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1.  Islet α-, β-, and δ-Cell Development Is Controlled by the Ldb1 Coregulator, Acting Primarily With the Islet-1 Transcription Factor 
Diabetes  2013;62(3):875-886.
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, β, 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-, β-, and δ-cell development through Isl1-dependent and potentially Isl1-independent control.
PMCID: PMC3581213  PMID: 23193182
2.  Elevation of transcription factor Islet-1 levels in vivo increases β-cell function but not β-cell mass 
Islets  2012;4(3):199-206.
A decrease in the expression of Islet-1 (Isl-1), an islet transcription factor, has been reported in several physiological settings of reduced β-cell function. Here, we investigate whether an increased level of Isl-1 in islet cells can enhance β-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 β 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 β cell function in vivo, and suggest that Isl-1 elevation could be beneficial to improve glucose homeostasis.
PMCID: PMC3442817  PMID: 22595886
Islet-1; glucose tolerance; insulin; transcription factor; β-cells
3.  Nkx2.2 and Arx genetically interact to regulate pancreatic endocrine cell development and endocrine hormone expression 
Developmental biology  2011;359(1):1-11.
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 (Nkx2.2null;ArxΔpanc) 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 Nkx2.2null;ArxΔpanc 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.
PMCID: PMC3192309  PMID: 21856296
Nkx2.2; Arx; transcriptional regulation; endocrine cell fate; ghrelin; PP; somatostatin
Severe congenital diarrhea occurs in about half of patients with ARX null mutations. The cause of this diarrhea is unknown. In a mouse model of intestinal Arx deficiency, the prevalence of a subset of enteroendocrine cells is altered, leading to diarrhea. Since polyalanine expansions within the ARX protein are the most common mutations found in ARX-related disorders, we sought to characterize the enteroendocrine population in human tissue of an ARX(GGC)7 mutation and in a mouse model of the corresponding polyalanine expansion (Arx(GCG)7).
Immunohistochemistry and quantitative RT-PCR were the primary modalities utilized to characterize the enteroendocrine populations. Daily weights were determined for the growth curves, and Oil-Red-O staining on stool and tissue identified neutral fats.
An expansion of seven alanines in the first polyalanine tract of both human ARX and mouse Arx altered enteroendocrine differentiation. In human tissue, cholecystokinin (CCK), glucagon-like peptide 1 (GLP-1), and somatostatin (SST) populations were reduced, while the Chromogranin A population was unchanged. In the mouse model, CCK and GLP-1 populations were also lost, though the SST-expressing population was increased. The ARX(GGC)7 protein was present in human tissue, while the Arx(GCG)7 protein was degraded in the mouse intestine.
ARX/Arx is required for the specification of a subset of enteroendocrine cells in both humans and mice. Due to protein degradation, the Arx(GCG)7 mouse recapitulates findings of the intestinal Arx null model, but is not able to further the study of the differential effects of the ARX(GCG)7 protein on its transcriptional targets in the intestine.
PMCID: PMC4308495  PMID: 25171319
(3-5): enteroendocrine dysgenesis; Arx; polyalanine
5.  Islet-1 is Required for the Maturation, Proliferation, and Survival of the Endocrine Pancreas 
Diabetes  2009;58(9):2059-2069.
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.
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.
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 β-cell function, was identified as a direct transcriptional target of Isl-1.
These results demonstrate the requirement for Isl-1 in the maturation, proliferation, and survival of the second wave of hormone-producing islet cells.
PMCID: PMC2731519  PMID: 19502415
6.  Arx Polyalanine Expansion in Mice Leads to Reduced Pancreatic α-Cell Specification and Increased α-Cell Death 
PLoS ONE  2013;8(11):e78741.
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 β- 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.
PMCID: PMC3827280  PMID: 24236044
7.  Pancreatic α-Cell Specific Deletion of Mouse Arx Leads to α-Cell Identity Loss 
PLoS ONE  2013;8(6):e66214.
The specification and differentiation of pancreatic endocrine cell populations (α-, β-, 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-β-like conversion through an intermediate bihormonal state. Furthermore, these Arx-deficient converted cells express β-cell markers including Pdx1, MafA, and Glut2. Surprisingly, short-term ablation of Arx in adult mice does not result in a similar α-to-β-like conversion. Taken together, these findings reveal a potential temporal requirement for Arx in maintaining α-cell identity.
PMCID: PMC3681972  PMID: 23785486
8.  Arx is required for normal enteroendocrine cell development in mice and humans 
Developmental Biology  2012;365(1):175-188.
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.
PMCID: PMC3322318  PMID: 22387004
Arx; transcription factor; glucagon; somatostatin; gastrin; CCK; secretin; serotonin; enteroendocrine cells; endocrine progenitors; specification; differentiation
9.  Nkx6.1 Controls a Gene Regulatory Network Required for Establishing and Maintaining Pancreatic Beta Cell Identity 
PLoS Genetics  2013;9(1):e1003274.
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.
Author Summary
Diabetes is a disease caused by the loss or dysfunction of insulin-producing beta cells in the pancreas. Recent studies suggest that modification of the beta cells' differentiation state is among the earliest events marking the progressive failure of beta cells in diabetes. Currently, very little is known about the factors that instruct cells to adopt beta cell characteristics and maintain the differentiated state of beta cells. We have discovered that a single transcription factor can instruct precursor cells of other endocrine cell types to change their identity and differentiate into beta cells. Conversely, inactivation of the transcription factor in endocrine precursors prevents their differentiation into beta cells and results in excess production of other endocrine cell types. When the factor is specifically inactivated in beta cells, beta cells lose their identity and adopt characteristics of other endocrine cell types, similar to what is seen in animal models of diabetes. Thus, we have identified a single factor that is both sufficient to program beta cells and necessary for maintaining their differentiated state. This factor could be an important target for diabetes therapy and could help reprogram other cell types into beta cells.
PMCID: PMC3561089  PMID: 23382704
10.  Expression analysis of the Islet-1 gene in the developing and adult gastrointestinal tract 
Gene expression patterns : GEP  2011;11(3-4):244-254.
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.
PMCID: PMC3065958  PMID: 21220053
Islet-1; stomach; intestine; gastrointestinal tract; development; transcriptional control; endocrine cell differentiation; somatostatin; gastrin; ghrelin
11.  Gut Endocrine Cell Development 
PMCID: PMC2905316  PMID: 20025933

Results 1-11 (11)