1. Volume 133, Issue 6, Pages 1989-1998.e3 (December 2007)
Prospective Identification of a Multilineage Progenitor in Murine Stomach Epithelium 
Xiaotan T. Qiao, Joshua W. Ziel, Wendy McKimpson, Blair B. Madison, Andrea Todisco, Juanita L. Merchant, Linda C. Samuelson, Deborah L. Gumucio 
Gastroenterology 
Volume 133, Issue 6, Pages e3 (December 2007)
DOI: /j.gastro
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2. Figure 1
The villin promoter is active in a rare subpopulation of cells in the adult gastric antrum. (A) In the antrum of a control wild-type mouse stained with X-gal, no β-gal–positive cells can be detected. (B) Widely spaced β-gal–positive cells are detected in a section from the antrum of an untreated Villinβ-gal/+ mouse. The inset reveals an oval-shaped cell with a large nucleus to cytoplasm ratio. (C) Cells are located in the lower third of the gland. (D) Rarely, 2 β-gal–positive cells are observed in a single sectioned gland. (E) Schematic views of gastric glands from the corpus and antrum are shown. Yellow cells indicate parietal cells, diagnostic for the corpus. The regions designated as pit, isthmus, and base are shown for each gland. (F) The relative distribution of β-gal cells within 1 entire mouse stomach is plotted as the percentage of cells at various positions along the gland axis in the corpus and antrum. This distribution was similar in the 3 control animals examined. Purple, pit; maroon, isthmus; yellow, base. (A and B) bar = 50 μm, (B) bar = 20 μm, (C), bar = 10 μm.
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3. Figure 2
Behavior of β-gal–positive cells in mouse models of metaplasia. (A and B) One-year-old Villinβ-gal/+/Cdx2 mice. (A) Widespread intestinal metaplasia in the antrum is detected by alkaline phosphatase staining (red). (B) X-gal staining in the same mouse reveals β-gal–positive cells at the surface, within the metaplastic region. The number and location of deep-gland β-gal–positive cells is similar to wild-type littermates. (C–E) Six-month-old Villinβ-gal/+/Ctox-7 mice. (C) Alkaline phosphatase staining (red) shows focally positive antral glands. (D) The number of β-gal–positive cells is greatly expanded. One gland contains 2 β-gal–positive cells distributed along the long axis of the gland (arrow). (E) Numerous β-gal–positive cells are detectable in the lower region of the antral glands. (F) IFNγ-treated mice. The number of β-gal–positive cells is increased in this model after only 2 weeks of treatment (compare with Figure 1B). (A–C) Bar = 50 μm, (D–F) bar = 20μm.
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4. Figure 3
β-gal–positive cells are located at or below the isthmus in isolated antral glands. (A–E) Note the distribution of β-gal–marked cells at variable distances in relation to the gland base. (F) Two β-gal–positive cells appear to be completing mitosis along the long axis of the gland. Dotted red lines in the inset surround two nuclei. (G and H) Two glands from INFγ-treated mice; β-gal–positive cells are distributed to 2 opposite sides of the gland tip. (I and J) Labeled cells from a 12.4KVil-EGFP mouse. (J) The 2 labeled cells in the gland both possess long cytoplasmic processes that are visible with the EGFP label.
Gastroenterology  , e3DOI: ( /j.gastro )
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5. Figure 4
Lineage tracing studies: single β-gal–positive cells clonally populate the gland with multiple gastric cell types. (A) Antral gland in a Villinβ-gal/+/ROSA26R control mouse treated with PBS. Few such fully stained glands are seen in control animals. (B and C) Fully labeled antral glands from Villinβ-gal/+/ROSA26R mice treated with INFγ. Labelled glands tend to cluster. (D and E) Whole-mount β-gal staining of the antrum in Villinβ-gal/+/ROSA26R mice treated with (D) PBS or (E) IFNγ and killed after 12 weeks. (D) The PBS-treated antrum shows no β-gal staining. The β-gal–positive region at the bottom is duodenum. (E) In an INFγ-treated mouse, large patches of contiguous β-gal–positive glands are visible on the ventral side of the antrum. (F–J) Fully stained glands are expressing gastric markers. (F) Anti–β-gal, (red); fluorescein isothiocyanate–conjugated GSII lectin, (green) (neck/gland cell marker). (G) Anti–β-gal, (red); fluorescein isothiocyanate–conjugated UAEI lectin, (green) (surface pit cell marker). (H and I) The section is stained with anti–β-gal (green) and antiserotonin (red), marking a common subtype of enteroendocrine cells. (I) Higher magnification of the region that is boxed in panel H. (J) A section from the junction of the corpus and antrum, costained with anti–β-gal (green) and anti-H+,K+ ATPase (red), a parietal cell marker. (D and E) Bar = 100 μm, (F) bar = 50 μm, (A–C and G–J) bar = 20 μm.
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6. Supplemental Figure 1
BrdU-labeling characteristics of β-gal–positive cells. (A, D, G, J, and M) BrdU staining (red) is shown. (B, E, H, K, and N) Signal for the fluorescein isothiocyanate–labeled anti–β-gal antibody (green) has been added to the BrdU (red) signal. (C, F, I, L, and O) DAPI channel (blue) has been added to the BrdU (red) and anti–β-gal (green). (A–C) This wild-type mouse was pulse labeled with BrdU 1 hour before death. β-gal–positive cells are located among the labeled cells (arrows), but the β-gal–positive cells themselves are not labeled, indicating that they are not cycling in the absence of stimulation. (D–I) Two examples of cells after 1 week of continuous BrdU labeling and 1 week of IFNγ treatment are shown. (D–F) β-gal–positive cell did not take up label (arrow). (G–I) β-gal–positive cell also is positive for the BrdU label (arrow). (J–O) Two examples are shown of label-retaining cells. These mice were given IFNγ and BrdU for 1 week and were killed after an additional week without treatment. In both cases, only 2 label-retaining cells are visible in the glands. In both cases, 1 label-retaining cell (white arrow) is positive for anti–β-gal staining whereas the other (yellow arrowhead) is not. (A–O) Bar = 20 μm.
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7. Supplemental Figure 2
The distribution of labeled cells is similar in 2 transgenic models driven by villin regulatory sequences. (A) β-gal–positive cells occupy the antrum of the 12.4KVil-LacZ mouse. Their distribution and location is similar to that seen in the Villinβ-gal/+ model (compare with Figure 1B). (B) Two weeks of IFNγ treatment causes amplification of β-gal–positive cells in the 12.4KVil-LacZ mouse antrum. (C) β-gal–positive cells in a 12.4KVil-LacZ mouse antrum are located deep in the gland and are triangular in shape. (D) Rare labeled cells are visible in an untreated 12.4KVil-EGFP mouse, here detected with a fluorescein isothiocyanate–conjugated anti-EGFP antibody. (E) IFNγ causes amplification of the marked cells in the 12.4KVil-EGFP model. (F) Labeled cells in 12.4KVil-EGFP mice are triangular and located in the bottom third of the glands. (A and D) Bar = 50 μm, (B and E) bar = 20 μm, (C and F) bar = 10 μm.
Gastroenterology  , e3DOI: ( /j.gastro )
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8. Supplemental Figure 3
A proposed life cycle of GPC-positive glands. GPC (yellow) are quiescent and reside in a niche that is located below the isthmus. (B) In response to specific triggers, such as INFγ, GPCs divide symmetrically along the long axis of the gland (see Figure 3J). (C) Recently divided GPCs migrate toward the gland base (see Figure 2B). (D–F) Arrival of 2 GPCs at the base of the gland may be a trigger for gland fission; GPCs segregate to opposite sides of the gland to be separated by fission (see Figures 1C, 3G, 3H, and 3J). Cycles of symmetric division and subsequent fission (yellow arrows) could explain clonal patches of GPC-containing glands seen in lineage tracing studies (see Figure 4E). (G) GPCs in recently divided glands migrate back up to a niche where they can receive triggers that promote asymmetric division. (H) Asymmetric division of GPCs (yellow) leads to rapid gland regeneration (blue). All cells of the regenerated gland are derived from a single GPC (designated in yellow).
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9. Supplemental Figure 4
β-gal–positive cells are present at the squamocolumnar junction and are visible in fetal life. (A) Several β-gal–positive cells are located on the initial columnar region at the mucocolumnar junction in this 12.4KVil-LacZ mouse. (B) A 12.4KVil-EGFP mouse shows similar labeled cells at the forestomach junction. (C) In an E16.5 mouse embryo, small triangular β-gal–positive cells are located next to the basement membrane of the immature antral epithelium. (A–C) Bar = 20 μm.
Gastroenterology  , e3DOI: ( /j.gastro )
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