Live Imaging of Drosophila Larval Neuroblasts

Summary

This protocol details a streamlined method used to conduct live cell imaging in the context of an intact larval brain. Live cell imaging approaches are invaluable for the study of asymmetric neural stem cell divisions as well as other neurogenic and developmental processes, consistently uncovering mechanisms that were previously overlooked.

Abstract

Stem cells divide asymmetrically to generate two progeny cells with unequal fate potential: a self-renewing stem cell and a differentiating cell. Given their relevance to development and disease, understanding the mechanisms that govern asymmetric stem cell division has been a robust area of study. Because they are genetically tractable and undergo successive rounds of cell division about once every hour, the stem cells of the Drosophila central nervous system, or neuroblasts, are indispensable models for the study of stem cell division. About 100 neural stem cells are located near the surface of each of the two larval brain lobes, making this model system particularly useful for live imaging microscopy studies. In this work, we review several approaches widely used to visualize stem cell divisions, and we address the relative advantages and disadvantages of those techniques that employ dissociated versus intact brain tissues. We also detail our simplified protocol used to explant whole brains from third instar larvae for live cell imaging and fixed analysis applications.

Introduction

Stem cells maintain a balance of differentiation and self-renewal to generate cellular diversity during early development and replace damaged cells in adult tissues. Regulation of this homeostasis prevents both the loss and over-expansion of the stem cell population, which can result in deleterious tissue degeneration or tumorigenesis.

Neuroblasts (NBs) are the widely studied neural stem cells of the Drosophila central nervous system (Figure 1A) that first form during embryonic stages^1,2. NBs undergo repeated rounds of asymmetric cell division (ACD) to produce two unequally fated ce....

Protocol

1. Preparation of Materials Required to Explant Third Instar Larval Brains

1. Prepare tools required for microdissection.
   1. Forge two dissecting tools (a scalpel and a hook) by first placing a single dissecting pin into each pin holder. Secure the pins tightly by hand or with pliers (Figure 1C).
   2. Use a pair of old forceps to bend one dissecting pin to an approximately 120° angle. Do this under a dissecting microscope to ensure that the top half of the pin lies flat when the pin holder is held in hand. This tool will be used as a scalpel to hold down or slice through tissue.
   3. Use a pair of old forceps to bend....

Results

Live imaging has elucidated a number of mechanisms that regulate NB ACD. Prior to image acquisition, it is necessary to determine the optimal imaging conditions. It is necessary to balance the frame capture rate with the duration of live cell imaging. For fine cellular analysis, for example, increased temporal and optical resolution may be required. When visualizing a single NB cell cycle (Figure 4A), the rate at which images are captured may be increased without introducing photodamage. Typically, singl.......

Discussion

Live cell imaging is an invaluable approach used to study the mechanisms that regulate ACD of stem cells, the differentiation of cell lineages, and the morphogenesis of complex tissues. Although research groups have historically employed a variety of techniques to visualize NB ACD, these approaches can be generally grouped into two categories that primarily differ with respect to whether the brain tissue is left intact or dissociated.

Imaging dissociated NBs has its advantages. For one, dissoc.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Lumox tissue culture dish 50 x 12 mm	Sarstedt	94.6077.410	A reusable culture dish with a gas-permeable and optically clear film base.
Schneider's S2 Medium	Invitrogen	21720-024
Gibco Antibiotic-Antimycotic (100x)	Invitrogen	15240-062	A supplement containing penicillin (10,000 U/ml), streptomycin (10,000 μg/ml), and amphotericin B (25 μg/ml) that is added to Schneider's S2 media to prevent bacterial and fungal contamination.
Glass coverslips, #1.5 22 x 22 mm	Fisher Scientific	12-541-B
Halocarbon oil 700	Sigma-Aldrich	H8898	A high viscosity inert oil that is clear and colorless.
Pair of nickel-plated pin holders, 17 cm long	Fine Science Tools	26018-17
Pair of Dumont #5 Forceps	Fine Science Tools	11252-20
Dissecting needle/probe with plastic handle	Fisher Scientific	08-965-A
Syringe filter, 0.22 μm pore size	Fisher Scientific	09-719A
Syringe, 5 ml	BD Biosciences	309646
Plastic transfer pipette	Fisher Scientific	S30467-1
Paraformaldehyde, 32% EM-grade	Electron Microscopy Sciences	15714	CAUTION: Formaldehyde is toxic; it should be handled in a fume hood. Follow MSDS guidelines for safe handling.
DAPI (4',6-diamidino-2-phenylindole, dihydrochloride)	Invitrogen	D1306	A dye that labels DNA and is excited by UV light.