1 Department of Marine Biosciences, Tokyo University of Marine Science and
Technology, 4-5-7 Konan, Minato-ku, Tokyo 108-8477, Japan
2 Department of Genetics, School of Medicine, Case Western Reserve University,
Cleveland, OH 44106, USA
3 Division of Developmental Biology, Cincinnati Children's Hospital Research
Foundation, Cincinnati, OH 45229, USA
* Author for correspondence (e-mail: Christopher.Wylie{at}cchmc.org)
Accepted 8 September 2005
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SUMMARY |
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Key words: Germ cells, FGF signaling, ERK
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Introduction |
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The FGF family of signaling ligands is known to control many aspects of
development, including growth, differentiation and migration
(Powers et al., 2000). FGF
signaling is complex, and involves four receptors (FGFR1-FGFR4). In
FGFR1-FGFR3, alternative splicing of the carboxy-terminal half of the Ig
domain III yields `IIIb' and `IIIc' forms of the receptor, which have distinct
expression patterns and ligand specificities. `IIIb' forms are thought to be
epithelially expressed and activated by mesenchymally produced FGFs, whereas
`IIIc' forms are usually mesenchymal and activated by epithelial FGFs.
Therefore, a total of seven receptors bind with varying affinities and
specificities to at least 22 distinct FGF ligands, four of which are thought
to be non-signaling (Ornitz and Itoh,
2001
).
FGF signaling is thought to be important in germ cell behavior during
migration. FGF2 has been reported to act as a mitogen for PGCs in vitro, and,
together with steel factor and LIF, causes cultured germ cells to change into
pluripotential permanent cell lines
(Matsui et al., 1992).
Furthermore, radiolabeled FGF2 binds to PGCs in culture, suggesting the
presence of FGF receptors (FGFRs) (Resnick
et al., 1998
). However, the FGFR combinations expressed by germ
cells, the ligands they respond to, and their individual or collective roles
in germ cell behavior are not known.
We show, using RT-PCR from flow-cytometry-purified germ cells, that germ
cells express the FGF receptors FGFR1-IIIc and FGFR2-IIIb during migration.
Antibodies specific to the di-phosphorylated forms of ERK1 and ERK2
(dp-ERK1/2) have been shown to be reliable markers of activity of the MAP
kinase pathway in the mouse embryo (Corson
et al., 2003). In addition, by using inhibitors of FGF signaling,
and of MEK1/2, which phosphorylates ERK1/2 in the MAP kinase pathway, we show
that di-phosphorylation of ERK1/2 in migrating germ cells is mediated, either
directly or indirectly, by FGF signaling.
Gain- and loss-of function experiments using embryo slices, together with time-lapse analysis of PGC migration, show that FGF signaling is required to maintain both motility and normal germ cell numbers during migration, but not for their migration to the genital ridges nor their proliferation during migration. This result was confirmed in vivo by examining the effects of a targeted mutation of FGFR2-IIIb, which causes reduced numbers of germ cells to colonize the genital ridges, and increased apoptosis of germ cells.
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Materials and methods |
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Chip analysis, RT-PCR and DNA sequencing
Gene expression profiles of E10.5 PGCs using the Affymetrix MG-U74Av2 were
reported previously (Molyneaux et al.,
2004). Chip analysis was performed using MicroArray Suite software
(Affymetrix) to statistically determine `presence' and `absense' calls. For
RT-PCR analysis, total RNA was isolated from FACS-purified E10.5 PGCs using
RNeasy Protect Mini Kit (Qiagen). Twenty-five ng of total RNA was reverse
transcribed using SuperScript First-Strand Synthesis System (Invitrogen). PCR
was performed in a 15-µl volume using Redmix Plus (2.0 mM MgCl2)
(GeneChoice) as a source of Taq, buffer and dNTPs. PCR reactions were
performed as described above. Products from PCR amplifications were cloned
into the pGEM-T easy vector (Takara) and subjected to DNA sequencing.
Primers used were as follows:
stella (Dppa3 - Mouse Genome Informatics), 5'-TGAGTTTGAACGGGACAGTG-3' (forward) and 5'-GATTTCCCAGCACCAGAAAA-3' (reverse);
steel (Kitl - Mouse Genome Informatics), 5'-AATGCACAACTGCCATCTCC-3' (forward) and 5'-AGGAATGCCTAGACTACTGGAAAA-3' (reverse);
Twist1, 5'-CCCCACTTTTTGACGAAGAA-3' (forward) and 5'-GATTTGCAGGCCAGTTTGAT-3' (reverse);
Odc, 5'-GCCATTGGGACAGGATTTGAC-3' (forward) and 5'-CATCATCTGGACTCCGTTACTGG-3' (reverse);
Fgfr1, 5'-CTTGACGTCGTGGAACGATCT-3' (common forward), 5'-CACGCAGACTGGTTAGCTTCAC-3' (IIIb reverse) and 5'-AGAACGGTCAACCATGCAGAG-3' (IIIc reverse);
Fgfr2, 5'-CCCATCCTCCAAGCTGGACTGCCT-3' (common forward), 5'-CAGAGCCAGCACTTCTGCATTG-3' (IIIb reverse) and 5'-CAGAACTGTCAACAATGCAGAGTG3' (IIIc reverse);
Fgfr3, 5'-CAAGTTTGGCAGCATCCGGCAGAC-3' (common forward), 5'-TCTCAGCCACGCCTATGAAATTGGTG-3' (IIIb reverse) and 5'-CACCACCAGCCACGCAGAGTGATG-3' (IIIc reverse); and
Fgfr4, 5'-TTCTGTTCCAGCCTTATGCCCC-3' (forward) and 5'-TGATGCCCCTTTCACCAAGATG-3' (reverse).
Embryo slice culture
Transverse slices from the hind gut regions of mouse embryos were cultured
and filmed as previously described
(Molyneaux et al., 2003).
Briefly, E9.5 embryos were manually sliced to 1.5- to 2-somites width by using
surgical blades. Slices were put on the Collagen IV-treated culture inserts
(Millipore) and incubated in DMEM/F-12 medium (Gibco BRL) with 0.04%
lipid-free BSA (Sigma) and 100 U/ml penicillin-streptomycin solution (Sigma).
To analyze the effect of FGF signaling, purified recombinant FGF2 (R&D
Systems) and FGF7 (Peprotech), and soluble inhibitors for FGF signaling [MEK
inhibitor U0126 (Cell signaling), FGFR inhibitor SU5402 (Calbiochem)] were
added at the indicated concentrations. Because these inhibitors were dissolved
in DMSO, the same amount of DMSO was added to control groups.
Time-lapse analysis of migrating germ cells
Slices were filmed using the Zeiss LSM 510 confocal system. Images were
captured every 7 minutes for 700 minutes. Movies were analyzed using NIH
image, as previously described (Molyneaux
et al., 2001). For counting PGCs in slices, the tissue was
optically sectioned in 15 µm steps (with a 5 µm overlap). PGCs were
counted in individual optical sections by using the overlay feature in the
Zeiss software to mark cell positions. Three to six independent repeat
experiments were performed to obtain means and standard errors of the mean
(s.e.m.), and Tukey's HSD test was used to identify significant differences
between controls and each treatment.
BrdU incorporation assay
Incorporation of bromodeoxyuridine (BrdU) into PGCs was assayed using the
Amersham Cell Proliferation Kit (RPN20). E9.5 embryo slices containing
migrating PGCs were treated with FGF2, FGF7 or FGFR inhibitor for 6 hours.
Slices were treated with 0.1% BrdU labeling reagent for an additional 2 hours
in the presence of FGF ligands and inhibitor. At the end of the culture
period, the slices were rinsed with PBS, fixed in 4% paraformaldehyde (PFA)
overnight at 4°C, rinsed with PBS containing 0.1% Triton X-100 (PBST),
then processed for frozen sectioning. In a single experiment, eight slices
dissected from three to four embryos were used for each treatment. Each
experiment was repeated three times to obtain means and the s.e.m. of the
percentages of BrdU-positive PGCs, and Tukey's HSD test was used to identify
significant differences between controls and each treatment. For the in utero
labeling of embryos, pregnant females at 11.5 days post coitus received an
i.p. injection of 500 µl of 100% BrdU labeling reagent and were pulsed for
4 hours. The harvested embryos were fixed in 4% PFA (overnight, 4°C), then
processed for frozen sectioning.
Immunofluorescence analysis on whole-mount embryo slices or frozen sections
Whole-mount dp-ERK1/2 staining was performed on PGC-containing embryo
slices dissected from E9.5 embryos. Slices were fixed in 4% PFA overnight at
4°C, and rinsed with PBST. Slices were blocked overnight in blocking
buffer (2% horse serum/2% IgG-free BSA in PBS), treated with a 1:100 diluted
anti-dp-ERK1/2 monoclonal antibody (Sigma) overnight, washed for 1 hour
(4x) in PBST, and treated with 15 µg/ml Cy5-conjugated anti-mouse IgG
antibody (Jackson ImmunoResearch Laboratories) in the blocking buffer
overnight in the dark. Tissues were washed as described and mounted in 75%
glycerol with 100 µg/ml DABCO (Sigma). All procedures were performed at
4°C.
The immunostaining of embryo slices or embryonic gonads with anti-OCT3/4 donkey IgG (1:100; R&D Systems), anti-BrdU mouse monoclonal (1:100; Amersham) and anti-cleaved caspase 3 rabbit IgG (1:1000; Cell Signaling) antibodies was performed on frozen sections. Samples were embedded in OCT medium (Triangle Biomedical Sciences) and 14-µm sections cut. Sections were washed in PBST for 20 minutes, blocked for 1 hour in the blocking buffer, and then incubated with diluted primary antibodies in the blocking buffer overnight. Slides were washed for 5 minutes (3x) with PBST and incubated with the appropriate 15 µg/ml Cy5-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories). Slides were washed as described above and mounted in glycerol with DABCO. All procedures were performed at room temperature. Images were captured using a Zeiss LSM 510 confocal system.
In order to obtain the density of germ cell numbers in the genital ridges, the area of the genital ridge was measured using the LSM software and multiplied by the thickness of image, to give the volume of genital ridges in z-stack confocal image series. Then, the number of germ cells was divided by the volume of genital ridge. A total of three independent litters were assayed to obtain the means and s.e.m. of the density of gonadal germ cells. Tukey's HSD test was used to identify significant differences between wild-type and Fgfr2-IIIb null embryos.
Alkaline phosphatase staining of germ cells
E11.5 embryos were fixed overnight in 4% PFA at 4°C, and washed
thoroughly in PBS. The genital ridges were excised and incubated for 2 hours
in permeabilization buffer (0.1% SDS, 1% Triton X-100 in PBS), then rinsed for
10 minutes (3x) in Tris-maleate (pH 9.0) buffer. The permeabilized
genital ridges were incubated in alkaline phosphatase buffer [0.4 mg/ml
Naphthol AS-MX phosphate (Sigma, N-5000), 1 mg/ml Fast Red TR salt (Sigma,
F2768) in Tris-maleate buffer] for 20 minutes. Samples were placed in 50%
glycerol/PBS and photographed using a dissecting microscope equipped with a
digital camera.
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Results |
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The oligonucleotides used on this chip do not distinguish between the two
splice variants Fgfr2-IIIb and Fgfr2-IIIc. To analyze
further the expression of FGF receptors, we dissected genital ridges, the
dorsal body wall, and the hind gut mesentery, as a single piece, from
OCT4PE:GFP embryos, in which the migrating germ cells express high
levels of GFP (Anderson et al.,
1999
). Tissues were dissociated, and separated by flow cytometry
into germ cell (GFP-positive) and somatic cell (GFP-negative) populations.
cDNA was made from the two cell populations. The purity of the preparations
was analyzed by the presence of the germ cell marker stella, and the somatic
cell markers steel and Twist1. As shown in
Fig. 1A, stella was found to be
present in PGC cDNA, but steel and Twist1 were absent. This
observation confirmed that signals from these RT-PCR experiments are reliable
in identifying genes expressed by migrating PGCs. Preparations were chosen for
further analysis in which the germ cell cDNA contained no detectable steel or
Twist1, and the somatic cells contained no detectable stella. PCR
primers were designed to identify all of the FGF receptors, including their
splice variants. As shown in Fig.
1B, germ cells were found to express Fgfr2-IIIb during
migration. In addition, we found that Fgfr1-IIIc was also expressed
by migrating germ cells. Products obtained from the RT-PCR amplifications were
of the predicted sizes (i.e. Ffgr1-IIIb, 323 bp; Fgfr1-IIIc,
343bp; Fgfr2-IIIb, 215 bp; Fgfr2-IIIc, 314 bp). PGC-derived
PCR products were sequenced to verified that these bands truly represented the
expression of both Fgfr2-IIIb and Fgfr1-IIIc in the PGCs
(data not shown).
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Analysis of the embryos under higher magnification (Fig. 2B-D) revealed cytoplasmic staining of PGCs in the absence of MEK inhibitor. Cells that were both dp-ERK1/2-positive, and GFP-positive, were scored as dp-ERK1/2-positive germ cells. In three independent preparations from E9.5, 49% of germ cells were positive for dp-ERK1/2 (Fig. 2C,I). The same proportion of PGCs was dp-ERK1/2 positive at E10.5 (data not shown). This suggests the ERK1/2 activation may be transient, either because it is cell cycle dependent, or because of a transient presence of signal. The pattern of dp-ERK1/2 staining and the percentage of dp-ERK1/2 positive PGCs in freshly isolated and fixed wild-type embryos was comparable with embryos cultured for four hours in DMSO, indicating that the dp-ERK1/2 staining seen in these experiments was not an artifact of the culture system. The staining was diminished by MEK inhibitor treatment. Only 12% of PGCs were dp-ERK1/2 positive in U0126-treated embryos (Fig. 2D,I).
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FGF signaling controls germ cell number during migration
The fact that germ cells continue to migrate out of the hind gut and into
the genital ridges in slice cultures from OCT4PE:GFP embryos, allows a
rapid functional assay for the roles of signaling pathways by the addition of
ligands, agonists and antagonists to the slices
(Molyneaux et al., 2001
;
Molyneaux, 2003
). As an
initial test of the role of FGF signaling, transverse slices from the hind gut
regions of E9.5 OCT4
PE:GFP embryos were cultured for 18 hours in
serum-free medium, either in the presence of FGF2 (0.1, 1, 10 ng/ml), FGF7 (1,
10, 100 ng/ml), or SU5402 (1, 5, 10 µM). FGF2 and FGF7, the high-affinity
ligands for FGFR1-IIIc and FGFR2-IIIb, respectively, are each thought not to
activate the other receptor. Effective doses for FGF2 and FGF7 are described
as 100-250 ng/ml and <1000 ng/ml, respectively, in the manufacture's
protocol, other published FGF studies have used a wide range of concentrations
for these ligands, from 1-1000 ng/ml
(Resnick et al., 1998
;
Weksler et al., 1999
;
Bridges et al., 2003
;
Kawase et al., 2004
). Because
FGF ligand-receptor interactions are influenced by many factors other than
ligand concentration, such as heparin and cell-surface proteoglycans, it is
impossible to determine what ligand concentrations in vitro will mimic
physiological conditions. Therefore, we estimated the lowest effective doses
of these reagents that would increase dp-ERK1/2 staining in our slice culture
system. We used dose ranges starting at these concentrations. FGF2 had no
effect on germ cell numbers in the dose range 0.1-10 ng/ml. Doses of 100 ng/ml
caused spreading of the slices and loss of morpholgy. FGF7 caused increased
germ cell numbers, when compared with controls, at all doses between 1 and 100
ng/ml. However, only the 100 ng/ml dose was significant at the P=0.05
level. The FGF receptor inhibitor SU5402 caused a dose-dependent decrease in
germ cell numbers (Fig. 3A).
These data suggest that FGF signaling, either directly on germ cells, or
indirectly via somatic cells, controls germ cell numbers during migration.
To test the role of FGF signaling on mitosis of migrating PGCs, embryo slices were exposed to BrdU for 2 hours, followed by a 6-hour incubation with FGF ligands or the inhibitors. Cells that were both BrdU positive, and GFP positive, were scored as BrdU-positive germ cells. The percentage of BrdU-positive PGCs was counted in eight embryo slices from each of two separate experiments for every treatment. As shown in Fig. 3B, levels of BrdU incorporation are not significantly affected by either the FGF ligands or the inhibitor.
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As shown in Fig. 5C, both the velocity and displacement of PGCs were significantly (P<0.05) increased by treatment with 10 ng/ml of FGF2. By contrast, they were significantly (P<0.05) decreased by 10 µM of SU5402. Neither of these parameters was affected by treatment with 100 ng/ml of FGF7 (Fig. 5C) or lower doses of SU5402 (e.g. 1 or 2.5 µM) (data not shown), even in slices where there were increased numbers (see Fig. 3A) or increased fragmentation of PGCs (see Fig. 4D). Indeed, most PGCs showed processes and migrated normally in lower doses of SU5402, whereas dying PGCs stopped process formation and migration just before fragmentation. We cannot obtain long enough trajectories from germ cells, which died in the middle of movie, for calculating values of velocity and displacement. Therefore, the data from dying germ cells were not included in the migratory properties.
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FGF2 treatment also caused the slices themselves to spread more rapidly than controls did on the substratum. This probably passively enhances the overall displacement of the germ cells during the culture period, in addition to their increased motility and process formation. However, the measurements of process formation (Fig. 5D) and velocity of individual germ cells (Fig. 5C) show that germ cell movement is enhanced by FGF2 treatment.
Targeted mutation of FGFR2-IIIb confirms a role for FGF signaling in maintaining germ cell numbers
To confirm in vivo the role suggested by slice culture experiments in
vitro, we examined the numbers and positions of germ cells in litters of E11.5
embryos containing targeted mutations in FGFR2-IIIb. Homozygous mutant embryos
are easy to score, because they lack fore and hind limbs
(Fig. 6A), as reported
previously (De Moerlooze et al.,
2000). The dorsal body wall, including the genital ridges, of each
embryo was dissected whole, fixed, and stained for alkaline phosphatase (AP),
whilst the corresponding anterior end was genotyped
(Fig. 6B). The most obvious
effect of loss of FGFR2-IIIb at E11.5, visible in AP-stained whole mounts, was
the dramatic reduction of ectopic germ cells
(Fig. 6C). Normally,
significant numbers of migrating germ cells do not reach the genital ridges,
instead they remain scattered, as ectopic germ cells, close to the genital
ridges, lateral to the midlines (see upper panel in
Fig. 6C). In the analysis of
six independent litters, which included 33 heterozygotes and 11 mutants, we
observed a 46±10% reduction of ectopic germ cells in
FGFR2-IIIb-/- embryos at E11.5
(Fig. 6I). Transverse sections
of AP-stained genital ridges suggested that the density of gonadal germ cells
was also reduced in FGFR2-IIIb-/- embryos
(Fig. 6D).
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To distinguish between the affects on proliferation and apoptosis of germ cells in the absence of FGFR2-IIIb, cell death and proliferation were examined in heterozygotes and mutants at E11.5 using cleaved caspase-3 staining (Fig. 6G) and BrdU incorporation assays (Fig. 6H). The percentage of cleaved caspase-3-positive or BrdU-positive germ cells was counted in four sections per embryo from each of three independent litters. Significant differences (P<0.05) in the numbers of cleaved caspase-3-positive cells were seen in heterozygous and null genital ridges [1.9±0.6% in heterozygotes (n=6) versus 7.3±0.8% in nulls (n=7); Fig. 6G,K]. However, similar proportions of BrdU-positive germ cells were seen in both heterozygous and null genital ridges (Fig. 6H,L). Our data show that E11.5 gonadal germ cells do not require FGFR2-IIIb to maintain normal rates of proliferation, but that the number of germ cells undergoing apoptosis in the genital ridges increases in the absence of this signaling. These in vivo results are consistent with the effects seen using gain- and loss-of-function experiments in slice cultures.
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Discussion |
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In this paper, we identified the FGFRs expressed by PGCs during migration
and focussed on the roles of these receptors in PGC development. It is
difficult to identify which FGF ligands are responsible for the activation of
these receptors in vivo, because the FGF protein family consists of at least
22 members (in humans), and FGFs within each subfamily have similar
receptor-binding properties and over-lapping patterns of expression
(Ornitz and Itoh, 2001).
Functional redundancy is therefore likely to occur in mice containing targeted
mutations in individual FGF genes. For example, FGF2 is known to be an
essential factor promoting PGC proliferation in vitro
(Matsui et al., 1992
;
Resnick et al., 1998
).
However, mice deficient for FGF2 or for both FGF1 and FGF2 are fertile
(Zhou et al., 1998
;
Miller et al., 2000
).
Whole-mount in situ hybridization studies for FGFs in mouse embryos showed
that FGF3, FGF4, FGF8, FGF10 and FGF17 are expressed in neighboring somatic
tissue along the migratory route of PGCs
(Wright and Mansour, 2003
;
Kawase et al., 2004
). It is
known that FGF4, FGF8 and FGF17 (epithelial FGFs), and FGF3 and FGF10
(mesenchymal FGFs), can activate FGFR1-IIIc and FGFR2-IIIb, respectively
(Powers et al., 2000
).
Therefore, these FGFs could act as the physiological ligands for migrating
PGCs in vivo. Although PGCs start expressing FGF4 and FGF8 after they
colonized the genital ridges (Kawase et
al., 2004
), no FGF ligands have been identified as being expressed
by migrating PGCs so far (Y.T. and C.W., unpublished).
It has been shown that FGF2 and FGF4 stimulate PGC proliferation in culture
(Matsui et al., 1992;
Resnick et al., 1998
;
Kawase et al., 2004
). However,
we show here that addition of FGF2 and FGF7 did not increase BrdU
incorporation in PGCs, and PGCs continued to divide and take up BrdU in the
absence of FGF signaling in cultured slices. In addition, BrdU incorporation
by PGCs was not affected in FGFR2-IIIb-deficient mice, although PGC number was
reduced in this mutant. These data suggest that FGF signaling via either
FGFR1-IIIc or FGFR2-IIIb does not stimulate mitogenic activity in PGCs in
vivo. It is not clear why the addition of FGF2 should cause different effects
in germ cells in embryo slices and in germ cells dissociated and cultured on
feeder layers. It may be that the feeder cells provide additional signals, or
that the cell contact phenomena are different, and modulate different
responses. Addition of FGF2 and FGF7, respectively, to embryo slice cultures
had different effects. FGF2 increased motility and process formation, whereas
FGF7 increased germ cell numbers but had no effect on motility. Addition of
the FGF receptor-blocking agent SU5402 caused a dose-dependent decrease in
germ cell numbers, and a decrease in motility at high doses but not low doses.
It is not clear why SU5402 did not affect motility at lower doses. It may be
that different threshold reductions in FGF receptor activity are required for
different FGF-mediated functions, or that different levels of the ligands for
each of the receptors are present. We conclude from this data that signaling
through the FGFR1-IIIc receptor, either in germ cells themselves or in
adjacent somatic cells, controls germ cell motility, whilst signaling through
the FGFR2-IIIb receptor controls germ cell survival. Genetic loss-of-function
data in FGFR2-IIIb embryos supports this proposed role in germ cell
survival.
Global knockouts of FGFR1 (both of the b and c forms) and FGFR1-IIIc mice
die late in gastrulation, too early for the effects on PGC migration to be
assessed, and thus they do not test the proposed role for FGFR1-IIIc in germ
cell migration. However, they do show defects in mesoderm cell migration and
specification (Yamaguchi et al.,
1994; Partanen et al.,
1998
). Furthermore, it is shown that FGFR1-/- cells
overexpress E-cadherin, and that this expression is accompanied by the
downregulation of mouse Snail
(Ciruna et al., 1997
). In the
present paper, we studied that the role of FGF signaling via FGFR1-IIIc by
adding its potential ligand FGF2, and the loss-of-function by adding the
global inhibitor of FGF/FGFR signaling SU5402. Germ cells extended exaggerated
processes when treated with FGF2, which correlated with an upregulation of
their migratory activity, as analyzed by time-lapse movies. The reverse
effects were seen following FGFR inhibitor treatment. These data strongly
suggest that FGFR1-IIIc signaling is required for PGC migration and/or the
establishment of cell-cell communications using pseudopodia.
The data here do not address whether FGFR1-IIIc- or FGFR2-IIIb-mediated events occur by direct ligand-receptor interaction on germ cells, on adjacent somatic cells, or on both. PGC-specific depletion of both receptors during migration by a conditional Cre/loxP gene targeting strategy will be required to examine the cell specificities of these pathways.
Defects in PGC migration underlie many human congenital disorders. For
example, extra-gonadal germ cell tumors are thought to arise from PGCs that do
not migrate correctly into the genital ridges and that fail to die
(Upadhyay and Zamboni, 1982;
Gobel et al., 2000
).
Therefore, it will be important to study the downstream targets of FGFR1-IIIc
and FGFR2-IIIb activation to understand the mechanisms that control process
formation and apoptosis of germ cells. In particular, the mechanism of cell
death of ectopic germ cells caused by the mis- or incomplete migration of PGCs
is not well understood to date. Future work will focus on trying to understand
how germ cell survival is controlled by FGF signaling.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/132/24/5399/DC1
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ACKNOWLEDGMENTS |
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