Spatiotemporal remodelling of the cloacal region determines the position of the anal opening in mouse embryos.

Conventional embryological models propose that the urorectal septum (URS) fuses with the cloacal membrane (CM) to form separate exits for the hindgut and urogenital sinus (UGS). However, previous studies have been re-evaluating this assumption, and the precise morphogenetic mechanisms determining the position of the anal opening remain unclear. To elucidate the spatial and temporal dynamics of cloacal development, we analysed mouse embryos from embryonic day (E)11.5 to E13.5 using serial midsagittal histological sections combined with three-dimensional reconstruction. We focused on the positional relationship between the URS and CM, as well as the internal remodelling events of the cloaca that lead to anal opening formation. Throughout development, the URS enlarged and shifted distally, but consistently remained dorsodistal to the CM without direct fusion. At E13.0, we identified an expanded space at the caudal end of the hindgut, distinct from the hindgut lumen. By E13.25, this space connected to the UGS via a duct-like structure, contributing to the separation of the UGS and hindgut. By E13.5, the CM ruptured and the anal opening emerged precisely at the junction between the hindgut lumen and the expanded space. Our findings demonstrate that the position of the anal opening is predetermined by cloacal internal space remodelling rather than fusion of the URS and CM. This study offers novel insights into normal anorectal development and the aetiology of congenital anorectal malformations.