<i>Neurog3</i> misexpression unravels mouse pancreatic ductal cell plasticity

<div><p>In the context of type 1 diabetes research and the development of insulin-producing β-cell replacement strategies, whether pancreatic ductal cells retain their developmental capability to adopt an endocrine cell identity remains debated, most likely due to the diversity of models employed to induce pancreatic regeneration. In this work, rather than injuring the pancreas, we developed a mouse model allowing the inducible misexpression of the proendocrine gene <i>Neurog3</i> in ductal cells <i>in vivo</i>. These animals developed a progressive islet hypertrophy attributed to a proportional increase in all endocrine cell populations. Lineage tracing experiments indicated a continuous neo-generation of endocrine cells exhibiting a ductal ontogeny. Interestingly, the resulting supplementary β-like cells were found to be functional. Based on these findings, we suggest that ductal cells could represent a renewable source of new β-like cells and that strategies aiming at controlling the expression of <i>Neurog3</i>, or of its molecular targets/co-factors, may pave new avenues for the improved treatments of diabetes.</p></div

Lineage tracing experiments unravel the conversion of ductal cells into endocrine cells upon the sole <i>Neurog3</i> misexpression.

<p>(<b>A</b>) Taking advantage of the β-galactosidase tracer, we monitored the fate of the ductal cells ectopically expressing <i>Neurog3</i>. X-Gal staining reveals β-galactosidase-positive cells (previously ductal cells) within the islet of Langerhans (outlined with red lines) of Tam-treated HNFN3OE mice. (<b>B</b>) Quantitative RT-PCR analyses confirm the presence of <i>β-galactosidase</i> mRNA in the transcriptome of islets isolated from Tam-treated animals (n = 6 animals for each condition). Statistics were performed using the Mann-Whitney test (<b>C-D</b>) Immunohistochemical analyses combining β-galactosidase and insulin detection. While control pancreata are negative for β-galactosidase (<b>C</b>), their Tam-treated counterparts display cells positive for both insulin and β-galactosidase (<b>D</b>), indicating duct-to-endocrine cell conversion. Note that the apparent staining in the exocrine tissue is artefactual and caused by the antibody used. (<b>E-F</b>) Accordingly, several glucagon⁺ (<b>E</b>) or somatostatin⁺ (<b>F</b>) cells are also found labeled with the ductal cell tracer, β-galactosidase.</p

Analysis of the <i>Neurog3</i> misexpression efficiency in HNFN3OE mice following short-term tamoxifen induction.

<p>(<b>A-B</b>) β-galactosidase activity assessment in the pancreata of HNFN3OE animals treated with vehicle (<b>A</b>) or Tam (<b>B</b>) for 2 weeks. A clear activity is noted solely in ductal cells. (<b>C</b>) Quantitative analysis of <i>Neurog3</i> transcript levels by qPCR (n = 6 animals for each condition) outlining a 2.5-fold increase in the pancreata of Tam-treated HNFN3OE animals compared to controls. Statistics were performed using the Mann-Whitney test (<b>D-E</b>) By means of immunohistochemical analyses using antibodies raised against Neurog3, Neurog3-expressing cells are detected within the ductal epithelium of HNFN3OE animals treated with Tam for only 2 weeks (<b>E</b>), whereas Neurog3⁺ cells cannot be detected in their vehicle-treated counterparts (<b>D</b>). For clarity, when required, the ductal lumen is outlined with yellow lines and islets with red lines.</p

Phenotypical analyses of islet cells from Tam-treated HNFN3OE pancreata.

<p>Representative pictures of immunohistochemical analyses performed on pancreas sections from untreated (<b>A, C, E, G, I, K</b>) and age-matched Tam-treated HNFN3OE mice (<b>B, D, F, H, J, L</b>) using the indicated antibody combinations. All insulin⁺ cells (preexisting and neogenerated) express the <i>bona fide</i> β-cell markers Nkx6.1 (<b>A-B</b>), NeuroD1 (<b>C-D</b>), Pdx1 (<b>E-F</b>), Rfx6 (<b>G-H</b>), Glut2 (<b>I-J</b>), and PC1/3 (<b>K-L</b>).</p

<i>Neurog3</i> misexpression in insulin⁺ cells does not alter β-cell function.

<p>(<b>A</b>) A combination of immunohistochemical detection and Salmon-Gal staining was used to assess β-galactosidase activity in InsCre::Neurog3OE pancreata. 80% of insulin-producing cells appeared positive for β-galactosidase. For clarity, Salmon-Gal staining was converted to a green labeling in the photographs. (<b>B</b>) Intraperitoneal glucose tolerance tests of adult InsCre::Neurog3OE animals (5-month old, n = 5) do not show any difference in glucose response comparing transgenic animals and their age-matched transgene-negative littermates (n = 4), indicating that maintained <i>Neurog3</i> misexpression in β-cells does not impair their ability to secrete insulin in response to glucose stimulation. Statistics were performed using the Mann-Whitney test.</p

<i>Neurog3</i> misexpression is maintained in newly-formed endocrine cells in adult Tam-treated HNFN3OE pancreata.

<p>The observation of islets in HNFN3OE animals treated with Tam for 10 months demonstrates that a clear majority of endocrine cells misexpress Neurog3 (<b>A-B</b>), this result being confirmed by qPCR analyses (<b>C</b>, n = 6 for each condition) Statistics were performed using the Mann-Whitney test.</p

The inactivation of <i>Pax4</i> in <i>Arx</i>-deficient glucagon-expressing cells does not impact β-like cell neogenesis.

<p>Representative photographs of immunohistochemical analyses performed on 6 month-old Glu-ArxKO/Pax4KO pancreata using the indicated antibody combinations. A clear loss of <i>Arx</i> was evidenced in Glu-ArxKO/Pax4KO glucagon⁺ cells (<b>A</b>), such cells not ectopically expressing <i>Pax4</i> (as seen in <b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003934#pgen.1003934.s002" target="_blank">Figures S2</a>-<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003934#pgen.1003934.s003" target="_blank">S3</a></b>). Interestingly, a number of insulin⁺ cells appeared Pax4⁻, such cells most likely deriving from Arx^−/Y Pax4^−/− glucagon⁺ cells (<b>A–C</b>). As noted in Glu-ArxKO and Dox+ IndGlu-ArxKO pancreata, an increase in islet size compared to controls (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003934#pgen-1003934-g001" target="_blank"><b>Figure 1</b></a>), caused by an insulin⁺ cell hyperplasia, was observed in Glu-ArxKO/Pax4KO pancreata (<b>A–I</b>). Non-β-cell endocrine hormone-expressing cells displayed a preferential localization at poles of the islets, adjacent to neighboring ducts (<b>E–I</b>), reminiscent of the phenotype of the sole inactivation of <i>Arx</i> in glucagon⁺ cells. (Each photograph is representative of at least three independent animals).</p

Quantitative analyses upon the dual inactivation of <i>Arx</i> and <i>Pax4</i> in glucagon-producing cells.

<p>(<b>A–E</b>) Quantitative comparison of the number of insulin- (<b>A</b>), glucagon- (<b>B</b>) and somatostatin- (<b>C</b>) expressing cells between 4 month-old Glu-ArxKO/Pax4KO animals and age-/sex-matched WT controls. A significant increase in the numbers of insulin- and somatostatin-expressing cells was observed in Glu-ArxKO/Pax4KO animals, whilst no significant variation in glucagon⁺ cells was noted. Interestingly, both islet count (<b>D</b>) and size (<b>E</b>) were significantly increased in these animals compared to their WT counterparts, suggesting a process of islet neogenesis in addition to an increased insulin⁺ cell mass. (<b>F</b>) 4 month-old Glu-ArxKO/Pax4KO animals (and age/sex-matched WT controls) were challenged with glucose. Double-mutant animals displayed an increased capacity to counteract the glucose bolus with a lower peak in glycemia, suggestive of a functional increased β-cell mass. n≥3 in all experiments, ** p<0.01, * p<0.05 using ANOVA. (<b>G</b>) Schematic detailing the consequences of <i>Arx</i> (and <i>Pax4</i>) inactivation triggered in α-cells. Following the inactivation of <i>Arx</i> (and <i>Pax4</i>), α-cells are converted into β-like cells (1–2). The resulting shortage in glucagon (and/or putative additional signals - 3) promotes the proliferation of duct-lining cells, some of which re-express the developmental factor Ngn3. Our results indicate that such Ngn3⁺ cells adopt an endocrine cell identity, the glucagon⁺ cell fate being clearly favored (4). Whether neo-generated somatostatin⁺ cells contribute to the supplementary β-like cell mass remains to be determined (“?”). Similarly, one could assume that Ngn3⁺ cells could directly give rise to β-like cells, but additional experiments and mouse lines would be required to address this question (“?”). Subsequently, neo-formed glucagon⁺ cells are, yet again, turned into β-like cells upon the inactivation of <i>Arx</i> (and <i>Pax4</i>) (5). Such repeated cycles of neogenesis/double conversion (3 to 5) eventually result in an islet hypertrophy caused by a β-like cell hyperplasia (6).</p

The conditional inactivation of <i>Arx</i> in glucagon-producing cells results in islet hypertrophy accompanied by islet neogenesis.

<p>(<b>A–I</b>) Representative photographs of insulin immunohistochemical analyses performed on pancreata of 3–9 month-old Glu-ArxKO (<b>D–F</b>) and on age-/sex-matched controls (<b>A–C</b>)). Note the dramatic augmentation in islet size and the substantial increase in islet numbers in mutant animals. Similar alterations were also noted in IndGlu-ArxKO animals treated with Dox for 3 (<b>G</b>) and 10 (<b>H–I</b>) months. (Each photograph is representative of at least three independent animals). (<b>J–K</b>) These results were confirmed by the means of optical projection tomography whereby 5 month-old WT controls (<b>J</b>) and age-matched Glu-ArxKO animals (<b>K</b>) were assayed for insulin expression (also see <b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003934#pgen.1003934.s006" target="_blank">Movie S1</a></b>). Quantification of insulin⁺ cell volume revealed a significant 2.71-fold increase in Glu-ArxKO pancreata compared to controls.</p

Localized cellular proliferation in the pancreatic ductal lining upon α-cell-specific <i>Arx</i> loss.

<p>(<b>A–I</b>) A 10-day BrdU pulse-chase was performed in 3 month-old IndGlu-ArxKO animals subjected to a 2 month-long Dox treatment. An increase in the number of proliferating cells was thereby noted, most of these being detected in the lining of the duct close to the non-β-cell cluster (<b>B–C, E–F, H–I</b> compared to <b>A, D, G</b>). Of note, the ductal epithelium was found thickened in mutant pancreata, suggesting an increased ductal cell number (<b>C, F, I</b>). (<b>J–L</b>) The investigation of cell proliferation in 13 month-old Glu-ArxKO animals also revealed increased numbers of proliferating cells (<b>K–L</b>) compared to controls (<b>J</b>), as assayed by the expression of the proliferation marker KI67. These were again mainly found in the ductal lining adjacent to the islets (<b>K–L</b>). For the purpose of clarity, islets are outlined in dashed yellow lines in selected photographs.</p