Establishment of intestinal identity and epithelial-mesenchymal signaling by Cdx2

Abstract

We demonstrate that conditional ablation of the homeobox transcription factor Cdx2 from early endoderm results in the replacement of the posterior intestinal epithelium with keratinocytes, a dramatic cell fate conversion caused by ectopic activation of the foregut/esophageal differentiation program. This anterior homeotic transformation of the intestine was first apparent in the early embryonic Cdx2-deficient gut by a caudal extension of the expression domains of several key foregut endoderm regulators. While the intestinal transcriptome was severely affected, Cdx2 deficiency only transiently modified selected posterior Hox genes and the primary enteric Hox code was maintained. Further, we demonstrate that Cdx2-directed intestinal cell fate adoption plays an important role in the establishment of normal epithelial-mesenchymal interactions, as multiple signaling pathways involved in this process were severely affected. We conclude that Cdx2 controls important aspects of intestinal identity and development, and that this function is largely independent of the enteric Hox code.

Figure 1. Conditional ablation of Cdx2 in mouse endoderm leads to abnormal intestinal growth and colon dysgenesis

(A–B) Whole mount β-galactosidase staining of E9.5 Cdx2^loxP/+, Foxa3Cre, R26R and Cdx2^loxP/loxP, Foxa3Cre, R26R embryos. (C–D) Immunohistochemistry for Cdx2 in E9.5 control and mutant embryos. (E–F) A pan endoderm marker, Foxa1, continues to be expressed in the Cdx2-deficient endoderm. (G) The distal intestine of the E12.5 mutant gut forms a blind-ended sac (arrow). (H) At E14.5, abnormal intestinal growth and terminal blockage are evident in the mutant posterior gut, with no colon formation. Arrow points to malformed cecum. (I–N) Confocal immunostaining for β-catenin (green) and Cdx2 (red) was performed on E14.5 anterior (I,J), medial (K,L) and posterior (M,N) intestinal sections. Nuclei were counterstained by DAPI in blue. Scale bars: 50 μm.

Figure 2. Initial intestinal differentiation is blocked in the Cdx2-deficient gut

(A–B) Severe intestinal shortening is evident in the E16.5 mutant duodenum. The colon fails to form in Cdx2 mutant embryos. Arrows point to cecum. d, duodenum; p, pancreas; s, stomach; sp, spleen. (C–D′) E18.5 mutant intestine is dilated due to terminal blockage. duo, duodenum; jej, jejunum; ile, ileum. (E–F) Alkaline phosphatase staining (red) of E16.5 control and mutant jejunum sections. (G–H) Confocal immunostaining for p63 (red) and DBA lectin (green) of E15.5 control and mutant jejunum sections. Nuclei were counterstained by DAPI in blue. (I–J) Alkaline phosphatase staining (red) of E18.5 control and Cdx2 mutant. (K–L) Alcian blue staining (blue) of E18.5 control and Cdx2 mutant jejunum sections demonstrates absence of goblet cells in mutant epithelium. (M–N) Confocal immunostaining of Cdx2 (green), lectin DBA (red) and chromogranin A (blue) demonstrates absence of lectin-positive intestinal mucosa as well as enteroendocrine cells in Cdx2 mutants. Arrowheads in M point to the rare enteroendocrine cells in the control intestine. Scale bars: 75 μm in E–F, I–L; 50 μm in G–H, I′, K′ and M–N.

Figure 3. Cdx2-deficiency affects villus morphogenesis and proliferation pattern

(A–B) Immunohistochemistry for Cdx2 on E16.5 control and mutant ileal sections. (C–J) H&E staining of position-matched E18.5 longitudinal sections of control and mutant duodenum, jejunum and ileum. The mutant duodenal epithelial folding is stunted (F). (K–M) Mosaic Cdx2 deletion in E16.5 mutant ileum is indicated by Cdx2 immunostaining (brown). Cdx2⁺ epithelia are contiguous to regions of Cdx2⁻ epithelia (K). Differentiated goblet cells (alcian blue) were observed only in the Cdx2⁺ epithelial cells (arrowhead in L and M), but not in the Cdx2⁻ epithelium. (N–O) Immunohistochemistry for Ki67 on E18.5 duodenal sections. (P–Q) BrdU incorporation followed by immunohistochemistry illustrates that proliferating cells are distributed throughout the stunted villi of Cdx2-deficient mice, in contrast to control BrdU⁺ cells that are localized to the intervillus region. (R) Quantification of BrdU⁺ cells located along the crypt-villus axis after 60 minutes of in vivo labeling. Scale bars: 75 μm in A–B; 100 μm in C–D, G–K; 50 μm in E–F, L–Q.

Figure 4. The Cdx2-deficient intestine lacks intestine-specific ultrastructural features

(A–D) Electron micrographs of E18.5 control and mutant anterior intestinal epithelium. Mutant cells lack brush border (D). (E–F) Electron micrographs of E18.5 control and mutant posterior intestinal epithelium. Double-ended arrows indicate the orientations of the mutant nuclei which are parallel to the luminal surface (F), as opposed to the perpendicular orientation in control cells (E). Blue arrowheads point to goblet cells in control tissue (E). (G–H) Mutant ileal cells contain abundant tonofilaments (yellow arrowheads in G) across intercellular junctions where desmosome-like structures (yellow arrowheads in H) are assembled. Scale bars: 2 microns in A–B, E–F; 500 nm in C–D, G–H.

Figure 5. The Cdx2-deficient posterior intestine activates esophageal genes

(A–B) The E18.5 control esophageal epithelium expresses both keratin 13 and p63 (green). Sections were counterstained by E-cadherin (red) and DAPI (blue) in B. (C–F) The E18.5 control ileal epithelium lacks keratin 13 and p63 staining, while the Cdx2 mutant ileal epithelium expresses both markers. (G) Confocal immunostaining for keratin 13 (green) and p63 on Cdx2 mutant ileum. (H) Western blot for Sox2, a foregut endoderm transcription factor. (I) Heat maps, generated for genes with at least 20-fold change in mutant ileum compared to control ileum. Scales of the heat map are log based. The blue and red brackets indicate significantly down- and up-regulated genes listed in J and K. (J) A partial list of intestinal genes (asterisks) that were extinguished in the mutant ileum. (K) A partial list of esophageal genes (asterisks) that are significantly activated in the mutant ileum. Scale bars: 50 μm in A–B, G; 75 μm in C–F.

Figure 6. Cdx2-deficiency replaces pro-intestinal factors with foregut regulators

(A–I) Quantitative RT-PCR analysis was performed on E12.5 and E14.5 control and mutant stomach (St.), proximal small intestine (P. Int.), distal small intestine (D. Int.) and cecum (C), using gene specific primers. *, p<0.05; #, p<0.01. Error bar shows S.E.M. (J) The top diagram illustrates tissue segments of the E14.5 gut, from anterior (A) to posterior (P) end, used in the qRT-PCR analysis. Diagram at the bottom summarizes the anteriorization event that occurred in the mutant intestinal domain. The foregut differentiation program is shown in red while intestinal differentiation is depicted in blue. (K) Western blots for Cdx1, HNF1α , HNF4α Wnt10a. (L) ChIP assay for Cdx2 occupancy of the HNF1α , HNF4α and Cdx1 promoters. No enrichment is detected in ChIP samples derived from Cdx2-mutant intestines. *, p<0.05.

Figure 7. Cdx2-deficiency affects selected posterior enteric Hox genes

(A–L) Quantitative RT-PCR analysis of E12.5 and E14.5 control and mutant stomach (St.), proximal small intestine (P. Int.), distal small intestine (D. Int.) and cecum (C), using gene specific primers. *, p<0.05; #, p<0.01. Error bar shows the S.E.M.

Figure 8. Cdx2-deficiency affects epithelial-mesenchymal signaling

(A) Heap map shows alterations in the expression of Wnt and Hedgehog genes in the Cdx2 mutant gut. Positive and negative fold changes are shown in red and green numbers, respectively. n=3 for each tissue type. (B) Western blots demonstrate upregulation of several Wnt targets in the Cdx2 mutant intestine. (C–F) Confocal immunostaining for smooth muscle actin (SMA). Sections were counterstained for E-cadherin (green) and DAPI (blue). (G) Expression of Cdx2 in midgut and hindgut endoderm directs these domains towards the intestinal cell fate. Cdx2 promotes intestinal identity via a feed-forward mechanism involving activation of pro-intestinal transcription factors, while a foregut/esophageal cell differentiation program is repressed by Cdx2 in the posterior gut (right panel). Removing Cdx2 from posterior endoderm (bottom left) leads to replacement of intestinal epithelial identity by ectopic foregut epithelial differentiation. Cdx2 is critical for the expression of signaling molecules, the epithelial-mesenchymal interaction, and the intestinal proliferation pattern.