The endo-lysosomal system drives lumen formation in a human epiblast model.

The formation of a central lumen in the epiblast is a critical step that occurs during implantation in the human embryo. Lumen formation is accompanied by highly dynamic and complex cargo trafficking in the endo-lysosomal system. However, our understanding of key players and machineries that control this critical trafficking process remains incomplete in the context of epiblast development. Here, we explored endo-lysosomal dynamics that are associated with the generation of the apicosome, the earliest stage of lumen formation in a model of human epiblast development based on human pluripotent stem cells. We uncovered a hybrid early/late endosome compartment as well as a previously unrecognized dynamics of late endosome and lysosome compartments in trafficking podocalyxin (PODXL), a sialomucin glycoprotein that helps to establish and maintain the open lumen, during apicosome formation. To gain molecular insight into these unique hybrid endosome and late endosome/lysosome machineries in PODXL traffic, we used APEX2-based spatial proteomics to identify PODXL-proximity partners during apicosome formation, and identified RAB35, a Rab small GTPase known to control PODXL traffic as well as early and late endosome dynamics, as a key player in controlling apicosome formation. Our results suggest that RAB35 limits excess apicosome formation by promoting the early to late endosome transition as well as lysosome formation, which help to reduce PODXL to a level necessary for single apicosome formation. Overall, this study reveals novel endo-lysosomal mechanisms that contribute to apical membrane morphogenesis in a human model of epiblast formation.