Female embryonic days 11.5, 13.5 and 16.5 (E11.5, E13.5 and E16.5) wild type (WT), ephrin-B2lacZ/+ (Z/+) and ephrin-B2 lacZ/lacZ (Z/Z) mice sections were dual labeled for ephrin-B2 and TUNEL immunohistochemistry. Anti-galactosidase antibody detected the ephrin-B2-gal fusion protein in Z/+ and Z/Z mice. WinSurf software was used for 3D reconstructions of the cloacal region.
In WT E11.5 females, the cloacal membrane (CM) is intact. The urorectal septum (URS) is partially separating the cloaca into urogenital sinus and hindgut; no vagina has developed. In contrast, Z/Z females had poor cloacal development. Z/+ embryos showed widened URS. Apoptosis in WT females occurred prominently in CM, dorsal cloaca, and hindgut. In contrast, apoptosis in Z/Z females was limited to the endoderm of the poorly formed cloaca. These changes progressed throughout E13.5 and finally at E16.5, Z/Z females exhibited persistent cloaca.
The 3D reconstruction of the cloacal region provides a better understanding of the mechanism by which altered ephrin-B2 signaling yields persistent cloaca via diminished apoptosis in Z/Z mice.