National Institute for Basic Biology


Naoto Ueno
Associate Professor:
Hiroshi Shibuya
Research Associates:
Makoto Nakamura
Makoto Mochii
JSPS Visiting Scientist:
Ken W. Y. Cho1)
Visiting Scientists:
Shinichi Higashijima2)
Fumihiko Hamada3)
Takeshi Suzuki4)
Kathy Neal5)
JSPS Postdoctoral Fellow:
Miho Shimizu
Graduate Students:
Hideyuki Nagaso6)
Masataka Nikaido6)
Yasuhiro Takatsu6)
Michiru Nishita6)
Katsura Sugawara
Satoru Yoshida
Yasunori Tomoyasu
Shinichi Nagai7)
Yayoi Nishida7)
Technical Staffs:
Chiyo Takagi
Takamasa Yamamoto
Shunichiro Iemura
Kiyokazu Morita
Yuko Takahashi
1) from University of California, Irvine
3) Osaka University
4) Medical School, Nagoya University
5) MRC, Mill Hill
6) Graduate School of Hokkaido University
7) Graduate School of Nagoya University

Early embryogenesis is regulated by a number of endocrine and paracrine factors that mediate cell-to-cell communications. Particularly, polypeptide growth factors are believed to be essential for the regulation of cell proliferation, differentiation and apoptosis in morphogenic events during early development. The most striking feature of growth factor is that their functions are extremely well conserved among animal species, which allows us to investigate developmental mechanisms governed by growth factors using multiple model animals, such as mouse, zebrafish, Xenopus, Drosophila, C. elegans etc. To understand molecular basis of morphogenesis, we focus on a group of growth factors Transforming growth factor-b (TGF-b) superfamily and investigate molecular mechanisms of their actions in early embryogenesis. Our goal is to unveil species-specific and conserved mechanism of early development.

I. The role of BMP in ectodermal pattern formation.

Bone morphogenetic proteins belonging to the TGF-b superfamily have critical roles in the determination of cell fate during early development. It is widely accepted that ventrally expressed BMPs are essential for epidermal differentiation. Overexpression BMP in dorsal blastomeres of Xenopus laevis results in the loss of neural tissues including head and eyes. To investigate whether BMP can determine epidermal differentiation by acting directly on ectoderm or acting through the conversion of mesodermal cell fate, we performed bead transplantation experiment using zebrafish as a model. BMP-soaked beads were transplanted into presumptive neural region of early gastrula (shield stage) embryo and marker gene expression was examined. The result indicated that BMP induces epidermal fate and inhibits neural fate, acting directly on ectoderm (Figure 1). We also found that action range of BMP in zebrafish ectoderm is rather restricted, denying the possibility of relay mechanism of signal propagation.

N eural induction takes place because BMP activity is inhibited by organizer factors emanated from Spemann organizer. Such organizer factors include Chordin, Noggin and Follistatin. Chordin and Noggin have been shown to bind BMPs directly and thus inhibit their epidermal inducing activity. We have been able to show that Follistatin can also bind BMPs directly albeit with a low affinity and high dissociation constant.

Figure 1.
Effect of BMP in ectodermal patterning. (A) BMP-soaked bead in neural region suppressed a neural marker otx-2 expression. (B) Control bead soaked in BSA does not affect otx-2 expression. (C) BMP-bead transplantation into shiled stage embryos does not perturb expression of mesodermal marker genes goosecoid (light brown) and no tail (dark brown). (D) BMP-bead ectopically induces an epidermal marker gata-3.

II. The role of XIAP and Dlx5 in BMP signaling

T AK1, a member of the MAP kinase kinase kinase family, and its activator, TAB1, participate in the bone morphogenetic protein (BMP) signaling pathway involved in mesoderm induction and patterning in early Xenopus embryos. However, the events leading from receptor activation to TAK1 activation remain to be identified. A yeast interaction screen was used to search for proteins that function in the pathway linking the receptors and TAB1-TAK1. The human X-chromosome-linked inhibitor of apoptosis protein (XIAP) was isolated as a TAB1-binding protein. XIAP associated not only with TAB1 but also with the BMP receptors in mammalian cells. Injection of XIAP mRNA into dorsal blastomeres enhanced the ventralization of Xenopus embryos in a TAB1-TAK1-dependent manner. Furthermore, a truncated form of XIAP lacking the TAB1-binding domain partially blocked the expression of ventral mesodermal marker genes induced by a constitutively-active BMP type I receptor. Taken together, we identified that XIAP participates in the BMP signaling pathway as a positive regulator linking the BMP receptors and TAB1-TAK1.

Recently, we isolated a murine homeobox-containing gene, distal-less 5 (mDlx5), as a BMP-inducible gene in osteoblastic MC3T3-E1 cells. Stable transfectants of MC3T3-E1 that overexpress mDlx5 mRNA showed increase in various osteogenic markers; a 4-fold increase in alkaline phosphatase activity, a 6-fold increase in osteocalcin production and appearance in mineralization of extracellular matrix. Furthermore, mDlx5 was induced orthotopically in mouse embryos treated with BMP-4 and in fractured bone of adult mice. Consistent with these observations, we also found that injection of mDlx5 mRNA in dorsal blastomeres enhanced the ventralization of Xenopus embryos. Our results indicated that mDlx5 is a target gene of the BMP signaling pathway and acts as an important regulator of both osteogenesis and dorso-ventral patterning of embryonic axis.

III. Eary pattern formation of the peripheral nervous system in Drosophila

Large mechanosensory bristles (macrochaetes) develop in fixed numbers at constant positions on the adult notum of Drosophila melanogaster. This pattern formation of macrochaetes has provided an ideal model system for studying two-dimensional patterning. Precise positioning of macrochaetes depends on the complex expression pattern of proneural genes in the wing disc. Prepattern genes are thought to regulate these expression of proneural genes. A proneural cluster of dorsocentral bristles forms adjacent to the dorsal side of wingless (wg)-expressing cells in the notum region of the wing imaginal disc. It has been shown that wg activity is required for these structures to form. However, the restriction of this proneural cluster to the dorsal posterior side of the wg expression domain in the anterior compartment of the wing imaginal disc has suggested that Wg signaling itself is insufficient to establish the dorsocentral proneural cluster. Some factor(s) from posterior side must participate in this action in cooperation with Wg signaling. We have examined the role of Dpp (Drosophila BMP2/4 homolog) signaling in dorsocentral bristle formation by either ectopically activating or conditionally reducing Dpp signaling. Ubiquitous activation of Dpp signaling in the notum region of the wing imaginal disc induced additional dorsocentral proneural cluster all along the dorsal side of the wg expression domain, and altered wg expression. Conditional loss-of-function of Dpp signaling during disc development resulted in the inhibition of dorsocentral proneural cluster formation and expansion of the wg expression domain. These results indicate that Dpp signaling has two indispensable roles in dorsocentral bristle formation, induction of the dorsocentral proneural cluster in cooperation with Wg signaling and restriction of the wg expression domain in the notum region of the wing imaginal disc.

IV. TGF-b family in nematode

Nematode C. elegans provides powerful genetical approaches to understand the role of TGF-b family ligands and their signaling mechanism. We have identified a new member of the TGF-b superfamily, CET-1, from C. elegans, which is expressed in the ventral nerve cord and other neurons. cet-1 null mutants have shortened bodies and male tail abnormal phenotype resembling sma mutants. Overexpression experiments demonstrated that cet-1 function requires wild type sma genes, suggesting that CET-1 functions as ligand in sma pathway. Interestingly, CET-1 appears to affect body length in a dose dependent manner. Heterozygotes for cet-1 displayed body lengths ranging between null mutant and wild type, and overexpression of CET-1 in wild type worms elongated body length close to lon mutants. Epistasis analysis concerning male sensory ray pattern revealed that mab-21 lies downstream and is negatively regulated by the cet-1/sma pathway. Our results show that cet-1 controls diverse biological processes during C. elegans development probably through different target genes.

Recently, almost entire genome sequence of C. elegans was published. The sequence information, therefore, allows us comprehensive approach in gene expression analysis. To identify the target genes of cet-1/sma pathway and understand the molecular basis of the regulation of body length, we performed a differential hybridization between wild-type and cet-1/sma mutants, using a high-density filter on which over 7,000 different cDNAs were arrayed. We are now analyzing functions of candidate genes whose expressions are up- or down-regulated in cet-1/sma mutants.

Publication List:
Iemura, S., Yamamoto, T. S., Takagi, C., Uchiyama, H., Natsume, T., Shimasaki, S., Sugino, H. and Ueno, N. (1998). Direct binding of follistatin to a complex of bone morphogenetic protein and its receptor inhibits ventral and epidermal cell fates in early Xenopus embryo. Proc. Natl. Acad. Sci. USA 95, 9337-9342.
Kurozumi, K. Nishita, M., Yamaguchi, K., Fujita, T., Ueno, N. and Shibuya, H. (1998) BRAM1, a BMP receptor-associated molecule involved in BMP signalling. Genes Cells 3, 257-264.
Mizuno, N. Mochii, M. Takagi, C., Takahashi, C. T., Eguchi, G. and Okada, T.S. (1998) A critical role for the optic vesicle in lens development; a reinvestigation of free lens formation in Cynops pyrrhogaster. Differentiation 63, 247-52.
Mochii, M., Mazaki, Y., Mizuno, N., Hayashi H. and Eguchi, G. (1998) Role of Mitf in differentiation and transdifferentiation of chicken pigmented epithelial cell. Dev. Biol. 193, 47-62.
Mochii, M., Ono, T., Matsubara, Y. and Eguchi, G. (1998) Spontaneous transdifferentiation of quail pigmented epithelial cell is accompanied by a mutation in the Mitf gene. Dev. Biol. 196, 145-159
Nikaido, M., Tada, M., Takeda, H., Kuroiwa, A. and Ueno, N. (1998) In vivo analysis using variants of zebrafish BMPR-IA: range of action and involvement of BMP in ectoderm patterning. Development 126, 181-190.
Shibuya, H., Iwata, H., Masuyama, N., Gotoh, Y., Yamaguchi, K., Irie, K., Matsumoto, K., Nishida, E. and Ueno, N. (1998). Role of TAK1 and TAB1 in BMP signaling in early Xenopus development. EMBO J. 17, 1019-1028.
Tomoyasu, Y. Nakamura, M. and Ueno, N. (1998) Role of Dpp signaling in prepattern formation of the dorsocentral mechanosensory organ in Drosophila melanogaster. Development 125, 4215-4224.
Wakamatsu, Y., Mochii, M., Vogel, K. S. and Weston J. A. (1998) Avian neural crest-derived neurogenic precursors undergo apoptosis on the lateral migration pathway. Development 125, 4205-4213.
Yamaguchi, K., Nagai, S., Ninomiya-Tsuji, J., Nishita, M., Tamai, K., Irie, K., Ueno, N., Nishida, E., Shibuya, H. and Matsumoto, K. (1999). XIAP, a cellular member of the inhibitor of apoptosis protein family, links the receptors to TAB1-TAK1 in the BMP signaling pathway. EMBO J. 18, 179-187.
Last Modified: 12:00, May 28, 1999