Biochemical and Biophysical Research Communications 271, 151–157 (2000)
doi:10.1006/bbrc.2000.2545, available online at http://www.idealibrary.com on

XSIP1, a Member of Two-Handed Zinc Finger
Proteins, Induced Anterior Neural Markers
in Xenopus laevis Animal Cap
Akira Eisaki,* Hiroki Kuroda,* Akimasa Fukui,* and Makoto Asashima* ,† ,1*Department of Life Science (Biology) and †CREST, Japan Science and Technology Corporation (JST),
University of Tokyo, 3-8-1 Komaba, Meguro, Tokyo 153-8902, Japan

Received March 6, 2000

We characterized Xenopus SIP1 (XSIP1), Smad interacting protein, from activin-treated animal caps bydifferential screening. The XSIP1 is very similar tomouse SIP1 in the protein coding region including thezinc finger domain and homeodomain. The expression
pattern was analyzed by RT-PCR and whole mount insitu hybridization. XSIP1 expression was initially restricted to the dorsal marginal zone in the late gastrula and was subsequently expressed at the lateraledge of neural plate and, in the tailbud stage, in theforebrain, neural tube, and eye. Overexpression of
XSIP1 at the animal caps resulted in activation ofanterior neural markers without mesodermal markers. Ectopic expression of XSIP1 induced enlargementof neural cells and disordered eye formation. In addition to abnormal head phenotypes, many embryos
were short-tailed. Our findings suggest that XSIP1 is atranscriptional repressor, which may be involved in
the activin-dependent signal pathway. © 2000 Academic
Press

Key Words: SIP1; neural induction; Xenopus laevis;
zinc finger.

Early embryonic differentiation is governed by two
principles. One is unequal distribution of maternal
information and the other is signaling molecules mediating between cell– cell communication. In Xenopus,
the Spemann organizer is formed in the dorsal mesoderm by maternal information and then secretes molecular signals, which play important roles in the induction and patterning of neural and mesodermal
tissue. Many of these signal molecules are expressed
especially in the organizer region and they are sorted
into two main signaling activities. One is the activin1
To whom correspondence should be addressed. Fax: ⫹81-3-54544370. E-mail: asashi@bio.c.u-tokyo.ac.jp.

dependent signal transudation pathway, involving, forexample chordin (1) and noggin (2); the other is theWnt signaling pathway exemplified by siamous (3) andnr3 (4). Activin, which is a member of the transforming
factor-␤ superfamily (TGF-␤), can induce several mesodermal and neural tissues in animal caps depending
on its concentration (5). On the other hand, BMPs,
which are also members of TGF-␤ superfamily, caninduce ventral formation through expression of signaling molecules in the ventral side and can also induce
ventral mesoderm (6). The TGF-␤ superfamily includes
activin and BMPs which can transmit their signals
through Smad proteins and TGF-␤ signaling plays very
important roles in Xenopus embryogenesis.
Because activin treatment of animal caps induce
neural tissues to form sandwiching animal caps, we
anticipated that certain molecules induced by activin
dependent signals suppress mesoderm formation andpromote neural formation. By differential screening,
we identified Xenopus SIP1 (XSIP1) as a candidate forthis neural induction molecule. The SIP1, a Smadinteracting protein, was identified as a gene encoding
two separated clusters of zinc fingers, one N-terminaland one C-terminal, and a homeodomain. Like othertwo-handed zinc finger/homeodomain proteins, SIP1
binds to different promoters, including the 5⬘-CACCT
sequence (7). SIP1 is a member of the ␦EF1 family andthe zinc fingers of these two genes are bound with two
hands to a two-target site (8). SIP1 binds to the SmadMH2 domain with its Smad binding domain (SBD) andmay act as a regulation factor of the immediate response gene for the activin-dependent signal pathway.
Here we analyze the expression and activity of the
XSIP1 gene. We show that XSIP1 expression demarcates the presumptive neural plate very early, by the
middle of gastrulation, and later defines the anterior
neural tube. Overexpression of XSIP1 induced enlargement of neural tissue in anterior region and some151

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FIG. 1. (A) Nucleotide sequence of XSIP1. Predicted ORF is boxed. (B) Amino acid sequence comparison between mouse SIP1 andXenopus XSIP1. Gray boxes represent identical or biochemically similar amino acids. The position of the C2H2 type zinc finger in STP1 isindicated by bold underlining, other zinc fingers in SIP1 are indicated by thin double underlining. The dotted line indicates the Smad binding
domain (SBD).

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FIG. 1—Continued
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FIG. 2. RT-PCR analysis was performed at various stages.
Stages according to Niewcoop and Faber (1956) are shown over thelanes. ODC (lower panel) is indicated as a loading control.

embryos failed to form eye vesicles. Ectopic expressionof XSIP1 induced anterior neural markers suggestingthat XSIP1 plays a role in early neurogenesis.
MATERIALS AND METHODS
Eggs and embryos. Xenopus eggs were obtained by injecting
adult males and females with human chorionic gonadotropin(Gestron) at a dose of 600 U. Fertilized eggs were dejellied by treatment with 4.5% cysteine hydrochloride in Steinberg’s solution (pH7.8) with kanamycin sulfate (100 mg/l; Banyu Pharmaceutical Co.),
then washed thoroughly with Steinberg’s solution (pH 7.4). Embryos
were transferred to culture dishes containing Steinberg’s solution
according to Nieukoop and Faber (12).
Differential screening. Animal caps were cut from the late blastula and treated with 100 ng/ml activin A (supplied by Dr. Yuzuru
Eto, Central Research Laboratories of Ajinomoto Co. Inc., Japan)
for 1 h, then cultured for 7–10 h in Steinberg’s solution. Poly(A)tailed RNA was extracted from 2000 animal caps cultured in
Steinberg’s solution for 7–10 h after activin A treatment. In brief,
total RNA was extracted according to the guanidine thiocyanate

method (10). Then Poly(A)-tailed RNA was extracted from total
RNA by binding to oligo d(T) cellulose. A cDNA library was constructed using this RNA as a template. The library, consisting ofapproximately 100,000 clones, was screened using 32 P-labeledprobes generated from stage-10 whole embryo cDNA. Negativeclones were sequenced and compared with known sequences, andnovel clones were examined for their mRNA localization in embryos by hole-mount in situ hybridization.
In vitro transcription and microinjection. Capped sense RNAs for
microinjection were synthesized (11) by using Ambion Megascript(Austin, TX) SP6 and T7 kits. The clone used was as follows: XSIP1
linearized by NotI and transcribed by Sp6; with embryos fertilized invitro, dejellied, cultured, and injected with solutions as described(12). Embryos were injected in the animal cap with 100 pg to 1 ng at
the 2-cell stage into both or one blastomere or at the 4-cell stage into
the left side blastomeres.
Whole mount in situ hybridization. Whole mount in situ hybridization was performed as described in Harland (13): Embryos obtained from albino females were used. Digoxigenin (DIG)-labeledantisense RNA corresponding to 4953 bp of XSIP was synthesized invitro. Antidigoxigenin antibodies were purchased from Boehringer
Mannheim. To prepare sections, embryos stained by whole mount insitu hybridization were dehydrated in ethanol, transferred to xylene
and embedded in paraffin and sectioned at 10 ␮m.
RT-PCR. Total RNA was isolated from various developmental
stages and mRNA was injected animal caps using ISOGEN (NIPPON
GENE). Two micrograms of total RNA were used as a template to
generate first strand cDNA. One tenth of this cDNA was used as atemplate in subsequent RT-PCR (Reverse Transcription-polymerase
chain reaction) analysis. The primers used were: XSIP (forward, 5⬘GGGAGCCTCACCTACTCTCCT-3⬘; reverse, 5⬘-ATCCGCCAGATCTCTTGCT-3⬘), ODC (14), ms-actin (15), F-spondin (16), XAG1 (17, 18),
NCAM (19) and X1HBOX6 (20).

FIG. 3. Whole mount in situ hybridization with an XSIP antisense probe. (A) Stage 9 embryo, vegetal view. XSIP was not detected. (Band C) Stage 10.5 embryo, dorsovegetal view and vegetal view. XSIP1 is detected in the dorsal ectoderm. (D) Stage 12 embryo, dorsal view.
(E) Stage 17 embryo, dorsal view. (F) Stage 26 embryo. Large white arrowhead and small white arrowhead indicate the telencephalon andmesencephalon. d, dorsal; v, ventral; b, blastopore.

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FIG. 4. XSIP1 overexpressing embryos. (A) Stage 38 embryo, uninjected control side. (B) Stage 38 embryo, XSIP1 mRNA (500 pg) injectedside. (C) Stage 38 embryo, XSIP1 mRNA (1 ng) injected side. (D) Stage 38 embryo, uninjected side. (E and F) Transverse section of the embryo
shown in B. The section levels are indicated in B. (G) Transverse section of the embryo shown in C. The section levels are indicated in C. l,lens; r, retina; nt, neural tube.

RESULTS AND DISCUSSION
Animal caps treated with activin A differentiatedinto mesodermal or endodermal tissues depending on
concentration, and treated animal caps mimic the organizer and induced a variety of tissues in other untreated animal caps with contact. For example, animal
caps, treated with 100 ng/ml activin A for 1 h and thencultured for 7–10 h, showed strong induction of neuraltissues into sandwiching animal caps (data not shown).
In order to isolate the genes responsible for activatingneural determination, we carried out differential
screening between activin A treated animal caps andlate blastula whole embryos. We isolated a number ofclones, up-regulated in animal caps treated with activin and cultured but not in whole embryo blastulas.
One clone had a two-handed zinc finger/homeodomainclass gene, but lacked the 5⬘ sequence. We screened thegastrula library again and isolated this clone (Fig. 1A).
In comparison with other vertebrate zinc finger genes,

this clone showed marked similarities to mouse SmadInteraction Protein 1 (SIP1), with 77% amino acid
identity (Fig. 1B), and to the vertebrate ␦-crystalline
enhancer binding protein (␦EF1), with 39% identity.
The homologies between mouse SIP1 and this gene
were 68%, at the nucleotide level, and 83%, at thebiochemically similar amino acid level, (Fig. 1B). Notably, these proteins share the same amino acids in thezinc finger domain and certain similarities in their
Smad binding domain (SBD). These findings suggestthat this clone is a homologue of SIP1 in Xenopus, andfor that reason the clone was named XSIP1.
The length of XSIP was 4529 bp and conceptual
translation of the 3642 bp ORF yielded a 1214 aminoacid sequence containing a two-handed zinc finger anda homeodomain. This sequence also contains 230 bp of
the 5⬘-untranslated region and 653 bp of the 3⬘untranslated region. The homeodomain has conserved
arginine and asparagine at the same position, which is155

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critical for DNA binding. However mSIP1, XSIP1 and␦EF1 do not contain these critical amino acids. Thissuggests they are not able to bind DNA directly andthat they are the same type of two-handed zinc finger/
homeodomain class gene.
To examine the XSIP1 gene expression pattern inembryos at various developmental stages, RT-PCR
analysis and whole mount in situ hybridization werecarried out (Fig. 2). We designed XSIP1 specific primers to discriminate another clone, which was for several of the first amino acids. This clone had almost thesame sequence (97% similarity to XSIP1) but there wasno effect on embryos with the injection of its mRNA(data not shown), suggesting that it might be a splicing
isoform. The single product was not detected from theunfertilized egg to the stage 8 embryo, and then first
appeared at stage 9 and its expression was maintained
through the progression of development, gradually increasing through the early neurula stage, and was thensustained in the tail-bud stage. ODC confirmed equalloading of PCR products in each lane.
To determine localization of XSIP1 expression, we
performed whole mount in situ hybridization using adigoxygenin-labeled XSIP1 sense and antisense RNAprobe on whole embryos. There was no detectable signal in stage 9 embryos (Fig. 3A), based on RT-PCR
detection. The first detectable expression of XSIP1
RNA was observed in the dorsal ectoderm at stage 10.5
(Figs. 3B and 3C). From the early neurula stage onwards, XSIP expression had entered a second phasewith restriction to the lateral edge of the neural plate(Fig. 3D), i.e., the presumptive dorsal neural tube andneural crest. Then, XSIP1 was expressed in the neuralplate and tube along the anteroposterior axis of thedeveloping central nervous system (CNS) (Fig. 3E).
After the tailbud stage, expression was seen mainly inthe telencephalon and diencephalon of the forebrain,
and more posteriorly along the neural tube and eye(Figs. 3F and 3G).
Since many zinc finger genes expressed in the earlygastrula through neurula stages induce neural genes
like those of the zinc family (21), we induced overexpression of XSIP1 by microinjection of synthesized
XSIP1 mRNA to test the neuralizing activity. XSIP1mRNA and control mRNA (pCS2) were transcribed invitro, then dissolved in Gurdon’s buffer (88 mM NaCl,
1 mM KCl, 15 mM Tris-HCl, pH 7.5). First, we injected
XSIP1 mRNA into one blastomere of 2-cell stage embryos to over express XSIP1 hemilaterally. In almost
all cases, the XSIP1 injected side was enlarged andshowed poor eye formation (Fig. 4B) or ocular defects(Figs. 4C and 4G), whereas the uninjected control side
was normal (Fig. 4A). The sections through the headregions of injected embryos showed neurals to be markedly thickened of the injection side (Figs. 4D and 4F).
Hyperplastic neural walls, as well as distorted eyes

FIG. 5. XSIP1 induced neural marker genes without mesoderm
induction in animal cap explants. Embryos were injected with 1 ng of
XSIP1 or control mRNA into the 2-cell stage animal pole. Animal
caps were explanted at stage 9 and cultured for 2 days. N-CAM,
general neural marker; XAG1, cement gland marker; F-spondin and
X1Hbox6, trunk/tail neural marker; ODC, internal positive control.

and neural tissues characterized the injected side. Particularly, no lens induction occurred on the injectedside and retinal pigment cells were variably diminished. In addition to the abnormal head phenotypes,
embryos injected with 1 ng of XSIP1 in both blastomeres developed short tails (Fig. 4G), probably due toinhibition of Xbra (7).
As has been noted, XSIP1 plays important roles inearly neural development. To clarify the effect of injected XSIP1 mRNA in molecular marker gene expression, after the injection of mRNA into the animal poleof 2-cell stage embryos, the animal caps from theseembryos at stage 9 were dissected and total RNA fromthe caps, which had been cultured for 2 days, wasextracted. We carried out RT-PCR analysis, demonstrating that XSIP1 mRNA injection caused upregulation of N-CAM (a general neural marker), and
XAG1 (cement gland marker), but no up-regulation of
F-spondin, XlHbox6 (trunk/tail neutral marker) or msactin (mesodermal marker) (Fig. 5). The structuresinduced by XSIP1 injection suggest that XSIP1 is anearly activin response gene. When animal caps arecultured in a medium containing activin A, neural tissue is secondarily induced, owing to primarily induced
mesodermal tissue, resulting in expression of N-CAM.
Mouse SIP1 interacts with Smads via the SBD and bybinding the Xbra-2 enhancer to regulate its expression
and down-regulating Xbra expression (8). XSIP1 is induced by activin A (data not shown), thereby inducing
the neural marker without mesodermal formation andsuppresses Xbra expression indicate the possibilities

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that XSIP1 makes neuroectoderm from neural rather
than mesodermal tissue. XSIP injected animal caps
expressed neural marker but did not form neural tissue (data not shown). This, it is probable that othersignals, which can induce neural tissue in animal capswith activin/Smad 2 signaling, cooperate with XSIP1
to induce the formation of neuroectoderm from neuraltissue.
ACKNOWLEDGMENTS
This work was supported by Grants-in-Aid for Scientific Researchfrom the Ministry of Education, Science, Sports and Culture of Japan, and by CREST (Core Research for Evolutional Science andTechnology) of the Japan Science and Technology Corporation. We
thank Dr. Yuzuru Eto of Central Research Laboratories of Ajinomoto
Co. Inc. for providing human recombinant activin A.

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