Developmental Biology 244, 267–277 (2002)
doi:10.1006/dbio.2002.0589, available online at http://www.idealibrary.com on

Axial Protocadherin Is a Mediator of Prenotochord
Cell Sorting in Xenopus
Hiroki Kuroda,* Masafumi Inui,† Kaoru Sugimoto,* Tadayoshi Hayata,*and Makoto Asashima* ,‡ ,1*Department of Life Sciences, ‡CREST Project, Graduate School of Arts and Sciences,
University of Tokyo, 3-8-1 Komaba, Meguro-ku, Tokyo, 153-8902, Japan;and †Department of Biological Science, Graduate School of Science,
University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-8654, Japan

Prenotochord cell sorting is regarded as one of the first cell sorting events in early chordate development. We recently
demonstrated that this sorting event occurs in vitro, although the mediator of this activity remains unidentified. Herein, we
report the isolation of a full-length cDNA clone of Axial protocadherin (AXPC), the homologue of human protocadherin-1(PCD1). AXPC encodes a transmembrane protein (AXPC) that is expressed exclusively in the notochord at the neurula stage
and in the pronephros, somites, heart, optic vesicle, otic vesicle, and distinct parts of the brain at the tailbud stage. Cell
dissociation and reaggregation assays and in vivo microinjection experiments demonstrated that cells overexpressing amembrane-tethered form of AXPC (MT-AXPC) acquired the same adhesive properties as prenotochord cells. Moreover,microinjection of either mRNA encoding the dominant negative form of AXPC (DN-AXPC) or morpholino oligonucleotides
interferes with the sorting activity of prenotochord cells and normal axis formation. This study suggests that AXPC isnecessary and sufficient for prenotochord cell sorting in the gastrulating embryo, and may also mediate sorting events laterin development. © 2002 Elsevier Science (USA)Key Words: cadherin; protocadherin; AXPC; PAPC; Xenopus; sorting; adhesive properties.

INTRODUCTION
The notochord is one of the most important signaling
centers in early chordate development. The anterioposterior and dorsoventral axes in vertebrates are regulated inpart by the organizer region of the embryo (Hamburger,
1988; Holtfreter, 1988; Spemann, 1938; Spemann and
Mangold, 1924). The majority of the organizer parts, together, form the embryological notochord, which inducescell identity along the dorsoventral (Hatta et al., 1991;
Krauss et al., 1993; Roelink et al., 1994; Yamada et al.,1991) and anterioposterior axes of the neural tube
(Hemmati-Brivanlou et al., 1990), and along the dorsoventral axis of the somites (Dietrich et al., 1993; Halpern et al.,
1993; Pourquie et al., 1993). The notochord also plays a rolein the specification of cardiac left–right orientation in
Zebrafish and Xenopus (Danos and Yost, 1996) and in the1
To whom correspondence should be addressed. Fax: ⫹81-35454-6993 (Office); ⫹81-3-5454-4330 (Lab). E-mail: asashi@bio.c.utokyo.ac.jp.

0012-1606/02 $35.00© 2002 Elsevier Science (USA)All rights reserved.

induction of pancreatic tissue following repression ofendodermal signaling in chick embryos (Hebrok et al.,
1998; Kim et al., 1997).
One of the most important roles of the notochord inchordate development may be in providing structural support for the embryo. This is achieved by elongating andstraightening the embryo from gastrula to tailbud stage
using a thread of notochord in the dorsal region derived
from a part of the Spemann’s Organizer (Adams et al., 1990;
Spemann, 1938). This dynamic movement from organizer
to notochord depends not only on convergent extension but
also on prenotochord cell sorting. When axial mesodermal
cells, including the prenotochord cells, and other fated cells
are brought together to form a mixed aggregate, the axial
mesodermal cells have to move to the central region of thehemisphere (Townes et al., 1955, Kuroda et al., 1999). We
propose that an adhesive molecule is required for thesorting of these axial mesodermal cells.
It is unlikely that similar cell types aggregate via heterophilic binding. We therefore propose that axial mesoderm
cell sorting is achieved via homophilic binding, and ad-

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vance the Ca 2⫹-dependent cadherins as candidates to mediate this activity. Cadherin family members play importantroles in specific cell– cell adhesion events. Their expression
appears to be tightly regulated during development andeach tissue or cell type shows a characteristic pattern of
cadherin expression (Nose et al., 1988; Takeichi, 1991).
Among the cadherin protein family, E-cadherin was firstdescribed as L-CAM in chicken (Gallin et al., 1983) and as
uvomorulin in mouse (Schuh et al., 1986). Soon after theidentification of E-cadherin, P-cadherin and N-cadherin
were characterized (Miyatani et al., 1989; Nose and Takeichi, 1986). There are approximately 50 different cadherins
expressed across both vertebrates and invertebrates (Nollet
et al., 2000). Generally, cadherins containing one signal
peptide (SP), four extracellular (EC) domains, one membrane proximal extracellular domain (MPEC), one transmembrane (TM) domain, one membrane proximal conserved domain (MPCD), and a catenin-binding domain(CBD) are known as “classical cadherins” (Fig. 1B, 1). Allother cadherins that do not follow this configuration are
called “protocadherins.” Recently, two novel protocadherin
genes were identified in the early Xenopus embryo (Kim etal., 1998). These were Paraxial protocadherin (PAPC), an
adhesion molecule expressed in Spemann’s organizer andparaxial mesoderm (somites) that mediates cell movement
during gastrulation, and a partial sequence of Axial protocadherin (AXPC), which is expressed in axial mesoderm
notochord. PAPC plays a role in paraxial mesodermal cellsorting and convergent extension during Xenopus gastrulation. When the partial AXPC was fused in frame with an
extracellular (EC) domain and transmembrane domain of
PAPC, the hybrid protein exhibited cell adhesion activity.
We therefore investigated AXPC as a candidate mediator of
prenotochord cell sorting.
We report here the isolation of a full-length AXPC clone.

FIG. 1. Structure of the AXPC protein. (A) Amino acid sequenceof AXPC. Regions highlighted in red and green demonstrate thesignal peptide and the transmembrane domain, respectively. Blackline indicates the position of the border between each domains. (B)Protein structures of the major cadherin family members, and thetwo protocadherins. 1, Classical cadherin structure. 2, Paraxial
protocadherin (PAPC, Kim et al., 1998). 3, Axial protocadherin(AXPC), this study. 4, Our constructed membrane-tethered form of
AXPC (MT-AXPC). 5, Our constructed soluble dominant-negative
form of AXPC (DN-AXPC). 6, Axial protocadherin fragment previously isolated by Kim et al. (1998). 7, The region of our isolatedmouse AXPC fragment. SP, signal peptide. EC, extracellular cadherin repeats. MPED, membrane-proximal extracellular domain.
TM, transmembrane domain. HYD, hydrophilic domain. MPCD,
membrane-proximal conserved domain. CBD, catenin-binding domain. CPD, cytoplasmic domain. (C) Amino acid alignment of
Xenopus AXPC (X-AXPC), mouse AXPC (M-AXPC), and the human homologue of AXPC, protocadherin-1 (H-PCD1). The identities between Xenopus–mouse, Xenopus– human and mouse–
human were 75, 74, and 96%, respectively.

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Prenotochord Cell Sorting in Xenopus

This is the homologue of human protocadherin-1. We
demonstrate that this molecule functions as a mediator of
prenotochord cell sorting, and show that AXPC may participate in the many sorting events of later stage embryos.

MATERIALS AND METHODS
Dissociation and reaggregation assay
Embryos at the two- or four-cell blastomere stage were coinjected with 10 nl of 1% Texas red-dextran-amine (TRDA) or 1%
fluorescein-dextran-amine (FDA) lineage tracers, and control (water) or protocadherin (AXPC or PAPC) mRNA (see Fig. 3A, Method2). At stage 9, 2 h before gastrulation, animal cap explants were
excised and transferred to Ca 2⫹- and Mg 2⫹-free Steinberg’s solution
containing 0.1% bovine serum albumin (CMFS-BSA). The cells of
the inner cell layer detached in approximately 10 min. The outerlayer cells were removed and transferred to CMFS-BSA supplemented with 1 ng/ml activin, incubated for 1 h, and washed threetimes in 100% Steinberg’s solution. These treatments were essentially as described by Green et al. (1994, 1997). Dissociated inner
layer cells from five TRDA-labeled animal caps were thoroughly
mixed with those from five FDA-labeled animal caps, transferred toa 1.5-ml tube containing Steinberg’s solution, and reaggregated bycentrifugation at 100g for 5 s. The cell mixture was washed again
and then incubated for 10 –72 h. Embryo staging was carried out
according to Nieuwkoop and Faber (1967).

Cloning of Full-Length AXPC and PAPC

FIG. 2. Expression of AXPC. (A) Temporal expression analyzed by
RT-PCR. Low levels of AXPC expression were detected before
gastrulation. Expression levels increased at stage 10, with highlevels of expression being maintained by stage 38. (B) AXPC was
expressed exclusively in the dorsal marginal zone at stage 10. V,
ventral marginal zone. D, dorsal marginal zone. L, lateral marginalzone. no, notochord. so, somite. (C) Whole-mount in situ hybridization analysis at late neurula (stage 18). AXPC is expressed in the
notochord region at stage 18. Ant., anterior. Post., posterior. (C⬘)
Section of (C) clearly shows the AXPC expression in notochord. (D)At stage 28, AXPC was expressed strongly in the pronephros region(pn) and weakly in the somites (so) and the heart (white arrowhead).
(D⬘) Enlarged view of (D) shows the expression of AXPC in the optic
vesicle (op), otic vesicle (ot), and some parts of central nervous
system (white asterisks). (E) AXPC expression in activin-treatedanimal caps. Animal caps were treated with 0 –500 ng/ml of activinfor 1 h, then cultured for 5 h. High AXPC expression was detected
in 50 or 100 ng/ml activin-treated animal caps, and low levels of
AXPC expression were detected in 10 or 500 ng/ml activin-treatedanimal caps. Note: Concentrations of 50 –100 ng/ml are known tomost effectively induce notochord in this animal cap assay(Asashima et al., 1990). (F) AXPC expressions in activin-treateddissociation-and-reaggregation animal caps (Fig. 3A, Method 1).

A stage 12 Xenopus gastrula cDNA library was screened withfragments of Xenopus AXPC and PAPC cDNA clones obtainedusing the following primers and cycling conditions: AXPC (60°C,
30 cycles; forward, 5⬘-ATCGTGGCTGTTGACTCTGG-3⬘; reverse, 5⬘-GTAATGAAGCTGAGCGTTGG-3⬘); PAPC (60°C, 30
cycles; forward, 5⬘-CGTCTAGCAGATACCAAGAATTC-3⬘; reverse, 5⬘-GCTAGTTAGGTGACAGGACAATG-3⬘). Full-length
cDNA clones for both AXPC and PAPC were cloned into pBluescript vector. The following primers and cycling conditions wereused to isolate partial mouse AXPC cDNA clones: M-AXPC (60°C,
30 cycles; forward, 5⬘-ACACTGGACTTATCACCGTGC-3⬘; reverse, 5⬘-TAGACGTCAATGACAACGCC-3⬘).

RT-PCR Assays
RT-PCR was performed by using the following primers and cycling
condition: AXPC (58°C, 28 cycles; forward, 5⬘-AAGGAAGCATT-

Lanes 1– 4 indicate AXPC expression in the reaggregate treatedwith activin for 1 h, reaggregated, and cultured for 5 h. Very highexpression levels of AXPC were detected in the reaggregates treatedwith 1 ng/ml activin. No expression was detected in the othersamples. Note: A concentration of 1 ng/ml induces notochord onlyin this dissociated and reaggregated animal cap assay (Kuroda et al.,1999). Lanes 5–9 show AXPC expression in dissociated cells with 1
ng/ml activin-treatment. Low AXPC expression was detected at 30(lane 6) and 45 min (lane 7) after the initiation of activin treatment,
and high AXPC expression was detected at 60 (lane 8) and 120 min(lane 9) after the initiation of activin treatment.

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Kuroda et al.

ATGGCCTCC-3⬘; reverse, 5⬘-TTGCTTCGCTCATACTCTGG-3⬘);
ODC (55°C, 25 cycles; forward, 5⬘-GTCAATGATGGAGTGTATGGATC-3⬘; reverse, 5⬘-TCCATTCCCTCTCCTGAGCAC-3⬘).

Gene Construction
The primers T3 and 5⬘-CCGGCCTCGAGGGACTTCTGCAAACCAGT-3⬘ were used to construct a membrane-tethered form of
AXPC (pCS2-MT-AXPC) (50°C, 25 cycles), and T3 and 5⬘-CCGGCCTCGAGAACAATTACTGGGGCATT-3⬘ were used to construct a dominant-negative secreted form of AXPC (pCS2-DN-AXPC)(50°C, 25 cycles). The mutant genes were cloned into the EcoRIXhoI
sites of pCS2⫹. Morpholino oligo, 5⬘-GCAGTTGCTGTCCTCTTCATTGTTA-3⬘, was designed by Gene Tools, LLC Co., and thecontrol morpholino oligo, 5⬘-AAACCCGGGTTTACG-3⬘ was provided by Gene Tools, LLC Co.

RESULTS
Cloning of AXPC cDNA in Xenopus and Mouse
An AXPC fragment was amplified by PCR using primers
designed to hybridize to the partial AXPC clone previously
described by Kim et al. (1998). This fragment was used toscreen a Xenopus gastrula-stage cDNA library. A full-length
AXPC clone was isolated (5643 bp, Accession No.
AB066361) with a single open reading frame encoding 1016
amino acids (Fig. 1A), including a signal peptide (SP), six
extracellular cadherin repeats (EC 1-6), a membrane-

FIG. 3. AXPC mediates homophilic cell sorting in cell dissociation and reaggregation assays. (A) Steps involved in the celldissociation and reaggregation procedure. Reaggregates were madeusing blastula ectodermal cells (animal cap cells). Method 1: toinduce particular tissue types. For example, if dissociated cells
were treated with 1 ng/ml of activin for 1 h, reaggregates differentiate into only notochord. Method 2: to examine the sorting
activity of cells. (B) Prenotochord cells show potent sorting activity. (a) The 3-day cultured reaggregate derived from the cells of
Method 1 in (A) differentiated into only notochord. Note: The
vacuolated structure is the typical feature of notochord tissue. (b)The reaggregate mixed with untreated cells using Method 2 in (A)did not show sorting, but (c) the red-labeled cells treated with 1
ng/ml of activin mimicked the prenotochord cells and demonstrated potent sorting activity. In this case, a vacuolated structure
could not be detected because the culture time was reduced (10 h).
The frequency of this sorting event was 100% (n ⫽ 8). (C)Dissociation and reaggregation assay using AXPC constructs (Fig.
1B, 4, 5) and morpholino oligo. (a) When reaggregates were madefrom cells injected with 500 pg of MT-AXPC mRNA (red) anduntreated cells (green), the frequency of a sorting event (FSE) was
87.5% (n ⫽ 8). The details of (b– h) are summarized in (D). (D)Table of the results in Fig. 3C, a– h. MI, microinjection of mRNA ormorpholino oligo in a four-cell stage embryo. TR, activin treatment
to dissociated cells. non, no injection and no treatment. n, total
number of experiments. So, the total number of reaggregates toshow sorting. FSE, frequency of the sorting event (%).

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Prenotochord Cell Sorting in Xenopus

proximal extracellular domain (MPED), a transmembrane
domain (TM), a hydrophilic domain (HYD), and a cytoplasmic domain (CPD) (Fig. 1B) as described by Nollet et al.
(2000). However, AXPC lacks a membrane-proximal conserved domain (MPCD) and a catenin-binding domain(CBD), both of which play a crucial role in lateral clustering,cytoskeletal interactions, and adhesion of cadherins (Stappert et al., 1994; Yap et al., 1998). A gene with this domain
structure (6 ECs and 1 MPED) has never been reported in
Xenopus. Full-length human protocadherin-1 (H-PCD1,
Sano et al., 1993) has a 72% amino acid identity withfull-length Xenopus AXPC, and was therefore regarded as
the human homologue of AXPC (Fig. 1C). We also isolateda partial mouse homologue of AXPC (M-AXPC) from embryonic day 7 mouse cDNA libraries by PCR using primers
designed to the mouse EST database sequence. M-AXPC
encodes a partial EC4, full EC5, and partial EC6 domain(663 bp, 221 aa; Fig. 1B, 7). Amino acid identities of theseregions of Xenopus–mouse, Xenopus– human, and mouse–
human were 75, 74, and 96%, respectively (Fig. 1C). The
identities of 72% in the full-length gene (Xenopus andhuman) and more than 74% in the partial sequence(Xenopus– human–mouse), indicating that AXPC must be aconserved gene throughout vertebrate evolution.

AXPC Expression and Morphogenesis
Temporal expression analyzed by RT-PCR (Fig. 2A) revealed a low abundance of transcripts from stage 1 toapproximately stage 9. After the start of gastrulation (stage

FIG. 4. Gain-of-function experiments using MT-AXPC mRNA
led to the acquisition of adhesive properties by cells around theprenotochord. (A) (a) Xnot expression in nontreated embryo at stage

13 and (a⬘) transversal view. (b) Xnot expression in 500 pg of
MT-AXPC mRNA-injected embryo at stage 13 and (b⬘) transversal
view. Xnot expressed region was expanded in the case of (b). (B) (a)Control embryos at stage 16 and (a⬘) transversal view stained byhematoxilin and eosin. (b) Microinjection of 500 pg of MT-AXPCmRNA into two dorsal blastmeres at the four-cell stage, which
inhibited normal neural formation and interfered with notochordformation at stage 16 (87.5%, n ⫽ 24), and (b⬘) transversal view of(b). (c– e), (c⬘– e⬘) Section in situ hybridization analysis using theposterior neural marker, HoxD1 (c), notochord marker, Chordin (d),and somite marker, MyoD (e). The control embryos and MT-AXPCinjected embryos were fixed, frozen, sectioned at stage 16, andhybridized with probes. Black arrowheads indicate markerdetected regions. no, notochord. so, somite. (C) Later effects of
MT-AXPC. The left panel shows a section of a stage 32 control
embryo, which had been injected with fluorescein-dextran amine(FDA) lineage tracer into two dorsal blastomeres at the four-cellstage. The green regions are cells derived from the dorsal sites offour-cell stage embryos. The right panel indicates a section of 500
pg of MT-AXPC mRNA and FDA coinjected embryo. Since manyembryos died by stage 30, we collected the surviving embryos atstage 30 and continued culturing of sibling embryos at stage 32.
Inhibition of the notochord separation from the neural tube,
somites, and endoderm was detected (57.9%, n ⫽ 19). nu, neuraltube; no, notochord; so, somite.

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10), the level of AXPC expression increased by one order ofmagnitude (Fig. 2A). AXPC was also expressed abundantly
in gastrula (stages 10 –12 in Fig. 2A), neurula (stages 14 and17 in Fig. 2A), and tailbud stages (stages 20, 25, 30, and 38 in
Fig. 2A). In addition, further RT-PCR analyses at gastrula(stage 10) showed that AXPC was expressed in the dorsal
marginal zone but not in the ventral or lateral marginal
zones (Fig. 2B). Thus, although we could not detect expression by in situ hybridization at early gastrula (data notshown), RT-PCR showed that AXPC gene expression must
be dorsal-specific at the beginning of zygotic expression.
RT-PCR provided us with a semiquantitative measure of
the AXPC expression, and whole-mount in situ hybridization detected a weak but clear specific expression of AXPCin the notochord at stage 14 (data not shown) and strong
expression at stage 18 (Figs. 2C and 2C⬘). At the late tailbud
stage (stage 28), AXPC was expressed strongly in the pronephros and also detectable in the somites, heart (Fig. 2D),
optic vesicles, otic vesicles, and some parts of the anterior
central nervous system (Fig. 2D⬘).
RT-PCR analyses using the potent mesoderm inducer
activin and intact animal caps or dissociated animal cap
cells revealed a more specific AXPC expression pattern inthe early stages (Figs. 2E and 2F). Activin used at 50 –100
ng/ml is known to strongly induce notochord tissue(Asashima et al., 1990, 1991, 1999; Green et al., 1990) as
well as many mesodermal and neural tissues. When we cut
animal caps at stage 9, treated them with 50 –100 ng/ml ofactivin for 1 h, and cultured them for 5 h, AXPC expressions
reached their maximum (Fig. 2E). Since this in vitro condition of 6 h culturing after stage 9 is representive of the lategastrula (stage 12–12.5) in vivo, AXPC must be expressed inprenotochord cells in stage 12–12.5 embryos. When dissociated animal cap cells are treated with activin for 1 h,washed, reaggregated, and then cultured for 3 days (Fig. 3A,
Method 1), the reaggregates differentiate into notochord
only with vacuole structure of the feature as notochord (Fig.
3B, a) (Kuroda et al., 1999). Thus, 0- to 5-h cultured
reaggregates after 1 h of treatment with 1 ng/ml are thought
to mimic the presumptive notochord. It is not known why
the activin response of dissociated animal cap cells is muchhigher than that of intact animal cap cells. One reason may
be that dissociated cells have more free receptors to bind
activin than intact animal caps. The other may be that
inhibitors, such as activin (Tanegashima et al., 2000) and
BMP4 (Sasai et al., 1995; Piccolo et al., 1996) are lost when
animal cap cells are dissociated. We detected high expression of the transcripts in reaggregates derived from cells
treated for 1 h and cultured for 5 h in this way (Fig. 2F, lane2). We could not detect AXPC expression in the conditions
designed to induce atypical epidermis (Fig. 2F, lane 1) andendodermal yolk-rich tissue (Fig. 2F, lanes 3 and 4). These
results also strongly indicate that AXPC is expressed in the
prenotochord cells. To determine when the expression of
AXPC is initiated by activin, cells were cultured in 1 ng/ml
activin for up to 2 h. AXPC expression was detected from 30
min of treatment (Fig. 2F, lane 6), suggesting that AXPC

FIG. 5. Loss-of-function experiment using dominant negative
DN-AXPC mRNA or morpholino antisense oligo. (A) Microinjection of 1 ng of DN-AXPC mRNA resulted in a rounded outward
form (left, 25.0%, n ⫽ 24) and loss of normal notochord formation(right, 16.7%, n ⫽ 24). (B) (a) Control wild-type embryos. (b– d) 10
ng of morpholino oligo-injected embryos. Twenty-three percent of
injected embryos developed normally (b). Fifty-nine percent of
injected embryos produced abnormal axis formation (c). Eighteen
percent of embryos produced the dominant negative-like effects,
unclosed neural tissue, and died by stage 30 (d). All embryos
injected with 20 ng of control morpholino developed normally (seetable).

was not only downstream of activin induction but also
must be expressed very early in the prenotochord region(Fig. 2E). Taken together with the in situ hybridization and
RT-PCR data, these results indicate that AXPC is expressed© 2002 Elsevier Science (USA). All rights reserved.

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Prenotochord Cell Sorting in Xenopus

FIG. 6. Model summarizing spatial regulation patterns in early gastrula (left figure) and early tailbud stage (right figure). The model of earlygastrula is shown in the dorsal view and above-below indicates anterior-posterior in future morphogenesis. Small circle indicates the yolk
endoderm. Green region indicates the PAPC expressed region, and the red region indicates the Xlim1 and Xnot expressed region. The modelof late stage is shown in the transversal view and above– below indicates dorsal–ventral. nu, neural tube; no, notochord; so, somite; pn,
pronephros tube; lp, lateral plate; end, endoderm. Red region indicates the Xlim1 expressed region (Chan et al., 2000).

in prenotochord at gastrula, notochord at neurula, and inseveral tissue types at the late tailbud stage.

AXPC Mediates Cell Sorting in Vitro
Functional analyses of AXPC were performed by using
two mutant constructs; a membrane-tethered form, whichlacks the CPD (Fig. 1B, 4, MT-AXPC), and a dominantnegative secreted form (Fig. 1B, 5, DN-AXPC). A modified
cell dissociation and reaggregation assay was used to determine whether AXPC mediates cell adhesion (Fig. 3A,
Method 2). From preliminary experiments, we knew thatfull-length AXPC did not work well as an adhesive mediator (data not shown). This was not unexpected because
PAPC is also an adhesive molecule, and if it has thecytoplasmic region intact, sorting activity goes down (Kimet al., 1998). The sorting activity of AXPC or PAPC, like
protocadherin molecules, may be negatively regulated by
the cytoplasmic region under normal conditions. Therefore,
we constructed MT-AXPC to study the sorting activity of
the AXPC molecule alone. Within reaggregates, cells injected with either lineage tracer alone were evenly interspersed (Fig. 3B, b), but if red cells were treated with 1 ng/mlof activin to induce notochord, red cells associated together

in the central region of the reaggregates during the 10-h
culture (Fig. 3B, c; Kuroda et al., 1999). The yellow region
indicates mixed red- and green-labeled cells.
The mutant AXPC constructs were also tested for celladhesion activity by using the method described in Fig. 3A,
Method 2. Cells coinjected with MT-AXPC mRNA andTRDA sorted into reaggregates after 10 h of incubation (Fig.
3C, a), as did cells treated with 1 ng/ml activin (Fig. 3B, c).
These aggregating cells could be regarded as having potent
adhesive properties. However, neither cells injected with
MT-AXPC mRNA alone nor those treated with activin
showed sorting (Fig. 3C, b). These results suggest that
MT-AXPC can induce the same adhesive properties inanimal cap cells as in presumptive notochord cells. The EC
domain of PAPC acts as an efficient dominant-negative
inhibitor of endogenous PAPC (Kim et al., 1998). This
property was exploited to block AXPC function in light of
its likely homophilic adhesive properties (Kim et al., 1998;
Brieher et al., 1996). Overexpression of DN-AXPC inhibited
sorting induced by MT-AXPC or activin in a specificmanner (Fig. 3C, c, d). This dominant-negative form waseffective only against AXPC because DN-AXPC did notinterfere with the sorting induced by FL-PAPC (Fig. 3C, g,h). From these results, we concluded that AXPC could© 2002 Elsevier Science (USA). All rights reserved.

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Kuroda et al.

mediate notochord-specific cell adhesion via its extracellular domains.

Gain-of-Function Experiments in Vivo
To investigate whether and how AXPC mediates cellsorting in vivo, two different types of AXPC mRNA, MTAXPC and DN-AXPC, were injected into the two dorsal
blastomeres at the four-cell stage. Xnot is a marker gene
expressed in the notochord region at neurula stage (Fig. 4A,a, a⬘; Gont et al., 1996). We detected much wider expressionof Xnot in the MT-AXPC-injected embryos (Fig. 4A, b, b⬘).
In Xenopus, the notochord cells have already separated
from the surrounding somitic mesoderm tissue by stage 16(Fig. 4B, a, a⬘). However, embryos injected with 500 pg of
MT-AXPC mRNA showed no evidence of a notochord
distinct from the surrounding somitic mesoderm tissue atstage 16 (Fig. 4B, b, b⬘). To address the fate of the notochordcells, somitic mesoderm cells, and neural cells, we conducted in situ hybridization on sections of the injected
embryos to detect expression of the posterior neuralmarker, HoxD1 (Kolm and Sive, 1992), notochord marker,
Chordin (Sasai et al., 1994), and paraxial mesoderm marker,
MyoD (Harvey, 1992). HoxD1 expression was weaker thanin the controls and located along the Chordin-expressing
region (Fig. 4B, c, c⬘, d, d⬘). MyoD was expressed at control
levels but was located more in paraxial regions (Fig. 4B, e,e⬘). It may be interpreted from these results that changes in
the location of notochord cells induced by the MT-AXPC
caused the abnormal formation of surrounding tissues.
More than half of the embryos injected with MT-AXPC did
not develop past stage 20, although some did continue
development after that stage. Figure 4C (left) shows acontrol embryo injected with FDA lineage tracer into two
dorsal blastomeres at the four-cell stage; clear spaces can beseen between the notochord and other tissues. Embryos
injected with MT-AXPC and the FDA tracer showed no
obvious separation of notochord from surrounding tissues(Fig. 4C, right). These data directly indicate that Xenopus
embryogenesis is strongly affected by the extracellulardomain of AXPC.

Loss-of-Function Experiments in Vivo
The failure of the notochord to correctly separate frommesodermal cells may be due to a marked change in celladhesion properties resulting from the experiments using
MT-AXPC. As mentioned, DN-AXPC was used in similar
experiments to examine the loss-of-function effects in vivo.
Some embryos injected with 1 ng of DN-AXPC mRNAappeared to be malformed and shortened after stage 14, andsections showed these embryos to lack normal notochordformation (Fig. 5A). The frequency of this inhibited phenotype
was 25% (6/24). We could not show rescue experiments
because coinjections of MT-AXPC (500 pg) and DN-AXPC (1ng) induced an exogastrulation phenotype. Exogastrulation is
the typical phenotype resulting from an excess of injectedmRNA injection. An antisense approach to inject morpholino
oligo and inhibit the translation of the DN-AXPC (Heasman etal., 2000) supported the data of the DN-AXPC-injected phenotypes (Fig. 5B). The phenotypic abnormalities observed in
the morpholino oligo-injected embryos were milder thanthose of DN-AXPC-injected embryos, but the efficiency of
inhibition was increased. More than half of the embryos did
not show straight axis formation (77%, Fig. 5B, c, d), and 18%
showed an unclosed neural tube and died by stage 30 (Fig. 5B,d). The injection volume of 10 ng is not toxic for normal
development as 20 ng was injected into control morpholinoinjected embryos and these developed normally (Fig. 5B,
table). These results suggest that loss-of-function of the isolated AXPC can cause abnormal axis formation and neuralinduction, but that the level of this abnormality was greater insome of the DN-AXPC-injected embryos. It is possible thatgenes homologous to AXPC are expressed at the protein level,
but are not inhibited by the morpholino oligo at the translational level. This is supported by the cell dissociation andreaggregation assays, where morpholino oligos inhibited sorting in response to MT-AXPC but not activin treatment (Fig.
3C, e, f). AXPC may be one of the adhesive molecules working
downstream of activin or in the prenotochord region. Since
prenotochord cell sorting is an important phenomenon, it is
not surprising that many genes with similar functions exist in
the prenotochord region.

DISCUSSION
Xenopus, Humans, and Mice Have the AXPC GeneIn this study, we isolated the full-length Xenopus AXPC
gene and a partial fragment of the mouse AXPC gene, andidentified human protocadherin-1 from sequenced databases as a homologue of AXPC. The sequence identities
between Xenopus–mouse, Xenopus– human, and mouse–
human AXPC were 75, 74, and 96%, respectively (Fig. 1C).
These scores are reasonable in terms of the evolutionary
distance between amphibians and mammals for these genes
to be homologues. The existence of high sequence identities for AXPC between Xenopus and other vertebrates
suggests that this molecule may be essential for notochordformation in early vertebrate development. As to whether
other animals also have this gene, the paraxial protocadherin gene is expressed in the somite region next to thenotochord in Xenopus (Kim et al., 1998, 2000), zebrafish(Yamamoto et al., 1998), and mice (Yamamoto et al., 2000);
therefore zebrafish must have the axial protocadherin gene.
We propose that all vertebrates may use this gene for
notochord formation, and isolating the homologues of thisgene is an important focus of future research.

Sorting Activity by AXPC
We demonstrated sorting to occur in reaggregates cultured for 10 h with MT-AXPC mRNA-expressing cells. This
time course is representative because the sorting event© 2002 Elsevier Science (USA). All rights reserved.

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Prenotochord Cell Sorting in Xenopus

caused by activin also happens at approximately 10 h after
treatment (Kuroda et al., 1999). Interestingly, although thesorting activity mediated by activin is continuously maintained, the activity induced by MT-AXPC later loses effectiveness, and cells cultured for more than 20 h did notexhibit potent sorting activity (data not shown). Thesefindings indicate two possibilities. One is that there is no
AXPC mRNA left in reaggregates cultured for 20 h with the
MT-AXPC-expressing cells, and AXPC is unlikely to induce
additional adhesive molecules. Initially, we expected that
AXPC would have some additional activities, as paraxial
protocadherin can induce the paraxial mesoderm marker,
myoD, and promote convergent-extension movements andcell shape changes (Kim et al., 1998). However, AXPC did
not show such additional activity. Therefore, all mRNA
must be translated and degraded between 10 and 20 h ofculture. The other possibility is that cell– cell binding in the
prenotochord requires another similar type of adhesive
molecule. We showed that DN-AXPC can readily interferewith the sorting activity mediated by both MT-AXPC andactivin (Fig. 3C, c, d), as well as in vivo (Fig. 5A). However,morpholino oligos of AXPC did not inhibit the activity
mediated by activin (Fig. 3C, e, f) and the effects due to themorpholino oligo were weaker than those seen with thedominant negative AXPC (Fig. 5). Consistent with our
results, mouse dominant negative paraxial protocadherin is
effective, but the null mutant of paraxial protocadherin has
no effect on mouse development (Yamamoto et al., 2000).
These molecules may have similar genes playing the samerole in the expressed region. If this is true, the mouse AXPC
knock out mutant might also be ineffective as a PAPC nullmutant.

Hypothesis of Early Morphogenesis Using theProtocadherin Model
Zygotic AXPC expression starts at stage 10 (Fig. 2A) and
we detected localized expression in the dorsal marginalzone at stage 10 (Fig. 2B), prenotochord region at gastrula(Figs. 2E and 2F), notochord region at stage 18 (Figs. 2C, C⬘),and pronephros region at stage 26 (Fig. 2D). This expression
pattern is quite similar to the LIM class homeobox gene
Xlim-1. Xlim-1 is expressed in Xenopus embryos in thedorsal lip at stage 10, notochord at neurula, and the pronephros and certain cells of the central nervous system at
tailbud stage (Taira et al., 1992, 1994a,b; Chan et al., 2000).
In preliminary experiments using the animal cap assay, we
have shown that some organizer genes, including Xlim1,
induce AXPC overexpression. Thus, Xlim1 is one of thestrongest candidates to induce AXPC in vivo and thereby
regulate cell sorting activity. In fact, AXPC and Xlim1
expression in the notochord and pronephros must require
tight regulation of sorting activity to form the tissue structure because prenotochord cells must separate from surrounding mesodermal cells, and pronephros tube must
separate from the lateral plate cells. Thus, the relationshipbetween Xlim1 and AXPC is clearly an important subject
for future studies.
At early gastrula, PAPC is strongly expressed while
AXPC is weakly expressed in the dorsal marginal zone (Kimet al., 1998, Fig. 2B). By midgastrula, PAPC expression isfound in the mesodermal mantle but is repressed in the
future notochord region, where Xlim1 is strongly expressed.
PAPC expression is induced by Vg1 (Zhang and King, 1996)
and VegT (Zhang et al., 1998), but inhibited by Xnot (Kim etal., 1998), while Xlim1 is probably upstream of AXPC.
Cerberus encodes a secreted factor expressed in anterior
endomesoderm that functions to inhibit the Wnt, Nodal,
and BMP signals that promote head formation (Bouwmeester et al., 1996, Picollo et al., 1999). Taken together,
these results suggest that multiple signaling pathways
regulate the spatial pattern of expression of the AXPC and
PAPC structural genes in the early gastrulating mesoderm.
In early-stage embryos, PAPC is expressed in the dorsal lip
region, but moves laterally due to inhibition of Xnot.
Xlim1, which is coexpressed with Xnot, induces AXPC
expression, perhaps initiating AXPC to induce the axial
mesodermal region via its potent sorting activity (Fig. 6).
Alternatively, Xenopus may utilize the AXPC sorting activity to induce pronephros formation along with otherstructures (Fig. 6). The AXPC isolated here will allow us totest whether regulatory cell interactions, such as thosehighlighted in this study, are conserved across the othertissues in Xenopus, and during the developments of otheranimals.

ACKNOWLEDGMENTS
Human recombinant activin A was kindly provided by Dr.
Yuzuru Eto, Ajinomoto Co., Kawasaki, Japan. This work was
supported in part by a Grant-in-Aid from the Ministry of Education,
Science, Sports and Culture of Japan, and CREST (Core Research
for Evolutional Science and Technology) of the Japan Science andTechnology Corporation. We thank Dr. Nishinakamura for teaching the method of in situ hybridization on section to us.

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© 2002 Elsevier Science (USA). All rights reserved.

Received for publication July 27, 2001
Revised December 14, 2001
Accepted December 20, 2001
Published online March 11, 2002