Abstract
Purpose :
Thyroid hormone receptor beta (thrb, trβ2) is required for vertebrate red cone development. We hypothesize that thrb is necessary for zebrafish to perceive red light. We constructed two thrb Crispr mutant strains: c.184_188delTATGGinsGTTCCC (6BP+1) frameshift indel and an in frame c.184_186delTAT single codon deletion (3BP) to test this hypothesis.
Methods :
Larval eyes at the ages of 5, 6, 7, and 12 days were isolated from spawns of mutant and het-mutant adults. Eyes were perfused with MEM plus 20mM L-aspartic acid to block inner retinal signals, isolating cone PIII ERG responses. PIII signals were recorded while exposing the eye to 9 wavelengths of light (330-650nm) at 7 different irradiances. Amplitudes were fit to a spectral model that calcuated the saturated signal amplitudes for red (Vr), green (Vg), blue (Vb), and UV (Vu) cones. Two-tailed t-tests and one-way ANOVA tests were conducted for statistical comparisons between mutants, heterozygotes (hets) and wild type (WT) fish.
Results :
The mean normalized peak amplitudes at 650, 610 and 570 nm for the 6BP+1 mutants was 0.09±0.03, significantly less than het-mutants (0.43±0.06, p<0.0001). There was no significant difference for the 3BP mutants (0.44±0.03), het (0.47±0.16), and WT (0.48±0.25) (p=0.635). The fractional red-cone signal saturation (Vr) in the 6BP+1 mutant was (0.03±0.009), significantly less than hets (0.456±0.018, p=0.00) or WT (0.57±0.06, p=0.00), with hets also significantly less than WT (p=0.007); and mutant Vb (0.33±0.03) was significantly higher than hets (0.22±0.03, p=0.03). Mutant and het Vu (0.43±0.03, 0.45±0.02) trended higher than WT (0.31±0.05), but was not significantly different (p=0.10). In the 3BP strain, mutant Vr (0.40±0.03) and het Vr (0.49±0.01) were significantly lower than WT (0.55±0.02, p=0.0005, 0.0004), and mutant Vb (0.36±0.07) was significantly higher than WT (0.16±0.05, p=0.0124).
Conclusions :
The 6BP+1 strain results were consistent with our hypothesis in that the response to wavelengths greater than 570 was lost in the absence of functional thrb. However, 3BP single codon deletion mutants maintained a red cone response, yet had a decreased red cone contribution to the overall signal and increased blue cone contribution as seen in the 6BP+1 mutant. This suggests thrb does not lose full function with the in frame 3BP deletion, but does with the frameshift 6BP+1 mutation.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.