Abstract
These protocols investigate the interaction of cytonemes with localized Wnt. Cell-niche signaling between naive or primed mouse embryonic stem cells (ESCs) and either Wnt-secreting trophoblast stem cells (TSCs) or Wnt signals tethered to microbeads can be scrutinized in vitro. This approach analyzes cytoneme reactivity during Wnt-interaction initiation, Ca2+ transients at Wnt-contacting cytonemes, and subsequent pairing between ESCs and Wnt-sources. This pairing interaction is crucial to synthetic embryogenesis; hence this protocol is effective for in vitro studies of developmental biology. For complete details on the use and execution of this protocol, please refer to Junyent et al. (2020, 2021a, 2021b).