Regulation populations
of gene expression of fetal, neonatal,
KEVIN A. ROTH, STEVEN M. COHN, MARIAN R. NEUTRA, AND JEFFREY
in gastric epithelial cell and adult transgenic mice DEBORAH C. RUBIN, I. GORDON
JEFFREY
F. TRAHAIR,
Departments of Pathology, Medicine, and Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, Missouri 63110; Child Health Research Institute, North Adelaide, South Australia 5006, Australia; Department of Pediatrics, Harvard Medical School and Childrens Hospital, Boston, Massachusetts 02115 Roth, Kevin A., Steven M. Cohn, Deborah C. Rubin, Jeffrey F. Trahair, Marian R. Neutra, and Jeffrey I. Gordon. Regulation of gene expression in gastric epithelial cell populations of fetal, neonatal, and adult transgenic mice. Am. J. Physiol.
263 (Gastrointest.
Liver
Physiol.
26): Gl86-
G197, 1992.-Little is known about lineage relationships and differentiation programs of various epithelial cells present in mousegastric units. We have previously usedrat liver fatty acid binding protein/human growth hormone (L-FABP/hGH) transgenesto define epithelial cell lineagesrelationships in the smallintestine of fetal and adult mice and to examine regulation of their terminal differentiation programs along the crypt-tovillus and duodenal-to-ilealaxes.We have now usedthesetransgenesto explore similar issuesin the stomach. Immunocytochemical studiesof fetal and adult transgenic L-FABP/hGH animals and their normal littermates revealed that the intact endogenousmouse L-FABP gene (Fabpl) is not expressedin gastric epithelium. Nucleotides -596 to +21 of the rat L-FABP gene direct “inappropriate” expression of hGH in the gastric epithelium as early as fetal day 15. From 1 to 13 mo, LFABP-5g6 to +=/hGH expressionoccursonly in surfacemucous cells of zymogenic and mucousgastric units; the reporter is not detectable in the enteroendocrine,parietal and chief cell populations of zymogenic glands. Electron microscopic immunocytochemistry revealed that hGH is directed to apical secretory granulesin surface and pit mucouscells expressingthe transgene.hGH levels vary widely amongsurface mucouscells both within singlepits and betweengastric units in a given animal. The heterogeneity noted in reporter expression suggeststhat there are marked differences in the regulatory environments of individual cellsof a singletype within a given gastric unit. This raisesthe possibility that cell differentiation programs in the stomachmay not be as tightly coupledto cellular translocation as in the small intestine. Finally, the lack of expressionof LFABP-5g6 to +=/hGH in gastrin- and serotonin-immunoreactive cellsof the stomachcontrasts with its efficient expression in comparablecell types located in the duodenum,providing a model system for examining differential regulation of geneexpression in terminally differentiated cell types represented in both gastric and intestinal epithelium. gastric epithelial cell lineages;cellular differentiation programs; stem cells DIVERSE EPITHELIAL CELL populations of the mouse stomach undergo continuous renewal. However, relatively little is known about their lineage relationships and differentiation pathways. The adult gastric epithelium maintains precise regional differences in cell type (17). The cephalic or proximal third of the mouse stomach, the forestomach, is lined with a keratinized stratified squamous epithelium. The distal two-thirds of the stomach is lined with a glandular epithelium containing numerous gastric units. Each unit is composed of three domains: 1) an upper pit region lined with mu-
THE
G186
0193-1857/92
cus-producing surface epithelial cells; 2) a narrow isthmus region containing immature cells and a zone of mitotic activity; and 3) a lower gland that is classified as zymogenic, mucous, or mucoparietal depending on its cellular composition. Zymogenic glands are characterized by the presence of enzyme-secreting zymogenic (chief) cells at their base. Acid-producing parietal cells as well as enteroendocrine cells are distributed throughout the zymogenic glands, whereas mucous neck cells are largely confined to the upper portion of the gland, close to the isthmus. Pure mucous glands lack zymogenic and parietal cells and are composed primarily of mucous cells of a type distinct from surface mucous cells. Some mucous glands also have parietal cell populations and are classified as mucoparietal. Both pure mucous and mucoparietal glands contain enteroendocrine cells. The distribution of zymogenic, mucoparietal, and pure mucous glands varies along the cephalocaudal axis of the adult mouse stomach (17). Leblond and colleagues (13, 15) used [3H]thymidine labeling methods and ultrastructural studies to examine gastric epithelial cell renewal in the mucous and zymogenic units of adult CD1 mice. Their data suggest that granule-free stem cells located in the isthmus region give rise to daughter cells that undergo a bipolar migration. In the mucous units of the pylorus, daughter cells in the isthmus produce two types of exocrine cell precursors. One type contains homogeneous dense mucous granules. They undergo successive rounds of division, move upward into the pit region, and then migrate to the surface. The lifespan of these surface mucous cells is -3 days. The other type of daughter cell possesses apical granules with dense cores, migrates downward, and ultimately gives rise to relatively long-lived glandular mucous cells (15). Similarly, in the zymogenic units, the rapidly proliferating undifferentiated cells in the isthmus are thought to give rise to immature forms of several cell types that undergo a complex bipolar migration (13). Surface mucous cells migrate upward to the luminal surface, whereas parietal cells generally migrate downward to the neck and base of the glands. Mucous neck cells also migrate downward and are believed to undergo successive transformation into mucozymogenic and then long-lived zymogenic cells. Enteroendocrine cells of the gastric epithelium presumably arise from stem cells in the isthmus and join the downward migration into the glands. The crypt-to-villus axis of the small intestinal epithelium represents a carefully studied system of cellular proliferation, differentiation, and bipolar migration (3,
$2.00 Copyright 0 1992 the American Physiological
Society
Downloaded from www.physiology.org/journal/ajpgi by ${individualUser.givenNames} ${individualUser.surname} (129.081.226.078) on December 21, 2018.
TRANSGENES
AS PROBES OF GASTRIC
22, 24, 32, 40) that can be used as a reference for analyses of gastric epithelial cell differentiation. Each small intestinal crypt in the adult mouse appears to be renewed by a single active multipotent stem cell (38-40). Daughter cells undergo amplification within a clearly definable zone of the crypt and subsequently differentiate into the four principal cell types that populate the small bowel epithelium (3, 22). Differentiation takes place during a remarkably well organized bipolar migration: enterocytes and goblet and enteroendocrine cells arise as they are rapidly translocated in vertical coherent bands to the apical extrusion zone located at the villus tip while Paneth cells differentiate during a downward migration to the crypt base (3, 32). We have previously used transgenic mice to examine the molecular mechanisms responsible for establishing and maintaining spatial and cell lineage-specific differences in gene expression among descendants of this multipotent stem cell. To do so, we linked portions of the 5’ nontranscribed domain of the rat liver fatty acid binding protein (L-FABP) gene (Fabpl) to several reporters including the human growth hormone (hGH) gene (5, 7, 26-28, 30, 35). This strategy permitted &acting elements to be mapped that regulate 1) developmental stage-specific activation and suppression of Fabpt transcription, 2) geographic differences in Fabpl expression along the duodenal-to-colonic axis, and 3) Fabpl expression in individual cell types during their migration/ differentiation along the crypt-to-villus axis. L-FABP/ hGH transgenes were found to be very good sensors of subtle differences in the regulatory environments present within and between gut epithelial cell lineages derived from a given crypt’s multipotent stem cell. They also could be used to operationally define differences in the differentiation programs of enterocytes and enteroendocrine cells that arise in epithelial cell populations of adjacent crypts (5, 8, 26-28, 35). Finally, expression of hGH in differentiating and differentiated exocrine cells, endocrine cells, and enterocytes provided a way of assessing how proteins are sorted in the apical and basolateral secretory pathways of these diverse cell types (37) In an earlier study (35), we noted that several pedigrees of young adult transgenic mice with nucleotides -596 to +21 of the rat L-FABP gene contained hGH mRNA in their stomach, a site that normally does not support expression of FabpZ.1 Addition of nucleotides -597 to 4,000 in either orientation silenced reporter expression, suggesting the presence of &acting suppressor elements in these 3.3 kb. We have now used immunocytochemical methods to examine the cellular patterns of L-FABP/hGH transgene expression in the gastric epithelium from late fetal life through adulthood. These analyses have allowed us to further analyze regl The silence of Fabpl in late gestation mouse gastric epithelium contrasts with what is reported to occur in developing and young adult rats (9-12, 34). Immunoreactive rat L-FABP appears at El9 in some surface mucous cells. During the first postnatal week, it is found in all surface mucous cells, parietal cells, somatostatin-producing enteroendocrine cells, and caveolated cells (21) but not in mucous neck or chief cells (11). By postnatal week 6-7, Fabpl expression is confined to caveolated cells and somatostatin cells (11, 12).
EPITHELIAL
DIFFERENTIATION
G187
ulation of regional differences in gene expression along the stomach-to-colonic axis of the gut, to compare the differentiation programs of specific epithelial cell populations in the stomach with those in intestine, and to test the ability of gastric epithelial cells to sort a foreign polypeptide hormone into regulated secretory granules. MATERIALS
AND
METHODS
Animals. Mice containing nucleotides -4,000 to +21 of the rat L-FABP gene linked to the hGH gene (L-FABP-4*000 to
+21/hGH) were derived from founder (Go) 46 (35). Obligate heterozygotes for the L-FABP-5g6 to +21/hGH transgenewere derived from founders Go13 and Go19 (35). Fetal mice were delivered between days 15 and 19 of gestation by cesareansection and subsequentlykilled. Adult micewere maintained under a strictly controlled light cycle (lights on from 0600 to 1800 h) and were fed a standard chow diet ad libitum. They were killed at l- 13 mo of ageby cervical dislocation. Transgenic mice were distinguished from their normal littermates by Southern blot analysis of EcoR I digestedcarcassDNA (35). Light microscopic immunocytochemical analyses. Immediately after death, the stomachwasseparatedfrom the esophagusat a point severalmillimeters proximal to the gastroesophageal junction. The gastropyloric junction wasinitially left intact and the small bowel were severedwithin the proximal jejunum (defined accordingto Ref. 35). The stomachand adjacentproximal small intestine were fixed by immersion in Bouin’s solution. Five micrometer paraffin-embedded tissue sections were prepared from the stomach along its cephalocaudalaxis. Alternatively, lo-pm cryostat sectionswere prepared from tissuesthat were cryoprotected in a 10% sucrosesolution preparedin phosphatebuffered saline (PBS). All morphologically distinct gastric segments (forestomach, zymogenic, mucoparietal and mucous zones; cf. Refs. 17 and footnote 2) were examined. Diluted primary antisera were applied overnight at 4°C to deparaffinized or cryostat sections after nonspecific protein binding sites were blocked with PBS containing 2% bovine serum albumin, 0.2% nonfat powderedmilk, and 0.3% Triton X-100. Antibody binding sites were subsequentlydetected either by gold-labeledsecondary antibodies and silver enhancement (Amersham, Arlington Heights, IL) and/or with fluorophore-labeledsecondary antibodies (Jackson Immunoresearch Laboratories, West Grove, PA). We have found (27) that deposition of silver around specific antibody-antigen complexes during the immunogold silver staining (IGSS) procedure destroys their antigenicity without precluding further immunostaining of other antigenswith other antisera. Thus colocalization studieswere performedusingtwo antisera raisedin a single speciesby sequential use of IGSS and fluorescencedetection (26-28).
The sourcesof the antisera used for these studiesand their 2 The distribution of zymogenic, mucoparietal, and pure mucous glands in the adult mouse stomach has a well-defined topology that is described in Lee et al. (17). The most caudal (distal) region of the glandular stomach is composed of gastric units with pure mucous glands and represents ~30% of the glandular mucosa. Just proximal to this zone is a region comprised of mucoparietal glands (forming -20% of the glandular mucosa). The fractional representation of parietal cells in these mucoparietal glands varies according to their position along the cephalic-to-caudal (proximal-to-distal) axis of the stomach: those that are more distal contain fewer parietal cells while those that are more proximal contain greater numbers distributed over a greater area of the gland-isthmus-pit axis. The zymogenic region occupies -50% of the glandular epithelium. Its distal border is defined by the mucoparietal “zone” while its proximal boundary is defined by a narrow band of mucous glands (the cephalic band). This band is interposed between the forestomach and the zymogenic region.
Downloaded from www.physiology.org/journal/ajpgi by ${individualUser.givenNames} ${individualUser.surname} (129.081.226.078) on December 21, 2018.
Downloaded from www.physiology.org/journal/ajpgi by ${individualUser.givenNames} ${individualUser.surname} (129.081.226.078) on December 21, 2018.
TRANSGENES
AS PROBES
OF GASTRIC
final dilutions (for IGSS) were as follows: rabbit and goat antiserotonin (1:4,000and l:l,OOO,respectively; Incstar, Stillwater, MN), rat anti-serotonin (1:2,000;EugeneTech, Allendale, NJ), rabbit anti-gastrin (1:2,000, Dako, Santa Barbara, CA), rabbit anti-somatostatin (1:2,000, Incstar), rabbit anti-rat-L-FABP (l:l,OOO, Ref. 35), rabbit anti-rat intrinsic factor (1500, Ref 16; kindly supplied by D. Alpers, Washington Univ.); goat antihGH (1:2,000, Refs. 20 and 35); and rabbit anti-hGH (1:2,000, Dako). The immunostaining characteristics and specificities of theseserahave been describedby the manufacturers and in our previous reports (27). Immunocytochemical studies were performed on multiple tissue sectionsprepared from 10 fetal [embryological days (E)15-181 and 42 adult (1-13 mo) transgenic mice derived from G,l3, 6 adult (4-12 mo) transgenic mice derived from G,19; 5 fetal (El7 or 18) and 12 adult (l-12 mo) transgenic mice derived from G,46, plus 16 fetal (E&19) and 6 adult (1-12 mo) nontransgeniclittermates of various pedigree members. Electron microscopic (EIM) analyses. Tissues from young adult maleL-FABP- 5g6to +21/hGH transgenic micewere examined. Samplesof gastric glandular mucosawere fixed in a solution containing freshly depolymerized paraformaldehyde (final concn = 2%), glutaraldehyde (2%), and Na cacodylate (0.1 M, pH 7.4) for 18 h at 23°C. They were dehydrated in ethanol with polyvinylpyrrolidone and embeddedin LR Gold resin at 4°C usingbenzoin methyl ether and ultraviolet light polymerization, according to the supplier’s instructions (Polysciences, Warrington, PA). Ultrathin sectionswere collected on nickel grids that had previously been coated with formvar and carbon (37). For EM immunocytochemistry, grids were floated section sidedown on drops of the solutions describedbelow, with subsequentrinsing on several drops of TBST [tris(hydroxymethyl)aminomethane (10 mM, pH 7.2), NaCl(500 mM), and Tween 20 (0.3%)]. Free aldehydeswere first quenchedwith PBS containing 10 mM NH&l, and nonspecific protein-binding sites were blocked with 5% nonimmune goat serum (diluted in TBST). Grids werethen incubated on dropsof rabbit anti-hGH serum (Dako), diluted 1:lOO in TBST, followed by anti-rabbit antibodies conjugatedto 10 nm colloidal gold (diluted 1:lOO in TBST). For control grids, anti-hGH serum was replaced by nonimmune rabbit serum. Sections of small intestine from the sametransgenicmice, previously shownto expresshigh levelsof hGH in enterocytes (37) were immunostained using the same reagentsand served as positive controls. All sections were examined and photographed with a JEOL 100 CX electron microscope. RESULTS
Endogenous mouse Fabpl gene is not expressed in gastric epithelium in late fetal life or adulthood. Light micro-
scopic immunocytochemical
studies of the gastric fore-
EPITHELIAL
DIFFERENTIATION
G189
stomach and glandular epithelium of ES-19 fetuses and l- to 1%mo-old normal C57BL/6 X LT/SV mice failed to detect any L-FABP at any developmental stage surveyed (data not shown). Analysis of the adjacent proximal small intestine confirmed that activation of the mouse Fabpl gene occurs coincident with initial cytodifferentiation of the small intestinal epithelium at El5 or 16 (8, 28) and that L-FABP is continuously expressed in villus, but not crypt, enterocytes throughout the first year of life (5). The presence of multiple (X00) copies of either the LFABP-4~000 to +21/hGH or L-FABP-5g6 to +21/hGH transgenes did not affect endogenous Fabpl expression in either fetal or adult transgenic mice (data not shown). Expression of L-FABP/hGH transgenes in fetal mouse gastric epithelium. Immunocytochemical surveys
of fetal mice containing L-FABP-5g6 to +21/hGH or to +21 /hGH transgenes revealed that the hGH reporter was present in epithelial cells located in the poorly differentiated glandular epithelium at the earliest time points examined (El5 for Go13-derived and El 7 for G,46-derived animals; see Fig. 1, A and B). Levels of the reporter varied considerably among cells. This mosaic pattern of expression was noted with both transgenes (e.g., Fig. 1B). There is little information available about the cellular populations represented in this epithelium at El5 and therefore we could only operationally define them as being either capable or incapable of supporting transgene expression. By the 18th day of gestation, hGH was readily detectable in a subpopulation of columnar epithelial cells of the glandular epithelium (Fig. 1, C and D) but was absent from the squamous epithelial cell population of the forestomach (Fig. 1E). The diffuse or apical staining of columnar epithelial cells with the anti-hGH serum presumably represented accumulation of the reporter in apical secretory granules. This pattern contrasts with the intense, concentrated staining of the supranuclear Golgi apparatus in El8 duodenal villus enterocytes that lack apical granules (cf. C and D with F in Fig. 1). Double labeling with anti-hGH and anti-somatostatin sera revealed that although somatostatin-producing enteroendocrine cells are present in the El7 or 18 glandular epithelium (Fig. lG), they do not support transgene expression (Fig. 1, H and‘l). Surveys of El 7 or 18 normal and L-FABp-4,OOO to +21/hGH transgenic mice also indicated that gastrin- and serotonin-producing enteroendocrine cells in the glandular epithelium failed to express L-FABp-4,000
Fig. 1. Immunocytochemical survey of reporter expression in gastric epithelium of fetal-L-FABP/hGH transgenic mice. A, B: sections of stomach from embryological (E)15 mouse containing L-FABP-5g6 to +21/hGH were incubated with rabbit anti-hGH serum. Antigen-antibody complexes were detected with gold-labeled goat anti-rabbit IgG with subsequent silver staining (IGSS) and light counterstaining with hematoxylin. Scattered hGH-positive epithelial cells are seen. B: similar pattern of gastric epithelial hGH expression was observed in fetal mice containing L-FABP-4*000 to +21/hGH. C: glandular portion of stomach of El8 L-FABP- 4yoooto +21/hGH transgenic mouse, stained for hGH as described above. Surface cells are stained. D-F: geographic and cellular distribution of transgene expression in an El8 L-FABP-4y000 to +21/hGH mouse. Sections were incubated with rabbit anti-hGH serum followed by Texas Red-labeled donkey anti-rabbit serum. D: surface cells in glandular stomach exhibit diffuse cytoplasmic staining for hGH. E: illustrates sharp border between hGH-negative forestomach squamous epithelium and glandular epithelium which contains hGH-positive surface epithelial cells. F: in contrast, duodenal hGH immunoreactivity is Golgi associated. G-I are from a single tangential section of El 7 L-FABP-4v000 to +21/hGH mouse stomach. Section was coincubated with rabbit anti-somatostatin and goat anti-hGH sera, followed by Texas Red-labeled donkey anti-rabbit and fluorescein-labeled donkey anti-goat sera. G: several somatostatin immunoreactive cells (indicated by arrows) and nerve terminals are seen. H: numerous hGH immunoreactive cells are observed, but somatostatin immunoreactive cells are devoid of hGH immunoreactivity. This lack of colocalization is confirmed in dual exposed photomicrograph shown in I. Multilabeling immunocytochemical studies also indicated that hGH was not expressed in gastrin- or serotonin-immunoreactive enteroendocrine cells present in gastric units at this developmental stage (data not shown). Bar, 25 pm.
Downloaded from www.physiology.org/journal/ajpgi by ${individualUser.givenNames} ${individualUser.surname} (129.081.226.078) on December 21, 2018.
--%I 5.;.
.
*
Downloaded from www.physiology.org/journal/ajpgi by ${individualUser.givenNames} ${individualUser.surname} (129.081.226.078) on December 21, 2018.
TRANSGENES
AS PROBES
OF GASTRIC
hGH. L-FABP immunoreactivity was absent from all gastric enteroendocrine cells at all gestational ages examined (E15-19). Expression of L-FABP-4y000 to +21/hGH, but not L-FABP-5g6 to +21/hGH, is silenced in glandular epitheZium during postnatal life. By the fourth postnatal week, expression of the L-FABP-4soo0 to +21/hGH transgene was almost completely silenced in the glandular stomach. Only a few scattered hGH-positive surface mucous cells were present in the pits and adjacent surface epithelium (data not shown). There was no obvious spatial organization of cells that contained detectable levels of reporter, i.e., they were not limited to any particular position within the gastric pits nor was their distribution confined to any one region of the zymogenic or mucous zones. No hGH was present in the keratinized squamous epithelium of the forestomach. By the end of the second month and for the entire first year of life, expression of this transgene was completely suppressed in all cellular populations of the glandular stomach (n = 12 animals surveyed). This finding agrees with the results of our earlier blot hybridization studies of hGH mRNA accumulation along the stomach-to-colonic axis of adult L-FABP-4y000 to +21/ hGH mice (35). One- to 13-mo-old L-FABP-5g6 to +21/hGH transgenic mice, in contrast, produced large quantities of hGH in certain gastric epithelial cell populations. Figure 2A illustrates the sharp border between the hGH-negative forestomach epithelium and the glandular stomach in which scattered surface epithelial cells contain the hGH reporter? hGH-positive cells were limited to the gastric pits, a region containing surface mucous cells (Fig. 2B). Marked heterogeneity in steady-state hGH levels occurred in epithelial cells located at comparable positions along the isthmus-pit axis of a single gastric unit (Fig. 2, B and C). 3 Lack of expression of L-FABP-5g6 to +21/hGH in the keratinized squamous epithelium of the distal esophagus and forestomach of the mouse is in stark contrast to the developmental pattern of expression of the mouse adenosine deaminase (ADA) gene (4). ADA levels are high in the keratinized squamous epithelium of the tongue, esophagus, and forestomach; representing 5-20% of the total soluble proteins at 1 mo of age. The concentration of ADA in the glandular stomach is 30-fold lower. The recently cloned mouse ADA gene (1, 18, 25) opens the way for identifying &-acting elements that promote transcription in the squamous epithelium of the forestomach and restrict expression in the glandular epithelium.
EPITHELIAL
DIFFERENTIATION
G191
Moreover, hGH expression in surface mucous cells varied dramatically among adjacent gastric units. In some areas of the zymogenic zone, many adjacent gastric units contained uniformly high concentrations of the reporter (Fig. 20). In other areas, large patches of wholly negative units contained single units in which all surface mucous cells were positive (Fig. 2E). These varying patterns were frequently encountered within a given animal and were not related to age. In contrast to surface mucous cells, other differentiated cell types present in zymogenic glands did not support L-FABP-5g6 to +21/hGH t ransgene expression in adult mice. Parietal cells did not contain levels of hGH detectable by light microscopic immunocytochemistry using the sensitive IGSS method (Fig. 2C). The necks of gastric glands in which most mucous neck cells are located were hGH negative (Fig. 20), although a few cells containing hGH were observed adjacent to parietal cells in positions consistent with mucous neck cells (Fig. 2C). Intrinsic factor is produced by chief cells in the mouse and rat (16). This cobalamin-binding protein thus provides an immunocytochemical marker for chief cells. The location of chief cells at the base of zymogenic glands contrasted sharply with the distribution of hGH positive surface mucous cells (Fig. 20). Such double-labeled preparations established that chief cells did not express L-FABP-5g6 to +21/hGH at any postnatal age examined (data not shown). Within the mucous zone of adult L-FABP-5g6 to +21/ hGH transgenic mice, transgene expression was confined to surface mucous cells (Fig. 2, E-G). As in the zymogenic zone, remarkable variability in hGH expression occurred within and between glands. Figure 2, E and F, shows isolated gastric units that are hGH positive, surrounded by hGH-negative gastric units. Other gastric units in the mucous zone contained heterogeneous levels of the reporter in their surface epithelial cell populations (Fig. 2G). This heterogeneity was not an insertion site effect, because it was noted in animals from two independent L-FABP-5g6 to +21/hGH pedigrees. Figure 2, H and I, illustrates transgene expression at the junction of the gastric glandular epithelium and the proximal duodenum. In this region where gastric and intestinal cell populations are sharply demarcated, the apical hGH accumulation in gastric surface mucous cells
Fig. 2. hGH expression in gastric epithelium of adult L-FABP- 5g6 to +21/hGH transgenic mice. A: section from squamous-glandular stomach transition zone of a 2mo-old transgenic (GJ3) mouse stained for hGH with IGSS method shows that expression of hGH is patchy and confined to glandular stomach. B, C: longitudinal (B) and cross (C) sections through zymogenic zones of 4- and ll-mo-old (respectively) transgenic (GJ3) mice show numerous hGH immunoreactive surface mucous cells near luminal surface. In these lightly hematoxylin- and eosin-counterstained sections, numerous plump pink parietal cells are visible but do not contain detectable levels of reporter. D: section from zymogenic zone of an ll-mo-old (G,l3) transgenic mouse, coincubated with rabbit anti-rat intrinsic factor and goat anti-hGH sera, and subsequently with Texas Red-labeled donkey anti-rabbit and fluorescein-labeled donkey anti-goat sera. Intrinsic factor (red) serves as a marker for chief cells at base of zymogenic glands. hGH immunoreactive cells (green) are confined to pit and surface epithelium. Parietal and mucous neck cells, which are not labeled by either antisera, are seen as a dark band between chief and surface epithelial cells. E-G: heterogeneous patterns of transgene expression in surface mucous cells. E: longitudinal section from pure mucous zone of a 7-mo-old (G,19) transgenic mouse. F: cross section from same zone in a 4mo-old (GJ3) transgenic mouse. G: pure mucous zone of a 2-mo-old (GJ3) transgenic mouse, showing a mixture of mucous units, some with wholly hGH-positive columns of surface epithelial cells. Other adjacent units exhibit a seemingly chaotic pattern of staining of surface epithelial cells during their migration up pit. These cellular patterns of transgene expression did not appear to correlate with transgenic pedigree (E, F) or age (D, G). H, I: section from gastroduodenal junction of a l-mo-old (G,l3) transgenic mouse incubated with rabbit anti-hGH serum and labeled with IGSS. H: darkfield microscopy of silver grain reveals diffuse cytoplasmic staining in gastric epithelium (left) and intense supranuclear Golgi associated staining in villus-associated duodenal enterocytes (right). I: a high magnification bright field photomicrograph of “junctional” villus illustrates 2 distinct patterns of intracellular hGH accumulation. Bar, 25 pm.
Downloaded from www.physiology.org/journal/ajpgi by ${individualUser.givenNames} ${individualUser.surname} (129.081.226.078) on December 21, 2018.
G192
TRANSGENES
AS PROBES OF GASTRIC
contrasted with the accumulation of hGH in the supranuclear (Golgi) region in enterocytes. hGH is concentrated in exocrine secretory granules of gastric mucous cells. Labeling of hGH in thin sections of adult zymogenic mucosa by EM immunogold cytochemistry confirmed the heterogeneous expression of the LFABP-5g6 to +21/hGH transgene in surface mucous cells. In these preparations, surface mucous cells can be identified by the presence of apical secretory granules with uniform dense content. In most randomly selected mucosal tissue blocks, no hGH was detectable. In some tissue samples, however, surface mucous cells showed hGH-specific immunoreactivity distributed throughout the matrix of each granule (Fig. 3A). hGH was also noted in a few cells with apical secretory granules containing dense cores, a morphological feature typical of mucous neck cells. hGH in these cells was concentrated in the dense cores of the apical granules (Fig. 3B). This finding suggests that the exocrine mucous cells that express hGH direct the hormone to their regulated secretory pathway. L-FABPjhGH transgenes remain silent in entero-endocrine cell populations of adult mouse glandular epithelium. Previous studies indicated that the intact endogenous
EPITHELIAL
DIFFERENTIATION
mouse Fabpl gene is only rarely expressed at detectable levels within adult duodenal enteroendocrine cell populations (27). Similarly, a survey of l- to 13-mo-old normal and transgenic mice failed to detect any immunoreactive L-FABP in gastric endocrine cells (data not shown). Double label immunocytochemical studies of duodenal and proximal jejunal sections prepared from adult L-FABP-5g6 to +21/hGH mice indicated that hGH is present in >80% of gastrin-immunoreactive cells and >30% of serotonergic cells (27, 28, 30). (Somatostatin immunoreactive cells are only rarely found in the proximal portion of the small bowel and therefore were not included in the double label survey.) In sharp contrast, multilabel studies of gastrin-, somatostatin-, and serotonin-producing enteroendocrine populations present in the gastric glandular epithelium of these same adult mice failed to detect any transgene expression in the mucous zone (Fig. 4) or in the zymogenic zone (data not shown). The highly sensitive EM immunogold labeling method also failed to detect hGH in any gastric enteroendocrine cell (data not shown) under labeling conditions that readily detected hGH in intestinal enteroendocrine cell granules (37).
Fig. 3. Immunolocalization of hGH in gastric mucous cells. Mucous cells in gastric epithelial surface, pits, and isthmus that expressed L-FABP- 5a6 ti +*r/hGH transgene were identified by anti-hGH immunogold labeling. Although morphological preservation was not optimal in these LR Gold-embedded tissues, apical mucous granules are clearly visualized; position of their limiting membranes is indicated by arrows. hGH is concentrated in apical secretory granules of all positive cells. A: hGH is distributed throughout homogeneous matrix of apical secretory granules in a surface mucous cell. B: in an isthmus mucous cell, apical secretory granules contain dense cores in an electron-lucent matrix. hGH is concentrated in these cores. Bar, 0.5 pm.
Downloaded from www.physiology.org/journal/ajpgi by ${individualUser.givenNames} ${individualUser.surname} (129.081.226.078) on December 21, 2018.
TRANSGENES
AS PROBES
OF GASTRIC
DISCUSSION
Our analysis of the expression of L-FABP/hGH transgenes in the gastric epithelium of fetal and I- to 13-moold transgenic mice has indicated that 1) differences in specific gastric epithelial cell types present during fetal and postnatal development can be operationally defined by their ability to support expression of L-FABP/hGH transgenes; 2) L-FABP+g6 to +21/hGH expression is largely confined to the surface mucous cells of zymogenic and mucous gastric units, but there is an unexpected degree of diversity in this cellular population both within and between gastric units; 3) mucous cells that express hGH direct this foreign protein into regulated apical secretory granules; and 4) the inability of this transgene to be expressed in gastric serotonergic and gastrin enteroendocrine subpopulations contrasts with its efficient expression in these cell types in the proximal small intestine. Temporal gradients and regional differences in gene expression talong stomach- to-colonic axis. Regional differ-
ences can be defined among the various renewing epithelial cell populations of the gastrointestinal tract based on temporal changes in transgene expression. We had previously examined L-FABP-5g6 to +21/hGH expression in the small and large bowel of fetal and 1- to 13-mo-old transgenic mice. Nucleotides -596 to +21 of the rat Fabpl are sufficient to recapitulate the appropriate temporal, cellular, and regional pattern of Fabpl activation in the fetal mouse intestinal epithelium with the exception that rare enteroendocrine cells (typically producing gastric inhibitory peptide) support transgene, but not Fabpl, expression (8, 28, 30). This contrasts with the situation in adult mice where “inappropriate” expression of L-FABP/ hGH (compared with Fabpl) occurs in many intestinal enteroendocrine cell subpopulations, in small intestinal crypt epithelial cells, and in the colonic epithelium from the cecum to the rectum (5, 27,35). With increasing age, a “wave” of extinction of transgene expression takes place in the colon but not in the small intestine (5). This silencing of transgene expression begins in the distal colon, proceeds to successively more proximal portions of the colon, and involves contiguous multicrypt patches with sharply demarcated boundaries. A comparable phenomenon occurs in mice containing the L-FABP-4y000 to +21/ hGH transgene, except that the “wave” of extinction moves more rapidly and involves individual crypts (5). In the present study, we demonstrated that &-acting suppressor element(s) located between nucleotides -4,000 to +21 also silenced L-FABP/hGH expression in the mouse stomach between late gestation and the first month of postnatal life. While the rate of extinction of transgene expression in the gastric epithelium was more rapid than in the colonic epithelium (complete in
of gene expression of fetal, neonatal,
KEVIN A. ROTH, STEVEN M. COHN, MARIAN R. NEUTRA, AND JEFFREY
in gastric epithelial cell and adult transgenic mice DEBORAH C. RUBIN, I. GORDON
JEFFREY
F. TRAHAIR,
Departments of Pathology, Medicine, and Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, Missouri 63110; Child Health Research Institute, North Adelaide, South Australia 5006, Australia; Department of Pediatrics, Harvard Medical School and Childrens Hospital, Boston, Massachusetts 02115 Roth, Kevin A., Steven M. Cohn, Deborah C. Rubin, Jeffrey F. Trahair, Marian R. Neutra, and Jeffrey I. Gordon. Regulation of gene expression in gastric epithelial cell populations of fetal, neonatal, and adult transgenic mice. Am. J. Physiol.
263 (Gastrointest.
Liver
Physiol.
26): Gl86-
G197, 1992.-Little is known about lineage relationships and differentiation programs of various epithelial cells present in mousegastric units. We have previously usedrat liver fatty acid binding protein/human growth hormone (L-FABP/hGH) transgenesto define epithelial cell lineagesrelationships in the smallintestine of fetal and adult mice and to examine regulation of their terminal differentiation programs along the crypt-tovillus and duodenal-to-ilealaxes.We have now usedthesetransgenesto explore similar issuesin the stomach. Immunocytochemical studiesof fetal and adult transgenic L-FABP/hGH animals and their normal littermates revealed that the intact endogenousmouse L-FABP gene (Fabpl) is not expressedin gastric epithelium. Nucleotides -596 to +21 of the rat L-FABP gene direct “inappropriate” expression of hGH in the gastric epithelium as early as fetal day 15. From 1 to 13 mo, LFABP-5g6 to +=/hGH expressionoccursonly in surfacemucous cells of zymogenic and mucousgastric units; the reporter is not detectable in the enteroendocrine,parietal and chief cell populations of zymogenic glands. Electron microscopic immunocytochemistry revealed that hGH is directed to apical secretory granulesin surface and pit mucouscells expressingthe transgene.hGH levels vary widely amongsurface mucouscells both within singlepits and betweengastric units in a given animal. The heterogeneity noted in reporter expression suggeststhat there are marked differences in the regulatory environments of individual cellsof a singletype within a given gastric unit. This raisesthe possibility that cell differentiation programs in the stomachmay not be as tightly coupledto cellular translocation as in the small intestine. Finally, the lack of expressionof LFABP-5g6 to +=/hGH in gastrin- and serotonin-immunoreactive cellsof the stomachcontrasts with its efficient expression in comparablecell types located in the duodenum,providing a model system for examining differential regulation of geneexpression in terminally differentiated cell types represented in both gastric and intestinal epithelium. gastric epithelial cell lineages;cellular differentiation programs; stem cells DIVERSE EPITHELIAL CELL populations of the mouse stomach undergo continuous renewal. However, relatively little is known about their lineage relationships and differentiation pathways. The adult gastric epithelium maintains precise regional differences in cell type (17). The cephalic or proximal third of the mouse stomach, the forestomach, is lined with a keratinized stratified squamous epithelium. The distal two-thirds of the stomach is lined with a glandular epithelium containing numerous gastric units. Each unit is composed of three domains: 1) an upper pit region lined with mu-
THE
G186
0193-1857/92
cus-producing surface epithelial cells; 2) a narrow isthmus region containing immature cells and a zone of mitotic activity; and 3) a lower gland that is classified as zymogenic, mucous, or mucoparietal depending on its cellular composition. Zymogenic glands are characterized by the presence of enzyme-secreting zymogenic (chief) cells at their base. Acid-producing parietal cells as well as enteroendocrine cells are distributed throughout the zymogenic glands, whereas mucous neck cells are largely confined to the upper portion of the gland, close to the isthmus. Pure mucous glands lack zymogenic and parietal cells and are composed primarily of mucous cells of a type distinct from surface mucous cells. Some mucous glands also have parietal cell populations and are classified as mucoparietal. Both pure mucous and mucoparietal glands contain enteroendocrine cells. The distribution of zymogenic, mucoparietal, and pure mucous glands varies along the cephalocaudal axis of the adult mouse stomach (17). Leblond and colleagues (13, 15) used [3H]thymidine labeling methods and ultrastructural studies to examine gastric epithelial cell renewal in the mucous and zymogenic units of adult CD1 mice. Their data suggest that granule-free stem cells located in the isthmus region give rise to daughter cells that undergo a bipolar migration. In the mucous units of the pylorus, daughter cells in the isthmus produce two types of exocrine cell precursors. One type contains homogeneous dense mucous granules. They undergo successive rounds of division, move upward into the pit region, and then migrate to the surface. The lifespan of these surface mucous cells is -3 days. The other type of daughter cell possesses apical granules with dense cores, migrates downward, and ultimately gives rise to relatively long-lived glandular mucous cells (15). Similarly, in the zymogenic units, the rapidly proliferating undifferentiated cells in the isthmus are thought to give rise to immature forms of several cell types that undergo a complex bipolar migration (13). Surface mucous cells migrate upward to the luminal surface, whereas parietal cells generally migrate downward to the neck and base of the glands. Mucous neck cells also migrate downward and are believed to undergo successive transformation into mucozymogenic and then long-lived zymogenic cells. Enteroendocrine cells of the gastric epithelium presumably arise from stem cells in the isthmus and join the downward migration into the glands. The crypt-to-villus axis of the small intestinal epithelium represents a carefully studied system of cellular proliferation, differentiation, and bipolar migration (3,
$2.00 Copyright 0 1992 the American Physiological
Society
Downloaded from www.physiology.org/journal/ajpgi by ${individualUser.givenNames} ${individualUser.surname} (129.081.226.078) on December 21, 2018.
TRANSGENES
AS PROBES OF GASTRIC
22, 24, 32, 40) that can be used as a reference for analyses of gastric epithelial cell differentiation. Each small intestinal crypt in the adult mouse appears to be renewed by a single active multipotent stem cell (38-40). Daughter cells undergo amplification within a clearly definable zone of the crypt and subsequently differentiate into the four principal cell types that populate the small bowel epithelium (3, 22). Differentiation takes place during a remarkably well organized bipolar migration: enterocytes and goblet and enteroendocrine cells arise as they are rapidly translocated in vertical coherent bands to the apical extrusion zone located at the villus tip while Paneth cells differentiate during a downward migration to the crypt base (3, 32). We have previously used transgenic mice to examine the molecular mechanisms responsible for establishing and maintaining spatial and cell lineage-specific differences in gene expression among descendants of this multipotent stem cell. To do so, we linked portions of the 5’ nontranscribed domain of the rat liver fatty acid binding protein (L-FABP) gene (Fabpl) to several reporters including the human growth hormone (hGH) gene (5, 7, 26-28, 30, 35). This strategy permitted &acting elements to be mapped that regulate 1) developmental stage-specific activation and suppression of Fabpt transcription, 2) geographic differences in Fabpl expression along the duodenal-to-colonic axis, and 3) Fabpl expression in individual cell types during their migration/ differentiation along the crypt-to-villus axis. L-FABP/ hGH transgenes were found to be very good sensors of subtle differences in the regulatory environments present within and between gut epithelial cell lineages derived from a given crypt’s multipotent stem cell. They also could be used to operationally define differences in the differentiation programs of enterocytes and enteroendocrine cells that arise in epithelial cell populations of adjacent crypts (5, 8, 26-28, 35). Finally, expression of hGH in differentiating and differentiated exocrine cells, endocrine cells, and enterocytes provided a way of assessing how proteins are sorted in the apical and basolateral secretory pathways of these diverse cell types (37) In an earlier study (35), we noted that several pedigrees of young adult transgenic mice with nucleotides -596 to +21 of the rat L-FABP gene contained hGH mRNA in their stomach, a site that normally does not support expression of FabpZ.1 Addition of nucleotides -597 to 4,000 in either orientation silenced reporter expression, suggesting the presence of &acting suppressor elements in these 3.3 kb. We have now used immunocytochemical methods to examine the cellular patterns of L-FABP/hGH transgene expression in the gastric epithelium from late fetal life through adulthood. These analyses have allowed us to further analyze regl The silence of Fabpl in late gestation mouse gastric epithelium contrasts with what is reported to occur in developing and young adult rats (9-12, 34). Immunoreactive rat L-FABP appears at El9 in some surface mucous cells. During the first postnatal week, it is found in all surface mucous cells, parietal cells, somatostatin-producing enteroendocrine cells, and caveolated cells (21) but not in mucous neck or chief cells (11). By postnatal week 6-7, Fabpl expression is confined to caveolated cells and somatostatin cells (11, 12).
EPITHELIAL
DIFFERENTIATION
G187
ulation of regional differences in gene expression along the stomach-to-colonic axis of the gut, to compare the differentiation programs of specific epithelial cell populations in the stomach with those in intestine, and to test the ability of gastric epithelial cells to sort a foreign polypeptide hormone into regulated secretory granules. MATERIALS
AND
METHODS
Animals. Mice containing nucleotides -4,000 to +21 of the rat L-FABP gene linked to the hGH gene (L-FABP-4*000 to
+21/hGH) were derived from founder (Go) 46 (35). Obligate heterozygotes for the L-FABP-5g6 to +21/hGH transgenewere derived from founders Go13 and Go19 (35). Fetal mice were delivered between days 15 and 19 of gestation by cesareansection and subsequentlykilled. Adult micewere maintained under a strictly controlled light cycle (lights on from 0600 to 1800 h) and were fed a standard chow diet ad libitum. They were killed at l- 13 mo of ageby cervical dislocation. Transgenic mice were distinguished from their normal littermates by Southern blot analysis of EcoR I digestedcarcassDNA (35). Light microscopic immunocytochemical analyses. Immediately after death, the stomachwasseparatedfrom the esophagusat a point severalmillimeters proximal to the gastroesophageal junction. The gastropyloric junction wasinitially left intact and the small bowel were severedwithin the proximal jejunum (defined accordingto Ref. 35). The stomachand adjacentproximal small intestine were fixed by immersion in Bouin’s solution. Five micrometer paraffin-embedded tissue sections were prepared from the stomach along its cephalocaudalaxis. Alternatively, lo-pm cryostat sectionswere prepared from tissuesthat were cryoprotected in a 10% sucrosesolution preparedin phosphatebuffered saline (PBS). All morphologically distinct gastric segments (forestomach, zymogenic, mucoparietal and mucous zones; cf. Refs. 17 and footnote 2) were examined. Diluted primary antisera were applied overnight at 4°C to deparaffinized or cryostat sections after nonspecific protein binding sites were blocked with PBS containing 2% bovine serum albumin, 0.2% nonfat powderedmilk, and 0.3% Triton X-100. Antibody binding sites were subsequentlydetected either by gold-labeledsecondary antibodies and silver enhancement (Amersham, Arlington Heights, IL) and/or with fluorophore-labeledsecondary antibodies (Jackson Immunoresearch Laboratories, West Grove, PA). We have found (27) that deposition of silver around specific antibody-antigen complexes during the immunogold silver staining (IGSS) procedure destroys their antigenicity without precluding further immunostaining of other antigenswith other antisera. Thus colocalization studieswere performedusingtwo antisera raisedin a single speciesby sequential use of IGSS and fluorescencedetection (26-28).
The sourcesof the antisera used for these studiesand their 2 The distribution of zymogenic, mucoparietal, and pure mucous glands in the adult mouse stomach has a well-defined topology that is described in Lee et al. (17). The most caudal (distal) region of the glandular stomach is composed of gastric units with pure mucous glands and represents ~30% of the glandular mucosa. Just proximal to this zone is a region comprised of mucoparietal glands (forming -20% of the glandular mucosa). The fractional representation of parietal cells in these mucoparietal glands varies according to their position along the cephalic-to-caudal (proximal-to-distal) axis of the stomach: those that are more distal contain fewer parietal cells while those that are more proximal contain greater numbers distributed over a greater area of the gland-isthmus-pit axis. The zymogenic region occupies -50% of the glandular epithelium. Its distal border is defined by the mucoparietal “zone” while its proximal boundary is defined by a narrow band of mucous glands (the cephalic band). This band is interposed between the forestomach and the zymogenic region.
Downloaded from www.physiology.org/journal/ajpgi by ${individualUser.givenNames} ${individualUser.surname} (129.081.226.078) on December 21, 2018.
Downloaded from www.physiology.org/journal/ajpgi by ${individualUser.givenNames} ${individualUser.surname} (129.081.226.078) on December 21, 2018.
TRANSGENES
AS PROBES
OF GASTRIC
final dilutions (for IGSS) were as follows: rabbit and goat antiserotonin (1:4,000and l:l,OOO,respectively; Incstar, Stillwater, MN), rat anti-serotonin (1:2,000;EugeneTech, Allendale, NJ), rabbit anti-gastrin (1:2,000, Dako, Santa Barbara, CA), rabbit anti-somatostatin (1:2,000, Incstar), rabbit anti-rat-L-FABP (l:l,OOO, Ref. 35), rabbit anti-rat intrinsic factor (1500, Ref 16; kindly supplied by D. Alpers, Washington Univ.); goat antihGH (1:2,000, Refs. 20 and 35); and rabbit anti-hGH (1:2,000, Dako). The immunostaining characteristics and specificities of theseserahave been describedby the manufacturers and in our previous reports (27). Immunocytochemical studies were performed on multiple tissue sectionsprepared from 10 fetal [embryological days (E)15-181 and 42 adult (1-13 mo) transgenic mice derived from G,l3, 6 adult (4-12 mo) transgenic mice derived from G,19; 5 fetal (El7 or 18) and 12 adult (l-12 mo) transgenic mice derived from G,46, plus 16 fetal (E&19) and 6 adult (1-12 mo) nontransgeniclittermates of various pedigree members. Electron microscopic (EIM) analyses. Tissues from young adult maleL-FABP- 5g6to +21/hGH transgenic micewere examined. Samplesof gastric glandular mucosawere fixed in a solution containing freshly depolymerized paraformaldehyde (final concn = 2%), glutaraldehyde (2%), and Na cacodylate (0.1 M, pH 7.4) for 18 h at 23°C. They were dehydrated in ethanol with polyvinylpyrrolidone and embeddedin LR Gold resin at 4°C usingbenzoin methyl ether and ultraviolet light polymerization, according to the supplier’s instructions (Polysciences, Warrington, PA). Ultrathin sectionswere collected on nickel grids that had previously been coated with formvar and carbon (37). For EM immunocytochemistry, grids were floated section sidedown on drops of the solutions describedbelow, with subsequentrinsing on several drops of TBST [tris(hydroxymethyl)aminomethane (10 mM, pH 7.2), NaCl(500 mM), and Tween 20 (0.3%)]. Free aldehydeswere first quenchedwith PBS containing 10 mM NH&l, and nonspecific protein-binding sites were blocked with 5% nonimmune goat serum (diluted in TBST). Grids werethen incubated on dropsof rabbit anti-hGH serum (Dako), diluted 1:lOO in TBST, followed by anti-rabbit antibodies conjugatedto 10 nm colloidal gold (diluted 1:lOO in TBST). For control grids, anti-hGH serum was replaced by nonimmune rabbit serum. Sections of small intestine from the sametransgenicmice, previously shownto expresshigh levelsof hGH in enterocytes (37) were immunostained using the same reagentsand served as positive controls. All sections were examined and photographed with a JEOL 100 CX electron microscope. RESULTS
Endogenous mouse Fabpl gene is not expressed in gastric epithelium in late fetal life or adulthood. Light micro-
scopic immunocytochemical
studies of the gastric fore-
EPITHELIAL
DIFFERENTIATION
G189
stomach and glandular epithelium of ES-19 fetuses and l- to 1%mo-old normal C57BL/6 X LT/SV mice failed to detect any L-FABP at any developmental stage surveyed (data not shown). Analysis of the adjacent proximal small intestine confirmed that activation of the mouse Fabpl gene occurs coincident with initial cytodifferentiation of the small intestinal epithelium at El5 or 16 (8, 28) and that L-FABP is continuously expressed in villus, but not crypt, enterocytes throughout the first year of life (5). The presence of multiple (X00) copies of either the LFABP-4~000 to +21/hGH or L-FABP-5g6 to +21/hGH transgenes did not affect endogenous Fabpl expression in either fetal or adult transgenic mice (data not shown). Expression of L-FABP/hGH transgenes in fetal mouse gastric epithelium. Immunocytochemical surveys
of fetal mice containing L-FABP-5g6 to +21/hGH or to +21 /hGH transgenes revealed that the hGH reporter was present in epithelial cells located in the poorly differentiated glandular epithelium at the earliest time points examined (El5 for Go13-derived and El 7 for G,46-derived animals; see Fig. 1, A and B). Levels of the reporter varied considerably among cells. This mosaic pattern of expression was noted with both transgenes (e.g., Fig. 1B). There is little information available about the cellular populations represented in this epithelium at El5 and therefore we could only operationally define them as being either capable or incapable of supporting transgene expression. By the 18th day of gestation, hGH was readily detectable in a subpopulation of columnar epithelial cells of the glandular epithelium (Fig. 1, C and D) but was absent from the squamous epithelial cell population of the forestomach (Fig. 1E). The diffuse or apical staining of columnar epithelial cells with the anti-hGH serum presumably represented accumulation of the reporter in apical secretory granules. This pattern contrasts with the intense, concentrated staining of the supranuclear Golgi apparatus in El8 duodenal villus enterocytes that lack apical granules (cf. C and D with F in Fig. 1). Double labeling with anti-hGH and anti-somatostatin sera revealed that although somatostatin-producing enteroendocrine cells are present in the El7 or 18 glandular epithelium (Fig. lG), they do not support transgene expression (Fig. 1, H and‘l). Surveys of El 7 or 18 normal and L-FABp-4,OOO to +21/hGH transgenic mice also indicated that gastrin- and serotonin-producing enteroendocrine cells in the glandular epithelium failed to express L-FABp-4,000
Fig. 1. Immunocytochemical survey of reporter expression in gastric epithelium of fetal-L-FABP/hGH transgenic mice. A, B: sections of stomach from embryological (E)15 mouse containing L-FABP-5g6 to +21/hGH were incubated with rabbit anti-hGH serum. Antigen-antibody complexes were detected with gold-labeled goat anti-rabbit IgG with subsequent silver staining (IGSS) and light counterstaining with hematoxylin. Scattered hGH-positive epithelial cells are seen. B: similar pattern of gastric epithelial hGH expression was observed in fetal mice containing L-FABP-4*000 to +21/hGH. C: glandular portion of stomach of El8 L-FABP- 4yoooto +21/hGH transgenic mouse, stained for hGH as described above. Surface cells are stained. D-F: geographic and cellular distribution of transgene expression in an El8 L-FABP-4y000 to +21/hGH mouse. Sections were incubated with rabbit anti-hGH serum followed by Texas Red-labeled donkey anti-rabbit serum. D: surface cells in glandular stomach exhibit diffuse cytoplasmic staining for hGH. E: illustrates sharp border between hGH-negative forestomach squamous epithelium and glandular epithelium which contains hGH-positive surface epithelial cells. F: in contrast, duodenal hGH immunoreactivity is Golgi associated. G-I are from a single tangential section of El 7 L-FABP-4v000 to +21/hGH mouse stomach. Section was coincubated with rabbit anti-somatostatin and goat anti-hGH sera, followed by Texas Red-labeled donkey anti-rabbit and fluorescein-labeled donkey anti-goat sera. G: several somatostatin immunoreactive cells (indicated by arrows) and nerve terminals are seen. H: numerous hGH immunoreactive cells are observed, but somatostatin immunoreactive cells are devoid of hGH immunoreactivity. This lack of colocalization is confirmed in dual exposed photomicrograph shown in I. Multilabeling immunocytochemical studies also indicated that hGH was not expressed in gastrin- or serotonin-immunoreactive enteroendocrine cells present in gastric units at this developmental stage (data not shown). Bar, 25 pm.
Downloaded from www.physiology.org/journal/ajpgi by ${individualUser.givenNames} ${individualUser.surname} (129.081.226.078) on December 21, 2018.
--%I 5.;.
.
*
Downloaded from www.physiology.org/journal/ajpgi by ${individualUser.givenNames} ${individualUser.surname} (129.081.226.078) on December 21, 2018.
TRANSGENES
AS PROBES
OF GASTRIC
hGH. L-FABP immunoreactivity was absent from all gastric enteroendocrine cells at all gestational ages examined (E15-19). Expression of L-FABP-4y000 to +21/hGH, but not L-FABP-5g6 to +21/hGH, is silenced in glandular epitheZium during postnatal life. By the fourth postnatal week, expression of the L-FABP-4soo0 to +21/hGH transgene was almost completely silenced in the glandular stomach. Only a few scattered hGH-positive surface mucous cells were present in the pits and adjacent surface epithelium (data not shown). There was no obvious spatial organization of cells that contained detectable levels of reporter, i.e., they were not limited to any particular position within the gastric pits nor was their distribution confined to any one region of the zymogenic or mucous zones. No hGH was present in the keratinized squamous epithelium of the forestomach. By the end of the second month and for the entire first year of life, expression of this transgene was completely suppressed in all cellular populations of the glandular stomach (n = 12 animals surveyed). This finding agrees with the results of our earlier blot hybridization studies of hGH mRNA accumulation along the stomach-to-colonic axis of adult L-FABP-4y000 to +21/ hGH mice (35). One- to 13-mo-old L-FABP-5g6 to +21/hGH transgenic mice, in contrast, produced large quantities of hGH in certain gastric epithelial cell populations. Figure 2A illustrates the sharp border between the hGH-negative forestomach epithelium and the glandular stomach in which scattered surface epithelial cells contain the hGH reporter? hGH-positive cells were limited to the gastric pits, a region containing surface mucous cells (Fig. 2B). Marked heterogeneity in steady-state hGH levels occurred in epithelial cells located at comparable positions along the isthmus-pit axis of a single gastric unit (Fig. 2, B and C). 3 Lack of expression of L-FABP-5g6 to +21/hGH in the keratinized squamous epithelium of the distal esophagus and forestomach of the mouse is in stark contrast to the developmental pattern of expression of the mouse adenosine deaminase (ADA) gene (4). ADA levels are high in the keratinized squamous epithelium of the tongue, esophagus, and forestomach; representing 5-20% of the total soluble proteins at 1 mo of age. The concentration of ADA in the glandular stomach is 30-fold lower. The recently cloned mouse ADA gene (1, 18, 25) opens the way for identifying &-acting elements that promote transcription in the squamous epithelium of the forestomach and restrict expression in the glandular epithelium.
EPITHELIAL
DIFFERENTIATION
G191
Moreover, hGH expression in surface mucous cells varied dramatically among adjacent gastric units. In some areas of the zymogenic zone, many adjacent gastric units contained uniformly high concentrations of the reporter (Fig. 20). In other areas, large patches of wholly negative units contained single units in which all surface mucous cells were positive (Fig. 2E). These varying patterns were frequently encountered within a given animal and were not related to age. In contrast to surface mucous cells, other differentiated cell types present in zymogenic glands did not support L-FABP-5g6 to +21/hGH t ransgene expression in adult mice. Parietal cells did not contain levels of hGH detectable by light microscopic immunocytochemistry using the sensitive IGSS method (Fig. 2C). The necks of gastric glands in which most mucous neck cells are located were hGH negative (Fig. 20), although a few cells containing hGH were observed adjacent to parietal cells in positions consistent with mucous neck cells (Fig. 2C). Intrinsic factor is produced by chief cells in the mouse and rat (16). This cobalamin-binding protein thus provides an immunocytochemical marker for chief cells. The location of chief cells at the base of zymogenic glands contrasted sharply with the distribution of hGH positive surface mucous cells (Fig. 20). Such double-labeled preparations established that chief cells did not express L-FABP-5g6 to +21/hGH at any postnatal age examined (data not shown). Within the mucous zone of adult L-FABP-5g6 to +21/ hGH transgenic mice, transgene expression was confined to surface mucous cells (Fig. 2, E-G). As in the zymogenic zone, remarkable variability in hGH expression occurred within and between glands. Figure 2, E and F, shows isolated gastric units that are hGH positive, surrounded by hGH-negative gastric units. Other gastric units in the mucous zone contained heterogeneous levels of the reporter in their surface epithelial cell populations (Fig. 2G). This heterogeneity was not an insertion site effect, because it was noted in animals from two independent L-FABP-5g6 to +21/hGH pedigrees. Figure 2, H and I, illustrates transgene expression at the junction of the gastric glandular epithelium and the proximal duodenum. In this region where gastric and intestinal cell populations are sharply demarcated, the apical hGH accumulation in gastric surface mucous cells
Fig. 2. hGH expression in gastric epithelium of adult L-FABP- 5g6 to +21/hGH transgenic mice. A: section from squamous-glandular stomach transition zone of a 2mo-old transgenic (GJ3) mouse stained for hGH with IGSS method shows that expression of hGH is patchy and confined to glandular stomach. B, C: longitudinal (B) and cross (C) sections through zymogenic zones of 4- and ll-mo-old (respectively) transgenic (GJ3) mice show numerous hGH immunoreactive surface mucous cells near luminal surface. In these lightly hematoxylin- and eosin-counterstained sections, numerous plump pink parietal cells are visible but do not contain detectable levels of reporter. D: section from zymogenic zone of an ll-mo-old (G,l3) transgenic mouse, coincubated with rabbit anti-rat intrinsic factor and goat anti-hGH sera, and subsequently with Texas Red-labeled donkey anti-rabbit and fluorescein-labeled donkey anti-goat sera. Intrinsic factor (red) serves as a marker for chief cells at base of zymogenic glands. hGH immunoreactive cells (green) are confined to pit and surface epithelium. Parietal and mucous neck cells, which are not labeled by either antisera, are seen as a dark band between chief and surface epithelial cells. E-G: heterogeneous patterns of transgene expression in surface mucous cells. E: longitudinal section from pure mucous zone of a 7-mo-old (G,19) transgenic mouse. F: cross section from same zone in a 4mo-old (GJ3) transgenic mouse. G: pure mucous zone of a 2-mo-old (GJ3) transgenic mouse, showing a mixture of mucous units, some with wholly hGH-positive columns of surface epithelial cells. Other adjacent units exhibit a seemingly chaotic pattern of staining of surface epithelial cells during their migration up pit. These cellular patterns of transgene expression did not appear to correlate with transgenic pedigree (E, F) or age (D, G). H, I: section from gastroduodenal junction of a l-mo-old (G,l3) transgenic mouse incubated with rabbit anti-hGH serum and labeled with IGSS. H: darkfield microscopy of silver grain reveals diffuse cytoplasmic staining in gastric epithelium (left) and intense supranuclear Golgi associated staining in villus-associated duodenal enterocytes (right). I: a high magnification bright field photomicrograph of “junctional” villus illustrates 2 distinct patterns of intracellular hGH accumulation. Bar, 25 pm.
Downloaded from www.physiology.org/journal/ajpgi by ${individualUser.givenNames} ${individualUser.surname} (129.081.226.078) on December 21, 2018.
G192
TRANSGENES
AS PROBES OF GASTRIC
contrasted with the accumulation of hGH in the supranuclear (Golgi) region in enterocytes. hGH is concentrated in exocrine secretory granules of gastric mucous cells. Labeling of hGH in thin sections of adult zymogenic mucosa by EM immunogold cytochemistry confirmed the heterogeneous expression of the LFABP-5g6 to +21/hGH transgene in surface mucous cells. In these preparations, surface mucous cells can be identified by the presence of apical secretory granules with uniform dense content. In most randomly selected mucosal tissue blocks, no hGH was detectable. In some tissue samples, however, surface mucous cells showed hGH-specific immunoreactivity distributed throughout the matrix of each granule (Fig. 3A). hGH was also noted in a few cells with apical secretory granules containing dense cores, a morphological feature typical of mucous neck cells. hGH in these cells was concentrated in the dense cores of the apical granules (Fig. 3B). This finding suggests that the exocrine mucous cells that express hGH direct the hormone to their regulated secretory pathway. L-FABPjhGH transgenes remain silent in entero-endocrine cell populations of adult mouse glandular epithelium. Previous studies indicated that the intact endogenous
EPITHELIAL
DIFFERENTIATION
mouse Fabpl gene is only rarely expressed at detectable levels within adult duodenal enteroendocrine cell populations (27). Similarly, a survey of l- to 13-mo-old normal and transgenic mice failed to detect any immunoreactive L-FABP in gastric endocrine cells (data not shown). Double label immunocytochemical studies of duodenal and proximal jejunal sections prepared from adult L-FABP-5g6 to +21/hGH mice indicated that hGH is present in >80% of gastrin-immunoreactive cells and >30% of serotonergic cells (27, 28, 30). (Somatostatin immunoreactive cells are only rarely found in the proximal portion of the small bowel and therefore were not included in the double label survey.) In sharp contrast, multilabel studies of gastrin-, somatostatin-, and serotonin-producing enteroendocrine populations present in the gastric glandular epithelium of these same adult mice failed to detect any transgene expression in the mucous zone (Fig. 4) or in the zymogenic zone (data not shown). The highly sensitive EM immunogold labeling method also failed to detect hGH in any gastric enteroendocrine cell (data not shown) under labeling conditions that readily detected hGH in intestinal enteroendocrine cell granules (37).
Fig. 3. Immunolocalization of hGH in gastric mucous cells. Mucous cells in gastric epithelial surface, pits, and isthmus that expressed L-FABP- 5a6 ti +*r/hGH transgene were identified by anti-hGH immunogold labeling. Although morphological preservation was not optimal in these LR Gold-embedded tissues, apical mucous granules are clearly visualized; position of their limiting membranes is indicated by arrows. hGH is concentrated in apical secretory granules of all positive cells. A: hGH is distributed throughout homogeneous matrix of apical secretory granules in a surface mucous cell. B: in an isthmus mucous cell, apical secretory granules contain dense cores in an electron-lucent matrix. hGH is concentrated in these cores. Bar, 0.5 pm.
Downloaded from www.physiology.org/journal/ajpgi by ${individualUser.givenNames} ${individualUser.surname} (129.081.226.078) on December 21, 2018.
TRANSGENES
AS PROBES
OF GASTRIC
DISCUSSION
Our analysis of the expression of L-FABP/hGH transgenes in the gastric epithelium of fetal and I- to 13-moold transgenic mice has indicated that 1) differences in specific gastric epithelial cell types present during fetal and postnatal development can be operationally defined by their ability to support expression of L-FABP/hGH transgenes; 2) L-FABP+g6 to +21/hGH expression is largely confined to the surface mucous cells of zymogenic and mucous gastric units, but there is an unexpected degree of diversity in this cellular population both within and between gastric units; 3) mucous cells that express hGH direct this foreign protein into regulated apical secretory granules; and 4) the inability of this transgene to be expressed in gastric serotonergic and gastrin enteroendocrine subpopulations contrasts with its efficient expression in these cell types in the proximal small intestine. Temporal gradients and regional differences in gene expression talong stomach- to-colonic axis. Regional differ-
ences can be defined among the various renewing epithelial cell populations of the gastrointestinal tract based on temporal changes in transgene expression. We had previously examined L-FABP-5g6 to +21/hGH expression in the small and large bowel of fetal and 1- to 13-mo-old transgenic mice. Nucleotides -596 to +21 of the rat Fabpl are sufficient to recapitulate the appropriate temporal, cellular, and regional pattern of Fabpl activation in the fetal mouse intestinal epithelium with the exception that rare enteroendocrine cells (typically producing gastric inhibitory peptide) support transgene, but not Fabpl, expression (8, 28, 30). This contrasts with the situation in adult mice where “inappropriate” expression of L-FABP/ hGH (compared with Fabpl) occurs in many intestinal enteroendocrine cell subpopulations, in small intestinal crypt epithelial cells, and in the colonic epithelium from the cecum to the rectum (5, 27,35). With increasing age, a “wave” of extinction of transgene expression takes place in the colon but not in the small intestine (5). This silencing of transgene expression begins in the distal colon, proceeds to successively more proximal portions of the colon, and involves contiguous multicrypt patches with sharply demarcated boundaries. A comparable phenomenon occurs in mice containing the L-FABP-4y000 to +21/ hGH transgene, except that the “wave” of extinction moves more rapidly and involves individual crypts (5). In the present study, we demonstrated that &-acting suppressor element(s) located between nucleotides -4,000 to +21 also silenced L-FABP/hGH expression in the mouse stomach between late gestation and the first month of postnatal life. While the rate of extinction of transgene expression in the gastric epithelium was more rapid than in the colonic epithelium (complete in
