ANChoR · Ancient signals, New models: Uncovering the regulation of multicellularity through the Hippo signaling pathway in Choanoflagellate Rosettes

Choanoflagellates are the closest living relatives of animals, and thus are a key source of information on animal origins. Some species of choanoflagellates, such as S. rosetta, exhibit facultative multicellularity through rosette colony formation. One critical pathway involved in multicellular development, conserved among metazoans, is Hippo signaling. This pathway plays a central role in regulating cell proliferation and organ size by regulating the activity of the key transcription factor Yorkie (or YAP/TAZ). In animals, Yorkie is regulated by mechanical stress, often mediated through the cytoskeleton, in a process known as mechanotransduction. Components of the Hippo pathway are conserved across all animals, but also in choanoflagellates. Although preliminary data show that the Hippo pathway sets colony size in S. rosetta, the upstream signals that modulate Hippo signaling and the full structure of the Hippo pathway in choanoflagellates remain unknown. This project aims to determine whether rosette colony size is regulated by mechanotransduction through Yorkie localization in S. rosetta. First, I will develop robust tools in this emerging model to monitor Yorkie localization and function, including genome editing, fluorescent fusion proteins, antibody production, and transcriptomic profiling. Next, I will explore mechanosensitivity by applying mechanical stress and cytoskeletal perturbations and monitoring gene expression and Yorkie localization. Finally, I will broadly characterize the choanoflagellate Hippo pathway by identifying all components using AirID – a proximity-labeling technique to identify protein–protein interactions – and performing tunable CRISPRi knockdowns of essential genes to evaluate their function while avoiding lethality.This work will provide key insights into the evolution of Hippo signaling and into the role of physical forces in the early evolution of animal multicellularity.