Bnip3 and AIF cooperate to induce apoptosis and cavitation during epithelial morphogenesis

Apoptosis is an essential step in cavitation during embryonic epithelial morphogenesis, but its mechanisms are largely unknown. In this paper, we used embryonic stem cell-differentiated embryoid bodies (EBs) as a model and found that Bnip3 (Bcl-2/adenovirus E1B 19-kD interacting protein), a BH3-only proapoptotic protein, was highly up-regulated during cavitation in a hypoxia-dependent manner. Short hairpin RNA silencing of Bnip3 inhibited apoptosis of the core cells and delayed cavitation. We show that the Bnip3 up-regulation was mediated mainly by hypoxia-inducible factor (HIF)-2. Ablation of HIF-2 alpha or HIF-1 beta, the common beta subunit of HIF-1 and -2, suppressed Bnip3 up-regulation and inhibited apoptosis and cavitation. We further show that apoptosis-inducing factor (AIF) cooperated with Bnip3 to promote lumen clearance. Bnip3 silencing in AIF-null EBs nearly blocked apoptosis and cavitation. Moreover, AIF also regulated Bnip3 expression through mitochondrial production of reactive oxygen species and consequent HIF-2 alpha stabilization. These results uncover a mechanism of cavitation through hypoxia-induced apoptosis of the core cells mediated by HIFs, Bnip3, and AIF.