Development and characterization of fluorescent ubiquitylation-based cell cycle indicator (FUCCI) for pancreatic beta-cells in zebrafish.

(A) Schematic diagrams of FUCCI constructs for pancreatic beta-cells. The S/G2/M reporter mAG-zGeminin(1/100) and the G1 indicator mKO2-zCdt1(1/190) are expressed under the zebrafish insulin promoter. For efficient selection of transgenic animals, an eye-marker cassette, cryaa:RFP or cryaa:CFP, was introduced into Tg(ins:mAG-zGeminin(1/100)) and Tg(ins:mCherry-zCdt1(1/190)), respectively [8]. (B) Tg(ins:mCherry-zCdt1(1/190),cryaa:CFP)^s948;Tg(ins:mAG-zGeminin(1/100),cryaa:RFP)^s947 larvae were examined at 4.5 dpf using fluorescence microscopy. A close up of the islet is shown in the inset. A majority of the beta-cells are Tg(ins:mCherry-zCdt1(1/190))⁺ indicating that they are in the G1 phase of the cell cycle. Only four beta-cells are Tg(ins:mAG-zGeminin(1/100))^{s947 +} indicating that they are in the S/G2/M phase of the cell cycle. Note that the animals are expressing the eye-marker, e.g., cryaa:CFP fluorescence can be observed through the GFP filter. (C) Time-lapse imaging of Tg(ins:mCherry-zCdt1(1/190))^s948;Tg(ins:mAG-zGeminin(1/100))^s947 larvae at 4 dpf. Arrowheads point to dividing Tg(ins:mAG-zGeminin(1/100))^{s947 +} beta-cells. (D) Tg(ins:mCherry-zCdt1(1/190))^s948;Tg(ins:mAG-zGeminin(1/100))^s947 larvae were incubated with EdU from 3 to 4 dpf. The white arrow points to a Tg(ins:mAG-zGeminin(1/100))^s947+ single-positive beta-cell. This cell exhibits high levels of EdU incorporation. The yellow arrow points to a Tg(ins:mCherry-zCdt1(1/190))^s948;Tg(ins:mAG-zGeminin(1/100))^s947 double positive beta-cell which exhibits low levels of EdU incorporation indicating that this cell entered S phase at the end of the EdU labeling period. (E) Confocal stacks of Tg(ins:mAG-zGeminin(1/100))^{s947 +} (green) beta-cells stained for Insulin (blue). The animals were fixed at 12 h intervals until 5 dpf. (Scale bar = 20 μm.). (F) The graph shows a quantification of the number of Tg(ins:mAG-zGeminin(1/100))^{s947 +} beta-cells. Error bars represent SEM; n = 13–15 larvae for each time point. B is a lateral view, anterior to the left and dorsal to the top. C–E show lateral views, anterior to the top and dorsal to the left.

Retinoic acid and Trazodone promote beta-cell proliferation without inducing hyperglycemia.

(A) Confocal images of Tg(ins:mAG-zGeminin(1/100))^{s947 +} beta-cells (green) stained for Insulin (blue). The animals were treated with 1 µM retinoic acid, 10 μM trazodone or 10 μM prednisolone in 1% DMSO (scale bar = 20 μm). (B) Dose–response curves for Tg(ins:mAG-zGeminin(1/100))^{s947 +} beta-cells showing the relationship between the concentration of the compounds and the number of proliferating beta-cells in larvae treated with retinoic acid, trazodone, or prednisolone in 1% DMSO from 3 to 4 dpf. Error bars represent SEM. *P<0.05, and ***P<0.005 compared to vehicle-treated controls; n = 16–20 larvae for each group. (C) Absolute glucose values in zebrafish larvae treated with retinoic acid, trazodone, or prednisolone in 1% DMSO. Error bars represent SEM. *P<0.05, and ***P<0.005 compared to vehicle-treated controls; n = 16–20 larvae for each group. (D) Blood glucose concentration in adult zebrafish treated with 30 μM prednisolone for 24 h. Error bars represent SEM. ***P<0.005 compared to vehicle-treated controls; n = 10 fish for each group.

RA and Prednisolone effectively increase beta-cell proliferation under feeding metabolism.

(A) Schematic diagram for assessment of beta-cell proliferation. At 21 dpf, after feeding from 5–21 dpf with Kyowa N-250 (Kyowa), Tg(ins:mAG-zGeminin(1/100))^s947 larvae were treated with 1 μM retinoic acid or 10 µM prednisolone in 1% DMSO. The number of Tg(ins:H2BGFP)⁺ cells was counted at 22 dpf. (B) Tg(ins:mAG-zGeminin(1/100))^{s947 +} beta-cells were examined at 22 dpf using an epifluorescence microscope. Scale bar = 200 μm. A close up of the islet examined using a confocal microscope is shown in the inset. Scale bar = 10 μm. (C) Quantification of proliferating beta-cells per larva at 22 dpf. Error bars represent SEM. *P<0.05, and ***P<0.005 compared to DMSO-treated controls; n = 13–16 animals for each group.

RA and Prednisolone but not Trazodone, enhance beta-cell regeneration.

(A) Schematic diagram for assessment of beta-cell regeneration. To examine beta-cell regeneration, we made use of the NTR/MTZ beta-cell ablation model. At 80 hpf, after ablating the beta-cells with MTZ from 50–80 hpf, Tg(ins:H2BGFP);Tg(ins:CFP-NTR) larvae were treated with the compounds for 48 h. The numbers of Tg(ins:H2BGFP)⁺ cells were counted at 128 hpf. (B) Confocal images of Tg(ins:H2BGFP)⁺ beta-cells in larvae treated with 1 μM retinoic acid, 10 μM trazodone, or 10 μM prednisolone in 1% DMSO at 128 hpf. Each image is a lateral view, anterior to the bottom and dorsal to the right. (C) Quantification of beta-cell regeneration per larva at 128 hpf, following treatment with hit compounds from 80–128 hpf. Error bars represent SEM. *P<0.05 compared to DMSO treated controls; n = 13–16 larvae for each group.

Schematic outline of the screening protocol used to identify compounds that promote beta-cell proliferation. The images show typical examples from the screen. Tg(ins:mAG-zGeminin(1/100))^s947 larvae were arrayed in 96-well plates and exposed to 10 μM of a compound in 1% DMSO from 3 to 4 dpf (i.e., when most beta-cells are in a resting phase (Fig. 1F)). Larvae were incubated in 1% DMSO as a negative control. Tg(ins:mAG-zGeminin(1/100))^s947+ beta-cells in 4 dpf anesthetized larvae were counted by eye under an inverted fluorescence microscope. Beta-cell proliferation can be easily quantified because mAG-zGeminin(1/100) labels the nuclei of proliferating beta-cells with bright fluorescence. Fluorescent image at the bottom panel is a lateral view, anterior to the left and dorsal to the top (Scale bar = 100 μm).

The hit compounds increase the number of beta-cells undergoing S-phase. (A) Tg(ins:Kaede) larvae were treated from 3 to 5 dpf with 1% DMSO, 1 μM retinoic acid, 10 μM trazodone, or 10 μM prednisolone in the presence of 2.5 mM EdU. The numbers of Tg(ins:Kaede)+(green) and EdU+ (red) beta-cells were increased in the animals treated with the hit compounds as compared to DMSO-controls. (B) Quantification of the number of Tg(ins:Kaede)+and EdU+ beta-cells. Retinoic acid (n = 18 animals) and prednisolone (n = 16 animals) significantly increased the number of EdU+ beta-cells compared to DMSO controls (n = 15 animals). Trazodone (n = 17 animals) only mildly increased the number of EdU+ beta-cell compared to DMSO controls consistent with its less potent effect on beta-cell proliferation (see Table 1); this effect was not statistically significant (N.S.) (p = 0.46). *p<0.05 and **p<0.01. Error bars represent SEM.