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• W2093252658 abstract "Zymogenic cells (ZCs), acid-producing parietal cells (PCs), and mucus-secreting pit cells are the principal epithelial lineages in the stomachs of adult mice and humans. Each lineage is derived from the multipotent gastric stem cell and undergoes perpetual renewal within discrete mucosal invaginations (gastric units). In this report, we analyze the molecular features of ZCs and their contributions to gastric epithelial homeostasis. GeneChip analysis yielded a dataset of 57 mRNAs encoding known proteins and 14 ESTs enriched in adult mouse ZCs. This dataset, obtained from comparisons of cellular populations purified by counterflow elutriation and lectin panning, was validated by real-time quantitative reverse transcription-PCR studies of the in vivo expression of selected genes using cells harvested from different regions of gastric units by laser capture microdissection. ZC-enriched mRNAs include regulators of angiogenesis (e.g. platelet-derived growth factors A and B). Because PCs are enriched in transcripts encoding other angiogenic factors (e.g. Vegfb), the contributions of these two lineages to vascular development was examined by performing quantitative three-dimensional imaging of the capillary networks that surround gastric units in two types of mice. In normal adult gnotobiotic FVB/N animals, network density is on average 2-fold higher in ZC- and PC-containing units located in the proximal (corpus) region of the stomach compared with units positioned in the distal (antral) region that lack these lineages (p < 0.01). Gnotobiotic transgenic mice with an engineered ablation of all ZCs and PCs have a 2-fold reduction in capillary network density in their corpus region gastric units compared with the corpus units of normal littermates (p < 0.01). These results support an emerging theme that angiogenesis in the adult mouse gut is modulated by cross-talk between its epithelial lineages and the underlying mesenchyme. Zymogenic cells (ZCs), acid-producing parietal cells (PCs), and mucus-secreting pit cells are the principal epithelial lineages in the stomachs of adult mice and humans. Each lineage is derived from the multipotent gastric stem cell and undergoes perpetual renewal within discrete mucosal invaginations (gastric units). In this report, we analyze the molecular features of ZCs and their contributions to gastric epithelial homeostasis. GeneChip analysis yielded a dataset of 57 mRNAs encoding known proteins and 14 ESTs enriched in adult mouse ZCs. This dataset, obtained from comparisons of cellular populations purified by counterflow elutriation and lectin panning, was validated by real-time quantitative reverse transcription-PCR studies of the in vivo expression of selected genes using cells harvested from different regions of gastric units by laser capture microdissection. ZC-enriched mRNAs include regulators of angiogenesis (e.g. platelet-derived growth factors A and B). Because PCs are enriched in transcripts encoding other angiogenic factors (e.g. Vegfb), the contributions of these two lineages to vascular development was examined by performing quantitative three-dimensional imaging of the capillary networks that surround gastric units in two types of mice. In normal adult gnotobiotic FVB/N animals, network density is on average 2-fold higher in ZC- and PC-containing units located in the proximal (corpus) region of the stomach compared with units positioned in the distal (antral) region that lack these lineages (p < 0.01). Gnotobiotic transgenic mice with an engineered ablation of all ZCs and PCs have a 2-fold reduction in capillary network density in their corpus region gastric units compared with the corpus units of normal littermates (p < 0.01). These results support an emerging theme that angiogenesis in the adult mouse gut is modulated by cross-talk between its epithelial lineages and the underlying mesenchyme. The human gastric epithelium is renewed continuously throughout life with an estimated 500,000 cells shed/min in adults (1Croft D.N. Pollock D.J. Coghill N.F. Gut. 1966; 7: 333-343Crossref PubMed Scopus (40) Google Scholar). Dysregulated renewal results in a number of disease states including gastric adenocarcinoma, the second most common cause of cancer-related deaths worldwide (2Murray C.J. Lopez A.D. Lancet. 1997; 349: 1269-1276Abstract Full Text Full Text PDF PubMed Scopus (3418) Google Scholar). The mouse has been used as a model organism to characterize the cellular and molecular features of this renewal and to decipher its regulation. The proximal third of the mouse stomach (forestomach) is lined with a squamous epithelium, while its distal two-thirds is lined with a glandular epithelium. The glandular epithelium contains thousands of tubular mucosal invaginations known as gastric units. In the central or corpus region of the stomach, each unit has a steady-state population of ∼200 epithelial cells representing three principal lineages (pit, parietal, and zymogenic) and two minor lineages (enteroendocrine, and caveolated) (3Karam S.M. Leblond C.P. Anat. Rec. 1992; 232: 231-246Crossref PubMed Scopus (117) Google Scholar). All of the lineages are derived from multipotent stem cells located in the mid-portion (isthmus) of each unit (Fig. 1, A-C) (4Bjerknes M. Cheng H. Am. J. Physiol. 2002; 283: G767-G777Crossref PubMed Scopus (135) Google Scholar, 5Karam S.M. Leblond C.P. Anat. Rec. 1993; 236: 259-279Crossref PubMed Scopus (323) Google Scholar). Karam and co-workers (5Karam S.M. Leblond C.P. Anat. Rec. 1993; 236: 259-279Crossref PubMed Scopus (323) Google Scholar, 6Karam S.M. Leblond C.P. Anat. Rec. 1993; 236: 297-313Crossref PubMed Scopus (188) Google Scholar, 7Karam S.M. Leblond C.P. Anat. Rec. 1993; 236: 280-296Crossref PubMed Scopus (167) Google Scholar, 8Karam S.M. Anat. Rec. 1993; 236: 314-332Crossref PubMed Scopus (168) Google Scholar, 9Karam S.M. Leblond C.P. Anat. Rec. 1993; 236: 333-340Crossref PubMed Scopus (129) Google Scholar, 10Karam S.M. Li Q. Gordon J.I. Am. J. Physiol. 1997; 272: G1209-G1220Crossref PubMed Google Scholar) used in vivo pulse labeling with [3H]thymidine followed by electron microscopic autoradiography to obtain morphologic descriptions of the presumptive multipotent stem cell and its immediate committed oligopotential daughters, as well as the pathways their descendants follow during terminal differentiation. Mucus-producing pit cells derived from one of the stem cell's committed daughters differentiate during a rapid (3-day) migration from the stem cell niche located in the middle portion of the unit (isthmus) through the upper portion of the unit (pit region) to the surface epithelium where they are removed by apoptosis or necrosis (Fig. 1A) (7Karam S.M. Leblond C.P. Anat. Rec. 1993; 236: 280-296Crossref PubMed Scopus (167) Google Scholar). In contrast, members of the zymogenic cell lineage differentiate during a downward migration from the isthmus through the neck region to base of the unit. During this passage, they undergo a series of morphologic transitions from pre-neck to neck to pre-zymogenic and finally to mature zymogenic cells (Fig. 1, A, B, and D) (6Karam S.M. Leblond C.P. Anat. Rec. 1993; 236: 297-313Crossref PubMed Scopus (188) Google Scholar). Zymogenic cells (ZCs), 1The abbreviations used are: ZC, zymogenic cell; PC, parietal cell; GEP, gastric epithelial lineage progenitor; n-LCM, navigated form of laser capture microdissection; qRT-PCR, real-time quantitative reverse transcriptase-PCR; DBA, Dolichos biflorus agglutinin; GSII, Griffonia simplifolia II lectin; AAA, Anguilla anguilla agglutinin; Igf, insulin-like growth factor; Igfbp, Igf-binding protein; Pdgf, platelet-derived growth factor; Pdgfr, Pdgf receptor; Vegf, vascular endothelial growth factor; PBS, phosphate-buffered saline; FITC, fluorescein isothiocyanate; EM, electron microscopy. also known as chief cells, are cleared at the base of the unit via necrosis and apoptosis or are phagocytosed by neighboring cells. The zymogenic lineage turns over every 190 days (6Karam S.M. Leblond C.P. Anat. Rec. 1993; 236: 297-313Crossref PubMed Scopus (188) Google Scholar). Unlike pit and zymogenic cells, acid-secreting parietal cells (PCs) differentiate within the isthmus (8Karam S.M. Anat. Rec. 1993; 236: 314-332Crossref PubMed Scopus (168) Google Scholar, 10Karam S.M. Li Q. Gordon J.I. Am. J. Physiol. 1997; 272: G1209-G1220Crossref PubMed Google Scholar) (Fig. 1. A, B, and E). PCs then migrate up through the pit region where they are eliminated by necrosis, exfoliation, or phagocytosis, or down to the base where they are removed by apoptosis or phagocytosis. The average lifespan of members of this lineage is 54 days (8Karam S.M. Anat. Rec. 1993; 236: 314-332Crossref PubMed Scopus (168) Google Scholar). We have embarked on a mouse gastric genome anatomy project (mG-GAP) (see Ref. 11Mills J.C. Syder A.J. Hong C.V. Guruge J.L. Raaii F. Gordon J.I. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13687-13692Crossref PubMed Scopus (70) Google Scholar, genome.wustl.edu/GSCGAP/, and www.scgap.org) to delineate the molecular features of these epithelial lineages with the goal of identifying new mediators/regulators of their renewal, new insights regarding their functions, and new perspectives regarding gastric physiology. To date, PCs and isthmal gastric epithelial progenitors (GEPs) have been characterized using a variety of functional genomics methods. Studies of PCs purified by lectin panning from the stomachs of normal adult mice belonging to the FVB/N inbred strain yielded a data base of 240 genes preferentially expressed in this lineage compared with all of the other mucosal lineages (11Mills J.C. Syder A.J. Hong C.V. Guruge J.L. Raaii F. Gordon J.I. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13687-13692Crossref PubMed Scopus (70) Google Scholar). Thirty-five percent of these genes encode proteins involved in various aspects of energy metabolism, a finding consistent with the fact that the proton pumps of PCs require high energy-generating capacity. PCs are also enriched in mRNAs encoding free radical scavengers that protect against potential damage from high rates of oxidative phosphorylation, proteins involved in various aspects of lipid metabolism (PCs maintain an elaborate tubulovesicular apparatus), and components of calcium signaling pathways (e.g. those involving calcineurin) (11Mills J.C. Syder A.J. Hong C.V. Guruge J.L. Raaii F. Gordon J.I. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13687-13692Crossref PubMed Scopus (70) Google Scholar). In another study, we generated a data base of 147 transcripts enriched in GEPs using an approach that did not physically disrupt the isthmal stem cell niche (12Mills J.C. Andersson N. Hong C.V. Stappenbeck T.S. Gordon J.I. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 14819-14824Crossref PubMed Scopus (90) Google Scholar). A substantial fraction of the transcripts encode products required for processing and cytoplasmic localization of mRNAs, including homologs of Drosophila genes needed for axis formation during oogenesis. These mRNA-targeting proteins may help gut epithelial progenitors establish differential communications with their neighbors (12Mills J.C. Andersson N. Hong C.V. Stappenbeck T.S. Gordon J.I. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 14819-14824Crossref PubMed Scopus (90) Google Scholar, 13Stappenbeck T.S. Mills J.C. Gordon J.I. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 1004-1009Crossref PubMed Scopus (123) Google Scholar). In this report, we present a molecular profile of ZCs retrieved from adult FVB/N mouse stomachs using counterflow elutriation and lectin panning. A GeneChip-derived dataset of transcripts enriched in ZCs compared with other gastric epithelial lineages was validated by real-time quantitative RT-PCR studies of laser capture-microdissected populations harvested from gastric cryosections. One finding from the analysis is that ZCs are a source of platelet-derived growth factors (Pdgf) A and B. Our previously published molecular profile of PCs (11Mills J.C. Syder A.J. Hong C.V. Guruge J.L. Raaii F. Gordon J.I. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13687-13692Crossref PubMed Scopus (70) Google Scholar) revealed that they are a source of another angiogenic factor, vascular endothelial growth factor B (Vegfb). The results of quantitative three-dimensional imaging studies of the capillary networks that surround gastric units in the proximal and distal glandular epithelium of adult germ-free transgenic mice with an engineered ablation of their PCs and ZCs, and in their normal germ-free littermates, provide direct evidence that these lineages contribute to the regulation of angiogenesis. Animals—Conventionally raised, specified pathogen-free normal FVB/N mice were maintained under a strict 12-h light cycle and fed a standard chow diet (Picolab rodent diet 20, Purina Mills). Members of a pedigree of germ-free FVB/N transgenic mice that express an attenuated diphtheria toxin A fragment (tox176) under the control of nucleotides from -1035 to +24 of the mouse gene encoding the β-subunit of H+/K+-ATPase (14Syder A.J. Oh J.D. Guruge J.L. O'Donnell D. Karlsson M. Mills J.C. Bjorkholm B.M. Gordon J.I. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 3467-3472Crossref PubMed Scopus (52) Google Scholar) and their germ-free normal littermates were maintained in plastic gnotobiotic isolators (15Hooper L.V. Mills J.C. Roth K.A. Stappenbeck T.S. Wong M.H. Gordon J.I. Sansonetti P. Methods in Microbiology. Vol. 31. Academic Press, London2002: 559-589Google Scholar). All experiments involving mice were conducted using protocols approved by the Animal Studies Committee of Washington University. Isolation of ZCs by Elutriation and Lectin Panning—For each cell preparation, 10 conventionally raised mice were sacrificed at 8 weeks of age. Their stomachs were rapidly removed and placed in sterile 150 mm NaCl, pH 7.4. Ligatures were positioned at the junction between the esophagus and forestomach and at the pylorus. A 2-mm incision was made in the forestomach, and the entire stomach was everted through the incision. Another ligature was placed at the junction between the forestomach and corpus, and 500 μl of a solution of 1.0 mg/ml Streptomyces griseus Pronase E (7 units/mg, Roche Applied Science) in medium A (70 mm NaCl, 5 mm KCl, 50 mm HEPES, 1 mm Na2HPO4, 0.5 mm NaH2PO4, 20 mm NaHCO3, 10 mm EDTA, 11 mm glucose, 5 mg/ml bovine serum albumin, pH 7.8) was instilled with a 27-gauge needle. Filled gastric sacs were placed in a flask containing 75 ml of medium A and incubated for 30 min at 37 °C (16Andersson N. Rhedin M. Peteri-Brunback B. Andersson K. Cabero J.L. Biochim. Biophys. Acta. 1999; 1451: 297-304Crossref PubMed Scopus (12) Google Scholar, 17Prinz C. Kajimura M. Scott D.R. Mercier F. Helander H.F. Sachs G. Gastroenterology. 1993; 105: 449-461Abstract Full Text PDF PubMed Scopus (279) Google Scholar). The everted sac was then put into 75 ml of medium B (medium A with 100 mm CaCl2, 15 mm MgCl2 and no EDTA, pH 7.4) and incubated for 30 min at 37 °C with constant gentle magnetic stirring. The medium, containing shed epithelial cells, was decanted and passed though a nylon mesh (60-μm-pore diameter, Millipore). Cells in the flow-through were recovered by centrifugation (150 × g, 5 min, 23 °C) and re-suspended in 5 ml of medium C (140 mm NaCl, 12 mm MgSO4, 10 mm CaCl2, 15 mm HEPES, 11 mm glucose, 0.5 mm dithiothreitol, 10 mg/ml bovine serum albumin, pH 7.4). Cellular viability was defined by staining an aliquot with a solution containing 0.1% Neutral Red, 0.1% Brilliant Cresyl Blue, and 0.01% Janus Green in PBS. Cellular concentration was adjusted to 1.5 × 107/ml medium C, and 5 × 107 cells were subjected to counterflow elutriation (JE-6 elutriator rotor with standard chamber (Beckman Instruments), injection flow rate 10 ml/min medium C containing 1 mg/ml bovine serum albumin). Details of the elutriation protocol are provided in Supplemental Table 1. Fractions 1-5 (Supplemental Table 1) were each subjected to Dolichos biflorus agglutinin (DBA)-streptavidin-conjugated magnetic bead panning (11Mills J.C. Syder A.J. Hong C.V. Guruge J.L. Raaii F. Gordon J.I. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13687-13692Crossref PubMed Scopus (70) Google Scholar) to recover PCs (PCs are the only gastric epithelial lineage in FVB/N mice that express GalNAcα(1-3)Gal-containing glycans, see Ref.18Falk P. Roth K.A. Gordon J.I. Am. J. Physiol. 1994; 266: G987-G1003Crossref PubMed Google Scholar). Lectin-panned cells from fractions 2-5(“PC+ population”) were pooled, and their viability and purity were defined with the vital dye mixture described above (Supplemental Tables 1 and 2) and by transmission EM. Cells in fractions 4 and 5 that remained after DBA panning (Supplemental Table 2) were pooled to generate a “ZC+” fraction. The purified ZC+ population, the purified PC+ population, and the cells in fractions 1-2 remaining after DBA panning (ZC-/PC- population) were each collected by centrifugation (150 × g, 5 min, 23 °C), re-suspended in RLT lysis buffer (Qiagen), and RNA was extracted (RNeasy mini kit with on-column DNase treatment, Qiagen). GeneChip Analysis—Equal amounts of ZC+, PC+, or ZC-/PC- RNA from each preparation (n = 6 preparations) were pooled (5 μg/cell population/preparation). Equal-sized aliquots of each RNA pool (9 μg) were used to generate biotinylated cRNA targets (11Mills J.C. Syder A.J. Hong C.V. Guruge J.L. Raaii F. Gordon J.I. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13687-13692Crossref PubMed Scopus (70) Google Scholar, 19Lee C.K. Weindruch R. Prolla T.A. Nat. Genet. 2000; 25: 294-297Crossref PubMed Scopus (895) Google Scholar). Each cRNA target, in turn, was used to interrogate a set of Mu11K GeneChips (Affymetrix) according to the manufacturer's recommendations. Data from each chip were scaled to an average target intensity of 150. A series of pairwise comparisons were performed (Fig. 2A) using proprietary GeneChip software (version 4.0). Real-time Quantitative (q)RT-PCR—Selected findings from the GeneChip study were independently validated by qRT-PCR analysis of the same RNAs employed for generating cRNA targets. Each 25-μl qRT-PCR reaction mixture contained cDNA (20Stappenbeck T.S. Hooper L.V. Manchester J.K. Wong M.H. Gordon J.I. Methods Enzymol. 2002; 356: 168-196Google Scholar), 12.5 μl of 2× SYBR Green master mixture (Applied Biosystems), 900 nm of gene-specific primers (Supplemental Table 3), and 0.25 units of UDP-N-glycosidase (Invitrogen). A melting curve (15Hooper L.V. Mills J.C. Roth K.A. Stappenbeck T.S. Wong M.H. Gordon J.I. Sansonetti P. Methods in Microbiology. Vol. 31. Academic Press, London2002: 559-589Google Scholar, 20Stappenbeck T.S. Hooper L.V. Manchester J.K. Wong M.H. Gordon J.I. Methods Enzymol. 2002; 356: 168-196Google Scholar) was used to define a temperature where the amplicon and not primer-dimers was the source of SYBR Green-bound fluorescence. All assays were performed in triplicate with an ABI Prism 7700 Sequence Detector (Applied Biosystems). Data were normalized to glyceraldehyde-3-phosphate dehydrogenase mRNA (ΔΔCT method). Navigated Laser Capture Microdissection (n-LCM)—Stomachs from conventionally raised 8-week-old mice (n = 5) were divided in half along their lesser and greater curvatures and rinsed quickly in PBS. Each half was imbedded in O.C.T. compound (Sakura Finetek, Torrance, CA) and frozen in Cytocool II (Richard-Allen Scientific, Kalamazoo, MI). Serial 7 μm-thick cryosections were cut, placed on Superfrost/Plus slides (Fisher Scientific), and stained with methyl green/eosin Y (20Stappenbeck T.S. Hooper L.V. Manchester J.K. Wong M.H. Gordon J.I. Methods Enzymol. 2002; 356: 168-196Google Scholar). Well oriented gastric units (i.e. having an uninterrupted epithelial column that extended from the base to the apex of the pit), located in the corpus region of the stomach, were subjected to n-LCM (PixCell II apparatus, 7.5-μm spot diameter; CapSure HS LCM caps, Arcturus, Mountain View, CA) as described under “Results.” Epithelial cells were recovered from three different areas: the lowest ∼25% (base of the gastric unit consisting mainly of ZCs); the top ∼25% (pit, predominantly pit cells); and the mesenchyme immediately below the gastric unit (n = 300 gastric units microdissected/cryosection, 4-5 cryosections sections/mouse). RNA was isolated from ∼5000 cells/fraction/mouse using the PicoPure RNA isolation kit (Arcturus) with on-column DNase treatment. Equal amounts of RNA from each area were pooled from three mice and the material used for qRT-PCR studies. Transmission EM—Cells recovered by elutriation/lectin panning were subjected to transmission EM analysis using protocols described in a previous publication (11Mills J.C. Syder A.J. Hong C.V. Guruge J.L. Raaii F. Gordon J.I. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13687-13692Crossref PubMed Scopus (70) Google Scholar). Three-dimensional Imaging Studies of the Gastric Microvasculature—8-week-old germ-free normal and tox176 mice were anesthetized, and 200 μl of a 20 mg/ml aqueous solution of fluorescein isothiocyanate (FITC)-labeled high molecular mass (2000 kDa) dextran (Sigma) was injected in their retro-orbital plexus using a 30-gauge needle attached to a 1-ml syringe. Three minutes after the 10-15-s infusion, animals were sacrificed and their stomachs were removed and perfused with fixation solution (0.5% paraformaldehyde, 15% picric acid, 0.1 m sodium phosphate buffer, pH 7.0). Each stomach was opened with an incision along its greater curvature, pinned on wax, and shaken at 4 °C for 12 h in fixation solution. Following three washes in ice-cold PBS (5 min/cycle), the stomach was incubated for 3 h in 10% sucrose/PBS (4 °C) and then overnight in 20% sucrose, 10% glycerol/PBS (4 °C). After freezing the tissue in O.C.T., 60 μm-thick cryosections were cut along the cephalocaudal axis. Sections were air-dried (2 h at room temperature in the dark), re-hydrated in ice-cold PBS (1 min), incubated overnight (4 °C) in 3% deoxycholic acid (Sigma), rinsed with deionized water (2 cycles, 5 min each, room temperature) followed by a PBS wash (5 min, room temperature), and stained with either Syto61 (1:1000 dilution in PBS, 1 h at room temperature, Molecular Probes) or FITC-DBA. Following three more PBS washes (5 min/cycle, room temperature), sections were mounted in 50% glycerol/PBS, viewed under a LSM 510 confocal microscope (Zeiss), and scanned at 3-μm-thick intervals. Scans were projected in three dimensions by taking 20 serial images, aligning them at 7-10° intervals, and then compiling/rotating them around the y axis using LSM 510 software. Purification of Zymogenic Cells from Adult FVB/N Mouse Stomachs—ZCs were purified from the stomachs of conventionally raised 8-week-old mice belonging to the FVB/N inbred strain. Mucosa from the corpus region of the stomach was dissociated by Pronase E digestion, and the cellular suspension was size-fractionated by counterflow elutriation. Five fractions were recovered, and each fraction was subjected to lectin panning with DBA-magnetic bead conjugates to remove parietal cells. This approach yielded two fractions enriched for ZCs (fractions 4 and 5 in Supplemental Tables 1 and 2). Histochemical and immunohistochemical analyses using a previously described panel of lineage-specific lectins and antibodies (11Mills J.C. Syder A.J. Hong C.V. Guruge J.L. Raaii F. Gordon J.I. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13687-13692Crossref PubMed Scopus (70) Google Scholar, 12Mills J.C. Andersson N. Hong C.V. Stappenbeck T.S. Gordon J.I. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 14819-14824Crossref PubMed Scopus (90) Google Scholar, 18Falk P. Roth K.A. Gordon J.I. Am. J. Physiol. 1994; 266: G987-G1003Crossref PubMed Google Scholar, 21Syder A.J. Guruge J.L. Li Q. Hu Y. Oleksiewicz C.M. Lorenz R.G. Karam S.M. Falk P.G. Gordon J.I. Mol. Cell. 1999; 3: 263-274Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar) disclosed that ZCs represented 71-73% of the cells in these fractions compared with 11% in the starting material (Supplemental Table 2, n = 6 independent preparations). Overall cell viability in the two fractions, which together composed the ZC+ population, was >90% as defined by vital dye exclusion (Supplemental Table 1). Transmission EM studies confirmed that the purified ZCs had retained the characteristic morphologic features of mature members of this lineage (Fig. 2B). A PC+ population obtained by combining the cells from fractions 2-5 that bound DBA contained 84 ± 1% PCs versus 21 ± 2% in the starting material (Supplemental Table 2 and Fig. 2C) with an overall viability of 91 ± 3% (n = 6 preparations). A third population was obtained by pooling DBA non-reactive cells from the two elutriated fractions that contained the smallest average cell size (fractions 1-2 in Supplemental Tables 1 and 2). This ZC-/PC- fraction contained an average of 50% pit cells, 20% neck cells, <1% PCs, <2% ZCs, and 27% of other cell types (GEPs, enteroendocrine cells, and mesenchymal components) (Supplemental Table 2 and Fig. 2D). GeneChip-based Dataset of Transcripts Enriched in Zymogenic Cells—Total cellular RNA was extracted from the ZC+, PC+, and ZC-/PC- populations obtained from each of the six preparations. Equivalent amounts of RNA from a given population/preparation were pooled. Two cRNA targets were then independently prepared from each pooled RNA sample (ZC+, PC+, or ZC-/PC-), and each cRNA was used to interrogate Affymetrix Mu11K GeneChips containing probe sets that recognize ∼11,000 mouse genes and ESTs. Duplicate comparisons of ZC+ versus PC+ RNAs yielded a dataset of 187 transcripts (156 encoding characterized proteins, 31 encoding unknown or hypothetical proteins) enriched in the ZC+ population (Supplemental Table 4). Duplicate comparisons of ZC+ versus ZC-/PC- RNAs identified 124 ZC+-enriched transcripts (95 encoding characterized proteins, 29 specifying unknown/hypothetical proteins, Supplemental Table 5). The union of these two datasets (Fig. 2A) produced a list of 57 known genes and 14 uncharacterized genes from what amounted to quadruplicate comparisons (Supplemental Table 6). A reciprocal analysis of PC+ versus ZC+ and PC+ versus ZC-/PC- datasets identified 83 transcripts enriched in PCs (data not shown). This list shared 93% identity (77 of 83 mRNAs) with our previously published datasets of transcripts enriched in PCs harvested by direct lectin panning (i.e. without an elutriation step, see Ref. 11Mills J.C. Syder A.J. Hong C.V. Guruge J.L. Raaii F. Gordon J.I. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13687-13692Crossref PubMed Scopus (70) Google Scholar). The similarity between the current and previous PC+ datasets indicated that the elutriation protocol used in the present study did not artifactually skew the transcriptional profiles of sorted cells. Verification of the Dataset—12 genes were culled from the ZC+ dataset for initial real-time qRT-PCR confirmation of their enriched expression in ZCs. Assays were performed on the same RNAs used to generate cRNA targets for the GeneChip study. 7 of the 12 genes were known to be expressed in ZCs: Pepf (pepsinogen F, see Ref. 22Blandizzi C. Lazzeri G. Colucci R. Carignani D. Tognetti M. Baschiera F. Del Tacca M. Eur. J. Pharmacol. 1999; 373: 71-84Crossref PubMed Scopus (13) Google Scholar); Cckar (cholecystokinin receptor A, binds cholecystokinin and is involved in regulating pepsinogen secretion) (22Blandizzi C. Lazzeri G. Colucci R. Carignani D. Tognetti M. Baschiera F. Del Tacca M. Eur. J. Pharmacol. 1999; 373: 71-84Crossref PubMed Scopus (13) Google Scholar); Amy2 (α-amylase); Try2/4 (trypsin 2/4); Gif (gastric intrinsic factor, intestinal absorption of cobalamin); Anpep (alanyl aminopeptidase N, also known as CD13, a metalloproteinase found in ZC vesicles, see Ref. 23Gorvel J.P. Rigal A. Sarles J. Maroux S. Cell Tissue Res. 1985; 239: 241-248Crossref PubMed Scopus (30) Google Scholar); and Spp1 (secreted phosphoprotein 1 or osteopontin, an adhesive glycoprotein implicated in mucosal barrier function, Ref. 24Qu H. Brown L.F. Dvorak H.F. Dvorak A.M. J. Histochem. Cytochem. 1997; 45: 21-33Crossref PubMed Scopus (28) Google Scholar). 5 of the 12 genes had not been previously reported to be transcribed in ZCs: Pdgfb (platelet-derived growth factor B, see below for discussion of functions); Fes (feline sarcoma oncogene, a non-receptor protein tyrosine kinase that co-localizes with Rab proteins and participates in both endocytic and exocytic pathways, see Refs. 25Greer P. Nat. Rev. Mol. Cell. Biol. 2002; 3: 278-289Crossref PubMed Scopus (189) Google Scholar and 26Zirngibl R. Schulze D. Mirski S.E. Cole S.P. Greer P.A. Exp. Cell Res. 2001; 266: 87-94Crossref PubMed Scopus (39) Google Scholar); Nucb2 (nucleobindin 2, a calcium-binding Golgi-associated protein whose expression has been correlated with differentiation and acquisition of polarity, Refs. 27Kawano J. Nakayama T. Takami Y. Kotani T. Sawaguchi A. Nagaike R. Oinuma T. Suganuma T. Histochem. Cell Biol. 2001; 115: 421-428Crossref PubMed Scopus (6) Google Scholar and 28Weiss T.S. Chamberlain C.E. Takeda T. Lin P. Hahn K.M. Farquhar M.G. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 14961-14966Crossref PubMed Scopus (50) Google Scholar); and Jun (c-Jun) and Jund1 (dimerize with one another or with JunB or Fos family members to form the AP-1 transcription factor that has pleiotropic effects on intracellular signaling, Refs. 29Patel A.R. Wang J.Y. Am. J. Physiol. 1999; 276: G441-G450PubMed Google Scholar and 30Li L. Liu L. Rao J.N. Esmaili A. Strauch E.D. Bass B.L. Wang J.Y. Gastroenterology. 2002; 123: 764-779Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). The qRT-PCR study confirmed that the levels of these 12 mRNAs were 10 ± 1 to 421 ± 97-fold higher in ZC+ versus PC+ RNA (Table I). The fo" @default.
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