Fig 1.
E17.5 tubular epithelium of bigenic pancreas expresses neither Pdx1Hi nor MafAMyc.
E17.5 bigenic and control pancreases were stained for E-cadherin (red, ABEF), Pdx1 (green, ABCD) and Myc (green, EF). Pdx1Hi, Pdx1Lo and Pdx1- cells are marked by arrows, triangles and arrowheads, respectively. In controls, tubular epithelial cells have Pdx1Lo expression while Pdx1Hi expression was only seen in endocrine cells. In bigenic pancreas only occasional Pdx1Hi cells and MafAMyc cells were seen indicating that Pdx1Lo expression was not sufficient for Pdx1tTA-dependent induction of MafAMyc expression (green, EF). DAPI (blue). Bar: 20 μm.
Fig 2.
Normal-appearing acinar and tubular epithelial cells in E17.5 bigenic pancreas.
H& E stained pancreatic sections from the E17.5 control and bigenic Pdx1tTA/+;tetOMafA mice (AB) show tubular epithelium and surrounding endocrine area (dashed line) with a lack of endocrine cells in the bigenic pancreas. Amylase (green, CD), insulin (red C-J), E-cadherin (green, GH), β-catenin (green, IJ), and DBA lectin (green EF) staining show a reduction in insulin+ cells in the bigenic pancreas but normal appearance of acinar and tubular epithelium. DAPI (blue). Bars: 50 μm.
Fig 3.
Endocrine differentiation of pancreas is impaired in Pdx1tTA/+;tetOMafA pancreas.
At E17.5 Isl1 (green, AB), Pax6 (green, CD) and Hb9 (green, EF), transcription factors implicated in endocrine differentiation and maturation, have severely reduced expression in Pdx1tTA/+;tetOMafA pancreas (ACE) compared to tetOMafA pancreas (BDF). This finding is consistent with their reduced number of insulin+ cells (red, A-F), and suggests that misexpression of the MafA transgene inhibits the entire endocrine differentiation program. DAPI (blue). Bar: 50 μm.
Fig 4.
Pancreas of Pdx1tTA/+;tetOMafA embryos show dramatic growth from E17.5 to P0.
Abdominal viscera were dissected from indicated genotypes at E17.5 and P0 (A-F). Continuous expression of MafAMyc transgene in Pdx1+ cells until E17.5 resulted in dramatic reduction in pancreatic mass (A). However, in P0 bigenic neonates there is significant growth of pancreatic tissue along the duct structures compared to E17.5 bigenic embryos as shown in B and D, which show higher magnification of the boxed areas in A and C.
Fig 5.
P1 transgenic pancreases contain significantly larger endocrine clusters compared to E17.5.
At P1 bigenic islets had relatively normal organization as compared to controls for expression of glucagon (green, AB, red, EF), somatostatin (green, CD), pancreatic polypeptide (green, EF), ghrelin (green, GH) and insulin (red, A-D, G-J). DBA-expressing branching ducts were observed in both neonates (green, IJ). 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 (KL). Total pancreatic area in Pdx1tTA/+;tetOMafA also shows enhanced growth (M). Blood glucose levels from each group of neonates (n = 8 for each group) show that bigenic neonates were significantly hyperglycemic at P1 (N). Mean ± s.e.m.
Fig 6.
Proliferation of DBA+ epithelial tubules and not insulin+ cells contributes to increased number of insulin+ cells in Pdx1tTA/+;tetOMafA pancreas at E19.5 and later.
Bigenic pancreas at both E17.5 and E19.5 showed proliferation (Ki67, red) of DBA+ (green, A-D) and insulin+ (green, E-H) 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 (I) in bigenic but not control pancreas whereas the proportion of insulin+ cells that were Ki67+ (J) 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.
Fig 7.
Tubular epithelial cells of bigenic pancreas express Sox9 and GLUT2 at E17.5.
At E17.5 both control and bigenic tubular epithelial cells express Sox9. Sox9 (green AB); DBA (green, CD); GLUT2 (green, EF); Insulin (red); DAPI (blue). The boxed areas in E and F are enlarged (GH: merged channels, IJ: 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.
Fig 8.
Retention of BrdU-labeled 1°MPC is not enhanced in E17.5 bigenic tubular epithelium.
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, AB) and Ki67 (red, CD) 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.
Fig 9.
Quantification of Neurog3 expressing cells indicates compensatory increase in endocrine differentiation in bigenic mice.
E19.5 pancreas of bigenic mice shows an increase in DBA+ cells but Neurog3+ cells (ACE) were comparable to controls (BDF). This equal Neurog3+ cell numbers is in contrast to the rare Neurog3+ cells seen in bigenics at E15.5 and E17.5 [17]. Quantification of Neurog3-expressing cells shows that the number of Neurog3-expressing cells normalized the pancreatic area for each section (G) 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.
Fig 10.
A schematic model depicting differential regulation of endocrine progenitors in control and bigenic pancreas.
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 [17] 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 (1), some of the 1°MPC progenitors (P) 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 (2), 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 Fig 8 support the second scenario.