A deletion in a photoreceptor-specific nuclear receptor mRNA causes retinal degeneration in the rd7 mouse

Abstract

The rd7 mouse, an animal model for hereditary retinal degeneration, has some characteristics similar to human flecked retinal disorders. Here we report the identification of a deletion in a photoreceptor-specific nuclear receptor (mPNR) mRNA that is responsible for hereditary retinal dysplasia and degeneration in the rd7 mouse. mPNR was isolated from a pool of photoreceptor-specific cDNAs originally created by subtractive hybridization of mRNAs from normal and photoreceptorless rd mouse retinas. Localization of the gene corresponding to mPNR to mouse Chr 9 near the rd7 locus made it a candidate for the site of the rd7 mutation. Northern analysis of total RNA isolated from rd7 mouse retinas revealed no detectable signal after hybridization with the mPNR cDNA probe. However, with reverse transcription-PCR, we were able to amplify different fragments of mPNR from rd7 retinal RNA and to sequence them directly. We found a 380-nt deletion in the coding region of the rd7 mPNR message that creates a frame shift and produces a premature stop codon. This deletion accounts for more than 32% of the normal protein and eliminates a portion of the DNA-binding domain. In addition, it may result in the rapid degradation of the rd7 mPNR message by the nonsense-mediated decay pathway, preventing the synthesis of the corresponding protein. Our findings demonstrate that mPNR expression is critical for the normal development and function of the photoreceptor cells.

Figure 1

Fundus photographs, ERGs, and histology of eyes from rd7/rd7 mice at 1 mo (a–c), 5 mo (d–f), and 16 mo (g–i) of age. At 1 mo of age, rd7 mice have white spots over the entire retina (a), but the scotopic and photopic (Inset) ERGs are normal (b), and light micrographs show photoreceptor and outer nuclear layer waves, whorls, and rosettes in the retina (c). At about 5 mo of age, the number of retinal spots is decreased (d), ERGs are still normal (e), and histology shows few retinal waves (f). By 16 mo of age, attenuated retinal vessels and mottled retinal pigment indicate retinal degeneration (g), ERGs are now 50% of normal (h), and the outer nuclear layer and outer segment have degenerated to half normal (i).

Figure 2

Maps of the rd7 region of mouse Chr 9. (a) Mapping of the rd7 locus. The first fraction between the markers is the number of recombinants/total in the 77–2C2a-special-rd7/rd7 x CAST/Ei backcross, and the second fraction is the number of recombinants/total in the B₆.Cg-rd7/rd7 X CAST/Ei intercross. (b) Composite map of mouse Chr 9 from the Encyclopedia of the Mouse Genome (www.informatics.jax.org/). The numbers to the right of the chromosome represent the cM distances from the centromere; the numbers to the left of the chromosome represent the chromosomal loci of the human orthologs of the mouse gene listed to the right. (c) Mapping of Pnr. The first fraction between the markers on the right is the number of recombinants/total from the cross (NFS/N X M. spretus) × M. spretus or C58/J, and the second fraction is the number of recombinants/total from the cross (NFS/N or C58/J X M. m. musculus) × M. m. musculus. Pml was typed only in the Mus spretus cross; Acra5 was typed only in the M. m. musculus cross.

Figure 3

Expression of mouse PNR mRNA. (a) Northern blot of adult mouse retinal RNAs hybridized with the mPNR cDNA probe. Lane 1, wild type; lane 2, (photoreceptorless) rd1/rd1; lane M, RNA molecular size standards (GIBCO/BRL). For loading control, ethidium bromide staining of the RNAs is shown (Right). (b) In situ hybridization of a mouse retina section with a mouse PNR probe. (Left) Sense probe; (Right) antisense probe.

Figure 4

Nucleotide sequence of the mouse PNR cDNA and its predicted amino acid sequence. The polyadenylation signal is underlined. The deleted region in the rd7 mouse PNR cDNA is boxed.

Figure 5

Northern blot of rd7 mouse retinal RNA hybridized with a mouse PNR cDNA probe. For loading control, ethidium bromide staining of the RNAs is shown (Right). Lane 1, wild type; lane 2, rd7/rd7; lane M, RNA molecular size standards.

Figure 6

Deletion in the rd7 mutant mouse PNR cDNA. (a) Representative sequence of a DNA fragment containing the site of the deletion in affected animals. The DNA fragment was obtained by RT-PCR by using primers MNR1 and MNR6. The arrow points to the site of the deletion. (b and c) Sequences of PNR fragments from unaffected animals obtained by RT-PCR by using primers MNR1 and MNR4 and MNR3 and MNR6, respectively. For primer sequences, see Table 1.