Developmental Genetics Laboratory, Cancer Research UK, 44 Lincoln's Inn Fields, London WC2A 3PX, UK
Accepted 16 April 2004
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SUMMARY |
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Key words: Receptor tyrosine phosphatase, Somitogenesis clock, Presomitic mesoderm, Notch signalling, Wnt, Convergent extension, Zebrafish
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Introduction |
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The periodic production of somites along the anteroposterior axis of the
vertebrate body involves a molecular oscillator, the `segmentation clock',
which can be visualised through the cyclic activation of a small set of
regulatory genes (for a review, see Maroto
and Pourquié, 2001). These oscillations result in dynamic
wave-like domains that sweep across the presomitic mesoderm (PSM) in a
posterior-to-anterior direction, narrowing as they approach its anterior end.
The oscillation becomes arrested in each cell as it passes from the presomitic
to the somitic region of the mesoderm. One temporal oscillation occurs in the
PSM for each somite that is formed, and mutations or treatments that perturb
oscillatory gene expression also disrupt segmentation
(Evrard et al., 1998
;
Henry et al., 2002
;
Holley et al., 2000
;
Hrabe Angelis et al., 1997
;
Jiang et al., 2000
;
Kusumi et al., 1998
;
Oates and Ho, 2002
;
Zhang and Gridley, 1998
).
In each cycle, these cycling genes are first expressed in the tailbud, and
expression is subsequently propagated through the posterior PSM. When it
reaches the anterior PSM, it becomes stabilized, and is localized to either
the rostral or caudal part of the future somite. Based on these observations
and others, the PSM has been subdivided into three different regions in which
the oscillator responds to different regulatory cues: the posterior
undetermined zone, the anterior committed zone (within which cycling is still
seen) and a differentiating anterior most zone, within which somite boundaries
and compartments are established (Gajewski
et al., 2003; Morales et al.,
2002
; Saga and Takeda,
2001
).
Most oscillatory genes are related to Notch signalling and dependent on
Notch signalling for their cyclic expression. In the mouse and chick, these
include lunatic fringe (lfng), which modulates the
efficiency of Notch signalling (Aulehla and
Johnson, 1999; Forsberg et
al., 1998
; McGrew et al.,
1998
), and various hairy-related genes [hairy1,
hairy2 and Hey/Hesr/HRT2 in chick
(Jouve et al., 2000
;
Leimeister et al., 2000
;
Palmeirim et al., 1997
);
Hes1, Hes7 and Hey1 in mouse (Bessho et al., 2001a;
Jouve et al., 2000
;
Nakagawa et al., 1999
)] that
are transcriptional targets of Notch signalling and encode basic
helix-loop-helix (bHLH) repressor proteins. In the zebrafish PSM, three genes
have so far been shown to have cyclic expression: the Notch ligand
deltaC (Jiang et al.,
2000
) and the hairy-related genes, her1 and
her7 (Henry et al.,
2002
; Holley et al.,
2000
; Oates and Ho,
2002
). Mutations in these cycling genes and other Delta/Notch
components result in defective somite segmentation: intersomitic clefts fail
to form or are late and irregular. In zebrafish, her1 and
her7 appear to cross regulate each other, and it has been proposed
that a negative feedback loop involving these genes constitutes the oscillator
(Henry et al., 2002
;
Holley et al., 2002
;
Lewis, 2003
;
Oates and Ho, 2002
).
In this study we present a novel regulator in the control of the
somitogenesis clock, RPTP, a member of the type IIB family of
receptor tyrosine phosphatase. We describe the cloning of zebrafish
RPTP
and its expression pattern during early zebrafish
development, and provide evidence that RPTP
is required for
normal oscillatory gene expression in the PSM. We show that RPTP
behaves as a positive regulator of her1 and her7 expression,
acting either upstream of or in parallel with Delta/Notch signalling. We also
find that RPTP
is required for convergent extension, a process
of cell-rearrangement during gastrulation, raising the possibility that
RPTP
functions in Notch and Wnt signalling.
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Materials and methods |
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Cloning of zebrafish RPTP and plasmid construction
Library screening
A chick RPTP cDNA was used to screen a zebrafish
ZapII cDNA library (Haddon et al.,
1998
) from which several positive cDNA clones were isolated, of
which the longest clone (clone 21) spanned sequence nucleotides 1621-4565.
5'-rapid amplification of cDNA ends (RACE)
The missing 5' sequence was obtained by reverse transcription-PCR
from 24 hpf embryo total RNA by using the 5'/3' RACE kit
(Boehringer) according to the manufacturer's protocol. Specific primers used
for 5'-RACE were: antisense 5'-RACE-A1,
5'-CCTTCTTGCCCTCGGTGTTGGCGAG-3' and antisense nested
5'-RACE-A2, 5'-CTCCTCAGTCTGAAACATGACCTCC-3'. The full-length
sequence of RPTP was deposited in the GenBank database under the
Accession Number AY555586.
Whole-mount in situ hybridisation and generation of riboprobes
Whole-mount in situ hybridisation was performed essentially as previously
described (Haddon et al.,
1998). For all experiments using multiple genotypes, hybridisation
was carried out in parallel and colour development allowed to run for the same
amount of time. The embryos were photographed using a Leica DC500 camera.
Digoxigenin-labelled RNA antisense probes were generated with a Stratagene RNA
transcription kit. Enzymes for linearization and transcription for probe
synthesis were as follows: RPTP
, EcoRI/T7; deltaC,
XbaI/T7; her1, XhoI/T3; her7, SpeI/T7;
mespa, EcoRI/T7; mespb, HindIII/T3; papC, ApaI/T3;
fgf8, EcoRV/SP6; ntl, HindIIII/T7; spt, EcoRI/T7;
dlx3, EcoRI/T7; hgg1, XhoI/T3.
Morpholino design and injection
Morpholinos (Genetools) were designed with sequences complementary to
RPTP cDNA in a location just upstream or covering the initiating
start codon based on the company's recommendations. The morpholino sequences
were: RPTPmo1, 5'-CGCAGGTATTCATTTTCCGTTGTTA-3';
RPTPmo2, 5'-GTTGGGAAAACAAGTCGAAATCATT-3'; 5-m (5-mispair
control oligonucleotide to RPTPmo1),
5'-CGgAGcTATTgATTTTCCcTTcTTA-3'; her1mo,
5'-CGACTTGCCATTTTTGGAGTAACCA-3'. Morpholinos were solubilised and
diluted as described by Nasevicius and Ekker
(Nasevicius and Ekker, 2000
)
and injected into one- or two-cell stage embryos at a total amount of 1-8
ng/embryo.
In vitro transcription and translation
To test the specificity and efficiency of the RPTP
morpholinos in knocking down the respective protein, we used in vitro
transcription and translation of RPTP
(TNT Coupled Reticulocyte Lysate
System, Promega) performed according to the manufacturer's protocol with the
following modifications: in a 25 µl reaction, 0.5 µg of
RPTP
cDNA and various amounts of morpholino antisense oligos
(25-250 nM) were added to the TNT mix, containing all of the required
components for in vitro transcription and translation, and incubated at
30°C for 90 minutes. Five microlitres from the reaction mix were resolved
by SDS/PAGE (NuPAGETM, 4-12% Bis-Tris Gel; Invitrogen), and
35S-labeled proteins were visualised by autoradiography.
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Results |
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To analyse the molecular function of RPTP during
somitogenesis, we used zebrafish, owing to the accessibility of its embryos
and the ease of its genetic manipulations. A partial zebrafish cDNA clone was
obtained by screening a zebrafish cDNA library with a chick RPTP
probe under low stringency. The missing 5' end was obtained by
5'RACE (see Materials and methods).
The predicted RPTP protein consists of a 740 amino acid extracellular
region, a single transmembrane domain and a 666 amino acid intracellular
region. The extracellular sequence contains a MAM (meprin/A5/PTPµ) domain,
an immunoglobulin-like domain and four fibronectin type III-like repeats,
characteristics of members of the RPTP type IIB family (or MAM domain
subfamily) of receptor tyrosine phosphatases (for a review, see
Stoker and Dutta, 1998).
Comparison of the derived amino acid sequence with other vertebrate receptor
tyrosine phosphatases clearly identifies the full-length clone as zebrafish
RPTP
, showing 73-78% homology to human, mouse and chick RPTP
(Aerne et al., 2003
;
Wang et al., 1996
;
Yoneya et al., 1997
).
Fig. 1A shows a schematic
representation of the zebrafish RPTP
protein domains.
|
RPTP morpholinos inhibit RPTP protein synthesis and disrupt segmentation
To examine the effects of reduced RPTP activity on segmentation, we
used two anti-RPTP
morpholinos (RPTPmo1 or
RPTPmo2) targeted to independent regions of the 5' end of the
RPTP
mRNA (Fig.
2A). Antisense morpholino oligos are specific inhibitors of
translation that act by binding to complementary sequences on mRNA and
inhibiting ribosome access (Nasevicius and
Ekker, 2000
; Summerton and
Weller, 1997
). In the absence of a specific antibody that
recognises the RPTP
protein, we tested the potency and specificity of
RPTP
morpholinos in an in vitro transcription and translation
system. Each RPTP
morpholino oligo inhibits protein translation
in a dose-dependent manner (Fig.
2B, lanes 5-8). Inhibition by unrelated or mispaired control
morpholinos is negligible, even at 250 nM
(Fig. 2B, lanes 3,4). These
data suggest that morpholino treatment significantly and specifically reduces
RPTP
protein levels. In the experiments described below, the phenotypic
effects of RPTPmo2 were indistinguishable from those of
RPTPmo1, whereas the mispaired control oligonucleotide did not
produce any phenotype.
|
|
Paraxial mesoderm specification and maturation is unaffected in RPTPmo embryos
The disruption of somitogenesis observed in the RPTPmo-injected
embryos could be due to interference with specification and maturation of the
PSM. Alternatively, processes during somite patterning, such as the
establishment of segment polarity or the timing and maintenance of the somite
oscillator, could be defective. To exclude some of these possibilities, we
analysed the integrity of the presomitic mesoderm by examining markers for
paraxial mesoderm formation (spadetail; spt) and maturation
(fgf8).
spt is required for the convergence of mesodermal cells towards
the dorsal side during gastrulation, and in the specification of cardiac and
presomitic mesoderm (Amacher et al.,
2002; Griffin and Kimelman,
2002
). Once cells of the paraxial mesoderm are formed, they
undergo a maturation process, which is determined by a gradient of
fgf8, with high levels in the posterior and low levels in the
anterior presomitic mesoderm. When fgf8 levels drop below a threshold
level, the segmentation clock slows down and somitogenesis is initiated
(Dubrulle et al., 2001
;
Dubrulle and Pourquié,
2004
; Sawada et al.,
2001
). In wild-type embryos, spt is expressed strongly in
adaxial and tailbud cells, and more weakly in presomitic and lateral mesoderm
cells. In RPTPmo embryos, the levels and pattern of spt
expression appear normal (Fig.
4), indicating that RPTP
is not required for
specification of presomitic mesoderm tissue. Similarly, the gradient and level
of fgf8 expression is not affected by morpholino treatment
(Fig. 4), arguing that the
disrupted segmentation seen in RPTPmo embryos is not due to impaired
mesoderm maturation.
|
Zebrafish papC (pcdh8 Zebrafish Information
Network), a rostral segment polarity marker, is expressed during segmentation
in four bilateral pairs of bands in the anterior paraxial mesoderm, and more
weakly and uniformly in the rest of the PSM
(Yamamoto et al., 1998). The
anteriormost bands are located at the anterior borders of the newest somite
formed (SI) and the forming somite (S0). Stronger, posterior bands are located
in successively less mature somite primordia (S-1, S-2)
(Fig. 5A). papC
expression in RPTPmo-injected embryos is very similar to that in
somite mutants of the Delta/Notch signalling pathway [e.g. after
eight (aei), a mutation in deltaD
(Jiang et al., 2000
);
Fig. 5D,G]. Expression is
strong but non-metameric in the region corresponding to newly formed and
nascent somites, with marked random variability of intensity from cell to
cell. Expression in the rest of the PSM is normal and diffuse.
|
Reduced RPTP activity also disrupts expression of markers of
caudal half-segments, such as myod and deltaC
(Fig. 3B,
Fig. 8F). Together, these
results show that RPTP
is required for the specification of
anteroposterior polarity within somites.
|
In wild-type embryos, cycling genes show dynamic patterns of expression in
the PSM, except at its anterior where somites are formed and expression
becomes stable and compartment specific. At high doses of injected morpholino
oligonucleotide, cyclic expression in the PSM is lost: expression of
deltaC, her1 and her7 is no longer dynamic, and only one
static pattern is observed (Fig.
6). For deltaC, expression in these embryos is moderate
in the posterior part of the PSM, relatively low in the middle part and high
in the anterior part of the PSM. her1 and her7, however,
show uniform expression throughout the PSM
(Fig. 6). Thus, dynamic
expression of all known cyclic zebrafish genes is disrupted in the treated
embryos, indicating that RPTP is directly involved in the
operation of the somitogenesis clock.
|
RPTP acts upstream or in parallel to Delta/Notch signalling and is required for transcriptional activation of both her1 and her7
To consider how RPTP affects the segmentation clock, we
analysed cyclic gene expression in RPTPmo embryos in more detail and
compared it with that in other known `clock arrested' embryos, e.g.
aei mutant and her1 morphant embryos
(Fig. 7A).
|
In her1mo embryos, levels of her7 expression are reduced
in the cycling PSM, although not as drastically as in RPTPmo embryos
(Fig. 7A). her1
levels, on the other hand, are greatly increased
(Fig. 7A), but this is probably
due to transcript stabilisation by the antisense oligonucleotide
(Gajewski et al., 2003;
Oates and Ho, 2002
). Indeed,
this increase is abolished by co-injection of RPTPmo, such that the
embryos resemble those injected with RPTPmo alone
(Fig. 7A). These results
indicate that RPTP
activity is needed for efficient transcription of both
her1 and her7, and suggest that RPTP
acts
upstream or in parallel to Delta/Notch signalling.
The situation in the more anterior PSM, where somite differentiation and
boundary formation would normally occur, is rather more complex. There,
RPTP activity is again needed for expression of her1
and her7, but deltaC expression is broadened into a single,
anterior stripe (Fig. 6,
Fig. 7A). Thus,
RPTP
is required for final, transient repression of
deltaC prior to its compartment-specific expression and formation of
the somite boundary.
Reduction of RPTP function affects convergent extension
Reduction of RPTP activity also results in shortened anteroposterior
and broadened mediolateral axes (Fig.
3A,B, Fig. 8). This
phenotype is characteristic of a failure of convergent extension, a process of
cell polarisation and intercalation that leads to lengthening and narrowing of
the embryonic body during gastrulation. An alteration in axial proportions is
confirmed by staining for distal-less3 (dlx3) and no
tail (ntl), which mark the boundaries of the neuroectoderm and
nascent notochord, respectively (Akimenko
et al., 1994
; Schulte-Merker
et al., 1992
). These markers reveal that the neural plate in
RPTPmo embryos is broader and shorter, and that the notochord is
wider and slightly undulated (Fig.
8A,B,D,E).
In addition, staining for a marker for the anteriormost prechordal plate,
hgg1 (hatching gland gene 1; ctlsb
Zebrafish Information Network) (Vogel and
Gerster, 1997) reveals that anterior migration of the prechordal
mesoderm is impaired in the treated embryos. hgg1 expression normally
lies rostral to dlx3, in the periphery of the neural plate; in
RPTP
morphants, hgg1 is located more caudally,
overlapping the edge of the broader neural plate
(Fig. 8B,E). Impairment of
convergence movements is further indicated by the presence of laterally
widened somites as shown by deltaC expression in
RPTPmo-injected embryos compared to the wild-type control
(Fig. 8C,F).
Defects in convergent extension might directly explain the lack of dynamic
expression in RPTPmo-treated embryos, e.g. if the segmentation clock
depends on novel neighbourhood relationships arising during cell
intercalation. This explanation seems unlikely because segmentation seems to
be more sensitive than convergent extension to reduced RPTP activity,
judged by the different levels of RPTPmo required to observe the
described phenotypes (Fig. 6).
Nevertheless, we tested this idea by examining cyclic gene expression in
embryos mutant for knypek (kny), which encodes a glypican
that promotes non-canonical Wnt signalling during convergent extension
(Topczewski et al., 2001
). The
body axis is shortened in kny mutant embryos, but segmentation
appears normal, and expression of cyclic genes (e.g. deltaC) is still
dynamic (Fig. 8G), indicating
that non-canonical Wnt signalling is not required for periodic gene expression
in the PSM.
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Discussion |
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RPTP is a regulator of the somitogenesis clock
Our experiments show that antisense-mediated reduction of RPTP
activity leads to the loss of oscillatory behaviour of cycling genes and to
severe reduction of both her1 and her7 transcription.
deltaC is also downregulated in the posterior PSM of RPTPmo
embryos. Thus, RPTP
appears to be required for effective Notch
signalling in the PSM. All these expression patterns resemble those in
zebrafish embryos defective for Delta-Notch signalling, consistent with
RPTP
acting upstream of, or in parallel with, this pathway.
her1 and her7 code for bHLH transcriptional repressors of
the Hairy/E(spl) family, genes encoding which are directly activated by Notch
signalling in a variety of developmental contexts, including segmentation
(Oates and Ho, 2002; Takke et
al., 1999). Hes1 and Hes7, her1 and her7
counterparts in mouse, negatively regulate their own expression both in
cultured cells and in vivo (Bessho et al.,
2003
; Hirata et al.,
2002
). Based on this observation, Lewis showed, using mathematical
modelling, that an auto-regulatory feedback loop involving her1 and
her7 provide a possible molecular basis for an intracellular
oscillator (Lewis, 2003
).
Reducing RPTP levels decreases her1/7 expression in the
cycling PSM, and this reduction is independent of her1/7 activity
(Fig. 6, Fig. 7A). Therefore,
RPTP
appears to be required to activate Notch target gene
transcription during cycling. This effect appears to be independent of effects
on Notch ligand expression because RPTPmo also reduces her1
expression in aei embryos (Fig.
7B). Overall levels of deltaC, her1 and her7,
and also mesp gene expression are much reduced in both the cycling
and anterior PSM (Fig. 5,
Fig. 6,
Fig. 7A,B). However, RPTP
is also needed for repression of deltaC during somite boundary
formation two anterior stripes in wild-type embryos become a single,
broad stripe perhaps because of the failure of anterior her
expression. This latter, indirect requirement for RPTP
may reflect
differing regulatory circuits operating in different regions of the posterior
PSM (Gajewski et al., 2003
;
Morales et al., 2002
;
Saga and Takeda, 2001
).
Nevertheless, it is still not clear to what extent Delta-Notch signalling
is required for the oscillation itself. For example, the first few somites are
still formed in zebrafish and mouse embryos defective in Notch signalling. One
possibility is that Notch signalling is required only to synchronise
neighbouring PSM cells (Jiang et al.,
2000), and that an upstream segmentation clock drives cyclic Notch
signalling.
One pathway that could account for the latter possibility is that of Wnt
signalling. Aulehla et al. (Aulehla et al.,
2003) showed recently that axin2, which encodes a
negative regulator of Wnt signalling, displays oscillating expression in the
mouse PSM, alternating with that of lfng and hes7. They
argue that wnt3a is necessary for cyclic expression of both
axin2 and the oscillating Notch signalling activity, but that Notch
signalling is not required for axin2 oscillation. This implies that
axin2 oscillations reflect cyclic Wnt signalling that is distinct
from, and possibly upstream of, cyclic Notch signalling in the mouse PSM.
It is not yet clear if cyclic Notch signalling in zebrafish embryos is
driven by an upstream Wnt clock. axin2 expression appears not to
cycle in the zebrafish PSM (B.A., unpublished), although Wnt signalling
components other than axin2 could be cycling in zebrafish and thereby
generate cyclic Wnt activity. In any case, RPTP, like Wnt3a in the mouse,
is required for Delta/Notch signalling.
RPTP is required for convergent extension during gastrulation
In addition to its role in segmentation, RPTP seems to be
required for convergent extension during gastrulation. RPTPmo embryos
have a shorter and broader body axis, a phenotype characteristic of convergent
extension mutants.
Convergent extension has been shown to depend on the so-called,
non-canonical Wnt signalling pathway (for a review, see
Tada et al., 2002). Unlike
canonical Wnt signalling, which targets the nucleus and directs changes in
gene transcription, non-canonical Wnt signalling is independent of
ß-catenin-mediated transcriptional activity, and directs morphogenetic
processes such as changes in cell shape and cell migration. How Wnt signalling
is translated into convergent extension movements during gastrulation is
poorly understood, but it clearly involves changes in the adhesive properties
of cells, e.g. via regulated decreases in the activity of cell adhesion
molecules such as cadherins (Kuhl et al.,
1996
; Marsden and deSimone,
2003
). Non-canonical Wnt signalling seems not to be required for
the segmentation clock, as we have shown that oscillator behaviour is normal
in kny mutants (Fig.
8G). Similarly, no role for Notch signalling in convergent
extension is known.
How might the dual effect of RPTP on the somite oscillator
and convergent extension be explained? The multiplicity of kinases in the
vertebrate genome implies that PTPs have a relatively broad range of substrate
specificities. One possibility, therefore, is that RPTP
affects factors
from independent pathways (e.g. Wnt and Notch) that regulate convergent
extension and somitogenesis. Alternatively, RPTP
might affect a single
pathway/component that impinges on both convergent extension and
somitogenesis. Human and mouse RPTP
have been shown to associate with
ß-catenin and to dephosphorylate ß-catenin both in vivo and in vitro
(Cheng et al., 1997
;
Wang et al., 1996
;
Yan et al., 2002
). Both these
processes could be modulated by RPTP
, e.g. by acting on tyrosine
phosphorylation levels of ß-catenin, which is crucial for both
instability of the ß-catenin/cadherin bond and for enhanced binding to
TBP and the Tcf complex (Piedra et al.,
2001
; Roura et al.,
1999
). Thus, RPTP
has the potential to promote adhesion and
negatively regulate ß-catenin-dependent transcriptional activity
(Balsamo et al., 1996
;
Balsamo et al., 1998
). It is
therefore possible that changes in RPTP
activity impinges both on
adhesion and migration processes during convergent extension movements, and on
Wnt-directed transcriptional regulation of the somite oscillator.
Clearly, further experiments are needed to pinpoint the targets of
RPTP activity in both processes. In any case, our study adds an
unexpected and novel component to the somitogenesis clock, which, until
recently, exclusively implicated members of the Delta/Notch signalling
pathway.
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ACKNOWLEDGMENTS |
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