E17.5 tubular epithelium of bigenic pancreas expresses neither Pdx1^Hi nor MafA^Myc.

<p>E17.5 bigenic and control pancreases were stained for E-cadherin (red, <b>ABEF</b>), Pdx1 (green, <b>ABCD</b>) and Myc (green, <b>EF</b>). Pdx1^Hi, Pdx1^Lo and Pdx1^- cells are marked by arrows, triangles and arrowheads, respectively. In controls, tubular epithelial cells have Pdx1^Lo expression while Pdx1^Hi expression was only seen in endocrine cells. In bigenic pancreas only occasional Pdx1^Hi cells and MafA^Myc cells were seen indicating that Pdx1^Lo expression was not sufficient for <i>Pdx1</i>^<i>tTA</i>-dependent induction of MafA^Myc expression (green, <b>EF</b>). DAPI (blue). Bar: 20 μm.</p

Endocrine differentiation of pancreas is impaired in <i>Pdx1</i>^<i>tTA/+</i><i>;tetO</i>^<i>MafA</i> pancreas.

<p>At E17.5 Isl1 (green, <b>AB</b>), Pax6 (green, <b>CD</b>) and Hb9 (green, <b>EF</b>), transcription factors implicated in endocrine differentiation and maturation, have severely reduced expression in <i>Pdx1</i>^<i>tTA/+</i><i>;tetO</i>^<i>MafA</i> pancreas (<b>ACE</b>) compared to <i>tetO</i>^<i>MafA</i> pancreas (<b>BDF</b>). This finding is consistent with their reduced number of insulin⁺ cells (red, <b>A-F</b>), and suggests that misexpression of the MafA transgene inhibits the entire endocrine differentiation program. DAPI (blue). Bar: 50 μm.</p

Normal-appearing acinar and tubular epithelial cells in E17.5 bigenic pancreas.

<p>H& E stained pancreatic sections from the E17.5 control and bigenic <i>Pdx1</i>^<i>tTA/+</i><i>;tetO</i>^<i>MafA</i> mice (<b>AB</b>) show tubular epithelium and surrounding endocrine area (dashed line) with a lack of endocrine cells in the bigenic pancreas. Amylase (green, <b>CD</b>), insulin (red <b>C-J</b>), E-cadherin (green, <b>GH</b>), β-catenin (green, <b>IJ</b>), and DBA lectin (green <b>EF</b>) staining show a reduction in insulin⁺ cells in the bigenic pancreas but normal appearance of acinar and tubular epithelium. DAPI (blue). Bars: 50 μm.</p

Quantification of Neurog3 expressing cells indicates compensatory increase in endocrine differentiation in bigenic mice.

<p>E19.5 pancreas of bigenic mice shows an increase in DBA⁺ cells but Neurog3⁺ cells (<b>ACE</b>) were comparable to controls (<b>BDF</b>). This equal Neurog3⁺ cell numbers is in contrast to the rare Neurog3⁺ cells seen in bigenics at E15.5 and E17.5 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142286#pone.0142286.ref017" target="_blank">17</a>]. Quantification of Neurog3-expressing cells shows that the number of Neurog3-expressing cells normalized the pancreatic area for each section (<b>G</b>) are comparable in both bigenic and control E19.5 pancreas but showed a trend to being reduced in bigenics when normalized to the DBA⁺ area. DAPI (blue). Mean ± s.e.m. n = 3 Bar: 100 μm.</p

Proliferation of DBA⁺ epithelial tubules and not insulin⁺ cells contributes to increased number of insulin⁺ cells in <i>Pdx1</i>^<i>tTA/+</i><i>;tetO</i>^<i>MafA</i> pancreas at E19.5 and later.

<p>Bigenic pancreas at both E17.5 and E19.5 showed proliferation (Ki67, red) of DBA⁺ (green, <b>A-D</b>) and insulin⁺ (green, <b>E-H</b>) cells. Quantification of the proportion of DBA⁺ cells or insulin⁺ cells that were Ki67⁺ showed significantly increased proliferation of DBA⁺ cells between E17.5 and E19.5 (<b>I</b>) in bigenic but not control pancreas whereas the proportion of insulin⁺ cells that were Ki67⁺ (<b>J</b>) at E19.5 compared to that at E17.5 increased in controls but not bigenic. The bigenic had significantly more replicating DBA⁺ cells at E19.5 than controls. N = 3 Mean ± s.e.m. Bar: 20 μm.</p

Retention of BrdU-labeled 1°MPC is not enhanced in E17.5 bigenic tubular epithelium.

<p>Images of E17.5 pancreases from bigenic and control pups from pregnant mothers receiving BrdU injections on both gestational days10.5 and 11.5, stained for BrdU (red, <b>AB</b>) and Ki67 (red, <b>CD</b>) with DBA (green). At this stage, only a few BrdU⁺ label-retaining cells remain in either E17.5 bigenic and control pancreas but many cells, including DBA⁺ tubular epithelium, are proliferating.</p

P1 transgenic pancreases contain significantly larger endocrine clusters compared to E17.5.

<p>At P1 bigenic islets had relatively normal organization as compared to controls for expression of glucagon (green, <b>AB</b>, red, <b>EF</b>), somatostatin (green, <b>CD</b>), pancreatic polypeptide (green, <b>EF</b>), ghrelin (green, <b>GH</b>) and insulin (red, <b>A-D</b>, <b>G-J</b>). DBA-expressing branching ducts were observed in both neonates (green, <b>IJ</b>). DAPI (blue). Bar: 50 μm. Quantification of these data show increases in the insulin⁺ and glucagon⁺ area of bigenic pancreas from E17.5 to P1 compared to controls (<b>KL</b>). Total pancreatic area in <i>Pdx1</i>^<i>tTA/+</i><i>;tetO</i>^<i>MafA</i> also shows enhanced growth (<b>M</b>). Blood glucose levels from each group of neonates (n = 8 for each group) show that bigenic neonates were significantly hyperglycemic at P1 (<b>N</b>). Mean ± s.e.m.</p

Tubular epithelial cells of bigenic pancreas express Sox9 and GLUT2 at E17.5.

<p>At E17.5 both control and bigenic tubular epithelial cells express Sox9. Sox9 (green <b>AB</b>); DBA (green, <b>CD</b>); GLUT2 (green, <b>EF</b>); Insulin (red); DAPI (blue). The boxed areas in E and F are enlarged (<b>GH</b>: merged channels, <b>IJ</b>: green channel showing GLUT2 expression). Higher GLUT2 staining intensity is seen in the bigenic tubular epithelial cells than in the controls. In bigenic pancreas GLUT2 staining intensity is comparable in insulin⁺ (marked by arrows) and insulin^- tubular epithelial cells (marked by arrowheads) whereas in control pancreas GLUT2 staining intensity is reduced in tubular epithelial cells than islets. Bar: 20 μm.</p

TWEAK treatment promotes proliferation of pancreatic duct and duct adjacent cells through its receptor Fn14.

<p>Pancreatic tissue of normal adult mice stained for Ki-67 (black) on day 3 after injection of (A) control Ig or (B) Fc-TWEAK, and costained with Ki-67 (red) and ductal epithelial marker CK (CK) (green) on day 4 after (C) control Ig or (D) Fc-TWEAK acute treatment. Scale bar  = 10 µm. (E–F) Quantification of the % Ki-67⁺ pancreatic (E) duct and (F) duct adjacent cells in mice treated with control Ig or Fc-TWEAK twice weekly. Data are shown as mean±SEM (n = 4). * p<0.05 for Fc-TWEAK vs control Ig treatment. (G–H) Quantification of the % Ki-67⁺ (G) duct and (H) duct adjacent cells in normal adult WT or Fn14 KO mice treated with control Ig or Fc-TWEAK twice per week. Data are shown as mean±SEM (n = 4) for percentage of Ki-67+ duct cells (E&G) or Ki-67+ duct adjacent cells per total duct cells (F&H); * p<0.05 for Fc-TWEAK treatment vs control Ig.</p

Chronic TWEAK treatment mimics the response to the partial pancreatectomy.

<p>Ductal structures with characteristics of regenerative foci were identified by H&E staining (A) at day 18 after TWEAK treatment twice weekly in all 4 treated animals. Inset of (A) is shown on serial sections stained with Ki-67 (B), CK (C), insulin (D) and glucagon (E). (F–H) Serial sections of area from another representative animal were stained with H&E (F), CD3 (G) F4/80 (H); isotype control (I) and Fn14 (J). The islets were labeled as “i”. Scale bar  = 50 µm in all panels.</p