Gastrointestinal (GI) endocrine cells represent the largest and most complex endocrine """"""""organ"""""""" in the mammalian body. I have used a combination of novel immunohistochemical techniques and transgenic mice to study the biologic properties of these cells. The patterns of transgene expression observed suggest several conclusions: 1) GI endocrine cell subpopulations occupy unique regulatory environments; 2) Regulatory environments include spatial, temporal, and luminal factors; 3) GI endocrine cell subpopulations are functionally interrelated; and 4) Enteroendocrine cells share a common endodermally derived stem cell origin with enterocytes, goblet cells, and Paneth cells. This proposal represents an expansion of these initial studies to an analysis of the regulation of neuroendocrine gene expression in the brain-gut axis. Many neuroendocrine peptides, including substance P and other tachykinins, are expressed in both the brain and GI tract. To begin to define the molecular sequences responsible for preprotachykinin gene expression in endocrine cells and neurons I have generated transgenic mice containing 865 nucleotides of the preprotachykinin 5' flanking region fused to a reporter gene, human growth hormone. Immunohistochemical studies indicate that this promotor directs reporter expression to endocrine cells; however, neuronal transgene expression is undetectable. Future constructs with preprotachykinin 5' nucleotide sequences fused either to hGH or to a nonbioactive hGH mutant reporter gene will be used to generate transgenic mice. Transgene expression in these mice will be analyzed by immunohistochemistry, in situ hybridization, RNA blot hybridization, and radioimmunoassay. Following the determination of the nucleotide sequences critical for endocrine and neuronal cell-specific preprotachykinin gene expression the hGH reporter will be replaced by the SV40 large T antigen gene. Expression of this multifunctional transforming protein in specific endocrine and/or neuronal cell populations will allow us to investigate the initiation of neuroendocrine neoplasms in vivo and to create neuroendocrine cell lines for the in vitro analysis of preprotachykinin gene expression. These investigations should yield important data on the cellular regulation of preprotachykinin gene expression in normal and neoplastic states.
