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

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

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

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

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

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

A schematic model depicting differential regulation of endocrine progenitors in control and bigenic pancreas.

<p>In control wild type (WT) pancreas (upper panel) Pdx1⁺ 1°MPC give rise to endocrine (Neurog3, N) progenitors that preferentially differentiate into α-cells during earlier stages of development and into β-cells at later. In lower panels the expression of MafA in 1°MPC prevents their expansion and differentiation [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142286#pone.0142286.ref017" target="_blank">17</a>] with few Neurog3⁺ or hormone⁺ cells at E17.5. However after release of this repression at E17.5, the hormone ⁺ cells increased by P1 even though there was no enhanced proliferation of the hormone⁺ cells at E17.5 or E19.5 as compared to controls but there were comparable numbers of Neurog3⁺ cells. Two scenarios are possible. In the first <b>(1)</b>, some of the 1°MPC progenitors (<b>P</b>) were retained in the tubular epithelium (D) and can resume differentiation and give rise to both α- and β-cells after E17.5 upon the release of MafA repression. In second <b>(2)</b>, 1°MPC are not retained in the bigenic tubular epithelium, but the E17.5 tubular epithelium itself (D) has the potential to differentiate into endocrine progenitors with competency to give rise to both α- and β-cells. Our results as presented in the <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142286#pone.0142286.g008" target="_blank">Fig 8</a></b> support the second scenario.</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

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

MafA Is Required for Postnatal Proliferation of Pancreatic β-Cells

<div><p>The postnatal proliferation and maturation of insulin-secreting pancreatic β-cells are critical for glucose metabolism and disease development in adults. Elucidation of the molecular mechanisms underlying these events will be beneficial to direct the differentiation of stem cells into functional β-cells. Maturation of β-cells is accompanied by increased expression of MafA, an insulin gene transcription factor. Transcriptome analysis of <i>MafA</i> knockout islets revealed MafA is required for the expression of several molecules critical for β-cell function, including <i>Glut2</i>, <i>ZnT8</i>, <i>Granuphilin</i>, <i>Vdr</i>, <i>Pcsk1</i> and <i>Urocortin 3</i>, as well as <i>Prolactin receptor</i> (<i>Prlr</i>) and its downstream target <i>Cyclin D2</i> (<i>Ccnd2</i>). Inhibition of <i>MafA</i> expression in mouse islets or β-cell lines resulted in reduced expression of Prlr and Ccnd2, and MafA transactivated the <i>Prlr</i> promoter. Stimulation of β-cells by prolactin resulted in the phosphorylation and translocation of Stat5B and an increased nuclear pool of Ccnd2 via Prlr and Jak2. Consistent with these results, the loss of MafA resulted in impaired proliferation of β-cells at 4 weeks of age. These results suggest that MafA regulates the postnatal proliferation of β-cells via prolactin signaling.</p></div