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First published online December 21, 2007
doi: 10.1242/10.1242/dev.011494
1 Department of Biological Sciences, Graduate School of Science, University of
Tokyo, Bunkyo-ku, Tokyo, Japan.
2 Graduate School of Life Science, University of Hyogo, 3-2-1 Kouto, Kamigori,
Ako-gun, Hyogo 678-1297, Japan.
Authors for correspondence (e-mails:
kirita{at}biol.s.u-tokyo.ac.jp;
htakeda{at}biol.s.u-tokyo.ac.jp)
Accepted 15 October 2007
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Medaka, fgfr1, Mesoderm, Prechordal plate, Hybrid sterility
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Naruse et al., 2004;
Wittbrodt et al., 2002). In
addition to the recent completion of the medaka draft genome
(Kasahara et al., 2007),
large-scale mutational screens using this organism have identified a number of
genetic loci that are required for normal embryonic development
(Furutani-Seiki et al., 2004).
Some medaka mutations also appear to be unique, ensuring that the medaka and
zebrafish are complementary model systems. Indeed, in our recent mutagenesis
screening, we isolated the medaka headfish (hdf) mutant
showing a severe defect in trunk-tail development, which is a phenotype that
has not so far been identified in zebrafish. Our subsequent analysis revealed
that this hdf mutant harbors a tryptophan-to-cysteine substitution at
a highly conserved amino acid position in the extracellular Ig-like domain II
(the ligand-binding domain) of fgf receptor 1 (fgfr1), and
is therefore the first fgf-receptor-related null mutant so far
described in fish (Yokoi et al.,
2007).
The gene fgf receptor encodes a cell surface tyrosine kinase
receptor that binds extracellular Fgfs and transduces the resulting signals
into the cytosol (Bottcher and Niehrs,
2005). Among the four Fgf receptor genes that have been
characterized, fgfr1 is thought to play a crucial role in early
vertebrate development, but its precise function has yet to be elucidated due
to the promiscuity of the Fgf receptor family and the large number of possible
ligands. In amphibians and teleosts, functional analyses of fgfr1
have mainly been performed by injection of RNA molecules encoding
dominant-negative forms of Fgfr1 (XFD)
(Amaya et al., 1991;
Amaya et al., 1993;
Carl and Wittbrodt, 1999;
Griffin et al., 1995;
Launay et al., 1996), but this
inevitably interferes with each of the Fgfr family members
(Ueno et al., 1992).
We previously reported that this hdf (fgfr1) mutant
exhibits a severe trunk-tail truncation with relatively normal head structures
(Yokoi et al., 2007). However,
this phenotype seemed less severe than expected, because both mesoderm
induction and neural patterning are known to depend upon Fgf signaling in fish
(Thisse and Thisse, 2005).
This may be due to the presence of other Fgfr species or maternally supplied
Fgfr1. Indeed, the ubiquitous expression of fgfr1 is detected in
medaka embryos from early cleavage (four-cell) to early gastrula stages
(Yokoi et al., 2007). As
gastrulation proceeds, fgfr1 expression is increased in the dorsal
margin, the presumptive anterior neural region and the underlying hypoblast
(Yokoi et al., 2007).
Furthermore, fgfr4 is also expressed during early gastrula, while
fgfr2 and fgfr3 begin to express from late gastrula stages
(H.Y., unpublished). To distinguish between genetic redundancy and maternal
effect, we have analyzed the function of Fgfr1 in mutant embryos in our
present study, in which both maternally and zygotically loaded gene products
are eliminated, i.e. maternal-zygotic (MZ) mutants.
As functional gene products are usually generated by a wild-type allele of
a heterozygous germ cell during oogenesis, the simplest way to obtain MZ
mutant embryos is to replace the wild-type host germ cells with the
corresponding homozygous mutants. Indeed, Schier and colleagues recently
reported the production of MZ mutant zebrafish by transplantation of mutant
germ cells into host embryos in which the endogenous germ cells had first been
eliminated by the morpholino-knockdown of the essential gene, dead
end (Ciruna et al., 2002).
In our current study, we utilize both this morpholino-knockdown method and a
novel method that we have ourselves developed for the production of MZ medaka
mutants using interspecific hybrids as the host embryos.
Interspecific hybrids are generally sterile (i.e. hybrid sterility) and
this is also the case for medaka. Among the several species that are closely
related to the genus Oryzias, the hybrid offspring from the Japanese
medaka (O. latipes) and Chinese Hainan medaka (O.
curvinotus) appeared to be healthy, although all are sterile as their
gonads are sexually differentiated but have severely impaired oogenesis and
spermatogenesis (Hamaguchi and Sakaizumi,
1992). Taking advantage of these characteristics, we have
developed a new method of germline replacement in which these hybrids are used
as a host for the production of MZ mutant offspring.
We herein report for the first time a detailed phenotypic analysis of medaka MZ mutants and reveal unexpected early roles for fgfr1 in fish development. We find that fgfr1-mediated signaling plays an essential role in the expansion and anterior migration of the axial mesoderm. By contrast, this signaling is not required for the dorsal convergence of the lateral mesoderm, and Fgfr1 is even dispensable for mesoderm induction in fish.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell transplantation experiments
Donor embryos were injected at the one-cell stage with 1.65% lysine fixable
tetramethylrhodamine-dextran (10 kDa; Molecular Probes). At the morula stage,
both the donors and hosts were dechorionated by hatching liquid
(Yasumasu et al., 1989). At
the mid-blastula stage [for primordial germ cell (PGC) transplantation], or at
the shield stage (for shield transplantation), the embryos were placed on
V-shaped grooves of a 1.5% agarose gel immersed in Yamamoto's Ringer's
solution (Yamamoto, 1956), and
then the cells were transplanted using a micromanipulator (Narishige M-152) in
combination with a microinjector (Narishige IM-6).
Genotyping of donor embryos
Donor embryos were fixed with 100% ethanol just after transplantation.
Genomic DNA was then extracted using a QIAamp DNA Micro Kit (Qiagen). Genomic
DNA fragments corresponding to the site of the mutation point were amplified
using the primer set F: 5'-GGAATGTACCCAAGTGTGAAAG and R:
5'-AGAAGAGAGACCCATGCCAC. The immunoglobulin II domain region of the
fgfr1 gene, including the G
C mutation site, was sequenced using
the Dynamic ET Terminator Cycle Sequencing Kit.
Whole-mount in situ hybridization
Embryos were fixed with 4% paraformaldehyde (PFA)/PBS overnight at room
temperature. Hybridization was performed with DIG-labeled RNA probes at
65°C overnight. Signals were detected by alkaline-phosphate
(AP)-conjugated anti-DIG Fab fragments (1:8000) and BM Purple (Boehringer
Mannheim).
Histological analysis
Ovaries were dissected, fixed in Bouin's solution and embedded in paraffin.
The specimens were then sectioned at 5 µm and stained with Hematoxylin. For
embryos, embedding was performed with a Technovit 8100 (Heraeus Kulzer,
Wehrheim).
Cell-tracing experiments
Two per cent DMNB-caged fluorescein-dextran (molecular weight 10,000,
Molecular Probes) was injected into the cytoplasm of one-cell-stage embryos,
which were then grown in the dark until the shield stage. To uncage the dye, a
beam of ultraviolet light, generated using a DAPI filter set, was directed for
5 seconds at the dorsal or lateral blastoderm margin. The locations of the
cells containing uncaged fluorescein-dextran were monitored and recorded at
the indicated periods. Images were quantitatively analyzed using NIH image
J.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
We first validated our method by transplanting cells from a wild-type
orange-red strain. Donor medaka embryos were injected at the one-cell stage
with rhodamine-dextran and the resulting blastula stage cells were then
transplanted from the deep layer of the blastoderm, where medaka PGCs tend to
reside (Kurokawa et al.,
2006), into the animal pole of the hybrid hosts. Transplanted
donor cells include both somatic cells and PGCs, which segregate during early
development. The former cells contribute to the anterior neural ectoderm,
whereas some of the latter reached the gonadal region. The successful transfer
of donor PGCs was thus assessed by the presence of rhodamine-dextran-labeled
cells in the gonadal mesoderm at the segmentation stages (52/174)
(Fig. 2C). The host embryos
that had been successfully transplanted were subsequently selected and raised
to adulthood (Fig. 2D,E).
Among the 20 females initially produced using this technique, 12 recovered
their fertility and produced donor-derived eggs. Histological sections also
revealed that the hybrid ovaries that had been transplanted with normal PGCs
contained both normally growing oocytes, possibly from the donor, and impaired
hybrid tissues (Fig. 2I-K). The
orange-red donor strain (b/b) lacks melanophores, which are present
in the skin of the hybrid host (B/B)
(Fukamachi et al., 2001).
Thus, we could confirm a successful germline replacement by determining the
presence of melanophores in the skin of the progeny
(Fig. 2F-H). Further
confirmation was obtained by analysis using genetic markers that can
discriminate between each strain via PCR-length polymorphisms
(Kimura et al., 2004)
(Fig. 2L).
We next transplanted germ cells derived from heterozygous hdf (fgfr1) parents. For the first five fertile host females, we retrospectively genotyped their donor embryos by sequencing an appropriate region of the fgfr1 gene. One donor embryo was genotyped as wild type (+/+), two as heterozygous (+/-) and two as homozygous (-/-). The two hybrid hosts transplanted with these homozygous PGCs eventually produced MZ offspring. In a later series of experiments, the donor embryos were allowed to develop further and their genotype was determined by an assessment of their external morphology.
In addition to our novel method, we also obtained MZ mutants using a
conventional knockdown method (Ciruna et
al., 2002) in which the host germ cells are eliminated by the
morpholino-knockdown of cxcr4, an essential gene for directing PGC
migration toward the gonads (Kurokawa et
al., 2006). The phenotypes of the MZ mutants obtained using these
two methods were found to be indistinguishable (data not shown). This
indicates that the environment of the hybrid host does not affect the later
development of their progeny, thus validating our new germline replacement
strategy.
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Fgfr1-mediated signaling is not required for mesoderm induction
We first examined whether the mesoderm is initially formed in the MZ
hdf (fgfr1) mutant. In the medaka wild-type embryo, the
expression of the pan-mesoderm marker no tail (ntl) is
initiated in the blastoderm margin during the late blastula to early gastrula
stages (Fig. 4A). During these
stages we cannot genotype these embryos using their external morphology, but
ntl expression remained detectable in all embryos obtained using any
combination of parental crosses (Fig.
4A-D). However, the levels of ntl expression,
particularly in the dorsal margin (fated to become the organizer, arrows in
Fig. 4A-D), were reduced in the
embryos that were thought to be zygotic or MZ mutants
(Fig. 4B,D). A similar tendency
was also observed with another early mesoderm marker, fgf8
(Fig. 4E-H), as well as with
the dorsal mesoderm marker chordin
(Fig. 4I-L).
We next examined the expression levels of sprouty4, a downstream target of Fgf signaling, in the margins of the embryos obtained by identical parental crosses. Approximately 50% of the hybrid host progeny with mutant germ cells (considered to be M mutants) exhibited almost normal levels of sprouty4 expression (Fig. 4O), whereas this expression was found to be undetectable in the remaining 50% (considered to be MZ mutants) (Fig. 4P). We also detected slight expression of sprouty4 in approximately 25% of the embryos derived from heterozygous hdf parents [considered to be zygotic (Z) mutants] (arrowheads in Fig. 4N), suggesting that maternal Fgfr1 is functional by at least this early gastrula stage. Hence, the early mesodermal markers appear to be activated without detectable levels of sprouty4 expression in the MZ mutant embryo. From these data, we reasonably contend that Fgfr1-mediated signaling is not required for initial mesoderm induction and its dorsoventral patterning in the medaka embryo.
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). The
embryos were incubated in the presence of SU5402 from the 32- to 64-cell to
early gastrula stages, and immediately subjected to in situ hybridization with
ntl or sprouty4 probes. As shown in
Fig. 4Q-S, the levels of
ntl expression were slightly reduced with increasing concentrations
of SU5402, but persisted at the 0.1 mg/ml concentration. By contrast,
sprouty4 expression was greatly reduced or undetectable at the 0.05
or 0.1 mg/ml concentrations, respectively
(Fig. 4T-V). SU5402 exposure at
a higher concentration than 0.1 mg/ml was found to cause a profound toxic
effect on the entire development of embryos and is therefore not likely to
give a physiological response (data not shown). These results further support
the idea that the mesoderm is initially formed in the absence of Fgf
signaling.
Expression analysis of anterior neural markers in the MZ hdf (fgfr1) mutant
We next examined the anterior neural patterning in wild-type, zygotic,
maternal and MZ hdf (fgfr1) mutant embryos at the bud (st.
18) and 12-somite (st. 23) stages. The markers used were krox20 for
rhombomere 3 and 5, pax2 for the MHB, and bf1 for the
telencephalon (Fig. 5A,E).
Krox20 expression in rhombomere 5 was found to be affected in all of
the mutants at the bud stage (Fig.
5A-D). Pax2 expression in the midbrain-hindbrain boundary
(MHB) was activated in all of the mutant types at the bud stage
(Fig. 5A-D). In the MZ mutant,
however, pax2 expression was found to decrease by the 12-somite stage
(red arrow in Fig. 5H) and to
have disappeared by the 16-somite stage (data not shown), whereas this
expression was maintained in both the zygotic and maternal mutants (red arrows
in Fig. 5F,G). The expression
domain of bf1 was normal in the zygotic and maternal mutants, but
smaller in size in the MZ mutant (black arrow in
Fig. 5H). Furthermore,
fgf8 (data not shown) and sprouty4 were expressed in the
anterior telencephalon and MHB in each type of mutant at the bud stage
(Fig. 5I-L). At the 12-somite
stage, however, the expression of these two genes, particularly in the MHB
region, became reduced or undetectable only in the MZ mutant
(Fig. 5M-P). Taken together,
these data show that in the MZ mutant, the forebrain and MHB structures are
formed but are not well maintained at later developmental stages, whereas the
anterior head patterning proceeds normally in both the zygotic and maternal
mutants.
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; Schier et al.,
1997; Shinya et al.,
2000). This prompted us to examine the expression of
goosecoid (gsc) in the prechordal plate in each of our
hdf (fgfr1) mutant types at the bud stage
(Fig. 6). The prechordal plate
is a group of cells that first involutes under the epiblast in the blastoderm
margin, then moves anteriorly and finally passes over the anterior end of the
neural plate (anterior neural ridge), reaching near to the animal pole
(Fig. 6A). As judged by the
gsc expression profile, this anterior limit of the prechordal plate
is posteriorly shifted in the MZ mutant embryos
(Fig. 6D). However, neither the
zygotic nor maternal hdf mutants show these phenotypes
(Fig. 6B,C). The severity of
the anterior neural phenotype in the MZ mutant was found to correlate with the
extent to which the prechordal plate underlies the anterior neural region
(data not shown). Moreover, this migration defect could already be observed as
early as the gastrula stage (Fig.
6E,F,E',F'). These findings suggest that the defects
in the anterior migration of the prechordal plate that are evident in the MZ
hdf (fgfr1) mutant represent the primary cause of the
anterior head malformation in these embryos.
Fgfr1 is required for the anterior movement of the axial mesoderm but is dispensable for the dorsal convergence movement of the lateral mesoderm during gastrulation
We speculated that the defective migration of the prechordal plate in the
MZ hdf (fgfr1) mutant could be due to a consequence of
impaired cell movement during gastrulation, such as epiboly and convergent
extension. We thus analyzed the process of epiboly in both the maternal (M)
and MZ embryos. The movement of epiboly, which drives blastoderm cells
vegetally, proceeds normally in the M mutant, and we thus used sibling M
mutants as controls in our subsequent analysis. The speed of epiboly movement
in the MZ mutant was also found to be normal until the stage at which
approximately 70% of the yolk sac is covered with blastoderm cells, i.e. 70%
epiboly (Fig. 7A,B,F,G).
However, as epiboly proceeded further, the extension of the embryonic midline
tissues in the MZ hdf mutant was observed to become severely
retarded, whereas the lateral and ventral margins moved vegetally at a normal
speed, resulting in an abnormal V-shaped margin centered by the midline
(Fig. 7C,D,H,I). Importantly,
the epiboly of the MZ mutant completed with the same timing as the M mutant,
leaving the axial margin arrested halfway along the animal-vegetal axis
(Fig. 7E,J). These findings
indicate that epiboly movement itself is normal in the MZ mutant embryo, with
the exception of the axial region.
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To trace the dorsal convergence movements in these embryos, we marked cells in the lateral blastoderm margin, at 90° from the dorsal embryonic shield, at the shield stage (Fig. 7N). During gastrulation, these marked mesoderm cells moved dorsally and anteriorly (white arrows in Fig. 7O,P), whereas those in the enveloping layer remained at the original angle and exhibited anterior expansion toward the animal pole (yellow arrows in Fig. 7O,P). Unexpectedly, labeled lateral cells in the MZ mutant also underwent normal convergence. This was confirmed by timecourse data obtained at the 6 hour (70% epiboly) and 12 hour (100% epiboly, st. 19) timepoints after labeling (Fig. 7R). These data thus indicate that Fgfr1-mediated signaling is specifically required for the expansion of the axial mesoderm along the anteroposterior axis, but is not required for the convergence movement of the lateral mesoderm.
Fgfr1 acts in a cell-autonomous manner during prechordal plate progenitor migration
As fgfr1 is broadly expressed until mid-gastrulation, we
postulated that the effects of Fgfr1 upon axial cells could be
non-cell-autonomous. To address this possibility, we transplanted prechordal
plate precursor cells (a group of cells that first involute in the dorsal
margin) obtained from MZ mutants into wild-type embryos at the shield stage
and vice versa (Fig. 8A). In
this series of experiments, we used MZ embryos obtained from female medaka
whose germ cells had been eliminated by the morpholino knockdown of
cxcr4 (Kurokawa et al.,
2006). At the bud stage (90% epiboly, st. 18), the migratory
ability of the precursor cells was assayed by the position of their anterior
limits (Fig. 8A). In most
control transplants (in which both the donor and host embryos were wild type),
the transplanted cells moved and distributed along the entire axial tissue,
including the anterior of the prechordal plate (transplants that showed
successful migration: 14/15) (Fig.
8B,E). However, when the MZ mutant cells were transplanted into
wild-type embryos, the anterior limit of the migrating cells tended to shift
posteriorly compared with the control transplants (successful migration: 3/16)
(Fig. 8C,F). Wild-type cells,
by contrast, migrated almost normally to the most anterior region in the
MZ-mutant background (successful migration: 12/12)
(Fig. 8D,G). Histological
sections confirmed that the transplanted cells had distributed in the
prechordal plate (Fig. 8H-S).
These results clearly show that Fgfr1 acts cell-autonomously in prechordal
plate precursor cells during their anterior migration.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). In their
report, the elimination of the host germline cells was achieved by morpholino
knockdown of dead end, an essential gene required for PGC
development, before the donor PGCs were transplanted. In our current method,
we avoid this step by taking advantage of interspecific hybrid sterility in
the medaka. We have compared the embryonic phenotypes of the MZ hdf
embryos obtained using interspecific hybrids and also that had derived from
cxcr4-MO-injected parents, and found no significant differences
between them. Hence both approaches produce comparable results in the medaka,
and we contend that our novel procedure using interspecific hybrids is a
simpler and faster method. Indeed, the resulting hybrid offspring also exhibit
a faster growth rate than normal, as a consequence of which the transplanted
hybrid females usually begin laying eggs at 5 to 6 weeks of age.
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; Amaya et al.,
1993; Carl and Wittbrodt,
1999; Griffin et al.,
1995; Launay et al.,
1996). However, in the Fgfr1-mutant mouse, initial
mesoderm formation occurs normally (Deng
et al., 1994; Yamaguchi et
al., 1994). This discrepancy could be due to the fact that XFD
interference has no specific preference for any type of Fgfr
(Ueno et al., 1992), or may be
due to the maternal contribution of Fgfr1 in mice, where maternal effects are
thought to be limited. However, in the MZ medaka mutant for fgfr1,
the early mesodermal markers ntl and fgf8 are initially
activated in the blastoderm margin, indicating that Fgfr1-mediated signaling
is dispensable for mesoderm induction in fish. Fgf signaling, mediated by
receptors other than Fgfr1, could conceivably compensate and participate in
this process, but this is not very likely for a number of reasons. First,
ntl is activated in MZ embryos in the absence of the induction of
sprouty4, a downstream target of Fgf signaling
(Minowada et al., 1999). More
importantly, ntl expression persists in wild-type embryos treated
with the chemical inhibitor SU5402, which is known to block nearly all types
of Fgfr. As mesoderm formation is also severely impaired in mouse and
zebrafish mutants for Nodal signaling
(Gritsman et al., 1999;
Zhou et al., 1993), this
pathway may play an essential role in mesoderm induction in these
organisms.
The MZ hdf (fgfr1) mutant reveals an early and essential role for Fgfr1 in prechordal plate migration
The MZ hdf (fgfr1) mutant displays severe anterior neural
defects that are never observed in its zygotic counterpart. This was found to
be attributable to a defective migration of the prechordal plate. In addition
to the organizing centers within the neural ectoderm, the underlying
prechordal plate plays a crucial role in anterior neural patterning, which
secretes signaling molecules such as Shh, Dickkoff1 and the Fgfs
(Niehrs et al., 2001;
Placzek and Briscoe, 2005;
Shinya et al., 2000). The role
of the prechordal plate in neural patterning has been clearly shown in
zebrafish one-eyed pinhead (oep) mutants in which Nodal
signaling is zygotically blocked. In this mutant, the prechordal plate fails
to form, and the anterior head structures are severely affected
(Schier et al., 1997;
Shinya et al., 2000). This
phenotype is very similar to the one we observe in the medaka MZ mutant for
hdf (fgfr1). However, expression and cell-tracing analyses
of these embryos have revealed that unlike the zebrafish oep mutant,
the medaka MZ fgfr1 mutant develops a (gsc-positive)
prechordal plate but its anterior migration, underneath the neural region,
fails to complete, leading to an arrest somewhere between the anterior neural
ridge and the MHB. As maternally provided proteins do not persist for long
after fertilization, until mid-gastrulation at most
(Yokoi et al., 2007), they
should function before gastrulation has completed. Consistently, the defect in
axial elongation, the earliest phenotype of the MZ embryo, becomes detectable
at the gastrula stage.
Interestingly, in spite of the early roles of fgfr1, both the maternal and zygotic medaka mutants for hdf (fgfr1) in our present study develop a normal anterior head, and this indicates that either maternally or zygotically derived Fgfr1 is sufficient for normal migration of the prechordal plate. Hence, the production of MZ mutants was the only way to uncover this early function of fgfr1 in lower vertebrates in which, unlike the mouse, maternally supplied materials sometimes play an essential role in early development.
There is now increasing evidence for the role of Fgf signaling in cell
movement or migration during early development in vertebrates. Examples of
this include the overexpression of Xsprouty2, a negative regulator of
Fgf signaling (Nutt et al.,
2001), and the knockdowns of neurotrophin receptor homolog (NRH)
and ankyrin repeat domain protein 5 (Anr5), both Fgf downstream targets
(Chung et al., 2005;
Chung et al., 2007), which
result in the inhibition of cell movement during convergent extension. In
addition, chimeric analyses of the Fgfr1-knockout mouse have shown
that these mutant cells fail to move away from the primitive streak
(Ciruna and Rossant, 2001;
Ciruna et al., 1997). Our
present analyses of MZ fgfr1 mutant medaka embryos further specify
the role of Fgfr1 in embryonic cell movement using genetic approaches and have
demonstrated the specific requirement for this factor during cell migration in
the axial mesoderm. The differentiation of the axial mesoderm must also be
impaired from the beginning, but judging from ntl and gsc
expression, the initial differentiation of this structure does take place in
the MZ mutant. Overall, the defective cell migration is most evident in the
prechordal plate, the precursors of which require the highest levels of cell
migration in order that the cells travel the considerable distance from the
margin to eventually underlie the anterior of the neural tube.
Our current transplantation study also provides evidence that
Fgfr1-mediated signaling acts in a cell-autonomous manner. Thus,
Fgfr1-mediated signaling appears to directly control the migratory activity of
axial cells in fish. In medaka, Fgf8 has been suggested to be a main ligand
for Fgfr1 during early development because of the phenotypic similarity
between zygotic hdf mutant and fgf8 morphant
(Yokoi et al., 2007). This
could be the case for prechordal plate migration, because fgf8
expresses in the early mesodermal lineage (H.Y., unpublished). However, it
remains to be elucidated whether this ligand directs a migratory pathway or
simply activates migratory behavior. Recent work demonstrated that prechordal
plate migration is directed by platelet derived growth factor (PDGF) and its
receptor (PDGFR) in Xenopus and zebrafish
(Liu et al., 2002;
Nagel et al., 2004). It is
thus possible that Fgfr1-mediated signaling cooperates with PDGF signaling in
anterior migration of axial mesodermal cells.
In conclusion, we have generated an MZ hdf (fgfr1) mutant in the medaka and have conducted phenotypic analysis, which has revealed the early cell-autonomous role of this gene during axial migration. Considering the advantages of using fish model systems in developmental studies, this hdf mutant could prove to be a valuable model with which to analyze the precise role of fgfr1-mediated signaling in early vertebrate development.
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ACKNOWLEDGMENTS |
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Footnotes |
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Present address: Institute of Neuroscience, University of Oregon, Eugene,
OR, USA
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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