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In an attempt to elucidate the molecular mechanisms underlying neuro-network formation in the developing brain, we analyzed 130 proteolytic cleavage peptides of membrane proteins purified from newborn mouse brains. We describe here the characterization of a membrane protein with an apparent molecular mass of 46 kDa, a member of the immunoglobulin superfamily of which the cDNA sequence was recently reported, encoding the mouse homologue of the human coxsackievirus and adenovirus receptor (mCAR). Western and Northern blot analyses demonstrated the abundant expression of mCAR in the mouse brain, the highest level being observed in the newborn mouse brain, and its expression was detected in embryos as early as at 10. 5 days post-coitus (dpc), but decreased rapidly after birth. On in situ hybridization, mCAR mRNA expression was observed throughout the newborn mouse brain. In primary neurons from the hippocampi of mouse embryos the expression of mCAR was observed throughout the cells including those in growth cones on immunohistochemistry. In order to determine whether or not mCAR is involved in cell adhesion, aggregation assays were carried out. C6 cells transfected with mCAR cDNA aggregated homophilically, which was inhibited by specific antibodies against the extracellular domain of mCAR. In addition to its action as a virus receptor, mCAR may function naturally as an adhesion molecule involved in neuro-network formation in the developing nervous system.

Cholecystokinin (CCK), a neuro-gut peptide, occurs not only in the nervous but also in the digestive system. As a first step in elucidating whether CCK gene expression and its physiological functions co-operate in these separate organs, transgenic mice were produced using CCK promoter that directs bacterial beta-galactosidase as a reporter gene. A new transgenic vector was constructed, inserting the SV40 poly A signal 5' to the CCK promoter to impede any transcription upstream of the transgene. A 2.4 kb.p. region upstream to the transcription start site of the mouse CCK gene was used as the promoter. Transgene expression was detected first at embryonic 13.5 days in the central nervous system and increased after birth. The distribution of cells expressing beta-galactosidase transgene agreed fairly well with that of in situ hybridization. In addition, the upregulation of CCK gene expression was clearly demonstrated by expressing beta-galactosidase after injury to the brain. These results indicated that the 2.4 kb.p. of the CCK gene promoter region was sufficient to direct appropriate tissue-specific gene expression in mice.