Graduate Student Dalhousie University Halifax, Nova Scotia, Canada
Abstract Body : Scleral ossicle bones can be seen in the eyes of many vertebrates, including reptiles, and their development is easily studied in the embryonic chicken eye. Previous research has described how these bones are induced through a corresponding set of papillae in the conjunctival epithelium of the eye. However, the induction of the conjunctival papillae is not yet understood. Interestingly, the early development of these conjunctival papillae involves a placode stage, which is a common feature of other epithelial appendages (e.g., feathers, hair, scales) and sensory organs (e.g., lens, olfactory, and otic placodes). A subset of this research has demonstrated that hydrocortisone treatment disrupts conjunctival placodes and papillae formation. The objective of my research was to refine the use of hydrocortisone to fully inhibit all conjunctival papillae in the developing chicken eye and to use this phenotype to and identify key factors in their development. Using stepwise adjustments, I established the precise timing (approximately HH28 and HH30) and dose (2mM) of hydrocortisone treatment to consistently prevent all conjunctival papillae development. I then assessed the effect of hydrocortisone treatment on apoptotic cell death, the developing eye vasculature, and mRNA expression of three placode-related genes (β-catenin, FGF20, EDA). Apoptotic cell death was assessed at HH34 using a combination of TUNEL assay and confocal microscopy. Fluorescein-conjugated lectin was circulated throughout the embryo to highlight the developing eye vasculature at HH34 and imaged using confocal microscopy. Gene expression was analyzed with qPCR and in situ hybridization at HH32 and HH34, approximately. This research did not reveal significant changes in apoptotic cell death or vasculature as a result of hydrocortisone treatment but did demonstrate a significant decrease in FGF20 expression at HH32, approximately 2.5 days after treatment. This research contributes to our understanding of the inductive structures responsible for this very unique set of bones and points to additional avenues of investigation into how and why some vertebrate eyes develop these bones and others do not.
