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. Author manuscript; available in PMC: 2013 Jun 6.
Published in final edited form as: Cell Metab. 2012 May 17;15(6):885–894. doi: 10.1016/j.cmet.2012.04.018

Figure 4. NECA enhances proliferation of β-cells, and not cell proliferation in general.

Figure 4

(A–C) Proliferation of α-cells was assessed by EdU incorporation in Tg(gcg:GFP)-expressing α-cells. Tg(gcg:GFP);Tg(ins:flag-NTR) larvae were treated with MTZ from 3–4 dpf for ablation of the β-cells, and subsequently treated with DMSO/NECA and EdU during β-cell regeneration from 4–6 dpf. (A) Confocal image of a DMSO-treated larva displaying one α-cell that had incorporated EdU (arrow). (B) Confocal image of a NECA-treated larva that does not show any EdU incorporation in α-cells, but substantial EdU incorporation in β-cells (positive for Tg(ins:flag-NTR) expression). (C) Quantification of EdU incorporation in α-cells (green bars) and β-cells (red bars) in the presence of DMSO or NECA. Note that the number of α-cells that incorporated EdU during treatment with NECA did not significantly increase, although incorporation of EdU in Tg(ins:flag-NTR)-expressing β-cells increased markedly in the same larvae. n = 11 larvae for each group.

(D–F) Proliferation of δ-cells was assessed by EdU incorporation in Tg(sst:RFP)-expressing δ-cells. Tg(sst:RFP);Tg(ins:flag-NTR) larvae were treated with MTZ from 3–4 dpf for ablation of the β-cells, and subsequently treated with DMSO/NECA and EdU during β-cell regeneration from 4–6 dpf. (D) Confocal image of a DMSO-treated larva displaying one δ-cell that had incorporated EdU (arrow). (E) Confocal image of a NECA-treated larva displaying one δ-cell that had incorporated EdU, and substantial EdU incorporation in β-cells (positive for Tg(ins:flag-NTR) expression). (F) Quantification of EdU incorporation in δ-cells (red bars) and β-cells (green bars) in the presence of DMSO or NECA. n = 10 larvae for each group.

(G) Quantification of proliferation in the gut and liver in Tg(ins:CFP-NTR) larvae following β-cell ablation from 3–4 dpf and subsequent treatment with DMSO/NECA from 4–6 dpf. The percentage of cells that incorporated EdU between 4–6 dpf during β-cell regeneration did not change with NECA treatment when compared to DMSO-treated controls. n = 10 larvae for each group. (H) The mitosis marker Phospho-Histone H3 (P-H3) co-labels a β-cell that expresses both insulin and Tg(neurod:GFP) (arrow) following β-cell ablation from 3–4 dpf and subsequent treatment with NECA from 4–6 dpf. Note that the β-cell that is positive for P-H3 is located in the periphery of the islet.

(I) Quantification of β-cell proliferation, as marked by co-localization of insulin and P-H3, following β-cell ablation from 3–4 dpf and subsequent treatment with DMSO/NECA from 4–6 dpf. n = 42 larvae for DMSO-treated; n = 48 larvae for NECA-treated. Error bars represent SEM. See also Figures S4 & 5.