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The distribution of complementary metabolic functions in hepatocytes along a portocentral axis is called liver zonation. Endothelial secreted Wnt ligands maintain metabolic zonation in the adult murine liver but whether those ligands are necessary to initiate zonation in the immature liver has been only partially explored. Also, numerous non-metabolic proteins display zonated expression in the adult liver but it is not entirely clear if their localization requires endothelial Wnts. Here we used a novel transgenic mouse model to compare the spatial distribution of zonated non-metabolic proteins with that of typical zonated metabolic enzymes during liver maturation and after acute injury induced by carbon tetrachloride (CCl4). We also investigated how preventing Wnt ligand secretion from endothelial cells affects zonation patterns under homeostasis and after acute injury. Our study demonstrates that metabolic and non-metabolic zonation are established non-synchronously during maturation and regeneration and require multiple endothelial Wnt sources.

The current paradigm of pancreatic neoplastic transformation proposes an initial step whereby acinar cells convert into acinar-to-ductal metaplasias, followed by progression of these lesions into neoplasias under sustained oncogenic activity and inflammation. Understanding the molecular mechanisms driving these processes is crucial to the early diagnostic and prevention of pancreatic cancer. Emerging evidence indicates that transcription factors that control exocrine pancreatic development could have either, protective or facilitating roles in the formation of preneoplasias and neoplasias in the pancreas. We previously identified that the homeodomain transcription factor Prox1 is a novel regulator of mouse exocrine pancreas development. Here we investigated whether Prox1 function participates in early neoplastic transformation using in vivo, in vitro and in silico approaches. We found that Prox1 expression is transiently re-activated in acinar cells undergoing dedifferentiation and acinar-to-ductal metaplastic conversion. In contrast, Prox1 expression is largely absent in neoplasias and tumors in the pancreas of mice and humans. We also uncovered that Prox1-heterozygosis markedly increases the formation of acinar-to-ductal-metaplasias and early neoplasias, and enhances features associated with inflammation, in mouse pancreatic tissues expressing oncogenic Kras. Furthermore, we discovered that Prox1-heterozygosis increases tissue damage and delays recovery from inflammation in pancreata of mice injected with caerulein. These results are the first demonstration that Prox1 activity protects pancreatic cells from acute tissue damage and early neoplastic transformation. Additional data in our study indicate that this novel role of Prox1 involves suppression of pathways associated with inflammatory responses and cell invasiveness.

The biliary tree is an essential component of transplantable human liver tissue. Despite recent advances in liver tissue engineering, attempts at re-creating the intrahepatic biliary tree have not progressed significantly. The finer branches of the biliary tree are structurally and functionally complex and heterogeneous and require harnessing innate developmental processes for their regrowth. Here we demonstrate the ability of decellularized liver extracellular matrix (dECM) hydrogels to induce the in vitro formation of complex biliary networks using encapsulated immortalized mouse small biliary epithelial cells (cholangiocytes). This phenomenon is not observed using immortalized mouse large cholangiocytes, or with purified collagen 1 gels or Matrigel. We also show phenotypic stability via immunostaining for specific cholangiocyte markers. Moreover, tight junction formation and maturation was observed to occur between cholangiocytes, exhibiting polarization and transporter activity. To better define the mechanism of duct formation, we utilized three fluorescently labeled, but otherwise identical populations of cholangiocytes. The cells, in a proximity dependent manner, either branch out clonally, radiating from a single nucleation point, or assemble into multi-colored structures arising from separate populations. These findings present liver dECM as a promising biomaterial for intrahepatic bile duct tissue engineering and as a tool to study duct remodeling in vitro.

Germline mutations in ATM (encoding the DNA-damage signaling kinase, ataxia-telangiectasia-mutated) increase Familial Pancreatic Cancer (FPC) susceptibility, and ATM somatic mutations have been identified in resected human pancreatic tumors. Here we investigated how Atm contributes to pancreatic cancer by deleting this gene in a murine model of the disease expressing oncogenic Kras (KrasG12D). We show that partial or total ATM deficiency cooperates with KrasG12D to promote highly metastatic pancreatic cancer. We also reveal that ATM is activated in pancreatic precancerous lesions in the context of DNA damage and cell proliferation, and demonstrate that ATM deficiency leads to persistent DNA damage in both precancerous lesions and primary tumors. Using low passage cultures from primary tumors and liver metastases we show that ATM loss accelerates Kras-induced carcinogenesis without conferring a specific phenotype to pancreatic tumors or changing the status of the tumor suppressors p53, p16Ink4a and p19Arf. However, ATM deficiency markedly increases the proportion of chromosomal alterations in pancreatic primary tumors and liver metastases. More importantly, ATM deficiency also renders murine pancreatic tumors highly sensitive to radiation. These and other findings in our study conclusively establish that ATM activity poses a major barrier to oncogenic transformation in the pancreas via maintaining genomic stability.

Pancreatic cancer occurs in the setting of a profound fibrotic microenvironment that often dwarfs the actual tumor. Although pancreatic fibrosis has been well studied in chronic pancreatitis, its development in pancreatic cancer is much less well understood. This article describes the dynamic remodeling that occurs from pancreatic precursors (pancreatic intraepithelial neoplasias (PanINs)) to pancreatic ductal adenocarcinoma, highlighting similarities and differences between benign and malignant disease. Although collagen matrix is a commonality throughout this process, early stage PanINs are virtually free of periostin while late stage PanIN and pancreatic cancer are surrounded by an increasing abundance of this extracellular matrix protein. Myofibroblasts also become increasingly abundant during progression from PanIN to cancer. From the earliest stages of fibrogenesis, macrophages are associated with this ongoing process. In vitro co-culture indicates there is cross-regulation between macrophages and pancreatic stellate cells (PaSCs), precursors to at least some of the fibrotic cell populations. When quiescent PaSCs were co-cultured with macrophage cell lines, the stellate cells became activated and the macrophages increased cytokine production. In summary, fibrosis in pancreatic cancer involves a complex interplay of cells and matrices that regulate not only the tumor epithelium but the composition of the microenvironment itself.

The liver has multiple functions that preserve homeostasis. Liver diseases are debilitating, costly and often result in death. Elucidating the developmental mechanisms that establish the liver’s architecture or generate the cellular diversity of this organ should help advance the prevention, diagnosis and treatment of hepatic diseases. We previously reported that migration of early hepatic precursors away from the gut epithelium requires the activity of the homeobox gene Prox1. Here, we show that Prox1 is a novel regulator of cell differentiation and morphogenesis during hepatogenesis. Prox1 ablation in bipotent hepatoblasts dramatically reduced the expression of multiple hepatocyte genes and led to very defective hepatocyte morphogenesis. As a result, abnormal epithelial structures expressing hepatocyte and cholangiocyte markers or resembling ectopic bile ducts developed in the Prox1-deficient liver parenchyma. By contrast, excessive commitment of hepatoblasts into cholangiocytes, premature intrahepatic bile duct morphogenesis, and biliary hyperplasia occurred in periportal areas of Prox1-deficient livers. Together, these abnormalities indicate that Prox1 activity is necessary to correctly allocate cell fates in liver precursors. These results increase our understanding of differentiation anomalies in pathological conditions and will contribute to improving stem cell protocols in which differentiation is directed towards hepatocytes and cholangiocytes.

Transcription factor expression fluctuates during β-cell ontogeny, and disruptions in this pattern can affect the development or function of those cells. Here we uncovered that murine endocrine pancreatic progenitors express high levels of the homeodomain transcription factor Prox1, whereas both immature and mature β-cells scarcely express this protein. We also investigated if sustained Prox1 expression is incompatible with β-cell development or maintenance using transgenic mouse approaches. We discovered that Prox1 upregulation in mature β-cells has no functional consequences; in contrast, Prox1 overexpression in immature β-cells promotes acute fasting hyperglycemia. Using a combination of immunostaining and quantitative and comparative gene expression analyses, we determined that Prox1 upregulation reduces proliferation, impairs maturation, and enables apoptosis in postnatal β-cells. Also, we uncovered substantial deficiency in β-cells that overexpress Prox1 of the key regulator of β-cell maturation MafA, several MafA downstream targets required for glucose-stimulated insulin secretion, and genes encoding important components of FGF signaling. Moreover, knocking down PROX1 in human EndoC-βH1 β-cells caused increased expression of many of these same gene products. These and other results in our study indicate that reducing the expression of Prox1 is beneficial for the expansion and maturation of postnatal β-cells.

Background: The assembly of distinct proteins into tight junctions results in the formation of a continuous barrier that regulates the paracellular flux of water, ions, and small molecules across epithelia. The claudin protein family encompasses numerous major structural components of tight junctions. These proteins specify the permeability characteristics of tight junctions and consequently, some of the physiological properties of epithelia. Furthermore, defective claudin expression has been found to correlate with some diseases, tumor progression, and defective morphogenesis. Investigating the pattern of claudin expression during embryogenesis or in certain pathological conditions is necessary to begin disclosing the role of these proteins in health and disease. Results: This study analyzed the expression of several claudins during mouse pancreas organogenesis and in pancreatic intraepithelial neoplasias of mouse and human origin. Conclusions: Our results underscored a distinctive, dynamic distribution of certain claudins in both the developing pancreas and the pancreatic epithelium undergoing neoplastic transformation. Developmental Dynamics 241:583–594, 2012. © 2012 Wiley Periodicals Inc.

During pancreas development, endocrine and exocrine cells arise from a common multipotent progenitor pool. How these cell fate decisions are coordinated with tissue morphogenesis is poorly understood. Here we have examined ductal morphology, endocrine progenitor cell fate and Notch signaling in Ngn3−/− mice, which do not produce islet cells. Ngn3 deficiency results in reduced branching and enlarged pancreatic duct-like structures, concomitant with Ngn3 promoter activation throughout the ductal epithelium and reduced Notch signaling. Conversely, forced generation of surplus endocrine progenitor cells causes reduced duct caliber and an excessive number of tip cells. Thus, endocrine progenitor cells normally provide a feedback signal to adjacent multipotent ductal progenitor cells that activates Notch signaling, inhibits further endocrine differentiation and promotes proper morphogenesis. These results uncover a novel layer of regulation coordinating pancreas morphogenesis and endocrine/exocrine differentiation, and suggest ways to enhance the yield of beta cells from stem cells.

MafA is a key transcriptional activator of islet beta cells, and its exclusive expression within beta cells of the developing and adult pancreas is distinct among pancreatic regulators. Region 3 (base pairs -8118 to -7750 relative to the transcription start site), one of six conserved 5' cis domains of the MafA promoter, is capable of directing beta-cell-line-selective expression. Transgenic reporters of region 3 alone (R3), sequences spanning regions 1 to 6 (R1-6; base pairs -10428 to +230), and R1-6 lacking R3 (R1-6(DeltaR3)) were generated. Only the R1-6 transgene was active in MafA(+) insulin(+) cells during development and in adult cells. R1-6 also mediated glucose-induced MafA expression. Conversely, pancreatic expression was not observed with the R3 or R1-6(DeltaR3) line, although much of the nonpancreatic expression pattern was shared between the R1-6 and R1-6(DeltaR3) lines. Further support for the importance of R3 was also shown, as the islet regulators Nkx6.1 and Pax6, but not NeuroD1, activated MafA in gel shift, chromatin immunoprecipitation (ChIP), and transfection assays and in vivo mouse knockout models. Lastly, ChIP demonstrated that Pax6 and Pdx-1 also bound to R1 and R6, potentially functioning in pancreatic and nonpancreatic expression. These data highlight the nature of the cis- and trans-acting factors controlling the beta-cell-specific expression of MafA.

Proteins Glis1 to -3 constitute a subfamily of Kruppel-like zinc finger transcriptional regulators that share a highly conserved five-C2H2-type zinc finger domain with members of the Gli and Zic subfamilies (6, 25, 27, 30-32, 35, 38, 39, 43, 56). Glis1 to -3 regulate gene transcription by binding specific DNA sequences referred to as Glis-binding sites (Glis-BS) in promoter regulatory regions of target genes (10, 30, 31). Although their precise physiological functions are still poorly understood, genetic studies have implicated Glis1 to -3 in several pathologies (7, 8, 24, 29, 33, 39, 45). Glis3 is abundantly expressed in the adult kidney, pituitary, pancreas, uterus, and thyroid gland (31, 45). During mouse embryonic development, Glis3 is expressed in a spatiotemporal pattern suggesting that Glis3 regulates gene expression at specific stages during development (31). Genetic alterations in the human GLIS3 gene have been linked to a rare syndrome characterized by neonatal diabetes and congenital hypothyroidism (NDH) (45, 50). Depending on the nature of the GLIS3 mutation, NDH patients can also display facial abnormalities, glaucoma, liver fibrosis, and polycystic kidney disease. Recently, a genome-wide association study identified the GLIS3 gene as a susceptibility locus for type 1 diabetes (8). These studies, together with evidence that Glis3 is expressed in pancreatic β cells, suggest that Glis3 has an important regulatory role in the pancreas. Although major advances have been made in understanding pancreatic development, many of the molecular mechanisms that regulate progenitor cell dynamics and cell differentiation are still not precisely understood (1, 18, 19, 21, 26, 28, 37). At approximately embryonic day 9 (E9) of mouse embryogenesis, the pancreas first appears from distinct ventral and dorsal anlagen as evaginations of the distal foregut endoderm (21, 36). The buds grow and initiate branching morphogenesis at about E11.5. Early multipotent pancreatic progenitors, marked by Pdx1, Ptf1a, Nkx2.2, and Cpa1 expression (12, 14, 41, 57), are the source of all differentiated cells of the exocrine, ductal, and endocrine cell lineages. Lineage determination is a complex process that involves many transcription factors and signaling pathways. Induction of Ngn3 marks the differentiation of pancreatic progenitors into proendocrine progenitors (15, 17, 22, 36, 51). Differentiation into the different endocrine cell lineages involves the induction of a combination of additional transcription factors, including Myt1, NeuroD, Isl1, Pax4, Pax6, and Arx (2, 13, 22, 34, 36, 37, 48, 49). Defects in the expression or activity of these transcription factors in mice and humans often result in abnormal pancreatic development and function that can lead to diabetes. To obtain greater insights into the physiological and molecular functions of Glis3, we recently generated Glis3zf/zf mutant mice that are deficient in Glis3 transactivating activity (24). In this study, we characterize the pancreatic phenotype of these mice and analyze the role of Glis3 in pancreatic development. We demonstrate that, in addition to cyst formation in the pancreatic ducts, Glis3zf/zf mutant mice develop neonatal diabetes that is associated with an almost total loss of β cells. We provide evidence which indicates that Glis3 plays a key role in cell lineage specification, particularly in the development of mature pancreatic β cells. We further identify Glis3 as a regulator of insulin 2 gene expression. Our study shows that Glis3 has multiple functions in the pancreas and suggests that Glis3 might provide a new therapeutic target to intervene in diabetes.

The large Maf family of basic leucine-zipper-containing transcription factors are known regulators of key developmental and functional processes in various cell types, including pancreatic islets. Here, we demonstrate that within the adult pancreas, MafB is only expressed in islet alpha-cells and contributes to cell type-specific expression of the glucagon gene through activation of a conserved control element found between nucleotides -77 to -51. MafB was also shown to be expressed in developing alpha- and beta-cells as well as in proliferating hormone-negative cells during pancreatogenesis. In addition, MafB expression is maintained in the insulin(+) and glucagon(+) cells remaining in mice lacking either the Pax4 or Pax6 developmental regulators, implicating a potentially early role for MafB in gene regulation during islet cell development. These results indicate that MafB is not only important to islet alpha-cell function but may also be involved in regulating genes required in both endocrine alpha- and beta-cell differentiation.

The pancreatic islet is necessary for maintaining glucose homeostasis. Within the pancreatic islet, the homeodomain protein Nkx2.2 is essential for the differentiation of all insulin-producing β cells and a subset of glucagon-producing α cells ( 1 ). Mice lacking Nkx2.2 have relatively normal sized islets, but a large number of cells within the mutant islet fail to produce any of the four major islet hormones. In this study we demonstrate that Nkx2.2 mutant endocrine cells have been replaced by cells that produce ghrelin, an appetite-promoting peptide predominantly found in the stomach. Intriguingly, normal mouse pancreas also contains a small population of ghrelin-producing cells, defining a new islet “ε” cell population. The expansion of ghrelin-producing cells at the expense of β cells may be a general phenomenon, because we demonstrate that Pax4 mutant mice display a similar phenotype. We propose that insulin and ghrelin cells share a common progenitor and that Nkx2.2 and Pax4 are required to specify or maintain differentiation of the β cell fate. This finding also suggests that there is a genetic component underlying the balance between insulin and ghrelin in regulating glucose metabolism.

Pancreatic beta cells play a central role in maintaining glucose homeostasis because they secrete insulin in response to increased level of blood glucose; failure of this capacity constitutes a major component of the pathogenesis of diabetes. The identification of key regulators of pancreatic beta-cell differentiation is relevant for the overall understanding of this process and for future experiments aimed at regenerating insulin-producing beta cells from pancreatic or embryonic stem cells. Several studies using transgenic or knockout mice have established that the development and function of pancreatic beta cells are controlled by several genes encoding specific transcription factors. By inactivating the homeobox gene Pax4, we previously demonstrated that its function is required for the formation of mature insulin-producing cells. Here, we show that during pancreas ontogeny, Pax4 is expressed in differentiating endocrine cells, including beta cells. Pax4 activity appears essential for appropriate initiation of beta-cell differentiation because loss of Pax4 prevents the expression of Pdx1, HB9 and insulin in beta-cell precursors. This role of Pax4 appears to be accomplished via its genetic interaction with another homeobox gene, Nkx2.2.

The mammalian pancreas contains two distinct cell populations: endocrine cells which secrete hormones into the bloodstream, and exocrine cells, which secrete enzymes into the digestive tract. The four endocrine cell types found in the adult pancreas-(alpha, beta, delta and PP-synthesize glucagon, insulin, somatostatin and pancreatic polypeptide, respectively. All of these endocrine cells arise from common multipotent precursors, which coexpress several hormones when they start to differentiate. Expression of some homeobox genes in the early developing pancreas has been reported. The Pax4 gene is expressed in the early pancreas, but is later restricted to beta cells. Inactivation of Pax4 by homologous recombination results in the absence of mature insulin- and somatostatin-producing cells (beta and delta, respectively) in the pancreas of Pax4 homozygous mutant mice, but glucagon-producing alpha cells are present in considerably higher numbers. We propose that the early expression of Pax4 in a subset of endocrine progenitors is essential for the differentiation of the beta and delta cell lineages. A default pathway would explain the elevated number of alpha cells in the absence of Pax4.

Oncogenic stimuli trigger the DNA damage response (DDR) and induction of the alternative reading frame (ARF) tumor suppressor, both of which can activate the p53 pathway and provide intrinsic barriers to tumor progression. However, the respective timeframes and signal thresholds for ARF induction and DDR activation during tumorigenesis remain elusive. Here, these issues were addressed by analyses of mouse models of urinary bladder, colon, pancreatic and skin premalignant and malignant lesions. Consistently, ARF expression occurred at a later stage of tumor progression than activation of the DDR or p16INK4A, a tumor-suppressor gene overlapping with ARF. Analogous results were obtained in several human clinical settings, including early and progressive lesions of the urinary bladder, head and neck, skin and pancreas. Mechanistic analyses of epithelial and fibroblast cell models exposed to various oncogenes showed that the delayed upregulation of ARF reflected a requirement for a higher, transcriptionally based threshold of oncogenic stress, elicited by at least two oncogenic ‘hits’, compared with lower activation threshold for DDR. We propose that relative to DDR activation, ARF provides a complementary and delayed barrier to tumor development, responding to more robust stimuli of escalating oncogenic overload. Oncogenic stimuli trigger the DNA damage response (DDR) and induction of the alternative reading frame (ARF) tumor suppressor, both of which can activate the p53 pathway and provide intrinsic barriers to tumor progression. However, the respective timeframes and signal thresholds for ARF induction and DDR activation during tumorigenesis remain elusive. Here, these issues were addressed by analyses of mouse models of urinary bladder, colon, pancreatic and skin premalignant and malignant lesions. Consistently, ARF expression occurred at a later stage of tumor progression than activation of the DDR or p16(INK4A), a tumor-suppressor gene overlapping with ARF. Analogous results were obtained in several human clinical settings, including early and progressive lesions of the urinary bladder, head and neck, skin and pancreas. Mechanistic analyses of epithelial and fibroblast cell models exposed to various oncogenes showed that the delayed upregulation of ARF reflected a requirement for a higher, transcriptionally based threshold of oncogenic stress, elicited by at least two oncogenic 'hits', compared with lower activation threshold for DDR. We propose that relative to DDR activation, ARF provides a complementary and delayed barrier to tumor development, responding to more robust stimuli of escalating oncogenic overload.

International audience; Neurogenin3(+) (Ngn3(+)) progenitor cells in the developing pancreas give rise to five endocrine cell types secreting insulin, glucagon, somatostatin, pancreatic polypeptide and ghrelin. Gastrin is a hormone produced primarily by G-cells in the stomach, where it functions to stimulate acid secretion by gastric parietal cells. Gastrin is expressed in the embryonic pancreas and is common in islet cell tumors, but the lineage and regulators of pancreatic gastrin(+) cells are not known. We report that gastrin is abundantly expressed in the embryonic pancreas and disappears soon after birth. Some gastrin(+) cells in the developing pancreas co-express glucagon, ghrelin or pancreatic polypeptide, but many gastrin(+) cells do not express any other islet hormone. Pancreatic gastrin(+) cells express the transcription factors Nkx6.1, Nkx2.2 and low levels of Pdx1, and derive from Ngn3(+) endocrine progenitor cells as shown by genetic lineage tracing. Using mice deficient for key transcription factors we show that gastrin expression depends on Ngn3, Nkx2.2, NeuroD1 and Arx, but not Pax4 or Pax6. Finally, gastrin expression is induced upon differentiation of human embryonic stem cells to pancreatic endocrine cells expressing insulin. Thus, gastrin(+) cells are a distinct endocrine cell type in the pancreas and an alternative fate of Ngn3+ cells.

alpha-L-Iduronidase is a lysosomal enzyme, the deficiency of which causes mucopolysaccharidosis I (MPS I); a canine MPS I colony has been bred to test therapeutic intervention. The enzyme was purified to apparent homogeneity from canine testis and found to consist of two electrophoretically separable proteins that had common internal peptides but differed at their amino termini. A 57-base oligonucleotide, corresponding to the most probable codons of the longest peptide, was used to screen a canine testis cDNA library. Three cDNAs were isolated, two of which lacked the 5'-end whereas the third was full-length except for a small internal deletion. The composite sequence encodes an open reading frame of 655 amino acids that includes all sequenced peptides. The amino terminus of the larger protein, glutamic acid 26, is at the predicted signal peptide cleavage site, whereas the amino terminus of the smaller protein is leucine 106. There are six potential N-glycosylation sites and a non-canonical polyadenylation signal, CTTAAA. A search of GenBank showed that the amino acid sequence of alpha-L-iduronidase has similarity to that of a bacterial beta-xylosidase. A full-length cDNA corresponding to the composite sequence was constructed (pcIdu) and inserted into the pSVL expression vector (pSVcIdu). Two days after Cos-1 cells were transfected with pSVcIdu, their intracellular and secreted level of alpha-L-iduronidase activity has increased 8- and 22-fold, respectively, over the endogenous activity. Fibroblasts of MPS I dogs, which have no alpha-L-iduronidase activity, lacked the normal alpha-L-iduronidase mRNA of 2.2 kilobases and contained instead a trace amount of a 2.8-kilobase species. Isolation and characterization of an expressible alpha-L-iduronidase cDNA represents the first step toward mutation analysis and replacement therapy.

The formation of adequate masses of endocrine and exocrine pancreatic tissues during embryogenesis is essential to ensure proper nutrition and glucose homeostasis at postnatal stages. We generated mice with pancreas-specific ablation of the 3-phosphoinositide-dependent protein kinase 1 (Pdk1) to investigate how signaling downstream of the phosphatidylinositol-3-OH kinase (PI3K) pathway controls pancreas development. Pdk1-conditional knock-out mice were born with conspicuous pancreas hypoplasia, and within a few weeks, they developed severe hyperglycemia. Our detailed characterization of the mutant embryonic pancreas also revealed distinct temporal, cell type-specific requirements of Pdk1 activity in the control of cell proliferation, cell survival, and cell size during pancreas development. These results thus uncover Pdk1 as a novel, crucial regulator of pancreatic growth during embryogenesis. In addition, we provide evidence that Pdk1 activity is required differently in mature pancreatic cell types, since compensatory proliferation and possible mTORC2 activation occurred in exocrine cells but not in β cells of the Pdk1-deficient postnatal pancreas.

We have previously reported that the loss of Arx and/or Pax4 gene activity leads to a shift in the fate of the different endocrine cell subtypes in the mouse pancreas, without affecting the total endocrine cell numbers. Here, we conditionally and ectopically express Pax4 using different cell-specific promoters and demonstrate that Pax4 forces endocrine precursor cells, as well as mature alpha cells, to adopt a beta cell destiny. This results in a glucagon deficiency that provokes a compensatory and continuous glucagon+ cell neogenesis requiring the re-expression of the proendocrine gene Ngn3. However, the newly formed alpha cells fail to correct the hypoglucagonemia since they subsequently acquire a beta cell phenotype upon Pax4 ectopic expression. Notably, this cycle of neogenesis and redifferentiation caused by ectopic expression of Pax4 in alpha cells is capable of restoring a functional beta cell mass and curing diabetes in animals that have been chemically depleted of beta cells.

Pax4-deficient mice have a severe gastrointestinal endocrine deficiency: they lack most pancreatic cells that produce insulin or somatostatin and various duodenal endocrine cell types. Remarkably, Pax4-deficient mice also have an overabundance of ghrelin-expressing cells in the pancreas and duodenum. Detailed analysis of the Pax4 nullizygous pancreas determined that the mutant islets are largely composed of a distinctive endocrine cell type that expresses ghrelin, glucagon, islet amyloid polypeptide (IAPP), and low levels of Pdx1. Lineage-tracing analysis revealed that most of these unique endocrine cells directly arose from Pax4-deficient progenitors. Previous in vitro work reported that Pax4 is a transcriptional repressor of islet amyloid polypeptide (IAPP) and glucagon. In this study, we expanded those results by showing that Pax4 is also a repressor of gherlin. Together, our data further support the notion that Pax4 activity is necessary to establish appropriate patterns of gene expression in endocrine progenitors of the digestive tract. Developmental Dynamics 237:51– 61, 2008. © 2007 Wiley-Liss, Inc.

Matricellular proteins mediate both tissue morphogenesis and tissue homeostasis in important ways because they modulate cell-matrix and cell-cell interactions. In this study, we found that the matricellular protein osteopontin (Opn) is a novel marker of undifferentiated pancreatic precursors and pancreatic ductal tissues in mice. Our analysis also underscored a specific, dynamic profile of Opn expression in embryonic pancreatic tissues that suggests the participation of this protein's function in processes involving cell migration, cell-cell interactions, or both. Surprisingly, our analysis of Opn-deficient pancreata did not reveal obvious alterations in the morphology or differentiation of these tissues. Therefore, in embryonic pancreatic tissues, it is possible that other proteins act redundantly to Opn or that this protein's function is dispensable for pancreas development. Finally, the maintenance of Opn expression in pancreatic tissues of adults argues for a possible function of this protein in injury and pathologic responses.

The transport of porphyrins across the mitochondrial membrane is important for cellular processes. The ABC transporter Abcb6 (an energy-requiring transporter) plays a key role not only in mitochondrial porphyrin transport but also in the upregulation of haeme biosynthesis. The movement of anionic porphyrins (for example, haem) across intracellular membranes is crucial to many biological processes, but their mitochondrial translocation and coordination with haem biosynthesis is not understood. Transport of porphyrins into isolated mitochondria is energy-dependent1,2,3, as expected for the movement of anions into a negatively charged environment. ATP-binding cassette transporters actively facilitate the transmembrane movement of substances. We found that the mitochondrial ATP-binding cassette transporter ABCB6 is upregulated (messenger RNA and protein in human and mouse cells) by elevation of cellular porphyrins and postulated that ABCB6 has a function in porphyrin transport. We also predicted that ABCB6 is functionally linked to haem biosynthesis, because its mRNA is found in both human bone marrow and CD71+ early erythroid cells (by database searching), and because our results show that ABCB6 is highly expressed in human fetal liver, and Abcb6 in mouse embryonic liver. Here we demonstrate that ABCB6 is uniquely located in the outer mitochondrial membrane and is required for mitochondrial porphyrin uptake. After ABCB6 is upregulated in response to increased intracellular porphyrin, mitochondrial porphyrin uptake activates de novo porphyrin biosynthesis. This process is blocked when the Abcb6 gene is silenced. Our results challenge previous assumptions about the intracellular movement of porphyrins and the factors controlling haem biosynthesis.

The large Maf family of basic leucine-zipper-containing transcription factors are known regulators of key developmental and functional processes in various cell types, including pancreatic islets. Here, we demonstrate that within the adult pancreas, MafB is only expressed in islet alpha-cells and contributes to cell type-specific expression of the glucagon gene through activation of a conserved control element found between nucleotides -77 to -51. MafB was also shown to be expressed in developing alpha- and beta-cells as well as in proliferating hormone-negative cells during pancreatogenesis. In addition, MafB expression is maintained in the insulin(+) and glucagon(+) cells remaining in mice lacking either the Pax4 or Pax6 developmental regulators, implicating a potentially early role for MafB in gene regulation during islet cell development. These results indicate that MafB is not only important to islet alpha-cell function but may also be involved in regulating genes required in both endocrine alpha- and beta-cell differentiation.

The development of the mammalian pancreas is governed by various signaling processes and by a cascade of gene activation events controlled by different transcription factors. Here we show that the divergent homeodomain transcription factor Prox1 is a novel, crucial regulator of mouse pancreas organogenesis. Loss of Prox1 function severely disrupted epithelial pancreas morphology and hindered pancreatic growth without affecting significantly the genesis of endocrine cells before E11.5. Conversely, the lack of Prox1 activity substantially decreased the formation of islet cell precursors after E13.5, during a period known as the “secondary transition”. Notably, this defect occurred concurrently with an abnormal increment of exocrine cells. Hence, it is possible that Prox1 contributes to the allocation of an adequate supply of islet cells throughout pancreas ontogeny by preventing exocrine cell differentiation of multipotent pancreatic progenitors. Prox1 thus appears to be an essential component of a genetic program destined to produce the cellular complexity of the mammalian pancreas.

The Pax-gene family encodes a group of transcription factors characterized by the presence of a highly conserved DNA-binding motif, the paired domain. Pax proteins are key regulators of vertebrate organogenesis since they play major roles in embryonic pattern formation, cell proliferation and cell differentiation (Chi and Epstein, 2002; Dahl et al., 1997; Dohrman et al., 2000; Epstein et al., 1994). Indeed, mutations in Pax genes lead to profound defects in organisms as diverse as flies, mice and humans (Chi and Epstein, 2002; Dahl et al., 1997). To date, nine mammalian Pax genes are known and these are grouped into five different subclasses according to their structural similarities. One of these subclasses comprises two close homologues, Pax4 and Pax6, that contain a second DNA-binding domain: the homeodomain (Dahl et al., 1997; Dohrman et al., 2000). Previous studies showed that Pax4 is a crucial regulator of mammalian pancreas development since lack of its activity prevents the formation of mature pancreatic insulin-producing (beta) cells (Dohrman et al., 2000; Sosa-Pineda et al., 1997; Wang et al., 2004). Presently, it is not yet clear how Pax4 is specifically required for the development of beta cells. Nonetheless, evidence gathered from recent studies has begun to unravel important aspects of the molecular function of Pax4 in pancreatic endocrine cells. Here, I will try to summarize the results of different efforts aimed at understanding how Pax4 is required for both, beta cell development and beta cell function.

In the presence of interferon-gamma (IFN-gamma), pancreatic ductal epithelial cells grow continuously, and islets undergo neogenesis. To determine whether these new islets are derived from conventional precursors, we tested whether IFN-gamma can complement the loss of transcription factors known to regulate pancreatic development. We analyzed the effect of a transgene on lethality in mice lacking the transcription factors Pax4, Pax6, or Pdx-1, by intercrossing such mice with transgenic mice whose pancreatic cells make IFN-gamma (ins-IFN-gamma mice). However, IFN-gamma expression did not rescue these mice from the lethal mutations, because no homozygous knockout mice carrying the IFN-gamma transgene survived, despite the survival of all other hemizygous gene combinations. This outcome demonstrates that the pathway for IFN-gamma regeneration requires the participation of Pax4, Pax6, and Pdx-1. We conclude that the striking islet regeneration observed in the ins-IFN-gamma NOD strain is regulated by the same transcription factors that control initial pancreatic development.

The mechanisms behind the cell-specific and compartmentalized expression of gut and pancreatic hormones is largely unknown. We hereby report that deletion of the Pax 4 gene virtually eliminates duodenal and jejunal hormone-secreting cells, as well as serotonin and somatostatin cells of the distal stomach, while deletion of the Pax 6 gene eliminates duodenal GIP cells as well as gastrin and somatostatin cells of the distal stomach. Thus, together, these two genes regulate the differentiation of all proximal gastrointestinal endocrine cells and reflect common pathways for pancreatic and gastrointestinal endocrine cell differentiation.

The functional unit of the endocrine pancreas is the islet of Langerhans. Islets are nested within the exocrine tissue of the pancreas and are composed of alpha-, beta-, delta- and gamma-cells. beta-Cells produce insulin and form the core of the islet, whereas alpha-, delta- and gamma-cells are arranged at the periphery of the islet and secrete glucagon, somatostatin and a pancreatic polypeptide, respectively. Little is known about the molecular and genetic factors regulating the lineage of the different endocrine cells. Pancreas development is known to be abolished in Pdx1-mutant mice and Pax4 mutants lack insulin-producing beta-cells. Here we show that the paired-box gene Pax6 is expressed during the early stages of pancreatic development and in mature endocrine cells. The pancreas of Pax6 homozygous mutant mice lack glucagon-producing cells, suggesting that Pax6 is essential for the differentiation of alpha-cells. As mice lacking Pax4 and Pax6 fail to develop any mature endocrine cells, we conclude that both Pax genes are required for endocrine fate in the pancreas.

Invasive pancreatic adenocarcinoma (PDAC) is one of the most lethal solid malignancies. PDAC arises from distinctive forms of neoplasia, with pancreatic intraepithelial neoplasia (PanIN) being the most common lesion. Mutational activation of Kras is nearly universal in PanINs and PDAC. We previously reported expression of the homeodomain transcription factor Prox1 in multipotent progenitors and in specific cell lineages of the mouse embryonic pancreas. We also uncovered that Prox1 controls growth and morphogenesis in the early developing pancreas (Wang et al., Dev.Dyn. 2005), and showed that the lack of its activity results in congenital ductal malformations, prominent loss of acinar cells, and gradual deterioration of the exocrine parenchyma (Westmoreland et al, Gastroenterology 2012). Prox1 expression persists in centroacinar and ductal cells in the adult mouse pancreas. In contrast, in mature pancreatic acinar cells Prox1 expression is never observed, except when these cells undergo acinar-to-ductal metaplastic conversion. Interestingly, different from other ductal transcription factors (e.g., Sox9, Pdx1, Hnf1β), Prox1 expression rapidly decays in developing PanINs whereas PDAC specimens from mice or humans completely lack Prox1 expression. This dynamic expression of Prox1 in the adult exocrine pancreas raised the possibility that Prox1 down-regulation could be necessary for pancreatic neoplastic transformation. We generated mice expressing both oncogenic Kras (KrasG12D) and normal Prox1 levels in the pancreas (KrasG12DPanc), and mice expressing KrasG12D which are also Prox1-happloinsufficient (Prox1ΔPanc/+;KrasG12DPanc), to investigate the effects of reducing the Prox1 dosage in PDAC formation. We found that Prox1-happloinsufficiency significantly increases the formation of both, PanINs and PDAC, in pancreatic tissues carrying oncogenic Kras. In addition, pancreatic tissues with combined Prox1-happloinsufficiency and KrasG12D expression also exhibited more prominent fibrosis, higher infiltration of inflammatory cells, and increased proliferation, in comparison to KrasG12DPanc mice. In line with these results, expression of Prox1 in Capan1 cells (which are Prox1-negative) using retroviruses significantly reduced CyclinD1 transcript levels and induced growth arrest. We are currently undertaking a combination of in vivo and in vitro approaches to uncover how Prox1 function counteracts Kras-driven transformation in pancreatic epithelial cells. Citation Format: Yiannis Drosos, Jianming Ye, Emin Kuliyev, Jerold Rehg, Anirban Maitra, Anna Means, Ramon Klein-Geltink, Gerard Grosveld, Beatriz Sosa-Pineda. Prox1-happloinsuficiency contributes to the transforming effects of Kras in the pancreas. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5017. doi:10.1158/1538-7445.AM2013-5017

Background & Aims: The exocrine portion of the pancreas functions in digestion and preserves pancreatic homeostasis. Learning how this tissue forms during embryogenesis could improve our understanding of human pancreatic diseases. Expression of the homeobox gene Prox1 in the exocrine pancreas changes throughout development in mice. We investigated the role of Prox1 in development of the exocrine pancreas in mice. Conclusions: Pancreas-specific deletion of Prox1 causes premature differentiation of acinar cells and poor elongation of epithelial branches; these defects indicate that Prox1 controls the expansion of tip progenitors in the early developing pancreas. During later stages of embryogenesis, Prox1 appears to regulate duct cell proliferation and morphogenesis. These findings identify Prox1 as an important regulator of pancreatic exocrine development. Methods: Mice with pancreas-specific deletion of Prox1 (Prox1ΔPanc) were generated and their pancreatic tissues were analyzed using immunohistochemistry, transmission electron microscopy, histologic techniques, quantitative real-time polymerase chain reaction, immunoblotting, and morphometric analysis. Results: Loss of Prox1 from the pancreas led to multiple exocrine alterations, most notably premature acinar cell differentiation, increased ductal cell proliferation, altered duct morphogenesis, and imbalanced expression of claudin proteins. Prox1ΔPanc mice also had some minor alterations in islet cells, but beta-cell development was not affected. The exocrine congenital defects of Prox1ΔPanc pancreata appeared to initiate a gradual process of deterioration that resulted in extensive loss of acinar cells, lipomatosis, and damage to ductal tissue in adult mice. Refereed/Peer-reviewed

Prox 1, a likely mouse homologue of the Drosophila homeobox gene prospero has been cloned and its expression pattern analyzed during development. In Drosophila, prospero is expressed in the developing CNS, lens-secreting cone cells of the eye, and midgut. In the mouse, Prox 1 is expressed in many of the same tissues: young neurons of the subventricular region of the CNS, developing eye lens and pancreas. Expression is also detected in the developing liver and heart, as well as transiently in the skeletal muscles. The similarities in protein sequence and expression patterns between the mouse and fly cognate genes suggest that Prox 1 may play, among others, a fundamental role in early development of the murine CNS.

Several genes are required during the early phases of liver specification, proliferation and differentiation1,2,3. Here we report that Prox1 is required for hepatocyte migration. Loss of Prox1 leads to formation of a smaller liver with a reduced population of clustered hepatocytes surrounded by a laminin-rich basal membrane.

Using a cDNA for an abundant Trypanosoma cruzi mRNA as probe, we have cloned and sequenced a gene which is organized in at least 20 nearly perfect tandem repeats of 940 base pairs. The 5' end of the mRNA has been sequenced by primer extension and found to contain a 35 nucleotide mini-exon (or spliced-leader) sequence that is ubiquitous in trypanosome mRNAs. This sequence, however, is not present in the tandem genomic repeats which encode the exon containing the major portion of the mRNA. Previous studies have shown that the 35-nucleotide sequence is encoded by a separate tandem gene family. One model to explain the formation of a segmented mRNA invokes priming of transcription by a small RNA which contains the leader sequence at its 5' end. However, northern blot analysis of total trypanosome RNA reveals a ladder of molecules larger than the mature mRNA, which appear to be faithful multimeric copies of the tandem gene. The discrete sizes of these RNAs correspond to those expected for partially processed precursors. These observations lend credence to the possibility of an alternative model where segmented mRNAs are generated by inter-molecular splicing.