Abstract
BackgroundTime-lapse imaging has proven highly valuable for studying development, yielding data of much finer resolution than traditional “still-shot” studies and allowing direct examination of tissue and cell dynamics. A major challenge for time-lapse imaging of animals is keeping specimens immobile yet healthy for extended periods of time. Although this is often feasible for embryos, the difficulty of immobilizing typically motile juvenile and adult stages remains a persistent obstacle to time-lapse imaging of post-embryonic development.ResultsHere we describe a new method for long-duration time-lapse imaging of adults of the small freshwater annelid Pristina leidyi and use this method to investigate its regenerative processes. Specimens are immobilized with tetrodotoxin, resulting in irreversible paralysis yet apparently normal regeneration, and mounted in agarose surrounded by culture water or halocarbon oil, to prevent dehydration but allowing gas exchange. Using this method, worms can be imaged continuously and at high spatial-temporal resolution for up to 5 days, spanning the entire regeneration process. We performed a fine-scale analysis of regeneration growth rate and characterized cell migration dynamics during early regeneration. Our studies reveal the migration of several putative cell types, including one strongly resembling published descriptions of annelid neoblasts, a cell type suggested to be migratory based on “still-shot” studies and long hypothesized to be linked to regenerative success in annelids.ConclusionsCombining neurotoxin-based paralysis, live mounting techniques and a starvation-tolerant study system has allowed us to obtain the most extensive high-resolution longitudinal recordings of full anterior and posterior regeneration in an invertebrate, and to detect and characterize several cell types undergoing extensive migration during this process. We expect the tetrodotoxin paralysis and time-lapse imaging methods presented here to be broadly useful in studying other animals and of particular value for studying post-embryonic development.Electronic supplementary materialThe online version of this article (doi:10.1186/s12861-016-0104-2) contains supplementary material, which is available to authorized users.
Highlights
Time-lapse imaging has proven highly valuable for studying development, yielding data of much finer resolution than traditional “still-shot” studies and allowing direct examination of tissue and cell dynamics
Some recent studies have used time-lapse imaging to capture relatively short time periods of post-embryonic processes [18,19,20,21], we are aware of no long-term longitudinal studies on invertebrates, and only a single recent study in a vertebrate system [10], that capture the full duration of processes such as regeneration or asexual reproduction
We have developed a set of protocols that overcome this challenge in naidid annelids, enabling us to perform low- and high-magnification time-lapse microphotography of adults undergoing head or tail regeneration
Summary
Time-lapse imaging has proven highly valuable for studying development, yielding data of much finer resolution than traditional “still-shot” studies and allowing direct examination of tissue and cell dynamics. A major challenge for time-lapse imaging of animals is keeping specimens immobile yet healthy for extended periods of time This is often feasible for embryos, the difficulty of immobilizing typically motile juvenile and adult stages remains a persistent obstacle to time-lapse imaging of post-embryonic development. The dynamic processes of animal development have traditionally been studied by examining select time points along a developmental trajectory and making inferences about intermediate steps between these data “still shots”. Often, these still shots must be collected from fixed tissue, precluding longitudinal studies of the same individual and requiring large sample sizes to account for inter-individual variability in the developmental process under study. While early embryos are usually static, movement becomes a major problem for imaging at later embryonic stages, when ciliary or muscular structures develop, and is a persistent impediment to imaging of typically motile juvenile and adult stages
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