We produced and characterized Olfactomedin 2 (Olfm2) knockout mice by replacing the Olfm2 gene with the LacZ gene. A previous study has shown that a mutation in OLFM2 is associated with primary open angle glaucoma in Japanese patients. Loss of Olfm2 resulted in no gross abnormalities. However, Olfm2 null mice showed reduced exploration, locomotion, olfactory sensitivity, abnormal motor coordination, and anxiety related behavior. The pattern of the Olfm2 gene expression was studied in the brain and eye using β-galactosidase staining. In the brain, Olfm2 was mainly expressed in the olfactory bulb, cortex, piriform cortex, olfactory trabeculae, inferior and superior colliculus. In the eye expression was detected mainly in retinal ganglion cells. In Olfm2 null mice, the amplitude of the first negative wave in the visual evoked potential test was significantly reduced as compared with wild-type littermates. Olfm2, similar to Olfm1, interacted with the GluR2 subunit of the AMPAR complexes and Olfm2 co-segregated with the AMPA receptor subunit GluR2 and other synaptic proteins in the synaptosomal membrane fraction upon biochemical fractionation of adult mice cortex and retina. Immunoprecipitation from the synaptosomal membrane fraction of Olfm2 null mouse brain cortex using the GluR2 antibody showed reduced levels of several components of the AMPAR complex in the immunoprecipitates including Olfm1, PSD95 and CNIH2. These results suggest that heterodimers of Olfm1 and Olfm2 interact with AMPAR more efficiently than Olfm2 homodimers and that Olfm2 plays a role in the organization of the AMPA receptor complexes. We also used zebrafish to study functions of olfactomedin domain-containing proteins. We continued the characterization of previously produced double null olfm1a-/-;olfm1b-/- zebrafish. To gain insight into possible molecular mechanisms of the observed phenotype, we analyzed the transcriptome profile of the mutant and wild-type larvae by RNA sequencing. There were 35741 different mRNA species identified in the heads of 3 days post fertilization olfm1 mutant or wild-type larvae. The levels of only 64 mRNA species with at least 499 counts of transcripts changed more than 2 fold between wild-type and olfm1 mutant (p<0.05). Genes important for retinal developments were examined and validated for their expression in the retina by in situ hybridization. There are two pax6 genes, pax6a and pax6b, in zebrafish, and both of them are expressed in the retinal ganglion and inner nuclear layers of the retina. Expression level of pax6b was dramatically reduced, while the level of pax6a expression was slightly reduced, in olfm1 mutant retina as compared with wild-type. The number of retinal ganglion cells was reduced by 30% in 7 days post fertilization larvae as compared with wild-type. These data suggest that olfm1 may play a role in differentiation of retinal ganglion cells.
