Acridine Orange dye for long-term staining and live imaging of cnidarian development and regeneration

As the sister phylum to Bilateria, Cnidaria, holds a phylogenetic position that is crucial in evolutionary studies of animal development. However, the analysis of cnidarian species, as well as other emerging models, is limited by the scarcity of tools that are readily available and applied with ease. Commercial fluorescent dyes are an accessible option to increase visibility when transgenic approaches are out of reach. In this study, the commercial dye Acridine Orange (AO) was found to be suitable for long-term staining and live imaging of the cnidarian model Nematostella vectensis. Temporary incubation in media containing AO resulted in broad cellular labeling. Individuals stained with AO as zygotes retained the dye throughout embryogenesis, metamorphosis, and for longer than three months of growth, at which point the signal became restricted to subsets of cells. In contrast, staining of zebrafish zygotes with AO resulted in signal decay within the first couple of days of development and preferential accumulation in the yolk sac. AO is retained in N. vectensis adults after amputation and inherited by new tissue during regeneration. Surprisingly, AO was not found suitable for cell tracking via microinjection, as the dye dissipated within minutes of being injected into the cytoplasm of zygotes and blastomeres. Surprisingly, the subcellular distribution of AO signal did not match the pattern expected from it use in mammalian cell lines, where green fluorescence is observed when associated with cellular DNA and red fluorescence when bound to RNA. Instead, long-lived green fluorescence was observed throughout the cell surface and/or in the cytoplasm of N. vectensis stained with AO, whereas red emission was similarly distributed but short lived. Expanding the use of AO as a visualization tool will facilitate future studies of cnidarians and other underexplored animal models.