Figure 2. Cyp27c1 expression is correlated with the presence of vitamin A2 in zebrafish and bullfrog RPE.
(A) Zebrafish were treated with TH or a vehicle control for three weeks, after which RPE was isolated and used to construct a cDNA library for transcriptome profiling by RNA-seq. Expression levels (in RPKM, Reads Per Kilobase of transcript per Million mapped reads) of individual transcripts from TH-treated RPE (y-axis) and vehicle-treated RPE (x-axis) are shown as dots, with significantly differentially expressed genes in black (quantile-adjusted conditional maximum likelihood [qCML] test, FDR < 0.05, n = 3).
(B) Dorsal and ventral thirds of bullfrog RPE were isolated and used to construct a cDNA library for transcriptome profiling by RNA-seq. Expression levels of individual transcripts from dorsal RPE (y-axis) and ventral RPE (x-axis) are shown as dots, with significantly differentially expressed genes in black (qCML test, FDR < 0.05, n = 3).
(C) Enrichment of the cyp27c1 transcript in cDNA samples used for RNA-seq was confirmed by quantitative real-time PCR (qRT-PCR). Expression was normalized to ribosomal protein rpl13a for zebrafish and rpl7a for bullfrog (two-sided Student's t-test, n = 2-3, *p < 0.005, error bars = s.e.m.).
(D) In situ hybridization of albino zebrafish treated with TH or vehicle control for three weeks. Top panels show cross-sections of the whole eye. Bottom panels show high-magnification images of the boxed regions from the top panels. The antisense probe for cyp27c1 localized exclusively to the RPE in TH-treated fish. No signal was observed with the cyp27c1 sense probe, but strong signal was observed in the RPE of TH-treated and control fish with an antisense probe against rpe65a, a gene that is expressed at high levels in RPE. Scale bars = 200 μm low power, 50 μm high power.
(E) Western blot with a rabbit polyclonal anti-Cyp27c1 antibody confirmed enrichment of Cyp27c1 protein in TH-treated zebrafish and dorsal bullfrog RPE. β-actin was used as a loading control.
(F) Immunohistochemistry of albino TH- and vehicle-treated zebrafish indicates induction of Cyp27c1 expression in the RPE of TH-treated fish (green), with DAPI used to counter-stain nuclei (blue). Scale bar = 50 μm.