Background/Purpose We observed that fibroblast growth factor receptors 1 and 2 (Fgfr1, Fgfr2) are expressed during abdominal wall development in mice and hypothesized that conditional mutation of these genes would result in abdomial wall defects. Methods Section in situ hybridizations were performed for Fgfr1 and Fgfr2 on wild-type embryos at embryonic day (E) 11.5 and E13.5. Conditional mutation of Fgfr1 and Fgfr2 was achieved with a tamoxifen inducible Cre at E8.5. Litters were harvested at E17.5, whole mount photographs were taken, and paraffin sections were generated and stained with hematoxylin and eosin. Results Fgfr1 was expressed in ectoderm, lateral plate mesoderm, and myoblasts, whereas Fgfr2 was expressed almost exclusively in the early dermis and ectoderm of the abdominal wall. Conditional mutation of both Fgfr2 alleles and one Fgfr1 allele resulted in omphalocele in 38.7% of mutants. Histologic examination in mutants demonstrated disruptions in dermal and muscle development. Conclusions Mutant embryos with omphalocele arising from mutation in Fgfr1 and Fgfr2 exhibit disruptions in the development of the secondary abdominal wall structures. These findings are consistent with a model of ventral abdominal wall development in which organization of the muscles and connective tissue (secondary abdominal wall structures) is influenced by positional information emanating from the primary abdominal wall.

Purpose-The generation of non-viable homozygous null mouse embryos from heterozygote null/+ breedings can be highly resource consuming, with only 25% of the embryos in the litter being null mutants. We hypothesized that 1) we could double the number of homozygous null mouse embryos in a litter without reducing litter size using Hypoxanthine-guanine phosphoribosyltransferase-Cre (Hprt-Cre) (which is active in the female germ line at the time of fertilization) and 2) these homozygous null mutants would be identical to mutants generated through traditional null/+ breedings.Methods-To test this hypothesis we used a conditional allele Fgfr2IIIb flox . This allele when recombined is identical to the Fgfr2IIIb null allele. An F1 generation of Fgfr2IIIb rec/+ ; Hprt Cre/+ females was created by mating Fgfr2IIIb +/+ ; Hprt cre′/cre females to a Fgfr2IIIb flox/flox male. The F1 females were then mated to a Fgfr2IIIb flox/flox male. F2 embryos were genotyped and the morphology and histology of the lungs, intestine, limbs and brain was analyzed.Results-The Hprt-Cre mating strategy results in 51% of pups being genotypic homozygous null embryos (85/166) versus 23% for the standard null/+ approach (38/167). These embryos did not express the Fgfr2IIIb transcript and were phenotypically identical to null embryos generated through standard null/+ breedings. Conclusions-TheHprt-Cre mating strategy increases the number of homozygous mutant embryos in a litter without decreasing litter size. Embryos generated through this approach are phenotypically identical to those from standard heterozygous breedings. We recommend this Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. , and cleft palate [9,10] have been described. Generation of homozygous null embryos through null/+ (n/+) heterozygous breedings can be highly resource consuming. When employing the n/+ breedings for one gene, a single homozygous null (n/n) embryo is generated for every 4 embryos. Thus, a litter of 10 will yield 2 or 3 homozygous null mutant embryos. Generating compound homozygous null mutants for 2 or 3 genes with this process becomes even less efficient as the frequency of homozygous null mutants decreases to 1 in 16 embryos and 1 in 64 embryos, respectively. Researchers also face increasing pressure to minimize the number of animals used while maintaining, or even increasing, the quality of basic science research. This pressure is driven in part by both the animal rights movement [11,12] To test the efficiency of the Hprt-Cre strategy, we determined the number of phenotypically null embryos in a litter as compared ...

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.

Introduction Intestinal atresias have long been hypothesized to result from either failure of recanalization of the intestinal lumen or in utero vascular accidents. Recent work in animal models is now calling for a reassessment of these widely held paradigms. Purpose In this review, we will examine the data that led to the original hypotheses and then evaluate more recent work challenging these hypotheses. Furthermore, we will discuss how defining the mechanism of atresia formation in animal models may provide insight into early intestinal development and the mechanism of lengthwise intestinal growth. Conclusion Such insight will be critical in developing regenerative therapies for patients with intestinal failure.

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.