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

BACKGROUND: The mechanism of intestinal atresia formation remains undefined. Atresia in fibroblast growth factor receptor 2IIIb (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 the downregulation 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 before atresia formation would prevent involution of the mesoderm and rescue normal intestinal development. METHODS: In situ hybridization was performed on control and Fgfr2IIIb(-/-) intestinal tracts for Shh or forkhead box protein F1 (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 fibroblast growth factor (FGF) 10 + SHH, or FGF10 with a SHH-coated bead. In situ hybridization was performed at E12.5 for Foxf1. RESULTS: SHH and Foxf1 expression were downregulated 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. CONCLUSIONS: 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.

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

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).

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

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.

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

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 (Fgfr2IIIb) have been shown to cause atretic defects of the duodenum in mice. 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 H&E, E-cadherin, or terminal deoxynucleotidyl transferase-mediated X-dUTP nick end labeling 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 because of apoptosis very early in development.

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

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 pharmacologic 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 used 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 embryonic day (E) 10.5, 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 hematoxylin-eosin 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.

A more efficient method to generate null mutants using Hprt-Cre with floxed alleles.

PURPOSE: The generation of nonviable 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 were analyzed. RESULTS: The Hprt-Cre mating strategy results in 51% of pups being genotypic homozygous null embryos (85/166) vs 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: The Hprt-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 approach to investigators using a model system that relies on the generation of homozygous null embryos.

Conditional mutation of fibroblast growth factor receptors 1 and 2 results in an omphalocele in mice associated with disruptions in ventral body wall muscle formation.

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 abdominal 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 Fgfr1and 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.

Humans, mice, and mechanisms of intestinal atresias: a window into understanding early intestinal 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.

Intersexuality and the cricket frog decline: historic and geographic trends.

Exposure to anthropogenic endocrine disruptors has been listed as one of several potential causes of amphibian declines in recent years. We examined gonads of 814 cricket frogs (Acris crepitans) collected in Illinois and deposited in museum collections to elucidate relationships between the decline of this species in Illinois and the spatial and temporal distribution of individuals with intersex gonads. Compared with the preorganochlorine era studied (1852-1929), the percentage of intersex cricket frogs increased during the period of industrial growth and initial uses of polychlorinated biphenyls (PCBs) (1930-1945), was highest during the greatest manufacture and use of p,p-dichlorodiphenyltrichloroethane (DDT) and PCBs (1946-1959), began declining with the increase in public concern and environmental regulations that reduced and then prevented sales of DDT in the United States (1960-1979), and continued to decline through the period of gradual reductions in environmental residues of organochlorine pesticides and PCBs in the midwestern United States (1980-2001). The proportion of intersex individuals among those frogs was highest in the heavily industrialized and urbanized northeastern portion of Illinois, intermediate in the intensively farmed central and northwestern areas, and lowest in the less intensively managed and ecologically more diverse southern part of the state. Records of deposits of cricket frog specimens into museum collections indicate a marked reduction in numbers from northeastern Illinois in recent decades. These findings are consistent with the hypothesis that endocrine disruption contributed to the decline of cricket frogs in Illinois.

Informed lay preferences for delivery of racially varied pharmacogenomics.

OBJECTIVES: To understand public perceptions and opinions of three options for prescribing medicine: individualized genetic testing, race-based prescription, and traditional prescription. METHODS: Focus groups in urban, suburban, and rural communities over-sampled for minority groups conducted from February through April, 2001 in Georgia. RESULTS: Group members (N = 102) identified individualized genetic testing as providing the best quality of care (60% of talk turns; 75% in postdiscussion anonymous survey), but stipulated the need for protection from the invasion of privacy, discrimination, and prohibitive cost. Most individuals chose genetic testing because it provided individualized attention, and African-Americans indicated they would choose genetic testing even if the costs were high. Overall, individuals were suspicious of race-based prescription. Analyses for degree of suspicion revealed a main effect for race and an interaction effect for race and gender. CONCLUSIONS: If issues of cost, discrimination, and privacy are addressed, lay individuals prefer genetic testing as the basis for prescription of medicines that exhibit racially patterned response variation.