The Parahox gene Pdx1 is required to maintain positional identity in the adult foregut.

The homeobox gene Pdx1 is a key regulator of pancreas and foregut development. Loss of Pdx1 expression results in pancreas agenesis and impaired development of the gastro-duodenal domain including Brunner’s glands. We previously demonstrated a key role for Pdx1 in maintaining the integrity and function of insulin-secreting beta cells in the adult pancreas. In the present study, we aimed to determine if expression of Pdx1 is required to maintain the cellular identity of the gastro-duodenal domain in adult mice. Immunohistological studies were performed in a mouse model in which expression of Pdx1 was conditionally repressed with the doxycycline-responsive tetracycline transactivator system. Mice in which Pdx1 was chronically repressed developed hamartomas in the gastro-duodenal domain. These lesions appeared to arise from ectopic foci of anteriorized cells, consistent with a localised anterior homeotic shift. They emerge with the intercalation of tissue between the anteriorized and normal domains and appear strikingly similar to lesions in the colon of mice heterozygous for another Parahox gene, Cdx2. Continuing expression of Pdx1 into adult life is required to maintain regional cellular identity in the adult foregut, specifically at the gastro-duodenal boundary. Loss of Pdx1 expression leads to anterior transformation and intercalary regeneration of ectopic tissue. We propose a model in which the posterior dominance of classical Hox genes is mirrored by the Parahox genes, providing further evidence of the functional conservation of the Parahox genes. These findings may have implications for further understanding the molecular basis of gastro-duodenal metaplasia and gastro-intestinal transformations such as Barrett’s esophagus.

Temporal restriction of pancreatic branching competence during embryogenesis is mirrored in differentiating embryonic stem cells.

To develop methods for the generation of insulin-producing ß-cells for the treatment of diabetes, we have used GFP-tagged embryonic stem cells (ESCs) to elucidate the process of pancreas development. Using the reporter Pdx1(GFP/w) ESC line, we have previously described a serum-free differentiation protocol in which Pdx1-GFP(+) cells formed GFP bright (GFP(br)) epithelial buds that resembled those present in the developing mouse pancreas. In this study we extend these findings to demonstrate that these cells can undergo a process of branching morphogenesis, similar to that seen during pancreatic development of the mid-gestation embryo. These partially disaggregated embryoid bodies containing GFP(br) buds initially form epithelial ring-like structures when cultured in Matrigel. After several days in culture, these rings undergo a process of proliferation and form a ramified network of epithelial branches. Comparative analysis of explanted dissociated pancreatic buds from E13.5 Pdx1(GFP/w) embryos and ESC-derived GFP(br) buds reveal a similar process of proliferation and branching, with both embryonic Pdx1(GFP/w) branching pancreatic epithelium and ESC-derived GFP(br) branching organoids expressing markers representing epithelial (EpCAM and E-Cadherin), ductal (Mucin1), exocrine (Amylase and Carboxypeptidase 1A), and endocrine cell types (Glucagon and Somatostatin). ESC-derived branching structures also expressed a suite of genes indicative of ongoing pancreatic differentiation, paralleling gene expression within similar structures derived from the E13.5 fetal pancreas. In summary, differentiating mouse ESCs can generate pancreatic material that has significant similarity to the fetal pancreatic anlagen, providing an in vitro platform for investigating the cellular and molecular mechanisms underpinning pancreatic development.

A protocol for removal of antibiotic resistance cassettes from human embryonic stem cells genetically modified by homologous recombination or transgenesis.

The first step in the generation of genetically tagged human embryonic stem cell (HESC) reporter lines is the isolation of cells that contain a stably integrated copy of the reporter vector. These cells are identified by their continued growth in the presence of a specific selective agent, usually conferred by a cassette encoding antibiotic resistance. In order to mitigate potential interference between the regulatory elements driving expression of the antibiotic resistance gene and those controlling the reporter gene, it is advisable to remove the positive selection cassette once the desired clones have been identified. This report describes a protocol for the removal of loxP-flanked selection cassettes from genetically modified HESCs by transient transfection with a vector expressing Cre recombinase. An integrated procedure for the clonal isolation of these genetically modified lines using single-cell deposition flow cytometry is also detailed. When performed sequentially, these protocols take approximately 1 month.

A mouse carrying the green fluorescent protein gene targeted to the Pdx1 locus facilitates the study of pancreas development and function.

The pancreatic and duodenal homeobox gene 1 (Pdx1) has multiple roles in the specification and development of foregut endoderm-derived tissues. We report the characterization of a mouse line in which the gene encoding green fluorescent protein (GFP) has been targeted to the Pdx1 locus, allowing the visualization of Pdx1 expressing cells. Analysis of GFP expression during development showed that the reporter faithfully reproduced the known expression pattern of Pdx1. We demonstrate the utility of this mouse line for the isolation of Pdx1(+) cells by fluorescence-activated cell sorting and for the real-time observation of Pdx1(+) cells in an ex vivo embryonic pancreas culture system. This mouse model should prove useful for the study of pancreas development and regeneration.

The homeodomain protein PDX1 is required at mid-pancreatic development for the formation of the exocrine pancreas.

The homeoprotein PDX1 is expressed throughout pancreatic development and is thought to play important roles at multiple stages. We describe the properties of a tet-off regulatory scheme to manage the expression of Pdx1 in utero. Cessation of Pdx1 expression at increasingly later gestational times blocked pancreatic development at progressive and morphologically distinct stages and provided the opportunity to assess the requirement for Pdx1 at each stage. Embryonic PDX1 is depleted below effective levels within 1 day of the initiation of doxycycline treatment of pregnant mice. We show that PDX1, which is necessary for early pancreatic development, is also required later for the genesis of acinar tissue, the compartment of the pancreas that produces digestive enzymes. Without PDX1, acini do not form; the precursor epithelium continues to grow and branch, creating a truncated ductal tree comprising immature duct-like cells. The bHLH factor PTF1a, a critical regulator of acinar development, is not expressed and cells producing digestive enzymes are rare. This approach should be generally applicable to study the in vivo functions of other developmental regulators with multiple, temporally distinct roles.

Conditional expression demonstrates the role of the homeodomain transcription factor Pdx1 in maintenance and regeneration of beta-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.

Progenitor cells in the adult pancreas.

The beta-cell mass in the adult pancreas possesses the ability to undergo limited regeneration following injury. Identifying the progenitor cells involved in this process and understanding the mechanisms leading to their maturation will open new avenues for the treatment of type 1 diabetes. However, despite steady advances in determining the molecular basis of early pancreatic development, the identification of pancreatic stem cells or beta-cell progenitors and the molecular mechanisms underlying beta-cell regeneration remain unclear. Recent advances in the directed differentiation of embryonic and adult stem cells has heightened interest in the possible application of stem cell therapy in the treatment of type 1 diabetes. Drawing on the expanding knowledge of pancreas development, beta-cell regeneration and stem cell research, this review focuses on progenitor cells in the adult pancreas as a potential source of beta-cells.

Experimental control of pancreatic development and maintenance.

To investigate the role of the HOX-like homeoprotein PDX1 in the formation and maintenance of the pancreas, we have genetically engineered mice so that the only source of PDX1 is a transgene that can be controlled by the application of tetracycline or its analogue doxycycline. In these mice the coding region for the tetracycline-regulated transactivator (tTA(off)) has replaced the coding region of the endogenous Pdx1 gene to ensure correct temporal and spatial expression of the regulatable transactivator. In the absence of doxycycline, tTA(off) activates the transcription of a bicistronic transgene encoding PDX1 and an enhanced green fluorescent protein reporter, which acts as a visual marker of transgene expression in living cells. Expression of the transgene-encoded PDX1 rescues the Pdx1-null phenotype; the pancreata of these mice develop and function normally. The rescue is conditional; doxycycline-mediated repression of the transgenic Pdx1 throughout gestation recapitulates the Pdx1 null phenotype. Moreover, application of doxycycline at mid-pancreogenesis blocks further development. Adult animals of the rescue genotype that were treated with doxycycline for 3 weeks shut off Pdx1 expression, decreased insulin production, and lost the ability to maintain glucose homeostasis. These results demonstrate the feasibility of controlling the formation of an organ during embryogenesis in utero and the maintenance of the mature organ through the experimental manipulation of a key developmental regulator.