The Role of Fibrillin-1 in Eye Development and Disease

The ciliary zonule of the human eye consists of a circumferential array of fibers that connect the ocular lens to the nonpigmented ciliary epithelium (NPCE) located at the inner wall of the eye. Zonular fibers consist of bundles of beaded filaments called microfibrils. Microfibrils are major structural elements of the extracellular matrix and are present in pure form in the ciliary zonule. Microfibrils are composed principally of fibrillin-1 (FBN-1); a large extracellular matrix glycoprotein. In humans, mutations in FBN1 underlie Marfan syndrome; a pleiotropic connective tissue disorder that profoundly affects the eye. Ocular manifestations include ectopia lentis (dislocated lenses), cataracts, glaucoma and axial myopia. The ocular phenotypes in Marfan syndrome suggest an important role for FBN-1 in eye development. In this report, I used mice as a model system to test the role of FBN1 in eye development and disease with an emphasis on understanding the role of FBN-1 in the synthesis of the ciliary zonule. Our lab has shown that the organization and composition of the mouse ciliary zonule is similar to humans. A recent proteomic study identified FBN-1 as the main component of the ciliary zonule, accounting for 60% to 70% of total zonule protein. In order to understand the role of FBN-1 in eye development, I had to first identify the cells in the eye responsible for expressing FBN-1 and other core components of the zonule (FBN-2, MFAP2, LTBP2 and ADAMTSL4). I used in situ hybridization on wild type mouse eye tissue to show that some zonule components (Fbn1, Fbn2, Ltbp2, Mfap2) are expressed by cells of the NPCE while others (Adamtsl4, Mfap2) are expressed by the lens. This suggests that the ciliary zonule is synthesized by both tissues. Immunofluorescence experiments on adult wild type mouse eyes using antibodies against core zonule proteins showed that the ciliary zonule is heterogeneous in composition and nonuniform along the length of fibers suggesting that the spatial distribution of proteins in the ciliary zonule could reflect the temporal expression of zonule components during eye development. The identification of NPCE cells as the likely source of FBN-1 in the zonule allowed me to test the contribution of Fbn1 directly. I used a conditional knockout approach to delete Fbn1 in the nasal and temporal regions of the mouse NPCE. Three-dimensional reconstructions of conditional Fbn1 knockout mouse eyes (Fbn1-NPCE) demonstrated that the zonule was produced in the nominal absence of its most abundant component. However, the Fbn1-NPCE mice developed ectopia lentis; the major ocular manifestation and diagnostic criterion for MFS in humans. Given the clinical importance of the disease, I decided to investigate the structural and biomechanical changes leading to ectopia lentis in the Fbn1-NPCE mouse model. Ultrastructural studies on FBN-1 deficient mouse eyes using scanning electron microscopy revealed that Fbn1-NPCE mice had significant changes in zonular fiber density and thickness. Biomechanical testing on wild type and Fbn1-NPCE mouse zonules showed that FBN-1 deficient zonular fibers were significantly weaker (~50%) than their wild type counterparts. Conditional deletion of Fbn1 in the mouse lens (Fbn1-lens) had no discernible effect on the structure or function of the ciliary zonule, suggesting that FBN-1 protein synthesized by the lens is not required for the stability of the ciliary zonule. Together, these data show that the ectopia lentis phenotype in Fbn1-NPCE mice is secondary to changes in ciliary zonule ultrastructure and biomechanical properties and that these effects may give some insights to the mechanism of ectopia lentis in Marfan syndrome. In addition to ectopia lentis, Fbn1-NPCE mice express other ocular phenotypes commonly associated with Marfan syndrome including cataracts, increased axial length and, in rare cases, glaucoma. The data presented in this report provide insights into the role of FBN-1 in eye development and the biology of the ciliary zonule. In addition, the development of the Fbn1-NPCE mouse model will serve as a platform for understanding the role of FBN-1 in ocular disease associated with Marfan and related syndromes