Chemical factors induce aggregative multicellularity in a close unicellular relative of animals

Regulated cellular aggregation is an essential process for development and healing in many animal tissues. In some animals and a few distantly related unicellular species, cellular aggregation is regulated by diffusible chemical cues. However, it is unclear whether regulated cellular aggregation was part of the life cycles of the first multicellular animals and/or their unicellular ancestors. To fill this gap, we investigated the triggers of cellular aggregation in one of animals’ closest unicellular living relatives—the filasterean Capsaspora owczarzaki. We discovered that Capsaspora aggregation is induced by chemical cues, as observed in some of the earliest branching animals and other unicellular species. Specifically, we found that calcium ions and lipids present in lipoproteins function together to induce aggregation of viable Capsaspora cells. We also found that this multicellular stage is reversible as depletion of the cues triggers disaggregation, which can be overcome upon reinduction. Our finding demonstrates that chemically regulated aggregation is important across diverse members of the holozoan clade. Therefore, this phenotype was plausibly integral to the life cycles of the unicellular ancestors of animals.We thank the Light Microscopy Center at the Indiana University for support in image acquisition and analysis (funding provided by the NIH grant NIH1S10OD024988-01) and the Advanced Light Microscopy Unit of the Center for Genomic Regulation for support on image acquisition. We also thank the Indiana University Nanoscale Characterization Facility, Electron Microscopy Center, and Laboratory for Biological Mass Spectrometry for use of their instruments. We want to special thank Claudio Scazzocchio for fruit-ful discussions and Sebastián R. Najle for feedback and support in early work on Capsaspora aggregation. We acknowledge Omaya Dudin for feedback and support in image acquisition and analysis, Michelle M. Leger for feedback on the manuscript, and Koryu Kin for support in immunocytochemistry experi-ments, image acquisition and analysis, and stimulating discussions. We also thank many people from the lab for insights and support that helped advance this project. This work was supported by a NIH grant (R35GM138376) to J.P.G. and grants BFU2017-90114-P from Ministerio de Economía y Competitividad, Agencia Estatal de Investigación (AEI), and Fondo Europeo de Desarrollo Regional and PID2020-120609GB-I00 by Ministerio de Ciencia e Innovación/AEI/10.13039/501100011033 and “European Regional Development Fund: A way of making Europe” to I.R.-T. N.R.-R. was supported by a “Formación del Profesorado Universitario (FPU13/01840)” predoctoral fellowship from Ministerio de Educación, Cultura y Deporte and R.Q.K. was supported by a NIH training grant (T32GM131994