Cyp1b1 Regulates Ocular Fissure Closure Through a Retinoic Acid–Independent Pathway

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.

Regional Gene Expression Profile Comparison Reveals the Unique Transcriptome of the Optic Fissure

“…S1). ISH of the genes that had demonstrated specific expression and/or critical functions in the OF, such as Bmpr1b, 48 Ntn1, 38 Smoc1, 39 Aldh1a3, 40 Cyp1b1, 42 and Vax2os, 37 showed specific hybridization signals in the OF region as expected and served as ISH technical controls (Figs. 3M-R).…”

“…This indicated that the expression of some OFspecific genes is also dynamically regulated during OF development. Among these 27 genes, Zfp503, 44 Cyp1b1, 42 Bmpr1b, 47 Smoc1, 39 and Lamb1 45 have been demonstrated to be essential for proper closure of the OF. How these OFspecific genes are dynamically regulated and whether their dynamic expression is important for proper OF closure is unclear.…”

Differences in neural crest sensitivity to ethanol account for the infrequency of anterior segment defects in the eye compared with craniofacial anomalies in a zebrafish model of fetal alcohol syndrome

“…As previously demonstrated, foxd3 marked a higher proportion of neural crest cells that entered the eye (Fig. A′–D′) (Williams et al, ). Foxd3‐ positive cells migrated between the optic cup and surface ectoderm and (arrows, Fig.…”

Cyp1b1 directs Srebp-mediated cholesterol and retinoid synthesis in perinatal liver; Association with retinoic acid activity during fetal development

“…The extensive Cyp1b1 expression at E9.5 parallels early development of both brain and liver [27,28]. Deficits in Cyp1b1 disrupt eye development in humans [29], mice [30] and zebrafish [31], but retinoid metabolism is not the source. The diversity of substrates provides many options for Cyp1b1 participation both in embryos and in the perinatal period.…”