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| Home Help Feedback Subscriptions Archive Search Table of Contents | ||||
First published online November 26, 2007
doi: 10.1242/10.1242/dev.000026
,*
1 Center for Transgene Technology and Gene Therapy, VIB, Herestraat 49, B-3000
Leuven, Belgium.
2 Department of Molecular and Cellular Medicine, KULeuven, Herestraat 49, B-3000
Leuven, Belgium.
3 Laboratory of Molecular Biology, Department of Molecular and Developmental
Genetics, VIB, Herestraat 49, B-3000 Leuven, Belgium.
4 Department of Human Genetics, KULeuven, Herestraat 49, B-3000 Leuven,
Belgium.
5 Division of Biochemistry, Department of Molecular Cell Biology, KULeuven,
Herestraat 49, B-3000 Leuven, Belgium.
* Authors for correspondence (e-mails: camila.esguerra{at}med.kuleuven.be; danny.huylebroeck{at}med.kuleuven.be)
Accepted 16 September 2007
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Alk4, E-cadherin, Gastrulation, Left-right asymmetry, Node, Smad
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Schier,
2003; Yost, 2003;
Raya and Belmonte, 2004). The
binding of Nodal to its receptor complex (Alk4/ActRIIB/co-receptor EGF-CFC)
activates Smad2/3, and phosphorylation of these Smads by Alk4 increases their
affinity for Smad4. The resulting Smad complexes accumulate in the nucleus and
participate in transcription of target genes by cooperating with various
activators, repressors and chromatin modulators
(Massagué, 2000;
van Grunsven et al.,
2005).
The best characterized Smad2/3 partners in Nodal-Activin signaling
(Stemple, 2000) are FoxHI
(Fast1) and Mixer (Hill, 2001;
Whitman, 2001; Attisano,
2001). Analysis of zebrafish mutants for FoxHI (schmalspur, sur)
(Pogoda et al., 2000) and
Mixer-like (bonnie and clyde, bon)
(Kikuchi et al., 2000)
revealed that the individual and combinatorial mutant phenotypes do not
represent all aspects of Nodal signaling
(Kunwar et al., 2003). This
could be due to several reasons, including the possibility that additional
players in Smad signaling remain to be identified. Nodal signaling studies in
fish have focused on the role of Smad2/FoxHI and identification of its
targets, whereas the situation is less clear with regard to Smad3
(complexes).
Within the context of an antisense screen in zebrafish using morpholino
oligomers (morpholinos, Mos) (Summerton
and Weller, 1997; Nasevicius
and Ekker, 2000), we identified Ttrap (TRAF and TNF
receptor-associated protein) (Pype et al.,
2000) as a regulator of embryogenesis. Human TTRAP interacts with
TNF receptor (TNFR) family members and TNFR-associated factors (TRAFs) and
inhibits NF-
B activation in TTRAP-overproducing cells
(Pype et al., 2000). TTRAP has
also been termed EAPII - ETS-associated protein II - revealing a possible dual
role of this protein within the cytoplasm and nucleus
(Pei et al., 2003). TTRAP
belongs to the family of divalent cation-dependent phosphodiesterases, with
highest homology to APE1, an endonuclease involved in DNA repair and
transcription factor activation
(Rodrigues-Lima et al.,
2001).
The in vivo role of Ttrap has not yet been described. We show that Ttrap
controls gastrulation movements and LR axis determination in zebrafish via
Smad3-mediated regulation of e-cadherin, which is known to be
regulated by the repressor snail and modulate cell movements (epiboly and
convergent extension) in fish embryos (Babb
and Marrs, 2004; Kane et al.,
2005; Shimizu et al.,
2005). We also uncovered a possible role for e-cadherin
in the organization of dorsal forerunner cells (DFCs) during formation of
Kupffer's vesicle (KV), a signaling center essential for establishing LR
asymmetry.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
Cloning of zebrafish ttrap, morpholinos and mRNA overexpression
A cDNA encoding full-length Ttrap (DQ524846) was isolated from a
5'cap-selected, normalized zebrafish embryo cDNA library by EST
screening. It is 24 bp longer at the 5' end than two other
ttrap cDNAs (BC083404, BC097117, GenBank). MOs and mRNAs were
injected into 1- to 2-cell-stage embryos. Plasmids containing cDNAs were
linearized, column purified and subjected to in vitro transcription (Ambion
mMessage mMachine High Yield Capped RNA kit), followed by poly(A)-tailing. For
DFC-specific knockdowns, embryos were injected into the yolk between 2 and 4
hpf with fluorescence-tagged MOs. The degree of fluorescence in DFCs and
forming KV were visually controlled using microscopy. For MOs used in this
study, see Table S1 in the supplementary material.
Antisense morpholino oligomer screen
Around 3000 antisense morpholino oligomers (MOs) targeting the putative
start codons and/or 5' untranslated regions (UTRs) of zebrafish mRNAs
were designed using GenBank cDNA sequences and 5' expressed sequence
tags (ESTs) generated from normalized, full-length (5' cap-selected)
cDNA libraries encompassing various developmental stages and adult tissues.
Targeted sequences were selected randomly. For all MOs that induced a
phenotype at one concentration, additional concentrations were tested in a
second round of screening (concentration range: 1-8 ng). A second MO
(non-overlapping with the first MO sequence) was designed for each gene that
was deemed `interesting' based on knockdown phenotype, protein structure, and
functional data, and tested again for phenotypic specificity. For the screen
in which TTRAP was initially identified as affecting vascular development,
MO-injected embryos were subjected to in situ hybridization analysis at 28 hpf
using a flk1 probe and subsequently in separate knockdown experiments
analyzed by live imaging to observe blood flow and outgrowth of vessels.
Whole-mount in situ hybridization analysis
In vitro transcription of digoxigenin-RNA probes and whole-mount in situ
hybridization were performed according to Hauptmann and Gerster
(Hauptmann and Gerster,
1994).
Microscopy and photodocumentation
Embryos were scored manually using light and fluorescence stereomicroscopy
[Stemi-2000C and Lumar V12 (Zeiss), and MZ100FLIII (Leica)]. Digital images
were captured using an AxioCam MRc5 and processed with Axiovision 4.5 Software
(Zeiss).
Whole-embryo qRT-PCR
For qRT-PCR, 15-20 embryos were pooled and RNA extracted (Tri-pure, Roche)
and purified (RNeasy RNA purification columns, Qiagen). RNA extraction on
single embryos was performed in a similar fashion. RT was performed using
MuMLV reverse transcriptase (Revert-aid, Fermentas), oligo-dT and random
primers. Real-time qPCR on single embryos was performed on ABI7000 using the
SYBRgreen amplification reagent (Eurogentec). For cdh1, we used PCR
primers: F, 5'-ATGATGTGGCGCCCACTTT-3' and R,
5'-CCGGTCGAGGTCTGTACTGAG-3'. PCR on whole-embryo cDNA was
performed with primers: F, 5'-TGCTCATTGCTCAGGTGACTTT-3' and R,
5'-TTCTTGTTTGCCCAGCTGTTC-3' to amplify a 251-bp region of
ttrap cDNA, and primers: F, 5'-GCCTTCCTTCCTGGGCATGG-3'
and R, 5'-CCAAGATGGAGCCACCGAT-3' for a 251-bp region of
β-actin cDNA.
Protein studies
To check wild-type and mutant TTRAP synthesis, 250 pg of mRNA made from
pCS2-huTTRAP or pCS2-huTTRAPT88A,T92A were injected into one-cell
embryos. To test the efficacy of knockdown in vivo, 80 pg mRNA from
pCDNA3-HA-zfttrap were injected either alone or together with 16 ng
TtrapSCMO or TtrapMO. Western blot analysis was carried
out on sonicated extracts from 20-30 pooled embryos; extracts were
immunoblotted and proteins detected using anti-TTRAP, anti-HA or anti-tubulin
antibodies.
Co-immunoprecipitation assays
2 µg MycAlk4-pCDNA3 or HA-Smad2/3/4-pCDNA3 were transfected into HEK293T
cells, together with 2 µg FlagTTRAP-pCS2 or FlagTTRAP-frame-shift-pCS2
(control). Co-immunoprecipitation studies of FlagTTRAP with Alk4 and Smads
were performed as described (Pype et al.,
2000). To test the interaction between TTRAP and Smad3, HEK293T
cells were transfected with 2 µg TTRAP-pCS3 or
TTRAPT88A,T92A-pCS3 together with either HA-Smad3-pCDNA3 or
FlagTTRAP-frame-shift-pCS2 (control).
Luciferase reporter assays
Reporter constructs were injected into the cytoplasm of one-cell embryos.
From a large collection of injected embryos, 15-20 (one set) were randomly
selected and re-injected with 16 ng TtrapMO and
controlMO. Embryos were allowed to develop to shield stage, lysed
(100 µl passive lysis buffer, Promega) and 10 µl lysate was aliquoted in
triplicate into 96-well plates. Lysates were incubated with two volumes
luciferin (Promega) and measured for luciferase activity. The readouts from
one triplicate set were averaged and treated as one data point. Fold induction
was calculated by dividing the mean value for TtrapMO by the mean
value for controlMO embryos. The pGL3 control assay was performed
three times and the ARE-luciferase assay eight times. Statistical analysis was
with the Student's unpaired t-test. The same conditions for
experiments +/- sqt or cyc RNA were used (see above), and
measurements performed in triplicate. Statistical analysis was carried out
using ANOVA (One-way analysis of variance).
Alk4 kinase assay
Strep-TTRAP-Myc-His was purified from HEK293T cells (Streptactin beads,
IBA, Göttingen) and incubated with 250 ng Alk4 (Upstate, Lake Placid) and
[
-32P]ATP for 10 minutes at 30°C. The reaction product
was separated by PAGE, blotted and exposed to film. For in vivo
phosphorylation, pStrepTTRAPMycHis was transfected into HEK293T cells with or
without an expression vector encoding constitutively active Alk4. TTRAP was
isolated using Ni-affinity purification under denaturing conditions.
Liquid chromatography mass spectrometry analysis
PhosphoTTRAP was reduced (10 mM DTT, 45 minutes, 60°C), alkylated (35
mM iodoacetamide, 30 minutes, 24°C; 15 mM DTT, 30 minutes, 24°C) and
separated from contaminating proteins by 10% tricine SDS-PAGE. The band of
interest was excised and the protein digested (trypsin, overnight, 37°C).
The resulting peptide mix was analysed by nanoLC-MS/MS, consisting of a
precursor 79(-) ion scan to signal the presence of putative phosphopeptides
and a product(+) ion scan to determine the phosphorylated residue. Nano
LC-MS/MS was performed on a Dionex Ultimate capillary liquid chromatography
system coupled to an Applied Biosystems 4000 QTRAP mass spectrometer. Peptides
were separated on a PepMap C18 column developed with a 30 minute linear
gradient (0.1% formic acid, 6% acetonitrile/water-0.1% formic acid, 40%
acetonitrile/water).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) and live analysis in the endothelium-specific transgenic
eGFP line Tg(fli1:egfp)y1 (Lawson
and Weinstein, 2002). In addition to vascular outgrowth defects,
zebrafish embryos with a MO-mediated knockdown of Ttrap (TtrapMO)
displayed pericardial edemas and abnormal blood circulation in trunk and tail
(not shown). These abnormalities were consistently associated with other gross
morphological defects, prompting us to titer MO doses in an attempt to
uncouple dysmorphology from vasculature defects. Closer inspection of Ttrap
morphants uncovered heart-looping defects, suggesting its involvement in LR
patterning.
Ttrap is essential for LR-axis determination and gastrulation
TtrapMO embryos (4 ng) displayed hallmarks of perturbed LR
patterning. Observation of live embryos and whole-mount in situ hybridization
(WISH) using cardiac myosin light chain 2 (cmlc2)
(Yelon et al., 1999) revealed
that 64% of TtrapMO embryos exhibited either reversed or no heart
looping and occasionally cardia bifida by 48 hpf
(Fig. 1;
Table 1). We also scored
TtrapMO embryos at 28 hpf, before morphological chamber
specification. In these embryos, the direction of heart jogging was also
randomized (Table 2). This
indicates that the heart looping defects were not simply due to improper
cardiac differentiation but rather to either a general defect in LR-axis
determination or failure of cardiac primordial cells to read LR positional
cues.
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). The
lack of a shield has also been reported for the homozygous Nodal mutant
squint (sqt) (Dougan et
al., 2003).
The germ ring defect was followed by incomplete convergent extension (CE)
movements as determined by analysis of paraxial protocadherin
(papc) (Yamamoto et al.,
1998) and myogenic differentiation (myod)
(Weinberg et al., 1996),
marking paraxial and adaxial mesoderm, respectively
(Fig. 2C-F). Epiboly was also
hampered in TtrapMO embryos, with some undergoing yolk cell lysis
between late gastrula stage (90% epiboly) and early somitogenesis (not shown).
This lysis was probably caused by constriction of marginal cells in their
attempt to achieve blastopore closure, despite their `regressed' position
relative to the vegetal pole (Fig.
2I). Of the embryos that did not undergo yolk cell lysis, some
managed to achieve full or partial (Fig.
2H) blastopore closure. TtrapMO embryos
(Fig. 2K) displayed a shortened
anterior-posterior axis, microcephaly, micropthalmia, and high incidence of
pericardial edemas at 24 hpf, and cardia bifida (not shown) by 48 hpf.
Together, these later phenotypes are similar to those resulting from CE
movement defects (Matsui et al.,
2005). The cardiac looping phenotype is unlikely to result from a
general perturbation of gastrulation movements because at a lower MO dose (4
ng), the looping defects and cardia bifida were still observed in normally
gastrulating embryos.
As depletion of Ttrap might also have randomized organ situs, we tested
markers normally expressed left of the midline and markers of visceral organs
(Fig. 3;
Table 3): bone
morphogenetic protein-4 (bmp4) for cardiac primordium
(Chen et al., 1997;
Schilling et al., 1999),
paired-like homeodomain transcription factor-2 (pitx2)
(Bisgrove et al., 1999;
Campione et al., 1999) and
southpaw (spaw) for left lateral plate mesoderm (LPM)
(Long et al., 2003),
forkhead box-A3 (foxA3) for liver, pancreas, gut
(Odenthal and Nusslein-Volhard,
1998; Alexander et al.,
1999), and lefty1 (lft1) for the left dorsal
diencephalon (Liang et al.,
2000) (Fig. 3).
These markers were either absent or visibly reduced in level of expression,
expressed bilaterally or on the right side in TtrapMO embryos
(Fig. 3A-E,F). LR-asymmetry
defects were therefore not restricted to the heart, but observed along the
entire rostral-caudal axis.
|
; Branford and Yost,
2004; Kishigami and Mishina,
2005). However, a direct involvement of Ttrap, at least in early
Bmp signaling, is less likely because dorsal-ventral patterning did not appear
to be affected in TtrapMO embryos, as evidenced by morphological
assessment and normal expression of chordin and bmp2b (not
shown).
|
The persistence of LR defects (despite normal gastrulation movements in
embryos injected with lower dose of TtrapMO), together with its
expression in the KV, suggests that Ttrap is involved in node formation and/or
function. However, defective shield formation in TtrapMO embryos
also implied that forerunner cell fate could be affected, but DFCs are still
present in TtrapMO embryos (see below). In addition, some
TtrapMO embryos still have a (less distinct) shield, which may be
sufficient to induce DFCs. Since Nodal signaling is important for node
function (Essner et al.,
2005), we addressed the role of Ttrap by exploiting a special
feature of zebrafish: at midblastula transition, the syncytium between yolk
and animal cells closes except for cytoplasmic bridges connected to DFCs, the
cells that will form KV (Cooper and
D'Amico, 1996; Essner et al.,
2005). These channels remain open until 
4 hpf. Between 2 and
4 hpf, MO injection results in DFC-specific knockdown
(Amack and Yost, 2004);
fluorescence-tagged MOs allow for visual control and selection of DFC-specific
(DFCMO) injected embryos.
TtrapDFCMO embryos gastrulated normally, yet still displayed
randomized heart looping or cardia bifida (48 hpf;
Table 4), and again asymmetry
markers were missing, expressed bilaterally or unilaterally on the opposite
side (Table 5). Analysis of
TtrapDFCMO embryos revealed that the KV was either absent or
smaller (Fig. 4G), and normal
in controlDFCMO embryos (Fig.
4F), even at 16 ng. We confirmed the KV phenotype using the node
marker chemokine receptor-4 (cxcr4a)
(Thisse et al., 2001)
(Fig. 4H,I). These findings
indicate that Ttrap plays a role in establishing LR asymmetry by regulating
the formation of KV.
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ALK4 phosphorylates TTRAP
TTRAP could serve as substrate for ALK4 kinase. Purified TTRAP was
incubated with human ALK4 and the reaction product analyzed by SDS-PAGE
followed by autoradiography. ALK4 phosphorylated TTRAP in vitro
(Fig. 6A). The band migrating
at the position of TTRAP was excised and analyzed by LC-MS/MS. One TTRAP
peptide was phosphorylated either on T88 and T92, or on T92 only
(Fig. 6B). T88 in TTRAP is
highly conserved across species, whereas T92 is exclusive to human, dog and
chicken (see Fig. S2 in the supplementary material). We tested in vivo
phosphorylation of Strep-TTRAP by co-expression with mouse Alk4 in HEK293T
cells, and affinity-purified TTRAP. In this preparation TTRAP-specific
peptides were reproducibly found in both the singly (T92) and doubly
phosphorylated form (T88/T92) (not shown).
|
|
) (or pGL3 as
negative control) was co-injected with TtrapMO into embryos, and
luciferase measured in lysates from pooled embryos at shield stage.
Co-injection resulted in fivefold higher Smad2/3 activity over
controlMO (mean value 4.6±2.5, P
0.01; Student's
unpaired t-test) (Fig.
7A). To test whether this increase is dependent on Nodal
signaling, we repeated the assay in the presence of Sqt or Cyc. The addition
(via RNA injection) of either ligand to TtrapMO embryos potentiated
ARE-luciferase tenfold above the activity detected with Sqt (or Cyc) but
without TtrapMO (Fig.
7B). Thus, Ttrap appears to negatively modulate Nodal signaling in
vivo.
We also tested whether expression of Smad3 targets was misregulated in
TtrapMO embryos. In contrast to Smad2, there is a striking paucity
for known Smad3 targets with regard to Nodal/Alk4 signaling. In
Xenopus, one reported target of both Smad2 and Smad3 is
Mix-2 (Yeo et al.,
1999). The fish mutant bon harbors a mutation in the
mixer-like gene and displays cardia bifida
(Chen et al., 1996;
Stainier et al., 1996;
Kikuchi et al., 2000). The
characterization of the bon promoter and its activation by Smad2/3
has not been reported. We observed an increase in bon staining in
TtrapMO embryos (Fig.
7C,D). We also tested whether Smad3 RNA injection would upregulate
bon. In zebrafish two smad3 genes exist, smad3a
(Dick et al., 2000) and
smad3b (Pogoda and Meyer,
2002). Because a gastrulation phenotype has been reported for
Smad3b RNA-injected embryos and smad3b is expressed in the tailbud
region (i.e. in the vicinity of KV), we focused on smad3b.
Importantly, Smad2 overproduction does not result in gastrulation defects
(Muller et al., 1999;
Dick et al., 2000). In embryos
overexpressing Smad3b (Smad3bOE), endogenous bon was
strongly upregulated (Fig.
7E,F; and data not shown).
To determine whether modulation of Smad3 activity by Ttrap depends on Alk4
signaling, we soaked embryos from dome stage onwards in the Alk4/5/7 inhibitor
SB431542 (Inman et al., 2002),
50 µM of which phenocopied the cyc;sqt double mutant by
shield stage (Feldman et al.,
1998) (not shown) and abolished bon expression in
TtrapMO embryos (Fig.
7G,H). Thus, the upregulation of bon in
TtrapMO embryos appears to depend on Alk signaling. Ttrap mRNA
levels are not affected by SB431542 (not shown). Morphological observation of
live Smad3bOE embryos and WISH for papc and myod
revealed CE and epiboly defects similar to TtrapMO embryos
(Fig. 7L-N). In addition,
overexpression of Smad3b in DFCs also induced a low percentage of
heart-looping defects (see Table S5 in the supplementary material). Since
targeting of mRNA to DFCs has not been previously reported, we initially
determined whether DFC-specific expression could be achieved, using eGFP RNA.
Fluorescence was detectable in the node at the time of KV formation, and all
embryos developed normally. However, in the majority of embryos, only part of
the KV was fluorescent, indicating that not all DFCs were targeted or
expressed eGFP. Therefore, DFC-RNA overexpression may not be as efficient as
DFC-MO injections (see Fig. S4 in the supplementary material). Nevertheless,
about 20% of DFC-Smad3b mRNA-injected embryos displayed heart-looping
defects.
If Ttrap knockdown increases Smad3 activity, then simultaneous reduction of
Smad3 in TtrapMO embryos should rescue the Ttrap knockdown
phenotype. Indeed, graded double knockdowns of Ttrap-Smad3b rescued up to 70%
of TtrapMO embryos with gastrulation and node formation defects
(Fig. 8A-C and Tables S6-S8 in
the supplementary material). Consistent with our co-immunoprecipitation data,
Ttrap-Smad2 double knockdowns did not rescue gastrulation defects in
TtrapMO embryos (see Table S9 in the supplementary material).
Single Smad2 knockdown resulted in a curved, shortened body axis, anterior
truncation, and loss of floorplate (see Fig. S5 in the supplementary
material), reminiscent of mutant sur
(Pogoda et al., 2000;
Sirotkin et al., 2000) and
similar to the phenotype described for Smad2 knockdown in Xenopus
(Rana et al., 2006).
|
|
;
Meno et al., 1999;
Cheng et al., 2000;
Feldman et al., 2002). We
tested whether Ttrap knockdown would alter expression of cyc, sqt
(Rebagliati et al., 1998a;
Rebagliati et al., 1998b) and
lft1. Ttrap was neither required for initiation nor maintenance of
cyc, sqt and lft1 expression at germ ring stage and 70%
epiboly (not shown). This is consistent with our data showing no detectable
interaction between TTRAP and Smad2, since expression of cyc, sqt and
lft1 is regulated primarily by Smad2/FoxHI
(Schier, 2003). Ttrap may
therefore play a role in distinguishing between Smad2 and Smad3 signaling.
To test whether Ttrap regulation of gastrulation movements is due to a
direct effect on cell motility or perturbation of mesendodermal cell fate, we
transplanted wild-type and TtrapMO cells sequentially from the
lateral margin into the germ ring of maternal-zygotic one-eyed
pinhead (MZoep) embryos (see Fig. S7 in the supplementary
material) (Schier et al.,
1997; Zhang et al.,
1998; Gritsman et al.,
1999). In contrast to wild-type cells, TtrapMO cells
neither internalized nor migrated to regions normally occupied by endodermal
progenitors (as observed for wild-type cells that continue to express
axial in MZoep mutants)
(Gritsman et al., 1999). Thus,
it appears that Ttrap function (at least its modulation of Smad3 activity) is
non cell-autonomous in this context. Moreover, MZoep mutants are not
rescued by Ttrap knockdown (not shown). This implies that Ttrap knockdown
cannot compensate for the lack of Smad2 activation in this Nodal-insensitive
oep background. This, and our reporter studies, supports our
hypothesis that the Ttrap-mediated increase in Smad3 activity is Nodal
dependent. In addition, the cell transplantation data implicate a role for
Smad3 in specific aspects of Nodal signaling.
|
;
Shimizu et al., 2005).
Because hab:cdh1 null mutants do not survive beyond
gastrulation, we again exploited DFC-specific knockdown to determine whether
cdh1 plays a role in node formation. We found randomized heart
looping, cardia bifida and smaller/absent KV in cdh1DFCMO embryos
(Table 6). Analysis of DFCs
using cas/sox32 (Dickmeis et al.,
2001; Kikuchi et al.,
2001) and prior to node formation revealed that DFCs are present
in TtrapDFCMO embryos (Fig.
9A,B). Moreover, at shield and 70-80% epiboly stages, no
significant difference in DFC number between TtrapMO and
controlMO embryos was detected (see Fig. S8, Table S10 and Fig. S9
in the supplementary material). However, TtrapMO DFCs do not always
converge at the midline to form one tight cluster of cells below the shield or
later in gastrulation. Rather, cells are more widely dispersed in a broad
stripe along the lateral axis of the embryo
(Fig. 9B; see Fig. S8, Table
S11, and Fig. S10 in the supplementary material), suggesting a defect in cell
migration. In line with our DFC data in TtrapMO embryos, DFCs are
still present in hab:cdh1 mutants
(Kane et al., 2005). The same
DFC defect was also observed in Smad3bOE embryos
(Fig. 9D) and may reflect an
inability of these cells to organize into KV.
|
0.01, Student's unpaired
t-test; not shown). This limited but consistent reduction may be
explained by a Ttrap-Smad3-mediated downregulation of cdh1 that is
localized only to regions where Nodal signaling is present and/or can be
sensed, and may therefore be partly masked by more general expression in the
rest of the blastoderm.
|
|
;
Grooteclaes and Frisch, 2000;
Comijn et al., 2001;
Hajra et al., 2002;
Eger et al., 2000), and
Snail is induced by TGFβ or Nodal
(Fujimoto et al., 2001;
Hajra et al., 2002;
Peinado et al., 2003;
Gotzmann et al., 2006;
Bennett et al., 2007).
At 60% epiboly, snail1a (snai1a) expression is restricted
to the blastoderm margin and presumptive paraxial mesoderm, but is excluded
from the dorsal most region of the shield (presumptive axial mesoderm)
(Hammerschmidt and Nüsslein-Volhard,
1993; Thisse et al.,
2001). DFCs are in close proximity to these dorsal cells and do
not express snai1a. We questioned whether the increase in Smad3
activity in TtrapMO embryos would be sufficient to cause ectopic
expression of snai1a in either axial mesoderm and/or DFCs, thereby
contributing to cdh1 downregulation. Knockdown of Ttrap resulted in
misexpression of snai1a in axial mesoderm (60% epiboly;
Fig. 10A,B) and DFCs (not
shown). To determine whether this expression is mediated by Smad3, we
performed Ttrap-Smad3 double knockdowns to test for reversion to the normal
snai1a domain. Double knockdown resulted in a 58% rescue of embryos
with ectopic snai1a in the axial mesoderm
(Fig. 10C,D;
Fig. 10E for graphical
depiction of rescue; data not shown). Curiously, neither the single Smad3b nor
Smad3a knockdown (and their combination) resulted in downregulation of
snai1a (not shown). Moreover, Smad3b knockdown did not result in any
visible gastrulation phenotype/s (our unpublished observations), despite the
presence of transcripts throughout early development
(Dick et al., 2000;
Pogoda and Meyer, 2002). By 24
hpf however, morphological defects similar to the Smad2 knockdown phenotype
such as head degeneration, absence of floorplate, and curved, shortened body
axis could be observed (see Fig. S6 in the supplementary material). The lack
of a phenotype before 24 hpf may be attributed to compensation by either
Smad2, Smad3a, and/or Smad2/3 maternal protein/s. Finally, we performed
Ttrap-Snail1a double-knockdowns to test for rescue of Ttrap-induced LR
defects. This resulted in a 62% rescue of heart looping defects (48 hpf; see
Table S12 and Fig. S11 in the supplementary material).
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|
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Kane et al., 2005;
Shimizu et al., 2005;
Ulrich et al., 2005). Our data
suggest that: (1) cdh1 is downstream of Alk4-Smad3 and that
cdh1 expression may, in part, be controlled by Ttrap through
attenuation of Smad3 activity; and (2) that knockdown of Ttrap increases Smad3
activity, which in turn downregulates cdh1, thereby leading to
impaired CE and epiboly. Our results also implicate snail as a potential link
between increase in Smad3 activity and downregulation of cdh1,
possibly via a snail-binding site in the cdh1 promoter.
Knockdown of Ttrap does not appear to affect mesodermal or endodermal cell
fate, because bon and cas expression persist in mesendoderm
and presumptive endoderm, respectively. This observation was also true for
meso/endodermal bhik, mix, ntl and gsc (not shown),
including in TtrapMO embryos with thickened germ ring. We therefore
propose that ttrap is primarily involved in cell migration in early
embryos. The results of the cell transplantations (both with wild-type and
with TtrapMO cells) into a defective Nodal signaling background
(MZoep) indicate a possible distinct function for Smad3 in directing
cell movement. TTRAP has been implicated in migration of cancer cells through
its interaction with ETS1/2 and FLI1 (Pei
et al., 2003). However, knockdown of Ets1 in fish did not yield
gastrulation defects (our unpublished observations), and fli1
expression only begins after gastrulation at 10 hpf
(Brown et al., 2000), making
fli1 an unlikely Ttrap target with respect to regulation of CE
movements and epiboly.
Potential role of Ttrap in Kupffer's vesicle formation
LR-axis determination in fish
(Kramer-Zucker et al., 2005)
and mouse, and the resulting laterality of heart and viscera, is initiated in
part by the action of monocilia residing within the node. This structure
consists of a `pit' of cells, with each cell protruding one monocilium, which
in mouse is posteriorly tilted at an angle of 60°
(Nonaka et al., 2005). The
first symmetry-breaking event occurs when these monocilia beat in vortical
fashion to direct unidirectional fluid flow, resulting in accumulation of
proteins left of the node. Physical models that can mimic such flow have shown
that even this small difference in Nodal flow is subsequently converted
through reaction-diffusion mechanisms involving Nodal/Lefty proteins into a
robust asymmetrical target gene expression
(Okada et al., 2005;
Hirokawa et al., 2006). This
results in activation of target genes in the LPM, which endow `leftness' to
this side of the embryo and activate asymmetrical differentiation of organ
primordia. However, the mechanism(s) by which these asymmetric signals are
translated into morphology is not well understood
(Shiratori and Hamada, 2006).
Genes expressed specifically in the right LPM also function in LR
determination, and other studies have also implicated the intact midline as
serving a barrier function between left- and right-sided factors
(Roessler and Muenke, 2001;
Tabin and Vogan, 2003;
Yost, 2003;
Raya and Belmonte, 2004;
Levin, 2005;
Raya and Belmonte, 2006).
Our findings suggest that Ttrap plays a role both in the earliest steps of
KV (node) formation and gastrulation. DFC-specific Ttrap knockdown embryos
gastrulate normally, yet DFC behavior is abnormal, resulting in a KV that is
strongly reduced in size, or absent. The laterality defects observed in
TtrapDFCMO embryos are consistent with LR defects obtained after
DFC ablation (Essner et al.,
2005). Moreover, we provide results implicating cdh1 as a
possible target of Ttrap and Smad3 for regulating gastrulation movements and
KV formation. E-cadherin (cdh1) is an established player in
mediating cell (de)adhesion/migration in embryos and invasive tumors. Our
results suggest that cdh1 may play a role in LR-axis determination.
It must be noted, however, that neither KV nor LR defects have been described
for cdh1 mutants to date. It therefore remains to be seen what the
precise role is for Cdh1 with regard to LR patterning and DFC migration. Our
data suggests that Ttrap regulates cdh1 via Smad3 as opposed to
Smad2. This is supported by a recent siRNA study on the
epithelial-to-mesenchymal transition (EMT) of proximal-tubule epithelial
cells, which showed a Smad3-dependent (and Smad2-independent) downregulation
of cdh1 following stimulation of cells with TGFβ
(Phanish et al., 2006).
Ttrap distinctly modulates Smad3 and not Smad2 activity
Smad2 and Smad3 share over 90% identity and a number of overlapping
functions, such as the co-regulation of the Nodal targets bon and
snail (Bennett et al.,
2007). Although our results indicate that bon and
snail can be regulated by Smad3, our data do not rule out
co-regulation by Smad2, and regulation of these genes most likely occurs via
cooperation between these two Smads. Nevertheless, there are
structural/functional differences between both Smads, several of which appear
to distinguish their actions in vitro (Yew
et al., 2004; Uemura et al.,
2005; Ju et al.,
2006) and in vivo (Dunn et
al., 2005; Wang et al.,
2006). In addition, the ratio of Smad2 versus Smad3 influences
their respective roles as effectors (Dunn
et al., 2004; Kim et al.,
2005). The functional differences between Smad2 and Smad3 may also
depend on their ability to associate with various co-factors that mediate
distinct responses to TGFβ (Attisano
et al., 2001). These co-factors include Fox proteins
(Nagarajan and Chen, 2000) or
the Smad-interacting protein Smicl, which primarily modulates Smad3 activity
during Nodal-dependent induction of chordin in the Spemann organizer
(Collart et al., 2005).
Clearly, additional Smad2- and Smad3-specific targets and co-factors remain to
be identified. Ttrap may be one such co-factor in Nodal-Alk4-Smad3 signaling.
Intriguingly, the Ttrap knockdown phenotype does not entirely mimic the Nodal
overexpression phenotype (Toyama et al.,
1995). In the latter study, Nodal mRNA injection into fish embryos
resulted in axis duplication and ectopic organizer formation, phenotypes not
observed in Ttrap knockdown embryos. Thus, Ttrap knockdown does not result in
a general over-activation of Nodal signaling, which would also encompass Smad2
activity.
The role of Ttrap as a co-factor for ETS
(Pei et al., 2003) suggests
that intranuclear Ttrap may play a similar role with regard to Smad3. This is
supported by the finding that Ttrap binds to SUMO proteins, which are
implicated in a number of cellular processes including transcription
(Hecker et al., 2006). In any
case, several lines of evidence support the hypothesis of Ttrap as modulator
of Alk4-dependent Smad3 activity: (1) the association between TTRAP and Smad3
is mutually exclusive with Alk4; (2) the high degree of overlap between
TtrapMO and Smad3bOE phenotypes; (3) the ARE-luciferase
data; and (4) the rescue of the Ttrap knockdown phenotype via Smad3 knockdown.
However, the functional mechanism underlying this modulation remains to be
investigated in detail.
The roles of Smad2/3 as effectors have been extensively characterized in
the mouse, including elegant studies that address the effects of changing
their ratio in vivo in Nodal-controlled mesoderm formation
(Dunn et al., 2004;
Dunn et al., 2005). Our data
show that Smad3 plays an important role in zebrafish in controlling cell
migration and/or (de)adhesion through Nodal-Alk4. Sqt is the most likely
ligand here because of its reported role in regulating gastrulation movements
and its expression in DFCs (Rebagliati et
al., 1998b; Feldman et al., 2000). Ttrap may serve as limiting
factor for Smad3, perhaps to maintain a balance between Smad2 (the DNA-binding
splice form) and Smad3 signaling, because both are capable of occupying the
same promoter sites of target genes for Nodal.
Using biochemical studies and phenotypic analysis in zebrafish, we have
uncovered a role for Ttrap as a novel player in TGFβ signaling in vivo.
Our findings suggest that this protein is essential for regulating Nodal
signaling, at least by limiting the early developmental activity of Smad3.
Given that extranuclear Ttrap and Alk4 also interact either directly or are
present together in a complex, it is possible that Alk4 itself, via a negative
feedback loop involving Ttrap and possibly other co-factors, functions to
regulate the level of its own signal transduction cascade. This type of higher
order regulation fits in the concept of self-enabled gene response cascades
(Massagué et al., 2005)
and has been observed in TGFβ-Smad signaling. Although further studies
are needed to elucidate the exact mechanism by which Ttrap modulates
Alk4-Smad3 activity, our data underscore the importance of tightly fine-tuning
TGFβ-Smad pathways in embryos.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/24/4381/DC1
|
ACKNOWLEDGMENTS |
|---|
|
Footnotes |
|---|
Present address: Stem Cell Institute Leuven (SCIL), KULeuven, Herestraat
49, B-3000 Leuven, Belgium 
Present address: Galapagos, Generaal De Wittelaan L11A3, B-2800 Mechelen,
Belgium
Present address: Laboratory for Molecular Virology and Gene Therapy,
Department of Molecular and Cellular Medicine, KULeuven, Kapucijnenvoer 33,
B-3000 Leuven, Belgium
¶ Present address: Department of Pharmaceutical Sciences, KULeuven,
Herestraat 49, B-3000 Leuven, Belgium
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REFERENCES |
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