Institut für Genetik, Universität zu Köln, Weyertal 121, 50931 Köln, Germany
* Author for correspondence (e-mail: mleptin{at}uni-koeln.de)
Accepted 24 November 2004
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SUMMARY |
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Key words: Signalling, FGF, Mesoderm, Drosophila
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Introduction |
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The transmission of the signal from an activated FGF receptor to
intracellular targets within cells is only partly understood. One protein that
is essential for signal transduction by the Drosophila FGF receptors
is the intracellular protein known as Dof, Heartbroken or Stumps
(Imam et al., 1999;
Michelson et al., 1998a
;
Vincent et al., 1998
), which
can physically interact with the receptor
(Battersby et al., 2003
;
Petit et al., 2004
;
Wilson et al., 2004
).
Mutations in dof specifically affect processes that depend upon FGF
signalling, and the protein is only present in cells that express FGF
receptors. A number of components shared with other receptor tyrosine kinase
(RTK) signalling pathways have also been shown to have a role in FGF
signalling (Casci et al., 1999
;
Gabay et al., 1997a
;
Gabay et al., 1997b
;
Gisselbrecht et al., 1996
;
Hacohen et al., 1998
;
Johnson Hamlet and Perkins,
2001
; Kramer et al.,
1999
; Perkins et al.,
1996
; Reich et al.,
1999
; Reichman-Fried et al.,
1994
). For example, Corkscrew (Csw) is important for the
FGF-dependent formation of heart precursors and the development of the
tracheal system (Gabay et al.,
1997a
; Johnson Hamlet and
Perkins, 2001
; Perkins et al.,
1996
). One function of Dof may be to recruit Csw to the signalling
complex (Petit et al., 2004
;
Wilson et al., 2004
). Other
proteins that associate with Dof have been identified
(Battersby et al., 2003
), but
their physiological significance in the activation of MAPK and the
morphogenesis of the mesoderm and the tracheae remains to be established.
Recently the Rac GTPases have been shown to be required for the development of
the tracheae, but it is not clear whether they act in the FGF pathway, as the
dynamic filopodia, which are promoted by FGF-signalling
(Ribeiro et al., 2002
;
Sato and Kornberg, 2002
), are
still present at the tips of tracheal branches with reduced Rac activity
(Chihara et al., 2003
).
FGF signalling is implicated in cell migration and morphogenetic movements
during gastrulation in many organisms, but the mechanisms and biochemical
pathways responsible for these cellular movements are poorly understood. In
Xenopus embryos Sprouty2 is induced in response to MAPK activation by
FGF signalling. Unlike Drosophila Sprouty, the Xenopus
protein does not block Ras/MAPK signalling, but regulates a process that is
not well defined to inhibit convergent extension movements
(Nutt et al., 2001). Thus, in
vertebrates the FGF-dependent pathways that regulate differentiation and
morphogenesis can be distinguished by their sensitivity to Sprouty2. FGF
signalling need not directly regulate the ability of cells to migrate. During
gastrulation in mouse embryos, FGF signalling has been shown to allow cells to
escape from the mass of the invaginated mesodermal primordium, by promoting
the downregulation of E-Cadherin during the epithelial to mesenchymal
transition (EMT), rather than by affecting the migratory properties of the
cells (Ciruna and Rossant,
2001
; Ciruna et al.,
1997
). In Drosophila, the mechanisms by which FGF
signalling leads to the dispersal of mesodermal cells over the ectoderm are
not known, nor is it known what additional cellular mechanisms participate in
this process. Integrins and cadherins are both expressed at this stage of
development, but surprisingly neither appears to be involved in the dispersal
of the mesoderm (Leptin et al.,
1989
; Oda et al.,
1998
). FGF signalling alone cannot be responsible for this
process, as in embryos mutant for components of the signalling system, a
considerable amount of spreading still occurs. To understand mesoderm
spreading, the potential roles for adhesion, intercalation, FGF-independent
migration or chemotactic behaviour need to be dissected. It has been proposed
that FGF signalling could be instructive, and direct migration of the
mesodermal cells dorsally (Gabay et al.,
1997b
; Gryzik and Müller,
2004
; Stathopoulos et al.,
2004
), but it is equally possible that the dispersal of the
mesoderm depends upon the induction of motility by FGF and the preferential
adhesion between mesoderm and ectodermal cells, rather than on a specific
directional cue (Stathopoulos et al.,
2004
; Wilson and Leptin,
2000
). In this paper, we investigate the specific requirement for
FGF signalling in the dispersal of the mesoderm cells over the ectoderm, and
assess the significance of the activation of MAPK within the mesoderm
following the invagination of the mesoderm anlage.
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Materials and methods |
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Identification of transgenic and homozygous mutant embryos
Unless stated otherwise, all crosses used stable lines with Balancer
chromosomes that carried a lacZ-transgene. The expression of
lacZ was controlled by the ftz-promoter, and can be detected in early
embryos from the stage of gastrulation. Embryos were double stained for
ß-Gal and the indicated markers, embedded in approximately 1 ml
unpolymerised araldite in 1 cm diameter dishes and sorted under a dissecting
microscope according to their ß-galactosidase staining pattern.
lacZ-positive and lacZ-negative embryos were matched in age
by various criteria. In the case of MAPK staining, the highly dynamic staining
pattern (in regions not affected by the mutations) provides an excellent
additional frame of reference. In the case of Twist-staining, we have a great
deal of experience in using the degree of germ band extension, the depth of
the dorsal folds and the appearance of the head fold as age markers
(especially as germ band extension is in its most rapid phase at the time that
is relevant for our analysis). In the case of the Eve-staining, the Eve
stripes provide a further marker that is very useful for early stages.
Age-matched embryos were then selected for sectioning, transferred to polymerisation moulds, photographed and sectioned. This allowed us to go back after sectioning and compare the phenotype in the sections with the age of the embryos. Several embryos covering various points during the time-span of interest were analysed in each case.
For rescue experiments, one parent usually carried a recombinant third chromosome with the htl mutation and the Twi-GAL4 transgene, the other parent carried a recombinant third chromosome with the htl mutation and the UAS-transgene. Thus, all homozygous mutant progeny expressed the transgene. Alternatively, one parent was homozygous for one of the transgenes (e.g. in cases where no insertions were available on the third chromosome), so that, again, all homozygous mutant progeny expressed the transgene. In each case, the recombinant chromosomes or the htl mutant chromosomes were kept over one of the marked balancers so that the mutant embryos could be identified by the absence of ß-Gal staining.
The `activated' kinase dead Htl transgene
A BglII-BamHI fragment encoding Htl was isolated from a
pSP73 cDNA clone, kindly provided by A. Michelson, and ligated into pAlter
that had been digested with BamHI. A recombinant pAlter.htl clone was
chosen with the 5' htl coding sequence juxtaposed to the
ampicillin resistance gene of the vector. A mutagenesis reaction was performed
with single-stranded DNA (ssDNA) derived from this clone with the following
oligonucleotides: 5'-GAAGTCTTCAGATCTTGGAAG-3',
5'-TGTCGCCGTGCGAATGGTGAAGGAAG-3' and
5'-GTGGTGTAATTACCTAGGCTAAAGAAATCAGGAT-3'. The resulting clone
pAlter.htl.BglII.K443A.AvrII was sequenced in its entirety. Subsequently, a
BglII-AvrII fragment encoding the transmembrane and
cytoplasmic domains of Htl was ligated in a three-way reaction to a
NotI-BglII fragment from pKS+-btl (M. Y. Zhu, R.W.
and M.L., unpublished) encoding the lambda cI dimerisation motif, and
a NotI-AvrII fragment of
Not-5'Flag-Dof-AvrII-2xHA-Asc.pNB40 (details available on request) to
provide HA epitope tags and a bacterial replication origin. Finally, a
NotI-AscI fragment of the resulting clone
pNB40.
htl.K443A-2xHA was ligated into a NotI-AscI
vector prepared from pUAST-
btl.3'Asc
(Wilson et al., 2004
) to
create pUAST-.
htl.K443A-2xHA. Transgenic flies were produced according
to standard procedures. The specific chromosome carrying an insertion was
identified by the segregation of the insertion from dominant markers on second
and third chromosomes, and sex linkage in the case of the X chromosome. To
confirm that the K443A mutation had abolished the kinase function of the
receptor, the receptor was expressed in S2 cells and whole cell extracts were
analysed for the presence of phosphotyrosine by western blotting (details
available on request).
Immunohistochemistry and microscopy
Standard procedures were followed to collect embryos, which were fixed over
30 minutes at 37°C using a phosphate-buffered saline solution containing
3.7% formaldehyde, or in the case of anti-diphospho-MAPK staining, 8%
formaldehyde. For immunohistochemistry, we used antibodies directed against
Twist (kindly provided by S. Roth, Cologne), Eve (courtesy of M. Frasch, Mount
Sinai), diphospho-MAPK (Sigma M8159), and ß-Galactosidase (Sigma G4644).
Proteins were detected in situ by using the coupled-peroxidase system
(Vectastain ABC Kit, Vector Laboratories). Two-colour staining was
accomplished in sequential steps, with the addition of Ni and Co in the second
detection reaction to produce a dark precipitate. Embryos were sorted and
embedded in Araldite, as previously described
(Leptin and Grunewald, 1990);
sections were cut at 8 µm using a Leica RM 2065 microtome. Photographs were
taken on a Zeiss Axioplan microscope at 400 x using a Kontron ProgRes
3008 digital camera, and the images were processed using Adobe Photoshop 5.5.
To analyse the data, a web page of the pictures was produced with the help of
Quicknailer 1.7.1 and Adobe PageMill 3.0, and a QuickTime movie of the images
from each embryo was created with GraphicConverter 4.4. Transmission electron
microscopy was performed essentially according to Tepass and Hartenstein
(Tepass and Hartenstein,
1994
), except that embryos were fixed in a 0.1 M phososphate (pH
7.2) buffer containing 1% osmiumtetraoxide and 2% glutaraldehyde.
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Results |
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A further experiment suggests that long-range chemotaxis is not responsible
for the dispersal of the mesoderm. A constitutively active form of Htl
expressed in the mesoderm of htl mutants rescues the ability of all
cells to move efficiently away from the site of invagination, although there
is no directional information in this situation
(Fig. 2H)
(Michelson et al., 1998a).
Thus, the role of FGF signalling in early mesoderm spreading appears to be
permissive, promoting the attachment and dispersal of the mesodermal cells
over the surface of the ectoderm, rather than being instructive or having a
chemotactic function that directs mesodermal cells towards a particular
location.
Specificity of receptor tyrosine kinase signalling during mesodermal spreading
Receptor tyrosine kinases (RTKs) share many downstream signalling elements,
and can replace each other in many assays. For example, the functions of the
FGF receptor Breathless during tracheal morphogenesis can be replaced in part
by constitutively active forms of other RTKs
(Lee et al., 1996;
Reichman-Fried et al., 1994
).
We examined whether this was also the case in the mesoderm by expressing
constitutively active forms (see Materials and methods) of Heartless,
Breathless, the PDGF and VEGF-related receptor (PVR), and the EGFR in
htl mutants under the control of Twist-Gal4. We initially tested the
function of these constructs in the mesoderm by scoring their effects on the
activation of MAPK in the primordia of the Eve-expressing heart precursors,
which have been shown to depend upon Htl activity
(Beiman et al., 1996
;
Gisselbrecht et al., 1996
;
Shishido et al., 1997
). All
constructs lead to the phosphorylation of MAPK in these cell clusters,
although there were differences in the extent of Eve expression
(Fig. 3). Similarly, all
constructs were able to activate Eve transcription in heart precursors, albeit
to different levels, and very inefficiently in the case of Breathless and PVR
(Fig. 3). These differences
mirrored the efficiency of the constructs in other assays for RTK activity
(e.g. inducing roughening of the eye, rescue of tracheal defects in
btl mutants (M. Y. Zhu, R.W. and M.L., unpublished)
(Freeman, 1996
;
Reichman-Fried et al., 1994
).
We assume this reflects differences in the signalling strength of the
receptors.
|
|
It was particularly surprising that the constitutively active EGFR was
unable to stimulate MAPK phosphorylation, as it is a very potent RTK in
Drosophila, and all the components necessary for signal transmission
are present in the early embryo. Indeed the EGFR shows high levels of activity
in the cells neighbouring the mesoderm
(Gabay et al., 1997a;
Gabay et al., 1997b
). This
suggests that MAPK activation by RTKs other than FGFRs is blocked in the
mesoderm. This inhibition appears to act on the MAPK cascade itself, because
expression of activated Raf or Ras in dof mutants did not result in
phosphorylation of MAPK (Fig.
4C and data not shown), even though at later stages activated Ras
is able to trigger the accumulation of diphospho-ERK
(Michelson et al., 1998a
).
More importantly, we also observed that even the constitutively active
FGF-receptors were not able to lead to early MAPK activation in all of the
invaginated mesodermal cells, although they are expressed in all cells. In
htl embryos expressing lambda-Htl, MAPK phosphorylation was evident
solely in the mesodermal cells that were in contact with the ectoderm, just as
observed in wild-type embryos. This indicates that activation of the FGF
receptor alone is not sufficient to promote MAPK activation within the
mesoderm at early stages of mesoderm spreading.
Our observation is in apparent contradiction to recent results showing that
expression of an Htl ligand throughout the mesoderm can trigger MAPK
activation in all cells (Stathopoulos et
al., 2004). We suspect that the activation of the wild-type
receptor by high levels of its ligand produces a stronger signal than the
constitutively active constructs, and that this high level of activity is
capable of overriding the requirement for contact with the ectoderm. In the
tracheal system, it is notable that there is a marked difference between the
effect of overexpressing the FGF ligand Branchless, and a constitutively
activated FGF receptor (R.W., E.V. and M.L., unpublished)
(Imam et al., 1999
; Michelson
et al., 1998; Sato and Kornberg,
2002
; Vincent et al.,
1998
). Nevertheless, the expression of lambda-Htl throughout the
mesoderm of htl mutant embryos reveals a difference in the
requirement for MAPK activation between those cells that make contact with the
ectoderm and those that do not. We suggest that contact with the ectoderm,
which in itself depends on FGF signalling, must provide an additional signal
that acts in conjunction with the activation of Heartless to trigger MAPK
phosphorylation. This is consistent with the fact that in pebble
mutant embryos, in which early contact of the mesoderm with the ectoderm does
not occur (Schumacher et al.,
2004
; Smallhorn et al.,
2004
), there is no MAPK activation at early stages
(Fig. 6F), although FGF signal
transduction as such at later stages is functional
(Schumacher et al., 2004
;
Smallhorn et al., 2004
). One
possible explanation for this might be that, because of the lack of proximity
of the mesoderm to the ectoderm, the FGF ligand cannot reach the mesodermal
cells. However, even when this potential problem is bypassed by expressing an
activated form of Htl in the early mesoderm of pebble mutants, no
activation of MAPK can be seen (data not shown). These results suggest that
the ectoderm has an important role in triggering the accumulation of activated
MAPK within the mesoderm, in addition to providing the Htl ligands.
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|
The role of Rho GTPases and their effectors
We tested whether molecules known to regulate the shape and migration of
cells in other contexts would influence mesoderm spreading. The generation of
protrusive force required for cell migration is thought to be due to the
activity of the small Rho family GTPases and their regulators and downstream
effectors, such as those involved in axon guidance or border cell migration
during oogenesis (Awasaki et al.,
2000; Bateman et al.,
2000
; Duchek et al.,
2001
; Garrity et al.,
1996
; Hing et al.,
1999
; Liebl et al.,
2000
; Newsome et al.,
2000
). The initial stages of mesoderm spreading were normal in
embryos lacking the function of Pak, dock, trio and myoblast
city (Fig. 6E, and data
not shown), although in embryos lacking the function of Pak we
observed that the adhesion of the mesoderm to the ectoderm was affected at
later stages (data not shown). An activated form of the Drosophila
myosin regulatory light chain, Spaghetti squash, which is one of the major
targets of Drosophila Rho-associated kinase
(Winter et al., 2001
), was
unable to rescue the defects in the mesoderm of dof mutant embryos
(not shown). These observations suggest that either these particular
regulatory molecules are not needed for the dispersal of the mesoderm, or that
they act together with other pathways in a redundant fashion. It is not
possible to generate embryos that completely lack RhoA function
(Magie et al., 1999
). When we
examined embryos with a reduced maternal dose of RhoA, we observed that the
shape of the invaginated mesoderm was abnormal, although it did not resemble
htl or dof mutants (Fig.
6B). By contrast, we observed strong defects in mesoderm spreading
when the function of the Rac GTPases was compromised. In Drosophila
there are three Rac-like proteins, Rac1, Rac2 and Mtl. All are provided
maternally, and single mutants in these genes are viable and fertile
(Hakeda-Suzuki et al., 2002
;
Ng et al., 2002
), and show
little or no defects in mesoderm spreading
(Fig. 6A and data not shown).
When we generated Rac1, Rac2 double mutant germ line clones we did not obtain
any eggs. However, when we analysed embryos derived from mothers with a
reduction in the maternal dose of Rac function, we found that the ability of
the mesoderm to spread was compromised. Defects became visible when the dose
of Rac1 and Rac2 was reduced (Fig.
6C), and were severe when the function of all three Rac-like
proteins was reduced simultaneously (Fig.
6D). Thus, a normal level of Rac activity appears to be important
for the mesoderm to be able to make contact with the ectoderm. To test whether
Rho family GTPases could act in a linear pathway downstream of FGF signalling,
we expressed activated forms of Rac1 and Cdc42 within the mesoderm of
htl and dof mutant embryos. Mesoderm spreading was not
rescued in these embryos (data not shown), suggesting that the htl
and dof mutant phenotype is unlikely to be due only to a lack of Rac
function per se, and that the FGF receptor and the Rac proteins do not
necessarily function exclusively in the same biochemical pathway.
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Discussion |
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The activation of MAPK within the mesoderm
As the mesoderm spreads out over the surface of the ectoderm, the
mesodermal cells that are in contact with the ectoderm accumulate high levels
of the active form of MAPK. The fact that this accumulation of active MAPK is
seen only in embryos with a functional FGF-signalling system in the mesoderm,
but not in htl or dof mutant embryos, indicates that it is
triggered by the FGF receptor. Htl and Dof are expressed throughout the
mesoderm, which suggests that the local activation of MAPK is induced by the
local availability of a ligand, consistent with the expression pattern of the
recently discovered ligands for Htl in the ectoderm
(Gryzik and Müller, 2004;
Stathopoulos et al., 2004
).
However, even a constitutively active form of Heartless expressed throughout
the mesoderm, which is able to rescue spreading in htl mutants, only
mediates MAPK activation at early stages in the cells directly apposed to the
ectoderm. We conclude that the presence of an activated form of the FGF
receptor is not sufficient to trigger MAPK activation in mesodermal cells.
This result may appear to contradict earlier studies showing the ability of
activated FGF-receptors to trigger MAPK activation throughout the mesoderm
(Michelson et al., 1998a), but
the embryos in these studies were not analysed during the phase of the
earliest contact of the mesoderm with the ectoderm, but rather at later
stages, just before the time when MAPK activation normally occurs in the heart
precursors in the dorsal region of the mesoderm. This phase of FGF-dependent
MAPK activation in the mesoderm clearly has different requirements from the
early phase, as is also shown by our results using other RTKs or downstream
effectors of the RTK signalling pathway. These experiments demonstrate that
signals from activated Raf cannot be transduced to MAPK in the cells during
the early phase, except in the presence of an activated FGF receptor. We
conclude that, in addition to the signal from an activated RTK via Raf, a
second event is necessary for MAPK to become phosphorylated. This event could
either generate a second positive signal, or it could lead to the release of a
negative, inhibitory signal (see Fig.
7A).
|
Both the establishment of mesoderm-ectodermal cell contact and the activation of MAPK require the kinase domain of the FGF receptor to be intact, which suggests that these events depend upon a substrate of the FGF receptors not recognised by other activated receptor tyrosine kinases. One possibility is that this substrate is Dof, which is specifically phosphorylated by an activated FGF receptor. In this situation, Dof would provide a unique function that cannot be substituted by other activated receptor tyrosine kinases. Alternatively, this substrate could be a second receptor that is activated upon contact of the mesoderm with the ectoderm, or a component that acts in, or on, a pathway triggered by the engagement of the mesoderm with the ectoderm (see Fig. 7A).
Several pathways promote contact between mesodermal and ectodermal cells
It seemed likely that the early morphogenetic activity might require
changes in subcellular architecture involving cytoskeletal regulators. Indeed,
the establishment of contact between the mesoderm and the ectoderm is affected
by mutations in the gene encoding the RhoGEF Pebble
(Schumacher et al., 2004;
Smallhorn et al., 2004
), and,
as shown here, a reduction in the level of Rho and Rac proteins within the
embryo. We do not know whether the Rho-family GTPases act downstream of or in
parallel with FGF signalling. The defects of htl mutants cannot be
rescued by the expression of an activated form of Rac or Cdc42. Thus, if Rac
acts downstream of the FGF receptor, it is not in a simple epistatic pathway
but requires the activation of other pathways as well. Alternatively, FGF
signalling may act in conjunction with a separate pathway that directs the
activity of the Rac proteins to promote contact between the mesoderm and the
ectoderm (see Fig. 7A).
FGF signalling is not essential for migration of mesodermal cells
Spreading of the mesoderm on the ectoderm leads to a redistribution of
mesodermal cells away from the site of invagination towards the dorsal edge of
the ectoderm. This is often considered to be a process of directed cell
migration. In this view, the graded distribution of activated MAPK levels in
the nuclei of the mesodermal cells is suggestive of a response to a
chemotactic signal originating from the target region
(Gabay et al., 1997b). Both
the expression pattern of the Htl ligands
(Gryzik and Müller, 2004
;
Stathopoulos et al., 2004
) and
the phenotypes of mutants in which the fate of the target region has been
changed (Wilson and Leptin,
2000
) are inconsistent with this view. The activation of Heartless
appears to be permissive for mesoderm spreading and we suggest that FGF
signalling functions primarily to promote the efficient interaction of the
entire mesodermal primordium with the surface of the ectoderm and that this
could act to impose order during the transition from an epithelial to a
mesenchymal state. Simple spatial constraints could lead to an apparently
directed migration. With the mass of mesodermal cells initially concentrated
near the site of invagination, the only direction available for migration is
away from this site. Hence, a signal-inducing motility would automatically
promote directional movement. The dispersal of the mesoderm mass in
dof mutants is noticeably improved by blocking cell division, and we
believe that this might be due to the smaller number of cells in the
mesodermal primordium having greater access to the surface of the
ectoderm.
Potential cellular mechanisms involved in dispersal of the mesoderm
These observations raise the questions of how mesodermal cells spread over
the surface of the ectoderm, and how activated MAPK accumulates in a graded
fashion. A number of possibilities can be envisioned to account for the
migration of the mesoderm. For example, the first cell that makes contact with
the ectoderm could crawl over the ectoderm and function as the `leading' cell
of the mesodermal sheet. The other cells of the mesoderm tube would make
contact with the ectoderm sequentially to follow the leading cell as it
migrates dorsally (Fig. 7B,
part i). In this case, the MAPK gradient would be explained by the
accumulation of the highest levels of activated MAPK in the cells that had
been in contact with the ectoderm for the longest period of time.
Alternatively, the cell that makes the initial contact with the ectoderm could
remain largely stationary, and other mesodermal cells would reach the ectoderm
by crawling over that cell (Fig.
7B, part ii). Once a mesodermal cell is in contact with the
ectoderm, motility of the cell would cease, as in the process of `boundary
capture' described for mesodermal cells in Xenopus reaching the
notochord during convergence movements
(Keller et al., 2000). In this
model, contact between the ectoderm and mesoderm would have an important role
in establishing the single cell layer of mesoderm that covers the surface of
the ectoderm at later stages. The MAPK gradient can be explained in this case
by a transient activation of MAPK, which is downregulated once motility
ceases, a model that is more consistent with known feedback mechanisms that
operate during signal transduction
(Freeman, 2000
;
Freeman and Gurdon, 2002
;
Perrimon and McMahon, 1999
;
Rebay, 2002
). This model
implies that the cells with the highest level of MAPK at the edge of the
mesoderm would have only just come into contact with the ectoderm (see
Fig. 7B, part ii). In order to
distinguish between the two mechanisms, cell labelling experiments will be
required.
FGF signalling is only one of many mechanisms that contribute to the establishment of the mesodermal cell layer. It is not essential for migration as such, but is clearly important for the orderly dispersal of mesodermal cells away from their site of invagination. Our results suggest that FGF-signalling facilitates cell spreading by promoting the apposition of the invaginated mesodermal epithelium against the ectoderm.
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ACKNOWLEDGMENTS |
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