Foxe3 is a winged helix/forkhead domain transcription factor necessary for mammalian and amphibian lens development. Human FOXE3 mutations cause anterior segment dysgenesis and cataracts. The zebrafish foxe3 cDNA was PCR amplified from 24 h post-fertilization (hpf) embryo cDNA. The zebrafish foxe3 gene consists of a single exon on chromosome 8 and encodes a 422 amino acid protein. This protein possesses 44% and 67% amino acid identity with the human FOXE3 and Xenopus FoxE4 proteins, respectively. A polyclonal antiserum was generated against a bacterial fusion protein containing the Foxe3 carboxyl terminus. The purified antiserum detects zebrafish Foxe3 on immunoblots, in embryo wholemounts, and frozen tissue sections. The zebrafish Foxe3 protein is first detected in the lens at 31hpf and is restricted to the nucleated cell population, including the epithelial and elongating fiber cells. Knockdown of Foxe3 protein using an antisense morpholino results in small lenses with multilayered epithelial cells and fiber cell dysmorphogenesis. The morphants posses normal retinas, although retinal cell proteins, including rhodopsin, are abnormally expressed in the morphant lens tissue. Functional interactions between foxe3 and pitx3 during lens development were assessed by RT-PCR and comparison of Foxe3 and Pitx3 protein expression in both foxe3 and pitx3 morphants. Immunoblots and immunohistochemistry reveal Pitx3 is expressed in the foxe3 morphant lens, while Pitx3 knockdown results in the elimination of Foxe3 expression. These data demonstrate that Foxe3 is necessary for lens development in zebrafish and that foxe3 lies genetically downstream of pitx3 in a zebrafish lens development pathway.

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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 hours 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.