1 Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, ON,
Canada
2 Department of Molecular and Medical Genetics, University of Toronto, ON,
Canada
Author for correspondence (e-mail:
rossant{at}mshri.on.ca)
Accepted 9 June 2003
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SUMMARY |
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Key words: Mouse, Embryo, MAPK, ERK, FGF Signaling
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Introduction |
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Although this RAS-MAPK pathway is not the only signaling cascade downstream
of RTKs, it is often the key pathway for many RTK-mediated cell fate
decisions. For example, in C. elegans and D. melanogaster,
mutations in ERK genes often give rise to phenotypes similar to those
generated by a loss of the RTKs themselves, and defects in various RTK
signaling pathways can be compensated by gain-of-function alleles of RAS, RAF,
MEK and/or ERK (Marshall,
1995). In vertebrates, numerous gain- and loss-of-function
experiments have implicated RTK signaling in various developmental processes,
including gastrulation, vasculogenesis, limb development, neural patterning
and placentation. Although these studies reveal general roles of fibroblast
growth factor (FGF), epidermal growth factor (EGF), vascular endothelial
growth factor (VEGF) and neurotrophin signaling in specific developmental
events, they do not reveal the precise location and timing of the signaling
interactions that occur during the genesis of the tissue or structure.
Although the expression domains of receptors and ligands identify potential
regions of signaling, they cannot reveal when ligands begin actively
signaling, the direction and distance across which ligands act, or the
intensity and duration of signaling. In order to view actual domains of RTK
signaling, a direct readout of RTK activation is needed.
Whole-mount immunohistochemistry with antibodies specific to the
di-phosphorylated forms of ERK1 and ERK2 (dp-ERK) has been used to map active
ERK signaling domains within Drosophila, Xenopus and zebrafish
embryos (Christen and Slack,
1999; Curran and Grainger,
2000
; Gabay et al.,
1997a
; Gabay et al.,
1997b
; Reich et al.,
1999
; Sawada et al.,
2001
; Shinya et al.,
2001
). Although other pathways, such as integrins, cytokines and
G-protein-coupled-receptors, can activate the RAS-MAPK pathway
(Belcheva and Coscia, 2002
;
Widmann et al., 1999
), the
majority of dp-ERK domains correspond to RTK signaling domains in these
embryos. Dp-ERK patterns are discrete and dynamic in embryos and correlate
largely with regions of FGF and EGF signaling. These studies have given
significant insight into RTK-MAPK signaling events guiding various
developmental processes in these species. Limited analysis of mouse embryos
indicated this approach would be feasible in the analysis of mouse
embryogenesis as well (Lai and Pawson,
2000
).
In this study, we have used whole-mount immunohistochemistry with phospho-ERK antibodies to map the spatial and temporal patterns of ERK activation during early postimplantation mouse development. In many cases, dp-ERK detection has enabled us to determine the timing, duration and intensity of receptor activation, to visualize gradients and boundaries of activation and to postulate the distribution of active ligand. This atlas of dp-ERK domains provides insight into how RTK signaling (FGF signaling in particular) is shaping the mouse embryo and how this signaling is regulated in vivo, and pinpoints regions where downstream target genes can be sought.
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Materials and methods |
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Primary antibodies
Two different primary antibodies against the diphosphorylated forms of ERK1
and ERK2 as well as a primary antibody against total ERK1 and ERK2 (Cell
Signaling Technology) were used in this study. The first phospho-ERK antibody
was a mouse monoclonal antibody (#M8159, Sigma) raised against a synthetic
diphosphorylated peptide corresponding to highly conserved residues around
Thr183/Tyr185 of ERK2. This antibody has been widely used to map dp-ERK
domains in Drosophila, Xenopus and zebrafish embryos
(Christen and Slack, 1999;
Curran and Grainger, 2000
;
Gabay et al., 1997a
;
Gabay et al., 1997b
;
Reich et al., 1999
;
Sawada et al., 2001
;
Shinya et al., 2001
). The
second phospho-ERK antibody was a rabbit polyclonal antibody raised against a
similar (if not identical) peptide (#9101, Cell Signaling Technology). The two
antibodies gave essentially the same staining patterns in mouse except in
regions where endogenous mouse immunoglobulins reside. Mouse monoclonal
antibody staining is problematic in such regions owing to the crossreactivity
of secondary anti-mouse antibodies to the endogenous immunoglobulins found in
the tissue. For this reason, the rabbit polyclonal antibody was used in the
majority of these studies.
Secondary antibodies
Detection methods included biotinylated secondary antibodies (Jackson
ImmunoResearch Laboratories) in conjunction with either Cy3-streptavidin
(Jackson ImmunoResearch Laboratories) or Vectastain HRP ABC Reagent (Vector
Laboratories). Because the HRP reaction catalyzed the formation of a
precipitate that could diffuse from the position of the primary antibody,
fluorophore-conjugated streptavidin gave better resolution of antigen
location. To visualize nuclei in fluorophore-labeled embryos, embryos were
incubated in Hoechst 33342 or YOYO-1 (Molecular Probes).
Sectioning
After imaging, whole-mount DAB-stained embryos were embedded in paraffin
wax, cut into 10 µm sections and counterstained with Toluidine Blue.
Whole-mount embryos labeled with Cy3 were sectioned transversely (50
µm sections) with glass needles and mounted in 80% glycerol between
coverslips.
Imaging
Smaller whole-mount embryos (<8.0 dpc) were photographed on a DMIRBE
compound microscope (Leica) with DIC optics and standard epifluorescence using
a CCD camera (Hamamatsu) and Openlab software (Improvision). Larger
whole-mount embryos were imaged on a MZFLIII stereoscope (Leica) with liquid
crystal filter (CRI) and the Hamamatsu-Openlab imaging system.
For higher resolution, a Zeiss LSM510 confocal or Delta Vision Olympus
deconvolution system was used. For confocal imaging, the pinhole was set wider
than 1 Airy unit and a series of thicker (5 µm) optical sections were
taken. In some instances, all the serial sections have been overlaid, using
the Zeiss confocal software, to give a composite confocal image. In other
instances, a single confocal section is presented.
Embryo culture
In order to verify that staining was specific to the phosphorylated form of
ERK and to determine which domains of dp-ERK staining were specifically due to
FGFR signaling, embryos were briefly treated with chemical inhibitors to MAPK
kinase (U0126, Cell Signaling Technology) or to FGFR (SU5402, CalBiochem)
prior to dp-ERK staining. Embryos were dissected out of the deciduas and
placed in 1-2 ml of RPMI with 1% BSA, pre-equilibrated at 37°C, 5%
CO2. A final concentration of 0.1% DMSO was added to each culture.
Controls contained DMSO only and experimental cultures included 50 µM U0126
(dissolved in DMSO) or 40 µM SU5402 (dissolved in DMSO). It was determined
empirically that smaller embryos required 30 minutes for the inhibitors
to penetrate and mediate a response while larger embryos required up to 90
minutes. Cultures were rotated gently every 10 minutes during the incubation
period at 37°C, 5% CO2.
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Results |
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5.0-8.0 dpc
The most intense, sustained dp-ERK domains in 5-8 dpc embryos were in
extra-embryonic tissues. Dp-ERK staining was first observed throughout the
extra-embryonic ectoderm (Exe) at 5.5 dpc
(Fig. 1B) and then became
restricted to a ring in the most proximal region of the Exe in subsequent days
(Fig. 1C-F). The intensity of
this dp-ERK domain as well as the cellular depth of the band decreased from 6
to 8 dpc and was barely detectable after 8.0 dpc. In the ectoplacental cone
(EPC), ERK signaling began throughout the EPC shortly after EPC formation
(
5.5 dpc) and was sustained in the central, diploid population of EPC
cells in subsequent days (6-8 dpc) (Fig.
1B-F).
In contrast to these regions of sustained signaling that were readily
detectable in every embryo, there were also regions of dp-ERK staining which
were not observed in all embryos isolated at a given stage (±0.5 dpc).
These seem to represent areas of signaling confined to a narrow window of
development. Such dynamic regions of ERK signaling were found in the distal
tip of the epiblast (5.5 dpc) (Fig.
1B), the allantoic bud (
7.5 dpc)
(Fig. 1D), blood island
mesoderm (
7.5 dpc) (Fig.
1D), headfold mesoderm (Fig.
1E) and heart primordia (early headfold to 4-somite)
(Fig. 1F). Scattered dp-ERK
staining was seen in mitotic cells in some regions of embryonic ectoderm and
mesoderm throughout development (see Fig.
1B-D). No significant dp-ERK staining was associated with the
primitive streak or newly forming somites at any stage of development.
8.0-10.5 dpc
As the complexity of the embryo increased so too did the complexity of the
dp-ERK patterns. The most prominent and consistently reproducible domains of
sustained signaling were the frontonasal process (9.0-10.5+ dpc), forebrain
(8.5-10+ dpc), midbrain-hindbrain boundary (8.5-10.5 dpc), branchial arches
(9.0-10.5+ dpc), foregut (8.5-9.0 dpc), limb buds (9.0-10.5+ dpc), liver
primordia (9.5-10.5+ dpc), tailbud (8.5-10.5+ dpc) and placenta (8-10.5+ dpc)
(Fig. 1G-J) (data not shown).
ERK activation appeared in the limb bud field just prior to outgrowth (9 dpc),
throughout the limb bud during early outgrowth (9-10 dpc), and in the distal
region of later limb buds (10.5+ dpc). Signaling was sustained in the
frontonasal process, the maxillary and mandibular components of the first
branchial arches, and (to a lesser degree) in the second and third branchial
arches during this period. In the CNS, dp-ERK staining was first observed in
the neural ectoderm of the anterior forebrain and across the
midbrain-hindbrain boundary at 8.5-9.0 dpc
(Fig. 1G) and later was most
prominent in axons and nerve tracts in these regions (9-10.5+ dpc).
In addition to regions of sustained ERK signaling, brief and dynamic pulses
of ERK activation were associated with blood vessel formation (8.0-10.5+ dpc),
somite remodeling (9.5 dpc), neural crest migration (9-9.5 dpc), as well
as during development of the ear primordia (9-9.5 dpc), eye primordia (9-10.5+
dpc), nasal pits (10-10.5 dpc) and peripheral nervous system (10-10.5+ dpc).
For example, at 8.5-9.0 dpc, dp-ERK was prevalent in the dorsal aorta (da) and
then in the newly forming intersomitic vessels (iv) sprouting from the dorsal
aorta (9 dpc) (Fig. 1H,
Fig. 2A-E). At later stages
(9.5-10.5+ dpc), dp-ERK staining in these established vessels was no longer
detectable (Fig. 2F); only the
most nascent blood vessels growing between the most caudal somites were dp-ERK
positive (Fig. 1I,J,
Fig. 2B-F). As a second
example, at 9.0 dpc, dp-ERK was prevalent in neural crest cells migrating away
from the neural tube (Fig.
3A-C) and then in newly formed ganglia and sensory nerve tracts at
10-10.5 dpc (Fig. 1J,
Fig. 3D-H).
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In order to determine which dp-ERK positive domains were activated by FGF
signaling, embryos were briefly cultured in an FGFR-specific inhibitor
(SU5402) prior to staining. SU5402 binds the ATP binding site of FGFR
receptors (Mohammadi et al.,
1997) and is used widely in developmental systems to specifically
block FGFR signaling (Maroon et al.,
2002
; Shinya et al.,
2001
). As seen in Fig.
4, dp-ERK staining was specifically diminished after SU5402
treatment in the extra-embryonic ectoderm (6-8 dpc), the heart primordia (8
dpc), limb buds, branchial arches, frontonasal process, midbrain-hindbrain
boundary and eye primordia, implicating FGFR-dependent ERK signaling in these
domains. By contrast, dp-ERK staining in the EPC
(Fig. 4D,E), mitotic cells of
the embryo proper (Fig. 4D) and
the heart ventricle (Fig. 4F)
were unaffected by the inhibitor, suggesting other FGFR-independent signaling
pathways are probably responsible for ERK activation in these regions. The
inhibitor-treated embryos were always compared with cultured controls because
culture, per se, altered dp-ERK patterns. Regions of transient, dynamic dp-ERK
(such as blood vessels) rapidly lost dp-ERK staining after brief culture and
could not be evaluated by these means.
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In addition to the contiguous domains of sustained ERK activation, dynamic,
punctate staining was often observed, scattered throughout embryonic regions
(e.g. Fig. 1A-D,G). Shown in
Fig. 6M-P are dp-ERK positive
cells along the apical side of the ectoderm of a gastrulating embryo (6.5
dpc). Double labeling embryos for dp-ERK and DNA revealed that over 85% of the
apparently sporadic dp-ERK positive cells had condensed chromosomes,
indicative of cells in mitosis. Only cells judged by chromosome morphology to
be in prophase to early anaphase were dp-ERK positive, consistent with
previous findings in cell culture in which ERK plays a role in entry into
mitosis and exit from anaphase (Shapiro et
al., 1998; Willard and Crouch,
2001
).
FGFR-ERK signaling shaping the mouse embryo
Detailed analysis of the dp-ERK patterns that occur during the development
of a tissue or organ can give insight into the signaling events shaping that
tissue. In some cases, dp-ERK patterns corresponded to regions where FGF
signaling is known to play specific roles. These patterns can be used to
further our understanding of the signaling processes in such tissues.
Signaling in limb bud
In the limb bud, dp-ERK was first observed in the surface ectoderm during
limb bud initiation (Fig. 7A).
As limb bud outgrowth continued, dp-ERK diminished in the ectoderm and
increased dramatically in the underlying mesenchyme
(Fig. 7E). By the time the
apical ectodermal ridge (AER) formed, a pronounced gradient of dp-ERK staining
was seen in the mesenchymal (Fig.
7B-D) and ectodermal (Fig.
7F,G) cells with the intensity strongest adjacent to the AER,
pointing to the AER as a putative FGF signaling source. In transverse
sections, dp-ERK staining was observed in some regions of the dorsal and
ventral surface ectoderm (Fig.
7F,G), suggesting there is FGF signaling from the underlying
mesenchyme or perhaps autocrine signaling within the surface ectoderm. A
gradient of dp-ERK was observed in the mesenchyme beneath the surface ectoderm
in some regions (e.g. Fig. 7F
ventral, Fig. 7G dorsal),
indicating the surface ectoderm may also be a putative source of FGF
signaling.
Genetic loss-of-function studies have evaluated the roles of FGF signaling
in limb bud initiation, outgrowth and patterning in the proximodistal
direction and have led to detailed models of FGF signaling in limb development
(Martin, 1998) (depicted in
Fig. 7H). FGF10 is thought to
signal from the lateral plate mesoderm to FGFR2 in the overlying surface
ectoderm to induce limb bud initiation (consistent with dp-ERK staining in the
surface ectoderm in Fig. 7A,H).
As outgrowth continues, FGF8 signals from the surface ectoderm to FGFR1 in the
underlying mesenchyme (consistent with dp-ERK staining in
Fig. 7E). By the time the AER
forms, FGF4 and FGF8 are thought to signal to FGFR1 and FGFR2b in the progress
zone mesenchyme beneath the AER (consistent with
Fig. 7B-D,F,G).
Beyond just confirming current models of limb bud development with FGF signaling playing an important role in proximodistal outgrowth and patterning, the FGFR-dependent dp-ERK staining in the dorsal and ventral surface ectoderm and underlying mesenchyme (Fig. 7F,G) suggests FGF signaling may also play a role in dorsoventral patterning.
Signaling in the extra-embryonic ectoderm
The correlation between previously suspected regions of signaling and
dp-ERK patterns in limb buds validated this approach of using phosphorylated
ERK as a readout of the active signaling during mouse embryogenesis. There are
many regions of FGFR-dependent dp-ERK staining, such as the extra-embryonic
ectoderm, where the precise role of FGF has not yet been elucidated.
In the extra-embryonic ectoderm, Fgfr2 is expressed throughout the
entire Exe (as shown in Fig.
8I,J, depicted by yellow in
Fig. 8F-H), but the
FGFR-dependent dp-ERK staining was more restricted. ERK activation was first
seen throughout the entire Exe at 5.5 dpc
(Fig. 8A,F), after the initial
formation of the tissue. The range of signaling gradually decreased as
development continued and the domain of activation became restricted to a ring
6-9 cells across at 6.0 dpc (white bracket,
Fig. 8B), 4-6 cells across at 7
dpc (white bracket, Fig. 8C)
and only 2-4 cells across by 7.5 dpc (white bracket,
Fig. 8D). At the early stages,
a gradient of dp-ERK with strongest staining in the proximal Exe was observed
(Fig. 8A,B), suggesting that
the receptor is activated by FGFs, possibly FGF4
(Niswander and Martin, 1992
),
derived from the adjacent epiblast. By later stages (7-8 dpc), the gradient of
dp-ERK had disappeared and the distal boundary of ERK activation was sharp
(Fig. 8C-E,H). In these older
embryos (7-8 dpc), dp-ERK staining was seen in both the peripheral and
internal layers of the extra-embryonic ectoderm
(Fig. 8E), suggesting
extra-embryonic mesoderm cells at the base of the chorion are the likely
source of FGF (see Fig.
8H).
FGF-dependent trophoblast stem cells (TS cells) have been derived from the
Exe at the stages the FGFR-dependent dp-ERK ring is evident (6-8 dpc)
(Tanaka et al., 1998;
Uy et al., 2002
). Tanaka et
al. proposed a model in which FGF4 signals from the epiblast to the overlaying
Fgfr2-expressing Exe to sustain this stem cell population in vivo
(Tanaka et al., 1998
). The
staining pattern is consistent with this and, thus, the dp-ERK ring may
demarcate the stem cell population in vivo.
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Discussion |
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Detailed analysis of the spatial and temporal pattern of dp-ERK within a given tissue yielded further insights into the specific role of RTK-ERK signaling in the region. In the limb buds, dp-ERK patterns were consistent with previously identified roles of FGF signaling from the lateral plate mesoderm to surface ectoderm (in order to induce limb bud initiation), from surface ectoderm to underlying mesenchyme (in order to stimulate outgrowth) and from AER to underlying mesenchyme (to promote proliferation, outgrowth, cell survival and patterning) (Fig. 7).
The parallels between dp-ERK patterns and current models of FGF signaling
in the limb bud validated this approach as a means to visualize signaling
processes and permit speculation on the roles of signaling in various, less
well studied regions. For example, in blood vessels, dp-ERK was seen
transiently in newly forming vessels but was attenuated as vessels matured,
suggesting a role of ERK signaling in blood vessel establishment but not
maintenance. Consistent with this, mutations in the Flk1 receptor
prevent blood vessel formation (Shalaby et
al., 1995), suggesting the transient ERK activation in nascent
blood vessels may be due to FLK1-ERK signaling. A brief pulse of
FGFR-dependent ERK activation in the heart primordia
(Fig. 1F;
Fig. 4B,E) indicated FGF
signaling may play a role in heart induction in mouse as it does in zebrafish
and chicken (Alsan and Schultheiss,
2002
; Reifers et al.,
2000
). In the EPC, dp-ERK staining began after the initial
formation of the tissue and persisted for several days in the central diploid
cells (Fig. 1) suggesting a
continued role of ERK signaling in the maintenance, patterning and/or
proliferation of the region. Similarly, patterns of ERK activation associated
with structures such as the eye, ear, branchial arches, extra-embryonic
ectoderm and peripheral nervous system provide insight into precisely when and
where signaling occurs during the genesis of these tissues.
Most dp-ERK domains correspond to regions of FGF signaling in
mouse
With more than 50 different RTKs in mouse and potential crosstalk with
other signaling pathways, it is striking that domains of dp-ERK were so
discrete. Many domains of dp-ERK correspond to regions of known or speculated
FGF signaling in mouse, and dp-ERK staining was abolished or attenuated in
most domains by an FGFR-specific inhibitor (Figs
4,
5). The majority of dp-ERK
domains in Xenopus and zebrafish are also FGFR-dependent
(Christen and Slack, 1999;
Curran and Grainger, 2000
;
Shinya et al., 2001
), and FGFs
induce particularly robust and sustained MAPK responses in cell culture
(Hadari et al., 1998
;
Marshall, 1995
). Although
RAS-ERK was described initially as a universal signaling cascade downstream of
all RTKs, some RTKs elicit only weak RAS-ERK responses, preferentially use
other pathways or even inhibit the RAS-ERK cascade
(Elowe et al., 2001
;
Schlessinger, 2000
). Thus, not
all domains of RTK signaling are scored in this assay. Instead, dp-ERK
staining in mouse embryos primarily reveals FGF signaling domains.
Although the majority of dp-ERK domains were FGFR dependent, not all
domains of FGFR signaling were dp-ERK positive. Loss-of-function studies
reveal FGFR1 is essential for mesoderm migration through the primitive streak
and somite formation (Ciruna et al.,
1997; Deng et al.,
1994
; Yamaguchi et al.,
1994
). Yet detailed examination of various stages failed to reveal
significant dp-ERK staining in these regions aside from sporadic mitotic cells
(see Fig. 6M-P, Fig. 2D). This lack of dp-ERK
staining was surprising as FGFR-dependent ERK signaling is prominent in the
primitive streak of Xenopus and zebrafish embryos
(Christen and Slack, 1999
;
Curran and Grainger, 2000
;
Shinya et al., 2001
), as well
as in the newly forming somites of zebrafish
(Sawada et al., 2001
).
However, weak dp-ERK staining was occasionally seen in newly forming somites
(in two embryos out of
80), suggesting transient FGFR1-ERK signaling may
occur in this region. Negative feedback inhibitors of the FGFR-ERK signaling
pathway, including Sprouty genes, Sef and MAP kinase phosphatase
genes, are highly expressed in these regions of the embryo
(Dickinson et al., 2002a
;
Klock and Herrmann, 2002
;
Lin et al., 2002
;
Minowada et al., 1999
) and may
account for decreased dp-ERK staining. Alternatively, FGFR1 may signal
preferentially through another pathway (such as PI3 kinase, PLC
or
perhaps other MAPKs) in these tissues.
Not all dp-ERK domains were eliminated by treatment with an FGFR inhibitor.
These FGFR-independent domains, including the EPC and sensory neurons, are
probably due to other signaling pathways that must also lead to sustained ERK
activation. Neurotrophins signaling through TRK receptors are essential for
sensory neuron survival, outgrowth and differentiation, and are likely
candidates for ERK signaling in nerve tracts and ganglia of the peripheral
nervous system (Crowley et al.,
1994; Farinas et al.,
2002
; Smeyne et al.,
1994
). EGF or HGF signaling may be responsible for dp-ERK staining
in the EPC (Patel et al.,
2000
; Sibilia and Wagner,
1995
).
Dp-ERK domains define regions where target genes of the signaling
pathways can be sought
The dp-ERK positive cells in a given region likely receive patterning and
differentiation cues that are different from those in adjacent dp-ERK negative
cells. Thus, spatial and temporal correlation of gene expression with a dp-ERK
domain indicates that the gene may be a downstream target of the signaling
pathway.
Previous studies have shown Lhx6 and Evx1 to be
downstream of FGF signaling in the branchial arch and limb, respectively
(Niswander and Martin, 1993;
Trumpp et al., 1999
), and
indeed expression patterns of these genes closely correlate with the
FGFR-dependent dp-ERK patterns. In the Exe, it is striking that expression of
trophoblast stem cell genes Eomes and Sox2 are expressed in
essentially identical spatial and temporal patterns as dp-ERK (see
Fig. 8)
(Ciruna and Rossant, 1999
;
Wood and Episkopou, 1999
),
suggesting they are downstream targets of FGF signaling in the Exe. Additional
studies are needed to test the relationship between FGF signaling and
expression of Eomes and Sox2. However, other T-box and HMG
transcription factors are known to be downstream of FGF signaling in other
tissues (Faber et al., 2001
;
LaBonne et al., 1995
;
Murakami et al., 2000
;
Smith et al., 1991
). These
genes, as well as additional unknown genes sharing similar spatial and
temporal expression patterns, are excellent candidate targets of FGF signaling
in the region.
Dp-ERK domains give insight into properties of ERK signaling in
vivo
From analysis of the spatial and temporal patterns of ERK activation, we
have gained insight into signaling processes as they occur in the
three-dimensional context of the mouse embryo.
Gradients versus sharp boundaries
During development, highly regulated growth factor signaling is known to
pattern fields of cells. Such signaling is not solely controlled by
availability of growth factor. A parallel set of inhibitory mechanisms is
often used to spatially and temporally restrict levels of signaling. In the
Exe, a gradient of FGFR-dependent ERK activation was seen throughout the
tissues at 5.5 dpc, gradually diminished in intensity
(Fig. 1B-F), and became
restricted to a ring two cells in diameter by 7.5 dpc
(Fig. 8D). FGFR inhibitors,
Sef and Sprouty genes, are induced by FGF signaling
(Chambers et al., 2000;
Furthauer et al., 2002
;
Ozaki et al., 2001
), and are
expressed in patterns consistent with a role in shaping the boundaries of this
dp-ERK domain in the Exe. Sef is expressed in a gradient from the
distal to proximal Exe (Lin et al.,
2002
), indicating it may play a role to attenuate signaling in the
distal Exe. Spry2, however, is transcriptionally induced in the same
region where dp-ERK is evident (Minowada
et al., 1999
) and may play a cell-autonomous role in modulating
the ERK response over time (Tefft et al.,
2002
). Sprouty genes and Sef are also expressed in the
limb bud and probably play a role in modulating ERK signaling in the region.
Additional studies will be needed to compare precisely domains of inhibitor
expression with dp-ERK staining and to test the role of inhibitors
functionally in defining boundaries (and intensities) of dp-ERK domains.
Signaling through cytoplasmic ERK
Although many aspects of ERK signaling in mouse were consistent with
findings in Drosophila, Xenopus and zebrafish, one striking
difference did exist: phosphorylated ERK appeared predominantly cytoplasmic in
the contiguous dp-ERK domains of the mouse, whereas dp-ERK is nuclear in
Drosophila and Xenopus embryos
(Curran and Grainger, 2000;
Gabay et al., 1997a
;
Gabay et al., 1997b
). It seems
unlikely that there was a technical problem with nuclear dp-ERK detection in
the mouse embryos because nuclear dp-ERK was easily observed in regions of
injury (Fig. 6).
One explanation for the apparent discrepancy is that the nuclear
localization of ERK may be transient in the mouse embryo. Phosphorylated ERK
may shuttle to the nucleus upon initial activation of the signaling pathway
but rapidly be dephosphorylated by nuclear MAPK phosphatases (MKP1 and MKP2)
(Volmat et al., 2001) or
become exported from the nucleus, such that only cytoplasmic dp-ERK is
detected. Confocal sectioning through the entire dp-ERK regions, in which
signaling was just beginning (such as the Exe at 5-5.5 dpc), however, failed
to reveal any evidence for nuclear dp-ERK. Furthermore, when we analyzed the
FGF-ERK signaling cascade in trophoblast stem cells derived from the Exe
(Tanaka et al., 1998
), only
sustained cytoplasmic dp-ERK was detected in cell culture (L.B.C. and J.R.,
unpublished). Taken together, this suggests phosphorylated ERK may not
translocate to the nucleus in endogenous domains of sustained signaling in
mouse embryos.
Biochemical analysis of the RAS-ERK signaling cascade in cell culture has
lead to current models of ERK-mediated transcription in which nuclear
translocation of phosphorylated ERK is important for activation of various
transcription factors (Pouyssegur et al.,
2002). However, ERK has numerous targets in the cytoplasm,
cytoskeleton and plasma membrane (Pearson
et al., 2001
). Cytoplasmic ERK may mediate a transcriptional
response in the mouse by phosphorylating cytosolic proteins involved in
transcriptional regulation, such as p90RSK, which in turn relay
signals to the nucleus. Sprouty and MAP kinase phosphatase genes are
transcriptionally induced by ERK (Camps et
al., 1998
; Chambers et al.,
2000
; Ozaki et al.,
2001
) and exhibit expression patterns correlating with subsets of
dp-ERK domains (Dickinson et al.,
2002a
; Dickinson et al.,
2002b
; Minowada et al.,
1999
). Thus, there is evidence for ERK-mediated transcription in
regions where only cytoplasmic dp-ERK is detected. Of interest, this
co-localization of the negative feedback inhibitors with dp-ERK domains
provides a means to control the duration and magnitude of MAPK activation in
each region, which is a parameter shown to be crucial for cell fate decisions
(Marshall, 1995
).
Cytoplasmic ERK also participates in the regulation of cytoskeletal
architecture in a manner that is independent of transcriptional regulation.
ERK has been implicated in cytoskeletal remodeling and focal adhesion assembly
required in cell motility (Fincham et al.,
2000; Klemke et al.,
1997
). Thus, cytoplasmic dp-ERK in sprouting blood vessels,
migrating neural crest and outgrowing sensory axons may be sufficient for the
roles of ERK in these cell populations.
In summary, this atlas of dp-ERK domains provides an overview of active ERK signaling regions in the mouse embryo. The timing, location, intensity, duration and magnitude of ERK activation in various regions yields insight into how signaling is shaping the mouse embryo, how this signaling is regulated in vivo, and where to look for downstream targets of the signaling cascades.
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ACKNOWLEDGMENTS |
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Footnotes |
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