Bark Anole Skeletal Preps

Today I prepared a Bark Anole embryo for a skeletal prep. I took it through a skeletal staining protocol which included the preparation of a 0.3% Alician Blue staining solution. I dropped the pH down to 1.0 - 1.5 in order to select for very negatively charged particles, as sulfated GAGs. I put the embryo in solution for 2 hours at room temperature. Then I left the embryo in 100% EtOH for one day.

After that, I took the embryo through a rehydration series of 100% EtOH -> 75% -> 50% -> 25%. Tomorrow, I will put it through 2 distilled water washes before preparing an Alizarin Red staining solution (to stain for bone). This will allow me to visualize some skeletal development. Pics from the early steps of the protocol are pictured below.

Bark Anole stained blue for cartilage.

Bark anole staining for cartilage.

Next, I placed the embryo in a 0.1% Alizarin Red solution to stain for calcium in developing bone. I used 0.5% KOH when making the Alizarin Red solution. The sample will sit at room temp for one day.

Bark anole lizard embryo in Alizarin Red Staining solution

After a few hours in the Alizarin Red/0.5% KOH, some of the tissue disintegrated, allowing for a better visualization of the hard tissues, like cartilage. I apologize for the quality of the photos. The blue staining overwhelms the camera's color profile and it can be hard to photograph.

Bark anole embryo, Alician Blue/Alizarin Red Staining

Bark anole head, jaw, eyes, iris

Bark Anole fingers

Bark anole lizard pelvic girdle

Bark anole tail; caudal vertebrae

After 1 day in Alizarin Red staining solution I could not detect any Alizarin stain for developing bone. The 0.5% KOH might have been too strong, as the specimen appears as if it might fall apart.

Bark anole lizard torso

Bark anole caudal vertebrae

Bark anole lizard pre-vertebral column

 In this picture, I can see a lighter blue "rod" like structure that is at the ventral side of the pre-vertebral column. I suspect this is the remaining notochord, which will disintegrate. While there is a bit of controversy about how the notochord changes during development, it appears it regresses except for in the intervertebral region, or the pulposus. According to Zhang's an Evo-Devo view of the origin of the backbone: "Sclerotomal cells migrate toward, and enwrap, the notochord, where they develop into vertebral bodies and intervertebral discs/ligaments at later developmental stages" (Zhang 2009).

caudal vertebrae

caudal vertebrae

Bark anole arm and ribs

The dorsal fissures are closed in this bark anole

After leaving the specimen in Alizarin Red for 2 days, it appears that there are no bone pre-cursors yet. I will be staining several more specimens from the Glor Lab at various stages of development. Final photos are below.

Bark anole embryo. Alcian blue stains for cartilage.

Dorsal view of the rostral bark anole embryo

Lateral view of a Anolis distichus skeletal prep

Bark Anole embryo. Ventral Side of the torso.

Dorsal side of the rib cage

Neural arches can be see to the top right of the speciman. Hemal arches below.

Another thing struck me about the Bark Anole skeletal prep. The rib-bearing pre-vertebrae are oddly distributed. There should be more rib protrusions between the upper rib-cage and the protrusions seen at the middle of the picture. This is very odd.

Bark Anole skeleton prep.

Below I am comparing the near-hatchling Green Anole embryo with the Day 14-28 Bark Anole embryo. While they are at different developmental stages, I still wanted to do a side-by-side comparison. In particular, I am drawn to the dorsal edge of the pre-vertebrae. In the Green Anole, there appears to be a fissure, where the two sides of the pre-vertebral structures have yet to meet and fuse. While the Bark Anole has not developed any bone yet, it appears that the dorsal edges of a cartilage pre-cursor have met and fused at the midline. This strikes me as odd and worthy of investigation.

Green Anole on Left. Bark Anole on Right.

Anolis carolinensis on Left. Anolis distichus on Right.

All embryos were treated ethically.  

Bark anole or Anolis distichus embryos were provided by Anthony Geneva from the Rich Glor lab in Rochester, NY.

  


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