ZFIN ID: ZDB-FIG-161018-3
Lu et al., 2016 - IGFBP1 increases β-cell regeneration by promoting α- to β-cell transdifferentiation. The EMBO journal   35(18):2026-44 Full text @ EMBO J.
ADDITIONAL FIGURES
EXPRESSION / LABELING:
Genes:
Antibody:
Fish:
Condition:
Knockdown Reagent:
Anatomical Terms:
Stage: Day 6
PHENOTYPE:
Fish:
Condition:
Knockdown Reagent:
Observed In:
Stage: Day 6

Fig. 3

Cellular mechanisms of Igfbp1a′s effect on βcell regeneration

A-C. Tg(ins:GFP);Tg(tp1:H2BmCherry);Tg(ins:FlagNTR) transgenics, with or without Tg(bactin:igfbp1a), were treated with MTZ from 3 to 4 dpf to ablate the β cells and were then allowed to regenerate from 4 to 6 dpf. Representative confocal images at 6 dpf of control (A) and Tg(bactin:igfbp1a) (B) larvae showing a modest number of ins+ tp1+ coexpressing cells, indicated by arrows, after 2 days of regeneration. Scale bars: 15 µm. (C) Quantification of the total number of β cells (green bars) at 6 dpf, and of β cells expressing Tg(tp1:H2BmCherry), that is, of ductal origin (yellow bars). ****< 0.0001, ns= nonsignificant. = 23 larvae in the control group, = 17 larvae in the Tg(bactin:igfbp1a) group.

D-F. Tg(ins:VenuszGeminin) was examined in control and Tg(bactin:igfbp1a) larvae at 6 dpf, after βcell ablation 3-4 dpf by using Tg(ins:FlagNTR). Representative confocal images (D, E) at 6 dpf of control and Tg(bactin:igfbp1a) larvae showing cell cycle activation of β cells in green. Scale bars: 15 µm. (F) Quantification of the total number of β cells with activated cell cycle at 6 dpf, ***< 0.001, = 32 larvae in the control group, = 41 larvae in the Tg(bactin:igfbp1a) group. The number of β cells with activated cell cycle is displayed together with the total number of β cells in experiments with the same setup, ****< 0.0001. = 39 larvae in the control group, = 33 larvae in the Tg(bactin:igfbp1a) group.

G-I. EdU was used as a marker for cell cycle progression. Tg(ins:H2BGFP);Tg(ins:FlagNTR) transgenics, with or without Tg(bactin:igfbp1a), were treated with MTZ from 3 to 4 dpf to ablate their β cells, and subsequently incubated with EdU during regeneration from 4 to 6 dpf. Representative confocal images (G, H) at 6 dpf of control and Tg(bactin:igfbp1a) larvae showing β cells in green and the β cells that had incorporated EdU in yellow (green and red overlap; arrowheads). Scale bars: 20 µm. (I) Quantification of the total number of β cells (green bars) at 6 dpf and of β cells that incorporated EdU (yellow bars) during βcell regeneration from 4 to 6 dpf. **< 0.01, ns = nonsignificant. = 16 in both the control and the Tg(bactin:igfbp1a) group.

J-L. Tg(ins:kaede);Tg(ins:CFPNTR) transgenics, with or without Tg(bactin:igfbp1a), were injected with a control morpholino or a morpholino that knocked down arx, which is necessary for αcell differentiation and thus glucagon expression (J, K). Scale bars: 20 µm. (L) Quantification of the total number of regenerating β cells at 4 dpf (after βcell ablation at 2-3 dpf). ****< 0.0001, *< 0.05, ns=nonsignificant. = 42, 20, 48 and 20, respectively.

Gene Expression Details
Gene Antibody Fish Conditions Stage Anatomy Assay
GFP s939Tg ; zf5Tg ; ki103Tg ; s950Tg chemical ablation: pancreatic B cell, chemical treatment by environment: metronidazole Day 6 regenerating tissue pancreatic B cell IFL
s939Tg ; zf5Tg ; s950Tg chemical ablation: pancreatic B cell, chemical treatment by environment: metronidazole Day 6 regenerating tissue pancreatic B cell IFL
s950Tg ; ki103Tg ; s960Tg chemical ablation: pancreatic B cell, chemical treatment by environment: metronidazole Day 6 regenerating tissue pancreatic B cell IHC
s960Tg ; s950Tg chemical ablation: pancreatic B cell, chemical treatment by environment: metronidazole Day 6 regenerating tissue pancreatic B cell IHC
Kaede s892Tg ; ki103Tg ; zf5Tg chemical ablation: pancreatic B cell, chemical treatment by environment: metronidazole Day 6 regenerating tissue pancreatic B cell IHC
s892Tg ; ki103Tg ; zf5Tg + MO3-arxa chemical ablation: pancreatic B cell, chemical treatment by environment: metronidazole Day 6 regenerating tissue pancreatic B cell IHC
mCherry s939Tg ; zf5Tg ; ki103Tg ; s950Tg chemical ablation: pancreatic B cell, chemical treatment by environment: metronidazole Day 6 pancreatic duct IFL
Day 6 regenerating tissue pancreatic B cell IFL
s939Tg ; zf5Tg ; s950Tg chemical ablation: pancreatic B cell, chemical treatment by environment: metronidazole Day 6 pancreatic duct IFL
Day 6 regenerating tissue pancreatic B cell IFL
Venus ki103Tg ; ki107Tg ; s950Tg chemical ablation: pancreatic B cell, chemical treatment by environment: metronidazole Day 6 regenerating tissue pancreatic B cell IFL
ki107Tg ; s950Tg chemical ablation: pancreatic B cell, chemical treatment by environment: metronidazole Day 6 regenerating tissue pancreatic B cell IFL
Antibody Labeling Details
Antibody Assay Fish Conditions Stage Qualifier Anatomy
Ab1-gcg IHC s892Tg ; ki103Tg ; zf5Tg chemical ablation: pancreatic B cell, chemical treatment by environment: metronidazole Day 6 glucagon secreting cell
IHC s892Tg ; ki103Tg ; zf5Tg + MO3-arxa chemical ablation: pancreatic B cell, chemical treatment by environment: metronidazole Day 6 Not Detected glucagon secreting cell
Phenotype Details
Fish Conditions Stage Phenotype
ki103Tg ; ki107Tg ; s950Tg chemical ablation: pancreatic B cell, chemical treatment by environment: metronidazole Day 6 pancreatic B cell cell cycle increased occurrence, abnormal
Day 6 pancreatic B cell regeneration increased occurrence, abnormal
s892Tg ; ki103Tg ; zf5Tg chemical ablation: pancreatic B cell, chemical treatment by environment: metronidazole Day 6 pancreatic B cell regeneration increased occurrence, abnormal
s892Tg ; ki103Tg ; zf5Tg + MO3-arxa chemical ablation: pancreatic B cell, chemical treatment by environment: metronidazole Day 6 glucagon secreting cell ab1-gcg labeling absent, abnormal
s892Tg ; ki103Tg ; zf5Tg + MO3-arxa chemical ablation: pancreatic B cell, chemical treatment by environment: metronidazole Day 6 pancreatic A cell absent, abnormal
s892Tg ; ki103Tg ; zf5Tg + MO3-arxa chemical ablation: pancreatic B cell, chemical treatment by environment: metronidazole Day 6 pancreatic B cell regeneration increased occurrence, abnormal
s939Tg ; zf5Tg ; ki103Tg ; s950Tg chemical ablation: pancreatic B cell, chemical treatment by environment: metronidazole Day 6 pancreatic B cell regeneration increased occurrence, abnormal
s950Tg ; ki103Tg ; s960Tg chemical ablation: pancreatic B cell, chemical treatment by environment: metronidazole Day 6 pancreatic B cell cell population proliferation increased occurrence, abnormal
Day 6 pancreatic B cell regeneration increased occurrence, abnormal
Acknowledgments:
ZFIN wishes to thank the journal The EMBO journal for permission to reproduce figures from this article. Please note that this material may be protected by copyright. Full text @ EMBO J.