[Show abstract][Hide abstract] ABSTRACT: Pancreatic ductal adenocarcinoma (PDAC) is believed to arise through a multistep model comprised of putative precursor lesions known as pancreatic intraepithelial neoplasia (PanIN). Recent genetically engineered mouse models of PDAC demonstrate a comparable morphologic spectrum of murine PanIN (mPanIN) lesions. The histogenesis of PanIN and PDAC in both mice and men remains controversial. The most faithful genetic models activate an oncogenic Kras(G12D) knockin allele within the pdx1- or ptf1a/p48-expression domain of the entire pancreatic anlage during development, thus obscuring the putative cell(s)-of-origin from which subsequent mPanIN lesions arise. In our study, activation of this knockin Kras(G12D) allele in the Elastase- and Mist1-expressing mature acinar compartment of adult mice resulted in the spontaneous induction of mPanIN lesions of all histological grades, although invasive carcinomas per se were not seen. We observed no requirement for concomitant chronic exocrine injury in the induction of mPanIN lesions from the mature acinar cell compartment. The acinar cell derivation of the mPanINs was established through lineage tracing in reporter mice, and by microdissection of lesional tissue demonstrating Cre-mediated recombination events. In contrast to the uniformly penetrant mPanIN phenotype observed following developmental activation of Kras(G12D) in the Pdx1-expressing progenitor cells, the Pdx1-expressing population in the mature pancreas (predominantly islet beta cells) appears to be relatively resistant to the effects of oncogenic Kras. We conclude that in the appropriate genetic context, the differentiated acinar cell compartment in adult mice retains its susceptibility for spontaneous transformation into mPanIN lesions, a finding with potential relevance vis-à-vis the origins of PDAC.
Proceedings of the National Academy of Sciences 12/2008; 105(48):18913-8. · 9.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: There are currently few therapeutic options for patients with pancreatic cancer, and new insights into the pathogenesis of this lethal disease are urgently needed. Toward this end, we performed a comprehensive genetic analysis of 24 pancreatic cancers. We first determined the sequences of 23,219 transcripts, representing 20,661 protein-coding genes, in these samples. Then, we searched for homozygous deletions and amplifications in the tumor DNA by using microarrays containing probes for approximately 10(6) single-nucleotide polymorphisms. We found that pancreatic cancers contain an average of 63 genetic alterations, the majority of which are point mutations. These alterations defined a core set of 12 cellular signaling pathways and processes that were each genetically altered in 67 to 100% of the tumors. Analysis of these tumors' transcriptomes with next-generation sequencing-by-synthesis technologies provided independent evidence for the importance of these pathways and processes. Our data indicate that genetically altered core pathways and regulatory processes only become evident once the coding regions of the genome are analyzed in depth. Dysregulation of these core pathways and processes through mutation can explain the major features of pancreatic tumorigenesis.
[Show abstract][Hide abstract] ABSTRACT: This article gives a brief overview of the common histologic subtypes seen in truncal sarcomas and discusses fundamental diagnostic and treatment principles. It also provides a general review of abdominal desmoids. For both truncal sarcomas and desmoids, recurrence rates are high, and definitive recommendations regarding optimal treating are lacking. A multidisciplinary approach to these entities therefore is critical to select appropriate therapeutic strategies for individual patients.
Surgical Clinics of North America 07/2008; 88(3):571-82, vi-vii. · 1.93 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Although the cell of origin for pancreatic cancer remains unknown, prior studies have suggested that pancreatic neoplasia may be initiated in progenitor-like cells. To examine the effects of oncogene activation within the pancreatic progenitor pool, we devised a system for real-time visualization of both normal and oncogenic KRAS-expressing pancreatic progenitor cells in living zebrafish embryos.
By using BAC transgenes under the regulation of ptf1a regulatory elements, we expressed either extended green fluorescent protein (eGFP) alone or eGFP fused to oncogenic KRAS in developing zebrafish pancreas.
After their initial specification, normal eGFP-labeled pancreatic progenitor cells were observed to actively migrate away from the forming endodermal gut tube, and subsequently underwent characteristic exocrine differentiation. In contrast, pancreatic progenitor cells expressing oncogenic KRAS underwent normal specification and migration, but failed to differentiate. This block in differentiation resulted in the abnormal persistence of an undifferentiated progenitor pool, and was associated with the subsequent formation of invasive pancreatic cancer. These tumors showed several features in common with the human disease, including evidence of abnormal Hedgehog pathway activation.
These results provide a unique view of the tumor-initiating effects of oncogenic KRAS in a living vertebrate organism, and suggest that zebrafish models of pancreatic cancer may prove useful in advancing our understanding of the human disease.
[Show abstract][Hide abstract] ABSTRACT: The mechanisms regulating pancreatic endocrine versus exocrine fate are not well defined. By analyzing the effects of Ptf1a partial loss of function, we uncovered novel roles for this transcription factor in determining pancreatic fates. In a newly identified hypomorphic ptf1a mutant, pancreatic cells that would normally express ptf1a and become exocrine cells, express the endocrine marker Isl1, indicating a cell fate switch. Surprisingly, a milder reduction of Ptf1a leads to an even greater increase of ectopic endocrine cells, suggesting that Ptf1a also plays a role in promoting endocrine development. We propose that low levels of Ptf1a promote endocrine fate, whereas high levels repress endocrine fate and promote exocrine fate.
Genes & Development 06/2008; 22(11):1445-50. · 12.64 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Although both endocrine and the exocrine pancreas display a significant capacity for tissue regeneration and renewal, the existence of progenitor cells in the adult pancreas remains uncertain. Using a model of cerulein-mediated injury and repair, we demonstrate that mature exocrine cells, defined by expression of an Elastase1 promoter, actively contribute to regenerating pancreatic epithelium through formation of metaplastic ductal intermediates. Acinar cell regeneration is associated with activation of Hedgehog (Hh) signaling, as assessed by up-regulated expression of multiple pathway components, as well as activation of a Ptch-lacZ reporter allele. Using both pharmacologic and genetic techniques, we also show that the ability of mature exocrine cells to accomplish pancreatic regeneration is impaired by blockade of Hh signaling. Specifically, attenuated regeneration in the absence of an intact Hh pathway is characterized by persistence of metaplastic epithelium expressing markers of pancreatic progenitor cells, suggesting an inhibition of redifferentiation into mature exocrine cells. Given the known role of Hh signaling in exocrine pancreatic cancer, these findings may provide a mechanistic link between injury-induced activation of pancreatic progenitors and subsequent pancreatic neoplasia.
[Show abstract][Hide abstract] ABSTRACT: The ability to regulate gene expression in a cell-specific and temporally restricted manner provides a powerful means to test gene function, bypass the action of lethal genes, label subsets of cells for developmental studies, monitor subcellular structures, and target tissues for selective ablation or physiological analyses. The galactose-inducible system of yeast, mediated by the transcriptional activator Gal4 and its consensus UAS binding site, has proven to be a highly successful and versatile system for controlling transcriptional activation in Drosophila. It has also been used effectively, albeit in a more limited manner, in the mouse. While zebrafish has lagged behind other model systems in the widespread application of Gal4 transgenic approaches to modulate gene activity during development, recent technological advances are permitting rapid progress. Here we review Gal4-regulated genetic tools and discuss how they have been used in zebrafish as well as their potential drawbacks. We describe some exciting new directions, in large part afforded by the Tol2 transposition system, that are generating valuable new Gal4/UAS reagents for zebrafish research.
[Show abstract][Hide abstract] ABSTRACT: Activating Kras mutations are a pervasive and characteristic feature of human pancreatic cancer. In order to examine the earliest in vivo effects of oncogenic Kras expression in the exocrine pancreas, we generated two lines of zebrafish expressing eGFP alone or eGFP fused to human Kras with an activating mutation in codon 12 (Kras G12V) driven by ptf1a regulatory elements using a BAC recombineering strategy (Park et al., 2008). In this review, we describe the techniques that we used to observe the effects of eGFP-Kras G12V expression in pancreatic progenitor cells of the zebrafish embryo, as well as techniques used to characterize malignant pancreatic tumors in the adult zebrafish. This zebrafish model of pancreatic neoplasia provides a unique view of the effects of oncogenic Kras in the embryonic pancreas and suggests that the zebrafish will be a useful model organism in which to study the biology of Kras-initiated pancreatic neoplasia.
Methods in Enzymology 02/2008; 438:391-417. · 2.19 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Symmetric cell divisions have been proposed to rapidly increase neuronal number late in neurogenesis, but how critical this mode of division is to establishing a specific neuronal layer is unknown. Using in vivo time-lapse imaging methods, we discovered that in the laminated zebrafish retina, the horizontal cell (HC) layer forms quickly during embryonic development upon division of a precursor cell population. The precursor cells morphologically resemble immature, postmitotic HCs and express HC markers such as ptf1a and Prox1 prior to division. These precursors undergo nonapical symmetric division at the laminar location where mature HCs contact photoreceptors. Strikingly, the precursor cell type we observed generates exclusively HCs. We have thus identified a dedicated HC precursor, and our findings suggest a mechanism of neuronal layer formation whereby the location of mitosis could facilitate rapid contact between synaptic partners.
[Show abstract][Hide abstract] ABSTRACT: We have adapted a novel multicistronic gene expression system involving viral peptides to the zebrafish. The viral 2A peptide allows production of multiple protein products from a single transgene. Based on highly inefficient peptide bond formation between glycine and proline residues within the 2A peptide, placement of 2A peptide sequence as a linker region between tandem cDNA's allows the stoichiometric translation of multiple unfused protein products. To test this system in zebrafish, we generated two different tandem reporter constructs employing eGFP and mCherry, separated by 2A peptide sequence. Using this system, we produced transgenic zebrafish in which fluorophores were produced as independent proteins from a single transcript. The successful application of this technology in zebrafish will be valuable for visually marking transgenic embryos and transgene-expressing cells, or in any situation where reliable expression of multiple transgenes is desired.
[Show abstract][Hide abstract] ABSTRACT: The transgenic enhanced green fluorescent protein (EGFP) expressing 'green' mouse (C57BL/6-TgN(ACTbEGFP)1Osb) is a widely used tool in stem cell research, where the ubiquitous nature of EGFP expression is critical to track the fate of single or small groups of transplanted haematopoietic stem cells (HSC). Our aim was to investigate this assumed ubiquitous expression by performing a detailed histological survey of EGFP expression in these mice.
Fluorescent microscopy of frozen tissue sections was used to perform a detailed histological survey of the pattern of EGFP expression in these mice. Flow cytometry was also used to determine the expression pattern in blood and bone marrow.
Three patterns of EGFP expression were noted. In most tissues there was an apparently stochastic variegation of the transgene, with individual cell types demonstrating highly variable rates of EGFP expression. Certain specific cell types such as pancreatic ductal epithelium, cerebral cortical neurones and glial cells and glomerular mesangial cells consistently lacked EGFP expression, while others, including pancreatic islet cells, expressed EGFP only at extremely low levels, barely distinguishable from background. Lastly, in the colon and stomach the pattern of EGFP expression was suggestive of clonal inactivation. Only cardiac and skeletal muscle showed near ubiquitous expression.
These findings raise questions regarding the 'ubiquitous' expression of EGFP in these transgenic mice and suggest caution in relying overly on EGFP alone as an infallible marker of donor cell origin.
[Show abstract][Hide abstract] ABSTRACT: Prior studies with transgenic zebrafish confirmed the functionality of the transcription factor Gal4 to drive expression of other genes under the regulation of upstream activator sequences (UAS). However, widespread application of this powerful binary system has been limited, in part, by relatively inefficient techniques for establishing transgenic zebrafish and by the inadequacy of Gal4 to effect high levels of expression from UAS-regulated genes. We have used the Tol2 transposition system to distribute a self-reporting gene/enhancer trap vector efficiently throughout the zebrafish genome. The vector uses the potent, hybrid transcription factor Gal4-VP16 to activate expression from a UAS:eGFP reporter cassette. In a pilot screen, stable transgenic lines were established that express eGFP in reproducible patterns encompassing a wide variety of tissues, including the brain, spinal cord, retina, notochord, cranial skeleton and muscle, and can transactivate other UAS-regulated genes. We demonstrate the utility of this approach to track Gal4-VP16 expressing migratory cells in UAS:Kaede transgenic fish, and to induce tissue-specific cell death using a bacterial nitroreductase gene under UAS control. The Tol2-mediated gene/enhancer trapping system together with UAS transgenic lines provides valuable tools for regulated gene expression and for targeted labeling and ablation of specific cell types and tissues during early zebrafish development.
[Show abstract][Hide abstract] ABSTRACT: Determining the functional attributes of pancreatic transcription factors is essential to understand how the pancreas is specified distinct from other endodermal organs, such as liver, stomach and duodenum, and to direct the differentiation of other cell types into pancreas. Previously, we demonstrated that Pdx1-VP16 was sufficient to convert liver to pancreas. In this paper, we characterize the functional ability of another pancreatic transcription factor, Ptf1a, in promoting ectopic pancreatic fates at early stages throughout the endoderm and later during organogenesis. Using the transthyretin promoter to drive expression in the early liver region/bud of transgenic Xenopus tadpoles, we find that Ptf1a-VP16 is able to convert liver to pancreas. Overexpression of the unmodified Ptf1a on the other hand has no effect in liver but is able to convert stomach and duodenum to pancreas. When overexpressed at earlier embryonic stages throughout the endoderm, Ptf1a activity is similarly limited, whereas Ptf1a-VP16 has increased activity. Interestingly, in all instances we find that Ptf1a-VP16 is only capable of promoting acinar cell fates, whereas Ptf1a promotes both acinar and endocrine fates. Lastly, we demonstrate that, similar to mouse and zebrafish, Xenopus Ptf1a is essential for the initial specification of both endocrine and exocrine cells during normal pancreas development.
[Show abstract][Hide abstract] ABSTRACT: In order to generate a zebrafish model of beta cell regeneration, we have expressed an Escherichia coli gene called nfsB in the beta cells of embryonic zebrafish. This bacterial gene encodes a nitroreductase (NTR) enzyme, which can convert prodrugs such as metronidazole (Met) to cytotoxins. By fusing nfsB to mCherry, we can simultaneously render beta cells susceptible to prodrug and visualize Met dependent cell ablation. We show that the neighboring alpha and delta cells are unaffected by prodrug treatment and that ablation is beta cell specific. Following drug removal and 36h of recovery, beta cells regenerate. Using ptf1a morphants, it is clear that this beta cell recovery occurs independently of the presence of the exocrine pancreas. Also, by using photoconvertible Kaede to cell lineage trace and BrdU incorporation to label proliferation, we investigate mechanisms for beta regeneration. Therefore, we have developed a unique resource for the study of beta cell regeneration in a living vertebrate organism, which will provide the opportunity to conduct large-scale screens for pharmacological and genetic modifiers of beta cell regeneration.
Mechanisms of Development 04/2007; 124(3):218-29. · 2.24 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In this issue of Cancer Cell, Guerra and colleagues provide important new insights regarding the ability of specific pancreatic cell types to generate invasive pancreatic cancer. First, they demonstrate that classical pancreatic "ductal" neoplasia can be induced by activation of oncogenic Kras in nonductal exocrine cells. Second, they show that, while Kras activation in immature acinar and centroacinar cells is readily able to induce ductal neoplasia, Kras-mediated tumorigenesis in mature exocrine pancreas requires the induction of chronic epithelial injury. The results shed new light on the "cell of origin" of pancreatic ductal cancer and demonstrate that chronic pancreatitis provides a permissive environment for Kras-induced pancreatic neoplasia.
Cancer Cell 04/2007; 11(3):211-3. · 23.89 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: It has been suggested that pancreatic acinar cells can serve as progenitors for pancreatic islets, a concept with substantial implications for therapeutic efforts to increase insulin-producing beta cell mass in patients with diabetes. We report what we believe to be the first in vivo lineage tracing approach to determine the plasticity potential of pancreatic acinar cells. We developed an acinar cell-specific inducible Cre recombinase transgenic mouse, which, when mated with a reporter strain and pulsed with tamoxifen, resulted in permanent and specific labeling of acinar cells and their progeny. During various time periods of observation and using several models to provoke injury, we failed to observe any chase of the labeled cells into the endocrine compartment, indicating that acinar cells do not normally transdifferentiate into islet beta cells in vivo in adult mice. In contrast, we observed a substantial role for replication of preexisting acinar cells in the regeneration of new acinar cells after partial pancreatectomy. These results indicate that mature acinar cells harbor a facultative acinar but not endocrine progenitor capacity.
Journal of Clinical Investigation 04/2007; 117(4):971-7. · 13.77 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Beta-catenin is an essential mediator of canonical Wnt signaling and a central component of the cadherin-catenin epithelial adhesion complex. Dysregulation of beta-catenin expression has been described in pancreatic neoplasia. Newly published studies have suggested that beta-catenin is critical for normal pancreatic development although these reports reached somewhat different conclusions. In addition, the molecular mechanisms by which loss of beta-catenin affects pancreas development are not well understood. The goals of this study then were; 1] to further investigate the role of beta-catenin in pancreatic development using a conditional knockout approach and 2] to identify possible mechanisms by which loss of beta-catenin disrupts pancreatic development. A Pdx1-cre mouse line was used to delete a floxed beta-catenin allele specifically in the developing pancreas, and embryonic pancreata were studied by immunohistochemistry and microarray analysis.
Pdx1-cre floxed beta-catenin animals were viable but demonstrated small body size and shortened median survival. The pancreata from knockout mice were hypoplastic and histologically demonstrated a striking paucity of exocrine pancreas, acinar to duct metaplasia, but generally intact pancreatic islets containing all lineages of endocrine cells. In animals with extensive acinar hypoplasia, putative hepatocyte transdifferention was occasionally observed. Obvious and uniform pancreatic hypoplasia was observed by embryonic day E16.5. Transcriptional profiling of Pdx1-cre floxed beta-catenin embryonic pancreata at E14.5, before there was a morphological phenotype, revealed significant decreases in the beta-catenin target gene N-myc, and the basic HLH transcription factor PTF1, and an increase of several pancreatic zymogens compared to control animals. By E16.5, there was a dramatic loss of exocrine markers and an increase in Hoxb4, which is normally expressed anterior to the pancreas.
We conclude that beta-catenin expression is required for development of the exocrine pancreas, but is not required for development of the endocrine compartment. In contrast, beta-catenin/Wnt signaling appears to be critical for proliferation of PTF1+ nascent acinar cells and may also function, in part, to maintain an undifferentiated state in exocrine/acinar cell precursors. Finally, beta-catenin may be required to maintain positional identity of the pancreatic endoderm along the anterior-posterior axis. This data is consistent with the findings of frequent beta-catenin mutations in carcinomas of acinar cell lineage seen in humans.