Biology

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Author: search.filters.author.Bely, Alexandra E.Subject: search.filters.subject.Regeneration

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    Long-term time-lapse live imaging reveals extensive cell migration during annelid regeneration
    (BioMed Central, 2016) Zattara, Eduardo E.; Turlington, Kate W.; Bely, Alexandra E.
    Background: 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. 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. Results: Here 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. Conclusions: Combining 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.
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    Transcriptome characterization via 454 pyrosequencing of the annelid Pristina leidyi, an emerging model for studying the evolution of regeneration
    (2012-06-29) Nyberg, Kevin G.; Conte, Matthew A.; Kostyun, Jamie L.; Forde, Alison; Bely, Alexandra E.
    Background: The naid annelids contain a number of species that vary in their ability to regenerate lost body parts, making them excellent candidates for evolution of regeneration studies. However, scant sequence data exists to facilitate such studies. We constructed a cDNA library from the naid Pristina leidyi, a species that is highly regenerative and also reproduces asexually by fission, using material from a range of regeneration and fission stages for our library. We then sequenced the transcriptome of P. leidyi using 454 technology. Results: 454 sequencing produced 1,550,174 reads with an average read length of 376 nucleotides. Assembly of 454 sequence reads resulted in 64,522 isogroups and 46,679 singletons for a total of 111,201 unigenes in this transcriptome. We estimate that over 95% of the transcripts in our library are present in our transcriptome. 17.7% of isogroups had significant BLAST hits to the UniProt database and these include putative homologs of a number of genes relevant to regeneration research. Although many sequences are incomplete, the mean sequence length of transcripts (isotigs) is 707 nucleotides. Thus, many sequences are large enough to be immediately useful for downstream applications such as gene expression analyses. Using in situ hybridization, we show that two Wnt/β-catenin pathway genes (homologs of frizzled and β-catenin) present in our transcriptome are expressed in the regeneration blastema of P. leidyi, demonstrating the usefulness of this resource for regeneration research. Conclusions: 454 sequencing is a rapid and efficient approach for identifying large numbers of genes in an organism that lacks a sequenced genome. This transcriptome dataset will be a valuable resource for molecular analyses of regeneration in P. leidyi and will serve as a starting point for comparisons to non-regenerating naids. It also contributes significantly to the still limited genomic resources available for annelids and lophotrochozoans more generally.