NATIONAL INSTITUTE FOR BASIC BIOLOGY
DIVISION OF BEHAVIOR AND NEUROBIOLOGY (ADJUNCT)
- Masatoshi Takeichi
- Research Associate:
- Akinao Nose
- Institute Research Fellow:
- Tomoko Tominaga
- Visiting Scientist:
- Tatsuo Umeda
How individual nerve cells find and recognize their targets during development
is one of the central issues in modern biology. The aim of our division is to
elucidate the molecular mechanism of axon guidance and target recognition by
using the simple and highly accessible neuro-muscular system of Drosophila.
The musculature of Drosophila embryos consists of 30 identifiable muscle fibers
per hemisegment. Each muscle fiber is innervated by a few motoneurons in a
highly stereotypic manner. The high degree of precision and previous cellular
manipulations of neuro-muscular connectivity suggest the presence of
recognition molecules on the surface of specific muscle fibers which guide the
growth cones of motoneurons.
By using an enhancer trap method, several genes have previously been identified
that are expressed in small subsets of muscle fibers prior to innervation, and
are thus good candidates for such recognition molecules. Two of them,
connectin and Toll, were shown to encode cell recognition molecules which
belong to the leucine-rich repeat (LRR) family. In paticular, connectin is
expressed not only on a subset of muscle fibers but also on the axons and
growth cones of the very motoneurons which innervate these muscles (Figure 1).
Its specific expression both in presynaptic motoneurons and postsynaptic
muscles, and its function as a homophilic cell adhesion molecule in vitro
strongly suggested that connectin play a role in the neuro-muscular
specificity. We are currently studying the function of connectin by molecular
genetic methods and also trying to clone novel genes implicated in the
I. Molecular genetic analysis of the function of connectin.
- To study the role of connectin in vivo, we misexpressed connectin on
muscle fibers that normally do not express the molecule by using Toll
promoter. As shown in Fig. 1, Toll is expressed on ventral muscles #6,
7, 14-17 and 28, a different subset of muscle fibers from those
expressing connectin. We used a 7 kb Toll upstream sequence sufficient
for the muscle expression to misexpress connectin on these muscle
fibers in P-element mediated transgenic flies.
- The analysis of the transgenic flies (Toll-connectin) showed that the
development of a motor nerve (SNb) is abnormal. After leaving the CNS,
SNb normally grows directly into the ventral muscles and makes synaptic
contact with the target muscles by stl7. In Toll-connectin, the SNb
axons often take abnormal trajectory and fail to make the right synaptic
contact by this stage. They often grow dorsally along another nerve
(ISN) or take an independent path below the ventral muscles. The results
suggest that ectopically expressed connectin on the ventral muscles (#6,
7, 14 and 28) prevent the SNb from growing into these muscle fibers,
pointing to connectin's role as a inhibitory signalling molecule in the
formation of neuro-muscular connectivity. Thus in addition to its
possible role as an attractive homophilic recognition molecule (for the
motoneurons and their target muscles both expressing the molecule),
connectin may serve as an inhibitory recognition molecule for other
motoneurons that do not express the molecule (probably via heterophilic
- We are currently trying to misexpress connectin in yet different
subsets of muscle fibers by using GAL4 system, to further analyse
connectin's role in vivo.
II. Cloning of novel genes implicated in the neuro-muscular
connectivity in Drosophila
- 1. Search for novel connectins. An interesting possibily is that
connectins constitute a LRR subfamily which are expressed on different
subsets of motoneurons and muscles. We are trying to isolate novel
connectins by using PCR and will study their expression pattern and
- 2. Cloning and characterization of other enhancer trap lines
- We are conducting molecular and genetic analysis of two other enhancer
trap lines that are expressed in specific subsets of muscles and/or
- rQ224 expresses the reporter gene (B-gal) in a small subset of
neurons including a motoneuron RP3 but not RP1. These two motoneurons
take the same peripheral pathway as they exit the CNS and send axons
via motor nerve SNb. However, despite the similarlity of their
trajectories, once they reach the target region, they show distinct
behaviors: the RP3 growth cone projects onto the cleft region between
muscle #6&7 while the RPI growth cone goes past 6&7 to innervate muscle
#13. Expression of rQ224 only in RP3 but not in RPI suggests its
possible role in such specific aspects of target recognition. The cDNA
cloning and partial sequence analysis showed that the ORF contains a
signal sequence. rQ224 product thus is probably a surface or secreted
molecule with potential roles in recognition. The sequencing of the
complete cDNA is now in progress. The other line AN34 expresses B-gal in
a single muscle fiber (#18) per hemisegment. The remarkable specificity
in its expression pattern (one out of 30 muscle fibers) makes it a good
candidate for the muscle target recognition molecule. The cDNA cloning
and sequencing revealed that AN34 protein shows extensive amino acid
similarity to rat F-spondin, a secreted molecule expressed at high
levels in the floor plate that has been shown to promote neural cell
adhesion and neurite extension in vitro.
- We are currently trying to isolate the 10ss-of-function mutants of
these two lines as well as the transgenic flies that ectopically express
these molecules (as described for connectin) to study their roles in the
- Redies, C., Engelhart, K. and Takeichi, M. (1993) Differential
expression of N- and R-cadherin in functional neuronal systems and other
structures of the developing chicken brain. J. Comparative Neurology
- Redies, C. and Takeichi, M. (1993) N- and R-cadherin expression in the
optic nerve of the chicken embryo. Glia 8, 161-171.
- Hamaguchi, M., Matsuyoshi, M., Ohnishi, Y., Gotoh, B., Takeichi, M. and
Nagai, Y. (1993) p60v-src causes tyrosine phosphorylation and
inactivation of the N-cadherin-catenin cell adhesion system. EMBO J. 12,
- Takeichi, M., Hirano, S., Matsuyoshi, N. and Fujimori, T. (1993)
Cytoplasmic control of cadherin-mediated cell-cell adhesion. Cold Spring
Harbor Quant. Biol. LVII, 327-334.
- Uemura, T., Shiomi, K., Togashi, S. and Takeichi, M. (1993) Mutation of
twins encoding a regulator of protein phosphatase 2A leads to pattern
duplication in Drosophila imaginal discs. Genes and Develop. 7, 429-440.
- Fujimori, T. and Takeichi, M. (1993) Disruption of epithelial cell-cell
adhesion by exogenous expression of a mutated non-functional N-cadherin.
Mol. Biol. Cell 4, 37-47.
- Oda, H., Uemura, T., Shiomi, K., Nagafuchi, A., Tsukita, S. and
Takeichi, M. (1993) Identification of a Drosophila homologue of
a-catenin and its association with the armadillo protein. J. Cell Biol.
- Redies, C. and Takeichi, M. (1993) Expression of N-cadherin mRNA during
development of the mouse brain. Develop. Dynamics 197, 26-39.
- Matsunami, H., Miyatani, S., Inoue, T., Copeland, N.G., Gilbert, D.J.,
Jenkins, N.A. and Takeichi, M. (1993) Cell binding specificity of mouse
R-cadherin and chromosomal mapping of the gene. J Cell Science 106,
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of hepatocyte growth factor on cadherinmediated cell-cell adhesion.
Cell Struct Funct. 181, 117-124.
- Oka, H., Shiozaki, H., Kobayashi, K., Inoue, M., Tahara, H., Kobayashi,
T., Takatsuka, Y., Matsuyoshi, N., Hirano, S., Takeichi, M., and Mori,
T. (1993) Expression of E-cadherin cell adhesion molecules in human
breast cancer tissues and its relationship to metastasis. Cancer Res.
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Kadowaki, T., Takeichi, M. and Mori, T. (1993) Correlation between
E-cadherin expression and invasiveness in vitro in a human esophageal
cancer cell line. Cancer Res. 53, 3421-3426.
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metastasis. Curr. Opinion Cell Biol. 5, 806-811.
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Shimamura, K. (1993) Dynamic control of cell-cell adhesion for
multicellular organization. C. R. Acad. Sci. Paris, Sciences de la
vie/Life sciences 316, 818-821.
- Steinberg, M.S. and Takeichi, M. (1993) Experimental specification of
cell sorting, tissue spreading and specific patterning by quantitative
differences in cadherin expression. Proc. Natl Acad. Sci. USA 91,