(Received for publication, June 23, 1995; and in revised form, September 28, 1995)
From the
We have previously cloned and sequenced a newt keratinocyte
growth factor receptor (KGFR) cDNA which exhibited a unique spatial and
temporal expression pattern in the regenerating newt limb. In this
report, we further characterize the biochemical and functional
properties of this newt KGFR. A stable Chinese hamster ovary
transfectant overexpressing the newt KGFR was capable of binding both I-fibroblast growth factor-1 (FGF-1) and
I-FGF-7 but not
I-FGF-2, indistinguishable
from the human KGFR. Scatchard analysis and cross-linking studies
further support the conclusion that FGF-1 and FGF-7 are the ligands for
the newt KGFR. In addition to their ability to bind to FGFs, both the
human and the newt KGFR are also capable of repressing differentiation
in mouse MM14 myoblasts. MM14 cells express FGFR1 and are repressed
from differentiation by FGF-1, FGF-2, and FGF-4 but not FGF-7.
Co-transfection of MM14 cells with either a human or newt KGFR
expression construct conferred a response to FGF-7 as determined by a
human
-cardiac actin/luciferase reporter construct. The response
to FGF-7 was similar to the endogenous FGF response as FGF-7 prevented
MM14 myoblasts from undergoing terminal differentiation. Thus, both the
human and the newt KGFRs transduce signals similar to those transduced
via the endogenous mouse FGFR1. Together these data indicate that this
newly isolated newt KGFR is a functional receptor as it binds two FGF
family members with high affinity and mediates signaling in skeletal
muscle myoblasts. Because the binding pattern of the newt KGFR is
similar to the pattern observed for its mammalian counterpart, it
emphasizes the strict conservation that this ligand/receptor system has
undergone through evolution.
Fibroblast growth factors (FGF) ()elicit a multitude
of different biological responses in a variety of mesodermal and
neuroectodermal derived cell types and are implicated in several
physiological and pathological processes (reviewed in (1) and (2) ). The distinct biological responses of cells to FGFs thus
far appear to be mediated by a family of transmembrane FGF receptors
(FGFRs). These include
FGFR1/flg(3, 4, 5) ,
FGFR2/bek(4, 6, 7) ,
FGFR3(8) , and FGFR4(9) . The four receptors constitute
a subclass of receptor tyrosine kinases that is characterized by three
(or two) extracellular immunoglobulin-like domains (Ig domains), a
membrane spanning region, and a cytoplasmic portion that contains a
tyrosine kinase domain(10) . FGFRs 1-3, but not FGFR4,
are subject to a high degree of alternative splicing of their primary
transcripts that results in a multitude of combinatorial splice
variants (reviewed in (11) ). Alternative splicing within the
tyrosine kinase domain of FGFR1 can result in a kinase-defective
molecule. Upon ligand binding this kinase-defective variant
oligomerizes with a kinase-containing FGFR resulting in a heterodimer
complex incapable of phosphorylation and activation of phospholipase
C
(12) . However, the majority of alternative splicing
events occur in the extracellular portion of the receptor. For example,
cDNAs for FGFR1 and FGFR2 which lack sequences corresponding to the
first Ig domain have been
isolated(5, 13, 14, 15, 16, 17, 18) .
Analysis of the genomic structures of FGFR1 and FGFR2 has revealed that
this domain is encoded by a single exon that is spliced out in the two
Ig domain forms of the receptor(19) . The absence of this Ig
domain does not appear to affect ligand binding to FGFR1 (13) or FGFR2(18, 20) .
Other splice variants in FGFR1 and FGFR2 arise in Ig domain III(13, 19) . The COOH-terminal half of this domain in FGFR1 is encoded by three alternative exons (IIIa, IIIb, and IIIc) that can result in the expression of a secreted receptor and two different transmembrane receptors, respectively. Exons IIIb and IIIc have homologs in the FGFR2 gene(19) . Their mutually exclusive splicing in the second half of Ig domain III results in the bek (IIIc) isoform (4, 6, 21) or the KGF receptor (KGFR) (IIIb) isoform(15, 17, 18, 22) . The bek receptor has been shown to bind with high affinity to FGF-1, FGF-2, FGF-4, and FGF-5 but not to FGF-7(4, 6, 23, 24) . In contrast, KGFR binds FGF-7 as well as FGF-1 but the binding to FGF-2 is significantly decreased(18, 25) . Recently, the genomic organization of Ig domain III of FGFR3 has revealed the existence of homologous IIIa, IIIb, and IIIc exons(26) . The original FGFR3 cDNA containing the IIIc exon is preferentially activated by FGF-1 and FGF-4, to a lesser extent by FGF-2, and has almost no response to FGF-5(27) . The IIIb splice variant of FGFR3, however, shows the most restricted ligand binding properties of any FGFR described so far, binding exclusively FGF-1(26) . The genomic organization of FGFR4 shows the presence of a single exon encoding the COOH-terminal half of the Ig domain III region (28) .
A role for FGFs in development is suggested by the unique expression patterns of several family members(29, 30, 31, 32) . Likewise, the FGFRs have recently been shown to have unique temporal and spatial expression patterns as well(33, 34, 35) . In addition, the targeted expression of a dominant negative FGFR1 in the epidermis (36) and FGFR2 in the lung (37) in transgenic mice results in disruptions in the normal developmental architecture of those tissues. FGFs and their receptors have been implicated in amphibian limb regeneration, a process which closely parallels normal limb development. Infusion of FGF into the distal stump of denervated newt limbs stimulates cell cycling over the depressed level normally observed after denervation(38) . Recently, FGF-1 (39) and two FGFRs, namely FGFR1 and FGFR2(22) , have been shown to be present in the regenerating limb blastemas of newts, with the latter displaying unique temporal and spatial expression patterns throughout the regeneration process. We further showed that KGFR is the FGFR2 variant that is expressed in the basal layer of the wound epithelium(40) . The present study was undertaken to explore the functionality of the newt homolog of KGFR and its potential relevance in amphibian limb regeneration. Here we present data showing that the newt KGFR possesses the ability to bind specific members of the FGF family in a manner indistinguishable from its human counterpart. Moreover, the expression of this receptor in mouse MM14 myoblast cells represses terminal differentiation mediated by FGF-7.
The EcoRI site of a 245-base pair EcoRI/HindIII
fragment of pHCA177CAT (45) containing the region of the human
-cardiac actin gene from -177 base pairs to +68 base
pairs was filled in and the insert cloned into the SmaI/HindIII site at the 5` end of the luciferase
gene in the pGL2 basic vector. The resultant plasmid is designated
-cardiac actin/luciferase reporter.
NIH/3T3 cells were maintained in Dulbecco's modified
Eagle's medium supplemented with 10% calf serum and
penicillin/streptomycin. A transformed NIH/3T3 cell line,
NIH/HKGFR(18) , which overexpresses the human KGFR was
maintained in the same media with the addition of G418 (750 µg/ml).
Mouse MM14 cells (47) were cultured on gelatin-coated plates in
growth medium consisting of Ham's F-10 supplemented with 0.8
mM CaCl, 100 units/ml penicillin, 5 µg/ml
streptomycin, and 15% horse serum. The concentration of FGF-2 was
increased from 5 to 40 ng/ml with increasing cell density.
The FGF-1 protein used in the skeletal muscle reporter gene activity assay was obtained from bovine brain as described elsewhere(48) .
For the qualitative binding assays, protein
assays were then performed on the solubilized cells as described by the
manufacturer (Bio-Rad). Incubation of the cells with the labeled FGFs
was carried out in the absence (total binding) or presence (nonspecific
binding) of 100-fold molar excess of unlabeled FGF. Mean cpm bound per
µg of total cell protein (±S.D.) was determined from
triplicate samples. Specific binding is defined as total binding minus
nonspecific binding. In the competition assays I-FGF-1
(2.3 ng/ml) or
I-FGF-7 (3.4 ng/ml) was added to the wells
in the presence of increasing amounts of unlabeled competitor.
Duplicate wells were used for each concentration of unlabeled
competitor. For the Scatchard analysis increasing amounts of
I-FGF were added to duplicate wells with nonspecific
binding counts obtained in parallel assays using 100-fold excess of
unlabeled FGF.
Figure 1: Newt FGFR2 constructs and qualitative binding assay of newt KGFR expressing cell line. A, illustration of the two newt FGFR2 receptor cDNA constructs. For both the bek and KGFR constructs the open boxes are the signal sequence, the filled boxes are the acidic domain, the hatched boxes represent the transmembrane domain, and the cross-hatched boxes are the kinase domain separated by a kinase insert. The loops represent the Ig-like domains. The line extending to the left of the signal sequence represents an extension of 19 amino acid residues to another in-frame methionine that was included in the cDNAs. The thicker line representing the carboxyl-terminal half of the proximal Ig-like domain highlights the area where two mutually exclusive exons (IIIb and IIIc) are spliced into the final transcript. The IIIb exon in newt KGFR is stippled, whereas the IIIc exon in newt bek is hatched. The small table shows the FGF specificity that these two exons impart onto the respective human FGFR2 receptors. B, graph showing the ability of the newt KGFR expressing cell line CHO/NKGFR (filled bar) to bind to the indicated iodinated FGFs compared to the vector transfected cell line CHO/Control (hatched bar).
In a qualitative binding assay the human KGFR showed a significant
increase in binding to I-FGF-1 and
I-FGF-7
relative to NIH/3T3 cells but was not capable of binding
I-FGF-2 over that which was observed in the parental cell
line (data not shown). These results confirmed the previously published
results of this human FGFR2 splice variant(18) . Likewise, the
newt KGFR expressing cell line, CHO/NKGFR, shows an increase in binding
to
I-FGF-1 and
I-FGF-7 but not
I-FGF-2 when compared to the expression vector
transfected cell line, CHO/Control (Fig. 1B). Thus, the
newt KGFR exhibits an FGF binding profile that is similar to its human
counterpart.
To determine the size of the newt KGFR expressed in the
CHO/NKGFR cell line, a cross-linking analysis was performed. The
predicted size of the newt KGFR based on the primary amino acid
sequence of the cDNA is 82 kDa. When either I-FGF-1 or
I-FGF-7 was cross-linked to the newt KGFR and analyzed by
7.5% SDS-PAGE, a predominant cross-linked product with an apparent
molecular mass of 165-170 kDa was detected (lanes 2, Fig. 2, A and B, respectively). Subtraction of
the molecular mass of the ligands yields an estimated molecular mass of
150 kDa for the expressed receptor. The difference between the apparent
and predicted molecular weights for newt KGFR is most likely due to an
extensive degree of N-linked glycosylation in the
extracellular domain. A cross-linked product is not observed in the
CHO/Control cell line with either of the labeled FGFs (lanes
1, Fig. 2, A and B). Cross-linkings
carried out in the presence of 100-fold excess of unlabeled FGFs
indicate that the receptor is specific for FGF-1 and FGF-7 as both of
these ligands are capable of competing with either
I-FGF-1 or
I-FGF-7 for the receptor (lanes 3 and 4 of Fig. 2, A and B). Interestingly, FGF-1 appears to be a more effective
competitor than FGF-7 for
I-FGF-7 which may reflect a
higher affinity of the receptor for FGF-1. Unlabeled FGF-2 is a much
less effective competitor for both
I-FGF-1 and
I-FGF-7 binding to the newt KGFR (lane 5, Fig. 2, A and B).
Figure 2:
Cross-linking analysis of the newt KGFR
expressing cell line CHO/NKGFR with labeled FGFs. The newt KGFR
expressing cell line CHO/NKGFR (lanes 2-5) and the
vector transfected cell line CHO/Control (lane 1) were
incubated with I-FGF-1 (A) or
I-FGF-7 (B) in the absence (lanes 1 and 2) or presence of a 100-fold molar excess of unlabeled
competitor: FGF-7 (lane 3), FGF-1 (lane 4), and FGF-2 (lane 5). The cells were incubated with the FGFs for 1 h at 4
°C after which they were cross-linked to cell surface receptors
with DSS (0.3 mM). The cross-linked products were then
analyzed by 7.5% SDS-PAGE. The molecular masses of the protein
standards (lane M) are in kDa.
To address the
specificity of the newt KGFR in a quantitative manner, a competition
assay was performed on CHO/NKGFR cells using I-FGF-1 and
I-FGF-7 as radiolabeled tracers in the presence of
increasing concentrations of unlabeled FGF-1, FGF-2, FGF-7, or EGF.
High affinity
I-FGF-1 binding to CHO/NKGFR cells was
effectively competed by FGF-1 and FGF-7 with similar efficiencies (50%
displacement at 35 ng/ml for FGF-1 and 60 ng/ml for FGF-7). However,
neither FGF-2 nor EGF was able to compete for
I-FGF-1
binding to newt KGFR on these cells (Fig. 3B). In a
similar manner, high affinity binding of
I-FGF-1 to
NIH/HKGFR cells was also competed by FGF-1 and FGF-7 (Fig. 3A). Although EGF again showed no specific
competition toward human KGFR, FGF-2 was capable of competing with
I-FGF-1 at higher concentrations. When
I-FGF-7 is used as the tracer a dramatic difference in
the pattern of competition can be seen with FGF-1 and FGF-7 in that
FGF-1 appears to be a more effective competitor than FGF-7 for binding
of
I-FGF-7 to both human KGFR (Fig. 4A)
and newt KGFR (Fig. 4B). This data is consistent with
the observations from the cross-linking analysis. The concentrations of
unlabeled FGF-1 and FGF-7 needed to achieve 50% displacement of the two
labeled tracers were consistently 3-4-fold higher for the newt
KGFR.
Figure 3:
Competition assay comparing human and newt
KGFRs with I-FGF-1 as the tracer. The human KGFR
expressing cell line NIH/HKGFR (A) and newt KGFR expressing
cell line CHO/NKGFR (B) were incubated with a constant amount
of
I-FGF-1 (2.3 ng/ml) in the presence of increasing
concentrations of unlabeled competitors: FGF-1 (open squares),
FGF-7 (open diamonds), FGF-2 (open circles), and EGF (open triangles). After washing the cells to remove unbound
ligands and solubilizing in 0.3 N NaOH, the samples were
counted on a Beckman
-counter. Each concentration of competitor
was carried out in duplicate and the average plotted as a percentage of
bound
I-FGF-1 in the presence of the lowest concentration
of competitor. The standard error of the mean for all samples never
exceeded 11% of the average.
Figure 4:
Competition assay comparing human and newt
KGFRs with I-FGF-7 as the tracer. The human KGFR
expressing cell line NIH/HKGFR (A) and newt KGFR expressing
cell line CHO/NKGFR (B) were incubated with a constant amount
of
I-FGF-7 (3.4 ng/ml) in the presence of increasing
concentrations of unlabeled competitors: FGF-1 (open squares),
FGF-7 (open diamonds), FGF-2 (open circles), and EGF (open triangles). After washing the cells to remove unbound
ligands and solubilizing in 0.3 N NaOH, the samples were
counted on a Beckman
-counter. Each concentration of competitor
was carried out in duplicate and the average plotted as a percentage of
bound
I-FGF-1 in the presence of the lowest concentration
of competitor. The standard error of the mean for all samples never
exceeded 10% of the average.
The results of the competition assay suggest that the newt
KGFR has a slightly lower affinity toward FGF-1 and FGF-7 relative to
its human counterpart. To more precisely determine the affinity of
FGF-1 and FGF-7 to the newt KGFR a Scatchard analysis was performed on
the CHO/NKGFR cell line. Fig. 5, A and B,
reveal that the newt KGFR binds to FGF-1 with a K of 660 pM and to FGF-7 with a K
of
860 pM. The affinity of the newt KGFR for FGF-7 is
approximately 4-fold lower than the reported affinity of the human KGFR
for this ligand(18) .
Figure 5:
Affinity of newt KGFR to mammalian FGFs.
Newt KGFR expressing CHO/NKGFR cells were incubated with increasing
concentrations of I-FGF-1 (A) and
I-FGF-7 (B) in the absence (total binding) or
presence (nonspecific binding) of a 100-fold excess of unlabeled self.
Specific binding is defined as total binding minus nonspecific binding.
Duplicate wells were used for each concentration of labeled FGF.
Saturation isotherms for both labeled FGFs are shown as insets to the corresponding Scatchard plots. The dissociation constants
for both FGF-1 and FGF-7 are indicated in the appropriate graphs. The
standard error of the mean for samples of both total and nonspecific
binding never exceeded 8% of the average. Receptor numbers per cell
obtained with FGF-1 and FGF-7 were 112,000 and 13,000,
respectively.
MM14 cells transfected
with the pBJ5 expression vector along with the -cardiac
actin/luciferase reporter construct exhibit a large increase in
luciferase activity, both in the absence and presence of FGF-7 (Fig. 6). These results verify that these cells do not express
endogenous receptors for FGF-7 and are not capable of responding to
this growth factor. As expected, when cultured in the presence of
either FGF-1 or FGF-2, the pBJ5 transfected cells are repressed from
differentiation as indicated by a low level of luciferase activity. The
ability of both FGF-1 and FGF-2 to repress differentiation in this
assay serves as an internal positive control for the competence of
these cells to respond to exogenously added FGF. The expression of
either human or newt KGFR by MM14 cells resulted in low levels of
luciferase activity when cultured in the presence of FGF-7. The data
are indicative of an acquired ability of these transfected cells to
repress differentiation mediated by FGF-7 (Fig. 6). In the
absence of exogenously added growth factor, both the human and newt
KGFR transfected cells showed an increase in luciferase activity. These
data suggest that the human as well as the newt KGFR isoform of FGFR2
is capable of mediating a signal within MM14 myoblasts that leads to
repression of differentiation. The ability of the ectopically expressed
KGFRs to function in this capacity is similar to or better than the
activity observed with the endogenous FGF receptors expressed by this
cell line.
Figure 6:
FGF-7 mediated repression of skeletal
muscle differentiation in MM14 cells expressing newt and human KGFR.
MM14 myoblasts were transfected with an -cardiac actin/luciferase
reporter plasmid, a CMV-LacZ plasmid, and the pBJ5 expression vector,
the newt KGFR expression plasmid pNKGFR, or the human KGFR expression
plasmid pHKGFR as described under ``Materials and Methods.''
The transfected cells were cultured either in the absence of any
exogenously added growth factor (filled bars) or in the
presence of FGF-1 (open bars), FGF-2 (stippled bars),
or FGF-7 (hatched bars). Cells were then harvested and assayed
for luciferase and
-galactosidase activities. Reporter gene
activity represents
-cardiac actin/luciferase activity of a given
cell extract divided by the CMV/
-galactosidase activity measured
for that extract. The value obtained for each set of transfected cells
cultured in the absence of growth factors was set at 1.0. The actual
values of the pNKGFR and pHKGFR transfectants cultured with no growth
factor were 72 and 62% that of the pBJ5 transfectant, respectively. We
showed that proliferating MM14 cells express FGF-7.
The
FGF-7 that the MM14 cells are expressing may be sufficient to activate
the transfected KGFRs and partially repress activation of the reporter
construct. The results shown are representative of three individual
experiments.
In this study, we report on the functional characterization of a newt FGFR2 variant which was previously isolated in our laboratory (22) . The construction of full-length cDNAs from the available overlapping cDNA clones resulted in receptors that lacked the first Ig domain and as a consequence the extracellular region of these receptors contain the acidic box followed by Ig domains II and III (Fig. 1A). The receptors differ only in the COOH-terminal half of Ig domain III where it is known that the alternative splicing of two different exons, IIIb and IIIc, takes place in a mutually exclusive fashion giving rise to the KGFR and bek variants, respectively. The newt KGFR possesses a unique spatial and temporal pattern of expression in the regenerating newt limb with the strongest expression observed predominantly in the basal layer of the wound epithelium(40) . Because the wound epithelium is a necessary component of the regenerate (51) and has been shown to express a number of molecules which are not expressed in skin epidermis(52, 53, 54) , we were interested in examining the functional capacity of the newt KGFR splice variant. Because no amphibian FGFs are available, mammalian FGFs were utilized as radiolabeled ligands in binding and functional assays. We were encouraged with this approach by the fact that radiolabeled bovine FGF-1 had been used successfully as a probe in in situ studies with axolotl limb blastemas (39) and because of the high degree of amino acid identity between human and newt KGFR(22) .
In qualitative binding assays using iodinated FGFs we found that a newt KGFR expressing CHO cell line, CHO/NKGFR, exhibited an increase in binding to FGF-1 and FGF-7 relative to a control cell line, CHO/Control (Fig. 1B). However, CHO/NKGFR showed no appreciable increase in binding to FGF-2. These results were indistinguishable from the binding of these FGFs to a human KGFR expressing cell line NIH/HKGFR (data not shown). These results combined with the high degree of amino acid sequence identity observed between the human and newt exon IIIb region (86%), strongly support our claim that this newt FGFR2 splice variant is a functional homolog of the human KGFR.
Cross-linking of either I-FGF-1 or
I-FGF-7 to receptors on the surface of CHO/NKGFR show a
predominant cross-linked product with an apparent molecular mass of 150
kDa. This cross-linked product was specific since the addition of a
100-fold molar excess of unlabeled FGFs prior to cross-linking competed
the radiolabeled ligand. Unlabeled FGF-1 and FGF-7 were able to
decrease binding of the two labeled FGFs; however, FGF-2 was not as
effective at competing for
I-FGF-1 or
I-FGF-7. These results support the qualitative binding
assay that the newt KGFR is specific for FGF-1 and FGF-7.
Interestingly, in the presence of
I-FGF-7, FGF-1 appears
to be a more effective competitor than FGF-7 for binding to the newt
KGFR. This could reflect the fact that the newt KGFR may have a higher
affinity for FGF-1 than FGF-7. Alternatively, this could be due to
differences in newt and mammalian FGFs. The size of the 150-kDa band is
greater than expected for a two Ig domain form of the newt KGFR based
on its primary amino acid sequence. Likewise, other investigators have
shown the two Ig domain form of FGFR2 cross-linked to labeled FGFs
migrating with an apparent size that is larger than predicted from
their primary amino acid sequences(17, 20) . This
difference in size is likely to be due to N-linked
glycosylation of the receptors as they have a number of potential N-linked glycosylation sites in their extracellular domain.
The newt KGFR has nine potential N-linked glycosylation sites,
whereas the two Ig domain form of human bek and KGFR have six
and seven, respectively. The cDNA encoding the newt KGFR that was
cloned into the pBJ5 expression vector does harbor an upstream in-frame
methionine which could code for an additional 19 amino acids (42) if translation were to start here. However, the amino
acids that follow this methionine do not exhibit an overall hydrophobic
character and would not be expected to function as a secretory signal
sequence. Interestingly, the position of this upstream methionine is
conserved in FGFR2 cDNAs isolated from other species, which may
indicate that it has some biological importance. At any rate, the
higher molecular weight exhibited by the newt KGFR suggests more
extensive post-translational modification. However, this modification
does not affect either the biochemical properties or functionality of
this receptor (see below).
The specificity of the newt KGFR to different FGFs was demonstrated in a more quantitative manner by competition assays using labeled FGF-1 and FGF-7 as tracers. Unlabeled FGF-1 and FGF-7 were effective in competing with the two labeled FGFs for binding to the newt KGFR, whereas FGF-2 was not an effective competitor at the concentrations used. The human KGFR exhibited the same specificity for binding to FGF-1 and FGF-7 and confirms the results obtained by others(18) . As previously observed in the cross-linking analysis, FGF-1 appears to be a more effective competitor than FGF-7 for binding of the newt KGFR to labeled FGF-7. The apparent higher affinity of the newt KGFR toward FGF-1 was confirmed by Scatchard analysis as FGF-1 and FGF-7 exhibited dissociation constants of 660 and 860 pM, respectively.
To address whether the
newt KFGR was capable of transducing a signal to the interior of a cell
after binding to its ligand, a functional assay was carried out in
mouse MM14 myoblast cells transiently transfected with the newt KGFR
expression vector and an -cardiac actin/luciferase reporter
plasmid. In the presence of exogenously added FGF-7, both the newt KGFR
and human KGFR were capable of transducing the signal required for
repressing differentiation in these cells. This was somewhat intriguing
because the KGFR isoform of FGFR2 is almost exclusively expressed in
cells of epithelial origin. In fact, FGFR1 is likely to be the only
FGFR expressed in MM14 cells (55) . (
)Thus, despite
the difference between the cytoplasmic portions of KGFR and FGFR1,
these two receptors converge to the same signaling pathway within MM14
cells.
In conclusion, we provide evidence that a newt KGFR previously isolated in our laboratory is a functional receptor in that it is capable of binding FGF-1 and FGF-7 with high affinity but not FGF-2 and is competent in transducing a proliferative signal in cells expressing it. These results when combined with the unique expression pattern observed in regenerating amphibian limbs indicate that KGFR may have a significant role within the wound epithelium early in the regeneration process. The appearance of the KGFR splice variant of FGFR2 in the basal layer of the wound epithelium during amphibian limb regeneration (40) is reminiscent of the expression of FGFR2 in the epidermis in a wound healing study(56) . In this study FGF-7 was shown to be induced 160-fold within the dermis of the skin injury, whereas FGF-1, FGF-2, and FGF-5 were induced only 2-10-fold, and FGF-3, FGF-4, and FGF-6 were undetected in normal and wounded skin. This suggests a possible paracrine role for the dermally expressed FGF-7 acting on the overlying epidermis during wound healing. In a similar fashion, an amphibian FGF-7 or FGF-7-like molecule may be involved in the regenerating amphibian limb. We hypothesize that this putative amphibian FGF-7 may act during the early stages of regeneration in establishing and/or maintaining the wound epithelium which is expressing KGFR.