Introduction The mechanism of intestinal atresia formation remains undefined. Atresia in Fgfr2IIIb−/− mutant mouse embryos is preceded by endodermal apoptosis and involution of the surrounding mesoderm. We have observed that involution of the atretic segment is preceded by down regulation of Sonic hedgehog (SHH) in the endoderm which is a critical organizer of the intestinal mesoderm. We hypothesized that supplementation of Fgfr2IIIb−/− intestinal tracts with exogenous SHH protein prior to atresia formation would prevent involution of the mesoderm and rescue normal intestinal development. Methods In situ hybridization were performed on control and Fgfr2IIIb−/− intestinal tracts for Shh or FoxF1 between embryonic (E) day 11.5 and E12.0. Control and Fgfr2IIIb−/− intestinal tracts were harvested at E10.5 and cultured in media supplemented with FGF10 + SHH, or FGF10 with a SHH-coated bead. In situs were performed at E12.5 for Foxf1. Results Shh and Foxf1 expression were down-regulated during intestinal atresia formation. Media containing exogenous FGF10 + SHH did not prevent colonic atresia formation (involution). A SHH protein point source bead did induce Foxf1 expression in controls and mutants. Discussion Shh and Foxf1 expression are disrupted in atresia formation of distal colon, thereby serving as potential markers of atretic events. Application of exogenous SHH (in media supplement or as a point source bead) is sufficient to induce Foxf1 expression but insufficient to rescue development of distal colonic mesoderm in Fgfr2IIIb−/− mutant embryos. Shh signal disruption is not the critical mechanism by which loss of Fgfr2IIIb function results in atresia formation.

Background Homozygous null mutation of the fibroblast growth factor receptor 2IIIb (Fgfr2IIIb) gene in mice results in 42% of embryos developing duodenal atresias. Retinaldehyde dehydrogenase 2 (Raldh2, a gene critical for the generation of retinoic acid) is expressed in the mouse duodenum during the temporal window when duodenal atresias form. Raldh2 is critical for the normal development of the pancreatoduodenal region; therefore, we were interested in the effect of a Raldh2 mutation on duodenal atresia formation. To test this, we rendered Fgfr2IIIb−/− embryos haploinsufficient for the Raldh2 and examined these embryos for the incidence and severity of duodenal atresia. Methods Control embryos, Fgfr2IIIb−/− mutants, and Fgfr2IIIb−/−; Raldh2+/− mutants were harvested at embryonic day 18.5, genotyped, and fixed overnight. Intestinal tracts were isolated. The type and severity of duodenal atresia was documented. Results A total of 97 Fgfr2IIIb−/− embryos were studied; 44 had duodenal atresias, and 41 of these presented as type III. In the 70 Fgfr2IIIb−/−; Raldh2+/− embryos studied, a lesser incidence of duodenal atresia was seen (15 of 70; P = .0017; Fisher exact test). Atresia severity was also decreased; there were 12 embryos with type I atresias, 3 with type II atresias, and 0 with type III atresias (P < 2.81E–013; Fisher exact test). Conclusion Haploinsufficiency of Raldh2 decreases the incidence and severity of duodenal atresia in the Fgfr2IIIb−/− model. The ability to alter defect severity through manipulation of a single gene in a specific genetic background has potentially important implications for understanding the mechanisms by which intestinal atresias arise.

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.

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.

Background Hprt-Cre doubles the prevalence of homozygous null embryos per litter versus heterozygous breedings without decreasing litter size. Resulting mutant embryos are genotypically and phenotypically equivalent between strategies. We set out to confirm the effectiveness of this approach with other alleles and hypothesized that it would increase efficiency in generating compound mutants. Materials and methods Null mutants for Cyp26b1, Pitx2, and Shh were generated with Hprt-Cre from conditional alleles as were double and triple allelic combinations of Fgfr2IIIb, Raldh2, and Cyp26b1. Embryos were genotyped and phenotyped by whole mount photography, histology, and immunohistochemistry. Results Fifty percent of Hprt-Cre litters were homozygous null for Cyp26b1 (15/29) and Pitx2 (75/143), with phenotypic and genotypic equivalence to mutants from standard heterozygous breedings. In multi-allele breedings, mutant embryos constituted half of litters without significant embryo loss. In contrast, Shh breedings yielded a smaller ratio of embryos carrying two recombined alleles (6 of 16), with a significant litter size reduction because of early embryonic lethality (16 live embryos from 38 deciduae). Conclusions Hprt-Cre can be used to efficiently generate large numbers of mutant embryos with a number of alleles. Compound mutant generation was equally efficient. However, efficiency is reduced for genes whose protein product potentially interacts with the Hprt pathway (e.g., Shh).