A, A Thrb2 cDNA, inserted at the start codon of Nrl, replaced both Nrl coding exons (grey boxes), creating the Nrl^b2 allele. Arrowhead, Nrl gene promoter, open box, 5′-non-coding exon; TGA, stop codon; pA, poly-adenylation site; loxP, residual loxP site after deletion of Neo selection gene.
B, Western blot analysis showing expression of TRβ2 in Nrl^b2/b2 mice. Arrowheads, specific bands for TRβ2 (~58 kDa) and NRL (29 – 35 kd). Thrb2^−/− lane, TRβ2-deficient control at E17.5. Numbers below lanes, signals quantified by densitometry relative to an arbitrary value of 1.0 in +/+ mice at the earliest age of detection; n.d., not detectable.
C, In situ hybridization showing ectopic expression of TRβ mRNA over the outer neuroblastic layer (ONBL) in Nrl^+/b2 and Nrl^b2/b2 mice at P7. Endogenous TRβ2 mRNA drops to low levels in the small cone population in +/+ neonates. Scale bar, 50 μm. INBL, inner neuroblastic layer.

A, Western blot analysis of cone opsins and rhodopsin in retina from 6 week old mice of the indicated Nrl genotypes. Thrb2^−/− lane, sample from TRβ2-deficient mice as a negative control for M opsin.
B, Western blot analysis of cone opsins and TRβ2 in retina from P14 mice of the indicated Nrl genotypes. The initial induction of M opsin is pronounced in Nrl^b2/b2 mice at this juvenile age. C, Quantitative PCR analysis of M opsin, S opsin and TRβ2 mRNA levels in retina of mice of the indicated Nrl genotypes at P17.

A, Representative electroretinogram traces for Nrl^b2/b2 and Nrl^−/− mice at around 8 weeks of age. Upper two rows, photopic cone responses to stimuli with wavelengths of 520 nm and 360 nm that optimally activate mouse M and S opsins, respectively. Lower row, scotopic rod responses. Enhanced M and S cone responses were detected in Nrl^b2/b2 mice but only enhanced S cone responses in Nrl^−/− mice. Families of traces are shown for light intensities of 0.5, 1.26, 2, 3.16 and 7.94 cd.s/m² at 520 nm and 0.0001, 0.00032, 0.001, 0.0032 and 0.0316 cd.s/m² at 360 nm for cones and 1×10⁻⁶, ×10⁻⁵, 10×⁻⁴, 10×⁻³ and 10×⁻² cd.s/m² for rods.
B, Intensity-response curves of average ERG responses for groups of 4 - 6 mice (means ± sem). Cone (top two rows) and rod (bottom row) ERG responses to varying stimulus intensities were determined for +/+, Nrl^+/b2 and Nrl^b2/b2 mice and separately, for +/+, Nrl^+/− and Nrl^−/− mice. Nrl^b2/b2 and Nrl^−/− mice were each compared with their own +/+ and heterozygous groups on comparable genetic backgrounds.

A, Retinal histology in 3 month old mice. The outer nuclear layer (ONL) in Nrl^+/b2 mice contains cones (large nuclei with dispersed chromatin) and rods (smaller, denser nuclei) in the same proportions as +/+ mice. Nrl^b2/b2 mice like Nrl^−/− mice lack rods, have excess cones and also display ONL folding and shortened outer segments (OS). INL, inner nuclear layer, IPL, inner plexiform layer, IS, inner segments, OPL, outer plexiform layer. Scale bar, 20 μm (same in B).
B, Higher magnification from images in panel A indicating cone nuclei (arrowheads) and a representative rod nucleus (r) in a +/+ mouse. Nrl^b2/b2 mice possess only cone-like nuclei.
C, Counts (mean ± SD) of cone and rod nuclei in ONL fields, determined on 3 μm thick plastic sections like those in panel A. Fields avoided grossly folded areas in Nrl^b2/b2 mice.

A, In situ hybridization analysis of cone opsin and rhodopsin mRNA in superior and inferior retinal regions in 8 week old mice. Nrl^b2/b2 mice overexpressed M and S opsins with a normal distribution trend whereas in Nrl^−/− mice, S opsin was strongly overexpressed in all retinal regions. Nrl^−/− mice showed a small increase in the density of M opsin-positive cells in superior regions compared to +/+ mice, partly due to ONL folding in Nrl^−/− mice. Scale bar (same in B), 50 μm.
B, In situ hybridization analysis of opsin mRNA in hypothyroid Nrl^b2/b2 mice at P17. On a hypothyroid Tshr^−/− background, M opsin expression was retarded and S opsin overexpression exacerbated in Nrl^b2/b2 mice.
C, Diagram of mouse eye section indicating location of superior and inferior retinal fields examined (boxes).

A, Double fluorescence analysis of cells positive for TRβ2 (@TRβ2 antibody, red), NrlpGFP (transgene, green), or both @TRβ2 and NrlpGFP (yellow or orange, arrows in merged image) in the outer neuroblastic layer of +/+ mouse retina at E16.5. Right panel, phase contrast image of the same field. Confocal microscope images represent a single 1 - 1.5 μm z-plane obtained from 10 μm thick mid-retinal cryosections (same in panels B, C, D, E). For orientation, the grey arrowhead indicates location of retinal pigmented epithelium. Scale bar, 14 μm.
B, Double fluorescence analysis of cells positive for TRβ2 and NrlpGFP in +/+ mouse retina during development at E14.5, E18.5, P2 and P8. Doubly-positive cells (yellow or orange) were most evident at E16.5 - E18.5.
C, Counts of cells positive for TRβ2, GFP or both TRβ2 and GFP, determined on single z-plane confocal images from experiments shown in A and B. Counts shown were determined in mid-retinal fields. Counts in superior and inferior fields gave similar results.
D, Verification of NrlpGFP transgene as a marker for cells expressing endogenous Nrl using an antibody against NRL protein (@Nrl) and direct fluorescence for NrlpGFP in +/+ embryos at E18.5. The @Nrl+ population included almost all GFP+ cells (yellowish and orange cells, merged image in left panel) and a few @Nrl+ cells that were negative for GFP (white arrows, middle panel). Scale bar, 10 μm (same in D).
E, Independent identification of cells that co-express TRβ2 (@bGal) and endogenous NRL protein (@Nrl) (yellow or orange cells in merged image, left panel). Analysis was performed on E18.5 embryos homozygous for a targeted insertion of lacZ in the TRβ2-specific exon of the Thrb gene.
F, Immunofluorescence analysis for co-expression of S opsin (@S opsin antibody, blue), NrlpGFP (direct fluorescence, green) and Thrb2p-lacZ transgenes (@bGal antibody, red, indicator for TRβ2) in +/+ mice at P0. Arrow, S opsin/GFP doubly-positive cell; arrowhead, S opsin/GFP/bGal triply-positive cell; asterisk, S opsin-positive cell with no detectable GFP or bGal.