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Live imaging reveals the progenitors and cell dynamics of limb regeneration.

ABSTRACT: Regeneration is a complex and dynamic process, mobilizing diverse cell types and remodelling tissues over long time periods. Tracking cell fate and behaviour during regeneration in active adult animals is especially challenging. Here, we establish continuous live imaging of leg regeneration at single-cell resolution in the crustacean Parhyale hawaiensis. By live recordings encompassing the first 4-5 days after amputation, we capture the cellular events that contribute to wound closure and morphogenesis of regenerating legs with unprecedented resolution and temporal detail. Using these recordings we are able to track cell lineages, to generate fate maps of the blastema and to identify the progenitors of regenerated epidermis. We find that there are no specialized stem cells for the epidermis. Most epidermal cells in the distal part of the leg stump proliferate, acquire new positional values and contribute to new segments in the regenerating leg.

SUBMITTER: Alwes F

PROVIDER: S-EPMC5079749 | biostudies-literature | 2016 Oct

REPOSITORIES: biostudies-literature

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Live imaging reveals the progenitors and cell dynamics of limb regeneration.

Alwes Frederike F Enjolras Camille C Averof Michalis M

eLife 20161025

Regeneration is a complex and dynamic process, mobilizing diverse cell types and remodelling tissues over long time periods. Tracking cell fate and behaviour during regeneration in active adult animals is especially challenging. Here, we establish continuous live imaging of leg regeneration at single-cell resolution in the crustacean <i>Parhyale hawaiensis</i>. By live recordings encompassing the first 4-5 days after amputation, we capture the cellular events that contribute to wound closure and ...[more]

PMID: 27776632

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Project description: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.

Dataset Information

Live imaging reveals the progenitors and cell dynamics of limb regeneration.

Publications

Live imaging reveals the progenitors and cell dynamics of limb regeneration.

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