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 cells: a self-renewing stem cell and a differentiating cell. ACD is guided by centrosomes, the non-membranous organelles that act as the microtubule-organizing centers of most cells³. During mitosis, NB centrosomes organize and orient the bipolar mitotic spindle along the apical-basal polarity axis. Upon cleavage of the dividing NB, apical fate determinants that specify the stem cell fate, and basal fate determinants that specify differentiation, are segregated into the unequal daughter cells.

In the larval central brain, two types of NBs may be distinguished by their number, position, transcription factor expression, and cell lineage (Figure 1A)^4-6. Type I NBs are the most abundant, and about 90 of them populate the anterior and posterior sides of each optic lobe of the brain⁷. These NBs express the transcription factor Asense (Ase), and they characteristically divide into one self-renewing NB and one smaller ganglion mother cell (GMC; Figure 1B). Each GMC undergoes a single terminal division to generate two neurons or glia (Figure 1B). In contrast, the eight Type II NBs that populate the posterior side of each optic lobe lack Ase expression⁵. They undergo ACD to produce one self-renewing NB and one intermediate neural progenitor (INP).

The INP, in turn, divides asymmetrically three to five times. Each of these divisions results in regeneration of the INP and the production of a single GMC⁴. Collectively, the specific NB identity and the temporal order of GMC birth gives rise to the astounding neuronal diversity of the adult central nervous system.

Understanding the cell biology that underlies NB ACD has been vastly improved through the use of live cell imaging techniques. Published protocols used by researchers to image live NBs vary widely. Overall, however, these methods may be grouped into two general categories distinguished by whether the larval brain is left intact or mechanically dissociated. Both techniques have distinct advantages and disadvantages depending on the researcher’s application.

Early reports revealing live NB cell divisions involve some degree of manual dissociation of the larval brain. These protocols detail smearing⁸ or teasing apart⁹ the brain in order to grow short-term primary cultures of the NBs on glass coverslips. To improve imaging, the round NBs are usually flattened on the coverslips with either glass slides⁸ or agarose pads⁹. Although flattened cells have improved optics, these techniques often lead to NB mitotic defects, including regression of the cleavage furrow and the inability to divide more than one time. Therefore, protocols that involve both manual dissociation and physical distortion of the larval brain tissue are generally only suited to very short-term (i.e., one cell cycle or less) applications. Likewise, semi-squashing or completely flattening intact brains between glass slides and coverslips limits the time one may spend imaging a given specimen to an approximately 30 min period¹⁰. Despite this limitation, this approach has been used successfully^11-14.

Recent efforts to image live dissociated NBs limit physical distortion of the isolated cells. These techniques are particularly useful for applications where it is necessary to sustain cell cultures for multiple cell division cycles. For example, dispersed cells from manually dissociated brains may be partially embedded in a clot made from the mixture of fibrinogen and thrombin¹⁵ for long-term imaging¹⁶. Alternatively, explanted brains may be first chemically dissociated with the enzyme collagenase, next manually disrupted by repeated passage through a pipet tip, and subsequently plated on poly-L-lysine-coated glass bottom dishes^17,18. Coating glass dishes with either fibrinogen or poly-L-lysine promotes the attachment and slight spreading of round cells, bringing them as close to the coverslip as possible without major physical distortion. In addition, these methods involve culturing the NBs in medium that may be readily exchanged for pharmacological perturbation experiments¹⁸. Overall, directly plating dispersed NBs improves imaging optics without sacrificing long-term imaging capabilities. Recently, a protocol describing the long-term culturing of dissociated NBs has been detailed¹⁹.

However, this approach is not without its limitations. There are several caveats to imaging live dissociated NBs. In a field of dispersed cells, for example, it is difficult to identify specific NB lineages that are spatially organized in the intact brain¹. Likewise, distinguishing the Type I versus Type II NBs within dissociated tissue is also challenging without the expression of lineage tracers or subtype-specific transgenes, such as worniu-GAL4, asense-GAL80²⁰. Although these transgenes are quite useful for some applications, they do limit researchers to those transgenes expressed under the control of the UAS enhancer element²¹ and require more complex genetic schemes. Studies that concern the spatial or temporal development of NBs, therefore, require in vivo imaging in the context of an intact tissue.

Moreover, studies of dissociated embryonic NBs indicate that the physical contact of adjacent cells is critical for the interphase localization of key polarity determinants^22,23. These studies show completely isolated NBs no longer maintain the invariant mitotic spindle axis. Instead, these cells display a more randomized spindle axis and wide angles of separation between successive GMC buds, perhaps due to the loss of apical centrosome positioning²². Although the relationship between the larval NBs and their neighboring cells has not been extensively studied, it is worth considering that the larval stem cell microenvironment may impinge upon cell polarity or other behaviors. To maintain the physiological context of larval NBs, we image ACD from whole brains.

Given the inherent limitations associated with imaging dissociated NBs, our lab and others have established protocols to image successive rounds of NB ACD from whole larval brains. Techniques to image intact brains often replace the rigid surface of glass on one side of the culture chamber with a flexible membrane to prevent tissue distortion or damage and allow for gas exchange. Some methods involve mounting explanted brains in a mixture of insect culturing medium, serum, and ascorbic acid with the fat bodies isolated from ten larvae²⁴. The isolated fat bodies are added because they secrete a mitogen known to stimulate NB cell division²⁵. This protocol preserves the integrity of the tissue for over 3 hr and is, therefore, amenable to long-term imaging^24,26-28. Recently, a protocol describing the long-term imaging of intact larval brains in the presence of ascorbic acid, serum, and fat bodies has been detailed²⁹. Importantly, because the tissue is neither exposed nor immobilized, this approach is less useful for applications where culturing medium is exchanged (e.g., drug studies, immunodepletion, etc.).

Our lab has simplified the whole mount preparation of live larval brains for long-term imaging^30,31. The main advantage to our protocol over others is its simplicity: our observations indicate neither serum, ascorbic acid, insulin, nor fat bodies are necessary additives to image successive rounds of NB ACD. Although additives, such as insulin, have been useful for the prolonged imaging of stem cell divisions³² and collective cell migration in Drosophila ovaries³³, they appear to be dispensable for NB ACD, as our imaging medium consists of a simple mixture of standard insect cell culturing medium supplemented with antibiotic-antimycotic to prevent contamination. Through the use of reusable gas-permeable or glass bottom culture dishes, we have been able to sustain several rounds of successive NB ACDs. The same explanted samples can readily be used for immunofluorescence and other procedures with fixed tissue. In this protocol, we detail the dissection of intact larval brains, as well as the preparation of brains for both live and fixed analysis.

Protocol

1. Preparation of Materials Required to Explant Third Instar Larval Brains Prepare tools required for microdissection. 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). 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 …

Representative 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

Lumox Tissue Culture Dish 50X12 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 mg/mL), and amphotericin B (25 mg/mL) that is added to Schneider's S2 media to prevent bacterial and fungal contamination.
Glass coverslips, #1.5 22X22 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
Minutien dissecting pins	Fine Science Tools	26002-10
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 mm pore size	Fisher Scientific	09-719A
Syringe 5 mL	BD Biosciences	309646
plastic transfer pipet	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.
Aqua/PolyMount; Polysciences, Inc.	Fisher Scientific	18606-20	A water-soluble mounting medium.