Olfactomedin 2 (Olfm2) is a secreted glycoprotein belonging to a family of olfactomedin domain-containing proteins. The human OLFM2 gene spans almost 83 kbp and consists of eight exons. Genetic data indicate that mutation of the OLFM2 gene leading to the Arg144Gln substitution in the protein sequence is a possible disease-causing mutation in Japanese patients with open-angle glaucoma. Two major mRNAs encode secreted proteins with a length of 454 and 478 amino acids. Olfm2 is more closely related to olfactomedin 1 (Olfm1) and olfactomedin 3 (Olfm3) than to any other family members. Olfm2 showed the most dynamic expression pattern as compared with Olfm1 and Olfm3 during mouse eye development and, similar to Olfm1, was expressed preferentially in the developing retinal ganglion cell layer. Among three Olfm2 substitutions tested (Thr86Met, Arg144Gln and Leu420Ser), only Leu420Ser completely blocked secretion of the protein. Olfm2 interacted with Olfm1 and Olfm3 but not with myocilin. Co-transfection of the Leu420Ser mutant with wild type Olfm1 and Olfm3 significantly inhibited secretion of Olfm1 and Olfm3. We concluded that highly conserved Olfm2 protein may play an important role in the course of retinal and eye development. Severe mutations in one of the closely related olfactomedin domain-containing proteins (Olfm1-3) may block secretion and probably activity of all three family members leading to more pronounced pathologies of the retina than knockouts of individual genes. We produced Olfm2 knockout mice. These mice are viable and are currently under investigation. In collaboration with Dr. J. F. Hejtmacik (National Eye Institute), we also investigated function of zinc-finger protein, znf513, in zebrafish eye development. It has been shown by Dr. Hejtmancik and coauthors that homozygous missense mutation in this gene (c.1015T>C, p.Cys339Arg) lead to retinitis pigmentosa in humans. znf513 mRNA is expressed in early developing zebrafish embryo brain and eyes. We showed that injection of znf513 morpholino oligonucleotides (znf513-MO) into zebrafish embryos reduced immunostaining for rod photoreceptor markers. By 5 days after fertilization, rod cells were almost completely absent from the retina of znf513-MO-injected larvae. We showed that rhodopsin and red opsin signals were completely absent from znf513-MO-treated larvae. Reduction of the green opsin and elimination of the red opsin in znf513-MO-treated retina suggests that remaining green/red cones are dysfunctional cone bodies. We concluded that znf513 has an important role in early retinal development.
