Wnt- and Glutamate-receptors orchestrate stem cell dynamics and asymmetric cell division

Abstract

13Wnt signalling regulates many aspects of cell biology. Wnt-pathway activation and its 14 downstream effects have been extensively studied, but the dynamic analysis of Wnt-ligands 15 on mammalian cellular membranes is obstructed by difficulties of visualization. We overcome 16 this using microbead-tethered Wnts presented to single embryonic stem cells, which undergo 17Wnt-mediated asymmetric cell division (ACD). Through live imaging and genetic editing, we 18show that knockout of Wnt co-receptor Lrp5 promotes cytoneme formation and Wnt-19 recruitment, which requires Lrp6 and β-catenin. Lrp5 facilitates ligand-retention at the 20 membrane, and alongside Lrp6 mediates Wnt-ligand stabilization and positioning. β-catenin 21 or Wnt co-receptor knockout causes misorientation at mitosis, and all but Lrp5 are required 22for Wnt-orientated ACD. Surprisingly, ionotropic glutamate receptor (iGluR) activity enables 23 initial Wnt-recruitment, positioning, and ultimately oriented ACD. Uniquely, we have 24 scrutinized the early Wnt ligand-membrane interaction, linking roles of Wnt-pathway 25 components and crosstalk with iGluRs in guiding cell fate determination by oriented ACD. 26 27