Zebrafish foxe3: Immunolocalization, Functional Characterization and Genetic Interactions During Eye Lens Development

Purpose: Identify and characterize the zebrafish (Danio rerio) ortholog of foxe3, a winged helix/forkhead domain transcription factor critical for mammalian ocular lens development. Methods: Both 5' and 3' RACE were used to amplify cDNA corresponding to the zebrafish foxe3 gene. A polyclonal antiserum was generated in rabbit against a bacterial fusion protein expressing the Foxe3 carboxyl terminus. Protein A-purified antiserum was used to detect zebrafish Foxe3 on immunoblots, in embryo whole-mounts and in frozen tissue sections. Foxe3 function during eye development was assessed by injection of antisense morpholino oligomers, which specifically prevented translation of the Foxe3 protein. The morphant eye phenotype was analyzed by histology and immunohistochemistry. Potential functional interactions between foxe3 and pitx3 during lens development were assessed by comparison of Foxe3 and Pitx3 protein expression in both foxe3 and pitx3 morphants. In addition, immunohistochemistry and RT-PCR were used to assess expression patterns of various lens development genes in the foxe3 and pitx3 morphants. Results: The zebrafish foxe3 gene, which consists of a single exon on chromosome 8, is capable of encoding a protein of 422 amino acids. The zebrafish protein possesses 44% and 67% identity with the human Foxe3 and Xenopus FoxE4 proteins, respectively. The Foxe3 protein is first detected in the zebrafish lens at 31 hpf and lens expression is restricted to the nucleated cell population including the epithelial and elongating fiber cells. Knockdown of Foxe3 protein expression using antisense morpholinos results in small lenses with multilayered epithelial cells and fiber cells that fail to elongate. alphaA-crystallin protein is expressed at near wild-type levels in the foxe3 morphant lens. While the morphants possses normal retinas, retinal cell proteins, including rhodopsin, are abnormally expressed in some morphant lens cells. Immunoblots and frozen section immunohistochemistry reveals that Pitx3 protein is expressed in the foxe3 morphant lens, while Pitx3 protein knockdown eliminates Foxe3 protein expression. Conclusions: Foxe3, which is expressed in the lens epithelial and elongating fiber cells, is necessary for lens development and growth in zebrafish. Foxe3 lies genetically downstream of pitx3 in a zebrafish lens development pathway.