BACKGROUND & AIMS:Pancreatitis is a major cause of morbidity and mortality and is a risk factor for pancreatic tumorigenesis. Upon tissue damage, an inflammatory response, made up largely of macrophages, provides multiple growth factors that promote repair. Here, we examine the molecular pathways initiated by macrophages to promote pancreas recovery from pancreatitis. METHODS:To induce organ damage, mice were subjected to cerulein-induced experimental pancreatitis and analyzed at various times of recovery. CD11b-DTR mice were used to deplete myeloid cells. Hbegff/f;LysM-Cre mice were used to ablate myeloid cell-derived heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF). To ablate EGFR specifically during recovery, pancreatitis was induced in Egfrf/f;Ptf1aFlpO/+;FSF-Rosa26CAG-CreERT2 mice followed by tamoxifen treatment. RESULTS:Macrophages infiltrating the pancreas in experimental pancreatitis make high levels of HB-EGF. Both depletion of myeloid cells and ablation of myeloid cell HB-EGF delayed recovery from experimental pancreatitis, resulting from a decrease in cell proliferation and an increase in apoptosis. Mechanistically, ablation of myeloid cell HB-EGF impaired epithelial cell DNA repair, ultimately leading to cell death. Soluble HB-EGF induced EGFR nuclear translocation and methylation of histone H4, facilitating resolution of DNA damage in pancreatic acinar cells in vitro. Consistent with its role as the primary receptor of HB-EGF, in vivo ablation of EGFR from pancreatic epithelium during recovery from pancreatitis resulted in accumulation of DNA damage. CONCLUSIONS:By using novel conditional knockout mouse models, we determined that HB-EGF derived exclusively from myeloid cells induces epithelial cell proliferation and EGFR-dependent DNA repair, facilitating pancreas healing after injury.

Objective: Homozygous loss-of-function mutations in the gene coding for the homeobox transcription factor (TF) PDX1 leads to pancreatic agenesis, whereas heterozygous mutations can cause Maturity-Onset Diabetes of the Young 4 (MODY4). Although the function of Pdx1 is well studied in pre-clinical models during insulin-producing beta-cell development and homeostasis, it remains elusive how this TF controls human pancreas development by regulating a downstream transcriptional program. Also, comparative studies of PDX1 binding patterns in pancreatic progenitors and adult beta-cells have not been conducted so far. Furthermore, many studies reported the association between single nucleotide polymorphisms (SNPs) and T2DM, and it has been shown that islet enhancers are enriched in T2DM-associated SNPs. Whether regions, harboring T2DM-associated SNPs are PDX1 bound and active at the pancreatic progenitor stage has not been reported so far. Methods: In this study, we have generated a novel induced pluripotent stem cell (iPSC) line that efficiently differentiates into human pancreatic progenitors (PPs). Furthermore, PDX1 and H3K27ac chromatin immunoprecipitation sequencing (ChIP-seq) was used to identify PDX1 transcriptional targets and active enhancer and promoter regions. To address potential differences in the function of PDX1 during development and adulthood, we compared PDX1 binding profiles from PPs and adult islets. Moreover, combining ChIP-seq and GWAS meta-analysis data we identified T2DM-associated SNPs in PDX1 binding sites and active chromatin regions. Results: ChIP-seq for PDX1 revealed a total of 8088 PDX1-bound regions that map to 5664 genes in iPSC-derived PPs. The PDX1 target regions include important pancreatic TFs, such as PDX1 itself, RFX6, HNF1B, and ME1S1, which were activated during the differentiation process as revealed by the active chromatin mark H3K27ac and mRNA expression profiling, suggesting that auto-regulatory feedback regulation maintains PDX1 expression and initiates a pancreatic TF program. Remarkably, we identified several PDX1 target genes that have not been reported in the literature in human so far, including RFX3, required for ciliogenesis and endocrine differentiation in mouse, and the ligand of the Notch receptor DLL1, which is important for endocrine induction and tip-trunk patterning. The comparison of PDX1 profiles from PPs and adult human islets identified sets of stage-specific target genes, associated with early pancreas development and adult beta-cell function, respectively. Furthermore, we found an enrichment of T2DM-associated SNPs in active chromatin regions from iPSC-derived PPs. Two of these SNPs fall into PDX1 occupied sites that are located in the intronic regions of TCF7L2 and HNF1B. Both of these genes are key transcriptional regulators of endocrine induction and mutations in cis-regulatory regions predispose to diabetes. Conclusions: Our data provide stage-specific target genes of PDX1 during in vitro differentiation of stem cells into pancreatic progenitors that could be useful to identify pathways and molecular targets that predispose for diabetes. In addition, we show that T2DM-associated SNPs are enriched in active chromatin regions at the pancreatic progenitor stage, suggesting that the susceptibility to T2DM might originate from imperfect execution of a beta-cell developmental program. (C) 2018 The Authors. Published by Elsevier GmbH.

Ptf1a and Pdx1 are critical transcription factors of early pancreatic development, as shown by loss of function studies where lack of each gene alone causes almost complete pancreas agenesis. Ptf1a is particularly interesting because it is linked to a recently reported signature gene expression profile associated with the multipotent condition. Few useful antibody reagents have been available for consistent and reliable immunohistochemical visualization of Ptf1a protein expression in the early developing pancreas in which the level of production of this critical regulator seems to be very low. We describe a novel rabbit antibody raised against the c-terminal portion of the mouse Ptf1a protein and report immunodetection, for the first time, as early as embryonic day (e) 8.5-e8.75 in the dorsal and ventral buds of the mouse pancreas as well as in the neural tube at e10.0. Detailed confocal analysis identifies an abundant triple-positive (Ptf1a(+)/Nkx6.1(+)/Pdx1(+)) putative early multipotent pancreatic progenitor cell that marks the e9.5 dorsal pancreas and e10.5 ventral pancreas. Furthermore, expression patterns of Nkx6.1 vs Ptf1a subsequently segregate during branching morphogenesis (trunk vs tip), ending up marking two distinct cell populations of progenitors at e12.5. From e15.5 (mouse) and in adult pancreas (mouse, rat, and human), the Ptf1a antibody marks only acinar cell nuclei, as expected for its subsequent role in committing/maintaining cells in this differentiated state. In summary, this antibody is a novel tool to further characterize important early steps of pancreas differentiation. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.

Pdx1 (IPF-1 in humans, which is altered in MODY-4) is essential for pancreas development and mature beta-cell function. Pdx1 is expressed dynamically within the developing foregut, but how its expression characteristics are linked to the various steps of organ specification, differentiation, and function is unknown. Deletion of a conserved enhancer region (Area I-II-III) from Pdx1 produced a hypomorphic allele (Pdx1(DeltaI-II-III)) with altered timing and level of expression, which was studied in combination with wild-type and protein-null alleles. Lineage labeling in homozygous Area I-II-III deletion mutants (Pdx1(DeltaI-II-III/DeltaI-II-III)) revealed lack of ventral pancreatic bud specification and early-onset hypoplasia in the dorsal bud. Acinar tissue formed in the hypoplastic dorsal bud, but endocrine maturation was greatly impaired. While Pdx1(-/-) (protein-null) mice have nonpancreatic abnormalities (e.g., distorted pylorus, absent Brunner's glands), these structures formed normally in Pdx1(DeltaI-II-III/DeltaI-II-III) and Pdx1(DeltaI-II-III/-) mice. Surprisingly, heterozygous (Pdx1(+/DeltaI-II-III)) mice had abnormal islets and a more severe prediabetic condition than Pdx1(+/-) mice. These findings provide in vivo evidence of the differential requirements for the level of Pdx1 gene activity in the specification and differentiation of the various organs of the posterior foregut, as well as in pancreas and gut endocrine cell differentiation.

Pancreas development begins with the formation of buds at specific sites in the embryonic foregut endoderm. We used recombination-based lineage tracing in vivo to show that Ptf1a (also known as PTF1-p48) is expressed at these early stages in the progenitors of pancreatic ducts, exocrine and endocrine cells, rather than being an exocrine-specific gene as previously described. Moreover, inactivation of Ptf1a switches the character of pancreatic progenitors such that their progeny proliferate in and adopt the normal fates of duodenal epithelium, including its stem-cell compartment. Consistent with the proposal that Ptf1a supports the specification of precursors of all three pancreatic cell types, transgene-based expression of Pdx1, a gene essential to pancreas formation, from Ptf1a cis-regulatory sequences restores pancreas tissue to Pdx1-null mice that otherwise lack mature exocrine and endocrine cells because of an early arrest in organogenesis. These experiments provide evidence that Ptf1a expression is specifically connected to the acquisition of pancreatic fate by undifferentiated foregut endoderm.

We used transgenesis to explore the requirement for downregulation of hepatocyte nuclear factor 6 (HNF6) expression in the assembly, differentiation, and function of pancreatic islets. In vivo, HNF6 expression becomes downregulated in pancreatic endocrine cells at 18. 5 days post coitum (d.p.c.), when definitive islets first begin to organize. We used an islet-specific regulatory element (pdx1(PB)) from pancreatic/duodenal homeobox (pdx1) gene to maintain HNF6 expression in endocrine cells beyond 18.5 d.p.c. Transgenic animals were diabetic. HNF6-overexpressing islets were hyperplastic and remained very close to the pancreatic ducts. Strikingly, alpha, delta, and PP cells were increased in number and abnormally intermingled with islet beta cells. Although several mature beta cell markers were expressed in beta cells of transgenic islets, the glucose transporter GLUT2 was absent or severely reduced. As glucose uptake/metabolism is essential for insulin secretion, decreased GLUT2 may contribute to the etiology of diabetes in pdx1(PB)-HNF6 transgenics. Concordantly, blood insulin was not raised by glucose challenge, suggesting profound beta cell dysfunction. Thus, we have shown that HNF6 downregulation during islet ontogeny is critical to normal pancreas formation and function: continued expression impairs the clustering of endocrine cells and their separation from the ductal epithelium, disrupts the spatial organization of endocrine cell types within the islet, and severely compromises beta cell physiology, leading to overt diabetes.