ABS 123: Dinosaur to Lizard Eyes: Exploring Scleral Ossicle Development in the Anolis sagrei

Nareem Al-Shurafa, Leah Green, Rida Osman, Michael Valdes, Krishi Desai, Doug Menke, James D Lauderdale

Van Wickle (2025) Volume 1, ABS 123

Introduction: The scleral ossicle, a ring of small bony structures in the front of the eye, helps maintain the eye’s mechanical stability to protect it from aging and environmental forces. In addition, scleral ossicles contribute to the shape of the eye by acting as a point of attachment for ciliary muscles. This decreases the stress on other structures and increases the eye’s ability to focus. Previously, scleral ossicles have been identified in various reptiles, fish, birds, and even in dinosaurs. However, they have only been rigorously studied in the domestic chicken, Gallus gallus. Due to this narrow scope, our understanding of ossicle development and the underlying genetic mechanisms controlling this process are lacking. Thus highlighting the need for studies on a broader range of organisms to gain a comprehensive understanding of scleral ossicle development.

The brown anole lizard, Anolis sagrei, is an attractive model for studying ossicle development and would establish a reptilian model for this research. The brown anole exhibits clear developmental stages, quick reproduction rates, and is easy to maintain in the lab, making it an ideal model to study the process of embryogenesis.

This project investigates the morphology and development of the scleral ossicles in the Anolis sagrei. To achieve this, we used Alizarin Red for bone visualization and micro-CT scans to visualize the scleral ossicles. For the first time, we determined how individual ossicle pieces fit together in this species, characterized any unique morphological features of each ossicle, and established the developmental timeline of ossicle formation. Additionally, we quantified the number of ossicles present at hatching and in adulthood. We predict that each ossicle will retain an identical shape to its corresponding counterpart in a matching embryo. Through this research, we hope to deepen our insights into ossicle structure, while also providing important data for vertebrate evolution.

Methods: We developed a staining protocol to visualize the ossicle ring in Anolis sagrei and assess ossicle count and morphology. The posterior eyes from adults and hatchlings were dissected to maintain the ossicle ring, incubated in 4% KOH for tissue clearing, and bleached with 30% H₂O₂. Samples were washed in PBS and fixed in ethanol. Alizarin Red S was used to stain calcium deposits overnight. Eyes were imaged under a compound microscope to assess ossicle count, overlap, and positioning. Each ossicle was photographed from anterior and posterior views to document morphology and dimensions. We also used peanut agglutinin (PNA) staining to visualize condensation development in the brown anole. We began with embryos in 100% ethanol. To prepare for wax infiltration, we gradually replaced the ethanol to xylene, and embedded the samples. Once solidified, the blocks were sectioned at 5 µm thickness. We lastly completed this procedure by staining with PNA.

Results: When looking at ossicle number, we can conclude that there are consistently 14 ossicles among all anoles and between left and right eyes. Ossicles 7, 9, and 14 were consistently at the front of the eye; 5, 8, and 11 toward the back. Overlap patterns were stable across individuals and eyes, though individual ossicle morphology varied among the same and different individuals. PNA staining revealed distinct, densely packed mesenchymal cells formed in the scleral region. This was consistent with Gallus gallus condensations in size and position. However, the stain lacks strong specificity, and better glycoprotein stains are needed.

Discussion: In regards to there being consistently 14 ossicles among all individuals, this was the first piece of data that showed us differences in ossicle development between species and helped establish Anolis sagrei as a model for reptilian ossicle development. The consistent numbering and overlap of specific ossicles across individuals and between left and right eyes indicates a stable developmental mechanism. Although shape varies, the interlocking pattern remains consistent, suggesting early patterning through signaling cues are likely. This, along with consistent condensations, aligns with the intramembranous ossification model. Similar condensation shape and positioning in Gallus gallus suggests a conserved mechanism among these two species.