Formation of duodenal atresias in fibroblast growth factor receptor 2IIIb−/− mouse embryos occurs in the absence of an endodermal plug

Abstract

Purpose Duodenal atresia in humans has been hypothesized to arise from a failure of the duodenal lumen to recanalize after formation of an endodermal plug. Recently, mutations in the Fibroblast Growth Factor Receptor 2 gene have been shown to cause atretic defects of the duodenum in mice (Fgfr2IIIb). However, work in rats suggests that murine species do not form an endodermal plug during normal duodenal development. These lines of data led us to hypothesize that mice are able to form a duodenal atresia in the absence of an endodermal plug. To test this hypothesis we examined duodenal development in wild-type and Fgfr2IIIb-/- embryos. Methods Paraffin sections were generated for either hematoxylin and eosin, E-cadherin or terminal deoxynucleotidyl transferase mediated X-dUTP nick end labeling (TUNEL) staining from Fgfr2IIIb-/- and wild-type embryos between Embryonic Days (E) 10.5 and E14.5. Sections were photographed and reconstructed into 3-dimensional display using Adobe Photoshop and Amira Visage software. Results Normal mouse duodenum does not form an endodermal plug, although a plug does form in the pyloric region of the stomach at E14.5. Fgfr2IIIb-/- embryos experience significant apoptosis in the duodenal region at E10.5, followed by the disappearance of the endoderm in the atretic precursor by E11.5. Thereafter, the mesoderm of the atretic precursor involutes over the next 2 days in the absence of further apoptosis. Interestingly, an endodermal plug was not observed at any point during the formation of a duodenal atresia. Conclusions These results suggest that duodenal atresia in the Fgfr2IIIb-/- model does not arise from persistence of an epithelial plug. Rather it appears to result from the loss of the endoderm due to apoptosis very early in development.

Haploinsufficiency of retinaldehyde dehydrogenase 2 decreases the severity and incidence of duodenal atresia in the fibroblast growth factor receptor 2IIIb−/− mouse model

“…In mice, formation of this defect is preceded by apoptosis within the endoderm at E 10.5, 12 followed by the disappearance of the endoderm in the atretic region (also termed the atretic precursor) by E 11.5 and involution of the mesoderm in the absence of further apoptosis. Formation of this defect goes to completion by E 13.5.…”

“…Formation of this defect goes to completion by E 13.5. 12,13 It is unclear where in this sequence of events alterations in retinoic acid signaling intervene to limit the severity of atresia defects. Based on our 3-dimensional reconstruction of a type I atresia, the lumen of the duodenum can be preserved.…”

“…8 In the Fgfr2IIIb model, the formation of colonic and duodenal atresia is preceded by an increase level of apoptosis in the endoderm. 12 Interestingly, unlike human duodenal development, normal development of the mouse duodenum proceeds without a solid core phase in the lumen. 12 These results suggest that in the Fgfr2IIIb mouse model, the formation of duodenal and colonic atresias result from the loss of endoderm during a critical phase in development, as opposed to a failure in recanalization of the lumen as proposed originally.…”

“…In this model, 93% of the atresias are type III defects; however, the penetrance ranges from 38% to 42%. 8,12 Therefore, if haploinsufficiency of Raldh2 were to increase the severity of the defects, we would expect to see a significant increase in atresia incidence. If haploinsufficiency of Raldh2 were to have the opposite effect, then we would expect both the severity and the incidence of atresias to decrease.…”

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

“…Animal models of intestinal atresia include ligation of vasculature or disruption of mesentery, electrocoagulation resulting in bowel obstruction during development, mutations in hedgehog protein signaling, or teratogen induction . In terms of genetic models, one of the best established mouse models of intestinal atresia is that of a Fibroblast growth factor receptor 2IIIb (Fgfr2IIIb) mutation …”