Abstract
The mechanisms that regulate cell lineage-specific and differentiation-dependent patterns of gene expression in the gastric units of the stomach are largely unknown. Transgenic mice were generated in order to identify cis-acting sequences that determine the zymogenic cell-specific pattern of expression of the mouse intrinsic factor (InF) gene and the parietal cell-specific pattern of expression of the mouse H+/K(+)-ATPase beta-subunit gene. Portions of the 5'-nontranscribed domains of each gene were linked to the human growth hormone (hGH) gene beginning at its nucleotide +3. RNA blot hybridization studies combined with multilabel immunocytochemical surveys using a panel of lineage-specific antibodies and lectins indicated that nucleotides -1035 to +24 of the mouse H+/K(+)-ATPase beta-subunit gene direct a pattern of reporter production which recapitulates the parietal cell-specific and developmental patterns of expression of the endogenous gene. Analysis of three mosaic founders containing H+/K(+)-ATPase beta-subunit-1035 to +24/hGH+3 revealed that they had monophenotypic gastric units: a given unit contained either a wholly hGH-positive or a wholly hGH-negative population of parietal cells. These latter findings provide very strong evidence that gastric units are monoclonal, i.e. they are supplied by stem cells having one genotype. Although some, but not all, parietal cells are apparently derived from the same committed progenitor as zymogenic cells, virtually all parietal cells in a given gastric unit, but none of its zymogenic cells, express InF-1029 to +55/hGH+3. This suggests that InF-1029 to +55 may contain cis-acting sequences which allow parietal cell expression in other species (e.g. humans) but lack additional elements which normally function in mice to suppress InF expression in this lineage. The absence of hGH in zymogenic cells also means that the transcriptional regulatory environments of parietal and zymogenic cells derived from the same precursor are distinguishable by InF-1029 to +55. H+/K(+)-ATPase beta-subunit-1035 to +24 and InF-1029 to +55 are the only two sequences reported to date that are able to direct foreign gene expression exclusively to a gastric epithelial cell lineage in transgenic mice. This ability to deliver gene products to parietal cells can now be exploited to identify factors that control their normal proliferation and differentiation programs and/or to specifically alter their biological properties.
Highlights
From the Departments of $Pathology and tMoEecular Biology and Pharmacology, Washington UniversitySchool of Medicine, St
The absence of hGH in zymogenic cells means that the transcriptional regulatoryenvironments of parietal and zymogenic cells derived from the same precursor are distinguishable by Intrinsic factor (InF)-102sto €€+/I(+-ATPaseP-subthe morphologic features of proliferating cells located in the stem cell zoneof mouse small intestinal crypts [11].The undifferentiated granule-free (? stem) cell appears to give rise to three other cell types: a granule-free pre-pit cell precursor, a granule-free pre-neckcellprecursor, and a pre-parietal cell unit-10s6to +24 and to are the onlytwose
Once neck cells enter the upper portion of the base of gastric units, they subclonedinto pBluescript I1 SK+(Stratagene, La Jolla, CA).A 2151-bp BarnHYEcoRI fragment containing the human growth hormone(hGH) gene beginning at itsnucleotide +3 [30]was recovered from pLFhGH7 [16] and placed into the BarnHI-EcoRIsite of the pBluescript plasmid which contained the 5’-nontranscribed domain of the mouse H+/K+ATPase P-subunit gene.A 3.2-kb EugI-EcoRI fragment from pHKATF”hGH1 was purified using agarose gel electroare transformed to pre-zymogenic cells
Summary
Refs. 19and 201, and for examining the point at which decisions about lineage allocation (commitment) are made as cells traverse the crypt-to-villusaxis (e.g. Refs. 15,16, and). 19and 201, and for examining the point at which decisions about lineage allocation (commitment) are made as cells traverse the crypt-to-villusaxis We have used transgenic mice to conduct analogous studies in the mouse stomach. The mouse P-subunit of H+/K+-ATPaseand intrinsic factor (1nF)l genes were used for these studies. Mouse parietal cells contain a H+/K+-ATPase(EC 3.6.1.3)which couples exchange of extracellular K+ and intracellular H+ to hydrolysis of ATP, yielding a transmembrane gradient of protons.The enzyme consists of a 100-kDa a-subunit which contains the catalytic site for ATP hydrolysis [22,23,24] and a 294-. Residue, heterogeneously glycosylated, P-subunit with unknown fbnction(s) [25, 26].
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