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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.

The meiotic sex chromosomes of the American marsupials Monodelphis dimidiata and Didelphis albiventris were studied with electron microscopy (EM) and with immunofluorescence localization of meiotic proteins SYCP1 and SYCP3, and proteins essential for meiotic sex chromosome inactivation (MSCI), gamma-H2AX and BRCA1. The chromatin of the non-synaptic X and Y chromosomes contains gamma-H2AX, first as foci and then as homogeneous staining at late stages. The thick and split X and Y axes are labelled with BRCA1 except at one terminus. The bulgings of the axes contain SYCP1 as well as the inner side of the dense plate. The evenly spaced and highly packed chromatin fibres of the conjoined XY body in these species have the same behaviour and the same components (gamma-H2AX in the chromatin, BRCA1 in the axes) as in the XY body of eutherian species. These observations and recent data from the literature suggest that XY body formation is ancestral to the metatherian-eutherian divergence.

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.

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