Proc Natl Acad Sci U S A 99(23): 14819-14824

Molecular characterization of mouse gastric epithelial progenitor cells

Departments of ^{Molecular Biology and Pharmacology and}^{Pathology and Immunology, Washington University School of Medicine, St. Louis, MO 63110}

^{To whom correspondence should be addressed. E-mail:}ude.ltsuw.loocelom@nodrogj.

Contributed by Jeffrey I. Gordon

Accepted 2002 Sep 20.

Abstract

The adult mouse gastric epithelium undergoes continuous renewal in discrete anatomic units. Lineage tracing studies have previously disclosed the morphologic features of gastric epithelial lineage progenitors (GEPs), including those of the presumptive multipotent stem cell. However, their molecular features have not been defined. Here, we present the results of an analysis of genes and pathways expressed in these cells. One hundred forty-seven transcripts enriched in GEPs were identified using an approach that did not require physical disruption of the stem cell niche. Real-time quantitative RT-PCR studies of laser capture microdissected cells retrieved from this niche confirmed enriched expression of a selected set of genes from the GEP list. An algorithm that allows quantitative comparisons of the functional relatedness of automatically annotated expression profiles showed that the GEP profile is similar to a dataset of genes that defines mouse hematopoietic stem cells, and distinct from the profiles of two differentiated GEP descendant lineages (parietal and zymogenic cell). Overall, our analysis revealed that growth factor response pathways are prominent in GEPs, with insulin-like growth factor appearing to play a key role. A substantial fraction of GEP transcripts encode products required for mRNA processing and cytoplasmic localization, including numerous homologs of Drosophila genes (e.g., Y14, staufen, mago nashi) needed for axis formation during oogenesis. mRNA targeting proteins may help these epithelial progenitors establish differential communications with neighboring cells in their niche.

Keywords: stem cells, growth factor signaling, protein turnover, mRNA localization , bioinformatics

Abstract

The mammalian gastrointestinal tract is lined by an epithelium that is constantly renewed. Although multipotent stem cells are known to fuel this renewal, the molecular properties of these cells are poorly understood.

Tritiated thymidine/EM autoradiographic lineage tracing studies have delineated the morphological features of the stem cell niche in the adult mouse stomach (1). The glandular epithelium is composed of tubular invaginations termed gastric units. In the corpus (central region) of the stomach, each unit contains an average of ≈200 cells, representing three predominant lineages: pit, parietal, and zymogenic. The multipotent stem cell (undifferentiated granule-free progenitor) resides in the unit's isthmus (ref. 1; Fig. Fig.11A). One of its committed daughters, the granule-free prepit cell precursor, produces mucus-secreting pit cells, which differentiate as they climb from the isthmus to the orifice of the unit (2). Another daughter, the granule-free preneck cell precursor, gives rise to pepsinogen-producing neck cells, which differentiate to zymogenic cells as they descend to the base of the unit (3). Unlike the pit and zymogenic lineages, acid-producing parietal cells (PCs) differentiate within the isthmus from granule-free preparietal cell progenitors and then migrate either up or down the unit (Fig. (Fig.11A and B; ref. 4).

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Fig 1.

Parietal cell ablation produces GEP amplification in tox176 mice. (A) Schematic representation of a gastric unit in the middle third of a normal adult mouse stomach. The unit contains four compartments: pit, isthmus, neck, and base. The multipotent stem cell in the isthmus gives rise to three principal epithelial lineages (pit, parietal, and zymogenic). Only PCs differentiate within the isthmal stem cell niche. They then undergo a bidirectional migration to the pit and base regions. (B) EM immunohistochemical study showing juxtaposition of two mitochondria-rich PCs and two granule-free GEPs in the isthmus of a normal adult mouse gastric unit. PCs are outlined by dashes. The boxed region in one GEP (and the higher power Inset) shows a portion of the nucleus labeled with goat anti-PCNA and 18-nm-diameter gold particle-conjugated donkey anti-goat Ig. (C and D) Multilabel study of a 16-week-old normal germ-free mouse (C) and an age-matched conventionally raised tox176 animal (D). Each mouse received an i.p. injection of BrdUrd 90 min before sacrifice. Purple, pit cells labeled with Alexafluor 647-AAA; red, neck cells tagged with biotinylated GSII and Alexafluor 594- streptavidin; green, isthmal S-phase progenitors detected with goat anti-BrdUrd and Alexafluor 488-donkey anti-goat Ig. Note marked expansion of S-phase cells (e.g., arrows) in PC-ablated tox176 gastric units. (Bars, 25 μm.)

We have shown that there is increased proliferation of isthmal gastric epithelial progenitors (GEPs) in adult transgenic mice with genetically engineered, mutant diphtheria toxin A fragment (tox176)-mediated ablation of PCs (Fig. (Fig.11C and D; refs. 5 and 6). Analysis of tox176 pedigrees with a mosaic pattern of transgene expression showed that GEP amplification occurs only in units lacking PCs (6), suggesting that PCs produce locally acting factors that regulate GEP census.

We reasoned that the increase in GEPs in tox176 mice offered an opportunity to identify molecular regulators of GEP biology, including those that mediate interactions with PCs, without having to disrupt the isthmal niche where they reside. Here, we present the results of a “dissection-free” approach that used a three-way comparison of gene expression profiles in the intact stomachs of (i) normal adult mice where GEPs represent <3% of the total epithelial population; (ii) tox176 adult mice where GEPs constitute ≈20% of the total; and (iii) embryonic day 18 (E18) mice where >90% of the developing gastric epithelium is composed of GEPs (7). Genes whose expression was increased in both ii and iii relative to i formed the GEP dataset. Several generally applicable experimental and computational methods, ranging from a new application of laser capture microdissection to an algorithmic approach for comparing the functional features of entire gene expression profiles, were used to validate and extend these results. The 147-member GEP dataset has prominent representation of genes involved in insulin-like growth factor (IGF) signaling, in regulating protein turnover (ubiquitin/proteosomal, sumoylation, and neddylation), and in controlling RNA processing and localization.

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Acknowledgments

We thank Sabrina Wagoner and Jaime Dant for technical assistance, David O'Donnell and Maria Karlsson for maintaining mice, Jung Oh for advice with qRT-PCR assays, and Diane Redmond for computer graphics. This work was supported by National Institutes of Health Grant DK58529. J.C.M. is a Howard Hughes Medical Institute physician-scientist postdoctoral fellow.

Acknowledgments

Abbreviations

AAA, Anguilla anguilla agglutinin
FR, fractional representation
GEP, gastric epithelial lineage progenitor
GO, Gene Ontology
GSII, Griffonia simplicifolia II
HSC, hematopoietic stem cell
IGF, insulin-like growth factor
PC, parietal cell
PCNA, proliferating cell nuclear antigen
qRT-PCR, quantitative RT-PCR

Abbreviations

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