1Department of Biology, Case Western Reserve University, 2Department of Genetics, Case Western Reserve University
Belu, M., Javier, M., Ayasoufi, K., Frischmann, S., Jin, C., Wang, K., et al. Upright Imaging of Drosophila Embryos. J. Vis. Exp. (43), e2175, doi:10.3791/2175 (2010).
1. Imaging Methodology
2. Representative Results
Because the embryos are left intact, this method can be used to take precise measurements of embryo size and distances between gene expression patterns. In Figure 1, we show a blastoderm stage embryos stained with the nuclear stain DAPI (blue), anti-Dorsal protein (red), sog mRNA (yellow) and sna mRNA (green). Morphological processes, such as invagination of the mesoderm (Figure 2A, B) and migration of mesodermal cells (Figure 3 and 4) can also be visualized using this method. Different Z-positions along the DV axis can be obtained by changing the focal plane, as shown in Figures 2-4.
Figure 1. Upright optical slice of an intact embryo in blastoderm stage. Double staining for mRNA and protein. Target mRNAs for in situ probes (sog and sna) and protein stained by primary antibody (anti-Dorsal) are indicated in the figure. DAPI was used to label the nuclei. Note that the expression of sog and sna are located apically in the cytoplasm, while the expression of Dorsal is nuclear. Strong signals from sog nascent transcripts that are located in nuclei can also be seen at this magnification.
Figure 2. Upright imaging of an intact gastrulating embryo. mRNA probes used and their respective colors are indicated in the figure. A) Note that in this stage, three genes stained with same fluor (sna, sog and dpp, in green), are expressed in spatially separated domains. The invaginating mesoderm expresses sna, the neuroblast layer expresses ind and the ectoderm expresses dpp, while sog expression is still present in ventral regions of the nervous system. B) In a deeper confocal plane of the same embryo, we note the base of the invaginating mesoderm strongly expresses sna, but its apical region expresses lower levels.
Figure 3. Upright imaging of an intact embryo with germ band extended. sog RNA(yellow); snail, ind and dpp mRNAs (green), Dorsal protein (red). Nuclei were stained with DAPI (blue). A) Focal plane near embryo head. B) Another focal plane in the trunk region of same embryo. Note mass of mesodermal cells before lateral migration (compare to Figure 4) lying close to the ventral midline on both sides of a germ-band extended embryo. Delaminating neuroblasts are labeled in green (ind and sna).
Figure 4. Upright imaging of an intact embryo during germ band extension. sog RNA(yellow); snail, ind and dpp mRNAs (green), Dorsal protein (red). A) Note the delaminating neuroblasts from the epithelium (arrow), stained both for ind and sna at this stage (green). Note also the restricted expression of sog to the ventral midline (yellow). Expression of dpp at this stage can be seen at lateral sides of the embryo (asterisk). B) Optical section in the same embryo shown in A near the end of the embryo length, which corresponds to mid-posterior region. The ventral midline cells are stained for sog.
Taking cross section images of Drosophila embryos can be challenging, since it requires either a careful hand dissection of slices 2 or the use of embedded media for microtome processing, which is usually detrimental for fluorescent stainings. Alternatively, Z-stacks made in a Confocal microscope can be used to make 3D reconstruction of cross-section images. However, it is time consuming to make several thin Confocal slices for an accurate 3D reconstruction and photobleaching becomes problematical. Another concern when imaging embryos that are mounted longitudinally is the light scattering that occurs when reaching deeper sections of the embryo, which also complicates taking precise measurements of fluorescent signals for the purpose of quantification of protein or mRNA expression levels1. Even with a two-photon confocal microscope, light scattering is still a concern due to the opacity of yolk material in the middle of the embryo, which makes selection of mounting media for optimal transparency of sample to be limited.
To circumvent these problems, a new technique called "End-on" imaging of positioning embryos vertically was recently developed to image both live and fixed samples 4. In this paper, we developed a new protocol for vertically mounted embryos that allows a simple preparation and visualization of fixed embryos or tissues of any size and shape that are stained with multiple in situ probes 3. Our method consists of using a mounting media with gelatinous consistency that is used to encase aligned embryos within it. Cut blocks of this mounting media containing embedded embryos can be positioned upright before imaging in any type of Confocal microscope.
By embedding the embryos into the jelly and positioning them vertically, we are able to take horizontal cross-sections using Confocal microscopy, in which cells along the dorsal-ventral axis of the embryo are presented simultaneously. This is a significant improvement for quantification purposes since problems of light scattering and photobleaching of fluorescent dyes that occurs in conventional Z-stack reconstruction of longitudinally mounted embryos are now eliminated. Thus, quantification of gene expression or protein levels along the dorsal-ventral axis is accurate, since cells located at opposite dorsal-ventral positions are imaged at the same time and at the same confocal Z-position. In addition, the described method allows us to obtain a complete 2-D image across the dorsal-ventral axis from a 3-D embryo without using complex computational unrolling methods to visualize cells at different positions along the DV axis 6.
Some of the critical steps include removing excess glycerol after aligning embryos, to avoid split between the two layers of jelly. However, if the sample is too dehydrated, the resulting image will be misshapen and have incorrect morphology. In addition, if the jelly medium around the embryo is cut at an angle rather than straight, the resulting image may appear less round and more ovular in shape, due to an incorrect positioning of the embryos at an angle other than 90 degrees. Finally, if the jelly is not cut closely enough to the embryo on the anterior/posterior ends, it is not possible to obtain a proper image because the objective's working distance would be mainly used to focus into the jelly and not the embryo.
Another important step that may be necessary when imaging late gastrulating embryo is to carefully sort the stages of interest under the scope before aligning them. Usually, embryos are staged according to morphological characteristics that can be most easily recognized when in a longitudinal position.
Among alternative modifications that can be made with this method is to include an anti-fade reagent (e.g. p-Phenylenediamine or PPD) into the second layer of jelly to help protect against photobleaching of fluorescent dyes.
No conflicts of interest declared.
The authors are especially indebted to Deborah Harris and Danielle MacKay. Support for this work was provided by the Department of Biology and the College of Arts and Sciences of CWRU, and by HHMI grant number 52005866 for support of undergraduate education in the biological sciences.
|Glycerin Jelly mounting media||Electron Microscopy Sciences||17998-10||Contains low concentration of phenol as preservative and should be used in hood or in well ventilated area. Recipe for making your own media is described in Zander, 1997.|
|Zeiss LSM 700 Confocal||Carl Zeiss, Inc.||The following objectives were used: Plan-Apo 20x 0.8 M27 (WD 0.55mm); EC Plan-Neofluar 40x 1.3 oil (WD 0.21mm); LD Plan-Neofluar 0.6 Korr 40X (WD 2.9mm).|
|Phosphate buffered saline with 0.1% Tween 20 (PBT)|
|Glass microscope slides||Fisher Scientific||12-544-7|
|Glass cover slips||Electron Microscopy Sciences||72204-02||Size 1 ½ (specific for the objectives used in this work).|
|Glass cover slips (for making supports)||Fisher Scientific||12-544-10||Use four coverslips glued with superglue.|
|Dissection microscope||M5 Wild Heerbrugg Wild Makroskop||M420|
|Razor blade||Steel back single edge industrial blades|
|Hypodermic needles||Fisher Scientific||1482610||26 Gauge|