Endocrine Cell Differentiation Research Articles

Differential effect of activin on mouse embryonic stem cell differentiation in insulin‐secreting cells under nestin‐positive selection and spontaneous differentiation protocols

Parallel to the importance of the development of cell therapies to treat diabetes is the production of sufficient numbers of pancreatic endocrine cells that function like primary islets. To increase the efficiency of endocrine pancreatic-like cell differentiation from mouse embryonic stem cells (ESCs), we applied activin-B to nestin-positive selection (protocol 1) and spontaneous differentiation (protocol 2) in different groups including: [A] activin-B, or [B] basic fibroblast growth factor (bFGF), and/or [C] activin-B+bFGF. The differentiated cells expressed most pancreatic-related genes. The number of insulin- and C peptide-positive cells, as well as dithizone-positive clusters in group A of protocol 1 was higher than in the other groups. Significant insulin concentrations in protocol 1 were produced when glucose was added to the medium, in comparison with protocol 2. Moreover, insulin release was increased significantly in group A of protocol 1 even with lower glucose. In conclusion, Addition of activin-B in a nestin-positive selection protocol increased the insulin-secreting cells in comparison with the same protocol with bFGF and/or spontaneous differentiation in presence of bFGF and/or activin-B alone. However, improvements of the current method are required to generate a sufficient source of true beta-cells for the treatment of diabetes mellitus.

Notch Signaling Is Required for Exocrine Regeneration After Acute Pancreatitis

The mechanisms for tissue regeneration and renewal after acute pancreatitis are not well understood but may involve activation of Notch signaling. To study the effect of Notch signaling ablation during acute experimental pancreatitis, we used a chemical and genetic approach to ablate Notch signaling in cerulein-induced pancreatitis in mice. Acute pancreatitis was induced by cerulein treatment in mice treated with the gamma-secretase inhibitor dibenzazepine or in conditional Notch1 knockout mice. Mice were characterized using immunohistologic, biochemical, and molecular methods. To investigate Notch and beta-catenin interaction, acinar 266-6 cells were analyzed using transfection and biochemical assays. Loss of Notch signaling results in impaired regeneration after acute pancreatitis with fewer mature acinar cells in dibenzazepine-treated and Notch1-deficient mice in the regenerative phase 3 days after induction. beta-catenin expression was increased and prolonged during exocrine regeneration. Crosstalk between Notch and beta-catenin-mediated signaling was identified, with Notch1-IC inhibiting beta-catenin-mediated transcriptional activity. This inhibition was dependent on a functional RAM domain. Inhibition of Notch signaling in vivo leads to impaired regeneration of the exocrine pancreas after acute pancreatitis. Our results suggest an interaction of Notch and Wnt signaling in pancreatic acinar cells, providing evidence for a role of these pathways in the regulation of the maturation process of acinar cells.

Pax-6 and c-Maf Functionally Interact with the α-Cell-specific DNA Element G1 in Vivo to Promote Glucagon Gene Expression

Specific expression of the glucagon gene in the rat pancreas requires the presence of the G1 element localized at -100/-49 base pairs on the promoter. Although it is known that multiple transcription factors such as Pax-6, Cdx-2/3, c-Maf, Maf-B, and Brain-4 can activate the glucagon gene promoter through G1, their relative importance in vivo is unknown. We first studied the expression of Maf-B, c-Maf, and Cdx-2/3 in the developing and adult mouse pancreas. Although Maf-B was detectable in a progressively increasing number of alpha-cells throughout development and in adulthood, c-Maf and Cdx-2/3 were expressed at low and very low levels, respectively. However, c-Maf but not Cdx-2/3 was detectable in adult islets by Western blot analyses. We then demonstrated the in vivo interactions of Pax-6, Cdx-2/3, Maf-B, and c-Maf but not Brain-4 with the glucagon gene promoter in glucagon-producing cells. Although Pax-6, Cdx-2/3, Maf-B, and c-Maf were all able to bind G1 by themselves, we showed that Pax-6 could interact with Maf-B, c-Maf, and Cdx-2/3 and activate transcription of the glucagon gene promoter. Overexpression of dominant negative forms of Cdx-2/3 and Mafs in alpha-cell lines indicated that Cdx-2/3 and the Maf proteins interact on an overlapping site within G1 and that this binding site is critical in the activation of the glucagon gene promoter. Finally, we show that specific inhibition of Pax-6 and c-Maf but not Cdx-2/3 or Maf-B led to decreases in endogenous glucagon gene expression and that c-Maf binds the glucagon gene promoter in mouse islets. We conclude that Pax-6 and c-Maf interact with G1 to activate basal expression of the glucagon gene.

Novel Effectors of Directed and Ngn3-Mediated Differentiation of Mouse Embryonic Stem Cells into Endocrine Pancreas Progenitors

The delineation of regulatory networks involved in early endocrine pancreas specification will play a crucial role in directing the differentiation of embryonic stem cells toward the mature phenotype of beta cells for cell therapy of type 1 diabetes. The transcription factor Ngn3 is required for the specification of the endocrine lineage, but its direct targets and the scope of biological processes it regulates remain elusive. We show that stepwise differentiation of embryonic stem cells using successive in vivo patterning signals can lead to simultaneous induction of Ptf1a and Pdx1 expression. In this cellular context, Ngn3 induction results in upregulation of its known direct target genes within 12 hours. Microarray gene expression profiling at distinct time points following Ngn3 induction suggested novel and diverse roles of Ngn3 in pancreas endocrine cell specification. Induction of Ngn3 expression results in regulation of the Wnt, integrin, Notch, and transforming growth factor beta signaling pathways and changes in biological processes affecting cell motility, adhesion, the cytoskeleton, the extracellular matrix, and gene expression. Furthermore, the combination of in vivo patterning signals and inducible Ngn3 expression enhances ESC differentiation toward the pancreas endocrine lineage. This is shown by strong upregulation of endocrine lineage terminal differentiation markers and strong expression of the hormones glucagon, somatostatin, and insulin. Importantly, all insulin(+) cells are also C-peptide(+), and glucose-dependent insulin release was 10-fold higher than basal levels. These data suggest that bona fide pancreas endocrine cells have been generated and that timely induction of Ngn3 expression can play a decisive role in directing ESC differentiation toward the endocrine lineage.

Rbp‐j regulates expansion of pancreatic epithelial cells and their differentiation into exocrine cells during mouse development

Notch signaling regulates cell fate determination in various tissues. We have reported the generation of mice with a pancreas-specific knockout of Rbp-j using Pdx.cre mice. Those mice exhibited premature endocrine and ductal differentiation. We now generated mice in which the Rbp-j gene was inactivated in Ptf1a-expressing cells using Ptf1a.cre mice. The timing of the Cre-mediated deletion in Rbp-j(f/f) Ptf1a.cre mice is 1 day later than that in Rbp-j(f/f) Pdx.cre mice. In Rbp-j(f/f) Ptf1a.cre mouse pancreases, at E13.5, the reduced Hes1 expression was accompanied by reduced epithelial growth, but premature endocrine cell differentiation was minimal. At E15.5, Pdx1 expression was repressed and acinar cell differentiation was reduced, but an increase in acinar cell proliferation was observed during the perinatal period. Our study indicates that, in addition to its role in preventing premature differentiation of early endocrine cells, Rbp-j regulates epithelial growth, Pdx1 expression, and acinar cell differentiation during mid-pancreatic development.

The retinoblastoma protein, RB, is required for gastrointestinal endocrine cells to exit the cell cycle, but not for hormone expression

Important functions of the RB family proteins include inhibition of cell cycle progression and regulation of terminal differentiation. We have examined the role of RB and the related protein, p107, in regulating cell cycle activity and differentiation of gastrointestinal endocrine cells, a relatively quiescent cell population, by conditionally disrupting the RB gene in neurogenin3 (Ngn3)-expressing cells in both p107+/+ and p107−/− mice. Endocrine cells in the small intestine, colon, pancreas, and stomach were present in normal numbers in RB and RB–p107 mutants except for an increase in serotonin cells and decrease in ghrelin cells in the antral stomach. Deletion of RB resulted in a dramatic increase in proliferating serotonin cells in the antral stomach and intestine, whereas other enteroendocrine cell types exhibited much lower cell cycle activity or remained quiescent. The related p107 protein appears dispensable for enteroendocrine differentiation and does not functionally compensate for the loss of RB. Our results suggest that RB is required for enteroendocrine cells, particularly serotonin cells, to undergo cell cycle arrest as they terminally differentiate. RB has relatively subtle effects on enteroendocrine cell differentiation and is not required for the expression of the normal repertoire of hormones in the gastrointestinal tract.

Loss of Myt1 function partially compromises endocrine islet cell differentiation and pancreatic physiological function in the mouse

Myelin transcription factor 1 ( Myt1) is one of the three vertebrate C2HC-type zinc finger transcription factors that include Myt1 ( Nzf1), Myt1L ( Png1), and Myt3 ( Nzf3, St18). All three paralogs are widely expressed in developing neuronal cells. Yet their function for mammalian development has not been investigated directly. Here we report that only Myt1 is expressed in the embryonic pancreas, in both endocrine progenitors and differentiated islet cells. Myt1 −/− animals die postnatally, likely due to confounding effects in multiple tissues. The endocrine tissues in the embryonic Myt1 −/− pancreas contained abnormal islet cells that expressed multiple hormones; although hormone levels were normal. We also created pancreas-specific Myt1 knockout mice. These mutant animals had no obvious physical defects from their wild-type littermates. Male mutant animals had reduced glucose-clearing abilities and abnormal multi-hormone-expressing cells present in their endocrine islets. In addition, they also had reduced Glut2 expression, and attenuated glucose-induced insulin secretion in the adult islets. Surprisingly, the expression of the Myt1 paralogs, Myt1l and Myt3, was induced in the embryonic Myt1 −/− pancreas. The consequences of Myt1 inactivation in the developing pancreas could be masked by activation of its paralogs, Myt1l and Myt3. These findings suggest Myt1 is involved in proper endocrine differentiation and function.

Intestinal Neurogenin 3 directs differentiation of a bipotential secretory progenitor to endocrine cell rather than goblet cell fate

Neurogenin 3 is essential for enteroendocrine cell development; however, it is unknown whether this transcription factor is sufficient to induce an endocrine program in the intestine or how it affects the development of other epithelial cells originating from common progenitors. In this study, the mouse villin promoter was used to drive Neurogenin 3 expression throughout the developing epithelium to measure the affect on cell fate. Although the general morphology of the intestine was unchanged, transgenic founder embryos displayed increased numbers of cells expressing the pan-endocrine marker chromogranin A. Accordingly, expression of several hormones and pro-endocrine transcription factors was increased in the transgenics suggesting that Neurogenin 3 stimulated a program of terminal enteroendocrine cell development. To test whether increased endocrine cell differentiation affected the development of other secretory cell lineages, we quantified goblet cells, the only other secretory cell formed in embryonic intestine. The Neurogenin 3-expressing transgenics had decreased numbers of goblet cells in correspondence to the increase in endocrine cells, with no change in the total secretory cell numbers. Thus, our data suggest that Neurogenin 3 can redirect the differentiation of bipotential secretory progenitors to endocrine rather than goblet cell fate.

Pancreas Development and β-Cell Differentiation of Embryonic Stem Cells

Embryonic stem (ES) cells may offer an unlimited cell source for the treatment of diabetes. However, a successful derivation of ES cells into islet-cells has proven to be more difficult than it was initially expected. Considering that the pancreas coordinates the global use of energy in the organism by secreting digestive enzymes and hormones, it is understandable that a sophisticated and tight regulation that lies on the pancreas itself to orchestrate its own tissue development and maturation. The complex process of endocrine cell differentiation can be better understood by analyzing the normal development of the pancreas. The proper detection of the signals provided in the pancreatic environment gives us a clue as to how the stem cells give rise to the whole pancreas. Careful and extensive screening of the natural or synthetic cytokines and growth factors and biochemical compounds that are essential in pancreatic development is required to properly mimic the process in vitro. Such a study would allow the researchers to achieve selective control of the differentiation and proliferation of the stem cells. The development and identification of the key molecules can provide us new insights into the pancreatic differentiation of the stem cells. We herein discuss the role of the microenvironment and transcriptional factors and cytokines, which have been recognized as important molecules during the major steps of the development of the pancreas. Finally, a more complete comprehension of the mechanisms that drive the pancreatic regeneration will provide us with new perspectives for future prophylactic and therapeutic interventions.

Glucose Is Necessary for Embryonic Pancreatic Endocrine Cell Differentiation

Mature pancreatic cells develop during embryonic life from endodermal progenitors, and this developmental process depends on activation of a hierarchy of transcription factors. While information is available on mesodermal signals controlling pancreas development, little is known about environmental factors, such as the levels of nutrients including glucose, that may control this process. Here, we studied the effects of glucose on pancreatic cells development. We used an in vitro model where both endocrine and acinar cells develop from early pancreatic and duodenal homeobox-1 (PDX1)-positive embryonic pancreatic progenitors. We first showed that glucose does not have a major effect on global pancreatic cell proliferation, survival, and acinar cell development. On the other hand, glucose controlled both alpha and beta cell development. Specifically, the surface occupied by insulin-positive cells was 20-fold higher in pancreases cultured in presence than in absence of glucose, and this effect was dose-dependent over the range 0.5-10 mm. Glucose did not appear to control beta cell development by activating the proliferation of early progenitors or beta cells themselves but instead tightly regulated cell differentiation. Thus, glucose did not modify the pattern of expression of Neurogenin3, the earliest marker of endocrine progenitor cells, but was necessary for the expression of the transcription factor NeuroD, a direct target of Neurogenin3 known to be important for proper pancreatic endocrine cell development. We conclude that glucose interferes with the pancreatic endocrine cells development by regulating the transition between Ngn3 and upstream NeuroD.

Nkx2.2 Regulates β-Cell Function in the Mature Islet

Nkx2.2 is a homeodomain transcription factor that is critical for pancreatic endocrine cell specification and differentiation in the developing mouse embryo. The purpose of this study was to determine whether Nkx2.2 is also required for the maintenance and function of the mature beta-cell in the postnatal islet. We have demonstrated previously that a repressor derivative of Nkx2.2 can functionally substitute for endogenous Nkx2.2 to fully restore alpha- and immature beta-cells in the embryonic islet; however, Nkx2.2 activator functions appear to be required to form a functional beta-cell. In this study, we have created transgenic mouse lines to express the Nkx2.2-repressor derivative in the mature beta-cell in the presence of endogenous Nkx2.2. The transgenic mice were assessed for beta-cell function, overall islet structure, and expression of beta-cell-specific markers. Using this transgenic approach, we have determined that the Nkx2.2-repressor derivative disrupts endogenous Nkx2.2 expression in adult mice and causes downregulation of the mature beta-cell factors, MafA and Glut2. Consistently, the Nkx2.2-repressor mice display reduced insulin gene expression and pancreatic insulin content and impaired insulin secretion. At weaning, the male Nkx2.2-repressor mice are overtly diabetic and all Nkx2.2-repressor transgenic mice exhibit glucose intolerance. Furthermore, the loss of beta-cell function in the Nkx2.2-repressor transgenic mice is associated with disrupted islet architecture. These studies indicate a previously undiscovered role for Nkx2.2 in the maintenance of mature beta-cell function and the formation of normal islet structure.

Calsenilin is required for endocrine pancreas development in zebrafish

Calsenilin/DREAM/Kchip3 is a neuronal calcium-binding protein. It is a multifunctional protein, mainly expressed in neural tissues and implicated in regulation of presenilin processing, repression of transcription, and modulation of A-type potassium channels. Here, we performed a search for new genes expressed during pancreatic development and have studied the spatiotemporal expression pattern and possible role of calsenilin in pancreatic development in zebrafish. We detected calsenilin transcripts in the pancreas from 21 somites to 39 hours postfertilization stages. Using double in situ hybridization, we found that the calsenilin gene was expressed in pancreatic endocrine cells. Loss-of-function experiments with anti-calsenilin morpholinos demonstrated that injected morphants have a significant decrease in the number of pancreatic endocrine cells. Furthermore, the knockdown of calsenilin leads to perturbation in islet morphogenesis, suggesting that calsenilin is required for early islet cell migration. Taken together, our results show that zebrafish calsenilin is involved in endocrine cell differentiation and morphogenesis within the pancreas.

MafB is required for islet β cell maturation

Pancreatic endocrine cell differentiation depends on transcription factors that also contribute in adult insulin and glucagon gene expression. Islet cell development was examined in mice lacking MafB, a transcription factor expressed in immature alpha (glucagon(+)) and beta (insulin(+)) cells and capable of activating insulin and glucagon expression in vitro. We observed that MafB(-/-) embryos had reduced numbers of insulin(+) and glucagon(+) cells throughout development, whereas the total number of endocrine cells was unchanged. Moreover, production of insulin(+) cells was delayed until embryonic day (E) 13.5 in mutant mice and coincided with the onset of MafA expression, a MafB-related activator of insulin transcription. MafA expression was only detected in the insulin(+) cell population in MafB mutants, whereas many important regulatory proteins continued to be expressed in insulin(-) beta cells. However, Pdx1, Nkx6.1, and GLUT2 were selectively lost in these insulin-deficient cells between E15.5 and E18.5. MafB appears to directly regulate transcription of these genes, because binding was observed within endogenous control region sequences. These results demonstrate that MafB plays a previously uncharacterized role by regulating transcription of key factors during development that are required for the production of mature alpha and beta cells.

TGF-β isoform signaling regulates secondary transition and mesenchymal-induced endocrine development in the embryonic mouse pancreas

Transforming growth factor-beta (TGF-β) superfamily signaling has been implicated in many developmental processes, including pancreatic development. Previous studies are conflicting with regard to an exact role for TGF-β signaling in various aspects of pancreatic organogenesis. Here we have investigated the role of TGF-β isoform signaling in embryonic pancreas differentiation and lineage selection. The TGF-β isoform receptors (RI, RII and ALK1) were localized mainly to both the pancreatic epithelium and mesenchyme at early stages of development, but then with increasing age localized to the pancreatic islets and ducts. To determine the specific role of TGF-β isoforms, we functionally inactivated TGF-β signaling at different points in the signaling cascade. Disruption of TGF-β signaling at the receptor level using mice overexpressing the dominant-negative TGF-β type II receptor showed an increase in endocrine precursors and proliferating endocrine cells, with an abnormal accumulation of endocrine cells around the developing ducts of mid–late stage embryonic pancreas. This pattern suggested that TGF-β isoform signaling may suppress the origination of secondary transition endocrine cells from the ducts. Secondly, TGF-β isoform ligand inhibition with neutralizing antibody in pancreatic organ culture also led to an increase in the number of endocrine-positive cells. Thirdly, hybrid mix-and-match in vitro recombinations of transgenic pancreatic mesenchyme and wild-type epithelium also led to increased endocrine cell differentiation, but with different patterns depending on the directionality of the epithelial–mesenchymal signaling. Together these results suggest that TGF-β signaling is important for restraining the growth and differentiation of pancreatic epithelial cells, particularly away from the endocrine lineage. Inhibition of TGF-β signaling in the embryonic period may thus allow pancreatic epithelial cells to progress towards the endocrine lineage unchecked, particularly as part of the secondary transition of pancreatic endocrine cell development. TGF-β RII in the ducts and islets may normally serve to downregulate the production of beta cells from embryonic ducts.

Pdx-1 Modulates Histone H4 Acetylation and Insulin Gene Expression in Terminally Differentiated α-TC-1 Cells

Islet-associated transcription factors play a critical role in regulating pancreatic endocrine cell differentiation and islet hormone gene expression. Both alpha- and beta-cells differentiate from a common endocrine precursor cell. Therefore, it is important to reveal the differential gene expression profiles between alpha- and beta-cells that can direct their terminal cell fates. alpha-TC-1 and beta-TC-1 are 2 terminally differentiated islet tumor cell lines derived from islets transformed by promoter-specific driven SV40 T antigen overexpression. In this study, we demonstrated that Pdx-1 is a potent transcriptional regulator of endogenous insulin gene expression in alpha-TC-1 cells. Reverse transcriptase-polymerase chain reaction and chromatin immunoprecipitation assays were used to analyze gene expression patterns and chromatin modifications in the insulin promoter. Differential transcription factor expression profiles of alpha-TC-1 and beta-TC-1 cells revealed that INSM-1 and Pdx-1 transcription factors were expressed exclusively in beta-TC-1 cells. Overexpression of Ad-Pdx-1 in alpha-TC-1 cells induced insulin gene expression. Chromatin immunoprecipitation assays in Ad-Pdx-1 alpha-TC-1 cells demonstrated Pdx-1 occupancy and the hyperacetylation of histone H4 in the insulin promoter region. Collectively, these experiments revealed that Pdx-1 is a potent transcriptional regulator that is capable of modulating histone H4 acetylation and activates the insulin gene in a terminally differentiated glucagonoma cell line.

Pancreatic Development and Disease

Pancreatic Development and Disease

Molecular regulation of pancreatic β-cell mass development, maintenance, and expansion

Pancreatic beta-cells are responsible for producing all of the insulin required by an organism to maintain glucose homeostasis. Defects in development, maintenance, or expansion of beta-cell mass can result in impaired glucose metabolism and diabetes. Thus, identifying the molecular regulators of these processes may provide new therapeutic targets for diabetes. Additionally, understanding the processes of beta-cell differentiation and proliferation may allow for in vitro cultivation of beta-cells in sufficient amounts to be transplanted into patients with diabetes. This review addresses many of the transcription factors and signaling pathways that play a role in early pancreatic development and endocrine cell (specifically beta-cell) differentiation, conditions that influence beta-cell mass development and molecular regulators of beta-cell proliferation and apoptosis that are responsible for maintaining and expanding beta-cell mass.

Rapid And Efficient in Vitro Generation of Pancreatic Islet Progenitor Cells from Nonendocrine Epithelial Cells in The Adult Human Pancreas

The absence of efficient and directed methods for the differentiation of adult pancreatic progenitor cell populations to pancreatic islet cells has raised doubts concerning the regeneration potential inherent in the adult pancreas. Relatively low levels of islet cell differentiation have been reported using adult pancreatic cells in vivo and in vitro. In the present study, we initially enriched for a nonendocrine epithelial component of the adult human pancreas and defined conditions that are permissive to islet cell differentiation in vitro. Sequential progression of cell differentiation in the permissive conditions allowed for incremental evaluation of changes occurring in the cell population. Optimization of the differentiation process, for the efficient production of islet endocrine cells, was accomplished by identifying specific factors and culture conditions that increased islet progenitor production 250-fold. Ultimately, 85% percent of the nonendocrine epithelial cells isolated from human pancreatic tissue and cultured in the optimized conditions for 8 days, readily re-expressed pancreatic duodenal homeobox-1 (Pdx1). Sixty-five percent of these Pdx1-expressing cells were capable of additional islet endocrine cell differentiation. This represents a significant advancement in the differentiation of an adult pancreatic progenitor cell population in vitro and suggests that the nonendocrine compartment of the human pancreas remains an important cell resource for the generation of transplantable islets to treat diabetes.

Novel Function of the Ciliogenic Transcription Factor RFX3 in Development of the Endocrine Pancreas

The transcription factor regulatory factor X (RFX)-3 regulates the expression of genes required for the growth and function of cilia. We show here that mouse RFX3 is expressed in developing and mature pancreatic endocrine cells during embryogenesis and in adults. RFX3 expression already is evident in early Ngn3-positive progenitors and is maintained in all major pancreatic endocrine cell lineages throughout their development. Primary cilia of hitherto unknown function present on these cells consequently are reduced in number and severely stunted in Rfx3(-/-) mice. This ciliary abnormality is associated with a developmental defect leading to a uniquely altered cellular composition of the islets of Langerhans. Just before birth, Rfx3(-/-) islets contain considerably less insulin-, glucagon-, and ghrelin-producing cells, whereas pancreatic polypeptide-positive cells are markedly increased in number. In adult mice, the defect leads to small and disorganized islets, reduced insulin production, and impaired glucose tolerance. These findings suggest that RFX3 participates in the mechanisms that govern pancreatic endocrine cell differentiation and that the presence of primary cilia on islet cells may play a key role in this process.

Regulation of pancreatic endocrine cell differentiation by sulphated proteoglycans

Epithelium-mesenchyme interactions play a major role in pancreas development. Recently, we demonstrated that embryonic pancreatic mesenchyme enhanced progenitor cell proliferation but inhibited endocrine cell differentiation. Here, we investigated the role played by sulphated proteoglycans, which are known to be essential to embryonic development, in this inhibitory effect. We first determined the expression of the genes encoding glypicans, syndecans and the main glycosaminoglycan chain-modifying enzymes in immature embryonic day (E) 13.5 and more differentiated E17.5 rat pancreases. Next, using an in vitro model of pancreas development, we blocked the action of endogenous sulphated proteoglycans by treating embryonic pancreases in culture with chlorate, an inhibitor of proteoglycan sulphation, and examined the effects on pancreatic endocrine cell differentiation. We first showed that expression of the genes encoding glypicans 1, 2, 3 and 5 and heparan sulphate 2-sulfotransferase decreased between E13.5 and E17.5. We next found that alteration of proteoglycan action by chlorate blocked the inhibitory effect of the mesenchyme on endocrine differentiation. Chlorate-treated pancreases exhibited a dramatic increase in beta cell number in a dose-dependent manner (169-and 375-fold increase with 30 mmol/l and 40 mmol/l chlorate, respectively) and in alpha cell development. Insulin-positive cells that developed in the presence of chlorate exhibited a phenotype of mature cells with regard to the expression of the following genes: pancreatic and duodenal homeobox gene 1 (Pdx1), proprotein convertase subtilisin/kexin type 1 (Pcsk1; previously known as pro-hormone convertase 1/3), proprotein convertase subtilisin/kexin type 2 (Pcsk2; previously known as pro-hormone convertase 2) and solute carrier family 2 (facilitated glucose transporter), member 2 (Slc2a1; previously known as glucose transporter 2). Finally, we showed that chlorate activated endocrine cell development by inducing neurogenin 3 (Neurog3) expression in early endocrine progenitor cells. We demonstrated that sulphated proteoglycans control pancreatic endocrine cell differentiation. Understanding the mechanism by which sulphated proteoglycans affect beta cell development could be useful in the generation of beta cells from embryonic stem cells.