Here we describe an effective method for studying dynamics of apoptotic cell clearance in vivo. This method employs live Drosophila embryos as a powerful model for monitoring phagocytosis of apoptotic cells using specific labeling of apoptotic cells and phagocytes.
The proper elimination of unwanted or aberrant cells through apoptosis and subsequent phagocytosis (apoptotic cell clearance) is crucial for normal development in all metazoan organisms. Apoptotic cell clearance is a highly dynamic process intimately associated with cell death; unengulfed apoptotic cells are barely seen in vivo under normal conditions. In order to understand the different steps of apoptotic cell clearance and to compare ‘professional’ phagocytes – macrophages and dendritic cells to ‘non-professional’ – tissue-resident neighboring cells, in vivo live imaging of the process is extremely valuable. Here we describe a protocol for studying apoptotic cell clearance in live Drosophila embryos. To follow the dynamics of different steps in phagocytosis we use specific markers for apoptotic cells and phagocytes. In addition, we can monitor two phagocyte systems in parallel: ‘professional’ macrophages and ‘semi-professional’ glia in the developing central nervous system (CNS). The method described here employs the Drosophila embryo as an excellent model for real time studies of apoptotic cell clearance.
The proper elimination of unwanted or aberrant cells through apoptosis and subsequent phagocytosis is crucial for embryonic development as well as for tissue homeostasis in the adult. Phagocytosis of apoptotic cells or apoptotic cell clearance is a highly dynamic process which proceeds in four steps: (1) recruitment of phagocytes to the apoptotic cell (‘find-me’), (2) recognition of the cell as a target for phagocytosis (‘eat-me’) and (3) engulfment, followed by (4) phagosome maturation and degradation of the apoptotic particle1-5. There are two types of phagocytes: ‘professional’ macrophages and immature dendritic cells, and the ‘non-professional’ tissue-resident neighboring cells, which are crucial for apoptotic cell clearance during metazoan development6,7 .
In Drosophila development, the elimination of superfluous cells through apoptosis occurs in three main phases, first in the mid to late embryo, then in the mid pupa, and again in the early adult. During embryogenesis apoptotic particles are removed by ‘professional’ phagocytes, macrophages, and by the ‘non-professional’ ectoderm and glia8,9 .
In our previous work we have identified a phagocytic receptor, Six-microns-under (SIMU), which is expressed exclusively in phagocytic cells during embryogenesis. Using the simu promoter we generated a specific marker for phagocytic cell populations (simu-cytGFP) which enables us to monitor macrophages, ectoderm and glia simultaneously in a live developing embryo8. In addition, by using specific markers for apoptotic cells in different stages of apoptosis, we are able to follow the dynamics of apoptotic cell clearance in vivo.
To follow the dynamics of apoptotic cell clearance in vivo two populations of cells must be labeled: phagocytic cells and apoptotic cells.
To mark phagocytic cell populations we use a Drosophila line containing the simu-cytGFP marker, which labels exclusively phagocytic cells in the embryo: macrophages, glia and ectoderm8. One can use different markers for phagocytic cells, including lines containing a hemocyte-specific Gal4 driver (crq-Gal4) or glia-specific Gal4 driver (repo-Gal4) and a genetically encoded fluorescent reporter under UAS control (uas-GFP).
To monitor apoptotic cells during apoptotic cell clearance we use different apoptosis/phagocytosis markers. Annexin V (Molecular Probes) serves as an early marker for apoptotic cells 10; Phiphilux (OncoImmunin) is a fluorogenic caspase-3 substrate, which is used as a later apoptosis marker, and LysoTracker (Molecular Probes) works as a phagosome marker. We inject these reagents using the microinjection system PicoPump PV 820.
1. Getting Ready
2. Embryo Preparation
3. Embryo Injection
4. Imaging
We image the embryos on an inverted confocal microscope with a 40X or 100X objective. We look for a nicely positioned embryo with the CNS in the middle, which shows strong GFP expression and a good labeling of apoptotic cells following injection.
For time lapse recordings of live embryos we usually choose 5 or 6 confocal slices (2 μm thick each). Afterwards, we make a projection of 3 slices (6 μm thickness) in order to observe whole cells.
We use markers for apoptotic cells which are stable and do not bleach easily. To avoid GFP bleaching we make recordings in intervals of 60 sec.
In some cases embryos may start rolling during the time of video recording. Therefore, we take a look once in a while at the recording in order to stop and start over if necessary.
Representative frames from a movie in which apoptotic cells are labeled with Annexin V, and glia, ectoderm and macrophages are labeled with simu-cytGFP are shown in Figure 2. Each frame is a projection of 3 slices 2 μm each.
The embryo of stage 15 is properly positioned showing the embryonic CNS in the middle with well labeled glia (g). Macrophages (m), which are mostly outside the CNS, show strong cytoplasmic GFP expression. Ectodermal cells are also labeled with cytoplasmic GFP (e). Many Annexin V positive cells are seen inside and outside the CNS. To follow the engulfment event is not easy. We highlighted one event with a white rectangle where a glial cell is engulfing an apoptotic particle. Note the probing behavior of the glial cell: touching the apoptotic particle a few times without engulfing it and then ending up with engulfment of the Annexin V labeled apoptotic cell (Figure 2).
Additional markers for different apoptotic stages could be used with the same procedure.
Figure 1. Schematic representation of embryo preparation for microinjections. A. A microscopic slide containing a piece of agar on which embryos are transferred after dechorionation. About 20 embryos are placed in line, close to the edge of the agar piece, with their ventral side up, for imaging the CNS. B. A coverslip containing a strip of heptane glue in the middle. C. The coverslip from B with embryos attached to the heptane glue strip.
Figure 2. Dynamic analysis of apoptotic cell clearance. Time-lapse recordings of apoptotic cell clearance in a stage 15 embryo. Glia (g), macrophages (m) and ectoderm (e) are labeled with simu-cytGFP (green). Apoptotic cells are marked with the fluorescent Annexin V (red). A. Selected frames from a representative movie are shown. A glial cell engulfing an apoptotic cell is depicted by a rectangle. B. Close up views of the marked rectangle.
Apoptotic cell clearance is a critical last stage of apoptosis, which is highly dynamic. Therefore real time studies of the process are of utmost importance. Here we describe a protocol that enables monitoring of apoptotic cell clearance in living developing Drosophila embryos. In this procedure, two populations of cells must be marked using specific markers: apoptotic cells and phagocytes. The simu-cytGFP reporter is suitable for monitoring phagocytic macrophages, glia and ectoderm simultaneously in the same embryo, which makes it possible to follow different phagocytic cell populations at the same time. This is a great advantage for studying ‘professional’ and ‘non-professional’ clearance concurrently, comparing specific aspects of the process such as rate, specificity and degradation ability. A disadvantage of this approach is that simu-cytGFP marker is expressed extremely strongly in macrophages and much weaker in glia, which could be tricky to adjust in the same movie. However, one can image apoptotic cell clearance by macrophages separately from this process in the CNS, by driving GFP expression with specific Gal4s (crq-Gal4 for macrophages and repo-Gal4 for glia).
Different markers for apoptotic cells reflecting specific molecular and morphological alterations are of great value for understanding the dynamics and reversibility of apoptosis and cell clearance. Moreover, real time recordings of apoptotic cell clearance using these markers add additional information about mutant phenotypes, which could not be achieved by using conventional immunohistochemistry techniques in fixed embryos.
The most difficult step of the described protocol, when imaging the developing CNS, is the extremely precise positioning of the embryos before injection. Even a slight move of the coverslip during attachment of the prepared embryos to the glue could change their position, which would not be appropriate for imaging the CNS. The embryo attaching step should be done with maximum attention.
Development of additional markers for specific stages in apoptosis and phagocytosis would be extremely desirable for finer dissection and possible manipulation of apoptotic cell clearance. The protocol presented here serves as a good basis for future development.
The authors have nothing to disclose.
This work was supported by a Marie Curie Reintegration Grant (IRG249084). We thank all members of the Kurant laboratory. We also thank E. Suss-Toby at the Interdepartmental Bioimaging facility for excellent technical support.
Reagent | |||
Annexin V | Molecular Probes | A35108 | |
PhiPhiLux G2D2 | OncoImmunin | A304R2G-5 | |
LysoTracker | Molecular Probes | L-7528 | |
Halocarbon oil 700 | Sigma | H8898 | |
Material | |||
Capillary tubing | FHC | 30-30-0 | |
Cell Strainer | SPL | 93100 | |
Paintbrush |