lor^p25bbtl mutants display increased labeling for rods. Ventral views of bright field images (A and D) and confocal immunofluorescent images of rod-specific labeling of eyes from WT (B and C) and a lor^p25bbtl mutant larvae (E and F) (dorsal is up). At 3 dpf, WT larvae display rod labeling first in the ventral retina (B) followed at 5 dpf, by sporadic labeling of individual cells in the central and dorsal retina (C). At 3 dpf, lor^p25bbtl mutant larvae display increased rod immunolabeling across the ventral and central retina (E) that is evenly distributed at 5 dpf (F). (G) Linkage analysis placed lor^p25bbtl between SSLP markers Z31583 and Z13481 on the MGH panel. The number of recombinants in 87 mutant larvae is shown (red). The interval containing the lor mutation and the tbx2b locus is shown.

tbx2b expression is reduced in lor mutant embryos. Sagittal sections of WT (A) and lor^p25bbtl mutant (C) embryos at 28 hours postfertilization (hpf) following in situ hybridization for tbx2b show labeling throughout the eye, but the lack of expression in the ventral retina near the choroid fissure (CF) and lens (L). Transverse sections of labeled WT (B) and lor^p25bbtl embryos (D) at 44 hpf. tbx2b expression is most intense at the dorsal retinal margin and cells adjacent to the developing outer nuclear layer (ONL) as well as the inner nuclear layer (INL) (optic nerve; ON). Note the dramatically reduced expression in the lor^p25bbtl embryos. (E) Amplification of tbx2b by RT-PCR from RNA extracted from WT and lor^p25bbtl embryos at 20 and 28 hpf demonstrates a reduction in the amount of tbx2b mRNA in the mutant.

Increased rod number and fewer UV cones in lor^p25bbtl and fby mutants. (A–C) Confocal images of eyes from 5-dpf WT (A), lor^p25bbtl (B), and fby (C) larvae, immunolabeled for UV opsin (green) and rods (red). Eyes from lor^p25bbtl and fby mutant larvae demonstrate a dramatic deficit in UV cones, with fby displaying a more severe phenotype; in this particular sample, no UV cones were detected. (D) Graph showing the average number of rods (red) and UV cones (green) per unit area quantified from confocal images of lor^p25bbtl mutant and WT retinas (mean + SD). (E and F) Expression analysis of opsins (E) and arrestins (F) by RT-PCR from RNA extracted from 5-dpf WT and lor^p25bbtl larvae. The RT-PCR product for UV opsin was reduced and products for rod opsin and rod arrestin were increased in lor^p25bbtl (red boxes).

Maintenance of the photoreceptor mosaic in tbx2b^p25bbtl mutant adults. Transverse (A and C) and tangential (B and D) histological sections through the ONL from WT and tbx2b^p25bbtl adult animals (rods are GFP⁺) (green), immunolabeled for UV opsin (red), and counterstained with DAPI (blue). (A and C) Tiering of the rod and cone photoreceptor cells is maintained although the UV cones (asterisks) in the tbx2b^p25bbtl retinas are diminished in number and appear to have a greater width than in WT retinas. The approximate plane of the sections in (B) and (D) are indicated [arrows in (A) and (C)]. (B) Immunolabeling of tangential sections for the UV opsin reveals the photoreceptor cell mosaic composed of rows of UV cones and blue cones (arrowheads). (D) In tbx2b^p25bbtl the regular arrangement of the rows of blue cones (gaps in labeling; arrowheads) and occasional UV cones are maintained although the width of the cones is greater and there is some distortion of the row mosaic.

tbx2b^p25bbtl acts cell-autonomously. Genetic chimeras were generated by blastula transplantation and allowed to develop to 80 hpf. Donor cells were labeled by rhodamine-dextran (red); tbx2b^p25bbtl deficient rods expressed the XOPS-GFP transgene. Chimeras were whole-mount immunolabeled for UV opsin (A and B) (blue) and for rods (A) (green). (A and B) Stacks of confocal images taken tangential to the photoreceptor cell layer and in the orthogonal plane (insets). (A) WT donor cells (red) located in the ONL of tbx2b^p25bbtl hosts frequently colabel for the UV opsin (inset). (B) tbx2b^p25bbtl mutant donor cells (red) in the ONL of WT hosts frequently differentiate as GFP⁺ rods and rarely label for the UV opsin. Inset shows dextran/GFP positive rods neighboring UV opsin-positive/dextran negative cone outer segments.

Persistent Nr2e3 expression in lor. (A–D) Transverse cryosections from 2- (A), 3- (B), and 4-dpf (C and D) WT and tbx2b^p25bbtl embryos immunolabeled for rods (4C12, green) and Nr2e3 (red), and counterstained with DAPI (blue); dorsal is up. (A) At 2 dpf, labeling for Nr2e3 is observed in the inner and outer retina before differentiation of photoreceptors. (B) At 3 dpf, Nr2e3 labeling is restricted to developing photoreceptors in the ONL. In the ventral retina, Nr2e3 nuclear labeling colocalizes with immunolabeling for rods (inset). (C and D) At 4 dpf, expression of Nr2e3 persists in differentiating rods in WT and tbx2b^p25bbtl retinas.

Reduced expression of UV opsin in lor^p25bbtl mutant larvae. Transverse retinal cryosections from WT (A–C, A′–C′) and lorp25bbtl (D–F, D′–F′) embryos at 5 dpf immunolabeled for rods (4C12, green) and the blue, UV, and green cone opsins (red). Nuclei were counter stained with DAPI (blue); dorsal is up. lor displays no labeling for UV cones in this particular section. Red opsin labeling was positive for lor^p25bbtl (data not shown).

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