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First published online 8 February 2006
doi: 10.1242/dev.02274
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1 Vertebrate Body Plan, Center for Developmental Biology, RIKEN Kobe,
Minatojima-Minami, Chuou-ku, Kobe 650-0047, Japan.
2 The Graduate School of Biological Sciences, Nara Institute of Science and
Technology, 8916-5 Takayama, Ikoma, Nara, 630-0101, Japan.
3 Applied Biological Science, Faculty of Science and Technology, Tokyo
University of Science, 641 Yamazaki, Noda-shi, Chiba 278-8510, Japan.
4 Kuju Agricultural Research Center, Kyushu University Graduate School of
Agriculture, Naoiri-gun Kuju-cho 878-0201, Japan.
* Author for correspondence (e-mail: ashimono{at}cdb.riken.jp)
Accepted 4 January 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Sfrp1, Sfrp2, Wnt signaling, Wnt antagonists, Embryonic patterning, Somitogenesis
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Perea-Gomez et al., 2002).
Wnt, a family of secreted glycoproteins, stimulates signals through the
seven transmembrane receptors of Frizzled
(Kawano and Kypta, 2003). Wnt
interactions with Frizzled and the co-receptor Lrp5/Lrp6 activate the
canonical Wnt/ß-catenin pathway, which leads to the stabilization of
ß-catenin as a transcriptional regulator in the nucleus
(Kawano and Kypta, 2003;
Kelly et al., 2004). A
proportion of Wnt molecules activate the non-canonical planar cell polarity
(PCP) pathways, which results in activation of Rho GTPases, or the
Wnt/Ca2+ pathway, which leads to intercellular Ca2+
release, and PKC and CamKII activation
(Kühl, 2002). Nineteen
Wnt genes have been identified in the human and murine genomes. Studies of
loss-of function mutations in the mouse, and knockdown or overexpression in
Xenopus, chicken and zebrafish, have revealed that Wnt proteins play
diverse roles during embryogenesis in vertebrates. Wnt signaling is required
for primitive streak formation (Liu et
al., 1999), mesoderm cell movement
(Heisenberg et al., 2000;
Ulrich et al., 2003),
generation of the tail organizer (Agathon
et al., 2003), posterior patterning and somitogenesis
(Takada et al., 1994;
Aulehla et al., 2003) (see also
http://www.stanford.edu/~rnusse/wntwindow.html).
Wnt signaling is inhibited by several secreted antagonists: Dkk, Wise, Wif
and Sfrp (Kawano and Kypta,
2003; Yamaguchi,
2001). Dickkopf (Dkk) and Wnt modulator in surface ectoderm (Wise)
interact with co-receptor Lrp6, leading to the inhibition of active
ligand-receptor complex formation
(Semënov et al., 2001;
Itasaki et al., 2003).
Secreted frizzled-related protein (Sfrp) and Wnt inhibiting factor (Wif)
possess a region related to the cysteine-rich domain (CRD) of Frizzled that
interacts with the Wnt ligand (Hsieh et
al., 1999; Kawano and Kypta,
2003). This inhibition of the ligand might regulate the degree of
active Wnt in tissues (i.e. regulation of ligand activity and gradient
generation). Regulation of Wnt signaling by secreted antagonists has been
implicated in anterior embryonic patterning. During mouse embryogenesis,
inhibition of Wnt activity by Dkk1 is necessary for anterior head
specification and limb patterning
(Mukhopadhyay et al., 2001).
In Xenopus, overexpression experiments revealed that Dkk1 and FrzB (a
member of the Sfrps) antagonize Wnt in the Spemann organizer, which is
required for head organizer activity
(Glinka et al., 1997;
Leyns et al., 1997;
Wang et al., 1997;
Niehrs, 1999).
The Sfrp gene family consists of five members in both the human and mouse
genomes (Kawano and Kypta,
2003), which are divided into Sfrp1 and FrzB subfamilies based on
amino acid sequence similarity. In addition, Crescent and
Sizzled are unique members found in Xenopus and zebrafish.
Sfrp1, Sfrp2 and Sfrp5 belong to the Sfrp1 subfamily
(Jones and Jomary, 2002),
whose members share homology in the CRD domain. Consequently, Sfrp1, Sfrp2 and
Sfrp5 suppress the canonical Wnt/ß-catenin signal, decrease
ß-catenin levels and downregulate target gene expression, such as
Myc expression, in cultured cells
(Suzuki et al., 2004). Tlc,
the zebrafish ortholog of Sfrp1 and Sfrp5, antagonizes Wnt8b in order to
establish the telencephalon (Houart et
al., 2002). In vitro data and Xenopus experiments suggest
that Sfrp1 (FrzA) interacts with Wnt1, Xwnt8 and Wnt2, but not with Wnt5a
(Xu et al., 1998;
Dennis et al., 1999).
Furthermore, Sfrp1 also interacts with Wnt7b, and is capable of attenuating
the non-canonical Wnt signaling pathway, leading to inhibition of axon
guidance (Rosso et al., 2005).
Thus, Sfrps exhibit a wide spectrum of functions with respect to regulation of
the activity of Wnt proteins in both the canonical and non-canonical pathways.
However, the functions of the Sfrps in embryogenesis have not been fully
elucidated.
In this study, Sfrp1 and Sfrp2 double mutant mice were generated to reveal the functions of Sfrp1 subfamily members during embryogenesis. The findings demonstrate that Sfrp1 and Sfrp2 exhibit highly redundant functions during embryogenesis. Furthermore, the results suggest that regulation of Wnt signaling by Sfrp1 and Sfrp2 is required for normal AP axis elongation and somitogenesis.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
Immunostaining
For whole embryo immunostaining against neurofilament, embryos were fixed
in 3.5% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) at 4°C
overnight. The embryos were hydrated in an ascending methanol series (25, 50,
75, 100%) in PBS containing 0.1% Tween-20 (PBT) before being re-hydrated in a
descending methanol series (75, 50, 25, 0%). Following treatment with PBT
containing 6% H2O2 and a wash in Tris-buffered saline
containing 0.1% Tween-20 (TBST), embryos were treated for blocking in TBST
containing 10% sheep serum. The embryos were incubated with a 2H3 monoclonal
neurofilament antibody (supernatant, 1/50 dilution) at 4°C overnight.
Embryos were then incubated with anti-mouse IgG antibody conjugated with HRP
(1/1000 dilution). Immunoreactivity was detected with diaminobenzidine.
Antibody staining against diphosphorylated ERK was conducted according to a
previous report (Corson et al.,
2003).
To detect non-phospho ß-catenin, embryos were fixed in 8% PFA in PBS.
Cryosections were subsequently generated according to a general protocol
(Wakamatsu et al., 1993).
Primary antibody against non-phospho ß-catenin (Upstate; mouse monoclonal
8E4) was used at a 1:400 dilution, with TBST containing 5% skim milk.
Immunoreactivity was detected with Alexa488-conjugated anti-mouse IgG antibody
(Molecular Probes) diluted 1:200 with TBST containing 3% BSA. Images were
captured on a BioRad Radiance 2100 Laser Scanning Confocal Microscope System
equipped with a Zeiss Axiovert, and processed with Adobe Photoshop CS.
In situ hybridization
In situ hybridization using digoxigenin (DIG)-labeled riboprobes was
performed on whole-mount embryos according to Wilkinson
(Wilkinson, 1992). Some
embryos were processed to generate cryosections. Double whole-mount in situ
hybridizations were conducted according to Wilkinson
(Wilkinson, 1992), with
modifications, using two differentially labeled probes: one labeled with DIG
and the other with fluorescein (FITC). After hybridization using two different
probes at same time, DIG-labeled RNA probe was detected with alkaline
phosphatase-conjugated anti-DIG antibody in color solution containing NBT
(Nitro-Blue Tetrazolium Chloride)/BCIP (5-Bromo-4-chloro-3-indolyl phosphate,
toluidine salt). Following the primary detection procedure, the antibody was
removed by washing with 0.1 M Glycine-HCl containing 0.1% Tween-20. A
secondary detection procedure was then performed with an alkaline
phosphatase-conjugated anti-FITC antibody in INT
[2-(4-Iodophenyl)-3-(4-nitrophenyl)-5-phenyltetrazolium chloride]/BCIP
solution.
Hoxa7 (BC036986) and Hoxd10 (BC048690) cDNA were obtained as IMAGE clones from Invitrogen. Axin2 (AK084644), Fgf17 (AK077555) and Nkd1 (AK082367) cDNA were obtained as FANTOM clones (RIKEN). A 1.5-kb fragment containing the 3' UTR of Hoxb2 cDNA was isolated from the E8.5 cDNA library (a gift from Dr H. Hamada, Osaka University). Images were captured with a Pixera Pro600ES digital camera, with a Zeiss Stemi2000-C stereomicroscope.
DiI cell labeling, whole-embryo and tail-bud culture
Whole-embryo culture and DiI
(1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine
perchlorate) labeling were performed according to the method of Shimono and
Behringer (Shimono and Behringer,
2003). DiI was injected at the primitive streak region in the tail
bud in order to label mesoderm cells. Labeled cells within the endoderm layer
and the neural plate did not spread during the shorter culture period.
Mesoderm cell labeling was also confirmed in cryosections of the cultured
embryos. Labeled embryos, which were selected based on a lateral view of the
labeling of three germ layers, were cultured for two hours. The images were
captured with a Zeiss AxioCam HRc digital camera with a Leica MZFLIII
fluorescence stereomicroscope. The culture was extended up to 22 hours to
confirm a contribution of the labeled cells to the pre-somitic mesoderm (PSM).
Embryos displaying labeled PSM cells were adopted (n). Tail-bud
culture was performed based on the method of Correia and Conlon
(Correia and Conlon,
2000).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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)
to generate conventional knockout mice (see Fig. S1D,E in the supplementary
material). Following germline transmission, mutant pups were obtained from the
intercross of heterozygous mutant mice with 129 and C57BL/6 mixed backgrounds.
Genotypes of the pups were determined by Southern hybridization or PCR
amplification at weaning, suggesting that mice carrying the homozygous
mutation in Sfrp1 (Sfrp1-/-) appeared normal
during embryogenesis (see Fig. S1C, Table S1 in the supplementary material)
(Bodine et al., 2004).
Similarly, most of the Sfrp2 homozygous mutant
(Sfrp2-/-) mice appeared normal and healthy (Fig. S1F,
Table S1 in the supplementary material), although hindlimb syndactyly occurred
at a lower frequency (3%; two out of 62 Sfrp2-/- pups).
The deleted exons encode the first methionine and the functional domain of the
protein. As a result, the mutant mice were regarded as functionally null. In
addition, expression of the transcript from the mutated alleles was
undetectable (data not shown).
Sfrp1 and Sfrp2 genes are functionally redundant
Functional redundancy between Sfrp1 and Sfrp2 has been
suggested on the basis of the similarity of their expression patterns during
embryogenesis. Overlapping expression of Sfrp1 and Sfrp2 is
evident in the forebrain, midbrain and hindbrain region and in the posterior
neural plate/tube at embryonic day (E) 8.5 and E9.5
(Leimeister et al., 1998). A
different study reported overlapping expression in the PSM
(Lee et al., 2000). Therefore,
Sfrp1-/-;Sfrp2+/- mice, which appeared normal
and healthy, were intercrossed to generate Sfrp1 and Sfrp2
double homozygous mutant mice
(Sfrp1-/-;Sfrp2-/-). No
Sfrp1-/-;Sfrp2-/- pups were recovered,
suggesting pre-natal lethality of this mutation (see Tables S1, S2 in the
supplementary material). Thus, Sfrp1 and Sfrp2 are
functionally redundant.
Sfrp1-/-;Sfrp2-/- embryos display severe shortening of the thoracic region
Sfrp1-/-;Sfrp2-/- embryos, which died at
around E16.5, exhibited edematous, craniofacial defects, limb outgrowth
defects and extra digits (Fig.
1A-E; see also Table S2 in the supplementary material). The limb
outgrowth defect is a typical phenotype induced by activated
Wnt/ß-catenin signaling, such as constitutively active ß-catenin
expression, Wnt overexpression and Wnt5a inactivation
(Topol et al., 2003;
Guo et al., 2004). Extra
digits were frequently observed in the anterior portion of the right hindlimb
(50% of embryos, n=16), but rarely in the left hindlimb
(Fig. 1F-I), a finding that was
correlated with extra stripes of Fgf8 expression domains on the
ventral surface of the hindlimb bud at E10.5
(Fig. 1J,K). Ectopic
Fgf8 expression has been associated with an upregulation of
Wnt/ß-catenin signaling in the apical ectodermal ridge during limb
morphogenesis (Mukhopaghyay at al.,
2001; Barrow et al.,
2003; Soshnikova et al.,
2003). Sfrp1 is expressed in the ventral body wall,
including the proximal region of the hindlimb bud, and Sfrp2 is
expressed in the limb mesenchyme at E10.5
(Leimeister et al., 1998).
Patterning in the forebrain, midbrain and hindbrain of
Sfrp1-/-;Sfrp2-/- embryos appeared normal up to
E9.5-E10.5. Although Sfrp1 and Sfrp2 are highly expressed in
the hindbrain (Leimeister et al.,
1998), rhombomere patterning was normal in
Sfrp1-/-;Sfrp2-/- embryos, as evidenced by
staining for Krox20 transcripts in rhombomeres 3 and 5, and
immunoreactivity staining with the 2H3 anti-neurofilament monoclonal antibody
(data not shown).
Gross morphology suggested that the entire AP body axis was shortened in
Sfrp1-/-;Sfrp2-/- embryos at E14.5-E16.5.
Moreover, cartilage staining revealed that the thoracic region was severely
shortened, and that the number of thoracic vertebrae was reduced from thirteen
to five (Fig. 1B-E). This
observation suggested that AP axis formation, especially in the thoracic
region, might be affected in Sfrp1-/-;Sfrp2-/-
embryos in a manner that correlates axis elongation with somite segmentation.
To elucidate the body axis at the marker level, expression of the following
Hox genes was examined at E9.25-E10.5: Hoxb2, whose anterior
expression boundary is located at rhombomere 3
(Rossel and Capecchi, 1999);
Hoxa7, which has an anterior boundary in the mesoderm at the
thirteenth somite (Li and Shiota,
1999); and Hoxd10, which is expressed up to the
twenty-seventh somite (Hérault et
al., 1998). Hoxb2 was expressed in the posterior region
from rhombomere 3 in Sfrp1-/-;Sfrp2-/- embryos,
as well as in the control (wild type,
Sfrp1-/-;Sfrp2+/+ and
Sfrp1-/-;Sfrp2+/-) embryos
(Fig. 2A,B). The anterior
boundary of Hoxa7 expression in the mesoderm was located around the
thirteenth somite in the Sfrp1-/-;Sfrp2-/-
embryos, a pattern similar to that of the control embryos at E9.25
(Fig. 2C,D). However, using
myogenin expression as the landmark in the double whole-mount in situ
hybridizations (Fig. 2E-H)
(Edmondson and Olson, 1989),
the expression boundary of Hoxd10 was shifted to the twenty-first
somite in Sfrp1-/-;Sfrp2-/- embryos at E10.5
(Fig. 2E,F). These observations
suggest that somites 11-23, which give rise to vertebrae in the thoracic
region, are fused, and/or reduced in number, during somite segmentation
(Fig. 2I).
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|
). Histological analysis showed randomized segmentation after
the eleventh somite in the region between the forelimb and the hindlimb
(Fig. 3C,D). This finding was
confirmed by marker analysis of Mox1
(Fig. 4A-D), which is expressed
in somites from the onset of segmentation and is gradually restricted to the
posterior half-somite of sclerotome lineage
(Candia et al., 1992), and of
Pax3 (Fig. 4E-H), a
marker for the dermatome lineage (Goulding
et al., 1991). Regular, albeit smaller, somites were generated in
a posterior region near the hindlimb, as well as around the forelimb level,
suggesting restoration of somite segmentation around the hindlimb level (Figs
3,
4). The somites maintained AP
identity within the region between the forelimb and the hindlimb, and the
abnormal somites expressed Uncx4.1 and Pax1, markers for the
sclerotome lineage (Mansouri et al.,
1997; Neubuser et al.,
1995) (Fig. 4I-K;
data not shown). Although myotome differentiation was delayed in the embryos,
as indicated by myogenin expression at E9.5
(Edmondson and Olson, 1989)
(Fig. 4L-N), these observations
reveal that cell differentiation occurs in somites with impaired segmentation.
Significantly, myogenin expression at E10.5 clearly demonstrated a reduced
number of somites between the forelimb and hindlimb in
Sfrp1-/-;Sfrp2-/- embryos (17 somites in
Sfrp1-/-;Sfrp2+/+ or
Sfrp1-/-;Sfrp2+/- embryos,
n=3; 13 somites in
Sfrp1-/-;Sfrp2-/- embryos,
n=3; Fig. 4R-T).
Moreover, the pre-somitic mesoderm (PSM) region was dramatically reduced in
the Sfrp1-/-;Sfrp2-/- embryos, as evidenced by
the expression pattern of the delta-like 1 gene (Dll1)
(Bettenhausen et al., 1995) at
E9.5 (Fig. 4O-Q). This
observation, in addition to the small somite around the forelimb level, is
suggestive of a defect in posterior embryonic patterning at an early stage.
The defect of posterior patterning was apparent as a reduction in the
posterior axis at E8.25, and appeared prior to small somite generation and
irregular and incomplete somite segmentation.
Sfrp1 and Sfrp2 are required for AP axis elongation in the thoracic region
Sfrp1-/-;Sfrp2-/- embryos were
indistinguishable from wild-type and control embryos at the late head-fold
stage. However, the Sfrp1-/-;Sfrp2-/- embryos,
which had begun to generate somites, were distinct from control embryos in the
posterior region in the increased thickness of the mesoderm layer (a bar in
the tail region, Fig. 5A-C).
The expression patterns of brachyury (T), Tbx6 and
Dll1 were examined in order to gain insight into the defect in
posterior axis extension. T expression in
Sfrp1-/-;Sfrp2-/- embryos suggested that the
primitive streak and the axial mesoderm (node and notochord) were generated
normally in the early somite stage (
three somites)
(Fig. 5A-C)
(Wilkinson et al., 1990).
Tbx6 and Dll1 were expressed in the PSM region (between the
arrow and arrowhead in Fig.
5D,E,G,H; data not shown) and in the paraxial mesoderm of the
primitive streak region (between the bar and arrow in
Fig. 5D,E,G,H; data not shown)
in wild-type and control embryos at E8.5
(Bettenhausen et al., 1995;
Chapman et al., 1996). In the
Sfrp1-/-;Sfrp2-/- embryos, Tbx6 and
Dll1 expression were observed with high intensity in the paraxial
mesoderm on both sides of the primitive streak (between the bar and arrow in
Fig. 5F,I; data not shown);
moreover, anterior extension of the expression domain was greatly reduced
(between the arrow and arrowhead in Fig.
5F,I; data not shown). Sections of embryos at somite stages 6/7
and 11 revealed that unusual Dll1 staining was predominantly due to
an increased number of paraxial mesoderm cells in the posterior embryonic
portion (Fig. 5K,L,N,O; data
not shown).
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), was not elevated in the posterior region of
Sfrp1-/-;Sfrp2-/- embryos in comparison with
control embryos (7.36±1.30% of anti-phospho-Histone H3-positive cells
in Sfrp1-/-;Sfrp2+/+ embryos; 6.48±1.63%
in Sfrp1-/-;Sfrp2-/- embryos; n=3) at
the 6 somite stage (data not shown). By contrast, the rate appeared to be
reduced in Sfrp1-/-;Sfrp2-/- embryos at the 11
somite stage (7.83±0.73% of anti-phospho-Histone H3-positive cells in
Sfrp1-/-;Sfrp2+/+ embryos; 3.76±0.88% in
Sfrp1-/-;Sfrp2-/- embryos; n=3; data
not shown). Thus, a reduction in cell proliferation may contribute to a
diminished posterior axis at a later stage. However, a reduction of the
posterior axis in conjunction with the increased thickness in the mesoderm
cell layer is evident in Sfrp1-/-;Sfrp2-/-
embryos at the end of the head-fold stage, when cell proliferation rate is
unaffected. Therefore, accumulation of Dll1-expressing cells appears
to be a consequence of cell migration defects in mesoderm cells along the AP
axis. Cell migration was directly evaluated via cell labeling with the lipophilic dye DiI. DiI was injected into the mesoderm cell population around the primitive streak region of control and Sfrp1-/-;Sfrp2-/- embryos. The DiI-labeled mesoderm cells migrated into the lateral region in the control embryos (Fig. 5P,Q; n=14). By contrast, migration of DiI-labeled mesoderm cells was greatly reduced in Sfrp1-/-;Sfrp2-/- embryos (Fig. 5R,S; n=5). Thus, Sfrp1 and Sfrp2 mediate posterior axis extension via regulation of cell migration in the paraxial mesoderm.
Wnt3a is expressed in the tail bud of developing embryos
(Yoshikawa et al., 1997).
Because Sfrp1 and Sfrp2 possess inhibitory activity against the Wnt pathway,
activation of Wnt signaling was examined in
Sfrp1-/-;Sfrp2-/- embryos at E8.5. Activation
of the Wnt/ß-catenin pathway was evaluated by antibody staining against
stabilized non-phospho ß-catenin. A higher staining intensity in the
cellular membrane and nucleus was observed in the hindgut and in the mesoderm
beneath the primitive streak in control embryos
(Fig. 6A,B,G-I). In
Sfrp1-/-;Sfrp2-/- embryos, a similar staining
intensity was ectopically detected in the tail bud region in the mesoderm and
neural ectoderm, as well as in the hindgut endoderm and the mesoderm beneath
the primitive streak (n=2; Fig.
6D-E,J-O). Thus, inactivation of Sfrp1 and Sfrp2
leads to activation of the Wnt pathway in the embryos.
Sfrp1 and Sfrp2 affect Notch oscillatory cycles in the PSM
Wnt and Fgf8 gradients play a role in the establishment of somite
segmentation boundaries (Aulehla et al.,
2003). A steeper gradient in a shorter PSM region could lead to
the generation of small somites. However, the shorter PSM is insufficient to
account for the randomized and incomplete segmentation in
Sfrp1-/-;Sfrp2-/- embryos. Coordinated somite
segmentation is regulated by the cyclic expression of Notch related genes,
such as Lfng and Hes7, in the PSM
(Saga and Takeda, 2001;
Bessho et al., 2001). Because
Wnt3a is required for oscillating Notch signaling activity in the PSM
(Aulehla et al., 2003), the
expression of Wnt3a in
Sfrp1-/-;Sfrp2-/- embryos was examined during
incomplete somite segmentation and following the restoration of regular somite
segmentation. The level of Wnt3a expression was normal in the tail
bud region of Sfrp1-/-;Sfrp2-/- embryos
displaying defective somite segmentation at E8.5 (see Fig. S2A-C in the
supplementary material). In addition, Fgf8 was normally expressed in
the tail bud of these embryos (see Fig. S2J-L' in the supplementary
material). Activation of Fgf signaling, visualized with an anti-diphospho-ERK
antibody (Corson et al.,
2003), was observed in posterior portion of the control embryos,
and showed reduced activation in anterior region of the PSM (see Fig. S2M,N in
the supplementary material). Similar staining patterns were observed in the
Sfrp1-/-;Sfrp2-/- embryos (Fig. S2O in the
supplementary material). However, Wnt3a expression decreased in the
tail bud at E9.5, when somite segmentation was restored (see Fig. S3J-L in the
supplementary material). In addition, Fgf8 expression was diminished
in the tail bud, which was consistent with the findings of a previous report
(Aulehla et al., 2003) (Fig.
S3G-I in the supplementary material). By contrast, other marker genes of the
tail bud region were normally expressed in
Sfrp1-/-;Sfrp2-/- embryos
(Maruoka et al., 1998;
Yamguchi et al., 1999a) (Fig. S3A-F,M-O in the supplementary material). Hence,
the defect is correlated with expression levels of Wnt3a. The
reduction of Wnt3a expression in the tail bud may reduce the
signaling activity elevated by Sfrp1 and Sfrp2 inactivation to levels that
have no effect on somite segmentation.
|
).
Therefore, expression of the Notch-related oscillator genes lunatic fringe
(Lfng) and Hes7 was examined in
Sfrp1-/-;Sfrp2-/- embryos at somite stages 10
to 13. Dynamic expression of Lfng, which is regulated by Notch
signaling, is required for the somite segmentation process
(Forsberg et al., 1998;
Evrard et al., 1998;
Cole et al., 2002;
Morales et al., 2002),which is
altered in Wnt3a mutants (Aulehla
et al., 2003). Oscillating expression of Lfng was
visualized via comparison between the half tail regions with and without
culture incubation; in addition, the expression pattern was precisely compared
between the explants according to Forsberg et al.
(Forsberg et al., 1998)
(Fig. 7G). All pairs of
explants derived from control embryos at E8.5 exhibited the oscillating
expression pattern (n=18; Fig.
7A,D,G). By contrast, the oscillations were perturbed in all pairs
of Sfrp1-/-;Sfrp2-/- tail explants
(n=7, Fig. 7B,C,E-G).
An extra stripe was observed in the anterior-most region of the PSM,
suggesting delayed somite segmentation (indicated by a dagger in
Fig. 7C,F; two out of seven
pairs of explants). Interestingly, cyclic expression of Lfng in the
Sfrp1-/-;Sfrp2-/- explants at E9.5 was
indistinguishable from that of control explants
(Fig. 7H,I; n=11 for
control, n=3 for Sfrp1-/-;Sfrp2-/-
explants). Consequently, the abnormal expression pattern of the oscillator
gene appears to be related to the defect in somite segmentation.
Hes7 expression is controlled by Notch signaling, and Hes7 protein
represses Lfng expression (Bessho
et al., 2001; Bessho et al.,
2003). Although normal oscillating cycles of Hes7 were
consistently observed in control explants
(Fig. 7J,M; n=6),
cyclic expression was affected in the
Sfrp1-/-;Sfrp2-/- embryos (three out of five
pairs of explants, Fig. 7L-O).
In a manner similar to that of Lfng expression, a strong extra stripe
frequently occurred in the anterior-most region of the PSM (indicated by a
dagger in Fig. 7L,N; two out of
five pairs of explants). Therefore, Sfrp1 and Sfrp2 affect
Notch oscillator cycles.
Axin2 and Nkd1, negative regulators of the Wnt pathway,
have been shown to exhibit an oscillatory expression pattern in the PSM
(Aulehla et al., 2003;
Ishikawa et al., 2004). On the
one hand, Dynamic expression of Axin2, a Wnt-driven oscillator
(Aulehla et al., 2003), was
observed in Sfrp1-/-;Sfrp2-/- embryo explants
(n=5), as well as in controls (n=19). On the other hand, the
majority of control explants (89%; total n=9) exhibited changes in
expression levels of Nkd1, an oscillator gene activated downstream of
Wnt signaling (Yan et al.,
2001) (Fig. 7S). By
contrast, most of the explants (75%; total n=8) derived from
Sfrp1-/-;Sfrp2-/- embryos retained higher
expression levels of Nkd1 (Fig.
7T,U). This observation indicates that the downstream target of
Wnt signaling is activated in the absence of Sfrp1 and
Sfrp2. In concert, the results derived from the explant cultures
suggest that inhibition of Wnt signaling by Sfrp1 and Sfrp2 is an essential
component of the somite segmentation process.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Sfrp1 and Sfrp2 possess redundant functions during embryonic development
It has been reported that Sfrp1 and Sfrp2 exert opposing effects on
ß-catenin stability in MCF-7 breast cancer cells: Sfrp1 decreased
ß-catenin stability, whereas Sfrp2 increased ß-catenin cellular
concentration (Melkonyan et al.,
1997). Moreover, Sfrp2 acts as an antagonist of Sfrp1 with respect
to Wnt inhibition (Yoshino et al.,
2001). The generation of mice that are null for both
Sfrp1 and Sfrp2 clearly reveals that these two proteins
compensate for one another during embryonic development. Overlapping
expression of the genes is observed from the early somite stage to the
organogenesis period. However, the expression pattern is not completely
coincident at later stages (Leimeister et
al., 1998). The fact that Sfrps are secreted antagonists, rather
than the differences between expression sites of the genes, could account for
the functional redundancy of Sfrp1 and Sfrp2.
|
). Sfrp1
is expressed in the anterior visceral endoderm (AVE) and the anterior
mesendoderm (AME), essential tissues for head formation in the mouse
(Shawlot et al., 1999),
whereas wider expression of Sfrp2 is detected in the embryonic
ectoderm at the primitive streak stage
(Mukhopadhyay et al., 2003).
Sfrp1 cDNA was isolated in a screen of differentially expressed genes
in wild-type and Lim1 (also referred as to Lhx1) mutant
AVE/AME at the late streak stage (Shimono
and Behringer, 1999; Shimono
and Behringer, 2000). Lim1 is necessary for head
organizer activity (Shawlot and Behringer,
1995; Shawlot et al.,
1999). In addition, Sfrp1 expression is reduced in the
AVE and AME of Lim1 homozygous mutant embryos (A.S., unpublished).
However, no defect was evident in anterior patterning in the
Sfrp1-/-;Sfrp2-/- embryos. Sfrp5 and
Dkk1 are expressed in the AVE, which may compensate for the loss of
Sfrp1 and Sfrp2 function in anterior patterning
(Mukhopadhyay et al., 2001;
Finley et al., 2003).
Sfrps and Wnts signaling
The results of this study provide genetic evidence for the in vivo function
of Sfrp genes in embryonic development. The limb defect is consistent
with the phenotype induced by Wnt/ß-catenin signal activation
(Mukhopaghyay at al., 2001;
Barrow et al., 2003;
Soshnikova et al., 2003). The
defect in posterior axis elongation in
Sfrp1-/-;Sfrp2-/- embryos at E8.25 appeared to
be associated with an abnormality in migration of the paraxial mesoderm cells.
In addition, we observed higher activation of the Wnt/ß-catenin pathway
in the posterior region of Sfrp1-/-;Sfrp2-/-
embryos. Mesoderm migration is known to be dependent on the function of
T, a downstream target of the Wnt/ß-catenin pathway
(Wilson et al., 1993;
Yamaguchi et al., 1999b).
Although hyperactivation of T expression was not detected in the
double homozygous mutant embryos, it is possible that moderate levels of
activation of expression affect mesoderm cell migration.
|
).
Wnt5a is not capable of association with Sfrp1 in vitro
(Xu et al., 1998;
Dennis et al., 1999), which
suggests that Wnt5a signaling is not directly regulated by Sfrp1 and Sfrp2.
Previous reports indicated that Wnt5a, which leads to activation of the
Wnt/Ca2+ pathway, plays a role in canonical Wnt/ß-catenin
pathway inhibition (Torres et al.,
1996; Topol et al.,
2003). Furthermore, the Wnt/Ca2+ pathway interacts with
the non-canonical PCP pathway
(Carreira-Barbosa et al.,
2003; Veeman et al.,
2003; Westfall et al.,
2003). Further analysis is required in order to elucidate the
relationship between the roles of Sfrp and Wnt5a. However, these observations
suggest that inactivation of Sfrp1 and Sfrp2 causes
misregulation of a wide spectrum of Wnt activities in the canonical and
non-canonical pathways.
Somitogenesis in Sfrp1-/-;Sfrp2-/- embryos
The segmentation clock in the PSM, which is generated by the expression of
oscillator genes, is needed for somite segmentation (Aulehla et al., 2004). A
number of oscillator genes, such as Lfng, Her1, chick
hairy1, chick hairy2, Hes1/Hes2 and Hes7, have been
identified as components of Notch signaling. In fact, mutations of Notch,
Dll1 and Dll3 (Notch ligands), and Lfng and Hes7 (Notch
effectors), all result in aberrant segmentation during somitogenesis
(Saga and Takeda, 2001;
Bessho et al., 2001). The
disruption of somite segmentation exhibited by
Sfrp1-/-;Sfrp2-/- embryos is
associated with altered Notch oscillations, which is supported by abnormal
Lfng and Hes7 expression cycles in
Sfrp1-/-;Sfrp2-/- embryos.
A Wnt3a gradient in the PSM is thought to function in somitogenesis
regulation. Expression of Notch-related oscillator genes is disrupted in
Wnt3a mutant embryos, which suggests that Wnt acts upstream of Notch
signaling (Aulehla et al.,
2003). Upregulation of the Wnt/ß-catenin pathway was
suggested by non-phospho ß-catenin staining in the tail bud of
Sfrp1-/-;Sfrp2-/- embryos at E8.5. In addition,
inactivation of Sfrp1 and Sfrp2 perturbed the oscillating
expression of Nkd1, an oscillator gene activated downstream of Wnt
signaling in the PSM (Ishikawa et al.,
2004). Moreover, evidence that LEF1/TCF, a downstream mediator of
Wnt signaling, activates expression of the Notch ligand Dll1 revealed
the connection between Wnt and Notch signaling
(Hofmann et al., 2004;
Galceran et al., 2004). These
data, and our observations, support the idea that inactivation of
Sfrp1 and Sfrp2 affects Wnt signaling activity in the PSM in
conjunction with the misregulation of oscillating Notch signaling, which
results in abnormal of Lfng and Hes7 expression.
Interestingly, dynamic expression of Axin2 in
Sfrp1-/-;Sfrp2-/- embryos was observed,
suggesting the presence of Axin2 oscillation. Rapid degradation of
the protein may exert an effect on the maintenance of the oscillatory
expression cycles even in the PSM of
Sfrp1-/-;Sfrp2-/- embryos
(Seidensticker and Behrens,
2000; Aulehla et al.,
2003). Thus, the intracellular oscillation cycle (Axin2
expression) does not correlate with the intercellular cycle (Notch
oscillators) in Sfrp1-/-;Sfrp2-/- embryos,
suggesting that Notch signaling is a non-cell autonomous signal that is
elevated in Wnt-receiving cells.
In summary, our findings clearly demonstrate the function of Sfrp1 and Sfrp2 in AP axis elongation and somitogenesisis. Because Sfrps regulate a wide spectrum of Wnt activities and pathways, these results suggest that coordinated regulation in the canonical and non-canonical Wnt pathways plays a significant role in embryonic patterning.
|
ACKNOWLEDGMENTS |
|---|
|
Footnotes |
|---|
Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/133/6/989/DC1
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