NATIONAL INSITUTE FOR BASIC BIOLOGY

National Institute for Basic Biology

Division of Behavior and Neurobiology

(Adjunct)

Professor:
Masatoshi Takeichi
Research Associates:
Akinao Nose
Kazuaki Tatei
Institute Research Fellow:
Emiko Shishido
JSPS Postdoctoral Fellow:
Takako Isshiki
Graduate Students:
Hiroki Taniguchi
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. We have previously isolated several enhancer trap lines that express the reporter gene b-galactosidase (b-gal) in small subsets of muscle fibers prior to innervation. By molecularly characterizing these lines, we are trying to identify genes that play roles in the specification of the muscles and neuromuscular connectivity. Previous studies showed that two of the lines are insertions in the connectin and Toll genes, that encode cell recognition molecules which belong to the leucine-rich repeat (LRR) family. We have been studying the function of these genes, and also characterizing other lines by molecular and genetic analysis.


I. Connectin can function as an attractive target recognition molecule

Connectin is expressed on a subset of muscle fibers (primarily lateral muscles) and on the axons, growth cones of the motoneurons which innervate these muscles (primarily SNa motoneurons) 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.

To study the role of Connectin in vivo, we ectopically expressed Connectin on all muscles by using MHC (myosin heavy chain) promoter (MHC-connectin) in the P-element mediated transformants. In MHC-connectin, SNa nerves were observed to send extra axon branches that form ectopic nerve endings on muscles 12, muscles they would never innervate in wild type. 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 and homophilic guidance molecule for SNa in vivo.


II. P750, a novel LRR cell surface molecule expressed on subsets of neurons and muscles

We have been conducting molecular and genetic analysis of other muscle enhancer trap lines. One of them, P750 expresses b-gal in subsets of neurons and muscles, including the RP5 motoneuron and its target, muscle 12. The cDNA cloning and sequencing revealed that P750 encodes a novel transmembrane protein that belongs to LRR family. It is interesting that three of the five muscle enhancer trap lines that we have thus far characterized contain LRRs. Within the LRR family, P750 was found to be most related to the Drosophila tartan gene that have been implicated in neural and muscular development. We have recently found that in the loss-of-function mutant of P750, the synaptic arborization pattern on muscle 12, a P750-expressing muscle, is abnormal, suggesting that this molecule play some roles in neuromuscular target recognition and/or stabilizaion of the synapses.


III. msh, a homeobox containing gene essential for neural and muscular development

Another line rH96 was found to be a P-element insertion in the muscle segment homeobox (msh) gene, that was previously cloned as a homeobox containing gene. By generating and analysing both loss-of-function and gain-of-function (ectopic expression) mutants, we showed that msh is essential for neural and muscular development. During CNS development, msh is specifically expressed in the dorsal neuroectoderm (Fig. 1A) and subsequently in many neuroblasts and their progeny derived from this region (Fig. 1B). We found that the loss of msh results in the failure of the proper differentiation of many neural and glial progeny derived from the dorsal neuroectoderm. Conversely, ectopic expression of msh in the entire neuroectoderm severely disrupts the formation of midline structure and differentiation of neuroblasts located in the ventral neuroectoderm. These results suggest that msh plays crucial roles in the dorso-ventral (DV) specification of the CNS. The vertebrate homologues of msh, Msxs are also know to be expressed in the dorsal portion of the spinal cord. Our work on msh raises a possiblity that this family of genes may plays a conserved role during DV patterning of the CNS.

Fig. 1.
The expression pattern of msh mRNA. Stage 8 (A) and stage 11 (B) whole mount Drosophila embryos hybridized in situ with msh riboprobe.


IV. M-spondin and G-spondin: a novel gene family of secreted molecules

By molecularly characterizing another enhancer trap line, AN34 which is also expressed in a subset of muscles and neurons, we identified a novel secreted protein, termed M-spondin, that is highly homologous 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. We found three regions that are highly conserved between M-spondin and F-spondin. One of them is a known repeating motif called thrombospondin type I repeats (TSRs). The other two domains (termed FS1 and FS2) are novel conserved sequences that we identified. By using PCR, we cloned two more genes that share similar overall structure with M-spondin and F-spondin in that they possessed FS1, FS2 and one to six TSRs. The idintification of these genes thus defines a novel gene family of secreted protein with potential roles in cell adhesion. One of the newly cloned genes, termed G-spondin, is expressed in a subset of glia that sit along the longitudinal axon tracts in the CNS. The specific expression pattern of G-spondin suggests that it may play a role in the guidance of specific axons.


Publication List:
Nose, A., Takeichi, M. and Goodman, C.S. (1996) Molecular genetic analysis of the role of Connectin in neuromuscular recognition in Drosophila. In Basic Neuroscience in Invertebrates (Japan Sci. Soc. Press), pp49-64.
Uemura, T., Oda, H., Kraut, R., Hayashi, S., Kataoka, Y. and Takeichi, T. (1996) Zygotic DE-cadherin expression is required for processes of dynamic epithelial cell rearrangement in the Drosophila embryo. Genes & Develop. 10, 659-671.
Kimura, Y., Matsunami, H. and Takeichi, M. (1996) Expression of cadherin-11 delineates boundaries, neuromeres and nuclei in the developing mouse brain. Develop. Dynamics 206, 455-462.
Uchida, N., Honjo, Y., Johnson, K.R., Wheelock, M.J. and Takeichi, M. (1996) The catenin/cadherin adhesion system is localized in synaptic junctions, bordering the active zone. J. Cell Biol. 135, 767-779.
Tanaka-Matakatsu, M., Uemura, T., Oda, H., Takeichi, M. and Hayashi, S. (1996) Cadherin-mediated cell adhesion and cell motility in Drosophila trachea regulated by the transcription factor Escargot. Development 122, 3697-3705.
Redies, C. and Takeichi, M. (1996) Cadherins in the developing central nervous system: An adhesive code for segmental and functional subdivisions. Develop. Biol. 180, 413-423.
Pai, L., Kirkpatrick, C., Blanton, J., Oda, H., Takeichi, M. and Peifer, M. (1996) a-Catenin and DE-cadherin bind to distinct regions of Drosophila Armadillo. J. Biol. Chemistry, in press.

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Last Modified: 12:00, June 27, 1997