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Brenda L Bohnsack, Bahaar Chawla; Cyp1b1 Regulates Ocular Fissure Closure and Subsequent Neural Crest Migration. Invest. Ophthalmol. Vis. Sci. 2014;55(13):4592.
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© ARVO (1962-2015); The Authors (2016-present)
CYP1B1 gene mutations are a common cause of congenital glaucoma, but the mechanism by which CYP1B1 regulates eye development is unknown. In these studies, we used zebrafish to study the role of cyp1b1 in the eye.
Morpholino oligonucleotides (MO) and mRNA injections were used with exogenous regulators and analogs of estradiol and retinoic acid (RA). Embryos were analyzed by time-lapse imaging, in situ hybridization, immunostaining, TUNEL assay, and histology.
Cyp1b1 was expressed in the retina with localization in the ocular fissures by 24 hours post fertilization (hpf). Cyp1b1 expression peaked at 36hpf and then disappeared in the dorsal and ventral retina by 60 and 72hpf, respectively. Cyp1b1 knockdown did not inhibit early (12-24hpf) NC migration into the craniofacial region. However, Cyp1b1 was required for later (24-60hpf) NC migration through the ocular fissures and iridocorneal angle formation. Cyp1b1 overexpression prevented closure of the inferior ocular fissure resulting in prominent colobomas (Fig). While Cyp1b1 overexpression did not inhibit NC migration, failure of fissure closure disrupted NC organization around the lens. Knockdown of Cyp1b1 significantly increased retinal and NC cell proliferation while overexpression did not affect the number of cells in S-phase. Increased or decreased Cyp1b1 inhibited retinal and NC survival. Additional studies assessed substrates and targets of Cyp1b1 in eye development. While Cyp1b1 regulation of eye development was not mediated by estradiol, Cyp1b1 overexpression partially rescued defects due to retinoic acid (RA) deficiency. However, Cyp1b1 did not regulate transcript expression of RA synthesis (raldh2) or degradation (cyp26c1) enzymes or pitx2, a known downstream target of RA in the periocular mesenchyme. In addition, alterations of Cyp1b1 did not significantly change transcript expression of genes known to be associated with colobomas such as vsx2, otx2, dvr1, gdf6, and pax6.
We demonstrated that cyp1b1 expression spatially and temporally correlated with the ocular fissures and regulated fissure closure. Further, Cyp1b1 had a non-cell autonomous effect on NC proliferation and migration. Although this effect is partially mediated by RA, additional downstream targets remain to be elucidated. These studies give insight into how mutations in CYP1B1 disrupt ocular NC development in the pathogenesis of congenital glaucoma.
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