Early development of the fruit fly, Drosophila melanogaster, is characterized by a number of cell shape changes that are well suited for imaging approaches. This article will describe basic tools and methods required for live confocal imaging of Drosophila embryos, and will focus on a cell shape change called cellularization.
The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging. Cell shape changes in the fly are analogous to those in higher organisms, and they drive tissue morphogenesis. So, in many cases, their study has direct implications for understanding human disease (Table 1)1-5. On the sub-cellular scale, these cell shape changes are the product of activities ranging from gene expression to signal transduction, cell polarity, cytoskeletal remodeling and membrane trafficking. Thus, the Drosophila embryo provides not only the context to evaluate cell shape changes as they relate to tissue morphogenesis, but also offers a completely physiological environment to study the sub-cellular activities that shape cells.
The protocol described here is designed to image a specific cell shape change called cellularization. Cellularization is a process of dramatic plasma membrane growth, and it ultimately converts the syncytial embryo into the cellular blastoderm. That is, at interphase of mitotic cycle 14, the plasma membrane simultaneously invaginates around each of ~6000 cortically anchored nuclei to generate a sheet of primary epithelial cells. Counter to previous suggestions, cellularization is not driven by Myosin-2 contractility6, but is instead fueled largely by exocytosis of membrane from internal stores7. Thus, cellularization is an excellent system for studying membrane trafficking during cell shape changes that require plasma membrane invagination or expansion, such as cytokinesis or transverse-tubule (T-tubule) morphogenesis in muscle.
Note that this protocol is easily applied to the imaging of other cell shape changes in the fly embryo, and only requires slight adaptations such as changing the stage of embryo collection, or using “embryo glue” to mount the embryo in a specific orientation (Table 1)8-19. In all cases, the workflow is basically the same (Figure 1). Standard methods for cloning and Drosophila transgenesis are used to prepare stable fly stocks that express a protein of interest, fused to Green Fluorescent Protein (GFP) or its variants, and these flies provide a renewable source of embryos. Alternatively, fluorescent proteins/probes are directly introduced into fly embryos via straightforward micro-injection techniques9-10. Then, depending on the developmental event and cell shape change to be imaged, embryos are collected and staged by morphology on a dissecting microscope, and finally positioned and mounted for time-lapse imaging on a confocal microscope.
1. Assemble Embryo Collection Cups
2. Make Apple Juice Agar Plates
Notes:
3. Add GFP Flies to the Embryo Collection Cups
4. Prepare a Mounting Chamber
Notes:
5. Dechorionate Embryos
Notes:
6. Stage and Mount Embryos
Notes:
7. Image Embryos
Note:
8. Alternative Method: Mount Embryos with “embryo-glue”
9. Representative Results:
If the embryos are healthy and the imaging is optimal, then cellularization should take 50-60 minutes, and the plasma membrane invaginations should ingress almost 40 microns. However, if the embryos are over-bleached, oxygen deprived or damaged by phototoxicity, then invagination will either slow or stop, particularly in the imaged area. Such deterioration of embryo health often results in altered development, and a failure to hatch as larvae. Thus, for a rigorous test to assay embryo health after imaging, keep your slides in a humidified chamber and watch for hatching the next day.
Figure 1. Workflow from embryo collection to imaging. The workflow of the protocol can be broken down into four main phases. In the first phase, all the individual supplies and components are prepared, and then the embryo collection cup, apple juice agar plate and GFP flies are put together to create the embryo-laying environment. In the second phase, the eggs and embryos that are laid on the apple juice agar plates are collected. In the third phase, the embryos are removed from the plate, staged and transferred to the mounting chamber. In the fourth phase, the mounted embryos are imaged on a confocal microscope.
Figure 2. Representative data from time-lapse imaging of cellularization. Embryos are mounted with dorsal (D) and ventral (V) sides clearly visible, and are imaged near their middle to follow the plasma membrane invaginations in cross-section. The embryo shown here expresses a GFP-Myosin-2 probe6, which concentrates at the tips of the plasma membrane invaginations. Thus, tracking the ingression of this front over time gives the rate at which the plasma membrane invaginates. The 0:00 minute time point corresponds to cellularization onset. Shortly after the 56:00 minute time point, gastrulation starts on the ventral side of the embryo. Bar is 40 microns.
Movie 1. Representative movie from time-lapse imaging of cellularization. This movie corresponds to Figure 2. To record the entire process of cellularization, imaging started in the prior mitotic cycle 13, capturing pseudocleavage furrow regression, and continued until gastrulation movements were seen on the ventral side of the embryo. Images were collected at one minute intervals. The intensities were increased post-acquisition to make it easier to see the gastrulation movements.
Click here for movie
Developmental event and timing* | Cell shape changes related to | A link to disease or human health | Recent references with live imaging |
Pseudo-cleavage furrow formation (4; 90 minutes pf) |
Cytokinesis | Polyploidy and cancer progression1 | Mavrakis et al., 20098 Cao et al., 20109 |
Cellularization (5; 130 minutes pf) |
Cytokinesis | Polyploidy and cancer progression1 | Cao et al., 200810 Sokac & Wieschaus, 200811 |
Ventral furrow formation; Mesoderm invagination (6; 180 minutes pf) |
Apical constriction; Epithelial-mesenchymal transition | Cancer metastasis2 | Fox & Peifer, 200712 Martin et al., 200913 |
Germband extension (7; 195 minutes pf) |
Convergent extension | Neural tube defects3 | Bertet et al., 200414 Blankenship et al., 200615 |
Tracheogenesis (11; 320 minutes pf) |
Epithelial tube formation and branching | Angiogenesis4 | Caussinus et al., 200816 Gervais & Casanova, 201017 |
Dorsal closure (14; 620 minutes pf) |
Apical constriction | Wound healing5 | Gorfinkiel et al., 200918 Solon et al., 200919 |
Table 1. Examples of cell shape changes imaged in living fly embryos
*The Bownes stage number and time post-fertilization (pf), when each event starts, are listed according to Campos-Ortega,1985.
Fly stock | Labels | Original reference |
Spider-GFP (95-1) | Plasma membrane | Morin et al., 200122 |
Resille-GFP (117-2) | Plasma membrane | Morin et al., 200122 |
GAP43-Venus | Plasma membrane | Mavrakis et al., 20098 |
Spaghetti Squash-GFP (Sqh-GFP) | Myosin-2 | Royou et al., 20026 |
E-cadherin-GFP (Ecad-GFP) | Cell-cell junctions | Oda et al., 200123 |
GFP-Moesin | F-actin | Kiehart et al., 200024 |
Utrophin-Venus (Utro-Venus) | F-actin | Sokac et al., unpublished results |
Table 2. Useful stocks for imaging cell shape change in fly embryos
The protocol described herein will permit the live, confocal imaging of a number of cell shape changes in the developing fly embryo. GFP stocks for imaging can be prepared by an individual lab (Table 2), but many such stocks are also publicly available from centers such as Bloomington Drosophila Stock Center at Indiana University (http://flystocks.bio.indiana.edu) and FlyTrap Stock Center at Yale University (http://flytrap.med.yale.edu). Once acquired these live imaging data can then be paired with quantitative analysis to thoroughly define protein functions, sub-cellular activities, and biophysical parameters that drive cell shape change and tissue morphogenesis.
The authors have nothing to disclose.
We gratefully acknowledge Eric Wieschaus, who provided the foundation on which this protocol was developed. Our work is supported by a Verna & Marrs McLean Department of Biochemistry and Molecular Biology Start-up Award, Baylor College of Medicine.
Material Name | Tipo | Company | Catalogue Number | Comment |
---|---|---|---|---|
Slides | Fisherbrand/Fisher Scientific | 12-550-343 | ||
Cover slips 25×25 | Fisher Scientific (Corning #2865-25) | 1 2-524C | ||
Squirt bottles (H2O) | Fisher Scientific | 02-897-11 | ||
50 ml Falcon tubes | Fisher Scientific (BD# 352070) | 14-432-22 | ||
Bulbs for small pipets, 1 mL | Fisherbrand/Fisher Scientific | 03-448-21 | ||
Scintillation vials with caps | VWR (Wheaton #986546) | 66021-533 | ||
Tri-Corn Beakers, 100 mL | Electron Microscopy Sciences | 60970 | ||
BD Falcon Petri dish 60x15mm | Fisher Scientific (BD# 351007) | 08757 100B | ||
BD Falcon Cell strainer | Fisher Scientific (BD#352350) | 08-771-2 | ||
Yellow pipet tips | Ranin | L200 | ||
Stainless steel mesh, 304, 12×24 | Small Parts | CX-0150-F-01 | ||
Glass 5¾ inch Pasteur Pipets | Fisherbrand/Fisher Scientific | 13-678-20B | ||
P4 Filter paper | Fisherbrand/Fisher Scientific | 09-803-6F | ||
Rubber bands | Office Max | A620645 | ||
Scotch double-sided tape, ½ inch | Office Max | A8137DM-2 | ||
Robert Simmons Expression paint brushes E85 round #2 | Jerry’s Artarama | 56460 | ||
Dumont #5 Forceps High Precision Inox | Electron Microscopy Services | 72701-DZ | ||
Razor blades | VWR (BD #214010) | 55411-050 | ||
Halocarbon Oil 27 | Sigma-Aldrich | H 8773 | ||
Heptane | Fisher Chemical/Fisher Scientific | H360-1 | ||
BD Bacto Agar | VWR (BD# 214010) | 90000-760 | ||
Sucrose | Sigma-Aldrich | S7903 | ||
p-Hydroxybenzoic acid | Sigma-Aldrich | H5501 | ||
Red Star Active Dry Yeast | LeSaffre | 15700 | ||
Paper towels, C-fold | Kleenex | |||
Heavy duty aluminum foil | Reynolds Wrap | |||
Bleach | Austin’s A-1 Commercial | |||
100% Apple juice | Ocean Spray or Tree Top |