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1.  Unlimited in vitro expansion of adult bi-potent pancreas progenitors through the Lgr5/R-spondin axis 
The EMBO Journal  2013;32(20):2708-2721.
Lgr5 marks adult stem cells in multiple adult organs and is a receptor for the Wnt-agonistic R-spondins (RSPOs). Intestinal, stomach and liver Lgr5+ stem cells grow in 3D cultures to form ever-expanding organoids, which resemble the tissues of origin. Wnt signalling is inactive and Lgr5 is not expressed under physiological conditions in the adult pancreas. However, we now report that the Wnt pathway is robustly activated upon injury by partial duct ligation (PDL), concomitant with the appearance of Lgr5 expression in regenerating pancreatic ducts. In vitro, duct fragments from mouse pancreas initiate Lgr5 expression in RSPO1-based cultures, and develop into budding cyst-like structures (organoids) that expand five-fold weekly for >40 weeks. Single isolated duct cells can also be cultured into pancreatic organoids, containing Lgr5 stem/progenitor cells that can be clonally expanded. Clonal pancreas organoids can be induced to differentiate into duct as well as endocrine cells upon transplantation, thus proving their bi-potentiality.
Unlimited in vitro expansion of adult bi-potent pancreas progenitors through the Lgr5/R-spondin axis
The establishment of conditions for long-term culture and expansion of adult, bi-potent pancreas progenitors may facilitate novel and tailored therapeutic approaches.
PMCID: PMC3801438  PMID: 24045232
beta cell; duct cell; pancreas; Wnt; stem cell
2.  In vitro expansion of single Lgr5+ liver stem cells induced by Wnt-driven regeneration 
Nature  2013;494(7436):247-250.
The Wnt target gene Lgr5 marks actively dividing stem cells in Wnt-driven, self-renewing tissues such as small intestine and colon1, stomach2 and hair follicles3. A 3D culture system allows long-term clonal expansion of single Lgr5+ stem cells into transplantable organoids that retain many characteristics of the original epithelial architecture2, 4, 5. A crucial component of the culture medium is the Wnt agonist Rspo16, the recently discovered ligand of Lgr57, 8. Here we show that Lgr5-LacZ is not expressed in healthy adult liver, yet that small Lgr5-LacZ+ cells appear near bile ducts upon damage, coinciding with robust activation of Wnt signaling. As shown by lineage tracing using a novel Lgr5-ires-CreERT2 knock-in allele, damage-induced Lgr5+ cells generate hepatocytes and bile ducts in vivo. Single Lgr5+ cells from damaged liver can be clonally expanded as organoids in Rspo1-based culture medium over multiple months. Such clonal organoids can be induced to differentiate in vitro and to generate functional hepatocytes upon transplantation into FAH−/− mice. These findings imply that previous observations on Lgr5+ stem cells in actively self-renewing tissues extend to damage-induced stem cells in a tissue with a low rate of spontaneous proliferation.
PMCID: PMC3634804  PMID: 23354049
3.  A Critical Role for the Wnt Effector Tcf4 in Adult Intestinal Homeostatic Self-Renewal 
Molecular and Cellular Biology  2012;32(10):1918-1927.
Throughout life, intestinal Lgr5+ stem cells give rise to proliferating transient amplifying cells in crypts, which subsequently differentiate into one of the five main cell types and migrate along the crypt-villus axis. These dynamic processes are coordinated by a relatively small number of evolutionarily conserved signaling pathways, which includes the Wnt signaling pathway. The DNA-binding proteins of the T-cell factor family, Tcf1/Tcf7, Lef, Tcf3/Tcf7l1, and Tcf4/Tcf7l2, constitute the downstream effectors of the Wnt signaling pathway. While Tcf4 is the major member active during embryogenesis, the role of these Wnt effectors in the homeostasis of the adult mouse intestinal epithelium is unresolved. Using Tcf1−/−, Tcf3flox, and novel Tcf4flox mice, we demonstrate an essential role for Tcf4 during homeostasis of the adult mouse intestine.
PMCID: PMC3347420  PMID: 22393260
4.  The Leukemia-Associated Mllt10/Af10-Dot1l Are Tcf4/β-Catenin Coactivators Essential for Intestinal Homeostasis 
PLoS Biology  2010;8(11):e1000539.
The leukemia-associated Mllt10/Af10 and its partner the histone methyltransferase Dot1l are identified as Tcf4/β-catenin co-activators and shown to be essential for Wnt-driven endogenous gene expression, intestinal development and homeostasis.
Wnt signaling maintains the undifferentiated state of intestinal crypt progenitor cells by inducing the formation of nuclear TCF4/β-catenin complexes. In colorectal cancer, activating mutations in Wnt pathway components cause inappropriate activation of TCF4/β-catenin-driven transcription. Despite the passage of a decade after the discovery of TCF4 and β-catenin as the molecular effectors of the Wnt signal, few transcriptional activators essential and unique to the regulation of this transcription program have been found. Using proteomics, we identified the leukemia-associated Mllt10/Af10 and the methyltransferase Dot1l as Tcf4/β-catenin interactors in mouse small intestinal crypts. Mllt10/Af10-Dot1l, essential for transcription elongation, are recruited to Wnt target genes in a β-catenin-dependent manner, resulting in H3K79 methylation over their coding regions in vivo in proliferative crypts of mouse small intestine in colorectal cancer and Wnt-inducible HEK293T cells. Depletion of MLLT10/AF10 in colorectal cancer and Wnt-inducible HEK293T cells followed by expression array analysis identifies MLLT10/AF10 and DOT1L as essential activators to a large extent dedicated to Wnt target gene regulation. In contrast, previously published β-catenin coactivators p300 and BRG1 displayed a more pleiotropic target gene expression profile controlling Wnt and other pathways. tcf4, mllt10/af10, and dot1l are co-expressed in Wnt-driven tissues in zebrafish and essential for Wnt-reporter activity. Intestinal differentiation defects in apc-mutant zebrafish can be rescued by depletion of Mllt10 and Dot1l, establishing these genes as activators downstream of Apc in Wnt target gene activation in vivo. Morpholino-depletion of mllt10/af10-dot1l in zebrafish results in defects in intestinal homeostasis and a significant reduction in the in vivo expression of direct Wnt target genes and in the number of proliferative intestinal epithelial cells. We conclude that Mllt10/Af10-Dot1l are essential, largely dedicated activators of Wnt-dependent transcription, critical for maintenance of intestinal proliferation and homeostasis. The methyltransferase DOT1L may present an attractive candidate for drug targeting in colorectal cancer.
Author Summary
The canonical Wnt pathway is a key regulatory pathway controlling intestinal cell proliferation, differentiation, and stem cell maintenance, and its deregulation leads to malignancies in the mammalian gut. A decade has passed since the discovery of the transcription factors TCF4-β-catenin as the downstream intestinal molecular effectors of Wnt, but few transcriptional activators essential and unique to the regulation of this transcription program have been found. In this study, using a proteomics approach, we identify the leukemia-associated Mllt10/Af10 and its partner the histone methyltransferase Dot1l as interactors with Tcf4/β-catenin in the mouse small intestinal epithelium. We demonstrate that Mllt10/Af10–Dot1l are recruited to Wnt target genes in intestinal epithelial cells and are essential to regulate expression of these targets. We also show a genetic link between the Wnt pathway and Mllt10/Af10-Dot1l in zebrafish and delineate their essential role in Wnt-driven endogenous gene expression. Finally, we demonstrate the physiological role of Mllt10/Af10-Dot1l in Wnt-driven intestinal development and homeostasis; depletion of Mllt10/Af10-Dot1l in zebrafish embryos mimics the Tcf4-depleted phenotype in which significant intestinal proliferation defects accompany a decrease in total number of intestinal cells. We conclude that the enzyme Dot1l may present an attractive candidate for drug targeting in colorectal cancer.
PMCID: PMC2982801  PMID: 21103407
5.  EuroDia: a beta-cell gene expression resource 
Type 2 diabetes mellitus (T2DM) is a major disease affecting nearly 280 million people worldwide. Whilst the pathophysiological mechanisms leading to disease are poorly understood, dysfunction of the insulin-producing pancreatic beta-cells is key event for disease development. Monitoring the gene expression profiles of pancreatic beta-cells under several genetic or chemical perturbations has shed light on genes and pathways involved in T2DM. The EuroDia database has been established to build a unique collection of gene expression measurements performed on beta-cells of three organisms, namely human, mouse and rat. The Gene Expression Data Analysis Interface (GEDAI) has been developed to support this database. The quality of each dataset is assessed by a series of quality control procedures to detect putative hybridization outliers. The system integrates a web interface to several standard analysis functions from R/Bioconductor to identify differentially expressed genes and pathways. It also allows the combination of multiple experiments performed on different array platforms of the same technology. The design of this system enables each user to rapidly design a custom analysis pipeline and thus produce their own list of genes and pathways. Raw and normalized data can be downloaded for each experiment. The flexible engine of this database (GEDAI) is currently used to handle gene expression data from several laboratory-run projects dealing with different organisms and platforms.
Database URL:
PMCID: PMC2963318  PMID: 20940178
6.  Epistasis of Transcriptomes Reveals Synergism between Transcriptional Activators Hnf1α and Hnf4α 
PLoS Genetics  2010;6(5):e1000970.
The transcription of individual genes is determined by combinatorial interactions between DNA–binding transcription factors. The current challenge is to understand how such combinatorial interactions regulate broad genetic programs that underlie cellular functions and disease. The transcription factors Hnf1α and Hnf4α control pancreatic islet β-cell function and growth, and mutations in their genes cause closely related forms of diabetes. We have now exploited genetic epistasis to examine how Hnf1α and Hnf4α functionally interact in pancreatic islets. Expression profiling in islets from either Hnf1a+/− or pancreas-specific Hnf4a mutant mice showed that the two transcription factors regulate a strikingly similar set of genes. We integrated expression and genomic binding studies and show that the shared transcriptional phenotype of these two mutant models is linked to common direct targets, rather than to known effects of Hnf1α on Hnf4a gene transcription. Epistasis analysis with transcriptomes of single- and double-mutant islets revealed that Hnf1α and Hnf4α regulate common targets synergistically. Hnf1α binding in Hnf4a-deficient islets was decreased in selected targets, but remained unaltered in others, thus suggesting that the mechanisms for synergistic regulation are gene-specific. These findings provide an in vivo strategy to study combinatorial gene regulation and reveal how Hnf1α and Hnf4α control a common islet-cell regulatory program that is defective in human monogenic diabetes.
Author Summary
The transcriptional activity of each gene is typically determined by multiple transcription factors. This concept has been well established in studies of single genes. However, transcription factors do not simply regulate single genes, they also control broad gene programs that underlie cellular function and disease. Understanding how combinations of transcription factors interact at the level of cellular regulatory programs remains a challenge. Humans with mutations in the genes encoding for the transcription factors Hnf1α and Hnf4α develop similar forms of diabetes that result from abnormal insulin secretion, suggesting that the two factors might have related functions in insulin-producing islet-cells. We now show that Hnf1α or Hnf4α bind to a common set of genes and that islet-cells from mice in which either Hnf1α or Hnf4α has been selectively disrupted show abnormal expression of similar genes. By comparing the gene expression defects of mice with mutations in either Hnf1a, Hnf4a, or both genes, we determined that Hnf1α and Hnf4α regulate common target genes through synergistic mechanisms. These results thus provide insight into a regulatory network that fails in human diabetes. Similar genetic strategies can also be employed to unravel how other transcription factors interact functionally in native cellular contexts.
PMCID: PMC2877749  PMID: 20523905
7.  Functional Targets of the Monogenic Diabetes Transcription Factors HNF-1α and HNF-4α Are Highly Conserved Between Mice and Humans 
Diabetes  2009;58(5):1245-1253.
The evolutionary conservation of transcriptional mechanisms has been widely exploited to understand human biology and disease. Recent findings, however, unexpectedly showed that the transcriptional regulators hepatocyte nuclear factor (HNF)-1α and -4α rarely bind to the same genes in mice and humans, leading to the proposal that tissue-specific transcriptional regulation has undergone extensive divergence in the two species. Such observations have major implications for the use of mouse models to understand HNF-1α– and HNF-4α–deficient diabetes. However, the significance of studies that assess binding without considering regulatory function is poorly understood.
We compared previously reported mouse and human HNF-1α and HNF-4α binding studies with independent binding experiments. We also integrated binding studies with mouse and human loss-of-function gene expression datasets.
First, we confirmed the existence of species-specific HNF-1α and -4α binding, yet observed incomplete detection of binding in the different datasets, causing an underestimation of binding conservation. Second, only a minor fraction of HNF-1α– and HNF-4α–bound genes were downregulated in the absence of these regulators. This subset of functional targets did not show evidence for evolutionary divergence of binding or binding sequence motifs. Finally, we observed differences between conserved and species-specific binding properties. For example, conserved binding was more frequently located near transcriptional start sites and was more likely to involve multiple binding events in the same gene.
Despite evolutionary changes in binding, essential direct transcriptional functions of HNF-1α and -4α are largely conserved between mice and humans.
PMCID: PMC2671044  PMID: 19188435
8.  Hnf1α (MODY3) Controls Tissue-Specific Transcriptional Programs and Exerts Opposed Effects on Cell Growth in Pancreatic Islets and Liver▿ †  
Molecular and Cellular Biology  2009;29(11):2945-2959.
Heterozygous HNF1A mutations cause pancreatic-islet β-cell dysfunction and monogenic diabetes (MODY3). Hnf1α is known to regulate numerous hepatic genes, yet knowledge of its function in pancreatic islets is more limited. We now show that Hnf1a deficiency in mice leads to highly tissue-specific changes in the expression of genes involved in key functions of both islets and liver. To gain insights into the mechanisms of tissue-specific Hnf1α regulation, we integrated expression studies of Hnf1a-deficient mice with identification of direct Hnf1α targets. We demonstrate that Hnf1α can bind in a tissue-selective manner to genes that are expressed only in liver or islets. We also show that Hnf1α is essential only for the transcription of a minor fraction of its direct-target genes. Even among genes that were expressed in both liver and islets, the subset of targets showing functional dependence on Hnf1α was highly tissue specific. This was partly explained by the compensatory occupancy by the paralog Hnf1β at selected genes in Hnf1a-deficient liver. In keeping with these findings, the biological consequences of Hnf1a deficiency were markedly different in islets and liver. Notably, Hnf1a deficiency led to impaired large-T-antigen-induced growth and oncogenesis in β cells yet enhanced proliferation in hepatocytes. Collectively, these findings show that Hnf1α governs broad, highly tissue-specific genetic programs in pancreatic islets and liver and reveal key consequences of Hnf1a deficiency relevant to the pathophysiology of monogenic diabetes.
PMCID: PMC2682018  PMID: 19289501
9.  Macrosomia and Hyperinsulinaemic Hypoglycaemia in Patients with Heterozygous Mutations in the HNF4A Gene 
PLoS Medicine  2007;4(4):e118.
Macrosomia is associated with considerable neonatal and maternal morbidity. Factors that predict macrosomia are poorly understood. The increased rate of macrosomia in the offspring of pregnant women with diabetes and in congenital hyperinsulinaemia is mediated by increased foetal insulin secretion. We assessed the in utero and neonatal role of two key regulators of pancreatic insulin secretion by studying birthweight and the incidence of neonatal hypoglycaemia in patients with heterozygous mutations in the maturity-onset diabetes of the young (MODY) genes HNF4A (encoding HNF-4α) and HNF1A/TCF1 (encoding HNF-1α), and the effect of pancreatic deletion of Hnf4a on foetal and neonatal insulin secretion in mice.
Methods and Findings
We examined birthweight and hypoglycaemia in 108 patients from families with diabetes due to HNF4A mutations, and 134 patients from families with HNF1A mutations. Birthweight was increased by a median of 790 g in HNF4A-mutation carriers compared to non-mutation family members (p < 0.001); 56% (30/54) of HNF4A-mutation carriers were macrosomic compared with 13% (7/54) of non-mutation family members (p < 0.001). Transient hypoglycaemia was reported in 8/54 infants with heterozygous HNF4A mutations, but was reported in none of 54 non-mutation carriers (p = 0.003). There was documented hyperinsulinaemia in three cases. Birthweight and prevalence of neonatal hypoglycaemia were not increased in HNF1A-mutation carriers. Mice with pancreatic β-cell deletion of Hnf4a had hyperinsulinaemia in utero and hyperinsulinaemic hypoglycaemia at birth.
HNF4A mutations are associated with a considerable increase in birthweight and macrosomia, and are a novel cause of neonatal hypoglycaemia. This study establishes a key role for HNF4A in determining foetal birthweight, and uncovers an unanticipated feature of the natural history of HNF4A-deficient diabetes, with hyperinsulinaemia at birth evolving to decreased insulin secretion and diabetes later in life.
HNF4A mutations were found to be associated with a considerable increase in birthweight and macrosomia, and were a cause of neonatal hypoglycaemia.
Editors' Summary
MODY, or maturity-onset diabetes of the young, is a particular subtype of diabetes; only a few percent of people with diabetes are thought to have this subtype. The condition comes about as a result of a mutation in one of six genes. Generally, people with MODY have high glucose (sugar) levels in the blood, and the typical symptoms of diabetes, such as increased thirst and urination, typically develop when the person is below the age of 25 y. Two of the genes that are known to cause MODY are mutant forms of HNF4A and HNF1A. The proteins that are encoded by these two genes control insulin levels produced by the pancreas; when these genes are mutated, not enough insulin is produced. Without enough insulin to control blood sugar, levels rise, leading to the symptoms of diabetes. However, MODY can be managed by many of the same interventions as other types of diabetes, such as diet, exercise, drug treatments, and insulin injections.
Why Was This Study Done?
Although the evidence shows that individuals who carry mutations in HNF4A and HNF1A do not produce enough insulin and therefore have higher glucose levels in their blood, there were some tantalizing suggestions from mouse experiments that this might not be the whole story. Specifically, the researchers suspected that during embryonic development, mutations in HNF4A or HNF1A might actually cause higher insulin levels. Too much insulin during development of a fetus is known to cause it to gain weight, resulting in a baby that is larger than the average size for its age. Larger babies are risky for both the baby and the mother. The researchers doing this study wanted to understand more precisely what the links were between the forms of MODY caused by HNF4A and HNF1A mutations, and birth-weight and blood-sugar levels.
What Did the Researchers Do and Find?
In this study, the researchers examined 15 families in which some family members had MODY caused by a mutation in HNF4A. They compared the birthweight for family members carrying the mutation (54 people) against the birthweight for those who did not (54 people). A similar comparison was done for 38 families in which some members had a different form of MODY, this time caused by a mutation in HNF1A. The results showed that the birthweight of family members who carried a mutation in HNF4A was, on average, 790 g higher than the birthweight of family members who didn't carry the mutation. Low blood-sugar levels at birth were also more common in people carrying the HNF4A mutation as compared to people who did not. However, the HNF1A mutation did not seem to be associated with greater birthweight or low blood-sugar levels at birth. Finally, in order to understand these findings further, the researchers created embryonic mice carrying mutations in the mouse equivalent of HNF4A. These embryos produced more insulin than normal mouse embryos and, after birth, were more likely to have low blood-sugar levels.
What Do These Findings Mean?
These findings show that there is a link between mutations in HNF4A, but not in HNF1A, and increased birthweight. The increase found in this study is quite substantial (a median weight of 4,660 g in the affected babies; a birthweight of more than 4,000 g is generally considered large). The results suggest that in human embryos with a mutated form of HNF4A, too much insulin is produced during development, causing faster growth and a higher chance of the baby being born with low blood-sugar levels. This is an unexpected finding, because later in life the HNF4A mutation causes lower insulin levels. Therefore, the biochemical pathways causing this type of MODY seem to be quite complicated, and further research will need to be done to fully understand them. Crucially, the research also suggests that pregnant women carrying HNF4A mutations should be closely followed to check their baby's growth and minimize the chance of complications. Doctors and families should also consider doing a genetic test for HNF4A if a baby has low blood-sugar levels and if there is a family history of diabetes; this would increase the chance of diagnosing MODY early.
Additional Information.
Please access these Web sites via the online version of this summary at 0040118.
In a related Perspective in PLoS Medicine, Benjamin Glaser discusses causes of type 2 diabetes mellitus in the context of this study's findings
The US National Institute of Diabetes and Digestive and Kidney Diseases has pages of information on different types of diabetes
Wikipedia has an entry on Maturity Onset Diabetes of the Young (MODY) (note that Wikipedia is an internet encyclopedia that anyone can edit)
Diabetes Research Department, Peninsula Medical School, Exeter, UK provides information for patients and doctors on genetic types of diabetes; the website is maintained by the research group carrying out this study
Information from the Centers for Disease Control and Prevention on diabetes and pregnancy
PMCID: PMC1845156  PMID: 17407387
10.  Genetic evidence that HNF-1α–dependent transcriptional control of HNF-4α is essential for human pancreatic β cell function 
Mutations in the genes encoding hepatocyte nuclear factor 4α (HNF-4α) and HNF-1α impair insulin secretion and cause maturity onset diabetes of the young (MODY). HNF-4α is known to be an essential positive regulator of HNF-1α. More recent data demonstrates that HNF-4α expression is dependent on HNF-1α in mouse pancreatic islets and exocrine cells. This effect is mediated by binding of HNF-1α to a tissue-specific promoter (P2) located 45.6 kb upstream from the previously characterized Hnf4α promoter (P1). Here we report that the expression of HNF-4α in human islets and exocrine cells is primarily mediated by the P2 promoter. Furthermore, we describe a G → A mutation in a conserved nucleotide position of the HNF-1α binding site of the P2 promoter, which cosegregates with MODY. The mutation results in decreased affinity for HNF-1α, and consequently in reduced HNF-1α–dependent activation. These findings provide genetic evidence that HNF-1α serves as an upstream regulator of HNF-4α and interacts directly with the P2 promoter in human pancreatic cells. Furthermore, they indicate that this regulation is essential to maintain normal pancreatic function.
PMCID: PMC151122  PMID: 12235114
11.  Hepatic Nuclear Factor 1-α Directs Nucleosomal Hyperacetylation to Its Tissue-Specific Transcriptional Targets 
Molecular and Cellular Biology  2001;21(9):3234-3243.
Mutations in the gene encoding hepatic nuclear factor 1-α (HNF1-α) cause a subtype of human diabetes resulting from selective pancreatic β-cell dysfunction. We have analyzed mice lacking HNF1-α to study how this protein controls β-cell-specific transcription in vivo. We show that HNF1-α is essential for the expression of glut2 glucose transporter and L-type pyruvate kinase (pklr) genes in pancreatic insulin-producing cells, whereas in liver, kidney, or duodenum tissue, glut2 and pklr expression is maintained in the absence of HNF1-α. HNF1-α nevertheless occupies the endogenous glut2 and pklr promoters in both pancreatic islet and liver cells. However, it is indispensable for hyperacetylation of histones in glut2 and pklr promoter nucleosomes in pancreatic islets but not in liver cells, where glut2 and pklr chromatin remains hyperacetylated in the absence of HNF1-α. In contrast, the phenylalanine hydroxylase promoter requires HNF1-α for transcriptional activity and localized histone hyperacetylation only in liver tissue. Thus, different HNF1-α target genes have distinct requirements for HNF1-α in either pancreatic β-cells or liver cells. The results indicate that HNF1-α occupies target gene promoters in diverse tissues but plays an obligate role in transcriptional activation only in cellular- and promoter-specific contexts in which it is required to recruit histone acetylase activity. These findings provide genetic evidence based on a live mammalian system to establish that a single activator can be essential to direct nucleosomal hyperacetylation to transcriptional targets.
PMCID: PMC86965  PMID: 11287626

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