Download Isolation of Xenopus FGF-8b and Comparison with FGF

Mol. Cells, Vol. 19, No. 3, pp. 310-317
Molecules
and
Cells
KSMCB 2005
Isolation of Xenopus FGF-8b and Comparison with FGF-8a
Sangwoo Shim1,†, Narina Bae1,†, Sang Yoon Park2, Won-Sun Kim2, and Jin-Kwan Han1,*
1
Department of Life Science and Division of Molecular and Life Sciences, Pohang University of Science and Technology,
Pohang 790-784, Korea;
2
Department of Life Science, Sogang University, Seoul 121-742, Korea.
(Received May 18, 2004; Accepted February 21, 2005)
The Xenopus FGF-8a and FGF-8b isoforms have been
reported to be neural crest and neuronal inducers,
respectively. However, cloning of Xenopus FGF-8b
(XFGF-8b) has not been reported previously and the
two isoforms do not seem to have been clearly distinguished in Xenopus experiments. Here, we describe the
cloning and expression of XFGF-8b and compare the
effects of the two isoforms. XFGF-8b has an 11 amino
acid insert in its N-terminal region compared with
XFGF-8a. Both isoforms are expressed in the anterior
neural regions of the early embryo, and in the apical
ectodermal ridge of limb buds and tips of growing digits in the larval stages. However, XFGF-8b is more
abundant than XFGF-8a throughout early development. The two isoforms are also regulated in similar
fashion by retinoic acid in early development. However,
although both XFGF-8a and XFGF-8b induce ectopic
neurogenesis, only XFGF-8a appears to be involved in
neural crest induction.
Keywords: Embryogenesis; FGF-8; In Situ Hybridization; Neural Crest; Neurogenesis; Retinoic Acid; Xenopus
laevis.
Introduction
Fibroblast growth factors (FGFs) are multifunctional peptide growth factors that influence diverse processes during
vertebrate development. At present, over 22 vertebrate
FGF genes have been identified (Ornitz and Itoh, 2001).
In Xenopus, at least seven members of the FGF family are
†
These first two authors contributed equally to this work.
* To whom correspondence should be addressed.
Tel: 82-54-279-2126; Fax: 82-54-279-2199
E-mail: [email protected]
present in the early embryos. These include FGF-2, FGF3, eFGF, FGF-8, FGF-9, FGF-10, and FGF-20 (Christen
and Slack, 1997; Isaacs, 1997; Koga et al., 1999; Yokoyama et al., 2000). FGF-8 was first identified as an androgen-induced growth factor in a mammary carcinoma
cell line (Tanaka et al., 1992). Alternative splicing of the
first exon of the FGF-8 gene generates eight potential
isoforms in the mouse (Crossley and Martin, 1995) and
four isoforms (FGF-8a, -8b, -8e, and -8f) in humans (Gemel et al., 1996; Tanaka et al., 1995). FGF-8 is present in
several regions of the developing mouse embryo that are
involved in outgrowth and patterning, including limbs,
elongating body axis, face, and midbrain/hindbrain boundary (MHB) (Crossley and Martin, 1995), and a similar
pattern is found in the chick (Sato et al., 2001; Vogel et
al., 1996). FGF-8 is an important signaling molecule in
vertebrate limb development (Crossley et al., 1996a; Martin, 1998) and in patterning of the central nervous system
(Chi et al., 2003; Crossley et al., 1996b; Meyers et al.,
1998). In Xenopus, only a homologue of the FGF-8a isoform, XFGF-8a, has been isolated, and it has been implicated in anteroposterior patterning and limb regeneration
(Christen and Slack, 1997).
Accumulating evidence suggests that different FGF-8
isoforms have different activities. FGF-8b has been
shown to have stronger transforming activity than FGF-8a
(MacArthur et al., 1995a), and in chick embryos, FGF-8a
and FGF-8b had different effects on midbrain development and gene expression (Sato et al., 2001). There is
also evidence that in Xenopus, XFGF-8a and XFGF-8b
are neural crest and neuronal inducers, respectively, and
that they act via distinct receptors (Hardcastle et al., 2000;
Monsoro-Burq et al., 2003). However, since the XFGF-8b
isoform has not been cloned and characterized, the two
isoforms do not seem to have been clearly distinguished
in experiments on Xenopus (Chalmers et al., 2002; HardAbbreviation: FGF, fibroblast growth factor.
Sangwoo Shim et al.
castle et al., 2000). To clarify the roles of the two isoforms, we isolated a Xenopus homologue of FGF-8b
(XFGF-8b) and compared the amino acid sequences, expression patterns and activities of the two isoforms.
Materials and Methods
Isolation of FGF-8b A Xenopus FGF-8 partial cDNA probe was
used to screen a Xenopus stage 10 cDNA library constructed in
the lambda ZAPII bacteriophage vector (Dr. Michael W. King).
Two positive clones were obtained and their inserts were isolated by in vivo excision to generate Xenopus FGF-8b. The
complete nucleotide sequence of a clone containing the fulllength XFGF-8b has been deposited in GenBank under accession No. AF461177.
Preparation of Xenopus embryos and retinoic acid treatment
Eggs were obtained from Xenopus laevis primed with 800 units
of human chorionic gonadotropin (Sigma). They were in vitro
fertilized using macerated testes, de-jellied in 2% cysteine solution (pH 7.8) and cultured in 0.33× Modified Ringer’s (MR)
(Godsave et al., 1988) to stage 8 and transferred to 0.1× MR.
Developmental stages of embryos were determined according to
Nieuwkoop and Faber’s normal table of development (1967).
All-trans retinoic acid (Sigma) was applied to dorsal marginal
zone (DMZ) explants from the early gastrula embryos or to
whole early gastrula embryos, which were cultured until sampled for RT-PCR analysis (Fig. 4).
In situ hybridization Whole-mount in situ hybridization using
digoxigenin-labeled riboprobes was carried out according to
Harland (1991). Alkaline phosphatase-coupled anti-digoxigenin
antibody and BM purple AP substrate (Boehringer-Mannheim)
were used to detect signals. An anti-sense in situ probe against
FGF-8b was generated by linearizing the pBS-KS-FGF-8b construct with HindIII and transcribing it with T3 RNA polymerase.
The FGF-8b sense probe was synthesized using T7 RNA polymerase on a BamHI-linearized template. No staining was seen in
the sense control. The probe for N-tubulin was generated by
cloning an RT-PCR fragment of N-tubulin (250 bp) into T-vector
and transcribing it in vitro.
Microinjection of RNA, and animal cap assays The coding
regions of XFGF-8a and XFGF-8b were PCR-amplified and subcloned into pCS2 vector. XFGF-8a and XFGF-8b mRNAs were in
vitro transcribed using an mMESSAGE mMACHINETM SP6 kit
(Ambion) with pCS2-XFGF-8a and pCS2-XFGF-8b as templates,
and microinjected with a Nanoliter Injector (WPI). Control and
injected embryos were analyzed by in situ hybridization or RTPCR analysis. For lineage tracing, GFP mRNA was co-injected
and embryos that displayed GFP signals only on the right side
were selected for in situ hybridization analysis. For animal cap
assays involving RNA microinjection, RNA was injected into the
animal poles of embryos at the two-cell stage and the embryos were
311
grown to stage 8 when their animal caps were removed and cultured
to an appropriate stage for sampling and RT-PCR analysis.
RT-PCR analysis For RT-PCR analysis, total RNA was extracted with TRI Reagent (Molecular Research Center) and
treated with RNase-free DNase I (Roche Molecular Biochemicals). It was transcribed with M-MLV reverse transcriptase
(Promega) at 37°C for 1 h and PCR products were analyzed on
2% agarose gels with ethidium bromide. For quantitative analysis, PCR products were collected and analyzed every 4 cycles.
Other PCR reactions were also amplified in the linear range by
empirically determining the appropriate number of PCR cycles
for each primer set. The PCR primers specific for XFGF-8b
were (forward) 5′-ACTTTCTACTCCTCCCCGTCTC-3′ and (reverse) 5′-CTCCCTCACATGCTGTGTAAAA-3′ (320 bp), and
the PCR primers that could distinguish XFGF-8a and XFGF-8b
were (forward) 5′-GCTGGTGCTACTGGGCTAAT-3′ and (reverse) 5′-CCTACCAGTTGTACAGCCGG-3′ (product of 205 bp
for XFGF8-a and of 238 bp for XFGF-8b) (Fig. 2B). Primers for
ornithine decarboxylase (ODC) (Bouwmeester et al., 1996),
EF1-α (Kengaku and Okamoto, 1995), OtxA (Lamb et al., 1993),
FoxD3 (Monsoro-Burq et al., 2003), Sox9 (Monsoro-Burq et al.,
2003), N-tubulin (http://www.xenbase.org) and muscle actin
(http://www.xenbase.org) were described previously.
Results
Isolation of XFGF-8b and comparison with XFGF-8a
We isolated a Xenopus homologue of FGF-8b (XFGF-8b)
by screening a stage 10 Xenopus cDNA library with a
partial fragment of Xenopus FGF-8a (XFGF-8a) cDNA as
probe. The full-length XFGF-8b (GenBank accession No.
AF461177) cDNA consisted of a 5′ untranslated region
(UTR) of 702 bp, an open reading frame (ORF) of 633 bp
and a 3′ UTR of 857 bp. The ORF encodes a protein of
211 amino acids with an insert of 11 amino acids in the Nterminal region compared to XFGF-8a (94.8% nucleotide
identity; Christen and Slack, 1997). It has 81.4, 81.4, 85.5,
82.5, and 89.2% identity at the amino acid level with its
counterparts in the mouse (Tanaka et al., 1992), human
(Gemel et al., 1996), chick (Crossely et al., 1996), zebrafish (Furthauer et al., 1997) and axolotl (Han et al., 2001),
respectively (Fig. 1). XFGF-8b contains a classical hydrophobic signal sequence suggestive of a secreted protein at its N-terminus also observed in other vertebrate
FGF-8s (Fig. 1). A cleavage site was predicted between
positions 22 and 23 of XFGF-8a (Leu-Gln-Ala ↓ Gln-Val)
and of XFGF-8b (Leu-Gln-Ala ↓ Gln-His) using the SignalP program. XFGF-8b contains two potential consensus
sequences for N(Asn)-linked glycosylation (NFT and
NYT at amino acid 31−33 and 137−139, respectively),
whereas XFGF-8a has only the latter (Fig. 1).
Expression pattern of XFGF-8b The spatial and tempo-
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Comparison of XFGF-8b with XFGF-8a
Fig. 1. Alignment of the predicted amino acid sequences of Xenopus FGF-8b with its homologues in mouse, human, chick, zebrafish
and axolotl. Dashes represent gaps inserted to maximize alignment, and identical and similar amino acids are shaded black and gray,
respectively. The predicted signal peptide and cleavage site are indicated by arrows and dotted arrows, respectively. NFT and NYT
above Asterisks mark the position of the N-glycosylation consensus sites. A motif conserved in most members of vertebrate FGFs [Gx-L-(x)9~10-C-x-F-x-E-(x)6-Y] is indicated by the heavy line.
ral expression pattern of XFGF-8b during early Xenopus
development was examined by whole-mount in situ
hybridization (Fig. 2). Since a full-length XFGF-8b
cDNA fragment was used to make the in situ probe and
the sequences of this probe largely overlapped with
XFGF-8a, we suspect that this XFer GF-8b expression
also includes XFGF-8a expression (Christen and Slack
1997, see Discussion). XFGF-8b mRNA was first detected
in the marginal zone of the pre-gastrula embryo and there
was a higher level of this mRNA on the dorsal side of the
embryo (Fig. 2B). As gastrulation proceeded, its circumblastoporal expression became more intense and
spread along the dorso-ventral axis (Figs. 2C and 2D).
During neurulation, this peri-blastoporal expression became localized to the anterior and posterior ends of the
body axis (Figs. 2E−2H). At the neural fold stage (stage
18), expression in the anterior neural region (Fig. 2F; data
not shown) became further restricted to three distinct domains comprising the two stripes of the future midbrain/hindbrain boundary (MHB), the anterior neural
ridge that is fated to form forebrain, and an epidermal
crescent-shaped region demarcating the outside of the
neural plate that forms a part of the gill (Figs. 2G and 2H).
These anterior expression domains persisted until the tailbud stage (stage 32) in the telencephalon, anterior and
posterior diencephalon, the midbrain/hindbrain boundary
(MHB), the branchial cleft and ear vesicle in addition to
the pronephros, and the tip of the tail bud (Fig. 2J). In the
larval stages, XFGF-8b was expressed in the apical ectodermal ridge (AER) of the developing hind limb buds
(Figs. 2K and 2L). When digits formed in the hind limb
buds, the XFGF-8b signal was restricted to the tips of the
growing digits (Fig. 2M).
We examined the temporal expression of XFGF-8b using the reverse transcriptase polymerase chain reaction
(RT-PCR) (Fig. 3A) and primers specific for the XFGF-8b
isoform (Fig. 3B). XFGF-8b transcripts were not present
in unfertilized eggs but they were expressed at a relatively
constant level throughout the early embryonic stages (Fig.
3A).
In order to further compare the expression levels of the
XFGF-8 isoforms in Xenopus, we designed a new primer
set to distinguish the isoforms by PCR (Fig. 3B) and used
it for quantitative RT-PCR on mRNAs isolated from stage
Sangwoo Shim et al.
A
B
C
D
A
E
F
G
H
B
I
K
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J
L
M
Fig. 2. Spatial expression of XFGF-8b (or -8b) during early
development analyzed by whole-mount in situ hybridization. A.
Blastula embryo (stage 8) with animal and vegetal views of the
same embryo (inset). B-D. Vegetal views of gastrula stage embryos (B, stage 10; C, stage 11; D, stage 11.5); dorsal side at top.
E-F. Mid-neurula embryo (stage 13) in posterior (E) and dorsal
(F) views. G-H. Neural fold stage embryo (stage 18) in anterior
(G) and dorsal (H) views. Anterior neural expression of XFGF8b is restricted to three distinct domains containing the prospective midbrain/hindbrain boundary (MHB) as two stripes (black
arrowheads), the anterior ridge of the neural plate as future
forebrain (black arrow), and an epidermal crescent-shaped region outside the neural plate (white arrow). I-J. Stage 32 embryo in lateral view (I) and a close-up view (J) of the head region of the same embryo. Anterior expression domains persist
into the telencephalon and anterior diencephalon (arrow head),
the posterior diencephalon (di), the midbrain/hindbrain boundary (MHB) (arrow), the branchial cleft (bc) and ear vesicle (e) in
addition to the pronephros (p) and the tip of the tail bud (tb). KM. Expression of XFGF-8b in the developing limbs of larval
stage Xenopus embryo. At stage 49 (K) and stage 51 (L), XFGF8b expression is seen in the apical ectodermal ridges (AER) of
the developing hindlimbs. At stage 54 (M), XFGF-8b is only
expressed in the growing tips of digits (arrows). Scale bars = 0.5
mm. D, dorsal; V, ventral; A, anterior; P, posterior.
26 embryos and tadpoles (stage 42). Since PCR was performed using the same primer pairs, quantitative difference in the amount of PCR products before saturation
should reflect quantitative difference in the amount of
mRNA present in the embryos. As shown in Fig. 3C, both
PCR products, the 238 bp product for XFGF-8b and the
205 bp product for XFGF-8a, were measured in the exponential phase of amplification up to PCR cycle 38, and the
C
D
Fig. 3. Expression of XFGF8a and XFGF8b analyzed by RTPCR. A. RT-PCR analysis of XFGF-8b expression during early
Xenopus development. ODC expression was used as a loading
control. B. Part of the cDNA and amino acid sequences showing
the differences between XFGF-8a and XFGF-8b, and the primer
sets used for RT-PCR. XFGF-8a lacks the boxed region. The
XFGF-8b-specific reverse PCR primer used in (A) is underlined
and shown by a dotted arrow, and the primers that can distinguish XFGF-8a and XFGF-8b and are used in C and D are underlined and indicated by arrows. Expected lengths of PCR
products for XFGF-8a and XFGF-8b are 205 and 238 bp, respectively. C. Comparison of the expression levels of XFGF8a
and XFGF8b by quantitative RT-PCR. The number above each
lane indicates the number of PCR cycles used. Lane M contains
DNA size markers. D. Comparison by RT-PCR of the expression
of XFGF-8a and XFGF-8b in anterior (A) and posterior (P)
halves dissected from stage 13 and stage 18 whole embryos (W).
EF-1α expression was used as loading control, and -RT indicates PCR of control embryos processed without reverse transcriptase to assay genomic contamination.
level of FGF-8b expression was much higher than that of
FGF-8a in both stages. No other isoforms were detected,
consistent with the absence of other isoform-specific sequences in the Xenopus EST database. The presence of
314
Comparison of XFGF-8b with XFGF-8a
A
B
Fig. 4. Absence of XFGF-8 isoform-switching by retinoic acid (RA) during early Xenopus embryogenesis. Retinoic acid treatment of
the dorsal marginal zone (DMZ) (A) and whole embryos (B) at the early gastrula stage are schematically represented, and the expression levels of the XFGF-8 isoforms are compared by RT-PCR. The effectiveness of the retinoic acid was confirmed by the reduction or
loss of the anterior neural marker OtxA. EF-1α expression was used as loading control. Lane M contains DNA size markers.
only two isoforms, and the predominant expression of
FGF-8b have also been observed in the chick (Sato et al.,
2001).
As shown in Figs. 2E−2H (see also Christen and Slack,
1998), expression of the XFGF-8 forms during the neurula stage was detected in domains located at the anterior
and posterior ends of the embryo. We tested whether the
expression of the two XFGF-8 isoforms was restricted
along this anteroposterior axis by dissecting mid-neurula
(stage 13) and late-neurula embryos (stage 18) into anterior and posterior halves, and assessing the levels of expression by RT-PCR. As shown in Fig. 3D, XFGF-8 isoforms were expressed in both halves and the higher level
of FGF-8b expression was seen in both regions and in
both stages. Taken together, these results demonstrate that
both transcripts are represented in regions where any
XFGF-8 expression is detected and that a higher level of
XFGF-8b expression is maintained both temporally and
spatially.
Absence of regulation of the XFGF-8 isoforms by retinoic acid Retinoic acid has been shown to differentially
regulate the FGF-8 isoforms in a prostate cancer cell line
(Brondani and Hamy 2000; Brondani et al., 2002). According to the proposed model (Brondani et al., 2002), in
the absence of retinoids, unliganded, phosphorylated retinoic acid receptor α (RARα) homodimers bind to their
unusual conformational binding sites and transactivate the
expression of the FGF-8b isoform. Upon addition of retinoids, on the other hand, liganded-RARα is no longer
able to transactivate FGF-8b expression, which is there-
fore shut down. At the same time RARα, either as a
liganded homodimer or as heterodimer with ligandedretinoic X receptor α (RXRα) binds to a known consensus retinoic acid response element (RARE) in the promoter and induces the expression of FGF-8a. We therefore tested whether retinoic acid affect the expression of
the XFGF-8 isoforms in Xenopus embryos. We explanted
dorsal marginal zones (DMZ) of early gastrula embryos
(stage 10.5), cultured in medium containing retinoic acid
(10−7 or 10−4 M) up to the neurula stage (stage 15) and
compared expression of the two isoforms (Fig. 4A). As
shown in Fig. 4A, no significant change in expression of
XFGF-8a or XFGF-8b was observed in response to retinoic acid treatment. We next treated whole embryos with
retinoic acid (10−6 or 10−5 M) from the early gastrula
(stage 10) to the late neurula (stage 17) stage, but the expression of XFGF-8 was again not affected (Figs. 4A and
4B). The efficacy of the retinoic acid on the embryos was
confirmed by the reduction or loss of the anterior neural
marker OtxA (Figs. 4A and 4B). The Xenopus homologues of RARα and RXRα that might have been involved in isoform-switching of XFGF-8 have been shown
to be maternally expressed in Xenopus (Blumberg et al.,
1992). Thus, isoform-switching of XFGF-8 by retinoic
acid does not appear to occur in early Xenopus embryogenesis, in contrast to the recent report in mammalian
cells.
Comparison of the effects of XFGF-8a and XFGF-8b
To compare the biological activities of the two XFGF-8
isoforms, we expressed them ectopically and analyzed the
Sangwoo Shim et al.
A
B
C
315
microinjected embryos. We first tested the neuronal inducing activities of the two isoforms (Chae et al., 2004).
We injected XFGF-8a and XFGF-8b mRNAs into one cell
of two-cell stage embryos and analyzed expression of Ntubulin, an early neuronal marker, by whole mount in situ
hybridization. As shown in Fig. 5A, both XFGF-8a and
XFGF-8b overexpression induced abundant ectopic Ntubulin that expanded into the non-injected region as well
as the ventral region of the embryos, consistent with previous result with XFGF-8b (Hardcastle et al., 2000). Next,
we examined neural crest induction. As previously reported, we injected mRNAs of the XFGF-8 isoforms into
the animal pole region of two-cell stage embryos, explanted animal caps at stage 8, incubated them until stage
15 and assayed for neural crest markers such as FoxD3
and Sox9 (Monsoro-Burq et al., 2003). RT-PCR analysis
(Fig. 5B) showed that FoxD3 and Sox9 were strongly induced in the XFGF-8a-injected animal caps whereas
XFGF-8b injection had little effect. This differential effect on neural crest markers was confirmed in two further
experiments as shown in Fig. 5C. We also confirmed that
both XFGF-8a and XFGF-8b induced ectopic neurogenesis as shown by N-tubulin expression without inducing
mesoderm as defined by muscle actin expression (Fig.
5B). These results suggest that XFGF-8a and XFGF-8b
may have redundant activities as neural inducers, whereas
only XFGF-8a is involved in neural crest induction.
Discussion
Fig. 5. Differential effects of XFGF-8a and XFGF-8b. A. Abundant ectopic induction of N-tubulin by both XFGF-8a and
XFGF-8b. One cell of two-cell stage embryos was injected with
XFGF-8a or XFGF-8b mRNAs (100 pg), fixed at stage 18-20
and analyzed for N-tubulin by whole mount in situ hybridization.
The right side was the injected side as determined by coinjection of GFP mRNA. Note that ectopic N-tubulin expression
expanded into the non-injected and ventral regions (bottom
panel). B. Induction of neural crest markers by XFGF-8a but not
by XFGF-8b. XFGF-8 isoform mRNAs (500 pg) were injected
into the animal pole region of two-cell stage embryos. Animal
caps were explanted at stage 8, incubated until stage 15 and
assayed for neural crest markers, FoxD3 and Sox9, by RT-PCR.
-RT indicates PCR of negative control embryos from stage 15
processed without reverse transcriptase. EF-1α was used as an
internal control. C. RT-PCR products indicative of neural crest
induction were quantified and their statistical significance was
assessed using ANOVA and the Newman-Keuls multiple comparison test. * = p < 0.05 vs. control AC. Values are means of
three independent experiments ± S.D.
In this report, we have described the cloning and expression of Xenopus homologues of FGF-8b. XFGF-8b was
expressed in anterior neural regions including prospective
forebrain, midbrain-hindbrain boundary (MHB) and branchial cleft as well as in the apical ectodermal ridge of limb
buds and the tips of growing digits in larval stages. This
localized expression of XFGF-8b did not differ much from
that of XFGF-8a (Christen and Slack, 1997). This may be
attributed to the lack of specificity of the two in situ probes
(the PstI fragment of XFGF-8a cDNA corresponding to
amino acids 1 to 130 for XFGF-8a (Christen and Slack,
1997) and the full-length coding sequence for XFGF-8b;
this study) that do not absolutely discriminate between the
two FGF-8 isoforms that have the same nu- cleotide sequence except for just 33 nucleotides. However, RT-PCR
analysis revealed the presence of the two isoforms in domains in the anterior and posterior halves of embryos at the
neurula stage (Fig. 3D). In addition, no major difference in
the spatial or temporal expression of the FGF-8 isoforms
was observed in the mouse in isoform-specific analysis by
in situ hybridization and immunohistochemistry (MacArthur et al., 1995b). The expression patterns of the XFGF-8
transcripts also overlap considerably with those of chick
(Sato et al., 2001; Vogel et al., 1996; Wall and Hogan,
316
Comparison of XFGF-8b with XFGF-8a
1995) and mouse (Crossley and Martin, 1995), pointing to
functional conservation of the FGF-8 family members in
vertebrate species, as well as functional redundancy of the
FGF-8 isoforms. We further revealed that XFGF-8b is more
abundantly expressed than XFGF-8a throughout early
Xenopus development, as also observed in the chick isthmus (Sato et al., 2001).
Accumulating evidence suggests that the FGF-8 isoforms have differential activities. Both in mouse and
chick (Lee et al., 1997; Liu et al., 1999; MacArthur et al.,
1995a), FGF-8b had stronger transforming activities and
more severe phenotypic defects than FGF-8a, and this can
be accounted for by the difference in the strength of their
signals (Sato et al., 2001). The alternative possibility that
there is a qualitative difference between the isoforms is
suggested by the fact that in a recent study XFGF-8a
proved to be a neural crest inducer (Monsoro-Burq et al.,
2003), while in another study, a putative XFGF-8b had
potent activity as a neuronal inducer (Hardcastle et al.,
2000). In the present study, we demonstrated that both
XFGF-8a and XFGF-8b induce ectopic neurogenesis,
whereas XFGF-8a seems to be mainly involved in neural
crest induction. It was previously shown that the effect of
XFGF-8a on neural crest induction is mediated by FGF
receptor type-1 (XFGFR1) but not by XFGFR-4a (Monsoro-Burq et al., 2003), whereas the effect of XFGF-8b on
neuronal differentiation is mediated mainly by FGFR-4a
but not as much by FGFR-1 (Hardcastle et al., 2000). Further analysis of ligand-receptor binding and activation
involving the XFGF-8 isoforms and XFGF receptors will
be required to elucidate the role of the isoforms in development.
The only difference in primary structure between
XFGF-8a and XFGF-8b is the insertion of a stretch of 11
amino acids that lies between the signal sequence and the
start of the conserved core polypeptide (Fig. 1). The insertion of this additional motif seems to enhance the average
hydrophobicity of the signal peptide (data not shown;
SOSUI signal program), and to produce a different Nterminally processed core polypeptide and to add an additional N-glycosylation site, NFT, to the secreted XFGF-8b
polypeptide (Fig. 1). The significance of these effects
remains unclear, but they may be related to the apparently
different activities of the XFGF-8 isoforms in vivo. As for
N-linked glycosylation, it has been shown to be important
in modulating receptor binding affinity and FGF activity,
on its own or by modulating N-terminal processing so as
to shift the conformation from a form with a lower affinity for its receptor to one with a higher affinity (Antoine
et al., 2001; Bellosta et al., 1993). In this connection, Nlinked glycosylation substantially reduces the activity of
FGF-2 (Duraisamy et al., 2001) and FGF-4 (Bellosta et
al., 1993) whereas it increases FGF-6 activity (Asada et
al., 1999). XFGF-8 should provide a useful experimental
model for investigating whether changes in glycosylation
can affect receptor specificity.
Acknowledgments We would like to thank Dr. Michael W.
King for providing the Xenopus embryo stage 10 cDNA library.
We also thank Dr. Jung-Ha Lee of Sogang University, and SunCheol Choi, Jae-Young Chang and other members of our laboratory for technical assistances and helpful comments. This research was supported by grants from the interdisciplinary research program of Korea Science and Engineering Foundation
(KOSEF) (R01-1999-00092 and 1999-1-207-002-3), the Advanced Basic Research Laboratory Program (R14-2002-01201001-0) of KOSEF, and the Brain Korea 21 Project.
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