DIVISION OF MOLECULAR & DEVELOPMENTAL BIOLOGY
 
Professor:
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Postdoctoral Fellows:



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Technical Assistant:

TAKADA, Shinji
KOSHIDA, Sumito
UTSUMI, Hideko
OHBAYASHI, Norihiko
KAWAMURA, Akinori
MUROYAMA, Yuko
KURATA, Tomoko
AKANUMA, Takashi
TAKADA, Ritsuko
YAMAGUCHI, Yoshifumi 1)
ODA, Ritsuko

1) Graduate School of Biostudies, Kyoto University
 

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.

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.

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.

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