1 School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ,
UK
2 CRUK Molecular Pharmacology Unit, Biomedical Research Centre, Level 5,
Ninewells Hospital, Dundee DD1 9SY, UK
* Author for correspondence (e-mail: a.munsterberg{at}uea.ac.uk)
Accepted 14 January 2005
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SUMMARY |
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Key words: FGF signalling, Chick, Somite, Tendon, Rib, Mkp3, ERK MAP kinase, Scleraxis
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Introduction |
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Signalling between different somite compartments results in further
patterning and it has been shown that growth factors from the myotome are
important for the specification of sclerotome derived cell lineages, including
ribs and tendons (Brent et al.,
2003; Huang et al.,
2003
; Tallquist et al.,
2000
). Detailed clonal analysis of avian somites has revealed the
origin of proximal and distal ribs (Evans,
2003
). Furthermore, ablation experiments suggested that the
mesenchymal cells at the rostral and caudal edges close to the dermomyotome,
which express the bHLH transcription factor scleraxis, contain distal
rib progenitors (Hirao and Aoyama,
2004
). Scleraxis marks a subpopulation of sclerotomal
cells and is involved in regulating gene expression within mesenchymal cell
lineages that give rise to cartilage and connective tissues
(Cserjesi et al., 1995
;
Schweitzer et al., 2001
). In
maturing somites, scleraxis marks axial tendon progenitors in the
dorsal sclerotome and it has been postulated that myotome-derived fibroblast
growth factor (FGF) signals are required to activate scleraxis
expression by an indirect mechanism (Brent
et al., 2003
). Recent work has implicated the Ets domain
transcription factors Pea3 and Erm in the regulation of scleraxis
expression (Brent and Tabin,
2004
). This demonstrates that close interactions between the
myotome and sclerotome are pivotal for the emergence of a discrete population
of scleraxis positive cells within the somite.
We were interested in investigating further the role of the FGF signalling
pathway in the specification of scleraxis positive somite
progenitors. We have previously identified a role for the dual specificity
phosphatase, MKP3, in negatively regulating the ERK MAP kinase pathway in limb
and early neural development (Eblaghie et
al., 2003) and this new study focuses on the function of this
pathway in somite patterning and cell specification.
Mitogen-activated protein kinase (MAP kinase) cascades are effectors for
many growth factor signals implicated in developmental processes, including
appendage outgrowth and organogenesis. The `classical' Ras/MAP kinase cascade
in which extracellular signal regulated kinases (ERK1 and ERK2) are activated
by phosphorylation of the T-X-Y motif within the activation loop of the kinase
is a major effector of signalling in mammalian cells
(Kouhara et al., 1997). The
developmental outcome of ERK signalling relies, at least in part, on the
competing actions of upstream activators and inhibitory MAP kinase
phosphatases (MKPs). Indeed, the level of FGF signalling has been suggested to
play a deterministic role in cell fate and survival in a number of different
systems (Hajihosseini et al.,
2004
; Partanen et al.,
1998
; Storm et al.,
2003
; Sato and Nakamura,
2004
; Tsang et al.,
2004
).
The dual-specificity MAP kinase phosphatase, MKP3 (also known as PYST1), is
a specific and potent regulator of the ERK class of MAP kinases
(Groom et al., 1996;
Muda et al., 1996
). This
specificity for the ERK 1/2 MAP kinases is mediated by specific
protein-protein interaction and subsequent ERK-dependent catalytic activation
of MKP3 (Camps et al., 1998
;
Muda et al., 1998
). We have
previously isolated the chicken and mouse orthologues of Mkp3 and
studied their embryonic expression. We found dynamic patterns of Mkp3
messenger RNA expression in important signalling centres and known sites of
FGF/FGF receptor signalling, which are associated with cell proliferation and
patterning in developing mouse and chick embryos
(Dickinson et al., 2002
;
Eblaghie et al., 2003
).
We demonstrate that in differentiating somites Mkp3 is expressed in scleraxis-positive progenitor cells. This expression is regulated by FGF signalling via the classical ERK MAP kinase pathway. During somite patterning, MKP3 regulates ERK MAP kinase activity by dephosphorylation demonstrating that this enzyme is part of a negative feedback loop controlling the levels of phosphorylated ERK (dpERK) in this tissue. Finally, we show that decreasing or increasing the levels of dpERK in chick somites, by mis-expression of human MKP3 or a constitutively active MEK1, respectively, results in loss of Mkp3 and scleraxis expression and affects rib formation. We propose that the tight regulation of dpERK levels is crucial for the activation of scleraxis expression and we show that FGF signalling is important for the specification of distal rib precursors in somites.
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Materials and methods |
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Immunohistochemistry and ISH on sections
To detect myosin heavy chain protein with MF-20 antibody (Developmental
Studies Hybridoma Bank) and transcripts for Mkp3, sprouty2 or
scleraxis, we performed in situ hybridisation with
immunohistochemistry in combination. We followed the method described
previously (Edom-Vovard et al.,
2001). The proteinase K step was omitted and replaced with 0.1%
Triton-X 100 15 minutes at room temperature. NBT/BCIP colour reaction was
enhanced with 10% polyvinylalcohol (Sigma). Scleraxis transcripts
were detected with Fast Red under a coverslip at 37°C. Sections were
incubated with MF-20, 1/1000 dilution overnight at 4°C and detected with
an `Alexa' secondary fluorescent antibody (488 nm, Molecular Probes).
FGF and pharmacological inhibitor beads
Heparin beads (Sigma H-5263) were soaked for 1 hour at room temperature in
recombinant FGF (R&D Systems) at the following concentrations: FGF4
(50µg/ml), FGF8 (1mg/ml) and FGF2 (400µg/ml). AG-1 X2 beads (BioRad)
were incubated in one of the following pharmacological inhibitors: SU5402,
from 5 to 10 mM; SB203580 and LY294002 both 20 mM, all from Calbiochem; and
PD184352, 20 mM (a gift from Philip Cohen, Dundee)
(Davies et al., 2000). All
compounds were dissolved in DMSO. Beads were soaked for 1 hour at room
temperature in the dark then washed twice in PBS and implanted adjacent to
cervical somites of HH13 embryos or adjacent to forelimb or flank level
somites of HH17 embryos. In addition, PD184352 or DMSO controls were diluted
1/10 with PBS and injected directly into thoracic somites. Embryos were fixed
in 4% paraformaldehyde and processed for in situ hybridisation 1, 5 or 24
hours after the operation.
Chick embryo manipulations and constructs
Fertile chicken White Leghorn eggs were obtained from Needle farm (Sussex)
and incubated at 37.5°C until the desired Hamburger-Hamilton stage was
reached. Electroporation was performed in ovo using a TSS20 Ovodyne
Electroporator (Intracel, UK). Expression plasmids for hMKP3-GFP,
hMKP3KIM-GFP, caMEK1 (=MKKE/E), sFREK:Fc and
dnFgfR1c have been described previously
(Eblaghie et al., 2003
;
Marics et al., 2002
;
Yang et al., 2002
;
Cowley et al., 1994
). Most
plasmids encoded GFP fusion proteins or produced GFP from an IRES.
Alternatively, a GFP expression plasmid was co-electroporated to mark
transfected somites. Positive electrode was platinum and negative electrode
was sharpened tungsten wire. Eggs were windowed and black ink was injected
underneath the blastoderm to visualise the embryos. DNA (3 mg/ml in water) was
injected underneath the myotome of flank somites at HH18-20, electrodes were
placed on either side of the embryo and 40 V were applied for 50 mseconds,
with 5 pulses spaced 500 mseconds apart. For RCAS-mediated gene expression,
concentrated virus was injected into presegmented mesoderm. RCAS-sFREK:Fc has
been described previously (Marics et al.,
2002
). The spread of infection was examined using a gag antisense
probe. Eggs were sealed and incubated for the times indicated and processed
for in situ hybridisation, Alcian Blue staining, western analysis or RNA
extraction.
Western blots
hMKP3-GFP encodes a fusion protein, caMEK1 was co-electroporated with a GFP
expression plasmid. GFP-labelled somites were pooled, protein was extracted
using standard protocols (NP-40 lysis buffer with protease and protein
phosphatase inhibitors, Roche), equal amounts were loaded on 10%
polyacrylamide gels. Primary antibodies were applied overnight at 4°C,
excess was washed and secondary antibodies coupled to HRP (Jackson
Laboratories) were applied for 1 hour at room temperature. Primary antibodies
used: dpERK (Cell Signaling); anti-GFP, (Clontech); and -tubulin
(SIGMA).
RT-PCR
RNA was harvested from GFP-expressing somites. cDNA was prepared using
random hexamer primers as described previously
(Münsterberg et al.,
1995). cDNA (1 µl) was used in PCR reactions, human MKP3-GFP
(30 cycles), chick Mkp3 (35 cycles), scleraxis (30 cycles) and GAPDH (25
cycles) (Münsterberg et al.,
1995
). Primers for chick scleraxis: forward,
5'-ACGTGAATTCCACACACACCGAACCACGGAC-3'; reverse,
5'-ACGTGAGCTCATTATACGAACTGCTCAGGC-3'. Primers specific for human
MKP3-GFP fusion: forward, 5'-ACGTCCATGGTAGATACGCTCAGACCCG-3';
reverse, 5'-ACGTAAGCTTTTACTTGTACAGCTCGTCC-3'. Specific primers for
chick Mkp3: forward,
5'-ACGTGCGGCCGCATGCTAGATACGTTCAGACCCGTC-3'; reverse,
5'-ACGTGAATTCTCACGTGGACTGCAGGGAGTCCACC-3'. Specificity of MKP3
primers was tested on 1 ng of plasmid template. Amplification conditions for
plasmid and cDNA: denaturation at 94°C for 5 minutes, followed by 30 to 35
cycles of 94°C for 30 seconds, annealing temperatures of 60°C and
55°C for both hMKP3-GFP and cMkp-3 and for scleraxis, respectively for 30
seconds and 72°C for 2 minutes. A 5 µl sample of the 30 µl reactions
was analysed on a 1-2% agarose gel.
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Results |
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This showed that FGFs known to act on mesenchymal cells were sufficient to induce ectopic Mkp3 transcripts via either ERK or PI3-kinase. Next, we wanted to investigate whether FGF signalling was necessary to regulate the expression of Mkp3 in the dorsal sclerotome. Application of a bead soaked in SU5402 led to a dramatic loss of Mkp3 transcripts beneath the bead (Fig. 2G). A bead soaked in LY294002 did not affect endogenous Mkp3 expression in contrast to ectopic Mkp3 expression, which was sensitive to PI3-kinase inhibition (Fig. 2I,E). By contrast, a bead soaked in the ERK MAP kinase inhibitor PD184352 did result in a significant loss of endogenous Mkp3 transcripts, while DMSO beads or pharmacological inhibitors that blocked other pathways had no effect (Fig. 2H; data not shown). In order to confirm in vivo the effect of PD184352, which inhibits MKK upstream of ERK, the somites beneath the bead were dissected, protein was extracted and analysed by western blot for the presence of dpERK. We observed the complete loss of dpERK in the presence of a PD184352 bead compared with readily detectable levels in untreated somites (Fig. 2F). Injection of PD184352 directly into somites resulted in complete loss of Mkp3 transcripts, while injection of DMSO had no effect (Fig. 2J,K). Thus, endogenous expression of Mkp3 was dependent on the presence of active, phosphorylated ERK MAP kinase and the residual expression observed in Fig. 2H is most likely due to the limited diffusibility of PD184352.
In somites, FGFs induce scleraxis, possibly by activating the Ets
domain transcription factor Pea3 (Brent and
Tabin, 2004). Therefore, we asked whether scleraxis
expression was dependent on dpERK and found that injection of PD184352
resulted in loss of detectable transcripts
(Fig. 2L,M). To corroborate
these findings, somites of HH18 embryos were electroporated with a plasmid
encoding a dominant-negative FGF receptor where the cytoplasmic tyrosine
kinase domain had been replaced with EYFP
(Fig. 2N). We also used a
vector encoding a secreted extracellular domain of FREK (cFGFR4) fused to the
immunoglobulin Fc domain, which mediates dimerisation
(Fig. 2O). Both of these mutant
receptors have previously been shown to inhibit FGF-mediated signalling
(Marics et al., 2002
;
Yang et al., 2002
), even
though they probably do not discriminate between different FGF receptors and
receptor isoforms. Expression of both these constructs by targeted
electroporation into somites resulted in loss of Mkp3 expression in
this tissue (Fig. 2N,O). By
contrast, electroporation of a GFP vector alone had no effect
(Fig. 2P). In addition, when we
expressed the murine orthologue of sprouty2, which has been shown to inhibit
the FGF MAPK pathway at the level of Ras and MEK
(Hanafusa et al., 2002
;
Sasaki et al., 2003
), we also
observed a loss of Mkp3 expression
(Fig. 2Q). Together, these
experiments show that somitic expression of Mkp3 depends on FGF
receptor-mediated signalling via phosphorylated ERK MAP kinase.
The response of Mkp3 to FGF beads over time illustrates a negative feedback interaction
To demonstrate that MKP3 is part of a negative-feedback loop regulating the
levels of dpERK in somites, we examined the dynamics of MKP3-dpERK
interactions in embryos. We looked at both the induction of Mkp3
expression and the levels of dpERK in response to FGF beads over time.
Exposure of HH17 somites to FGF2, FGF4 or FGF8 resulted in high-level ectopic
expression of Mkp3 within 1 hour, reaching a maximum at 5 hours
(Fig. 3A,B; data not shown).
Transcripts were seen in all somite regions
(Fig. 3F). Interestingly, after
an exposure of 24 hours, we noted a clear loss of endogenous Mkp3
transcripts in the vicinity of the beads. Ectopic expression immediately
around the bead was still detectable, suggesting that active FGF was released
from beads at these later time points (Fig.
3C). We next examined how the initial induction and following loss
of Mkp3 transcripts correlated with levels of active ERK by western
analysis of dissected somites at the same time points
(Fig. 3D). After exposure for 1
hour to FGF4, the levels of dual phosphorylated ERK were significantly
increased compared with control somites. However, after a 5 hour exposure the
levels of dpERK were reduced to the same levels as in control somites, most
probably owing to the increased levels of MKP3 protein produced from the
ectopic transcripts within the somite at this point. After 24 hours, dpERK was
no longer detected, even though the levels in untreated somites had increased.
This correlated extremely well with the apparent inhibition of Mkp3
expression by FGFs after 24 hours. Interestingly, increased levels of
scleraxis transcripts were only detected after 5 hours of exposure to
an FGF bead and these were more restricted to the sclerotome compartment
(Fig. 3E,G). This is consistent
with observations by others (Brent et al.,
2003; Brent and Tabin,
2004
) and in addition indicates differences between Mkp3
and scleraxis transcriptional regulation and the competence of cells
to express these genes.
|
|
Ectopic MKP3 within somites results in loss of scleraxis positive progenitor cells
We next investigated how manipulating the levels of active ERK MAP kinase
influenced somite patterning and in particular the specification of
scleraxis-positive progenitor cells. Electroporation of the hMKP3-GFP
fusion protein into somites of HH18 embryos resulted in high levels of
hMKP3-GFP expression throughout the dermomyotome, myotome and dorsal
most part of the sclerotome, as visualised by detection of GFP and MyoD in
sections (Fig. 4E). In situ
hybridisation using the chick Mkp3 probe, which crossreacted with
human transcripts, also demonstrated high-level expression
(Fig. 4F). In agreement with
RT-PCR results, we found that expression of hMKP3-GFP led to a loss of
scleraxis expression (Fig.
4G). The same result was obtained after expression of chick
Mkp3 from a different expression vector (data not shown). Expression
of EGFP alone had no effect on scleraxis
(Fig. 6F). To exclude the
possibility that expression of the MKP3 phosphatase at high levels could
affect non-specifically other phospho-proteins required for scleraxis
expression we used a mutant of MKP3 that does not contain the conserved
kinase-interaction motif `KIM' (Nichols et
al., 2000). This mutant protein has a normal basal activity in
vitro but is unable to undergo catalytic activation in response to ERK2 or to
inactivate ERK2 upon transfection into mammalian cells
(Karlsson et al., 2004
).
Electroporation of the KIM mutant did not affect scleraxis expression
in any of the embryos (Fig.
4H), even when expressed at high levels
(Fig. 4I). These experiments
suggest that active ERK MAP kinase, the levels of which are regulated by MKP3,
is required for scleraxis expression in somitic progenitor cells.
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Discussion |
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Transcriptional regulation of scleraxis depends on negative feedback loops that control the level of dpERK
This study uncovered a function of the dual specificity ERK MAP kinase
phosphatase, MKP3, for the specification of mesenchymal progenitors in the
somite sclerotome. We showed that Mkp3 was expressed in a
twin-striped pattern, which closely matched the emergence of
scleraxis transcripts along the anteroposterior somite edges
(Fig. 1). This pattern
suggested a link between the modulation of FGF signalling by MKP3 and
scleraxis expression. We demonstrated that somitic expression of
Mkp3 in the dorsal sclerotome was itself dependent on active ERK MAP
kinase. This implied that FGF signalling in dorsal sclerotome cells is
modulated by a negative feedback loop, which involves MKP3 and ERK MAP kinase.
Indeed, we found that the levels of Mkp3 transcripts detected in
response to FGF beads can cycle between extensive overexpression after a short
exposure to complete loss of endogenous Mkp3 message after 24 hours
(Fig. 3A-C; data not shown). We
showed by western blot analysis that this dynamic response correlated with an
increase in active dpERK protein after 1 hour and the loss of dpERK after 24
hours (Fig. 3D). This suggests
that high levels of MKP3 protein resulted in depletion of dpERK from somites,
which then lead to a loss of further endogenous Mkp3 transcription.
This was confirmed by RT-PCR analysis (Fig.
4C). In mouse embryos, implanting an FGF4 bead into the primitive
streak had similar effects after 24 hours because of expansive Spry2
expression (Davidson et al.,
2000) and it is likely that in chick somites sprouty and other
negative regulators impact upon this feedback loop. Interestingly, the
expression of Mkp3 immediately around the bead was not affected by
the feedback mechanism (Fig.
3C), as an ERK MAP kinase independent pathway, i.e. PI3-kinase
(Fig. 2E) could also regulate
Mkp3 expression in response to FGF beads
(Kawakami et al., 2003
;
Echevarria et al., 2005
).
MKP3 is a cell-autonomous modifier of FGF signalling and this strongly
suggested that FGFs signal to the dorsal sclerotome to control
scleraxis expression. We also show that dpERK is localised in the
region where scleraxis positive cells are found
(Fig. 4D). It is possible that
other signalling pathways cooperate with ERK MAP kinase and converge to
control the spatiotemporal expression of Mkp3 in developing somites
(Moreno and Kintner, 2004;
Rintelen et al., 2003
;
Tsang et al., 2004
). For
example, our results using caMEK1, which led to a loss of MyoD, would be
consistent with the idea that a MyoD-dependent factor contributes to the
expression of Mkp3 and scleraxis
(Fig. 5F). However, MyoD was
also lost after application of an FGF bead
(Fig. 5G), which induced
ectopic expression of Mkp3 and scleraxis, and this argues
against a MyoD-dependent mechanism. Furthermore, scleraxis is
expressed in MyoD- and Myf5-null mice
(Tajbakhsh et al., 1996
;
Brent et al., 2005
). In mouse,
FGF4 is important for scleraxis expression
(Brent et al., 2005
). We show
that Fgf4 expression is lost after caMEK1 treatment
(Fig. 5H); this could provide a
simple explanation for the loss of scleraxis under these conditions.
However, scleraxis expression depended on ERK activity
(Fig. 2L,
Fig. 4C,G,
Fig. 6A), which was directly
stimulated in somites after caMEK1 electroporation
(Fig. 4E). Therefore, loss of
MyoD and Fgf4 could not explain the loss of
scleraxis transcription.
We have focused here on MKP3, which is one of many negative regulators of
the MAP kinase transduction pathway. Other members of this group include Sef,
Spred and Sprouty. Expression of Sef and Sprouty is induced by activation of
the MAP kinase cascade itself, and as MKP3 regulates MAP kinase activity
directly it could be pivotal in controlling these antagonists, which act at
different levels in the pathway (Ozaki et
al., 2001; Furthauer et al.,
2002
; Tsang et al.,
2002
; Yusoff et al.,
2002
; Kovalenko et al.,
2003
). This suggests that MKP3 acts at a crucial level in the FGF
signal transduction cascade regulating all downstream events that depend on
ERK MAP kinase, including activation of transcription factors and
phosphorylation of cytoplasmic targets. Furthermore, other signalling
cascades, including the retinoic acid and Wnt/ß-catenin pathways, are
able to regulate the expression of Mkp3 in other tissues
(Moreno and Kintner, 2004
;
Tsang et al., 2004
). This
provides a possible role for other pathways, yet to be described in somites,
to limit Mkp3 expression to the dorsal sclerotome and would explain
how FGF responsive genes can be restricted.
In this study, overexpression experiments in chick embryos demonstrated the
close interdependence of dpERK and MKP3, which established a tightly
controlled level of active ERK MAP kinase in cells exposed to FGFs. There seem
to be discrepancies between the FGF bead experiments and caMEK1
electroporation. Mkp3 was expressed when high levels of dpERK were
present in response to a bead (Fig.
3A). Equally, caMEK1 induced an increase of dpERK but led to a
loss of Mkp3 transcription (Fig.
5A). The most likely explanation for these apparently conflicting
results is our finding that the bead-mediated upregulation of Mkp3
can go through ERK MAP kinase and PI3-kinase
(Fig. 2D,E). By contrast,
expression of scleraxis is probably dependent on dpERK only, and we
and others have found that scleraxis is upregulated in response to an
FGF bead after 5 hours (Fig.
3E) (Brent and Tabin,
2004). Electroporation of hMKP3-GFP led to loss of dpERK and
concomitant loss of scleraxis
(Fig. 4A,C,G). We propose that
scleraxis can only be induced at a certain level of dpERK, set by the
MKP3-ERK feedback loop. This is consistent with the delayed induction of
scleraxis by FGF beads relative to Mkp3
(Fig. 3E), which correlates
with a specific level of dpERK (Fig.
3D) and with the observed loss of scleraxis when dpERK
levels are either too high (Fig.
6D) or too low (Fig.
4C,G; Fig. 6A). In
Drosophila, the puckered gene, a member of the same family
as Mkp3, functions in a negative feedback loop to modulate JNK MAP
kinase activity. In both puckered overexpression and loss-of-function
experiments, a similar defect in dorsal closure resulted, reminiscent of our
data (Martin-Blanco et al.,
1998
). In addition, negative-feedback regulation has been proposed
to confer multistability on ERK MAP kinase activity
(Markevich et al., 2004
) and
the response of scleraxis to an intermediate level of dpERK would be
in agreement with this model. Our finding that MKP3 is capable of inhibiting
all downstream effectors involved in scleraxis induction, presumably
by depleting active ERK MAP kinase, suggests that within the somite, the
MKP3-dpERK negative feedback loop is crucial for establishing specific signal
strength.
The function of MKP3 and scleraxis in distal rib specification
This work demonstrated a functional importance of MKP3 for the correct
expression of scleraxis. This in turn is pivotal for the
specification of cells in the dorsal sclerotome. Scleraxis marks both
tendon and rib progenitors in the sclerotome of thoracic somites. Based on our
data, we speculate that scleraxis functions in the specification of distal rib
chondrocytes at an early stage for the following reasons. First, scleraxis has
been shown to increase aggrecan expression and stimulate chondrogenesis in
cell culture (Liu et al.,
1997). Second, scleraxis is transiently co-expressed with
sox9, a chondrogenic marker in rib primordia, in mouse at 12.5 dpc
(Asou et al., 2002
;
Brent et al., 2005
). However,
after 13.5 dpc expression diverges, which we interpret as scleraxis acting at
an early stage of rib specification. Similarly, in chick, scleraxis
is no longer expressed in condensing ribs and the expression pattern is
consistent with an early requirement for scleraxis in rib development
(Brent et al., 2003
). Third,
when we altered the signal strength of ERK MAP kinase, distal ribs did not
form (Fig. 6). Of the somitic
markers analyzed, only scleraxis expression was consistently lost
under these conditions. Fourth, in electroporated embryos, one rib was
typically affected, consistent with the loss of scleraxis expression
from the anterior border of one somite and the posterior border of the next
(Fig. 6D). Thus, we speculate
that one domain of scleraxis formed by two successive somites, gives
rise to one rib structure. This is in agreement with experiments demonstrating
that the distal rib arises through a process involving resegmentation
(Aoyama and Asamoto, 2000
).
The mechanism leading to rib loss remains to be investigated and could
involve a failure of progenitor cells to become specified. For example,
scleraxis may act together with BMP signalling and forkhead
transcription factors in cell specification
(Buchberger et al., 1998;
Kramer et al., 2000
;
Sudo et al., 2001
).
Alternatively, progenitor cells may undergo apoptosis or they might fail to
migrate. In this context, it is interesting to note that the migration of
cells from the lateral somite into the somatopleure is dependent on the
regulation of paxillin by ERK activity
(Ishibe et al., 2004
).
Interestingly, in mouse, the expression of Fgf8, scleraxis and
Mkp3 is different suggesting a species-specific change in the
function of MKP3. In chick, Fgf8 transcripts are found in the central
myotome but in mice they colocalise with scleraxis at the rostral and
caudal somite edges, indicating that FGF8 signals in an autocrine fashion
(Crossley and Martin, 1995).
In mouse, Mkp3 is not expressed in the dorsal sclerotome but in the
dermomyotome/myotome (Dickinson et al.,
2002
; Klock and Herrmann,
2002
). Thus, it would be interesting to investigate whether
Mkp3 knockout mice have normal ribs and tendons.
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ACKNOWLEDGMENTS |
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