Catherine Lee May

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.

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 β-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 β-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.

Gut Endocrine Cell Development

Endocrine cells are scattered throughout the gastrointestinal mucosa from the stomach to the colon and constitute one of the largest endocrine systems in the body. Enteroendocrine cells labeled by chromogranin A comprise about 1% of all epithelial cells in the gastrointestinal tract and are surrounded by mucus, chief, and parietal cells in the stomach, and enterocyte, goblet and Paneth cells in the intestine (Figure 1). The enteroendocrine system consists of at least 15 different cell types that can be classified based on their main hormonal products and on the ultrastructure of their secretory granules [1]. A given enteroendocrine cell secretes one or more hormone or hormone-like substance, which is released directly into the lamina propria and diffuses into the capillaries. These hormones include gastrin, histamine, serotonin, cholecystokinin (CCK), somatostatin and glucagon-like peptides (GLP1 and 2) [1]. Although enteroendocrine cells are very scarce, they are essential regulators of digestion, gut motility, appetite, and metabolism. Figure 1 Illustration of the major cell types found in the gastrointestinal epithelium. Gastric epithelium contains mucus, chief, Parietal, and endocrine cells, whereas enterocyte, goblet, endocrine, and Paneth cells are found in the intestinal epithelium. Intestinal ... The development of enteroendocrine cells is a fascinating biological problem: how is the relative proportion of the individual subtypes maintained? How are so many different cell types specified from a common precursor? And how is the endocrine compartment maintained in the gastrointestinal epithelium with its rapid and life-long turnover? However, the study of the mechanisms of enteroendocrine cell differentiation is not only an “academic exercise” but of relevance to human health and disease on multiple levels. First, deficiencies in enteroendocrine cell specification or survival contribute to human diseases, as exemplified by congenital malabsorptive diarrhea, discussed in detail below, which is due solely to deficient enteroendocrine cells. Second, several enteroendocrine cells play a role the control of glucose homeostasis and thus diabetes, and there relatedness to pancreatic beta-cells makes transdifferentiation of enteroendocrine cells an interesting possibility. Therefore, lessons learned from the study of normal gut endocrine cell development promise to be instructive to

Foxl1-Expressing Mesenchymal Cells Constitute the Intestinal Stem Cell Niche

Background & Aims Intestinal epithelial stem cells that express leucine-rich repeat-containing G-protein coupled receptor 5 (Lgr5) and/or B cell specific Moloney murine leukemia virus integration site 1 (Bmi1) continuously replicate and generate differentiated cells throughout life. Previously, Paneth cells were suggested to constitute an epithelium-intrinsic niche that regulates the behavior of these stem cells. However, ablating Paneth cells has no effect on the maintenance of functional stem cells. Here, we show definitively that a small subset of mesenchymal subepithelial cells expressing the winged-helix transcription factor forkhead box l1 (Foxl1) are a critical component of the intestinal stem cell niche. Methods We genetically ablated Foxl1+ mesenchymal cells in adult mice using 2 separate models by expressing either the human or simian diphtheria toxin receptor under Foxl1 promoter control. Conclusions Killing Foxl1+ cells by diphtheria toxin administration led to an abrupt cessation of proliferation of both epithelial stem- and transit-amplifying progenitor cell populations that was associated with a loss of active Wnt signaling to the intestinal epithelium. Therefore, Foxl1-expressing mesenchymal cells constitute the fundamental niche for intestinal stem cells.

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.

Pancreatic α-Cell Specific Deletion of Mouse Arx Leads to α-Cell Identity Loss

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.

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.

Jagged1 is a competitive inhibitor of Notch signaling in the embryonic pancreas

Pancreatic endocrine cells originate from precursors that express the transcription factor Neurogenin3 (Ngn3). Ngn3 expression is repressed by active Notch signaling. Accordingly, mice with Notch signaling pathway mutations display increased Ngn3 expression and endocrine cell lineage allocation. To determine how the Notch ligand Jagged1 (Jag1) functions during pancreas development, we deleted Jag1 in foregut endoderm and examined postnatal and embryonic endocrine cells and precursors. Postnatal Jag1 mutants display increased Ngn3 expression, alpha-cell mass, and endocrine cell percentage, similar to the early embryonic phenotype of Dll1 and Rbpj mutants. However, in sharp contrast to postnatal animals, Jag1-deficient embryos display increased expression of Notch transcriptional targets and decreased Ngn3 expression, resulting in reduced endocrine lineage allocation. Jag1 acts as an inhibitor of Notch signaling during embryonic pancreas development but an activator of Notch signaling postnatally. Expression of the Notch modifier Manic Fringe (Mfng) is limited to endocrine precursors, providing a possible explanation for the inhibition of Notch signaling by Jag1 during mid-gestation embryonic pancreas development.

Islet-1 regulates Arx transcription during pancreatic islet α-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.