Pancreatic Endocrine Development Research Articles

Differentiation of human pluripotent stem cells into two distinct NKX6.1 populations of pancreatic progenitors

BackgroundThe expression of a specific combination of transcription factors (TFs) in the multipotent progenitor cells (MPCs) is critical for determining pancreatic cell fate. NKX6.1 expression in PDX1+ MPCs is required for functional β cell generation. We have recently demonstrated the generation of a novel population of human pluripotent stem cell (hPSC)-derived MPCs that exclusively express NKX6.1, independently of PDX1 (PDX1−/NKX6.1+). Therefore, the aim of this study was to characterize this novel population to elucidate its role in pancreatic development.MethodsThe hPSCs were exposed to two differentiation protocols to generate MPCs that were analyzed using different techniques.ResultsBased on the expression of PDX1 and NKX6.1, we generated three different populations of MPCs, two of them were NKX6.1+. One of these NKX6.1 populations coexpressed PDX1 (PDX1+/NKX6.1+) which is known to mature into functional β cells, and an additional novel population did not express PDX1 (PDX1−/NKX6.1+) with an undefined role in pancreatic cell fate. This novel population was enriched using our recently established protocol, allowing their reorganization in three-dimensional (3D) structures. Since NKX6.1 induction in MPCs can direct them to endocrine and/or ductal cells in humans, we examined the coexpression of endocrine and ductal markers. We found that the expression of the pancreatic endocrine progenitor markers chromogranin A (CHGA) and neurogenin 3 (NGN3) was not detected in the NKX6.1+ 3D structures, while few structures were positive for NKX2.2, another endocrine progenitor marker, thereby shedding light on the origin of this novel population and its role in pancreatic endocrine development. Furthermore, SOX9 was highly expressed in the 3D structures, but cytokeratin 19, a main ductal marker, was not detected in these structures.ConclusionsThese data support the existence of two independent NKX6.1+ MPC populations during human pancreatic development and the novel PDX1−/NKX6.1+ population may be involved in a unique trajectory to generate β cells in humans.

Single-Cell Gene Expression Analysis of a Human ESC Model of Pancreatic Endocrine Development Reveals Different Paths to β-Cell Differentiation.

SummaryThe production of insulin-producing β cells from human embryonic stem cells (hESCs) in vitro represents a promising strategy for a cell-based therapy for type 1 diabetes mellitus. To explore the cellular heterogeneity and temporal progression of endocrine progenitors and their progeny, we performed single-cell qPCR on more than 500 cells across several stages of in vitro differentiation of hESCs and compared them with human islets. We reveal distinct subpopulations along the endocrine differentiation path and an early lineage bifurcation toward either polyhormonal cells or β-like cells. We uncover several similarities and differences with mouse development and reveal that cells can take multiple paths to the same differentiation state, a principle that could be relevant to other systems. Notably, activation of the key β-cell transcription factor NKX6.1 can be initiated before or after endocrine commitment. The single-cell temporal resolution we provide can be used to improve the production of functional β cells.

Sonic hedgehog pathway suppression and reactivation accelerates differentiation of rat adipose-derived mesenchymal stromal cells toward insulin-producing cells

Background aimsSonic hedgehog (Shh) is an intercellular signaling molecule that regulates pancreas development in mammals. Manipulation of Shh signaling pathway can be used as reliable approach to improve the generation of functional insulin-producing cells (IPCs) from mesenchymal stromal cells (MSCs). MethodsIn the present study, a novel differentiation protocol was used to produce IPCs from adipose tissue–derived MSCs (ATDMSCs) based on sequential inhibition and reactivation of Shh pathway. ATDMSCs were differentiated into IPCs via a 14-day basic protocol using 1% insulin transferrin selenium (ITS) and 1% nicotinamide in Dulbecco's Modified Eagle's Medium medium. A mixture of 0.25 µmol/L cyclopamine + 64 ng/mL basic fibroblast growth factor at day 3 of differentiation and 150 ng/mL recombinant Shh at day 11 of differentiation were used, respectively, to promote sequential inhibition and reactivation of Shh pathway. Insulin granule formation, glucose-stimulated insulin secretion and gene expression pattern related to the pancreatic endocrine development and function were analyzed in manipulated and unmanipulated IPCs. ResultsIPCs obtained after Shh manipulation secreted higher amounts of insulin in vitro. This phenotype was accompanied by increased expression of both genes critical for β-cell function and transcription factors associated with their mature phenotype including Pdx1, MafA, Nkx2.2, Nkx6.1, Ngn3, Isl1 and insulin at day 14 of differentiation. ConclusionsOur findings indicated that the early inhibition and late reactivation of Shh signaling pathway during the differentiation of ATDMSCs improved the functional properties of IPCs, a novel method that could be considered as an alternative approach for cell-based therapy for type 1 diabetes.

Identification of a small molecule that facilitates the differentiation of human iPSCs/ESCs and mouse embryonic pancreatic explants into pancreatic endocrine cells.

Pancreatic beta-like cells generated from human induced pluripotent stem cells (hiPSCs) or human embryonic stem cells (hESCs) offer an appealing donor tissue source. However, differentiation protocols that mainly use growth factors are costly. Therefore, in this study, we aimed to establish efficient differentiation protocols to change hiPSCs/hESCs to insulin (INS)+ cells using novel small-molecule inducers. We screened small molecules that increased the induction rate of INS+ cells from hESC-derived pancreatic and duodenal homeobox 1 (PDX1)+ pancreatic progenitor cells. The differentiation protocol to generate INS+ cells from hiPSCs/hESCs was optimised using hit compounds, and INS+ cells induced with the compounds were characterised for their in vitro and in vivo functions. The inducing activity of the hit compounds was also examined using mouse embryonic pancreatic tissues in an explant culture system. Finally, RNA sequencing analyses were performed on the INS+ cells to elucidate the mechanisms of action by which the hit compounds induced pancreatic endocrine differentiation. One hit compound, sodium cromoglicate (SCG), was identified out of approximately 1250 small molecules screened. When SCG was combined with a previously described protocol, the induction rate of INS+ cells increased from a mean±SD of 5.9±1.5% (n=3) to 16.5±2.1% (n=3). SCG induced neurogenin 3-positive cells at a mean±SD of 32.6±4.6% (n=3) compared with 14.2±3.6% (n=3) for control treatment without SCG, resulting in an increased generation of endocrine cells including insulin-producing cells. Similar induction by SCG was confirmed using mouse embryonic pancreatic explants. We also confirmed that the mechanisms of action by which SCG induced pancreatic endocrine differentiation included the inhibition of bone morphogenetic protein 4 signalling. SCG improves the generation of pancreatic endocrine cells from multiple hiPSC/hESC lines and mouse embryonic pancreatic explants by facilitating the differentiation of endocrine precursors. This discovery will contribute to elucidating the mechanisms of pancreatic endocrine development and facilitate cost-effective generation of INS+ cells from hiPSCs/hESCs. The RNA sequencing data generated during the current study are available in the Gene Expression Omnibus ( www.ncbi.nlm.nih.gov/geo ) with series accession number GSE89973.

Genetic evidence that Nkx2.2 acts primarily downstream of Neurog3 in pancreatic endocrine lineage development.

Many pancreatic transcription factors that are essential for islet cell differentiation have been well characterized; however, because they are often expressed in several different cell populations, their functional hierarchy remains unclear. To parse out the spatiotemporal regulation of islet cell differentiation, we used a Neurog3-Cre allele to ablate Nkx2.2, one of the earliest and most broadly expressed islet transcription factors, specifically in the Neurog3+ endocrine progenitor lineage (Nkx2.2△endo). Remarkably, many essential components of the β cell transcriptional network that were down-regulated in the Nkx2.2KO mice, were maintained in the Nkx2.2△endo mice - yet the Nkx2.2△endo mice displayed defective β cell differentiation and recapitulated the Nkx2.2KO phenotype. This suggests that Nkx2.2 is not only required in the early pancreatic progenitors, but has additional essential activities within the endocrine progenitor population. Consistently, we demonstrate Nkx2.2 functions as an integral component of a modular regulatory program to correctly specify pancreatic islet cell fates.

In Vivo Differentiation of Mesenchymal Stem Cells into Insulin Producing Cells on Electrospun Poly-L-Lactide Acid Scaffolds Coated with Matricaria chamomilla L. Oil.

This study examined the in vivo differentiation of mesenchymal stem cells (MSCs) into insulin producing cells (IPCs) on electrospun poly-L-lactide acid (PLLA) scaffolds coated with Matricaria chammomila L. (chamomile) oil. In this interventional, experimental study adipose MSCs (AMSCs) were isolated from 12 adult male New Zealand white rabbits and characterized by flow cytometry. AMSCs were subsequently differentiated into osteogenic and adipogenic lines. Cells were seeded onto either a PLLA scaffold (control) or PLLA scaffold coated with chamomile oil (experimental). A total of 24 scaffolds were inserted into the pancreatic area of each rabbit and placement was confirmed by ultrasound. After 21 days, immunohistochemistry analysis of insulin-producing like cells on protein levels confirmed insulin expression of insulin producing cells (IPSCs). Real-time polymerase chain reaction (PCR) determined the expressions of genes related to pancreatic endocrine development and function. Fourier transform infrared spectroscopy (FTIR) results confirmed the existence of oil on the surface of the PLLA scaffold. The results showed a new peak at 2854 cm(-1) for the aliphatic CH2 bond. Pdx1 expression was 0.051 ± 0.007 in the experimental group and 0.009 ± 0.002 in the control group. There was significantly increased insulin expression in the scaffold coated with chamomile oil (0.09 ± 0.001) compared to control group (0.063 ± 0.009, P≤0.05). Both groups expressed Ngn3 and Pdx1 specific markers and pancreatic tissue was observed at 21 days post transplantation. The pancreatic region is an optimal site for differentiation of AMSCs to IPCs. Chamomile oil (as an antioxidant agent) can affect cell adhesion to the scaffold and increase cell differentiation. In addition, the oil may lead to increased blood glucose uptake in pathways in the muscles, liver and fatty tissue of a diabetic animal model by some probable molecular mechanisms.

β-Cell-Specific Mafk Overexpression Impairs Pancreatic Endocrine Cell Development.

The MAF family transcription factors are homologs of v-Maf, the oncogenic component of the avian retrovirus AS42. They are subdivided into 2 groups, small and large MAF proteins, according to their structure, function, and molecular size. MAFK is a member of the small MAF family and acts as a dominant negative form of large MAFs. In previous research we generated transgenic mice that overexpress MAFK in order to suppress the function of large MAF proteins in pancreatic β-cells. These mice developed hyperglycemia in adulthood due to impairment of glucose-stimulated insulin secretion. The aim of the current study is to examine the effects of β-cell-specific Mafk overexpression in endocrine cell development. The developing islets of Mafk-transgenic embryos appeared to be disorganized with an inversion of total numbers of insulin+ and glucagon+ cells due to reduced β-cell proliferation. Gene expression analysis by quantitative RT-PCR revealed decreased levels of β-cell-related genes whose expressions are known to be controlled by large MAF proteins. Additionally, these changes were accompanied with a significant increase in key β-cell transcription factors likely due to compensatory mechanisms that might have been activated in response to the β-cell loss. Finally, microarray comparison of gene expression profiles between wild-type and transgenic pancreata revealed alteration of some uncharacterized genes including Pcbd1, Fam132a, Cryba2, and Npy, which might play important roles during pancreatic endocrine development. Taken together, these results suggest that Mafk overexpression impairs endocrine development through a regulation of numerous β-cell-related genes. The microarray analysis provided a unique data set of differentially expressed genes that might contribute to a better understanding of the molecular basis that governs the development and function of endocrine pancreas.

Progranulin Stimulates Proliferation of Mouse Pancreatic Islet Cells and Is Overexpressed in the Endocrine Pancreatic Tissue of an MEN1 Mouse Model.

Progranulin (PGRN) promotes cell growth and cell cycle progression in several cell types and contributes to tumorigenesis in diverse cancers. We have recently reported PGRN expression in islets and tumors developed in an MEN1 transgenic mouse. Here we sought to investigate PGRN expression and regulation after exposure to hypoxia as well as its effects on pancreatic islet cells and neuroendocrine tumors (NETs) in MEN1(+/−) mice. Gene and protein expression were analyzed by quantitative polymerase chain reaction, immunohistochemistry, and Western blot. We also investigated PGRN expression in samples from patients carrying pancreatic NETs associated or not with the multiple endocrine neoplasia 1 syndrome, using enzyme-linked immunosorbent assay and immunohistochemistry analysis. Progranulin is upregulated in tumors and islets of the MEN1 mouse as well as in the serum of patients with pancreatic NETs associated with glucagonoma syndrome. In normal mice islets and pancreatic tumors, PGRN expression was strongly potentiated by hypoxia. Progranulin promotes cell proliferation in islet cells and βTC-6 cells, a process paralleled by activation of the mitogen-activated protein kinase signaling cascade. Our findings identify PGRN as an effective inducer of pancreatic islet cell proliferation and a possible important factor for pancreatic endocrine tumor development.

The Role of ARX in Human Pancreatic Endocrine Specification.

The in vitro differentiation of human embryonic stem cells (hESCs) offers a model system to explore human development. Humans with mutations in the transcription factor Aristaless Related Homeobox (ARX) often suffer from the syndrome X-linked lissencephaly with ambiguous genitalia (XLAG), affecting many cell types including those of the pancreas. Indeed, XLAG pancreatic islets lack glucagon and pancreatic polypeptide-positive cells but retain somatostatin, insulin, and ghrelin-positive cells. To further examine the role of ARX in human pancreatic endocrine development, we utilized genomic editing in hESCs to generate deletions in ARX. ARX knockout hESCs retained pancreatic differentiation capacity and ARX knockout endocrine cells were biased toward somatostatin-positive cells (94% of endocrine cells) with reduced pancreatic polypeptide (rarely detected), glucagon (90% reduced) and insulin-positive (65% reduced) lineages. ARX knockout somatostatin-positive cells shared expression patterns with human fetal and adult δ-cells. Differentiated ARX knockout cells upregulated PAX4, NKX2.2, ISL1, HHEX, PCSK1, PCSK2 expression while downregulating PAX6 and IRX2. Re-expression of ARX in ARX knockout pancreatic progenitors reduced HHEX and increased PAX6 and insulin expression following differentiation. Taken together these data suggest that ARX plays a key role in pancreatic endocrine fate specification of pancreatic polypeptide, somatostatin, glucagon and insulin positive cells from hESCs.

Dermatoglyphic meta-analysis indicates early epigenetic outcomes & possible implications on genomic zygosity in type-2 diabetes.

Dermatoglyphic studies, particularly those arising from the Dutch Hunger Winter Families Cohort, indicate an involvement of prenatal epigenetic insults in type-2 diabetes. However, the exact orchestration of this association is not fully understood. Herein is described a meta-analysis performed based on a belief that such an approach could shed some light as to the role of genetic & epigenetic influences in the etiology of type-2 diabetes. The study incorporated reports identified from PubMed, Medline, & Google Scholar databases for eligible case-control studies that assessed dermatoglyphics in type-2 diabetes cases relative to controls. Over 44,000 fingerprints & 2300 palm prints from around 4400 individuals were included in the analysis. Decreased loops patterns [OR= 0.76; 95% CI= (0.59, 0.98)], increased non-loop patterns [OR= 1.31; 95% CI= (1.02, 1.68)], and reduced absolute finger ridge counts [OR= -0.19; 95% CI= (-0.33, -0.04)] were significant findings among the diabetic group. These results are indicative of mild developmental deviances, with epigenetic insults significantly linked to early gestation wherein critical events &signaling pathways of the endocrine pancreas development are witnessed. Further, the increased loop patterns with decreased non-loop patterns were deemed as possible indicators of decreased genomic heterozygosity with concurrently increased homozygosity in the diabetic group, linked to reduced buffering capacities during prenatal development. Epigenetic insults primarily during the 1 st trimester, to a lesser extent between the early-to-mid 2 ndtrimester, but least likely linked to those beyond the mid-second trimester are evident in type-2 diabetes. It is recommended that future research aimed at expounding the prenatal origins of T2DM, as well as developing novel therapeutic methods, should focus on the early stages of endocrine pancreatic development.

Initial Cell Seeding Density Influences Pancreatic Endocrine Development During in vitro Differentiation of Human Embryonic Stem Cells

Human embryonic stem cells (hESCs) have the ability to form cells derived from all three germ layers, and as such have received significant attention as a possible source for insulin-secreting pancreatic beta-cells for diabetes treatment. While considerable advances have been made in generating hESC-derived insulin-producing cells, to date in vitro-derived glucose-responsive beta-cells have remained an elusive goal. With the objective of increasing the in vitro formation of pancreatic endocrine cells, we examined the effect of varying initial cell seeding density from 1.3 x 104 cells/cm2 to 5.3 x 104 cells/cm2 followed by a 21-day pancreatic endocrine differentiation protocol. Low density-seeded cells were found to be biased toward the G2/M phases of the cell cycle and failed to efficiently differentiate into SOX17-CXCR4 co-positive definitive endoderm cells leaving increased numbers of OCT4 positive cells in day 4 cultures. Moderate density cultures effectively formed definitive endoderm and progressed to express PDX1 in approximately 20% of the culture. High density cultures contained approximately double the numbers of PDX1 positive pancreatic progenitor cells and also showed increased expression of MNX1, PTF1a, NGN3, ARX, and PAX4 compared to cultures seeded at moderate density. The cultures seeded at high density displayed increased formation of polyhormonal pancreatic endocrine cell populations co-expressing insulin, glucagon and somatostatin. The maturation process giving rise to these endocrine cell populations followed the expected cascade of pancreatic progenitor marker (PDX1 and MNX1) expression, followed by pancreatic endocrine specification marker expression (BRN4, PAX4, ARX, NEUROD1, NKX6.1 and NKX2.2) and then pancreatic hormone expression (insulin, glucagon and somatostatin). Taken together these data suggest that initial cell seeding density plays an important role in both germ layer specification and pancreatic progenitor commitment, which precedes pancreatic endocrine cell formation. This work highlights the need to examine standard culture variables such as seeding density when optimizing hESC differentiation protocols.

Conditional deletion of p53 and Rb in the renin-expressing compartment of the pancreas leads to a highly penetrant metastatic pancreatic neuroendocrine carcinoma

Efforts to model human pancreatic neuroendocrine tumors (PanNETs) in animals have been moderately successful, with minimal evidence for glucagonomas or metastatic spread. The renin gene, although classically associated with expression in the kidney, is also expressed in many other extrarenal tissues including the pancreas. To induce tumorigenesis within rennin-specific tissues, floxed alleles of p53 and Rb were selectively abrogated using Cre-recombinase driven by the renin promoter. The primary neoplasm generated is a highly metastatic islet cell carcinoma of the pancreas. Lineage tracing identifies descendants of renin-expressing cells as pancreatic alpha cells despite a lack of active renin expression in the mature pancreas. Both primary and metastatic tumors express high levels of glucagon; furthermore, an increased level of glucagon is found in the serum, identifying the pancreatic cancer as a functional glucagonoma. This new model is highly penetrant and exhibits robust frequency of metastases to the lymph nodes and the liver, mimicking human disease, and provides a useful platform for better understanding pancreatic endocrine differentiation and development, as well as islet cell carcinogenesis. The use of fluorescent reporters for lineage tracing of the cells contributing to disease initiation and progression provides an unique opportunity to dissect the timeline of disease, examining mechanisms of the metastatic process, as well as recovering primary and metastatic cells for identifying cooperating mutations that are necessary for progression of disease.

Control of Cell Identity in Pancreas Development and Regeneration

The endocrine and exocrine cells in the adult pancreas are not static, but can change their differentiation state in response to injury or stress. This concept of cells in flux means that there may be ways to generate certain types of cells (such as insulin-producing β-cells) and prevent formation of others (such as transformed neoplastic cells). We review different aspects of cell identity in the pancreas, discussing how cells achieve their identity during embryonic development and maturation, and how this identity remains plastic, even in the adult pancreas.

Smad signaling pathways regulate pancreatic endocrine development

Expansion of the pancreatic endocrine cell population occurs during both embryonic development and during post-natal pancreatic growth and regeneration. Mechanisms of the expansion of endocrine cells during embryonic development are not completely understood, and no clear mechanistic link has been established between growth of the embryonic endocrine pancreas and the islet cell replication that occurs in an adult animal. We found that transforming growth factor-beta (TGF-β) superfamily signaling, which has been implicated in many developmental processes, plays a key role in regulating pancreatic endocrine maturation and development. Specifically, the intracellular mediators of TGF-β signaling, smad2 and smad3, along with their inhibitor smad7, appear to mediate this process. Smad2, smad3 and smad7 were all broadly expressed throughout the early embryonic pancreatic epithelium. However, during later stages of development, smad2 and smad3 became strongly localized to the nuclei of the endocrine positive cells, whereas the inhibitory smad7 became absent in the endocrine component. Genetic inactivation of smad2 and smad3 led to a significant expansion of the embryonic endocrine compartment, whereas genetic inactivation of smad7 led to a significant decrease in the endocrine compartment. In vitro antisense studies further corroborated these results and supported the possibility that interplay between the inhibitory smad7 and the intracellular mediators smad2/3 is a control point for pancreatic endocrine development. These results should provide a better understanding of the key control mechanisms for β-cell development.

New findings in pancreatic and intestinal endocrine development to advance regenerative medicine

We highlight some of the major recent advances that have been made towards understanding the mechanisms that control endocrine differentiation and cell identity in the pancreas and intestine. Notch signaling plays a complex role in the fate choice between endocrine, duct, and acinar lineages in the developing pancreas. New approaches to dissecting the role of mesenchymal cells in the developing endocrine pancreas reveal inhibitory signals from the endothelium. Epigenetic mechanisms represent another layer of control over pancreatic development and β cell identity. Further details on the transcriptional control of enteroendocrine cell development have emerged and revealed a surprising role for FoxO1 in restraining insulin expression in the gut. Incremental progress is being made in the field of directed differentiation of embryonic stem cells to pancreatic β cells and the first reported differentiation of human embryonic stem cells into intestinal organoids containing enteroendocrine cells represents a major breakthrough. Greater knowledge of the fundamental processes controlling endocrine development in the pancreas and intestine has the potential to advance the field of regenerative medicine by providing a pathway to successfully create cell types of clinical interest.

Perinatal programming of obesity: an introduction to the topic

Perinatal programming of obesity: an introduction to the topic

Endocrine pancreatic development: impact of obesity and diet

During embryonic development, multipotent endodermal cells differentiate to form the pancreas. Islet cell clusters arising from the pancreatic bud form the acini tissue and exocrine ducts whilst pancreatic islets form around the edges of the clusters. The successive steps of islet differentiation are controlled by a complex network of transcription factors and signals that influence cell differentiation, growth and lineage. A Westernized lifestyle has led to an increased consumption of a high saturated fat diet, and an increase in maternal obesity. The developing fetus is highly sensitive to the intrauterine environment, therefore any alteration in maternal nutrition during gestation and lactation which affects the in-utero environment during the key developmental phases of the pancreas may change the factors controlling β-cell development and β-cell mass. Whilst the molecular mechanisms behind the adaptive programming of β-cells are still poorly understood it is established that changes arising from maternal obesity and/or over-nutrition may affect the ability to maintain fetal β-cell mass resulting in an increased risk of type 2 diabetes in adulthood.

Relative roles of the different Pax6 domains for pancreatic alpha cell development

BackgroundThe transcription factor Pax6 functions in the specification and maintenance of the differentiated cell lineages in the endocrine pancreas. It has two DNA binding domains, the paired domain and the homeodomain, in addition to a C-terminal transactivation domain. The phenotype of Pax6-/- knockout mice suggests non-redundant functions of the transcription factor in the development of glucagon-expressing α-cells as this cell type is absent in the mutants. We ask the question of how the differentiation of pancreatic endocrine cells, in particular that of α-cells, is affected by selective inactivation of either one of the three major domains of Pax6.ResultsThe Pax6Aey18 mutant mouse line, in which the paired domain is inactivated, showed a phenotype similar to that of Pax6-/- knockout mice with a near complete absence of glucagon-positive α-cells (0-4 cells/section; ≤1% of wt), reduced β-cell area (74% of wt) and disorganized islets. The proportion of ghrelin-positive ε-cells was expanded. In Pax6Sey-Neu mutants, which lack the transactivation domain, α-and β-cells where reduced to 25 and 40% of wt, respectively. We also studied two mouse lines with mutations in the homeodomain, Pax64Neu and Pax6132-14Neu. Neighboring amino acids are affected in the two lines and both point mutations abolish DNA binding of the classical P3 homeodomain target sequence. The pancreatic phenotype of the two mutants however was divergent. While Pax64Neu homozygotes showed a reduction of α- and β-cells to 59 and 61%, respectively, pancreatic endocrine development was unaltered in the Pax6132-14Neu mutant strain.ConclusionsWe show that inactivation of the Pax6 paired domain leads to a more severe phenotype with regards to the differentiation of pancreatic α-cells than the loss of the transactivation domain. The analysis of two different homeodomain mutants suggests that the binding of Pax6 to P3 homeodomain consensus sequences is not required for α-cell development. It rather seems that the homeodomain has a modulating role in Pax6 function, possibly by facilitating a PH0-like binding confirmation on paired domain target genes like proglucagon. This function is differentially affected by the two homeodomain mutations analyzed in this study.

Effect of forkhead box O1 (FOXO1) on beta cell development in the human fetal pancreas

Recent studies have demonstrated that in adult murine beta cells the forkhead box O1 (FOXO1) transcription factor regulates proliferation and stress resistance. However, the role of FOXO1 during pancreatic development remains largely unknown. The present study aimed to characterise the expression of the FOXO1 transcription factor in the early to mid-gestation human fetal pancreas and to understand its role in islet cell development. Human (8-21 week fetal age) pancreases were examined using immunohistological, quantitative RT-PCR and western blotting. Isolated human (18-21 week) fetal islet epithelial cell clusters were treated with insulin or glucose, or transfected with FOXO1 small interfering RNA (siRNA). Nuclear and cytoplasmic FOXO1 were widely produced during human fetal endocrine pancreatic development, co-localising in cells with the transcription factors pancreatic and duodenal homeobox 1 (PDX-1) and neurogenin 3 (NGN3) as well as cytokeratin 19 (CK19), insulin and glucagon. Treatment with exogenous insulin (50 nmol/l) induced the nuclear exclusion of FOXO1 in both cytokeratin 19 (CK19)(+) (p < 0.01) and insulin(+) cells (p < 0.05) in parallel with increased phospho-Akt (p < 0.05) production. siRNA knockdown of FOXO1 significantly increased the number of NGN3(+) (p < 0.01) and NK6 homeobox 1 (NKX6-1)(+) (p < 0.05) cells in parallel with increases in insulin gene expression (p < 0.03) and C-peptide(+) cells (p < 0.05) and reduced levels of hairy and enhancer of split 1 (HES1) (p < 0.01). Our results indicate that FOXO1 may negatively regulate beta cell differentiation in the human fetal pancreas by controlling critical transcription factors, including NGN3 and NKX6-1. These data suggest that the manipulation of FOXO1 levels may be a useful tool for improving cell-based strategies for the treatment of diabetes.

Expression of glucose-dependent insulinotropic polypeptide in the zebrafish

In mammals, glucose-dependent insulinotropic polypeptide (GIP) is synthesized predominately in the small intestine and functions in conjunction with insulin to promote nutrient deposition. However, little is known regarding GIP expression and function in early vertebrates like the zebrafish, a model organism representing an early stage in the evolutionary development of the compound vertebrate pancreas. Analysis of GIP and insulin (insa) expression in zebrafish larvae by RT-PCR demonstrated that although insa was detected as early as 24 h postfertilization (hpf), GIP expression was not demonstrated until 72 hpf, shortly after the completion of endocrine pancreatic development but prior to the commencement of independent feeding. Furthermore, whole mount in situ hybridization of zebrafish larvae showed expression of GIP and insa in the same tissues, and in adult zebrafish, RT-PCR and immunohistochemistry demonstrated GIP expression in both the intestine and the pancreas. Receptor activation studies showed that zebrafish GIP was capable of activating the rat GIP receptor. Although previous studies have identified four receptors with glucagon receptor-like sequences in the zebrafish, one of which possesses the capacity to bind GIP, a functional analysis of these receptors has not been performed. This study demonstrates interactions between the latter receptor and zebrafish GIP, identifying it as a potential in vivo target for the ligand. Finally, food deprivation studies in larvae demonstrated an increase in GIP and proglucagon II mRNA levels in response to fasting. In conclusion, the results of these studies suggest that although the zebrafish appears to be a model of an early stage of evolutionary development of GIP expression, the peptide may not possess incretin properties in this species.