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First published online 28 November 2007
doi: 10.1242/dev.009266
1 Cancer and Developmental Biology Laboratory, Center for Cancer Research,
National Cancer Institute-Frederick, NIH. Frederick, MD 21702, USA.
2 Department of Pharmacology, Graduate School of Medicine, Kyoto University,
Sakyo, Kyoto, 606-8501, Japan.
* Author for correspondence (e-mail: tyamaguchi{at}ncifcrf.gov)
Accepted 16 September 2007
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Wnt3a, β-catenin, Gastrulation, Mesoderm, Segmentation, Somitogenesis
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
The morphogenetic process of gastrulation continuously drives the movement of
PSM progenitors from the streak to the posterior end of the PSM. Cells in the
posterior PSM remain in an undetermined and immature state, and become
anteriorly displaced as new cells are added to the posterior PSM. When PSM
cells reach a prescribed position in the anterior PSM, they undergo a dramatic
transition in gene expression, initiating a segmentation program that
determines where and when a morphological segment boundary will form. The
rhythmic formation of a new boundary in the anterior PSM, every 2 hours, leads
to the formation of somites. Thus the coordinated addition of new mesodermal
cells to the posterior PSM, coupled with the cleaving of new somites from the
anterior PSM, is critical for the maintenance of the PSM and, ultimately, for
the rapid growth and posterior extension of the body axis that occurs during
vertebrate embryogenesis (Aulehla and
Herrmann, 2004; Dubrulle and
Pourquie, 2004; Pourquie,
2001; Saga and Takeda,
2001).
The bHLH transcription factor Mesp2, under the control of the Notch
signaling pathway, plays an important role in the segmentation program.
Mesp2 is expressed in a segmental prepattern in the anterior PSM
prior to the formation of overt boundaries, and is required for segment
polarity and boundary formation (Saga et
al., 1997). The prevailing `clock and wavefront', or `clock and
gradient' models postulate that segment boundaries are positioned along the
anterior-posterior (AP) axis by gradients of fibroblast growth factor 8 (Fgf8)
and/or Wnt3a and an opposing gradient of retinoic acid (RA), which together
define a boundary determination front in the anterior PSM. The periodicity of
boundary formation is thought to be controlled by an oscillating segmentation
clock driven by the Wnt and Notch signaling pathways
(Aulehla and Herrmann, 2004;
Pourquie, 2003;
Rida et al., 2004). The
molecular mechanisms linking these signaling pathways to the clock and to
boundary formation, are not well understood.
Feedback suppressor loops in the Wnt and Notch pathways are considered
central molecular components of the segmentation clock. Notch activity
oscillates in the PSM, driving periodic expression of its target genes lunatic
fringe (Lfng) and Hes7
(Bessho et al., 2001;
Morimoto et al., 2005). The
glycosyltransferase Lfng, and the transcriptional repressor Hes7, function as
negative regulators of Notch signaling and are required for proper
segmentation (Bessho et al.,
2003; Evrard et al.,
1998). Similarly, the Wnt target genes Axin2 and
Nkd1 encode negative regulators of Wnt signaling, oscillate in the
PSM, and are thought to function as integral components of the clock to
periodically suppress Wnt signaling
(Aulehla et al., 2003;
Ishikawa et al., 2004).
Oscillating genes in both the Wnt and Notch pathways depend upon
Wnt3a (Aulehla et al.,
2003; Nakaya et al.,
2005), however the significance of Wnt-centered feedback loops for
the clock remains unclear because mutations in Axin2 or Nkd1
do not lead to somite or mesodermal phenotypes
(Li et al., 2005;
Yu et al., 2005).
Wnt3a controls gene expression by stabilizing cytosolic levels of
β-catenin, the central player in the canonical Wnt/β-catenin
pathway. Stabilized β-catenin can then translocate to the nucleus, bind
to Tcf/Lef transcription factors, and activate target genes
(Stadeli et al., 2006;
Willert and Jones, 2006).
Embryos lacking Wnt3a display posterior axis truncations
(Takada et al., 1994). This is
due, at least in part, to changes in the expression of the target genes
T (Brachyury), a T-box transcription factor gene necessary
for mesoderm formation, and Dll1, which encodes a Notch ligand
required for segmentation (Arnold et al.,
2000; Aulehla et al.,
2003; Galceran et al.,
2001; Galceran et al.,
2004; Hofmann et al.,
2004; Nakaya et al.,
2005; Yamaguchi et al.,
1999). Although a few important target genes have been validated,
the transcriptional network activated by Wnt3a in vivo remains largely
unresolved. Since functional redundancy between Wnt ligands may confound a
full understanding of the role of Wnts and their target genes in these
processes, we have turned to conditional loss and gain of function alleles of
β-catenin to determine the precise role that Wnt3a and β-catenin
play in mesoderm formation and segmentation.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), T-cre
(Perantoni et al., 2005), and
BATlacZ mice (Nakaya et al.,
2005), were described previously. Transgenic mice were generated
in the Transgenic Core Facility, NCI-Frederick, by pronuclear injection
following standard procedures. All animal experiments were performed in
accordance with the guidelines established by the NCI-Frederick Animal Care
and Use Committee.
Half-embryo explant cultures
PSM/somite explants were dissected from E9 outbred NIH Swiss embryos, and
bisected down the midline as previously described
(Correia and Conlon, 2000).
One half was immediately fixed whereas the remaining half was cultured in 10%
FBS in DMEM for 1 hour.
In situ hybridization
Single and double whole-mount in situ hybridization (WISH) was performed as
previously described (Biris et al.,
2007). Embryos were photographed on a Leica stereoscope or a Zeiss
Axiophot compound microscope. Unless indicated otherwise, at least four mutant
embryos were examined with each probe, and all yielded similar results.
Gene expression profiling
Total RNA was isolated with TRIzol reagent (Invitrogen) as previously
described (Baugh et al., 2003)
from microdissected node and primitive streak regions of E7.75-E8 wild-type
and Wnt3a mutant embryos. Protocols for synthesis of cDNA and cRNA were
performed using the Affymetrix Two-Cycle Target Labeling Kit (Affymetrix,
Santa Clara, CA) according to the manufacturer's recommendations. Array
hybridizations (GeneChip Mouse Genome 430 2.0, Affymetrix) were performed in
triplicate per genotype. Subsequent washing, staining, and array scanning were
carried out according to Affymetrix protocols. Statistical analysis was
performed on probe-intensity level data using BRB ArrayTools (v3.2). Class
comparison analysis was conducted using a random-variance F-test
(P
0.001). Hierarchical clustering was carried out for
statistically significant genes using normalized log2 of the signal
values.
Expression constructs, transfections, cell culture and luciferase reporter assays
Luciferase reporter constructs were generated by cloning the
Ripply2 enhancer fragment (2.1 kb; SacI-XhoI) with
and without the Ripply2 promoter (1.1 kb;
XhoI-NcoI) into pGL4.10[luc2] or
pGL4.23[luc2/minP] vectors (Promega), respectively. Two deletion
constructs were generated, one consisting of the 1.1 kb Ripply2
promoter lacking the proximal putative Tbx6 binding site and E-box, and a
second containing three repeats of this 44 bp region upstream of a minimal
promoter. HEK293 cells were seeded at 0.55x105 cells per well
in 24-well plates and grown to 70% confluency. A total of 400 ng DNA
containing the reporter plasmid (200 ng) and empty vector were co-transfected
with or without expression vectors, pCS2-3X FLAG Tbx6 (10 ng), p3X FLAG-CMV
Mesp2 (50 ng), and pCDNA3 
N-β-catenin-myc (150 ng) using Fugene 6
(Roche). Cells were lysed 48 hours after transfection and luciferase activity
was measured using the Dual Luciferase Assay Kit (Promega) as per
manufacturer's recommendation. For each condition, 10 ng pGL4.74[hRluc/TK]
Vector (Promega) was used as an internal control to normalize for transfection
efficiency. Fold change was calculated as a ratio of the luciferase vector
containing Ripply2 regulatory elements relative to empty luciferase
vector for identical experimental conditions, normalized to a control
condition minus expression vectors. The reported values consist of one
experiment but are representative of at least three independent
experiments.
Electrophoretic mobility shift assay
Double-stranded DNA oligonucleotides were end labeled and protein-DNA
complexes were analyzed using the DIG Gel Shift Assay Kit (Roche) according to
the manufacturer's instructions. A 3 x Flag-Tbx6 protein was made using
the TNT Reticulocyte Lysate System (Promega) in vitro. Binding reactions were
incubated at room temperature for 15 minutes and the protein-DNA complexes
were analyzed on 6% DNA retardation gels (Invitrogen). Oligonucleotide
sequences used in the EMSA assays are as follows:
5'-CGTTCACACCCGCGCGCGGCCCGCGGCGCC-3' (wt Tbx6 BS);
5'-CGTTGATATCCGCGCGCGGCCCGCGGCGCC-3' (mut Tbx6 BS).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Huelsken et al.,
2000), we conditionally inactivated
Ctnnb1tm2Kem (Brault et
al., 2001) in the PS on E7.5 (one day after gastrulation begins)
using the T promoter to express Cre recombinase
(Perantoni et al., 2005).
Conditional Ctnnb1 loss of function (LOF) mutants, hereafter referred
to as T-Cre;Ctnnb1flLOF/, gastrulated normally but
displayed posterior truncations (Fig.
1B) that were similar but more severe than that observed in
Wnt3a-/- mutants
(Takada et al., 1994). Head
structures were normal but mutants lacked a PS, posterior mesoderm and obvious
somites. Loss of β-catenin activity was confirmed by monitoring the
expression of the BATlacZ transgene, an in vivo reporter of
β-catenin/Tcf function (Nakaya et
al., 2005). Reporter activity was down-regulated specifically in
the posterior embryo, with residual activity only detectable in surface
ectoderm (Fig. 1B). Conversely,
conditional stabilization of β-catenin
[Ctnnb1lox(ex3)
(Harada et al., 1999),
hereafter referred to as Ctnnb1flGOF/+] in
streak-derived mesoderm through the T-Cre-mediated deletion of the
phosphorylation/degradation domain of β-catenin, led to a grossly
enlarged PSM, at the apparent expense of somites, and impaired embryo turning
(Fig. 1C). BATlacZ
expression appeared upregulated and anteriorized in the
T-Cre;Ctnnb1flGOF/+ mutants, confirming the enhanced
activity of β-catenin.
Mutants were examined at earlier developmental stages (E8.2-8.5) to assess
segmentation phenotypes. Somites were not observed in the anterior paraxial
mesoderm of T-Cre;Ctnnb1flLOF/ embryos
(Fig. 1I), although they were
clearly distinguishable in controls (Fig.
1D). Histological analyses confirmed that the mutant anterior
paraxial mesoderm was unsegmented, thickened and disorganized, but small,
incompletely epithelialized somites were observed in posterior regions where
the PSM would normally lie (cf. Fig.
1L,M with Fig.
1G,H). The BATlacZ reporter was specifically
downregulated in the mesoderm (Fig.
1J) and posterior streak (Fig.
1I,K), but remained easily detectable in the streak ectoderm and
posterior neural tube. Similar small disorganized somites were also found in
T-Cre;Ctnnb1flGOF/+ mutants at early somite stages, but
were only detectable anteriorly, rostral to the enlarged PSM
(Fig. 1N-Q). As expected, the
β-catenin reporter was upregulated in the streak and anteriorized in the
elongated PSM (Fig. 1N,O). At
these stages, the somites generally formed anterior to the BATlacZ
expression domain in both the wild-type and
T-Cre;Ctnnb1flGOF/+ embryos
(Fig. 1D,F,N).
|
; Herrmann et al.,
1990; Hrabe de Angelis et al.,
1997; Sun et al.,
1999). All four genes were absent from the
T-Cre;Ctnnb1flLOF/ PS and PSM, but were upregulated
and anteriorly expanded in T-Cre;Ctnnb1flGOF/+ embryos.
These results suggest that β-catenin is necessary and sufficient for the
specification of posterior PSM fates and for the maintenance of the PS.
Furthermore, they demonstrate that Fgf8, which is thought to be
important for the determination of segment boundary position, lies downstream
of β-catenin. Anterior paraxial mesoderm was present in both LOF and GOF
mutants, as indicated by the continued expression of the somite markers
Mox1 (Fig. 2A-C) and
Paraxis (not shown). However, neither Mox1 nor
Paraxis (also known as Tcf15 - MGI) were expressed in a
segmental fashion in the anterior paraxial mesoderm of
T-Cre;Ctnnb1flLOF/ embryos. Coupled with the
histological observations that small somites were only found posteriorly, the
data indicate that segmentation is delayed but ultimately proceeds in the
absence of β-catenin.
Oscillating gene expression in conditional Ctnnb1 mutants
To determine whether the somitogenesis defects were due to abnormal cycling
of the segmentation clock, we examined embryos for the expression of
oscillating target genes of the Wnt, Fgf and Notch signaling pathways. As
expected from analyses of Wnt3a mutants
(Aulehla et al., 2003;
Nakaya et al., 2005), the
Wnt3a target gene Axin2 was not expressed in the
T-Cre;Ctnnb1flLOF/ streak and PSM, although easily
detected in the head (see Fig. S1 in the supplementary material). Analysis of
oscillating genes associated with the Fgf signaling pathway
(Dequeant et al., 2006) such
as Dusp6/Mkp3 (Fig.
2J,K), which encodes an extracellular signal-related kinase (ERK)
phosphatase, and the FGF pathway inhibitor Spry2 (not shown), also
revealed little to no expression in the
T-Cre;Ctnnb1flLOF/ PSM. Similarly, the expression
of the oscillating Notch target genes Lfng and Hes7 were not
observed in the T-Cre;Ctnnb1flLOF/ PSM
(Fig. 2M,N,Q,R). These results
demonstrate that β-catenin is upstream of all signaling pathways known to
oscillate in the PSM.
|
)], then stabilization of β-catenin should disrupt the
molecular oscillations associated with the clock. Examination of
Axin2 expression revealed that it was indeed upregulated and
anteriorized in the paraxial mesoderm of
T-Cre;Ctnnb1flGOF/+ embryos (see Fig. S1 in the
supplementary material), consistent with it being a Wnt3a/β-catenin
target gene. The Fgf pathway genes Dusp6/Mkp3
(Fig. 2L) and Spry2
(not shown) were also similarly upregulated and anteriorized in
T-Cre;Ctnnb1flGOF/+ embryos. Remarkably, stripes of
Lfng and Hes7 mRNA were still observed in the enlarged PSM
(Fig. 2O,S), demonstrating that
the oscillations of the Notch pathway persist despite the highly stabilized,
non-cycling expression of β-catenin and Axin2. Although no more
than two or three domains of Lfng or Hes7 expression were
observed in the wild-type PS and PSM (Fig.
2M,Q), four to seven stripes were observed in the elongated
T-Cre;Ctnnb1flGOF/+ PSM, depending upon embryo age and PSM
size (Fig. 2O,S, and data not
shown). It is formally possible that the continued periodicity observed in
these embryos is attributable to the presence of the wild-type Ctnnb1
allele. However, examination of Lfng expression in embryos that only
express the stabilized form of β-catenin (i.e.
T-Cre;Ctnnb1flGOF/), revealed persistent pairs of
Lfng stripes in the anterior PSM
(Fig. 2P). These results show
that the oscillating Notch pathway is surprisingly robust in the presence of
stabilized β-catenin, and suggest that Wnt/β-catenin signaling
defines a cellular state that is permissive, but not instructive, for the
segmentation clock.
Segmental gene expression occurs in Wnt3a and Ctnnb1 mutants
The abnormal somites observed in conditional Ctnnb1 mutants
prompted us to examine mutants for segment polarity defects because the
establishment of segment polarity correlates with proper boundary formation
(Pourquie, 2001). Expression
of the paired-type homeobox gene Uncx4.1 is restricted to the
posterior halves of segmented somites
(Mansouri et al., 1997).
Initial analyses indicated that Uncx4.1 was absent in Ctnnb1
mutants compared with wild-type embryos
(Fig. 2Q-S); however, a
detailed examination using higher contrast chromogens revealed surprising
results. Despite our demonstration that periodic expression of Fgf, Notch and
Wnt target genes was arrested in T-Cre;Ctnnb1flLOF/
mutants, Uncx4.1 was still expressed in a segmented pattern in the
mutant somites at E8.5 (Fig.
3C,D). Uncx4.1 stripes were notably absent from the
anterior-most paraxial mesoderm, and were compressed and posteriorly shifted,
demonstrating that segmentation proceeded but was delayed. These stripes of
Uncx4.1 mRNA were no longer detectable by E9-9.5 (not shown)
suggesting that β-catenin is required for the maintenance of the
epithelial segment boundary. Interestingly, stabilization of β-catenin in
T-Cre;Ctnnb1flGOF/+ embryos resulted in an anterior shift
of the Uncx4.1 spatial domain
(Fig. 3E,F,J). Uncx4.1
was expressed as two distinct stripes anteriorly, but was fused and
disorganized posteriorly.
To compare segment polarity in Wnt3a and Ctnnb1 mutants,
we assessed the expression of Uncx4.1 together with Tbx18, a
marker of the anterior half-somite (Kraus
et al., 2001). Segment polarity was well preserved in the anterior
Wnt3a-/- somites (Fig.
3H), but was aberrant and less well defined in
T-Cre;Ctnnb1flLOF/ mutants
(Fig. 3I). Nevertheless,
interspersed, fuzzy stripes of Tbx18 and Uncx4.1 were still
detected posteriorly, indicating that rudimentary polarized gene expression
and segment borders can form in the absence of β-catenin. By contrast,
the caudal limit of the Uncx4.1 expression domain was shifted
anteriorly in T-Cre;Ctnnb1flGOF/+ embryos, whereas the
somitic expression of Tbx18 was almost completely repressed
(Fig. 3J).
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mutants (not shown), although it
was easily detected in the wild-type 10 ss (somite-stage) littermate (cf.
Fig. 3L,P with
Fig. 3K,O). Notably,
Mesp2 expression and PS and PSM cells, were rescued in
Wnt3a-/- mutants by stabilized β-catenin
(n=4) (Fig. 3N,R). The
Wnt3a-/-; T-Cre;Ctnnb1flGOF/+ double mutant
phenocopies the T-Cre;Ctnnb1flGOF/+ mutant
(Fig. 3M,Q), demonstrating that
Ctnnb1 is epistatic to Wnt3a, and that Mesp2 lies
downstream of both genes. In addition, the data provides formal genetic proof
that β-catenin mediates the mesoderm-inducing activity of Wnt3a.
Because the loss of Mesp2 expression in E8.5 mutants may simply be
due to the absence of posterior mesoderm, we examined the position of
Mesp2 transcripts in younger Wnt3a-/- embryos
(0-4 ss), which still possess posterior mesoderm. Although the Mesp2
stripes always lay adjacent to the anterior end of the wild-type node
(Fig. 3S), Mesp2
expression was posteriorized in Wnt3a-/- mutants, abutting
the posterior-most end of the node (Fig.
3T). Examination of T-Cre;Ctnnb1flLOF/
embryos revealed that Mesp2 was generally absent (5/6 embryos)
(Fig. 3U). In the remaining
E8.2 embryo, the Mesp2 stripe was undefined and further posteriorized
than in Wnt3a-/- embryos (not shown). Remarkably, multiple
anteriorized stripes of Mesp2 expression, reminiscent of the multiple
Lfng and Hes7 stripes, were observed in the expanded PSM of
T-Cre;Ctnnb1flGOF/+ embryos
(Fig. 3V). Wnt3a and
β-catenin presumably control the spatiotemporal activation of
Mesp2 indirectly, since Notch and Tbx6 are known to directly activate
Mesp2 (Yasuhiko et al.,
2006).
Identification of Ripply2, a putative boundary determination gene
Mesp2 is thought to function in the anterior PSM to arrest the segmentation
clock by activating Lfng, and thereby suppressing Notch activity
(Morimoto et al., 2005). The
ectopic stripes of Lfng, Hes7, and Mesp2 mRNA observed in
the T-Cre;Ctnnb1flGOF/+ PSM indicates that the cycling
Notch clock failed to arrest despite the expression of Mesp2 and
Lfng. These observations suggest that genes in addition to
Mesp2 may be required for segment boundary determination. In a
genome-wide microarray screen designed to identify the in vivo target genes of
Wnt3a during gastrulation and somitogenesis (unpublished), we identified a
RIKEN EST (C030002E08) that was differentially expressed (P<0.001)
in Wnt3a null mutants compared with the wild type
(Fig. 4A). This cDNA represents
a putative isoform of the recently described Zebrafish Ripply2
(Kawamura et al., 2005). The
closely related family member Ripply1 functions in fish to regulate
somitogenesis by binding to the transcriptional corepressor Groucho and
repressing the zebrafish Mesp2 homolog, mesp-b.
Two-color whole-mount in situ hybridization (WISH) demonstrated that Ripply2 mRNA was expressed in the anterior PSM in one or two stripes in prospective somites S0 and S-I, overlapping with intense anterior PSM expression of components of the Notch pathway, including Dll1 (Fig. 4B), Lfng (not shown), Hes7 (Fig. 4C) and Mesp2 in S-I (see below). To examine the temporal aspects of Ripply2 and Mesp2 expression in the anterior PSM, half-embryo culture experiments were performed. These experiments revealed that Ripply2 expression was dynamic and was periodically activated in S-I after Mesp2 was activated there (Fig. 4D,E). Similarly, Ripply2 overlapped, but was expressed out of phase with, Hes7 (not shown).
|
embryos
(Fig. 4H), indicating that
Wnt3a and β-catenin are required for the maintenance of Ripply2
expression. Again, similarly to Mesp2 expression, weak, posteriorized
expression was observed in one (n=3) E8.2
T-Cre;Ctnnb1flLOF/ embryos (not shown). However, in
contrast to the multiple stripes of Mesp2 expression observed in
T-Cre;Ctnnb1flGOF/+ embryos
(Fig. 3V), Ripply2 was
strikingly absent (n=4, Fig.
4I). These results are consistent with the microarray data and
together, suggests that Ripply2 is exquisitely sensitive to the
levels of Wnt3a/β-catenin signaling.
Tbx6, Mesp2 and the Wnt pathway regulate Ripply2
To investigate the molecular mechanisms underlying the control of
Ripply2 expression, we compared the human and mouse Ripply2
loci to identify potential regulatory elements. Two conserved regions were
found, a putative promoter adjacent to the initiator codon, and a putative
enhancer 6 kb upstream (Fig.
5A). Both fragments were tested for their ability to drive
expression of a lacZ reporter in vivo. Analysis of transgenic founder
embryos revealed that the combined enhancer and promoter fragments (Rip2EP)
drove expression in the anterior PSM and the anterior halves of the newly
formed somites (Fig. 5A1). The
distal enhancer element (Rip2E) directed reporter expression in the PSM and
throughout posterior somites (Fig.
5A2), whereas expression from the promoter element (Rip2P) was
largely restricted to a stripe in the anterior PSM and the anterior halves of
posterior somites (Fig. 5A3).
We conclude that these fragments contain cis-acting regulatory
elements sufficient to drive appropriate Ripply2 expression in the
anterior PSM in vivo.
Since Ripply2 expression overlapped with the expression of several
components of the Notch pathway, we hypothesized that transient
Ripply2 expression in S-I is activated by Notch signaling. To address
this, we asked whether the activated Notch intracellular domain (NICD), or
transcription factors such as Tbx6 or Mesp2 that function downstream or in
parallel with Notch (Yasuhiko et al.,
2006), could activate the Ripply2 regulatory elements in
luciferase reporter assays in vitro. Although NICD (not shown) or Mesp2 had
minimal activity on Ripply2 regulatory elements, Tbx6 activated the
promoter (Fig. 5B2), and
strongly stimulated the combined enhancer and promoter elements 26-fold
(Fig. 5B1). Expression of T had
similar activity to Tbx6 (not shown). Coexpression of NICD and Tbx6 resulted
in a modest increase in transcriptional activation of the Ripply2
promoter (not shown); however, strong synergistic activation was observed when
Tbx6 and Mesp2 were coexpressed (Fig.
5B1,2). Since the same qualitative effect was observed with the
Ripply2 promoter or the combined promoter/enhancer, subsequent
experiments focused on the promoter.
An E-box, capable of binding Mesp2
(Nakajima et al., 2006), and a
near-consensus putative Tbx6 binding site (BS)
(White and Chapman, 2005), are
conserved in the proximal mouse and human Ripply2 promoters
(Fig. 5C). Electrophoretic
mobility shift assays (EMSA) confirmed that the putative Tbx6 BS specifically
bound FLAG-Tbx6 (Fig. 5D). A 44
bp deletion that removed the E-box and Tbx6 BS (Rip2PTbx6luc)
strongly diminished the ability of Tbx6, or Tbx6 and Mesp2 together, to
activate the Ripply2 promoter
(Fig. 5B2,3). Multimerizing the
44 bp region was sufficient to restore the synergistic activation of
Ripply2 by Tbx6 and Mesp2 to levels observed in the full promoter
construct (Fig. 5B4). Since
Tbx6 and Mesp2 expression overlaps only in S-I
(Yasuhiko et al., 2006), we
suggest that they function together to control the periodic activation of
Ripply2 in S-I.
Our genetic analyses suggest that elevated Wnt signaling represses Ripply2. To test whether Wnt signaling can suppress the ability of Tbx6 and Mesp2 to activate the Ripply2 promoter, we cotransfected stabilized β-catenin with Tbx6 and Mesp2 and assessed luciferase activity. Consistent with the in vivo data, expression of activated β-catenin reduced Mesp2/Tbx6-mediated activation of the enhancer/promoter and the promoter construct (Fig. 5B).
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). This is
supported by recent studies identifying additional cyclic Wnt target genes
(Dequeant et al., 2006).
Although our genetic studies demonstrate that Wnt3a and β-catenin are
necessary in the PSM for oscillating gene expression in the Wnt, Fgf and Notch
pathways, the continued periodic expression of Notch target genes in the
presence of a stabilized form of β-catenin that is refractory to
proteolytic degradation does not support a role for β-catenin as a core
component of the oscillator. Moreover, we can find little evidence for striped
expression of Wnt/β-catenin reporters in wild-type PSM, either at the
protein or RNA level (Nakaya et al.,
2005) (not shown), suggesting that β-catenin activity itself
does not oscillate. Since only a small subset of Wnt/β-catenin target
genes oscillate, out of a much larger number of target genes
(Dequeant et al., 2006;
Lickert et al., 2005;
Morkel et al., 2003) (our
unpublished data), it appears that additional regulatory inputs are required
for the oscillation of select Wnt target genes. The functional significance of
oscillating Wnt target gene expression is not currently well understood. We
conclude that the Wnt3a/β-catenin pathway plays a permissive, and not
instructive, role in the regulation of oscillating clock genes.
|
). However, normal segmentation of the anterior-most 7-10
somites is a feature common to virtually all Notch pathway mutants
(Rida et al., 2004), including
Mesp2 mutants (Saga et al.,
1997). These observations are difficult to explain if Notch
signaling drives the segmentation clock. Studies of zebrafish segmentation
have led to the alternative proposal that Notch functions to synchronize
oscillations between neighboring PSM cells
(Horikawa et al., 2006;
Jiang et al., 2000). This
hypothesis nicely accounts for the normal formation of the anterior somites in
embryos lacking Notch pathway activity because it does not require Notch to
drive the oscillator. Our observation that segmental Uncx4.1
expression becomes progressively indistinct and fused posteriorly in both LOF
and GOF Ctnnb1 mutants, is consistent with Notch functioning in the
coupling of oscillators. The fact that partial segment boundaries and
polarized segmental gene expression occur in the
T-Cre;Ctnnb1flLOF/ mutants, despite the absence of
oscillating gene expression in the Wnt or Fgf/Notch pathways, suggests that
boundary formation in the anterior PSM can proceed independently of
oscillating gene expression in the posterior PSM. Alternatively, it remains
possible that an additional component(s) of an oscillating clock mechanism
remains to be discovered, although this seems unlikely given the recent
comprehensive, genome-wide survey of oscillating gene expression in the PSM
(Dequeant et al., 2006).
Examination of Mesp2 and Ripply2 expression in the
Wnt3a and Ctnnb1 mutants demonstrates that
Wnt3a/β-catenin signaling regulates segment boundary determination. Mesp2
is known to participate in this process
(Morimoto et al., 2005;
Nomura-Kitabayashi et al.,
2002; Saga et al.,
1997; Takahashi et al.,
2003; Takahashi et al.,
2000). The elongated PSM, segmentation defects, ectopic
Mesp2 expression and complete absence of Ripply2 transcripts
in the T-Cre;Ctnnb1flGOF/+ embryos are consistent with the
segmentation phenotype and ectopic mesp-b expression observed in
zebrafish Ripply1 knockdowns
(Kawamura et al., 2005).
Interestingly, Ripply1 mRNA overexpression also caused segmentation
defects, and downregulated mesp-b expression in a Groucho-dependent
manner, suggesting that Ripply1 represses mesp-b transcription.
Similar results have been obtained with Xenopus Ripply orthologs
(Chan et al., 2006;
Kondow et al., 2006). Our
demonstration that Ripply2 expression follows Mesp2
expression in S-I is consistent with a role for Mesp2 in the activation of
Ripply2, as well as a reciprocal role for Ripply2 in the repression
of Mesp2. The synergistic activation of the Ripply2 promoter
by Mesp2 and Tbx6 indicates a direct role for Mesp2 in Ripply2
activation. This is supported by recent complementary studies, which
demonstrated that Mesp2 alone can directly bind and activate the
Ripply2 enhancer (Morimoto et
al., 2007). Moreover, analyses of Ripply2 null mutants
are consistent with Ripply2 negatively regulating Mesp2. Together,
the data strongly suggest that segment boundary determination is regulated by
an Mesp2-centered negative feedback loop in which Mesp2 and Tbx6 activate
Ripply2, and Ripply2, in turn, represses Mesp2.
Although segment boundaries form in conditional T-Cre;Ctnnb1
mutants, the small somites appear incompletely epithelialized.
Wnt/β-catenin signaling has been implicated in the control of somite
epithelialization in chick through the activation of Paraxis
(Linker et al., 2005).
Paraxis expression is unaffected by the
T-Cre;Ctnnb1flLOF/ mutation (not shown), indicating
that the epithelial defects are not dependent upon Paraxis. In
addition to the regulation of Wnt target gene transcription, β-catenin
has a well-characterized role in cell adhesion
(Nelson and Nusse, 2004).
Future studies will address the potential role that β-catenin, localized
to adherens junctions, may play in somite epithelialization.
Previous studies have implicated graded Fgf8 signals in the positioning of
the boundary determination front (Dubrulle
et al., 2001), and have suggested that Fgf8 functions downstream
of Wnt3a (Aulehla et al.,
2003). Our data are consistent with these suggestions, because the
Fgf8 gradient is modestly reduced in Wnt3a mutants (not
shown), absent in the Ctnnb1 LOF embryos and greatly expanded in the
Ctnnb1 GOF mutants. Interestingly, Fgf8 mRNA is still
expressed as a gradient in the T-Cre;Ctnnb1flGOF/+ PSM
(Fig. 2I). Since the
stabilization of β-catenin in these mutants anteriorly extended the
Fgf8 gradient but had little to no effect upon the gradient itself,
we conclude that Wnt/β-catenin signaling is not instructive for
establishment of the Fgf8 gradient and therefore indirectly controls
Fgf8 expression in the streak. Although conditional T-Cre;
Fgf8fl/ mutants do not display segmentation phenotypes
(Perantoni et al., 2005), the
expression of other Fgfs presumably compensate. Experiments designed
to test Fgf redundancy during PSM specification and somitogenesis are
ongoing.
Wnt3a-dependent transcriptional networks coordinate mesoderm formation and segmentation
Our data suggest that Wnt3a controls posterior development by stimulating
the canonical β-catenin/Tcf pathway in multipotent PS stem cells,
initiating a cascade of gene expression that links mesoderm fate specification
to the oscillatory segmentation clock and segment boundary formation
(Fig. 6). The
Wnt3a/β-catenin target genes Dll1, T/Tbx6, and additional
unidentified target genes are critical for understanding how the
Wnt/β-catenin signaling pathway regulates the spatiotemporal expression
of segment boundary determination genes. Wnt3a activates the Tbox
transcription factor gene T in the PS
(Yamaguchi et al., 1999).
T, together with the Notch pathway
(White et al., 2005), in turn
activates Tbx6 in the PS and PSM
(Hofmann et al., 2004) to
specify mesoderm fates. Wnt3a and Tbx6 then synergistically activate the Notch
ligand Dll1 in the PS and PSM
(Galceran et al., 2004;
Hofmann et al., 2004), to
define a PSM domain that is permissive for oscillating gene expression in the
Notch pathway. Tbx6 functioning together with the activated Notch pathway
activates the segment boundary determination gene Mesp2 in the
anterior PSM (Yasuhiko et al.,
2006). We show here that Tbx6 and Mesp2 subsequently activate
Ripply2 in the anterior PSM. Ripply2 then functions as a feedback
suppressor, bound to the transcriptional repressor Groucho, to repress
Mesp2 (Kawamura et al.,
2005; Morimoto et al.,
2007). Thus Tbx6 emerges as a major regulator of posterior
development downstream of Wnt3a, functioning to integrate the Wnt and Notch
pathways during boundary formation.
Additionally, we have shown that stabilized Wnt/β-catenin signaling anteriorly extends the Tbx6 and Dll1-positive PSM, allowing additional cycles of the segmentation clock and ectopic activation of Mesp2. Despite the elevated Tbx6 and Mesp2 expression, stabilized β-catenin signaling repressed Ripply2. Since Wnt/β-catenin signaling activates target gene transcription, we suggest that Wnt signaling is indirectly repressing the Mesp2/Tbx6-mediated activation of Ripply2 by activating a currently unknown transcriptional repressor. Repression of Ripply2 in posterior PSM presumably ensures that the segmentation program initiates only in the anterior PSM. Future studies will address the nature of this unknown Wnt target gene(s). Given the demonstrated links between Wnt3a and mesoderm formation, segmentation, left-right and AP patterning, and axial elongation, we suggest that Wnt3a is the principal organizer of mammalian trunk and tail development.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/1/85/DC1
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Arnold, S. J., Stappert, J., Bauer, A., Kispert, A., Herrmann, B. G. and Kemler, R. (2000). Brachyury is a target gene of the Wnt/beta-catenin signaling pathway. Mech. Dev. 91,249 -258.[CrossRef][Medline]
Aulehla, A. and Herrmann, B. G. (2004).
Segmentation in vertebrates: clock and gradient finally joined.
Genes Dev. 18,2060
-2067.
Aulehla, A., Wehrle, C., Brand-Saberi, B., Kemler, R., Gossler, A., Kanzler, B. and Herrmann, B. G. (2003). Wnt3a plays a major role in the segmentation clock controlling somitogenesis. Dev. Cell 4,395 -406.[CrossRef][Medline]
Baugh, L. R., Hill, A. A., Slonim, D. K., Brown, E. L. and
Hunter, C. P. (2003). Composition and dynamics of the
Caenorhabditis elegans early embryonic transcriptome.
Development 130,889
-900.
Bessho, Y., Sakata, R., Komatsu, S., Shiota, K., Yamada, S. and
Kageyama, R. (2001). Dynamic expression and essential
functions of Hes7 in somite segmentation. Genes Dev.
15,2642
-2647.
Bessho, Y., Hirata, H., Masamizu, Y. and Kageyama, R.
(2003). Periodic repression by the bHLH factor Hes7 is an
essential mechanism for the somite segmentation clock. Genes
Dev. 17,1451
-1456.
Biris, K. K., Dunty, W. C., Jr and Yamaguchi, T. P. (2007). Mouse Ripply2 is downstream of Wnt3a and is dynamically expressed during somitogenesis. Dev. Dyn. 236,3167 -3172.[CrossRef][Medline]
Brault, V., Moore, R., Kutsch, S., Ishibashi, M., Rowitch, D. H., McMahon, A. P., Sommer, L., Boussadia, O. and Kemler, R. (2001). Inactivation of the beta-catenin gene by Wnt1-Cre-mediated deletion results in dramatic brain malformation and failure of craniofacial development. Development 128,1253 -1264.[Abstract]
Chan, T., Satow, R., Kitagawa, H., Kato, S. and Asashima, M. (2006). Ledgerline, a novel Xenopus laevis gene, regulates differentiation of presomitic mesoderm during somitogenesis. Zool. Sci. 23,689 -697.[CrossRef][Medline]
Chapman, D. L. and Papaioannou, V. E. (1998). Three neural tubes in mouse embryos with mutations in the T-box gene Tbx6. Nature 391,695 -697.[CrossRef][Medline]
Correia, K. M. and Conlon, R. A. (2000). Surface ectoderm is necessary for the morphogenesis of somites. Mech. Dev. 91,19 -30.[CrossRef][Medline]
Dequeant, M. L., Glynn, E., Gaudenz, K., Wahl, M., Chen, J.,
Mushegian, A. and Pourquie, O. (2006). A complex oscillating
network of signaling genes underlies the mouse segmentation clock.
Science 314,1595
-1598.
Dubrulle, J. and Pourquie, O. (2004). Coupling
segmentation to axis formation. Development
131,5783
-5793.
Dubrulle, J., McGrew, M. J. and Pourquie, O. (2001). FGF signaling controls somite boundary position and regulates segmentation clock control of spatiotemporal Hox gene activation. Cell 106,219 -232.[CrossRef][Medline]
Evrard, Y. A., Lun, Y., Aulehla, A., Gan, L. and Johnson, R. L. (1998). lunatic fringe is an essential mediator of somite segmentation and patterning. Nature 394,377 -381.[CrossRef][Medline]
Galceran, J., Hsu, S. C. and Grosschedl, R.
(2001). Rescue of a Wnt mutation by an activated form of LEF-1:
regulation of maintenance but not initiation of Brachyury expression.
Proc. Natl. Acad. Sci. USA
98,8668
-8673.
Galceran, J., Sustmann, C., Hsu, S. C., Folberth, S. and
Grosschedl, R. (2004). LEF1-mediated regulation of
Delta-like1 links Wnt and Notch signaling in somitogenesis. Genes
Dev. 18,2718
-2723.
Haegel, H., Larue, L., Ohsugi, M., Fedorov, L., Herrenknecht, K. and Kemler, R. (1995). Lack of beta-catenin affects mouse development at gastrulation. Development 121,3529 -3537.[Abstract]
Harada, N., Tamai, Y., Ishikawa, T., Sauer, B., Takaku, K., Oshima, M. and Taketo, M. M. (1999). Intestinal polyposis in mice with a dominant stable mutation of the beta-catenin gene. EMBO J. 18,5931 -5942.[CrossRef][Medline]
Herrmann, B. G., Labeit, S., Poustka, A., King, T. R. and Lehrach, H. (1990). Cloning of the T gene required in mesoderm formation in the mouse. Nature 343,617 -622.[CrossRef][Medline]
Hofmann, M., Schuster-Gossler, K., Watabe-Rudolph, M., Aulehla,
A., Herrmann, B. G. and Gossler, A. (2004). WNT signaling, in
synergy with T/TBX6, controls Notch signaling by regulating Dll1 expression in
the presomitic mesoderm of mouse embryos. Genes Dev.
18,2712
-2717.
Horikawa, K., Ishimatsu, K., Yoshimoto, E., Kondo, S. and Takeda, H. (2006). Noise-resistant and synchronized oscillation of the segmentation clock. Nature 441,719 -723.[CrossRef][Medline]
Hrabe de Angelis, M., McIntyre, J., 2nd and Gossler, A. (1997). Maintenance of somite borders in mice requires the Delta homologue DII1. Nature 386,717 -721.[CrossRef][Medline]
Huelsken, J., Vogel, R., Brinkmann, V., Erdmann, B., Birchmeier,
C. and Birchmeier, W. (2000). Requirement for beta-catenin in
anterior-posterior axis formation in mice. J. Cell
Biol. 148,567
-578.
Ishikawa, A., Kitajima, S., Takahashi, Y., Kokubo, H., Kanno, J., Inoue, T. and Saga, Y. (2004). Mouse Nkd1, a Wnt antagonist, exhibits oscillatory gene expression in the PSM under the control of Notch signaling. Mech. Dev. 121,1443 -1453.[CrossRef][Medline]
Jiang, Y. J., Aerne, B. L., Smithers, L., Haddon, C., Ish-Horowicz, D. and Lewis, J. (2000). Notch signalling and the synchronization of the somite segmentation clock. Nature 408,475 -479.[CrossRef][Medline]
Kawamura, A., Koshida, S., Hijikata, H., Ohbayashi, A., Kondoh, H. and Takada, S. (2005). Groucho-associated transcriptional repressor ripply1 is required for proper transition from the presomitic mesoderm to somites. Dev. Cell 9, 735-744.[CrossRef][Medline]
Kondow, A., Hitachi, K., Ikegame, T. and Asashima, M. (2006). Bowline, a novel protein localized to the presomitic mesoderm, interacts with Groucho/TLE in Xenopus. Int. J. Dev. Biol. 50,473 -479.[Medline]
Kraus, F., Haenig, B. and Kispert, A. (2001). Cloning and expression analysis of the mouse T-box gene Tbx18. Mech. Dev. 100,83 -86.[CrossRef][Medline]
Li, Q., Ishikawa, T. O., Miyoshi, H., Oshima, M. and Taketo, M.
M. (2005). A targeted mutation of Nkd1 impairs mouse
spermatogenesis. J. Biol. Chem.
280,2831
-2839.
Lickert, H., Cox, B., Wehrle, C., Taketo, M. M., Kemler, R. and
Rossant, J. (2005). Dissecting Wnt/beta-catenin signaling
during gastrulation using RNA interference in mouse embryos.
Development 132,2599
-2609.
Linker, C., Lesbros, C., Gros, J., Burrus, L. W., Rawls, A. and
Marcelle, C. (2005). beta-Catenin-dependent Wnt signalling
controls the epithelial organisation of somites through the activation of
paraxis. Development
132,3895
-3905.
Mansouri, A., Yokota, Y., Wehr, R., Copeland, N. G., Jenkins, N. A. and Gruss, P. (1997). Paired-related murine homeobox gene expressed in the developing sclerotome, kidney, and nervous system. Dev. Dyn. 210,53 -65.[CrossRef][Medline]
Morimoto, M., Takahashi, Y., Endo, M. and Saga, Y. (2005). The Mesp2 transcription factor establishes segmental borders by suppressing Notch activity. Nature 435,354 -359.[CrossRef][Medline]
Morimoto, M., Sasaki, N., Oginuma, M., Kiso, M., Igarashi, K.,
Aizaki, K., Kanno, J. and Saga, Y. (2007). The negative
regulation of Mesp2 by mouse Ripply2 is required to establish the
rostro-caudal patterning within a somite. Development
134,1561
-1569.
Morkel, M., Huelsken, J., Wakamiya, M., Ding, J., van de
Wetering, M., Clevers, H., Taketo, M. M., Behringer, R. R., Shen, M. M. and
Birchmeier, W. (2003). Beta-catenin regulates Cripto- and
Wnt3-dependent gene expression programs in mouse axis and mesoderm formation.
Development 130,6283
-6294.
Nakajima, Y., Morimoto, M., Takahashi, Y., Koseki, H. and Saga,
Y. (2006). Identification of Epha4 enhancer required for
segmental expression and the regulation by Mesp2.
Development 133,2517
-2525.
Nakaya, M. A., Biris, K., Tsukiyama, T., Jaime, S., Rawls, J. A.
and Yamaguchi, T. P. (2005). Wnt3a links left-right
determination with segmentation and anteroposterior axis elongation.
Development 132,5425
-5436.
Nelson, W. J. and Nusse, R. (2004). Convergence
of Wnt, beta-catenin, and cadherin pathways. Science
303,1483
-1487.
Nomura-Kitabayashi, A., Takahashi, Y., Kitajima, S., Inoue, T., Takeda, H. and Saga, Y. (2002). Hypomorphic Mesp allele distinguishes establishment of rostrocaudal polarity and segment border formation in somitogenesis. Development 129,2473 -2481.[Medline]
Perantoni, A. O., Timofeeva, O., Naillat, F., Richman, C.,
Pajni-Underwood, S., Wilson, C., Vainio, S., Dove, L. F. and Lewandoski,
M. (2005). Inactivation of FGF8 in early mesoderm reveals an
essential role in kidney development. Development
132,3859
-3871.
Pourquie, O. (2001). Vertebrate somitogenesis. Annu. Rev. Cell Dev. Biol. 17,311 -350.[CrossRef][Medline]
Pourquie, O. (2003). The segmentation clock:
converting embryonic time into spatial pattern.
Science 301,328
-330.
Rida, P. C., Le Minh, N. and Jiang, Y. J. (2004). A Notch feeling of somite segmentation and beyond. Dev. Biol. 265,2 -22.[CrossRef][Medline]
Saga, Y. and Takeda, H. (2001). The making of the somite: molecular events in vertebrate segmentation. Nat. Rev. Genet. 2,835 -845.[CrossRef][Medline]
Saga, Y., Hata, N., Koseki, H. and Taketo, M. M.
(1997). Mesp2: a novel mouse gene expressed in the presegmented
mesoderm and essential for segmentation initiation. Genes
Dev. 11,1827
-1839.
Stadeli, R., Hoffmans, R. and Basler, K. (2006). Transcription under the control of nuclear Arm/beta-catenin. Curr. Biol. 16,R378 -R385.[CrossRef][Medline]
Sun, X., Meyers, E. N., Lewandoski, M. and Martin, G. R.
(1999). Targeted disruption of Fgf8 causes failure of cell
migration in the gastrulating mouse embryo. Genes Dev.
13,1834
-1846.
Takada, S., Stark, K. L., Shea, M. J., Vassileva, G., McMahon,
J. A. and McMahon, A. P. (1994). Wnt-3a regulates somite and
tailbud formation in the mouse embryo. Genes Dev.
8, 174-189.
Takahashi, Y., Koizumi, K., Takagi, A., Kitajima, S., Inoue, T., Koseki, H. and Saga, Y. (2000). Mesp2 initiates somite segmentation through the Notch signalling pathway. Nat. Genet. 25,390 -396.[CrossRef][Medline]
Takahashi, Y., Inoue, T., Gossler, A. and Saga, Y.
(2003). Feedback loops comprising Dll1, Dll3 and Mesp2, and
differential involvement of Psen1 are essential for rostrocaudal patterning of
somites. Development
130,4259
-4268.
Tam, P. P. and Beddington, R. S. (1987). The formation of mesodermal tissues in the mouse embryo during gastrulation and early organogenesis. Development 99,109 -126.[Abstract]
White, P. H. and Chapman, D. L. (2005). Dll1 is a downstream target of Tbx6 in the paraxial mesoderm. Genesis 42,193 -202.[CrossRef][Medline]
White, P. H., Farkas, D. R. and Chapman, D. L. (2005). Regulation of Tbx6 expression by Notch signaling. Genesis 4
5 ss
wild-type (D-F), T-Cre;Ctnnb1flLOF/
