Interaction of Wnt and caudal-related genes in zebrafish posterior body formation

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Developmental Biology

Interaction of Wnt and caudal-related genes in zebrafish posterior

body formation

Takashi Shimizu, Young-Ki Bae, Osamu Muraoka, Masahiko Hibi*

Laboratory for Vertebrate Axis Formation, Center for Developmental Biology, RIKEN, 2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe,

Hyogo 650-0047, Japan

Received for publication 9 October 2004, revised 2 December 2004, accepted 7 December 2004

Available online 13 January 2005

Abstract

Although Wnt signaling plays an important role in body patterning during early vertebrate embryogenesis, the mechanisms by which

Wnts control the individual processes of body patterning are largely unknown. In zebrafish, wnt3a and wnt8 are expressed in overlapping

domains in the blastoderm margin and later in the tailbud. The combined inhibition of Wnt3a and Wnt8 by antisense morpholino

oligonucleotides led to anteriorization of the neuroectoderm, expansion of the dorsal organizer, and loss of the posterior body structure–a

more severe phenotype than with inhibition of each Wnt alone–indicating a redundant role for Wnt3a and Wnt8. The ventrally expressed

homeobox genes vox, vent, and ved mediated Wnt3a/Wnt8 signaling to restrict the organizer domain. Of posterior body-formation genes,

expression of the caudal-related cdx1a and cdx4/kugelig, but not bmps or cyclops, was strongly reduced in the wnt3a/wnt8 morphant

embryos. Like the wnt3a/wnt8 morphant embryos, cdx1a/cdx4 morphant embryos displayed complete loss of the tail structure, suggesting

that Cdx1a and Cdx4 mediate Wnt-dependent posterior body formation. We also found that cdx1a and cdx4 expression is dependent on Fgf

signaling. hoxa9a and hoxb7a expression was down-regulated in the wnt3a/wnt8 and cdx1a/cdx4 morphant embryos, and in embryos with

defects in Fgf signaling. Fgf signaling was required for Cdx-mediated hoxa9a expression. Both the wnt3a/wnt8 and cdx1a/cdx4 morphant

embryos failed to promote somitogenesis during mid-segmentation. These data indicate that the cdx genes mediate Wnt signaling and play

essential roles in the morphogenesis of the posterior body in zebrafish.

D 2004 Elsevier Inc. All rights reserved.

Keywords: Wnt; caudal-related gene; fgf; hox; Tail; Posterior body; Segmentation; Zebrafish

Introduction

Wnts are secreted glycoproteins that play important roles

in body patterning, cell proliferation/differentiation, and

tumorigenesis. Wnts exhibit their functions through two

distinct signaling cascades: the canonical and non-canonical

pathways (reviewed in (Huelsken and Birchmeier, 2001;

Moon et al., 2002; Veeman et al., 2003)). The canonical Wnt

pathway induces nuclear accumulation of h-catenin and

activates transcription through a complex of h-catenin and

the transcription factors Tcf and Lef. wnt8, which activates

the canonical pathway, is expressed in the ventro-lateral

0012-1606/$ - see front matter D 2004 Elsevier Inc. All rights reserved.

doi:10.1016/j.ydbio.2004.12.007

* Corresponding author. Fax: +81 78 306 3136.

E-mail address: [email protected] (M. Hibi).

mesoderm during gastrulation in Xenopus and zebrafish

(Christian et al., 1991; Kelly et al., 1995). Inhibition of

Wnt8 function by a dominant-negative construct in Xenopus

or MOs in zebrafish leads to the anteriorization of neuro-

ectoderm, reduction of the posterior body, expansion of

dorsal axial tissues such as the prechordal plate, and a

concomitant reduction of paraxial tissues (Christian and

Moon, 1993; Erter et al., 2001; Lekven et al., 2001),

indicating a role for Wnt8 in the posteriorization of

neuroectoderm, and the formation of paraxial mesoderm

and the tail. In addition to Wnt8, Wnt3a functions in body

patterning during early embryogenesis. wnt3a-deficient

mouse embryos show severe reduction or loss of the somite

structures and the posterior body structure (Takada et al.,

1994). However, the roles of wnt8 and wnt3a in the

embryogenesis of other vertebrate species remain to be

279 (2005) 125–141

T. Shimizu et al. / Developmental Biology 279 (2005) 125–141126

elucidated. In contrast to the Wnt canonical pathway, the

Wnt non-canonical pathway is involved in the convergent

extension cell movements during gastrulation in zebrafish

and Xenopus (reviewed in (Myers et al., 2002b; Tada et al.,

2002; Veeman et al., 2003)).

Inhibition of theWnt canonical pathway causes expansion

of the dorsal organizer and its derivatives, such as the

prechordal plate and notochord (Hashimoto et al., 2000;

Lekven et al., 2001), and can rescue the reduction or loss of

dorsal organizer formation in zebrafish bozozok mutants

(Fekany-Lee et al., 2000; Hashimoto et al., 2000), suggesting

a role for Wnt in restricting the organizer domain. bozozok,

which is expressed in the Nieuwkoop center/blastula organ-

izer center, encodes a transcriptional repressor-type homeo-

domain protein Dharma/Nieuwkoid (Koos and Ho, 1998;

Yamanaka et al., 1998) that represses the expression of the

ventrally expressed homeobox genes, vox, vent, and ved,

which also encode transcriptional repressors (Imai et al.,

2001; Kawahara et al., 2000a,b; Shimizu et al., 2002). Vox,

Vent, and Ved function redundantly to repress the expression

of the dorsal organizer gene on the ventral side, and the

repression of vox, vent, and ved byBozozok on the dorsal side

plays an important role in restricting the dorsal organizer

domain (Shimizu et al., 2002). Although it was recently

reported that Vox and Vent mediates the activity of Wnt8 to

repress the dorsal organizer (Ramel and Lekven, 2004), it has

not yet been made clear whether Ved or other Wnts are

involved in the restriction of the organizer.

Zebrafish tail is established through a combination of

coordinated cell movements and cell proliferation during and

after gastrulation (Kanki and Ho, 1997). The cells from the

ventral and dorsal margin meet at the vegetal pole at the end

of gastrulation and form the tailbud. In the tailbud, the

posterior bud cells that are derived from the ventral

blastoderm dsubductT beneath the anterior bud cells, which

are derived from the dorsal region. The posterior tailbud cells

further move laterally and anteriorly, and form the presomitic

mesoderm (PSM). It is reported that the ventral marginal

blastoderm of early gastrula zebrafish embryos functions as

the tail organizer, and Bmp, Nodal, and Wnt8, which are co-

expressed in the ventral marginal blastoderm, are responsible

for the function of the tail organizer (Agathon et al., 2003).

However, it has been proposed that BMP signaling is

involved in the movements of tail progenitor cells (Myers

et al., 2002a). In the Nodal signal-defective mutants, such as

maternal-zygotic one-eyed pinhead (MZoep), the posterior

somites are often normally formed. In contrast, inhibition of

Wnt signaling by either Wnt8 MOs or misexpression of

Dickkopf1 (Dkk1) consistently reduces the formation of the

tail (Hashimoto et al., 2000; Lekven et al., 2001). Similar to

Wnt inhibition, inhibition of FGF signaling in Xenopus and

zebrafish elicits truncation of the posterior body structure

(Amaya et al., 1993; Draper et al., 2003; Griffin et al., 1995,

1998), suggesting that Wnt and FGF signaling function in

parallel or in a linear cascade for formation of the posterior

body. In zebrafish, the T-box genes no tail (ntl) and spadetail

(spt) are at least partly involved in the FGF-mediated

formation of the posterior body (Amacher et al., 2002;

Griffin et al., 1995, 1998). The non-canonical Wnt pathway

in collaboration with ntl functions in the morphogenetic

movements of the posterior body (Marlow et al., 2004).

Although tbx6 is suggested to mediate BMP and Wnt

signaling for the posterior body formation (Szeto and

Kimelman, 2004), the mechanisms by which the canonical

Wnt pathway regulates the formation of the posterior body in

zebrafish are largely unknown.

In mouse and Xenopus, caudal-related genes, the cdxs,

are proposed to function downstream of Wnt and FGF

signaling. Three cdx genes (cdx1, cdx2, and cdx4 in mouse)

are homeobox genes related to the Drosophila gene,

caudal, which is required for the formation of the posterior

body in Drosophila (reviewed in (Lohnes, 2003)). A

dominant repressor form of Xcad3, a Xenopus ortholog of

Cdx4, inhibits posterior body formation in Xenopus

embryos (Isaacs et al., 1998). The heterozygotes of cdx2-

deficient mice show a reduced tail structure, and this tail-

defect phenotype is enhanced in a combinational mutation

in the cdx1 and cdx2 genes (Subramanian et al., 1995; van

den Akker et al., 2002). In zebrafish, a point or insertional

mutation in the cdx4/kugelig gene leads to reduction of the

posterior body structure (Davidson et al., 2003; Golling et

al., 2002). All of these data indicate the involvement of the

cdx genes in the posterior body formation during vertebrate

embryogenesis. Cdx proteins bind to specific DNA ele-

ments and regulate the expression of target genes, including

hox cluster genes, which are expressed in the posterior body

(Charite et al., 1998; Isaacs et al., 1998; Subramanian et al.,

1995). The expression of the cdx genes is regulated by

multiple signaling pathways, such as the retinoic acid, Wnt,

and FGF pathways (Haremaki et al., 2003; Houle et al.,

2000, 2003; Ikeya and Takada, 2001; Isaacs et al., 1998;

Lickert et al., 2000; Northrop and Kimelman, 1994;

Pownall et al., 1996; Prinos et al., 2001; Shiotsugu et al.,

2004). wnt3a-null or hypomorphic mutant (vestigial) mice

show a reduced or loss of expression of cdx1, but not cdx2

or cdx4 in the tail region (Ikeya and Takada, 2001; Prinos et

al., 2001). However, the cdx1-deficient embryos show only

anterior homeotic transformation of the anterior cervical

and thoracic vertebrate column, and do not exhibit

abnormalities in posterior body formation (Subramanian et

al., 1995), suggesting that some system other than the

wnt3a-cdx1 cascade is also involved in the formation of the

posterior body.

In this study, we demonstrate that Wnt3a (referred as

Wnt3l in Zebrafish Information Network) and Wnt8

function redundantly in the restriction of the dorsal

organizer domain and in the formation of the posterior

body in zebrafish. Wnt3a and Wnt8 restrict the dorsal

organizer domain by regulating the ventrally expressed

homeobox genes, and they control posterior body formation

through regulation of the caudal-related genes, cdx1a and

cdx4. Cdx1a/4 and Fgf signaling cooperatively regulate the

T. Shimizu et al. / Developmental Biology 279 (2005) 125–141 127

expression of the posterior hox genes. These findings, taken

collectively, indicate that the Wnt-Cdx cascade is required

for morphogenesis of posterior body.

Material and methods

Fish embryos

Wild-type zebrafish (Danio rerio) embryos were obtained

from natural crosses of fish with the AB/India genetic

background.

Constructs, RNA synthesis, and transcription detection

The zebrafish wnt3a cDNA was isolated by expression

cloning, which we carried out with a modified pCS2+ vector

as described previously (Bae et al., 2003; Yamanaka et al.,

1998). Briefly, bacteria transformants containing 200–300

cDNA clones were pooled, and 5V-capped RNA was

synthesized in vitro from each pool. One to two nanograms

of the 5V-capped RNAwas injected into one- or two-cell-stage

embryos, and the effects were evaluated by morphological

inspection. We obtained wnt3a as a positive clone that

elicited dorsalization and posteriorization of the embryos.

The sequence ofwnt3a was identical to that ofwnt3a that was

recently reported (Buckles et al., 2004) (wnt3l in Zebrafish

Information Network). The zebrafish cdx1a cDNA was

isolated from a zebrafish gastrula library by low-stringency

hybridization using the homeobox gene pnx (Bae et al.,

2003). Although the gene displays a stronger sequence

similarity to Xenopus cdx2, we named the gene cdx1a based

on the genomic mapping data (Leonard I. Zon, personal

communication). The cdx4 and hoxa9a cDNAs were isolated

by PCR with primers based on the published sequence

(Davidson et al., 2003). The nucleotide sequence for cdx1a

was deposited in the database under Accession Number

AB067733. The pCS2+ or modified pCS2+ plasmids

containing the wnt3a and cdx1a cDNA were used to

synthesize the capped RNA, and pBluescript SK+ (Stra-

tagene) containing wnt3a and pGEMT-Easy (Promega)

containing cdx1a, cdx4, or hoxa9a were used to synthesize

the anti-sense riboprobes, as described previously (Shimizu

et al., 2002). The expression plasmids for wnt8 (open

reading frame 1) (Lekven et al., 2001), fgf3 (Koshida et al.,

2002), fgf8 (Furthauer et al., 1997), dominant negative

Xenopus FGF receptor 1 (XFD) (Hongo et al., 1999) are

published. The expression plasmid for constitutively active

Ras was constructed by inserting RasV12 cDNA into

pCS2+. The detection of otx2 (Mori et al., 1994), goosecoid

(Stachel et al., 1993), chordino (Miller-Bertoglio et al.,

1997), spadetail (Griffin et al., 1998; Nojima et al., 2004),

floating head (Talbot et al., 1995), dickkopf1 (Hashimoto et

al., 2000), vox (Kawahara et al., 2000a), ved (Shimizu et al.,

2002), wnt8 (Lekven et al., 2001), bmp2b, bmp4 (Nikaido

et al., 1997), cyclops (Rebagliati et al., 1998b), fgf3 (Shinya

et al., 2001), fgf8 (Furthauer et al., 1997), myoD (Weinberg

et al., 1996), papc (Yamamoto et al., 1998), and her1

(Sawada et al., 2000) has also been published. Whole-mount

in situ hybridization was performed as described (Shimizu et

al., 2002). BM purple (Roche) was used as a substrate for

alkaline phosphatase.

Morpholino oligonucleotides

The antisense morpholino oligonucleotides (MOs) were

generated by Gene Tools (LLC, Corvallis, OR, USA). For

wnt3a, the sequences were: wnt3aMO (recognizes the

splicing donor of the first intron), 5V-TGTTTTTACC-GAACGTCCAGCTTCA-3V; wnt3amisMO, 5V-TGTTaT-TACCcAAgGTCgAGgTTCA-3V (lower case letters

indicate mis-paired bases); wnt3aatgMO (binds to the

translational initiation site), 5V-GTTAGGCTTAAACTGA-CACGCACAC-3V. The sequences for the MOs for cdx1a

were: cdx1aMO, 5V-GTCCAGCAGGTAGCTCACGGA-CATT-3V and cdx1amisMO, 5V-GTgCAcCAGGTAcCT-CACcGAgATT-3V. The wnt8-1MO, wnt8-2MO, voxMO,

ventMO, vedMO, and cdx4MO were previously published

(Imai et al., 2001; Lekven et al., 2001; Shimizu et al., 2002)

(Davidson et al., 2003).The MOs were dissolved in and

diluted with 1� Danieau’s buffer and injected into the yolk

of one-cell-stage embryos (Nasevicius and Ekker, 2000).

SU5402 treatment

SU5402 (Calbiochem) at 10 mg/ml in DMSO was used

as a stock solution. Embryos were incubated with 200 AMSU5402 in the embryonic medium from the shield stage for

the indicated periods.

Cell proliferation analysis and TUNEL assay

Cell proliferation analysis in the posterior body was

carried out as reported (Marlow et al., 2004). Fixed embryos

were first stained by whole-mount in situ hybridization with

the paraxial protocadherin (papc) probe and Fast Red,

followed by immunochemistry with mouse anti-phosphohi-

stone H3 (Sigma) and an FITC-labeled anti-mouse IgG

secondary antibody. Images were taken by a Zeiss Pascal

laser scanning inverted microscope. Apoptotic cells were

identified by the TUNEL method using the DeadEnd

Fluorometric TUNEL System (Promega). Apoptotic cells

were visualized with alkaline phosphatase (AP)-conjugated

anti-fluorescein antibody and BM purple.

Results

Redundant role of Wnt3a and Wnt8 in body patterning

We obtained zebrafish wnt3a cDNA from a gastrula

cDNA library using an expression cloning strategy. In the


screening, we found that the injection of wnt3a RNA into

one-cell-stage embryos led to dorsalization and posteriori-

zation of the embryos (data not shown), a phenotype that is

indicative of hyper-activation of the Wnt canonical path-

way in zebrafish (Kelly et al., 1995). The sequence of

zebrafish wnt3a was identical to that of wnt3a that was

recently reported (Buckles et al., 2004). wnt3a is expressed

in the tail and dorsal neural tube from the early

segmentation stage (Buckles et al., 2004; Krauss et al.,

1992) (Fig. 1A). In addition, we found that wnt3a was first

detectably expressed in the blastoderm margin from the

early gastrula stage, and the expression in the blastoderm

margin continued during gastrulation and was confined to

the tailbud at the bud stage (Fig. 1A). This expression

pattern is similar to that of wnt8 (Kelly et al., 1995; Lekven

et al., 2001), suggesting a functional redundancy of Wnt3a

and Wnt8 in early embryogenesis.

To reveal the function of Wnt3a, we prepared two types

of antisense morpholino oligonucleotides (MOs) against the

translational initiation site (wnt3aatgMO, Table 1) and the

splicing donor site of the first intron (wnt3aMO) of the

wnt3a gene. Injection of wnt3aatgMO and wnt3aMO

inhibited the translation of GFP-tagged Wnt3a protein and

the splicing out of the first intron, respectively (Fig. S1).

However, injection of either wnt3aatgMO or wnt3aMO

alone elicited no significant abnormalities in early embryo-

genesis (Table 1), consistent with a recent publication

(Buckles et al., 2004). We next injected MOs against both

Fig. 1. Redundant role of Wnt3a and Wnt8 in early embryogenesis. (A) Expressio

stage (13 hpf); e, 17-somite stage (17 hpf). a, animal pole view with dorsal to the r

with anterior to the left. (B) Phenotype of the wnt3aMO- and/or wnt8MO-injected

were classified into three categories. Class I embryos had a ventrally bent tail str

enlarged head, and a short and twisted tail (number of the somites 20–30; average

structure (less than twenty somites; average 13.7 (n = 8)). The percentage of e

neuroectoderm marker otx2 (a, c, e) and the paraxial mesodermal marker spadeta

embryos injected with both wnt3aMO and wnt8MOs (wnt8-1MO and wnt8-2M

Arrowheads (a, c, e) indicate the posterior limits of the otx2 expression.

Wnt3a and Wnt8 into the embryos. The zebrafish wnt8

locus encodes a bicistronic mRNA, and the inhibition of

both of the open reading frames of wnt8 by MOs (wnt8-

1MO and wnt8-2MO, referred as wnt8MOs hereafter)

elicited anteriorization of the neuroectoderm and reduction

of the posterior body, as reported (Lekven et al., 2001)(Fig.

1B, Table 1). The coinjection wnt8MO and wnt3aMO or

wnt3aatgMO, but not the control MO (wnt3amisMO), led

to enlargement of the head and reduction or truncation of the

posterior body; these effects were more severe than those

induced by the injection of wnt8MOs alone (Fig. 1B and

Table 1). The expression of otx2, which marks the forebrain

and midbrain, expanded more posteriorly in the wnt3a/wnt8

morphant embryos than in the wnt8 morphant embryos

(Figs. 1Ca, c, and e). The expression of spadetail (spt),

which marks the prospective paraxial mesoderm, but not no

tail (ntl), was reduced in the wnt3a/wnt8 morphant

embryos. The extent of the reduction was greater than in

the wnt8 morphant embryos at the mid-to-late gastrula stage

(Figs. 1Cb, d, and data not shown), indicating that Wnt3a

and Wnt8 are dispensable for mesoderm induction but are

required for patterning of the mesoderm. The phenotypes of

embryos injected with both wnt3aMO and wnt8MOs

(wnt3a/wnt8 morphant embryos) were similar to those of

the embryos injected with a large amount of dickkopf1

(dkk1) RNA, which is a specific inhibitor of the Wnt

canonical pathway (Glinka et al., 1998; Hashimoto et al.,

2000; Shinya et al., 2000). All of these data indicate that

n of wnt3a. a, shield stage; b, 80% epiboly stage; c, bud stage; d, 8-somite

ight; b, dorsal view; c, d, lateral view with dorsal to the right; e, lateral view

pharyngula-stage (24 hpf) embryos. The phenotypes of the injected embryos

ucture and a normal number of somites (30–34). Class II embryos had an

27.4 (n = 13)). Class III embryos had an enlarged head and a truncated tail

mbryos in each class is shown in Table 1. (C) Expression of the anterior

il (spt) (b, d) at the 90% epiboly stage in wild-type embryos (wt, a, b), and

O, c, d) or wnt8MOs alone (e). Lateral views with dorsal to the right.

Table 1

Effects of wnt3aMO and wnt8MO on early embryogenesis

Morpholinos Dose (ng) Class I (%) Class II (%) Class III (%) Wild type (%) n

wnt3aMO 4.2 0 0 0 100 26

8.4 44 0 0 56 41

wnt3amisMO 8.4 0 0 0 100 36

wnt8-1MO + wnt8-2MO 2.1 + 2.1 31 69 0 0 36

4.2 + 4.2 22 78 0 0 46

wnt3aMO + wnt8-1MO + wnt8-2MO 2.1 + 2.1 + 2.1 0 44 56 0 39

4.2 + 4.2 + 4.2 0 10 90 0 80

wnt3amisMO + wnt8-1MO + wnt8-2MO 4.2 + 4.2 + 4.2 22 75 3 0 69

wnt3aatgMO + wnt8-1MO + wnt8-2MO 2.1 + 2.1 + 2.1 0 5 95 0 41

wnt3aMO (recognizing the splicing donor of the first intron), the control MO for wnt3aMO (wnt3amisMO), wnt3aatgMO (recognizing the translational

initiation site), wnt8-1MO, and wnt8-2MO were injected into one-cell-stage embryos in the indicated combinations, and the embryos were classified into three

categories (Classes I–III) by morphological inspection (Fig. 1B) at 24 hpf.


Wnt3a and Wnt8 function redundantly but play a major role

in body patterning in early embryogenesis. Therefore, we

further investigated the wnt3a/wnt8 morphant embryos,

mainly to elucidate the function of canonical Wnt signaling

in body patterning, especially in dorsalization and posterior

body formation.

Interaction between Wnt3a/Wnt8 and ventrally expressed

homeobox genes

In the wnt3a/wnt8 morphant embryos, the expression

patterns of goosecoid (gsc), floating head (flh) and

chordino (din) (Miller-Bertoglio et al., 1997; Stachel et

al., 1993; Talbot et al., 1995; Thisse et al., 1994), which

are expressed in the organizer region, were expanded

ventrally at the shield stage (Fig. 2A), and the notochord

was morphologically expanded (data not shown), indicat-

ing that the organizer and its derivatives were expanded

from the early gastrula stage. In contrast to these markers,

dkk1 expression in the dorsal organizer was strongly

reduced in the wnt3a/wnt8 morphant embryos (Figs.

2Ad, h). The promoter of zebrafish dkk1 contains a Tcf/

Lef-binding site, which is a target for the Wnt canonical

pathway (Shinya et al., 2000); therefore, dkk1 expression

might require the zygotic activation of Wnt signaling. dkk1

seems to be dispensable for the formation of axial

mesendoderm in the reduction or absence of Wnt3a and

Wnt8 function.

We and others have previously demonstrated that the

ventrally expressed homeobox genes vox, vent, and ved

function redundantly to restrict the domains of the dorsal

organizer and its derivatives (Imai et al., 2001; Kawahara et

al., 2000a,b; Shimizu et al., 2002). We therefore investigated

the relationship between the wnt genes and the ventrally

expressed homeobox genes. The expression of vox and ved is

excluded from the most dorsal blastoderm margin and the

dorsal yolk syncytial layer (Kawahara et al., 2000a; Melby et

al., 2000; Shimizu et al., 2002). vox and ved expression was

reduced, and the vox- and ved-negative domains were

expanded ventrally at the shield stage in the wnt3a/wnt8

morphant embryos (Figs. 2Ba, b, e, and f). Similarly, the

expression of wnt3a and wnt8 was reduced in the vox/vent/

ved morphant embryos from the mid-to-late gastrula stage

(Figs. 2Bc, d, g, and h), indicating reciprocal regulations

between wnt3a/wnt8 and vox/vent/ved during gastrulation.

To further elucidate the functional relationship between these

genes, we co-injected wnt3a/8MO and ved RNA (Fig. 2C).

Misexpression of ved elicited ventralization: the expansion of

ventral tail, loss of notochord, and defects in head formation

(Shimizu et al., 2002) (Fig. 2Cb). Injection of a low dose (100

pg) of ved RNA with wnt3a/8MO suppressed the head

formation (Fig. 2Cc). Co-injection of a high dose (500 pg) of

ved RNAwithwnt3a/8MO increased lethality of the embryos

before the pharyngula period, but the survived embryos had

defects in head and notochord formation (data not shown),

suggesting that ved functions downstream of wnt3a/wnt8 in

the restriction of the organizer. The data also imply that ved,

together with vox and vent, functions downstream of Wnt3a/

Wnt8 to restrict the dorsal organizer domain. Coinjection of

the high dose of ved RNAwith wnt3a/8MO rescued the tail

formation incompletely, suggesting that Wnt signaling

functions through factors in addition to ved-related genes in

posterior body formation.

Caudal-related genes cdx1a and cdx4 are regulated by

Wnt3a/Wnt8 signaling

We next examined the expression of genes that are

reported to be involved in the posterior body formation, in

the wnt3a/wnt8 morphant embryos. The expression of

bmp2b and bmp4 in the ventro-lateral region was reduced,

whereas the expression in the blastoderm margin (80%

epiboly stage, data not shown) and later the tailbud (bud

stage, Fig. 3A) was not significantly affected in the wnt3a/

wnt8 morphant embryos. The expression of cyclops (cyc)

in the axial mesendoderm was expanded in these embryos

(Fig. 3A). These changes in the bmp and cyc expression

were probably due to the expansion of the dorsal

organizer and/or dorsalization of the embryos. The

expression of these genes in the blastoderm margin and

tailbud was not significantly affected in the wnt3a/wnt8

morphant embryos.

Fig. 2. Interaction between the Wnt3a/8 signaling and the ventrally expressed homeobox genes vox, vent, and ved for restriction of the dorsal organizer domain.

(A) Expression of organizer genes in the wnt3a/wnt8 morphant embryos. The embryos received 4.2 ng each of wnt3aMO, wnt8-1MO, and wnt8-2MO (wnt3a/

8MO, wnt3a/wnt8 morphant embryos) and were fixed at the shield stage. The expression of goosecoid (gsc), floating head (flh), and chordino (din) was

expanded ventrally in the wnt3a/wnt8 morphant embryos compared with wild-type embryos (wt) or embryos receiving an injection of wnt3aMO (4.2 ng) alone

(data not shown). In contrast, the expression of dickkopf1 (dkk1) was strongly reduced in the wnt3a/wnt8 morphant embryos. a–c, e–f animal pole views with

dorsal to the right. d,h, Dorsal views. (B) Expression of vox and ved in the wnt3a/wnt8 morphant embryos, and expression of wnt3a and wnt8 in the vox/vent/

ved morphant embryos. The vox/vent/ved triple morphant embryos were prepared by injecting 0.3 ng each of voxMO, ventMO, and vedMO, as published

previously (Shimizu et al., 2002). a, b, e, f, shield stage, animal pole views with dorsal to the right. c, d, g, h, 80% epiboly stage, lateral views with dorsal to the

right. Arrowheads indicate the dorsal limit of the vox and ved expression. The expression of vox and ved was reduced on the dorsal side in the wnt3a/wnt8

morphant embryos. The expression of wnt3a and wnt8 in the blastoderm margin was reduced in the vox/vent/ved morphant embryos. (C) Ved is epistatic to the

Wnt3a/Wnt8 signaling. ved RNA (100 pg) was injected alone (b) or with wnt3aMO and wnt8MOs (c). The co-injection of ved RNA suppressed the head

enlargement but did not suppress the reduction in posterior body observed in the wnt3a/wnt8 morphant embryos.


We next examined the expression of the caudal-related

genes cdx1a and cdx4. In wild-type embryos, both cdx1a

and cdx4 were expressed in the blastoderm margin from the

early gastrula stage (Davidson et al., 2003) (data not shown

for cdx1a), in the posterior third of the embryo, which

includes mesoderm and ectoderm, at the mid-to-late gastrula

stages, and in the posterior somite/PSM at the segmentation

period (cdx4 was also expressed in the posterior neuro-

ectoderm, Figs. 3Ba–c, and h–j). In the wnt3a/wnt8

morphant embryos, the expression of both cdx1a and

cdx4 was reduced from the mid-to-late gastrula stage (Figs.

3Bd and k). The cdx1a expression was barely detectable at

the mid-segmentation stage (17 h post fertilization, hpf, Fig.

3Bf). Similarly, the cdx4 expression was strongly reduced in

the somite/PSM at the mid-segmentation stage, but the

expression in the posterior neuroectoderm was relatively

preserved, compared with the reduction in the somite/PSM

(Figs. 3Bl and m). Reduced expression of cdx1a and cdx4

was also observed in the embryos that received the dkk1

RNA injection (Figs. 3Bg and n). Misexpression of wnt3a

by plasmid injection led to the expansion of cdx1a and

cdx4, but did not elicit ectopic expression in the anterior

region of the embryo (Figs. 3Bo and p). These findings

indicate that cdx1a and cdx4 are regulated by Wnt3a/Wnt8

signaling in the zebrafish, but that other signaling pathways

may also be involved in their regulation.

Fig. 3. Tailbud expression of caudal-related genes cdx1a and cdx4, but not bmps or cyclops is dependent on the Wnt3a/Wnt8 signaling. (A) Expression of

bmp2 and bmp4 at the bud stage, and expression of cyclops (cyc) at the 80% epiboly stage in the wild-type (wt, a, b, c) and wnt3a/wnt8 morphant (wnt3a/8MO,

d, e, f) embryos. Expression of bmp2b and bmp4 in the ventro-lateral domains but not in the tailbud was reduced in the wnt3a/wnt8 morphant embryos.

Expression of cyc in the axial mesoderm was slightly expanded in the wnt3a/wnt8 morphant embryos. a, b, d, e, lateral views with dorsal to the right. c, f,

dorsal views. (B) Expression of cdx1a and cdx4 in the wnt3a/wnt8 morphant embryos and dkk1 RNA (25 pg)-injected embryos, and in embryos misexpressing

wnt3a. Expression of cdx1a and cdx4 was reduced in the wnt3a/wnt8 morphant embryos, compared with wild-type embryos, at the bud (a, d, h, k), 8-somite

(13 hpf, b, e, i, l), and 17-somite (17 hpf, c, f, j, m) stages. Expression of cdx1a and cdx4 was also reduced in the dkk1 RNA-injected embryos at 17 hpf (g, n).

Note that the expression of cdx4 in the posterior neuroectoderm (arrows) was less reduced than in the paraxial mesoderm (arrowheads) in the wnt3a/wnt8

morphant embryos. Ectopic expression of cdx1a and cdx4 was detected in the embryos injected with 6 pg of pCS2 + wnt3a plasmid DNA at the bud stage (o,

p, arrows). a, d, h, k, o, p, lateral views with dorsal to the right. b, e, i, l, tailbud views. c, f, g, l, m, n, lateral views with anterior to the left.



Cdx1a and Cdx4 function redundantly in posterior body

formation

To reveal the role of cdx1a and cdx4 in posterior

body formation, we inhibited the function of Cdx1a and

Cdx4 by MOs. Injection of cdx1aMO specifically

inhibited the translation of GFP-tagged Cdx1a protein

(Fig. S1), and cdx4MO used in this study was identical to

that previously published (Davidson et al., 2003). As

published, embryos that received the cdx4MO displayed

truncation of the posterior body, similar to embryos that

have a point or insertional mutation in the cdx4 gene

(kugelig/kgg) (Davidson et al., 2003; Golling et al.,

2002)(Fig. 4A, Table 2), while the cdx1aMO-injected

embryos did not show any significant abnormalities in

posterior body formation (Fig. 4A, Table 2). Coinjection of

cdx1aMO and cdx4MO into the embryos (cdx1a/cdx4

morphant embryos) elicited a severe truncation of the

posterior body, compared with the injection of cdx4MO

alone (Fig. 4A, Table 2), indicating redundant roles for

cdx1a and cdx4 in posterior body formation. The truncation

of posterior body structures was similar in the wnt3a/wnt8

and cdx1a/cdx4 morphant embryos (Fig. 4B). However, in

contrast to the wnt3a/wnt8 morphant embryos, the cdx1a/

cdx4 morphant embryos did not show expansion of the

organizer/axial mesendoderm (expansion of gsc), expansion

of the anterior neuroectoderm (head enlargement and

expansion of otx2), or reduction of the paraxial mesoderm

(reduction of spt) (Figs. 4B and C). These data indicate that,

although cdx1a and cdx4 are regulated by the Wnt3a and

Wnt8 signaling, they are involved only in the posterior body

formation and not in the other aspects of Wnt-mediated

body patterning.

Interaction between wnts, cdxs, and FGF signaling

The caudal-related gene Xcad3 is regulated by FGF

signaling in Xenopus embryos (Haremaki et al., 2003;

Northrop and Kimelman, 1994; Pownall et al., 1996). In this

study, we also examined regulation of the cdx genes by FGF

signaling. In embryos treated with SU5402 (Figs. 5C and

D), a specific inhibitor of the FGF receptor (Mohammadi et

al., 1997), or receiving an injection of a dominant-negative

Xenopus FGF receptor, XFD (Figs. 5E and F), the

expression of both cdx1a and cdx4 was reduced at the

early segmentation stage. In contrast, misexpression of fgf8

by a plasmid injection elicited expansion of the cdx1a and

cdx4 expression (Figs. 5I and J), indicating regulation of the

cdx genes by FGF signaling. Inhibition of both Wnt3a/Wnt8

by MOs and FGF signaling by SU5402 further reduced the

cdx1a and cdx4 expression (Figs. 5G and H), indicating that

the Wnt and FGF signaling cooperatively regulate the

expression of cdx1a and cdx4.

We next examined fgf expression in wnt3a/wnt8 and

cdx1a/cdx4 morphant embryos (Fig. 6). The expression of

fgf3 and fgf8 in the posterior region was reduced from the

bud stage in both the wnt3a/wnt8 and cdx1a/cdx4

morphant embryos. It remains to be elucidated whether

the Wnt/Cdx cascade directly controls the expression of

fgf3 and fgf8 or the reduction of fgf3 and fgf8 expression

is a secondary consequence of the reduction in posterior

tissue.

Regulation of the posteriorly expressed hox genes by

Wnt3a/8 and Cdx1a/4

Cdx proteins are known to bind directly to the regulatory

elements of the hox genes, which are located 5V to the hox

gene cluster and expressed posteriorly (Charite et al., 1998;

Isaacs et al., 1998; Subramanian et al., 1995), and the

expression of hoxa9a and hoxb7a is reduced in zebrafish

cdx4 mutant embryos (Davidson et al., 2003). We examined

the expression of hoxa9a and hoxb7a in the wnt3a/wnt8 and

cdx1a/cdx4 morphant embryos. hoxa9a expression was

reduced in the wnt3a/wnt8 morphant embryos and abro-

gated in the cdx1a/cdx4 morphant embryos (Figs. 7A, B,

and C), indicating that hoxa9a functions downstream of

Wnt3a/Wnt8 and Cdx1a/Cdx4. The misexpression of Wnt3a

by plasmid injection led to a slight ventral expansion of the

hoxa9a expression, but that was abrogated by the co-

injection of cdx1aMO and cdx4MO (Figs. 7D and E),

indicating that Cdx1a and Cdx4 are required for the Wnt-

mediated hoxa9a expression. We examined the role of FGF

signaling in Cdx-mediated hoxa9a expression. Misexpres-

sion of cdx1a RNA induced the expansion of hoxa9a

expression, but SU5402 inhibited Cdx1a-induced hoxa9a

expression (Figs. 7F–H). Expression of XFD similarly

inhibited Cdx1a-induced hoxa9a expression, but this was

restored by co-expression of constitutively active Ras

(CAras) (Figs. 7K–N), further implying that Ras-mediated

Fgf signaling is involved in Cdx-mediated hoxa9a expres-

sion. The misexpression of fgf8 by plasmid injection

induced the expansion of hoxa9a, while the co-injection

of cdx1aMO and cdx4MO inhibited FGF8-mediated

hoxa9a expression (Figs. 7I and J). These data indicate

that both Cdx1a/Cdx4 and FGF signaling are required for

the expression of hoxa9a. We obtained similar results for

hoxb7a (data not shown).

Wnt3a/Wnt8 and Cdx1a/Cdx4 are required for

morphogenesis of the posterior body

We examined cell proliferation and cell death in the

wnt3a/wnt8 and cdx1a/cdx4 morphant embryos by immu-

nohistochemistry using anti-phosphohistone H3 antibody

and TUNEL, respectively. We did not observe a significant

reduction in the cell proliferation in the PSM of the wnt3a/

wnt8 and cdx1a/cdx4 morphant embryos (Figs. 8Aa–d). The

mitotic indices (MIs) for wild-type (MI = 4.5; n = 4

embryos; 492 cells), wnt3a/wnt8 morphant embryos (MI =

5.4; n = 3 embryos; 553 cells), and cdx1a/cdx4 morphant

(MI = 3.6; n = 4 embryos; 745 cells) were not significantly

Fig. 4. Cooperation of Cdx1a and Cdx4 in formation of the posterior body. (A) Phenotypes of cdx1aMO and/or cdx4MO-injected pharyngula-stage (24 hpf)

embryos. The phenotypes were classified into three categories. In Class I embryos, the tail was extended but its size was reduced, and the number of somites

was not changed (30–34 somites). Class II embryos had a truncated tail structure (number of the somites 20–30, average 25.2 (n = 10)). Class III embryos had

no evident structure protruding from the yolk (no tail structure; less than twenty somites, average 10.3 (n = 12)). The percentage of embryos in each class is

shown in Table 2. (B) Comparison between the wnt3a/wnt8 and cdx1a/cdx4 morphant embryos. The phenotypes of wild-type (non-injected) control embryos

(wt, a, d), embryos receiving an injection of 4.2 ng of wnt3aMO and wnt8MOs (wnt3a/8MO, b, e), and embryos receiving an injection of 2.1 ng of cdx1aMO

and cdx4MO (cdx1a/4MO, cdx1a/cdx4 morphant embryos, c, f), at the bud stage (a–c, lateral views with dorsal to the right) and 17 hpf (d–f, lateral views, with

anterior to the left). Neither the wnt3a/wnt8 nor the cdx1a/cdx4 morphant embryos showed strong morphological abnormalities at the bud stage. At 17 hpf (17-

somite stage for the wild-type), both the wnt3a/wnt8 and cdx1a/cdx4 morphant embryos displayed a truncated tail, whereas only the wnt3a/wnt8 morphant

embryos had an enlarged head and eyes. (C) Expression of goosecoid (gsc) (a) at the shield stage, and otx2 (b) and spadetail (spt) (c) at the 90% epiboly stages

in the cdx1a/cdx4 morphant embryos. Expression of these genes was not significantly affected in the cdx1a/cdx4 morphant embryos, compared with the wild-

type embryos (Fig. 1). a, animal pole view with dorsal to the right. b, c lateral views with dorsal to the right.


different. We did not detect increased cell death in the

posterior body of wnt3a/wnt8 morphant embryos, but found

a slight increase in that of cdx1a/cdx4 morphant embryos at

7- to 8-somite stage (13 hpf). This increased cell death may

be partly involved in the reduction of the posterior body in

the cdx1a/cdx4 morphant embryos.

The wnt3a/wnt8 and cdx1a/cdx4 morphant embryos had

reduced numbers of somites (Figs. 1 and 4), which

prompted us to investigate somite formation in these

embryos. At 13 hpf (the 7-to-8-somite stage), the wnt3a/

wnt8 morphant embryos had a narrower paraxial meso-

derm than wild type, but a normal number of somites. At

this stage, the number of somites in the cdx1a/cdx4

morphant embryos was also normal (Figs. 8Ba–c). Both

the wnt3a/wnt8 and cdx1a/cdx4 morphant embryos showed

a reduced but normal pattern of her1 expression, which is

known to oscillate in the PSM (Sawada et al., 2000), at 13

hpf (Figs. 8Bg–i). However, at 17 hpf (the 17-somite stage

for wild-type embryos), the wnt3a/wnt8 and cdx1a/cdx4

morphant embryos showed no generation of her1 expres-

sion in the PSM or further segmentation (Figs. 8Bj–l),

resulting in a reduced number of somites (average number

of somites, wild type 17.6 (n = 10); wnt3a/wnt8 morphant

14 (n = 10); cdx1a/cdx4 morphant 10.4 (n = 8)). The

number of somites was not increased at 24 hpf in these

morphant embryos (wnt3a/wnt8 morphant 13.7 (n = 8);

cdx1a/cdx4 morphant 10.3 (n = 12)), indicating that

segmentation stopped by 17 hpf. wnt3a/wnt8 and cdx1a/

cdx4 morphant embryos had a shortened or no presomitic

mesodermal area at 17 hpf, which was evidenced by the

reduction or loss of adaxial myoD expression posterior to

the formed somites (Figs. 8Bd–f). These data indicate that

Table 2

Inhibition of cdx1a and cdx4 by MOs

Morpholinos Dose (ng) Class

I (%)

Class

II (%)

Class

III (%)

Wild

type

(%)

n

cdx1aMO 1 0 0 0 100 50

2.1 0 0 0 100 48

cdx1amisMO 2.1 0 0 0 100 20

cdx4MO 0.2 100 0 0 0 53

1 17 83 0 0 48

2.1 7 93 0 0 44

cdx1aMO +

cdx4MO

0.2 + 0.2 13 87 0 0 40

1 + 1 0 0 100 0 47

2.1 + 2.1 0 0 100 0 32

cdx1amisMO +

cdx4MO

2.1 + 2.1 0 90 10 0 99

cdx1aMO, the control MO for the cdx1aMO (cdx1amisMO), and cdx4MO

were injected in the indicated combinations, and the embryos were

classified into three categories (Class I–III) by morphology (Fig. 4A) at

24 hpf.


Wnt3a/Wnt8 and Cdx1a/Cdx4 are required for the for-

mation of posterior somites.

Fig. 5. Cooperation of FGF and Wnt signaling for the cdx1a and cdx4

expression. Expression of cdx1a and cdx4 at the 3-somite stage in wild-type

embryos (wt, A, B), SU5402 (an inhibitor of FGF receptors)-treated

embryos (C, D), a dominant negative Xenopus FGF receptor 1 (XFD) RNA

(500 pg)-injected embryos (E, F), SU5402-treated wnt3a/wnt8 morphant

embryos (G, H), and embryos receiving an injection of 6 pg of pCS2 + fgf8

(I, J). The cdx1a and cdx4 expression was slightly reduced in the SU5402-

treated embryos, and more strongly reduced in the embryos defective in

both Wnt3a/Wnt8 and FGF signaling. In contrast, expression of cdx1a and

cdx4 was expanded anteriorly in the fgf8-misexpressing embryos. Lateral

views with dorsal to the right.

Discussion

Role of canonical Wnt signaling in restriction of the

organizer domain

The wnt3a/wnt8 morphant embryos displayed reduced

paraxial mesoderm and expanded axial mesendoderm

(Figs. 1 and 8). Since the wnt3a/wnt8 morphant embryos

showed a dorsalized phenotype (expansion of chordino

and reduction of bmp2b in the lateral region, Figs. 2 and

3), the ventralization of mesoderm is not the cause of the

reduction of paraxial mesoderm. The expression of flh,

which represses the fate of the paraxial mesoderm and is

required for the establishment of the axial mesendoderm

(Amacher and Kimmel, 1998; Talbot et al., 1995), was

expanded at the shield stage (Fig. 2), and the expression of

spadetail, which is required for the paraxial mesoderm

formation (Ho and Kane, 1990; Kimmel et al., 1989), was

reduced at the mid-to-late gastrula stage in the wnt3a/wnt8

morphant embryos (Fig. 1), indicating that axial mesoderm

rather than paraxial mesoderm expands at the early

gastrula stage in these embryos. We found that the

expression of vox and ved was regulated by the redundant

activity of Wnt3a and Wnt8, and that misexpression of ved

inhibited the expansion of the axial mesoderm in the

wnt3a/wnt8 morphant embryos (Fig. 2). These data

suggest that vox, vent, and ved function downstream of

Wnt signaling to restrict the dorsal organizer/axial mesen-

doderm domain. This is consistent with a recent report

showing that vox and vent are required for Wnt8-mediated

repression of axial mesendoderm (Ramel and Lekven,

2004). Our data indicate that ved and wnt3a also

participate in the restriction of the organizer and its

derivatives. Both wnt8 and the ventrally expressed homeo-

box genes vox, vent, and ved are negatively regulated by

the transcriptional repressor Bozozok/Dharma (Fekany et

al., 1999; Kawahara et al., 2000a; Melby et al., 2000;

Shimizu et al., 2002). It is possible that Bozozok/Dharma

inhibits the expression of the Wnt signal in the dorsal

organizer domain and thereby represses the expression of

vox, vent, and ved. Alternatively, the Wnt signal may be

required for the ventro-lateral expression of vox, vent, and

ved, with Bozozok/Dharma repressing their expression in

the dorsal organizer independently of the Wnt signaling.

Fig. 6. Regulation of fgf3 and fgf8 expression by Wnt3a/Wnt8 and Cdx1a/Cdx4. (A–F) Expression of fgf3 and fgf8 in the tailbud (bud stage) was reduced in the

wnt3a/wnt8 (B, E) and cdx1a/cdx4 (C, F) morphant embryos (A, D, wild-type control). Note that in the wnt3a/wnt8 morphant embryos, fgf3 expression in the

forebrain was expanded posteriorly, and in the hindbrain it was shifted posteriorly. Similarly, fgf8 expression in the midbrain–hindbrain boundary (MHB) was

shifted posteriorly in the wnt3a/wnt8 morphant embryos. These changes in the fgf3 and fgf8 expression were probably due to anteriorization of the

neuroectoderm in the wnt3a/wnt8 morphant embryos. Lateral views with dorsal to the right. (G–L) Reduction of fgf3 and fgf8 expression in the tail region of

the wnt3a/wnt8 (H, K) and cdx1a/cdx4 (I, L) morphant embryos (G, J, control). 13 hpf (8-somite stage for wild-type embryos). Posterior (tail) views with

dorsal to the top. (M–R) Reduction or loss of fgf3 and fgf8 expression in the tail of wnt3a/wnt8 (N, Q) and cdx1a/cdx4 (O, R) morphant embryos (M, P,

control). 17 hpf (17-somite stage for wild-type embryos). Lateral views with anterior to the left.


Further studies on the regulation of wnt3a/wnt8 and the

ventrally expressed homeobox genes will be required to

address this issue.

The wnt3a/wnt8 morphant embryos displayed reduced

paraxial mesoderm, whereas vox/vent/ved morphant

embryos have normal or expanded paraxial mesoderm

(Shimizu et al., 2002), suggesting that Wnt-dependent but

Vox/Vent/Ved-independent regulation of paraxial mesoderm

formation. spadetail is one candidate gene that has been

suggested to mediate Wnt3a/Wnt8 signaling in paraxial

mesoderm formation.

Redundant role of Wnt3a and Wnt8 in posterior body

formation

In mouse, wnt3a plays an essential role in posterior

body formation (Takada et al., 1994). However, in

zebrafish, inhibition of Wnt3a alone by MOs did not

elicit any significant abnormalities in posterior body

formation (Table 1), suggesting wnt3a is dispensable for

this process in zebrafish. There are several possible

explanations for the different roles played by Wnt3a in

the mouse versus zebrafish. First, Wnt3a activity may not

have been completely inhibited by the MO, but some

activity was retained, resulting in a wnt3a hypomorphic

phenotype. Second, there might be another wnt3a gene

(or another coding frame) in zebrafish, as there is for

wnt8 (Lekven et al., 2001). Third, in zebrafish, Wnt3a

may function redundantly with Wnt8, with the loss of

Wnt3a producing the phenotype only in the absence of

Wnt8 activity. We favor the third explanation, as the

wnt3a/wnt8 morphant embryo displayed a phenotype

similar to that of embryos injected with a high amount

of dkk1 RNA (Fig. 3) (Hashimoto et al., 2000), which is

thought to inhibit most canonical Wnt signaling activity.

If there are unidentified canonical Wnt(s) expressed in

early embryogenesis, it is likely that they only make a

small contribution to the posterior body formation in

zebrafish.

We sought the genes that function downstream of

Wnt3a/Wnt8 in posterior body formation. There is a set

of genes that are reported to be involved in posterior body

formation, including the wnt8, bmps, and nodal-related

genes, which confer the tail-organizing activity in zebrafish

(Agathon et al., 2003). Slack and colleagues have proposed

that Bmp initiates the program of tail formation in Xenopus

embryos (Beck and Slack, 1998, 1999; Beck et al., 2001).

We found a reduction of bmp2b and bmp4 in the ventro-

lateral region but not in the tailbud in the wnt3a/wnt8

morphant embryos at the bud stage. The expression of cyc

was not reduced, but rather was expanded in the axial

mesendoderm (Fig. 3), and another nodal-related gene,

squint, is not expressed in the tailbud later than the mid-

gastrula stage (Erter et al., 1998; Rebagliati et al., 1998a).

These data indicate that bmps or nodal-related genes do not

function downstream of Wnt3a/Wnt8 in the prospective

posterior body region. In contrast, we detected a reduction

in cdx1a and cdx4 expression from the mid-gastrula stage

Fig. 7. Involvement of Wnt, Cdx1a/4, and FGF in hoxa9a expression. Expression of hoxa9a at the 3-somite stage (A for the wild-type embryos). Expression of

hoxa9a was reduced in the wnt3a/wnt8 morphant embryos (B) and it was not detected in the cdx1a/cdx4 morphant embryos (C). Although 6 pg of pCS2 +

wnt3a plasmid injection led to increased hoxa9a expression (D), no hoxa9a expression was detected in the wnt3a-injected cdx1a/cdx4 morphant embryos (E).

The hoxa9a expression was expanded in the cdx1a RNA (25 pg)-injected embryos (F), but was reduced in the SU5402-treated cdx1a RNA-injected embryos

(H), as in the SU5402-treated non-injected embryos (G). The hoxa9a expression was also expanded in the 6 pg of pCS2 + fgf8-injected embryos (I), but was

not detected in the embryos receiving injections of both pCS2 + fgf8- and cdx1a/4MO (J). The hoxa9a expression was reduced in the XFD RNA (500 pg)-

injected (K), and XFD (500 pg) and cdx1a RNA (25 pg)-injected embryos (L). Co-injection of RNA for a constitutively active Ras (RasV12, CAras, 250 pg)

with XFD (500 pg) and cdx1a RNA (25 pg) restored the expression of hoxa9a (N). (M) Expression of hoxa9a in the CAras RNA-injected embryos. Lateral

views with dorsal to the right.


(Fig. 3), indicating that the cdx genes function downstream

of Wnt3a/Wnt8.

Regulation of cdx1a and cdx4 by Wnt3a/Wnt8

cdx1, but not cdx2 or cdx4, is down-regulated in wnt3a-

deficient mouse embryos, which have defects in the

posterior body (Ikeya and Takada, 2001), suggesting that

Cdx1 has a role in posterior body formation in mouse.

However, cdx1-null mouse embryos do not have defects in

the posterior body (Subramanian et al., 1995). Here, we

showed that the expression of cdx1a and cdx4 is under the

control of Wnt3a and Wnt8 signaling (Fig. 3), and that

Cdx1a and Cdx4 were required for posterior body for-

mation. These data raise the question of whether the role of

the Wnt canonical pathway in regulating the cdx genes is

different between mouse and zebrafish. One explanation is

that wnt8, wnt3, or other wnt(s) function redundantly with

wnt3a to regulate cdx1 and cdx4 in mouse, as we found in

zebrafish. Alternatively, other signals, such as FGF signal-

ing, might regulate cdx1a and cdx4. The mouse cdx1 gene

has a Tcf/Lef-binding site in its promoter region, which is

involved in cdx1’s expression in the intestine and posterior

body (Lickert et al., 2000; Prinos et al., 2001). Similarly, we

found Tcf/Lef-binding sites in the promoter region of

zebrafish cdx1a and cdx4 (data not shown). Thus, cdx1a

and cdx4 may be regulated by the canonical Wnt signaling

directly, and further experiments, such as promoter analysis

in transgenic fish and chromatin immunoprecipitation

analysis, will clarify this point. In the case of cdx4, its

expression in the posterior paraxial mesoderm was strongly

reduced but retained in the posterior neuroectoderm (Fig. 3),

indicating that the posterior neuroectoderm expression was

regulated by signals other than Wnt3a and Wnt8. We found

that FGF signaling was also involved in the expression of

cdx1a and cdx4 and that the inhibition of the both Wnt and

FGF signaling strongly reduced the cdx1a and cdx4

expression (cdx4 expression was reduced in the posterior

neuroectoderm) (Fig. 5). These data indicate that early

cooperation between Wnt and FGF signals controls the cdx

genes in the posterior body. Xcad3, which is a Xenopus

ortholog of cdx4, contains combinatory sites for Ets and

Tcf/Lef binding in its intronic regulatory elements (Harem-

aki et al., 2003), which are required for FGF-mediated

Xcad3 expression. It is tempting to speculate that the Wnt

and FGF signals are integrated into a common transcrip-

tional complex that controls the expression of cdx1a and

cdx4.

The wnt3a/wnt8 and cdx1a/4 morphant embryos dis-

played a similar phenotype: posterior body truncation and

Fig. 8. The Wnt-Cdx cascade is involved in morphogenesis of the posterior body. (A) Cell proliferation and cell death was not strongly impaired in the in

the wnt3a/wnt8 (c, f) and cdx1a/cdx4 morphant embryos (d, g) (b, e, control embryos). The morphant embryos displayed the Class III phenotype. The

embryos were fixed at 17 hpf and stained with the paraxial mesoderm marker papc probe (red) and with anti-phosphohistone H3 antibodies (green). (Aa)

Schematic representation of the stained embryos and (Ab–d) confocal images of representative embryos. Note that the number of phosphoH3-positive cells

was not significantly different among the wild-type, wnt3a/wnt8-morphant, and cdx1a/cdx4 morphant embryos. (Ae–g) The embryos were fixed at 13 hpf

and subjected to Tunel staining. (B) Impairment of the posterior somite formation. Expression of myoD at 13 hpf (a–c, gata2 was also stained) and 17 hpf

(d–f). Expression of her1 at 13 hpf (g–i) and 17 hpf (j–l). (a–f) dorsal views and (g–l) posterior views. her1 expression was reduced but displayed normal

oscillation in the wnt3a/wnt8 and cdx1a/cdx4 morphant embryos at the early segmentation stage, which had normal anterior somites. At the mid-to-late

segmentation periods, her1 expression was strongly reduced or abrogated in the wnt3a/wnt8 and cdx1a/cdx4 morphant embryos, and segmentation stopped

for the posterior somites.


reduction of the genes expressed in the posterior body,

strongly indicating that wnt3a/wnt8 and cdx1a/cdx4 function

in a linear cascade in posterior body formation (Fig. 4).

However, the reduction of cdx1a/cdx4 expression in the

wnt3a/wnt8 morphant embryos may not explain all the

phenotypes of the posterior body observed in them. The

wnt3a/wnt8 morphant embryos, but not the cdx1a/cdx4

morphant embryos, showed expanded chordino expression

and reduced bmp expression (Figs. 2 and 3). The Bmp

ventralizing signal is involved in the movements of the

ventral cells to the ventral tail (posterior) region (Myers et al.,

2002a). Consistent with this, some of the wnt3a/wnt8

morphant embryos had defects in ventral tail fin formation

or had a curled and shortened tail (Classes I and II, Fig. 1,

Table 1), which are also observed in mutant embryos

defective in Bmp signaling (Connors et al., 1999; Dick et

al., 2000; Hild et al., 1999; Kishimoto et al., 1997; Mintzer et

al., 2001; Mullins et al., 1996). Therefore, it is likely that the

reduction in Bmp signaling also contributed to the abnormal

tail phenotypes of the wnt3a/wnt8 morphant embryos. Since

the expression of cdx1a and cdx4 was not reduced in snail

house/bmp7 mutant embryos (data not shown), Bmp signal-

ing is not likely to be involved in cdx expression.

Furthermore, wnt3a/wnt8 morphant but not cdx1a/cdx4

morphant embryos showed reduction of the spt expression

(Fig. 4). Therefore, the T-box genes spt and possibly tbx6

(Szeto and Kimelman, 2004) are also likely involved in Wnt-

but not Cdx-mediated posterior body formation.

FGF signaling is required for Cdx-mediated hox expression

Cdx is a transcriptional activator that binds to specific

elements in some of the hox cluster genes (Charite et al.,

1998; Isaacs et al., 1998; Subramanian et al., 1995). We first

speculated that Cdx1a/4 does not require other signals to

activate the downstream hox genes. The expression of

hoxa9a and hoxb7a is reduced in cdx4/kugelig mutant

embryos and was abrogated in the cdx1a/cdx4 morphant

embryos (Davidson et al., 2003)(Fig. 7), further confirming

that the expression of these hox genes strictly depends on

Cdx1a/Cdx4 function. Unexpectedly, we found that FGF

signaling was required for the Cdx-dependent hoxa9a

expression, but that FGF was not sufficient to induce hoxa9a

(Fig. 7). Therefore, we find that the Cdx1a/Cdx4 and FGF

signals are integrated to elicit expression of the hox genes.

Tail formation by the Wnt-Cdx cascade

Our data demonstrate that the Wnt-Cdx cascade plays an

important role in posterior body formation. To address how


the Wnt-Cdx cascade controls the morphogenesis of the

posterior body, we investigated the behavior of the

progenitor cells for the posterior body (Fig. 8). We did not

find a significant reduction in cell proliferation in the PSM

in either wnt3a/wnt8 or cdx1a/cdx4 morphant embryos (Fig.

8). We found a slight increase of cell death in the posterior

body in the cdx1a/cdx4 morphant embryos, but not in the

wnt3a/wnt8 morphant embryos (Fig. 8), suggesting that

apoptosis is partly involved in the loss of the posterior body

in the cdx1a/cdx4 morphant embryos. However, the

dramatic reduction of posterior body structures in the

wnt3a/wnt8 and cdx1a/cdx4 morphant embryos cannot be

explained by cell proliferation and cell death. We considered

the possibility that abnormal cell fate changes might take

place during gastrulation in these embryos. We found no

morphological abnormalities during gastrulation, and tail-

bud formation appeared normal in the wnt3a/wnt8 and

cdx1a/cdx4 morphant embryos (Fig. 4). However, the

expression domains of fgf3 and fgf8 were reduced in the

tailbud of the wnt3a/wnt8 and cdx1a/cdx4 morphant

embryos (Fig. 6), suggesting that the functional (but not

morphological) tailbud might be smaller in these morphants.

A portion of the tailbud progenitor cells might not

contribute to the functional tailbud in the wnt3a/wnt8 and

cdx1a/cdx4 morphant embryos. It is also possible that cell

movements associated with the posterior body formation

may be partly defective in the wnt3a/wnt8 and cdx1a/cdx4

morphant embryos, as reported in knypek and pipetail/wnt5

mutant embryos, which have defects in the non-canonical

Wnt signals (Marlow et al., 2004). Precise fate mapping and

tracing of the tail bud progenitor cells should answer this

question.

Somite formation by Wnts and Cdxs

It was previously reported that wnt3a plays an important

role in the segmentation clock in the mouse (Aulehla et al.,

2003). axin2, which encodes a negative regulator of the Wnt

canonical pathway, shows an oscillating expression pattern

in the PSM. Wnt3a is proposed to be involved in the

oscillation of Notch activity in the PSM. We found that the

wnt3a/wnt8 and cdx1a/cdx4 morphant embryos showed

normal oscillation of her1 expression at the early segmen-

tation stage, but failed to generate her1 expression at the

mid-segmentation stage (Fig. 8). These data suggest that

Cdx1a and Cdx4 mediate the Wnt3a/Wnt8 signaling to

control the segmentation clock in the PSM during the

process of posterior segmentation.

FGF activity in the PSM is proposed to determine the

position of the wave front where the oscillating genes stop

their sweep and the somite boundary is established (Holley

and Takeda, 2002; Saga and Takeda, 2001; Sawada et al.,

2001). In this model (the wave front model), high FGF

activity is required for the wave of oscillating genes to

sweep, and the wave stops where the FGF activity is

reduced to the threshold level. The expression of fgf3 and

fgf8 was reduced in the wnt3a/wnt8 and cdx1a/cdx4

morphant embryos (Fig. 6). The FGF activity in the PSM

might be lower than the threshold level in these embryos,

and might result in the disturbance of the segmentation

clock. A similar role in establishing the wave front has also

been proposed for Wnt3a in mouse (Aulehla and Herrmann,

2004; Aulehla et al., 2003), and the redundant activity of

Wnt3a and Wnt8 may also contribute to the oscillation of

gene expression in zebrafish.

In summary, the redundant activity of Wnt3a/Wnt8

controls a number of downstream genes, which function

in the individual processes of body patterning. Ventrally

expressed homeobox genes and the cdx genes mediate

the Wnt signals for the restriction of the organizer and

posterior body formation, respectively. Our data shed

light on the mechanisms by which a given signaling

molecule controls several different processes of vertebrate

development.

Acknowledgments

We thank Drs. R. T. Moon, B. Thisse, C. Thisse, D. J.

Grunwald, H. Okamoto, H. Takeda, M. Nikaido, N.

Ueno, M. Mishina, M. Halpern, and M. Rebagliati for

providing the DNA samples, Drs. C. Thorpe, R. T. Moon,

and L. I. Zon for sharing unpublished results, and Ms. C.

Hirata-Fukae and H. Akiyama for fish care and technical

assistance. This work was supported by Grants-in-Aid for

Scientific Research from the Ministry of Education,

Science, Sports and Technology (15570184 to T. S.,

and 13138204 to M. H.) and by a grant from RIKEN (to

M. H.).

Appendix A. Supplementary data

Supplementary data associated with this article can be

found, in the online version, at doi:10.1016/j.ydbio.

2004.12.007.

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Documents

Interaction of Wnt and caudal-related genes in zebrafish posterior body formation