| The zebrafish (Danio rerio) provides a rare opportunity to visualize and quantify in real time the microscopic development of the living eye, one of the most exquisite functional systems in biology. Development of the vertebrate ocular lens begins with an epithelial placode in the cephalic surface ectoderm that normally produces epidermal or neural cells that scatter light and are not refractile or transparent. Cells of the developing lens must undergo exquisite specializations to achieve the high index of refraction, glass-like transparency, and almost perfect symmetry required for a functional element in the optical pathway. The zebrafish has unique differences from the mammal in regard to early lens development and crystallin protein expression that may relate to the aquatic environment in which it lives. Rather than forming a hollow, fluid filled lens vesicle that pinches off from the surface ectoderm, the developing zebrafish lens forms as a solid mass of cells that delaminate to separate the lens from the cornea. This dissertation will present the first systematic, high-resolution, quantitative, real-time, three-dimensional description of lens development in a living vertebrate from the placode stage at 16 hours postfertilization (hpf) through 4 days postfertilization (dpf). Live-cell imaging will establish a fate map for the lens placode and provide a direct and progressive view of the establishment of primary fibers, an anterior epithelium, and secondary fibers during development of the symmetric, transparent lens. Immunohistochemistry will demonstrate primary fiber cell differentiation, anterior epithelial cell differentiation, and progressive delamination of the lens mass from the surface ectoderm that does not involve apoptosis as a primary mechanism. Quasielastic laser light scattering and proteomics will demonstrate similarities in biophysical properties and protein expression between zebrafish lens and cornea. Size exclusion chromatography and shotgun proteomics will characterize protein expression in the zebrafish lens during maturation and aging to identify novel crystallins and demonstrate the importance of alpha crystallin in the aging lens. Based on these results, eye organogenesis and lens cell differentiation appear to be well conserved among vertebrates despite the lack of a lens vesicle stage in zebrafish. |
