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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.
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