1 Institute of Molecular and Cell Biology, 61 Biopolis Drive, Proteos, 138673
Singapore
2 Department of Biological Sciences, National University of Singapore, 10 Kent
Ridge Crescent, 119260 Singapore
Author for correspondence (e-mail:
vlad{at}imcb.a-star.edu.sg)
Accepted 9 June 2005
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SUMMARY |
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Key words: T-box, Cell adhesion, Zebrafish, Frizzled 7, Dishevelled, DIX domain, ß-catenin
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Introduction |
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In the zebrafish ectoderm, individual cells lose their independence and
integrate their behaviour to achieve a coherent movement over the yolk as a
sheet (Concha and Adams, 1998),
while the underlying mesodermal cells migrate as individuals or groups of
cells. This is in contrast to Xenopus, where the ectoderm is tightly
coupled to the mesoderm (Shih and Keller,
1994
). Optimal cell adhesion is essential for proper cell movement
(Nelson and Nusse, 2004
).
Cell-surface adhesion molecules such as cadherins control cell-cell adhesion
and influence cell migration. Disparity in cell adhesion properties among
neighbouring cells is known to lead to loss of integrity and cell sorting
within the ectodermal sheet (Concha and
Adams, 1998
).
The embryonic ectoderm has the potential to acquire either epidermal or
neural fates. According to the default model, neural induction depends on the
suppression of bone morphogenetic protein (Bmp, vertebrate homologue of Dpp)
signalling by organizer-derived inhibitors
(Munoz-Sanjuan and Brivanlou,
2002). Mass cell movements during gastrulation, especially that of
convergent extension (CE), eventually establish the neural plate, which can be
differentiated from the remaining epidermal ectoderm by early neural markers
such as sox19 (Vriz and
Lovell-Badge, 1995
). CE movement is most extensively investigated
at the molecular level within the context of mesoderm formation
(Myers et al., 2002
). As such,
the molecular mechanism governing dorsal movement of the overlying ectoderm
leading to the formation of the neural plate is not well understood.
In Xenopus, Wnt/ß-catenin has been demonstrated to inhibit
Bmp (Baker et al., 1999) and is
required for the expression of secreted Bmp antagonists
(Wessely et al., 2001
). In the
zebrafish, Wnt/ß-catenin-dependent bozozok (boz) is
sufficient to suppress expression of bmp
(Fekany-Lee et al., 2000
). This
is consistent with the role of ß-catenin-dependent Wnt signalling, or
canonical Wnt signalling, in fate determination
(Moon et al., 2002
).
ß-Catenin-independent Wnt signalling, or non-canonical Wnt signalling,
has been demonstrated to influence morphogenetic cell movement (Tada et al.,
2000; Heisenberg et al., 2000
)
by controlling Dishevelled (Ds)-mediated polarity in a manner reminiscent of
the Drosophila planar-cell-polarity (PCP) pathway. However, in
addition to its function in the Wnt signalling pathway, ß-catenin also
binds to the cytoplasmic domain of type 1 cadherins and plays an essential
role in the structural organization and function of cadherins in the actin
cytoskeleton (Jamora et al.,
2003
). Different cadherins have been shown to stimulate directly
the differentiation of stem cells into specific tissues
(Larue et al., 1996
). As such,
there is a possible convergence of Wnt, ß-catenin and cadherin signalling
in adhesion, morphogenetic movement and differentiation.
Transcription factors of the T-box family (Tbx) play important roles in
vertebrate development (Smith,
1999; Wilson and Conlon,
2002
). Brachyury (T) and the zebrafish
orthologue no tail (ntl) are known to be essential for the
specification of axial mesoderm (Smith,
1999
). In addition, T, Tbx16/spadetail
(spt) and Xbra have been shown to be required for mesodermal
cell movement (Wilson and Beddington,
1997
; Ho and Kane,
1990
; Kwan and Kirschner,
2003
). A number of human disorders have been linked to mutations
in T-box genes, confirming their medical importance
(Packham and Brook, 2003
).
Recently, a knockout of TBX2 in mice resulted in abnormal development of the
heart (Harrelson et al.,
2004
).
Ntl is shown to function in parallel to Wnt5 signalling in the
morphogenesis of the posterior body
(Marlow et al., 2004) and Xbra
is shown to regulate Wnt11 expression during gastrulation
(Tada and Smith, 2000
). The
Drosophila Tbx2-related opto-motor blind (omb) is regulated by the
Wingless (Wg), Decapentaplegic (Dpp) (Grimm
and Pflugfelder, 1996
) and Hedgehog (Hh)
(Kopp and Duncan, 1997
)
signalling pathways; Xenopus Tbx2 is known to function within the
Sonic hedgehog (Shh) pathway (Takabatake
et al., 2002
); in chick, Tbx2 is known to function through both
Shh and Bmp signalling (Suzuki et al.,
2004
).
Zebrafish tbx2b was previously named tbx-c. Using a
dominant-negative (dn) approach, it was shown to function downstream of
Tgfß signalling, of ntl and of floating head
(flh) in the late-phase specification of the notochord and
development of motoneurons (Dheen et al.,
1999). In this report, we show that neural development is
dependent on early Tbx2b activity during gastrulation. Using a combination of
antisense morpholino oligonucleotide (MO) gene `knockdown'
(Nasevicius and Ekker, 2000
)
and in vivo analysis of chimaeric embryos generated by cell transplantation or
single blastomere injection, we demonstrate that, during neural plate
formation and neuronal specification, Tbx2b functions within the context of
Wnt signalling to mediate cell migration.
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Materials and methods |
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Whole-mount in situ hybridization, immunohistochemistry, western blot and TOP-Flash assay
Whole-mount in situ hybridization and whole-mount immunohistochemistry with
anti-fluorescein-POD were performed according to established techniques.
Proteins were extracted from 20 embryos/lane for western blot, and detected
with chemiluminescent detection kit (Cell Signalling Technology, USA).
TOP-flash assay was performed as described
(Korinek et al., 1997).
Dominant negative Fz7-EGFP/BAC construct
Dn-fz7 targeting construct was generated from fz7 cDNA by
replacing the stop codon with an EGFP-kanamycin cassette using overlapping
amplicons and primer extension. Primer sequences were as follows:
5'UTRF, 5'-AGG AAA CCG CAC TCT GTT CA-3'; fz7R,
5'-TAC CGT CGT CTC GCC CTG GTT-3'; fz7EGFPF, 5'-GGC
GAG ACG ACG GTA ATG GTG AGC AAG GGC-3'; kanR, 5'-TCA GAA GAA CTC
GTC AAG AAG GCG ATA GAA-3'; kan3'UTRF, 5'-CTT CTT GAC GAG
TTC TTC TGA GAA AAA GAG CGA TCG TTT TCG-3'; and 3'UTRR,
5'-ACA AGT CCC CGG TTA AAA CAA GTG TTG G-3'. The resultant
dnfz7-EGFP construct was co-transformed with pGETrec into E.
coli DH10B carrying a BAC of the intronless fz7 gene. Homologous
recombination was then induced (Narayanan
et al., 1999).
Transplantation
Donor embryos were injected at the one-cell stage with fluorescein dextran
pre-mixed with MO or Danieau's solution. About 20 cells from each donor were
transplanted into wild-type host embryo of the same stage. Chimaeric embryos
were grown to 10 and 24 hpf. Location of transplanted cells at 10 hpf was
visualized by a combination of immunohistochemistry with anti-fluorescein-POD
and sox19 whole-mount in situ hybridization. Images were taken using
the Olympus AX70 (Olympus, Japan) microsope fitted with CCD camera.
Fluorescent and bright-field images were superimposed with Photoshop 5.5
(Adobe) software.
Injections at 16 cell stage
Dechorionated 16-cell stage embryos were transferred to a Petri dish with
moulded agar (1.5% agarose in egg water) injection wells and one central
blastomere was injected with not more than 200 pl of reagents. Injected
embryos were allowed to develop to 4 hpf, and then were inspected under a UV
dissecting microscope. Embryos with labelled clones in the centre of the
blasdoderm, when viewed from the animal pole, that represented a perfect cone
when viewed from the side, were allowed to develop further. In this way only
ectodermal derivatives were labelled.
Real time RT-PCR
Total DNA-free RNA (1 mg) was subjected to oligo(dT)15 primed RT with
PowerScript Reverse Transcriptase (Clontech) according to the manufacturer's
recommendations, and 1/20 of the reaction mix was used for PCR. Real-time
semi-quantitative PCR assay was carried out using gene-specific primers for
tbx2b (forward, 5'-AGG AAC CCG TTC TTG AGC AGC-3';
reverse, 5'-AGG CCG CTT GGC AAT CCG GTG-3'), EF1 (forward,
5'-AGA CTG GTG TCC TCA AGCC TG-3'; reverse, 5'-TGA AGT TGG
CAG CCT CCA TGG-3') and fz7 (forward, 5'-TCA CTG TGG CTC
TAC AAA CGA CC-3'; reverse, 5'-TGC ACT TCG AGA CCG GCG
TCC-3') in a DNA Engine Opticon System (MJ Research, USA). SYBR Green
was used as the reporter for real-time PCR. Briefly, HotStart Taq (Qiagen)
activation for 15 minutes, four segment amplification and quantification
program repeated 35 times [95°C for 30 seconds; 62°C for 10 seconds
with a single fluorescence measurement; 72°C for 30 seconds, melting curve
program (78°C to 95°C)]. Serial dilutions of cDNA were used as
standards for semi-quantitation. Correction for inefficiencies in RNA input or
reverse transcriptase was performed by normalizing to EF1
amplification. The comparative C(T) method, calculating relative expression
levels compared to control, was used for quantification.
Hanging-drop culture
Hanging-drop cultures were carried out with animal caps from embryos at 50%
epiboly as previously described (Steinberg
and Takeichi, 1994). Fluorescent images were obtained with a Zeiss
Axioplan2 equipped with Zeiss AxioCam HRc CCD camera (Zeiss, Germany).
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Results |
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The initial functional analysis on the role of tbx2b in
development was performed with a dn-Tbx2b lacking the C-terminal
transactivation domain (Dheen et al.,
1999). For this study, a MO-mediated loss-of-function approach
(targeted gene `knockdown') was adopted. Two anti-tbx2b MOs were
designed (see Materials and methods). Both straddle the start codon of
tbx2b, but offset by 10 bases along the 5'-UTR. To establish
their efficacy, the two MOs were injected into one-cell stage embryos
(pan-embryonic injection). At the same concentration, tbx2b MOv2 was
more effective in producing a phenotype than tbx2b MOv1 and was used
for all experiments in this study (henceforth referred to as tbx2b
MO).
When co-injected with tbx2b MO pan-embryonically, translation of a
myc-tagged tbx2b containing the target site for
tbx2b MO was blocked in a dose-dependent manner
(Fig. 1B). The midline axial
mesoderm of the morphants, as analyzed and compared with embryos obtained from
overexpression of dn-Tbx2b with the marker sonic hedgehog
(shh), had malformed notochord and lacked the floor plate
(Fig. 1C). In the most severely
affected morphants, only remnants of the notochord were present
(Fig. 1D). This is reminiscent
of the mutant flh, and is in agreement with our previous study
(Dheen et al., 1999). These
results suggest that the phenotype observed with tbx2b MO was indeed
specific to the `knockdown' of tbx2b.
Pan-embryonic `knockdown' of tbx2b with tbx2b MO delayed
the onset and progression of gastrulation by up to 2 hours and produced
phenotypes at 24 hpf in a dose-dependent manner. In its most severe form (at 1
pmol/embryo), tbx2b morphants display phenotypes similar to
boz/ embryos
(Fekany-Lee et al., 2000)
short AP axis, absent or malformed notochord, fused somites, small
and/or cyclopic eyes, and small brain (Fig.
1F-G). Cross-sections of the morphants at the level of the eyes
showed that the neural tube was severely malformed: the ventricle was absent
and the eyes were underdeveloped (Fig.
1H,I). Injections of a sense MO up to 10 pmol showed no
phenotype.
The disorganized forebrain of the tbx2b morphant supported a role for Tbx2b in neural development. However, the severity of the malformation rendered analyses of pan-embryonic morphants ambiguous. To circumvent this, we analyzed the behaviour of Tbx2b-deficient cells in chimaeric embryos. The chimaeras were generated by two complementary approaches aiming to target ectodermal derivatives cell transplantation and single-blastomere injection.
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In both transplantation and single-blastomere injection experiments, there
were reduced numbers of Tbx2b morphant cells compared with control. This could
arise from an increase in cell death, from a decrease in cell proliferation or
from cell loss. Staining of tbx2b morphants with Acridine Orange and
TUNEL assay showed no significant increase in apoptosis (not shown). We also
failed to observe cell loss because of sloughing during gastrulation, although
we could not rule out this possibility later in development. Therefore the
reduction in cell count was probably the result of impairment in cell
proliferation. This is consistent with reports implicating human TBX2 in cell
cycle control and oncogenesis (Jacobs et
al., 2000; Prince et al.,
2004
).
sox19 encodes the early pan-neural transcription factor
(Vriz and Lovell-Badge, 1995).
It is expressed in the neural plate at 10 hpf
(Fig. 2F). In tbx2b
morphants the neural plate was broader, with a pronounced gap in expression
around the midline (Fig. 2G,
arrow). We asked if this was likely to be due to an early defect in
specification, and thus focused on at early patterning. The expression
patterns of bmp2 and fz7 in control and morphant 5 hpf
embryos were similar (Fig.
2H-K), although expression in the morphants appeared to be more
punctate than in control embryos. This could be the result of the delayed
epiboly in tbx2b morphants. In addition, during gastrulation,
transplanted donor cells from tbx2b morphants continue to express
sox19 in wild-type hosts at 5 hpf
(Fig. 2L-N). These results
suggest that early fate specification is not affected by the loss of Tbx2b
function; instead, dorsal convergence cell movement was delayed in the
morphants.
When 1/16 injected embryos were stained for both fluorescein and
sox19 at 10 hpf, control labelled cells were found in the neural
plate and epidermis (Fig. 3A).
By contrast, Tbx2b morphant cells were excluded from the neural plate
(Fig. 3B). This suggested that
the lack of labelled cells in the 24 hpf morphants observed in previous
experiments was due to the absence of labelled cells in the neural plate at 10
hpf. 1/16 injection of tbx2b mRNA led to the appearance of labelled
cells with normal morphology at the midline at 10 hpf, and in the notochord at
24 hpf, in about 10% of the injected embryos (n=30,
Fig. 3C,D). Although this
result is consistent with the role of Tbx2b in the formation of axial
mesodermal structures (Dheen et al.,
1999) it is nevertheless interesting and surprising because the
clonal population derived from the injected blastomere were ectodermal in
origin. This apparent respecification of fate across germ layers by
overexpression of tbx2b needs to be further investigated.
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The `exclusion' phenotype points to aberrant cell migration within the embryonic ectoderm, suggesting that cell adhesion could be compromised. The surface of embryos undergoing gastrulation is smooth (Fig. 4A). By contrast, pan-embryonic injections of tbx2b MO (Fig. 4B) caused rounding of cells on the surface of morphants. As a result, the morphants acquired a `rough' phenotype, suggesting that cell adhesion in vivo was affected. Currently, we do not know if there were other cell polarity defects associated with this `rough' phenotype.
To test whether Tbx2b-depleted cells exhibited altered cell adhesion
properties, cell re-association experiments were performed. Texas Red-labelled
animal caps of 5 hpf embryos from wild-type or MO-depleted embryos were
dissociated and mixed with fluorescein-labelled wild-type cells and cultured
as hanging-drop. After 24 hours, the two control-derived populations formed a
single aggregate (Fig. 4D),
suggesting similar surface adhesion properties. By contrast, cells that were
co-injected with fluorescein and tbx2b MO
(Fig. 4E) formed shells around
small aggregates of control cells. This is consistent with idea that
aggregated control cells have higher level of the same cadherin than do
morphant cells in the outer shells
(Steinberg and Takeichi,
1994).
Cadherins are essential for cell adhesion. Indeed, supporting the
observation from hanging-drop culture, the level of cadherins failed to
increase during gastrulation in tbx2b morphants
(Fig. 4G). This suggests that
the deficiency in cadherins could be the proximal cause of the defects in cell
adhesion and migration observed in vivo. It is known that during epiboly,
cell-cell adhesion plays a crucial role in maintaining the integrity of the
ectodermal sheet (Concha and Adams,
1998). Although Xbra is known to inhibit cell migration,
it was shown to do so by inhibiting adhesion to fibronectin instead of
affecting the level of cadherin directly
(Kwan and Kirschner, 2003
).
The mutation in parachute (pac) eliminates N-cadherin.
Similar to tbx2b morphants, this mutant is characterized by abnormal
anterior neural tube (Lele et al.,
2002
). Indeed, cells transplanted from
pac/ into wild-type hosts failed to populate
the anterior CNS by 24 hpf (9/9). By contrast, cells from heterozygous
siblings were found in the CNS of hosts (10/10).
To demonstrate that migration of cells deficient in Tbx2 is also affected, morphant cells were co-transplanted with control cells into the ectoderm of wild-type embryos and observed in vivo (Fig. 4H-I). The morphant cells lagged behind control cells when migrating to the dorsal side of the embryo at 50% (Fig. 4J) and 80% epiboly (not shown). In addition, by the end of gastrulation, these cells did not express sox19 and remained outside the neural plate at 10 hpf (Fig. 4L). Thus, an essential aspect of neural plate formation is the requirement of Tbx2b for optimal cell adhesion and consequent cell movement into the dorsal ectoderm destined to develop as the neural plate.
The appearance of `exclusion' phenotype from 1/16 injection of tbx2b MO in neural plate stage embryos provided a quick assay to screen for genes that are potentially functioning in the same pathway as tbx2b (Table 1). Reagents (MOs or mRNAs) were injected in the 1/16 manner together with a fluorescein tracer, and the number of embryos exhibiting the `exclusion' phenotype at 10 hpf was scored. A percentage similar to or higher than that obtained from tbx2b MO suggests a potential correlation of function. MOs and mRNAs used in the screen were previously tested in pan-embryonic injections for efficacy and viability (data not shown). Where available, MOs were tested to phenocopy the respective mutants (see Materials and methods). The assay allowed us to narrow down quickly on a potential pathway.
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fz7 is expressed in the ectoderm during gastrulation (Fig. 5B); thus, it is present in the ectodermal cells at the time when they acquire neural fates. Ectopic overexpression (by intracellular injection of DNA construct into one-cell stage embryos) of a dn-Fz7-EGFP/BAC (with the EGFP inserted into the C terminus to replace the stop codon) caused the loss of tbx2b expression (Fig. 5C). Real-time PCR analysis of tbx2b- and fz7-morphants (MOs injected at 0.5 pmol and 0.2 pmol) showed that whereas fz7 MO downregulates fz7 and tbx2b transcription at 5 hpf, tbx2b MO has no effect on the transcription of fz7 and its target mRNA (Fig. 5E).
Further analysis showed that fz7 morphants displayed `rough'
phenotype during gastrulation (Fig.
4C), fz7-deficient cells failed to aggregate with control
cells in hanging-drop cultures (Fig.
4F), fz7 morphants failed to upregulate cadherins
(Fig. 4G), and transplanted
fz7-deficient cells did not co-migrate with control cells
(Fig. 4K,M). A 1/16
co-injection of tbx2b mRNA with fz7 MO led to the appearance
of morphologically undifferentiated labelled cells in the eyes and forebrain
at 24 hpf (Fig. 5D), suggesting
that Tbx2b is able to rescue the cell movement, but not the fate specification
function of Fz7. Owing to the pleiotropic nature of Fz7 function, attempts to
rescue the `exclusion' phenotype of fz7 MO by co-injection of a
fz7 mRNA without the MO target site were unsuccessful. Together,
these results suggest that Tbx2b may function as a downstream effector of
Fz7-mediated cell movement during neural plate formation
(Djiane et al., 2000).
Is the cell movement defect arising from Tbx2b `knockdown' the direct cause
of neuronal specification failure in these cells later in development? To
answer this, cells from shield stage (6 hpf) of Tbx2b morphant donors were
transplanted directly into the dorsal side of homochronic wild-type hosts at
two locations: one that gives rise to the eyes and the other to the hindbrain
by 24 hpf (Kimmel et al.,
1990; Moens and Fritz,
1999
). Whereas wild-type cells transplanted into wild-type hosts
ended up in appropriate positions and differentiated according to neighbouring
tissues (Fig. 5F-G), in both
cases all transplanted morphant cells ended up in the intended locations at 24
hpf but could not acquire the typical morphology of cells in these tissues,
suggesting that they were unable to differentiate properly (not shown).
However, owing to the delay in the onset and progression of gastrulation in
morphants, the donors were not morphologically equivalent to wild-type shield
stage embryos (they resembled 4 hpf embryos). To confirm that the failure in
specification was not due to the delay in development, transplantation was
repeated with cells from morphant donors at shield stage irrespective of the
actual age (8 hpf). Although a few transplanted cells managed to regain
the typical morphology of cells in these tissues (arrowheads,
Fig. 5H,I), the majority did
not. This result suggests that Tbx2b may have a later role in neuronal
differentiation. Similarly, we demonstrated that in the absence of Fz7,
overexpression of tbx2b could only rescue the cell movement defect
but not the subsequent differentiation of transplanted cells
(Fig. 5D). Altogether, these
results suggest that, independent of its requirement in cell adhesion and cell
movement, Tbx2b is necessary, but insufficient, for neuronal differentiation
in the late neuroectoderm. tbx2b is expressed at high levels in
sensory cells and other neuronal lineages later in development, and is
therefore likely to play a role in their differentiation
(Dheen et al., 1999
).
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Discussion |
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Pan-embryonic injection of tbx2b MO led to a delay in epiboly and
malformed anterior forebrain at 24 hpf. In order to separate analyses in
ectoderm from mesoderm, we focused on the behaviour of Tbx2b-depleted
ectodermal cells in an otherwise normal wild-type embryo. We demonstrated that
cells deficient in Tbx2b were consistently excluded from the neural plate at
10 hpf, and did not develop as neural cells by 24 hpf. Embryonic cells are
pluripotent up to the early gastrula stages
(Ho and Kimmel, 1993).
Therefore, alteration of cell movement can cause changes in fate specification
by virtue of exposure to local signalling cues prevalent in the `adopted'
position. Conversely, a failure in fate specification can cause a change in
cell movement behaviour. Either mechanism could lead to exclusion of cells
from the neural plate. However, analysis of expression of bmp2, fz7
and sox19 suggest that early fate specification is not affected by
`knockdown' of Tbx2b. Indeed, the neural plate is induced in the Tbx2b
morphant. Our results thus indicate that Tbx2b functions in cell migration in
the ectoderm during the formation of the neural plate.
It has been shown that strong intercellular adhesion plays a crucial role
in maintaining the integrity of the gastrulating ectodermal sheet
(Concha and Adams, 1998). In
zebrafish null mutant for N-cadherin (pac), neuroectodermal cell
adhesion is altered, leading to compromised convergent cell movements during
neurulation (Lele et al.,
2002
). We show that cells depleted of Tbx2b have reduced
cadherins, and this altered cell adhesion is likely to be the cause of
migration defects leading to their exclusion from the neural plate in
chimeras. As ß-catenin binds tightly to the cytoplasmic domain of type 1
cadherins (Jamora et al.,
2003
), loss of cadherins in Tbx2b-depleted cells may lead to
defects in ß-catenin stabilization and localization, with subsequent
consequences in differentiation. As such, there is a possible convergence of
Tbx2b, Wnt, ß-catenin and cadherin signalling in adhesion, morphogenetic
movement and differentiation.
Although spt (Ho and Kane,
1990), T (Wilson and
Beddington, 1997
), ntl
(Conlon and Smith, 1999
) and
Tbx5 (Hatcher et al.,
2004
) have been implicated in cell movement, the roles
demonstrated for them are confined within the context of mesoderm. Still,
these studies showed that in chimeras with neighbouring wild-type and
T-box-deficient cells, the movement of wild-type cells in their normal
developmental context resulted in the exclusion of T-box-deficient cells
similar to what we demonstrate here. It would seem that this is a common
phenomenon and may indicate a general functional mechanism for T-box proteins
in cell migration during development.
Interestingly, overexpression of Tbx2b in the ectoderm led to the
appearance of cells in the notochord, a mesodermal structure. Although it is
known that Tbx2b is required for the specification of axial mesodermal
structures at the expense of lateral mesodermal tissues
(Dheen et al., 1999), what we
demonstrate here is its ability to transfate cells from ectoderm to dorsal
mesoderm. We do not yet know the precise mechanism of this phenomenon.
However, we show here that a low level of Tbx2b is needed for proper cell
adhesion and cell movement within the ectodermal sheet. It is possible that
dorsal mesoderm has higher levels of cell adhesion, and the affected cells
move into the underlying mesoderm during gastrulation. Analysis with
maternal-zygotic one-eyed-pinhead (MZoep) mutant cells
showed that internalization and mesendoderm formation in zebrafish can be
attained autonomously by single cells
(Carmany-Rampey and Schier,
2001
). If so, it opens the possibility that differential
activation of Tbx2b is a mechanism by which systematic differences in cell
surface properties and cell segregation behaviour is modulated for the purpose
of long-term effects on cell fate specification. More analysis is needed to
better understand this observation.
The exclusion phenotype provided a rapid assay for screening signalling
pathways that Tbx2b might be operating within. The screen suggests that Tbx2b
functions within the context of Wnt signalling, specifically through the
receptor Fz7. Zebrafish Fz7, like its homologue in Xenopus, has a
broad range of functions from fate specification to cell movement, but it is
the cell movement requirement for Fz7 that is mediated by Tbx2b. As
Drosophila Omb also acts within the Wnt context in multiple
developmental situations (Grimm and
Pflugfelder, 1996), this developmental link may be conserved in
evolution. Although Fz7 is shown to regulate tbx2b transcriptionally,
a full analysis of the detailed molecular mechanism is beyond the scope of
this study. Preliminary analysis of the 2.5 kb 5' genomic sequence of
tbx2b identified numerous Tcf-binding sites (not shown), hinting at
the possibility of regulation of tbx2b transcription via
ß-catenin/Tcf.
The functional roles of the canonical and non-canonical Wnt pathways were
thought to be quite different (Moon et
al., 1997). In vertebrates, Wnt-dependent morphogenetic cell
movements were linked to the non-canonical pathway (Tada et al., 2000;
Heisenberg et al., 2000
),
which directly controls Ds-mediated cell polarity
(Wallingford et al., 2000
).
The canonical Wnt pathway was associated with the initiation of CE movements,
but not movement per se (Moon et al.,
1997
). Although ß-catenin acts in both signalling and
adhesion in insects and vertebrates (Moon
et al., 2002
), the involvement of the canonical Wnt pathway in
cell movement has been shown only in invertebrates
(Korswagen, 2002
).
In the Xenopus blastula, ß-catenin was proposed to regulate
CE and mediate cell fate via two parallel pathways involving Nodal-related 3
and Siamois, respectively (Kuhl et al.,
2001). In zebrafish, ß-catenin modulates Nodal signalling, to
regulate both cell fate and cell movement
(Myers et al., 2002
); and
Boz, to repress bmp2b
(Leung et al., 2003
). In this
study, the ability of Fz7 to mediate Wnt signalling via the DIX-domain of Ds
to effect cell adhesion and cell movement during gastrulation suggests that Ds
may not function in a strictly modular manner. The current notion that cell
movement events are mediated by the non-canonical pathway through the DEP- and
PDZ-domains of Ds may need to be looked at afresh.
It has been shown recently that Tbx2 is required for patterning the
atrioventricular canal and for morphogenesis of the outflow tract during heart
development in mice (Harrelson et al.,
2004). Cell migration plays an important role in heart formation
in teleosts (Glickman and Yelon,
2002
) and amniotes (Hatcher et
al., 2004
). Although tbx2b is expressed in the developing
heart and the morphants display cardiac defects later in development (not
shown), it remains to be seen if Tbx2b plays a role in the migration of
cardiac cells.
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ACKNOWLEDGMENTS |
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Footnotes |
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REFERENCES |
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