Figure 1.
Arx is expressed in enteroendocrine precursors, downstream of Neurog3.
(A) Real time RT-PCR analysis of Neurog3, ChgA, Arx and Pax4 expression in different intestinal regions of 8 weeks old wild-type mice (n = 3). (B) Real time RT-PCR analyses of Neurog3 and Arx expression in 8–10 weeks old Villin-Cre;Neurog3f/f (KO) mice and control Villin-Cre;Neurog3f/+ (Ctr) mice. Arx 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) In situ hybrization and Immunofluorescence on cross sections of wild-type embryonic pancreas (p) and intestine (i). Blue arrows point to cells expressing Arx, Pax4 or Neurog3 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.
Figure 2.
Arx is expressed in early differentiating GLP1-, GIP-, CCK- and Gastrin-expressing cells in the adult small intestine.
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.
Figure 3.
Hormone expression in Arx-deficient intestine.
(A) Real time RT-PCR analyses of various intestinal hormones mRNAs in Arx-deficient and control small intestine and colon at 2 days postpartum (n = 5). Glp1, Gip, Cck, Pyy, Nts and Sct mRNA levels are significantly reduced in Arx mutant mice, whereas Sst and Ghrl 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 Arx-deficient duodenum while the number of Serotonin-cells (5HT) is unchanged. Student's T-test *p<0.05, **p<0.01, ***p<0.001.
Figure 4.
Normal goblet cell differentiation in Arx-deficient mice.
(A) Periodic Acid Schiff (PAS) staining showing PAS+ goblet cells in wild type and Arx-deficient newborn intestine. (B) mRNA quantification of the goblet cell marker Muc2 and Gfi1, a key TF regulating goblet cell specification, in Arx mutant intestine at P2. The expression of Muc2 and Gfi1 is not statistically different between Arx-deficient intestines (n = 5) and controls (n = 5).
Figure 5.
Short-term lineage tracing of Arx-deficient cells and Pax4-expressing cells.
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. Arx-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.
Figure 6.
Hormone expression in Pax4-deficient intestine.
(A) Real time RT-PCR analyses of various intestinal hormones mRNAs in Pax4-deficient and control small intestine and colon at 2 days postpartum (n = 4). Gip, Nts, Gast, Sct and Tph1 mRNA levels decrease significantly in Pax4 mutant small intestine, Glp1 and Ghrl 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.
Figure 7.
Expression of transcription factors in Arx- and Pax4-deficient intestines.
Real time PCR analyses in (A) Arx- and (B) Pax4- (n = 4) deficient (n = 5) and control (n = 5) small intestine and colon at 2 days postpartum. (A) Pdx1 and Foxa1/a2 expression are increased in Arx mutant colon and small intestine, respectively. (B) Arx is significantly upregulated in Pax4 mutants. Student's T-test *p<0.05, **p<0.01, ***p<0.001.
Figure 8.
Model of enteroendocrine subtype specification during small intestine development: roles of Arx and Pax4.
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.