DIVISION OF MOLECULAR NEUROBIOLOGY |
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JST Technical Staffs: |
NODA, Masaharu
SHINTANI, Takafumi
SAKUTA, Hiraki
HIYAMA, Takeshi
TAKEUCHI,Yasushi
FUKADA, Masahide
TAKAHASHI, Hiroo (Oct. ’03~)
SUZUKI, Ryoko
TAKAO, Motoharu (Apr. ’03~)
FUJIKAWA, Akihiro 1)
KATO, Akira 1) (~ Sept.’03)
NAKAMURA, Takahiro 1)
YAMAMOTO, Yasunori 1) (Apr.’03~)
TAMURA, Hiroshi
OHKAWARA, Takeshi
TAKAHASHI, Hiroo (~ Sept. ’03)
IHARA, Masaru
TORIUMI, Shigeru (~ Sept. ’03)
ETANI, Kazuma (Apr. ’03~)
SHIMIZU, Hidetada (Apr. ’03~)
TANAKA, Rumi (Apr. ’03~)
YONEHARA, Keisuke (Apr. ’03~)
YUASA, Junichi 2) (~Mar. ’03)
WATAKABE, Ikuko (~Feb. ’03)
KODAMA, Akiko
AYABE, Yuko (~Sept. ’03)
MIZOGUCHI, Masae
GOTOH, Megumi
YAMADA, Kaoru
MATSUI, Mie (~Sept. ’03)
AYABE, Yuko (Oct. ’03~) |
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1) CREST, JST 2) PREST,
JST |
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We have been studying the molecular
and cellular mechanisms underlying the development of the vertebrate
central nervous system. It covers all the major events including the
patterning of the nervous system, neuronal differentiation, axonal
navigation and targeting, synapse formation and plasticity, and neuronal
regeneration, mainly in the visual system. The scope of our interests
also encompasses various functions of the matured brain, including
sensation, behavior, learning and memory.
I. Regional specification in the retina and topographic retinotectal projection
Topographic maps are a fundamental feature of neural networks in the
nervous system. Understanding the molecular mechanisms by which topographically
ordered neuronal connections are established during development has
long been a major challenge in developmental neurobiology. The retinotectal
projection of lower vertebrates including birds has been used as a
readily accessible model system. In this projection, the temporal
(posterior) retina is connected to the rostral (anterior) part of
the contralateral optic tectum, the nasal (anterior) retina to the
caudal (posterior) tectum, and likewise the dorsal and ventral retina
to the ventral and dorsal tectum, respectively. Thus, images received
by the retina are precisely projected onto the tectum in a reversed
manner.
Since 1992, we have been devoting our efforts to searching for molecules
with asymmetrical distribution in the embryonic chick retina, and
to characterization of their roles in the topographic retinotectal
projection. In 1996, we first identified two winged-helix transcriptional
regulators, CBF-1 and CBF-2, expressed in the nasal
and temporal retina, respectively. Misexpression experiments in the
retina using a retroviral vector showed that these two transcription
factors determine the regional specificity of the retinal ganglion
cells, namely, the directed axonal projections to the appropriate
tectal targets along the anteroposterior axis. Secondly, we identified
a novel retinoic acid-generating enzyme, RALDH-3, which is specifically
expressed in the ventral region of the retina, together with a dorsal-specific
enzyme RALDH-1.
Furthermore, we recently identified a novel secretory protein, Ventroptin,
which has BMP-4 neutralizing activity. Ventroptin is expressed in
the retina with a ventral high-dorsal low gradient at early stages.
This expression pattern is complementary to that of BMP-4.
At later stage (E6~), a nasal high-temporal low gradient expression
pattern of it is also detected. Ventroptin thus shows a double-gradient
expression profile along the dorsoventral and nasotemporal axes. Misexpression
of Ventroptin altered expression patterns of several topographic
genes and projection of the retinal ganglion-cell axons to the tectum
along the both axes.
In this year of 2003, we revealed that misexpression of CBF-1 represses the expression of EphA3 and CBF-2, and
induces that of SOHo-1, GH6, ephrin-A2 and ephrin-A5. CBF-1 controls ephrin-A5 by a DNA binding-dependent mechanism, ephrin-A2 by a DNA
binding-independent mechanism, and CBF-2, SOHo-1, GH6 and EphA3 by dual mechanisms (Fig. 1A). BMP-2 expression begins double-gradiently in the retina from E5 instead
of BMP-4 in a complementary pattern to the Ventroptin expression. Ventroptin antagonizes BMP-2 as well as BMP-4. CBF-1 interferes
in BMP-2 signaling and thereby induces expression of ephrin-A2.
Our data suggest that CBF-1 is located at the top of the
gene cascade for the regional specification along the nasotemporal
(NT) axis in the retina (Fig. 1B) and distinct BMP signals play pivotal
roles in the topographic projection along both axes.
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Fig.1. The molecular mechanisms by which CBF-1
controls the expression of topographic molecules. (A) Schematic
representation of modes of actions of CBF-1. You can see that
one Eph-ephrin system is controlled as a set by each mode of
CBF-1 action. |
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(B) Expressional regulation of the topographic
molecules along the NT axis by CBF-1. EphA3 and ephrins
are directly implicated in the control of axon guidance. CBF-1
and Ventroptin repress expression of BMP-2
by inhibiting BMP signaling as an interrupter and antagonist,
respectively, and induce ephrin-A2 expression. CBF-1
represses the transcription of negative regulators, X
and Y. SOHo-1 and GH6 inhibit the
expression of EphA3. CBF-1 also represses
CBF-2 expression, however, its downstream target genes
have not been identified yet. |
Currently, with respect to the other identified molecules, we are
conducting misexpression experiments using chick embryos and generating
knockout and transgenic mice to elucidate the molecular functions.
We expect that our studies will lead to dissection of the molecular
mechanism underlying the retinal patterning and topographic retinotectal
projection, and ultimately to uncovering the basic principles for
establishing complicated but extremely precise neural networks.
II. Axonal morphogenesis and behavior
During development, cells undergo dynamic morphological changes by
rearrangements of the cytoskeleton including microtubules. However,
molecular mechanisms underlying the microtubule remodeling between
orientated and disorientated formations are almost unknown. We found
that novel subtypes of collapsin response mediator proteins (CRMP-As)
in addition to the originals (CRMP-Bs), which occur from the alternative
usage of different first coding exons, are involved in this conversion
of microtubule patterns.
Overexpression of CRMP2A and CRMP2B in chick embryonic
fibroblasts induced orientated and disorientated patterns of microtubules,
respectively. Moreover, sequential overexpression of another subtype
overcame the effect of the former expression of the countersubtype.
Overexpression experiments in cultured chick retinae showed that CRMP2B
promoted axon branching and suppressed axon elongation of ganglion
cells, while CRMP2A blocked these effects when co-overexpressed (Fig.
2). Our findings suggest that the opposing activities of CRMP2A and
CRMP2B contribute to the cellular morphogenesis including neuronal
axonogenesis through remodeling of microtubule organization.
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Fig.2. Antagonistic effects of CRMP2A and –2B
on axon morphology. Retinae from chick E6 embryos were electroporated
with IGG vector to express CRMP2B (A) and CRMP2B + CRMP2A (B),
and cultured for 2 days. |
III. Physiological roles of protein tyrosine phosphatase receptor type Z (Ptprz)
Protein tyrosine phosphorylation plays crucial roles in various biological
aspects including all stages of brain development. In 1994, we found
that PTPz/RPTPb/Ptprz,
a nervous system-rich receptor-type PTP, is expressed as a chondroitin
sulfate proteoglycan in the brain. Ptprz is expressed from
the early developmental stage to the adulthood in neurons as well
as astrocytes. This suggests that this molecule plays variegated roles
in the brain development and brain function.
We found in 1996 that Ptprz binds pleiotrophin (PTN)/HB-GAM and midkine
(MK), closely related heparin-binding growth factors which share many
biological activities. The chondroitin sulfate portion of Ptprz is
essential for the high affinity binding (Kd = ~0.25 nM) to these growth
factors, and removal of chondroitin sulfate chains results in a marked
decrease of binding affinity (Kd = ~13 nM).
To identify the substrate molecules of Ptprz, we recently developed
the yeast substrate-trapping system. This system is based on the yeast
two-hybrid system with two essential modifications: Conditional expression
of v-src to tyrosine-phosphorylate the prey proteins and screening
using a substrate-trap mutant of PTP as bait. Using this system, we
successfully isolated a number of candidate clones for substrate molecules
(ex. GIT1/Cat-1) and continuously-interacting molecules (ex. PSD-95/SAP90)
for Ptprz. We are now continuing efforts to characterize these candidate
clones.
In addition, to know the physiological roles of Ptprz in vivo,
we generated Ptprz-deficient mice in which the Ptprz gene
was replaced with the LacZ gene in 1997. We are currently
studying the phenotype of Ptprz-deficient mice biochemically,
anatomically, physiologically and ethologically.
We reported this year that mice deficient in Ptprz do not
show mucosal damage by VacA, although VacA is incorporated into the
gastric epithelial cells to the same extent as in wild-type mice (Fig.
3). The vacuolating cytotoxin VacA produced by Helicobacter pylori causes massive cellular vacuolation in vitro and gastric
tissue damage in vivo, leading to gastric ulcers, when administered
intragastrically. Primary cultures of gastric epithelial cells from Ptprz+/+ and Ptprz-/- mice
also showed similar incorporation of VacA, cellular vacuolation and
reduction in cellular proliferation, but only Ptprz+/+ cells showed marked detachment from a reconstituted basement membrane
24 h after treatment with VacA. VacA bound to Ptprz, and the levels
of tyrosine phosphorylation of Git1, a Ptprz substrate, were higher
after treatment with VacA, indicating that VacA behaves as a ligand
for Ptprz. Furthermore, PTN, an endogenous ligand of Ptprz, also induced
gastritis specifically in Ptprz+/+ mice when administered
orally. Taken together, these data indicate that erroneous Ptprz signaling
induces gastric ulcers.
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Fig.3. Pathological analyses of gastric tissues
treated with VacA. (A,a) Stereomicroscopic
appearance of the inside of stomachs from Ptprz+/+
mice 48 h after administration of VacA (500 mg
per kg body weight). (A,b) Diagrammatic representation
of a showing the gastric ulcer (red area with arrowheads). (B)
Appearance of the inside of stomachs from Ptprz-/-
mice treated equally. (C,D) Gastric sections stained with hematoxylin
and eosin. Loss of epithelial cells and gastric gland structure
(arrowheads) was observed in the mucosal layer in Ptprz+/+
mice (C) but not Ptprz-/-
mice (D). The lower panels are enlargements.
Scale bars: 500 mm. M, Mucosa; Sm,
Submucosa; Mp, Muscularis propia. |
IV. Na+-level sensing in the brain
Sodium-level sensing system in the brain is essential for the regulation
of the sodium and water balance in body fluids. Previously, we demonstrated
that Nax (also called Nav2/NaG)
gene is expressed in the circumventricular organs and Nax-deficient
mice ingest salt in excess. In Nax-deficient
mice, c-fos expression in the subfornical organ (SFO) and
organum vasculosum laminae terminalis (OVLT) was markedly elevated
as compared with wild-type mice. Stimulation of the SFO/OVLT of
wild-type animals by infusion of a hypertonic sodium solution into
intracerebroventricule (ICV) leads to avoidance of salt intake.
In contrast, Nax-deficient mice did not show
such aversion.
We showed that Nax is a sodium channel which is sensitive
to the increase of extracellular sodium level. Entry of sodium ions
occurred in response to a rise of the extracellular sodium concentration
(C1/2= 159 mM). In contrast, these responses
were not observed in Nax-immunonegative cells or neurons
of Nax-deficient mutant origin. Transfection
of Nax cDNA conferred the sodium sensitivity
on Nax-deficient cells. All of the GABA-immunopositive
neurons isolated from the SFO responded to the extracellular sodium
increase. Based on these findings, we proposed that GABAergic inhibitory
neurons expressing Nax control the activity
of the SFO and suppress the salt-intake behavior of animals under
thirst conditions.
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Publication List:
Asahi, M., Tanaka, Y., Izumi, T., Ito, Y., Naiki, H., Kersulyte, D., Tsujikawa, K., Saito, M., Sada, K., Yanagai, S., Fujikawa, A., Noda, M. and Itokawa, Y. (2003) Helicobacter pylori CagA containing ITAM-like sequences localized to lipid rafts negatively regulates VacA-induced signaling in vivo. Helicobacter, 8, 1-14.
Fujikawa, A., Shirasaka, D., Yamamoto, S., Ota, H., Yahiro, K., Fukada, M., Shintani, T., Wada, A., Aoyama, N., Hirayama, T., Fukamachi, H. and Noda, M. (2003) Mice deficient in protein tyrosine phosphatase receptor type Z are resistant to gastric ulcer induction by VacA of Helicobacter pylori. Nature Genet., 33, 375-381.
Nakayama, M., Kimura, M., Wada, A., Yahiro, K., Ogushi, K., Niidome, T., Fujikawa, A., Shirasaka, D., Aoyama, N., Kurazono, H., Noda, M. and Hirayama, T. (2003) Helicobacter pylori VacA activates the p38/ATF-2-mediated signal pathway in AZ-521 cells. J. Biol. Chem., (Online in Nov.)
Sakaguchi, N., Muramatsu, H., Ichihara-Tanaka, K., Maeda, N., Noda, M., Yamamoto, T., Michikawa, M., Ikematsu, S., Sakuma, S. and Muramatsu, T. (2003) Receptor-type protein tyrosine phosphatase z as a component of the signaling receptor complex for midkine-dependent survival of embryonic neurons. Neurosci. Res., 45, 219-224.
Takahashi, H., Shintani, T., Sakuta, H. and Noda, M. (2003) CBF-1 controls the retinotectal topographical map along the anteroposterior axis through multiple mechanisms. Development, 130, 5203-5215.
Tanaka, M., Maeda, N., Noda, M. and Marunouchi, T. (2003) A chondroitin sulfate proteoglycan PTPz/RPTPb regulates the morphogenesis of Purkinje cell dendrites in the developing cerebellum. J. Neurosci., 23, 2804-2814.
Watanabe, U., Shimura, T., Sako, N., Kitagawa, J., Shingai, T., Watanabe, E., Noda, M. and Yamamoto, T. (2003) A comparison of voluntary salt-intake behavior in Nax-gene deficient and wild-type mice with reference to peripheral taste inputs. Brain Res., 967, 247-256.
Yuasa-Kawada, J., Suzuki, R., Kano, F., Ohkawara, T., Murata, M. and Noda, M. (2003) Axonal morphogenesis controlled by antagonistic roles of two CRMP subtypes in microtubule organization. Eur. J. Nuerosci., 17, 2329-2343.
Ohyama, K., Ikeda, E., Kawamura, K., Maeda, N. and Noda, M. (2004) Receptor-like protein tyrosine phosphatase z/RPTPb is expressed on tangentially aligned neurons in early mouse neocortex. Develop. Brain Res., 59, 34-47.
Shintani, T., Kato, A., Yuasa-Kawada, J., Sakuta, H., Takahashi, M., Suzuki, R., Ohkawara, T., Takahashi, H. and Noda, M. (2004) Large-scale identification and characterization of genes with asymmetric expression patterns in the developing chick retina. J. Neurobiol., 148, 121-127. |
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