Wnt2bb-triggered neighbor cell proliferation is essential for cloaca formation by inducing apoptosis in the cloaca through cell competition.

Cloaca serves as the only opening for the digestive, urinary, and reproductive tracts in multiple species. Apoptosis is crucial for shaping the cloaca, yet the underlying mechanisms remain largely unclarified. Here we find that Wnt2bb-triggered neighbor cell proliferation is essential for zebrafish cloaca formation by inducing apoptosis in the cloaca, likely through mechanical cell competition. wnt2bb is expressed robustly in the cloaca throughout its formation. wnt2bb inactivation downregulates the nuclear accumulation of β-catenin and the expression of downstream target cyclin D1, severely suppressing peri-cloacal cell proliferation. The resulting decline in local cell density is associated with a biosensor-indicated reduction in mechanical compression on the cloacal cells, which prevents their competitive elimination and eventually causes cloacal obstruction. Moreover, palbociclib, a CDK4/6 inhibitor, not only inhibits peri-cloacal cell proliferation, but also abolishes apoptosis in the cloaca, generating similar defects in wild-type embryos. Overexpression of constitutively active β-catenin exclusively in the cells surrounding the cloaca, but not within the cloaca itself, is sufficient to rescue the cloacal obstruction. Thus, we uncover an important role of Wnt2bb-regulated cell competition during cloaca development.