Differentiation Of Exocrine Cells Research Articles

On the Origin of Pancreatic Cancer: Molecular Tumor Subtypes in Perspective of Exocrine Cell Plasticity.

Pancreatic ductal adenocarcinoma (PDAC) is a devastating type of cancer. While many studies have shed light into the pathobiology of PDAC, the nature of PDAC’s cell of origin remains under debate. Studies in adult pancreatic tissue have unveiled a remarkable exocrine cell plasticity including transitional states, mostly exemplified by acinar to ductal cell metaplasia, but also with recent evidence hinting at duct to basal cell transitions. Single-cell RNA sequencing has further revealed intrapopulation heterogeneity among acinar and duct cells. Transcriptomic and epigenomic relationships between these exocrine cell differentiation states and PDAC molecular subtypes have started to emerge, suggesting different ontogenies for different tumor subtypes. This review sheds light on these diverse aspects with particular focus on studies with human cells. Understanding the “masked ball” of exocrine cells at origin of PDAC and leaving behind the binary acinar vs duct cell classification may significantly advance our insights in PDAC biology.

Activation of Pancreatic Stellate Cells Is Beneficial for Exocrine but Not Endocrine Cell Differentiation in the Developing Human Pancreas.

Pancreatic stellate cells (PaSCs) are non-endocrine, mesenchymal-like cells that reside within the peri-pancreatic tissue of the rodent and human pancreas. PaSCs regulate extracellular matrix (ECM) turnover in maintaining the integrity of pancreatic tissue architecture. Although there is evidence indicating that PaSCs are involved in islet cell survival and function, its role in islet cell differentiation during human pancreatic development remains unclear. The present study examines the expression pattern and functional role of PaSCs in islet cell differentiation of the developing human pancreas from late 1st to 2nd trimester of pregnancy. The presence of PaSCs in human pancreata (8–22 weeks of fetal age) was characterized by ultrastructural, immunohistological, quantitative RT-PCR and western blotting approaches. Using human fetal PaSCs derived from pancreata at 14–16 weeks, freshly isolated human fetal islet-epithelial cell clusters (hIECCs) were co-cultured with active or inactive PaSCs in vitro. Ultrastructural and immunofluorescence analysis demonstrated a population of PaSCs near ducts and newly formed islets that appeared to make complex cell-cell dendritic-like contacts. A small subset of PaSCs co-localized with pancreatic progenitor-associated transcription factors (PDX1, SOX9, and NKX6-1). PaSCs were highly proliferative, with significantly higher mRNA and protein levels of PaSC markers (desmin, αSMA) during the 1st trimester of pregnancy compared to the 2nd trimester. Isolated human fetal PaSCs were identified by expression of stellate cell markers and ECM. Suppression of PaSC activation, using all-trans retinoic acid (ATRA), resulted in reduced PaSC proliferation and ECM proteins. Co-culture of hIECCs, directly on PaSCs or indirectly using Millicell® Inserts or using PaSC-conditioned medium, resulted in a reduction the number of insulin+ cells but a significant increase in the number of amylase+ cells. Suppression of PaSC activation or Notch activity during the co-culture resulted in an increase in beta-cell differentiation. This study determined that PaSCs, abundant during the 1st trimester of pancreatic development but decreased in the 2nd trimester, are located near ductal and islet structures. Direct and indirect co-cultures of hIECCs with PaSCs suggest that activation of PaSCs has opposing effects on beta-cell and exocrine cell differentiation during human fetal pancreas development, and that these effects may be dependent on Notch signaling.

Pancreatic development: one cell at a(pseudo)time.

Gaining a comprehensive understanding of the principles guiding lineage differentiation during organogenesis is a challenge. In this issue, Yu et al (2019) tackle this venture by taking advantage of single‐cell transcriptomic analyses and multiple mouse genetic tools. Their work provides new insights into pancreatic endocrine and exocrine cell differentiation landscapes and identifies new pathways regulating pancreatic lineage allocation in vivo .

Lnx2 ubiquitin ligase is essential for exocrine cell differentiation in the early zebrafish pancreas

The gene encoding the E3 ubiquitin ligase Ligand of Numb protein-X (Lnx)2a is expressed in the ventral-anterior pancreatic bud of zebrafish embryos in addition to its expression in the brain. Knockdown of Lnx2a by using an exon 2/intron 2 splice morpholino resulted in specific inhibition of the differentiation of ventral bud derived exocrine cell types, with little effect on endocrine cell types. A frame shifting null mutation in lnx2a did not mimic this phenotype, but a mutation that removed the exon 2 splice donor site did. We found that Lnx2b functions in a redundant manner with its paralog Lnx2a. Inhibition of lnx2a exon 2/3 splicing causes exon 2 skipping and leads to the production of an N-truncated protein that acts as an interfering molecule. Thus, the phenotype characterized by inhibition of exocrine cell differentiation requires inactivation of both Lnx2a and Lnx2b. Human LNX1 is known to destabilize Numb, and we show that inhibition of Numb expression rescues the Lnx2a/b-deficient phenotype. Further, Lnx2a/b inhibition leads to a reduction in the number of Notch active cells in the pancreas. We suggest that Lnx2a/b function to fine tune the regulation of Notch through Numb in the differentiation of cell types in the early zebrafish pancreas. Further, the complex relationships among genotype, phenotype, and morpholino effect in this case may be instructive in the ongoing consideration of morpholino use.

RNA Profiling and Chromatin Immunoprecipitation-Sequencing Reveal that PTF1a Stabilizes Pancreas Progenitor Identity via the Control of MNX1/HLXB9 and a Network of Other Transcription Factors

Pancreas development is initiated by the specification and expansion of a small group of endodermal cells. Several transcription factors are crucial for progenitor maintenance and expansion, but their interactions and the downstream targets mediating their activity are poorly understood. Among those factors, PTF1a, a basic helix-loop-helix (bHLH) transcription factor which controls pancreas exocrine cell differentiation, maintenance, and functionality, is also needed for the early specification of pancreas progenitors. We used RNA profiling and chromatin immunoprecipitation (ChIP) sequencing to identify a set of targets in pancreas progenitors. We demonstrate that Mnx1, a gene that is absolutely required in pancreas progenitors, is a major direct target of PTF1a and is regulated by a distant enhancer element. Pdx1, Nkx6.1, and Onecut1 are also direct PTF1a targets whose expression is promoted by PTF1a. These proteins, most of which were previously shown to be necessary for pancreas bud maintenance or formation, form a transcription factor network that allows the maintenance of pancreas progenitors. In addition, we identify Bmp7, Nr5a2, RhoV, and P2rx1 as new targets of PTF1a in pancreas progenitors.

Impairment of Rat Fetal Beta-Cell Development by Maternal Exposure to Dexamethasone during Different Time-Windows

AimGlucocorticoids (GCs) take part in the direct control of cell lineage during the late phase of pancreas development when endocrine and exocrine cell differentiation occurs. However, other tissues such as the vasculature exert a critical role before that phase. This study aims to investigate the consequences of overexposure to exogenous glucocorticoids during different time-windows of gestation for the development of the fetal endocrine pancreas.MethodsPregnant Wistar rats received dexamethasone acetate in their drinking water (1 µg/ml) during the last week or throughout gestation. Fetuses and their pancreases were analyzed at day 15 and 21 of gestation. Morphometrical analysis was performed on pancreatic sections after immunohistochemistry techniques and insulin secretion was evaluated on fetal islets collected in vitro.ResultsDexamethasone given the last week or throughout gestation reduced the beta-cell mass in 21-day-old fetuses by respectively 18% or 62%. This was accompanied by a defect in insulin secretion. The alpha-cell mass was reduced similarly. Neither islet vascularization nor beta-cell proliferation was affected when dexamethasone was administered during the last week, which was however the case when given throughout gestation. When given from the beginning of gestation, dexamethasone reduced the number of cells expressing the early marker of endocrine lineage neurogenin-3 when analyzed at 15 days of fetal age.ConclusionsGCs reduce the beta- and alpha-cell mass by different mechanisms according to the stage of development during which the treatment was applied. In fetuses exposed to glucocorticoids the last week of gestation only, beta-cell mass is reduced due to impairment of beta-cell commitment, whereas in fetuses exposed throughout gestation, islet vascularization and lower beta-cell proliferation are involved as well, amplifying the reduction of the endocrine mass.

A Novel Stepwise Differentiation of Functional Pancreatic Exocrine Cells from Embryonic Stem Cells

Cultivation of functional pancreatic cells isolated from adult mammalian pancreas remains difficult. We developed a differentiation protocol that gradually induced the formation of mouse pancreatic exocrine cells from embryonic stem cells (ESCs). This process mimicked in vivo pancreatic development by directing cells through definitive endoderm (DE), gut tube endoderm, and pancreatic progenitor cells to differentiated cells that expressed pancreatic exocrine enzymes. Mouse ESCs were cultured in hanging drops to form embryoid bodies. Treatment of embryoid bodies with activin A induced the formation of DE cells that expressed marker mRNAs Goosecoid and Mixl1 and that were double-positive with Foxa2 and Sox17 proteins. Subsequent treatment of the DE cells by retinoic acid induced the formation of gut tube endoderm cells that expressed the specific marker Hnf1b. Expression of Goosecoid and Mixl1 was downregulated during this period. Fibroblast growth factor 7 (FGF7) promoted differentiation of PDX1-expressing pancreatic progenitor cells that also expressed Foxa2 mRNA, an endodermal marker, suggesting derivation from the DE cells. Exocrine cell differentiation was induced with FGF7, glucagon-like peptide-1, and nicotinamide. The differentiated cells expressed mature pancreatic exocrine cell mRNAs, such as Amylase, Elastase, and Carboxypeptidase A. Additionally, they produced pancreatic elastase, amylase, carboxypeptidase A, and chymotrypsin proteins that were identified in cytoplasmic granules by immunocytochemistry. Active amylase was released into the medium. Moreover, FGF7 was associated with differentiation of pancreatic exocrine cells. The findings reported here offer a novel and effective process to develop pancreatic exocrine cells from ESCs.

THE HEXOSAMINE BIOSYNTHESIS PATHWAY IS ESSENTIAL FOR PANCREATIC BETA CELL DEVELOPMENT

Pancreatic exocrine and endocrine cells develop during embryonic life from endodermal progenitors. This process depends on activation of a hierarchy of transcription factors. While information is available regarding the mesodermal signals controlling pancreas development, little is known about the role of environmental factors such as nutrients— including glucose—that also may impact development. Previously, we showed that glucose plays an important and specific role in beta cell development by activating the transition of Neurogenin3‐positive endocrine progenitors into beta cells. Here, we examined the implication of glucose metabolism and more precisely the role of the hexosamine biosynthesis pathway (HBP) to understand the mechanisms by which glucose regulates beta cell development. We have established an in vitro model of endocrine and exocrine cells development from E13.5 rat pancreases in a manner that replicates in vivo pancreas development perfectly. Using this model, we tested the effect of selective inhibitors and activators of the HBP. If the HBP has a modest effect on cell proliferation and exocrine cell differentiation, beta cell development is tightly controlled by the HBP. Specifically, HBP activators increase beta cell development, whereas inhibitors repress such development. Importantly, both the HBP and glucose control the same steps in beta cell development.

Exocrine Pancreas Development and Trypsin Expression in Cultured European Sea Bass (Dicentrarchus labrax) Larvae

The ontogenesis and formation stages of exocrine pancreas in European sea bass (D. labrax) larvae were investigated from hatching to 40 days after hatching (DAH). Histological and enzymatical techniques were used to explain the functional development of the pancreas in D. labrax with the expression of trypsinogen activity. The incipient pancreas appeared as a lamination of the dorsal wall of the digestive tract. It was observed that the primary visible indication of exocrine cell differentiation was polarization. The first zymogen granules and pancreas with exocrine polyhedral cells appeared on 6 DAH and became abundant as a compact structure located dorsal and slightly posterior to the liver. At the same time, firstly, anus and then mouth were opened, and total lengths of larvae were determined as 3.47±0.26 mm. Until larval metamorphosis, the pancreas became diffuse, spreading throughout the mesentery enclosure, the stomach, the upper intestine and the pyloric caeca. On the other hand, zymogen granules were more numerous and larger, and a greater quantity of material was carried by the ducts, indicating an increased cellular activity. The specific activity of trypsin was determined as early as after hatching (42.54±6.8 mU/mg protein-1) at 4.28±0.2 mm total length of larvae and increased immediately during the following days especially after exogenous feeding. The highest tryptic activity was detected on 30 DAH as 122.45±11.76 mU/mg protein-1. It is concluded that exocrine pancreas organogenesis is the main critical step of the zymogen granules and trypsin activity is present as early as after hatching and continuously increasing with larval period of D. labrax.

E2F-1-Deficient NOD/SCID Mice Developed Showing Decreased Saliva Production

The non-obese diabetic mouse (NOD) is the most characterized model used to study insulin-dependent type 1 diabetes mellitus (IDDM) and Sjoögren's syndrome (SS). In a previous report, we found NOD.E2f1(-/-) mice show a greater progressive development to IDDM and SS compared to NOD mice. Our previous data indicated a progressive decrease in regulatory T cells (CD4(+)CD25(+)) and a decrease in the systemic secretion systems for insulin, and saliva was associated with the progression of IDDM and SS. Therefore, to define the mechanism of early-onset IDDM SS in E2F-1 deficient NOD mice required further investigation by producing E2F-1 deficient NOD/SCID mice in which the T and B cells do not develop. The purpose here was to analyze the essential function of the E2F-1 molecule in the development of IDDM and SS; and the dysfunction of the pancreas islet and salivary gland in the NOD background using NOD/SCID mice. We produced NOD/SCID.E2f1(-/-) mice using homologous recombination; determined diabetes development; measured saliva and insulin production; and performed a histological analysis. The deficient mice showed a decreasing volume of saliva; no infiltration of lymphocytes into salivary glands; no development of diabetes; and no protein localization of FGFR-2b in the ducts of the salivary gland that regulates submandibular gland proliferation and morphogenesis. Therefore, we considered a deficiency in E2F-1 induces a decrease in regulatory T cells and an increase in auto-reactive T cells; however, the E2F-1 deficiency is not associated with T and B cells-independent dysfunction of pancreatic beta cell in insulin secretion. Further, the E2F-1 deficiency is associated with T and B cells-independent dysfunction of the salivary gland exhibits a decrease in saliva production volume. We suggest E2F-1 may be also associated with the differentiation of exocrine cells in the duct where FGFR-2b is expressed in the salivary gland. The E2F-1 deficient NOD/SCID mouse model is useful for showing the development of the salivary gland; and is also useful for various experiments in humanized mice.

Identification of known and novel pancreas genes expressed downstream of Nkx2.2 during development

BackgroundThe homeodomain containing transcription factor Nkx2.2 is essential for the differentiation of pancreatic endocrine cells. Deletion of Nkx2.2 in mice leads to misspecification of islet cell types; insulin-expressing β cells and glucagon-expressing α cells are replaced by ghrelin-expressing cells. Additional studies have suggested that Nkx2.2 functions both as a transcriptional repressor and activator to regulate islet cell formation and function. To identify genes that are potentially regulated by Nkx2.2 during the major wave of endocrine and exocrine cell differentiation, we assessed gene expression changes that occur in the absence of Nkx2.2 at the onset of the secondary transition in the developing pancreas.ResultsMicroarray analysis identified 80 genes that were differentially expressed in e12.5 and/or e13.5 Nkx2.2-/- embryos. Some of these genes encode transcription factors that have been previously identified in the pancreas, clarifying the position of Nkx2.2 within the islet transcriptional regulatory pathway. We also identified signaling factors and transmembrane proteins that function downstream of Nkx2.2, including several that have not previously been described in the pancreas. Interestingly, a number of known exocrine genes are also misexpressed in the Nkx2.2-/- pancreas.ConclusionsExpression profiling of Nkx2.2-/- mice during embryogenesis has allowed us to identify known and novel pancreatic genes that function downstream of Nkx2.2 to regulate pancreas development. Several of the newly identified signaling factors and transmembrane proteins may function to influence islet cell fate decisions. These studies have also revealed a novel function for Nkx2.2 in maintaining appropriate exocrine gene expression. Most importantly, Nkx2.2 appears to function within a complex regulatory loop with Ngn3 at a key endocrine differentiation step.

The Hexosamine Biosynthesis Pathway Is Essential for Pancreatic Beta Cell Development

Pancreatic exocrine and endocrine cells develop during embryonic life from endodermal progenitors. This process depends on activation of a hierarchy of transcription factors. Although information is available regarding the mesodermal signals controlling pancreas development, little is known about the role of environmental factors such as nutrients, including glucose, that also may impact development. Previously, we showed that glucose plays an important and specific role in beta cell development by activating the transition of Neurogenin3-positive endocrine progenitors into beta cells. Here, we examined the implication of glucose metabolism and more precisely the role of the hexosamine biosynthesis pathway (HBP) to understand the mechanisms by which glucose regulates beta cell development. We have established an in vitro model of endocrine and exocrine cells development from embryonic day 13.5 rat pancreases in a manner that replicates in vivo pancreas development perfectly. Using this model, we tested the effect of selective inhibitors and activators of the HBP and found that the HBP has a modest effect on cell proliferation and exocrine cell differentiation. On the other hand, beta cell development is tightly controlled by the HBP. Specifically, HBP activators increase beta cell development, whereas inhibitors repress such development. Importantly, both the HBP and glucose control the same steps in beta cell development.

Conditional ablation of Notch signaling in pancreatic development

The role of the Notch signaling members Notch1, Notch2 and Rbpj in exocrine pancreatic development is not well defined. We therefore analyzed conditional pancreas-specific Rbpj and combined Notch1/Notch2 knockout mice using Ptf1a(+/Cre(ex1)) mice crossed with floxed Rbpj or Notch1/Notch2 mice. Mice were analyzed at different embryonic stages for pancreatic exocrine and endocrine development. The absence of Rbpj in pancreatic progenitor cells impaired exocrine pancreas development up to embryonic day 18.5 and led to premature differentiation of pancreatic progenitors into endocrine cells. In Rbpj-deficient pancreata, amylase-expressing acini and islets formed during late embryonic and postnatal development, suggesting an essential role of Rbpj in early but not late development. Contrary to this severe phenotype, the concomitant inactivation of Notch1 and Notch2 only moderately disturbed the proliferation of pancreatic epithelial cells during early embryonic development, and did not inhibit pancreatic development. Our results show that, in contrast to Rbpj, Notch1 and Notch2 are not essential for pancreatogenesis. These data favor a Notch-independent role of Rbpj in the development of the exocrine pancreas. Furthermore, our findings suggest that in late stages of pancreatic development exocrine cell differentiation and maintenance are independent of Rbpj.

Analyses of pancreas development by generation of gfp transgenic zebrafish using an exocrine pancreas-specific elastaseA gene promoter

In contrast to what we know on development of endocrine pancreas, the formation of exocrine pancreas remains poorly understood. To create an animal model that allows observation of exocrine cell differentiation, proliferation, and morphogenesis in living animals, we used the zebrafish elastaseA ( elaA) regulatory sequence to develop transgenic zebrafish that display highly specific exocrine pancreas expression of GFP in both larvae and adult. By following GFP expression, we found that the pancreas in early development was a relatively compact organ and later extended posterior along the intestine. By transferring the elaA:gfp transgene into slow muscle omitted mutant that is deficient in receiving Hedgehog signals, we further showed that Hedgehog signaling is required for exocrine morphogenesis but not for cell differentiation. We also applied the morpholino knockdown and toxin-mediated cell ablation approaches to this transgenic line. We showed that the development of exocrine pancreas is Islet-1 dependent. Injection of the diphtheria toxin A ( DTA) construct under the elastaseA promoter resulted in selective ablation of exocrine cells while the endocrine cells and other endodermal derivatives (liver and intestine) were not affected. Thus, our works demonstrated the new transgenic line provided a useful experimental tool in analyzing exocrine pancreas development.

Prox1 activity controls pancreas morphogenesis and participates in the production of “secondary transition” pancreatic endocrine cells

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.

Dissecting the role of glucocorticoids on pancreas development.

To determine whether glucocorticoids are involved in pancreas development, glucocorticoid treatment of rat pancreatic buds in vitro was combined with the analysis of transgenic mice lacking the glucocorticoid receptor (GR) in specific pancreatic cells. In vitro treatment of embryonic pancreata with dexamethasone, a glucocorticoid agonist, induced a decrease of insulin-expressing cell numbers and a doubling of acinar cell area, indicating that glucocorticoids favored acinar differentiation; in line with this, expression of Pdx-1, Pax-6, and Nkx6.1 was downregulated, whereas the mRNA levels of Ptf1-p48 and Hes-1 were increased. The selective inactivation of the GR gene in insulin-expressing beta-cells in mice (using a RIP-Cre transgene) had no measurable consequences on beta- or alpha-cell mass, whereas the absence of GR in the expression domain of Pdx-1 (Pdx-Cre transgene) led to a twofold increased beta-cell mass, with increased islet numbers and size but normal alpha-cell mass in adults. These results demonstrate that glucocorticoids play an important role in pancreatic beta-cell lineage, acting before hormone gene expression onset and possibly also modulating the balance between endocrine and exocrine cell differentiation.

Retinoic acid signaling is essential for pancreas development and promotes endocrine at the expense of exocrine cell differentiation in Xenopus

How and when the vertebrate endoderm is first subdivided into discrete progenitor cell populations that will give rise to the different major organs, including pancreas and liver, are only poorly understood. We have used Xenopus laevis as a model system to characterize these events, since it is particularly suited to study the early embryonic patterning in vertebrates. Our experimental results support the notion that retinoic acid (RA) functions as an essential endodermal patterning signal in Xenopus and that it acts as early as during gastrulation. As a result of RA treatment, the expression of Sonic Hedgehog (Shh), a known inhibitor of pancreas development in other vertebrate systems, is negatively regulated in the dorsal prepancreatic endoderm. Furthermore, RA is found to promote endocrine at the expense of exocrine differentiation in the dorsal pancreas, correlating with a specific inhibition of Notch signaling activities in this territory. Conversely, RA enhances exocrine marker gene expression in the ventral pancreas.

Glucagon-like peptide 1 receptor signaling influences topography of islet cells in mice.

Glucagon-like peptide 1 (GLP-1) amplifies glucose-induced insulin release in vivo and in vitro. Activation of GLP-1 receptor (GLP-1R) signaling leads to differentiation of exocrine cells towards a beta-cell phenotype in vitro and stimulation of islet cell proliferation in vitro and in vivo, suggesting a potential role for GLP-1 in the modulation of islet growth and differentiation. To determine whether basal levels of GLP-1R signaling are essential for islet development, we examined islet cell composition and topography in GLP-1R-/- mice. Total beta-cell volume and number are not altered, but the topography of beta cells is markedly different in GLP-1R-/- mice compared with GLP-1R+/+ controls. The distribution of beta cells is shifted from large to small and medium-sized islets in the absence of GLP-1R signaling (large islets: 50 +/- 3% in GLP-1R+/+ vs 28 +/- 4% in GLP- 1R-/-, P < 0.01 and medium islets: 32 +/- 2% in GLP- 1R+/+ vs 48 +/- 3% in GLP-1R-/-, P < 0.001). Furthermore, GLP-1R-/- islets exhibit abnormalities in cell topography, with two to threefold more centrally located alpha cells detected in GLP-1R-/- islets. These alterations in alpha- and beta-cell topography indicate that basal levels of GLP-1 signaling in the normal rodent are involved in the normal cellular organization of the endocrine pancreas.

Role of cell division in branching morphogenesis and differentiation of the embryonic pancreas.

A new culture system for the embryonic pancreas enables the formation of a branched organ in vitro. In such cultures, each terminal branch originates as a small bud and the number of buds and of terminal branches increases progressively with the expansion of the culture. However buds can also be resorbed during growth. The normal labelling index of cells in incipient buds ("tips") is greater than between buds ("dips") suggesting that budding may be driven by a local increase of cell division. Consistent with this, treatments that reduce cell division repress the formation of buds and branches. It is not possible to initiate budding in isolated endodermal epithelium by treatment with fibroblast growth factor, although this does increase the degree of differentiation of exocrine cells. Cultures in which cell division is completely inhibited by aphidicolin treatment will produce more endocrine cells than usual and inhibit the differentiation of exocrine cells. Consistent with this it is found that in untreated cultures the division of endocrine precursors cannot be detected by BrdU labelling whereas the division of exocrine precursors is frequent. It is concluded that cell division is necessary for bud formation in the embryonic pancreas and that the growth factors required for this normally come from the mesenchyme. Cell division is also necessary for exocrine differentiation. Endocrine cells, however, can arise from undifferentiated progenitors without cell division.

Conservation of PDX-1 structure, function, and expression in zebrafish.

Development of the mammalian pancreas has been studied extensively in mice. The stages from budding of the pancreatic anlaga through endocrine and exocrine cell differentiation and islet formation have been described in detail. Recently, the homeodomain transcription factor PDX-1 has been identified as an important factor in the proliferation and differentiation of the pancreatic buds to form a mature pancreas. To evaluate the possibility of using zebrafish as a model for the genetic analysis of pancreas development, we have cloned and characterized PDX-1 from this organism. The deduced sequence of zebrafish PDX-1 contains 246 amino acids and is 95% identical to mammalian PDX-1 in the homeodomain. We also cloned zebrafish preproinsulin complementary DNA as a marker for islet tissue. By in situ hybridization we demonstrate that PDX-1 and insulin are coexpressed during embryonic development and in adults, although PDX-1 expression appears to be biphasic. Insulin expression apparently begins before 44 hpf, the earliest stage examined in this study. Additionally, very high levels of PDX-1 expression were observed in the pyloric caeca, the accessory digestive organs that also are derived from the proximal region of the intestine in teleosts. Finally, our data show that the evolutionary conservation of zebrafish PDX-1 extends to its DNA binding properties. Zebrafish PDX-1 was equally as effective as mouse PDX-1 in stimulating insulin gene transcription, and maximum promoter activation was dependent on the presence of four intact A elements. The demonstration of this capability suggests that transcriptional regulatory mechanisms that control pancreatic development and insulin gene expression have been conserved among vertebrates.