Develop. Growth Differ. (2000) 42, 87–93

Cloning a novel developmental regulating gene,
Xotx5: Its potential role in anterior formation in
Xenopus laevis
Hiroki Kuroda,1 Tadayoshi Hayata,1 Akira Eisaki1 and Makoto Asashima1,2*
1

Department of Life Sciences (Biology) and 2CREST Project, Department of Life Sciences,
Graduate School of Arts and Sciences, The University of Tokyo, 3-8-1 Komaba, Meguro-ku,
Tokyo, 153-8902, Japan.

The vertebrate Otx gene family is related to otd, a gene contributing to head development in Drosophila. In
Xenopus, Xotx1, Xotx2, and Xotx4 have already been isolated and analyzed. Here the cloning, developmental
expression and functions of the additional Otx Xenopus gene, Xotx5 are reported. This latter gene shows agreater degree of homology to Xotx2 than Xotx1 and Xotx4. Xotx5 was initially expressed in Spemann’sorganizer and later in the anterior region. Ectopic expression of Xotx5 had similar effects to other Xotx genes
in impairing trunk and tail development, and especially similar effects to Xotx2 in causing secondary cement
glands. Taken together, these findings suggest that Xotx5 stimulates the formation of the anterior regions andrepresses the formation of posterior structures similar to Xotx2.
Key words:

head inducer, homeobox, orthodenticle, Xenopus laevis, Xotx2.

Introduction
Two major and distinct classes of homeobox-containing
genes have been shown to exert key developmental
functions throughout the Metazoa: the HOX genes,
which are related to the Drosophila antennapedia gene,and those related to the Drosophila gene paired. A new
family of paired-class homeobox genes related to the
Drosophila orthodenticle gene has been described(reviewed in Finkelstein & Boncinelli 1994; Boncinelli &Mallamaci 1995). As for the Drosophila gene, theexpression pattern of this gene family is strictly related
to the development of anterior head structures.
Among the family members, the most conserved
genes in vertebrates are Otx1 and Otx2 (Simeone etal. 1993; Bally-Cuif & Wassef. 1994; Li et al. 1994;
Pannese et al. 1995; Kablar et al. 1996). Mouse knockout experiments have recently confirmed the hypothesis that these genes are possibly involved in anterior
brain patterning as Otx2–/– mice lack forebrain and midbrain regions (Acampora et al. 1995; Matsuo et al.
1995; Ang et al. 1996), while Otx1–/– mice show several brain abnormalities (Acampora et al. 1996; Suda

*Author to whom all correspondence should be addressed.
E-mail: asashi@bio.c.u-tokyo.ac.jp
Received 27 September 1999; revised 25 October 1999;
accepted 25 October 1999.

et al. 1996). After two distinct genes (Otx1 and Otx2)
were first characterized in the mouse, a few homologs
have been described in zebrafish (Zotx1, Zotx2, andZotx3; Li et al. 1994; Mori et al. 1994), in chicken (Cotx2;
Bally-Cuif et al. 1995) and in Xenopus (Xotx1, Xotx2,
and Xotx4; Pannese et al. 1995; Kablar et al. 1996).
These findings suggest that the occurrence Otx genes
may not have been exhausted yet. Both Xenopus andzebrafish have had three types of orthodenticle-likegenes isolated. Sequencing analyses have shown that
Xotx1 is similar to Xotx4, Zotx1 and Zotx3, while Xotx2
is similar to Zotx2. Here the cloning of the Xenopus
fourth orthodenticle-like gene is reported, and named
Xotx5.
Activin A has very potent mesoderm-inducing activity
on Xenopus presumptive ectoderm called animal cap
and induces various mesodermal tissues in a dosedependent manner (Asashima et al. 1990; Ariizumi etal. 1991a,b). In addition, cells dissociated from animal
caps have several prospective cell fates dependent on
distinct activin A concentration thresholds (Green et al.
1994); for example, a concentration of 1 ng/mL activin
A induced only notochord development (Kuroda et al.
1999). In the present study, Xotx5 was cloned using
reaggregates made from 1 ng/mL treated animal cap
dissociated cells and the degenerated oligonucleotide
reverse transcription–polymerase chain reaction(RT-PCR) method.

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H. Kuroda et al.

Materials and Methods

RT-PCR

Embryos and activin A

The oligos used for PCR amplification were as
follows: Xotx5 primers 59-GTGGCTGTGTTCATTCTTTT39 and 59-GCTCCTCCATCTTGTTGTTG-39; Xotx2
primers 59-GGATGGATTTGTTGCACCAGTC-39 and59-CACTCTCCGAGCTCACTTCTC-39; XAG primers
59-CTGACTGTCCGATCAGAC-39 and 59-GAGTTGCTTCTCTGGCAT-39; XCG primers 59-GGTGATGTTACTTCCCCAGAGCAG-39 and 59-GGGAAGTAACATCAAACAAAGCAACCA-39; EF1 a primers 59-GACAAGAGAACCATCGAGAA-39 and 59-GTGCCGGTGATCATGTTCTT-39. The annealing temperatures were
58°C (Xotx5 and Xotx2) and 60°C (XAG, XCG, andEF1 a), and 23 cycles (EF1 a), 25 cycles (Xotx5, Xotx2,
XAG, and XCG) were performed. The RT-PCR products
were resolved on a 2% SeaKem GTG agarose gel(Takara, Kyoto, Japan).

Xenopus laevis eggs were obtained from females byinjecting them with 400 units of human chorionicgonadotropin (Gestron; Denka Seiyaku Co., Kawasaki,
Japan), fertilized in vitro with minced testis, andcultured in 13 Steinberg’s solution (58 mM sodium
chloride, 0.67 mM potassium chloride, 0.34 mM calcium
nitrate, 0.83 mM magnesium sulfate, 100 mg/L kanamycin sulfate and 4.6 mM timza base; pH 7.4). Staging
was according to Nieuwkoop and Faber (1967).
The jelly coat was removed with 3% cysteine hydrochloride in Steinberg’s solution (pH 7.8), and thevitelline membrane was manually removed with fineforceps. All operations were carried out under sterile
conditions. Human recombinant activin A was dissolved
at concentrations of 0.05–100 ng/mL in 13 Steinberg’s
solution containing 0.1% bovine serum albumin (BSA;
Sigma Chemical Co., St Louis, MO, USA).

Cell dissociation and reaggregation
The cell dissociation and reaggregation method was
performed as described by Kuroda et al. (1999).

Cloning
Total ribonucleic acid (RNA) was isolated from fivereaggregated cells treated with 1 ng/mL of activin A.
Full-length cDNA was synthesized using a Clontech
Smart PCR cDNA synthesis kit (Clontech, CA, USA).
Using SMART II oligonucleotide 59-AAGCAGTGGTAACAACGCAGAGTACGCGGG-39 and degenerateoligonucleotide primer 59-CKNCKRTTYTKRAACCANAC-39 corresponding to a conserved homeodomain region N-VWF(Q/K)NRR-C of homeobox genes,
cDNA was PCR amplified. Amplified fragments were
subcloned in pGEM-T EASY Vector (Promega,
Madison, WI, USA).

Library screening
Using Xotx5 primers 59-CGATTAGTTTAGTGGAGTGG39 and 59-GAGGCTGTTTGATGTAGGAC-39, approximately 2 3 107 plaque-forming units (p.f.u.) of thephargemid library prepared from stage 12 Xenopus
embryos were screened using PCR.

Whole-mount in situ hybridization
Whole-mount in situ hybridization was performed onstaged alvino embryos as described by Harland(1991). We used Xotx2 plasmid T7TSMC19/1 (Pannese
et al. 1995).

Histological examination
Reaggregates were fixed with Bouin’s fluid for 3 h,dehydrated with ethanol, embedded in paraffin wax,
sectioned at 6 µm and stained with hematoxylin andeosin (HE).

Embryo microinjections
Capped synthetic RNA was generated using in vitrotranscription of full coding sequences of Xotx5 andXotx2. We used Xotx2 plasmid T7TSMC19/1 (Pannese
et al. 1995). All RNA was resuspended in 88 mM NaCl,
5 mM Tris pH 7.5 and injected into a single cell of 1-cellembryos or two blastomeres of 8-cell embryos. Injectedamounts ranged between 0.2 and 1.6 ng. This quantity is not toxic (Rebagliati & Melton 1987). Injected
embryos were allowed to develop until uninjected control embryos reached stage 9 for cutting animal caps
or stages 36–38 for observing mRNA overexpression
embryos.

Results

Cloning and sequencing
Using the degenerate PCR method, four novel homeobox genes were amplified (Table 1). One of the four
PCR products of the novel homeobox genes was 60%
similar to Xotx2 DNA sequencing and was named
Xotx5. A cDNA library prepared from stage 12 X. laevis
embryos was screened using two primers corresponding to Xotx5, and the Xotx5 full-length clone(2.7 kb) was purified. Figure 1(A) shows the coding
region and the deduced peptide sequence of Xotx5.
Overall, homeodomain, 59-domain, or 39-domain
sequence similarity between Xotx5 and the other Xotx

Fourth Otx family gene in Xenopus

89

Table 1. Xenopus homeobox gene isolated using degenerate
polymerase chain reaction
Genes

Number of isolates

Previously known genes
Xlim1
goosecoid
Mix.1
Mixer
Hbox10
milk
Xotx2
Xhox3
Mix.2
Xlim3
Xnot
Xotx1
Xnot2
Novel genes
Xotx5
Rat DRG11-like geneZebrafish Arx-like gene
Newt Hoxc-10-like gene

12
1
1
1

Total

135

8
7
6
4
3
3
3
2
2
2
2
2
1

genes was less than 75, 98, 76, or 68% at the peptide
level (Fig. 1B). Xenopus laevis is a tetraploid animal.
Although Xotx2 was the most similar gene to Xotx5, asimilarity of 68% in the 39-domain was too low to regard
Xotx5 as an Xotx2 duplicated gene.

Xotx5 expression in normal embryos
Reverse transcription–polymerase chain reaction
analysis (Fig. 2) detected the presence of low abundance transcripts from stage 1 to approximately stage
9, when the abundance of Xotx5 transcripts increased.
Using whole-mount in situ hybridization, Xotx5 transcripts were confined to dorsal internal regions of themarginal zone at stage 11 (Fig. 3B). As part of thisregion differentiates into notochord in the future, it might
have been possible to isolate Xotx5. At this stage Xotx2and Xotx5 expression domains were similar but not
identical. Although Xotx2 transcripts were present in theprechordal plate, bottle cells and presumptive rostralbrain (Panesse et al. 1995; Fig. 3A), Xotx2 was slightly
localized at the lateral side (Fig. 3A), but Xotx5 wasmore localized at the center of the Xotx2 expressedregions (Fig. 3B). Xotx5 expression was confined to thecement gland-forming region (Fig. 3C,D) at stages
19–25, and the forebrain, optic vesicle and otocyst atstage 28 (Fig. 3E).

Xotx5 overexpression phenotype
Synthetic Xotx5 mRNA was microinjected into theanimal hemisphere of fertilized eggs and two

Fig. 1. (A) Sequence of the Xenopus Xotx5 coding region. The
deduced peptide sequence of the Xotx2 gene product is also
shown using the one-letter amino acid code. The homeodomain
is underlined. (B) Peptide sequence similarity between Xotx5 andthe other Xotxs. HD, homeodomain; aa, amino acids.

Fig. 2. Temporal expression pattern of Xotx5. Reverse
transcription–polymerase chain reaction (RT-PCR) analysis was
performed using early Xenopus embryos. The number indicatesthe developmental stage of the embryos. Xotx5 expressedmaternally, increased at the early gastrula stage (stage 10), anddecreased at stage 45. The EF1 a is the internal positive control
and RT2 is the negative control.

90

H. Kuroda et al.

blastomeres of 8-cell stage embryos to examine thefunction of Xotx5. Embryos injected with Xotx5 between
400 pg and 1.6 ng into the animal hemisphere of fertilized eggs display posterior deficiencies, shortened
tails and additional cement glands (Fig. 4B,D,E). Larger
ectopic cement gland sections revealed the presenceof columnarized cells containing pigment granules(compare Fig. 4, panels D,E). The frequency of thisphenotype increased with increasing amounts ofinjected Xotx5 (Table 2). When embryos were injectedwith 800 pg of Xotx5 at the two-blastomere stage of
8-cell embryos, only embryos injected at the ventral
animal blastomeres displayed additional cement
glands (Table 2). When Xotx5 was injected into thevegetal ventral blastomeres, a different class of phenotype exhibited secondary axes (Fig. 4C; Table 2). No
embryo injected at the dorsal blastomeres displayedabnormal phenotypes (Table 2). As these findings
resemble the Xotx2 overexpression phenotype(Table 2), Xotx5 may be a coworker of Xotx2.
To examine whether Xotx5 acts as an activator or
repressor, the carboxy-terminal domains following
Xotx5 homeodomains were replaced either by the VP16
activator domain (Friedman et al. 1988) or the engrailed
repressor domain (Han & Manley 1993). Embryos
injected with the fusion constructs did not display
strange phenotypes (data not shown).

Upstream and downstream of Xotx5
Fig. 3. Localized Xotx5 expression. (A) Whole-mount in situ
hybridization, dorsal view at stage 11. An arrowhead indicatesblastopore. (B) Dorsal view at stage 11. (C) Anterior view at stage
19. (D) Anterior view at stage 25. (E) Head region view at stage
28. fb, forebrain; ov, optic vesicle; ot, otocyst.

Xotx2 is able to activate the cement gland markers XCG
and XAG (Gammill & Sive 1997). Therefore, examination of whether Xotx5 also induced cement glandformation in isolated ectoderm was also undertaken.

Fig. 4. Phenotypes of embryos
injected with Xotx5. (A) Normal
embryo at stage 36. (B) The embryos
shown were injected with 1.6 ng of
Xotx5 at the animal pole of 1-cellembryos. White lines indicate section
places. (C) The embryo injected with
0.8 ng of Xotx5 at two ventral vegetal
blastomeres at the 8-cell stage hadpartial additional axis formation. (D)Transverse section through the trunkof an Xotx5-injected embryo. (E)Transverse section through the headof an Xotx5-injected embryo. Note thatthe normal cement gland shows characteristic columnar cells with dark pigmentation. CG, normal cement gland;CG’, additional cement gland.

Fourth Otx family gene in Xenopus

Table 2.

91

External phenotypes of injected embryos

RNA injection
(ng)

n

Stage

Secondaryaxis (%)

Secondary cement
gland (%)

Posteriordefects (%)

Shorttail (%)

Minor
defects (%)

Normalembryos (%)

Xotx5
1.6
0.8
0.4
1.6
0.8
0.8
0.8
0.8
0.8
0.8

40
43
48
48
23
30
22
24
24
24

1C ani.
1C ani.
1C ani.
1C veg.
4C dor.
4C ven.
8C dor.-ani.
8C dor.-veg.
8C ven.-ani.
8C ven.-veg.

0
0
0
0
0
0
0
0
0
20.8

17.5
48.3
2.1
0
8.7
0
0
0
66.7
0

40
34.9
18.7
37.5
13
50
0
0
8.3
29.2

32.5
14
6.3
6.3
13
26.7
0
4.2
0
0

17.5
11.6
8.3
8.3
17.4
13.3
0
0
16.7
8.3

0
23.3
66.7
45.8
39.1
6.7
100
95.8
12.5
45.8

Xotx2
1.6
0.8
0.4
0.2
0.8

22
21
24
24
23

1C ani.
1C ani.
1C ani.
1C ani.
8C ven.-veg.

0
0
0
0
0

63.6
42.9
12.5
8.3
0

90.1
57.1
29.2
4.2
26.1

0
28.6
16.7
0
21.7

0
4.8
0
0
0

4.5
0
50
88
56.4

Water

50

1C ani.

0

0

0

0

2

98

RNA, ribonucleic acid; ani., animal region; veg., vegetal region; dor., dorsal region; ven., ventral region.

Animal caps were removed from Xotx5-injected
embryos which were incubated until sibling embryos
reached at stage 20 or 30. Inductions of XCG and XAG
were detected in Xotx5-injected and incubated animal
caps until sibling embryos reached at stage 20 (Fig.
5A), and cement gland formation was detected in
Xotx5-injected and incubated animal caps until siblingembryos reached stage 30 (Fig. 5B). These findings
suggested Xotx5 also induced XCG and XAG expression and cement gland formation similar to Xotx2.
As Xotx5 was cloned using reaggregates made from
activin A-treated animal cap dissociated cells and thedegenerated oligonucleotide RT-PCR method, we
examined whether activin A induced Xotx5 expression.
Animal caps treated or untreated with activin A wereincubated until sibling embryos reached stage 12.
Although induction of Xotx5 was detected in bothanimal caps, Xotx5 expression in animal caps treatedwith 50 or 100 ng/mL of activin A was higher than theothers (Fig. 5C). In reaggregates made from animal cap
dissociated cells, induction of Xotx5 was detected inreaggregates treated with 1 ng/mL of activin A. Thesefindings suggested activin A induces Xotx5 expression.

Discussion
Xotx5, a novel Xenopus homeobox gene of the bicoidclass homologous to Otx2 was examined. The Xotx5
peptide sequence was most similar to Xotx2. Its functions resembled Xotx2. Its expression patterns at theearly embryo stages were a little different from Xotx2.

Fig. 5. Reverse transcription–polymerase chain reaction(RT-PCR) analysis of Xotx5 upstream and downstream. (A) Xotx5
induced cement gland marker expression, XAG and XCG, in isolated animal caps. Animal caps isolated from Xotx5-injected
embryos, and developed until sibling embryos reached stage
20. (B) Xotx5 induced large cement glands (arrowhead) in animal
caps. Animal caps isolated from Xotx5-injected embryos developed until sibling embryos reached stage 30. (C) Activin A
induced Xotx5 expression in animal caps and reaggregates.
Animal caps treated with 5–100 ng/mL of activin A or untreateddeveloped until sibling embryos reached stage 12.

Fourth otx family gene in X. laevis
Otx1 and Otx2 (Simeone et al. 1993; Bally-Cuif &Wassef. 1994; Li et al. 1994; Pannese et al. 1995;

92

H. Kuroda et al.

Kablar et al. 1996) were first isolated as orthodenticle
family genes in vertebrates. After two distinct genes(Otx1 and Otx2) were first characterized in the mouse,a few homologs have been described in zebrafish(Zotx1, Zotx2, and Zotx3; Li et al. 1994; Mori et al.
1994), in chicken (Cotx2; Bally-Cuif et al. 1995) and in
Xenopus (Xotx1, Xotx2, and Xotx4; Pannese et al. 1995;
Kablar et al. 1996). Reported here is the cloning of the
Xenopus fourth orthodenticle-like gene, which wasnamed Xotx5. In addition, Otx family genes have also
been described in Branchiostoma (BfOtx, GenBank
2828715), in Lampetra (LjOTXA, GenBank 3650205;
LjOtxB, GenBank 3650207), and in Petromyzon(PmOtx, GenBank 3941721). Among the several Otx
family genes that have been reported, the relationshipbetween known otx family 39-domain peptide
sequences was clarified (Figs 1,6). The Otx family peptide has the following domains: 59-domain, homeodomain and 39-domain. As the 39-domain has much
more variety than the 59-domain and homeodomain, the39-domain was regarded as the functional domain. The
chordate Otx family is divided into two types: 39-shortforms or 39-long forms (Fig. 6). Otx2, Zotx2, Xotx2,
Xotx5, LjOtxA, LjOtxB and PmOtx belong to the 39-shortforms, while Otx1, Zotx1, Zotx3, Xotx1, Xotx4 and BfOtx
belong to the 39-long forms. There are two reports about
the difference between short and long forms. Mouseknock-out experiments have shown that Otx2–/– micelack forebrain and midbrain regions (Acampora et al.
1995; Matsuo et al. 1995; Ang et al. 1996), while Otx1–/–
mice show several brain abnormalities (Acampora etal. 1996). In Xenopus, Xotx1 overexpression inhibits tailorganizer activity, while Xotx2 overexpression is able
to turn a tail organizer into a head organizer(Andreazzoli et al. 1997). In the present study, Xenopus
showed not only two types of the 39-long forms isolated
from the otx family (Xotx1 and Xotx4), but also two typesof the 39-short forms isolated from the otx family (Xotx2and Xotx5). In future studies, if additional Otx family
genes are isolated, this novel gene should be divided
on the point of the 39-domain sequence of Xenopus otx
family genes.

Potential role of Xotx5 in anterior formation in
X. laevis
Xotx5 expression was analyzed in normal Xenopus
embryos. Reverse transcription–polymerase chain
reaction analysis (Fig. 2) showed that maternal transcripts of this gene are already detectable in unfertilized
eggs. Directly after the midblastula transition, when
zygotic transcription starts, Xotx5 expression increases.
Whole-mount in situ hybridization experiments revealed
early localized expression in the dorsal internal regionsof the marginal zone at stage 11 (Fig. 3B). At stages
19 (Fig. 3C) and 25 (Fig. 3D) the major expression siteof Xotx5 was the cement gland. At stage 28 (Fig. 3E)
the expression domains were the forebrain, optic
vesicle and otocyst. These expression patterns imply
that Xotx5 has a role in anterior formation. Indeed
microinjection experiments lend further support tothe hypothesis of the role of Xotx5 in patterning theanterior–posterior axis of the embryo. A major class of
shortened embryos with reduced trunk–tail structures
and additional cement gland was observed (Fig. 4B).
Xotx2 can activate XCG and XAG to form the cement
gland (Gammill & Sive 1997). In the present study Xotx5
was also able to activate XAG and XCG (Fig. 5A). Thesefindings show Xotx5 could, similar to Xotx2, inhibit
posterior formation at the early gastrula stage andinduce the cement gland at the late gastrula to neurula
stage. Xotx5 was probably also involved in forebain,
optic vesicle and otocyst formation at the tail-bud
stage.
Moreover, 20.8% of Xotx5-injected embryos at thevegetal ventral blastomeres exhibited secondary axes
phenotypes (Fig. 4C; Table 2). Although these secondary axes of Xotx5-injected embryos were not as complete as Xwnt8-injected embryos (Steinbeisser et al.
1993), Xotx5 was able to induce partial secondary
axes. Xotx1 and Xotx2 were also able to induce partial
secondary axes (Table 2; Andreazzoli et al. 1997), but
the score was lower than Xotx5. Thus, Xotx59s ability
to induce secondary axes may be more intense thanthe other otx family genes.

Acknowledgements
Fig. 6. Relationships between 13 chordate otx family genes. The
tree is inferred from the 39-domain peptide sequence. Bf,
Branchiostoma floridae; Lj, Lampetra japonica; Pm, Petromyzon
marinus.

We thank Dr Maria Pannese and Dr Edoardo Boncinelli
for the gift of the Xotx2 plasmid. This work was
supported in part by a Grant-in-Aid from the Ministryof Education, Science and Culture of Japan, andFourth Otx family gene in Xenopus

CREST (Core Research for Evolutional Science andTechnology) of the Japan Science and Technology
Corporation. Human recombinant activin A was kindly
provided by Dr Yuzuru Eto, Ajinomoto Co., Kawasaki,
Japan.

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