Sfrp1, Sfrp2, and Sfrp5 regulate the Wnt/beta-catenin and the planar cell polarity pathways during early trunk formation in mouse.

Abstract

The secreted frizzled-related protein gene family encodes proteins that regulate Wnt signaling. Msx1 in situ hybridization of 9.5 days post coitus mouse embryos showing normal neural tube development in an Sfrp1; Sfrp2 double mutant (left) but severe neural tube defects in a Looptail (Lp/+); Sfrp1; Sfrp2 triple mutant (right). These findings suggest that Sfrps regulate the Wnt planar cell polarity pathway. See Satoh et al. in this issue.

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ARTICLE
Sfrp1, Sfrp2, and Sfrp5 Regulate the Wnt/b-Catenin
and the Planar Cell Polarity Pathways During Early Trunk
Formation in Mouse
Wataru Satoh,1,2Makoto Matsuyama,1Hiromasa Takemura,1,2Shinichi Aizawa,1
and Akihiko Shimono1*
1Vertebrate Body Plan, Center for Developmental Biology, RIKEN Kobe, Minatojima-Minami, Chuou-ku, Kobe, Japan
2The Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, Nara, Japan
Received 23 July 2007; Revised 30 November 2007; Accepted 2 December 2007
Summary: Sfrp is a secreted Wnt antagonist that directly
interacts with Wnt ligand. We show here that inactiva-
tion of Sfrp1, Sfrp2, and Sfrp5 leads to fused somites
formation in early-somite mouse embryos, simultane-
ously resulting in defective convergent extension (CE),
which causes severe shortening of the anteroposterior
axis. These observations indicate the redundant roles of
Sfrp1, Sfrp2, and Sfrp5 in early trunk formation. The
roles of the Sfrps were genetically distinguished in
terms of the regulation of Wnt pathways. Genetic analy-
siscombiningSfrpsmutants
revealed the involvement of Sfrps in CE through the reg-
ulation of the planar cell polarity pathway. Furthermore,
Dkk1-deficient embryos carrying Sfrp1 homozygous and
Sfrp2 heterozygous mutations display irregular somites
and indistinct intersomitic boundaries, which indicates
that Sfrps-mediated inhibition of the Wnt/b-catenin
pathway is necessary for somitogenesis. Our results
suggest that Sfrps regulation of the canonical and non-
canonical pathways is essential for proper trunk forma-
tion. genesis 46:92–103, 2008.
andLoop-tailmice
V V
C 2008 Wiley-Liss, Inc.
Key words: Sfrp;
polarity pathway; convergent extension; somitogenesis
Wnt/b-cateninpathway; planarcell
INTRODUCTION
Wnt is a family of secreted glycoproteins (Prud’homme
et al., 2002; http://www-leland.stanford.edu/ ~ rnusse/
wntwindow.html). The Wnt family activates different
intercellular signaling cascades through its receptor,
Frizzled. These cascades are classified as either the ca-
nonical Wnt/b-catenin pathway or the noncanonical
Wnt pathways, which include the Wnt/Ca21and the pla-
nar cell polarity (PCP) pathways (Widelitz, 2005).
Secreted Frizzled-related protein (Sfrp) possesses a
structure similar to the cysteine-rich domain (CRD) of
Frizzled and directly interacts with Wnt ligands (Kawano
and Kypta, 2003). This ligand interaction theoretically
suggests that Sfrp can regulate the canonical and nonca-
nonical pathways (Kawano and Kypta, 2003). The Sfrp
gene family consists of five members in the mouse and
human genomes (Jones and Jomary, 2002). Crescent and
Sizzled are unique members in Xenopus, zebrafish, and
chicken (Jones and Jomary, 2002). In particular, Sfrp1,
Sfrp2, and Sfrp5 comprise a subfamily (referred to here-
after as Sfrps), which is suggestive of the similar molecu-
lar function of Sfrps. Indeed, Sfrps inhibit the Wnt/b-cat-
enin pathway in vitro; moreover, Sfrps demonstrate dis-
tinct characteristics in comparison to Sfrp3 and Sfrp4
probably due to ligand specificity (Galli et al., 2006;
Suzuki et al., 2004). Furthermore, a double homozygous
mutation of Sfrp1 and Sfrp2 (Sfrp12/2 Sfrp22/2)
results in cell migration abnormalities in the presomitic
mesoderm (PSM) and incomplete somite segmentation
albeit only in the thoracic region (Satoh et al., 2006).
This observation suggested functional redundancy of
Sfrp1, Sfrp2, and Sfrp5; therefore, the role of Sfrps dur-
ing embryonic development remained unclear. More-
over, how Sfrps control mesoderm cell movement and
somitogenesis with respect to regulation of Wnt path-
ways was unknown.
The canonical Wnt pathway plays critical roles with
respect to the somite segmentation process. Wnt3a is
thought to determine the segmentation boundary
through its own protein gradient and/or via direct activa-
tion of Fgf8 expression (Aulehla and Herrmann, 2004).
Current address for Hiromasa Takemura: Laboratory of Animal Experi-
ments for Regeneration, Institute for Frontier Medical Sciences, Kyoto Uni-
versity, 53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan.
Published online 6 February 2008 in
Wiley InterScience (www.interscience.wiley.com).
DOI: 10.1002/dvg.20369
This article contains supplementary material available via the Internet at
http://www.interscience.wiley.com/jpages/1526–954X/suppmat.
*Correspondence to: Akihiko Shimono, Vertebrate Body Plan, Center for
Developmental Biology, RIKEN Kobe, Minatojima Minami, Chuou-ku, Kobe
650-0047, Japan.
E-mail: ashimono@cdb.riken.jp
' 2008 Wiley-Liss, Inc.genesis 46:92–103 (2008)

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Furthermore, somite formation is coupled to oscillatory
Notch-related gene expression in the PSM. In this con-
text, Wnt/b-catenin signaling activates Dll1 expression
and modulates Notch signaling (Galceran et al., 2004;
Hofmann et al., 2004). The canonical Wnt pathway is
essential for primitive streak and mesoderm formation at
earlier stages (Liu et al., 1999). Moreover, mesoderm
migration is dependent on the function of T, a down-
stream target of the canonical pathway (Wilson et al.,
1993; Yamaguchi et al., 1999a). In contrast, studies in
Xenopus and zebrafish demonstrated that the PCP path-
way is required for convergent extension (CE), which is
the process consisting of simultaneous and interdepend-
ent mediolateral narrowing and anteroposterior elonga-
tion of embryonic tissue (Keller, 2002; Torban et al.,
2004). Recent studies involving mice carrying mutations
of PCP pathway components, including double homozy-
gous mutant mice of Dvl1 and Dvl2 as well as Loop-tail
(Lp) mice (characterized by the Stbm/Vangl2 mutation),
afforded evidence pertaining to regulation of CE by the
PCP pathway in higher vertebrates (Wang et al., 2006;
Ybot-Gonzalez et al., 2007).
In this study, Sfrp1, Sfrp2, and Sfrp5 triple homozy-
gous mutant embryos were generated to reveal the
redundant roles in embryonic development. Moreover,
crossing compound mutant mice with Dkk1-deficient
mice, a mouse line displaying a defective canonical Wnt
pathway, and with Lp mice, a strain exhibiting a defec-
tive noncanonical pathway, permitted genetic manipula-
tion of the balance of Sfrps regulation in the canonical
and noncanonical Wnt pathways. We provide here
genetic evidence regarding Sfrps regulation of the Wnt/
b-catenin and PCP pathways in association with somito-
genesis and CE during early trunk formation.
RESULTS
Generation of Sfrp1, Sfrp2, and Sfrp5 Compound
Mutant Mice
To elucidate the role of Sfrps in embryogenesis, single
Sfrp1, Sfrp2, and Sfrp5 knock-out (KO) mouse lines
were generated initially; these murine lines were viable
and fertile (Satoh et al., 2006) (Fig. S1a,b; see the Supple-
mentary Data). The expression pattern during early
embryogenesis is suggestive of the redundant role of
Sfrps. In streak stages embryos, overlapping expression
of Sfrp1 and Sfrp5 is observed in the anterior visceral
endoderm (AVE) (Finley et al., 2003; Hoang et al., 1998).
At later stages, e.g., embryonic day (E) 8.5, expression of
both Sfrp1 and Sfrp2 occurs in the neuroectoderm along
the anteroposterior (a-p) axis (Leimeister et al., 1998),
whereas simultaneous Sfrp1, Sfrp2, and Sfrp5 expres-
sion is apparent in the neural tube caudal to the hind-
brain (Finley et al., 2003) (Fig. 1a).
To identify the redundant roles, the following five ge-
notypes were produced by crosses between mutant
mice and, subsequently, compound mutant mice: (a)
Sfrp12/2 Sfrp52/2, (b) Sfrp22/2 Sfrp52/2, (c)
Sfrp11/2 Sfrp21/2 Sfrp52/2, (d) Sfrp11/2 Sfrp22/
2 Sfrp52/2, and (e) Sfrp12/2 Sfrp21/2 Sfrp52/2.
These compound mutant mouse lines (a–e) were viable
and fertile. No obvious abnormalities in gross morphol-
ogy, with the exception of hindlimb syndactyly and tail
kinks during embryogenesis in a minor population of
those compound mutant mice carrying both mutated
Sfrp2 alleles as previously reported (Cox et al., 2006;
Satoh et al., 2006), were observed. In contrast, highly
specific spatial and temporal expression patterns of
Sfrps are observed in many developing organs (Lei-
meister et al., 1998). Hence, the long-range effect of Sfrp
as a soluble factor could compensate for the functions of
other Sfrps in those mutants.
Subsequently, to revel redundant roles of Sfrp1,
Sfrp2, and Sfrp5 in embryonic development, embryos
carrying the triple homozygous mutation of Sfrp1,
Sfrp2, and Sfrp5 (Sfrp12/2 Sfrp22/2 Sfrp52/2)
were obtained by intercrosses of Sfrp12/2 Sfrp21/2
Sfrp52/2
mice.
Sfrp12/2
embryos were lethal at around E12.5 and characterized
by a severe a-p axis elongation defect in the trunk (data
not shown, Fig. 1b,c). In Sfrp12/2 Sfrp22/2 Sfrp52/2
embryos at E9.5, an open neural tube was observed
consistently (Fig. 1b). The neural tissue possessed dor-
sal–ventral patterning as indicated by the expressions of
Msx1 (Robert et al., 1989) in the roof and Shh (Echelard
et al., 1993) in the floor plate of the neural tube (Fig.
1b). At these stages, embryonic growth was delayed,
probably due to abnormalities in embryonic turning and
umbilical cord formation. These observations suggest
that Sfrp1, Sfrp2, and Sfrp5 are functionally redundant
during early trunk formation.
Sfrp22/2
Sfrp52/2
Somitogenesis and a-p Axis Elongation Defects in
Sfrp1, Sfrp2, and Sfrp5 Triple Knock-Out Embryos
Sfrp12/2 Sfrp22/2 Sfrp52/2 embryos at E8.5
exhibited compression of the somites along the a-p axis
and lateral expansion (Fig. 1c, between arrowheads), a
phenotype more severe than that observed in Sfrp12/2
Sfrp22/2 embryos (Fig. 1c). The Sfrp12/2 Sfrp22/2
Sfrp52/2 embryos display the fused stripes of the
Uncx4.1 expression domain, a marker occurring in the
posterior half of the somite (Mansouri et al., 1997;
Fig. 1c). In addition, histological analysis revealed fused
small somites in E8.5 Sfrp12/2 Sfrp22/2 Sfrp52/2
embryos (Fig. 1d, arrows).
To identify a defect in the expression of cyclic gene
during somitogenesis, Lfng expression in Sfrp12/2
Sfrp22/2 Sfrp52/2 embryos was examined at E8.25,
prior to the point where delayed embryonic growth
became apparent. Lfng, which is a direct target of Notch
signaling, encodes a glycosyltransferase that acts as a
Notch signaling inhibitor; furthermore, Lfng expression
oscillates in the PSM (Cole et al., 2002; Dale et al., 2003;
Morales et al., 2002). The expression patterns at E8.25
were typically classified into three phases, which
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THE ROLES OF SFRPS IN EARLY TRUNK FORMATION

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appeared with similar frequencies in control embryos
(phase I, 28%; phase II, 38%; phase III, 34%; Fig. 1e). In
the PSM of Sfrp12/2 Sfrp22/2 Sfrp52/2 embryos at
E8.25, which corresponds to the 8–11 somites stage, the
asymmetric stripe of Lfng expression was frequently
observed in the anterior-most region of the PSM in
Sfrp12/2 Sfrp22/2 Sfrp52/2 embryos (Fig. 1e, aster-
isks). In addition, the appearance frequency of phase I
FIG. 1.
defect. Arrow, midbrain-hindbrain boundary. Sections of the embryos (inset) generated at the broken line. Arrowhead, floor plate; arrow,
notochord; Fl, forelimb bud. (c) Compression of somites along the a-p axis and lateral spreading (between arrowheads) in Sfrp12/2
Sfrp22/2 Sfrp52/2 embryos at E8.5. (Inset) Higher magnification of Uncx4.1 expression domains in Sfrp12/2 Sfrp22/2 Sfrp52/2
embryos. (d) Horizontal section of control and Sfrp12/2 Sfrp22/2 Sfrp52/2 embryos at E8.5. Arrows, fused somites. A, anterior; P, poste-
rior; Sc, spinal cord; PSM, presomitic mesoderm. (e) The oscillation cycles of Lfng are altered in the PSM of Sfrp12/2 Sfrp22/2 Sfrp52/2
embryos at the stage corresponding to 8–11 somites (lower panels) in comparison with control embryos (upper panels). A fraction indicates
the appearance frequency of each phase.
(a) Expression of Sfrp1, Sfrp2, and Sfrp5 at E8.5. (b) Sfrp12/2 Sfrp22/2 Sfrp52/2 embryos at E9.5 show open neural tube
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in Lfng oscillatory cycles was significantly increased
(58%; 15 of 26 embryos), which suggests altered oscilla-
tory expression of Lfng in a manner consistent with
observations in double homozygous Sfrp12/2 Sfrp22/2
embryos (Satoh et al., 2006; Fig. 1e).
The defect in a-p axis elongation as well as the open
neural tube in Sfrp12/2 Sfrp22/2 Sfrp52/2 embryos
appeared to be related to CE. Regionalization along the
a-p axis at E8.5–9, which was monitored by regional
markers in the brain and spinal cord, occurred in the
neural tissue (Fig. S2a–f), although the rhombomere
appeared to be affected and compressed along the a-p
axis (Fig. S2d). Furthermore, the primitive streak was
maintained in E8.5 Sfrp12/2 Sfrp22/2 Sfrp52/2
embryos, which was indicated by markers expression
(Fig. S2g–i). Interestingly, the Sfrp12/2 Sfrp22/2
Sfrp52/2 embryos displayed ectopic colonization of
axial mesoderm characterized by Shh expression (Fig.
S2j, arrowheads), which is suggestive of abnormality in
distribution of the axial mesoderm.
The defect of axial mesoderm distribution and noto-
chord lengthening is the evidence of disruption of CE
(Wang et al., 2006). Therefore, we observed the expres-
sion of Shh in the Sfrp12/2 Sfrp22/2 Sfrp52/2
embryos at earlier stages to determine when axial meso-
derm distribution became defective. At the late head-fold
stages, when Shh expression was distributed as a stripe
along the a-p axis in control embryos, the expression
was laterally expanded in the midline of the embryo
(Fig. 2c,d; arrow). At the late bud stage, Shh expression
was widely distributed in the axial region of Sfrp12/2
Sfrp22/2 Sfrp52/2 embryos in comparison with that
in control embryos, even before morphological abnor-
malities in the embryos became apparent (Fig. 2a,b;
between arrowheads).
Next, the axial elongation abnormalities were directly
monitored with respect to cells derived from the ante-
rior end of the primitive streak or the node at the late
bud/early head-fold stage. To conduct this analysis, cells
were labeled with 1, 10-dioctadecyl-3, 3, 30, 30-tetrame-
thylindocarbocyanine perchlorate (DiI) (Fig. 2e,g; t 5 0);
and then the embryos were cultured for 16 h (Fig. 2f,h;
t 5 16). The labeled axial tissue including the axial mes-
oderm expanded along the a-p axis in control embryos
after the culture (Fig. 2e,f, arrowheads; n 5 3). In con-
trast, expansion was greatly reduced in Sfrp12/2
Sfrp22/2 Sfrp52/2 embryos; additionally the labeled
cells were laterally distributed (Fig. 2g,h, arrowheads;
n 5 3). These observations suggest that Sfrp1, Sfrp2,
and Sfrp5 are functionally redundant during CE as well
as during somitogenesis.
Genetic Analysis of Sfrps Function in Canonical
and Noncanonical Wnt Pathways
The fused somites may be caused by an abnormality in
the canonical Wnt pathway during somitogenesis in
Sfrp12/2 Sfrp22/2 (Satoh et al., 2006) and Sfrp12/2
Sfrp22/2 Sfrp52/2 embryos. However, Sfrps theoreti-
cally regulate the canonical and noncanonical pathways;
consequently, the requirement of Sfrps with respect to
Wnt pathway regulation in terms of CE and somitogene-
sis is difficult to distinguish. To differentiate Sfrp regula-
tion of the Wnt pathways, the balance of the signaling
regulation was genetically manipulated using compound
mutant mice crossed with Lp mice, a strain displaying a
defective noncanonical pathway, and with Dkk1-defi-
cient mice, a mutant mouse line exhibiting a defective
canonical Wnt pathway.
Sfrps Regulate the PCP Pathway
Lp is a mouse line carrying a mutation in Stbm/Vangl2
(also known as Ltap), which encodes a membrane pro-
tein (Torban et al., 2004). Lp/1 Sfrp12/2 Sfrp21/2
Sfrp52/2 embryos at E14.5 were obtained from crosses
between Sfrp12/2 Sfrp21/2 Sfrp52/2 female and
Lp/1 Sfrp12/2 Sfrp52/2 male mice. The Lp heterozy-
gous mutation led to a local neural tube closure defect,
spina bifida, in approximately 13% of the embryos char-
acterized by the Sfrp12/2 Sfrp52/2 background (total
n 5 16) as well as in the wild type background as
reported previously (Lu et al., 2004). The frequency of
spina bifida increased to 62% in Lp/1 Sfrp12/2
Sfrp21/2 Sfrp52/2 embryos (total n 5 13) (Fig.
S3a,b). Thus, Sfrps genetically interact with Stbm/
Vangl2.
In addition, Lp/1 Sfrp12/2 Sfrp22/2 Sfrp51/2
embryos (at E9.5) were obtained by crossing Sfrp12/2
Sfrp21/2 females with Lp/1 Sfrp12/2 Sfrp21/2
Sfrp52/2 males. The embryos consistently displayed
FIG. 2.
of CE. (a–d) Shh expression in control (a,c) and Sfrp12/2 Sfrp22/2
Sfrp52/2 (b,d) embryos at the late bud (a,b) and late head fold
stage (c,d). (e–h), DiI-labeling assay of the node in control (e,f) and
Sfrp12/2 Sfrp22/2 Sfrp52/2 embryos (g,h) at the early bud stage.
A, anterior; P, posterior; Hf, head fold.
Sfrp12/2 Sfrp22/2 Sfrp52/2 embryos exhibit disruption
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THE ROLES OF SFRPS IN EARLY TRUNK FORMATION

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craniorachischisis (n 5 17), which was not observed in
the Sfrp12/2 Sfrp22/2 Sfrp51/2 (n 5 17) or in the
Lp/1 Sfrp12/2 Sfrp21/2 Sfrp51/2 embryos (n 5
41) (Fig. 3a). An abnormality in somitogenesis was appa-
rent in Lp/1 Sfrp12/2 Sfrp22/2 Sfrp51/2 embryos
in a region behind the forelimb, similarly found in
Sfrp12/2 Sfrp22/2 Sfrp51/2 embryos (Fig. 3d,
bracket). The Sfrp12/2 Sfrp22/2 Sfrp51/2 embryos
were morphologically indistinguishable from Sfrp12/2
Sfrp22/2 embryos at E9.5 (see Fig. 3). On the basis of
FIG. 3.
Stbm/Vangl2. Lp/1 Sfrp12/2 Sfrp22/2
Sfrp51/2 embryos at E9.5 display cra-
niorachischisis (a). T (b), Dll1 (c), and
Uncx4.1 (d) expression suggests that
defective a-p axis elongation in Lp/1
Sfrp12/2 Sfrp22/2 Sfrp51/2 embryos
was enhanced in the trunk in compari-
sonto that observed in
Sfrp22/2
Sfrp51/2
dorsal view). Arrow, anterior boundary of
the PSM; arrowhead, the tail bud end. Fl,
forelimb bud.(e)
revealed wide distribution of axial meso-
derm in the midline of Lp/1 Sfrp12/2
Sfrp22/2 Sfrp51/2 embryos at the late
head fold stage (upper, frontal view;
lower, lateral view).
Sfrps genetically interact with
Sfrp12/2
embryos (Inset,
Shh expression
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SATOH ET AL.