Intestinal Neurod1 expression impairs paneth cell differentiation and promotes enteroendocrine lineage specification

Abstract

Transcription factor Neurod1 is required for enteroendocrine progenitor differentiation and maturation. Several earlier studies indicated that ectopic expression of Neurod1 converted non- neuronal cells into neurons. However, the functional consequence of ectopic Neurod1 expression has not been examined in the GI tract, and it is not known whether Neurod1 can similarly switch cell fates in the intestine. We generated a mouse line that would enable us to conditionally express Neurod1 in intestinal epithelial cells at different stages of differentiation. Forced expression of Neurod1 throughout intestinal epithelium increased the number of EECs as well as the expression of EE specific transcription factors and hormones. Furthermore, we observed a substantial reduction of Paneth cell marker expression, although the expressions of enterocyte-, tuft- and goblet-cell specific markers are largely not affected. Our earlier study indicated that Neurog3+ progenitor cells give rise to not only EECs but also Goblet and Paneth cells. Here we show that the conditional expression of Neurod1 restricts Neurog3+ progenitors to adopt Paneth cell fate, and promotes more pronounced EE cell differentiation, while such effects are not seen in more differentiated Neurod1+ cells. Together, our data suggest that forced expression of Neurod1 programs intestinal epithelial cells more towards an EE cell fate at the expense of the Paneth cell lineage and the effect ceases as cells mature to EE cells.

Figure 1

Generation of a conditional Neurod1 mouse line at the ROSA26 locus. (A) In ROSA26 loci, the insertion of a loxP-PGK-neo-3xpA (STOP)-loxP sequence upstream of a knock-in Neurod1-IRES-eGFP gene prevents Neurod1 from being transcribed. Cre-mediated deletion of the STOP sequence upon crossing with a driver-mice (e.g, Villin-Cre mice), allows the ROSA26 promoter to drive expression of the Neurod1-IRES-eGFP bi-cistronic fusion transcript. PCR primers used for verification of the indicated locations of recombined clone are shown by arrows of different colors, e.g. red arrows (G1:G2 primers); green arrows (S1:S2 and S1:NDrev); and blue and purple arrows. (B) Expression of Neurod1 message is relatively high in Vil-cre;^LSLND mice. Relative mRNA levels of Neurod1, EYFP or EGFP (normalized to β-actin) from indicated cells were shown. Values are mean ± SD (n = 3); *P ≤ 0.0001. (C) Representative double immunostaining for EYFP/EGFP (green) and Neurod1 (red) of duodenum tissues from Vil-cre; ROSA-YFP and Vil-cre;^LSLNeurod1 mice. Bar graph shown summarized the %GFP+ cells stained for Neurod1 from indicated mouse intestine. p = 0.0082 as indicated. Scale bars = 50 µm. Red arrows pointed to nuclear Neurod1 staining. (D) Table summarized the % Neurod1+ cells along intestinal tract. ±represent standard deviation from at least three experimental replicates. The p values between each pair are: 0.45 (Duod to Colon), 0.72 (Ileum to Colon) and 0.33 (Duod to Ileum). These p values are >0.05, indicating no significant difference among the three testing groups. (E) Expression of Neurod1 protein in Vil-cre;^LSLNd1 mice. Equal amounts of total protein from cell lysates of the duodenum of Vil-cre;YFP and Vil-cre;^LSLNeurod1 mice (top panel: 60 ug protein/lane; bottom panel: 20 ug protein/lane) were separated on SDS-PAGE followed by immune-blotting with Neurod1 antibody (top panel, black arrow) or Actin antibody (bottom panel).

Figure 2

Expansion of EECs with conditional Neurod1 expression in Villin⁺ cells. (A) Representative double immunostaining for EYFP/EGFP (green) and ChgA (red) of Crypt and Villus compartments from duodenum and colon samples of Vil-cre;^LSLEYFP and Vil-cre;^LSLNeurod1 mice. Bar graph on the right summarized the % of ChgA+ cells in the intestines between two mouse strains as indicated. The p values shown in the graph were: 0.0005 for Duodenum and 0.0001 for Colon. Nuclei were stained with Dapi. Scale bars = 50 µM. (B,C) Neurod1 in Villin+ cells increased the expression of gastrointestinal hormones (B) and EEC-specific transcription factors (C). Bar graph represents normalized (normalized to β-actin) mRNA levels of the indicated genes. Values are mean ± SD (n = 3). In B, P value of all hormone pairs ≤0.001; in C, *P = 0.21, the rest P ≤ 0.001.

Figure 3

The effects of Neurod1 expression in Villin+ cells on the differentiation of non-EEC cell lineages. Neurod1 in Villin+ cells did not affect marker gene expression for goblet cells and enterocytes. A reduction of Paneth cell marker expression was observed. Bar graph represents normalized (normalized to β-actin) mRNA levels of the indicated genes. Values are mean ± SD (n = 3), **P ≤ 0.0001; No substantial differences were seen of exogenous Neurod1 expression on other lineage marker expression including enterocyte, goblet and tuft cells (P ≥ 0.05).

Figure 4

Conditional Neurod1 expression in different EEC differentiation stages. Targeted Neurod1 expression in Neurod1+, Neurog3+ and Villin+ cells were driven by Neurod1-cre, Neurog3-cre and Vil-cre mice respectively. (A) Duodenum sections from WT, Neurod1-cre^LSLNeurod1, Neurog3-cre^LSLNeurod1 and Vil-cre^LSLNeurod1 were stained with antibodies against ChgA (red) and Neurod1(green). DAPI (blue) stains for nuclei. Scale bar = 50 µM. (B) The number of ChgA⁺ cells in the intestines of indicated Cre-^LSLNeurod1 mice were quantified as shown in the bar graph. Error bars represent standard deviation from at least three experimental replicates. *P = 0.007; **P = 0.004, ***P ≤ 0.0001.

Figure 5

Ectopic expression of Neurod1 in Neurog3+ cells in the intestine prevents Neurog3+ progenitor cells from becoming Paneth cells. Co-Immunofluorescent staining detected EGFP and the Paneth cell marker, lysozyme (Lyz), or goblet cell marker (Muc2) in the duodenum tissues of either Neurog3-cre-^LSLNeurod1 (A) or Tamoxifen-inducible Neurog3-creER^T2;^LSLNeurod1. (B) Scale bars represent 50 µm. Bar graphs on the left summarized the % GFP+ intestinal epithelial cells stained for EEC (ChgA), Goblet (Muc2) and Paneth (Lyz) cells. Error bars represent standard deviation from at least three experimental replicates. P values were indicated in the graph.