Abstract
Purpose:
A classic example of adaptive plasticity is the spectral reprogramming of the photoreceptors of aquatic organisms in response to changes in their light environment. This reprogramming, known as the rhodopsin-to-porphyropsin switch, red-shifts the sensitivity of photoreceptors and is mediated by the oxidation of retinal to 3,4-didehydroretinal. Despite 75 years of research, the enzyme mediating this switch has eluded identification.
Methods:
We leveraged two different biological models to discover the enzyme that generates 3,4-didehydroretinal. First, we exploited a unique adaptation of the bullfrog eye: the dorsal, but not the ventral, retina of adult bullfrogs contains 3,4-didehydroretinal, which originates from the retinal pigment epithelium (RPE). Second, the exposure of zebrafish to thyroid hormone induces the conversion of retinal to 3, 4-didehydroretinal. We therefore used RNA-Seq to compare the transcriptomes of RPE from thyroid hormone-treated and control zebrafish, and of dorsal and ventral bullfrog RPE, to identify differentially expressed enzymes. Expression was further assessed using western blot, in situ hybridization, and immunohistochemistry. The function of candidate genes was assayed in vitro in HEK-293 cells, and in zebrafish null mutants generated using TALENs. For all experiments, retinoids were measured using HPLC.
Results:
The differential expression analysis converged on a single markedly up-regulated gene encoding the cytochrome P450 family member, Cyp27c1, which was significantly enriched in dorsal bullfrog RPE relative to ventral bullfrog RPE, and in thyroid hormone-treated RPE relative to control RPE (by 39- and 36-fold respectively, FDR = 0.05, n=3). Induction of Cyp27c1 protein in both species was confirmed by western blot, and in situ hybridization and immunohistochemistry localized Cyp27c1 expression to the RPE. Introduction of Cyp27c1 into HEK-293 cells was sufficient to induce the conversion of retinal into 3,4-didehydroretinal in vitro. Furthermore, Cyp27c1 null mutant zebrafish fail to produce 3,4-didehydroretinal in response to thyroid hormone, indicating that Cyp27c1 is necessary for this reaction in vivo.
Conclusions:
Taken together, these data confirm that the long-sought 'terminal ring dehydrogenase' that mediates the rhodopsin-to-porphyropsin switch is the cytochrome P450 family member Cyp27c1.