Major ß cell-specific functions of NKX2.2 are mediated via the NK2-specific domain.

The consolidation of unambiguous cell fate commitment relies on the ability of transcription factors (TFs) to exert tissue-specific regulation of complex genetic networks. However, the mechanisms by which TFs establish such precise control over gene expression have remained elusive-especially in instances in which a single TF operates in two or more discrete cellular systems. In this study, we demonstrate that ß cell-specific functions of NKX2.2 are driven by the highly conserved NK2-specific domain (SD). Mutation of the endogenous NKX2.2 SD prevents the developmental progression of ß cell precursors into mature, insulin-expressing ß cells, resulting in overt neonatal diabetes. Within the adult ß cell, the SD stimulates ß cell performance through the activation and repression of a subset of NKX2.2-regulated transcripts critical for ß cell function. These irregularities in ß cell gene expression may be mediated via SD-contingent interactions with components of chromatin remodelers and the nuclear pore complex. However, in stark contrast to these pancreatic phenotypes, the SD is entirely dispensable for the development of NKX2.2-dependent cell types within the CNS. Together, these results reveal a previously undetermined mechanism through which NKX2.2 directs disparate transcriptional programs in the pancreas versus neuroepithelium.

Pancreatic ß cell dedifferentiation as a mechanism of diabetic ß cell failure.

Diabetes is associated with ß cell failure. But it remains unclear whether the latter results from reduced ß cell number or function. FoxO1 integrates ß cell proliferation with adaptive ß cell function. We interrogated the contribution of these two processes to ß cell dysfunction, using mice lacking FoxO1 in ß cells. FoxO1 ablation caused hyperglycemia with reduced ß cell mass following physiologic stress, such as multiparity and aging. Surprisingly, lineage-tracing experiments demonstrated that loss of ß cell mass was due to ß cell dedifferentiation, not death. Dedifferentiated ß cells reverted to progenitor-like cells expressing Neurogenin3, Oct4, Nanog, and L-Myc. A subset of FoxO1-deficient ß cells adopted the α cell fate, resulting in hyperglucagonemia. Strikingly, we identify the same sequence of events as a feature of different models of murine diabetes. We propose that dedifferentiation trumps endocrine cell death in the natural history of ß cell failure and suggest that treatment of ß cell dysfunction should restore differentiation, rather than promoting ß cell replication.

Groucho co-repressor proteins regulate ß cell development and proliferation by repressing Foxa1 in the developing mouse pancreas.

Groucho-related genes (GRGs) are transcriptional co-repressors that are crucial for many developmental processes. Several essential pancreatic transcription factors are capable of interacting with GRGs; however, the in vivo role of GRG-mediated transcriptional repression in pancreas development is still not well understood. In this study, we used complex mouse genetics and transcriptomic analyses to determine that GRG3 is essential for ß cell development, and in the absence of Grg3 there is compensatory upregulation of Grg4Grg3/4 double mutant mice have severe dysregulation of the pancreas gene program with ectopic expression of canonical liver genes and Foxa1, a master regulator of the liver program. Neurod1, an essential ß cell transcription factor and predicted target of Foxa1, becomes downregulated in Grg3/4 mutants, resulting in reduced ß cell proliferation, hyperglycemia, and early lethality. These findings uncover novel functions of GRG-mediated repression during pancreas development.

Regulation of ß-cell death by ADP-ribosylhydrolase ARH3 via lipid signaling in insulitis.

BACKGROUND: Lipids are regulators of insulitis and ß-cell death in type 1 diabetes development, but the underlying mechanisms are poorly understood. Here, we investigated how the islet lipid composition and downstream signaling regulate ß-cell death. METHODS: We performed lipidomics using three models of insulitis: human islets and EndoC-ßH1 ß cells treated with the pro-inflammatory cytokines interlukine-1ß and interferon-γ, and islets from pre-diabetic non-obese mice. We also performed mass spectrometry and fluorescence imaging to determine the localization of lipids and enzyme in islets. RNAi, apoptotic assay, and qPCR were performed to determine the role of a specific factor in lipid-mediated cytokine signaling. RESULTS: Across all three models, lipidomic analyses showed a consistent increase of lysophosphatidylcholine species and phosphatidylcholines with polyunsaturated fatty acids and a reduction of triacylglycerol species. Imaging assays showed that phosphatidylcholines with polyunsaturated fatty acids and their hydrolyzing enzyme phospholipase PLA2G6 are enriched in islets. In downstream signaling, omega-3 fatty acids reduce cytokine-induced ß-cell death by improving the expression of ADP-ribosylhydrolase ARH3. The mechanism involves omega-3 fatty acid-mediated reduction of the histone methylation polycomb complex PRC2 component Suz12, upregulating the expression of Arh3, which in turn decreases cell apoptosis. CONCLUSIONS: Our data provide insights into the change of lipidomics landscape in ß cells during insulitis and identify a protective mechanism by omega-3 fatty acids. Video Abstract.

ßlinc1 encodes a long noncoding RNA that regulates islet ß-cell formation and function.

Pancreatic ß cells are responsible for maintaining glucose homeostasis; their absence or malfunction results in diabetes mellitus. Although there is evidence that long noncoding RNAs (lncRNAs) play important roles in development and disease, none have been investigated in vivo in the context of pancreas development. In this study, we demonstrate that ßlinc1 (ß-cell long intergenic noncoding RNA 1), a conserved lncRNA, is necessary for the specification and function of insulin-producing ß cells through the coordinated regulation of a number of islet-specific transcription factors located in the genomic vicinity of ßlinc1. Furthermore, deletion of ßlinc1 results in defective islet development and disruption of glucose homeostasis in adult mice.

Retinoic acid signaling within pancreatic endocrine progenitors regulates mouse and human ß cell specification.

Retinoic acid (RA) signaling is essential for multiple developmental processes, including appropriate pancreas formation from the foregut endoderm. RA is also required to generate pancreatic progenitors from human pluripotent stem cells. However, the role of RA signaling during endocrine specification has not been fully explored. In this study, we demonstrate that the disruption of RA signaling within the NEUROG3-expressing endocrine progenitor population impairs mouse ß cell differentiation and induces ectopic expression of crucial δ cell genes, including somatostatin. In addition, the inhibition of the RA pathway in hESC-derived pancreatic progenitors downstream of NEUROG3 induction impairs insulin expression. We further determine that RA-mediated regulation of endocrine cell differentiation occurs through Wnt pathway components. Together, these data demonstrate the importance of RA signaling in endocrine specification and identify conserved mechanisms by which RA signaling directs pancreatic endocrine cell fate.

Islet Regeneration: Endogenous and Exogenous Approaches.

Both type 1 and type 2 diabetes are characterized by a progressive loss of beta cell mass that contributes to impaired glucose homeostasis. Although an optimal treatment option would be to simply replace the lost cells, it is now well established that unlike many other organs, the adult pancreas has limited regenerative potential. For this reason, significant research efforts are focusing on methods to induce beta cell proliferation (replication of existing beta cells), promote beta cell formation from alternative endogenous cell sources (neogenesis), and/or generate beta cells from pluripotent stem cells. In this article, we will review (i) endogenous mechanisms of beta cell regeneration during steady state, stress and disease; (ii) efforts to stimulate endogenous regeneration and transdifferentiation; and (iii) exogenous methods of beta cell generation and transplantation.

Fluctuation localization imaging-based fluorescence in situ hybridization (fliFISH) for accurate detection and counting of RNA copies in single cells.

Quantitative gene expression analysis in intact single cells can be achieved using single molecule-based fluorescence in situ hybridization (smFISH). This approach relies on fluorescence intensity to distinguish between true signals, emitted from an RNA copy hybridized with multiple oligonucleotide probes, and background noise. Thus, the precision in smFISH is often compromised by partial or nonspecific probe binding and tissue autofluorescence, especially when only a small number of probes can be fitted to the target transcript. Here we provide an accurate approach for setting quantitative thresholds between true and false signals, which relies on on-off duty cycles of photoswitchable dyes. This fluctuation localization imaging-based FISH (fliFISH) uses on-time fractions (measured over a series of exposures) collected from transcripts bound to as low as 8 probes, which are distinct from on-time fractions collected from nonspecifically bound probes or autofluorescence. Using multicolor fliFISH, we identified radial gene expression patterns in mouse pancreatic islets for insulin, the transcription factor, NKX2-2 and their ratio (Nkx2-2/Ins2). These radial patterns, showing higher values in ß cells at the islet core and lower values in peripheral cells, were lost in diabetic mouse islets. In summary, fliFISH provides an accurate, quantitative approach for detecting and counting true RNA copies and rejecting false signals by their distinct on-time fractions, laying the foundation for reliable single-cell transcriptomics.

Animal Models of Pancreas Development, Developmental Disorders, and Disease.

The pancreas is a glandular organ responsible for diverse homeostatic functions, including hormone production from the endocrine islet cells to regulate blood sugar levels and enzyme secretion from the exocrine acinar cells to facilitate food digestion. These pancreatic functions are essential for life; therefore, preserving pancreatic function is of utmost importance. Pancreas dysfunction can arise either from developmental disorders or adult onset disease, both of which are caused by defects in shared molecular pathways. In this chapter, we discuss what is known about the molecular mechanisms controlling pancreas development, how disruption of these mechanisms can lead to developmental defects and disease, and how essential pancreas functions can be modeled using human pluripotent stem cells. At the core of understanding of these molecular processes are animal model studies that continue to be essential for elucidating the mechanisms underlying human pancreatic functions and diseases.

Comprehensive characterisation of compartment-specific long non-coding RNAs associated with pancreatic ductal adenocarcinoma.

OBJECTIVE: Pancreatic ductal adenocarcinoma (PDA) is a highly metastatic disease with limited therapeutic options. Genome and transcriptome analyses have identified signalling pathways and cancer driver genes with implications in patient stratification and targeted therapy. However, these analyses were performed in bulk samples and focused on coding genes, which represent a small fraction of the genome. DESIGN: We developed a computational framework to reconstruct the non-coding transcriptome from cross-sectional RNA-Seq, integrating somatic copy number alterations (SCNA), common germline variants associated to PDA risk and clinical outcome. We validated the results in an independent cohort of paired epithelial and stromal RNA-Seq derived from laser capture microdissected human pancreatic tumours, allowing us to annotate the compartment specificity of their expression. We employed systems and experimental biology approaches to interrogate the function of epithelial long non-coding RNAs (lncRNAs) associated with genetic traits and clinical outcome in PDA. RESULTS: We generated a catalogue of PDA-associated lncRNAs. We showed that lncRNAs define molecular subtypes with biological and clinical significance. We identified lncRNAs in genomic regions with SCNA and single nucleotide polymorphisms associated with lifetime risk of PDA and associated with clinical outcome using genomic and clinical data in PDA. Systems biology and experimental functional analysis of two epithelial lncRNAs (LINC00673 and FAM83H-AS1) suggest they regulate the transcriptional profile of pancreatic tumour samples and PDA cell lines. CONCLUSIONS: Our findings indicate that lncRNAs are associated with genetic marks of pancreatic cancer risk, contribute to the transcriptional regulation of neoplastic cells and provide an important resource to design functional studies of lncRNAs in PDA.

GATA4 and GATA6 regulate pancreatic endoderm identity through inhibition of hedgehog signaling.

GATA4 and GATA6 are zinc finger transcription factors that have important functions in several mesodermal and endodermal organs, including heart, liver and pancreas. In humans, heterozygous mutations of either factor are associated with pancreatic agenesis; however, homozygous deletion of both Gata4 and Gata6 is necessary to disrupt pancreas development in mice. In this study, we demonstrate that arrested pancreatic development in Gata4(fl/fl); Gata6(fl/fl); Pdx1:Cre (pDKO) embryos is accompanied by the transition of ventral and dorsal pancreatic fates into intestinal or stomach lineages, respectively. These results indicate that GATA4 and GATA6 play essential roles in maintaining pancreas identity by regulating foregut endodermal fates. Remarkably, pancreatic anlagen derived from pDKO embryos also display a dramatic upregulation of hedgehog pathway components, which are normally absent from the presumptive pancreatic endoderm. Consistent with the erroneous activation of hedgehog signaling, we demonstrate that GATA4 and GATA6 are able to repress transcription through the sonic hedgehog (Shh) endoderm-specific enhancer MACS1 and that GATA-binding sites within this enhancer are necessary for this repressive activity. These studies establish the importance of GATA4/6-mediated inhibition of hedgehog signaling as a major mechanism regulating pancreatic endoderm specification during patterning of the gut tube.

The novel enterochromaffin marker Lmx1a regulates serotonin biosynthesis in enteroendocrine cell lineages downstream of Nkx2.2.

Intestinal hormone-producing cells represent the largest endocrine system in the body, but remarkably little is known about enteroendocrine cell type specification in the embryo and adult. We analyzed stage- and cell type-specific deletions of Nkx2.2 and its functional domains in order to characterize its role in the development and maintenance of enteroendocrine cell lineages in the mouse duodenum and colon. Although Nkx2.2 regulates enteroendocrine cell specification in the duodenum at all stages examined, it controls the differentiation of progressively fewer enteroendocrine cell populations when deleted from Ngn3(+) progenitor cells or in the adult duodenum. During embryonic development Nkx2.2 regulates all enteroendocrine cell types, except gastrin and preproglucagon. In developing Ngn3(+) enteroendocrine progenitor cells, Nkx2.2 is not required for the specification of neuropeptide Y and vasoactive intestinal polypeptide, indicating that a subset of these cell populations derive from an Nkx2.2-independent lineage. In adult duodenum, Nkx2.2 becomes dispensable for cholecystokinin and secretin production. In all stages and Nkx2.2 mutant conditions, serotonin-producing enterochromaffin cells were the most severely reduced enteroendocrine lineage in the duodenum and colon. We determined that the transcription factor Lmx1a is expressed in enterochromaffin cells and functions downstream of Nkx2.2. Lmx1a-deficient mice have reduced expression of Tph1, the rate-limiting enzyme for serotonin biosynthesis. These data clarify the function of Nkx2.2 in the specification and homeostatic maintenance of enteroendocrine populations, and identify Lmx1a as a novel enterochromaffin cell marker that is also essential for the production of the serotonin biosynthetic enzyme Tph1.

Nkx2.2 repressor complex regulates islet ß-cell specification and prevents ß-to-α-cell reprogramming.

Regulation of cell differentiation programs requires complex interactions between transcriptional and epigenetic networks. Elucidating the principal molecular events responsible for the establishment and maintenance of cell fate identities will provide important insights into how cell lineages are specified and maintained and will improve our ability to recapitulate cell differentiation events in vitro. In this study, we demonstrate that Nkx2.2 is part of a large repression complex in pancreatic ß cells that includes DNMT3a, Grg3, and HDAC1. Mutation of the endogenous Nkx2.2 tinman (TN) domain in mice abolishes the interaction between Nkx2.2 and Grg3 and disrupts ß-cell specification. Furthermore, we demonstrate that Nkx2.2 preferentially recruits Grg3 and HDAC1 to the methylated Aristaless homeobox gene (Arx) promoter in ß cells. The Nkx2.2 TN mutation results in ectopic expression of Arx in ß cells, causing ß-to-α-cell transdifferentiation. A corresponding ß-cell-specific deletion of DNMT3a is also sufficient to cause Arx-dependent ß-to-α-cell reprogramming. Notably, subsequent removal of Arx in the ß cells of Nkx2.2(TNmut/TNmut) mutant mice reverts the ß-to-α-cell conversion, indicating that the repressor activities of Nkx2.2 on the methylated Arx promoter in ß cells are the primary regulatory events required for maintaining ß-cell identity.

Epigenetic modifications and long noncoding RNAs influence pancreas development and function.

Insulin-producing ß cells within the pancreatic islet of Langerhans are responsible for maintaining glucose homeostasis; the loss or malfunction of ß cells results in diabetes mellitus. Recent advances in cell purification strategies and sequencing technologies as well as novel molecular tools have revealed that epigenetic modifications and long noncoding RNAs (lncRNAs) represent an integral part of the transcriptional mechanisms regulating pancreas development and ß cell function. Importantly, these findings have uncovered a new layer of gene regulation in the pancreas that can be exploited to enhance the restoration and/or repair of ß cells to treat diabetes.

High Spatial Resolution Imaging of Mouse Pancreatic Islets Using Nanospray Desorption Electrospray Ionization Mass Spectrometry.

Nanospray Desorption Electrospray Ionization mass spectrometry imaging (nano-DESI MSI) enables ambient imaging of biological samples with high sensitivity and minimal sample pretreatment. Recently, we developed an approach for constant-distance mode MSI using shear force microscopy to precisely control the distance between the sample and the nano-DESI probe. Herein, we demonstrate the power of this approach for robust imaging of pancreatic islets with high spatial resolution of ∼11 µm. Pancreatic islets are difficult to characterize using traditional mass spectrometry approaches due to their small size (∼100 µm) and molecular heterogeneity. Nano-DESI MSI was used to examine the spatial localization of several lipid classes including phosphatidylcholine (PC), phosphatidylethanolamine (PE), sphingomyelin (SM), phosphatidylinositol (PI), and phosphatidylserine (PS) along with fatty acids and their metabolites (e.g., prostaglandins) in the individual islets and surrounding tissue. Several lipids were found to be substantially enhanced in the islets indicating these lipids may be involved in insulin secretion. Remarkably different distributions were observed for several pairs of Lyso PC (LPC) and PC species differing only by one double bond, such as LPC 18:1 vs LPC 18:0, PC 32:1 vs PC 32:0, and PC 34:2 vs PC 34:1. These findings indicate that minor variations in the fatty acid chain length and saturation have a pronounced effect on the localization of PC and LPC species in pancreatic islets. Interestingly, oxidized PC species observed experimentally were found to be specifically localized to pancreatic islets. These PCs are potential biomarkers for reactive oxygen species in the islets, which could be harmful to pancreatic beta cells. The experimental approach presented in this study will provide valuable information on the heterogeneity of individual pancreatic islets, which is difficult to assess using bulk characterization techniques.

Unique functions of Gata4 in mouse liver induction and heart development.

Gata4 and Gata6 are closely related transcription factors that are essential for the development of a number of embryonic tissues. While they have nearly identical DNA-binding domains and similar patterns of expression, Gata4 and Gata6 null embryos have strikingly different embryonic lethal phenotypes. To determine whether the lack of redundancy is due to differences in protein function or Gata4 and Gata6 expression domains, we generated mice that contained the Gata6 cDNA in place of the Gata4 genomic locus. Gata4(Gata6/Gata6) embryos survived through embryonic day (E)12.5 and successfully underwent ventral folding morphogenesis, demonstrating that Gata6 is able to replace Gata4 function in extraembryonic tissues. Surprisingly, Gata6 is unable to replace Gata4 function in the septum transversum mesenchyme or the epicardium, leading to liver agenesis and lethal heart defects in Gata4(Gata6/Gata6) embryos. These studies suggest that Gata4 has evolved distinct functions in the development of these tissues that cannot be performed by Gata6, even when it is provided in the identical expression domain. Our work has important implications for the respective mechanisms of Gata function during development, as well as the functional evolution of these essential transcription factors.

Genetic evidence that Nkx2.2 and Pdgfra are major determinants of the timing of oligodendrocyte differentiation in the developing CNS.

In the central nervous system (CNS), oligodendrocyte maturation and axonal myelination occur on a predictable schedule, but the underlying timing mechanisms are largely unknown. In the present study, we demonstrate that Nkx2.2 homeodomain transcription factor is a key regulator for the timing of oligodendrocyte differentiation during development. Whereas induced expression of Nkx2.2 in early oligodendrocyte precursor cells (OPCs) causes precocious differentiation of oligodendrocytes, conditional ablation of Nkx2.2 temporally delays oligodendrocyte maturation. Moreover, Nkx2.2 can directly bind to the promoter of platelet-derived growth factor receptor alpha (Pdgfra) and repress its gene expression. Genetic ablation of Pdgfra mimics the effect of Nkx2.2 overexpression in accelerating OPC differentiation in the developing spinal cord. Together, our findings strongly suggest that Nkx2.2 functions as a major 'switch' to turn off Pdgfra signaling in OPCs and initiate the intrinsic program for oligodendrocyte differentiation.

Regulation of Neurod1 contributes to the lineage potential of Neurogenin3+ endocrine precursor cells in the pancreas.

During pancreatic development, transcription factor cascades gradually commit precursor populations to the different endocrine cell fate pathways. Although mutational analyses have defined the functions of many individual pancreatic transcription factors, the integrative transcription factor networks required to regulate lineage specification, as well as their sites of action, are poorly understood. In this study, we investigated where and how the transcription factors Nkx2.2 and Neurod1 genetically interact to differentially regulate endocrine cell specification. In an Nkx2.2 null background, we conditionally deleted Neurod1 in the Pdx1+ pancreatic progenitor cells, the Neurog3+ endocrine progenitor cells, or the glucagon+ alpha cells. These studies determined that, in the absence of Nkx2.2 activity, removal of Neurod1 from the Pdx1+ or Neurog3+ progenitor populations is sufficient to reestablish the specification of the PP and epsilon cell lineages. Alternatively, in the absence of Nkx2.2, removal of Neurod1 from the Pdx1+ pancreatic progenitor population, but not the Neurog3+ endocrine progenitor cells, restores alpha cell specification. Subsequent in vitro reporter assays demonstrated that Nkx2.2 represses Neurod1 in alpha cells. Based on these findings, we conclude that, although Nkx2.2 and Neurod1 are both necessary to promote beta cell differentiation, Nkx2.2 must repress Neurod1 in a Pdx1+ pancreatic progenitor population to appropriately commit a subset of Neurog3+ endocrine progenitor cells to the alpha cell lineage. These results are consistent with the proposed idea that Neurog3+ endocrine progenitor cells represent a heterogeneous population of unipotent cells, each restricted to a particular endocrine lineage.

A retrotransposon insertion in the 5' regulatory domain of Ptf1a results in ectopic gene expression and multiple congenital defects in Danforth's short tail mouse.

Danforth's short tail mutant (Sd) mouse, first described in 1930, is a classic spontaneous mutant exhibiting defects of the axial skeleton, hindgut, and urogenital system. We used meiotic mapping in 1,497 segregants to localize the mutation to a 42.8-kb intergenic segment on chromosome 2. Resequencing of this region identified an 8.5-kb early retrotransposon (ETn) insertion within the highly conserved regulatory sequences upstream of Pancreas Specific Transcription Factor, 1a (Ptf1a). This mutation resulted in up to tenfold increased expression of Ptf1a as compared to wild-type embryos at E9.5 but no detectable changes in the expression levels of other neighboring genes. At E9.5, Sd mutants exhibit ectopic Ptf1a expression in embryonic progenitors of every organ that will manifest a developmental defect: the notochord, the hindgut, and the mesonephric ducts. Moreover, at E 8.5, Sd mutant mice exhibit ectopic Ptf1a expression in the lateral plate mesoderm, tail bud mesenchyme, and in the notochord, preceding the onset of visible defects such as notochord degeneration. The Sd heterozygote phenotype was not ameliorated by Ptf1a haploinsufficiency, further suggesting that the developmental defects result from ectopic expression of Ptf1a. These data identify disruption of the spatio-temporal pattern of Ptf1a expression as the unifying mechanism underlying the multiple congenital defects in Danforth's short tail mouse. This striking example of an enhancer mutation resulting in profound developmental defects suggests that disruption of conserved regulatory elements may also contribute to human malformation syndromes.