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Tara Stach, Jakub Famulski, Andrew Waskiewicz, Ordan Lehmann; The superior ocular fissure: a novel finding in early eye development. Invest. Ophthalmol. Vis. Sci. 2013;54(15):3362.
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© ARVO (1962-2015); The Authors (2016-present)
To determine the cause of a novel ocular developmental anomaly through integrated human and zebrafish model organism approaches.
Human genetic analyses encompassing Sanger and exomic next generation sequencing were combined with zebrafish morpholino oligonucleotide (MO) inhibition, in situ hybridization, confocal microscopy and immunofluorescence.
Five patients with superiorly positioned iris/lens/retinochoroidal colobomata were identified over a 6 year period, and in one the molecular basis was defined [biallelic CYP1B1 mutations: R368H & A287X]. Zebrafish have a transient superior fissure, readily visible in the dorsal retina using light microscopy as well as with anti-Laminin immunofluorescence. Loss of Bone Morphogenetic Protein (BMP) signaling, as seen in zebrafish gdf6a-/- mutants, causes a persistent superior colobomata phenotype. Since cyp1b1’s expression is restricted to the vertical axis of the developing retina, and it synthesizes retinoic acid (RA), the contribution of RA to superior fissure closure was explored. MO inhibition of cyp1b1 results in an increased duration of an open superior fissure [3.5 fold increased incidence in cyp1b1 MO embryos], and loss of the RARE transgenic reporter of RA-dependent ocular signalling. Cyp1b1’s retinal expression is dependent on gdf6a, as reduced expression is observed in zebrafish gdf6a-/- mutants and gdf6a morphants have a 40 fold increased incidence of delayed fissure closure. Notably, RA supplementation rescues these phenotypes [2.7 fold decrease in incidence in RA treated gdf6a morphants]. The superior fissure lies at the expression interface of two forkhead box genes that specify nasal (foxg1) and temporal (foxd1) retinal identity. MO inhibition of Foxd1 results in superior fissure twinning whilst inhibition of Foxg1 shifts the superior fissure nasally.
These studies identify a novel superior fissure in the developing retina conserved between species separated by 450 million years of evolutionary time. Its location is specified by the forkhead transcription factors foxd1 and foxg1, whilst its closure is dependent on CYP1B1, BMP and RA signaling. Although these findings run contrary to current knowledge of early eye development, they provide opportunities to enhance understanding of initial steps in vertebrate retinal development.
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