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

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 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

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

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

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

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

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