Suppression of Ptf1a activity induces acinar-to-endocrine conversion

Abstract

Pluripotent embryonic cells become progressively lineage restricted during development in a process that culminates in the differentiation of stable organ-specific cell types that perform specialized functions. Terminally differentiated pancreatic acinar cells do not have the innate capacity to contribute to the endocrine β cell lineage, which is destroyed in individuals with autoimmune diabetes. Some cell types can be reprogrammed using a single factor, whereas other cell types require continuous activity of transcriptional regulators to repress alternate cell fates. Thus, we hypothesized that a transcriptional network continuously maintains the pancreatic acinar cell fate. We found that postembryonic antagonism of Ptf1a, a master regulator of pancreatic development and acinar cell fate specification, induced the expression of endocrine genes including insulin in the exocrine compartment. Using a genetic lineage tracing approach, we show that the induced insulin+ cells are derived from acinar cells. Cellular reprogramming occurred under homeostatic conditions, suggesting that the pancreatic microenvironment is sufficient to promote endocrine differentiation. Thus, severe experimental manipulations may not be required to potentiate pancreatic transdifferentiation. These data indicate that targeted postembryonic disruption of the acinar cell fate can restore the developmental plasticity that is lost during development.

Figure 1. pt f1a 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 immuno-fluorescence. Scale bars = 20μm. (A–B′) Confocal projections of pancreatic tissue at 36 hpf. (C–H′) Confocal sections of pancreatic tissue at 60 (C–E′) and 84 hpf (F–H′). Dotted lines delineate Tg(ptf1a:eGFP) expressing tissue. (A–B′) Tg(ptf1a:eGFP) expressing cells initially co-express Nkx6.1 and Prox1. (C–D′) By 60 hpf, N kx6.1 (C,C′, arrow s) and Prox1 (D,D′) expression becomes excluded from the Tg(ptf1a:eGFP) expression domain. (E–F′) Co-expression 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 (F′, arrow head ), adjacent to Tg(ptf1a:eGFP) negative intra-pancreatic cells (F, arrow s). (G–H′) The duct marker Nkx6.1 (G,G′) and the endocrine marker Isl1 (H,H′) 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,H′, arrows).

Figure 2. Engrailed-Ptf1a exhibits dose-dependent antagonism of wild-type Ptf1a activity

(A) A stable cell line (293-3xRBPJ-Luc) was generated in which Luciferase was placed under the control of RBPJ binding sites. (B) Normalized luminescence of 293-3xRBPJ-Luc cells that were transfected with Ptf1a and / or Ptf1aEN (mean +/− SD). *p<0.05; **p<0.01.

Figure 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 perm its heat-inducible expression of Engrailed -Ptf1a. (B–K′) Confocal sections through pancreata marked with immuno-fluorescence (D–E′) or fluorescent transgenes (F–K′). Scale bars = 20μm. (B–E′) Tg(ela3l:cre); (hsp70l:loxP:mCherrySTOP:loxP:H2B-GFP) embryos were heat-shocked at 60 hpf (B,B′) or 4.5 dpf (C–E′) and analyzed 24 h post heat-shock. (F–I′) elastase:cre-Ptf1aEN (F,F′,H,H′,J,J′) and Tg(ela3l:cre) negative control embryos (G,G′,I,I′,K,K′) were heat-shocked at 4.5 dpf and analyzed at 5.5 dpf (H–I′) or 6.5 dpf (F–G′, J–K′). (B,B′) Tg(ela3l:cre) did not mark pancreatic cells by 60 hpf (dotted outline). (C,C′) By 4.5 dpf, Tg(ela3l:cre) marked a large fraction of pancreatic cells (dotted outline). Cells that were marked by Tg(ela3l:cre) at 4.5 dpf co-expressed the acinar marker Elastase (D,D′, arrow ) but never co-expressed the endocrine marker Insulin (E,E′) at 5.5 d pf. (F,F′) elastase:cre-Ptf1aEN induction caused down-regulation of Tg(elastase:eGFP) expression in a subset of acinar cells (F′, inset). Some acinar cells that expressed low levels of Tg(elastase:eGFP) co-expressed Tg(insulin:d sRED) (F, inset). (G,G′) Tg(insulin:d sRED) expression was neverdetected in Tg(ela3l:cre) negative controls. (H–I′) elastase:cre-Ptf1aEN expression induced expression of the endocrine progenitor marker Tg(pax6b:eGFP) in cells outside of the principal islet (arrow s, H,H′). 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 extra-pancreatic duct (arrow head s, I,I′). (J–K′) elastase:cre-Ptf1aEN also induced Tg(gcga:GFP) expression in the exocrine compartment (J,J′), whereas Tg(gcga:GFP) expression was restricted to the principal islet in Tg(ela3l:cre) negative controls (K,K′).

Figure 4. 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 perm its expression of H2B-GFP in cells that activate the insulin promoter. (B–E″) Confocal sections through pancreata marked with fluorescent transgenes and TOPRO to mark DNA. Scale bars = 20μm. (B–B″) Tg(ela3l:cre); Tg(insulin:loxP:mCherrySTOP:loxP:H2B-GFP) did not mark any pancreatic cells in wild-type animals. (C–C″) elastase:cre-Ptf1aEN expression induced scattered H2B-GFP positive cells throughout the pancreatic domain (arrows), which were not induced in Ptf1aEN negative controls (D–D″). (E–E″) elastase:cre-Ptf1aEN expression induced isolated H2B-GFP positive cells at 14 dpf (arrow).