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Centroacinar Cells Are Progenitors That Contribute to Endocrine Pancreas Regeneration

View Article: PubMed Central - PubMed

ABSTRACT

Diabetes is associated with a paucity of insulin-producing β-cells. With the goal of finding therapeutic routes to treat diabetes, we aim to find molecular and cellular mechanisms involved in β-cell neogenesis and regeneration. To facilitate discovery of such mechanisms, we use a vertebrate organism where pancreatic cells readily regenerate. The larval zebrafish pancreas contains Notch-responsive progenitors that during development give rise to adult ductal, endocrine, and centroacinar cells (CACs). Adult CACs are also Notch responsive and are morphologically similar to their larval predecessors. To test our hypothesis that adult CACs are also progenitors, we took two complementary approaches: 1) We established the transcriptome for adult CACs. Using gene ontology, transgenic lines, and in situ hybridization, we found that the CAC transcriptome is enriched for progenitor markers. 2) Using lineage tracing, we demonstrated that CACs do form new endocrine cells after β-cell ablation or partial pancreatectomy. We concluded that CACs and their larval predecessors are the same cell type and represent an opportune model to study both β-cell neogenesis and β-cell regeneration. Furthermore, we show that in cftr loss-of-function mutants, there is a deficiency of larval CACs, providing a possible explanation for pancreatic complications associated with cystic fibrosis.

No MeSH data available.


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RNA-Seq of the CAC transcriptome. A: PNCs (arrows) form the entire pancreatic duct (green) in 5 dpf larvae. gb, gallbladder. Scale bar = 50 μm. B: In the adult pancreas, CACs (arrows) represent a subset of the ductal epithelium (green), which contains mostly non-Notch-responsive cells (arrowheads). CACs are the only epithelial pancreas cells that remain Notch responsive. Scale bar = 20 μm. Anti-2F11 (green), Tp1:hmgb1-mCherry (red), DAPI (blue). C: Differential gene expression (fold change >1.5) between CACs and non-CACs from two separate preparations. Expression of 236 genes was enriched in CACs in both preparations. D: Genes most highly enriched in CACs vs. non-CACs. P, probability of a gene being equally expressed in CAC and non-CAC samples. E: qPCR for cftr, clcn1b, and her15 from an independent cell preparation confirms their enrichment in CACs (**P ≤ 0.005, t test). F: Functional annotation clustering of genes upregulated in CACs. DAVID analysis of biological process, cellular component, and molecular function gene ontology terms reveals significant enrichment (enrichment score >1.3) of four clusters of terms associated with the identified 236 CAC markers.
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Figure 1: RNA-Seq of the CAC transcriptome. A: PNCs (arrows) form the entire pancreatic duct (green) in 5 dpf larvae. gb, gallbladder. Scale bar = 50 μm. B: In the adult pancreas, CACs (arrows) represent a subset of the ductal epithelium (green), which contains mostly non-Notch-responsive cells (arrowheads). CACs are the only epithelial pancreas cells that remain Notch responsive. Scale bar = 20 μm. Anti-2F11 (green), Tp1:hmgb1-mCherry (red), DAPI (blue). C: Differential gene expression (fold change >1.5) between CACs and non-CACs from two separate preparations. Expression of 236 genes was enriched in CACs in both preparations. D: Genes most highly enriched in CACs vs. non-CACs. P, probability of a gene being equally expressed in CAC and non-CAC samples. E: qPCR for cftr, clcn1b, and her15 from an independent cell preparation confirms their enrichment in CACs (**P ≤ 0.005, t test). F: Functional annotation clustering of genes upregulated in CACs. DAVID analysis of biological process, cellular component, and molecular function gene ontology terms reveals significant enrichment (enrichment score >1.3) of four clusters of terms associated with the identified 236 CAC markers.

Mentions: During development, endocrine cells, ductal cells, and CACs originate from ductal PNCs, which are the only Notch-responsive cells in the larval pancreas (Fig. 1A) (10,12). CACs are the only PNC-derived cells that remain Notch-responsive in adults (12) (Fig. 1B); hence, we hypothesized that CACs may function as adult progenitors. To explore this possibility, we sought to characterize the CAC transcriptome using RNA-seq. To flow sort adult CACs, we dissociated pancreata from Tp1:GFP adult fish and used two methods to remove contaminating Notch-responsive endothelial cells: a CD105 allophycocyanin-conjugated antibody to label endothelial cells red (method 1) and a second transgene, fli:dsRed, which drives red fluorescent protein expression in endothelial cells (method 2) (Supplementary Fig. 1A). Using either red fluorescent blood vessel marker (bv), we collected four populations from dissociated adult pancreata: green fluorescent protein (GFP)+/bv−, GFP+/bv+, GFP−/bv+, and GFP−/bv− (Supplementary Fig. 1B). As expected, qPCR revealed high expression of the CAC marker sox9b in GFP+ populations and of the vascular marker cdh5 in bv+ populations (Supplementary Fig. 1C).


Centroacinar Cells Are Progenitors That Contribute to Endocrine Pancreas Regeneration
RNA-Seq of the CAC transcriptome. A: PNCs (arrows) form the entire pancreatic duct (green) in 5 dpf larvae. gb, gallbladder. Scale bar = 50 μm. B: In the adult pancreas, CACs (arrows) represent a subset of the ductal epithelium (green), which contains mostly non-Notch-responsive cells (arrowheads). CACs are the only epithelial pancreas cells that remain Notch responsive. Scale bar = 20 μm. Anti-2F11 (green), Tp1:hmgb1-mCherry (red), DAPI (blue). C: Differential gene expression (fold change >1.5) between CACs and non-CACs from two separate preparations. Expression of 236 genes was enriched in CACs in both preparations. D: Genes most highly enriched in CACs vs. non-CACs. P, probability of a gene being equally expressed in CAC and non-CAC samples. E: qPCR for cftr, clcn1b, and her15 from an independent cell preparation confirms their enrichment in CACs (**P ≤ 0.005, t test). F: Functional annotation clustering of genes upregulated in CACs. DAVID analysis of biological process, cellular component, and molecular function gene ontology terms reveals significant enrichment (enrichment score >1.3) of four clusters of terms associated with the identified 236 CAC markers.
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Figure 1: RNA-Seq of the CAC transcriptome. A: PNCs (arrows) form the entire pancreatic duct (green) in 5 dpf larvae. gb, gallbladder. Scale bar = 50 μm. B: In the adult pancreas, CACs (arrows) represent a subset of the ductal epithelium (green), which contains mostly non-Notch-responsive cells (arrowheads). CACs are the only epithelial pancreas cells that remain Notch responsive. Scale bar = 20 μm. Anti-2F11 (green), Tp1:hmgb1-mCherry (red), DAPI (blue). C: Differential gene expression (fold change >1.5) between CACs and non-CACs from two separate preparations. Expression of 236 genes was enriched in CACs in both preparations. D: Genes most highly enriched in CACs vs. non-CACs. P, probability of a gene being equally expressed in CAC and non-CAC samples. E: qPCR for cftr, clcn1b, and her15 from an independent cell preparation confirms their enrichment in CACs (**P ≤ 0.005, t test). F: Functional annotation clustering of genes upregulated in CACs. DAVID analysis of biological process, cellular component, and molecular function gene ontology terms reveals significant enrichment (enrichment score >1.3) of four clusters of terms associated with the identified 236 CAC markers.
Mentions: During development, endocrine cells, ductal cells, and CACs originate from ductal PNCs, which are the only Notch-responsive cells in the larval pancreas (Fig. 1A) (10,12). CACs are the only PNC-derived cells that remain Notch-responsive in adults (12) (Fig. 1B); hence, we hypothesized that CACs may function as adult progenitors. To explore this possibility, we sought to characterize the CAC transcriptome using RNA-seq. To flow sort adult CACs, we dissociated pancreata from Tp1:GFP adult fish and used two methods to remove contaminating Notch-responsive endothelial cells: a CD105 allophycocyanin-conjugated antibody to label endothelial cells red (method 1) and a second transgene, fli:dsRed, which drives red fluorescent protein expression in endothelial cells (method 2) (Supplementary Fig. 1A). Using either red fluorescent blood vessel marker (bv), we collected four populations from dissociated adult pancreata: green fluorescent protein (GFP)+/bv−, GFP+/bv+, GFP−/bv+, and GFP−/bv− (Supplementary Fig. 1B). As expected, qPCR revealed high expression of the CAC marker sox9b in GFP+ populations and of the vascular marker cdh5 in bv+ populations (Supplementary Fig. 1C).

View Article: PubMed Central - PubMed

ABSTRACT

Diabetes is associated with a paucity of insulin-producing β-cells. With the goal of finding therapeutic routes to treat diabetes, we aim to find molecular and cellular mechanisms involved in β-cell neogenesis and regeneration. To facilitate discovery of such mechanisms, we use a vertebrate organism where pancreatic cells readily regenerate. The larval zebrafish pancreas contains Notch-responsive progenitors that during development give rise to adult ductal, endocrine, and centroacinar cells (CACs). Adult CACs are also Notch responsive and are morphologically similar to their larval predecessors. To test our hypothesis that adult CACs are also progenitors, we took two complementary approaches: 1) We established the transcriptome for adult CACs. Using gene ontology, transgenic lines, and in situ hybridization, we found that the CAC transcriptome is enriched for progenitor markers. 2) Using lineage tracing, we demonstrated that CACs do form new endocrine cells after β-cell ablation or partial pancreatectomy. We concluded that CACs and their larval predecessors are the same cell type and represent an opportune model to study both β-cell neogenesis and β-cell regeneration. Furthermore, we show that in cftr loss-of-function mutants, there is a deficiency of larval CACs, providing a possible explanation for pancreatic complications associated with cystic fibrosis.

No MeSH data available.


Related in: MedlinePlus