EPITHELIAL-CELL DIFFERENTIATION IN NORMAL AND TRANSGENIC MOUSE INTESTINAL ISOGRAFTS

Transgenes consisting of segments of the rat liver fatty acid-binding protein (L-FABP) gene's 5' non-transcribed domain linked to the human growth hormone (hGH) gene (minus its regulatory elements) have provided useful tools for analyzing the mechanisms that regulate cellular and spatial differentiation of the continuously renewing gut epithelium. We have removed the jejunum from normal and transgenic fetal mice before or coincident with, cytodifferentiation of its epithelium. These segments were implanted into the subcutaneous tissues of young adult CBY/B6 nude mouse hosts to determine whether the bipolar, migration-dependent differentiation pathways of gut epithelial cells can be established and maintained in the absence of its normal luminal environment. Immunocytochemical analysis of isografts harvested 4-6 wk after implantation revealed that activation of the intact endogenous mouse L-FABP gene (fabpl) in differentiating enterocytes is perfectly recapitulated as these cells are translocated along the crypt-to-villus axis. Similarly, Paneth and goblet cells appear to appropriately differentiate as they migrate to the crypt base and villus tip, respectively. The enteroendocrine cell subpopulations present in intact 4-6-wk-old jejunum are represented in these isografts. Their precise spatial distribution along the crypt-to-villus axis mimics that seen in the intact gut. A number of complex interrelationships between enteroendocrine subpopulations are also recapitulated. In both "intact" and isografted jejunum, nucleotides -596 to +21 of the rat L-FABP gene were sufficient to direct efficient expression of the hGH reporter to enterocytes although precocious expression of the transgene occurred in cells located in the upper crypt, before their translocation to the villus base. Inappropriate expression of hGH occurred in a high percentage (> 80%) of secretin, gastrin, cholecystokinin, and gastric inhibitory peptide producing enteroendocrine cells present in the intact jejunum of 4-6-wk-old L-FABP-596 to +21/hGH transgenics. Addition of nucleotides -597 to -4,000 reduced the percentage of cells co-expressing this reporter four- to eightfold in several of the subpopulations. Jejunal isografts from each transgenic pedigree studied contained a lower percentage of hGH positive enteroendocrine cells than in the comparably aged intact jejunum. Together our analyses suggest that (a) the complex program of enteroendocrine cell differentiation can be expressed in the absence of extracellular luminal contents; (b) L-FABP/hGH transgenes appear to be useful reporters of a subtle heterogeneity in the regulatory environments of enteroendocrine cell subpopulations and a sensitive marker of the fact that luminal contents can regulate gene expression within and between subpopulations; (c) in contrast to enteroendocrine cells, transgene expression in enterocytes does not appear to be modulated by luminal factors; and (d) transgenic intestinal isografts represent a general and powerful tool not only for assessing the influence of extracellular factors on cis-acting elements contained in specific genes during epithelial differentiation but also for mapping these elements.