Stimulation of Activin Receptor II Signaling Pathways Inhibits Differentiation of Multiple Gastric Epithelial Lineages

Qiutang Li, Sherif M. Karam, Katherine A. Coerver, Martin M. Matzuk and Jeffrey I. Gordon

Department of Molecular Biology and Pharmacology (Q.L., J.I.G.) Washington University School of Medicine St. Louis, Missouri 63110
Department of Anatomy (S.M.K.) Faculty of Medicine Kuwait University Safat 13110, Kuwait
Departments of Pathology, Molecular and Human Genetics, and Cell Biology (K.A.C., M.M.M.) Baylor College of Medicine Houston, Texas 77030


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Activins are TGFß family members known to mediate a variety of developmental events. We examined the effects of activins on the self-renewing epithelial lineages present in gastric units of the adult mouse stomach. These lineages are descended from multipotent stem cells located in the midportion of each unit. The stem cell and its immediate descendants can be identified by their morphological features. Studies of knockout mice lacking activins A or B, and/or activin type II receptors (ActRII) revealed that ActRII-mediated signaling is not required for normal gastric epithelial morphogenesis or homeostasis. Mice homozygous for a null allele of the {alpha}-inhibin gene (inham1/m1) develop gonadal sex cord stromal tumors that secrete large amounts of activins A and B. Analysis of inham1/m1 mice, with or without gonads, established that supraphysiological levels of activins block differentiation of preparietal to acid-producing parietal cells, differentiation of neck cells to pepsinogen-producing zymogenic cells, and terminal differentiation of mucus-producing pit cells. ActRII mRNA is normally present in pit, parietal, and zymogenic cells. inham1/m1actRIIm1/m1 compound homozygotes develop activin-secreting gonadal tumors but have no abnormalities in their gastric epithelium, indicating that persistent stimulation of ActRII-dependent signaling pathways produces pleiotrophic effects on gastric epithelial differentiation. When a lineage-specific promoter is used to ablate mature parietal cells with an attenuated diphtheria toxin A fragment in transgenic mice, there is increased proliferation of the multipotent gastric stem cell and its committed daughters and subsequent development of gastric neoplasia. Parietal cell loss in inham1/m1mice is not associated with this proliferative response. These different responses to parietal cell loss suggest that stimulation of ActRII-dependent signaling pathways in inham1/m1 animals affects the proliferative activity of the stem cell and its immediate descendents. This finding may have therapeutic significance.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The adult mouse gastric epithelium undergoes continuous self-renewal throughout the animal’s life. Renewal occurs in tubular mucosal invaginations known as gastric units. In the central, or corpus, region of the stomach, each gastric unit contains an average steady state population of ~200 epithelial cells, representing five lineages, all derived from a multipotent stem cell (1, 2). These lineages give rise to pit, parietal, zymogenic, enteroendocrine, and caveolated cells. A remarkable feature of the gastric unit is its capacity to maintain an accurate steady state census of its component cell types despite marked differences in their rates of differentation, migration, and lifespan. In addition, the distinctive morphological features of committed lineage precursors permit quantitative analysis of their responses to changes in the census of their progeny, as well as their responses to genetically engineered changes in expression of potential mediators of epithelial cell proliferation, differentiation, migration, or death programs.

Figure 1Go outlines what is currently known about the organization of gastric units in the corpus region. Proliferation is largely confined to the centrally positioned isthmus. In vivo pulse labeling with [3H]thymidine followed by electron microscopic radioautography has identified a presumptive multipotent isthmal stem cell with an estimated turnover time of ~3 days (2). Three of the stem cell’s immediate descendants are known: a granule-free pre-pit cell precursor, a granule-free pre-neck cell precursor, and a granule-free pre-parietal cell precursor (2, 7). Mucus-producing pit cells differentiate during a 3-day upward migration through the upper portion of the gastric unit (the pit) to the surface epithelium where they undergo an apoptotic or necrotic death (4). Members of the zymogenic cell lineage differentiate during a downward migration from the isthmus through the neck and base regions of the gastric unit (5). Differentiation involves the following sequence: granule-free pre-neck cell precursor->pre-neck cell->neck cell->pre-zymogenic cell->zymogenic cell. Zymogenic cells have a lifespan of ~190 days and die by necrosis or apoptosis. The parietal cell lineage is the only one of the three principal gastric epithelial lineages that completes its terminal differentiation within the stem cell zone (isthmus). Preparietal cells are converted to mature acid-producing parietal cells in 1 day (3, 8). Mature parietal cells then undergo a bipolar migration away from the isthmus (3). Half migrate up the pit where they are eliminated by necrosis, exfoliation, or phagocytosis. An equal number of parietal cells move down through the neck to the base were they are removed by apoptosis or phagocytosis (3). Cellular lifespan averages 54 days (3).



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Figure 1. Summary of Gastric Epithelial Differentiation in Gastric Units Located within the Corpus of the Normal Adult Mouse Stomach

Cellular intermediates in the differentiation programs of the pit, parietal, zymogenic, enteroendocrine, and caveolated cell lineages have been identified based on [3H]thymidine-electron microscopy autoradiographic studies (2–7). The immediate descendants of the multipotent isthmal stem cell include the granule free (GF) pre-pit cell precursor, GF pre-parietal cell precursor, and GF pre-neck cell precusor as well as less well characterized precursors of the enteroendocrine and caveolated cell lineages. The bold black arrows indicate the direction of migration of various members of a given lineage.

 
The results of a recent lineage ablation experiment suggest that parietal cells are able to influence the proliferation and/or differentiation programs of isthmal lineage precursors and their differentiated descendants. Studies (8, 9, 10) had shown that nucleotides -1035 to +24 of the noncatalytic ß-subunit gene of mouse H+/K+ATPase (ß-subunit-l035 to +24 1) can direct expression of foreign gene products to preparietal and parietal cells in transgenic mice. Transcription is lineage specific: ß-subunit-l035 to +24/reporter transgenes are silent in both precursors and differentiated members of the pit and zymogenic lineages. When ß-subunit-l035 to +24 was used to express an attenuated diphtheria toxin A chain (tox176) in transgenic mice, mature parietal cells were completely ablated (10). Ablation was accompanied by a 4-fold increase in the proliferative activity and steady state number of isthmal stem cells and granule-free pre-pit and pre-neck cell precursors. Parietal cell ablation also produced a block in terminal differentiation of the zymogenic lineage and a 2- to 3-fold increase in the fractional representation of pit cells (10).

If parietal cells are important regulators of gastric epithelial homeostasis, then it is important to identify factors that regulate their differentiation and survival. Studies in knockout mice have indicated that activins may play such a role (11). Activins are members of the transforming growth factor-ß (TGFß) family. Family members are known to modulate cellular proliferation and differentiation programs in other organs of the developing and adult mouse (12, 13). These disulfide-linked dimers initiate their signal transduction cascades by interacting with two transmembrane serine/threonine kinases, type I and type II receptors. Ligand binding to type II receptors appears to function upstream of type I receptors in a sequential kinase cascade (14, 15). Activins and inhibins share a common ß-subunit. Inhibins are {alpha}ß-heterodimers while activins are composed of various combinations of closely related ß-subunits [activin A = ßAßA homodimers; activin B = ßBßB homodimers; activin AB = ßAßB heterodimers (14)]. Mice homozygous for an activin ßA null allele or compound homozygotes for activin ßA and activin ßB null alleles die of craniofacial abnormalities within 1 day after birth (16, 17), well before completion of gastric unit morphogenesis (7). Mice homozygous for a null allele of the inhibin {alpha}-subunit (inham1/m1) develop gonadal sex cord-stromal tumors derived from granulosa/Sertoli cells (11, 18). The tumors produce large quantities of activins A and B, resulting in a wasting syndrome with anemia, loss of hepatocytes due to massive apoptosis, and loss of parietal cells by unknown mechanisms (11, 18). Removal of the gonads before development of tumors prevents these changes (11).

Two type II activin receptors (ActR) have been identified: ActRII and ActRIIB (19, 20, 21). The majority of ActRII-deficient mice live to adulthood (22). Crossing ActRIIm1/m1 and inham1/m1 animals to generate compound homozygotes does not prevent development of activin-producing gonadal tumors but does prevent the weight loss, anemia, hepatocyte depletion, and parietal cell loss (23). This finding establishes a role for ActRII-mediated signaling in the pathogenesis of this syndrome.

In the current study, we have performed detailed analyses of epithelial proliferation and differentiation programs in the stomachs of inham1/m1 mice with and without gonads, actRIIm1/m1 mice, inham1/m1 actRIIm1/m1 compound homozygotes, actßAm1/m1 mice, actßBm1/m1mice, actßAm1/m1 actßBm1/m1 compound homozygotes, and actßBm1/m1actRIIm1/m1 compound homozygotes. The results, coupled with an analysis of the cellular patterns of expression of ActRII, indicate that activin stimulation of ActRII-mediated signaling pathways affects the differentiation programs of multiple gastric epithelial lineages and the proliferative status of the multipotent isthmal stem cell.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
{alpha}-Inhibin-Deficient Mice with Gonadal Sex Cord-Stromal Tumors Have Abnormalities in the Differentiation Programs of Multiple Gastric Epithelial Lineages
Figure 2Go, A-D, shows the distribution of pit, parietal, neck, and zymogenic cells in gastric units located in the corpus region of the stomach of 8-week-old mice homozygous for wild-type Inha and ActRII alleles. Parietal cells can be readily identified by their distinctive morphology (Fig. 2AGo), by their production of GalNAc-containing glycoconjugates recognized by Dolicos biflorus agglutinin (Fig. 2BGo), and by their synthesis of the noncatalytic ß-subunit of H+/K+ ATPase (Fig. 2CGo). Parietal cells are evident in the pit, isthmus, neck, and base compartments of these normal gastric units. Pit cells elaborate fucosylated glycoconjugates detectable with Anguilla anguilla and Ulex europeaus agglutinins. They also produce glycoconjugates with ganglioside GM1 epitopes that are recognized by cholera toxin B subunit (Fig. 2CGo). Neck cells synthesize GlcNAcß1,4-containing glycoconjugates that react with Griffonia simplifolica agglutinin II (GSII) (Fig. 2DGo). As neck cells migrate through the neck region they accumulate pepsinogen (Fig. 2DGo). Terminal differentiation of neck to zymogenic cells is associated with loss of GSII reactivity, continued production of pepsinogen, and induction of intrinsic factor synthesis. Zymogenic cells are confined to the base of these units (Fig. 2Go, B and D). Table 1Go provides quantitative data on the average number of lineage precursors and their fully differentiated descendants per longitudinal section of a gastric unit in mice homozygous for wild-type Inha and ActRII alleles.



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Figure 2. Multilabel Immunohistochemical Analysis of Gastric Epithelial Differentiation in inham1/m1 Mice with and without Testicular Stromal Tumors

Panels A-D, Sections of gastric units from an 8-week-old wild-type mouse. Panel A, Toluidine blue-stained, epon-embedded, 0.5 µm thick section showing the base of a gastric unit. Differentiated parietal cells are evident (e.g. open arrows). Zymogenic cells (e.g. closed arrows) contain numerous dense secretory granules. Panel B, Section incubated with (i) FITC-labeled Dolicos biflorus agglutinin (DBA) to visualize parietal cells (green) distributed throughout the gastric unit and (ii) rabbit anti-intrinsic factor (InF) plus Cy-3-labeled donkey anti-rabbit Ig to visualize differentiated zymogenic cells (red) at the base of the unit. Panel C, Section incubated with FITC-labeled Cholera toxin B-subunit (CTB), rabbit antibodies to the ß-subunit of H+/K+ ATPase, and Cy3-donkey anti-rabbit Ig. Pit cells that produce ganglioside GM1-containing glycoconjugates recognized by CTB appear green. Parietal cells appear red-orange. Multilabel studies indicated that >98% of the parietal cells coexpress the ß-subunit of H+/K+ ATPase and glycoconjugates recognized by DBA (data not shown). Panel D, Multilabel study using FITC-labeled GSII, rabbit antipepsinogen, and Cy3-donkey anti-rabbit Ig. As GSII-positive neck cells (green) migrate down through the neck toward the base of gastric units, they accumulate pepsinogen (GSII- and pepsinogen-positive cells appear yellow-green). As cells complete their terminal differentiation, production of pepsinogen is sustained while expression of GlcNAcß1,4-containing glycoconjugates recognized by GSII is extinguished (pepsinogen-positive, GSII-negative zymogenic cells appear red). Panels E–H, Sections of gastric units from an 8-week-old mouse homozygous for a null allele of Inha. Sections shown in panels E–H were processed exactly as in panels A–D, respectively. Panel E, Base region of gastric units from an {alpha}-inhibin-deficient mouse showing increased numbers of caveolated cells (closed arrows), increased numbers of neck cells (e.g. open arrows), and the absence of mature zymogenic cells. The closed arrowhead points to cell in M-phase. Panel F, Section showing complete loss of InF-positive zymogenic cells and markedly reduced numbers of DBA-positive parietal cells. Panel G, Section showing loss of ganglioside GM1-containing glycoconjugates in pit cells. Scattered parietal cells are present that produce the ß-subunit of H+/K+ ATPase. Panel H, GSII-positive neck cells are increased but do not contain detectable levels of immunoreactive pepsinogen. InF- and pepsinogen-positive zymogenic cells are absent at the base (see panel D for comparison). Panels I–K, Castration precludes production of supraphysiological levels of activins by sex cord stromal tumors and blocks the development of gastric epithelial pathology. Sections were prepared from a male inham1/m1 mouse that had been castrated at 1 month of age and then killed 2 months later. Sections were processed exactly as in panels B–D and F–H. Panel I, DBA-positive parietal cells (green) and InF-positive zymogenic cells (red) are present in normal numbers (compare with panel B). Panel J, CTB-positive pit cells are present (green) as are ß-subunit of H+/K+ ATPase-positive parietal cells (orange) (compare with panel C). Panel K, Pepsinogen is normally expressed in a subpopulation of GSII-positive neck cells (yellow-green) and in GSII-negative/InF-positive mature zymogenic cells (red). Bars = 25 µm.

 

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Table 1. Epithelial Cell Populations in the Gastric Units of Mice with Wild-Type and Null Alleles of the Inha and ActRII Genes

 
By 8 weeks of age, mice homozygous for a null allele of the {alpha}-inhibin gene (inham1/m1) have developed activin-secreting gonadal tumors and exhibit abnormalities in all three principal gastric epithelial lineages. There is a block in the differentiation of parietal cells: the number of mature parietal cells is reduced 4-fold while preparietal cell number is not significantly different from normal age- and gender-matched littermates (Table 1Go and Fig. 2FGo). There is also a complete and discrete block in the differentiation program of the zymogenic cell lineage: pre-neck and neck cell numbers are increased while their prezymogenic and zymogenic descendants are entirely absent (Table 1Go and Fig. 2Go, E, F, and H). The loss of mature parietal and zymogenic cells is not due to an increased apoptosis, or to increased necrosis, or to increased phagocytosis by neighboring cells (Table 1Go plus data not shown).

A statistically significant 2- to 2.5-fold increase in the steady state number of pre-pit and pit cells per unit is also evident at 8 weeks of age in inham1/m1 mice (Table 1Go). This can also be viewed as an increase in their fractional representation: even though parietal and zymogenic cells are lost, the total cellular census of gastric units remains equivalent to that in age-matched wild-type littermates (Table 1Go). While conversion of pre-pit to pit cells is not blocked in inham1/m1 units, terminal differentiation of this lineage is altered as judged by reduced accumulation of {alpha}-L-fucose-containing glycoconjugates that react with Anguilla anguilla agglutinin and reduced levels of ganglioside GM1 epitopes that react with cholera toxin B subunit (compare panels C and G in Fig. 2Go).

As noted in the Introduction, the multipotent isthmal stem cell also gives rise to the enteroendocrine and caveolated cell lineages (6). Enteroendocrine and caveolated cells are much less abundant than pit, parietal, or neck/zymogenic cells in wild-type gastric units (Table 1Go). The two cell types have a similar long lifespan [average = 100 days for enteroendocrine cells (6)]. The function of caveolated cells is unknown although they appear to be a source of nitric oxide (24).

While the total number of enteroendocrine cells is not increased in 8-week-old inham1/m1 mice, there is a marked increase in precaveolated and mature caveolated cells (Fig. 2EGo and Table 1Go). Like parietal cells, caveolated cells normally complete their differentiation within the isthmus before undergoing a bipolar migration (Ref. 6; cf Fig. 1Go). Immature caveolated cells are readily apparent in inham1/m1 gastric units (data not shown), suggesting partial inhibition of caveolated cell differentiation.

More than 95% of male inham1/m1 mice die by 12 weeks of age while >95% of female mice succumb by 17 weeks (11, 18). The changes in gastric epithelial cell biology noted at 8 weeks of age in male and female inham1/m1 mice persist and, in some cases, become more exaggerated in females that survive to 15 weeks of age. Mature parietal cells are no longer detectable. The blockade in terminal differentiation of the zymogenic cell lineage is manifested by an even greater augmentation of pre-neck cells (Table 1Go). Caveolated cells are more numerous (Table 1Go). The steady state population of isthmal-based lineage precursors is increased further to 3-fold above that of age-matched wild-type littermates (Table 1Go) without a detectable change in proliferation (defined by the number of 5'-bromo-2'-deoxyuridine (BrdU)-positive (S-phase) or M-phase cells in the isthmus; e.g. Table 1Go). These latter findings suggest that gastric epithelial differentiation may be inhibited at very early stages.

Two observations indicate that the alterations in parietal, zymogenic, pit, and caveolated cell differentiation are related to the gonadal sex cord-stromal tumors that develop in male and female inham1/m1 mice. First, the evolution of these gastric epithelial abnormalities corresponds to the time course of progression of the gonadal tumors. For example, there are no qualitative or quantitative abnormalities in the epithelial cell lineages of 4-week-old male and female inham1/m1 animals (data not shown), a time when only microscopic foci of gonadal neoplasia are present (11, 18). Second, when inha-/- mice are gonadectomized at 4 weeks of age and examined 8 weeks later, there are no detectable qualitative or quantitative abnormalities in parietal, zymogenic, pit, or caveolated cell differentiation (controls = age-matched Inha+/+ and inha+/m1 littermates) (e.g. Fig. 2Go, I–K).

Signaling through ActRII Is Required to Produce the Gastric Epithelial Abnormalities in inham1/m1 Mice with Gonadal Tumors
Autonomous production of activins by the gonadal tumors leads to >10-fold elevations in the serum levels of activins A and B (11). The observed rescue of gastric epithelial abnormalities by gonadectomy raises two questions. What is the normal role of activins in regulating gastric epithelial proliferation and differentiation programs in the developing and adult stomach? Do the gastric epithelial lineage abnormalities noted in adult {alpha}-inhibin-deficient mice reflect persistent stimulation of ActRII-mediated signaling pathways by high circulating levels of gonadally derived activins?

Gastric Unit Morphogenesis and Epithelial Differentiation in Mice that Lack Activin A, Activin B, and/or ActRII
Gastric unit morphogenesis in the mouse is not completed until the third postnatal week (7). At embryonic day 12.5 (E12.5), the stomach is lined with a simple undifferentiated epithelial monolayer. At E18, nascent gastric units appear as short solid epithelial infoldings, ~90% of whose cells have the morphological appearance of adult isthmal stem cells, their granule-free committed daughters, plus more differentiated pre-neck, pre-pit, and pre-parietal cell descendants (7). From P1-P7, immature cells decrease to ~20% of the total as differentiated pit, neck, and parietal cells appear (7). Between P15 and P21, the multipotent stem cell and its descendants are assembled into a distinct proliferative zone (the isthmus) and cellular migration/differentiation programs become compartmentalized (7).

The cellular patterns of accumulation of mRNAs encoding the {alpha}, ßA, and ßB subunits of activins, the activin type IA and IB receptors, and the activin type II and IIB receptors have been examined in the developing (fetal) stomach by in situ hybridization (25, 26). {alpha}- and ßA-subunit mRNAs are not detectable. ßB mRNA is confined to the epithelium. ActRIA mRNA is restricted to the mesenchyme while ActRIB mRNA is located in the epithelium. ActRII and ActRIIB mRNAs are both present at low levels in the mesenchymal and epithelial layers.

actßBm1/m1 mice survive to the adult stage (16). Our single and multilabel immunohistochemical surveys of 3-, 4-, and 10-month-old mice failed to disclose any abnormalities in the proliferative status of isthmal-based precursors or in the terminal differentiation programs of any gastric epithelial lineage (Fig. 3Go, A–C, plus data not shown).



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Figure 3. The Effects of Knocking Out the ActßA, ActßB, ActRII, and Inha Genes, Singly or in Various Combinations, on Gastric Epithelial Morphogenesis and Homeostasis

Panels A–C, Knocking out the ActßB gene has no detectable effects on differentiation of the parietal or zymogenic cell lineages or on isthmal precursor proliferation. Panel A, Section from a 3-month-old wild-type (ActßB+/+) mouse that received BrdU 1.5 h before death. The section was incubated with FITC-GSII (to detect neck cells as green), plus goat anti-BrdU and Cy3-donkey anti-goat Ig (to detect S-phase cells as orange). Panel B, Section from an activin B-deficient littermate of the mouse shown in panel A that was processed in an identical fashion. Results are indistinguishable from those obtained from the wild-type mouse. Panel C, Another section obtained from the activin B-deficient animal shown in panel B but incubated with FITC-DBA to visualize parietal cells (green) and rabbit anti-InF plus Cy3-donkey anti-rabbit Ig to mark zymogenic cells (orange-red). Identical results were obtained when a section from the wild-type littermate was stained with these reagents (see Fig. 2BGo). Panels D and E, actßAm1/m1 mice die within 24 h after birth (postnatal day 0; P0) without detectable histological abnormalities in their gastric epithelium. Panel D, Hematoxylin and eosin-stained section of a P0 wild-type (ActßA+/+) stomach. At this stage of development, gastric buds are composed predominantly of lineage precursors (see text). Scattered parietal cells are evident (e.g. arrow). Panel E, Hematoxylin and eosin- stained section from a P0 activin A-deficient littermate (arrow points to parietal cell). Panel F, 3-month-old male mouse homozygous for a null allele of the ActRII gene shows no abnormalities in the differentiation programs of its parietal or zymogenic cell lineages (green and red, respectively). Panels G–I, ActRII-deficient inham1/m1 mice with gonadal sex cord stromal tumors. The results of multilabel immunohistochemical studies of a 3-month-old inham1/m1actRIIm1/m1 male compound homozygote are shown. Sections in panels G–I were processed exactly as in Fig. 2Go, B, D, and C, respectively. In panel G, DBA-positive parietal cells appear green while InF-positive zymogenic cells are red. In panel H, pepsinogen-negative/GSII-positive neck cells are green, pepsinogen-positive/GSII-positive neck cells are yellow, and pepsinogen-positive/GSII-negative zymogenic cells are red. In panel I, CTB-positive pit cells are green, and H+/K+ ATPase ß-subunit-positive parietal cells are orange. Zymogenic and parietal cell differentiation are normal, but there is a reduction in ganglioside GM1 production in pit cells (compare with Fig. 2CGo). Bars = 25 µm.

 
actßAm1/m1 mice and actßAm1/m1 actßBm1/m1 compound homozygotes die within 24 h after birth. These animals have no detectable histological abnormalities in their gastric epithelium while they are alive (Fig. 3Go, D and E, and data not shown).

Northern blot studies of RNAs prepared from the corpus of adult ActRII+/+ mouse stomachs revealed ActRII and ActRIIB mRNAs (data not shown). Surveys of 15-week-old actRIIm1/m1 mice disclosed no quantitative or qualitative abnormalities in their gastric epithelium (Table 1Go and Fig. 3FGo). As expected from these results, 3-month-old actßBm1/m1 actRIIm1/m1 compound homozygotes had no detectable defects in gastric epithelial proliferation or differentiation programs (data not shown). Together, our findings establish that ActRII-mediated signaling is not essential for normal gastric unit morphogenesis or for maintenance of normal epithelial homeostasis. The data also indicate that two of the receptor’s ligands, activins A and B, are dispensible.

Removal of a Functional ActRII Rescues the Gastric Epithelial Abnormalities Associated with High Circulating Levels of Activins A and B in inham1/m1 Animals
inham1/m1 actRII m1/m1 compound homozygotes develop bilateral gonadal sex cord-stromal tumors and have serum levels of activins A and B equal to or greater than those documented in inham1/m1 animals (23). Nonetheless, the majority of these animals survive past 15 weeks (males) or 18 weeks (females). Quantitative morphological and multilabel immunohistochemical analysis of gastric units in 12-week-old inha m1/m1 actRII m1/m1 compound homozygotes revealed no block in parietal or zymogenic cell differentiation, no augmentation in the number of caveolated cells, and no increase in isthmal lineage precursors (Table 1Go and Fig. 3Go, G–I). There was no increase in the fractional representation of pit cells (Table 1Go), although terminally differentiated pit cells had reduced production of ganglioside GM1-containing glycoconjugates (compare Fig. 3IGo with Fig. 2Go, C and J).

ActRII mRNA Is Present in Parietal, Zymogenic, and pit Cells
The results obtained from inha m1/m1actRII m1/m1 compound homozygotes suggested that activins originating from the gonadal tumors of {alpha}-inhibin- deficient mice signal through an ActRII-dependent pathway to produce the observed block in terminal differentiation of the parietal and zymogenic cell lineages, as well as the other abnormalities enumerated above. At least two mechanisms can be envisioned. 1) Members of the parietal, zymogenic, pit, and caveolated cell lineages express ActRII and the downstream effectors of the signaling pathway activated by its ligands. Therefore, these lineages are equally vulnerable to the effects of supraphysiological levels of activins. 2) Only the parietal cell lineage expresses ActRII and members of its downstream signal transduction pathway. In scenario 2, the primary effect of constitutive ActRII-dependent signaling would be to block parietal cell differentiation. The resulting loss of mature parietal cells would then produce secondary effects on the zymogenic and pit cell lineages, analogous to the situation that occurs when parietal cells are ablated by an attenuated diphtheria toxin A fragment (tox176) in transgenic animals (see Introduction).

To explore these possibilities, we defined the cellular distribution of ActRII mRNA in the gastric epithelium of 15-week-old wild-type mice. Northern blots of total cellular stomach RNA were probed with a cDNA derived from the 3'-nontranslated region of ActRII mRNA. The results confirmed that the probe reacted with a unique mRNA of the expected size (data not shown). 33P-labeled antisense and sense riboprobes, derived from the same 3'-nontranslated region, were subsequently incubated with serial sections prepared from the corpus of the stomach. ActRII mRNA is present in epithelial cells distributed throughout the length of the gastric unit and is not detectable in the muscularis layer (Fig. 4AGo). Pretreatment of the stomach sections with RNAse A before application of the antisense probe reduced the signal intensity to a level equivalent to that observed with the sense strand probe (Fig. 4BGo). High-power views of sections counterstained with hematoxyin and eosin show that ActRII mRNA is present in terminally differentiated zymogenic, parietal, and pit cells (Fig. 4Go, C–E).



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Figure 4. In situ Hybridization Analysis of ActRII mRNA Accumulation in the Gastric Epithelium of 15-Week-Old Inha+/+ActRII+/+ Mice

Panel A, Section of the corpus incubated with a 33P-labeled anti-sense ActRII riboprobe. Note the pronounced labeling of the glandular epithelium and the absent labeling of the underlying muscularis. Cells located in the isthmus and neck regions, as well as mature parietal scattered throughout the unit, have lower levels of ActRII mRNA than mature zymogenic cells present in the base region. Panel B, Control experiments. An adjacent section was incubated with a sense strand riboprobe. Another adjacent section was treated with RNAse A before application of the antisense riboprobe (inset). Panels A and B were photographed under dark field with epilumination. Panels C–E, High-power views of the base (C), neck (D), and pit (E) regions of gastric units. Sections were counterstained with hematoxylin and eosin. ActRII mRNA is present in mature zymogenic cells (e.g. closed arrows in panel C), parietal cells (e.g. open arrows in panel D), and pit cells (e.g. closed arrowheads in panel E). Bars = 25 µm.

 
The Proliferative Response of Isthmal Lineage Progenitors to Parietal Cell Loss Is Different In inham1/m1 Mice Compared with Transgenic Mice with Diphtheria Toxin-Mediated Lineage Ablation
inham1/m1 mice containing gonadal tumors were compared with age- and gender-matched transgenic mice that had undergone diphtheria toxin A fragment (tox176)-mediated ablation of their mature parietal cells (10). There were three notable differences. First, unlike inham1/m1 mice, loss of mature parietal cells in the ß-subunit/tox176 transgenics is accompanied by a marked increase in proliferation, both within and outside of the isthmus. The number of S-phase cells is increased >10-fold relative to nontransgenic littermates (Fig. 5Go, A–C). This proliferative response involves isthmal precursors that expand into the ‘former’ neck and base regions (Fig. 5CGo). Cells with the morphological appearance of the gastric stem cell and its granule-free committed daughters form adenomas and ultimately invade the muscularis layer of the stomach between the sixth through twelfth months of life (Q. Li, A. Syder, R. G. Lorenz, S. M. Karam, and J. I. Gordon, manuscript in preparation). Neither augmented proliferation of isthmal lineage precursors nor gastric neoplasia occur in inham1/m1 mice during their 3- to 4-month lifespan (n = 20). The lack of a proliferative response to parietal cell loss in inham1/m1 mice suggests that activation of ActRII pathways may serve to suppress cell division among this precursor population. Second, complete loss of zymogenic cells occurs in inham1/m1 mice even when there is a small population of mature parietal cells present (Table 1Go), whereas in the tox176 transgenic mice complete loss of parietal cells is not associated with a complete loss of zymogenic cells. Third, there was no increase in the number of caveolated cells in the transgenic mice.



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Figure 5. Comparison of S-Phase Cells in the Gastric Units of inham1/m1 with Sex Cord Stromal Tumors and Transgenic Mice with Diphtheria Toxin-Mediated Ablation of Mature Parietal Cells

Mice (12 weeks old) were injected with BrdU 1.5 h before death. Tissue sections were incubated with FITC-DBA (to detect parietal cells as green), plus goat anti-BrdU and Cy3-donkey anti-goat Ig (to detect S-phase cells as orange). Panel A, inham1/m1 mouse. BrdU-positive cells are confined to the isthmus. A few scattered DBA-positive parietal cells are still present at this age. Panel B, Normal FVB/N mouse. Panel C, ß-Subunit-1035 to +24/tox176 transgenic littermate of the mouse shown in panel B. Diphtheria toxin-mediated ablation of mature parietal cells is associated with a marked increase in the number of proliferating cells with morphological features of lineage precursors (see text).

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Stimulation of ActRII-mediated signaling pathways by high levels of circulating activins inhibits the differentiation programs of four of the five self-renewing epithelial cell lineages present in the corpus of the adult mouse stomach. The loss of mature parietal and zymogenic cells reflects a block in conversion of preparietal to parietal cells and neck to prezymogenic cells. The presence of ActRII mRNA in both lineages does not permit us to definitively determine whether the blockade in terminal differentiation of neck cells is a primary effect of constitutive activation of ActRII-mediated signaling pathways or a secondary effect of the loss of mature parietal cells. However, the absence of mature zymogenic cells in young (8-week-old) inham1/m1 mice even when some mature parietal cells are present argues for some primary effect(s) of activins on the zymogenic cell lineage.

Conversion of pre-pit to pit cells is not blocked by persistent stimulation of activin type II receptors. Moreover, removal of ActRII from inhibin-deficient mice does not fully restore a normal terminal differentiation program to their pit cell lineage, in contrast to the complete rescue associated with removal of their activin-secreting gonadal tumors. The different responses of the pit, parietal, and zymogenic cell lineages to supraphysiological levels of activins A and B cannot be correlated with any discernible differences in their levels of ActRII mRNA. The different responses emphasize the importance of defining the downstream effectors of ActRII-dependent signaling in the various gastric epithelial lineages (e.g. Mad proteins; Ref.15).

The absence of any detectable abnormalities in gastric epithelial proliferation, differentiation, or death programs in actRIIm1/m1 mice with normal circulating levels of activins suggests that signal transduction pathways involving this receptor are not essential for completion of gastric unit morphogenesis or for maintaining normal epithelial homeostasis once these units are fully formed. It is possible that other receptors function in an analogous way to overcome loss of ActRII in these knockout mice. Alternatively, ActRII-dependent signaling pathways may normally be silent in the gastric epithelium or opposed by signals derived from other pathways. Since targeted disruption of ActRII blocks the gastric epithelial pathology produced by supraphysiological levels of activins A and B, there are apparently no other receptors in the gastric epithelium that are functionally equivalent to ActRII in terms of their ability to inhibit differentiation when persistently stimulated by these ligands.

Even though ActRII does not appear to be required for normal gastric epithelial morphogenesis and homeostasis, there are several reasons why this epithelium may be a good model for deciphering the contributions of (potential) components of ActRII-dependent signaling pathways to the regulation of cellular differentiation programs in adult mice. The response of the parietal and zymogenic cell lineages is pronounced and readily definable in inham1/m1 animals. The response appears to principally involve an alteration in differentiation. Such a response is quite distinct from the massive, activin-induced, p53-independent apoptotic response of hepatocytes (Refs. 11 and 27; and W. Shou and M. M. Matzuk, manuscript in preparation). Activins have been shown to impede differentiation in erythroid lineages [e.g. erythroid differentiation factor (28)]. However, unlike erythropoiesis, there is a physically well organized continuum of cellular proliferation and differentiation in normal and inham1/m1 gastric units that can facilitate an analysis of the interrelationships between execution of a given lineage’s differentiation program and accumulation of putative downstream components of ActRII-signaling cascades. The absence of a pathological response to high circulating levels of activins in the self-renewing intestinal epithelium is remarkable given the common themes shared by crypt-villus and gastric units: a multipotent stem cell, functionally anchored in a distinct proliferative compartment, giving rise to lineages that differentiate during an orderly migration (29). As such, the crypt-villus unit serves as a reference control that may help define factors required for activins A and B to produce their effects on epithelial differentiation programs.

Finally, the ActRII-mediated response to high levels of circulating activins appears to involve the multipotent stem cell and committed lineage progenitors located within the isthmus of gastric units. This conclusion is based largely on the fact that a proliferative response to loss of mature parietal cells is not manifested by the stem cell or its immediate daughters in inham1/m1 mice but is observed when parietal cells are ablated using an attenuated diphtheria toxin A fragment and a lineage-specific promoter. In the latter case, the eventual result is a gastric neoplasia composed of cells that resemble lineage precursors (Q. Li, A. Syder, R. G. Lorenz, S. M. Karam, and J. I. Gordon, manuscript in preparation). If persistent stimulation of ActRII-dependent signaling pathways affects the proliferative activity/potential of the multipotent isthmal stem cell and and its immediate daughters, such pathways may have therapeutic importance in the setting of gastric neoplasia where stimulation may prove useful in suppressing growth. Further evaluation of this possibility will require an analysis of the expression of ActRII and its downstream effectors in these neoplasms and whether progression of tumorigenesis is accompanied by mutations in this receptor and/or its effectors (30, 31, 32).


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Mice
inham1/m1 mice, actßAm1/m1 mice, actßBm1/m1mice, actRIIm1/m1 mice, and three types of compound homozygotes — inham1/m1actRIIm1/m1, actßAm1/m1actßBm1/m1, and actßBm1/m1 actRIIm1/m1 were generated as described (16, 17, 18, 22, 23). Mice containing a transgene composed of nucleotides -l035 to +24 of the ß-subunit of H+/K+ ATPase linked to an attenuated diphtheria toxin fragment A (tox176) are described in Ref. 10. Mice were maintained in a specific pathogen-free state and given a standard chow diet ad libitum.

Single and Multilabel Light Microscopic Immunohistochemical Studies
inham1/m1 mice were killed at 1, 2, 3, and 4 months of age or were gonadectomized when they were 1 month old and then killed 2 months later (n = 2–4 mice per time point). actRIIm1/m1 mice were killed at 3 and 12 months (n = 3). inham1/m1actRIIm1/m1 compound homozygotes were studied at 3 and 5 months (n = 4). actßAm1/m1 mice were examined within 24 h after birth (n = 2). actßBm1/m1 animals were killed at 3, 4, and 10 months of age (n = 1–2 mice per time point). actßAm1/m1actßBm1/m1 compound homozygotes were surveyed at postnatal day 1 (n = 2). actßBm1/m1actRIIm1/m1 compound homozygotes were killed at 3 months (n = 3). Control littermates homozygous or heterozygous for wild type Inha, ActßA, ActßB, and ActRII alleles were killed at similar time points (at least two animals per time point). ß-Subunit-l035 to +24/tox176 transgenic mice and their normal littermates were examined at 3 months (n = 5 per group). Some animals received an intraperitoneal injection of 5'-bromo-2'-deoxyuridine (BrdU; 120 mg/kg) and 5'-fluoro-2'-deoxyuridine (12 mg/kg) 90 min before death.

Stomachs were removed, opened along their cephalocaudal axis, and fixed in Bouin’s solution. Five micron-thick sections were cut from the middle of the stomach. Immunohistochemical analyses were conducted using methods described previously (8, 10). The following antisera were diluted in PBS-blocking buffer (8) and incubated overnight at 4 C with gastric sections: 1) rabbit anti-rat intrinsic factor [InF; specificity = zymogenic cells (9); final dilution = 1:1000]; 2) rabbit anti-rat pepsinogen [a generous gift of Michael Samloff, UCLA, Los Angeles, CA; subset of neck cells and zymogenic cells (9); 1:500]; 3) rabbit anti-ß-subunit of rat H+/K+ ATPase [kindly supplied by Michael Caplan, Yale University, New Haven, CT; parietal cells (9); 1:1000]; and 4) goat anti-BrdU (Ref. 9; 1:1000). Antigen-antibody complexes were detected using indocarbocyanine (Cy3)-labeled sheep or donkey anti-rabbit or anti-goat Igs (Jackson Immunoresearch, West Grove, PA; 1:500). No epithelial cell staining was observed when primary antibodies were omitted. Glycoconjugate production was assessed in various epithelial cell lineages using protocols described in Ref. 33 and the following fluorescein isothiocyanate (FITC)-tagged lectins: Dolicus biflorus agglutinin; Griffonia simplifolica II; cholera toxin B subunit;Anguilla anguilla agglutinin; and Ulex europeaus agglutinin type I (see Ref. 33 for sources).

Quantitative Light and Electron Microscopic Morphological Studies of Epithelial Cell Populations Present in Gastric Units
inham1/m1 mice and their normal littermates were killed at 1, 2, and 3–4 months of age; actRIIm1/m1 mice and their normal littermates were killed at 3–4 months; and inham1/m1actRIIm1/m1 compound homozygotes were killed at 3 and 4 months (n = 2–3 mice per genotype/time point). Tissue fragments (~1 mm3) from the corpus of each stomach were fixed in 0.1 M sodium cacodylate containing 2% paraformaldehyde, 2.5% glutaraldehyde, and 0.2% tannic acid, washed in the cacodylate buffer (pH 7.4), postfixed for 1 h at 4 C in 1% osmium tetroxide, dehydrated, and embedded in Poly/Bed 812 (Polyscience, Niles, IL). Semithin sections (0.5 µm thick) were stained with 0.1% toluidine blue for light microscopy. Three tissue blocks from each mouse were used to prepare the 0.5 µm thick sections. Fifteen to twenty longitudinally oriented gastric units in a section were selected to identify epithelial cells having morphological features similar to those described in adult C57BL/6 and FVB/N mice (1, 8). At least one section from each of the three blocks prepared per animal per time point per group was examined. All cells per longitudinally sectioned unit were counted. Cellular identity was confirmed by examining adjacent 0.1 µm thick sections with an electron microscope after the sections had been stained with uranyl acetate and lead nitrate.

In Situ Hybridization
A 640-bp ActRII cDNA, derived from the 3'-untranslated region of ActRII mRNA (19), was subcloned into pBluescript SK+ (Stratagene, La Jolla, CA). The specificity of this cDNA for ActRII mRNA has been established in previous RNA blot hybridization studies of ActRII+/+ and actRIIm1/m1 mice (22, 23).[33P]UTP-labeled antisense and sense riboprobes with identical specific activities were synthesized using the plasmid as template plus T3 or T7 RNA polymerases, respectively. In situ hybridizations were performed using paraformaldehyde-fixed and protease K-digested sections prepared from the stomachs of 15-week-old normal littermates and 33P-labeled riboprobes (5 x 106 dpm/100 µl hybridization solution) according to Ref. 34. Control tissue sections were pretreated with RNAse A before application of the antisense RNA probe (34). Experiments were repeated three times with similar results (n = 2 animals per experiment).


    ACKNOWLEDGMENTS
 
We thank Lisa Roberts, Pei Wang, W. Shou, and Glory Alexander for technical assistance.


    FOOTNOTES
 
Address requests for reprints to: Jeffrey I. Gordon, Department of Molecular Biology and Pharmacology, Box 8103, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, Missouri 63110.

This work was supported by grants from the NIH (DK-33487, CA-60651, HD-32067) and the Kuwait Foundation for the Advancement of Sciences (KFAS 95–07-02). K.A.C. is a student in the Medical Scientist Training Program (NIH Grant EY07102).

1 Abbreviations used include: ß-subunit-1035 to +24, nucleotides -1035 to +24 of the mouse gene encoding the ß-subunit of H+/K+ ATPase; Inha, mouse gene encoding the {alpha}-subunit of inhibin; inham1/m1; mice homozygous for a null allele (mutant allele number 1) of the Inha gene; ActRII, mouse gene encoding the type II activin receptor; InF, intrinsic factor. Back

Received for publication September 12, 1997. Accepted for publication November 5, 1997.


    REFERENCES
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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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