DIVISION OF MORPHOGENESIS |
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Professor:
Associate Professor:
Research Associates:
Technical Staffs:
NIBB Research Fellows:
Postdoctoral Fellows:
Graduate Students: |
UENO, Naoto
KINOSHITA, Noriyuki
NAKAMURA, Makoto
TAKAHASHI, Hiroki
TAKAGI, Chiyo
YAMAMOTO, Takamasa
HYODO, Akiko
TERASAKA, Chie
MOROKUMA, Yoshie
YAMADA, Shigehiro
TANABE, Fumio
HYODO-MIURA, Junko
TAKEUCHI, Masaki
KITAYAMA, Atsushi
MOROKUMA, Junji
HOTTA, Kohji
IIOKA, Hidekazu 1)
MIYAKOSHI, Akira 1)
CHUNG, Hyeyoung 2)
KUJIRAOKA, Masahiro 1)
YOSHIKANE, Nami 3) |
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1) Graduate University for Advanced Studies, SOKENDAI
2) University of Tokyo
3) Nara Institute of Science and Technology |
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The complex morphogenesis of organisms
is achieved by consecutive cell-to-cell interactions during development.
Recent studies suggest that growth factors play crucial roles in controlling
such intercellular communications in a variety of organisms. In addition
to secretory factors, transcription factors which act cell-autonomously
are thought to be essential for the determination of cell fates. Our
main interest is to know how pattern formation and morphogenesis during
development is regulated by these growth factors and transcription
factors. We address this problem using several model animals, including
frog, fly and ascidian, employing embryology, genetics, molecular
and cellular biology, and biochemistry. In addition, we have recently
introduced genome-wide approaches to elucidate precise genetic program
controlling early development.
I. Gastrulation movement regulated by Wnt signaling
Gastrulation is one of the most important processes during morphogenesis
of early embryo, involving dynamic cell migration and change in embryo
shape. Almost all animals undergo gastrulation to form the gut. In
spite of its importance, the mechanism underlying the event has just
begun to be studied at molecular level. During Xenopus gastrulation,
mesodermal cells migrate to the inside of the embryo and move on the
blastocoel roof. One of the important mechanisms for this process
is convergent extension. As convergent extension begins, cells are
polarized and aligned mediolaterally, followed by the intercalation
of these cells. As described above, one of the Wnt signaling pathways,
called Wnt/JNK (c-Jun N-terminal kinase) pathway, is shown to be important
for the regulation of convergent extension. The pathway is highly
conserved among species and initially found to be essential for the
establishment of planar cell polarity (PCP) of Drosophila
wing hair.
We have previously demonstrated that Xenopus prickle (Xpk),
a Xenopus homologue of a Drosophila PCP gene, is an
essential component for gastrulation cell movement. Both gain-of-function
(GOF) and loss-of-function (LOF) of Xpk severely perturbed
gastrulation and caused spina bifida embryos without affecting
mesodermal differentiation. We also demonstrated that XPK binds to Xenopus Dsh as well as to JNK. This suggests that XPK plays
a pivotal role in connecting Dsh function to JNK activation. To understand
the molecular mechanism, we identified proteins which binds to XPK
by yeast two-hybrid screening. One of XPK binding protein was found
to be a member of the Ste20 kinase family and named as Xenopus prickle-interacting kinase, XPIK. Developmental expression pattern
of XPIK is reminiscent of that of XPIK, suggesting functional interaction
between XPK and XPIK. GOF and LOF of XPIK resulted in perturbation
of gastrulation, leading to shortened or spina bifida embryo.
Furthermore, we have found that XPIK is not only sufficient to activate
JNK in embryo, but also required for full activation of JNK by Dishevelled.
These suggest that XPIK also plays an essential role in connecting
extracellular Wnt signal to JNK activation through Dishevelled and
XPIK.
In addition, we have been attempting to identify novel regulatory
components controlling gastrulation cell movements by an expression
cloning method based on morphology of dorsal marginal zone explant
(Keller's explant) and of embryo. After 1,500 clones were examined
by overexpression in the dorsal region of embryos, approximately 5%
of clones were found to perturb normal gastrulation cell movements.
Functional relevance of identified genes to cell movements is currently
under investigation.
II. Regulation of actin cytoskeletal dynamics
during Xenopus gastrulation
Because gastrulation movements are accompanied by dynamic changes
in cell polarity, morphology, and motility, it is very likely that
actin cytoskeleton is carefully regulated. Thus, we analyzed the regulatory
mechanism of actin cytoskeletal dynamics during this process. Among
several factors implicated in the regulation of the actin cytoskeleton,
we decided to focus on myristoylated alanine-rich C kinase substrate
(MARCKS). MARCKS is an actin-binding, membrane-associated protein
previously implicated in the regulation of F-actin dynamics. It has
been investigated mainly in tissue culture cells and biochemical analyses. MARCKS knock-out mice has been reported to show neural tube
defects. Yet, its molecular mechanism has not been elucidated. We
demonstrate that MARCKS is essential for gastrulation movements and
neural tube closure in Xenopus embryos. Cell biological analyses
revealed that MARCKS knock-down with Morpholino oligo (MO) significantly
reduced cortical actin formation and caused defects in cell polarity,
adhesion, motility and protrusive activity, leading to these developmental
defects. We also showed that the Wnt pathway dramatically promoted
the formation of lamelipodia- and filopodia-like protrusions and MARCKS
is required for this activity. These findings show that the Wnt signaling
pathway regulates cortical actin dynamics and MARCKS is requisite
for the Wnt function. We conclude that MARCKS is essential for dynamic
morphogenetic movements during embryogenesis.
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Figure 1 MARCKS is required for convergent extension
movements. MARCKS Mo or control Mo, Rhodamine dextran
(RhDx), and Venus mRNA were co-injected into one of the two
dorsal blastomeres. Venus mRNA alone was injected into the other
dorsal blastomere. In the absence of MARCKS Mo, red
cells and non-red cells were polarized and intercalated. In
contrast, MARCKS Mo-injected cells were not polarized
and did not participate in the intercalation. |
III. Genetic screening for novel DPP/BMP
signaling components utilizing Drosophila model system
Drosophila is one of the ideal model organisms
to dissect signal transduction pathway by genetic methods. We have
carried out dominant suppressor screening for a transgenic mutant
fly that expresses activated DPP/BMP type-I receptors in wing imaginal
discs. We isolated 19 suppressor mutants, Suppressor of constitutively
activated Dpp singnaling (Scad). Alleles of punt, Mad,
shn and dCrebA were found in isolated Scad mutants.
Most of the Scad mutants encode a nuclear protein suggesting these
molecules regulates DPP signaling at nuclear level.
We now focus to study one of the mutants Scad67. Scad67 was also isolated by a Mexican group and named as tonnalli (tna).
We also isolated vertebrate homologs of Scad67/tna, TONAS-1
and TONAS-2. The most characteristic feature of these proteins is
the existence of a single SP-RING finger motif in the middle. The
SP-RING motif was originally found in the PIAS family SUMO-E3 ligase
proteins. We found that TONAS has SUMO-E3 ligase activity and TONAS
facilitates specific SUMO-2/3 conjugation to TONAS itself. TONAS also
shows strong activity of nuclear body formation in cultured cells.
TONAS effectively recruits transcriptional regulators including PML,
CBP and P300 to the nuclear body.
It has been shown that the Trithorax group componets are essential
factors in ATP-dependent chromatin remodeling complex. Our results
suggest a role for Scad67/TNA and TONAS in the connection of the Trithorax/SWI/SNF
chromatin remodeling complex to CBP/P300, the relocation of these
protein complexes into the nuclear substructure, and the regulation
of gene expression.
IV. Brachyury downstream
notochord differentiation in the ascidian embryo
Ascidians, urochordates, are one of the three chordate groups, and
the ascidian tadpole is thought to represent the most simplified and
primitive chordate body plan. It contains a notochord, which is a
defining characteristic of chordate embryo composed of only 40 cells.
To understand the morphogenesis in this simple system, we have focused
on a gene, Brachyury, which is known to play an important
role in the notochord development. In ascidian, Brachyury is expressed exclusively in the notochord and the misexpression of
the Brachyury gene (Ci-Bra) of Ciona intestinalis is sufficient to transform endoderm into notochord. This gene encodes
a sequence-specific activator that contain a T-box DNA-binding domain,
and in vertebrates, it is initially expressed throughout the presumptive
mesoderm and gradually restricted to the developing notochord and
tailbud. The phenotype of the Brachyury mutants in mice and
zebrafish revealed that this gene is essential for notochord differentiation.
Our goal is to elucidate the down stream pathway of this important
gene in ascidian in order to set the stage for understanding not only
the formation and function of the notochord but how this important
structure has evolved. We conducted the subtractive hybridization
screens to identify potential Brachyury target genes that
are induced upon Ci-Bra overexpression. Out of 501 independent
cDNA clones that were induced, 38 were specifically expressed in notochord
cells. We characterized of subcellular-localizations of the 20 GFP
fusion gene products in the notochord cell. These products observed
after electroporating the embryos at one-cell stage with GFP fusion
gene containing notochord specific promoter. They showed various subcellular
localizations in the notochord cells of the tadpole tails. In addition
to investigate the actual functions of the genes during the notochord
formation, functional analyses were performed by injecting of antisence
morphollino oligos.
V. Comprehensive analysis of developmentally regulated genes using
cDNA microarray
In order to examine the global expression profile during early development
of Xenopus laevis, we have collected massive EST sequences
from three normalized cDNA libraries of early gastrula, neurula and
tailbud stage. To date, more than 100,000 ESTs (~30% of Xenopus
laevis ESTs registered in the public database) were produced.
Using these cDNA clones, we generated the NIBB 40k cDNA macroarray
and 4.6k non-redundant cDNA microarray. With the DNA arrays, we conducted
a series of large-scale screening of the genes which expressions are
regulated by some transcription factors and signaling factors (Xnr-1,
FAST1, FGF etc.). Consequently, we have been able to isolate lots
of the candidate genes that encode a variety of signal transduction
and transcription regulatory components, and also cytoskeletal components,
suggesting dynamic cellular changes in the early development of Xenopus
laevis. This also proves DNA array to be an effective screening
assay for novel genes which function during early development process
of Xenopus laevis.
Adding the EST sequences, the assembled sequences and their annotation
information are available through the web at NIBB Xenopus laevis
EST database XBD2 (Fig.2). XDB2 also provides the whole mount in situ hybridization images as the spatial expression pattern
(Fig.3).
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Figure 2 XDB2 (http://xenopus.nibb.ac.jp). |
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Figure 3 Whole mount in situ hybridization
images. |
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Publication List:
Azumi, K., Takahashi, H., Miki, Y., Fujie, M., Usami, T., Ishikawa, H., Kitayama, A., Satou, Y., Ueno, N. & Satoh, N. (2003) Construction of a cDNA microarray derived from the ascidian Ciona intestinalis. Zoological Science. 20, 1223-1229.
Carreira-Barbosa, F., Concha, M. L., Takeuchi, M., Ueno, N., Wilson, S. W. and Tada, M. (2003) Prickle 1regulates cell movements during gastrulation and neuronal migration in zebrafish. Development 130, 4037-4046.
Hotta, K., Takahashi, H., Ueno, N. & Gojobori, T. (2003) A genome-wide survey of the Genes for Planar Polarity signaling or Convergent Extention-related genes in Ciona intestinalis and comparisons of evolutionary conserved signaling components. Gene. 317, 165-185.
Kinoshita, N., Iioka, H., Miyakoshi, A. and Ueno, N. (2003) PKCd is essential for Dishevelled function in a noncanonical Wnt pathway that regulates Xenopus convergent extension movements. Genes Dev. 17, 1663-1676.
Kurata, T. and Ueno, N. (2003) Xenopus Nbx, a novel NK-1 related gene essential for neural crest formation. Dev. Biol. 257, 30-40.
Ohkawara, B., Yamamoto, T. S., Tada, M. and Ueno, N. (2003) Role of glypican 4 in the regulation of convergent extension movements during gastrulation in Xenopus laevis. Development 130, 2129-2138.
Suzuki, M., Ueno, N. and Kuroiwa A. (2003) Hox proteins functionally cooperate with the GC box-binding protein system through distinct domains. J. Biol. Chem. 278, 30148-30156.
Takeuchi, M., Nakabayashi, J., Sakaguchi, T., Yamamoto, S. T., Takahashi, H., Takeda, H. Ueno, N. (2003) Prickle-related gene in vertebrates is essential for gastrulation cell movements. Curr. Biol., 15, 674-679. |
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