Homeodomain Transcription Factor PDX-1 Research Articles

Senescence caused by inactivation of the homeodomain transcription factor Pdx1 in adult pancreatic acinar cells in mice.

In this study, we used tamoxifen-inducible Elastase-Cre-mediated inactivation of pancreatic and duodenal homeobox1 (Pdx1), an indispensable gene during embryonic pancreatogenesis, to investigate the role of Pdx1 in adult pancreatic exocrine tissue. We found that Pdx1 depletion in approximately 50% of acinar cell mass did not show any macroscopic phenotype. Lineage tracing experiments revealed that the percentage of Pdx1-depleted cells did not change initially but gradually decreased, while the proliferation of Pdx1-preserved cells increased. Electron microscopic analysis showed the emergence of round-shaped mitochondria with less cristae, dilated ER lumen and increased number of autophagosomes but no apoptosis. Instead, Pdx1-depleted acinar cells became senescent. These findings indicate that intracellular stress caused by Pdx1 inactivation triggers the senescence-associated secretory phenotype to maintain organ homeostasis in this model.

Identification of dynamic undifferentiated cell states within the male germline

The role of stem cells in tissue maintenance is appreciated and hierarchical models of stem cell self-renewal and differentiation often proposed. Stem cell activity in the male germline is restricted to undifferentiated A-type spermatogonia (Aundiff); however, only a fraction of this population act as stem cells in undisturbed testis and Aundiff hierarchy remains contentious. Through newly developed compound reporter mice, here we define molecular signatures of self-renewing and differentiation-primed adult Aundiff fractions and dissect Aundiff heterogeneity by single-cell analysis. We uncover an unappreciated population within the self-renewing Aundiff fraction marked by expression of embryonic patterning genes and homeodomain transcription factor PDX1. Importantly, we find that PDX1 marks a population with potent stem cell capacity unique to mature, homeostatic testis and demonstrate dynamic interconversion between PDX1+ and PDX1− Aundiff states upon transplant and culture. We conclude that Aundiff exist in a series of dynamic cell states with distinct function and provide evidence that stability of such states is dictated by niche-derived cues.

A Pdx-1-Regulated Soluble Factor Activates Rat and Human Islet Cell Proliferation.

The homeodomain transcription factor Pdx-1 has important roles in pancreas and islet development as well as in β-cell function and survival. We previously reported that Pdx-1 overexpression stimulates islet cell proliferation, but the mechanism remains unclear. Here, we demonstrate that overexpression of Pdx-1 triggers proliferation largely by a non-cell-autonomous mechanism mediated by soluble factors. Consistent with this idea, overexpression of Pdx-1 under the control of a β-cell-specific promoter (rat insulin promoter [RIP]) stimulates proliferation of both α and β cells, and overexpression of Pdx-1 in islets separated by a Transwell membrane from islets lacking Pdx-1 overexpression activates proliferation in the untreated islets. Microarray and gene ontology (GO) analysis identified inhibin beta-B (Inhbb), an activin subunit and member of the transforming growth factor β (TGF-β) superfamily, as a Pdx-1-responsive gene. Overexpression of Inhbb or addition of activin B stimulates rat islet cell and β-cell proliferation, and the activin receptors RIIA and RIIB are required for the full proliferative effects of Pdx-1 in rat islets. In human islets, Inhbb overexpression stimulates total islet cell proliferation and potentiates Pdx-1-stimulated proliferation of total islet cells and β cells. In sum, this study identifies a mechanism by which Pdx-1 induces a soluble factor that is sufficient to stimulate both rat and human islet cell proliferation.

303. Deletion of Pdx1 in Mouse Pancreatic Ducts by CRISPR-Cas9 Mediated Gene Editing

Introduction: The homeodomain transcription factor Pdx1 is crucial for pancreas formation. Pdx1-expressing cells are first observed at embryonic day 8.5 (E8.5), prior even to the earliest indication of morphogenesis, in endodermal cells designated to give rise to the pancreas. In adult mouse, Pdx1 protein is transiently expressed when pancreas is injured, such as Langerhans islets damage in alloxan induced diabetic mice, implying Pdx1 may be necessary for the neogenetic formation of β-cells from mature ducts. In order to study the role of pdx1 in the pancreatic duct for pancreatic regeneration, most investigators use the Cre-lox system to generate duct-specific Pdx1 deletions in mice. This method is able to delete Pdx1specifically from tissue or cells with or without a tamoxifen-inducible (cre-ERT2) system; however, several caveats and limitations exist with tamoxifen-induced depletion. In addition, the cre-ERT2 system may be leaky, resulting in constitutive rather than inducible activation in some cells. To overcome these flaws from the Cre-ERT2/lox system, we decided to use a novel CRISPR-Cas9 gene editing technique to delete pdx1 in pancreatic ducts. Methods: We previously used a mouse short Sox9 promoter (468 bp) to generate a recombinant adeno-associated virus (AAV) carrying a reporter GFP (AAV6-Sox9-GFP), and this recombinant virus construct was infused into the pancreatic ducts, through the common bile/pancreatic duct, to specifically tag the duct cells. In order to address pancreatic duct specific deletion of Pdx1 after alloxan ablation of beta-cells, we first to applied the CRISPR-Cas9 gene editing technique on the HEK293-CMV-GFP cell line in vitro (Figure 2Figure 2) and ROSA26 LSL tomato reporter mice ex vivo (Figure 3Figure 3), and then employed AAV6 mediated CRISPR-SaCas9 under the control of the mouse sox9 short promoter, coupled with paired guide RNAs (gRNAs) flanking the Pdx1 exon2 (Figure 4Figure 4) through pancreatic duct infusion into the pancreas. RESULTS AND CONCLUSIONS: These CRISPR-SaCas9 and gRNAs AAV viruses were co-infused into pancreases of C57BL/6J mice 2 days after alloxan treatment. One week after AAV6-SaCas9+gRNAs infusion, it appeared that only the Pdx1 in pancreatic ductal cells were deleted. Comparatively, PBS or AAV6-CMV-GFP viral infused C57BL/6J mice and ROSA26 LSL tomato reporter mice expressed fluorescence in the entire pancreas as a result of the deleted loxP sites that removed the stop cassette, the Pdx1 is not deleted. Thus, we first report here a highly efficient and specific ablation of Pdx1 in adult mouse pancreatic ducts using the CRISPR-Cas9 technique. Figure 1Figure 1. Construction of AAV CRISPR-SaCas9 and gRNA vectors. Figure 2Figure 2. Deletion of ZsGeen in HEK293-CMV-GFP cells using SacCas9 and gRNA in vitro. Figure 3Figure 3. Valiation of SaCas9 and gRNA in vivo deletion of loxP sites in ROSA26-LSL tomato mice pancreas. Figure 4Figure 4. Deletion of Pdx1 exon2 in alloxan treated C57 pancreatic ducts.View Large Image | Download PowerPoint SlideView Large Image | Download PowerPoint SlideView Large Image | Download PowerPoint SlideView Large Image | Download PowerPoint Slide

Diabetes Susceptibility Genes Pdx1 and Clec16a Function in a Pathway Regulating Mitophagy in β-Cells.

Mitophagy is a critical regulator of mitochondrial quality control and is necessary for elimination of dysfunctional mitochondria to maintain cellular respiration. Here, we report that the homeodomain transcription factor Pdx1, a gene associated with both type 2 diabetes and monogenic diabetes of the young, regulates mitophagy in pancreatic β-cells. Loss of Pdx1 leads to abnormal mitochondrial morphology and function as well as impaired mitochondrial turnover. High-throughput expression microarray and chromatin occupancy analyses reveal that Pdx1 regulates the expression of Clec16a, a type 1 diabetes gene and itself a key mediator of mitophagy through regulation of the E3 ubiquitin ligase Nrdp1. Indeed, expression of Clec16a and Nrdp1 are both reduced in Pdx1 haploinsufficient islets, and reduction of Pdx1 impairs fusion of autophagosomes containing mitochondria to lysosomes during mitophagy. Importantly, restoration of Clec16a expression after Pdx1 loss of function restores mitochondrial trafficking during mitophagy and improves mitochondrial respiration and glucose-stimulated insulin release. Thus, Pdx1 orchestrates nuclear control of mitochondrial function in part by controlling mitophagy through Clec16a. The novel Pdx1-Clec16a-Nrdp1 pathway we describe provides a genetic basis for the pathogenesis of mitochondrial dysfunction in multiple forms of diabetes that could be targeted for future therapies to improve β-cell function.

Tshz1 Regulates Pancreatic β-Cell Maturation.

The homeodomain transcription factor Pdx1 controls pancreas organogenesis, specification of endocrine pancreas progenitors, and the postnatal growth and function of pancreatic β-cells. Pdx1 expression in human-derived stem cells is used as a marker for induced pancreatic precursor cells. Unfortunately, the differentiation efficiency of human pancreatic progenitors into functional β-cells is poor. In order to gain insight into the genes that Pdx1 regulates during differentiation, we performed Pdx1 chromatin immunoprecipitation followed by high-throughput sequencing of embryonic day (e) 13.5 and 15.5 mouse pancreata. From this, we identified the transcription factor Teashirt zinc finger 1 (Tshz1) as a direct Pdx1 target. Tshz1 is expressed in developing and adult insulin- and glucagon-positive cells. Endocrine cells are properly specified in Tshz1-null embryos, but critical regulators of β-cell (Pdx1 and Nkx6.1) and α-cell (MafB and Arx) formation and function are downregulated. Adult Tshz1+/− mice display glucose intolerance due to defects in glucose-stimulated insulin secretion associated with reduced Pdx1 and Clec16a expression in Tshz1+/− islets. Lastly, we demonstrate that TSHZ1 levels are reduced in human islets of donors with type 2 diabetes. Thus, we position Tshz1 in the transcriptional network of maturing β-cells and suggest that its dysregulation could contribute to the islet phenotype of human type 2 diabetes.

Glucose regulates protein kinase CK2 in pancreatic β-cells and its interaction with PDX-1

The pancreatic duodenal homeodomain transcription factor PDX-1 plays a pivotal role in the development of the pancreas and the maintenance of glucose homeostasis by pancreatic β-cells. Recently, we found that the highly conserved, ubiquitously expressed tetrameric Ser/Thr protein kinase CK2, which is formed by two catalytic subunits (α and/or α′) and two non-catalytic subunits (β), phosphorylates PDX-1. So far, only little is known about CK2 in pancreatic β-cells and how this enzyme is regulated in these cells. In the present study, we found that (i) CK2 binds to PDX-1, (ii) the binding between CK2 and PDX-1 is regulated by glucose, (iii) glucose modulates the subcellular localization of PDX-1 and CK2 and (iv) the kinase activity is also regulated by glucose.Our novel data indicate that CK2 is a co-factor of PDX-1 in response to glucose in pancreatic β-cells.

Pdx-1 Activates Islet α- and β-Cell Proliferation via a Mechanism Regulated by Transient Receptor Potential Cation Channels 3 and 6 and Extracellular Signal-Regulated Kinases 1 and 2

The homeodomain transcription factor Pdx-1 has important roles in pancreatic development and β-cell function and survival. In the present study, we demonstrate that adenovirus-mediated overexpression of Pdx-1 in rat or human islets also stimulates cell replication. Moreover, cooverexpression of Pdx-1 with another homeodomain transcription factor, Nkx6.1, has an additive effect on proliferation compared to either factor alone, implying discrete activating mechanisms. Consistent with this, Nkx6.1 stimulates mainly β-cell proliferation, whereas Pdx-1 stimulates both α- and β-cell proliferation. Furthermore, cyclins D1/D2 are upregulated by Pdx-1 but not by Nkx6.1, and inhibition of cdk4 blocks Pdx-1-stimulated but not Nkx6.1-stimulated islet cell proliferation. Genes regulated by Pdx-1 but not Nkx6.1 were identified by microarray analysis. Two members of the transient receptor potential cation (TRPC) channel family, TRPC3 and TRPC6, are upregulated by Pdx-1 overexpression, and small interfering RNA (siRNA)-mediated knockdown of TRPC3/6 or TRPC6 alone inhibits Pdx-1-induced but not Nkx6.1-induced islet cell proliferation. Pdx-1 also stimulates extracellular signal-regulated kinase 1 and 2 (ERK1/2) phosphorylation, an effect partially blocked by knockdown of TRPC3/6, and blockade of ERK1/2 activation with a MEK1/2 inhibitor partially impairs Pdx-1-stimulated proliferation. These studies define a pathway by which overexpression of Pdx-1 activates islet cell proliferation that is distinct from and additive to a pathway activated by Nkx6.1.

British Society for Gene and Cell Therapy (BSGCT) Conference AbstractsRoyal HollowayUniversity of London17–19 April, 2013

British Society for Gene and Cell Therapy (BSGCT) Conference AbstractsRoyal HollowayUniversity of London17–19 April, 2013

Intestinal Pdx1 mediates nutrient metabolism gene networks and maternal expression is essential for perinatal growth in mice

The homeodomain transcription factor Pdx1 is essential for pancreas formation and functions in pancreatic islets cells to regulate genes involved in maintenance of glucose homeostasis. In order to investigate a role for Pdx1 in intestinal cells, we analyzed the functions and networks associated with genes differentially expressed by Pdx1 overexpression in human Caco-2 cells. In agreement with previous results for intestine isolated from mice with Pdx1 inactivation, functional analysis of genes differentially expressed with Pdx1 overexpression revealed functions significantly associated with nutrient metabolism. Similarly, network analysis examining the interactions among the differentially expressed genes revealed gene networks involved in lipid metabolism. Consistent with defects in maternal nutrient metabolism, mouse pups born to dams with intestine-specific Pdx1 inactivation are underweight and fail to thrive in the neonatal period compared to pups born to control dams. We conclude that Pdx1 mediates lipid metabolism gene networks in intestinal cells and that maternal expression is essential for perinatal growth in mice.

Phosphoproteomic Identification of a PDX-1/14-3-3ε Interaction in Pancreatic Beta Cells

Glucose-dependent activation of the homeodomain transcription factor PDX-1 leads to its phosphorylation, to an increase in DNA binding capacity, and to NLS dependent translocation into the nucleus. To uncover unknown mediators of PDX-1 activation, PDX-1 interacting proteins were analysed by pull-down from (32)P-labelled, glucose-stimulated MIN6 cells. Recovered proteins were analysed by 2D gel electrophoresis and mass spectrometry. We identified 14-3-3ε as a novel PDX-1 binding protein and confirmed the interaction in vivo by Fluorescence Resonance Energy Transfer (FRET) analysis. We propose that 14-3-3ε interacts directly with PDX-1 to regulate its cellular distribution in pancreatic beta cells.

Biphasic Induction of Pdx1 in Mouse and Human Embryonic Stem Cells Can Mimic Development of Pancreatic β-Cells

Embryonic stem (ES) cells represent a possible source of islet tissue for the treatment of diabetes. Achieving this goal will require a detailed understanding of how the transcription factor cascade initiated by the homeodomain transcription factor Pdx1 culminates in pancreatic beta-cell development. Here we describe a genetic approach that enables fine control of Pdx1 transcriptional activity during endoderm differentiation of mouse and human ES cell. By activating an exogenous Pdx1VP16 protein in populations of cells enriched in definitive endoderm we show a distinct lineage-dependent requirement for this transcription factor's activity. Mimicking the natural biphasic pattern of Pdx1 expression was necessary to induce an endocrine pancreas-like cell phenotype, in which 30% of the cells were beta-cell-like. Cell markers consistent with the different beta-cell differentiation stages appeared in a sequential order following the natural pattern of pancreatic development. Furthermore, in mouse ES-derived cultures the differentiated beta-like cells secreted C-peptide (insulin) in response to KCl and 3-isobutyl-1-methylxanthine, suggesting that following a natural path of development in vitro represents the best approach to generate functional pancreatic cells. Together these results reveal for the first time a significant effect of the timed expression of Pdx1 on the non-beta-cells in the developing endocrine pancreas. Collectively, we show that this method of in vitro differentiation provides a template for inducing and studying ES cell differentiation into insulin-secreting cells.

Two conserved domains in PCIF1 mediate interaction with pancreatic transcription factor PDX-1

PCIF1 is a TRAF and POZ domain containing nuclear factor that interacts with and inhibits transactivation of pancreatic homeodomain transcription factor PDX-1. Here, we demonstrate interaction of endogenous PDX-1 and PCIF1 in MIN6 insulinoma cells. Within PCIF1, the TRAF and POZ domains are both required for physical and functional interaction with the C-terminus of PDX-1, whereas the C-terminal domain of PCIF1 directs its nuclear localization. A human PDX-1 mutation associated with diabetes, E224K, disrupts the ability of PCIF1 to inhibit PDX-1 transactivation, suggesting that the interaction between PDX-1 and PCIF1 is required for normal glucose homeostasis. Inhibition of transactivation occurs by a mechanism distinct from the classical role of POZ domains to recruit co-repressors and histone deacetylases. Understanding the functional roles of PCIF1 domains may have application to therapeutic β-cell replacement strategies involving PDX-1 for the treatment of diabetes.

Interactions with p300 enhance transcriptional activation by the PDZ-domain coactivator Bridge-1

Transcriptional coactivators are essential mediators of signal amplification in the regulation of gene expression in response to hormones and extracellular signals. We previously identified Bridge-1 (PSMD9) as a PDZ-domain coregulator that augments insulin gene transcription via interactions with the basic helix-loop-helix transcription factors E12 and E47, and that increases transcriptional activation by the homeodomain transcription factor PDX-1. In these studies, we find that transcriptional activation by Bridge-1 can be regulated via interactions with the histone acetyltransferase and nuclear receptor coactivator p300. In transfection assays, transcriptional activation by Bridge-1 is increased by the inhibition of endogenous histone deacetylase activity with trichostatin A, indicating that the transcriptional activation function of Bridge-1 can be regulated by histone modifications. The exogenous expression of p300 enhances the transcriptional activation by Bridge-1 in a dose-dependent manner. In contrast, the sequestration of p300 by the overexpression of the adenoviral protein E1A, but not by an E1A mutant protein that is unable to interact with p300, suppresses the transcriptional activation by Bridge-1. We demonstrate that p300 and Bridge-1 proteins interact in immunopre-cipitation and glutathione-S-transferase (GST) pull-down assays. Bridge-1 interacts directly with multiple regions within p300 that encompass C/H1 or C/H2 cysteine- and histidine-rich protein interaction domains and the histone acetyltransferase domain. Deletion or point mutagenesis of the Bridge-1 PDZ domain substantially reduces transcriptional activation by Bridge-1 and interrupts interactions with p300. We propose that p300 interactions with Bridge-1 can augment the transcriptional activation of regulatory target genes by Bridge-1.

Development of a single-cassette system for spatiotemporal gene regulation in mice

The tetracycline-regulated gene expression system has been widely used in mice to turn a transgene on and off in a target organ, but with only limited success. We developed an advanced system in which a Tet-off regulation unit was integrated into the ROSA26 locus and became active after Cre-mediated excision of the neo r gene. We examined the utility of this system through regulable expression of the homeodomain transcription factor pdx-1 and enhanced green fluorescent protein. The resulting mice showed strict tetracycline-regulable gene expression in all the organs where the neo r gene had been removed. When combined with organ-specific Cre recombinase transgenic mice, our system allows us to manipulate the gene expression in an organ-specific and temporal manner. This Tet-off system should serve as an efficient tool to analyze the roles of genes in complex biological systems, such as embryogenesis, metabolism, immune system, etc.

Conditional Expression Demonstrates the Role of the Homeodomain Transcription Factor Pdx1 in Maintenance and Regeneration of β-Cells in the Adult Pancreas

The homeodomain transcription factor Pdx1 is essential for pancreas development. To investigate the role of Pdx1 in the adult pancreas, we employed a mouse model in which transcription of Pdx1 could be reversibly repressed by administration of doxycycline. Repression of Pdx1 in adult mice impaired expression of insulin and glucagon, leading to diabetes within 14 days. Pdx1 repression was associated with increased cell proliferation predominantly in the exocrine pancreas and upregulation of genes implicated in pancreas regeneration. Following withdrawal of doxycycline and derepression of Pdx1, normoglycemia was restored within 28 days; during this period, Pdx1(+)/Ins(+) and Pdx(+)/Ins(-) cells were observed in association with the duct epithelia. These findings confirm that Pdx1 is required for beta-cell function in the adult pancreas and indicate that in the absence of Pdx1 expression, a regenerative program is initiated with the potential for Pdx1-dependent beta-cell neogenesis.

Pdx1-related homeodomain transcription factors are distinctly expressed in mouse adult pancreatic islets

Complex gene networks are responsible for the proper operation of the endocrine pancreas. A central member of such networks, the homeodomain transcription factor Pdx1, belongs to the ParaHox gene cluster, an array of Hox-like homeobox genes. With a combination of mRNA in situ hybridisation and immunodetection, we have found that the rest of ParaHox cluster genes, Cdx1, Cdx2/3, and Cdx4, and Gsh1 and Gsh2, are all expressed in specific islet cell types of the endocrine pancreas. To our knowledge, this is the first report that locates ParaHox genes other than Pdx1 and Cdx2/3 in a place as to be involved in the pancreatic transcriptional regulatory networks, potentially regulating glucagon–insulin homeostasis.

In vitro induction of adult hepatic progenitor cells into insulin-producing cells

Organ-specific stem cells are the natural progenitors in tissue regeneration and possess plasticity to differentiate into specialized cells in adult tissues. Small hepatocytes (SHCs) identified in the adult liver are one such cell type. Here we show that SHCs, which are capable of self-renewal and differentiation into hepatocytes, can be induced to generate insulin-producing cells under appropriate culture conditions. These differentiated cells express pancreatic β cell differentiation-related transcripts and hepatocyte differentiation-related transcripts, as shown by reverse-transcription PCR/nested PCR. In addition, enforced expression of the homeodomain transcription factor Pdx1 in these cells contributes to enhancement of insulin release in response to insulin secretagogues. These results indicate that the SHCs described here have the ability to differentiate into insulin-producing cells, and further support the idea that engineering to generate insulin-secreting cells could provide a useful resource for future therapies for diabetes mellitus.

Gastric PDX-1 expression in pancreatic metaplasia and endocrine cell hyperplasia in atrophic corpus gastritis

The homeodomain transcription factor PDX-1 plays a key role in endocrine and exocrine differentiation processes of the pancreas. PDX-1 is also essential for differentiation of endocrine cells in the gastric antrum. The role of PDX-1 in the pathogenesis of endocrine cell hyperplasia and pancreatic metaplasia in corpus and fundus gastritis has not been evaluated. By immunohistochemistry and double-immunofluorescence, we investigated the expression of PDX-1 in 10 tissue specimens with normal human gastric mucosa, nonatrophic and atrophic gastritis and in pancreatic metaplasia, respectively. In normal corpus mucosa and in nonatrophic corpus gastritis, PDX-1 was mainly absent. In pancreatic metaplasia, PDX-1 was found in metaplastic cells and in adjacent gastric glands. In contrast to normal gastric corpus mucosa, PDX-1 could be strongly detected in the cytoplasm of the parietal cells surrounding metaplastic areas. Furthermore, PDX-1 expression was found in hyperplastic endocrine cells and in the surrounding gastric glands in chronic atrophic gastritis. Hyperplastic endocrine cells coexpressed the β-subunit of the gastric H,K-ATPase. We conclude that PDX-1 represents a candidate switch factor for glandular exocrine and endocrine transdifferentiation in chronic gastritis and that an impaired parietal cell differentiation might play a key role in disturbed gastric morphogenic processes.

Gastric PDX-1 expression in pancreatic metaplasia and endocrine cell hyperplasia in atrophic corpus gastritis

The homeodomain transcription factor PDX-1 plays a key role in endocrine and exocrine differentiation processes of the pancreas. PDX-1 is also essential for differentiation of endocrine cells in the gastric antrum. The role of PDX-1 in the pathogenesis of endocrine cell hyperplasia and pancreatic metaplasia in corpus and fundus gastritis has not been evaluated. By immunohistochemistry and double-immunofluorescence, we investigated the expression of PDX-1 in 10 tissue specimens with normal human gastric mucosa, nonatrophic and atrophic gastritis and in pancreatic metaplasia, respectively. In normal corpus mucosa and in nonatrophic corpus gastritis, PDX-1 was mainly absent. In pancreatic metaplasia, PDX-1 was found in metaplastic cells and in adjacent gastric glands. In contrast to normal gastric corpus mucosa, PDX-1 could be strongly detected in the cytoplasm of the parietal cells surrounding metaplastic areas. Furthermore, PDX-1 expression was found in hyperplastic endocrine cells and in the surrounding gastric glands in chronic atrophic gastritis. Hyperplastic endocrine cells coexpressed the β-subunit of the gastric H,K-ATPase. We conclude that PDX-1 represents a candidate switch factor for glandular exocrine and endocrine transdifferentiation in chronic gastritis and that an impaired parietal cell differentiation might play a key role in disturbed gastric morphogenic processes.