Drosophila hemocytes disperse over the entirety of the developing embryo. This protocol demonstrates how to mount and image these migrations using embryos with fluorescently labelled hemocytes.
Preparation
Procedimento
Representative Result:
This protocol describes how to mount Drosophila embryos for live imaging of hemocytes on the ventral side of the embryo. If done correctly it will be easy to generate either stills or movies of hemocytes. The major determinant is the microscope used to image the hemocytes (in particular the objective lens), but the nature of the images acquired will also depend upon the stage of development, the temperature the embryos were raised at and the Gal4 and uas lines used.
Higher levels of fluorescent protein expression will enable hemocytes to be imaged with greater ease, therefore it is important to be able to see hemocytes when the embryos are at stage 2.11 of the protocol (Figure 2 contains examples of clear hemocytes within embryos, taken with a camera fitted to a dissection microscope). Therefore increased numbers of Gal4 and uas constructs enable a greater signal-to-noise ratio. Furthermore this reduces the need for high laser intensities or increased exposure times when imaging, which in turn enables hemocyte behavior to be followed for longer periods of time.
Very high levels of GFP expression will show up fine details of hemocyte morphology, particularly the thin sheet-like lamellae that surround the circular cell body (Figure 4A-B). Circular regions excluding GFP represent phagosomes (Figure 4A-C). Finger-like filopodia can also be seen emerging from the lamellae (Figure 4B). Two Gal4 drivers remain sufficient to see these processes (Figure 4C), particularly if one or more is srp-Gal4 (see discussion), however slower scanning speeds or greater laser power on the confocal microscope may be required. As expression levels decrease it becomes more difficult to image the protrusions of hemocytes; nonetheless it is still possible to track the migration of hemocytes under these conditions as the cell body remains obvious even when protrusions are less clear (Figure 4D).
At earlier stages of development (up to stage 13) hemocytes migrate in close contact to one another and it is often hard to distinguish individual cells. By the end of stage 13 hemocytes have formed a single line down the ventral midline (Figure 5A), then, becoming more motile, migrate laterally to the edges of the ventral nerve cord (Figure 5B). The actin cytoskeleton within the dynamic protrusions of hemocytes can be directly observed through expression of GMA (Figure 5C) or cherry-moesin.
Mounting the embryos in this way allows gas exchange and prevents dehydration and embryos remain viable following imaging. If the embryo is damaged during mounting it is generally obvious as the embryo content will leak through its vitelline membrane. If an embryo does start to dehydrate then this can often been seen by deformations in the vitelline membrane. Occasionally an embryo will roll during the course of a timelapse movie, however this only tends to be problematic for longer timescale movies. Lastly, mounting several embryos at once gives the experimentalist the best chance of obtaining an embryo in the perfect orientation for their experiment.
Figure 1. Forceps for manipulation of dechorionated embryos.
The tips of watchmakers forceps (size number 5) should be bent inwards in order to fashion a tool to scoop up embryos as shown here. The outer surface of the bent region is also useful to manipulate embryos when positioning on the Petriperm membrane as they possess no sharp edges that could puncture the embryo.
Figure 2. Representative images of embryos that will yield good live imaging results.
Images of dechorionated embryos in voltalef oil (at stage 2.11 of the protocol) taken on a fluorescent dissecting microscope. Lateral views of stage 13 (A) and stage 15 (B) srp-Gal4,uas-GFP;crq-Gal4,uas-GFP embryos. Lateral view of a stage 15 srp-Gal4,uas-GFP/+;crq-Gal4,uas-GFP/uas-N17Rac embryo (C) in which hemocytes have failed to migrate out of the head, demonstrating what embryos look like when hemocytes are not apparent along their migratory routes. Lateral view of a stage 17 srp-Gal4,uas-GFP;crq-Gal4,uas-GFP embryo showing the convoluted structure of the gut at this stage of development (D); the onset of muscle contraction prevents live imaging of embryos beyond this stage of development. Ventral views of stage 13 (E) and stage 14 (F) srp-Gal4,uas-red stinger embryos showing dispersal of hemocytes with fluorescently labeled nuclei. Observation of hemocytes by fluorescence at stage 2.11 of the protocol is a prerequisite to obtain excellent images; anterior is to the right for all images.
Figure 3. Mounting of embryos on a Petriperm/Lumox dish.
Two 18x18mm coverslips (thickness 1) are stuck to the bottom face of the Petriperm dish using a small drop of oil, separated by about 1cm as shown. Embryos are then lined up ventral side up with their long (anterior-posterior) axis parallel to the edges of the coverslips and covered with a small drop of oil. Once the oil has spread to fill the gap between the two coverslips a third coverslip (18x18mm thickness 1) is gently placed on top of the oil-covered embryos using the two previously adhered coverslips as a bridge to prevent the embryos from being squashed. This coverslip is then glued to the two coverslip bridges using two small drops of nail polish. Once set, the embryos can be imaged on an upright or inverted microscope with the objective lens focusing down through the coverslip (as opposed to through the Petriperm membrane).
Figure 4. Representative results from live imaging of GFP labeled hemocytes.
Z-projections of hemocytes on the ventral side of a stage 14 srp-Gal4,uas-GFP;crq-Gal4,uas-GFP embryo (A-B). (A) is a lower magnification image such as used to monitor hemocyte developmental migrations in timelapse movies. (B) is a higher magnification still of hemocytes on the ventral midline, showing fine details of their morphology. (C) is a single 1 m slice of hemocytes on the ventral midline in a stage 14 srp-Gal4,uas-GFP/+;crq-Gal4,uas-GFP/+ embryo, revealing that lower copy numbers of Gal4 drivers and uas constructs are also sufficient to generate good images. (D) displays a z-projection of hemocytes in a stage 14 crq-Gal4,uas-GFP embryo. Here hemocyte protrusions are less obvious due to lower expression of GFP but it is still possible to make movies and track hemocyte migration with this combination of Gal4 driver and uas construct. Images were taken on a Leica LSM510 confocal microscope; anterior is up in all images; the rings at the periphery of images are caused by vitelline membrane autofluorescence.
Figure 5. Representative results from live imaging of GMA expressing hemocytes.
Z-projections of hemocytes on the ventral midline of stage 13 (A) and stage 14 (B) srp-Gal4,uas-GMA embryos, taken from timelapse movies to show developmental migrations of hemocytes. Detailed information on actin dynamics can be obtained by higher magnification imaging of GMA expressing hemocytes (C). GMA consists of GFP fused to the actin-binding domain of moesin and labels actin filaments. Anterior is up in all images; images were taken on a confocal microscope.
The most important elements of this procedure are the selection of healthy embryos with clearly labeled hemocytes and to mount them carefully without damaging them. Once the embryos are in the halocarbon oil they are resistant to dehydration and once mounted can be imaged for several hours. In our hands we can image hemocytes for three hours, with negligible dehydration of the embryo or obvious photo-damage, taking a z-stack of images every three minutes on our Zeiss LSM510 confocal microscope with a 40X objective. Since hemocytes are highly dynamic there may a trade-off between spatial and temporal resolution: if a very high-resolution image is needed the hemocyte and structures within it may move during the scan. The precise details of how images are subsequently collected will be determined by the experimental questions to be addressed.
We typically use uas-GFP or uas-GMA3 to label hemocytes; uas-GMA is particularly useful since, in addition to marking hemocytes, it provides a read-out of actin filament dynamics. Other uas constructs may be used to probe hemocyte behavior: for example uas-cherry constructs9 can be exploited as alternative fluorophores or uas-tau-GFP10 could be used to visualize the microtubules within a hemocyte. Uas constructs with a nuclear label may be particularly valuable for automated tracking of hemocyte movements (Figure 2E-F). Two-color imaging is also possible (e.g. one can label both the actin and microtubule cytoskeletons using uas-cherry-moesin and uas-tau-GFP, respectively, under the control of a hemocyte-specific Gal4 driver). As previously mentioned, a key determinant in live imaging of hemocytes is the Gal4 driver employed: as regards to current hemocyte-specific promoters srp-Gal42 > crq-Gal411 > pxn-Gal44, with only srp-Gal4 sufficient to label hemocytes when heterozygous, whereas at least two copies of crq-Gal4 or pxn-Gal4 are needed to observe hemocytes live. Nonetheless optimal imaging with srp-Gal4 requires homozygosity (or the presence of an alternative Gal4 driver).
By following hemocytes live in this way it is possible to study their developmental dispersal from the head8 and observe how they interact with the cues that pattern this process, deposit matrix and engulf apoptotic corpses. Hemocytes also represent an interesting system with which to probe the machinery of cell migration; by examining the migration of hemocytes in embryos that lack components of the actin or microtubule cytoskeletons it is possible to understand their in vivo function more clearly (e.g. Rho GTPases4, 12). Exploitation of uas constructs to label these cytoskeletons provides yet more detailed information on their regulation.
We have modified this technique to probe hemocyte behavior in a variety of contexts such as their responses to laser-induced wounds4 and injection of fluorescently labeled bacteria13. Furthermore this method can easily be adapted to image other cell types by choosing different Gal4 drivers. For example we have previously imaged dorsal closure using epithelial Gal4 drivers14, 15. The application of this technique to other cell types is limited by the strength of the Gal4 driver, the position of the cells to be imaged within the embryo and the type of microscope used for imaging; multiphoton confocal microscopes enable the user to image cell behavior deep within the embryo (e.g. germ cell transepithelial migration16), whereas conventional confocal microscopes are unable to reach these depths (this is why we image along the ventral midline where hemocytes are very superficial, trapped between the developing ventral nerve cord and epidermis).
The strength of this protocol is that it provides an environment to maintain embryos in a healthy condition for live imaging. While a degree of manual dexterity is required to position the embryos, it is an easy technique to master and quickly gives reproducible results and can easily be modified using different Gal4 drivers and uas constructs to probe many different aspects of hemocyte biology. Additionally, the protocol is not restricted to hemocytes since the use of alternative Gal4 drivers enables the behavior of other tissue types to be analyzed.
This protocol has been developed through our work within and in collaboration with the laboratories of Paul Martin and Antonio Jacinto. We thank the Bloomington Stock Centre for its excellent service and the Drosophila community for continuing to share fly lines. BS is currently funded by a BBSRC project grant. WW is funded by a Wellcome Trust Career Development Fellowship.
Material Name | Tipo | Company | Catalogue Number | Comment |
---|---|---|---|---|
Cell strainer | BD Falcon | 352350 | 70μm pores | |
Halcarbon oil 700 | Sigma | H8898 | ||
Lumox/Petriperm dish | Sarstedt | 96077305 |