The transcriptional landscape of mouse beta cells compared to human beta cells reveals notable species differences in long non-coding RNA and protein-coding gene expression
© Benner et al.; licensee BioMed Central Ltd. 2014
Received: 27 March 2014
Accepted: 10 July 2014
Published: 22 July 2014
Insulin producing beta cell and glucagon producing alpha cells are colocalized in pancreatic islets in an arrangement that facilitates the coordinated release of the two principal hormones that regulate glucose homeostasis and prevent both hypoglycemia and diabetes. However, this intricate organization has also complicated the determination of the cellular source(s) of the expression of genes that are detected in the islet. This reflects a significant gap in our understanding of mouse islet physiology, which reduces the effectiveness by which mice model human islet disease.
To overcome this challenge, we generated a bitransgenic reporter mouse that faithfully labels all beta and alpha cells in mouse islets to enable FACS-based purification and the generation of comprehensive transcriptomes of both populations. This facilitates systematic comparison across thousands of genes between the two major endocrine cell types of the islets of Langerhans whose principal hormones are of cardinal importance for glucose homeostasis. Our data leveraged against similar data for human beta cells reveal a core common beta cell transcriptome of 9900+ genes. Against the backdrop of overall similar beta cell transcriptomes, we describe marked differences in the repertoire of receptors and long non-coding RNAs between mouse and human beta cells.
The comprehensive mouse alpha and beta cell transcriptomes complemented by the comparison of the global (dis)similarities between mouse and human beta cells represent invaluable resources to boost the accuracy by which rodent models offer guidance in finding cures for human diabetes.
KeywordsBeta cell Alpha cell Pancreatic islet Insulin Glucagon Ucn3 Crh Diabetes lncRNA RNA-seq Prolactin receptor Prlr Growth hormone receptor Ghr Ciliary neurotrophic factor receptor Cntfr Islet amyloid polypeptide Iapp Amylin Ets1
Pancreatic beta and alpha cells are clustered together in pancreatic islets to ensure tight coordination of the secretion of insulin and glucagon, whose opposing actions on hepatic glucose metabolism are essential for glucose homeostasis. Yet, despite the functional juxtaposition of insulin and glucagon, pancreatic beta and alpha cells derive from a common progenitor population that during embryonic development is first uniquely defined by the expression of the basic helix-loop-helix transcription factor neurogenin 3 (Neurog3) . Under the influence of lineage-specific sets of transcription factors, Neurog3+ early endocrine progenitors undergo stepwise differentiation along parallel lineages to develop into mature alpha and beta cells [2–5]. It follows that upon completion of their differentiation and maturation, alpha and beta cells, by virtue of the secretion of their signature hormones glucagon and insulin, have adapted to functionally opposing roles in glucose metabolism. Insulin acts on most peripheral tissues to facilitate the clearance of glucose from the general circulation by inhibiting hepatic gluconeogenesis and promoting hepatic glycogen synthesis and glucose uptake in skeletal muscle and adipocytes [6, 7]. Furthermore, insulin conveys important metabolic feedback to hypothalamic feeding centers regarding the energy state of the body . In contrast, the hepatic actions of glucagon, which include the stimulation of glycogenolysis, provide an essential counter-regulatory mechanism to ensure stable glycemic control . The serious consequences of the absolute insulin deficiency that occur secondary to autoimmune attack of the beta cells in type 1 diabetes as well as the relative insulin deficiency characteristic of type 2 diabetes illustrate the critical importance of insulin. However, the view that hyperglucagonaemia contributes significantly to the progression of both major forms of diabetes has gained acceptance . It follows that a comprehensive understanding of both the alpha and the beta cell and how they regulate glucose homeostasis through the coordination of their activity quite possibly will hold the key to therapeutic interventions aimed at curbing the current diabetes epidemic.
The intimate clustering of beta and alpha cells in pancreatic islets subserves the tight coordination of the secretion of insulin and glucagon, but has long complicated the process of obtaining highly pure populations of alpha and beta cells from isolated islets. To overcome this challenge, we have developed a reporter strain that fluorescently marks all beta cells by the nuclear expression of mCherry under control of the mouse insulin 1 (Ins1) promoter and crossed it to a GFP reporter line that labels alpha cells. Bitransgenic offspring of a cross between both lines then enabled the collection of purified populations of alpha and beta cells from the same islets. These tools enabled the first transcriptome-wide comparison of murine alpha and beta cells by RNA-seq, which revealed over 2500 genes that are differentially expressed (p-value < 1 × 10-7, false discovery rate (FDR) < 0.1%) between both populations, with insulin the second-most significantly enriched in the beta cell fraction. Next generation sequencing combines the tremendous benefits in sensitivity and dynamic range of techniques traditionally used to assess expression of targeted sets of genes, such as quantitative PCR, with the scale of microarray and the distinct benefit that no a priori sequence information is required, which enables novel transcript discovery. We take advantage of our data to conduct the first transcriptome-wide assessment of the similarities and differences that exist between rodent and human beta cells. These data provide an unprecedented global view of the core beta cell transcriptome that is conserved across species, while simultaneously highlighting marked differences in the expression of receptors and associated long non-coding RNAs (lncRNAs) between beta cells of both species. An unbiased and comprehensive comparison between mouse and human beta cells is an invaluable resource that can facilitate the translation of preclinical findings in rodent models towards therapeutic strategies aimed at alleviating or curing diabetes.
Generation and validation of mIns1-H2b-mCherry and S100b-eGFP reporter lines
Whole transcriptome analysis of highly enriched mouse beta and alpha cells
A comprehensive comparison of the transcriptomes of alpha and beta cells revealed 2547 genes that were differentially expressed between beta and alpha cells. A total of 1075 genes were significantly (p-value < 1 × 10-7, FDR < 0.1%) enriched in beta cells. The Insulin 2 (Ins2) and Ins1 genes are the second- and third most significantly enriched genes in beta cells compared to alpha cells, after the beta cell-specific transcription factor Mafa (Figure 2E; Additional file 2), which serves as a potent confirmation of the high purity of the beta cell population obtained by the use of our mIns1-H2b-mCherry reporter mouse. Other genes that were highly significantly enriched in mouse beta cells are genes encoding the prolactin receptor (Prlr) and the peptide hormone Urocortin3 (Ucn3) (Figure 2E). Conversely, 1472 genes were significantly (p-value < 1 × 10-7, FDR < 0.1%) enriched in the alpha cell fraction, including the mouse alpha cell-specific transcription factors Arx, Irx1, Mafb, as well as glucagon (Gcg) (Additional file 3). It is no surprise that Ins2 and Ins1 are the most abundantly detected transcripts in beta cells, comprising on average 13.0% and 5.1%, respectively, of all mapped reads in the beta cell libraries (Figure 2F). In the alpha cell-enriched libraries, Gcg is responsible for on average 15.7% of all mapped reads. Expression of other islet hormones, including pancreatic polypeptide (Ppy), somatostatin (Sst), peptide YY (Pyy) and islet amyloid polypeptide (Iapp), is between 6.5 and 33-fold lower than the expression of Gcg, which confirms the successful enrichment of alpha cells (Figure 2G). While the abundance of Ins2/Ins1 and Gcg transcripts is expected in beta- and alpha cell-enriched libraries, respectively, the fraction of reads that maps to insulin is lower than for human beta cells, where upwards of 38% of all mapped reads in FACS-purified beta cells were assigned to INS [15, 16]. As a further validation of our FACS purification strategy, expression of known mouse beta cell-specific genes in the alpha cell fraction, including Ins1, Ins2, Ucn3 [17, 18] and Mafa , was on average less than 2.39% of the expression detected in beta cells (Figure 2H-K). Conversely, transcripts encoding the alpha cell-transcription factors Arx1, Irx1 and Mafb and Gcg, which are all highly significantly enriched in alpha cells (Additional file 3), are detectable in the beta cell fraction at on average less than 1.49% of their expression in alpha cells (Figure 2L-O). We compared our alpha and beta cell transcriptomes to a previous study that contrasted FACS-purified MIP-GPF + beta cells to transcriptomes of intact islets . Their effort was somewhat limited by the aforementioned mosaicism of the MIP-GFP reporter line , which reduced the contrast between the transcriptomes of MIP-GFP + beta cells and total islets . This manifested as the unremarkable enrichment of key beta cell markers including Ins2, Ins1, Mafa and others in this study compared to our work (Additional file 4).
Functional validation of alpha and beta cell transcriptomes
Differences between mouse and human islet cells
Comprehensive comparison of mouse and human beta cell transcriptomes
The small subset of 156 genes that is significantly (p < 1 × 10-7, FDR < 0.1%) and robustly (>10-fold) enriched in mouse beta cells, but still detectable in human beta cells, constitutes less than 1.5% of the common beta cell transcriptome (Figure 5A). This cluster includes Iapp, which is more than 30-fold reduced in mouse over human beta cells (Figure 5B, F), perhaps in adaptation to the amyloid properties of human but not mouse IAPP that contribute to the onset and progression of type 2 diabetes . The strongly disparate relative expression of the gene encoding IAPP in mouse and human islets is in contrast to other hormones such as INS/Ins2 and Ucn3, which are expressed at similar levels between mouse and human beta cells (Figure 5A, B). We further validated the relatively low expression of Iapp message by demonstrating that IAPP peptide content is reduced almost 28-fold in human islets compared to mouse, while INS and GCG content is similar in islets from both species (Figure 5G; Additional file 10). A similarly small set of 149 genes that is detectably expressed in beta cells of both species, but robustly and significantly enriched in human beta cells includes Gad2 (Figure 5B, H), which is a major auto-antigen in human type 1 diabetes [40, 41].
Notable differences in cell surface receptors between human and mouse beta cells
Several of the most notable gene expression differences between mouse and human beta cells concern cell surface receptors of the class I helical cytokine receptor family . Prlr has long been implicated in beta cell mass expansion in adaptation to the increased metabolic demand of pregnancy in rodents in response to PRL or placental lactogens [43, 44]. Indeed, Prlr is abundantly and selectively expressed in mouse beta cells (Figures 2E, 5A, B; Additional file 2). In contrast, while Prlr is detectable in FACS-purified human beta cells and is associated with a human PDX1 ChIP-Seq signature (Figure 5I), it is expressed at approximately 15-fold lower RPKM values than mouse beta cells (Figure 5B, Additional file 9) and is significantly depleted compared to co-purified human non-beta cells (not shown). Similarly, related receptor genes for growth hormone (Ghr) (Figure 5J) and ciliary neurotrophic factor (Cntfr) (Figure 5K) are robustly expressed by mouse beta cells, but are virtually undetectable in human beta cells.
Novel transcript discovery
The interleukin-1 receptor locus in beta cells
The interleukin-1 receptor type 1 (Il1r1/CD121a) locus aptly recapitulates all aspects of our study. Il1r1 is the most abundantly expressed cell surface receptor on mouse beta cells by a considerable margin with an RPKM value > 200. Expression of Il1r1 is significantly enriched in mouse beta over alpha cells (p < 1.9 × 10-15) and the mouse Il1r1 promoter is associated with strong transcription factor binding by the beta cell transcription factors PDX1, NKX6.1 and NEUROD1 (Figure 7A), validating the beta cell as the source of Il1r1 expression. In sharp contrast, IL1R1 expression in human islet populations, while detectable, is significantly lower in human beta cells in comparison to both mouse beta cells (Figure 5A, B) and human alpha cells (Figure 7A, B). The high levels of Il1r1 message in mouse beta cells translate into abundant cell surface expression of IL1R1/CD121a on mCherry + beta cells as measured by FACS (Figure 6C) and confocal microscopy (Figure 7D, E). An additional notable feature unique to the mouse Il1r1 locus is a lncRNA positioned about 30 kb upstream of the Il1r1 transcription start site. This lncRNA demonstrates strong beta cell enrichment similar to the adjacent Il1r1 transcript and is associated with significant transcription factor binding by PDX1, NKX6.1, MAFA and NEUROD1 (Figure 7A). The corresponding region of the human IL1R1 locus shows no human PDX1 binding or evidence for the expression of a similar lncRNA in islet cells (Figure 7B). Moreover, expression of this mouse-specific lncRNA upstream of Il1r1 is the most significantly up regulated of all transcripts, coding and non-coding, following overnight stimulation of mouse islets with glucose (Figure 7F; Additional file 11). A repeat of 13 consensus binding sites for the transcription factor V-Ets Avian Erythroblastosis Virus E26 Oncogene Homolog 1 (ETS1) (Figure 6F) that is absent from the human IL1R1 locus may contribute to both the expression of this lncRNA by mouse but not human beta cells and the robust glucose-induced up regulation of its expression, as Ets1 expression is significantly up regulated by glucose (Figure 7G).
To ensure tight coordination of the secretion of insulin and glucagon, pancreatic beta and alpha cells are intimately colocalized in the pancreas, which has long complicated the process of obtaining highly pure populations of alpha and beta cells from isolated islets. We successfully overcame this problem by generating a mIns1-H2b-mCherry beta cell reporter line that enabled us to achieve over 98% beta cell purity, which is markedly higher than previous studies that used antibody-based strategies to purify beta cells from dissociated human islet suspensions [15, 16, 46]. The fact that both mouse insulin genes rank as the second and third most significantly enriched genes in mouse beta over alpha cells - trailing only the key beta cell-specific transcription factor Mafa - underscores our high quality of beta cell enrichment. Further confirmation that our transcriptomes faithfully recapitulate known expression patterns in alpha and beta cells follows from the fact that expression of key alpha and beta cell transcription factors as well key genes involved in glucose sensing, stimulus secretion coupling and insulin exocytosis generally adheres closely to the literature [2–5]. It is noteworthy that many additional genes involved in glycolysis, membrane depolarization, calcium entry and insulin exocytosis are not, or only modestly enriched in mouse beta over alpha cells, suggestive of similarities in the triggering of hormone release by either cell type as was recently reported .
Importantly, our transcriptome data now offer the opportunity for the systematic comparison of gene expression by mouse and human beta cells. We identified a core set of 9906 genes that are shared between mouse and human beta cells. It is no surprise that this set contains many of the key genes required for the stimulus-appropriate release of insulin such as G6pc2, Ero1lb, Pcsk1 and Glp1r. We also uncovered significant transcriptome differences between mouse and human beta cells that are accompanied with species-specific enrichment of beta cell-specific transcription factors, and sometimes associated with species-specific lncRNAs. We minimized the potential caveats that can stem from differences in islet isolation, dissociation, FACS sorting and library preparation between laboratories by focusing only on robustly (10-fold enrichment) and significantly differentially expressed (p < 1 × 10-7, FDR < 0.1) genes. Further validation of transcriptome differences between mouse and human beta cell transcriptomes was obtained by demonstrating that species-specific enrichment of gene expression correlates well with species-specific enrichment of ChIP signatures for the beta cell specific transcription factors Pdx1  and Nkx6.1 [48, 49]. Among the more striking examples are Prlr, Ghr and Cntfr, three related receptors that respond to structurally similar ligands , that are abundantly and selectively expressed by mouse beta cells, with only nominal expression in human islets. Co-stimulation of CNTF and EGF promotes acinar to beta cell transdifferentiation in diabetic mice  and GH and PRL are potent inducers of mouse beta cell proliferation  and implicated in the expansion of maternal beta cell mass during pregnancy in mice [43, 44]. However, efforts to use PRL or GH to induce human beta cell proliferation in vitro have been largely unsuccessful [52, 53] and expansion of human beta cell mass during pregnancy is modest compared to mouse and driven by neogenesis instead of beta cell proliferation . While there are clearly differences between mouse and human beta cells that prevent the latter from entering the cell cycle , the lack of GHR and relative absence of PRLR are not conducive to robust responses to their potential mitogenic ligands.
The most abundantly expressed cell surface receptor in mouse beta cells, measured by gene expression, is Il1r1, which responds to the pro-inflammatory cytokines IL-1α and IL-1β. Importantly, the human islet is not devoid of IL1R1 transcript, in line with a body of literature that established that mouse and human beta cells respond to sustained IL-1β stimulation with a reduction in function and an increase in apoptosis [56–58]. The robustly elevated Il1r1 expression in mouse islets could betray a more prominent role for locally produced IL-1β in beta cell glucotoxicity in mouse islets and potentially render mouse beta cells more sensitive to IL-1β-mediated pro-inflammatory insults. This observation is important, as local inflammation in the islet precipitated by obese conditions is thought to contribute to beta cell failure and exacerbate diabetes .
Several of the genes that displayed markedly higher expression in mouse over human beta cells, such as Il1r1 and Pparg, were flanked by mouse-specific novel lncRNAs. We therefore leveraged our transcriptome data to identify 145 novel lncRNAs that are enriched for significant transcription factor binding of key beta cell transcription factors and are co-regulated with the nearest protein-coding gene in line with previous observations [15, 60, 61]. As lncRNAs are not bound by the constraints of protein-coding genes, they tend to display less sequence conservation and stability over longer phylogenetic distances . Indeed, comparison of the set of 145 mouse lncRNAs with human  revealed evidence for a human orthologous lncRNA for only two. It appears that the repertoire of islet lncRNAs is dynamic and has changed considerably since the divergence of primates and rodents, in line with the general notion that the lncRNA repertoire across vertebrates is dynamic, even between closely related species [60, 63, 64]. Their generally high plasticity and poor sequence conservation notwithstanding, lncRNAs are emerging as an important and novel transcriptional regulatory mechanism that is likely to significantly impact beta cell fate and function. Indeed, we observed that in response to stimulation with glucose, the quintessential beta cell trigger, quite a few lncRNAs were up- or down regulated by 10-fold or more. The arduous task of attributing function to the beta cell-enriched lncRNAs that have emerged from ours and similar studies [15, 20] is now underway.
Rodents are commonly used to study the islets of Langerhans, with the ultimate goal of improving the outlook of diabetic patients and despite considerable differences in islet architecture and innervation between rodent and primate islets. Our comprehensive beta and alpha cell transcriptomes will greatly enhance our understanding of normal islet physiology and yield significant new leads to direct the behavior of its two primary endocrine constituents. Importantly, our data leveraged against recently published transcriptomes for FACS-purified human islet populations will now facilitate routine cross comparison between mouse and human alpha and beta cell transcriptomes. This is an invaluable resource for all with an interest in islet physiology and disease that stands to improve the translatability of rodent studies  by ensuring that potential therapeutic targets identified by preclinical experiments on rodents are similarly expressed by human beta cells.
Generation of a mIns1-H2b-mCherry beta cell reporter line
Animals were maintained on a 12-h light/12-h dark cycle with free access to water and standard rodent how. DNA encoding a fusion between human histone-2b and monomeric Cherry (H2b-mCherry) was ligated in the Spe I site of the mouse insulin 1 promoter (pBS.MIP1(-SpeI); generously gifted by Dr. Mark Magnuson). The resulting mIns1-H2b-mCherry reporter construct (Figure 1A) was used for pronuclear injection by the Salk transgenic core, leading to two founder lines. One founder line demonstrated bright nuclear expression of mCherry in only a small minority of beta cells and was discarded. The other founder line was determined to have the transgenic cassette inserted at two separate loci, one of which was silent and was bred out. No fertility or viability issues were noted and reporter expression was only observed in the pancreas. Although no effect of transgene copy number was observed, only hemizygous offspring were used. The mIns1-H2b-mCherry reporter line was crossed to S100b-eGFP mice (The Jackson Laboratory, Bar Harbor, ME; strain 005621) . Glucose tolerance tests were carried out as previously described . All procedures were approved by the Salk Institute for Biological Studies Institutional Animal Care and Use Committee.
Islet isolation and FACS sorting
Islet isolation was carried out as previously described . Islets from mIns1-H2b-mCherry × S100b-eGFP bitransgenic animals were pooled by sex in two replicate groups of 10 or 11 animals. In preparation for sorting, islets were hand-picked into a 15 ml conical tube and allowed to sediment before excess media was aspirated. Islets were dissociated at 37°C by adding 0.25% trypsin-EDTA (Invitrogen) aided by regular but gentle mechanical dissociation with a p200 pipette. Dissociated islet cells were washed in HBSS containing 10% FBS and sorted at the Salk Institute Flow Cytometry core on a FACS Vantage SE DiVa (Becton-Dickinson, Franklin Lakes, NJ) using 488 and 568 excitation lines for eGFP and mCherry, respectively. Sorted cells were collected directly in Trizol. Flow cytometric staining for Il1r1/CD121a-APC (Biolegend, San Diego, CA) was conducted on dissociated mCherry + beta cells at 5 microgram/ml in 100 microliter HBSS + 10% FBS. For glucose stimulation, islets from 3 month old male C57Bl6 mice (Harlan, Indianapolis, IN) were cultured overnight in RPMI containing 11 mM glucose, before glucose deprivation for 2 hours in RPMI containing 2.8 mM glucose followed by 12 h incubation with 2.8 mM or 16.8 mM glucose. One hundred islets were used per replicate.
RNA isolation and library prep
RNA was isolated from Trizol by a chloroform extraction, assisted by phase lock tubes. RNA was precipitated by isopropanol and cleaned up over an RNEasy microcolumn (Qiagen, Valencia, CA) per the manufacturer's instructions, taking great care to avoid carry over of ethanol following the column washes. Following elution in 30 microliter elution buffer, RNA quality was verified by BioAnalyzer (Agilent, Santa Clara, CA). Indexed sequencing libraries were constructed using the TruSeq RNA sample Prep Kit v2 (Illumina Inc. San Diego, CA) and sequenced at 50 cycles, single read on an Illumina HiSeq 2000 platform.
RNA-Seq and ChIP-Seq analysis
Sequencing reads from two beta cell populations and two alpha cell populations were mapped using STAR  to the mouse genome version mm9 (NCBI build 37). Over 30 million reads were sequenced for each library with over 83% of sequenced reads aligning uniquely (>93% alignment overall). Bedtools  was used to create count tables of the sorted bam files using reads aligning to RefSeq defined exons. DESeq  was used for statistical comparison. See Additional file 12 for a count table with all genes in our alpha and beta transcriptomes. Raw RNA-seq sequence files of human beta and non-beta cells  and ChIP-Seq data for mouse and human PDX1 , mouse and human NKX6.1 [48, 49], and mouse MAFA and NEUROD1 , were remapped using STAR (RNA-seq) or bowtie2 (ChIP-Seq) to their respective genomes (hg19, mm9). Normalized genome browser tracks were prepared using HOMER (http://homer.salk.edu)  and uploaded into the University of California Santa Cruz genome browser to generate browser plots. ChIP-Seq peaks were determined using HOMER. Individual read alignments, transcripts, and ChIP-Seq peak positions for each human sequencing experiment were converted to the mouse genome using the UCSC liftOver tool. Homologene was used to map gene identifiers between mouse and human. To discover species-specific PDX1 peaks, human PDX1 ChIP-Seq read alignments were converted to the mm9 genome. Peaks were found for both species with respect to the mm9 genome and differentially bound peaks were defined by peak regions containing 4-fold more reads in one species relative to the other. Instances of the PDX1 motif were found using HOMER. Peak positions were converted between mm9 and hg19 genomes using the liftOver tool and scanned independently to identify species-specific motifs. The MEME Suite package MCAST  was used for the identification of statistically significant consensus binding sites for the four transcription factors found to be differentially-regulated by glucose (Ets1, Pax6, Pdx1, Myc). The full mouse transcriptome was scanned using default parameters. The consensus site motifs used were taken from the JASPAR Database .
Novel transcript discovery
To identify novel lncRNA, RNA-Seq libraries from all replicates of alpha and beta cells were combined into a single meta-experiment to maximize sensitivity. Two approaches where used to identify transcripts. First, Cufflinks  was used with default settings to identify transcripts de novo. Second, the findPeaks program in homer was used with “-style histone” and “-minDist 1500” to identify regions of continuous high read density that Cufflinks missed. This approach yielded 71,730 predicted transcripts, which were used to confirm the absence or presence of transcripts predicted by other studies. Because many of the 71,730 transcripts found by HOMER and Cufflinks were short fragments of RNA found within introns or immediately upstream and downstream of known transcripts, we adopted stringent filtering criteria to identify novel, high-quality lncRNAs. Novel transcripts must (1) not overlap with any known RefSeq or UCSC gene exon, (2) have a total transcript length greater than 3 kb, (3) expression in either alpha or beta cell experiment > 1 RPKM. (4) and not overlap with known rRNA loci. We also excluded transcripts with a PhastCon score > 0.4 which likely reflect mapping artifacts to pseudogenes instead of actual novel transcripts.
Islet peptide content determination
Islets were sonicated in 300 μl of KREBS ringer buffer, then centrifuged 10 min at 8000 K. The supernatant was transferred to a fresh tube. Stock samples were stored at -20 C. For each assay the samples were diluted in the assay buffer supplied with each individual kit. Human and mouse insulin, glucagon and IAPP islet peptide content were measured with commercially available RIA or ELISA kits (EMD Millipore, Billerica, MA) per the manufacturer's instructions. As cross-reactivity for mouse IAPP had not been determined by EMD Millipore, a separate rat/mouse IAPP (American Peptide Company, Sunnyvale, CA) standard curve was run in equivalent doses and shown to be superimposable with the human IAPP curve supplied with the ELISA, confirming 100% cross-reactivity with mouse IAPP (Additional file 10). Mouse and human islet IAPP samples were each quantified according to their homologous standard curve.
Immunofluorescence was carried out as previously described  using commercial antisera against glucagon (guinea pig anti-glucagon at 1:7000; EMD Millipore, Billerica MA), somatostatin (sheep anti-somatostatin at 1:1000; American Research Products Inc., Waltham MA), insulin (chicken anti-insulin at 1:1000; Abcam) and IL1r1/CD121a (armenian hamster anti-mouse Il1r1/CD121a-APC at 1:100; Biolegend, San Diego, CA). Rabbit antisera against UCN3 (#7218, 1:2000) and CRH (rc70, 1:5000) were generated in house. Secondary antibodies were raised in donkey against each of these host species and were obtained from Jackson Immuno Research Laboratories (West Grove, PA) conjugated to Alexa Fluor-405, -488, -649 or Cy3 and used at 1:600. Dapi was applied as a nuclear stain at 1 microgram/ml and slides were embedded with Prolong Gold antifade reagent (Life Technologies, Carlsbad, CA). All imaging was carried out on Zeiss LSM780 confocal microscopes at The Waitt Advanced Biophotonics Center Core facility of the Salk Institute.
Human donor islets were obtained through the Integrated Islet Distribution Program (IIDP) and declared exempt from approval by the Institutional Review Board of the Salk Institute for Biological Studies.
CB is the director of the Razzavi Newman Integrated Genomics and Bioinformatics Core at the Salk Institute for Biological Studies and is an expert bio-informatician, who wrote Homer (Hypergeometric Optimization of Motif Enrichment), a suite of tools for Motif Discovery and next-generation sequence analysis. MOH is a Staff Scientist at the Salk Institute's Clayton Foundation Laboratories for Peptide Biology and will continue to grow his research program at the University of California, Davis in the fall of 2014. His group studies the role of Corticotropin Releasing Hormone (CRH) and the related peptide hormone Urocortin3 in the pancreas. The direct actions of these and other peptide hormones on the pancreas add a novel layer of complexity to the intricate network of signaling molecules that in concert governs beta cell mass and endocrine output of the pancreas.
Images were acquired at the Waitt Advanced Biophotonics Center, whose support is gratefully acknowledged. Cell sorting was done at the Salk Institute Flow Cytometry Core Facility. The mouse insulin 1 promoter was a generous gift of Dr. Marc Magnuson at Vanderbilt University. Christopher Cowing-Zitron is acknowledged for his assistance with bioinformatics analysis of the glucose-stimulated islet libraries. MOH is a recipient of a Career Development Award of the Juvenile Diabetes Research Foundation (JDRF). These studies were funded by grants 17-2012-424 and 2-2013-54 from the JDRF, P01-DK026741 from the NIH/NIDDK and the Clayton Medical Research Foundation, Inc. The authors have no conflicts to declare.
Sequencing data sets described in this work have been deposited in the Gene Expression Omnibus (GEO) repository under accession number GSE54973 and GSE58384.
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