Figure 6

Notch-responsive endocrine progenitors are absent in pdx1 morphants. (A-D) Confocal image projections of Tg(NeuroD:EGFP)nl1 embryos immunostained for green fluorescent protein (GFP) and insulin. Optical sections that obscure the posterior pancreatic tail have not been included. (E-J) Native enhanced green fluorescent protein (EGFP) expression in the pancreas of living TgBAC(NeuroD:EGFP)nl1 embryos at 5.5 days post fertilization (dpf), with overlay of concurrently acquired brightfield image. Shown are uninjected control embryos (A-D, E, F), hb9 morphants (G,H) and pdx1 morphants (I,J) treated either with vehicle (dimethylsulfoxide (DMSO)) (A,C,E,G,I) or 100 mM N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT) (B,D,F,H,J) from 3 to 5 dpf. DAPT treatment in control embryos leads to the induction of EGFP expression in the pancreatic tail (A,B,E,F) and of Ins in individual of these EGFP+ cells (B, D). In hb9 morphants the patterns of EGFP expression in DMSO-treated and DAPT-treated embryos are similar to that in control embryos (E-H), while pdx1 morphants show decreased EGFP+ cells in the principal islet and virtually no posterior EGFP+ cells are induced by DAPT (I,J). All embryos lateral view, anterior to the left. Scale bar = 10 μM. (K) Quantitation of NeuroD:EGFP cells induced in the pancreatic tail following DAPT treatment in control and morphant embryos. Graphed are the mean and SD for (n) number of embryos. P values were determined using one-way analysis of variance (ANOVA) with Bonferroni's post test, ***P < 0.001.