hepatocyte growth factor stimulates chemotactic response in mouse embryonic limb myogenic cells in...

170 K.K.H. LEE ET AL.

© 1999 WILEY-LISS, INC.

Hepatocyte Growth Factor Stimulates ChemotacticResponse in Mouse Embryonic Limb Myogenic CellsIn Vitro

K.K.H. LEE,1* C.C. WONG,1 S.E. WEBB,1 M.K. TANG,1 A.K.C. LEUNG,1P.F. KWOK,1 D.Q. CAI,2 AND K.M. CHAN2

1Department of Anatomy, Faculty of Medicine, The Chinese University ofHong Kong, Shatin, Hong Kong, People�s Republic of China

2Department of Orthopaedics and Traumatology, Prince of Wales Hospital,Faculty of Medicine, The Chinese University of Hong Kong, Shatin, HongKong, People�s Republic of China

ABSTRACT In this study we investigate the influence of Hepatoctye Growth Factor (HGF) onthe motility of embryonic forelimb myoblasts. Using Blindwell chemotactic chambers, it was foundthat HGF at concentrations of 1–50 ng/ml dramatically enhanced the ability of myogenic cells tomigrate. This stimulatory effect was elicited in a dose-dependent fashion and the effect was re-versed with the addition of HGF neutralizing antibodies. A checkerboard analysis was performedand it revealed that HGF’s effect on limb myoblast motility was through both chemokinesis andchemotaxis. HGF was also examined for its ability to stimulate myogenic cell proliferation, usingMF20 antibody as the myogenic marker. At all concentrations tested, HGF did not stimulate anoverall increase in the numbers of MF20-positive myoblasts in culture. To examine the chemoki-netic effect of HGF on cell migration in the limb, cells were isolated from the proximal regions ofthe limb (areas rich in myogenic cells), exposed to HGF, labeled with DiI and transplanted into11.5 day mouse forelimbs. After 36 h of culture, it was found that DiI-labeled limb cells, pre-treated with HGF, migrated significantly further in the limb than labeled cells that have not beenexposed to HGF. The chemotactic effect of HGF was also investigated by implanting beads loadedwith and without HGF into the 11.5 day limb. Proximal to the beads, DiI-labeled limb cells werealso transplanted. It was found that HGF was able to chemotactically attract and direct the mi-gration of DiI-labeled limb cells. Immunohistological staining was performed with HGF antibodiesto determine the distribution of HGF in the 11.5 day mouse forelimb. It was found that HGF wasstrongly expressed by the apical ectodermal ridge (AER), the ectoderm and the mesenchyme di-rectly beneath the AER. Positive staining was also obtained for the myogenic regions. However,the pattern was heterogeneous—punctuated with myogenic cells expressing and not expressingHGF. J. Exp. Zool. 283:170�180, 1999. © 1999 Wiley-Liss, Inc.

HGF is a multifunctional cytokine capable ofexerting major effects on cell proliferation, cell mi-gration and organogenesis. HGF is an effector ofcells expressing the Met tyrosine kinase receptorand has been demonstrated to stimulate hepato-cytes, epidermal melanocytes, keratinocytes, re-nal tubular epithelium, gastric epithelium, biliaryepithelium and vascular endothelium to prolifer-ate in vitro (Matsumoto et al., ’91; Morimoto etal., ’91; Takahashi et al., ’93). It is also known asScatter Factor because of its ability to induce avariety of epithelial cells in explants to scatter,by breaking the junctions between cells, duringculture. Simultaneously, it is also capable of stimu-lating the epithelial cells to become highly motile(Stoker et al., ’87; Hartmann et al., ’92). During

Grant sponsor: Mainline; Grant number: 2040607; Grant sponsor:Direct; Grant number: 2040512.

*Correspondence to: Dr. Kenneth Lee, Department of Anatomy, TheChinese University of Hong Kong, Shatin, Hong Kong, People’s Re-public of China. E-mail: [email protected]

Received 3 March 1998; Accepted 3 June 1998.

embryogenesis, HGF plays a major role in the de-velopment of the nervous system (Ebens et al.,’96; Yamamoto et al., ’97), kidney (Woolf et al.,’95), liver (Schmidt et al., ’95) mammary gland(Niranjan et al., ’95) limb (Bladt et al., ’95) andneural crest cells (Takayama et al., ’96).

In early embryos, HGF is expressed by mesen-chymal cells in the proximal regions of the limb,but not by mesenchymal cells in the flank (Bladtet al., ’95; Thèry et al., ’95; Yang et al., ’96;Takayama et al., ’96). There is now increasing evi-

JOURNAL OF EXPERIMENTAL ZOOLOGY 283:170–180 (1999)

HGF AND MYOBLAST MIGRATION 171

dence to show that HGF plays a crucial role inthe establishment of the skeletal musculature inavian and mammalian limbs (Bladt et al., ’95;Thèry et al., ’95; Brand-Saberi et al., ’96; Heymannet al., ’96). The skeletal musculature of the limbis formed by myogenic precursor cells that migrateinto the limb from the ventro-lateral edge of thedermomyotome (Christ et al., ’77; Lee and Sze,’93; Bober et al., ’94; Sze et al., ’95; Daston et al.,’96). These precursor cells specifically express thec-met receptor when they are located in theventro-lateral edge of the dermomyotome. Corre-spondingly, HGF is expressed by the proximal limbmesenchyme, which is located directly adjacentto the somites (Bladt et al., ’95; Yang et al., ’96).Bladt et al. (’95) reported that targeted disrup-tion of the c-met-encoding receptor tyrosine kinasegene in mice inhibited the migration of myogenicprecursor cells from the somite. These authorsproposed that interactions between the c-met re-ceptor and HGF, through some form of paracrinemechanism, induce myogenic precursors to ‘scat-ter’ from the somites and migrate into the proxi-mal limb mesenchyme. This view is supported byrecent findings that beads impregnated with HGFand transplanted into the flank mesenchyme in-duces Pax-3 expressing myogenic cells to emigratefrom the flank somites (Brand-Saberi et al., ’96).Similar results also have been produced by trans-planting limbs, whose proximal regions expressHGF, ectopically into the flank regions (Hayashiand Ozawa, ’95). Consequently, it has been pro-posed that somitic cells in the flank cannot in-vade the flank somatopleure, even through theyexpress the c-met receptor, because they are nor-mally not exposed to HGF. In contrast, migrationoccurs at the limb level because proximal limbmesenchyme is capable of producing HGF (Brand-Saberi et al., ’96).

In the limb, myogenic cells retain their inva-sive property and actively migrate in a proximo-distal direction to establish the limb’s skeletalmuscle pattern (Brand-Saberi et al., ’89; Lee andEde, ’89). Growth factors such as FGF-2 and -4(Webb et al., ’97) and PDGF-AB and -BB (Venka-tasubramanian and Solursh, ’84; Webb and Lee,’97) have been demonstrated to be important inmaintaining this directed migration. According tothe literature, not much is known regarding howHGF interacts with limb myogenic cells to con-trol cell migration. HGF must be important be-cause it has been reported that, in day 10.5 and11.5 mouse limbs, myogenic cells specifically ex-press the c-met receptor while the surrounding

mesenchymal cells express HGF (Yang et al., ’96).Therefore, we have investigated the ability of HGFto chemotactically stimulate myogenic cell to mi-grate in vitro; whether the chemotactic stimulationis by chemokinesis or chemotaxis; and, whetherHGF can enhance myogenic cell proliferation.

MATERIALS AND METHODSIsolation of limb tissues

E11.5 day mouse embryos were obtained fromICR pregnant females (Chinese University ofHong Kong). The presence of a vaginal plug wasdesignated as day 0.5. The mice were killed bycervical dislocation and the embryos were removedfrom the decidua in pre-warmed Dublbecco’s phos-phate-buffered saline (Sigma, St. Louis, MO) plus0.4% bovine serum albumin (Gibco BRL, GrandIsland, NY). The forelimbs were then excised fromthe embryos and divided into proximal and distalportions.

Preparation of limb cell suspensionThe cells used in the chemotactic assays were

obtained from the proximal region of day 11.5 fore-limbs. The distal region was not used because itcontained very few myoblasts (Webb et al., ’97).The proximal tissues were dissociated in 0.5%trypsin and 0.25% pancreatin at 37°C for 15 min.The enzymatic reaction was stopped with 10% foe-tal calf serum and then the tissues were mechani-cally dissociated using a Pasteur pipette. The cellsuspension was filtered through a 21 µm nylonmesh to give a single cell suspension. Cell con-centration was established with a haemocytometerand viability was assessed with trypan blue dye.

Chemotactic assayThe chemotactic assays were performed in

Blindwell chemotactic chambers (Costar Corpora-tion, Cambridge, MA) as described by Liu et al.(’95). Briefly, the Blindwell chemotactic chamberis composed of two compartments: an upper com-partment for holding 50µl a cell suspension and alower compartment for holding the chemotacticfactors of interest. A nucleopore polycarbonate fil-ter (8.0 µm pore size, Costar Corporation), is in-serted between the two compartments. Prior touse, the filters are sterilized by UV- irradiationand coated with 100 µg/ml of fibronectin solution(Gibco BRL). If the substance placed in the lowerchambers is a chemoattractant, cells will migratethrough the pores in the filters and accumulateon the underside of the filter.


Between 1 and 50 ng/ml of HGF (R&D System,Inc., Minneapolis, MN) suspended in DMEM wasintroduced into the lower compartment of theBlindwell chamber while a suspension of limb cellsin DMEM medium (1 × 106 cells/ml) was added tothe upper compartment. The chambers were in-cubated at 37°C in 5% CO2 for 5 hr. All of the cul-ture media used were serum-free. At the end ofthe incubation period, the chambers were dis-mantled and the upper surface of each filter waswiped with sterile gauze to remove cells that hadattached but had not migrated to the lower sur-face. Immunohistochemistry was performed withmonoclonal anti-sarcomere myosin antibody (MF-20, Developmental Studies Hybridoma Bank, Bal-timore, MD) to identify the myogenic cells. Thefilters were first fixed with 70% ethanol for 30min, then MF-20 antibody (diluted 1:300 in PBS)was added and viewed with a mouse ABC kit (Vec-tor Laboratories, Burlingame, CA) using metalenhanced DAB as the substrate (Amersham In-ternational). In all specimens examined, we didnot detect the presence of MF-20 positive myo-blasts despite the presence of numerous cells onthe lower surface of the filter. However, this wasnot surprising because early myoblasts are noto-riously difficult to detect because they do not ex-press any known antigenic marker. We overcamethis problem by culturing the cells on the filtersfor a further 24 hr to allow the early myoblasts todifferentiate. Prior to culture, the filter surfacedirectly exposed to the upper chamber was wipedclean of cells.

A checkerboard analysis also was performed todistinguish whether HGF was inducing myoblastsmigration by chemotaxis and/or chemokinesis. Theprocedure involved adding different concentrationsof HGF (1–50 ng/ml) to both the upper and lowercompartments of the chemotactic chambers (Ven-katasuramanian and Solursh, ’84).

Immunostaining for the presence ofHGF protein

Day 11.5 forelimbs were fixed overnight in 4%paraformaldehyde. The specimens were then de-hydrated, cleared in xylene and embedded in par-affin wax. The tissues were sectioned at 7 µm andmounted on gelatin-coated slides. For immunohis-tochemistry, the sections were first blocked usingrabbit serum suspended in PBS for 30 min atroom temperature. Goat primary polyclonal anti-serum against recombinant human HGF (20 µg/ml in PBS, R&D System) was then applied to eachslide. Incubation was performed at room tempera-

ture overnight. The antibody staining was viewedusing an anti-goat ABC kit (Vectastain) with DABas the substrate.

HGF neutralization assayTo determine whether the chemotactic effect on

myogenic cells was attributed to HGF rather thansomething else present in the culture medium,various concentrations of anti-human HGF neu-tralizing antibody (R&D System) were mixed withHGF suspended in DMEM and introduced intothe lower compartments of the chemotactic cham-bers HGF antibody (0.5–5 µg/ml) were pre-mixedwith 50 ng/ml HGF peptide (concentration whichexerted the strongest chemotactic effect) for 1 hrbefore they were used in the migration assays.

Tto demonstrate that any reduction in myoblastmigration was due to the neutralization of HGFrather than a toxic side effect of the anti-HGF,the neutralizing antibody was mixed with PDGF-BB (a known chemoattractant of limb myoblasts,Webb and Lee, ’97). PDGF-BB at 10 ng/ml (con-centration with the strongest chemotactic effect)was mixed with 5 µg/ml anti-HGF and introducedinto the chemotactic chambers. If there is no dif-ference in the number of myoblasts responding toPDGF-BB plus anti-HGF and PDGF-BB alone,this will suggest that the anti-HGF antibody isnot cytotoxic to limb myoblasts.

Effect of HGF on myoblasts proliferationA suspension of limb cells (4 × 105 cells/ml of

DMEM ) was prepared from the proximal regionsof day 11.5 fore-limbs according to methods de-scribed above. These cells were plated onto gela-tin-coated coverslips inserted into 24-well culturedishes. The cells were cultured in the presence ofDMEM plus 1% FBS, in the absence or presenceof HGF (1–50 ng/ml). The cultures were incubatedat 37°C and 1% CO2 for 48 hr. After incubation,the cultures were fixed in acetone for 10 min,stained with MF-20 antibody and the number ofmyoblasts present were counted.

Capacity of HGF to influence chemokinesisand chemotaxis in the limb

To investigate the chemokinetic effect of HGFon limb cell migration, a suspension of proximallimb cells (5 × 106 cells/ml of DMEM) was pre-pared from 11.5 day forelimbs. Half of these cellswere exposed to 50 ng/ml of HGF for 3 hr whilethe other half was left untreated and used as thecontrol. All of the cells were then labeled with 5%DiI (Molecular Probe, Eugene, OR) and 10 µl of


the labeled cells were injected into the proximalregions of whole 11.5 day forelimbs. To study thechemotactic effects of HGF, a suspension of DiI-labeled limb cells (5 × 106 cells/ml of DMEM) wasprepared. The labeled cells were injected into theproximal region of 11.5 forelimbs. Distally, ap-proximately 500–700 µm from the injected cells,an Affi-Gel Blue bead (150–200 µm), was im-planted into the limb. The experimental beadswere loaded with 500 ng/ml of HGF while controlbeads were loaded with PBS. All of the experi-mental limbs were inserted into 25 ml bottles con-taining 3 ml of DMEM/F12 medium supplementedwith 10% foetal calf serum. Six limbs were placedinto each bottle. The cultures were then gassed with5% CO2, 40% O2 balanced in N2, maintained on aroller incubator rotating at 30 rpm and kept at 37°Cfor 36 hr. Following culture, the limbs were fixed in4% paraformaldehye, cleared in 30% sucrose andexamined under a confocal microscope.

RESULTSEffect of HGF on myogenic cell migrationPrior to experimentation, the average numbers

of myogenic cells present in the proximal and dis-tal regions of 11.5 day forelimbs were quantified.Tissues from these regions were dissociated intosingle cells and cultured for 24 hr. The number ofmyogenic cells present was identified using MF-20 antibody. It was found that there were 3.5times more myoblasts in the proximal than thedistal regions. Hence, only the proximal regionswere used in our studies. Blindwell chemotacticchambers were used to investigate the effect ofHGF on myogenic cell motility. HGF at concen-trations of 1–50 ng/ml were tested and the extentof migration was determined by counting the num-ber of myogenic cells found on the underside ofnucleopore filters, removed from the Blindwellchambers after 5 hr incubation. MF-20, the earli-est known antibody marker for myogenic cells,was used to positively identify the presence ofmyogenic cells on the filters. Initially, in all of thespecimens examined, none contained the presenceof MF-20 positive myoblasts despite the presenceof numerous cells on the filter. However, this wasnot surprising because early myogenic cells arenotoriously difficult to detect because they expressvery few known antigenic markers. We overcamethis problem by culturing the cells found on thelower surface of the filters for an additional 24 hrto allow the early myoblasts to differentiate. Un-der such conditions, it was found that on average

3.2 MF-20 positive myoblasts per mm2 migrated inresponse to the culture medium alone (Figs. 1A and2). In contrast, in the presence of 1, 5, 10 and 50ng/ml of HGF there was a dramatic and significantincrease in the number myogenic cells present onthe filters (Figs. 1B–D and 2). For example, in thepresence of 50 ng/ml of HGF, an average of 77.5myogenic cells were found per mm2 surface area ofthe filters—a 25-fold increase in myogenic cell mi-gration as compared with the controls.

Because we did not determine the number ofmyogenic cells directly after the chemotactic as-says and the filters containing the migrant cellshad to be cultured for a further 24 hr for myo-genic cell identification, it was important to provethat the increase of myogenic cells number ob-served in the presence of HGF could not have beenattributed to the ability of the peptide to stimu-late myoblast proliferation. Hence, 11.5 day proxi-mal limb cells were cultured in the presence of 0,1, 5, 10 and 50 ng/ml of HGF for 48 hr. Therewere five replicates in each treatment. No signifi-cant difference was observed in the numbers ofMF-20-positive myoblasts between the experimen-tal and control groups (Fig. 3).

HGF neutralization and myoblast migrationTo demonstrate the increased myoblast migra-

tion detected in the presence of HGF was attrib-uted to HGF and not some other peptide presentin the culture medium, HGF antibody was usedto neutralize the HGF. HGF at 50 ng/ml was se-lected for neutralization because this concentra-tion stimulated the greatest increase in myoblastmigration. HGF antibody at 0.5, 1 and 5 µg/mlwas used to neutralize 50ng/ml of HGF. In theabsence of anti-HGF, 77.5 myoblasts/mm2 mi-grated in response to HGF. However, in the pres-ence of anti-HGF, myoblast migration decreasedsignificantly in a dose-dependent manner (Fig.1E–H), such that in the presence of 5 µg/ml anti-HGF, the number of myoblasts found per mm2 offilter was 2.8 (Table 1).

To confirm the decrease in myoblast migrationobserved in the presence of anti-HGF was attrib-uted to HGF neutralization and not a toxic side ef-fect of the antibody, the ability of myoblasts tomigrate in the presence of another chemoattractantwas examined. As a chemoattractant 10 ng/ml ofPDGF-BB was used and this was mixed with 5 µg/ml anti-HGF. In the presence of PDGF-BB alone,there was an average of 34.4 myoblasts found permm2 of the nucleopore filters. When anti-HGF wasadded along with PDGF-BB, the average number


Figure 1.


found was 35.7 myoblasts per mm2 filter. Mann-Whitney U-test demonstrated that there was no sig-nificant difference in the number of myoblastsbetween the two groups (Table 1).

Checkerboard analysisA checkerboard analysis, using different concen-

trations of HGF in the upper and lower compart-ments of the Blindwell chemotactic chambers, wasperformed to distinguish the extent to which myo-blast migration to HGF was attributed to chemo-taxis, chemokinesis or a combination of both. Theresults show evidence that HGF can exert bothchemotaxis and chemokinesis on limb myogeniccells (Table 2). In the column 0 HGF, upper cham-ber, the number of trans cells goes from 3.2 to73.5 with increasing concentrations of HGF, sup-plying evidence for chemotaxis. The diagonals ofthe checkerboard table, where the concentrationof HGF in the upper and lower compartments arethe same, provide evidence for chemokinesis. How-ever, in row 0 HGF, lower chamber, the increasein cell migration is small as the concentration ofHGF in the upper chambers is increased (Table

2). This suggests that the chemokinetic compo-nent is small compared with chemotaxis.

Distribution of HGF in the limbThe distribution pattern of HGF peptide in the

11.5 day fore-limbs was determined using HGFantibody. The AER and ectoderm were stainedvery intensely for HGF, as well as the soft con-nective tissues directly beneath the AER (Fig. 4A).HGF staining was detected in the myogenic re-gions, but the staining pattern was heterogeneous,with HGF-positive cells punctuating the myogenicregions (Fig. 4B). In addition, significantly moremyogenic cells were in the proximal than the dis-tal myogenic regions expressing HGF.

Ability of HGF to influence chemokinesisand chemotaxis in the limb

The chemotactic effects of HGF on cell migra-tion in the limb were investigated. Dissociatedcells were obtained from the proximal regions ofthe limb (area enriched with myogenic cells), la-beled with DiI and transplanted into 11.5 daymouse limbs. Beads loaded with and without HGFwere transplanted distal to the implanted cells.

Fig. 2. Myogenic cell migration in the presence of a HGFgradient. Sample size per treatment is 5. *Significantly differ-ent from control as determined by one-way ANOVA analysis.1Concentration of HGF introduced into the lower compartmentof a Blindwell chemotactic chamber.

TABLE 1. Effects of combining HGF antibody with HGF orPDGF-BB on myogenic cell migration†

Conc. of growth Conc. of anti-HGF (µg/ml)factors 0 0.5 1 5

HGF 77.5 ± 11.0* 56 ± 3 42 ± 2 2.8 ± 1.0(50 ng/ml)PDGF-BB 34.4 ± 7.0 ND1 ND 35.7 ± 3.0(10 ng/ml)†Results are expressed as mean ± s.e.m. of 4 experiments.*Denotes the number of myogenic cells that have migrated throughthe filters in the Blindwell chamber.1ND, experiment not done.

Fig. 1. Effects of HGF and HGF neutralizing antibody onmyogenic cell migration in vitro. In the presence of HGF (A–D), migration increased in a dose-dependent fashion. The ad-dition of HGF neutralizing antibodies to the chemotacticassays blocks the stimulatory effects of HGF (50 µg/ml) onmyogenic cells (E–H). Sample size per treatment is four.MF20-positive myogenic cells (arrows); pores in the nucleoporefilter (arrowhead). Bar = 50 µm.

Fig. 3. Effects of HGF on myogenic cell proliferation.Sample size per treatment is five. One-way ANOVA analysisrevealed no significance between the samples.


It was found that, in all six of the controls, beadsloaded with PBS were unable to attract the mi-gration of DiI-labeled cells (Fig. 4C). In contrast,HGF-impregnated beads were able to chemotac-tically draw DiI-labeled cells to migrate towardthe beads (Fig. 4D). This was observed in three ofsix experimental limbs.

In the chemokinetic studies, dissociated proxi-mal limb cells were exposed to HGF for 3 hr, la-beled with DiI and then transplanted into 11.5day mouse limbs. The limbs were then culturedfor 36 hr. In all six of the control specimens ex-amined, numerous DiI labeled cells were foundat the transplantation site. Some of these cellshave spread both laterally and distally from thesite. However, the distances traveled, comparedwith cells treated with HGF, were relatively shortand in the order of 20–30 cell length (Fig. 4E).Limb cells treated with HGF, prior to transplan-tation, were able to migrate a greater distancethan nontreated cells. In four of six HGF treatedlimbs examined, streams of DiI-labeled cells werefound migrating distally from the transplantationsite (Fig. 4F).

DISCUSSIONThe limb bud is not a closed embryonic system.

During development, it is invaded by a variety ofcells that include endothelial cells, myogenic cells,neural crest cells and nerve fibers (Beddingtonand Martin, ’89; Krenn et al., ’91; Brand-Saberiet al., ’95). The myogenic cells are derived fromthe somites (Christ et al., ’77; Newman et al., ’81;Lee and Sze, ’94; Sze et al., ’95), and it is nowrecognized that the migration of these cells intothe limb depends on interactions between c-metreceptors and HGF, expressed by somitic cells andthe proximal limb mesoderm respectively (Bladtet al., ’95; Brand-Saberi et al., ’96; Heymann etal., ’96; Yang et al., ’96). In situ hybridization stud-ies of normal and mutant mouse limbs also have

suggested HGF may be important for myogenesis,as it was revealed that, in the muscle-forming re-gions, the myoblasts expressed c-met receptorswhile the mesenchymal cells surrounding the myo-blasts expressed HGF (Yang et al., ’96). This sug-gests the possibility of a paracrine relationshipbetween the myoblasts and the mesenchymalcells. In this present study, we used Blindwellchemotactic chambers to examine the effects ofHGF on myogenic cell migration, as this processplays a fundamental role in limb myogenesis. Ourresults demonstrated that HGF, at concentrationsas low as 1 ng/ml, can dramatically enhance themotility of limb myoblasts. This migration in-creased in a dose-dependent fashion as the con-centration of HGF is raised. We have performeda neutralization assay using HGF antibodies andhave demonstrated a substantial reduction inmyogenic cell migration when HGF is neutralized.The test confirms that the dramatic increase myo-blast motility observed in the presence of HGF isattributed solely to HGF rather than the myo-blasts responding to the possible presence of otherchemoattractive factors in the culture medium. Wehave already reported that FGF-2 and FGF-4 canstimulate a significant increase in myoblast mo-tility in vitro (Webb et al., ’97); however, comparedwith HGF (at same concentration) the latter ismuch more potent. Moreover, we have found thatHGF, compared with PDGF-AB and PDGF-BB(Webb and Lee, ’97) at similar concentrations,stimulated more myogenic cells to migrate. Thesefindings suggest that HGF may play an evengreater role in regulating myogenic cell movementthan either FGF or PDGF isoforms. Interestingly,HGF can also chemotactically stimulate satellitecells to migrate and HGF expression is up-regu-

TABLE 2. Checkerboard analyses using variousconcentrations of HGF*

Lower chamber Upper chamberHGF (ng/ml) 0 1 10 50

0 3.2 ± 1.1 5.1 ± 0.6 12.9 ± 2.3 16.2 ± 5.41 7.2 ± 1.8 10.4 ± 0.5 19.1 ± 1.6 32.5 ± 6.75 24.3 ± 6.7 13.8 ± 3.5 30.0 ± 4.9 20.9 ± 2.4

10 35.4 ± 9.4 36.5 ± 3.7 53.6 ± 1.2 52.4 ± 9.350 73.5 ± 9.2 113.9 ± 0.5 126.6 ± 4.4 94.1 ± 5.5*Numbers in bold italic indicate myogenic cells migrating in theabsence of a gradient. Results are expressed as mean ± s.e.m. of 4experiments.

Fig. 4. A: Immunohistological staining showing that HGFis expressed in the myogenic regions (m), progress zone (pz),ectoderm, apical ectodermal ridge and soft connective tissue.Cartilage (c). Bar = 300 µm. B: Higher magnification of myo-genic regions in (A) showing the presence of HGF-positivemyogenic cells (arrows). Bar = 20 µm. C: Chemotaxis analy-sis showing that a bead (b) loaded with PBS is unable toattract the migration of DiI-labeled limb cells from their graftsite (g). D: In contrast, a bead (b) loaded with HGF can chemo-tactically attract and direct the migration of DiI-labeled limbcells (short arrows). E: Chemokinesis analysis revealed thatDiI-labeled limb cells transplanted into 11.5 day forelimbs,without HGF pretreatment, remain localized at the graft site(g). F: HGF pre-treatment, prior to transplantation, stimu-lated the migration of DiI-labeled limb cells (short arrow) ex-amined 36 hr after culture. White dots show the outline ofthe cultured limbs. D-P, Distal-proximal axis; b, location oftransplanted beads. Bar = 300 µm.


Figure 4.


lated in regenerating skeletal muscles (Jennischeet al., ’93; Bischoff, ’97).

There are three possible means limb myogeniccells can respond to HGF: by chemotaxis (di-rected migration along a concentration gradi-ent); by chemokinesis (nondirected increase incell migration in the absence of a gradient); ora combination of both these processes. To dis-tinguish between the three processes, we per-formed a checkerboard assay. By varying theconcentrations of HGF introduced into the up-per and lower wells of our Blindwell chemotac-tic chambers, we discovered that HGF couldinduce both chemotaxis and chemokinesis on thelimb myogenic cells. Takayama et al. (’96) havealso demonstrated the ability of HGF to inducechemotaxis in transgenic embryos over express-ing HGF. They showed that ectopic expression ofHGF in the neural tube can induce inappropriateformation of skeletal muscle in the CNS. Conse-quently, the authors proposed that the defect wasbrought about by HGF acting as a chemotacticagent to guide met-expressing somitic cells towardthe neural tube. In the limb, myogenic cells mi-grate specifically in a proximo-distal fashion toestablish the skeletal musculature of the limb(Brand-Saberi et al., ’89; Lee and Ede, ’89). Thisdirected migration also might be facilitatedthrough chemotaxis, as we have revealed thatHGF is expressed strongly in the AER and thedistal mesenchyme while the myogenic cells arelocated proximally. Myokai et al. (’95) also havefound in the chick that concomitant with the es-tablishment of the AER, the distal limb mesen-chyme expresses HGF intensely with a gradienthigher in the progress zone. In addition, we alsohave demonstrated that beads loaded with HGFcan chemotactically attract and direct the migra-tion of proximal limb cells in the limb. BesidesHGF, many other growth factors are present inthe limb, such as FGFs, PDGFs and TGFβ, whichcan induce chemotaxis in limb myoblasts andthese peptides could collaborate with HGF to con-fer directionality to migrating myogenic cells inthe limb (Venkatasubramanian and Solursh, ’84;Lucas and Caplan, ’88; Webb et al., ’97; Webb andLee, ’97). Chemotaxis probably does not play arole in directing the migration of somitic cells(myogenic precursors) into the limb despite theideal conditions for chemotaxis to operate (i.e.,somitic cells expressing the c-met receptor whilethe adjacent limb mesenchyme expressing HGF(Bladt et al., ’95). Heymann et al. (’96) have dem-onstrated that HGF-impregnated beads cannot

elicit a chemotactic response in somitic cells whentransplanted into the flank of chick embryos. How-ever, the addition of FGF-2 can and these authorssuggested that HGF’s main function is to delami-nate the dermomyotome so that the myogenic pre-cursors can invade the limb bud. It has now beenestablished, from studies on the splotch mutant,that the pax-3 gene is vital for initiating the mi-gration of limb myogenic precursor cells from thedermomyotome (Bober et al., ’94; Epstein et al.,’96). In addition, pax-3 acts upstream of the c-met receptor gene in these cells. These findingsfurther highlight the importance of HGF in limbmyogenesis since HGF is the ligand for the c-metreceptor.

HGF also could participate in myogenesis bysustaining the capacity of myogenic cells to mi-grate in the limb through chemokinesis, becausewe have demonstrated that DiI-labeled limb mes-enchymal cells pretreated with HGF migrated fur-ther in the limb than untreated specimens.Moreover, Yang et al. (’96) established that thereis a considerable spatial overlap between limbmyoblasts expressing the c-met receptor and HGF,allowing some form of autocrine mechanism to beset up. Indeed, the establishment of a HGF/metautocrine signaling loop is often involved in in-ducing diverse tumorigenesis in a wide variety oftissue types (Takayama et al., ’97).

Besides the ability of HGF to influence cell mi-gration, it can also stimulate an increase in theproliferation of a variety of cell types. Melanocytes,keratinocytes, renal tubular epithelium, gastricepithelium, biliary epithelium, vascular endothe-lium and hepatocytes can all be stimulated byHGF (Matsumoto et al., ’91; Morimoto et al., ’91;Takahashi et al., ’93). We were initially troubledby these findings because in our chemotactic as-says, we did not examine the myoblasts directlyafter experimentation because the myogenic cellscould not be identified by immunohistochemistry.We had to extend the culture period for an addi-tional 24 hr before the myogenic cells could bedetected with MF20 antibody. Therefore, it wasimportant to determine whether the increase inmyogenic cell numbers observed in presence ofHGF was attributed to an increase in myogeniccell proliferation or migration. Our proliferationtests revealed that HGF did not increase the num-bers of MF20-positive myoblasts, so the increasethat we had obtained from the chemotactic assayscould only have been derived from an increase inactive cell migration. HGF can apparently stimu-late quiescent skeletal muscle satellite cells in vitro


to precociously enter the cell cycle (Allen et al., ’95).In sum, it appears that HGF in the limb plays apositive role in regulating myogenic cell migration,but probably plays little role in controlling myo-genic cell proliferation.

ACKNOWLEDGMENTSThe authors thank Paul Sze and Jenny Hou for

their technical assistance.

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