The homeodomain-containing transcription factors Arx and Pax4 control enteroendocrine subtype specification in mice

Abstract

Intestinal hormones are key regulators of digestion and energy homeostasis secreted by rare enteroendocrine cells. These cells produce over ten different hormones including GLP-1 and GIP peptides known to promote insulin secretion. To date, the molecular mechanisms controlling the specification of the various enteroendocrine subtypes from multipotent Neurog3(+) endocrine progenitor cells, as well as their number, remain largely unknown. In contrast, in the embryonic pancreas, the opposite activities of Arx and Pax4 homeodomain transcription factors promote islet progenitor cells towards the different endocrine cell fates. In this study, we thus investigated the role of Arx and Pax4 in enteroendocrine subtype specification. The small intestine and colon of Arx- and Pax4-deficient mice were analyzed using histological, molecular, and lineage tracing approaches. We show that Arx is expressed in endocrine progenitors (Neurog3(+)) and in early differentiating (ChromograninA(-)) GLP-1-, GIP-, CCK-, Sct- Gastrin- and Ghrelin-producing cells. We noted a dramatic reduction or a complete loss of all these enteroendocrine cell types in Arx mutants. Serotonin- and Somatostatin-secreting cells do not express Arx and, accordingly, the differentiation of Serotonin cells was not affected in Arx mutants. However, the number of Somatostatin-expressing D-cells is increased as Arx-deficient progenitor cells are redirected to the D-cell lineage. In Pax4-deficient mice, the differentiation of Serotonin and Somatostatin cells is impaired, as well as of GIP and Gastrin cells. In contrast, the number of GLP-1 producing L-cells is increased concomitantly with an upregulation of Arx. Thus, while Arx and Pax4 are necessary for the development of L- and D-cells respectively, they conversely restrict D- and L-cells fates suggesting antagonistic functions in D/L cell allocation. In conclusion, these finding demonstrate that, downstream of Neurog3, the specification of a subset of enteroendocrine subtypes relies on both Arx and Pax4, while others depend only on Arx or Pax4.

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;Neurog3^f/f (KO) mice and control Villin-Cre;Neurog3^f/+ (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.