Functional clarification of the bombesin-like peptide system.

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1. Generation and analysis of mice lacking NMB-R and GRP-R : We obtained male double-knocked out mice as well as male wild-type mice by mating female NMB-R +/-, GRP-R +/- mice and male NMB-R +/- mice. The mutant mice did not show great difference as compared with wild-type mice. The possible alterations observed were the points already noticed in the GRP-R or NMB-R single mutant mice.2. Development of the chick embryos over-expressing NMB-R, GRP-R or BRS-3 : We constructed the retrovirus vectors containing cDNA coding for each subtype of mouse receptpr with EGFP cDNA fused at its C-terminal end. Infection of virus particles prepared using these vectors to the brain region of the chick embryos, resulted in the strong and wide-spread expression of EGFP. We are currently analyzing the phenotypes of these embryos by histological methods.3. Analysis of bombesin systems in birds : In order to elucidate the bombesin like peptides system in birds, we successfully cloned two bombesin-like peptide receptors from chick brain by using RT-PCR with degenerate primers. We named these clones as chGRP-R which showed high similarity with GRP-R of other species, and chBRS-3.5 which showed similarity with BRS-3 and BB4. In situ hybridization analysis showed that chGRP-R was expressed in the hypothalamus and in discrete regions of forebrain, and chBRS-3.5 was widely expressed in the forebrain including striatum. None of these two receptors was expressed in the cerebellum. These results demonstrated that the bombesin-like peptides system exists also in birds and this suggests that this system plays an important role in birds like in mammals.