Age-related islet inflammation marks the proliferative decline of pancreatic beta-cells in zebrafish
Figures

Beta-cell proliferation declines with age.
(a) 3D-rendering of a primary islet from Tg(ins:Fucci-G1);Tg(ins:Fucci-S/G2/M) animals at 3 mpf showing nuclear Tg(ins:Fucci-G1) (red) and Tg(ins:Fucci-S/G2/M) (green) expression. (b) Quantification of percentage of Tg(ins:Fucci-S/G2/M)-positive and Tg(ins:Fucci-G1)-negative (green-only) beta-cells at 35 dpf (n = 5), 3 mpf (n = 9) and 1 ypf (n = 10) animals. Each dot represents one animal. Horizontal bars represent mean values (one-way ANOVA, *p<0.05). (c, d, e) Confocal projection of whole-mount islets from Tg(ins:Fucci-G1);Tg(ins:Fucci-S/G2/M) animals at 35 dpf, 3 mpf and 1 ypf. Anterior to the top. Scale bar 50 µm. (f) Ex vivo live-imaging of beta-cells from Tg(ins:nlsRenilla-mKO2);Tg(ins:GCaMP6s) animals at 3 mpf. Beta-cells (red) were stimulated with 2.5 (basal) mM D-Glucose, (f’) 11 mM D-glucose, (f’’) 16.7 mM D-glucose and (f’’’) depolarized using 30 mM KCl while monitoring GCAMP6s-fluorescence (green). Scale bar 10 µm.

Beta-cell proliferation declines with age in secondary islets.
(a,b) Confocal projection of whole-mount secondary islets from Tg(ins:Fucci-G1);Tg(ins:Fucci-S/G2/M) animals at 35 dpf, 3 mpf and 1 ypf. Scale bar 20 µm. (c) Quantification of percentage of Tg(ins:Fucci-S/G2/M)-positive and Tg(ins:Fucci-G1)-negative (green-only) beta-cells from 35 dpf (n = 8 fish, secondary islets = 16), 3 mpf (n = 8 fish, secondary islets = 70) and 1 ypf (n = 8 fish, secondary islets = 76) animals. Error bars show s.e.m. (one-way ANOVA, *p<0.05).

Transcriptome profiling of younger and older beta-cells.
(a) Schematic showing isolation and FAC-sorting of beta-cells from Tg(ins:nlsRenilla-mKO2) animals at 3 mpf and 1 ypf followed by high-throughput mRNA-Sequencing. (a’) Heatmap depicting differentially regulated genes among the beta-cells at 1 ypf and 3 mpf involved in beta-cell proliferation, function and inflammation (asterisks denote genes vaildated by single-cell RT-qPCR). (b) Volcano plot representing the distribution of genes that were differentially regulated in beta-cells from 1 ypf and 3 mpf (1.5-log2fold change, p<0.05). (c) The biological categories of enriched genes in beta-cells at one ypf (1.5-log2fold change, p<0.05) based on literature survey. (c’) Unbiased gene-ontology analysis using DAVID of genes enriched in beta-cells at 1 ypf (p<0.05). (d) Gene expression analysis was carried out using single-cell RT-qPCR. Violin plots denote expression distribution of the candidate genes. The Y-axis shows -log10(Ct) values of transcript levels in single beta-cells. The X-axis shows gene names and the respective developmental stages. The percentage values under each violin plot denote the proportion of beta-cells with detectable transcript levels. The cycle threshold for detectable gene expression was set as Ct = 40. The value −1.6 (-log10(40)) on the Y-axis represents undetectable expression as measured by single-cell RT-qPCR (see Materials and methods). Each dot represents one beta-cell. Significance testing for differences in proportion of cells with detectable gene expression at each stage was performed using Pearson’s Chi-Square test (**p<0.01, ***p<0.001).

Fluorescent activated cell sorting of beta-cells.
(a) Fluorescent activated cell sorting (FACS) of RFP-positive and calcein-positive beta-cells from Tg(ins:nlsRenilla-mKO2) animals.

An inflammation reporter reveals heterogeneous activation of NF-kB signaling in beta-cells with age.
(a) The images show single confocal planes from islets of 5 dpf larvae. The tnfrsf1b coding sequence was expressed under the control of the insulin promoter. The plasmid was injected in Tg(NF-kB:GFP) embryos at the one-cell-stage, leading to mosaic and stochastic expression of the construct in beta-cells. The Tg(NF-kB:GFP) reporter expresses GFP (green) under the control of six tandem repeats of NF-kB DNA-binding sites. Beta-cells were labelled using an insulin antibody (red). Arrows indicate GFP-positive beta-cells. Scale bar 5 µm. (b) The graph shows the percentage of GFP-positive and insulin-positive cells in uninjected controls (n = 5) and tnfrsf1b injected animals (n = 6) at 5 dpf. Horizontal bars represent mean values. (c–e) Confocal stack of islets from Tg(NF-kB:GFP) animals at 1 mpf, 3 mpf and 1 ypf. Beta-cells were labeled using an insulin antibody (red). NF-kB:GFP reporter expression is shown in green. Scale bars 20 µm. (c’–e’) Insets show high magnification single planes of the confocal stacks (corresponding to the regions shown using white dotted-lines in the top panels). Scale bar 10 µm. (f–g) Beta-cells from 3 mpf Tg(NF-kB:GFP) animals were labeled with TSQ (Zn2+ labeling dye) and analyzed using FACS. The graph shows GFP intensity (along the X-axis) and the distribution of beta-cells at 3 mpf and 1 ypf. Horizontal lines indicate the division point between GFPlow and GFPhigh levels. Percentage values represent proportion of cells with GFPlow or GFPhigh expression.

Activation of NF-kB signaling in beta-cells of the secondary islets with age.
(a,b,c) Confocal stack of secondary islets from Tg(NF-kB:GFP) animals at 1 mpf, 3 mpf and 1 ypf. Beta-cells were labeled using an insulin antibody (red). NF-kB:GFP reporter expression is shown in green. Scale bars 20 µm. (a’,b’,c’) Insets show high-magnification single planes of the confocal stacks corresponding to the regions outlined using white dotted-lines in the top panels. Scale bar 10 µm. (d) Graph showing the total normalized GFP fluorescence intensity of the secondary islets from 3 mpf (n = 9 fish, secondary islets = 32) and 1 ypf (n = 8, secondary islets = 30) animals. Each dot represents one islet (two-tailed t-test, *p<0.05).

Fluorescent activated cell sorting of NF-kB:GFPhigh and NF-kB:GFPlow beta-cells.
(a) Confocal image of dissociated cells from dissected islets of 3 mpf Tg(ins:nlsRenilla-mKO2) animals that were labeled with TSQ (Zn2+ labeling dye). Islets were incubated with TSQ after dissociation and imaged using a confocal microscope. Beta-cells show RFP expression (red) while cells rich in Zn2+ are labeled with TSQ (cyan). TSQ strongly labels all beta-cells and weakly labels some unknown endocrine cells. (b) Fluorescent activated cell sorting (FACS) of live TSQ-positive GFPhigh and GFPlow cells from Tg(NF-kB:GFP) animals at 3 mpf and 1 ypf. Dead cells were labelled using far-red stain DRAQ7.

NF-kB:EGFP mRNA levels in beta-cells increase with age.
(a) Graph showing the relative fold change increase in EGFP mRNA levels in beta-cells from 1 ypf compared to 3 mpf animals, as measured using RT-qPCR (n=5 biological replicates from three fish each, 1000 cells for each condition). Error bars show SD (two-tailed paired t-test, *p<0.05). (b) The graph shows the fluorescence levels of individual FAC-sorted beta-cells from Tg(nF-kB:GFP);Tg(ins:mCherry) animals as log10(GFP intensity) (along the Y-axis) and Expression threshold (Et) values of GFP mRNA (along X-axis) measured using single-cell RT-qPCR. Line indicates the correlation between GFP fluorescence intensity and GFP-mRNA expression levels in single cells. Each dot represents one beta-cell. R2 = 0.28.

Immune cells infiltrate the islet during development and persist throughout adult life.
(a) Confocal images of pancreata from 15, 21 and 45 dpf animals. Beta-cells were labeled using an insulin antibody (grey), leukocytes were labeled using an L-plastin antibody (magenta) and Tg(ptf1a:GFP) marks the acinar cells (green). Immune cells are present within the islet at 45 dpf (arrows). (b) Confocal images of whole islets from Tg(tnfα:GFP) animals at 1 ypf. Islets were labeled using TSQ (Zn2+ labeling dye) (blue), leukocytes were labelled with an L-plastin antibody (magenta) and Tg(tnfα:GFP) marks cells expressing tnfα (green). Scale bars 20 µm. (b’) Insets show high-magnification single planes from the confocal stacks (corresponding to the area marked using a white dotted-line in b). Scale bar 10 µm. (c–c’) Confocal image of a one ypf islet showing a single plane. The TgBAC(tnfα:GFP) line marks the tnfα-positive cells (green), whereas Tg(mpeg1:mCherry) marks the macrophages (red). The L-plastin antibody marks all leukocytes (grey) and TSQ (Zn2 +labeling dye) was used to mark the islet (n = 5). Scale bar, 10 µm. (d) Confocal images showing islets at five dpf. The tnfα cDNA was expressed under the insulin promoter. The plasmid was injected in Tg(NF-kB:GFP) embryos at the one-cell-stage and the islets were analyzed at 5 dpf. Beta-cells were labeled with an insulin antibody (red). Tg(NF-kB:GFP) reporter expression is shown in green. (e) The graph shows the percentage of GFP and insulin double-positive cells in un-injected controls (n = 7) and ins:tnfα injected animals (n = 8) at five dpf. Horizontal bars represent mean values (two-tailed t-test, *p<0.05).

Immune cells infiltrate the islet during development.
(a,a’) Confocal image of a juvenile islet. Tg(ins:CFP-NTR) line marks the beta-cells with CFP (green), leukocytes were labelled with the L-plastin antibody (blue) and Tg(mpeg1:mCherry) marks the macrophages (red). (b) Quantification of the percentage of TgBAC(tnfa:GFP) and L-plastin double-positive cells over the total number of L-plastin-positive cells in the islets of TgBAC(tnfa:GFP) animals at 3 mpf (n = 5) and 1 ypf (n = 5). Horizontal bars represent mean values (two-tailed t-test, p>0.05). (c) Quantification of the total number of TgBAC(tnfα:GFP)-positive and L-plastin double-positive cells at 3 mpf (n = 5) and 1 ypf (n = 5). Horizontal bars represent mean values (two-tailed t-test, p>0.05). (d) Quantification of the total number of L-plastin positivecells at 3 mpf (n = 5) and 1 ypf (n = 5). Horizontal bars represent mean values (two-tailed t-test, p>0.05).

NF-kB:GFPhigh beta-cells proliferate less than their neighbors.
(a) Schematic showing the EdU (5-ethynyl-2′-deoxyuridine) incorporation assay. Tg(NF-kB:GFP) animals were incubated in EdU at 3 mpf for two consecutive nights and fed during each day. (b) EdU incorporation assay was performed to mark the proliferating beta-cells in Tg(NF-kB:GFP) animals at 3 mpf. The confocal image (single plane) shows an overview of a section through the islet. Beta-cells were labeled with an insulin antibody (red), a GFP antibody (green) and EdU (blue). Arrowheads point to EdU-positive beta-cells. (b’–b’’) The insets show higher magnification images with and without the EdU channel. EdU incorporation can be observed in some of the GFPlow cells (white arrow-heads). (c) An insulin-positive cell was first located by going through individual sections in the confocal z-stack. The optical section containing the largest area of the nucleus was chosen as the center of the cell. A region-of-interest (ROI) was drawn around the nucleus and the fluorescence intensities of the GFP and DAPI channels were recorded. The normalized GFP intensity was calculated as a ratio of mean GFP intensity and mean DAPI intensity for each ROI. The average total normalized GFP-intensity of each islet was set as a threshold for dividing the cells into GFPhigh and GFPlow populations. The graph shows the percentage of EdU and insulin double-positive cells among the GFPhigh and GFPlow populations. Each dot represents one islet (n = 9). Horizontal bars represent mean values (two-tailed t-test, *p<0.05). (d) The graph shows the percentage of PCNA and insulin double-positive cells among the GFPhigh and GFPlow populations. Each dot represents one islet (n = 13). Horizontal bars represent mean values (two-tailed t-test, *p<0.05). See also Figure 5—figure supplement 2 for representative PCNA antibody staining.

The GFP fluorescence of the transgenic reporterTg(NF-kB:GFP)remains stable for 72 hours in beta-cells.
(a,b,c) Beta-cells from Tg(NF-kB:GFP);Tg(ins:mCherry) animals at 3 mpf were FAC-sorted as single cells in 384-well plates and followed over 72 hr. NF-kB:GFPhigh cells at 24, 48 and 72 hr post-FAC-sorting. Scale bar 5 µm. (a’,b’,c’) NF-kB:GFPlow cells at 24, 48 and 72 hr post-FAC-sorting. Scale bar 5 µm. (d) Quantification showing the mean GFP fluorescence intensity of GFPhigh and GFPlow cells (n = 7 GFPhigh cells and n = 5 GFPlow cells).

Proliferating cell nuclear antigen (PCNA) antibody staining shows that NF-kB:GF Phighbeta-cells proliferate less than NF-kB:GFP low beta-cells.
(a) Islets were stained for PCNA to mark the proliferating beta-cells in Tg(NF-kB:GFP) animals at 3 mpf. The confocal image (single plane) shows an overview of a section through the islet. The sections were stained with an insulin antibody (red), a GFP antibody (green) and PCNA (blue). (a’–a’’) The insets show higher magnification images. PCNA can be observed in some of the GFPlow cells (white arrow-heads).

Socs2 is enriched in NF-kB:GFPhigh cells and inhibits beta-cell proliferation in a cell-autonomous manner.
(a) Schematic showing the sorting of beta-cells from the double transgenic line Tg(ins:mCherry);Tg(NF-kB:GFP) at 3 mpf into GFPhigh and GFPlow cells using FACS. (b) Bulk RT-qPCR was performed on the GFPhigh and GFPlow beta-cells (n = 3 to 4 biological replicates, n = 3 animals per replicate, 1000 cells per condition). Candidate genes significantly enriched in beta-cells at 1 ypf were chosen to be compared between the GFPhigh and GFPlow populations at 3 mpf. The graph shows relative fold-change between GFPhigh and GFPlow cells. The expression of all genes was normalized to β-actin expression before calculating fold-change. socs2 shows higher expression in the GFPhigh cells. Error bars, SD (two-tailed paired t-test, *p<0.05). (c) Schematic showing the method for mosaic overexpression of candidate genes in beta-cells. The socs2 coding sequence is linked to nuclear-CFP using a T2A sequence. The entire construct was expressed under the insulin promoter. This construct was injected in one-cell-stage-embryos from Tg(ins:Fucci-G1);Tg(ins:Fucci-S/G2/M) animals leading to mosaic and stochastic expression of socs2 in beta-cells during islet development. Control animals were injected with plasmid containing only nuclear-CFP sequence (See Figure 6—figure supplement 2). (d) Confocal projections showing mosaic expression of socs2-T2A-CFP (blue) at 23 dpf (blue). Proliferating beta-cells are marked by Tg(ins:Fucci-S/G2/M) expression (green) and absence of Tg(ins:Fucci-G1) expression (red). Anterior to the left. Scale bar 10 µm. (d’) Insets show higher magnification single planes from the confocal stacks (white dotted-line in d) with separate channels. The proliferating beta-cells are CFP-negative (yellow arrowheads), whereas some of the non-proliferating cells are CFP-positive (white arrowheads) (e) Quantification of the percentage of Tg(ins:FUCCI-S/G2/M)-positive and Tg(ins:FUCCI-G1)-negative (green only) beta-cells. The socs2 expressing β-cells exhibit reduced cell-cycle progression compared to wild-type neighbors (n = 9). Horizontal bars represent mean values (two-tailed t-test, *p<0.05).
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Figure 6—source data 1
This spreadsheet contains the Relative Fold Change between NF-kB:GFPhigh and NF-kB:GFPlow beta-cells used to generate the bar plots and average data shown in Figure 6b.
- https://doi.org/10.7554/eLife.32965.018

Fluorescent activated cell sorting of NF-kB:GFPhigh and NF-kB:GFPlowbeta-cells.
(a) Contour plot showing FACS of live RFP-positive GFPhigh and GFPlow cells from Tg(NF-kB:GFP);Tg(ins:mCherry) animals at 3 mpf. Live cells were labeled with calcein. (b) Dot plot shows FACS of live RFP-positive GFPhigh and GFPlow cells from Tg(NF-kB:GFP);Tg(ins:mCherry) animals at 3 mpf.

Mosaic expression of candidate genes in beta-cells to study their effect on proliferation.
(a,b,c) Confocal images showing mosaic expression of nuclear-CFP (a), CFP-T2A-spry4 (b) and CFP-T2A-rapgef4 (c) at 23 dpf. Tg(ins:Fucci-G1) expression is shown in red, Tg(ins:Fucci-S/G2/M) expression in green and CFP expression in blue. Anterior to the top. Scale bar 20 µm. (a’,b’,c’) Quantification of the percentage of Tg(ins:FUCCI-S/G2/M)-positive and Tg(ins:FUCCI-G1)-negative (green only) beta-cells among the CFP-positive and CFP-negative sub-populations for each experiment shown in the left-hand panels. Horizontal bars represent mean values (two-tailed t-test, p>0.05).

A schematic summarizing our model.
Beta-cell proliferation declines with age together with a concurrent increase in NF-kB signaling. The activation of NF-kB signaling is heterogeneous among beta-cells and correlates with their proliferative heterogeneity. In particular, beta-cells with higher NF-kB activity proliferate less compared to neighbors with lower activity, and express higher levels of socs2, which can inhibit beta-cell proliferation. Furthermore, the crosstalk with tnfα-positive immune cells in the islet provides a potential source of inflammation and NF-kB activation in beta-cells.
Tables
Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
---|---|---|---|---|
Gene (Danio rerio) | flag-tnfrsf1b | synthesized from GenScript | ||
Gene (Danio rerio) | tnfα | Dharmacon | MDR1734-202796946 | ZGC tnfa cDNA (CloneId:8148192) |
Gene (Danio rerio) | cpf-T2A | synthesized from GenScript | ||
Genetic reagent (Danio rerio) | Tg(ins:FUCCI-G1)s948 | PMID: 23791726 | ||
Genetic reagent (Danio rerio) | Tg(ins:FUCCI-S/G2/M)s946 | PMID: 23791726 | ||
Genetic reagent (Danio rerio) | Tg(NF-kB:GFP) | PMID: 21439961 | ||
Genetic reagent (Danio rerio) | TgBAC(tnfα:GFP) | PMID: 25730872 | ||
Genetic reagent (Danio rerio) | Tg(ins:nlsRenilla-mKO2) | PMID: 28939870 | ||
Genetic reagent (Danio rerio) | Tg(ins:gCaMP6s; cryaa:mCherry) | PMID: 28939870 | ||
Genetic reagent (Danio rerio) | Tg(ins: loxP:mCherrySTOP:loxP:H2B-GFP) | PMID: 21497092 | ||
Genetic reagent (Danio rerio) | Tg(mpeg1:mCherry) | PMID: 21084707 | ||
Genetic reagent (Danio rerio) | Tg(ins:CFP-NTR) | PMID: 17326133 | ||
Antibody | anti-insulin | Dako | A0564 | guinea pig (1:200) |
Antibody | anti-EGFP | Abcam | ab13970 | chicken (1:500) |
Antibody | anti-PCNA | Dako | M0879 | mouse (1:500) |
Antibody | anti-L-plastin | Biozol | LS-C210139-250 | rabbit (1:1000) |
Antibody | Alexa Fluor488, 568 and 647 secondaries | Molecular Probes | (1:300) | |
Recombinant DNA reagent | ins:Flag-tnfrsf1b;cryaa:RFP (plasmid) | This paper | cloned into ins:MCS2;cryaa:RFP | |
Recombinant DNA reagent | ins:tnfα;cryaa:CFP (plasmid) | This paper | cloned by replacing mCherry-zCdt1 with tnfα in ins:mCherry-zCdt1;cryaa:CFP | |
Recombinant DNA reagent | ins:CFP-T2A-socs2;cryaa:RFP (plasmid) | This paper | cloned into ins:MCS2;cryaa:RFP | |
Recombinant DNA reagent | ins:CFP-T2A-rapgef4;cryaa:RFP (plasmid) | This paper | cloned into ins:MCS2;cryaa:RFP | |
Recombinant DNA reagent | ins:CFP-T2A-spry4;cryaa:RFP (plasmid) | This paper | cloned into ins:MCS2;cryaa:RFP | |
Recombinant DNA reagent | ins:mAG-zGeminin;cryaa:RFP (plasmid) | PMID: 23791726 | ||
Recombinant DNA reagent | ins:MCS2;cryaa:RFP (plasmid) | PMID: 28939870 | ||
Recombinant DNA reagent | ins:mCherry-zCdt1;cryaa:CFP (plasmid) | PMID: 23791726 | ||
Software, algorithm | edgeR package | PMID:19910308 | ||
Other | TSQ (N-(6-Methoxy-8-Quinolyl)-p-Toluenesulfonamide) | ThermoFisher | M-688 | 30 µM |
Additional files
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Supplementary file 1
List of genes differentially expressed from RNA-Seq of beta-cells at 3 mpf and 1 ypf (log2FC ± 1.5).
- https://doi.org/10.7554/eLife.32965.020
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Supplementary file 2
List of primer sequences of genes validated using single-cell RT-qPCR and bulk RT-qPCR.
- https://doi.org/10.7554/eLife.32965.021
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Transparent reporting form
- https://doi.org/10.7554/eLife.32965.022