Figure 1.
Construction of the Lrit3 knock-out allele and genotyping.
(A) Wild-type (wt) Lrit3 allele comprises 4 exons. In the knock-out (ko) construction, exons 3 and 4 were replaced by a selection cassette with only 21 bp of exon 3 still remaining. For genotyping, mLrit3_ex4aF and mLrit3_ex4aR were designed to only amplify the wt allele, whereas mLrit3_ex3F and mLrit3_CasR were designed to only amplify the ko allele. (B) After migration on 2% agarose gel, Lrit3+/+ mice exhibited a single fragment at the expected length of 602 bp, Lrit3nob6/nob6 exhibited a single fragment at the expected length of 377 bp and Lrit3nob6/+ mice exhibited both fragments. Legends: WT: wild-type allele; Mut: mutant allele.
Figure 2.
wt and ko Lrit3 cDNAs and RT-PCR.
(A) ko cDNA comprised the remaining 21 bp of exon 3 and only the first 8 bp of the selection cassette which leads to a premature stop codon. mLrit3_RT_ex2F and mLrit3_RT_ex3R were designed to only amplify the wt cDNA whereas mLrit3_RT_ex2F and mLrit3_RT_CasR were designed to only amplify the supposed ko cDNA. (B) After amplification and migration on agarose gel, Lrit3+/+ mice exhibited a single fragment at 539 bp, Lrit3nob6/nob6 mice exhibited a 443 bp fragment and Lrit3nob6/+ mice exhibited both fragments. Legends: WT: wild-type cDNA; Mut: mutant cDNA. (C) Sequence of the ko LRIT3 protein. This 206 amino acid protein are supposed to lack its Ig-like, Serine-rich, fibronectin III, transmembrane and PDZ-binding domains.
Figure 3.
Dark-adapted ERG series were obtained from representative Lrit3+/+ (black line), Lrit3nob6/+ (blue line) and Lrit3nob6/nob6 (red line) littermates. (A) At 6 weeks of age. The scale indicates 100 ms and 200 µV. Values to the left of the row of waveforms indicate flash intensity in log cd.s/m2. (B) Amplitude of the major components of the dark-adapted ERG with increasing flash intensity at 6 weeks of age. The b-wave component is absent in Lrit3nob6/nob6 mice and therefore this data is not plotted. (C) Implicit time of the major components of the dark-adapted ERG with increasing flash intensity at 6 weeks of age. The b-wave component is absent in Lrit3nob6/nob6 mice and therefore this data is not plotted. (D) At 6 months of age. The scale indicates 100 ms and 200 µV. Values to the left of the row of waveforms indicate flash intensity in log cd.s/m2. (E) Amplitude of the major components of the dark-adapted ERG with increasing flash intensity at 6 months of age. The b-wave component is absent in Lrit3nob6/nob6 mice and therefore this data is not plotted. (F) Implicit time of the major components of the dark-adapted ERG with increasing flash intensity at 6 months of age. The b-wave component is absent in Lrit3nob6/nob6 mice and therefore this data is not plotted.
Figure 4.
Cone-mediated ERG series were obtained from representative Lrit3+/+ (black line), Lrit3nob6/+ (blue line) and Lrit3nob6/nob6 (red line) littermates. (A) At 6 weeks of age. The scale indicates 100 ms and 100 µV. Values to the left of the row of waveforms indicate flash intensity in log cd.s/m2. (B) Amplitude of the a-wave with increasing flash intensity at 6 weeks of age. (C) Amplitude of the b-wave with increasing flash intensity at 6 weeks of age. (D) Implicit time of the a-wave with increasing flash intensity at 6 weeks of age. (E) Implicit time of the b-wave with increasing flash intensity at 6 weeks of age. (F) At 6 months of age. The scale indicates 100 ms and 100 µV. Values to the left of the row of waveforms indicate flash intensity in log cd.s/m2. (G) Amplitude of the a-wave with increasing flash intensity at 6 months of age. (H) Amplitude of the b-wave with increasing flash intensity at 6 months of age. (I) Implicit time of the a-wave with increasing flash intensity at 6 months of age. (J) Implicit time of the b-wave with increasing flash intensity at 6 months of age.
Figure 5.
The number of head movements per minute was obtained in scotopic conditions with spatial frequencies from 0.063 to 0.75-Wallis statistical test in representative Lrit3+/+ (black box), Lrit3nob6/+ (blue box) and Lrit3nob6/nob6 (red box) littermates. The star indicates a significant test (p<0.05). (A) At 6 weeks of age. (B) At 6 months of age.
Figure 6.
Retinal anatomy of Lrit3nob6/nob6 mice.
Retinal sections of representative Lrit3+/+, Lrit3nob6/+ and Lrit3nob6/nob6 littermates were compared by light microscopy at 6 weeks and 6 months of age. Scale bar, 40 µm.
Figure 7.
SD-OCT retinal nuclear layers measurements.
(A) ONL, INL and IPL+GCL+NFL thickness were obtained by SD-OCT and compared using Kluskal-Wallis statistical test in representative Lrit3+/+ (black box), Lrit3nob6/+ (blue box) and Lrit3nob6/nob6 (red box) littermates. The star indicates a significant test (p<0.05). (B) At 6 weeks of age. (C) At 6 months of age.