FIGURE SUMMARY
Title

Suppression of Ptf1a activity induces acinar-to-endocrine conversion

Authors
Hesselson, D., Anderson, R.M., and Stainier, D.Y.
Source
Full text @ Curr. Biol.

ptf1a Expression Becomes Restricted to Differentiated Acinar Cells during Early Larval Development Embryos express Tg(ptf1a:eGFP) in the pancreatic domain. Expression of pancreatic markers was analyzed by immunofluorescence. Scale bars represent 20 μm. (A–B2) Confocal projections of pancreatic tissue at 36 hours postfertilization (hpf). (C–H2) Confocal sections of pancreatic tissue at 60 (C–E2) and 84 hpf (F–H2). Dotted lines delineate Tg(ptf1a:eGFP)-expressing tissue. (A–B2) Tg(ptf1a:eGFP)-expressing cells initially coexpress Nkx6.1 and Prox1. (C–D2) By 60 hpf, Nkx6.1 (C and C2, arrows) and Prox1 (D and D2) become excluded from the Tg(ptf1a:eGFP) expression domain. (E–F2) Coexpression of Tg(ptf1a:eGFP) with the acinar cell marker Elastase (Ela) is detected by 84 hpf. Elastase-positive granules are detected on the apical surface of acinar cells (F2, arrowhead), adjacent to Tg(ptf1a:eGFP)-negative intrapancreatic cells (F, arrows). (G–H2) The duct marker Nkx6.1 (G and G2) and the endocrine marker Isl1 (H and H2) appear to be restricted to Tg(ptf1a:eGFP)-negative ductal and islet cells respectively by 84 hpf. Isl1 is also expressed in the pancreatic mesenchyme (H and H2, arrows).

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.

Genetic Lineage Tracing of Acinar to insulin+ Cell Fate Conversion (A) Tg(ela3l:cre);Tg(insulin:loxP:mCherrySTOP:loxP:H2B-GFP) lineage tracing. Expression of Cre in acinar cells excises the mCherry-STOP cassette and permits expression of H2B-GFP in cells that activate the insulin promoter. (B–E3) Confocal sections through pancreata marked with fluorescent transgenes and TO-PRO to mark DNA. Scale bars represent 20 μm. (B–B3) Tg(ela3l:cre);Tg(insulin:loxP:mCherrySTOP:loxP:H2B-GFP) did not mark any pancreatic cells in wild-type animals. (C–D3) elastase:cre-Ptf1aEN expression induced scattered H2B-GFP-positive cells throughout the pancreatic domain (arrows) (C–C3), which were not induced in Ptf1aEN-negative controls (D–D3). (E–E3) elastase:cre-Ptf1aEN expression induced isolated H2B-GFP-positive cells at 14 dpf (arrow). See also Figure S2.

The hsp70l Regulatory Sequence Induces Transgene Expression throughout the Pancreas (Related to Figure 3)

Confocal sections through Tg(ptf1a:eGFP); Tg(hsp70l:loxP:mCherrySTOP:loxP:H2B-GFP) (A-A′′′) and control (B-B′′′) pancreata marked with TOPRO at 72 hpf. Scale bars represent 20 m. Embryos were heat shocked at 60 hpf.

(A-A′′′) All eGFP expressing cells (A′′) co-expressed mCherry (outlined tissue in A′′′).

(B-B′′′) Nontransgenic control embryos did not exhibit red fluorescence in the pancreatic domain (outlined tissue in B′′′).

elastase:cre-Ptf1aEN Expression Does Not Induce an Inflammatory Response (Related to Figure 4)

(A and B) Confocal sections through Tg(mpx:GFP) expressing pancreata marked with TOPRO at 2 hr post heat shock. Pancreatic granulocyte marked with arrow. Scale bars represent 20 μm.

(C) Quantification of pancreatic expressing granulocytes per animal after heat-shock treatment (mean ± SD, n = 10 animals for each time point).

Acknowledgments
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