Figure 1.
Construction of the GhrlLCA acceptor and Ghrl:Cre-eGFP exchange alleles.
(a) Schematic of the Ghrl:Cre-eGFP knock-in allele generated using recombinase mediated cassette exchange (RMCE). First, a floxed Ghrelin (GhrlLCA) 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 GhrlLCA 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.
Figure 2.
Reporter expression accurately recapitulates ghrelin expression at e18.5.
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 Ghrl:Cre allele in any of the tissues studied (d–f). Scale bar, all panels 50 µm, insets 20 µm.
Figure 3.
Ghrelin-labeled cells mark a subpopulation of glucagon- and Ppy-expressing cells in neonates.
Representative images showing immunofluorescence of islet hormones and direct tomato reporter fluorescence on cryosections of Ghrl-Cre;R26-Tomato (TOM) P0 pancreas (a-d). Reporter fluorescence co-localized with a significant number of glucagon-expressing cells (a) and Ppy-expressing cells (b). No significant co-localization was detected with insulin- (c) and somatostatin-expressing cells (d). DAPI staining was used to visualize the nuclei. Scale bars: low magnification = 50 µm, insets = 10 µm.
Figure 4.
Two distinct populations of alpha and PP cells can be distinguished in the adult endocrine pancreas based on their origin.
We analyzed pancreata from 8 week old (8w) Ghrl-Cre;R26-TOM 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.
Figure 5.
Ghrelin lineage-labeled cells are frequently found in the pancreatic exocrine and ductal compartments in neonates.
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 Ghrl-Cre;R26-TOM P0 pancreas in the indicated genetic backgrounds. DAPI staining was used to visualize the nuclei. Scale bar: all panels = 50 µm, insets = 10 µm.
Figure 6.
Ghrelin lineage-labeled cells are committed to the endocrine lineage and do not differentiate into chief nor parietal cells in the stomach.
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.
Figure 7.
Schematic summarizing the lineage tracing experiments to define the fate of ghrelin-labeled cells in the pancreas of Ghrl-Cre;R26-TOM mice.
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.