Nkx6.1 controls a gene regulatory network required for establishing and maintaining pancreatic Beta cell identity.

All pancreatic endocrine cell types arise from a common endocrine precursor cell population, yet the molecular mechanisms that establish and maintain the unique gene expression programs of each endocrine cell lineage have remained largely elusive. Such knowledge would improve our ability to correctly program or reprogram cells to adopt specific endocrine fates. Here, we show that the transcription factor Nkx6.1 is both necessary and sufficient to specify insulin-producing beta cells. Heritable expression of Nkx6.1 in endocrine precursors of mice is sufficient to respecify non-beta endocrine precursors towards the beta cell lineage, while endocrine precursor- or beta cell-specific inactivation of Nkx6.1 converts beta cells to alternative endocrine lineages. Remaining insulin(+) cells in conditional Nkx6.1 mutants fail to express the beta cell transcription factors Pdx1 and MafA and ectopically express genes found in non-beta endocrine cells. By showing that Nkx6.1 binds to and represses the alpha cell determinant Arx, we identify Arx as a direct target of Nkx6.1. Moreover, we demonstrate that Nkx6.1 and the Arx activator Isl1 regulate Arx transcription antagonistically, thus establishing competition between Isl1 and Nkx6.1 as a critical mechanism for determining alpha versus beta cell identity. Our findings establish Nkx6.1 as a beta cell programming factor and demonstrate that repression of alternative lineage programs is a fundamental principle by which beta cells are specified and maintained. Given the lack of Nkx6.1 expression and aberrant activation of non-beta endocrine hormones in human embryonic stem cell (hESC)-derived insulin(+) cells, our study has significant implications for developing cell replacement therapies.

Impaired enteroendocrine development in intestinal-specific Islet1 mouse mutants causes impaired glucose homeostasis.

Enteroendocrine cells secrete over a dozen different hormones responsible for coordinating digestion, absorption, metabolism, and gut motility. Loss of enteroendocrine cells is a known cause of severe congenital diarrhea. Furthermore, enteroendocrine cells regulate glucose metabolism, with the incretin hormones glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) playing critical roles in stimulating insulin release by pancreatic ß-cells. Islet1 (Isl1) is a LIM-homeodomain transcription factor expressed specifically in an array of intestinal endocrine cells, including incretin-expressing cells. To examine the impact of intestinal Isl1 on glycemic control, we set out to explore the role of intestinal Isl1 in hormone cell specification and organismal physiology. Mice with intestinal epithelial-specific ablation of Isl1 were obtained by crossing Villin-Cre transgenic animals with mice harboring a Isl1(loxP) allele (Isl1(int) model). Gene ablation of Isl1 in the intestine results in loss of GLP-1, GIP, cholecystokinin (CCK), and somatostatin-expressing cells and an increase in 5-HT (serotonin)-producing cells, while the chromogranin A population was unchanged. This dramatic change in hormonal milieu results in animals with lipid malabsorption and females smaller than their littermate controls. Interestingly, when challenged with oral, not intraperitoneal glucose, the Isl-1 intestinal-deficient animals (Isl1(int)) display impaired glucose tolerance, indicating loss of the incretin effect. Thus the Isl1(int) model confirms that intestinal biology is essential for organism physiology in glycemic control and susceptibility to diabetes.

Separation of telomere protection from length regulation by two different point mutations at amino acid 492 of RTEL1.

RTEL1 is an essential DNA helicase that plays multiple roles in genome stability and telomere length regulation. A variant of RTEL1 with a lysine at position 492 is associated with short telomeres in Mus spretus , while a conserved methionine at this position is found in M. musculus, which has ultra-long telomeres. In humans, a missense mutation at this position ( RTEL1 M492I ) causes a fatal telomere biology disease termed Hoyeraal-Hreidarsson syndrome (HHS). We previously described a M. musculus mouse model termed 'Telomouse', in which changing methionine 492 to a lysine (M492K) shortened the telomeres to their length in humans. Here, we report on the derivation of a mouse strain carrying the M492I mutation, termed 'HHS mouse'. The HHS mouse telomeres are not as short as those of Telomice but nevertheless they display higher levels of telomeric DNA damage, fragility and recombination, associated with anaphase bridges and micronuclei. These observations indicate that the two mutations separate critical functions of RTEL1: M492K mainly reduces the telomere length setpoint, while M492I predominantly disrupts telomere protection. The two mouse models enable dissecting the mechanistic roles of RTEL1 and the different contributions of short telomeres and DNA damage to telomere biology diseases, genomic instability, cancer, and aging.

Arx is required for normal enteroendocrine cell development in mice and humans.

Enteroendocrine cells of the gastrointestinal (GI) tract play a central role in metabolism, digestion, satiety and lipid absorption, yet their development remains poorly understood. Here we show that Arx, a homeodomain-containing transcription factor, is required for the normal development of mouse and human enteroendocrine cells. Arx expression is detected in a subset of Neurogenin3 (Ngn3)-positive endocrine progenitors and is also found in a subset of hormone-producing cells. In mice, removal of Arx from the developing endoderm results in a decrease of enteroendocrine cell types including gastrin-, glucagon/GLP-1-, CCK-, secretin-producing cell populations and an increase of somatostatin-expressing cells. This phenotype is also observed in mice with endocrine-progenitor-specific Arx ablation suggesting that Arx is required in the progenitor for enteroendocrine cell development. In addition, depletion of human ARX in developing human intestinal tissue results in a profound deficit in expression of the enteroendocrine cell markers CCK, secretin and glucagon while expression of a pan-intestinal epithelial marker, CDX2, and other non-endocrine markers remained unchanged. Taken together, our findings uncover a novel and conserved role of Arx in mammalian endocrine cell development and provide a potential cause for the chronic diarrhea seen in both humans and mice carrying Arx mutations.

Telomouse-a mouse model with human-length telomeres generated by a single amino acid change in RTEL1.

Telomeres, the ends of eukaryotic chromosomes, protect genome integrity and enable cell proliferation. Maintaining optimal telomere length in the germline and throughout life limits the risk of cancer and enables healthy aging. Telomeres in the house mouse, Mus musculus, are about five times longer than human telomeres, limiting the use of this common laboratory animal for studying the contribution of telomere biology to aging and cancer. We identified a key amino acid variation in the helicase RTEL1, naturally occurring in the short-telomere mouse species M. spretus. Introducing this variation into M. musculus is sufficient to reduce the telomere length set point in the germline and generate mice with human-length telomeres. While these mice are fertile and appear healthy, the regenerative capacity of their colonic epithelium is compromised. The engineered Telomouse reported here demonstrates a dominant role of RTEL1 in telomere length regulation and provides a unique model for aging and cancer.

Nkx2.2 and Arx genetically interact to regulate pancreatic endocrine cell development and endocrine hormone expression.

Nkx2.2 and Arx are essential pancreatic transcription factors. Nkx2.2 is necessary for the appropriate specification of the islet alpha, beta, PP and epsilon cell lineages, whereas Arx is required to form the correct ratio of alpha, beta, delta and PP cells. To begin to understand the cooperative functions of Nkx2.2 and Arx in the development of endocrine cell lineages, we generated progenitor cell-specific deletions of Arx on the Nkx2.2 null background. The analysis of these mutants demonstrates that expansion of the ghrelin cell population in the Nkx2.2 null pancreas is not dependent on Arx; however, Arx is necessary for the upregulation of ghrelin mRNA levels in Nkx2.2 mutant epsilon cells. Alternatively, in the absence of Arx, delta cell numbers are increased and Nkx2.2 becomes essential for the repression of somatostatin gene expression. Interestingly, the dysregulation of ghrelin and somatostatin expression in the Nkx2.2/Arx compound mutant (Nkx2.2(null);Arx(Δpanc)) results in the appearance of ghrelin+/somatostatin+ co-expressing cells. These compound mutants also revealed a genetic interaction between Nkx2.2 and Arx in the regulation of the PP cell lineage; the PP cell population is reduced when Nkx2.2 is deleted but is restored back to wildtype numbers in the Nkx2.2(null);Arx(Δpanc) mutant. Moreover, conditional deletion of Arx in specific pancreatic cell populations established that the functions of Arx are necessary in the Neurog3+ endocrine progenitors. Together, these experiments identify novel genetic interactions between Nkx2.2 and Arx within the endocrine progenitor cells that ensure the correct specification and regulation of endocrine hormone-producing cells.

Islet-1 regulates Arx transcription during pancreatic islet alpha-cell development.

Aristaless related homeodomain protein (Arx) specifies the formation of the pancreatic islet α-cell during development. This cell type produces glucagon, a major counteracting hormone to insulin in regulating glucose homeostasis in adults. However, little is known about the factors that regulate Arx transcription in the pancreas. In this study, we showed that the number of Arx(+) cells was significantly reduced in the pancreata of embryos deficient for the Islet-1 (Isl-1) transcription factor, which was also supported by the reduction in Arx mRNA levels. Chromatin immunoprecipitation analysis localized Isl-1 activator binding sites within two highly conserved noncoding regulatory regions (Re) in the Arx locus, termed Re1 (+5.6 to +6.1 kb) and Re2 (+23.6 to +24 kb). Using cell line-based transfection assays, we demonstrated that a Re1- and Re2-driven reporter was selectively activated in islet α-cells, a process mediated by Isl-1 in overexpression, knockdown, and site-directed mutation experiments. Moreover, Arx mRNA levels were up-regulated in islet α-cells upon Isl-1 overexpression in vivo. Isl-1 represents the first known activator of Arx transcription in α-cells, here established to be acting through the conserved Re1 and Re2 control domains.

LIM domain-binding 1 maintains the terminally differentiated state of pancreatic ß cells.

The recognition of ß cell dedifferentiation in type 2 diabetes raises the translational relevance of mechanisms that direct and maintain ß cell identity. LIM domain-binding protein 1 (LDB1) nucleates multimeric transcriptional complexes and establishes promoter-enhancer looping, thereby directing fate assignment and maturation of progenitor populations. Many terminally differentiated endocrine cell types, however, remain enriched for LDB1, but its role is unknown. Here, we have demonstrated a requirement for LDB1 in maintaining the terminally differentiated status of pancreatic ß cells. Inducible ablation of LDB1 in mature ß cells impaired insulin secretion and glucose homeostasis. Transcriptomic analysis of LDB1-depleted ß cells revealed the collapse of the terminally differentiated gene program, indicated by a loss of ß cell identity genes and induction of the endocrine progenitor factor neurogenin 3 (NEUROG3). Lineage tracing confirmed that LDB1-depleted, insulin-negative ß cells express NEUROG3 but do not adopt alternate endocrine cell fates. In primary mouse islets, LDB1 and its LIM homeodomain-binding partner islet 1 (ISL1) were coenriched at chromatin sites occupied by pancreatic and duodenal homeobox 1 (PDX1), NK6 homeobox 1 (NKX6.1), forkhead box A2 (FOXA2), and NK2 homeobox 2 (NKX2.2) - factors that co-occupy active enhancers in 3D chromatin domains in human islets. Indeed, LDB1 was enriched at active enhancers in human islets. Thus, LDB1 maintains the terminally differentiated state of ß cells and is a component of active enhancers in both murine and human islets.

Arx polyalanine expansion in mice leads to reduced pancreatic α-cell specification and increased α-cell death.

ARX/Arx is a homeodomain-containing transcription factor necessary for the specification and early maintenance of pancreatic endocrine α-cells. Many transcription factors important to pancreas development, including ARX/Arx, are also crucial for proper brain development. Although null mutations of ARX in human patients result in the severe neurologic syndrome XLAG (X-linked lissencephaly associated with abnormal genitalia), the most common mutation is the expansion of the first polyalanine tract of ARX, which results primarily in the clinical syndrome ISSX (infantile spasms). Mouse models of XLAG, ISSX and other human ARX mutations demonstrate a direct genotype-phenotype correlation in ARX-related neurologic disorders. Furthermore, mouse models utilizing a polyalanine tract expansion mutation have illustrated critical developmental differences between null mutations and expansion mutations in the brain, revealing context-specific defects. Although Arx is known to be required for the specification and early maintenance of pancreatic glucagon-producing α-cells, the consequences of the Arx polyalanine expansion on pancreas development remain unknown. Here we report that mice with an expansion mutation in the first polyalanine tract of Arx exhibit impaired α-cell specification and maintenance, with gradual α-cell loss due to apoptosis. This is in contrast to the re-specification of α-cells into ß- and δ-cells that occurs in mice null for Arx. Overall, our analysis of an Arx polyalanine expansion mutation on pancreatic development suggests that impaired α-cell function might also occur in ISSX patients.

Pancreatic α-cell specific deletion of mouse Arx leads to α-cell identity loss.

The specification and differentiation of pancreatic endocrine cell populations (α-, ß-, δ, PP- and ε-cells) is orchestrated by a combination of transcriptional regulators. In the pancreas, Aristaless-related homeobox gene (Arx) is expressed first in the endocrine progenitors and then restricted to glucagon-producing α-cells. While the functional requirement of Arx in early α-cell specification has been investigated, its role in maintaining α-cell identity has yet to be explored. To study this later role of Arx, we have generated mice in which the Arx gene has been ablated specifically in glucagon-producing α-cells. Lineage-tracing studies and immunostaining analysis for endocrine hormones demonstrate that ablation of Arx in neonatal α-cells results in an α-to-ß-like conversion through an intermediate bihormonal state. Furthermore, these Arx-deficient converted cells express ß-cell markers including Pdx1, MafA, and Glut2. Surprisingly, short-term ablation of Arx in adult mice does not result in a similar α-to-ß-like conversion. Taken together, these findings reveal a potential temporal requirement for Arx in maintaining α-cell identity.

Islet α-, ß-, and δ-cell development is controlled by the Ldb1 coregulator, acting primarily with the islet-1 transcription factor.

Ldb1 and Ldb2 are coregulators that mediate Lin11-Isl1-Mec3 (LIM)-homeodomain (HD) and LIM-only transcription factor-driven gene regulation. Although both Ldb1 and Ldb2 mRNA were produced in the developing and adult pancreas, immunohistochemical analysis illustrated a broad Ldb1 protein expression pattern during early pancreatogenesis, which subsequently became enriched in islet and ductal cells perinatally. The islet-enriched pattern of Ldb1 was similar to pan-endocrine cell-expressed Islet-1 (Isl1), which was demonstrated in this study to be the primary LIM-HD transcription factor in developing and adult islet cells. Endocrine cell-specific removal of Ldb1 during mouse development resulted in a severe reduction of hormone⁺ cell numbers (i.e., α, ß, and δ) and overt postnatal hyperglycemia, reminiscent of the phenotype described for the Isl1 conditional mutant. In contrast, neither endocrine cell development nor function was affected in the pancreas of Ldb2(-/-) mice. Gene expression and chromatin immunoprecipitation (ChIP) analyses demonstrated that many important Isl1-activated genes were coregulated by Ldb1, including MafA, Arx, insulin, and Glp1r. However, some genes (i.e., Hb9 and Glut2) only appeared to be impacted by Ldb1 during development. These findings establish Ldb1 as a critical transcriptional coregulator during islet α-, ß-, and δ-cell development through Isl1-dependent and potentially Isl1-independent control.

Elevation of transcription factor Islet-1 levels in vivo increases ß-cell function but not ß-cell mass.

A decrease in the expression of Islet-1 (Isl-1), an islet transcription factor, has been reported in several physiological settings of reduced ß-cell function. Here, we investigate whether an increased level of Isl-1 in islet cells can enhance ß-cell function and/or mass. We demonstrate that transgenic mice with Isl-1 overexpression display improved glucose tolerance and enhanced insulin secretion without significant changes in ß cell mass. From our microarray study, we identify approximately 135 differentially expressed genes in the islets of Isl-1 overexpressing mice that have been implicated to function in numerous biological processes including protein trafficking, metabolism and differentiation. Using real-time PCR we have confirmed upregulation of Caps2, Sec14l4, Slc2a10, P2rx7, Afamin, and Neurogenin 3 that may in part mediate the observed improved insulin secretion in Isl-1 overexpressing mice. These findings show for the first time that Isl-1 is a key factor in regulating adult ß cell function in vivo, and suggest that Isl-1 elevation could be beneficial to improve glucose homeostasis.

Expression analysis of the Islet-1 gene in the developing and adult gastrointestinal tract.

LIM-homeodomain genes encode a family of proteins defined by the cysteine-rich protein/protein interacting (Lin-11, Isl-1, and Mec-3) LIM domain and a highly conserved DNA-binding domain. Studies in several organisms have shown that these transcriptional regulators control multiple aspects of embryonic development and are responsible for the pathogenesis of several human diseases. Here we report the expression of Islet-1 (Isl-1) in the gastrointestinal epithelium in developing and adult mice. At embryonic day (E) 9.5-10.5, Isl-1 expression was first detected in the ventral gastric mesenchyme, and expression in the dorsal mesenchyme initiated a few days later. Isl-1 expression was first observed in the gastric epithelium at E13.5 and at E14.5 was restricted to the posterior half of the stomach. In the mature stomach, Isl-1 expression was detected only in subsets of enteroendocrine cells. Furthermore, Isl-1 expression in the intestinal epithelium was first detected at E15.5 and was restricted to subpopulations of enteroendocrine cells in adult mice. These expression analyses suggest that Isl-1 might have an early broad role in stomach and intestinal cells and a secondary role in terminal differentiation and/or maintenance of mature enteroendocrine subtypes in the gastrointestinal epithelium.

The role of Islet-1 in the endocrine pancreas: Lessons from pancreas specific Islet-1 deficient mice.

Recently, we have reported the LIM-homeodoman (HD) transcriptional regulator, Islet-1 (Isl-1) as a key regulator for pancreatic islets after the secondary transition and into early postnatal stages in mice. Previously, the role of Isl-1 had only been examined during early pancreas development in vivo and cell lines. These early studies concluded that Isl-1 is required for the differentiation of early endocrine cells, and hormone gene expression is regulated by Isl-1 in cell culture. However, it was not clear from these studies whether the regulation of hormone gene transcription by Isl-1 was a direct transcriptional event. In addition, the function of Isl-1 during the formation of principle hormone producing endocrine cells had not been investigated since Isl-1 null animals die prior to the formation of these cells. Using pancreas-specific inactivation of Isl-1 in mice, we have elucidated the role of Isl-1 during maturation, proliferation and survival of the endocrine pancreas after the secondary transition. We have also identified MafA, a potent Insulin gene regulator, as the first direct target of Isl-1 in ß-cells.

Islet-1 is required for the maturation, proliferation, and survival of the endocrine pancreas.

OBJECTIVE: The generation of mature cell types during pancreatic development depends on the expression of many regulatory and signaling proteins. In this study, we tested the hypothesis that the transcriptional regulator Islet-1 (Isl-1), whose expression is first detected in the mesenchyme and epithelium of the developing pancreas and is later restricted to mature islet cells, is involved in the terminal differentiation of islet cells and maintenance of islet mass. RESEARCH DESIGN AND METHODS: To investigate the role of Isl-1 in the pancreatic epithelium during the secondary transition, Isl-1 was conditionally and specifically deleted from embryonic day 13.5 onward using Cre/LoxP technology. RESULTS: Isl-1-deficient endocrine precursors failed to mature into functional islet cells. The postnatal expansion of endocrine cell mass was impaired, and consequently Isl-1 deficient mice were diabetic. In addition, MafA, a potent regulator of the Insulin gene and beta-cell function, was identified as a direct transcriptional target of Isl-1. CONCLUSIONS: These results demonstrate the requirement for Isl-1 in the maturation, proliferation, and survival of the second wave of hormone-producing islet cells.

Defining pancreatic endocrine precursors and their descendants.

OBJECTIVE: The global incidence of diabetes continues to increase. Cell replacement therapy and islet transplantation offer hope, especially for severely affected patients. Efforts to differentiate insulin-producing beta-cells from progenitor or stem cells require knowledge of the transcriptional programs that regulate the development of the endocrine pancreas. RESEARCH DESIGN AND METHODS: Differentiation toward the endocrine lineage is dependent on the transcription factor Neurogenin 3 (Neurog3, Ngn3). We utilize a Neurog3-enhanced green fluorescent protein knock-in mouse model to isolate endocrine progenitor cells from embryonic pancreata (embryonic day [E]13.5 through E17.5). Using advanced genomic approaches, we generate a comprehensive gene expression profile of these progenitors and their immediate descendants. RESULTS: A total of 1,029 genes were identified as being temporally regulated in the endocrine lineage during fetal development, 237 of which are transcriptional regulators. Through pathway analysis, we have modeled regulatory networks involving these proteins that highlight the complex transcriptional hierarchy governing endocrine differentiation. CONCLUSIONS: We have been able to accurately capture the gene expression profile of the pancreatic endocrine progenitors and their descendants. The list of temporally regulated genes identified in fetal endocrine precursors and their immediate descendants provides a novel and important resource for developmental biologists and diabetes researchers alike.