Ghrelin Expression in the Mouse Pancreas Defines a Unique Multipotent Progenitor Population

<div><p>Pancreatic islet cells provide the major source of counteractive endocrine hormones required for maintaining glucose homeostasis; severe health problems result when these cell types are insufficiently active or reduced in number. Therefore, the process of islet endocrine cell lineage allocation is critical to ensure there is a correct balance of islet cell types. There are four endocrine cell types within the adult islet, including the glucagon-producing alpha cells, insulin-producing beta cells, somatostatin-producing delta cells and pancreatic polypeptide-producing PP cells. A fifth islet cell type, the ghrelin-producing epsilon cells, is primarily found during gestational development. Although hormone expression is generally assumed to mark the final entry to a determined cell state, we demonstrate in this study that ghrelin-expressing epsilon cells within the mouse pancreas do not represent a terminally differentiated endocrine population. Ghrelin cells give rise to significant numbers of alpha and PP cells and rare beta cells in the adult islet. Furthermore, pancreatic ghrelin-producing cells are maintained in pancreata lacking the essential endocrine lineage regulator Neurogenin3, and retain the ability to contribute to cells within the pancreatic ductal and exocrine lineages. These results demonstrate that the islet ghrelin-expressing epsilon cells represent a multi-potent progenitor cell population that delineates a major subgrouping of the islet endocrine cell populations. These studies also provide evidence that many of hormone-producing cells within the adult islet represent heterogeneous populations based on their ontogeny, which could have broader implications on the regulation of islet cell ratios and their ability to effectively respond to fluctuations in the metabolic environment during development.</p> </div

Ghrelin lineage-labeled cells are frequently found in the pancreatic exocrine and ductal compartments in neonates.

<p>Ghrelin-lineage labeled Tom+ cells predominantly localize to the islet periphery (a). Approximately 50% of the embryos analyzed contained ghrelin-labeled cells within the amylase+ acini clusters (b). This phenomenon is preferentially observed in juxta-duodenal regions of the ventral pancreas. (c) Ghrelin-lineage labeled cells that co-expressed ductal markers were also identified in the same animals. In Neurog3^−/− pancreata (d–f), which are almost completely devoid of endocrine hormone-producing cells, we could detect a small number of ghrelin-expressing and lineage labeled cells (d). We also detected ghrelin descendants in the exocrine (e) and ductal tissue (f) of Neurog3^−/− embryos. (g–j) In Nkx2.2^−/− mice, a subset of Ppy- (g) and glucagon-expressing cells (h) are still derived from ghrelin-expressing cells. Ghrelin-labeled cells are also detected in the exocrine and ductal compartment of Nkx2.2^−/− animals (i–j). The frequency and magnitude of lineage labeling of these cells were not significantly different from wild type pancreas. a, d and e are representative images of at least 3 pancreata. b, c, f, g, h, i, j are representative image of at least 3 embryos in which we detected labeled cells localized to the exocrine and ductal pancreatic tissue. All images are from <i>Ghrl-Cre;R26-TOM</i> P0 pancreas in the indicated genetic backgrounds. DAPI staining was used to visualize the nuclei. Scale bar: all panels = 50 µm, insets = 10 µm.</p

Two distinct populations of alpha and PP cells can be distinguished in the adult endocrine pancreas based on their origin.

<p>We analyzed pancreata from 8 week old (8w) <i>Ghrl-Cre;R26-TOM</i> mice. In the adult, we could not detect ghrelin expression by immunofluorescence but ghrelin-lineage labeled cells localized primarily to the periphery of the islet. (a–b) Adjacent images of the same islet of Langerhans showing co-localization of the ghrelin-lineage labeled cells with glucagon (b) but not with insulin (a). White boxes in b shows two distinct population of alpha cells, one derived from a ghrelin-expressing cell (magnified in b”) and one independent of the ghrelin lineage (magnified in b). (c–d) Adjacent images show that ghrelin-expressing cells contribute to the PP (magnified in d’) but not to the delta lineage. (e) Immunofluorescence analysis with glucagon and Ppy antibodies confirm that two independent populations of alpha and PP cells are derived from ghrelin-expressing cells. (f) Quantitative analysis of the total number of ghrelin-labeled cells per endocrine area at the two stages studied, P0 and 8w pancreas. (g) Fraction of ghrelin-labeled cells within a given population of hormone producing cells at P0 and 8w. The percentage of ghrelin cells labeled with the reporter at P0 is indicative of the Cre recombination efficiency. No ghrelin cells were detected at 8w. Three mice were examined per time point. Error bars show mean ± SEM (p<0.05 (*) Student’s unpaired t-test). DAPI staining was used to visualize the nuclei. Scale bar: low magnification = 50 µm, B, B’ and inset in E = 10 µm, d’ = 20 µm.</p

Schematic summarizing the lineage tracing experiments to define the fate of ghrelin-labeled cells in the pancreas of <i>Ghrl-Cre;R26-TOM</i> mice.

<p>Ghrelin-expressing cells are rare in the adult pancreas; however cells that formerly expressed ghrelin or their progeny contribute to the final set of endocrine cells in the islet, specifically within the alpha and PP cells. Interestingly, not all α and PP cells are descendants of ghrelin-expressing cells, suggesting that two distinct populations of glucagon- and PP-expressing cells inhabit the adult pancreas. Moreover, we identified Neurog3-independent ghrelin-expressing cells that have the potential to contribute to exocrine and ductal tissue.</p

Reporter expression accurately recapitulates ghrelin expression at e18.5.

<p>Tom+ cells co-express ghrelin in pancreas (a), stomach (b) and duodenum (c). Note that the pancreatic ghrelin-producing epsilon population only represent 5–10% of the total pancreatic endocrine population at this stage of development. No leakiness of the reporter was detected in the absence of the <i>Ghrl:Cre</i> allele in any of the tissues studied (d–f). Scale bar, all panels 50 µm, insets 20 µm.</p

Ghrelin lineage-labeled cells are committed to the endocrine lineage and do not differentiate into chief nor parietal cells in the stomach.

<p>Representative images showing immunofluorescence for gastric cell markers and direct reporter fluorescence on cryosections and whole mounts of stomach from 6w Ghrl-Cre;R26-TOM mice. Whole mount stomach shows reporter fluorescence in the corpus and in the pyloric antrum but not in the fundus (a–a’). Although, almost all ghrelin-expressing cells were labeled with TOM, not all TOM+ cells co-localize with ghrelin, indicating ghrelin-expressing cells differentiate into other cell types (b). The tomato reporter is expressed in gastrin-expressing G cells (c), as well as a large number of somatostatin-expressing D cells in the corpus of the stomach (d), but not serotonin-expressing cells (e). All TOM+ cells were also Chromogranin A+, indicative of an endocrine commitment of the ghrelin-expressing cells in the stomach (f). Ghrelin-labeled cells did not colocalize with pepsin C, a marker for chief cells (f), or with DBA, a marker of parietal cell (g). (h) Schematic representation of the lineage tracing experiments defining the fate of ghrelin-labeled cells in the stomach. G, gastrin producing cells; P, parietal cells; EE, enteroendocrine cells; C, chief cells; X/A, ghrelin-producing X/A-like cells; D, somatostatin producing cells. Scale bar, low magnification panels = 50 µm, insets = 20 µm.</p

Construction of the Ghrl^LCA acceptor and Ghrl:Cre-eGFP exchange alleles.

<p>(a) Schematic of the Ghrl:Cre-eGFP knock-in allele generated using recombinase mediated cassette exchange (RMCE). First, a floxed Ghrelin (Ghrl^LCA) allele was created by inserting a positive/negative selection cassette flanked by the Lox71 and Lox2272 modified lox sites into the ghrelin locus by homologous recombination. The Lox71 and Lox2272 sites allow for directional and irreversible Cre mediated recombination while suppressing intramolecular recombination. An exchange cassette vector containing the complimentary Lox66 and Lox2272 sites and a Cre-eGFP coding sequence spanning exons 2 and 3 (Ghrl:Cre-eGFP) was then created and inserted into the genome using RMCE. (b) Insertion of the Ghrl^LCA allele was confirmed by Southern blot using either ScaI digested (5′ recombination) or NdeI digested (3′ recombination) genomic DNA. (c) Correct exchange of the Ghrl:Cre-eGFP allele in clones that survived both positive and negative selection was confirmed by PCR analysis. All seven tested clones had correct allele exchange.</p

Nkx6.1 and Isl1 function as antagonistic transcriptional regulators of the <i>Arx Re1</i> enhancer.

<p>Immunofluorescence staining of pancreata from <i>Ngn3-Cre;Z/EG</i> mice at e14.5 (A) and e16.5 (B) for Nkx6.1, Arx, and GFP shows that the majority of progeny of Ngn3-expressing cells (GFP⁺) co-express Arx and Nkx6.1 at e14.5 (arrowheads in A), while the Arx⁺ and Nkx6.1⁺ domains are distinct at e16.5 (arrowheads in B point to GFP⁺Arx⁺Nkx6.1⁻ cells). (C) Schematic of the <i>Arx</i> locus; asterisks indicate phylogenetically-conserved Nkx6.1 binding motifs and numbers indicate the distance from the transcriptional start site. Nkx6.1 binds to site C (<i>Re1</i> element) in the <i>Arx</i> locus in chromatin from Min6 cells (D) and FACS-sorted GFP⁺ cells (E) from e15.5 pancreata of <i>Neurog3</i>^eGFP embryos analyzed by ChIP with antibodies against Nkx6.1 or control immunoglobulin G (IgG). Mouse <i>glucagon</i> promoter and intergenic primers were used as positive (+) and negative (−) controls, respectively. (F) Co-transfection of αTC1–6 cells with the <i>Arx Re1</i> enhancer-luciferase construct, the <i>CMV-Renilla</i> expression construct, and with or without the <i>CMV-Nkx6.1</i> expression construct. Lane one (M) represents basal luciferase expression of the minimal promoter. Luciferase activity was quantified relative to the expression of the minimal promoter. Activity of the <i>Re1</i> enhancer is repressed by Nkx6.1. (G) Co-transfection of αTC1–6 cells with the <i>Arx Re1</i> enhancer-luciferase construct, <i>CMV-Renilla</i>, and with different concentration of <i>CMV-Nkx6.1</i> and <i>CMV-Isl1</i>, as indicated. Nkx6.1 prevents activation of the <i>Arx Re1</i> enhancer by Isl1 in a dose-dependent manner (lanes 2–7). Luciferase activity was quantified relative to the expression of the <i>Re1</i> enhancer. Increasing concentrations of Isl1 are not sufficient to restore baseline activity of the <i>Re1</i> enhancer in the presence of 200 ng of <i>CMV-Nkx6.1</i> (lanes 8–12). Scale bar = 50 µm. Error bars represent S.E.M; *p<0.05, **p<0.01, ***p<0.001.</p

<i>Nkx6.1</i> is required for beta cell specification downstream of Ngn3.

<p>(A, B) Schematic of the alleles and transgenes for <i>Nkx6.1</i> inactivation and lineage tracing; Triangles, <i>loxP</i> sites. Immunofluorescence staining of pancreata at e15.5 (C, D) or postnatal day (P) 2 (E–P). Recombined, GFP⁺ cells are restricted to the endocrine compartment (antibody against the pan-endocrine marker Chromogranin A, Chga) in control (E) and <i>Nkx6.1^f/−;Ngn3-Cre;Z/EG</i> mice (F). The insets show higher magnifications and arrowheads point to GFP⁺ cells expressing Ngn3 (C, D) or hormones (G–P). Quantification of hormone⁺GFP⁺ (Q), Ki67⁺GFP⁺ (R), or TUNEL⁺GFP⁺ (S) co-positive cells as a percentage of all GFP-expressing cells in pancreata of <i>Nkx6.1^f/−;Ngn3-Cre;Z/EG</i> and <i>Ngn3-Cre;Z/EG</i> mice at P2 (n = 4). Loss of <i>Nkx6.1</i> in endocrine precursors favors alternative, non-beta endocrine cell fate choices over beta cell fate. Horm, hormones; Ins, insulin; Glc, glucagon; Som, somatostatin; PP, pancreatic polypeptide; Ghr, ghrelin; endo, endocrine. Scale bar = 50 µm. Error bars represent S.E.M; *p<0.05, **p<0.01.</p

Loss of <i>Nkx6.1</i> in endocrine precursors results in activation of non-beta endocrine genes.

<p>(A–L) Immunofluorescence staining of pancreata from <i>Ngn3-Cre;Z/EG</i> and <i>Nkx6.1^f/−;Ngn3-Cre;Z/EG</i> mice at postnatal day (P) 2 shows reduced Pdx1 (A, B), absence of MafA (C, D), and ectopic expression of Arx (E, F), Brn4 (G, H), glucagon (Glc; I, J), and somatostatin (Som; K, L) in <i>Nkx6.1</i>-deficient, recombined, insulin⁺GFP⁺ cells. Arrowheads point to insulin⁺ cells ectopically expressing non-beta endocrine markers. Ins, insulin. Scale bar = 50 µm.</p