Protocol for fluorescent live-cell staining of tardigrades.

Tardigrades are microscopic organisms with exceptional resilience to environmental extremes. Most protocols to visualize the internal anatomy of tardigrades rely on fixation, hampering our understanding of dynamic changes to organelles and other subcellular components. Here, we provide protocols for staining live tardigrade adults and other postembryonic stages, facilitating real-time visualization of structures including lipid droplets, mitochondria, lysosomes, and DNA.

The tardigrade Hypsibius exemplaris dramatically upregulates DNA repair pathway genes in response to ionizing radiation.

Tardigrades can survive remarkable doses of ionizing radiation, up to about 1,000 times the lethal dose for humans. How they do so is incompletely understood. We found that the tardigrade Hypsibius exemplaris suffers DNA damage upon gamma irradiation, but the damage is repaired. We show that this species has a specific and robust response to ionizing radiation: irradiation induces a rapid upregulation of many DNA repair genes. This upregulation is unexpectedly extreme-making some DNA repair transcripts among the most abundant transcripts in the animal. By expressing tardigrade genes in bacteria, we validate that increased expression of some repair genes can suffice to increase radiation tolerance. We show that at least one such gene is important in vivo for tardigrade radiation tolerance. We hypothesize that the tardigrades' ability to sense ionizing radiation and massively upregulate specific DNA repair pathway genes may represent an evolved solution for maintaining DNA integrity.

Expression patterns of FGF and BMP pathway genes in the tardigrade Hypsibius exemplaris.

A small number of conserved signaling pathways regulate development of most animals, yet we do not know where these pathways are deployed in most embryos. This includes tardigrades, a phylum with a unique body shape. We examined expression patterns of components of the BMP and FGF signaling pathways during embryonic segmentation and mesoderm development of the tardigrade Hypsibius exemplaris. Among the patterns examined, we found that an FGF ligand gene is expressed in ectodermal segment posteriors and an FGF receptor gene is expressed in underlying endomesodermal pouches, suggesting possible FGF signaling between these developing germ layers. We found that a BMP ligand gene is expressed in lateral ectoderm and dorsolateral bands along segment posteriors, while the BMP antagonist Sog gene is expressed in lateral ectoderm and also in a subset of endomesodermal cells, suggesting a possible role of BMP signaling in dorsal-ventral patterning of lateral ectoderm. In combination with known roles of these pathways during development of common model systems, we developed hypotheses for how the BMP and FGF pathways might regulate embryo segmentation and mesoderm formation of the tardigrade H. exemplaris. These results identify the expression patterns of genes from two conserved signaling pathways for the first time in the tardigrade phylum.