Abstract
Axial pattern formation is sustained in the mammalian gut epithelium despite rapid and continuous renewal of its four principal cell lineages. The mouse and rat liver fatty acid-binding protein (L-FABP) genes (Fabpl) represent an excellent model for understanding the mechanisms that determine differentiation-dependent, cell lineage-specific, and distinct regional patterns of expression along the crypt-to-villus and duodenal-to-ileal axes of the gut, as well as within the liver acinus. We have used transgenic mice to map cis-acting elements in rat Fabpl that control these patterns of gene expression. Seven transgenes were analyzed, representing sequential deletions of the 5'-nontranscribed domain of Fabpl linked to the human growth hormone (hGH) gene beginning at its nucleotide +3 (L-FABP/hGH+3). Several pedigrees of mice containing each one of the L-FABP/hGH+3 transgenes were examined at the end of their 8th and 20th weeks of postnatal life using immunocytochemical and RNA hybridization analyses. A remarkably compact sequence spanning nucleotides -132 to +21 of Fabpl is sufficient to establish and maintain a distribution of reporter mRNA and protein in villus-associated enterocytes located along the duodenal-to-ileal axis of the gut that resembles the pattern of expression of the endogenous Fabpl gene. L-FABP-132 to +21/hGH+3 is also expressed in surface and pit mucous cells of gastric units and in enterocytes located in the colonic homologs of small intestinal villi, the surface epithelial cuffs. This pattern of transgene expression in the stomach and colon recapitulates that of the intact endogenous donor rat Fabpl but not that of mouse Fabpl, which is silent in these proximal and distal segments of the gastrointestinal tract. Analysis of mice containing L-FABP-4000 to +21/hGH+3, L-FABP-1600 to +21/hGH+3, L-FABP-596 to +21/hGH+3, L-FABP-246 to +21/hGH+3, and L-FABP-186 to +21/hGH+3 indicate that Fabpl's cephalocaudal gradient is influenced by cis-acting suppressors of cecal and colonic expression located between nucleotides -4000 and -1600 and by cis-acting activators of cecal and colonic expression located between nucleotides -597 and -351. L-FABP-132 to +21/hGH+3 is precociously activated in proliferating and nonproliferating epithelial cells located in intestinal crypts. The suppressor(s) of L-FABP accumulation in crypt epithelial cell populations are not represented between nucleotides -4000 and +21, indicating that different cis-acting sequences regulate regional and differentiation-dependent patterns of Fabpl expression.(ABSTRACT TRUNCATED AT 400 WORDS)
Highlights
Our studies suggest that the cephal- hGH’3 transgenes more susceptible to this phenomenon and ocaudal gradients of expression of both genes are affected better reporters of variations in the transcriptional by additional positive and negative cis-acting elements located regulatory environments of the descendants of adjacent upstream of these minimal domains
The Potential Significance of Villus Striping in L-FABPI Each of the seven L-FABP/hGH+3 transgenes examined in hGW3 Transgenic Mice-It will be important to determine the present report is activated inproliferating crypt cells, yet the lineage-specific and regional patterns of expression of light microscopic studies indicate that L-FABP/hGH+3 candidate regulators of Fabp transcription in the gut epithe- expression is sustained in both enterocytes and enteroendolium and in the liver a ~ i n u sI.f~a known transcription factor crine cells during their subsequent migration/differentiation
Nucleotides -184 to +28 of Fabpi may lack cisacting elements that bind to positive factors produced in Acknowledgments-We thank David O’Donnell and Cecelia Latham for superb technical assistance
Summary
Enterocytes, goblet cells, and most enteroendocrine cells mi- ferences in the differentiation programs of each epithelial cell grate upward in vertical coherent bands from each crypt to lineage and that establishment and maintenance of regionthe apical extrusion zones of adjacent small intestinal villi. specific anddifferentiation-dependentpatterns of Fabpl, Each villus is supplied by several monoclonal crypts (Wright Fabpi, and Ilbp expression in enterocytes do not require and Irwin, 1982; Schmidt et al, 1985; Roth et al, 1991b). Control experimentsemploying Northern blots of total cellular RNA prepared from transgenic mouse tissues established that these hybridization and washing stringencies resulted in the reaction of both. The concentration of hGH or L-FABP mRNA was determined by comparing the intensity of the hybridization signal produced by a sample of tissue RNA with the intensities of the signals produced by the reference mRNA standards present in the same filter These measurements were made using a DEPC-treated phosphate-buffered saline, fixed for 5 min in 4% paraformaldehyde, washed again in DEPC-treated phosphate-buffered saline, and acetylated by incubation for 10 min at room temperature in a solution of acetic anhydride, 0.1 M triethanolamine HC1, pH 8.0. No specific staining was observed in any of these control reactions (data notshown)
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