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Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Developmental Biology

Live-Cell Imaging of Drosophila melanogaster Third Instar Larval Brains

Published: June 23rd, 2023

DOI:

10.3791/65538

1Department of Biology, University of Washington

Here, we discuss a workflow to prepare, dissect, mount, and image live explant brains from Drosophila melanogaster third instar larvae to observe the cellular and subcellular dynamics under physiological conditions.

Drosophila neural stem cells (neuroblasts, NBs hereafter) undergo asymmetric divisions, regenerating the self-renewing neuroblast, while also forming a differentiating ganglion mother cell (GMC), which will undergo one additional division to give rise to two neurons or glia. Studies in NBs have uncovered the molecular mechanisms underlying cell polarity, spindle orientation, neural stem cell self-renewal, and differentiation. These asymmetric cell divisions are readily observable via live-cell imaging, making larval NBs ideally suited for investigating the spatiotemporal dynamics of asymmetric cell division in living tissue. When properly dissected and imaged in nutrient-supplemented medium, NBs in explant brains robustly divide for 12-20 h. Previously described methods are technically difficult and may be challenging to those new to the field. Here, a protocol is described for the preparation, dissection, mounting, and imaging of live third-instar larval brain explants using fat body supplements. Potential problems are also discussed, and examples are provided for how this technique can be used.

Asymmetric cell division (ACD) is the process by which subcellular components such as RNA, proteins, and organelles are partitioned unequally between daughter cells1,2. This process is commonly seen in stem cells, which undergo ACD to give rise to daughter cells with different developmental fates. Drosophila NBs divide asymmetrically to produce one NB, which retains its stemness, and one ganglion mother cell (GMC). The GMC undergoes further divisions to produce differentiating neurons or glia3. Asymmetrically dividing NBs are abundant in the developing brains of third-instar la....

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NOTE: Figure 1 shows the materials required to perform this study.

1. Considerations and preparations for the experiment

  1. Prevent the larvae from overcrowding.
    NOTE: The quality of explant larval brains is directly related to the health and quality of the larvae prior to dissection. Larvae that are malnourished from overcrowding will generally yield lower-quality brains30.
    1. Ensure that no more t.......

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Dissection and imaging of central brain lobe NBs expressing Pins::EGFP and Cherry::Jupiter
To showcase this protocol, larvae expressing UAS-driven Cherry::Jupiter13 and endogenously tagged Pins::EGFP16 (w; worGal4, UAS-cherry::jupiter/CyO; Pins::EGFP/TM6B, Tb) were imaged for 4 h using the described protocol using multi-well imaging slides (Figure 5C,D). Additional data were taken from larvae expressing U.......

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This protocol outlines one approach for the imaging of live explant brains from Drosophila melanogaster larvae. The protocol described here allows for explant brains to be observed for 12-20 h under the right experimental conditions. Special consideration must be given to the preparation of samples and the design of the desired experiments. As mentioned above, one of the most critical factors that determines the quality of the dissected tissue is the health of the larvae. To achieve the highest quality possible,.......

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This research is supported by R35GM148160 (C. C.) and a National Institutes of Health (NIH) Training Grant T32 GM007270 (R. C. S)

....

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Name Company Catalog Number Comments
0.22 µm polyethersulfone (PES) Membrane Genesee 25-231 Vacuum-driven filters
Agar Genesee 20-248 granulated agar
Analytical Computer Dell NA Intel Xeon Gold 5222 CPU with two 3.80 GHz processors running Windows 10 on a 64-bit operating system
Bovine Growth Serum HyClone SH30541.02
Chambered Imaging Slides Ibidi 80826
Confocal Microscope Nikon NA
Custom-machined metal slide NA NA See Cabernard and Doe 2013 (Ref. 34) for specifications
Dissection Dishes Fisher Scientific 5024343 3-well porcelain micro spot plate
Dissection Forceps World Precision Instruments Dumont #5
Dissection Microscope Leica NA
Dissection Scissors Fine Science Tools (FST) 15003-08
Embryo collection cage Genesee 59-100
Flypad with access to CO2 to anesthetize adult flies Genesee 59-172
Gas-permeable membrane YSI 98095 Gas-permeable membrane
Glass Cover Slides Electron Microscopy Sciences 72204-03 # 1.5; 22 mm x 40 mm glass coverslips
Imaris Oxford Instruments NA Alternatives: Fiji, Volocity, Aivia
Imaris File Converter Oxford Instruments NA
Instant Yeast Saf-Instant NA
Molasses Genesee 62-117
Petri dish Greiner Bio-One 628161 60 mm x 15 mm Petri dish
Petroleum Jelly Vaseline NA
Schneider's Insect Medium with L-glutamine and sodium bicarbonate liquid Millipore Sigma S0146
SlideBook acquisition software 3i NA
Vacuum-Driven Filtration Unit with a 0.22 µµm PES membrane filter Genesee Scientific, GenClone 25-231

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