DIVISION OF BEHAVIOR AND NEUROBIOLOGY (ADJUNCT)


                              NATIONAL INSTITUTE FOR BASIC BIOLOGY

DIVISION OF BEHAVIOR AND NEUROBIOLOGY (ADJUNCT)

Professor:: 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 neuro-muscular connectivity.

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 interaction) .

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 function.

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 motoneurons.

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 neuro-muscular development.

Publication List:

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 333, 398-416.

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, 307-314.

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. 121, 1133-1140.

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, 401-409.

Watabe, M., Matsumoto, K., Nakamura, T. and Takeichi, M. (1993) Effect 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. 53, 1696-1701.

Doki, Y., Shiozaki, H., Tahara, H., Inoue, M., Oka, H., Iihara, K., 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.

Takeichi, M. (1993) Cadherins in cancer: Implication for invasion and metastasis. Curr. Opinion Cell Biol. 5, 806-811.

Takeichi, M., Watabe, M., Sibamoto, S., Ito, F., Oda, H., Uemura, T. and 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, 206-209.