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biology articles imaging

Mitotic waves and gastrulation in a fly embryo

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 (cycles of nuclei divisions) and gastrulation in the fruit fly Drosophila melanogaster. Also on Wikimedia Commons.

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.

Foe and Odell 1989 Nuclear cycles
Cycles of nuclear divisions in early Drosophila embryogenesis (Foe and Odell 1989).

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 nuclei 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.

Foe and Odell 1989 Gastrulation
Developmental events during the gastrulation of Drosophila (Foe and Odell 1989).

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 simultaneously. 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. While 3D renderings for this type of data look great, I decided to flatten the image to 2D.

First, I ran Despeckle to reduce some noise (it’s low but helps smoothen the image). Second, I ran Subtract background... (with rolling=20) to remove out-of-focus information. Fly embryos have a dense yolk sac inside that can blur the fluorescence. Finally, 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 maxima values. It’s a practical way to visualize 3D data in 2D.

Lastly, we can color-code each fluorescent marker differently for better visualization. Some colors fit well together, others don’t. To map pixel intensities to color gradients in ImageJ we use lookup tables (or LUTs). Here, I chose a purple-yellow gradient named mpl-inferno for the DNA signal, a perceptually uniform colormap I like a lot, and a standard grayscale for the membranes (gray is great).

The end

That’s it, I hope you enjoy the video! I’ve uploaded a copy to Wikimedia Commons for eternity. Feel free to use it and if you need additional info, please contact me.

Categories
biology notes

Platynereis or Spotify?

Every time I open Spotify, I see the pattern of engrailed expression in an early Platynereis larva. Once you see it, there is no turning back!

Platynereis or Spotify?
The similarity between engrailed expression in Platynereis and the Spotify logo. A) Whole-mount in situ hybridization of engrailed in a 48h larva of the annelid Platynereis dumerilii (Prud’homme et al. 2003). B) Illustration of engrailed expression pattern in Platynereis. C) Spotify logo. D) Illustration of adapted Spotify logo.

Reference

Prud’homme, B., de Rosa, R., Arendt, D., Julien, J.-F., Pajaziti, R., Dorresteijn, A. W. C., Adoutte, A., Wittbrodt, J., & Balavoine, G. (2003). Arthropod-like expression patterns of engrailed and wingless in the annelid Platynereis dumerilii suggest a role in segment formation. Current Biology: CB, 13(21), 1876–1881. https://doi.org/10.1016/j.cub.2003.10.006

Categories
imaging biology notes

A mitotic wave

A mitotic wave traveling through an early #Drosophila #embryo #FlyFriday

3D HisGap cleavage cover
Early syncytial embryo of the fly Drosophila melanogaster. Nuclei (blue) are dividing in a wave from posterior to anterior. Membrane components (white) are already organized around the nuclei. The image is a frame from a timelapse acquired under lightsheet microscopy and rendered in 3D.
Categories
imaging articles code

ImageJ macro to synchronize and combine image stacks

The embryos I study rarely develop in perfect synchrony. That means that when I film them under the microscope some embryos will be younger—or older—than others.

ImageJ macro with Drosophila embryo
Using an ImageJ macro to help me analyze movies of Drosophila embryos.

For this reason, I often need to synchronize the recordings to make sure they all begin at the same embryonic stage. When the movies are synchronized I can combine them side-by-side, and it becomes much easier to compare and spot differences between two embryos.

ImageJ macros save time

Combining movies in Fiji/ImageJ is straightforward using the Combine... command. But synchronizing is way harder. It depends on human classification and involves some calculations and stack juggling that can (and will) become tedious.

To help me out, I wrote a small ImageJ macro available here: SyncAndCombineStacks.ijm. Follow below to see how it works.

Combined movies without syncing

That’s what unsynchronized movies look like. I combined them fresh off the microscope without any synchronization:

Two embryos of the fruit fly Drosophila melanogaster. Both were acquired in the same microscopy session. The top embryo is older than the bottom embryo.

Combined movies after syncing

Here are the same two movies now synchronized by the embryonic stage:

The same two embryos are now synchronized.

How it works

The macro performs the hard work. It calculates how many frames to trim from each stack. Then it duplicates the selected range of frames common to both stacks. Finally, it combines the synchronized recordings into a single image stack. All you need to do is to select the corresponding frames between the two stacks.

Step-by-step instructions

Here are the instructions step-by-step:

  1. Open both image stacks in ImageJ.
  2. Adjust the contrast if needed (before running the macro).
  3. Select a reference frame in the top stack (e.g. stage easy to recognize).
  4. Select the correspondent frame in the bottom stack.
  5. Run the macro and fill in the dialog parameters.
  6. Click OK, wait a few seconds, and check if the synchronization is good. Otherwise, re-run with different parameters.

Screencast

I’ve also recorded a small screencast:

Note! The macro does not touch the original stacks, but it outputs an RGB Color stack. There are a couple of reasons for that. Converting to RGB avoids contrast issues when the stacks have different pixel intensities. It also prevents quirks in video players that can’t handle 16-bit movies. But if you need to perform image analyses on the final stack, remove this option. I may add a checkbox for that in the future.

Categories
biology imaging notes

The blastopore of bryozoan embryos

This is a bryozoan embryo exhibiting its blastopore. These animals are discreet but ubiquitous in oceans and lakes all over the world.

Bryozoan embryo during gastrulation revealing its blastopore.
Embryo of the bryozoan Membranipora membranacea under confocal microscopy.

What we see is the DNA inside the nucleus of the cells of the embryo. The color gradient indicates if the nuclei are closer (yellow) or further away (purple) from the microscope camera.

The embryonic cells are arranged in a circle and form a central opening that we call the blastopore. This opening, in bryozoans, will become the mouth of the animal after the embryo develops.

You can follow the process on video or learn more details in the paper.

What about our mouth, where does it come from?

Categories
biology imaging notes

Fruit fly embryo under lightsheet microscopy

A short video that I made about the embryonic development of the likeable Drosophila, also known as fruit fly or vinegar fly, won an honorable mention in the Small World in Motion.

A single embryo imaged from four different angles.

The details on the techniques I used and the video on its full resolution are available for download and re-use on the Wikimedia Commons.

Categories
imaging biology notes

The surface of a brachiopod embryo

Brachiopod embryo showing its surface and blastopore.
Embryo of the brachiopod Novocrania anomala at the gastrula stage showing its outer surface and the blastopore at the bottom. Cell membranes were stained (F-actin) and the original image stack was converted to a 3D animation using Fiji/ImageJ.
Categories
biology notes

Understanding the evolution of cleavage patterns in early embryonic development

Video summary about our work on bryozoan development and the evolution of cleavage patterns published in BMC Biology!

The video was produced by Research Square. Also available on Vimeo.

Categories
biology imaging notes

Bryozoan embryos viewed from the animal and vegetal poles

The first 24 hours in 1.5 minutes of bryozoan embryos.

Categories
notes biology imaging

Bryozoan embryos viewed from the animal pole