Expression of transcription factors in <i>Arx-</i> and <i>Pax4</i>-deficient intestines.

<p>Real time PCR analyses in (A) <i>Arx-</i> and (B) <i>Pax4-</i> (n = 4) deficient (n = 5) and control (n = 5) small intestine and colon at 2 days <i>postpartum</i>. (A) <i>Pdx1</i> and <i>Foxa1</i>/<i>a2</i> expression are increased in Arx mutant colon and small intestine, respectively. (B) <i>Arx</i> is significantly upregulated in Pax4 mutants. Student's T-test *p<0.05, **p<0.01, ***p<0.001.</p

Model of enteroendocrine subtype specification during small intestine development: roles of Arx and Pax4.

<p>Gast-, GIP-, Nts-, Sct-, CCK- and GLP1-expressing cells arise from endocrine progenitors expressing Neurog3 then Pax4 and Arx. Upon Arx inactivation these progenitors are reallocated into Sst-expressing cells while the differentiation of Gast-, GIP-, Nts-, Sct-, CCK- and GLP1-expressing-cells is impaired. Sst- and Serotonin (5-HT)-expressing cells are generated from progenitors expressing Neurog3 then Pax4. Inactivation of Pax4 leads to the up-regulation of Arx and the differentiation of these progenitors into GLP1-expressing cells, while the differentiation of Sst-, Serotonin (5-HT)- Gast-, GIP- and Nts-expressing cells is impaired. Key transcription factors controlling intestinal cell destiny are also indicated.</p

Arx is expressed in early differentiating GLP1-, GIP-, CCK- and Gastrin-expressing cells in the adult small intestine.

<p>Co-immunostaining with Arx and intestinal hormones antibodies on sections of adult small intestine. Arx is strongly expressed in GLP1⁺, GIP⁺, CCK⁺, and selected Ghrl⁺ cells located in the crypts (B), but not in Sst⁺ or Serotonin⁺ (5-HT) cells. Arx expression level decreases in enteroendocrine cells in the villi (A). Scale bar 10 µm.</p

Hormone expression in <i>Pax4</i>-deficient intestine.

<p>(A) Real time RT-PCR analyses of various intestinal hormones mRNAs in <i>Pax4</i>-deficient and control small intestine and colon at 2 days <i>postpartum</i> (n = 4). <i>Gip</i>, <i>Nts</i>, <i>Gast</i>, <i>Sct</i> and <i>Tph1</i> mRNA levels decrease significantly in Pax4 mutant small intestine, <i>Glp1</i> and <i>Ghrl</i> expressions increase in both the small intestine and colon. (B) Quantification of GLP1⁺ cells in Pax4^+/+ (n = 3) and Pax4^−/− P1 ileum (n = 3). GLP1-expressing cells are more abundant in Pax4 mutant ileum. Student's T-test *p<0.05, **p<0.01, ***p<0.001.</p

Arx is expressed in enteroendocrine precursors, downstream of Neurog3.

<p>(A) Real time RT-PCR analysis of <i>Neurog3</i>, <i>ChgA</i>, <i>Arx</i> and <i>Pax4</i> expression in different intestinal regions of 8 weeks old wild-type mice (n = 3). (B) Real time RT-PCR analyses of <i>Neurog3</i> and <i>Arx</i> expression in 8–10 weeks old Villin-Cre;Neurog3^f/f (KO) mice and control Villin-Cre;Neurog3^f/+ (Ctr) mice. <i>Arx</i> expression is completely lost in absence of Neurog3 (n = 5). (C–D) Immunofluorescence on sections of wild-type adult duodenum (C,) and jejunum (D). In C, Arx⁺ cells (red arrows) are localized in the crypt and are ChgA-negative (ChgA⁺ cells, green arrows). In D, Partial overlapping expression of Arx and Neurog3 in the adult mouse intestine is illustrated. Yellow, green and red arrows point to double-labeled, single Neurog3⁺ and single Arx⁺ cells, respectively. (E) <i>In situ</i> hybrization and Immunofluorescence on cross sections of wild-type embryonic pancreas (p) and intestine (i). Blue arrows point to cells expressing <i>Arx</i>, <i>Pax4</i> or <i>Neurog3</i> transcripts. Arx and Pax4 expressions are detected 24 h after Neurog3 expression in enteroendocrine precursors. The red arrow points to an Arx expressing cell. p., proximal; d., distal; duo., duodenum; jej., jejunum; ile., ileum; col., colon; SI, small intestine; p, pancreas; I, intestine. Values are means ± SD. Scale bars (C, left panel) 50 µm, (C right panel, D) 10 µm. ND, Not Detected.</p

Short-term lineage tracing of <i>Arx</i>-deficient cells and <i>Pax4</i>-expressing cells.

<p>Co-immunodetection of beta-gal and intestinal hormones in the adult duodenum of Arx heterozygous females (A) and of Pax4 heterozygous mice (B). (A) The beta-gal protein was never detected in GLP1-, GIP- or CCK-cells. <i>Arx</i>-deficient cells, which express the beta-gal instead of Arx, can differentiate into Sst- or Serotonin- (5HT-) expressing cells. In Pax4 heterozygous mice (B), the beta-gal is expressed in the crypts and can be detected in all endocrine cell types. beta-gal is not expressed in endocrine cells located in the villi.</p

Hormone expression in <i>Arx</i>-deficient intestine.

<p>(A) Real time RT-PCR analyses of various intestinal hormones mRNAs in <i>Arx</i>-deficient and control small intestine and colon at 2 days <i>postpartum</i> (n = 5). <i>Glp1</i>, <i>Gip</i>, <i>Cck</i>, <i>Pyy</i>, <i>Nts</i> and <i>Sct</i> mRNA levels are significantly reduced in Arx mutant mice, whereas <i>Sst</i> and <i>Ghrl</i> expression are increased in the small intestine. (B) Quantification of Sst⁺ and Ghrl⁺ cells in Arx^+/+ (n = 3) and Arx⁻ P1 duodenum (n = 3). Both Sst and Ghrl-expressing cell numbers increase in <i>Arx</i>-deficient duodenum while the number of Serotonin-cells (5HT) is unchanged. Student's T-test *p<0.05, **p<0.01, ***p<0.001.</p

Normal goblet cell differentiation in <i>Arx</i>-deficient mice.

<p>(A) Periodic Acid Schiff (PAS) staining showing PAS⁺ goblet cells in wild type and <i>Arx</i>-deficient newborn intestine. (B) mRNA quantification of the goblet cell marker <i>Muc2</i> and <i>Gfi1</i>, a key TF regulating goblet cell specification, in Arx mutant intestine at P2. The expression of <i>Muc2</i> and <i>Gfi1</i> is not statistically different between <i>Arx</i>-deficient intestines (n = 5) and controls (n = 5).</p

<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

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