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First published online 25 June 2008
doi: 10.1242/dev.020107
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1 Department of Biological Science, Graduate School of Science, Hiroshima
University, Kagamiyama 1-3-1, Higashi-Hiroshima, Hiroshima, 739-8526
Japan.
2 Division of Biological Science, Graduate School of Science, Nagoya University,
Furo-cho, Chikusa-ku, Nagoya, 464-8602 Japan.
3 Ludwig Institute for Cancer Research, Royal Melbourne Hospital, Parkville, VIC
3050, Australia.
Author for correspondence (e-mail:
yutaka{at}hiroshima-u.ac.jp)
Accepted 27 May 2008
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Zebrafish, Endoderm, Migration, Gastrulation, Sdf1 (Cxcl12), Cxcr4a
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). During internalization, the mesodermal and endodermal cells
move via the blastopore beneath the ectoderm. It is well known that after
internalization, the migration of mesodermal cells is controlled by
convergence and extension (C&E) movements, which narrow the field of
mesodermal cells mediolaterally and elongate it along the anterior-posterior
axis (Myers et al., 2002;
Montero and Heisenberg, 2004).
Recent comprehensive studies in Xenopus and zebrafish have
demonstrated that the Wnt/planar cell polarity (PCP) signaling pathway is
implicated in C&E movements (Myers et
al., 2002). The internalization of endoderm is coincident with
that of mesoderm and, after its migration, it gives rise to the epithelial
lining of the digestive tract as well as associated organs
(Warga and Nüsslein-Volhard,
1999). Although two studies concerning endoderm movements have
been reported (Ober et al.,
2004; Matsui et al.,
2005), the molecular mechanisms underlying endodermal cell
movement during gastrulation remain largely unexplored in vertebrates.
In zebrafish, a Sox-related transcription factor, Casanova (Cas; Sox32 -
ZFIN), is essential for endodermal cell specification
(Kikuchi et al., 2001;
Fukuda and Kikuchi, 2005) and
functions to control its downstream target, sox17
(Alexander and Stainier, 1999).
cas-expressing endodermal cells are distributed around the blastoderm
margin in a salt-and-pepper pattern before gastrulation and this distribution
is maintained during the migration process
(Kikuchi et al., 2001;
Warga and Nüsslein-Volhard,
1999). After specification, internalized endodermal and mesodermal
cells form the hypoblast layer underneath the epiblast layer, which consists
of ectodermal cells. A detailed study using zebrafish embryos has revealed
that within the hypoblast, the endodermal cells are internal, and are located
next to the extra-embryonic yolk syncytial layer (YSL), whereas the mesodermal
cells form a layer between the endoderm and the ectoderm
(Warga and Nüsslein-Volhard,
1999). Endodermal cells form a noncontiguous monolayer, which is
overlaid by a more coherent multilayer of mesodermal cells
(Warga and Nüsslein-Volhard,
1999) (see also Fig. S1 in the supplementary material). Time-lapse
video-microscopy has further shown that following internalization, endodermal
cells change their morphology and become flattened with numerous filopodial
processes (Warga and
Nüsslein-Volhard, 1999). Since endodermal cells migrate
separately from mesodermal cells within the hypoblast layer, the zebrafish
gastrula provides a useful model system in which to study endodermal cell
migration.
Chemokines are a large family of proteins with crucial roles not only in
the immune response, but also during various developmental processes such as
cell migration and the growth cone guidance of neuronal axons
(Kucia et al., 2004). The
chemokines, which are small secreted molecules, act through their receptors,
which belong to the superfamily of G protein-coupled receptors (GPCRs)
(Kucia et al., 2004). One such
chemokine, stromal cell-derived factor 1 (Sdf1; Cxcl12 - ZFIN), and its
receptor, Cxcr4, are well characterized in various biological processes
(Busillo and Benovic, 2007). A
major function of Sdf1/Cxcr4 signaling is to regulate the movement of cells,
as Sdf1 attracts cxcr4-expressing cells. In zebrafish, the movement
of various types of cells, such as primordial germ cells, olfactory placodal
precursors, lateral line primordial cells and slow muscle cells, is regulated
by Sdf1/Cxcr4 signaling during development
(David et al., 2002;
Li et al., 2004;
Raz and Reichman-Fried, 2006;
Chong et al., 2007;
Miyasaka et al., 2007).
Moreover, a recent report has shown that Sdf1/Cxcr4 signaling regulates
mesendodermal cell migration during Xenopus gastrulation
(Fukui et al., 2007). In
Xenopus embryos, Sdf1
in the ectodermal layer attracts the
Cxcr4-expressing mesendodermal cells
(Fukui et al., 2007). However,
the molecular mechanisms underlying mesendodermal cell migration regulated by
Sdf1/Cxcr4 signaling have not been analyzed at single-cell resolution.
In this study, we have focused on the molecular mechanisms underlying the control of endoderm migration by Sdf1/Cxcr4 signaling during gastrulation in zebrafish. We show that sdf1a- and sdf1b-expressing mesodermal cells control the movement of cxcr4a-expressing endodermal cells. The directional migration of endodermal cells during gastrulation is inhibited by knockdown of either cxcr4a or sdf1a/sdf1b (collectively referred to as sdf1) using morpholino antisense oligonucleotides. We also show that misexpressed Sdf1 acts as a chemoattractant for cxcr4a-expressing endodermal cells. To further analyze endoderm migration at single-cell resolution, we generated an endoderm-specific transgenic line carrying an EGFP reporter driven by a sox17 promoter fragment, Tg(sox17:EGFP). Using this system, we found that the Sdf1/Cxcr4a signaling pathway regulates both the formation and orientation of the filopodial processes in endodermal cells. The accumulation of phosphoinositide 3,4,5-trisphosphate (PIP3), which is known to occur at the leading edge of migrating cells, is not observed at the filopodia of endodermal cells. Taken together, our results suggest that sdf1-expressing mesodermal cells, which overlie the endodermal layer, guide the cxcr4a-expressing endodermal cells to the dorsal side of the embryo during gastrulation, possibly through a PIP3-independent pathway in zebrafish.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). Embryos
were incubated in 1/3 Ringer's solution (39 mM NaCl, 0.97 mM KCl, 1.8 mM
CaCl2, 1.7 mM HEPES, pH 7.2) at 28.5°C and staged according to
Kimmel et al. (Kimmel et al.,
1995). The cass4 mutant allele
(Kikuchi et al., 2001) was
used and genotyping of homozygote cas mutant embryos was performed as
described previously (Kikuchi et al.,
2001).
To generate the Tg(sox17:EGFP) line, a 5.0 kb region of the
sox17 gene promoter was cloned as described previously
(Reim et al., 2004), and then
subcloned into pEGFP-1 (Clontech) to generate pSox17:EGFP. The DNA fragment
carrying the sox17 promoter and EGFP was isolated and
diluted to 100 ng/µl in distilled water containing 0.1% Phenol Red. The
resulting DNA solution was then injected into the blastomere of one-cell stage
zebrafish embryos. Embryos showing fluorescent signals were raised to sexual
maturity and founder fish were subsequently selected by the expression of
EGFP-fluorescence in their progeny. Only one stable transgenic line was
obtained using this procedure, designated Tg(sox17:EGFP).
Whole-mount in situ hybridization, β-galactosidase staining and histological analysis
Whole-mount in situ hybridization was performed using conventional nitro
blue tetrazolium (NBT)/5-bromo-4-chloro-3-indolyl phosphate (BCIP)
precipitation by alkaline phosphatase
(Westerfield, 1995). cDNA
fragments for cxcr4a (Chong et
al., 2001), sdf1a
(Doitsidou et al., 2002),
sdf1b (Li et al.,
2004), sox17
(Alexander and Stainier, 1999),
foxa3 (Odenthal and
Nüsslein-Volhard, 1998), ceruloplasmin (cp)
(Korzh et al., 2001),
insulin (ins), pdx1
(Milewski et al., 1998),
hatching gland gene 1 (hgg1; ctsl1b - ZFIN)
(Thisse et al., 1994),
distal-less 3 (dlx3)
(Akimenko et al., 1994), no
tail (ntl) (Schulte-Merker
et al., 1994) and myoD (myod1 - ZFIN)
(Weinberg et al., 1996) were
utilized as templates for the antisense probes. β-Galactosidase staining
and double staining with whole-mount in situ hybridization were performed as
described previously (Mizoguchi et al.,
2006).
For histology, the NBT/BCIP-stained in situ embryos were dehydrated,
embedded in Technovit 7100 (Heraeus Kulzer, Wehrheim, Germany) and cut at 10
µm. The sectioned in situ samples were imaged by confocal laser-scanning
reflection microscopy, a newly developed technique that visualizes NBT/BCIP
staining by reflection of the confocal laser beam
(Jékely and Arendt,
2007). The reflection images were taken with an Olympus FV1000D
confocal microscope using 473 nm and 635 nm lasers, and a 40x
oil-immersion lens.
mRNA and morpholino injections
The pCS2+ vector carrying cDNA fragments encoding cas, nuclear
lacZ (nlacZ), sdf1a and sdf1b were used in
this study. For pleckstrin homology (PH)-mCherry
mRNA synthesis, a gene encoding the PH domain of protein kinase B
(Watton and Downward, 1999)
was fused to the N-terminus of the mCherry gene (Clontech)
(Shaner et al., 2004) to
generate pCS2-PH-mCherry. Capped mRNA was synthesized using the SP6
mMESSAGE mMACHINE system (Ambion). Injection of nlacZ mRNA (200 pg)
or co-injection of cas (50 pg) and nlacZ (200 pg) mRNAs was
performed into one blastomere at the 16-cell stage. PH-mCherry mRNA
(250 pg) was injected into one-cell stage embryos.
Injections of morpholino antisense oligonucleotides (MOs) (Gene Tools) were
carried out as described (Nasevicius and
Ekker, 2000). We designed two MOs to block translation initiation
of cxcr4a mRNA (cxcr4aMO1,
5'-CGATGTGTCCGTAATAAGCCATCTC-3', sequence complementary
to the predicted start codon is underlined; cxcr4aMO2,
5'-CCTTCAGTCTCCAGCAAGTCTTCAG-3') and a control MO for
cxcr4aMO1 with five mispaired bases (misMO1,
5'-CcATGTcTCCcTAATAAcCCATgTC-3', lowercase letters indicate
mispaired bases). For the double knockdown of sdf1a and
sdf1b (sdf1), we co-injected the sdf1aMO
(5'-CTACTACGATCACTTTGAGATCCAT-3')
(Doitsidou et al., 2002) and
the sdf1bMO (5'-CGCTACTACTTTGCTATCCATGCCA-3')
(Knaut et al., 2003). All MOs
were injected into one-cell stage embryos, 5 ng each. Injection of
cas mRNA (2 pg) or co-injection of cas mRNA (2 pg) and
cxcr4aMO1 (0.1 ng) with dextran plus Alexa Fluor 594
(dextran-Alexa594; Invitrogen) as a lineage tracer was performed into one
blastomere at the 128-cell stage.
Live imaging and confocal time-lapse imaging
Tg(sox17:EGFP) transgenic embryos were dechorionated at the
one-cell stage using 0.2 mg/ml pronase in 1/3 Ringer's solution and were
raised to the three-somite stage or for 24 hours post-fertilization (hpf). The
embryos were oriented in 3% methylcellulose/1/3 Ringer's solution.
Low-magnification images of Tg(sox17:EGFP) transgenic embryos were
obtained with an Olympus SZX-RFL3 microscope and Penguin 600CL cooled CCD
camera (Pixera).
Confocal time-lapse imaging was performed on an Olympus FV1000 confocal microscope using a 60x or a 100x oil-immersion lens. Tg(sox17:EGFP) embryos were dechorionated at the one-cell stage also using 0.2 mg/ml pronase in 1/3 Ringer's solution and were raised to 90% epiboly. These embryos were mounted in 0.7% low-melting-temperature agarose in 1/3 Ringer's solution. We recorded 60 z-stacks (1.25 µm steps) at 30-second intervals and analyzed the images using ImageJ software (NIH). The orientation of filopodial processes was analyzed using Origin8 software (LightStone, Japan).
Cell transplantation
nlacZ (2 ng), sdf1a (3 ng) or sdf1b (2 ng) mRNA
was injected together with dextran-Alexa594 into donor wild-type embryos and
sdf1MOs were injected into host Tg(sox17:EGFP) embryos at
the one-cell stage. Cells were extracted from donor embryos at the dome stage
(4.3 hpf) and injected into the ventrolateral side, close to the margin, of
shield-stage (6 hpf) hosts using standard methods
(Westerfield, 1995). The
time-lapse images were taken with an Olympus FV1000D confocal microscope using
a 20x lens.
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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; Li et al.,
2004; Raz and Reichman-Fried,
2006; Chong et al.,
2007; Miyasaka et al.,
2007), we next examined the expression pattern of cxcr4a,
sdf1a and sdf1b during gastrulation. We confirmed that, as
previously reported (Chong et al.,
2001), cxcr4a is maternally expressed and its zygotic
expression is restricted to a subset of blastomeres around the margin at
shield stage (6 hpf) (data not shown). The restricted expression of
cxcr4a is maintained during gastrulation and disappears by the
three-somite stage (Chong et al.,
2001) (data not shown). As this characteristic salt-and-pepper
expression pattern of cxcr4a is remarkably similar to that of both
cas and sox17, we next examined whether cxcr4a is
expressed in endodermal cells. The salt-and-pepper expression pattern of
cxcr4a was completely abolished in cas mutants, whereas its
expression in other tissues was unaffected in cas mutants
(Fig. 1A,B). Moreover, an
ectopic expression pattern of cxcr4a was observed in cas
mRNA-misexpressing cells (Fig.
1C,D). These results clearly demonstrate that cxcr4a is
expressed in endodermal cells and is dependent on Cas.
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). Consistent with these published data, sdf1a- and
sdf1b-expressing mesodermal cells in the animal-lateral region were
found to move towards the dorsal midline from 80% epiboly to tail bud (TB)
stages (Fig. 1E,I,J,K,O,P; red
dashed lines). The sox17-expressing endodermal cells also moved
towards the dorsal midline during late gastrulation
(Fig. 1Q-S; red dashed lines),
and this dorsal movement behavior coincided with that of the sdf1a-
and sdf1b-expressing mesodermal cells, suggesting that the movements
of endodermal cells might be regulated by mesodermal cells.
Endoderm migration is delayed by the inhibition of cxcr4a or sdf1
To investigate the function of Sdf1/Cxcr4a signaling in the movement of the
endodermal cells, we knocked down the corresponding genes by injecting
specific MOs. The sox17-expressing endodermal cells of the
animal-lateral region moved dorsally in misMO1-injected embryos at late
gastrulation (Fig. 2Aa;
arrowheads). By contrast, the dorsal migration of endodermal cells was delayed
in cxcr4aMO1-, cxcr4aMO2- or sdf1MOs-injected
embryos (Fig. 2Ab-Ac;
arrowheads). Throughout this study, we compared the phenotypes of non-injected
and misMO1-injected embryos, and observed no differences between them (data
not shown).
In order to visualize the movements of the endodermal cells in live
zebrafish embryos, we generated the Tg(sox17:EGFP) transgenic line.
Although the endodermal expression of sox17 begins soon after the
onset of gastrulation (Alexander and
Stainier, 1999), the earliest detectable EGFP fluorescence in the
endodermal cells of the transgenic embryos is from the 60% epiboly (7 hpf)
stage, and this fluorescence continues until 2 days post-fertilization (see
Movies 1 and 2 in the supplementary material; data not shown). Using this
transgenic line, the delay in the endodermal dorsal migration caused by
knockdown of cxcr4a or sdf1 could be clearly seen during
early somitogenesis (Fig.
2Ba-Bd) and this lead to a bifurcation of the gut in the foregut
region at 24 hpf (Fig. 2Ca-Cd;
arrows). Since Sdf1a and Sdf1b are two distinct ligands in zebrafish, we
compared the phenotypes of single- and double-knockdown embryos. Although the
affect of sdf1b knockdown on the dorsal migration of endodermal cells
was a little stronger than that of sdf1a knockdown, single-knockdown
embryos showed only a mild phenotype compared with the double-knockdown
embryos (see Fig. S2 in the supplementary material).
|
)],
cp (marker of liver), pdx1 (marker of pancreas) and
ins (marker of β-cells in pancreas). The anterior part of the
gut was lost from cxcr4aMO1-, cxcr4aMO2- or
sdf1MOs-injected embryos, as assessed by foxa3
(Fig. 2Db-Dd; arrows).
Consistent with this abnormal foxa3 expression pattern, liver was not
present in these morphant embryos, as assessed by cp
(Fig. 2Ea-Ed; arrowheads).
Moreover, the expression of pdx1 and ins revealed that the
pancreas in these morphant embryos is small and improperly assembled, and that
the endocrine cells of the pancreas do not cluster
(Fig. 2Fa-Gd: black arrowheads
in Fa-Fd; white arrowheads in Ga-Gd). Together, the results of these knockdown
experiments suggest that Sdf1/Cxcr4a signaling is involved in the regulation
of endodermal cell movement. This idea is further supported by our observation
that the abnormalities in endoderm migration and organ formation are
indistinguishable between the cxcr4a and sdf1 knockdown
embryos. Despite the defects in endodermal cell migration, both the levels of
sox17 expression and the number of sox17-expressing cells at
the 60% epiboly stage were found to be normal in misMO1-, cxcr4aMO1-
or sdf1MOs-injected embryos by in situ analyses using sox17
as a probe (data not shown), indicating that early endoderm specification is
unaffected by suppression of Sdf1/Cxcr4a signaling.
Sdf1 acts as a chemoattractant for cxcr4a-expressing endodermal cells
It is now well established that Sdf1 acts as a chemoattractant for
cxcr4-expressing cells (Busillo
and Benovic, 2007). To test the impact of zebrafish Sdf1 on the
migration of endodermal cells, we examined the consequences of transplanting
nlacZ-, sdf1a- or sdf1b-expressing cells into
sdf1MOs-injected embryos. The EGFP-expressing endodermal
cells were found to be attracted by the cluster of sdf1a- or
sdf1b-expressing cells (Fig.
3; see Movies 3, 4 and 5 in the supplementary material),
indicating that both zebrafish Sdf1a and Sdf1b function as a chemoattractant
for cxcr4a-expressing endodermal cells. A higher dose of
sdf1a mRNA (3 ng) was required to achieve an effect on the endodermal
cells as compared with sdf1b mRNA (2 ng), suggesting that the
chemoattractant effect of Sdf1b for endodermal cells is stronger than that of
Sdf1a, although it is difficult to quantify such differences. Moreover, a
higher density of EGFP-expressing endodermal cells was observed
around the transplanted sdf1-expressing cells
(Fig. 3G,J; see Movies 4 and 5
in the supplementary material). These transplantation experiments were
repeated at least five times for each of the three genes (nlacZ,
sdf1a and sdf1b), and we obtained consistent results for each
experiment. We have already shown that sdf1a and sdf1b are
expressed in mesodermal cells and that cxcr4a is expressed in
endodermal cells. The endodermal cells occupy the deepest cell layer next to
the extra-embryonic YSL, and the mesodermal cell layer lies above the
endodermal cells, both within the hypoblast layer (see Fig. S1 in the
supplementary material) (Warga and
Nüsslein-Volhard, 1999). These data, combined with the
results of our MO-knockdown experiments, suggest that sdf1-expressing
mesodermal cells might attract endodermal cells during gastrulation.
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|
). The
shape and position of the prechordal plate and neural plate, the gap between
prechordal plate and notochord, and body length were found to be normal in
Sdf1/Cxcr4a signaling-inhibited embryos
(Fig. 4), thereby indicating
that the Sdf1/Cxcr4a signaling pathway does not govern the C&E movements
of either mesodermal or ectodermal cells during gastrulation.
To test whether Sdf1/Cxcr4a signaling directly regulates endodermal cell
movements, we knocked down the function of Cxcr4a specifically within
endodermal cells. This was achieved by injecting cxcr4aMO1 together
with cas mRNA, because cas overexpression causes marginal
blastomere cells to adopt an endodermal fate
(Kikuchi et al., 2001). Using
this endoderm-specific knockdown method, the influence of Cxcr4a function in
mesoderm and ectoderm could be completely excluded. The endodermal cells
exhibited a significant delay in their migration following the co-injection of
cxcr4aMO1 and cas mRNA as compared with an injection of
cas mRNA alone (Fig.
5). The expression pattern and level of sdf1a and
sdf1b were unchanged in these experiments (data not shown). These
data therefore suggest that the Sdf1/Cxcr4a signaling pathway
cell-autonomously regulates the movements of the endodermal cells without
affecting the C&E movements of the mesoderm and the ectoderm.
The Sdf1/Cxcr4a signaling pathway controls the morphology of endodermal cells
After involution, endodermal cells gradually flatten and extend their
filopodial processes, and then form a noncontiguous inner layer of cells
adjacent to the yolk (Warga and
Nüsslein-Volhard, 1999). To examine whether the shape of the
endodermal cells is altered when Sdf1/Cxcr4a signaling is inhibited, we
carefully observed the shape and movement of individual endodermal cells in
the lateral region of zebrafish embryos by confocal laser microscopy.
Consistent with this previous report, the endodermal cells that we tracked
exhibited many filopodial processes during their migration in live
Tg(sox17:EGFP) transgenic embryos
(Fig. 6A; see Movie 6 in the
supplementary material). We found that the formation of these filopodia was
suppressed when the function of either cxcr4a or sdf1 was
inhibited (Fig. 6B,C; see
Movies 7 and 8 in the supplementary material). Our statistical analyses
indicated that endodermal cells in either cxcr4a or sdf1
knockdown embryos exhibited a more rounded shape than their counterparts in
misMO1-injected embryos (Fig.
6D). We next examined the orientation of the filopodial processes
in the endodermal cells and found that more than 70% of the filopodia were
oriented in the direction of migration (from 0° to ±90°) in the
misMO1-injected embryos (Fig.
6F). By contrast, the number of filopodial processes occurring in
the direction of migration was reduced in the cxcr4aMO1- or
sdf1MOs-injected embryos (Fig.
6F). In addition to the formation of filopodia, trajectory
analyses of endodermal cells showed that they migrate almost straight towards
the dorsal side in the misMO1-injected embryos
(Fig. 6G), whereas they make
many turns, and move in different (sometimes reverse) directions in both the
cxcr4a and sdf1 knockdown embryos
(Fig. 6H,I). These results
suggest that the reduced number and randomized orientation of filopodial
processes that follow the inhibition of Sdf1/Cxcr4a signaling result in a
suppression of the directional migration of endodermal cells. Furthermore, we
found that the total speed of migration of endodermal cells is not
significantly different when Sdf1/Cxcr4a signaling is inhibited (data not
shown). Therefore, it appears that the reduced level of directional migration
is the major contributor to an overall delay in endodermal cell migration to
the dorsal midline during gastrulation.
|
).
Locally activated PI3K produces PIP3 at the leading edges of the
chemotactic cells, and this accumulation of PIP3 leads to F-actin
polymerization at these leading edges
(Sasaki and Firtel, 2006). We
observed that F-actin is strongly localized at the plasma membrane and
filopodia of endodermal cells in WT embryos (data not shown). We further found
that co-injection of mRNA encoding a dominant-negative form of PI3K
(dnPI3K) (Montero et al.,
2003) and cas mRNA does not affect the migration of
endodermal cells during gastrulation (see Fig. S3 in the supplementary
material). We next tested whether PIP3 accumulates in mesendodermal
cells by expressing the Pleckstrin homology (PH) domain of protein kinase B,
which binds with high affinity to this molecule
(Watton and Downward, 1999;
Montero et al., 2003). The
PH-mCherry fusion protein was strongly localized at the plasma membrane in
mesodermal cells (Fig. 7A),
whereas the localization of this exogenous product in endodermal cells was
predominantly cytoplasmic (Fig.
7B-D). Taken together, these results suggest that the Sdf1/Cxcr4a
signaling pathway controls the formation and orientation of the filopodial
processes of endodermal cells, possibly through a PIP3-independent
pathway, and that an abnormal filopodia phenotype results in the impairment of
the directional migration of endodermal cells. |
DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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14-24% reduction at the 90% epiboly stage by in
situ hybridization using sox17 as a probe (data not shown). These
data suggest that Sdf1/Cxcr4a signaling might be involved in the
proliferation, but not specification, of endodermal cells. Since it is well
known that Sdf1/Cxcr4 signaling promotes the growth of primary tumors
(Luker and Luker, 2006),
excess Sdf1 secreted from the transplanted cells might enhance the
proliferation of endodermal cells. It will be interesting to analyze the role
of Sdf1/Cxcr4a signaling in the proliferation of endodermal cells during
gastrulation. In Sdf1/Cxcr4a signaling-inhibited embryos, the anterior part of gut tube and liver are lost, and the pancreas is small and improperly assembled (Fig. 2). It appears that the delay of endodermal cell migration caused by inhibition of Sdf1/Cxcr4a signaling results in defects in endodermal organ patterning. Another possible explanation is that Sdf1/Cxcr4a-dependent movement might be necessary for the differentiation or patterning of the endodermal organs at the somitogenesis stage. Further analyses will be required to elucidate the function of Sdf1/Cxcr4a signaling in endodermal organ formation.
|
). Having first adopted a tear-drop shape, the endodermal
cells gradually flatten, lose their polar appearance and develop filopodial
processes (Warga and
Nüsslein-Volhard, 1999). Our confocal time-lapse analysis of
Tg(sox17:EGFP) transgenic embryos is consistent with this report. We
show that endodermal cells in the zebrafish gastrula have many filopodial
processes during dorsal migration, and that their morphology is regulated by
Sdf1/Cxcr4a signaling. Recently, the chemokine Apelin and its receptor Agtrl1b
(Aplnrb - ZFIN) were shown to regulate the protrusion of filolamellipodia and
the movement of lateral mesodermal cells during zebrafish gastrulation
(Scott et al., 2007;
Zeng et al., 2007). Together,
these results suggest that chemokine-GPCR signaling controls the movements of
mesendodermal cells through regulation of their morphology. We propose that
endodermal cells develop filopodia so that they can assess the correct
direction in which to migrate. This idea is supported by the fact that more
than 70% of the filopodial processes are orientated in the direction of
migration. It will be interesting to analyze the roles of the filopodial
processes that are oriented independently of the direction of migration.
|
).
We found that the distribution of endodermal cells at 60% epiboly (identified
by in situ hybridization with a cas riboprobe) is wild-type in
sdf1/cxcr4a knockdown embryos (data not shown), suggesting
that the random walk upon internalization is independent of Sdf1/Cxcr4a
signaling. Moreover, we found that the movement and directional migration of
endodermal cells in late gastrulae is impaired but not completely lost as a
result of inhibition of Sdf1/Cxcr4a signaling. There are two possible
explanations for this. One is that another factor shares redundancy of
function with either Sdf1 or Cxcr4a. Alternatively, the migration of
endodermal cells might be controlled by additional systems acting in concert
with Sdf1/Cxcr4a signaling. To date, two signaling pathways, Wnt/PCP signaling
and Vascular endothelial growth factor C (Vegfc) signaling, have been
implicated in the regulation of endodermal cell migration
(Ober et al., 2004;
Matsui et al., 2005).
Genetic and molecular evidence has accumulated to demonstrate that the
Wnt/PCP signaling pathway regulates the C&E movements of mesodermal cells
during vertebrate gastrulation (Myers et
al., 2002). Recently, the role of Wnt/PCP signaling in C&E
movements in endodermal cell migration was also reported in zebrafish embryos
(Matsui et al., 2005).
Overexpression of a dominant-negative form of Dishevelled or combinatorial
inhibition of Wnt ligands leads to a split gut and bilateral endodermal
organs, suggesting that the Wnt/PCP signaling pathway is required for
migration of endodermal cells toward the dorsal midline
(Matsui et al., 2005). It is
possible that the requirement of Wnt/PCP signaling for endodermal cell
migration occurs during two developmental stages: gastrulation and
somitogenesis. We found that the migration of endodermal cells is marginally
delayed when the Wnt/PCP signaling pathway is inhibited exclusively in
endodermal cells during gastrulation (T.M. and Y.K., unpublished). In addition
to gastrulation, wnt4a and wnt11-related begin to be
strongly expressed during somitogenesis in the neural ectoderm and
mesendoderm, and the knockdown of three Wnt ligands (wnt4a,
wnt11-related and wnt11) inhibits the migration of endodermal
cells toward the dorsal midline (Matsui et
al., 2005). These data, combined with our unpublished
observations, suggest that Wnt/PCP signaling is partly involved in endodermal
cell migration during gastrulation and is required for this process during
somitogenesis.
A previous study revealed that the Vegfc signaling pathway regulates the
migration of endodermal cells in zebrafish embryos
(Ober et al., 2004). Knockdown
experiments have demonstrated that Vegfc signaling is required for two
distinct steps during endoderm development, the first being the initial
differentiation of the dorsal endoderm, and the second involving the
coalescence of the anterior endoderm with the dorsal midline
(Ober et al., 2004).
vegfc knockdown embryos show a splitting of the anterior gut tube, in
the most severe cases accompanied by a duplication of the liver and pancreatic
bud (Ober et al., 2004). Taken
together, we speculate from the current evidence that either the Wnt/PCP or
Vegfc signaling pathway partially compensates for the movement and directional
migration of endodermal cells in sdf1/cxcr4a knockdown
embryos.
Roles of PI3K and PIP3 in endodermal cell migration
Sdf1 stimulation through Cxcr4 locally activates Ras at the leading edge of
the migrating cell (Busillo and Benovic,
2007). PI3K is well established as one of the downstream effectors
of Ras signaling (Sasaki and Firtel,
2006). Moreover, PIP3 accumulation by PI3K activity
occurs exclusively at the leading edge, and its local accumulation leads to
the assembly of a branched network of actin filaments
(Sasaki and Firtel, 2006).
Recent reports have shown that the migration of the primordial germ cells
(PGCs) is guided by Sdf1 in zebrafish (Raz
and Reichman-Fried, 2006). Zebrafish PGCs exhibit a uniform
distribution of PIP3 on their membrane and depletion of
PIP3 in these cells has no effect on their ability to migrate in
the correct direction (Dumstrei et al.,
2004). In addition, a more recent study has revealed that cellular
calcium is required for proper migration and formation of bleb-like
protrusions in PGCs (Blaser et al.,
2006). These results suggest that the PIP3-independent
mechanisms could regulate the directional migration of these cells. In
addition, Dictyostelium cells in which PIP3 production is
reduced remain responsive to directional cues
(Sasaki and Firtel, 2006). In
our current study, we find that the migration of endodermal cells is regulated
by Sdf1 from mesodermal cells and that F-actin is localized to the filopodial
processes in migrating endodermal cells. However, the migration of endodermal
cells is not affected by misexpression of dnPI3K, and a significant
accumulation of PIP3 at the filopodia is not observed in endodermal
cells as compared with their mesodermal counterparts. These data suggest that
F-actin polymerization might be controlled in a PIP3-independent
manner in endodermal cells. Future analyses of the F-actin polymerization
mechanisms that regulate Sdf1/Cxcr4a signaling in the filopodia of endodermal
cells should further our understanding of the molecular events underlying
their directional migration.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/15/2521/DC1
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ACKNOWLEDGMENTS |
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Footnotes |
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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A/p2, where
A=area and p=perimeter. Note that the differences between the misMO1 and
cxcr4aMO1 values, as well as the differences between the misMO1 and
sdf1MOs values, are statistically significant
(*P<0.01, Student's t-test). Error bars
represent the standard error. (E) Schematic representation of the
methods used to measure the angles of the filopodial processes relative to the
direction of migration of the endodermal cells. ±180° indicates
opposing the direction of migration. (F) Rose diagrams representing the
orientation of filopodial processes relative to the direction of migration.
The orientation of the filopodia from 4-5 randomly chosen cells was measured
at two consecutive time points (time interval, 10 minutes) from 9-10
independent movies in misMO1- (n=100 cells), cxcr4aMO1-
(n=99 cells) and sdf1MOs- (n=87 cells) injected
embryos. (G-I) The migration tracks of 10-11 individual endodermal
cells over a period of 30 minutes. The direction of migration is to the right
and the cell position was determined every 30 seconds. Scale bars: 20
µm.