Molecular Genetics Of Early Eye Development

National Eye Institute

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Abstract

In collaboration with Dr. Faklers laboratory (Institute of Physiology, University of Freiburg, Freiburg, Germany), we continue the investigation of the effects of Olfm1 and Olfm2 null mutations on the composition and activity of the AMPA receptor complex. Elimination of Olfm1 leads to profound changes in the composition of the AMPA receptor complex in both brain and retina, and the physiological consequences of these changes are currently under investigation. Our previous data indicated that Olfm1 may stimulate axon regeneration in vitro and utilizing the in vivo ONC model, we explored this further. After intravitreal injection of adeno-associated viral vector encoding Olfm1, we demonstrate a moderate stimulatory effect of Olfm1 on optic nerve regeneration in rodents. Olfm1 is now being tested in combination with a number of other proteins and factors that stimulate optic nerve regeneration. The CRISPR technology was used to produce zebrafish null mutations in several genes encoding proteins of the AMPA receptor complex. Zebrafish with null mutations in these genes as well as with double null mutations in olfm1 and other genes encoding protein of the AMPA receptor complex are currently being analyzed. Collaboration with Dr. S.-Y. Chen (University of Georgia, Athens, GA) led to the identification of Olfm2 as a novel factor mediating smooth muscle cell phenotypic modulation. Olfm2 may serve as a potential target for therapeutic intervention in treating vascular remodeling following injury. The compound heterozygous mutations in the subunit of chaperonin containing TCP-1 (CCT), encoded by CCT2, lead to the Leber congenital amaurosis (LCA). We produced a cct2 mutant line of zebrafish to investigate the role of CCT2 mutations in LCA in vertebrates. The cct2 mutation (L394H-7del) led to the synthesis of a mutated cct protein with the L394H replacement and deletion of 7 amino acid residues (positions 395- 401). The homozygous cct2- L394H-7del mutant exhibited a small eye phenotype at 2 days post fertilization (dpf) and was embryonically lethal after 5 dpf. In homozygous cct2- L394H-7del mutants, the retinal ganglion cell differentiation was attenuated, retinal cell cycle was delayed as a result of S-phase elongation, and the neural retinal cell death was significantly increased at 2 dpf compared with wild-type. Injection of RNA encoding wild-type human CCT rescued the small eye phenotype, reduced retinal cell death, and restored the levels of cct protein and the major client protein G1 that were significantly reduced in the homozygous cct2-L394H-7del mutant compared with wild-type. These results indicate that cct2 plays an essential role in retinal development by regulating the cell cycle. We concluded that the retinal pathology observed in the homozygous cct2-L394H-7del mutants resembles the retinal pathology of human LCA patients.