National Insitute for Basic Biology

DIVISION OF BEHAVIOR AND NEUROBIOLOGY

(ADJUNCT)

Professor:
Masatoshi Takeichi
Research Associate:
Akinao Nose
Kazuaki Tatei
Institute Research Fellow:
Tomoko Tominaga
Graduate Student:
Tatsuo Umeda
Visiting Scientist:
Takeshi Umemiya (from Kyoto University)
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 neuromuscular 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 neuromuscular 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 (primarily lateral muscles) but also on the axons and growth cones of the very motoneurons which innervate these muscles (primarily SNa motoneurons, see Figure 1) and on several associated glial cells. When coupled with its ability to mediate homophilic cell adhesion in vitro, these results led to the suggestion that Connectin functions as an attractive signal for SNa pathfinding and targeting. We are currently studying the function of Connectin by molecular genetic methods and also trying to clone novel genes implicated in the neuromuscular connectivity.

Figure 1

Fig. 1. Ectopic expression of Connectin.
Schematic diagram showing wild type and ectopic Connectin expression on embryonic muscles and motor nerves. In wild type, Connectin is expressed on a subset of motoneurons (SNa and SNc, shown in red) and muscles (primarily lateral muscles, shown in yellow). In Toll-connectin, Connectin is ectopically expressed on a subset of ventral muscles (also shown in yellow). In MHC-connectin, Connectin is ectopically expressed on all muscles.

I. Molecular genetic analysis of the function of Connectin

To study the role of Connectin in vivo, we first ectopically expressed Connectin on muscle fibers that normally do not express the molecule by using Toll enhancer. Toll is expressed on ventral muscles 6, 7, 14-17 and 28. These muscle fibers do not normally express Connectin, and are innervated by SNb motoneurons. We used a 7 kb Toll upstream sequence sufficient for the muscle expression to express Connectin on these muscle fibers in P-element mediated transgenic flies (Figure 1).

The analysis of the transformants (Toll-connectin) showed that the development of SNb is abnormal. The SNb growth cones change their morphology and their trajectory as they encounter ectopic Connectin-positive ventral muscles, displaying "bypass", "detour" and "stall" phenotypes. Moreover, SNb synapse formation is prevented by Connectin expression on ventral muscles. These results revealed a repulsive function for Connectin during motoneuron growth cone guidance and synapse formation.

In Toll-connectin, we did not observe any abnormality in the SNa motoneurons To further study the possible role of Connectin as an attractive guidance molecule for SNa, we then ectopically expressed Connectin on all muscles by using MHC (myosin heavy chain) promoter (MHC-connectin, see Figure 1). In MHC-connectin, SNa nerves were observed to send an extra axon branch that forms an ectopic nerve endings on muscle 12, a muscle they would never innervate in wild type (Figure 2). This phenoype was highly penetrant and was observed in over 60% of the segments examined. Furthermore, the ectopic innervation on muscle 12 was dependent on the Connectin expression on SNa. These results showed that Connectin functions as an attractive homophilic guidance molecule for SNa in vivo.

Figure 2

Fig. 2. Connectin functions as an attractive guidance molecule.
In wild type, muscle 12 is innervated by SNb motoneurons (short arrow). Connectin-positive SNa motoneurons innervate more distal muscles. Ectopic Connectin expression on muscle 12 in MHC-connectin changed SNa trajectories: SNa sends an extra axon branch that forms an ectopic nerve ending on muscle 12 (large arrow).

Taken together, the results suggest that Connectin plays bifunctional roles during motoneuronal pathfinding and targetting. One is an attractive and homophilic function, and another is repulsive and perhaps heterophilic function. Single recognition molecule having bifunctional roles may be an effective and economical way to establish precise patterns of neural connection.

II. Cloning of novel genes implicated in the neuromuscular connectivity in Drosophila

We are conducting molecular and genetic analysis of two other enhancer trap lines that are expressed in specific subsets of muscles and/or motoneurons. One of the line, AN34 expresses the reporter gene (ß-gal) in a single muscle fiber18 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 encodes a putative secreted protein with extensive amino acid similarity to rat F-spondin. F-spondin is a secreted molecule expressed at high levels in the floor plate and has been shown to promote neural cell adhesion and neurite extension in vitro. These results strongly suggest that AN34 is involed in motoneuronal gidance and/or targeting.

Another line, rQ224 expresses ß-gal in a small subset of neurons including an identified motoneuron RP3. The cDNA cloning and sequence analysis showed that it encodes a type II membrane protein homologous to vertebrate dopamine beta-hydroxylase, an enzyme involved in catecholamine synthesis. rQ224 thus may be involved in specific synaptic functions.

We are currently trying to isolate the loss-of-function mutants of these two lines as well as the tranagenic flies that ectopically express these molecules (as described for Connectin) to study their roles in the neuromuscular development and function.

Publication List:

Takaishi, K., Sasaki, T., Kato, M., Yamochi, W., Kuroda, S., Nakamura, T., Takeichi, M. and Takai, Y. (1994) Involvement of Rho p21 small GTP-binding protein and its regulator in the HGF-induced cell motility. Oncogene 9, 273-279.
Shibamoto, S., Hayakawa, M., Takeuchi, K., Hori, T., Oku, N., Miyazawa, K., Kitamura, N., Takeichi, M. and Ito, F. (1994) Tyrosine phosphorylation of b-catenin and plakoglobin enhanced by hepatocyte growth factor and epidermal growth factor in human carcinoma cells. Cell Adhesion & Commun. 1, 295-305.
Uchida, N., Shimamura, K., Miyatani, S., Copeland, N.G., Gilbert, D.J., Jenkins, N.A. and Takeichi, M. (1994) Mouse aN-catenin, two isoforms, specific expression in the nervous syiem and chromosomal localization of the gene. Develop. Biol. 163, 75-85.
Shiomi, K., Takeichi, M., Nishida, Y., Nishi, Y. and Uemura, T. (1994) Alternative cell fate choice induced by low-level expression of a regulator of protein phosphatase 2A in the Drosophila peripheral nervous system. Development 120, 1591-1599.
Uchiyama, N., Hasegawa, M., Yamashina, T., Yamashita, J., Shimamura, K. and Takeichi, M. (1994) Immunoelectron microscopic localization of E-cadherin in dorsal root ganglia, dorsal root and dorsal horn of postnatal mice. J Neurocytol. 23, 460-468.
Shimamura, K., Hirano, S., McMahon, A.P. and M. Takeichi. (1994) Wnt1-dependent regulation of local E-cadherin and aN-catenin expression in the embryonic mouse brain. Development 120, 2225-2234.
Watabe, M., Nagafuchi, A., Tsukita, S. and Takeichi, M. (1994) Induction of polarized cell-cell association and retardation of growth by activation of the E-cadherin-catenin adhesion system in a dispersed carcinoma line. J. Cell Biol. 127, 247-256.
Tong, K.I., Yau, P., Overduin, M., Bagby, S., Porumb, T., Takeichi, M. and Ikura, M. (1994) Purification and spectroscopic characterization of a recombinant amino-terminal polypeptide fragment of mouse epithelial cadherin. FEBS Ietters 352, 318-322.
Nose, A., Takeichi, M. and Goodman, C.S. (1994) Ectopic expression of connection reveals a repulsive function during growth cone guidance and synapse formation. Neuron 13, 525-539.
Oda, H., Uemura, T., Harada, Y., Iwai, Y. and Takeichi, M. (1994) A Drosophila homolog of cadherin associated with Armadillo and essential for embryonic cell-cell adhesion. Develop. Biol. 165, 716-726.
Hatta, M. and Takeichi, M. (1994) Complex cell type-specific transcriptional regulation by the promoter and an intron of the mouse P-cadherin gene. Develop. Growth & Differ. 36, 509-519.