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First published online 13 August 2008
doi: 10.1242/dev.020396
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1 Department of Anatomy and Developmental Biology, University College London,
Gower Street, London, WC1E 6BT, UK.
2 Max Planck Institute of Molecular Cell Biology and Genetics,
Pfotenhauserstrasse 108, 01307 Dresden, Germany.
* Author for correspondence (e-mail: m.tada{at}ucl.ac.uk)
Accepted 8 July 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: Gastrulation, Cell movement, Wnt, S1P, Pdgf, Zebrafish
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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; Solnica-Krezel,
2005).
In Xenopus, once the mesoderm has involuted, the most anterior
dorsal mesodermal cells migrate directionally toward the animal pole across
the blastocoel roof of the ectoderm using fibronectin as a substrate
(Nagel and Winklebauer, 1999).
In zebrafish, the first internalised axial hypoblast cells that become fated
to the prechordal plate move as a cohesive sheet of cells toward the animal
pole of the embryo using the epiblast as a substrate
(Montero et al., 2005;
Ulrich et al., 2003). The
cohesive property of prechordal plate progenitor cells is thought to provide a
mechanism for effective directed migration
(Ulrich et al., 2005), and is
reminiscent of the collective migration seen in border cells in
Drosophila and in lateral line progenitor cells in zebrafish
(reviewed by Lecaudey and Gilmour,
2006; Rorth,
2007). However, the mechanism underlying this process remains
poorly understood, in particular is collective migration really important or
can cells migrate as individuals?
The identification of directional cues for the anterior migration of
prechordal plate progenitor cells has fascinated developmental biologists for
several decades. The best characterised is platelet-derived growth factor
(Pdgf) in Xenopus, as Pdgf signalling is required for the orientation
of cells toward the animal pole and for their directionality
(Nagel et al., 2004). By
contrast, Pdgf and its key intracellular transducer PI3K are necessary for
cell polarisation and motility, but not for directionality, in zebrafish
(Montero et al., 2003).
Despite the migration defects in embryos with compromised Pdgf pathway
activities, prechordal plate progenitor cells are largely still capable of
undergoing directed migration in Xenopus and zebrafish, suggesting
the existence of another cue(s) in this process.
What are the genetic pathways involved in the regulation of coherence of
cells during anterior migration of the presumptive prechordal plate?
E-cadherin and Rab5-mediated endocytosis regulate the cell coherence of
prechordal plate progenitors, and these are mediated by Wnt11
(Ulrich et al., 2005), a
member of the non-canonical Wnt/planar cell polarity (PCP) pathway (reviewed
by Tada et al., 2002;
Seifert and Mlodzik, 2007).
Indeed, silberblick (slb)/wnt11 mutant embryos
exhibit reduced anterior migration of prechordal plate progenitor cells
(Ulrich et al., 2005). The
expression of E-cadherin (cdh1) in the presumptive
prechordal plate is regulated by snail genes that are required for
anterior migration of the prechordal plate in zebrafish
(Blanco et al., 2007).
Furthermore, Liv1 (Slc39a6 - Zebrafish Information Network), a zinc
transporter, controls nuclear localisation of Snail, and is required for
anterior migration of the prospective prechordal plate during zebrafish
gastrulation (Yamashita et al.,
2004). Moreover, the secreted Wnt antagonist Dkk1 modulates
anterior migration of mesodermal cells by interacting with Knypek/Glypican4, a
cofactor for Wnt11, in zebrafish (Caneparo
et al., 2007).
The bioactive lipid, sphingosine-1-phosphate (S1P), is a signalling
molecule that acts through binding to a family of seven-pass transmembrane,
G-protein-coupled receptors (S1PRs) that have been implicated in the
regulation of cytoskeletal rearrangements, cell motility and cell adhesion in
a variety of cell types (reviewed by
Spiegel and Milstien, 2003).
In addition to the role for S1P as a ligand, it acts as a second messenger
within the cell, and its intracellular levels are regulated by the balance
between its production by Sphingosine kinases and its degradation by S1P
lyases (reviewed by Alvarez et al.,
2007). However, little is known about the functions of S1P and its
receptors in regulating directed cell migration in the embryo.
In this study, we sought to search for a novel genetic pathway that
controls the collective migration of prechordal plate progenitor cells at the
onset of zebrafish gastrulation. In our morpholino (MO)-based screen, we have
identified miles apart (mil) as a genetic suppressor of
defective migration of prechordal plate cells in slb/wnt11 mutant
embryos. mil was initially isolated as a heart mutant with a cardia
bifida phenotype, and it encodes a S1P receptor, Edg5, also called S1pr2
(Kupperman et al., 2000). We
analysed the cell behaviour of the presumptive prechordal plate based on DIC
time-lapse movies of the living zebrafish gastrula, and found that, in
slb embryos injected with mil-MO, the cells migrated with
increased motility but decreased directionality, without restoring the
coherence of cell migration in slb embryos. Furthermore, we showed
that Mil controls cell motility through the Pdgf/PI3K pathway but modulates
individual cell behaviours underlying cell coherence separately from this
pathway. These results highlight the unexplored role of the motility and
coherence of individual cells, regulated by the Mil/S1P signal, in the
directed migration of prechordal plate progenitors.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). The
allele we used was homozygous slbtx226, which had been
selected for slb carriers that consistently showed the defective
movement of the prechordal plate, and slb mutant embryos were
obtained from in-crossing of the homozygotes. The embryos were kept at
28.5°C for time-lapse analysis or at 31°C for in situ
hybridisation.
Microinjections and constructs
The sequences of morpholinos used in this study were: mil-MO,
5'-CCGCAAACAGAGCCGAACTAGTCAT-3' (ATG-MO)
(Matsui et al., 2007); and
edg1-MO, 5'-TTAGGTCATCCATGGTTTGCACTGG-3' (ATG-MO).
mil-MO and edg1-MO were, respectively, injected at
concentrations of 4.3 ng/nl and 6.2 ng/nl. mil-MO injection
phenocopied the cardia bifida condition in the mil mutant (87.5%,
n=56). To validate the specificity of the edg1-MO, RNA
encoding GFP tagged with amino acid sequences that include the sequence
corresponding to the edg1-MO, or irrelevant sequences (ILK) as a
negative control, was co-injected with edg1-MO, and GFP
expression was examined at dome stages. GFP expression from the
construct containing the sequence corresponding to the edg1-MO was
suppressed (GFP-positive: 0%, n=19), whereas GFP
expression was retained in the negative control (GFP-positive: 100%,
n=36). Together with the ability of the edg1-MO to cancel
the edg5-morphant phenotype when co-injected (see
Fig. 6B,E,H to compare with
Fig. 1B,F,I), these data
validate the specificity of the edg1-MO.
For overexpression studies, the following constructs were used:
pCS2-dn-PI3K, pCS2-p110CAAX and pCS2-PH-GFP [all described previously by
Montero et al. (Montero et al.,
2003)]. As for the mil construct, a PCR-amplified
full-length fragment from a gastrula library was cloned into pCS2+, and RNA
for injection was made after linearisation with NotI. All of the RNAs
were synthesised in vitro essentially as described
(Smith, 1993).
In situ hybridisation
Antisense RNA probes were synthesised with a digoxigenin RNA-labelling kit
(Roche), using plasmids containing cDNA for ntl
(Schulte-Merker et al., 1994),
hgg1 (Thisse et al.,
1994), dlx3 (Akimenko
et al., 1994), hlx1
(Fjose et al., 1994) and
sprouty4 [(Furthauer et al.,
2004) originally published as sprouty2]. Whole-mount in
situ hybridisation was carried out as described previously
(Barth and Wilson, 1995).
Time-lapse imaging of embryos
For time-lapse imaging, embryos were manually dechorionated at the shield
stage and mounted in 0.8% low-melting-point agarose (Sigma) in embryo medium.
For DIC observation, images were taken at 1 frame per minute for 1 hour, with
approximately 18 z-levels spaced 3 µm apart, using a 40x
water immersion lens on an Axioplan 2 (Zeiss) compound microscope and a
Hamamatsu Orca ER digital camera. Two-photon microscopy was performed as
described by Montero et al. (Montero et
al., 2003).
Cell movement analysis
The positions of five randomly chosen cells were plotted every minute,
using OpenLab5.0 software (Improvision). The `speed' of a cell was measured as
µm/minute using gross length of migration. The `persistence' of a cell was
calculated by the quotient of net migration per gross migration over every 14
minutes. Assuming that cells change their relative positions more dynamically
in less coherent conditions, the `coherence-1' of migration of a
group of cells was defined as the rate of change in cell-cell distance per
length of cell migration. Here, the change of distance between two cells was
determined, then normalised for each cell by dividing by its net migration
(see Fig. S1 in the supplementary material). This calculation was applied to
all 10 combinations among five cells, every 9 minutes. For each experimental
condition, three to five embryos from independent experiments were analysed.
To test the significance between two mean values, Welch's t-test
based on an unequal variance was applied and described as
`mean±s.e.m.'.
Analysis of cell shape
Approximately 25 cells at the leading edge from three to five embryos were
randomly chosen from time-lapse images, and, of those, cells with a
distinctive cellular protrusion were used for analysis with ImageJ software.
The `length' and `angle' of the largest protrusion were determined by assuming
a line from the centre of the nucleus to the tip of the protrusion: the length
being its length in µm, and the angle being measured from the direction of
general migration (i.e. the upper-vertical axis). Here, the mean and standard
deviation of angles are calculated in cosine, as measured anticlockwise from
the upper-vertical axis. White dots indicate the central position of the
nucleus, and green lines show the length and angle of a protrusion. Red `fans'
represent the average length and angle (in degrees) of the protrusions,
including their standard deviations (respectively depicted by its height and
open angle). To test the significance in the length between two mean values,
Welch's t-test based on an unequal variance was applied and described
as `mean±s.e.m.'.
Cell culture
Cell culture experiments were carried out according to Montero et al.
(Montero et al., 2003) with
slight modification. In brief, 50 embryos injected with 100 pg
cyclops RNA together with the RNA(s) indicated in each experiment
were manually dechorionated at the dome stage, and dissociated in L-15 medium
containing 1xtrypsin-EDTA (Biowhittaker). After stopping the enzymatic
reaction by the addition of chick serum, cells were harvested by
centrifugation at 150 g for 2 minutes and re-suspended in 2.5
ml of fresh L-15 medium, containing 1 mg/ml insulin, 0.3 mg/ml L-glutamine,
100 U/ml penicillin and 100 µg/ml streptomycin. Cells were then cultured
for 1 hour at 25°C on a plastic Petri dish coated with 50 µg/ml
fibronectin (Sigma), prior to treatment with 50 ng/ml Pdgf-AA (Sigma).
Fluorescence intensity analysis
The grey value along a line of 3-pixel weight was measured using ImageJ
software. The x-axis of the graph represents the distance, while the
y-axis indicates the relative signal intensity (as normalised by
total intensity), along the line.
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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; Ulrich et al.,
2005). In order to identify genetic cascades that regulate
anterior migration of the prospective prechordal plate, we have undertaken a
candidate approach in which morpholino antisense oligonucleotides (MOs) are
injected into slb homozygous embryos as a sensitised background to
detect either enhancement or suppression of the slb phenotype. During
the course of this approach, we identified miles apart (mil)
as a suppressor of the slb phenotype. mil was originally
isolated as a mutant exhibiting a bifurcated heart, and encodes a
sphingosine-1-phosphate (S1P) receptor, Edg5
(Kupperman et al., 2000). When
the position of the anterior prechordal plate was visualised with respect to
the anterior edge of the neural plate at the end of gastrulation, the
presumptive prechordal plate of the slb embryo was located more
posteriorly than that of the WT embryo
(Fig. 1A,C,E,G)
(Heisenberg et al., 2000).
Strikingly, the deficit in anterior movement of the slb prechordal
plate was largely rescued by injection of mil-MO
(Fig. 1C,D,G,H; 65.4%,
n=52). When WT embryos were injected with mil-MO, the shape
of the prospective prechordal plate was slightly flatter and wider, and its
positioning was slightly more anterior than in WT embryos
(Fig. 1A,B,E,F). In addition,
the anteroposterior length of the posterior prechordal plate was increased in
embryos with compromised Mil functions in WT and slb embryos
(Fig. 1I-L; compare with
Fig. 1A-D and Fig. S2 in the
supplementary material). In contrast to the prechordal plate, slb
embryos exhibit a shorter and thicker notochord than do WT embryos, implying
defective C&E movements in the presumptive notochord
(Heisenberg et al., 2000), but
this defect was not rescued by injection of mil-MO
(Fig. 1O,P).
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).
Despite the fact that axial mesodermal cells migrated faster in the
mil morphant, anterior movement of lateral mesodermal cells was
unchanged when compared to the WT embryo (90.3%, n=31;
Fig. 1M,N). These results
suggest that Mil activity is required only for prechordal plate progenitor
cells to undergo directed anterior migration, and not for other populations of
mesodermal cells.
Prechordal plate cells can migrate individually if their motility is increased and their coherence is decreased
In order to explore the cellular mechanisms by which the abrogation of Mil
function restores the reduced migration of prechordal plate progenitor cells
in slb embryos, we tracked the cells based on DIC time-lapse movies
at the onset of gastrulation, and analysed the cell behaviours that underlie
collective migration. We extracted three parameters: first, speed, by the
total length of their paths over the time; second, persistence, by the gained
distance over the total length of their paths; and third, coherence, by
changes in relative distances between pairs of cells over time for all the
combinations of five cells (a total of 10 combinations per time point).
Cell tracking analysis revealed that the reduced migration of slb
hypoblast cells is closely associated with a lower persistency in directed
migration and a reduced coherence of cell migration
(Fig. 2A-F,J; see Movies 1 and
2 in the supplementary material) (Ulrich
et al., 2005; Witzel et al.,
2006). Surprisingly, when Mil function is compromised,
slb hypoblast cells migrated faster than did WT cells, but more
randomly and with more space between the cells, thus maintaining a lower cell
cohesion than WT cells (Fig.
2G-I,J; see Movie 3 in the supplementary material). Occasionally,
we observed that an isolated single cell popped out but then travelled back
into the group of cells (Fig.
2H; observed in four movies out of five). These results indicate
that slb cells with compromised Mil function have acquired more
motility, but that their directionality and coherence remain as low as
slb cells. Consistent with this, leading edge cells of the
slb embryo with compromised Mil function formed longer lamellipodia
than did both slb cells and WT cells, but were unable to stabilise
their processes toward the direction of movement when compared with WT cells
(Fig. 3A-C).
|
; Ulrich et al.,
2005) (Fig. 4A and
Movie 8 in the supplementary material), slb cells with compromised
Mil function underwent internalisation as one or two layers of cells
(Fig. 4B and Movie 9 in the
supplementary material). Similarly, abrogation of Mil function in the WT
embryo leads to the scattered cell behaviour phenotype during internalisation,
as seen in the slb embryo with compromised Mil function (see Movie 10
in the supplementary material). Consistent with the observation from DIC
movies (Fig. 2G,H), there was
more space in between the hypoblast cells in slb embryos injected
with mil-MO than in WT embryos, confirming that prechordal plate
progenitor cells migrate individually in slb embryos when Mil
function is compromised. Despite the fact that slb cells with compromised Mil activity retain a lower coherence of cell migration to the direction of motion, they can gain the net distance as efficiently as WT cells due to their acquisition of greater motility (Fig. 5). In this extreme circumstance, cells appear be capable of migrating individually rather than in a cluster of coherent cells.
Edg1 and Mil/Edg5 reciprocally regulate directed migration of the presumptive prechordal plate
It has been shown that the two structurally related S1P receptors, Edg1
(also called S1pr1) and Edg5, are positive and negative regulators,
respectively, of cell migration in lymphocytes and endothethial cells (e.g.
Yamaguchi et al., 2003). To
test this possibility in directed migration of the prospective prechordal
plate, we first injected WT embryos with edg1-MO. Opposite to the
mil-morphant phenotype, directed migration of the anterior and
posterior prechordal plate was perturbed in edg1 morphants
(Fig. 1B,F,J and
Fig. 6A,D,G). Consistent with
their opposing activities in cultured cells, the reduced migration of
edg1-morphant prechordal plate cells was rescued by co-injection of
mil-MO (Fig. 6B,E,H).
Next, we tested whether edg1 is an enhancer of the slb
phenotype as opposed to mil being identified as a suppressor. When
edg1 function was compromised, slb embryos exhibited a more
posteriorly displaced prechordal plate but the notochord was unaffected
(Fig. 1C,G,K,
Fig. 6C,F,I, data not shown).
These results suggest that Edg1 and Mil act in a mutually antagonistic manner
to regulate directed migration of the presumptive prechordal plate, and that
Edg receptors control directed migration of prechordal plate progenitors but
not of other mesodermal cells.
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;
Nagel et al., 2004;
Symes and Mercola, 1996).
Therefore, we investigated a possible interaction of the Mil/S1P signal with
the Pdgf/PI3K pathway in directed migration of prechordal plate progenitor
cells. We hypothesised that Mil controls directed migration by negatively
regulating the Pdgf/PI3K pathway, as inhibition of the Pdgf/PI3K pathway leads
to a reduction in cell migration (Montero
et al., 2003), and this phenotype is the opposite of the
mil morphant. To test this hypothesis, we first investigated process
formation in response to Pdgf in dissociated mesendodermal cells from
zebrafish blastulae. Although mesendodermal cells formed lamellipodia-like
long processes when treated with Pdgf, Pdgf treatment of cells overexpressing
mil RNA resulted in a reduction in the frequency and length of
processes (Fig. 7A,B). Next, to
test whether Edg5 inhibits the intracellular effecter PI3K, we monitored the
membrane localisation of PH-GFP induced by Pdgf, and of membrane-RFP as a
reference. Pdgf-induced membrane localisation of PH-GFP was inhibited by the
presence of mil RNA (Fig.
7C-F). These results indicate that the Mil/S1P signal might
control cell polarisation through the Pdgf/PI3K pathway.
Mil modulates coherence of cell migration independently from Wnt11 and PI3K
The possible interaction of Mil with the Pdgf signal, as well as with
Wnt11, inspired us to identify which parameters (coherence, directionality and
motility) are regulated by Mil through or in parallel with these pathways in
the directed migration of prechordal plate progenitors. Because Mil can
modulate cell adhesion underlying coherence of cell migration in a
fibronectin-dependent manner (Matsui et
al., 2007), we hypothesised that the reason why slb cells
(when Mil function is compromised) can move faster than WT cells even though
they retain a lower coherence of cell migration might be because Mil can
modulate cell cohesion independently of Slb/Wnt11 activity. To test this, we
injected WT embryos with mil-MO and analysed the cell behaviour of
prechordal plate progenitors. Cells with compromised Mil function migrated
persistently but less coherently than did WT cells (see Fig. S1 and Movie 4 in
the supplementary material; see also Fig.
8C). If Mil modulates cell cohesion through PI3K, prechordal plate
progenitor cells with increased PI3K activity would lead to phenotypes similar
to that of mil-morphant cells. To test this possibility, we expressed
RNA encoding a constitutively active form of PI3K (p100CAAX) in the WT embryo
to examine the cell behaviour of migrating prechordal plate progenitors. In
contrast to abrogation of Mil function, the expression of p100CAAX
RNA did not cause cells to change their cohesive properties in WT embryos
(Montero et al., 2003) (see
Fig. S4 and Movie 5 in the supplementary material; see also
Fig. 8C). Conversely, a
reduction in the motility of WT cells expressing dominant-negative (dn)-PI3K
was not due to a lower coherence of cell migration (see Fig. S4 and Movie 6 in
the supplementary material; see also Fig.
8C). These results suggest that Mil regulates the coherence of
cell migration independently of Wnt11 activity and PI3K.
Mil regulates cell motility through the Pdgf/PI3K pathway
We further tested whether Mil-mediated cell motility is dependent upon the
Pdgf/PI3K pathway. As the phenotype caused by dn-PI3K shows reduced migration
and is opposite apparently to that of the mil morphant, we performed
epistasis analysis of the interaction between the two signals by examining
details of the cell behaviours based on DIC time-lapse movies. Prechordal
progenitor cells expressing dn-PI3K migrated much slower than did WT cells,
while their directionality was largely unaffected
(Montero et al., 2003)
(Fig. 8A,C; see also Movie 6 in
the supplementary material). The reduced motility of cells expressing dn-PI3K
was not reversed by knocking down Mil function, which suggests that Mil
regulates cell motility through PI3K (see Fig. S4 and Movie 7 in the
supplementary material; see also Fig.
8C). However, these cells migrated much less persistently than did
cells with reduced PI3K activity only. The lower persistency of cell movement,
when both PI3K and Mil functions are compromised, correlated with the
randomised orientation of lamellipodia of leading edge cells with respect to
their direction of motion (Fig.
8C). These results prompt us to propose that Mil controls the
directed migration of prechordal plate progenitor cells in both a
Pdgf/PI3K-dependent and -independent manner.
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Ulrich et al., 2005).
Cases for which collective migration in the embryo have been well
documented are lateral line primodia in zebrafish and border cells in
Drosophila. In contrast to the mesenchymal nature of prechordal plate
progenitor cells in zebrafish, lateral line primodia cells undergo collective
migration as a sheet of epithelia, such that only a few front-line cells lead
their migration while the rest of the cells follow the leader cells
(Haas and Gilmour, 2006). By
contrast, border cells in Drosophila undergo collective migration as
mesenchymal cells by changing their position within the cluster in the absence
of leader cells (Prasad and Montell,
2007). There is no evidence that leader cells exist within
prechordal plate progenitor cells, but we observed that cells located at the
leading edge retain their position at least for the duration of the time-lapse
movies (e.g. Movies 1 and 8 in the supplementary material). This suggests that
prechordal plate progenitor cells are capable of migrating as individual
cells, and that this might be similar to the traditional case of individual
cell migration during chemotaxis in Dictyostelium (reviewed by
Dormann and Weijer, 2006;
Franca-Koh et al., 2006).
Prechordal plate progenitor cells can migrate as isolated single cells in
extreme situations. Interestingly, single cells do not travel alone far away
from the group of prechordal plate progenitors and in all cases they come back
to the group. This raises the possibility that the isolated cell might lose
its identity as a presumptive prechordal plate progenitor; for example,
goosecoid expression could have been lost. In MZoep embryos, a single
WT cell can internalise but is incapable of migrating anteriorly
(Carmany-Rampey and Schier,
2001), presumably its anterior mesendodermal cell identity has
been lost. Alternatively, the isolated cell might lose motility through the
loss of a community effect that normally maintains cells as a group. However,
it is difficult to distinguish between these possibilities, as it is
technically challenging to visualise cell identity in the living embryo in
such a short period. Also, it will be interesting to test whether or not cells
with higher motility and lower directionality can migrate individually when
they are isolated from surrounding cells with lower motility and lower
coherence.
Does Mil/S1P mediate the non-canonical Wnt/PCP pathway?
We have identified mil/edg5 as a suppressor of the
slb/wnt11 phenotype in this study. This raises the intriguing
possibility that Mil might act in the non-canonical Wnt/PCP pathway. However,
several lines of evidence do not support this possibility. First, although
abrogation of Mil function with MOs restores the anterior migration of
prechordal plate progenitors in slb embryos, it fails to rescue
defects in the slb presumptive notochord. Second, while injection of
dkk1-MO facilitates anterior migration of the prechordal plate in WT
embryos, as in the mil morphants, it cannot rescue the defective
anterior migration of the slb prechordal plate
(Caneparo et al., 2007),
suggesting that Dkk1-regulated migration is dependent upon Slb/Wnt11 function
in this context. Indeed, it has been shown that Dkk1 acts in the non-canonical
Wnt pathway by physically and functionally interacting with Knypek/Glypican4,
a known co-factor of Wnt11 (Caneparo et
al., 2007; Topczewski et al.,
2001). Third, the cardia bifida phenotype caused by
mil-MO injection is still observed in the slb homozygous
background (data not shown). These results support the notion that the Mil
signal acts independently of the non-canonical Wnt pathway.
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12/13 is
activated by EDG5 (Gonda et al.,
1999). Importantly, abrogation of 12/13 function
leads to impaired mediolateral elongation of mesendodermal cells undergoing
convergence of the lateral mesoderm and extension of the notochord during
zebrafish gastrulation (Lin et al.,
2005). However, it implies that 12/13 is
unlikely to mediate EDG5/S1P signalling directly, as embryos with altered
EDG5/EDG1 activities exhibit little defect in mesodermal cells undergoing
convergence and extension. It remains to be investigated which heteromeric G
protein mediates signalling downstream of Mil/S1P in the directional migration
of prechordal plate progenitors. In addition, how different G protein-coupled
receptors (GPCRs) regulate the movements of distinct cell populations during
gasrulation needs to be addressed, as another GPCR Agtrl1b predominantly
mediates the movements of the lateral mesoderm of the zebrafish gastrula
(Scott et al., 2007;
Zeng et al., 2007).
Does S1P act as a directional cue or together with directional cue(s)?
The best candidate for a directional cue is Pdgf, but it appears that Pdgf
regulates anterior migration of the lateral mesoderm as well as of the
anterior axial mesoderm in Xenopus and zebrafish
(Nagel et al., 2004) (data not
shown), whereas the Mil/S1P signal is more specific to prechordal plate
progenitor cells in zebrafish. Do Edg receptors regulate directional migration
cell-autonomously within the prospective prechordal plate? Considering the
fact that the expression of both mil and edg1 is ubiquitous
during gastrulation (Kupperman et al.,
2000) (data not shown), the localisation of the bioactive lipid
S1P might be restricted to either the presumptive prechordal plate or the
overlying neurectoderm. Although it will be challenging to visualise the short
life of the bioactive lipid in the embryo, the temporal and spatial
localisation of its producing and degrading enzymes, such as Sphingosine
kinase and S1P lyase, respectively, might allow us to speculate where a
potential gradient of the bioactive lipid is, as in the case for the gradient
of retinoic acid in the zebrafish hindbrain
(White et al., 2007).
Alternatively, S1P receptors might be required cell-non-autonomously in the
overlying neurectoderm, as it appears that there is correlation of tissue
movements between the presumptive prechordal plate and the hypothalamus (see
Fig. S3 in the supplementary material). To clarify the apparent
interdependency of the two tissue movements, we will need to manipulate the
movement of one tissue to see possible alteration in the other tissue. This
will require the use of a transgenic approach rather than a transplantation
one, as transplanted cells normally disperse in the host and thereby lose
coherence of cell movement, which might mediate the interaction of two
distinct populations.
|
).
Importantly, embryonic fibroblasts from sgpl1-/- mice in
cultures showed reduced migration in response to Pdgf
(Schmahl et al., 2007),
indicating that the ability of cells to inactivate active S1P is required for
the proper response to Pdgf signalling to mediate cell motility. Moreover,
Pdgf stimulates the production of S1P by activating and translocating
Sphingosine kinase, an S1P-producing enzyme, to the membrane in lymphocytes
(Hobson et al., 2001). This
further suggests a possible auto-regulatory loop between Pdgf and S1P signals.
Therefore, it will be fascinating to investigate this issue in directed
migration in the gastrulating embryo.
Recent studies in medaka revealed that maternal-zygotic fgfr1
mutants exhibit defective migration of the axial mesoderm but not of the
lateral mesoderm (Shimada et al.,
2008), and this phenotype is reminiscent of the
edg1-morphant phenotype, raising the possibility that the Mil/S1P
signal might also co-operate with the Fgf signal. It will be intriguing to
test this possibility in zebrafish, as Fgf is capable of activating PI3K as
well as Pdgf. Furthermore, the question of how prechordal plate cells acquire
greater motility and/or can sense directional cues more efficiently than the
other mesodermal cells remains unsolved.
How does the Mil/S1P signal regulate cell motility, directionality and coherence?
We demonstrated that overexpression of mil RNA inhibits
Pdgf-induced cell polarisation in dissociated mesendodermal cells by
interfering with Pdgf-mediated activation of PI3K, and that the facilitated
migration of the mil morphant is blocked totally by dn-PI3K. These
results indicate that Edg5 can act upstream of PI3K to negatively regulate the
Pdgf pathway. However, cell-tracking analysis of mil morphants and of
embryos with increased or reduced PI3K activity revealed that coherence of
cell migration mediated by Mil is likely to be independent of PI3K activity
(Fig. 9). By contrast, the
directionality of migrating cells is totally lost in the mil
morphants when PI3K is blocked, implying that Pdgf and Edg signalling might
co-operate to mediate the directionality of prechordal plate progenitor cells
(Fig. 9). This is different
from the mode of regulation of directionality in Xenopus, as blocking
the Pdgf signal is sufficient to lose the directionality of anterior axial
mesodermal cells (Nagel et al.,
2004). Taken together, these results suggest that Mil modulates
cell motility through the Pdgf/PI3K pathway, but that it modulates coherence
independently of the pathway.
Does Mil modulate cell cohesion passively as a consequence of increased
cell motility or instructively by unknown mechanisms? We favour the latter
possibility for several reasons. First, abrogation of Mil function leads to
the reduced coherence of cell migration in WT embryos, whereas WT embryos with
increased or decreased PI3K activity do not show any changes in coherence of
cell migration. This implies that cell motility and coherence are
independently regulated. Second, when E-cadherin-mediated cell cohesion was
decreased in slb embryos, slb embryos with compromised Mil
function acquired more motility than WT embryos. Moreover, even
E-cadherin-morphant cells, when Mil function was compromised, were able to
migrate as efficiently as WT cells (data not shown), suggesting the presence
of a Mil mediator of cell coherence that functions independently of
E-cadherin-dependent cell adhesion. Third, abrogation of Mil function in the
WT embryo leads to the scattered cell behaviour phenotype during
internalisation, as seen in the slb embryo with compromised Mil
function (see Movie 10 in the supplementary material). This supports the
notion that Mil modulates cell cohesive properties independently of Slb
activity (Fig. 9). Fourth, the
mil cardia bifida phenotype might be explained by the modulation of
adhesive properties through the interaction with fibronectin
(Matsui et al., 2007). Whether
or not Mil modulates the cell cohesive property underlying the directed
migration of collective cells in the embryo will require further
investigation.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/18/3043/DC1
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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