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J Biol Chem, Vol. 275, Issue 13, 9581-9586, March 31, 2000
From the Terry Fox Cancer Research Laboratories, Division of Basic
Medical Sciences, Faculty of Medicine, Memorial University of
Newfoundland, St. John's, Newfoundland A1B 3V6, Canada
Previously, we cloned a variant form
of the type 1 fibroblast growth factor receptor (FGFR1), FGFR-VT Fibroblast growth factors
(FGFs)1 represent a family of
related polypeptides known to stimulate a variety of cellular
activities (reviewed in Ref. 1), including mesoderm differentiation in the Xenopus embryo (2). Their effects are mediated by high affinity transmembrane FGF receptors (FGFRs) containing intrinsic tyrosine kinase activity (reviewed in Ref. 3). Like other receptor tyrosine kinases, FGFR signal transduction is initiated by ligand binding and results in activation of several well characterized intracellular signaling pathways, including the protein kinase C (PKC)
and Ras/mitogen-activated protein kinase pathways (4, 5).
Four FGFR genes have been described to date, FGFR1-FGFR4, along with a
number of alternately spliced variants (reviewed in Refs. 3 and 6).
Previously, we cloned from Xenopus embryos an alternately
spliced isoform of the FGFR1 that contains a deletion of
Val423-Thr424 in the juxtamembrane region (7).
We demonstrated that this site could be phosphorylated by PKC.
Furthermore, in a functional assay, activation of PKC by the phorbol
ester phorbol 12-myristate 13-acetate significantly reduced the
activity of the VT-containing isoform (VT+) in Xenopus
oocytes while having little effect on the deletion isoform (VT In the blastula stage Xenopus embryo, cells located in the
equatorial region (marginal zone) are induced to differentiate into
mesoderm in response to signal(s) from neighboring vegetal cells
(reviewed in Refs. 8 and 9). Use of a dominant negative form of the
FGFR1 to block FGF activity has provided evidence that FGF/FGFR
signaling is required for normal development of the mesoderm (10). The
current view is that FGF does not act as the initial inducing signal(s)
but rather as a competence factor in the responding cells and that its
activity is required for the full range of responses leading to
mesoderm formation (9, 11). In this report, we examine the role of the
VT+ and VT Embryos and Microinjections--
Xenopus laevis were
purchased from Nasco. Eggs were artificially inseminated, and embryos
were cultured as described under Godsave et al. (12);
embryonic stages were determined according to Nieuwkoop and Faber (13).
Stage 8 blastulae were dissected into animal, vegetal, and marginal
zone regions as described (14). cRNA was transcribed from FGFRSP64T
constructs (7) using the SP6 Ribomax system (Promega). 4.6 nl
containing diethyl pyrocarbonate-treated H2O or 650 pg/nl
cRNA was microinjected into stage 1 embryos. Embryos were cultured at
room temperature until they reached the stage required for the assays
described below.
RNase Protection and Reverse Transcription-Polymerase Chain
Reaction (RT-PCR)--
Probe preparation, RNA extraction, and RNase
protection analysis were performed as described (7). RT-PCR
analysis was performed as described in Ref. 15 using forward
(5'-GGGCTGCTTTTGTGTCCGCAAT-3') and reverse
(5'-CATTGATGAGCTGGAGTCCCCTG-3') primers that bracket the VT region and
generate 156- and 162-bp fragments for the FGFR-VT Protein Analysis--
For expression analysis of injected FGFR
cRNA, 0.5 µCi of [35S]methionine was co-injected into
each embryo. Protein extraction, immunoprecipitation, and
SDS-polyacrylamide gel electrophoresis analysis were performed as in
Ref. 18. The anti-Xenopus FGFR1 used for immunoprecipitation
was a polyclonal antibody raised against a synthetic C-terminal
peptide (18).
Mesoderm Induction Assays--
Recombinant Xenopus
FGF-2 was expressed and purified according to Kimelman et
al. (19). Animal cap explants were excised from injected embryos
and treated with FGF-2 as described (18). Explants were cultured for
various times then extracted for RNA analysis or scored for mesoderm
induction as in Ref 2.
Whole Mount in Situ Hybridization--
Whole mount in
situ hybridization with digoxygenin (Roche Molecular
Biochemicals)-labeled Xenopus Brachyury (Xbra) or
Xenopus chordin cRNA was performed as described (20), using
maleic acid buffer. The cDNAs for Xenopus Brachyury and
chordin were kindly provided by Dr. R. T. Moon
(University of Washington).
Previously, we demonstrated that the activity of the VT+ and VT Embryos were dissected into three regions representing the three germ
layers: animal (presumptive ectoderm), marginal zone (presumptive
mesoderm), and vegetal (presumptive endoderm). RNase protection assays
of these three regions revealed that the VT Although we had previously reported that the VT+ isoform was the major
form expressed throughout development and that no change in the ratio
of VT+ to VT Our previous work demonstrated that these two FGFR1 isoforms arise by
alternate use of a 5' splice donor site during transcription (7). In
Xenopus embryos, however, zygotic transcription does not
begin until midblastula transition (21). This occurs during stage 8, but the precise timing of this developmental event cannot be determined
by either the number of cell divisions or the time after fertilization
(Ref. 21; reviewed in Ref. 22). Instead, an increase in elongation
factor 1- We investigated the possibility that FGF itself was involved in this
switch in expression pattern, since Musci et al. (24) and
Friesel and Dawid (25) have reported that FGFR1 mRNA levels in
animal cap explants were regulated by FGF. We cultured blastula stage
animal cap explants with FGF-2 in a standard mesoderm induction assay
(2) and determined VT+ and VT
The VT+ and VT
Isoforms of the Fibroblast Growth Factor
Receptor Type 1 Are Differentially Expressed in the Presumptive
Mesoderm of Xenopus Embryos and Differ in Their Ability to
Mediate Mesoderm Formation*
ABSTRACT
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INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
,
from Xenopus embryos (Gillespie, L. L., Chen, G., and
Paterno, G. D. (1995) J. Biol. Chem. 270, 22758-22763). This isoform differed from the reported FGFR1 sequence
(FGFR-VT+) by a 2-amino acid deletion,
Val423-Thr424, in the juxtamembrane region.
This deletion arises from the use of an alternate 5' splice donor site,
and the activity of the VT+ and VT forms of the FGFR1 was regulated
by phosphorylation at this site. We have now investigated the
expression pattern and function of these two isoforms in mesoderm
formation in Xenopus embryos. Cells within the marginal
zone are induced to form mesoderm during blastula stages. RNase
protection analysis of blastula stage embryos revealed that the VT+
isoform was expressed throughout the embryo but that the VT isoform
was expressed almost exclusively in the marginal zone. The ratio of
VT+:VT transcripts in the marginal zone indicated that the VT+ form
was predominant throughout blastula stages except for a brief interval,
coinciding with the start of zygotic transcription, when a dramatic
increase in VT expression levels was detected. This increase could be
mimicked in part by treatment of animal cap explants with FGF-2.
Overexpression of the VT+ isoform in Xenopus embryos
resulted in development of tadpoles with severe reductions in trunk and
tail structures, while embryos overexpressing the VT isoform
developed normally. A standard mesoderm induction assay revealed that a
10-fold higher concentration of FGF-2 was required to reach 50%
induction in VT+-overexpressing animal cap explants compared with those
overexpressing the VT isoform. Furthermore, little or no expression
of the panmesodermal marker Brachyury (Xbra)
was detected in VT+-overexpressing embryos, while VT-overexpressing
embryos showed normal staining. This demonstrates that VT+
overexpression had a negative effect on mesoderm formation in
vivo. These data are consistent with a model in which mesoderm
formation in vivo is regulated, at least in part, by the
relative expression levels of the VT+ and VT isoforms.
INTRODUCTION
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ABSTRACT
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EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
). We
speculated that differential expression of these two isoforms might
represent an important mechanism for regulating FGFR activity in the
Xenopus embryo (7).
isoforms in mesoderm formation in Xenopus and
show that the two isoforms are differentially expressed in the
presumptive mesoderm and that they differ in their ability to mediate
mesoderm formation in vitro and in vivo.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
and FGFR-VT+ gene
products, respectively. Histone H4 was used as an input control with
forward and reverse primers as described (16). EF1
was amplified
using 5'-CCTGAATCACCCAGGCCAGATTGGTG-3' and
5'-GAGGGTAGTCTGAGAAGCTCTCCACG-3', as forward and reverse primers, respectively. The [32P]CTP-labeled PCR products were
analyzed in the linear range for amplification, determined empirically
(16) to be 19 cycles for histone H4, 22 cycles for EF1, and 25 cycles for FGFR, and visualized on a 6% polyacrylamide/6 M
urea gel by autoradiography. Quantitation by densitometry was performed
as in Ref. 17.
RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
isoforms could be differentially regulated by phosphorylation (7). One
obvious question is whether these two isoforms function differently in
mesoderm formation in Xenopus embryos. Induction to form
mesoderm takes place in the marginal zone cells of the blastula stage
embryo, so we began by examining the spatial expression pattern of the
VT+ and VT isoforms during this stage of development.
isoform was expressed
predominantly in the marginal zone with very little detectable message
in the animal and vegetal regions (Fig.
1B). In contrast, only small
differences in VT+ expression were observed in the three regions (Fig.
1B).
View larger version (30K):
[in a new window]
Fig. 1.
FGFR-VT expression is spatially and
temporally restricted during blastula stage. A,
schematic illustrating the 261-nt probe that corresponds to the
sequence of the VT+ isoform as well as the VT+ and VT protected
fragments produced by RNase digestion for the analysis shown in
B. Digestion of probe:VT+ hybrids results in a 162-nt
protected fragment, while digestion of probe:VT hybrids results in
digestion of the 6-nt single strand loop encoding the VT
(black box), producing two protected fragments of
107 and 49 nt. The size in nt is listed below each fragment.
B, RNase protection analysis of FGFR-VT+ and FGFR-VT
mRNA in Xenopus blastulae. Stage 8 blastulae were
dissected into animal, vegetal, and marginal zone regions (as
illustrated in the schematic diagram shown above
lanes 5-7), as described (14). Total RNA was
isolated from each region, and RNase protection analysis was performed
using a 32P-labeled 261-nt probe, as in Ref. 7. A
representative experiment is shown. Lane 1, probe;
lane 2, digested probe; lanes 3-7, protected
fragments from in vitro transcribed FGFR-VT+ cRNA, in
vitro transcribed FGFR-VT cRNA, marginal zone cells
(M), animal cells (A), and vegetal cells
(V), respectively. The positions of the undigested probe
(arrow), the VT+ protected fragments (square
brackets), and VT 107-nt protected fragment
(arrow) are indicated; the 49-nt VT protected fragment is
not shown. C, RT-PCR analysis of the VT+ and VT temporal
expression pattern in marginal zone cells. Blastula stage embryos were
collected at the following postfertilization times: 4.5 h (stage
7), 5.0 h (stage 8), 5.5 h (stage 8), and 6.0 h (stage
8). Marginal zones were dissected, and total RNA was isolated as in
B. RT-PCR was performed as described under "Experimental
Procedures," and the VT+ and VT products, which differ in size by 6 nt, were analyzed by autoradiography on a 6% polyacrylamide, 6 M urea sequencing gel. A representative experiment is
shown. Amplification products of VT+ cDNA (lane 1) and
VT cDNA (lane 2) mark the position of the VT+ and VT
products (arrows) representing the two isoforms in the
marginal zone cells (lanes 3-6). Each marginal zone sample
was also amplified using primers for histone (H4), as an input control,
and for elongation factor 1-, as a measure of zygotic transcription.
Dev Time, development time.
isoforms was detected (7), the time intervals used in
that study were large in order to cover a broad range of developmental
stages. In light of the results in Fig. 1B, we decided to
reexamine the temporal VT+ and VT expression patterns in the marginal
zone, using shorter time intervals and focusing on the stages when
mesoderm induction is known to take place. Blastula stage embryos were
collected at 0.5-h time intervals, and the VT+ and VT expression
levels in the marginal zone were analyzed by RT-PCR. For this purpose,
we employed primers that bracket the VT region and generate VT+ and
VT products that can be distinguished on a sequencing gel. Our
analysis revealed that in early blastulae (4.5 h postfertilization;
late stage 7), the VT+ isoform was the major form in marginal zone
cells (Fig. 1C, lane 3), consistent
with our previous findings (7). However, 30 min later (stage 8), a
dramatic increase in the level of VT relative to VT+ was observed,
such that the VT isoform became predominant (Fig. 1C,
lane 4). This was quickly followed by a decrease
in VT expression to initial levels, with the VT+ isoform remaining
predominant at all subsequent time points examined (Fig. 1C,
lanes 5 and 6).
expression, one of the earliest transcripts to be
expressed by the embryonic genome (23), has been frequently used to
indicate that zygotic transcription has begun. We measured elongation
factor 1- levels in our marginal zone samples and determined that
expression levels began to increase as early as 5 h (Fig.
1C, lane 4). This demonstrates that
the increase in VT expression takes place concurrent with the onset of zygotic transcription.
expression levels at various times
after the addition of FGF-2. VT expression levels in FGF-2-treated explants increased within 0.5 h, compared with untreated control explants (Fig. 2, lanes
1 and 2). At all subsequent time intervals tested, VT expression levels in FGF-treated explants remained the
same as control levels (Fig. 2, lanes 3-8). VT+
expression levels, on the other hand, remained relatively constant
throughout. These data demonstrate that a temporary increase in the
expression level of the VT isoform can be triggered by FGF. It is
clear, however, that FGF induction alone cannot account for the large increase in VT levels observed in vivo (compare Fig.
1C, lane 4, with Fig. 2,
lane 2). The additional factor(s) responsible for
the increase in VT expression in the marginal zone remain to be
determined.
View larger version (34K):
[in a new window]
Fig. 2.
FGF can stimulate an increase in VT
expression. Animal cap explants were cultured with (+) or without
() 100 ng/ml FGF-2, and at the time indicated above each
lane, RNA was extracted. RT-PCR analysis was performed as in
Fig. 1C. The experiment was performed on four separate
occasions, and the increase in VT expression ranged from 2- to
3-fold. A representative experiment is shown. The positions of the VT+
and VT isoforms as well as the H4 input control are indicated.
The rapid and transient switch to VT expression in the presumptive
mesoderm suggests that these two FGFR1 isoforms have distinct roles in
early development. We investigated this possibility by examining the
effects of overexpressing each isoform. cRNA was microinjected into
fertilized eggs, and FGFR-injected embryos were compared with control
H2O-injected embryos for their ability to develop into
normal tadpoles. While embryos
overexpressing the VT isoform developed normally (Figs. 3A
and 4C), less than 10% of
those overexpressing the VT+ isoform developed into normal tadpoles
(Figs. 3A and 4B). VT+-injected embryos began to
develop abnormally at 10-12 h postinjection: gastrulation was
incomplete, leaving an enlarged blastopore with protruding yolk plug
(not shown). Despite this, embryos continued to develop, albeit
abnormally. The obvious effect was a reduction in trunk and tail
structures in the resulting tadpoles (Fig. 4B). This
differential effect was not due to differential stability or
translation of the cRNAs, since equivalent levels of VT+ and VT RNA
(Fig. 3B) and protein (Fig. 3C) were detectable
at 24 h, long past the stage when abnormalities first became
apparent in the VT+-overexpressing embryos.
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The VT+ abnormalities were similar to those reported by Amaya et
al. (10) in embryos overexpressing a dominant negative FGFR1
(XFD). These authors showed that XFD inhibited endogenous FGFR
signaling and that overexpression in embryos caused severe posterior
truncations resulting from a reduction in posterior mesoderm
development, as well as deficiencies in gastrulation movements. This
similarity suggested that the VT+ phenotype may result from a
deficiency in mesoderm formation. To test this, we investigated the
effect of VT+ and VT overexpression on mesoderm formation in
vitro and in vivo.
First, we measured the FGF-2 dose-response curve in explants from
embryos microinjected with either VT+ or VT cRNA and compared it with
the curve for explants from H2O-injected embryos. Our results demonstrate that overexpression of the VT+ isoform reduced the
level of mesoderm induction by FGF-2; overexpressing explants required
a 2-fold higher concentration of FGF than control explants to achieve
50% induction (Fig. 5A).
Explants overexpressing the VT isoform, on the other hand, reached
50% induction at a 5-fold lower concentration than control explants
(Fig. 5A). Thus, overexpression of VT+ decreased sensitivity
to FGF, while overexpression of VT dramatically increased
sensitivity. Examination of the VT and VT+ expression levels in these
explants revealed that the sensitivity to FGF was directly correlated
with the relative expression levels of the two isoforms (Fig.
5B). The ratio of VT to VT+ in explants from VT+-injected
embryos was half that of control explants (Fig. 5B,
lanes 1 and 3), as was the proportion
of induced VT+ explants at virtually every concentration of FGF tested
(Fig. 5A). Explants from VT-injected embryos, on the other
hand, had the highest ratio of VT to VT+ (Fig. 5B,
lane 5) and the highest sensitivity to FGF (Fig.
5A). In fact, 30% of the latter were induced in the absence
of added FGF-2 (Fig. 5A). This autoinduction may result from
interaction of overexpressed VT with maternally derived FGF present
in animal cap explants (9).
|
), VT
), or VT+-injected
(
) embryos were cultured in the presence of the indicated
concentration of FGF-2 for 72 h. Mesoderm induction was scored by
morphological criteria as described in Ref. From the results of the dose-response curves, one would predict that
overexpressing the VT+ isoform would result in a decreased level of
mesoderm formation in vivo. To test this hypothesis, we
examined the expression of an early panmesodermal marker
Xbra, which is normally expressed throughout the presumptive
mesoderm of the early gastrula stage embryo (26). Furthermore, FGFR
signaling is required for Xbra expression (reviewed in Ref.
27). Staining for Xbra was not detectable or was very faint
in VT+-injected embryos (Fig.
6A). VT-injected embryos, on
the other hand, expressed levels similar to those of
H2O-injected controls (Fig. 6A). Expression of
chordin, a dorsal lip marker involved in neural development (28, 29), was unaffected in VT+ embryos (Fig. 6B),
demonstrating that lack of Xbra expression in VT+-injected
embryos was not the result of a nonspecific inhibition of
transcription. Thus, the VT+ isoform can function to negatively
regulate mesoderm formation in Xenopus embryos.
|
In this study, we have shown that while the VT+ isoform was predominant
in the presumptive mesoderm during most of blastula stages, a brief but
dramatic increase in VT mRNA expression occurred during this time
period (Fig. 1C), coinciding temporally with mesoderm
induction in vivo (9). It would be important to determine how this transient burst in VT expression affects known FGF signaling pathways in the embryo, such as mitogen-activated protein kinase and
phospholipase C
. Labonne and Whitman (30) reported that mitogen-activated protein kinase activity was first detectable during
midblastula and peaked by midgastrula. Phosphorylation of phospholipase
C, on the other hand, occurred over a period of 1.5 h during
early blastula to midblastula stages (18). While activation of these
pathways persisted over a longer time interval than that reported in
this study, it is possible that the VT protein has a longer half-life
than its cognate mRNA. We are attempting to generate antibodies
that can distinguish between the VT+ and VT isoforms and would enable
investigation of isoform-specific signaling pathways.
We have also shown that the VT+ and VT isoforms differ significantly
in their ability to mediate mesoderm induction. This difference in
isoform function could be due to PKC activity. PKC is known to be
activated during mesoderm induction by FGF (4), and activated PKC
caused a substantial reduction in FGF signaling through the VT+, but
not the VT, isoform (7). The differential activity of these two FGFR
isoforms means that two tissues in the embryo, one expressing
predominantly VT and the other predominantly VT+, could be exposed to
the same, low concentration of FGF, and only the tissue expressing high
levels of VT would be induced. Our data suggest then that mesoderm
formation in vivo is dependent not only on the local
concentration of FGF but also on the relative expression levels of the
VT+ and VT isoforms in the responding tissue. This hypothesis
provides a possible mechanism for restricting mesoderm induction to
marginal zone cells, although all cells in the blastula stage embryo
have been shown to express FGFRs (11, 14). While much of the work on
mesoderm induction has focused on analysis of potential inducers, such
as eFGF, activin, and Vg1 (reviewed in Refs. 9, 31, and 32), our
results demonstrate that regulated expression of receptors and receptor isoforms also plays a critical role.
| |
ACKNOWLEDGEMENTS |
|---|
We thank Corinne Mercer for expert technical assistance.
| |
FOOTNOTES |
|---|
* This work was supported by grants from the Natural Sciences and Engineering Research Council of Canada (to L. L. G. and G. D. P.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 709-737-6293;
Fax: 709-737-7010; E-mail: lgillesp@morgan.ucs.mun.ca.
| |
ABBREVIATIONS |
|---|
The abbreviations used are: FGF, fibroblast growth factor; FGFR, FGF receptor; PCR, polymerase chain reaction; RT-PCR, RT, reverse transcription-PCR; PKC, protein kinase C; nt, nucleotide(s).
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REFERENCES |
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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 H. R. Burgar, H. D. Burns, J. L. Elsden, M. D. Lalioti, and J. K. Heath Association of the Signaling Adaptor FRS2 with Fibroblast Growth Factor Receptor 1 (Fgfr1) Is Mediated by Alternative Splicing of the Juxtamembrane Domain J. Biol. Chem., February 1, 2002; 277(6): 4018 - 4023. [Abstract] [Full Text] [PDF]
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