Lack of discreet colocalization of epithelial apoptosis to the atretic precursor in the colon of the Fibroblast growth factor receptor 2IIIb mouse and staining consistent with cellular movement suggest a revised model of atresia formation

Abstract

Background: Colonic atresias in the Fibroblast growth factor receptor 2IIIb (Fgfr2IIIb) mouse model have been attributed to increased epithelial apoptosis and decreased epithelial proliferation at embryonic day (E) 10.5. We therefore hypothesized that these processes would colocalize to the distal colon where atresias occur (atretic precursor) and would be excluded or minimized from the proximal colon and small intestine.

Results: We observed a global increase in intestinal epithelial apoptosis in Fgfr2IIIb ^-/- intestines from E9.5 to E10.5 that did not colocalize to the atretic precursor. Additionally, epithelial proliferations rates in Fgfr2IIIb ^-/- intestines were statistically indistinguishable to that of controls at E10.5 and E11.5. At E11.5 distal colonic epithelial cells in mutants failed to assume the expected pseudostratified columnar architecture and the continuity of the adjacent basal lamina was disrupted. Individual E-cadherin-positive cells were observed in the colonic mesenchyme.

Conclusions: Our observations suggest that alterations in proliferation and apoptosis alone are insufficient to account for intestinal atresias and that these defects may arise from both a failure of distal colonic epithelial cells to develop normally and local disruptions in basal lamina architecture.

Keywords: E-cadherin; EMT; basal lamina; embryonic development; gene expression; laminin; mice.

Figure 1:. 3D reconstruction of intestinal epithelium apoptotic cells using TUNEL stain.

Three-dimensional reconstruction of intestinal tracts from control and Fgfr2IIIb mutant embryos aged E9.5-E12.5. Note that for E9.5-E10.5 the epithelial cells from the entirety of the intestinal tract are present, whereas only the cecum to distal colon are visualized in the E11.5-E12.5 reconstructions. Pan-epithelial apoptosis is present in both the control and null embryos at E9.5 (A and A’). At E10.5, there is a decrease in apoptosis globally, but a greater decrease in the distal colon of the null embryo (B and B’). By E11.5, apoptosis is largely absent outside of the developing cecal region (C and C’). At E12.5, little to no apoptosis is seen (D and D’). Note the shortened intestinal tract in D’, indicative of the distal colonic atresia and loss of the distal colon. Abbreviations: Prox= proximal, Dist= distal, SI= small intestine, Co = colon

Figure 2:. Pooled TUNEL counts from all epithelial cells in 50-100 μm sections of colon.

Evaluation of TUNEL positive cells within all the epithelial cells found in 50-100μm segments of colon across embryonic ages E9.5-E12.5 in wild type and Fgfr2IIIb−/− mutant intestines. TUNEL counts obtained using imaged 5μm sections. Pooling the apoptotic data across the epithelial cells allows for general synopsis of apoptosis within the colon.

Figure 3:. Frequency distribution of apoptotic cells in 5μm sections.

A high level of apoptotic variability occurs within the colon, with the greatest inconsistency in cell death across 5μm sections occurring in null embryos at E9.5-E10.5. Frequency distribution plots show the percentage of TUNEL positive stained epithelial cells per 5μm segment (with bins set at 10%: 0-10%, 10-20%, etc) across all 5μm sections counts from the colon at each stage.

Figure 4:. 3D reconstruction of PHH3 staining in epithelial cells of colon.

Three-dimensional reconstruction of colon epithelial tissue from control and Fgfr2IIIb mutant embryos aged E10.5-E11.5. Images reconstructed from 5μm PHH3 stained and images sections of tissue where the mesenchyme in the image was cropped out before reconstruction. There discreet staining found across the length of the epithelium in both the wild type and mutant conditions. Abbreviations: Prox= proximal, Dist= distal, Co = colon

Figure 5:. Epithelial cell proliferation in the colon at E10.5 and E11.5.

Mean percentage of PHH3 positive stained epithelial cells averaged across 5pm stained sections of colon on E10.5-E11.5 wild type and Fgfr2IIIb −/− mutant intestines. Using a Fisher’s exact test, there was no statistically significant difference found between conditions with a P-value 0.2429 at E10.5 and 0.518 at E11.5. n=3 embryos for E10.5 intestine and n=5 embryos for E11.5 intestine.

Figure 6:. E-cadherin staining of distal colon.

E-cadherin staining by immunohistochemistry on E11.5. Fgfr2IIIb−/− mutant and wild type distal colon. Within the wild type condition, there is a discreet staining of this epithelial cell marker at the cell borders and the epithelium retains a pseudostratified columnar organization (A-C). The Fgfr2IIIb−/− mutant the distal colon display a lack of pseudostratified columnar architecture often co-occurring with a loss of lumen (D-F), at times a diffuse pattern of E-cadherin staining (D,F), rare E-cadherin positive staining cells in the mesenchyme (indicated by the arrow in E), and near the termination of the atresia greatly diminished E-cadherin staining (F). Scale bar is 50 μm.

Figure 7:. Laminin and E-cadherin staining.

Laminin staining of extracellular matrix (red), E-cadherin staining of epithelial cells (green), and DAPI stain for nuclear material (blue) of the proximal colon of an E11.5 wild type intestine (A), distal colon of an E11.5 wild type intestine (B), the proximal colon of an E11.5 Fgfr2IIIb−/ −intestine (C), the distal colon at the atretic precursor region of an E11.5 Fgfr2IIIb−/ −intestine (D). Red arrow indicates an example section of intact laminin staining in each image. White arrows indicate breaks in laminin. Abbreviations: mes= mesenchyme, epi= epithelium. Scale bar is 25 μm.

Figure 8:. Cellular identity/cellular movement markers.

Immunostaining of cellular identity proteins in E11.5 wild type and Fgfr2IIIb−/ − intestine. Staining for β-catenin, alpha smooth muscle actin, and vimentin (red), E-cadherin staining of epithelial cells (green), and DAPI stain for nuclear material (blue). E-cadherin is an epithelial cell marker, alpha smooth muscle actin and vimentin are mesenchymal cell markers. β-catenin is upregulated during epithelial to mesenchymal transition. Abbreviations: Co= colon. SI= small intestine. Scale bar is 100 μm.