45.05 Epithelial-specific Mutation of Fgfr 2IIIb is Sufficient to Cause Atresia in Mouse Embryos

A. P. Rogers¹, A. Kowalkowski¹, A. Reeder¹, P. F. Nichol^{1 1}University Of Wisconsin,Pediatric Surgery,Madison, WI, USA

Introduction:
Intestinal atresia can lead to chronic health issues including intestinal dysmotility, short gut syndrome, and, in extreme cases, intestinal failure. Understanding the mechanisms that lead to atresia is critical to developing preventative strategies for this defect and its sequelae. Previous work hypothesized that intestinal atresia results from persistence of a developmental epithelial plug, vascular accident, or mechanical obstruction. Fibroblast growth factor receptor 2IIIb (Fgfr2IIIb) is a transmembrane receptor expressed only in the intestinal epithelium during early intestinal organogenesis. Homozygous mutation of Fgfr2IIIb throughout the developing embryo results in intestinal atresia of the distal colon in 100% of all embryos. However, atresias arising from epithelial-specific mutation of Fgfr2IIIb during intestinal development have never been shown. We hypothesized that conditional mutation of Fgfr2IIIb within the developing intestinal epithelium is sufficient to induce intestinal atresia. We modified the existing mouse model to directly test this hypothesis.

Methods:
The epithelial specific Cre driver Sonic Hedgehog Cre (ShhCre) was used to selectively mutate Fgfr2IIIb in the developing intestinal epithelium by mating Fgfr2IIIb^LoxP/+; Shh^EGFPCre/+ males to Fgfr2IIIb^LoxP/LoxP females. For lineage analysis Shh^EGFPCre/+ breedings were performed on a Rosa^LacZ background with or without Fgfr2IIIb^LoxP alleles. TUNEL and BrdU staining was performed at embryonic age (E)10.5, in situ hybridization for Fgfr2IIIb was performed at E11.5. Lineage analysis was performed at E13.5. Incidence of colonic atresia was determined.

Results:
ShhCre eliminated Fgfr2IIIb expression specifically from the intestinal epithelium in Fgfr2IIIb^LoxP/r; Shh^EGFPCre/+ mouse embryos resulting in distal colonic atresias in all homozygous conditional mutants by E13.5 (40/40) where as control littermates did not form atresias (0/155) (P<0.01). TUNEL staining was increased in the proximal colon, but largely absent from the distal colon at E10.5. BrdU labeling was diminished throughout the colon at this same time point. Lineage analysis demonstrated that atresias were fully formed by E13.5 and featured an absence of both colonic epithelium and surrounding mesenchyme.

Conclusion:
This data supports our hypothesis that epithelial-specific mutation of Fgfr2IIIb is sufficient to induce distal colonic atresias in mice. ShhCre eliminates expression of the Fgfr2IIIb conditional alleles from the intestinal epithelium; a tissue layer separate from the one that gives rise to the vasculature of the intestine. Thus, our findings also suggest that disruptions in epithelial development alone are sufficient to induce atresias and point to a potential mechanism separate from vascular accidents in the etiology of these defects.