Analysis of cell identity, morphology, apoptosis and mitotic activity in a primary neural cell culture system in Drosophila
© Moraru et al.; licensee BioMed Central Ltd. 2012
Received: 21 November 2011
Accepted: 3 May 2012
Published: 3 May 2012
In Drosophila, most neurogenetic research is carried out in vivo. Mammalian research demonstrates that primary cell culture techniques provide a powerful model to address cell autonomous and non-autonomous processes outside their endogenous environment. We developed a cell culture system in Drosophila using wildtype and genetically manipulated primary neural tissue for long-term observations. We assessed the molecular identity of distinct neural cell types by immunolabeling and genetically expressed fluorescent cell markers. We monitored mitotic activity of cell cultures derived from wildtype and tumorous larval brains. Our system provides a powerful approach to unveil developmental processes in the nervous system and to complement studies in vivo.
KeywordsDrosophila Neurogenesis Notch Primary brain culture Neural cells
Neurogenesis in Drosophila is a biphasic process consisting of an embryonic and a postembryonic period of neurogenesis. During embryogenesis primary neurons are generated, that form the functional larval central nervous system (CNS). Subsequently, during postembryonic neurogenesis secondary neurons are generated, which build up the adult brain during larval and pupal stages. During embryonic stages neural precursor cells termed neuroblasts (NBs) divide asymmetrically in a stem cell-like fashion thereby self-renewing and producing smaller ganglion mother cells (GMCs). The GMCs have a limited mitotic potential and divide only once more, to generate a pair of neurons and/or glial cells. At the end of embryogenesis, NBs undergo a quiescent phase and only a subset of NBs enter mitosis again to generate secondary neurons during larval development, reviewed in [1–3].
Depending on their mode of proliferation, larval NBs can be further subdivided into Type I and Type II NBs. In contrast to Type I NBs, in which the GMCs divide once to form two postmitotic cells, Type II NBs give rise to an intermediate progenitor that can divide multiple times. Therefore, Type II lineages are substantially larger than Type I lineages [4–6]. A third type of neurogenesis occurs in the developing optic lobe, where NBs derive from neuroepithelial precursors. Neuroepithelial cells initially divide symmetrically to increase the pool of precursor cells. Later, during larval development, neuroepithelial cells gradually transform to NBs and switch to an asymmetric division mode .
In contrast from what we know about mammalian neural stem cell behavior, most knowledge about precursor cells in the Drosophila nervous system is based on findings in vivo. It has been difficult to study defined neural Drosophila cells in vitro over a longer culture period . Dissociation of neural tissue into individual cells allows studying how neural precursors, differentiating neurons and glial cells behave outside their natural environment and therefore, to determine what aspects are controlled by either extrinsic or intrinsic cues. Many processes in the development of the brain depend on signals from neighboring cells and the correct environmental context. For instance, the reactivation of NBs in early larval stages is regulated through paracrine signals, Drosophila insulin like peptides that derive from glia cells [9, 10]. In contrast, an intrinsic cascade of transcription factors regulates the temporal identity of NBs and the fate of their progenies. This process occurs independently of the cellular environment, since the temporal cascade is not altered in isolated NBs in culture .
Development and neurogenesis in the CNS underlie tightly controlled molecular mechanisms, to ensure that the correct number and types of neurons are generated at different developmental stages. There are two main mechanisms to terminate NBs proliferation in the larval CNS. In the central abdomen pro-apoptotic proteins are activated at advanced larval stages to remove the dividing NBs by cell death once the neuronal lineage is complete . Thoracic and central brain NBs, instead, proliferate two days longer into pupal stages and then make a final symmetric division before exiting the cell cycle . An intriguing question is whether the termination of NBs proliferation is solely controlled by intrinsic factors or whether extrinsic mechanisms have any role in this process.
We investigate the behavior of neural cells outside their endogenous environment in a newly developed primary cell culture system for larval CNS cells. We use cell type specific molecular markers in order to identify neural precursors, differentiating neurons and glial cells in vitro. Genetically controlled expression of fluorescent markers allows us to identify specific subtypes of cultured brain cells. We further investigate the mitotic activity of brain cells after dissociation. We show distinct labeling techniques to identify mitotically active cells and cells that undergo apoptosis in vitro. Finally, we demonstrate that our primary culture system can maintain proliferating optic lobe precursor cells.
Results and discussion
Identifying neuronal and glial cells in primary cell culture
Finally, the homeodomain transcription factor Reversed polarity (Repo) is exclusively expressed in glial cells in the larval brain  (Figure 1I). We found cells in primary culture that express Repo suggesting a glial cell fate (Figure 1K). In summary, commonly used molecular markers for whole mount experiments can also be used to determine cellular identity and morphology in our primary cell culture system.
Identifying and tracing neuronal lineages in primary cell culture
We also tested whether we can use the GAL4/UAS system to trace neuronal lineages in unfixed cultured neurons. We were able to visualize GFP positive neurons with axonal extensions in the living primary culture. This suggests that the brain dissociation protocol can be used in combination with the GAL4/UAS system to perform lineage analysis in fixed culture and live imaging experiments (Figure 2G, G’, G’’).
We observed neurite extensions in GH146-Gal4/UAS-mCD8::GFP and MB247-Gal4/UAS- mCD8::GFP primary cell cultures. These cells are likely primary neurons of embryonic origin. To address if secondary neurons are able to form neurite extensions, we generated labeled clones induced during larval stages. While secondary neurons in vivo can have long extensions (Figure 2H), we found that mCD8::GFP labeled cells in culture are able to form small cellular extensions (Figure 2I). We next addressed how neurite extensions behave over time. We cultured wildtype brain cells and visualized cell extensions with an antibody against the cell adhesion molecule Neuroglian (Nrg). After dissociation and plating cells on a Concanavalin A coated slide, we observed many cells with a neuronal morphology showing neurite extensions (Figure 2J). After 5 h cells still show neurite extensions; however, after 25 h, cellular projections are largely diminished (Figure 2J’, J”). Hence, initial outgrowth of extensions is established but not maintained over longer time periods.
Identifying mitotically active cells in primary cell culture
Antibodies against phosphorylated Histone3 (PH3) are widely used to identify mitotically active cells in Drosophila and other model organisms. PH3 immuno-positive cells are found in areas of the developing larval brain (Figure 3C) where mitosis occurs. Similarly, in primary cell culture we can use anti-PH3 antibodies to visualize mitotic active cells, such as NBs and GMCs (Figure 3D).
The thymidine homolog BrdU can be used to identify cells that go through S-phase and thus actively replicate DNA. Exposure of third instar larval brains to short pulses of BrdU, results in BrdU incorporation in dividing NBs and clusters of progeny cells in the central brain and thorax, as well as in the proliferation centers of the optic lobes (Figure 3E). BrdU incorporation was found in small groups of cells in culture (Figure 3F, arrow). We conclude that molecular labeling techniques to identify mitotically active cells in whole mount larval brains can also be applied to study neurogenesis in primary neural cell culture.
Neural precursors reveal mitotic activity and apoptosis during several days in culture
Symmetric dividing optic lobe precursors are maintained in culture
In the larval brain, symmetric divisions are found in the optic proliferation centers, where neuroepithelial cells proliferate (Figure 6C). We therefore dissociated larval brains and applied BrdU to cell culture, where optic neuroepithelial cells were genetically labeled by Histone2B::RFP (c855a- Gal4 driven UAS- H2B::mRFP). We found that at both time points about half of all BrdU + cells were also expressing Histone2B::RFP (56% at 15 h, n = 62; 49% at 45 h n = 49) (Figure 6D, E). These results suggest that half of the number of dividing cells are of optic lobe origin and are symmetrically dividing neuroepithelial cells. The other symmetric dividing cells may be GMCs and/or intermediate progenitor cells of the Type II NB lineages. Although intermediate progenitors divisions are asymmetric, based on cell fate determinant distributions, they appear symmetrically based on cell size.
Notch misexpression leads to increased mitotic activity that is maintained in cell culture
The developing brain of Drosophila is an impacting model system, to understand how neuronal cells are specified and how they are instructed to build a functional neuronal network. In vivo studies in the developing brain have to deal with great cellular complexity and, in particular, it is difficult to distinguish between cell-autonomous and cell non-autonomous factors. To complement in vivo studies we have established a primary cell culture system, which allows us to study normal and genetically manipulated neural lineages in vitro. We were able to examine neural mitotic activity and apoptosis for six days after larval brain dissociation. Interestingly, in the living animal, neurogenesis in the brain ceases during metamorphosis and, virtually, no mitotic activity can be observed 4 days (96 h) after puparium formation [34, 35]. Primary cell cultures derived from tumorous brains maintain an increased pool of mitotically active precursor cells. This may be a first step towards establishing a first stable cell line of neural precursor cells in Drosophila. Our cell culture system now facilitates experiments to analyze the contribution of extrinsic and intrinsic factors that control neurodevelopmental processes, such as proliferation and the termination of mitotic activity.
Materials and methods
Flies were kept at standard laboratory conditions and raised on corn-meal medium at 25°C, 12:12 h light:dark cycle. We used the following lines: w 1118 , canton S (CS), GH146- Gal4, pdf- Gal4, MB247- Gal4, UAS-CD8::GFP, pcna- GFP, w; c855a-Gal4 (Bloomington Drosophila Stock Center, Indiana, USA), UAS-Histone2B::mRFP1 (from Y. Bellaiche). For clonal induction, we used hs- FLP, tub- Gal4, UAS-mCD8::GFP/CyO, FRT82B, tub- Gal80/TM6B and w; FRT82B (from B. Bello). For Notch misexpression experiment we used insc-Gal4; tub-Gal80ts (Bloomington) and UAS-Notch intra (from S. Bray).
In vitro primary cell culture of larval brains
Primary cell cultures were obtained from wildtype and transgenic wandering third instar larvae. The cell culture protocol was adapted and modified after . The larvae were collected in PBS and their surface-sterilization achieved by immersing the larvae twice into 70% ethanol and three times into sterile water. Larval brains were dissected on sterile Petri dishes, in culture medium which is 90% L-Glutamine-supplemented Schneider`s medium (Invitrogen, Lucerne 6, Switzerland), 10% heat-inactivated Fetal Bovine Serum (HyClone Laboratories, Logan, UT, USA), 0.1% penicillin G (50–unit/ml)/streptomycin sulfate (50 μg/ml) (Gibco). The brain lobes and ventral nerve cord were collected in a saline similar to Rinaldini solution (800 mg NaCl, 20 mg KCl, 5 mg NaH2PO4H2O, 100 mg NaHCO3, 100 mg glucose, in 100 ml distilled water) and kept on ice, until the desired number of brains was collected. Dissected brains were washed three times in Rinaldini-like solution and then incubated in 0.5 mg sterile-filtered Collagenase solution type I (Sigma-Aldrich, Buchs, Switzerland)/ml Rinaldini-like solution, for 1 h, at room temperature. To remove the collagenase solution, three washes with culture medium were performed. A total of 10 μl of culture medium/brain were added and dissociation of brains into single cells was achieved by repeatedly flushing the brains through the pipette tip. For all of the protocol`s steps, we used siliconized Eppendorfs (Fisher Scientific, Wohlen, Switzerland) and pipette tips (VWR International, Dietikon, Switzerland). The cell suspension was plated into 96-well plates (40 μl cell suspension/well) (BD Falcon, Allschwil, Switzerland) and supplemented with culture medium (160 μl/well). Dissociated cells do not adhere and are free floating in culture medium. To achieve cell adhesion for cell differentiation experiments we cultured brain cell suspension on UV-treated 8-wells diagnostic Teflon slides (Thermo Scientific, Allschwil, Switzerland), coated with 15 μg sterile-filtered Concanavalin A/ml H2O (Sigma-Aldrich). A total of 40 μl cell suspension was placed in one well of the slide and supplemented with 40 μl culture medium. All cultures were kept in sterile wet boxes in an incubator (OKT Germany GmbH, Potsdam, Germany) at 25°C.
Immunofluorescent stainings and antibodies
For whole mount brain immunofluorescent labeling, larval brains were dissected in PBS (Gibco, Invitrogen). Brains were fixed for 30 min in 4% formaldehyde PBS solution (Sigma-Aldrich). Brains were washed in PBS-Triton 0.3% (Acros Organics, Basel, Switzerland) three times for 10 min. Non-specific binding sites in the tissue were blocked with 10% Normal Goat Serum (NGS) (Vector Laboratories, Servion, Switzerland) diluted in PBST for 30 min. Primary and secondary antibodies were diluted in PBST and 5% NGS. Primary antibodies were incubated overnight at 4°C and secondary antibodies at room temperature for 2 h. Brains were washed four times for 15 min. Brain samples were mounted in Vectashield mounting medium with DAPI (Vector Laboratories).
Primary brain culture immunofluorescent labeling was performed in a wet box, on 15 μg Concanavalin A/ml H2O-coated eight-well diagnostic Teflon slides (Thermo Scientific). A total of 40 μl cell suspension was placed in one well of the slide and allowed to settle down for 15 min. During this period cells start to adhere to the coated surface in order to perform immunostainings. The cells were fixed for 15 min in 4% formaldehyde and washed with PBST three times, every 2 min. 10% NGS treatment was applied for 30 min. Immunofluorescent labeling was performed as described for whole mount larval brains.
If not stated otherwise, antibody dilutions apply for immunofluorescent labelings of whole mount brains and cell cultures: rat and mouse anti-Elav (1:30, Developmental Studies Hybridoma Bank, DSHB, Iowa City, Iowa, USA), mouse anti-Pros (1:10, DSHB), mouse anti-Repo (1:10, DSHB), mouse anti-BrdU (1:200 for brains and 1:300 for primary brain culture staining, DSHB), mouse anti-FasII (1:30 on brains and 1:10 on cells, DSHB), sheep anti-GFP (1:1000, AbD, Serotec, Kidlington, UK), mouse anti-PH3 (1:1000, Cell Signaling Technology, Allschwil, Switzerland), rabbit anti phospho-Histone H3 (1:1000, Upstate Biotechnology, Lake Placid, NY, USA), rabbit anti Cleaved Caspase3 (1:200 for brains and 1:300 for primary brain culture staining, Cell Signaling Technology), rat anti-Deadpan (8:10, gift of C.Q. Doe), guinea pig anti-Deadpan (1:500, gift of J. Skeath), mouse anti-Discs large (1:100, DSHB), mouse anti-Neuroglian/BP104 (1:30, DSHB). Fluorescent labeled secondary antibodies raised in Donkey or Goat were used (Molecular Probes, Lucerne, Switzerland and Jackson ImmunoResearch, West Grove, PA, USA) at a dilution of 1:300 for whole brains and 1:400 for cultured cells.
BrdU labeling, clonal induction and Notch misexpression
Dissected L3 larval brains were incubated in 15 μg BrdU/ml of culture medium . For cell culture experiments, 15 μg of BrdU/ml culture medium was added. DNA was denatured in 2 N HCl for 30 min in whole brains and for 15 min in cultured cells. BrdU incorporation was detected by immunofluorescent labeling as described above.
To induce GFP labeled clones we used the MARCM (mosaic analysis with a repressible cell marker) system . Larvae with genotype hs- FLP; tubP- GAL4, UAS- mCD8::GFP/+; FRT82B, tub- GAL80/FRT82B were heat shocked for 1 h at 37°C and brains were dissociated at wandering third instar.
For Notch misexpression embryos of genotype w; insc-Gal4/+; tub-Gal80ts/UAS-Notch intra developed at 18°C and freshly hatched larvae were transferred to 29°C to induce Notch gene expression in NBs. Notch misexpression cultures were prepared from wandering third instar larvae and incubated at 25°C.
Microscopy and image processing
A Leica TCS SP5 confocal microscope was used to collect Z stacks with optical sections at 0.5 to 1.5 μm intervals. Whole brains were imaged with a 20X objective and cultured cells were imaged with a 63X oil-immersion objective. To follow GFP tagged proteins in neurons we used DIC microscopy and the LAS AF software in our confocal. The Cell Counter plugin of ImageJ was used to analyze mitotic cells and apoptotic cells in single sections. Images were processed in ImageJ and Adobe Photoshop.
These authors contributed equally to this work
After larval hatching
After puparium formation
Central nervous system
Ganglion mother cell
We thank the Developmental Studies Hybridoma Bank (DHSB) and the Bloomington Stock Center, B. Bello, K. Matthews, S. Bray, C. Doe, Y. Bellaiche and J. Skeath for antibodies and fly stocks. Special thanks go to our colleagues in the Department of Biology at the University of Fribourg and to the Sprecher lab for fruitful discussions. BE is supported by the Swiss University Conference (SUK/CUS). This work was funded by a grant of the Swiss National Science Foundation (PP00P3_123339), the Novartis Foundation for Biomedical research and the Velux foundation (to S.G.S.).
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