Pancreatic Transcription Factors Containing Protein Transduction Domains Drive Mouse Embryonic Stem Cells towards Endocrine Pancreas

Protein transduction domains (PTDs), such as the HIV1-TAT peptide, have been previously used to promote the uptake of proteins into a range of cell types, including stem cells. Here we generated pancreatic transcription factors containing PTD sequences and administered these to endoderm enriched mouse embryonic stem (ES) cells under conditions that were designed to mimic the pattern of expression of these factors in the developing pancreas. The ES cells were first cultured as embryoid bodies and treated with Activin A and Bone morphogenetic protein 4 (BMP4) to promote formation of definitive endoderm. Cells were subsequently plated as a monolayer and treated with different combinations of the modified recombinant transcription factors Pdx1 and MafA. The results demonstrate that each transcription factor was efficiently taken up by the cells, where they were localized in the nuclei. RT-qPCR was used to measure the expression levels of pancreatic markers. After the addition of Pdx1 alone for a period of five days, followed by the combination of Pdx1 and TAT-MafA in a second phase, up-regulation of insulin 1, insulin 2, Pdx1, Glut2, Pax4 and Nkx6.1 was observed. As assessed by immunocytochemistry, double positive insulin and Pdx1 cells were detected in the differentiated cultures. Although the pattern of pancreatic markers expression in these cultures was comparable to that of a mouse transformed β-cell line (MIN-6) and human islets, the expression levels of insulin observed in the differentiated ES cell cultures were several orders of magnitude lower. This suggests that, although PTD-TFs may prove useful in studying the role of exogenous TFs in the differentiation of ES cells towards islets and other pancreatic lineages, the amount of insulin generated is well below that required for therapeutically useful cells

The role of ARF6 in biliary atresia

Background & Aims: Altered extrahepatic bile ducts, gut, and cardiovascular anomalies constitute the variable phenotype of biliary atresia (BA). Methods: To identify potential susceptibility loci, Caucasian children, normal (controls) and with BA (cases) at two US centers were compared at >550000 SNP loci. Systems biology analysis was carried out on the data. In order to validate a key gene identified in the analysis, biliary morphogenesis was evaluated in 2-5-day post-fertilization zebrafish embryos after morpholino-antisense oligonucleotide knockdown of the candidate gene ADP ribosylation factor-6 (ARF6, Mo-arf6). Results: Among 39 and 24 cases at centers 1 and 2, respectively, and 1907 controls, which clustered together on principal component analysis, the SNPs rs3126184 and rs10140366 in a 3' flanking enhancer region for ARF6 demonstrated higher minor allele frequencies (MAF) in each cohort, and 63 combined cases, compared with controls (0.286 vs. 0.131, P = 5.94 x 10-7, OR 2.66; 0.286 vs. 0.13, P = 5.57 x 10-7, OR 2.66). Significance was enhanced in 77 total cases, which included 14 additional BA genotyped at rs3126184 only (p = 1.58 x 10-2, OR = 2.66). Pathway analysis of the 1000 top-ranked SNPs in CHP cases revealed enrichment of genes for EGF regulators (p<1 x 10-7), ERK/MAPK and CREB canonical pathways (p<1 x 10-34), and functional networks for cellular development and proliferation (p<1 x 10-45), further supporting the role of EGFR-ARF6 signaling in BA. In zebrafish embryos, Mo-arf6 injection resulted in a sparse intrahepatic biliary network, several biliary epithelial cell defects, and poor bile excretion to the gall bladder compared with uninjected embryos. Biliary defects were reproduced with the EGFR-blocker AG1478 alone or with Mo-arf6 at lower doses of each agent and rescued with arf6 mRNA. Conclusions: The BA-associated SNPs identify a chromosome 14q21.3 susceptibility locus encompassing the ARF6 gene. arf6 knockdown in zebrafish implicates early biliary dysgenesis as a basis for BA, and also suggests a role for EGFR signaling in BA pathogenesis

Activation of PyMT in β Cells Induces Irreversible Hyperplasia, but Oncogene-Dependent Acinar Cell Carcinomas When Activated in Pancreatic Progenitors

It is unclear whether the cellular origin of various forms of pancreatic cancer involves transformation or transdifferentiation of different target cells or whether tumors arise from common precursors, with tumor types determined by the specific genetic alterations. Previous studies suggested that pancreatic ductal carcinomas might be induced by polyoma middle T antigen (PyMT) expressed in non-ductal cells. To ask whether PyMT transforms and transdifferentiates endocrine cells toward exocrine tumor phenotypes, we generated transgenic mice that carry tetracycline-inducible PyMT and a linked luciferase reporter. Induction of PyMT in β cells causes β-cell hyperplastic lesions that do not progress to malignant neoplasms. When PyMT is de-induced, β cell proliferation and growth cease; however, regression does not occur, suggesting that continued production of PyMT is not required to maintain the viable expanded β cell population. In contrast, induction of PyMT in early pancreatic progenitor cells under the control of Pdx1 produces acinar cell carcinomas and β-cell hyperplasia. The survival of acinar tumor cells is dependent on continued expression of PyMT. Our findings indicate that PyMT can induce exocrine tumors from pancreatic progenitor cells, but cells in the β cell lineage are not transdifferentiated toward exocrine cell types by PyMT; instead, they undergo oncogene-dependent hyperplastic growth, but do not require PyMT for survival

Impaired Spleen Formation Perturbs Morphogenesis of the Gastric Lobe of the Pancreas

Despite the extensive use of the mouse as a model for studies of pancreas development and disease, the development of the gastric pancreatic lobe has been largely overlooked. In this study we use optical projection tomography to provide a detailed three-dimensional and quantitative description of pancreatic growth dynamics in the mouse. Hereby, we describe the epithelial and mesenchymal events leading to the formation of the gastric lobe of the pancreas. We show that this structure forms by perpendicular growth from the dorsal pancreatic epithelium into a distinct lateral domain of the dorsal pancreatic mesenchyme. Our data support a role for spleen organogenesis in the establishment of this mesenchymal domain and in mice displaying perturbed spleen development, including Dh +/−, Bapx1−/− and Sox11−/−, gastric lobe development is disturbed. We further show that the expression profile of markers for multipotent progenitors is delayed in the gastric lobe as compared to the splenic and duodenal pancreatic lobes. Altogether, this study provides new information regarding the developmental dynamics underlying the formation of the gastric lobe of the pancreas and recognizes lobular heterogeneities regarding the time course of pancreatic cellular differentiation. Collectively, these data are likely to constitute important elements in future interpretations of the developing and/or diseased pancreas

Dicer Is Required for Maintaining Adult Pancreas

Dicer1, an essential component of RNA interference and the microRNA pathway, has many important roles in the morphogenesis of developing tissues. Dicer1 null mice have been reported to die at E7.5; therefore it is impossible to study its function in adult tissues. We previously reported that Dicer1-hypomorphic mice, whose Dicer1 expression was reduced to 20% in all tissues, were unexpectedly viable. Here we analyzed these mice to ascertain whether the down-regulation of Dicer1 expression has any influence on adult tissues. Interestingly, all tissues of adult (8–10 week old) Dicer1-hypomorphic mice were histologically normal except for the pancreas, whose development was normal at the fetal and neonatal stages; however, morphologic abnormalities in Dicer1-hypomorphic mice were detected after 4 weeks of age. This suggested that Dicer1 is important for maintaining the adult pancreas

Dicer Is Required for Maintaining Adult Pancreas

Dicer1, an essential component of RNA interference and the microRNA pathway, has many important roles in the morphogenesis of developing tissues. Dicer1 null mice have been reported to die at E7.5; therefore it is impossible to study its function in adult tissues. We previously reported that Dicer1-hypomorphic mice, whose Dicer1 expression was reduced to 20% in all tissues, were unexpectedly viable. Here we analyzed these mice to ascertain whether the down-regulation of Dicer1 expression has any influence on adult tissues. Interestingly, all tissues of adult (8–10 week old) Dicer1-hypomorphic mice were histologically normal except for the pancreas, whose development was normal at the fetal and neonatal stages; however, morphologic abnormalities in Dicer1-hypomorphic mice were detected after 4 weeks of age. This suggested that Dicer1 is important for maintaining the adult pancreas

Sox9-Haploinsufficiency Causes Glucose Intolerance in Mice

The HMG box transcription factor Sox9 plays a critical role in progenitor cell expansion during pancreas organogenesis and is required for proper endocrine cell development in the embryo. Based on in vitro studies it has been suggested that Sox9 controls expression of a network of important developmental regulators, including Tcf2/MODY5, Hnf6, and Foxa2, in pancreatic progenitor cells. Here, we sought to: 1) determine whether Sox9 regulates this transcriptional network in vivo and 2) investigate whether reduced Sox9 gene dosage leads to impaired glucose homeostasis in adult mice. Employing two genetic models of temporally-controlled Sox9 inactivation in pancreatic progenitor cells, we demonstrate that contrary to in vitro findings, Sox9 is not required for Tcf2, Hnf6, or Foxa2 expression in vivo. Moreover, our analysis revealed a novel role for Sox9 in maintaining the expression of Pdx1/MODY4, which is an important transcriptional regulator of beta-cell development. We further show that reduced beta-cell mass in Sox9-haploinsufficient mice leads to glucose intolerance during adulthood. Sox9-haploinsufficient mice displayed 50% reduced beta-cell mass at birth, which recovered partially via a compensatory increase in beta-cell proliferation early postnatally. Endocrine islets from mice with reduced Sox9 gene dosage exhibited normal glucose stimulated insulin secretion. Our findings show Sox9 plays an important role in endocrine development by maintaining Ngn3 and Pdx1 expression. Glucose intolerance in Sox9-haploinsufficient mice suggests that mutations in Sox9 could play a role in diabetes in humans

Predicting Pancreas Cell Fate Decisions and Reprogramming with a Hierarchical Multi-Attractor Model

Cell fate reprogramming, such as the generation of insulin-producing β cells from other pancreas cells, can be achieved by external modulation of key transcription factors. However, the known gene regulatory interactions that form a complex network with multiple feedback loops make it increasingly difficult to design the cell reprogramming scheme because the linear regulatory pathways as schemes of causal influences upon cell lineages are inadequate for predicting the effect of transcriptional perturbation. However, sufficient information on regulatory networks is usually not available for detailed formal models. Here we demonstrate that by using the qualitatively described regulatory interactions as the basis for a coarse-grained dynamical ODE (ordinary differential equation) based model, it is possible to recapitulate the observed attractors of the exocrine and β, δ, α endocrine cells and to predict which gene perturbation can result in desired lineage reprogramming. Our model indicates that the constraints imposed by the incompletely elucidated regulatory network architecture suffice to build a predictive model for making informed decisions in choosing the set of transcription factors that need to be modulated for fate reprogramming