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Molecular Characterization of Mouse Gastric Epithelial Progenitor Cells

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Molecular Characterization of Mouse Gastric Epithelial Progenitor Cells Molecular characterization of mouse gastric epithelial progenitor cells Jason C. Mills*†, Niklas Andersson*, Chieu V. Hong*, Thaddeus S. Stappenbeck*, and Jeffrey I. Gordon*‡ Departments of *Molecular Biology and Pharmacology and †Pathology and Immunology, Washington University School of Medicine, St. Louis, MO 63110 Contributed by Jeffrey I. Gordon, September 20, 2002 The adult mouse gastric epithelium undergoes continuous renewal transgene expression showed that GEP amplification occurs only in discrete anatomic units. Lineage tracing studies have previously in units lacking PCs (6), suggesting that PCs produce locally disclosed the morphologic features of gastric epithelial lineage acting factors that regulate GEP census. progenitors (GEPs), including those of the presumptive multipo- We reasoned that the increase in GEPs in tox176 mice offered tent stem cell. However, their molecular features have not been an opportunity to identify molecular regulators of GEP biology, defined. Here, we present the results of an analysis of genes and including those that mediate interactions with PCs, without pathways expressed in these cells. One hundred forty-seven tran- having to disrupt the isthmal niche where they reside. Here, we scripts enriched in GEPs were identified using an approach that did present the results of a ‘‘dissection-free’’ approach that used a not require physical disruption of the stem cell niche. Real-time three-way comparison of gene expression profiles in the intact quantitative RT-PCR studies of laser capture microdissected cells stomachs of (i) normal adult mice where GEPs represent Ͻ3% retrieved from this niche confirmed enriched expression of a of the total epithelial population; (ii) tox176 adult mice where selected set of genes from the GEP list. An algorithm that allows GEPs constitute Ϸ20% of the total; and (iii) embryonic day 18 quantitative comparisons of the functional relatedness of auto- (E18) mice where Ͼ90% of the developing gastric epithelium is matically annotated expression profiles showed that the GEP composed of GEPs (7). Genes whose expression was increased profile is similar to a dataset of genes that defines mouse hema- in both ii and iii relative to i formed the GEP dataset. Several topoietic stem cells, and distinct from the profiles of two differ- generally applicable experimental and computational methods, entiated GEP descendant lineages (parietal and zymogenic cell). ranging from a new application of laser capture microdissection Overall, our analysis revealed that growth factor response path- to an algorithmic approach for comparing the functional features ways are prominent in GEPs, with insulin-like growth factor ap- of entire gene expression profiles, were used to validate and pearing to play a key role. A substantial fraction of GEP transcripts extend these results. The 147-member GEP dataset has prom- encode products required for mRNA processing and cytoplasmic inent representation of genes involved in insulin-like growth localization, including numerous homologs of Drosophila genes factor (IGF) signaling, in regulating protein turnover (ubiquitin͞ (e.g., Y14, staufen, mago nashi) needed for axis formation during proteosomal, sumoylation, and neddylation), and in controlling oogenesis. mRNA targeting proteins may help these epithelial RNA processing and localization. progenitors establish differential communications with neighbor- ing cells in their niche. Materials and Methods Mice. Conventionally raised FVB͞N tox176 transgenic mice (6) stem cells ͉ growth factor signaling ͉ protein turnover ͉ mRNA localization ͉ were maintained in microisolator cages in a specified pathogen- bioinformatics free state. Nontransgenic, germ-free FVB͞N mice were raised in plastic gnotobiotic isolators (8). he mammalian gastrointestinal tract is lined by an epithelium Tthat is constantly renewed. Although multipotent stem cells GeneChip Comparisons. Whole stomachs were excised and RNA are known to fuel this renewal, the molecular properties of these extracted (midi-RNeasy kit; Qiagen, Valencia, CA). Duplicate cells are poorly understood. cRNA targets were prepared from pooled RNAs (see below). Each Tritiated thymidine͞EM autoradiographic lineage tracing stud- cRNA was hybridized to a set of Mu11K GeneChips (Affymetrix, ies have delineated the morphological features of the stem cell niche Santa Clara, CA). Overall fluorescence across each GeneChip set in the adult mouse stomach (1). The glandular epithelium is was scaled to a target intensity of 150 and pairwise comparisons composed of tubular invaginations termed gastric units. In the performed using Affymetrix MICROARRAY SUITE software (V.4.0). corpus (central region) of the stomach, each unit contains an Ϸ SYBR-Green-Based Real-Time Quantitative RT-PCR (qRT-PCR). Assays average of 200 cells, representing three predominant lineages: pit, CELL BIOLOGY parietal, and zymogenic. The multipotent stem cell (undifferenti- were performed in triplicate as described (9), using the gene- ated granule-free progenitor) resides in the unit’s isthmus (ref. 1; specific primers listed in Table 1, which is published as support- Fig. 1A). One of its committed daughters, the granule-free prepit ing information on the PNAS web site, www.pnas.org. Normal- ⌬⌬ cell precursor, produces mucus-secreting pit cells, which differen- ization was to 18S rRNA ( CT method; ref. 9). tiate as they climb from the isthmus to the orifice of the unit (2). Another daughter, the granule-free preneck cell precursor, gives Navigated Laser Capture Microdissection (n-LCM). Stomachs were ϭ rise to pepsinogen-producing neck cells, which differentiate to removed from tox176 mice (n 4), flushed with PBS, and zymogenic cells as they descend to the base of the unit (3). Unlike divided in half along the cephalocaudal axis, and each half- the pit and zymogenic lineages, acid-producing parietal cells (PCs) stomach was rinsed with OCT compound. Half-stomachs were differentiate within the isthmus from granule-free preparietal cell placed in a cryomold, overlaid with OCT compound, and frozen progenitors and then migrate either up or down the unit (Fig. 1 A (Cytocool II, Richard-Allan Scientific, Kalamazoo, MI). Cryo- and B; ref. 4). We have shown that there is increased proliferation of isthmal Abbreviations: AAA, Anguilla anguilla agglutinin; FR, fractional representation; GEP, gastric epithelial progenitors (GEPs) in adult transgenic mice gastric epithelial lineage progenitor; GO, Gene Ontology; GSII, Griffonia simplicifolia II; with genetically engineered, mutant diphtheria toxin A fragment HSC, hematopoietic stem cell; IGF, insulin-like growth factor; PC, parietal cell; PCNA, (tox176)-mediated ablation of PCs (Fig. 1 C and D; refs. 5 and proliferating cell nuclear antigen; qRT-PCR, quantitative RT-PCR. 6). Analysis of tox176 pedigrees with a mosaic pattern of ‡To whom correspondence should be addressed. E-mail: [email protected]. www.pnas.org͞cgi͞doi͞10.1073͞pnas.192574799 PNAS ͉ November 12, 2002 ͉ vol. 99 ͉ no. 23 ͉ 14819–14824 Downloaded by guest on September 24, 2021 age, GEPs account for 10% of the total gastric unit epithelial cell census, and by 20 weeks 25% (Ϸ20-fold higher than in age- matched normal littermates; Fig. 1 C and D; ref. 5). Guided by the strategy outlined in the Introduction, we isolated RNA from the intact stomachs of conventionally raised 16-week-old tox176 mice (equal amounts of RNA pooled from five stomachs), age-matched normal germ-free mice (n ϭ 4), and normal embryonic day 18 animals (n ϭ 31). Because tox176 mice lose the acid barrier to microbial colonization, they inevitably develop a mild diffuse chronic gastritis associated with bacterial over- growth. Because a comparison of gene expression in tox176 versus normal adult germ-free stomachs would likely yield a dataset substantially enriched for GEP-associated mRNAs but ‘‘contaminated’’ by immune response genes, we referenced the ‘‘tox176 versus normal’’ dataset to the ‘‘E18 versus normal’’ dataset (where E18 represents a GEP-enriched, gastritis-free state). Duplicate cRNA targets, independently generated from each RNA, were used to probe GeneChips representing Ϸ11,000 mouse genes and EST clusters. Genes with enhanced expression in duplicate comparisons between tox176 and normal stomachs and in duplicate comparisons between E18 and normal stomachs were culled, yielding a list of 147 genes and 6 uncharacterized ESTs with quadruplicate ‘‘Increased’’ calls by GeneChip soft- ware (see Table 2, which is published as supporting information on the PNAS web site). Initial Validation of the GEP Dataset. The three RNAs used for the GeneChip comparisons were used as templates for qRT-PCR analysis of the expression of seven genes, selected from the GEP Fig. 1. Parietal cell ablation produces GEP amplification in tox176 mice. dataset based on their known roles in regulating proliferation, (A) Schematic representation of a gastric unit in the middle third of a normal adult differentiation, and polarity in other systems. They encoded mouse stomach. The unit contains four compartments: pit, isthmus, neck, and proliferating cell nuclear antigen (PCNA; ref. 10), retinoblas- base. The multipotent stem cell in the isthmus gives rise to three principal epithelial lineages (pit, parietal, and zymogenic). Only PCs differentiate within toma binding protein 7 (together with RbAP48 increases histone the isthmal stem cell niche. They then undergo a bidirectional migration to the pit acetyltransferase activity; ref. 11), colony stimulating
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