SOX2 redirects the developmental fate of the intestinal epithelium toward a premature gastric phenotype

Abstract

Various factors play an essential role in patterning the digestive tract. During development, Sox2 and Cdx2 are exclusively expressed in the anterior and the posterior parts of the primitive gut, respectively. However, it is unclear whether these transcription factors influence each other in determining specification of the naïve gut endoderm. We therefore investigated whether Sox2 redirects the fate of the prospective intestinal part of the primitive gut. Ectopic expression of Sox2 in the posterior region of the primitive gut caused anteriorization of the gut toward a gastric-like phenotype. Sox2 activated the foregut transcriptional program, in spite of sustained co-expression of endogenous Cdx2. However, binding of Cdx2 to its genomic targets and thus its transcriptional activity was strongly reduced. Recent findings indicate that endodermal Cdx2 is required to initiate the intestinal program and to suppress anterior cell fate. Our findings suggest that reduced Cdx2 expression by itself is not sufficient to cause anteriorization, but that Sox2 expression is also required. Moreover, it indicates that the balance between Sox2 and Cdx2 function is essential for proper specification of the primitive gut and that Sox2 may overrule the initial patterning of the primitive gut, emphasizing the plasticity of the primitive gut.

Figure 1

Ectopic expression of Sox2 severely affects the intestinal tract. (A) Macroscopic appearances of the digestive tracts from stomach until rectum isolated at E18.5 of a non-transgenic embryo (top) and double transgenic embryo treated with doxycycline (bottom), showing that Sox2 induction leads to dilated and fluid-filled intestines. Scale bar, 2 mm. (B) IHC using an antibody against Sox2 on cross-sections of the duodenum, jejunum, ileum, and colon reveals specific nuclear staining in the epithelium of double transgenic animals throughout the intestinal tract, whereas Sox2 is absent in the control. Scale bar, 50 μm. (C) IHC using an antibody against Ki67 shows an increased number of cycling cells in the double transgenic embryos, compared with control intestine. Moreover, proliferating cells were randomly distributed throughout the intestinal epithelium of the double transgenic animals, whereas proliferation is restricted to the prospective crypt compartment at the base of villi in control intestines. Scale bar, 20 μm.

Figure 2

Sox2 affects the normal differentiation of intestinal epithelium. Cross-sections of the duodenum at E18.5 of controls and double transgenic embryos, which received doxycycline. IHC using antibodies against Mucin2 (A) and synaptophysin (B) showed a reduced number of goblet cells and enteroendocrine cells, respectively, in the double transgenic animals (positive cells are indicated by arrows). Scale bar, 20 μm (A and B). (C) Analysis of the expression level of marker genes of goblet cells (Muc2), enteroendocrine cells (ChgA), and enterocytes (Lct) by qPCR showed a significant reduction of expression in the small intestine of double transgenic embryos at E18.5. Additionally, qPCR was used to determine the expression levels of two genes specific to the intestinal brush border, i.e. members of the solute carrier family (Slc), Slc2a2 and Slc5a1, which are involved in glucose transport. Both are down-regulated in double transgenic embryos. (D) PAS staining revealed the absence of the intestinal brush border in Sox2-induced animals compared with the controls (arrows indicate the positive lining of the brush border). Scale bar, 25 μm.

Figure 3

Transcriptome analysis reveals upregulation of gastric cell-specific transcripts by Sox2. (A) OmniViz Treescape showing the hierarchical clustering of Affymetrix probe sets that matched the selection query. Gene expression levels compared with the geometric mean are indicated in red for upregulated genes and in blue for downregulated genes. The color intensity correlates with the degree of change. Con, control; DT, double transgenic. (B) Sox2-upregulated and Sox2-downregulated gene lists were compared based on their associated GO term fractional representations with previously described GO term profiles of several other gastrointestinal cells and tissues (Doherty et al., 2008). ‘Mature intestinal crypt cells’ refers to an expression profile of genes derived from β-catenin deleted mice (Fevr et al., 2007), which causes crypt cells to mature. ‘Hyperplastic intestinal cells’ refers to PTEN-deficient intestinal cells, which become hyperplastic (He et al., 2007).

Figure 4

Sox2 induces stomach-like cells in the intestinal environment. Cross-sections of the stomach and duodenum at E18.5 of controls and double transgenic embryos, which received doxycycline. IHC using markers for basal cells (p63) (A) and parietal cells (H⁺/K⁺ ATPase4β) (B) showed positive staining in the stomach. Ectopically expressed Sox2 induced the appearance of basal cells and parietal cells in duodenum of double transgenic animals (arrows), whereas control duodenum is devoid of staining. Immunofluorescence for GSII lectin (C), a marker for the mucous neck cells in the stomach, showed positive cells in the double transgenic intestine (arrows), while no expression was found in the control. Scale bar, 20 μm (A–C). (D) Analysis of the expression of marker genes for basal cells (p63), suprabasal cells (Ker13), parietal cells (H⁺/K⁺ ATPase4α), and gastric pit/surface-cell mucin (Muc5ac) by qPCR showed an increase in the small intestine of double transgenic animals. No significant change in the expression level of the stomach-specific mesenchymal marker Barx1 was detected. Expression of the Gastrin hormone was reduced in double transgenic animals. (E) Nuclei of normal intestinal epithelium are oriented toward the basal membrane, whereas the nuclei of the double transgenic intestinal epithelial cells are positioned more apically, shown by EM. Bar diagram represents the quantification of the distance of the nuclei to the apical border in control and double transgenic intestines. Scale bar, 6 μm.

Figure 5

Sox2 and Cdx2 are co-expressed. IHC on sequential sections of E18.5 duodenum showed Sox2 expression only in double transgenic intestines (A), and Cdx2 expression throughout the epithelium in both control and double transgenic embryos (B). Confocal microscopy (C) of control and double transgenic animals showed that Cdx2 and Sox2 are co-expressed in the same intestinal epithelial cells in Sox2 expression embryos. Individual images of DAPI (blue), Cdx2 (green), and Sox2 (red) stainings are shown as inserts. Scale bar, 50 μm.

Figure 6

Sox2 affects the expression of Cdx2 target genes by inhibiting Cdx2 binding to target genes. (A) The expression levels of the Cdx2 target genes Hnf4α, Heph, Cdx1, and Mep1α are significantly downregulated in the small intestine of double transgenic animals at E18.5. (B) IHC with an antibody against Hnf4α on cross-sections of duodenum at E18.5 of control and Sox2 overexpressing animals showed a dramatic loss of staining in the double transgenic animals. Scale bar, 20 μm. (C) ChIP assay showed a dramatic loss of binding of Cdx2 to Cdh17 and Hnf4α in the double transgenic animals. Amylase served as a negative control.