Storage cell proliferation during somatic growth establishes that tardigrades are not eutelic organisms

ABSTRACT Tardigrades, microscopic ecdysozoans renowned for their resilience to extreme environments, have long been thought to maintain a constant cell number after completing embryonic development, a phenomenon known as eutely. However, sporadic reports of dividing cells have raised questions about this assumption. In this study, we investigated whether tardigrades truly exhibit a fixed cell number during somatic growth using the model species Hypsibius exemplaris . Comparing hatchlings to adults, we observed an overall increase in the number of storage cells, a tardigrade cell type involved in nutrient storage. To assess cell proliferation, we monitored DNA replication via the incorporation of the thymidine analog 5-ethynyl-2’-deoxyuridine (EdU). A significantly higher number of storage cells incorporated EdU while animals were still growing. Starvation halted both animal growth and storage cell proliferation, linking the two processes. Additionally, we found that EdU incorporation in storage cells is associated with molting, a critical process in tardigrade post-embryonic development, since it involves cuticle renewal to enable further growth. Finally, we show that hydroxyurea, a drug that slows down DNA replication progression, strongly reduces the number of EdU + cells and results in molting-related fatalities. Our data not only provide a comprehensive picture of replication events during tardigrade growth but also highlight the critical role of proper DNA replication in tardigrade molting and survival. This study definitively challenges the notion of eutely in tardigrades, offering promising avenues for exploring cell cycle, replication stress, and DNA damage management in these remarkable creatures as genetic manipulation techniques emerge within the tardigrade research field. SIGNIFICANCE Tardigrades, microscopic invertebrate animals renowned for their resilience in extreme conditions, have traditionally been considered eutelic, implying little to no somatic cell proliferation during their growth. However, a few isolated reports challenged this notion. In this study, using the emerging model Hypsibius exemplaris , we provide unequivocal molecular evidence of DNA replication and proliferation in a specific tardigrade cell type called ‘storage cells’, primarily involved in nutrient storage, throughout the animal’s growth. Furthermore, we demonstrate that this proliferation is associated with the timing of cuticle molting, and we highlight the critical role of proper DNA replication in tardigrade molting and survival. Our research definitively resolves the long-standing controversy surrounding tardigrade eutely, opening up uncharted territories in tardigrade research.