Skip to main content
. 2022 Oct 28;66:101624. doi: 10.1016/j.molmet.2022.101624

Figure 1.

Figure 1

Gut FoxO1 ablation expands the EEC progenitor pool. (A) Schematic representation of ACTB-tdTomato-EGFP; Neurog3-Cre: FoxO1fl/fl mouse model. The mTmG reporter mice contain single copy of the transgene integrated into the ROSA 26 locus. In the cell expressing Neurog3, Cre mediated excision removed the mTomato transgene. CAG promoter drives expression of membrane bound EGFP. Therefore, the cells which have expressed Neurog3 are distinctive as EGFP positive cells from tdTomato positive cells. (B) Schematic lineage tree differentiation diagram of gut cells. Lgr5+ ISCs differentiate into absorptive cells and secretory cells, which EEC cells were differentiated from. Neurog3 expression determines the EEC lineage, therefore cells at this stage are named as EEC progenitor. NeuroD is also common transient regulator of EEC differentiation. Chromogranin A (ChgA) widely expresses in differentiated EECs. Mature EECs are usually classified based on their hormone production: K cells (Gastric inhibitory peptide, Gip), δ cells (Somatostatin, Sst), X cells (Ghrelin, GHRL), L cells (Glucagon-like peptide 1, GLP1), I cells (Cholecystokinin, Cck), N cells (Neurotensin, Nts), S cells (Secretin, SEC), and Enterochromaffin (EC) cells (Serotonin, 5-HT coded by Tph1). (C, D) Representative images of GFP and RFP immunofluorescence in intestinal duodenum sections of TG-WT (C) and TG-NFKO (D) mice. (E) Representative FACS plot of TG-WT gut epithelial cells. P4: RFP+; P5: RFP+/GFP+; P6: GFP+. (F–H) qPCR of ChgA (F), Tph1 (G), and FoxO1 (H) using mRNA from sorted P4, P5, and P6 cells from TG-WT gut epithelial cells 96-hr after DBZ treatment. N = 3 for each group. (I–L) Time course of induction of P5 and P6 population after DBZ treatment. White and red circles indicate TG-WT and TG-NFKO, respectively. 3 × 105 cells were analyzed in each experiment, and each group consists of biological triplicates. Merged FACS plots of three samples from TG-WT (K) and TG-NFKO (L) following 96-hr of DBZ treatment are shown. (M, N) Representative GFP and RFP immunofluorescence images in intestinal duodenum sections of TG-WT (M) and TG-NFKO (N) after 96-hr DBZ treatment. ISC: Intestinal stem cell, EEC: Enteroendocrine cell, TG-WT: ACTB-tdTomato,-EGFP; Neurog3-Cre (−): TG-NFKO, ACTB-tdTomato-EGFP; Neurog3-Cre (+): FoxO1fl/fl. Data are presented as means ± SD. Scale bar = 50 μm ∗ = p < 0.05; ∗∗ = p < 0.01; ∗∗∗ = p < 0.001.