Using directed evolution to study the origins of multicellular development - ABSTRACT Little is known about how novel multicellular developmental programs arise in evolution, largely because multicellularity arose deep in the past and early steps have been lost to extinction. The PI has circumvented this constraint by creating a new model system, the Multicellularity Long Term Evolution Experiment (MuLTEE), to directly study the origin of multicellularity and development. Over the last 7,500 generations, multicellular ‘snowflake yeast’ have evolved from small, dysregulated clusters of cells into groups with synchronous cell division, three transcriptionally-distinct cell types, and have increased their biomechanical strength by over 10,000x, evolving to be as strong and tough as wood. Through a combination of synthetic biology, mathematical modeling and biophysical analysis, we have gained fundamental insight into the evolutionary and cell biological dynamics through which this occurs. Specifically, we have identified general biophysical mechanisms underpinning the origin of multicellular heritability, which set the stage for genetic systems regulating development and differentiation. We have shown how novel multicellular traits arise as an emergent behavior of cell-cell interactions, and have identified the mechanisms underpinning novel multicellular adaptation (both genetic and epigenetic). We have also shown how the process of multicellular evolution can entrench cells in a multicellular state, preventing future reversion to unicellularity. Building upon this progress, we will pursue three key research directions over the next five years, during which the MuLTEE should reach ~15,000 generations: 1) We will examine the origin of cellular differentiation from an initially homogenous ancestor. Preliminary data shows that snowflake yeast have evolved to generate three putative cell types. We will examine the regulation and functional significance of these cell types using single-cell transcriptomics, spatiotemporal imaging, and genetic reconstruction. 2) We will examine the evolutionary dynamics of synchronized cell division during development, and test the hypothesis that, by increasing developmental consistency, this trait potentiates further multicellular innovation. 3) We will investigate the evolution of whole genome duplication in the MuLTEE, exploring how polyploidy and aneuploidy arises, is maintained for thousands of generations, and is leveraged to fuel rapid multicellular adaptation. The MuLTEE is, and will likely remain, the only experiment of its kind, using long-term directed evolution to prospectively explore the evolution of multicellularity and cellular differentiation. The proposed work thus will provide unique insights into the interplay between physical, developmental, and evolutionary processes that shape the emergence of biological complexity, with important implications for understanding the dynamics of cellular evolution in disease contexts like tumorigenesis and microbial biofilms.