Nicole King (UC Berkeley, HHMI) 2: Choanoflagellate colonies, bacterial signals and animal origins

Nicole King introduces her lab's motivation to understand animal origins, focusing on the genomic innovations and interspecies interactions that contributed to the transition to multicellularity. She explains that choanoflagellates, as the closest living relatives of animals, provide insights into the biology of our last common ancestor. The talk will focus on the choanoflagellate species S. rosetta and its ability to transition from single-celelled to simple multicellularity, and how this process is regulated, potentially offering insights into animal multicellularity.

A quick review of choanoflagellate biology highlights their spheroid cell body, apical collar of microvilli, and flagellum that creates water currents for swimming and capturing bacteria, their primary prey. Some choanoflagellates can form multicellular colonies, reminiscent of marine invertebrate embryos, raising questions about cell interactions and regulation. S. rosetta, in particular, cycles through various morphologies, including single cells, rosette colonies, chain colonies, and attached cells, suggesting environmental influence on differentiation.

Rosette colonies in S. rosetta form through repeated cell division, with cells remaining attached, mirroring animal embryogenesis. These cells adhere via fine intercellular bridges, believed to be the result of incomplete cytokinesis, and through an extracellular matrix (ECM) that covers the cells and fills the colony's interior. The combination of these two mechanisms contributes to the structural integrity of the rosette.

Initially, S. rosetta cultures in the lab rarely formed rosettes, leading to research frustration. While preparing the choanoflagellate for genome sequencing, an undergraduate, Rick Zuzow, treated cultures with antibiotics to remove bacterial DNA. Surprisingly, one antibiotic cocktail led to a bloom of rosette development, while another completely abolished it. This led to the hypothesis that environmental bacteria regulate rosette formation, which was confirmed by adding environmental bacteria to non-rosette forming cultures.

Further investigation identified a single bacterial species, *Algoriphagus machipongonensis*, from the original environmental sample, as capable of inducing rosette development. This bacterium was co-isolated with S. rosetta, is a sufficient prey source, and belongs to the Bacteroidetes group, which are abundant in guts and diverse environments. This interaction highlights the potential for bacteria to influence eukaryote biology and is a key to understanding animal origins.

The speaker emphasizes the historical importance of bacteria in the context of animal origins. Early multicellular eukaryotes evolved in environments teeming with bacteria. The first animals likely engaged in bacterivory, meaning interactions with bacteria were an obligate part of their life history. Furthermore, development in many living animals is regulated by bacterial signals, though studying this in complex systems is challenging.

Through collaboration with Jon Clardy, a bioactive molecule, RIF-1 (rosette-inducing factor 1), was identified from *Algoriphagus*. RIF-1 is a novel sulfonolipid, a class not previously known for signaling, and is incredibly potent, inducing rosette development at femtomolar concentrations. While RIF-1 is significant, it's not the complete story, as it only induces about 5% of cells to form rosettes. Further research revealed other bioactive sulfonolipids, some inactive, and other classes of lipids like LPEs that synergize with RIFs.

The bioassay has also been used to survey a wide range of bacteria, finding many diverse species across the bacterial tree that induce rosette formation, using different bioactive molecules. The research also extends to bacteria from the vertebrate gut system, with findings that bacteria from the cecum and colon induce rosette development. The ultimate goal is to understand how choanoflagellates sense these bacterial signals and whether these mechanisms are conserved, offering insights into interactions between animals and their commensal bacteria.