Dataset Information

LarvaSPA, A Method for Mounting Drosophila Larva for Long-Term Time-Lapse Imaging.

ABSTRACT: Live imaging is a valuable approach for investigating cell biology questions. The Drosophila larva is particularly suited for in vivo live imaging because the larval body wall and most internal organs are transparent. However, continuous live imaging of intact Drosophila larvae for longer than 30 min has been challenging because it is difficult to noninvasively immobilizeimmobilizing larvae for a long time. Here we present a larval mounting method called LarvaSPA that allows for continuous imaging of live Drosophila larvae with high temporal and spatial resolution for longer than 10 hours. This method involves partially attaching larvae to the coverslip using a UV-reactive glue and additionally restraining larval movement using a polydimethylsiloxane (PDMS) block. This method is compatible with larvae at developmental stages from second instar to wandering third instar. We demonstrate applications of this method in studying dynamic processes of Drosophila somatosensory neurons, including dendrite growth and injury-induced dendrite degeneration. This method can also be applied to study many other cellular processes that happen near the larval body wall.

SUBMITTER: Ji H

PROVIDER: S-EPMC7241436 | biostudies-literature | 2020 Feb

REPOSITORIES: biostudies-literature

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LarvaSPA, A Method for Mounting Drosophila Larva for Long-Term Time-Lapse Imaging.

Ji Hui H Han Chun C

Journal of visualized experiments : JoVE 20200227 156

Live imaging is a valuable approach for investigating cell biology questions. The Drosophila larva is particularly suited for in vivo live imaging because the larval body wall and most internal organs are transparent. However, continuous live imaging of intact Drosophila larvae for longer than 30 min has been challenging because it is difficult to noninvasively immobilizeimmobilizing larvae for a long time. Here we present a larval mounting method called LarvaSPA that allows for continuous imagi ...[more]

PMID: 32176208

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Project description:Geographic atrophy (GA), the late stage of age-related macular degeneration, is a major cause of visual disability whose pathophysiology remains largely unknown. Modern fundus imaging and histology revealed the complexity of the cellular changes that accompanies atrophy. Documenting the activity of the disease in the margins of atrophy, where the transition from health to disease occurs, would contribute to a better understanding of the progression of GA. Time-lapse imaging facilitates the identification of structural continuities in changing environments. In this retrospective pilot study, we documented the long-term changes in atrophy margins by time-lapse imaging of infrared scanning laser ophthalmoscopy (SLO) and optical coherence tomography (OCT) images in 6 cases of GA covering a mean period of 32.8 months (range, 18-72). The mean interval between imaging sessions was 2.4 months (range, 1.4-3.8). By viewing time-lapse sequences we observed extensive changes in the pattern of marginal hyperreflective spots, which associated fragmentation, increase and/or disappearance. Over the entire span of the follow-up, the most striking changes were those affecting hyperreflective spots closest to margins of atrophy, on the non-atrophic side of the retina; a continuum between the successive positions of some of the hyperreflective spots was detected, both by SLO and OCT. This continuum in their successive positions resulted in a subjective impression of a centrifugal motion of hyperreflective spots ahead of atrophy progression. Such mobilization of hyperreflective spots was detected up to several hundred microns away from atrophic borders. Such process is likely to reflect the inflammatory and degenerative process underlying GA progression and hence deserves further investigations. These results highlight the interest of multimodal time-lapse imaging to document cell-scale dynamics during progression of GA.Clinical trial registrationclinicaltrials.gov, identifier: NCT04128150 and NCT04129021.

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.

Project description:The Drosophila CNS midline glia (MG) are multifunctional cells that ensheath and provide trophic support to commissural axons, and direct embryonic development by employing a variety of signaling molecules. These glia consist of two functionally distinct populations: the anterior MG (AMG) and posterior MG (PMG). Only the AMG ensheath axon commissures, whereas the function of the non-ensheathing PMG is unknown. The Drosophila MG have proven to be an excellent system for studying glial proliferation, cell fate, apoptosis, and axon-glial interactions. However, insight into how AMG migrate and acquire their specific positions within the axon-glial scaffold has been lacking. In this paper, we use time-lapse imaging, single-cell analysis, and embryo staining to comprehensively describe the proliferation, migration, and apoptosis of the Drosophila MG. We identified 3 groups of MG that differed in the trajectories of their initial inward migration: AMG that migrate inward and to the anterior before undergoing apoptosis, AMG that migrate inward and to the posterior to ensheath commissural axons, and PMG that migrate inward and to the anterior to contact the commissural axons before undergoing apoptosis. In a second phase of their migration, the surviving AMG stereotypically migrated posteriorly to specific positions surrounding the commissures, and their final position was correlated with their location prior to migration. Most noteworthy are AMG that migrated between the commissures from a ventral to a dorsal position. Single-cell analysis indicated that individual AMG possessed wide-ranging and elaborate membrane extensions that partially ensheathed both commissures. These results provide a strong foundation for future genetic experiments to identify mutants affecting MG development, particularly in guidance cues that may direct migration. Drosophila MG are homologous in structure and function to the glial-like cells that populate the vertebrate CNS floorplate, and study of Drosophila MG will provide useful insights into floorplate development and function.

Dataset Information

LarvaSPA, A Method for Mounting Drosophila Larva for Long-Term Time-Lapse Imaging.

Publications

LarvaSPA, A Method for Mounting Drosophila Larva for Long-Term Time-Lapse Imaging.

Similar Datasets

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