Last year, I published a snapshot of a mitotic wave in a fruit fly embryo. Here’s the video of that same embryo going through cleavage (nuclei divisions) and gastrulation (cell movements):
Mitotic waves
What you see at the beginning of the movie are the cycles of synchronous nuclei divisions. They happen in waves from the posterior to the anterior side (from right to left). Fly embryos undergo fourteen cleavage cycles after fertilization, but the movie only starts on the tenth. After each cycle, the embryo gets more packed with nuclei until they cover its entire surface.
At this point, the embryo is still a syncytium, that is, a single cell with many nuclei (yeah, that’s how flies do it). But on the 14th division cycle, about 11s into the movie, the embryo cellularizes, each nucleus being encapsulated by cell membranes. It’s a curious process, though not visible in this video.
Gastrulation
Once cells form, the embryo begins to gastrulate. Gastrulation in flies is complicated, and many events happen at the same time. Cells move, invaginate, disappear, flow around the embryo, and start dividing again, again, and again. This movie shows about 3 hours of development, but embryogenesis continues for 24 hours post-fertilization until the embryo hatches out of the egg as a larva.
Sample
The fly in the video is a transgenic line containing two types of fluorescent proteins. One is a green fluorescent protein (GFP) which is attached to a histone (H2A), a protein that binds to DNA in the cell nucleus. The other is a variant red fluorescent protein known as mCherry, which is fused to a protein that attaches to cell membranes (GAP43). In this way, we can see where the chromatin and cell membranes are located in the embryo during development (and learn something from it).
Acquisition
After collecting embryos, I glued them sideways on a tiny glass coverslip attached to the sample holder of the microscope, a Zeiss Z.1 Lightsheet. The scope has lasers to excite the fluorescent proteins, which in turn emit light at different wavelengths. Filters allow us to capture the individual signals at the same time. This is crucial for fast acquisition and makes two-color time-lapses possible.
I set the microscope to acquire 30 slices spaced by 3 µm, from the outer surface of the left side to the middle section of the body. One timepoint was taken every 35 s.
Processing
After the recording, I performed some image processing steps using Fiji/ImageJ.
Denoise and subtract background
First, I ran Despeckle to reduce camera noise (it’s low but helps to smoothen the image). Then, Subtract background... (with rolling=20) to remove out-of-focus information. Fly embryos have a dense yolk sac inside that can blur the fluorescence.
Project to 2D
While 3D renderings for such data look great, I decided to flatten it to 2D, to visualize all the information in the same image. For that, I did a maximum intensity projection using the Z Project... command. This looks through the 30 slices in each pixel position, and only keeps the maximum value for each pixel. It’s a practical way to visualize 3D data in 2D.
Color code
Finally, we can color code each fluorescent marker differently for better visualization. Some colors match perfectly, others don’t. To map pixel intensities to color gradients in ImageJ, we use lookup tables (or LUTs). For the DNA signal, I chose a purple-yellow gradient called mpl-inferno, a perceptually uniform colormap that I like a lot. For the cell membranes I picked a standard grayscale (gray is great!).
The end
That’s it, I hope you enjoy the video! I’ve uploaded a copy to Wikimedia Commons for eternity. You are welcome to use it and if you need more info, please contact me.
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