Visualization of the development of Drosophila embryos with a Polycomb-GFP fusionprotein

The movies presented here are meant as an addendum to our article
 
Dietzel, S., H. Niemann, B. Brückner, C. Maurange and R. Paro (1999).
The nuclear distribution of Polycomb during Drosophila melanogaster development shown with a GFP fusion protein. Chromosoma , 108(2):83-94. ( Abstract)

If you are not coming from there anyway, you might want to check out the introduction page .
The download section is at the end of this page. Maybe you want to start a download and read this page in parallel.


Introduction

In the four movies presented here, PC-GFP is used as a nuclear marker to follow the development of the embryo over time, starting shortly before blastoderm stage. During blastoderm the large fluorescent nuclei can be seen as bright dots on a black background. In the later stages with more and smaller nuclei the dots tend to fuse at the given low resolution. The autofluorescent chorion (the outer shell of the egg) and in later stages the autofluorescent guts are also visible. Until nuclear cycle 14 all nuclei in the Drosophila embryo divide synchronously in a syncytium where no new cell membranes are formed. Thus, at this stage the embryo is one multinuclear cell. When the nuclear membrane breaks down during mitosis, the nuclear content thus can leak unrestricted into the embryonic cytoplasm. In the movies, it can be observed in these stages that the bulk of the PC-GFP seems to disappear and then reappear during mitosis. This is due to the sudden distribution of the fluorescent molecules in a much larger volume, thus the signal to noise ratio decrease dramatically.
For furher information on Drosophila embryonic development see Foe et al. (in: The Development of Drosophila melanogaster, 1993, M. Bate and A. M. Arias. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp 149-300).

Legends

In all movies the embryo is viewed laterally, the ventral side left  and anterior towards the top of the image. Image size is 0.5 mm from top to bottom. The stack of four (or five) confocal sections shown in each frame was recorded at one time point (that is, of course, consecutively, one section after the other, starting with the one at the bottom. The scanning of one image stack took 55 seconds). The bottom section is relatively close to the center of the embryo, the top section is grazing through the outer edge of the embryo.
The first two movies show the embryo from the blastula stage until hatching of the larvae which can be observed at the end of these movies. The hatching of the larva at the end of the movie demonstrates that the observation conditions, e.g. phototoxicity, were not lethal. The waiting time between the recording of two stacks  is 15 minutes in these movies (excluding scanning time). The first movie contains 126 frames, the total duration is thus about 33 hours, the second movie has 130 frames, thus spanning about 34 hours. During the later stages of embryonic development the embryo starts to move within the egg. Since it took about 14 seconds to record one image (frame averaged), the images are somewhat blurred due to that motion . The movement of the larvae also caused the rotation of the egg towards the end of movie 1.
The third movie focuses on early stages. It starts before blastoderm and ends after germband retraction. Recording conditions were as above except that there was a variable waiting time between stacks: 5 minutes between the first 121 stacks, thereafter 30 minutes (each including 55 seconds scanning time). The larger intervals start at the beginning of germband retraction. The 140 frames thus add up to 120x5 +19x30 min = 19 hours 30 minutes. Nuclei become visible when they get to the surface of the embryo to form the blastoderm (10th frame of the movie). Cycle 14 can be easily distinguished from earlier cycles by the longer duration and the lack of mitotic waves thereafter. Then one can count backwards through the mitotic waves to determine when the nuclei reached the surface: It is during cycle 10 (in agreement with the literature).
The fourth movie starts before blastoderm and stops somewhat after pole cell movement begins. Delay between frames is 6 minutes (including scanning) and 34 frames are shown, adding up to 3 hours 18 minutes. As in movie 3, the nuclei are visible first during cycle 10.

Methods:

Embryos were mounted in paraffin (1-3) or voltalef (4) oil on a coverslip. The chorion was not removed and the chorion extensions were used to orient the embryos under a binocular. Then the coverslip was mounted on the inverted Leica TCS NT microscope of the ZMBH . Excitation wavelength was 488 nm,  a dichroic mirror at 500 nm and a bandpass 500 - 550 nm were used. Confocal sections were recorded at a low magnification (20x dry lens) so that the complete embryo could be imaged. The recording was performed at room temperature, around 22 Centigrade (72 F). The scanning of one image stack took 55 seconds for 1-3 and 60 seconds for 4). The left section is relatively close to the center of the embryo, the right section is grazing through the outer edge of the embryo. The images were transferred from the microscope to a Mac, further processed in NIH Image (e.g. reduced in size by a factor of 2) and saved as Quicktime Movies.


Download section

All movies are in Quicktime Format. You can download a Quicktime player for Macintosh or Windows from Apple's Web site if you need to, but  if you have a newer Version of these operating systems, chances are that you already have one, even if you don't know it.

Warning: These files are relatively large. You certainly do not want to download them via modem if you have to pay your phone bill by the minute. I offer two options for the download:

Option 1: Download via Web page. All four movies are embedded in this Web page . You will need a Browser with Quicktime Plug-in installed to see them. This is certainly the more convenient option if you have a fast internet connection to our site or if you want to download them all anyway. The combined file size of the movies is about 24 Mbytes.

Option 2: Download only those movies you are interested in from the table below. If you do not have a Quicktime Plug-in installed, your browser should ask you what to do with the files and offer the option  to save them to disk.
 
Movie 1 
on_e8unten_klein4.mov
5274 Kbytes
This movie is also available at Chromosoma's Web site.

Movie 2 
on_e9li.mov
7242 Kbytes

Movie 3 
on_e10li_kleinclean.mov
7835 Kbytes

Movie 4 
epcho_e8_4bis8bw.mov
4338 Kbytes



Last Update: 4. September 2000
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