF in the limb bud, FGF-2 bead application to the flank of chick embryos. Thus, moving from the proximal mesenchyme in younger buds to the our data suggest that the AER maintains SF/HGF expression distal mesenchyme in older buds, reflects the domains of active in the limb bud mesenchyme via FGF signalling. Although we myogenic cell migration as maintained by the SF/HGF- could induce SF/HGF expression in the limb bud mesenchyme mediated c-met signalling pathway. Thus, the expression by application of FGF-2 protein, we cannot rule out the control of SF/HGF represents an actual directing influence on possibility that other FGFs, namely FGF-8 which is expressed, myogenic precursor cells in the limb bud. This is in line with as is FGF-2, throughout the AER, are the natural inducers of the observation that migrating myogenic precursor cells SF/HGF in the limb bud. This is not easy to determine because express c-met and Pax-3, the latter being considered to be FGF functions are highly redundant (reviewed in Martin, involved in c-met expression control (Bladt et al., 1995; Epstein 1998). et al., 1996; Daston et al., 1996). Moreover, our results are We next examined whether the anterior bias of SF/HGF supported by the findings of Dietrich et al. (1999) who found expression in the limb bud from stage 19 onwards is due to evidence that, in the mouse, SF/HGF expression coincides with signals from the ZPA. We found that, after excision of the ZPA, the routes of migrating muscle precursor cells, as identified by SF/HGF expression in the limb bud is generally more intense Lbx 1 expression (Dietrich et al., 1998), and by the observation and that its expression domain is extended towards the of Brand-Saberi and Christ (1992) that myogenic cell posterior margin of the limb bud mesenchyme. This indicates migration ceases after stage 25, which is the latest stage that, in the normal limb bud, SF/HGF expression is restricted showing SF/HGF expression. Interestingly, Zhi et al. (1996) to the anterior portion by inhibitory signals from the posterior found that muscle precursor cells detaching from the most limb bud mesenchyme. We next tested whether factors caudal somite adjacent to the wing region (somite 21) expressed in the posterior limb bud mesenchyme can influence sometimes do not take part in wing muscle formation, or they SF/HGF expression. Surprisingly, we found that anterior remain in the proximal mesenchyme and are restricted to application of SHH enhanced SF/HGF expression at the proximal muscle blastemas. This may reflect the absence of anterior margin of the limb bud mesenchyme, whereas BMP- SF/HGF from the posterior limb bud mesenchyme from stage 2 was observed to downregulate SF/HGF expression in the 19 onwards. Consistent with our model, we found that after vicinity of the bead. This indicates that BMP-2, but not SHH, SF/HGF injection into the limb bud mesenchyme, the inhibits SF/HGF expression in the posterior limb bud expression of MyoD, which is a marker for differentiating and mesenchyme. These results seem to be hard to reconcile, as stationary myoblasts, is downregulated, suggesting that SHH is thought to induce BMP-2 expression in the limb SF/HGF keeps myogenic cells in an undifferentiated, motile mesenchyme, BMP-2 thus being a putative downstream target state. of the SHH signalling pathway. It should be noted that, after Taken together, we conclude from these results that the SHH bead application, SF/HGF was upregulated exclusively expression of SF/HGF in the limb bud mesenchyme is in the anterior marginal limb bud mesenchyme, but not in the necessary for myogenic cell migration. We hypothesize that the immediate vicinity of the more proximomedially located bead. dynamic SF/HGF expression domain shifting from proximal- Laufer et al. (1994) observed that SHH can induce BMP-2 to-distal positions during limb bud outgrowth indirectly guides expression only when applied close to the AER, and not when 4892 M. Scaal and others applied to proximomedial positions in the limb bud. They formation are regulated by the same signalling mechanisms found, moreover, that FGF-4 from the AER is required to that pattern the limb skeleton. induce the competence of the mesoderm to respond to SHH, and that shh and FGF-4 expression are linked via a positive We appreciate the help of Professor Ursula Abbott and Dr Jacky feedback loop. Therefore, an explanation for the above results Pisenti, in providing us with limbless-mutant embryos. We thank could be that SHH released from the implanted bead induced Ulrike Pein and Lidia Koschny for excellent technical assistance, FGF-4 expression in the anterior marginal ectoderm, which in Professor Frank Stockdale for critical comments on the manuscript, and Drs Moises Mallo and Benoit Kanzler for helpful support. This turn induced upregulation of SF/HGF in the underlying work was supported by the Deutsche Forschungsgemeinschaft, DFG- mesenchyme. BMP-2 application, however, could exert its grant Br 957/5-1. inhibitory function without being counteracted against by FGF signalling from the ectoderm. Yet, this model still does not explain the conflicting functions of SHH and BMP-2 which REFERENCES are, in normal limb buds, coexpressed in the posterior limb bud mesenchyme. Thus, these data remain ambiguous and will Birchmeier, C. and Birchmeier, W. (1993). Molecular aspects of epithelial- need further investigation. mesenchymal interactions. Ann. Rev. Cell Biol. 9, 511-540. Taken together, our results suggest that the AER maintains Bladt, F., Riethmacher, D., Isenmann, S., Aguzzi, A. and Birchmeier, C. SF/HGF expression in the distal limb bud mesenchyme via (1995). Essential role for the c-met receptor in the migration of myogenic FGF signalling, and that the anterior bias of the SF/HGF precursor cells into the limb bud. Nature 376, 768-771. Bober, E., Franz, T., Arnold, H. H., Gruss, P. and Tremblay, P. (1994). Pax- expression domain is due to an inhibitory influence in the 3 is required for the development of limb muscles: a possible role for the posterior mesenchyme, probably mediated by BMP-2. migration of dermomyotomal muscle progenitor cells. Development 120, 603-612. SF/HGF is a mediator between limb pattern Brand-Saberi, B., Krenn, V. and Christ, B. (1989). The control of directed formation and myogenic cell migration myogenic cell migration in the avian limb bud. Anat. Embryol. 180, 555- 566. We showed that SF/HGF is required for motilizing and Brand-Saberi, B. and Christ, B. (1992). A comparative study of myogenic directing myogenic cell migration in the limb bud, and that its cell invasion of the avian wing and leg bud. Europ. J. Morphol. 30, 169- expression is controlled by the AER and the ZPA. 180. In limbless embryos, which never form a functional AER Brand-Saberi, B., Müller, T. S., Wilting, J., Christ, B. and Birchmeier, C. (1996). Scatter factor/hepatocyte growth factor (SF/HGF) induces (Fallon et al., 1983), we found that muscle precursor cells emigration of myogenic cells at interlimb level in vivo. Dev. Biol. 179, 303- detach from the lateral tips of the dermomyotomes at limb 308. level, similar to the wild-type situation. However, in the wing Cann, G. M., Lee, J. W. and Stockdale, F. E. (1999). Sonic hedgehog bud rudiments of limbless specimens at stage 20 or later stages, enhances somite cell viability and formation of primary slow muscle fibers in avian segmented mesoderm. Anat. Embryol. 200, 239-252. no Pax-3-positive cells were found. Thus, obviously, the Carrington, J. L. and Fallon, J. F. (1988). Initial limb budding is independent limbless gene product is not involved in the recruitment of of apical ectodermal ridge activity; evidence from a limbless mutant. myogenic precursor cells from the somite. Development 104, 361-367. From our results, we speculate that myogenic cell migration Chevallier, A., Kieny, M. and Mauger, A. (1977). Limb-somite relationship: is a two-step process. In a first step, myogenic precursor cells origin of the wing musculature. J. Embryol. Exp. Morph. 41, 245-258. Christ, B., Jacob, H. J. and Jacob, M. (1974). Über den Ursprung der de-epithelialize, detach from the lateral dermomyotomes and Flügelmuskulatur. Experimentelle Untersuchungen an Wachtel- und populate the limb field mesenchyme. In a second step, during Hühnerembryonen. Experientia 30, 1446-1448. limb bud outgrowth, these cells migrate actively towards their Christ, B., Jacob, H. J. and Jacob, M. (1977). Experimental analysis of the distal destinations in the limb bud mesenchyme. SF/HGF is origin of wing musculature in avian embryos. Anat. Embryol. 150, 171-186. Christ, B., Jacob, H. J. and Jacob, M. (1978). Zur Frage der regionalen involved in both steps, but with different roles. In the early Determination der frühembryonalen Muskelanlagen. Experimentelle phase, it is involved in the epitheliomesenchymal transition of Untersuchungen an Wachtel- und Hühnerembryonen. Verh. Anat. Ges. 72, the myogenic precursor cells and their subsequent motilization. 353-357. In the later phase, it is needed to keep the cells undifferentiated Cooper, C. S., Park, M., Blair, D. G., Tainsky, M. A., Huebner, K., Croce, and motile and guide them during their migration within the C. M. and Van de Woude, G. F. (1984). Molecular cloning of a new transforming gene from a chemically transformed human cell line. Nature limb bud mesenchyme. In a different context, this latter 311, 29-34. property of SF/HGF may also have an implication regarding Daston, G., Lamar, E., Olivier, M. and Goulding, M. (1996). Pax-3 is carcinoma metastasis formation, since we show that SF/HGF necessary for migration but not differentiation of limb muscle precursors in acts not only on the detachment of epithelial and epithelia- the mouse. Development 122, 1017-1027. Davis, R. L., Weintraub, H. and Lassar, A. B. (1987). Expression of a single derived cells, but also on the motile phase of individually transfected cDNA converts fibroblasts to myoblasts. Cell 51, 987-1000. migrating cells. Dietrich, S., Schubert, F. R., Healy, C., Sharpe, P. T. and Lumsden, A. After ZPA removal, we observed a shift of Pax-3-expressing (1998) Specification of the hypaxial musculature. Development 125, 2235- cells towards the posterior margin of the operated limb bud. 2249. This is consistent with the idea that the presence of SF/HGF Dietrich, S., Abou-Rebyeh, F., Brohmann, H., Bladt, F., Sonnenberg- Riethmacher, E., Yamaai, T., Lumsden, A., Brand-Saberi, B. and in the mesenchyme is needed to make way for myogenic cell Birchmeier, C. (1999). The role of SF/HGF and c-Met in the development migration, as the SF/HGF expression pattern is, in a similar of skeletal muscle. Development 126, 1621-1629. fashion, extended to the posterior margin of the ZPA excised Dugina, V. B., Alexandrova, A. Y., Lane, K., Bulanova, E. and Vasiliev, J. limb bud. M. (1995). The role of the microtubular system in the cell response to HGF/SF. J. Cell Sci. 108, 1659-1667. Our data therefore suggest a SF/HGF-mediated role of the Epstein, J. A., Shapiro, D. N., Cheng, J., Lam, P. Y. P. and Maas, R.L. ZPA in patterning of premuscle masses. This strongly supports (1996). Pax 3 modulates expression of the c-Met receptor during limb the hypothesis that early stages of limb muscle pattern muscle development. Proc. Natl. Acad. Sci USA 93, 4213-4218. SF/HGF is involved in early limb muscle development 4893

Fallon, J. F., Frederick, J. M., Carrington, J. L., Lanser, M. R. and in the limbless mutant: Polarized gene expression in the absence of SHH Simandl, B. K. (1983). Studies on a limbless mutant in the chick embryo. and an AER. Dev. Biol. 179, 339- In Limb Development and Regeneration Part A (ed. J. F. Fallon and A. I. Parr, B. A. and McMahon, A. P. (1995). Dorsalizing signal Wnt-7a required Caplan), pp. 33-34. New York: Liss. for normal polarity of D-V and A-P axes of mouse limb. Nature 374, 350- Francis, P. H., Richardson, M. K., Brickell, P. M. and Tickle, C. 353. (1994).Bone morphogenetic proteins and a signalling pathway that controls Prahlad, K. V., Skala, G., Jones, D. G. and Briles, W. E. (1979). Limbless: patterning in the developing chick limb. Development 120, 209-218. A new genetic mutant in the chick. J. Exp. Zool. 209, 427-434. Franz, T., Kothary, R., Surani, M. A. H., Halata, Z. and Grim, M. (1993). Riddle, R. D., Johnson, R. L., Laufer, E. and Tabin, C. (1993). Sonic The Splotch mutation interferes with muscle development in the limbs. Anat. hedgehog mediates the polarizing activity of the ZPA. Cell 75, 1401-1416. Embryol. 187,153-160. Saunders, J. W. Jr. (1948). The proximo-distal sequence of origin of limb Giordano, S., Zhen, Z., Medico, E., Gaudino, G. and Comoglio, P. M. parts of the chick wing and the role of the ectoderm. J. Exp. Zool. 108, 363- (1993). Transfer of motogenic and invasive response to scatter 404. factor/hepatocyte growth factor by transfection of human met Saunders, J. W. Jr. and Gasseling, M. T. (1968). Ectodermal-mesenchymal protooncogene. Proc. Natl. Acad. Sci. USA 90, 694-653. interactions in the origin of limb symmetry. In Epithelial-mesenchymal Goulding, M., Lumsden, A. and Paquette, A. J. (1994). Regulation of Pax- interactions (ed. R. Fleischmajer, R. E. Billingham), pp. 78-79. Baltimore. 3 expression in the dermomyotome and its role in muscle development. Schmidt, C., Bladt, F., Goedecke, S., Brinkmann, V., Zschiesche, W., Development 120, 957-971. Sharpe, M., Gherardi, E. and Birchmeier, C. (1995). Scatter Gumpel-Pinot, M., Ede, D. A. and Flint, O. P. (1984). Myogenic cell factor/hepatocyte growth factor is essential for liver development. Nature movement in the developing avian limb bud in presence and absence of the 373, 699-702. apical ectodermal ridge (AER). J. Embryol. Exp. Morph. 80, 105-125. Stoker, M., Gherardi, E., Perryman, M. and Gray, J. (1987). Scatter factor Hamburger, V. and Hamilton, H. (1951). A series of normal stages in the is a fibroblast-derived modulator of epithelial cell motility. Nature 327, 239- development of the chick embryo. J. Morph. 88, 49-92. 242. Heymann, S., Koudrova, M., Arnold, H. H., Köster, M. and Braun, T. Takayama, H., LaRochelle, W., Anver, M., Bockman, D. E. and Merlino, (1996). Regulation and function of SF/HGF during migration of limb muscle G. (1996). Scatter factor/hepatocyte growth factor as a regulator of skeletal precursor cells in chicken. Dev. Biol. 180, 655-578. muscle and neural crest development. Proc. Natl. Acad. Sci. USA 93, 5866- Laufer, E., Nelson, C. E., Johnson, R. L., Morgan, B. A. and Tabin, C. 5871. (1994). Sonic hedgehog and Fgf-4 act through a signalling cascade and Thery, C., Sharpe, M. J., Batley, S. J., Stern, C. D. and Gherardi, E. (1995). feedback loop to integrate growth and patterning of the developing limb bud. Expression of HGF/SF, HGF1/MSP, and c-met suggests new function Cell 79, 993-1003. during early chick development. Dev. Genet. 17, 90-101. Martin, G.R. (1998). The roles of FGFs in the early development of vertebrate Wachtler, F., Christ, B. and Jacob, H. J. (1982). Grafting experiments on limbs. Gen. Dev. 12, 1571-1586. determination and migratory behaviour of presomitic, somitic and Matsumoto, K., Matsumoto, K., Nakamura, T. and Kramer, R. H. (1994). somatopleural cells in avian embryos. Anat. Embryol. 164, 369-378. Hepatocyte growth factor/scatter factor induces tyrosine phosphorylation of Weidner, K. M., Behrens, J., Vanderkerckhove, J. and Birchmeier, W. focal adhesion kinase (p125FAK) and promotes migration and invasion by (1990). Scatter factor: molecular characteristics and effect on the oral squamous cell carcinoma cells. J. Biol. Chem. 269, 31807-31813. invasiveness of epithelial cells. J. Cell Biol. 111, 2097-2108. Matsumoto, K. and Nakamura, T. (1996). Emerging multipotent aspects of Weidner K.M., Sachs M., Birchmeier, W. (1993). The Met receptor tyrosine hepatocyte growth factor. J. Biochem. 119, 591-600. kinase transduces motility, proliferation and morphogenic signals of scatter Myokai, F., Washio, N., Asahara, Y., Yamaai, T., Tanda, N., Ishikawa, factor/hepatocyte growth factor in epithelial cells. J. Cell Biol. 121, 145- T., Aoki, S., Kurihara, H., Murayama, Y., Saito, T., Matsumoto, K., 154. Nakamura, T., Noji, S. and Nohno, T. (1995). Expression of the Weintraub, H., Tapscott, S. J., Davis, R. L., Thayer, M. J., Adam, M. A., hepatocyte growth factor gene during chick development. Dev. Dyn. 202, Lassar, A. B. and Miller, A. D. (1989). Activation of muscle-specific genes 80-90. in pigment, nerve, fat, liver, and fibroblast cell lines by forced expression of Nakamura, T., Nishizawa, T., Hagija, M., Seki, T., Shimonishi, M., MyoD. Proc. Natl. Acad Sci. USA 86, 5434-5438. Sugimura, A., Tashiro, K. and Shimizu, S. (1989). Molecular cloning and Yang, X. M., Vogan, K., Gros, P. and Park, M. (1996). Expression of the expression of human hepatocyte growth factor. Nature 342, 440-443. met receptor tyrosine kinase in muscle progenitor cells in somites and limbs Nieto, M. A., Patel, K. and Wilkinson, D. G. (1996). In situ hybridization is absent in Splotch mice. Development 122, 2163-2171. analysis of chick embryos in whole mount and tissue sections. Methods Cell Zhi, Q., Huang, R., Christ, B. and Brand-Saberi, B. (1996). Participation Biol. 51, 219-235. of individual brachial somites in skeletal muscles of the avian distal wing. Noramly, S., Pisenti, J., Abbott, U. and Morgan, B. (1996). Gene expression Anat. Embryol. 194, 327-339.