Fig. 3
Inducible genetic blockade of insulin signaling promotes β cell formation. (A) Schematic of genetic insulin signaling blockade strategy. In double transgenic embryos, heat shock induces Cre expression and recombination of the mCherry-switch-dnIRS2-GFP transgene as well as expression of the recombined dnIRS2-GFP transgene. (B) Immunoblot of pi-Akt in Cre-negative control versus Cre-positive embryos showing that induction of dnIRS2-GFP effectively blocks insulin signaling. (C,D) Confocal planes of 54 hpf control and HOT:dnIRS2 islets stained for GFP (green), Insulin (red), and Glucagon (white). Embryos were heat shocked repeatedly, at 10, 24, 28, 32, 36, and 48 hpf; dnIRS2-GFP was induced ubiquitously, but only in Cre-positive HOT:dnIRS2 embryos. (E) Quantification of Insulin+ β cells and Glucagon+ α cells in control (gray, n=8) and dnIRS2-expressing HOT:dnIRS2 (red, n=6) groups. (F–G′) Merged and single channel confocal projections of 24 hpf control and HOT:dnIRS2 pancreatic endoderm stained for GFP (green) and Pdx1 (red). dnIRS2-GFP is induced ubiquitously in HOT:dnIRS2 embryos, which show expanded Pdx1 protein expression in the principal islet (arrows) and adjacent ventral endoderm (arrowheads). Student t-test was used in B and two-way ANOVA was used in E to determine significance.
Reprinted from Developmental Biology, 409(2), Ye, L., Robertson, M.A., Mastracci, T.L., Anderson, R.M., An insulin signaling feedback loop regulates pancreas progenitor cell differentiation during islet development and regeneration, 354-69, Copyright (2016) with permission from Elsevier. Full text @ Dev. Biol.