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A zebrafish ortholog of human lengsin was identified by EST analysis of an adult lens cDNA library. During zebrafish development, lengsin transcription is first detected at 24 h post-fertilization (hpf). Immunolocalization, using polyclonal antiserum generated against a Lengsin bacterial fusion protein, detects lens-specific protein in whole-mount embryos at 30 hpf. Lengsin expression in zebrafish follows the temporal expression of the αA- αB1- and βB1-crystallin proteins in the lens. At 72 hpf, Lengsin is localized to a subpopulation of differentiating secondary fiber cells, while no expression is detected in the lens epithelial cells or central lens fibers. In the adult lens, Lengsin is restricted to a narrow band of cortical fibers and co-localizes with actin at the lateral faces of these interdigitating cells. Stable transgenic lines, using a 3 kb lengsin genomic fragment to regulate EGFP expression, recapitulate the Lengsin temporal and spatial expression patterns. Lengsin function in zebrafish lens formation was examined by antisense morpholino-mediated translation and mRNA splice inhibition. At 72 hpf, the lengsin morphant lenses are reduced in size and exhibit separations within the cortex due to defects in secondary fiber morphogenesis. The location of the morphant lens defects correlates with the Lengsin protein localization at this age. These results demonstrate Lengsin is required for proper fiber cell differentiation by playing roles in either cell elongation or the establishment of cell interactions.