Name: Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations (Video) | JoVE Journal
Uploaded: 2010-02-12T18:30:00.000+00:00
Duration: 8 min 35 s
Description: Learn live imaging of hemocyte migration in Drosophila melanogaster embryos using fluorescent labeling and mounting techniques. This protocol enables researchers to study embryonic macrophage dynamics in developmental immunology.

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5 minutes

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.

Preparation
<ol>
	<li>Obtain appropriate Drosophila lines containing a hemocyte-specific Gal4 driver (e.g. srp-Gal42) and a genetically encoded fluorescent reporter under UAS control (e.g. uas-GFP). Flies homozygous for srp-Gal4,uas-GMA3 or crq-Gal4,uas-GFP4, 5 are particularly useful for imaging purposes (n.b. GMA is GFP fused to the actin-binding domain of moesin); see below for a discussion of the range of Gal4 drivers and uas constructs available (the Bloomington Stock Centre contains a wide variety).</li>
	<li>Typically genetic crosses are carried out such that mutant alleles are balanced using fluorescent balancers CTG or TTG6, with Gal4 drivers and uas constructs carried on alternative homologous chromosomes. This makes it possible to select homozygous mutants on the basis of absence of CTG/TTG-associated GFP fluorescence (this is done at stage 2.11).</li>
	<li>Amplify stocks and place flies in a laying cage with an apple juice agar plate7. The flies need at least two days to acclimatize to the laying cage before enough embryos begin to be laid. In general twenty flies of each sex should be sufficient to generate enough embryos for imaging but it should be noted that different lines have differing degrees of fertility. We use 55mm Petri dishes that fit into the bottom of a plastic beaker, punctured at its base to allow airflow. The exact means of embryo collection is unimportant, but the timings are critical in order to collect correctly staged embryos.</li>
	<li>Collect embryos from an overnight apple juice agar plate maintained at 25°C or from a timed plate. For the latter we typically allow the flies to lay on a pre-warmed plate for 4 hours, before removing the plate and placing it at 18°C for 15-16 hours prior to mounting of embryos; this provides embryos from late stage 12 through to stage 15 of development. An overnight plate contains a greater diversity of stages but offers the advantage of higher levels of fluorescent reporter expression in hemocytes due to a longer period of time at 25°C as the Gal4-UAS system is temperature sensitive.</li>
</ol>
Procedure
<ol>
<li>Dislodge embryos from the apple juice agar plate using a small amount of water and a soft-tipped paintbrush. Dislodged embryos can be seen easily with the naked eye.</li>
<li>Transfer embryos to a cell strainer (Fisher) or home-made basket7 by pouring water from the apple juice agar plate into the basket held over a beaker to collect waste water.</li>
<li>Repeat step 2.2 until you are satisfied you have enough embryos transferred from the apple juice agar plate.</li>
<li>Wash embryos in cell strainer/basket using water.</li>
<li>Place cell strainer/basket in the Petri dish lid of the apple juice agar plate and add enough neat bleach to suspend embryos in the cell strainer/basket.</li>
<li>Follow dechorionation of the embryos on a dissection microscope under brightfield: dechorionation is complete when the dorsal appendages have dissolved, which should occur within two minutes.</li>
<li>Remove cell strainer/basket containing embryos from the bleach and wash off residual bleach using water. All traces of bleach should be removed before proceeding to step 2.8. One trick to assess whether all the bleach has been removed is to blot off residual water on blue-colored laboratory tissues - if there is residual bleach the blue color will be bleached white/pink.</li>
<li>Blot off remaining water using laboratory tissue/mediwipes applied to the underside of the cell strainer/basket.</li>
<li>Place a droplet of water in a Petri dish lid. With a fine paintbrush, collect all the dechorionated embryos from the embryo basket and resuspend them in the droplet. Next dry the embryos by aspirating water using a micropipette or carefully absorbing it with a laboratory tissue/mediwipes.</li>
<li>Once the embryos have been dried, add a drop of voltalef oil to cover all the embryos. Put a second small drop of oil adjacent to the droplet containing the embryos. N.B. we have been unable to find a UK-based supplier of voltalef oil; halocarbon oil 700 (Sigma) may be used instead. </li>
<li>Under a fluorescent dissection microscope select appropriately staged embryos of the desired genotype using a pair of watchmakers forceps (number 5) from the oil droplet. These forceps should be bent inwards (Figure 1) in order to scoop up the embryos without puncturing their vitelline membrane. Transfer selected embryos to the second oil droplet. It is important that you are able to see fluorescent hemocytes on the dissecting microscope in order to be able to collect good images on the confocal microscope (Figure 2). We typically mount stage 13/14 embryos to image lateral migration of hemocytes on the ventral midline or stage 15 embryos to image the motility of hemocytes following dispersal over the embryo.</li>
<li>Stick two coverslips (18x18mm, thickness 1) to the underside of a Petriperm/Lumox dish (Sarstedt), using 2 small drops of voltalef oil, leaving approximately 1cm between them (Figure 3); these will be used to support a coverslip placed over the embryos, so as not to crush them. Petriperm dishes (50mm diameter) contain a hydrophobic, gas-permeable membrane. We find that the dishes become easier to use once they have been used several times (dishes can be wiped with 70% ethanol and reused).</li>
<li>Under brightfield on the dissection microscope, pick up selected embryos one by one with the bent forceps and line them ventral side up and parallel to the edge of the coverslips (Figure 3). It is possible to align up to 15 embryos in this way, depending on your dexterity and your patience. It is important to manipulate the embryos gently as both the embryos and Petriperm dish membrane are fragile and can be easily ruptured.</li>
<li>Once the embryos are aligned add a small drop of oil and let it spread to form a homogenous layer between the two coverslips. After the oil has spread (this may take a few minutes) check that the embryos are still ventral side up. If the embryos have rolled slightly, reposition them again with the forceps.</li>
<li>Finally, using tweezers (number 3) place a coverslip (18x18mm, thickness 1) over the embryos, resting upon the two previously adhered coverslips. Glue this coverslip to the coverslip supports using nail polish (Figure 3).</li>
<li>Take the Petriperm dish with mounted embryos to the confocal or wide-field microscope and mount the Petriperm dish on the stage using an appropriate adapter. Either an upright or inverted microscope may be used, with the objective lens focusing through the coverslip (as opposed to through the membrane).</li>
</ol>
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.
<img src="/files/ftp_upload/1696/1696fig1.jpg" alt="Tweezer tips under microscope, examining material structure, microscopy analysis, magnification study." data-altupdatedat="1788235205000" data-alt="Figure 1"> 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.
<img src="/files/ftp_upload/1696/1696fig2.jpg" alt="Embryo development stages; fluorescent microscopy images; stages st13 to st17; cellular dynamics." data-altupdatedat="1788235205000" data-alt="Figure 2"> 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.
<img src="/files/ftp_upload/1696/1696fig3.jpg" alt="Embryo mounting diagram with Petri dish, coverslips, oil; process for microscopy." data-altupdatedat="1788235205000" data-alt="Figure 3"> 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).
<img src="/files/ftp_upload/1696/1696fig4.jpg" alt="Microscopy images showing cellular structures at 10 µm scale, essential for cell biology studies." data-altupdatedat="1788235205000" data-alt="Figure 4"> 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.
<img src="/files/ftp_upload/1696/1696fig5.jpg" alt="Microscopic images of cellular structures during development stages; magnification 10 μm; research data." data-altupdatedat="1788235205000" data-alt="Figure 5"> 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.

<ol><li>Brand, A. H., Perrimon, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Targeted+gene+expression+as+a+means+of+altering+cell+fates+and+generating+dominant+phenotypes%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">Targeted gene expression as a means of altering cell fates and generating dominant phenotypes.</a> Development. 118, 401-415 (1993).</li>
<li>Bruckner, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+PDGF%2FVEGF+receptor+controls+blood+cell+survival+in+Drosophila%5BTitle%5D)+AND+%22Dev+Cell%22%5BJournal%5D)">The PDGF/VEGF receptor controls blood cell survival in Drosophila.</a> Dev Cell. 7, 73-84 (2004).</li>
<li>Dutta, D., Bloor, J. W., Ruiz-Gomez, M., VijayRaghavan, K., Kiehart, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Real-time+imaging+of+morphogenetic+movements+in+Drosophila+using+Gal4-UAS-driven+expression+of+GFP+fused+to+the+actin-binding+domain+of+moesin%5BTitle%5D)+AND+%22Genesis%22%5BJournal%5D)">Real-time imaging of morphogenetic movements in Drosophila using Gal4-UAS-driven expression of GFP fused to the actin-binding domain of moesin.</a> Genesis. 34, 146-151 (2002).</li>
<li>Stramer, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Live+imaging+of+wound+inflammation+in+Drosophila+embryos+reveals+key+roles+for+small+GTPases+during+in+vivo+cell+migration%5BTitle%5D)+AND+%22J+Cell+Biol%22%5BJournal%5D)">Live imaging of wound inflammation in Drosophila embryos reveals key roles for small GTPases during in vivo cell migration.</a> J Cell Biol. 168, 567-573 (2005).</li>
<li>Wood, W., Jacinto, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Drosophila+melanogaster+embryonic+haemocytes%3A+masters+of+multitasking%5BTitle%5D)+AND+%22Nat+Rev+Mol+Cell+Biol%22%5BJournal%5D)">Drosophila melanogaster embryonic haemocytes: masters of multitasking.</a> Nat Rev Mol Cell Biol. 8, 542-551 (2007).</li>
<li>Halfon, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((New+fluorescent+protein+reporters+for+use+with+the+Drosophila+Gal4+expression+system+and+for+vital+detection+of+balancer+chromosomes%5BTitle%5D)+AND+%22Genesis%22%5BJournal%5D)">New fluorescent protein reporters for use with the Drosophila Gal4 expression system and for vital detection of balancer chromosomes.</a> Genesis. 34, 135-138 (2002).</li>
<li>Sullivan, W., Ashburner, M., Hawley, R. S. Drosophila protocols. , Cold Spring Harbor Laboratory Press, Cold Spring Harbor. NY. (2000).</li>
<li>Tepass, U., Fessler, L. I., Aziz, A., Hartenstein, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Embryonic+origin+of+hemocytes+and+their+relationship+to+cell+death+in+Drosophila%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">Embryonic origin of hemocytes and their relationship to cell death in Drosophila.</a> Development. 120, 1829-1837 (1994).</li>
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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...

<table><tbody><tr><td>Cell strainer</td><td>BD Biosciences</td><td>352350</td><td>70&amp;#x03BC;m pores</td></tr><tr><td>Halcarbon oil 700</td><td>Sigma-Aldrich</td><td>H8898</td><td></td></tr><tr><td>Lumox/Petriperm dish</td><td>Sarstedt Ltd</td><td>96077305</td><td></td></tr></tbody></table>

live imaging of drosophila melanogaster embryonic hemocyte migrations

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Many studies address cell migration using in vitro methods, whereas the physiologically relevant environment is that of the organism itself. Here we present a protocol for the mounting of Drosophila melanogaster embryos and subsequent live imaging of fluorescently labeled hemocytes, the embryonic macrophages of this organism. Using the Gal4-uas system1 we drive the expression of a variety of genetically encoded, fluorescently tagged markers in hemocytes to follow their developmental dispersal throughout the embryo. Following collection of embryos at the desired stage of development, the outer chorion is removed and the embryos are then mounted in halocarbon oil between a hydrophobic, gas-permeable membrane and a glass coverslip for live imaging. In addition to gross migratory parameters such as speed and directionality, higher resolution imaging coupled with the use of fluorescent reporters of F-actin and microtubules can provide more detailed information concerning the dynamics of these cytoskeletal components.

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.

Many studies address cell migration using in vitro methods, whereas the physiologically relevant environment is that of the organism itself. Here we ...

This video demonstrates a procedure for mounting drosophila embryos to image hemo cytes. Their embryonic macrophages. Embryos are laid by flies on apple juice agar plates in a laying cage overnight.The next day the agri plate is removed and the embryos are washed from the agri plate to a cell strainer following additional washes to remove debris. The cell strainer containing embryos is submerged in bleach to coate the embryos. After washing off the bleach, the embryos are transferred to a droplet of water.The droplet of water is then aspirated and the embryos are covered in hello carbon oil. Appropriately staged embryos with visible he cytes are transferred to a Petri per membrane between two affixed cover slips and carefully aligned in oil. A third cover slip is then placed over the embryos and glued into place with nail varnish.The motility of hemo cytes within the embryo can now be imaged through the uppermost cover slip on a microscope. Hi, I'm Jennifer Zaed from Brian Strm laboratory at the rental division of Seoul and Molecular biophysics at Kings College London. And I'm Ian Evans from the Wood Lab in the Department of Biology and Biochemistry at the University of Bath.Today we'll show you a procedure for mounting Josephs bryo to live image, the hemo site, the embryonic macrophages of this organism. We use this procedure to study the developmental dispersal and wind response to these important immune cells and the cytoskeletal machinery they utilize to carry out these processes. So Let's get started.Begin this procedure by obtaining appropriate TROs OAL lines containing hemo cyte specific gal four drivers and genetically encoded fluorescent reporters under UAS control. Here, serpent gal four is used to drive expression of a fluorescent red nuclear marker from UAS Red Stinger. For a discussion of the range of recommended GAL four drivers and UAS constructs, please see the accompanying written protocol place 20 drosophila of each sex in a laying cage.The laying cage is comprised of a 55 milliliter apple juice agri plate fitted to the bottom of a plastic tube with gauze covering the other end to allow airflow. Alternatively, a simple plastic peaker can be used with holes. Perforating the base, allow the flies to acclimatize to the laying cage for at least two days after the flies have acclimatized change to a new prewarm apple juice grate and allow the flies to lay overnight at 25 degrees Celsius.For information regarding the collection of more discreetly staged embryos, please see the accompanying written protocol. Once the flies have incubated for the appropriate amount of time for laying, use a wash bottle to apply approximately three milliliters of water to the plate. Then using a soft tipped paintbrush, dislodge embryos from the apple juice, aate dislodged embryos can be easily seen with the naked eye holding a cell strainer over a beaker.Pour the water from the apple juice aate into the cell strainer to collect the embryos. Then add more water to the apple juice.Aate. Repeat the straining process until the desired number of embryos has been collected.Next, using a wash bottle, wash the embryos in the cell strainer using approximately 10 milliliters of water. Once the embryos have been washed, place the cell strainer in the lid of the apple juice Aerate. Add neat bleach enough to suspend embryos in the cell strainer to coate the embryos.Transfer the cell strainer and petri dish lid to a dissection microscope to follow the decoration of the embryos. Under brightfield illumination, decoration is complete when the dorsal appendages have dissolved, which should occur within two minutes. After decoration is complete, remove the cell strainer containing the embryos from the bleach, again, holding the strainer over a beaker.Use a wash bottle to gently but thoroughly wash off residual bleach. Apply blue colored laboratory tissues to the underside of the cell strainer to blot off the remaining water. If the blue color changes to white or pink residual bleach is present, continue washing ensuring that all traces of bleach have been removed before proceeding to the next step.Removing excess water from the cell strainer makes it easier to pick up embryos with a paintbrush in the next step. Once all the bleach has been removed from the embryos, place a droplet of water in a Petri dish lid with a fine paintbrush. Collect all the coated embryos from the strainer and resuspend them in the droplet.Next, apply a chem wipe to the outside of the cell strainer to dry the embryos. Once the embryos have been dried, add a drop of halo carbon oil, 700 to cover all the embryos. Then place a second small drop of oil adjacent to the droplet containing the embryos.Inspect the embryos under a fluorescent dissection microscope. Identify appropriately staged embryos of the desired genotype to image lateral migration of he cytes. On the ventral midline.Choose stage 13 to 14 embryos. In this example, he cytes are identified by the fluorescent nuclei and are visible in the head and along the ventral nerve cord and developing dorsal vessel to image the motility of hemos following dispersal over the embryo, choose stage 15 embryos. At this stage, hemos will have dispersed over the entire embryo.However, three parallel lines of hemos should be visible on the ventral side of the embryo following lateral migration from the midline. Once the embryos have been selected, use a pair of curved number five watchmakers forceps to scoop up the selected embryos. Taking care not to puncture vial membranes.Then place the embryos in the second drop of oil on the underside of a 50 millimeter Petri perm lumax dish. Place two small drops of Halo Caron oil 700 about one centimeter apart. Apply a cover slip to each drop under brightfield illumination on a dissection microscope.Carefully transfer up to 15 embryos using curved forceps. Aligning the embryos ventral side up and parallel to the edge of the cover. Slips once the embryos are aligned at a small drop of oil and allow it to spread to form a homogenous layer between the two cover slips.After the oil has spread, this may take a few minutes. Check that the embryos are still ventral. Side up if the embryos have rolled slightly.Reposition them again with the forceps. Finally using number three tweezers. Place a third cover slip over the embryos.Bridging the two previously adhered cover slips. Glue this cover slip to the cover. Slip supports using nail polish.The embryos are now ready for imaging. This SRP gal four UAS Red Stinger embryo was mounted ventral side up and time-lapse images were acquired on a Leica M 2 0 5 fluorescent dissection microscope. He cytes are visualized by their red labeled nuclei.The movie begins at stage 12 to 13 of development when the he cytes leave the head and migrate down the ventral midline. After about one hour, he cytes begin migrating off the midline to disperse to lateral positions along the ventral surface. For the remainder of development.The hemo cytes are highly active, migrating throughout the embryo. We have to show you how to monitor Joseph embryo to follow the movement or the hemo side live in vivo. When doing this procedure, it's important to handle the embryos carefully, especially on the petro perm dish, to avoid damaging either once the embryos are in oil, they're relatively resistant to dehydration, but care should be taken not to bleach the embryos for too long when removing the corion.Lastly, by mounting several embryos, you will give yourself the best possible chance to obtain an embryo in the perfect orientation for live imaging. So that's it. Thanks for watching and good luck with your experiments.

live-imaging-of-drosophila-melanogaster-embryonic-hemocyte-migrations

Research

JoVE Journal

Biology

Here we describe a protocol to examine the migration of glial cells into the developing Drosophila eye using live microscopic analysis paired with GFP tagged glial cells.

Live Imaging of Glial Cell Migration in the Drosophila Eye Imaginal Disc

Source: Hiroyasu, A., et al. Extraction of Hemocytes from Drosophila melanogaster Larvae for Microbial Infection and Analysis. J. Vis. Exp. (2018)This video demonstrates high-resolution confocal imaging of Drosophila melanogaster hemocytes infected with Coxiella burnetii to visualize pathogen localization in 3D, enabling detailed analysis of intracellular infection dynamics and host-pathogen interactions.

High-Resolution Confocal Imaging of Pathogen-Infected Drosophila Hemocytes

Source: Hellerman, M. B., et al. Live-imaging of the Drosophila Pupal Eye. J. Vis. Exp. (2015)The video demonstrates a live-imaging technique to study eye patterning in a Drosophila melanogaster pupa. Green fluorescent protein (GFP)-tagged markers enable visualization of cellular organization and differentiation during the formation of the developing eye structure.

Live Imaging of the Drosophila Pupal Eye Using Fluorescence Microscopy

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.

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

This paper demonstrates the use of a fast scanning confocal microscope to image cell behavior directly through the puparium. By leaving the pupal case intact, this method allows observation and measurement of dynamic cell processes at a stage of Drosophila development that is difficult to study directly.

Imaging Through the Pupal Case of Drosophila melanogaster

This protocol presents an efficient method for imaging the live Drosophila pupal eye neuroepithelium. This method compensates for tissue movement and uneven topology, enhances visualization of cell boundaries through the use of multiple GFP-tagged junction proteins, and uses an easily-assembled imaging rig.

Live-imaging of the Drosophila Pupal Eye

Drosophila blood cells, or hemocytes, cycle between resident sites and circulation.  In the larva, resident (sessile) hemocytes localize to inductive microenvironments, the Hematopoietic Pockets, while circulating hemocytes move freely in the hemolymph. The goal of this protocol is the standardized isolation and quantification of these two, behaviorally distinct but interchanging, hemocyte populations.

Assaying Blood Cell Populations of the Drosophila melanogaster Larva

This method demonstrates how to visualize pathogen invasion into insect cells with three-dimensional (3D) models. Hemocytes from Drosophila larvae were infected with viral or bacterial pathogens, either ex vivo or in vivo. Infected hemocytes were then fixed and stained for imaging with a confocal microscope and subsequent 3D cellular reconstruction.

Extraction of Hemocytes from Drosophila melanogaster Larvae for Microbial Infection and Analysis

Here we present a protocol for live-imaging wound repair and the associated inflammatory response at high spatio-temporal resolution in vivo. This method utilizes the pupal stage of Drosophila development to enable long-term imaging and tracking of specific cell populations over time and is compatible with efficient RNAi-mediated gene inactivation.

Long-term In Vivo Tracking of Inflammatory Cell Dynamics Within Drosophila Pupae

Light sheet-based fluorescence microscopy is the most valuable tool in developmental biology. A major issue in comparative studies is ambient variance. Our protocol describes an experimental framework for simultaneous live imaging of multiple specimens and, therefore, addresses this issue pro-actively.

Simultaneous Live Imaging of Multiple Insect Embryos in Sample Chamber-Based Light Sheet Fluorescence Microscopes

Here, we provide a detailed protocol to perform live imaging of the asymmetric division of germline stem cells (GSCs) in the Drosophila ovarian niche. We use a transgenic line that ubiquitously expresses a green fluorescent protein (GFP) fusion of the spectrosome protein Par-1.

Extended Live Imaging of Female Drosophila melanogaster Germline Stem Cell Niches

Learn live imaging of hemocyte migration in Drosophila melanogaster embryos using fluorescent labeling and mounting techniques. This protocol enables researchers to study embryonic macrophage dynamics in developmental immunology.

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

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