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M. Aose, T. M. S. Greiling, H. R. Djajadi, T. H. Linbo, D. W. Raible, J. I. Clark; The Formation of a Second Lens in the Mutant Zebrafish, Occhiolino. Invest. Ophthalmol. Vis. Sci. 2010;51(13):2348.
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Analysis of abnormal lens development in Occhiolinomutant zebrafish.
Zebrafish were treated with N-ethyl-N-nitrosourea (ENU) and offspring screened for eye phenotypes. Zebrafish were euthanized in 0.4mM tricaine; fixed in 4% paraformaldehyde/5% sucrose/phospate buffered saline (PBS); the fixed fish were oriented in 1.5% agarose/5% sucrose/PBS and cryoprotected in 30% sucrose/PBS; then the agarose blocks were mounted in OCT and frozen in isopentane cooled in liquid nitrogen. The fish were cryosectioned for histology and immunofluoresence. H&E staining, BrdU labeling (proliferation) TUNEL labeling (apoptosis) and immunohistochemistry were performed using selected antibodies. For 2-photon imaging, the Occhiolino zebrafish were crossed with the Q01 transgenic fish which express CFP fused to Gap43, a membrane-targeting sequence driven by an EF1α promoter and a hexamer of the DF4 pax6 enhancer.
The Occhiolino mutant was identified in an F3 screen for mutations affecting zebrafish nervous system development. The eye of the Occhiolino mutant developed normally until 3 days post fertilization (dpf), when visual function was established. After 3 dpf, the epithelial cells in the Occhiolino lens lost spherical symmetry and the ability to proliferate and migrate. The abnormal epithelium became multilayered and a new lens mass formed, sometimes appearing as a second lens between the first lens and the cornea. BrdU and TUNEL labeling demonstrated the cell-cycle was abnormal in the cells of the second lens cell mass. The first lens ruptured and was displaced posteriorly, pushing into the developing retina, occasionally contacting the extra cellular matrix (ECM) of Bruch’s membrane. The retina appeared to expand and decrease the pupil diameter. The first lens was smaller, and the proteins different than a normal lens. Markers for epithelial mesenchymal transition (EMT) were abnormal in the mutant lens.
The phenotype of Occhiolinomutant zebrafish resulted from abnormal proliferation and migration of the lens epithelium.
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