One of the research interests of this
laboratory is to understand molecular mechanism how a cell signaling
molecule, including members of Wnt, BMP and FGF families, regulates
different developmental events. A number of evidence indicated that
each signal is involved in many aspects of the vertebrate development.
For instance, we have revealed that Wnt-3a, a members of Wnt family,
plays essential roles in a number of aspects of the mouse development,
including somite development, neural crest formation and neural development.
However, cellular and molecular mechanisms how a cell signaling molecule
regulates these different events. Thus, we are focusing on precise
functional analysis of cell-to-cell signals and identification of
target genes induced by these signals.
Another interest is to understand molecular mechanism of development
of the vertebrate trunk, especially somite. One of interesting features
of the vertebrate trunk development is that it proceeds gradually.
To understand how metameric structures of somites are gradually generated
in an anterior to posterior order along the both sides of embryonic
body axis and how each somite are characterized differently along
the anterio-to-posterior axis, we are also trying genetical approach
with the zebrafish.
I. Roles of Wnt signals during neural development
The Wnt family of genes that encode cysteine rich secreted proteins
consists of at least 17 members in the vertabrate. It has already
been shown that some of them are expressed and play important roles
during neural development. For instance, we showed that Wnt-1 and
Wnt-3a, which are expressed in the most dorsal region within the developing
central nervous system, direct specification of the dorsal interneurons.
Analysis of mouse embryos lacking both Wnt1 and Wnt3a and culture
of explants from the neural plate indicated that these Wnt signals
promote generation of the most dorsal subclass of the interneuron,
called D1 and D2, at the expense of that of more ventral subclass,
called D3.
Wnt signaling is also implicated in the control of cell growth and
differentiation during CNS development from studies of mouse and chick
models, but its action at the cellular level has been poorly understand.
In vitro stem cell culture is a powerful tool for examining the effect
of an external signal on neural stem cells. In vitro clonal analysis
has shown that, in a serum-free defined medium supplemented with FGF-2,
single cells derived from the embryonic or adult brain proliferate
and form floating spherical colonies, called neurospheres. Single
cells derived from neurospheres self-renew to generate a new neurosphere
or differentiate into neurons or glia depending on the culture condition,
indicating that they have characteristics of stem cells.
Thus, to understand function of Wnt signaling on the neural stem cells,
we examined the in vitro function of Wnt signaling in embryonic neural
stem cells, dissociated from neurospheres derived from E11.5 mouse
telencephalon. Conditioned media containing active Wnt-3a proteins
were added to the neural stem cells and its effect on regeneration
of neurospheres and differentiation into neuronal and glial cells
was examined. Wnt-3a proteins inhibited regeneration of neurospheres,
but promoted differentiation into MAP2-positive neuronal cells (FIG.
1). Wnt-3a proteins also increased the number of GFAP- positive astrocytes
but suppress the number of oligodendroglial lineage cells expressing
PDGFR or O4. These results indicate that Wnt-3a signaling can inhibit
the maintenance of neural stem cells, but rather promote the differentiation
of neural stem cells into several cell lineages.
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Fig.1. Inhibition of neurosphere formation and
promotion of neural cell differentiation in the presence of
Wnt3a CM.
A: Cells dissociated from the E11.5 mouse forebrain were cultured
for 2 days in low attachment multiwell plates under various
conditions: with Wnt3a conditioned medium (Wnt-3a CM) (A), with
immunoabsorbed Wnt-3a CM (B), with control conditioned medium
(control CM) (C), and without conditioned medium (D). Many large
spheres were generated in the culture with control CM (D). In
contrast, treatment with Wnt-3a CM significantly increased the
number of differentiated cells and almost wiped out neurospheres
(A). This effect was dependent on Wnt-3a concentration (data
not shown). These effects of Wnt-3a CM were almost eliminated
by immunoabsorption, indicating that these effects are due to
the Wnt-3a protein (B). Total 10 fields were counted under phase-contrast
microscope using 100x magnification.
B: Dissociated cells from the primary neurospheres obtained
from the E11.5 telencephalon were cultured for two days on poly
D-lysine coated coverslips in the presence of Wnt3a CM and FGF2.
Then, these cells were continuously cultured for an additional
four days with Wnt3a CM (A-C) or control CM (D-F), but without
FGF2. After 4 days in culture, cells were immunostained for
Nestin or MAP2. Immunofluorescence microscopy for Nestin (red)
(A, D), MAP2 (green) (B, E) and DAPI (blue) (C, F). |
II. Functional Analysis of molecular targets of
Wnt signaling during development
Wnt-3a is also expressed in the primitive streak ectoderm during gastrulation
and in the tailbud in later development of the mouse. For dissecion
of the complex developmental events regulated by Wnt-3a signaling
in these regions, it is important to identify genes regulated by this
signal. It has already been demonstrated that T (Brachyury) is a direct
target of Wnt-3a in the anterior primitive ectoderm, which is fated
to give rise to the paraxial mesoderm, suggesting that Wnt-3a modurates
a balance between mesodermal and neural cell fates via T.
To gain more insight into roles of Wnt signaling during embryogenesis,
we searched for potential target genes of this signaling by an induction
gene trap screening in mouse ES cells. In at least three ES cell clones
among 794 clones screened, expression of beta-geo reporter genes was
dramatically changed in response to the conditioned medium of Wnt-3a
expressing cells. The expression analysis of the reporter genes in
embryos generated from these ES cell clones revealed that the spatiotemporal
expression patterns of these reporter genes were well correlated to
those of several Wnt genes. These results suggested that an induction
gene trap approach is effective for screening of target genes of Wnt
signaling during embryogenesis.
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Fig. 2. An example of the expression of trapped
genes which are repressed by Wnt signal in ES cells. In vivo
expression and in vivo function of this gene have been examined
by generating mice carrying this trapped allele. |
III. Genetical approaches for revealing molecular mechanism of trunk
development in zebrafish
To identify genes involved in several aspects during early embryogenesis
of the vertebrate, we have performed screening of zebrafish mutants
induced by ENU, a chemical mutagen. Until now, we have screened 630
of F2 families and found a number of mutants whose phenotypes are
different from those already reported. For instance, some of these
mutants displayed defects in the somite and tailbud development. Cloning
of genes that are responsible for these defects is in progress.
To complement a forward genetical approach, we have also screened
genes expressed in the tailbud and presomitic mesoderm, in which somite
progenitors exist. Until now, we have identified 50 genes that are
expressed preferentially in these regions. To examine developmental
roles of these genes, functional analysis of these genes has been
performed by injecting morpholino anti-sense oligonucleotides.
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Fig. 3. Zebrafish mutant embryos which display
abnormal shape of the tailbud (B) and a segmentation defect
of somites (C). |
Publication List:
Fujino T, Asaba H, Kang MJ, Ikeda Y, Sone H, Takada S, Kim DH, Ioka
RX, Ono M, Tomoyori H, Okubo M, Murase T, Kamataki A, Yamamoto J,
Magoori K, Takahashi S, Miyamoto Y, Oishi H, Nose M, Okazaki M, Usui
S, Imaizumi K, Yanagisawa M, Sakai J, Yamamoto TT. (2003) Low-density
lipoprotein receptor-related protein 5 (LRP5) is essential for normal
cholesterol metabolism and glucose-induced insulin secretion. Proc
Natl Acad Sci U S A. 100, 229-234
Muroyama Y. Kondoh H. Takada S. Wnt proteins promote neuronal differentiation
in neural stem cell culture. Biochem. Biophys. Res. Commun.
In press
Nakagawa S, Takada S, Takada R, Takeichi M. (2003) Identification
of the laminar-inducing factor: Wnt-signal from the anterior rim induces
correct laminar formation of the neural retina in vitro. Dev Biol.
260, 414-425.
Oishi I, Suzuki H, Onishi N, Takada R, Kani S, Ohkawara B, Koshida
I, Suzuki K, Yamada G, Schwabe GC, Mundlos S, Shibuya H, Takada S,
Minami Y. (2003) The receptor tyrosine kinase Ror2 is involved in
non-canonical Wnt5a/JNK signalling pathway. Genes Cells.
8, 645-654 |