Fig. 4

NECA Enhances Proliferation of β Cells, and Not Cell Proliferation in General

(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) colabels 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 colocalization 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 and S5.