Cell fate and differentiation of the developing ocular lens.

The generation of a highly symmetric, refractile, transparent lens from a sheet of undifferentiated cranial epithelial cells is one of the most remarkable processes in development. Highly coordinated cellular proliferation, polarization, migration, and elongation lead to the reorganization of the surface cells in the lens placode to a functional optical element in the visual pathway, the ocular lens. The symmetry necessary for refraction of light and image formation in the visual system requires a high degree of precision in specifying the fates of individual cells and their spatial relationships in the adult lens. A functional visual system is especially important for species such as humans and zebrafish, in which vision is the primary sensory pathway for seeking food. Developmental defects in the lens are extremely rare, suggestive of unique intersecting, developmental pathways that are protected against the deleterious effects of cell and molecular aging. In mammalian, avian, and teleost species, lens formation begins as cells of the surface ectoderm become columnar to create the lens placode overlying the optic primordium in the embryonic head.1–5 The placode invaginates to form a lens pit that deepens and pinches off from the surrounding surface ectoderm as a hollow, fluid-filled vesicle in mammalian and avian lenses. Differentiation continues based on a spatial polarization of the lens vesicle: cells in the anterior hemisphere of the lens vesicle form the anterior epithelium, and cells in the posterior hemisphere elongate and differentiate as primary fiber cells to obliterate the vesicle space. Polarization of the vesicle and cellular differentiation are thought to be influenced by extrinsic transcription and growth factors from the aqueous and vitreous humors such as FGF, IGF-1, insulin, and TGFβ.6–8 In contrast to mammals or avians, the earliest stages of lens development in zebrafish occur without the formation of a lens vesicle.9–12 Although described as a major difference between mammalian and teleost development, our hypothesis is that the processes of cell differentiation that determine cell fates in lens are similar in zebrafish and mammalian development. However, in the absence of a vesicle stage, the expected correspondence between cells in the anterior vesicle and the lens epithelium and the posterior vesicle and the primary fibers did not exist. Previous work using live-cell imaging demonstrated that cells in the zebrafish placode thickened and underwent progressive delamination that resembled epithelial-to-mesenchymal transition.1 During delamination, the solid, multilayered lens cell mass was reorganized into a spherical structure that underwent cell differentiation to form an anterior epithelium and posterior elongating primary fibers, similar to the mammalian lens.1,12 To determine the fates of cells in the developing zebrafish lens, live-cell imaging combined with immunohistochemistry can be used to define how and when zebrafish primary fiber cells differentiate during lens development. In this report, two-photon live-embryo imaging was used to follow individual fluorescently labeled cells in real-time to develop a fate map of the zebrafish lens placode. Cells were followed from the placode stage at 16 hours postfertilization (hpf) until obvious morphologic differentiation into epithelium or fiber cells had occurred by approximately 28 hpf. We determined that cells destined to become epithelial or primary fiber cells followed a predictable but separate path of migration during development. Cells in the peripheral lens placode migrated to the anterior lens mass and differentiated into anterior epithelium. Cells in the central lens placode migrated to the posterior lens mass and differentiated into primary fiber cells. Although a living fate map of the mammalian lens placode has never been constructed, results in the zebrafish confirm the widely accepted mammalian model: cells in the placode periphery become epithelium, and cells in the central placode become primary fibers. Immunohistochemistry demonstrated that stages of proliferation were similar to those of the mammal and that apoptosis was not a primary mechanism in delamination. We conclude that despite the lack of a zebrafish lens vesicle, eye organogenesis appears to be well conserved among vertebrates.