Assessment of glucose handling in control and CPTD6 mice.

<p>(<b>A</b>) Urine glucose levels were tested weekly using Diastix (Bayer) test sticks from week 0 (before first injection) to termination of the experiment (week 5). CPTD6 mice presented with glucose in their urine when tested 3 weeks post first injection. None of the control animals (tamoxifen- nor oil-injected controls) showed glucose in their urine at any time. (<b>B</b>) Fasting blood glucose levels were normal in all three experimental groups in the week prior to the tamoxifen/oil injections (white bars). When tested again in the final week of the experiment (start of week 5) CPTD6 mice presented with hyperglycaemia (black bars) (<b>C</b>). A glucose tolerance test performed in the week before injection revealed slight glucose intolerance in CPTD6 mice compared to the control animals. (<b>D</b>) Glucose intolerance of the CPTD6 mice was exacerbated post injection. Data are shown as mean ± SEM.</p

Expression analysis of key transcription factors.

<p>Immunohistochemistry was performed in control (<b>A</b>, <b>C</b>, <b>E</b>, <b>G</b>, <b>I, K</b>) and CPTD6 (<b>B</b>, <b>D</b>, <b>F</b>, <b>H</b>, <b>J, L</b>) mice. (<b>A</b>, <b>B</b>) Nuclear expression of the pancreatic islet marker Islet 1 (Isl1) is retained in CPTD6 mice, showing that the islet cells are present. (<b>C,D</b>) Expression of Nkx2.2 is also unaffected in CPTD6 mice. (<b>E, F</b>) In contrast expression of Nkx6.1 is reduced in Pax6 deficient cells. (<b>G, H</b>) The nuclear expression of the transcription factor Pdx1 is lost in CPTD6 mice. Images are representative of at least four animals from each group. Scale bars, 50 µm.</p

Upregulation of Ghrelin expression in CPTD6 islets.

<p>Immunohistochemistry for ghrelin (<b>B, </b><b>E, H, K, N, Q</b>) was performed in combination with insulin (<b>A</b>, <b>D</b>), glucagon (<b>G, J</b>), and somatostatin (<b>M, P</b>). Merged images with DAPI nuclear counter-staining are shown (<b>C, F, I, L, O, R</b>) on pancreata from control (<b>A</b>, <b>B, C, G, H, I, M, N, O</b>) and CPTD6 (<b>D, E, F, J, K, L, P, Q, R</b>) mice. Levels of insulin and glucagon expression are markedly reduced or lost in the majority of islet cells of CPTD6 mice in comparison with the controls, while the number of somatostatin positive cells appears more moderately reduced. Very few, if any, ghrelin positive cells can be found in the islets of control adult mice (<b>B, C</b>, <b>H, I, N, O</b>)<b>.</b> In contrast, strong staining is observed in many cells of the CPTD6 pancreata, located predominantly around the periphery of the islets. Images are representative of at least four animals from each group. Scale bars, 50 µm. (S, T, U, V). Quantification of the relative numbers of hormone expressing cells in the islets of Langerhans of control and CPTD6 animals shown as the percentage of immunofluorescence positive cells per total number of DAPI positive cells. (S) Insulin producing cells (control 72% >19% CPTD6), (T) Glucagon producing cells (control 11.4% >5.3% CPTD6) (U) Somatostatin producing cells (control 11.6% >9.5% CPTD6), and (V) Ghrelin producing cells (control 0.6% >20.6% CPTD6).</p

Expression analysis of pancreatic endocrine hormones.

<p>Images show immuno-stainings for insulin (<b>A</b>, <b>B</b>), glucagon (<b>C</b>, <b>D</b>), and somatostatin (<b>E, F</b>) in sections through the pancreas of control (<b>A</b>, <b>C</b>, <b>E</b>) and CPTD6 (<b>B</b>, <b>D</b>, <b>F</b>) mice sacrificed 5 weeks post-injections. Insulin expression is almost completely abolished in CPTD6 islets compared with control islets (<b>A, B</b>)<b>.</b> The number of cells staining positive for glucagon is also decreased in the CPTD6 islets compared to controls (<b>C, D</b>). Somatostatin expression is more moderately reduced in the CPTD6 islets with most CPTD6 islets showing fewer positive cells compared to the controls. (<b>E, F</b>). Images are representative of at least four animals from each group. Scale bar, 50 µm.</p

Experimental procedure used to investigate the role of Pax6 in the adult mouse pancreas.

<p>(<b>A</b>) Timeline of the experiment. Mice were bred to obtain the various experimental and control genotypes. After a pre-experimental period of 6 months (indicated by the period to the left of timepoint = 0 weeks) all mice received daily injections, with tamoxifen or oil only, on 5 consecutive days during week 1. Blood glucose level and glucose tolerance measurements were carried out in the week prior to the injections and in the final week (week 5). All mice were sacrificed at the end of week 5 and tissues were fixed for immunohistochemistry (<b>B</b>) Schematic representation of the combinations of genotypes and treatments (oil or tamoxifen injection) of all animals used in the study. Animals were sorted into control (oil only control or Tamoxifen control) or CPTD6 (CreER™-floxed Pax6-Tamoxifen-Diabetic) groups. (<b>C</b>) Graph showing male mouse body weights from 4 weeks pre-treatment until 5 weeks post-treatment, with a time-line of the procedure shown at the top. All animals exhibited healthy body weight prior to treatment. During the 5 day period of daily injections all animals lost some weight, but recovered in the week following the injections. While control animals continued to gain weight and were back to pre-treatment body weight at termination, CPTD6 animals began to lose weight again from the second week post-treatment onwards until termination of the experiment. Data are shown as mean ± SEM (Standard error of the mean). (<b>D</b>) Immunohistochemistry for Pax6 was used to assess the efficiency of the Cre-mediated deletion of <i>Pax6,</i> by staining for the presence or absence of Pax6. Control animals show nuclear Pax6 immuno-reactivity in the pancreatic islet cells. The CPTD6 islets show absence of Pax6 immuno staining in the large majority of cells, indicating successful Cre-mediated inactivation of the Pax6 gene. Scale bar, 50 µm.</p

Glucose sensing, hormone processing and the exocrine compartment.

<p>(<b>A</b>, <b>B</b>) Expression of the glucose transporter Glut2 (Slc2a2), localised at the endocrine cell surface, is lost in CPTD6 mice. (<b>C, D</b>) Cytoplasmic expression of the prohormone processing factor PC1/3 (Pcsk1) is absent in CPTD6 mice. (<b>E</b>, <b>F</b>). Expression of the processing factor PC2 (Pcsk2) and (<b>G, H</b>) the exocrine enzyme amylase are not affected.</p

The parapineal and habenular defects are coupled in <i>sox2</i> morphants.

<p>(<b>A–C</b>) In control embryos, the left-sided parapineal projects towards the left habenula. As a result, the left habenula has denser neuropils than the right, as judged by phalloidin staining. (<b>D–F</b>) In <i>sox2</i> morphants with left-sided parapineal projections, the left habenula is larger than the right. (<b>G–I</b>) Morphants with right-sided parapineal organs display reverse habenular asymmetries, whereas (<b>J–L</b>) morphants with bilateral parapineal projections have symmetric habenulae. (<b>M</b>) The average volume of the left (blue bars) and right (red bars) habenular neuropils, as judged by the volume of phalloidin-positive areas within the habenulae. y-axis show volume in µm³. (<b>N</b>) Average asymmetry index in control (purple bar) and <i>sox2</i> morphants (orange bars). 3D reconstructions of confocal images at 4 dpf, arrows show parapineal projections and blue lines surround the habenular neuropils, error bars represent ± standard error, (<b>M</b>) * = p-value <0.05 and ** = p-value <0.001 (Wilcoxon test).</p

A model for pineal cell-fate determination.

<p>Cell-fate determination has two phases: a prepatterning phase, followed by a determination phase. Sox2 (Sox2 levels or Sox2 along with a partner protein (X)) is important during the prepatterning phase, where is inhibits cells from adopting a PhR fate. In contrast, the Notch and BMP pathways are important during the determination fate, where BMP induces the PhR fate and Notch inhibits the PN fate. A yet-to-be-identified modulator (Y) is responsible for inducing the PN fate.</p

Sox2 expression within the pineal anlage is downregulated with differentiation.

<p>(<b>A–D</b>) Sox2 expression overlaps with expression of <i>flh</i>, a marker for pineal precursors, at 8 ss. (<b>E–H</b>) As pineal cells start differentiating, Isl1 is upregulated whereas Sox2 is downregulated. Yellow arrows show cells that have both Isl1 and Sox2 expression at low levels. (<b>I–L</b>) Sox2 is absent from the fully differentiated pineal cells. Scale bars = 25 µm, optical sections from confocal microscopy, insets show a three times magnified view of the image, (<b>E,I,M</b>) GFP expression of <i>Tg(flh:GFP)</i> (<b>F,J,N</b>) immunofluorescence for Isl1, (<b>G,K,O</b>) immunofluorescence for Sox2, (<b>H,L,P</b>) merged images with <i>Tg(GFP:flh)</i> in green, Isl1 in blue and Sox2 in red, developmental stages are shown at the beginning of each row.</p

Sox2 inhibits the PhR cell fate.

<p>(<b>A–C</b>) <i>Tg(aanat2:GFP)</i> drives GFP expression in the pineal PhRs. (<b>B</b>) Isl1 labels the pineal cells. (<b>D–F</b>) Knockdown of <i>sox2</i> results in increased number of PhRs. (<b>G</b>) The average number of PhRs in control (purple bar) and <i>sox2</i> morphants (orange bar). (<b>H–J</b>) <i>Tg(elavl3:GFP)</i> drives GFP expression specifically in the PNs. (<b>K–M</b>) The knockdown of <i>sox2</i> does not affect the number of PNs. (<b>N</b>) The average number of PNs in controls (purple bar) and <i>sox2</i> morphants (orange bar) does not significantly differ. Confocal maximum projections of 28 hpf embryos, scale bars = 25 µm, error bars represent ± standard error, ** = p-value <0.001 (MWU test). See also <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087546#pone.0087546.s004" target="_blank">Figure S4</a></b>.</p