Beginner's look at Embryology

25 May 2012

I mainly study the green anole lizard, including at times, developing embryos. The golden anole egg is about the size of a large jelly bean. It is soft (though firm) and leathery. It is made in part by fibers of protein enclosing a yolk sac. The egg rotates as it passes through an ovary, winding a fabric-like sheath of protein 360 degrees around the egg.

The architecture of the egg is remarkable, in that it has to serve two functions simultaneously. It has to keep moisture in and it has to let gas diffuse out. During development, veins develop a network through the yolk, bringing nutrients to the developing embryo, as arteries help move metabolic wastes, like gasses away from the embryo. Normally arteries carry oxygen-rich blood to the systemic part of the body and veins carry depleted blood back to the heart, but in the early stages of development this is reversed. The biophysical environment, if disturbed, can result in quick destabilization.

Anolis carolinensis lizard egg

To dissect the eggs, I first fill a petri dish with PBS, or phosphate-buffered solution. The PBS helps to control osmosis. Putting the egg into plain water can cause diffusion that can be destructive to the cells in the embryo. A budding embryologist quickly learns that even the smallest forces, like the movement of water molecules, are enough to harm an embryo. 

While I am dissecting, I am constantly recalibrating my awareness to fit the tiny world of the embryo, where even the smallest movements I make can have enormous effects. This mindset has very much affected the way that I see biology. Effects from processes at one scale ripple through to smaller scales, sometimes creating strange and marvelous patterns. 

Biophysics and Embryos
Amniotic eggs evolved in and are "optimal" for a certain physical environment. It is vital that internal hydrostatic pressure is maintained during development. When the hydrostatic pressure changes, deformities in the embryo frequently occur. Sometimes I open up an egg that looks a little withered. When I find the embryo inside, part of its body is collapsed (most noticeable in the eyes or head). The blood flow is reduced and sometimes appears completely coagulated. The force of blood flowing is probably necessary to counter the attractive qualities of blood elements, such as fibrinogens, which are sticky proteins that help your blood clot. The fluidic pressure and blood flow has to be above a certain level to keep the “pipeworks” of the embryo clear.

This shows that development requires the right physical features in order to create a normal embryo. And it also implies that the current “developmental program” we see now must, itself, have evolved under the same conditions. In fact, small scale forces are a source of cheap energy for embryos. Imagine how much energy would be required if every molecule had to be “pushed” around the embryo instead of passively diffusing. Things like cohesion (Van der Waals forces) and Brownian motion (particles vibrating, bumping into each other, scattering and diffusing) are all mechanisms of development.

The cleidoic egg is a contained microcosm capable of self-organization and generating complex adult organisms. Every time I pick up a golden brown egg and am ready to dissect it, I am reminded of our ancient vertebrate past. The egg is a symbol of so many things, like life and fertility, but also the addition of novel innovations from unending evolutionary processes. 

The anole egg has an inner sac, as well, which holds the yolk in. I can peel the outer shell off and by shining a light through the egg, I can sometimes see where the embryo is positioned. 
 

Anole lizard egg with the outer shell removed

Opening the Egg
The moment you open the egg is filled with anxiety. With the first hole, the internal pressure is shot and the yellow yolk comes torpedoing out. Somewhere in the murky dish there could be an embryo floating around. Or it could still be lodged within a partially deflated shell. For the fledging biologist this is always a moment of terror, when you don’t know if the embryo will shoot out of a tiny hole, shredding it to pieces. Although the outer shell is leathery, it is still very strong and hard to rip. If you try ripping off patches, the pressure along a strand, if pressed up against the embryo is enough to tear it in two. So the shell very much becomes the enemy, as does your own forceps. Even the slightest jerk of your hand looks exaggerated under the microscope. One small jerk can destroy the embryo.

I always believe that patience trumps talent, so instead of risking a decapitated embryo, I instead unwrap the outer eggshell in strips. I try to put my forceps between the outer shell and the inner shell before I tear a tiny hole in it. Then I start to remove the outer shell. 

What you can see below is the yolk still contained inside an inner sac. Now, I have a much more controlled attempt at removing the embryo, as well as having a visual on its location. I make sure to choose a side opposite from the embryo before I pierce the inner sac. 

Inner sac of an anole lizard egg

At this point, I still need to be careful that there are no strands of shell that can harm the embryo. Sometimes those strands can tear through the embryo, like piano wire, when you pull them apart. In the picture below, you can see a ring of tissue around the middle of the yolk; this could rip the embryo. It's vital to pay attention to how your movements may affect the embryo.

Another difficulty in extracting embryos is- what do you grab onto? While you need a little bit of force to remove the embryo from the surrounding tissues (which don't tear so easily) the embryos are delicate and prone to damage. For instance, you can't grab a limb bud and pull in the other direction. Oftentimes, you only have a thin translucent layer of tissue which you can use to hold onto the embryo, like part of the inner sac, or tissue near the umbilicus. That doesn't make it any less challenging though!

Anole lizard egg yolk

The embryo is tucked away in the middle pocket of the yolk, above. After I remove the outer shell and the thinner membrane surrounding the embryo, the yolk will begin seeping out. 

 Most of the time it looks like an explosion of yolk and tissue. The PBS becomes cloudy and it may take a while to sift through the mess to find the actual embryo. Can you spot the embryo below? Look for the circular/spiral of the tail. This embryo is pretty small, though some are so small its almost impossible to find them in the yolk, as there aren't even visible signs of vasculature. Red blood cells atleast give you a rough idea of where the embryo is.

Anole lizard embryo and vasculature

This next one was particularly neat and orderly, but they are rarely so.
You can see within the tangle of veins where the embryo is. The black spot is the embryo's eye.
 

Anole lizard embryo wrapped in yolk

The red branches of the vascular network surrounding the embryo show me where it is. I approach carefully, using my forceps to gently remove the yellow, and sometimes stringy, yolk that surrounds it. Piece by piece I make my way towards the middle. 

There also another inner sac around the embryo, as well. This sac also presents difficulty, as it is kind of "pressurized." That means when I open it, at the point of pressure release, tissue near it will be sucked out quickly. With younger embryos, the thin, translucent inner sac is enough to tear the head off of an embryo, which can happen once you open the sac. In a split second, the embryo gets sucked out of the hole you make, possibly destroying it.

Secondly, it is very hard to hold the sac with the tweezers. Its like trying to pick up an ice cube with chopsticks... the sac evades many attempts at being pinched. 

Anole lizard embryo in inner sac

 
I have learned how to grab the sac, near the top of the head, and quickly rip it open (like a bag of potatoe chips), by tearing the sac in opposite directions. If done right, the embryo will pop out right out, very cleanly. If you pierced the inner sac but didn't manage to get the embyro out, it will sometimes cling tightly to the embryo, making it very difficult to find a place to grab it (much like finding the end of a tape roll). 

Anole lizard embryo wrapped tightly in sac and vasculature

Oftentimes the embryo is still encompassed by an innermost sac, and you have to tediously clean the embryo one stroke at a time, trying to differentiate between the transulucent sac and the near-translucent embryo. As always, patience is a virtue! 

With the embryo below, I partially removed the sac. The caudal part of the embryo is out, while the sac is stuck around the embryo's head and neck. This is a tricky situation, as I can either reach under the "chin" or to the back of the neck. Directly under where the future jawbone will be, is the beating heart. Even with steady hands, tearing the sac from there is a challenge.

Anole lizard embryo, with sac partially removed

Once I’ve cleared most of the yolk, I now I have the challenge of pinching off the umbilical cord, the last remaining connection the embryo has to its yellowy nutritional source. This technique can be quite difficult, because the forceps barely fit between the tail and the heart to pinch off the umbilicus. Ever so gently I pinch the forceps and remove the last of the yolk. 

Anole lizard embryo

Now the embryo looks similar to what you’d see in a book. It takes a bit of effort and delving through the tiny world of an egg to get it out. After all the yolk is removed, I am done. The embryos are left overnight in 4% Paraformaldehye in PBS at 4 degrees Celsius. Then I will take them through a dehydration serious of 25%, 50%, 75% and 100% Methanol, for 5 minutes each. After that, they are stored in a -20 degrees Celsius freezer.

Anole lizard embryo

The embryo becomes a frozen biological capsule. The molecules floating around in the once active embryo are frozen as well (from the PFA), allowing us to probe what genes were being expressed in what areas (in a specific developmental time window) in the embryo. Embryos can be used for in situ hydrizations (ISH), like the mouse embryo below.

Anolis carolinensis in situ hybridization (ISH) of lunatic fringe gene

Every where purple is expressed shows where the gene product, lunatic fringe, is. This gives us an idea of what occurs in development. For example, at 9.5 days old (or dpc, days post coitum) the lunatic fringe gene is being expressed in the neural tube, the head and the eye. Technologies, like ISH, allow molecular biologists to analyze the molecular components of developmental processes. 

Just to show you how big these embryos are, here is a comparison next to my finger. Four embryos are sitting in "netwells." Older embryos will be a just bit bigger, but you get the point.
 

Anole lizard embryos in netwells for in situ hybridization (ISH)

I would like to add that all embryos were treated ethically according to an IUCAC protocol, and have not yet developed central nervous systems or brains to process sensory information. 

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