Figure 4. Cyp27c1 is necessary for vitamin A₂ production and red-shifting of photoreceptors in vivo.

(A) TALENs were used to generate cyp27c1 mutant zebrafish. Two independent alleles are shown, cyp27c1^Δ1 and cyp27c1^Δ2. Both alleles contain frame-shift mutations resulting in premature stop codons at amino acids 151 and 150, respectively.

(B) Western blot with rabbit polyclonal anti-Cyp27c1 antibody shows that Cyp27c1 protein is detected in the RPE of wild-type fish but not in the RPE of cyp27c1^Δ1/Δ2 fish after three weeks of TH treatment. β-actin was used as a loading control.

(C) Wild-type and cyp27c1^Δ1/Δ2 siblings were treated with TH or vehicle control for three weeks, and retinas and RPE from three animals were pooled and analyzed for retinoid content using HPLC. A TH-driven switch to vitamin A₂ was observed in wild-type fish, but not in cyp27c1^Δ/1Δ2 mutants.

(D) The spectral sensitivity of red cones from TH-treated cyp27c1^Δ1Δ2 fish and their wild-type siblings was assessed using single-cell suction electrode recording. The photoreceptors were exposed to a series of dim flashes at 560, 600, 660, and 700 nm, and their response amplitudes were used to calculate flash sensitivity (y-axis) as a function of wavelength (x-axis). No significant difference was observed between vehicle-treated cyp27c1^Δ/1Δ2 and wild-type red cones. However, TH-treated wild-type red cones were significantly more sensitive to 660 and 700 nm light, and less sensitive to 560 nm light relative to cyp27c1^Δ/1Δ2 red cones (Student's t-test, *p < 0.0005, n = 12-21, error bars = s.e.m.). Fitted curves were calculated using a model that incorporates both vitamin A₁- and A₂-based pigment. This model predicts 100% vitamin A₁ in vehicle-treated wild-type and cyp27c1^Δ/1Δ2 fish, 99.7% vitamin A₁ in TH-treated cyp27c1^Δ/1Δ2 fish, and 95.5% vitamin A₂ in TH-treated wild-type siblings.

(E) Physiological parameters of the red cones are indicated ± s.e.m. These include wavelength of peak sensitivity (λ_max), dark current (I_dark), estimated half-saturation light intensity at λ_max (I_o), dim flash response time to peak (T_peak), recovery time constant (τ_rec; estimated from a single-exponential fit to the tail of the response), and integration time (T_int; estimated as the time integral of the normalized flash response).

See also Figure S3.