Abstract
PurposeMutations in the CYP1B1 gene are the most commonly identified genetic causes of primary infantile-onset glaucoma. Despite this disease association, the role of CYP1B1 in eye development and its in vivo substrate remain unknown. In the present study, we used zebrafish to elucidate the mechanism by which cyp1b1 regulates eye development.MethodsZebrafish eye and neural crest development were analyzed using live imaging of transgenic zebrafish embryos, in situ hybridization, immunostaining, TUNEL assay, and methylacrylate sections. Cyp1b1 and retinoic acid (RA) levels were genetically (morpholino oligonucleotide antisense and mRNA) and pharmacologically manipulated to examine gene function.ResultsUsing zebrafish, we observed that cyp1b1 was expressed in a specific spatiotemporal pattern in the ocular fissures of the developing zebrafish retina and regulated fissure patency. Decreased Cyp1b1 resulted in the premature breakdown of laminin in the ventral fissure and altered subsequent neural crest migration into the anterior segment. In contrast, cyp1b1 overexpression inhibited cell survival in the ventral ocular fissure and prevented fissure closure via an RA-independent pathway. Cyp1b1 overexpression also inhibited the ocular expression of vsx2, pax6a, and pax6b and increased the extraocular expression of shha. Importantly, embryos injected with human wild-type but not mutant CYP1B1 mRNA also showed colobomas, demonstrating the evolutionary and functional conservation of gene function between species.ConclusionsCyp1b1 regulation of ocular fissure closure indirectly affects neural crest migration and development through an RA-independent pathway. These studies provide insight into the role of Cyp1b1 in eye development and further elucidate the pathogenesis of primary infantile-onset glaucoma.
Highlights
We observed that cyp1b1 was expressed in a specific spatiotemporal pattern in the ocular fissures of the developing zebrafish retina and regulated fissure patency
Cyp1b1 overexpression inhibited the ocular expression of vsx[2], pax6a, and pax6b and increased the extraocular expression of shha
Embryos injected with human wild-type but not mutant CYP1B1 mRNA showed colobomas, demonstrating the evolutionary and functional conservation of gene function between species
Summary
Zebrafish eye and neural crest development were analyzed using live imaging of transgenic zebrafish embryos, in situ hybridization, immunostaining, TUNEL assay, and methylacrylate sections. Zebrafish (Danio rerio) were raised in a breeding colony under a 14-hour light/10-hour dark cycle as previously described.[10,11,17,18] Embryos were maintained at 28.58C and staged as previously described.[19] The transgenic strains Tg(sox10:EGFP) and Tg(foxd3:GFP) were gifts from Thomas Schilling and Mary Halloran. The strains were crossed into the casper (royÀ/À, nacreÀ/À) background as indicated.[20,21,22] The Tg(RARE:mCHERRY) transgenic strain was generated using Gateway cloning techniques as previously described.[23] Animal protocols were performed in accordance with the guidelines for the humane treatment of laboratory animals established by the University of Michigan Committee on the Use and Care of Animals (IACUC, protocol 10205) and the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research
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