自由論題［研究論文］SKL Tagging

A Method for Generating Dominant-negative Inhibitorsof the Homeobox Transcription Factor Superfamily
and Smads

SKL タグトラップ法

二量体型転写因子および Smad における機能阻害法の構築

Yuka Sato
Master Program, Graduate School of Media and Governance, Keio University
佐藤 友香
慶應義塾大学大学院政策・メディア研究科修士課程

Shinya Matsukawa
Postdoctoral Fellow, Axial Pattern Dynamics, RIKEN Center for Developmental Biology
松川 晋也
理化学研究所多細胞システム形成研究センター体軸動態研究チーム研究員

Hiroki Kuroda
Associate Professor, Faculty of Environment and Information Studies, Keio University
黒田 裕樹
慶應義塾大学環境情報学部准教授

Abstract:

Peroxisome targeting signal 1 (PTS1) is a C-terminal tripeptide with aconsensus sequence such as serine-lysine-leucine (SKL). Using this molecularmechanism, we developed a novel dominant-negative assay, designated the SKLtag trap assay, to negatively regulate dimeric transcription factor, as typified by
the homeobox transcription factor superfamily. We tested this method on the
homeobox protein Siamois, a well-studied dorsalizing factor in vertebrates, with
Vent1 and Vent2 as ventralizing factors. In both cases, protein functions wereeffectively blocked by the SKL-tag trap assay. Moreover, we showed that thetechnical feasibility of the SKL-tag trap assay was confirmed for Smad transcription
factors. Taken together, we conclude that the SKL-tag trap assay can be used for theeffective way to inhibit function of dimeric, trimeric, and oligomeric transcriptional
factors in amphibian embryos.

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ペルオキシソーム移行シグナル 1 (PTS1) とはタンパク質の C 末端に存在するセリン—リシン—ロイシンの 3 アミノ酸 (SKL) のことである。我々は、二量体型転写因子の働きを阻害する初めての手法として SKL タグトラップ法を構築することにした。脊椎動物の背側化を導くもしくは腹側化を導くホメオボッ
クス分子、それぞれ Saimois もしくは Vent1 と Vent2 についてこの手法を試みた結果、いずれの場合においてもその有効性が確認された。さらに、この手法
は転写因子 Smad に対しても効力を示すことが判明した。以上より、SKL タ
グトラップ法は両生類胚において有効な二量体、三量体、さらには多量体形成
型転写因子の機能を阻害する手法であると言える。

Keywords: SKL motif, homeobox, dorsalization, ventralization, Xenopus laevis
SKL モチーフ、ホメオボックス、背側化、腹側化、アフリカツメガエル

1 Introduction
In eukaryotic cells proteins destined for single-membrane subcellularorganelles, peroxisomes, which contain digestive enzymes for breaking down
toxic materials in cells, have a carboxy (C)-terminal peroxisome targeting
signal consisting of the tripeptide serine (S)-lysine (K)-leucine (L), which iswell known as the most effective signal peptide in PTS1. This C-terminal
signal sequence allows the protein to bind to the transporter protein PEX5
and to be translocated into the peroxisome through PEX13-14 protein channel(Fig. 1) (Brocard et al., 2006).
Xenopus laevis is a genus of African frogs that are commonly known
as the African clawed frogs and broadly used as an invaluable tool to study
vertebrate embryology and development because of the following features.
Embryos tolerate extensive manipulation such as single cell or tissuetransplantations. It is very easy to inject of a range of materials such as
DNAs, mRNAs, proteins, and whole nuclei into whole embryo or specificcells. Cell fate of each early embryonic cell in Xenopus is known, allowing
targeted gene knock-out, knockdown and overexpression studies eggs andembryos provide abundant source for high-throughput biochemical studies.
The homeobox gene Siamois is expressed in a localized fashion in
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the blastula chordin- and noggin-expressing (BCNE) center of amphibian
blastula and works as a central player of dorsalizing activity of Xenopus
laevis embryos (Lemaire et al., 1995; Carnac et al., 1996; Laurent et al.,
1997; Ishibashi et al., 2008). Siamois homologue has never been reported
in mammalians. However, human organizer cells express DUXO (DUX of
the Organizer), a novel member of the double-homeobox (DUX) family oftranscription factors, and both of DUXO’s homeodomains share very highsimilarity with the amino acid sequence of the Xenopus laevis proteins
Siamois (Sharon et al., 2012). Overexpression of DUXO upregulates theprominent organizer markers such as goosecoid and cerberus1, suggestingthat DUXO is working as dorsalizing factor in mammalian and can besubstitute for Siaomis (Sharon et al., 2012). Other homeobox genes Vent1 andVent2 are widely expressed at the ventral side of gastrula embryos under thecontrol of the BMP (bone morphogenetic protein) signaling and have a role ofventralizing factor (Sander et al., 2007). Interestingly, the putative homologof the Xenopus Vent2 gene, VENTX is isolated in human and has a normal
and malignant myelopoiesis (Rawat et al., 2010). Smad transcription factors
are working as the main cytoplasmic regulator of the TGFβ signaling. Fiveof the mammalian Smads—Smad1, Smad2, Smad3, Smad5, and Smad8—act
as receptor-regulated Smads (R-Smad). Smads 1, 5, and 8 are substrates forthe receptors targeted by ventralizing factors, BMPs, and Smads 2 and 3 forreceptors targeted by mesoderm inducers such as activin and Nodal. Smad4,
also referred to as Co-Smad, serves as a common partner for all R-Smads.
Smad6 and Smad7 are inhibitory Smads (I-Smad) that interfere with Smadreceptor or Smad-Smad interactions.
In this study, we introduce a novel and simple approach for inhibiting
homeobox transcription factor superfamily proteins. Homeobox proteinsare dimerized by hydrogen bonds to attain biological activity in nucleus(Kuroiwa, 1987). The phenomena described above prompted us to investigate
the following idea named as the SKL-tag trap assay. Artif icially, SKL-

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conjugated homeobox proteins may dimerize with endogenous homeobox
protein. If so, target homeobox superfamily molecules would be trapped by
PEX5 (Lanyon-Hogg et al., 2010), and as a result they should shuttle into
peroxisome and never function in nucleus (Fig. 1). We find that this is indeed
the case and also adaptable for other transcription factors such as Smads.
PTS are observed in all eukaryotes, so the SKL-tag trap method could be
widely applicable to inhibit the function of oligomeric transcriptional factors.
In order to block the function of target gene, zinc-finger nucleases (ZFNs),
transcription activator-like effector nucleases (TALENs), and CRISPR/
Cas methods have been developed as powerful tool to make gene knockout
creatures (Gaj et al., 2013), and RNA interference technique (RNAi) andantisense morpholino oligomer (MO) as gene knockdown (Flynt et al.,2011; Heasman, 2002). However, these methods are not so effective whenfunctionally interchangeable proteins are existed. On the other hand, theSKL-tag trap assay is also able to inhibit such interchangeable proteins by
dimerization.

Fig. 1 Schematic representation of the SKL-tag trap assay
I: Normal pathway of homodimer-typed transcriptional factors such as the homeobox
transcriptional factor superfamily proteins. II: Scheme of the SKL-tag trap assay.
An experimentally generated carboxy-terminal SKL-conjugated subunit may bindsubunits in the complementary endogenous heterodimeric protein via the formationof a hydrogen bond. The resultant protein complex should then be trapped by PEX-5,
resulting in its continuous shuttling to the peroxisome.
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2 Experimental procedures
To conjugate the SKL motif to the carboxy-terminal end of a target
protein, Siamois, five nucleotides were added to the 5’ end of the forward
primer, followed by a stop codon sequence, while four nucleotides wereadded to the reverse primer (Fig. 2). In the cases for conjugating the SKL
motif to Vent1, Smad1, Smad2, and Smad4, nine nucleotides were added
to the 5’ end of the forward primer. Plasmids pRN3-Siamois, pCS2-Vent1,
pCS2-Smad1, pCS2-Smad2, and pCS2-Smad4 were used for creating
SKL-tag mutants. The primers 5’-AATTATGACTTCTTCGGCCGCG-3’ and5’-TACTGTTTGGGTAGGGCTCTCTAT-3’ were used for constructing Siamois-SKL.
The forward primer 5’-AGTAAACTATAAGCCTCTAGAACTATAGTGAGTCG-3’
was used for constructing Vent1-SKL, Smad1-SKL, Smad2-SKL, andSmad4-SKL. The reverse primers 5’-CATATACTGAGCCCCAAAGAGTG-3’,
5’-ATCCATTAACGAGACCGAGGAG-3’, 5’-GGACATGCTTGAGCAGCGG-3’, and5’-GTCCAGAGGCTGAGGATCAGC-3’ were used for constructing Vent1-SKL,
Smad1-SKL, Smad2-SKL, and Smad4-SKL, respectively. The underlined
sequences in the primers were used for adding SKL. pUC57-Vent2-SKL wasFig. 2 Schematic diagram of SKL conjugation
An SKL conjugate method to the carboxy-terminal end of target molecules. Only asingle long-range polymerase chain reaction is required. Primer regions are indicatedby arrow and box. The sequence of the arrow region is exactly same as the sequencein plasmid. The box region has the sequence of Serine-Lysine-Leucine (SKL). This ismerely an example of gene construction way to conjugate SKL, many other ways canbe applicable for it as we noted about gene construction for Vent1-SKL, Smad1-SKL,
Smad2-SKL, and Smad4-SKL.

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used for constructing Vent2-SKL. We performed long-length polymerase
chain reaction involving these primers and high-fidelity DNA polymerase(KOD-plus Neo, Toyobo, Japan). The 5’ ends of the DNA fragments were
phosphorylated using T4 polynucleotide kinase, followed by self-ligation
with T4 DNA ligase. The mRNAs used for microinjection experiments weretranscribed from linearized plasmids by using mMessage mMachine kit(Ambion). Note that the lengths of plasmid that we used for inverse PCR inthis study were 3 to 5 kbps.

3 Results and Discussions
3.1 The SKL-tag trap assay blocks activity of Siamois
Only one polymerase chain reaction is required to conjugate the three
SKL amino acids at the carboxy-terminal end of a target protein. To evaluate
whether the mutated molecules constructed by this simple method actuallyfunctions, we used our experimental model Xenopus Siamois, a homeobox
transcription factor superfamily that functions as a strong dorsalizing factor
during Xenopus embryogenesis.
Siamois proteins, which belong to the homeobox transcription factor
superfamily, are known to be essential for dorsal formation during vertebrate
embryonic development (Ishibashi et al., 2008). In the Xenopus species,
Siamois is expressed on the dorsal-animal side of blastula embryos prior toany other BCNE players (Kuroda et al., 2004). Therefore, ectopic activationof Siamois (Fig. 3A) can easily induce complete secondary axis containing
head structure (Fig. 3B-E, I). On the other hand, most embryos injected withmRNA encoding SKL-tagged Siamois (Siamois-SKL) did not have secondary
axis formation (Fig. 3F, G, I), and only one embryo had an incomplete
secondary axis (Fig. 3H, I). Next, we tried to examine whether Siamois-SKL
works as an inhibitor of the endogenous Siamois. If the nuclear localization
step in Siamois were to be inhibited using the SKL-tag trap assay, the anterior
structure would fail to develop. MOs are used as loss-of-functional methods
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to inhibit translation of target gene (Heasman, 2002). As reported (Ishibashi
et al., 2008), microinjection of MOs targeting Siamois mRNA (Siamois-MO)
into dorsal side (Fig. 4A) caused anterior defects and ventralization (Fig. 4B,
C, E, G). Interestingly, almost same phenotypical effects were observed in
Siamois-SKL mRNA-injected embryos (Fig. 4D, F, G). These results alsosuggest that Siamois can dimerize with Twin, which is thought as multiple
protein for Siamois (Ishibashi et al., 2008), in endogenous condition.

Fig. 3 Siamois-SKL does not have dorsalizing activity(A) Experimental procedure of microinjection and embryo culture. mRNAs were
injected into single site of ventral vegetal blastomere at 8-cell stage. (B) Control
embryo at stage 38. (C) Control embryo at stage 18. (D) Siamois mRNA-injected
embryo at stage 38. (E) Siamois mRNA-injected embryo at stage 18. (F) SiamoisSKL mRNA-injected embryo at stage 38. (G) Siamois-SKL mRNA-injected embryo
at stage 18. (H) Weak secondary axis formation observed in Siamois-SKL mRNAinjected embryo at stage 38. (I) Graph to show the histogram of D-H.

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3.2 The SKL-tag trap assay blocks activity of Vent1 and Vent2
Vents were originally isolated as the downstream molecules ofventralizing signal dependent on BMPs, and positive feedback loop of Vent/
BMP is thought as the major ventralization mechanism of vertebrate early
embryogenesis (Onichtchouk et al., 1996). We next asked whether the
SKL-tag trap assay is adaptable for Vent1 and Vent2. As shown in Fig. 5,
we designed SKL-tagged Vent1 and Vent2 (Vent1-SKL and Vent2-SKL) to
block BMP signaling. Then we found that ventralization activity by Vent2
was completely diminished in the case of Vent2-SKL (Fig. 5A, B, C, D).
Moreover, a small secondary axis formation, which is generally defined as a

Fig. 4 Siamois-SKL mimics Siamois-MO(A) Experimental procedure of microinjection and embryo culture. mRNAs were
injected into two sites of dorsal blastomere at 8-cell stage. (B) Control embryo atstage 38. (C, E) Siamois-MO-injected embryo at stage 38. (D, F) Siamois-SKL
mRNA-injected embryo at stage 38. (G) Graph to show the histogram of C-F.
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dorsalized effect, was observed by single ventral injection of Vent1-SKL or
Vent2-SKL mRNAs (Fig. 5E, F, G), and coinjection of Vent1-SKL and Vent2SKL mRNAs improved the quality of ectopic secondary axis structure (Fig.
5H, I). These results suggest that the SKL-tagging should be an effective
dominant negative way for the homeobox transcriptional factor superfamily.

Fig. 5 Vent1-SKL and Vent2-SKL have dorsalizing activity(A) Experimental procedure of microinjection and embryo culture. mRNAs were
injected into two sites of dorsal blastomere at the 4-cell stage. (B) Control embryo atstage 38. (C) Vent2 mRNA-injected embryo. (D) Vent2-SKL mRNA-injected embryo.
No obvious effect was observed. (E) Experimental procedure of microinjection andembryo culture. mRNAs were injected into single site of ventral vegetal blastomere
at 8-cell stage. (F) Vent1-SKL mRNA-injected embryo. White arrowhead indicatesweak secondary axis formation. (G) Vent2-SKL mRNA-injected embryo. (H) Vent1SKL and Vent2-SKL mRNAs-coinjected embryo. Siamois-MO-injected embryo atstage 38. (I) Graph to show the histogram of F-H.

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3.3 The SKL-tag trap assay can target other transcription factors
Is the SKL-tag trap assay effective for the other dimer-typed oroligomer-typed transcription factors? In order to answer this question,
Smad family is one of the best candidates for evaluating this assay because
Smads can move into nucleus as heterotrimer or homotrimer when activated(Conidi et al., 2013). We added SKL at the carboxy-terminal regions of
Smad1, Smad2, and Smad4 by PCR (Smad1-SKL, Smad2-SKL, and Smad4SKL) and then injected each mRNA into the 2-cell stage embryos (Fig. 6A).
Surprisingly, strong toxicity was observed in the case of Smad1-SKL mRNA
(Fig. 6B, C, F), and severe posteriorization was induced in only a few survived
embryos. Usually, the BMP/Smad1 signal is required for ventralization andepidermalization in ectoderm region, so loss-of-function of Smad1 should
induce dorsalization or neuralization in epidermis. Interestingly, Smad1-MOin zebrafish embryos strongly upregulates transcription of ERK (Zhang etal., 2014). Strong posteriorization and toxicity are observed when ERK was
activated under the control of FGF signal or Xnr3 (Hou et al., 2007; Yokota etal., 2003). Therefore, phenotypical effects observed in Smad1-SKL mRNAinjected embryos may be explained by overexpression of ERK. In the caseof Smad2-SKL, most of embryos lost head structure and had shorten body
axis (Fig. 6D, F). This level of shorten axis was not induced by microinjectionof Smad1-SKL mRNA. Smad2 is required for mesoderm induction by Nodal
signaling, so this result might be quite reasonable. Interestingly, in the case of
Smad4-SKL, anterior defects were observed as the main phenotypical effects(Fig. 6E, F). Especially, optic structure was diminished in many embryos(Fig. 6E). Smad4 is usually working with Smad1 or Smad2 (Eivers et al.,2008), but Smad1 and Smad2 can be still working without Smad4 because
R-Smad such as Smad1 and Smad2 can form a symmetric homotrimer(Conidi et al., 2013). Therefore, these obvious anterior defects were inducedby combination of moderate inhibitions of the BMP/Smad1 and Nodal/
Smad2 signaling.
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Fig. 6 SKL-conjugation is effective for Smad proteins(A) Experimental procedure of microinjection and embryo culture. mRNAs were
injected into two sites of animal side at 2-cell stage. (B) Control embryo at stage 38.
(C) Smad1-SKL mRNA-injected embryo. (D) Smad2-SKL mRNA-injected embryo.
(E) Smad4-SKL mRNA-injected embryo. (F) Graph to show the histogram of C-E.

3.4 Conclusions
In the present study, we have demonstrated a novel, yet simple and very
effective technique for generating dominant-negative proteins by conjugating
the SKL motif to the carboxy-terminal end of transcriptional factors. In thissystem, we used only one long-range polymerase chain reaction requiring
two primers, each having several additional nucleomers, for adding the SKL
motifs (Fig. 2). It may be quite reasonable way to add several nonsense
amino acids between C-terminal region of target protein and SKL as linker,
but at least in the cases of Siamois, Vents, and Smads it was not required. It
must be next way when simple conjugation of SKL does not work for target
protein. We did not perform typical control experiment such as conjugationof not-SKL tripeptide instead of SKL because we could not find proper
tripeptide for it. One possible way is to use alanine (A), which is the simplest
and uncharged asymmetric amino acid, instead of K in SKL, meaning to use

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SAL instead of SKL, because a positively charged residue (K) is the mostsignificant feature for keeping PTS activity (Brocard et al., 2006). However,this type of experiment may be not required because it is well known
that C-terminal protein-tags with leucine at the C-terminal region such as
Calmodulin-tag (KRRWKKNFIAVSAANRFKKISSSGAL) and Myc-tag(EQKLISEEDL) do not make any functional effect on target proteins.
The phenotypical observation using African clawed frog has long history
and is thought as one of the most reliable methods to evaluate the effectof the BMP, the Wnt, and the Nodal signaling (De Robertis and Kuroda,
2004). In this study, obvious low-Wnt, high-BMP, or low-Nodal effects
were respectively induced by overexpression of Siamois-SKL, Vent1-SKL
and Vent2-SKL, or Smad2-SKL. Since many studies have been focused on
Nodal, BMP, and Wnt proteins in general, the methods described in this study
will be, at a minimum, useful to cell biologists who study these molecules.
Furthermore, it may also be applicable in studies involving different
molecules in various organisms. The uses of this SKL-tag trap assay should,
therefore, allow researchers in molecular and cellular biology to readily
generate new tools for inhibiting the activities of target dimeric, oligomeric,or polymeric transcriptional factors.

Acknowledgments
We thank Prof. Tatsuo Michiue for providing DNA constructs; Minami Ito, Shoko Mori,
and Nana Saito for providing technical assistance.

References
Brocard C., Hartig A., “Peroxisome targeting signal 1: is it really a simple tripeptide?” Biochimica
et Biophysica Acta, 1763, 2006, pp. 1565-1573.
Carnac G., Kodjabachian L., Gurdon J.B., Lemaire P., “The homeobox gene Siamois is a target of
the Wnt dorsalisation pathway and triggers organiser activity in the absence of mesoderm.”
Development, 122, 1996, pp. 3055-3065.
Conidi A., Van den Berghe V., Huylebroeck D., “Aptamers and their potential to selectively target

KEIO SFC JOURNAL Vol.16 No.1 2016

215

自由論題
aspects of EGF, Wnt/β-catenin and TGFβ-smad family signaling.” International Journal of
Molecular Sciences, 14, 2013, pp. 6690-6719.
De Robertis E.M., Kuroda H., “Dorsal-ventral patterning and neural induction in Xenopus
embryos.” Annual Review of Cell and Developmental Biology, 20, 2004, pp. 285-308.
Eivers E., Fuentealba L. C., De Robertis E. M., “Integrating positional information at the level of
Smad1/5/8.” Current Opinion in Genetics & Development, 18, 2008, pp. 304-310.
Flynt A. S., Lai E. C., “RNAi in Xenopus: look before you leap.” Genes & Development, 25,2011, pp. 1105-1108.
Gaj T., Gersbach C. A., Barbas C. F. 3rd., “ZFN, TALEN, and CRISPR/Cas-based methods forgenome engineering.” Trends in Biotechnology, 31, 2013, pp. 397-405.
Heasman J., “Morpholino oligos: making sense of antisense?” Developmental Biology, 243,
2002, pp. 209-214.
Hou S., Maccarana M., Min T.H. Strate I., Pera E.M., “The secreted serine protease xHtrA1
stimulates long-range FGF signaling in the early Xenopus embryo.” Developmental Cell,
13, 2007, pp. 226-241.
Ishibashi H., Matsumura N., Hanafusa H., Matsumoto K., De Robertis E.M., Kuroda H.,“Expression of Siamois and Twin in the blastula Chordin/Noggin signaling center isrequired for brain formation in Xenopus laevis embryos.” Mechanisms of Development,
125, 2008, pp. 58-66.
Kuroda H., Wessely O., De Robertis E.M., “Neural induction in Xenopus: requirement for
ectodermal and endomesodermal signals via Chordin, Noggin, beta-Catenin, and Cerberus.”
PLoS Biology, 2, 2004, e92.
Kuroiwa A. “Homeotic genes and the homeobox.” Seikagaku, 59, 1987, pp. 45-68.
Lanyon-Hogg T., Warriner S.L., Baker A., “Getting a camel through the eye of a needle: theimport of folded proteins by peroxisomes.” Biology of the Cell, 102, 2010, pp. 245-263.
Laurent M.N., Blitz I.L., Hashimoto C., Rothbächer U., Cho K.W., “The Xenopus homeobox
gene twin mediates Wnt induction of goosecoid in establishment of Spemann’s organizer.”
Development, 124, 1997, pp. 4905-4916.
Lemaire P., Garrett N., Gurdon J.B., “Expression cloning of Siamois, a Xenopus homeobox gene
expressed in dorsal-vegetal cells of blastulae and able to induce a complete secondary axis.”
Cell, 81, 1995, pp. 85-94.
Onichtchouk D., Gawantka V., Dosch R., Delius H., Hirschfeld K., Blumenstock C., Niehrs
C., “The Xvent-2 homeobox gene is part of the BMP-4 signalling pathway controlling[correction of controling] dorsoventral patterning of Xenopus mesoderm.” Development,
122, 1996, pp. 3045-3053.
Pauli A., Montague T. G., Lennox K. A., Behlke M. A., Schier A. F., “Antisense OligonucleotideMediated Transcript Knockdown in Zebrafish.” PLoS One, 10, 2015, e0139504.
Rawat V.P., Arseni N., Ahmed F., Mulaw M.A., Thoene S., Heilmeier B., Sadlon T., D’Andrea R.J.,
Hiddemann W., Bohlander S.K., Buske C., Feuring-Buske M., “The vent-like homeobox
gene VENTX promotes human myeloid differentiation and is highly expressed in acute
myeloid leukemia.” Proceedings of the National Academy of Sciences of the United Statesof America, 107, 2010, pp. 16946-16951.
Sander V., Reversade B., De Robertis E.M., “The opposing homeobox genes Goosecoid andVent1/2 self-regulate Xenopus patterning.” EMBO Journal, 26, 2007, pp.2955-2965.
Sharon N., Mor I., Zahavi E., Benvenisty N., “DUXO, a novel double homeobox transcription
factor, is a regulator of the gastrula organizer in human embryonic stem cells.” Stem Cell
Research, 9, 2012, pp. 261-269.

216

SKL Tagging
Yokota C., Kofron M., Zuck M., Houston D.W., Isaacs H., Asashima M., Wylie C.C., Heasman J.,“A novel role for a nodal-related protein; Xnr3 regulates convergent extension movements
via the FGF receptor.” Development, 130, 2003, pp. 2199-2212.
Zhang C., Lv J., He Q., Wang S., Gao Y., Meng A., Yang X., Liu F., “Inhibition of endothelial
ERK signalling by Smad1/5 is essential for haematopoietic stem cell emergence.” Nature
Communications, 5, 2014, pp. 1-13.

〔受付日 2016. 2. 25〕〔採録日 2016. 6. 9 〕

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