|
Fig. 3
Inhibition of Ptf1a Activity Induces Ectopic Endocrine Gene Expression (A) elastase:cre-Ptf1aEN, heat-inducible overexpression of Engrailed-Ptf1a in an elastase3l:Cre-restricted domain. Expression of Cre in acinar cells excises the EBFP2-STOP cassette and permits heat-inducible expression of Engrailed-Ptf1a. (B–K2) Confocal sections through pancreata marked with immunofluorescence (D–E2) or fluorescent transgenes (F–K2). Scale bars represent 20 μm. (B–E2) Tg(ela3l:cre);(hsp70l:loxP:mCherrySTOP:loxP:H2B-GFP) embryos were heat shocked at 60 hpf (B and B2) or 4.5 days postfertilization (dpf) (C–E2) and analyzed 24 hr after heat shock. (B and B2) Tg(ela3l:cre) did not mark pancreatic cells by 60 hpf (dotted outline). (C and C2) By 4.5 dpf, Tg(ela3l:cre) marked a large fraction of pancreatic cells (dotted outline). (D–E2) Cells that were marked by Tg(ela3l:cre) at 4.5 dpf coexpressed the acinar marker Elastase (D and D2, arrow) but did not coexpress the endocrine marker Insulin (E and E2) at 5.5 dpf. (F–I2) elastase:cre-Ptf1aEN (F, F2, H, H2, J, and J2) and Tg(ela3l:cre)-negative control embryos (G, G2, I, I2, K, and K2) were heat shocked at 4.5 dpf and analyzed at 5.5 (H–I2) or 6.5 dpf (F–G2 and J–K2). (F and F2) elastase:cre-Ptf1aEN induction caused downregulation of Tg(elastase:eGFP) expression in a subset of acinar cells (F2, inset). Some acinar cells that expressed low levels of Tg(elastase:eGFP) coexpressed Tg(insulin:dsRED) (F, inset). (G and G2) Tg(insulin:dsRED) expression was not detected in Tg(ela3l:cre)-negative controls. (H–I2) elastase:cre-Ptf1aEN expression induced expression of the endocrine progenitor marker Tg(pax6b:eGFP) in cells outside of the principal islet (H and H2, arrows). In Tg(ela3l:cre)-negative controls at this stage, Tg(pax6b:eGFP) was expressed in the principal islet and in endocrine cells that differentiated in the extrapancreatic duct (I and I2, arrowheads). (J–K2) elastase:cre-Ptf1aEN also induced Tg(gcga:GFP) expression in the exocrine compartment (J and J2), whereas Tg(gcga:GFP) expression was restricted to the principal islet in Tg(ela3l:cre)-negative controls (K and K2). See also Figure S1.