Volume 123, Issue 6, Pages 1889-1903 (December 2002) Gastric hyperplasia in mice with targeted disruption of the carbonic anhydrase gene Car9  Marta Ortova Gut, Seppo Parkkila, Zdeòka Vernerová, Elvira Rohde, Jan Závada, Michael Höcker, Jaromír Pastorek, Tuomo Karttunen, Adriana Gibadulinová, Zuzana Závadová, Klaus–Peter Knobeloch, Bertram Wiedenmann, Jan Svoboda, Ivan Horak, Silvia Pastoreková  Gastroenterology  Volume 123, Issue 6, Pages 1889-1903 (December 2002) DOI: 10.1053/gast.2002.37052 Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 1 Targeted disruption of Car9 gene. (A) Targeting strategy: top, genomic structure of Car9 locus showing exons 1–9; middle, pCar9-neo targeting vector of 6.8 kilobases with pgk-neo cassette replacing 14 base pairs in exon 1; bottom, structure of the gene after homologous recombination. The 4.0-kilobase XbaI (X) and 6.5-kilobase EcoRI (E) fragments present in the wild-type allele are shown above, and the 2.0-kilobase XbaI and 8.3-kilobase EcoRI fragments present in the mutated allele are shown below. Positions of the restriction sites for BamHI (B), HindIII (H), and the probes used to distinguish between the wild-type and the mutant alleles after digestion with EcoRI are indicated. (B) Southern blot genotyping of the offspring from a heterozygous mating. DNA from tails of 3-week-old mice was digested with EcoRI and hybridized with the 3' probe. Bands correspond to wild-type (6.4 kilobases) and mutated (8.3 kilobases) allele, respectively. (C) Analysis of mRNA expressed from the targeted Car9 gene. Position of the primers on Car9 genomic locus is schematically illustrated above the figure, showing the RT-PCR amplification products from wild-type (lanes 1 and 7) and mutant (lanes 2, 4, 6, and 8) stomach mRNAs as well as the PCR amplicons from the mutated genomic DNA (lanes 3 and 5). (D) Western blot analysis of the protein extracts from the wild-type Car9+/+ (lane 1) and mutant Car9−/− (lane 2) mouse stomach epithelium. Extracts from NIH3T3 cells transfected with the full-length Car9 cDNA (lane 3) and from mock-transfected NIH3T3 cells (lane 4) served as controls. Blot was processed with anti–CA IX rabbit antiserum raised against a full-length recombinant GST-mCA IX protein. Gastroenterology 2002 123, 1889-1903DOI: (10.1053/gast.2002.37052) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 2 Histologic analysis of Car9+/+ and Car9−/− mouse stomachs during fetal development. Paraffin sections from the (A–C) wild-type and (D–F) mutant mouse stomachs were stained with H&E. Gastric epithelia show normal morphology at embryonic days (A and D) E15.5 and (B and E) E17.5, whereas the mutant stomach at (F) postnatal day P0.5 shows the first signs of the mucosa thickening when compared with (C) age-matched normal stomach. (Original magnification 400×.) Gastroenterology 2002 123, 1889-1903DOI: (10.1053/gast.2002.37052) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 3 Organization of the stomach mucosa in adult Car9+/+ and Car9−/− stomachs. Sections of the stomachs obtained from the 1.5-year-old (A) wild-type and (B and C) mutant littermates were stained with H&E. Mucosa of both wild-type and mutant stomachs contains mucus-producing surface cells (M), parietal cells (PA), and chief cells (CH) underlined by muscularis mucosae (MM). (C) The mutant epithelium is expanded and shows distorted architecture with numerous cysts. (Original magnification 100×.) Gastroenterology 2002 123, 1889-1903DOI: (10.1053/gast.2002.37052) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 4 Statistical analysis of the number of cells in stomachs of 25- to 35-week-old Car9+/+ versus Car9−/− mice. As quantified by the Mann–Whitney rank test, the mutant mouse stomach mucosa showed (A) increased total numbers of cells, (B) decreased absolute numbers of chief cells, and (C) increased absolute numbers of parietal cells. However, loss of CA IX led to (D) decreased proportion of both pepsinogen-positive chief cells and (E) H+/K+-ATPase positive parietal cells. Regression analysis showed significant correlation between the total cell numbers and the numbers of parietal cells in gastric units (F) and showed a significant difference between the slopes of regression lines corresponding to gastric units of Car9+/+ and Car9−/− mice, respectively. Gastroenterology 2002 123, 1889-1903DOI: (10.1053/gast.2002.37052) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 5 Gastric pH and acid secretion in 16- to 19-week-old Car9−/− and Car9+/+ mice. All measurements were performed 30 minutes after histamine stimulation. (A) Relative stomach weight calculated per gram of body weight, (B) pH of gastric secretions, and (C) acid content expressed as microequivalents normalized to gram of wet stomach weight. The genotypes are below the chart. ■, Car9+/+ mice; □, Car9−/− mice. Median values are indicated by horizontal bars, and probability values are shown inside the charts. Mann–Whitney rank test showed a significant difference in the relative stomach weight but not in pH or acid secretion. Gastroenterology 2002 123, 1889-1903DOI: (10.1053/gast.2002.37052) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 6 Immunohistochemical localization of CA IX in Car9+/+ and Car9−/− mouse stomachs. Stomach sections were stained with the anti–CA IX rabbit antiserum raised against the mouse CA IX. Glandular stomach epithelium of the wild-type mouse (A and B) shows strong positive staining confined predominantly to the glandular and pit cells and (B) located at the basolateral membranes. (C) The staining is absent from the mutant Car9−/− stomach corpus, as well as from (D) the wild-type Car9+/+ forestomach. (Original magnification 200×.) Gastroenterology 2002 123, 1889-1903DOI: (10.1053/gast.2002.37052) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 7 PCNA distribution in the stomach epithelium of Car9−/− mice. The proliferative zone (PZ) in the (A) Car9+/+ mouse stomach mucosa is confined to the isthmus of the gland where the epithelial cells are clearly positive for PCNA. (B) In the stomach mucosa of Car9−/− mice, the proliferative zone is markedly expanded. Magnified areas show distinct nuclear PCNA-specific staining. (Original magnification 100×.) Gastroenterology 2002 123, 1889-1903DOI: (10.1053/gast.2002.37052) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 8 Distribution of PAS staining in stomach corpus of 25- to 35-week-old Car9+/+ and Car9−/− mice. The PAS staining is confined to the mucus-producing pit region (M) of the (A) wild-type mice. (B) In the mutant mice, the positive reaction extends toward the deep glandular layer of the stomach corpus epithelium. (Original magnification 100×.) Gastroenterology 2002 123, 1889-1903DOI: (10.1053/gast.2002.37052) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 9 Distribution of (A and C) pepsinogen C–positive cells and (B and D) α subunit H+/K+-ATPase–positive cells in stomachs of 25- to 35-week-old (A and B) Car9+/+ and (C and D) Car9−/− mice analyzed by immunohistochemistry. Pepsinogen C–positive chief cells are predominantly distributed in the basal parts of the gastric glands in both wild-type and mutant mice, but their number is reduced in CA IX–deficient mucosa. Parietal cells expressing α subunit of H+/K+-ATPase are scattered in the gastric glands in both cases, but their proportion is lower in Car9−/− mouse epithelium. (Original magnification 200×.) Gastroenterology 2002 123, 1889-1903DOI: (10.1053/gast.2002.37052) Copyright © 2002 American Gastroenterological Association Terms and Conditions