Formation of intestinal atresias in the Fgfr2IIIb−/− mice is not associated with defects in notochord development or alterations in Shh expression

Abstract

Purpose The etiology of intestinal atresia remains elusive but has been ascribed to a number of possible events including in utero vascular accidents, failure of recanalization of the intestinal lumen and mechanical compression. Another such event that has been postulated to be a cause in atresia formation is disruption in notochord development. This hypothesis arose from clinical observations of notochord abnormalities in patients with intestinal atresias as well as abnormal notochord development observed in a pharmacological animal model of intestinal atresia. Atresias in this model result from in utero exposure to Adriamycin, wherein notochord defects were noted in up to 80% of embryos that manifested intestinal atresias. Embryos with notochord abnormalities were observed to have ectopic expression of Sonic Hedgehog (Shh) which in turn was postulated to be causative in atresia formation. We were interested in determining whether disruptions in notochord development or Shh expression occurred in an established genetic model of intestinal atresia and utilized the Fibroblast Growth Factor Receptor 2IIIb homozygous mutant (Fgfr2IIIb−/−) mouse model. These embryos develop colonic atresias (100% penetrance) and duodenal atresias (42% penetrance). Methods Wild-type and Fgfr2IIIb−/− mouse embryos were harvested at E10.5, E11.5, E12.5 and E13.5. Whole mount in situ hybridization was performed on E10.5 embryos for Shh. Embryos at each time point were harvested and sectioned for H&E staining. Sections were photographed specifically for the notochord and resulting images reconstructed in 3-D using Amira software. Colons were isolated from wild-type and Fgfr2IIIb−/− embryos at E10.5, then cultured for 48 hours in matrigel with FGF10 in the presence or absence of exogenous SHH protein. Explants were harvested, fixed in formalin and photographed. Results Fgfr2IIIb−/− mouse embryos exhibit no disruptions in Shh expression at E10.5, when the first events in atresia formation are known to occur. Three-dimensional reconstructions failed to demonstrate any anatomical disruptions in the notochord by discontinuity or excessive branching. Culture of wild-type intestines in the presence of Shh failed to induce atresia formation in either the duodenum or colon. Cultured Fgfr2IIIb−/− intestines developed atresias of the colon in either the presence, or absence, of Shh protein. Conclusions Although disruptions in notochord development can be associated with intestinal atresia formation, in the Fgfr2IIIb−/− genetic animal model neither disruptions in notochord development nor the presence of exogenous Shh protein are causative in the formation of these defects.

Exogenous Sonic hedgehog protein does not rescue cultured intestine from atresia formation

“…Fixed samples were dissected and dehydrated to 100% MeOH through a series of escalating Methanol/PBS-Tween steps and stored at −20°C. The in situ hybridization protocol has been published elsewhere (10) and included incubation with antisense riboprobes, at 68°C for Shh and 70°C for Foxf1 (constructs kindly provided by H. Hamada and Y. Saijoh). Photographs were taken using a dissecting light microscope.…”

“…In this set of experiments, the base culture media was supplemented with FGF10 (PeproTech, Rocky Hill, NJ) and SHH (R&D Systems, Minneapolis, MN) to a final concentration of 500 ng/mL each. Media-supplemented organ culture were conducted 5 times following previously published protocols (10), with 12 normal ( Fgfr2IIIb+/− ) littermate controls and 8 mutant ( Fgfr2IIIb−/−) tracts.…”

Utility and limits of Hprt-Cre technology in generating mutant mouse embryos

“…For double and triple compound mutants Fgfr2IIIb flox/flox ; Raldh2 flox/flox or Fgfr2IIIb flox/flox ; Raldh2 flox/flox ; Cyp26b1 flox/flox males were bred to Fgfr2IIIb rec/+ ; Hprt Cre/+ females to generate Fgfr2IIIb rec/rec ; Raldh2 rec/+ or Fgfr2IIIb rec/rec ; Raldh2 rec/+ ; Cyp26b1 rec/+ embryos. All embryos were genotyped by polymerase chain reaction [6–9]. …”

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 …”

“…Null embryos in this model have been shown to form a number of congenital defects . Of note for this study, these mutant mice present with distal colonic atresia with 100% penetrance across all homozygous mutant embryos . Given the anatomic precision and fidelity of colonic atresia formation in this model, we focused on this distal colonic region as a discreet location where cellular differences during organogenesis would be of interest.…”