NATIONAL INSTITUTE FOR BASIC BIOLOGY

DIVISION OF MOLECULAR NEUROBIOIOGY

Professor:
Masaharu Noda

Research Associates:
Nobuaki Maeda
Shinji Hirano

Graduate Students:
Hiroki Hamanaka
Jun-ichi Yuasa

Visiting Fellow:
Haruyuki Matsunaga *

Technical Stafff:
Tomoko Mori
Shigemi Ohsugi

( * from Environmental Health Science Laboratory, Sumitomo Chemical C'o., Ltd:)

The principal interest of this division is molecular mechanisms that govern the development of the vertebrate central nervous system. Our efforts are currently focused on projects to reveal the molecular bases of neuronal cell migration and neuronal network formation.

I. Molecular basis of neuroblast migration

In the developing central nervous system, migration of post-mitotic neurons is the key process through which laminated and/or nucleated structures form out of the uniform neuroepithelium. Thus, to establish the functional neuronal network, the course of neuroblast migration must be precise with the combination of radial and tangential movement. Although many adhesion molecules are known to be expressed in the neural tissue, the molecular basis of neuroblast migration remains to be elucidated.

T6 1 antigen is a good candidate of the T6 1 antigen is a good candidate of the molecule which is responsible for the neuroblast migration in the radial direction. Monoclonal antibody T61 was originally characterized by its inhibitory activity on neurite outgrowth from chick retinal explants in vitro. However, it turned out recently that neuroblast migration was inhibited when the T61 producing hybridoma was injected in the ventricle of the developing midbrain of chick embryos. As far as we know, this is the first example in which an antibody inhibited neuroblast migration in vivo. molecule which is responsible for the neuroblast migration in the radial direction. Monoclonal antibody T61 was originally characterized by its inhibitory activity on neurite outgrowth from chick retinal explants in vitro. However, it turned out recently that neuroblast migration was inhibited when the T61 producing hybridoma was injected in the ventricle of the developing midbrain of chick embryos. As far as we know, this is the first example in which an antibody inhibited neuroblast migration in vivo.

The expression of T61 antigen is restricted to the neural tissue, and the distribution within the tissue is rather uniform. Immunocytechemistry of primary culture cells showed that only neuronal cells express the antigen. The finding that T61 antigen is concentrated at the filopdia of growth cone is consistent with the presumptive function of this molecule in the cell migration. The T61 detects several bands including a major 440kD band by Western blotting and immunoprecipitation. These results suggest that T61 antigen is a novel molecule. Screening of (RAMDA)gt 11 library prepared from chick brain with T61 gave rise to many positive signals with various intensity. Nucleotide sequence analysis revealed that these clones are derived from several different mRNAs. We are now trying to identify the authentic cDNA clone from them.

II. Proteoglycan and brain development

Proteoglycans have been recognized to play important roles in the regulation of cell growth, differentiation and adhesion. The vertebrate brain also contains many kinds of proteoglycans, which are considered to be involved in the neuronal migration, axon guidance and axonal outgrowth. 6B4 proteoglycan is a brain specific large chondroitin sulfate proteoglycan with a 300-kDa core protein. This proteoglycan has the soluble and membrane-bound forms, and its expression is dynamically regulated during development of the brain. In the adult rat hindbrain, 6B4 proteoglycan is selectively expressed around the neurons constituting the cerebellar mossy fiber system. In the early postnatal animals, the expression of 6B4 proteoglycan is highly correlated with the synapse formation of this system, suggesting that 6B4 proteoglycan plays important roles in the cerebellar circuit formation.

To investigate this possibility, we developed an in vitro reconstitution system of cerebellar circuits, in which slices of cerebellum and pontine nuclei were cocultured. The latter is a major precerebellar nuclei from which many mossy fibers project to the cerebellum in vivo. During long-term culture (2 3 weeks), Purkinje cells developed well-arbored dendrites and projected to the deep cerebellar nuclei as revealed by anti-InsP3 receptor monoclonal antibody staining (Fig. 1A). On the other hand, the pontine fibers projected to the cerebellar granule cell layer as shown by Dil staining (Fig. 1B). Such a projection pattern is very similar to that observed in vivo. We are now studying the function of 6B4 proteoglycan in the fiber projection and the neural circuit formation using this system.

Publication List:

Noda, M. (1993) Structure and function of sodium channels. In Molecular Basis of lon channels and Receptors Involved in Nerve Exitation, Synaptic Transmission and Muscle Contractions. Annals of The New York Academy of Sciences. Vol. 707, pp. 20-37.