Expression profiling identifies novel gene targets and functions for Pdx1 in the duodenum of mature mice

Abstract

Transcription factor pancreatic and duodenal homeobox 1 (Pdx1) plays an essential role in the pancreas to regulate its development and maintain proper islet function. However, the functions of Pdx1 in mature small intestine are less known. We aimed to investigate the intestinal role of Pdx1 by profiling the expression of genes differentially regulated in response to inactivation of Pdx1 specifically in the intestinal epithelium. Pdx1 was conditionally inactivated in the intestinal epithelium of Pdx1(flox/flox);VilCre mice. Total RNA was isolated from the first 5 cm of the small intestine from mature Pdx1(flox/flox);VilCre and littermate control mice. Microarray analysis identified 86 probe sets representing 68 genes significantly upregulated or downregulated 1.5-fold or greater in Pdx(flox/flox);VilCre mice maintained under standard conditions. Ingenuity Pathway Analysis revealed that functions of the differentially expressed genes are significantly associated with metabolism of nutrients including lipids and iron. Network analysis examining the interactions among the differentially expressed genes further supports the notion that Pdx1 may modulate metabolism of lipids and iron from mature intestinal epithelium. Following forced oil feeding, Pdx1(flox/flox);VilCre mice showed diminished lipid staining in the duodenal epithelium and decreased serum triglyceride levels, indicating reduced lipid absorption compared with control duodenal epithelium. Blood samples from Pdx1(flox/flox);VilCre mice have significantly lower mean values for mean corpuscular volume and mean corpuscular hemoglobin, consistent with iron deficiency. The absence of nonheme iron in the villous epithelium and lamina propria of Pdx1(flox/flox);VilCre duodenum indicates that the duodenal epithelium lacking Pdx1 may have defects in importing iron through enterocytes, resulting in iron deficiency in Pdx1(flox/flox);VilCre mice.

Fig. 1.

Hierarchical clustering of the differentially expressed genes in mature duodenum to pancreatic and duodenal homeobox 1 (Pdx1) inactivation. Each column represents an individual sample from Pdx1^flox/flox;VilCre (n =4) and littermate control Pdx1^flox/flox mice (n =4). Each of the 86 probe sets detecting significant differential expression 1.5-fold or greater is depicted in each row. Some of the genes exhibiting significant changes in expression were detected by multiple probe sets. Dendrogram on the top shows that the samples are separated into two clusters corresponding to genotype, Pdx1^flox/flox;VilCre (K) or control Pdx1^flox/flox (C). Dendrogram on the right indicates that the genes exhibiting significant differential expression are clustered into 2 groups according to expression patterns, downregulated (top) or upregulated (bottom), in response to intestinal epithelium-specific Pdx1 inactivation. Levels of gene expression are indicated by standardized intensities shown in the continuous color map, where red depicts upregulation, whereas blue represents downregulation.

Fig. 2.

Representative network emphasizes the role of Pdx1 in modulating metabolism of lipids and iron in mature intestinal epithelium. Genes significantly upregulated in expression by 1.5-fold or greater are in red, and genes significantly downregulated are in green. This network scored high statistical significance with right-tailed Fisher's Exact Test and contained a high fraction of differentially expressed genes. Genes in gray did not show significant changes in expression or exhibited significant changes ≤1.5-fold. Genes or chemicals in white were not included on Affymetrix Mouse Genome 430 2.0 Array chip.

Fig. 3.

Pdx1^flox/flox;VilCre duodenal epithelium appears to have decreased lipid absorption compared with control duodenal epithelium. At 7 h after oral gavage of vegetable oil, fat droplets (red) in the duodenal epithelium of 2-mo-old littermate control Pdx1^flox/flox (A and C) and Pdx1^flox/flox;VilCre (B and D) mice were visualized by Oil Red O staining. A and B: representative images from matched locations within the first 5 cm (segment 1) of control and Pdx1^flox/flox;VilCre small intestine, respectively. C and D: images from matched locations within segment 2 (5–10 cm) of small intestine from littermate control and Pdx1^flox/flox;VilCre mice, respectively. Serum triglyceride levels were measured immediately (time 0) and 7 h following forced oil feeding of 3-mo-old littermate control Pdx1^flox/flox (○) and Pdx1^flox/flox;VilCre (■) mice (E).

Fig. 4.

Pdx1 inactivation restricted to the intestinal epithelium alters iron metabolism in mature mice. Average red blood cell size (mean corpuscular volume, MCV) (A) and hemoglobin amount per red blood cell (mean corpuscular hemoglobin, MCH) (B) were measured in the blood samples from 3- and 6-mo-old Pdx1^flox/flox;VilCre (n = 8) and littermate control Pdx1^flox/flox mice (n = 3). *P < 0.005. The presence of nonheme iron (blue) in the mucosa was visualized by Perls Prussian blue stain of duodenal sections from 6-mo-old littermate control Pdx1^flox/flox (C, E, and F) and Pdx1^flox/flox;VilCre (D) mice. Diffuse blue nonheme iron staining was observed in the villous epithelium (C and E), whereas concentrated iron staining was mainly found in the lamina propria (F). C and D: representative views taken at comparable location in the duodenum of littermate control and Pdx1^flox/flox;VilCre mice. E and F: higher magnification of control duodenal mucosa showing the presence of nonheme iron in the epithelial cells and lamina propria. Lu, lumen; Br, Brunner's glands.