I stained the embryo for 2 days in Alcian Blue, followed by 2 days in Alizarin Red. Then I let the embryo destain for several days. Below you can see the lizard embryo in a 50 ml conical tube.
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| Anolis carolinensis embryo skeletal prep |
I was really surprised by what I found! Initially, I hypothesized that the yellow chunks I saw on the embryo were hardened pieces of yolk and/or some sort of mold that was growing on the embryo (since the egg was not discovered for sometime until after the embryo was inanimate). HOWEVER, upon staining for calcium these yellow dots turned pink! These dots are both superficial (on the scales) and deep (found adjacent to bone and underneath deep muscle).
What are these "deposits" in the lizard embryo? I am not really sure if the pathology formed pre or post mortem. The fact that there are deposits deep to the bone makes me think that this process began while the embryo was still animate.
Below is the curve of the forming vertebral column. You can see one of these pink dots.
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| Vertebrae in an Anole lizard embryo skeletal prep (left lateral view) |
Below is the hip joint, covered in a cluster of 5+ dots. (The vertical long blue bone is the femur.)
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| Lizard Embryo vertebrae (right lateral view) |
Below you can see the upper thigh. The lower leg still has scales on; however if the KOH doesn't remove it soon, I will. Doing a skeletal prep on a tiny embryo is a ridiculously difficult task requiring a lot of patience.
Even on full grown lizards, it is easy to make a mistake while dissecting and remove a whole arm, or break (autotomize) the tail. The anole tail has fracture planes built into it, which allow an anole to escape if caught by a predator (called self-autotomy).
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| Green anole tail autotomy. Note the cup-shaped bone reminants from the tail break. |
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| Close up of caudal vertebrate autotomy, or fracture planes in Anolis carolinensis |
When dissecting, the tails tend to break rather easily, especially if the supporting muscles around the vertebrae have been removed. This is but another challenge to doing a skeletal prep on an anole. An autotomized lizard is below.
Hopefully, the KOH will make it easier to remove the scales, revealing the lower leg.
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| Anole lizard embryo femur and vertebrae (right lateral view) |
I dissected the leg a bit more, to reveal the tibia, fibula and patella.
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| Femur, patella and proximal tibia of an anole lizard embryo |
Here is the foot. You can faintly see long metatarsal bones below.
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| Right foot: metacarpals of an anole lizard embryo |
Below, you can see where the bones of the hand, the metacarpals, are forming. I left some of the scales on the hand; however after more staining with the 0.1% Alizarin Red + 0.2% KOH, some of this tissue should disintegrate off, revealing the finger bones beneath.
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| Anole lizard embryo hand and metacarpals |
I did notice a lower concentration of cartilage staining overall and I wonder if this because it needs an extra day in Alcian blue. The bones of the hand below appear clear. I am currently restaining the embryo with more Alcian Blue; the cartilage should pick up some of the stain.
Under a thin layer of scales, you can faintly see some red staining on the outline of the jawbone.
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| Anolis carolinensis embryo skeletal prep (left lateral view) |
Below (in pink) you can see what is presumably an embryonic lizard's clavicle. It is very slim and after more cartilage staining, should articulate with the scapula of the back (dorsal) and the sternum of the (ventral) front.
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| Shoulder girdle of an anole lizard embryo (left lateral view) |
Here is the radius and ulna of the forearm. Near the ends you can see a bit more red, indicating the beginning of calcium matrix deposition.
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| Left Radius and Ulna of a lizard embryo (left lateral view) |
Below is a side view of the back of the head. You can see (pink) skull bones.
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| Jaw and skull of a green anole embryo (left lateral view) |
Here is a closer shot of those spots, which appear superficial to the vertebrae (in pink). They are clustered in groups and under the microscope appear as bright pink. I wonder if the Alizarin Red is binding non-specifically to some minerals. The stain is supposed to target Osteogenic calcium deposits, which indicate extracellular matrix ossification.
After restaining with Alcian Blue a bit, the cartilage color should look more brilliant. Following that, I will do another round of Alizarin Red, wherein the white dots below will probably take on a redder hue. Notice the leg below. One of the "deposits" is very close to the femur, deep under the skin. I have no idea what could be causing these presumably post-mortem embryonic deposits.
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| Anole lizard embryo (right lateral view) |
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| Anole lizard embryo (right lateral view) |
After a second round of Alician Blue, I notice the lamellae of the toe pads took on an interesting pattern. There are intermittent stripes of blue.
Anoles, like geckoes, have lamellae (containing microscopic setae) that allow them to adhere to the surface of leaves very strongly, through molecular forces like the van der Waals force. It appears that there are cartiliage-like strips on the toe pad.
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| Anole lizard embryo foot |
What is interesting is their location on the toes. Their location varies on each toe. Some have two parallel strips and some only have one.
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| Lizard embryo toes |
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| Anole lizard embryo lamellae |
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| Anolis carolinensis embryo lamellae and toes |
All embryos were treated ethically according to IUCAC protocol.
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