|Molecular Vision 2000;
Received 19 September 2000 | Accepted 27 October 2000 | Published 31 October 2000
Further genetic analysis of two autosomal dominant mouse eye defects, Ccw and Pax6coop
Mary F. Lyon,1 D.
Bogani,1 Y. Boyd,1 P. Guillot,1 J. Favor2
1MRC Mammalian Genetics Unit, Harwell, Didcot, Oxfordshire, UK; 2GSF National Research Center for Environment and Health, Institute of Mammalian Genetics, Neuherberg, Germany
Correspondence to: Mary F. Lyon, MRC Mammalian Genetics Unit, Harwell, Didcot, Oxon OX11 0RD, UK; Phone: 01235 834393; FAX: 01235 834776; email: email@example.com
Purpose: The work forms part of a major project to study the genetics of mouse cataract mutants found during the course of mutagenesis experiments. The long-term aim is to find the underlying gene mutation in each cataract mutant. Here we report further studies of the mutant cataract and curly whiskers (Ccw), previously mapped to Chromosome 4, and also investigations of the corneal opacity (Coop) mutant, which is shown to involve a mutation in the Pax6 gene.
Methods: For Ccw, the methods included mapping relative to microsatellite markers and histological studies. For the Coop mutant, breeding methods were used to show that Coop was allelic with Pax6. The Pax6 coding region in the mutant was then sequenced.
Results: The Ccw locus was mapped to approximately position 45cM on the consensus map of Chr 4. Histologically, progressive degeneration of the lens was seen. In the Coop mutant, a base-pair change C->T was found at position 1033 in the Pax6 gene, which created a stop codon leading to premature termination of translation, and to a truncated Pax6 protein.
Conclusions: The phenotype in Ccw/+ heterozygotes involves a new type of lens degeneration in the mouse. On the basis of the phenotype and the locus position, no candidate gene has yet been identified. The Pax6coop mutant differs in phenotype from known null alleles of Pax6, implying that it is a hypomorph.
Previously, we have reported genetic analysis of various autosomal dominant mouse congenital cataracts generated during the course of a mutagenesis program . The cataract total opacity-3, To3, involved a mutation in the Lim2 gene coding for the MP19 lens membrane protein [2,3] and the nuclear opacity-2, No2, cataract showed a mutation in the Gja8 gene coding for the lens-specific gap junction protein connexin 50 . The loci of other cataract genes have been mapped, but the genes have not yet been identified. Included among these other cataracts was the mutant termed cataract and curly whiskers, Ccw, which arose spontaneously. The gene concerned was mapped to Chromosome 4, and shown to be lethal at an early embryonic stage when homozygous . Here we report further genetic mapping of the Ccw locus, and some details of the histology of the lens of Ccw/+ heterozygotes.
We further report the analysis of an ethylnitrosourea (ENU)-induced mutant causing corneal opacity in heterozygotes and show that this defect is due to a new mutation in the Pax6 gene.
The mutants were generated at the Institute of Mammalian Genetics, Neuherberg, Germany  and were renamed after their transfer to the MRC Mammalian Genetics Unit, Harwell, UK. The Ccw mutant (originally C-34) was found in an irradiation experiment, but is thought to have arisen spontaneously . The corneal opacity mutant (originally ENU-4002) was found after treatment of strain DBA/2 spermatogonia with 80 mg/kg ENU , and was given the symbol Coop, which will be used in this paper. However, demonstration of a mutation in the Pax6 gene means that it should in future be designated Pax6coop.
The stocks were maintained by crossing to either of the inbred strains C3H/HeH or 102/ElH. Although Coop was found after treatment of a DBA/2 male, it had been crossed to strain C3H for >10 generations at the time of study. All animals described here were maintained at the MRC Mammalian Genetics Unit, Harwell, UK under the guidance issued by the Medical Research Council in "Responsibility in the use of animals for medical research" (July 1993) and Home Office Project Licences 30/000875 and 30/1517.
To identify homozygotes for Coop, Coop/+ heterozygotes were crossed together. A search was conducted for homozygotes by phenotype among liveborn young, and also among fetuses in females dissected when pregnant. To test for allelism with Pax6, Coop/+ females were crossed with males carrying the Pax6 deletion Del(2)Sey4H83H (abbreviated to Sey4H), and were dissected when pregnant.
For examination of Ccw/+ animals, whole eyes of animals of various ages were fixed in 10% neutral formal-saline, embedded in paraffin wax, sectioned and stained with hematoxylin and eosin.
Total RNA was extracted from 12.5 day and 13.5 day homozygous mutant and normal mouse embryos and first strand cDNA synthesized by random hexamer (N)6 priming and reverse transcription using Superscript IITM reverse transcriptase (Life Technologies, Gaithersburg, MD). Primers were designed to amplify overlapping 500-600 bp fragments of the coding region of the mouse Pax6 gene (GenBank accession X63963). Polymerase chain reactions were performed, and amplified fragments of the expected size directly sequenced following a dideoxy nucleotide chain termination method with a ABI BigDyeTM Terminator Cycle Sequencing DNA sequencing kit (Perkin-Elmer, Warrington, Cheshire, UK) following the manufacturer's instructions. A sequencing thermal profile of 30 cycles of 10 s at 96 °C, 5 s at 50 °C and 4 min at 60 °C was applied. Sequencing reactions were run on an ABI 377 automated DNA sequencer (Perkin-Elmer) and visualized using the software ABI Prism 377 XL collection (Perkin Elmer). Forward and reverse sequences of the same amplified DNA fragment were aligned and a consensus sequence obtained for each sample. Sequence comparison was performed using BLAST 2 Sequences at NCBI.
Females heterozygous for Ccw were crossed with wild type males of the species Mus spretus, and Ccw/+ heterozygous female offspring were then backcrossed to males of strain C3H/HeH. The offspring were scored for curly whiskers as a marker of the Ccw phenotype, and for microsatellite markers in the region of Chr 4 in which the Ccw locus was known to lie. Among 113 offspring scored, there were no crossovers between Ccw and the marker D4Mit231 (Figure 1). The order of markers and numbers of recombinants were D4Mit162 - 16/113 - D4Mit81 - 7/113 - (D4Mit231, Ccw) - 4/113 - D4Mit175 - 7/113 - D4Mit146, giving recombination percentages D4Mit162 - 14.2±3.3 - D4Mit81 - 6.2±2.3 - (D4Mit231, Ccw) - 3.5±1.7 - D4Mit175 - 6.2±2.3 - D4Mit146. This placed the Ccw locus approximately at position 45cM on the consensus map, and agrees with our previous finding that Ccw lies distal to Tyrp1 (38.0cM) and proximal to misty, m, (46.1cM) .
Histology of Ccw/+ lenses
Histological studies showed that the lens of Ccw/+ animals developed fairly normally, but then underwent degeneration, leading to total dissolution of the lens. The Ccw/+ phenotype could first be identified by the curly whiskers at about 10 days of age. At this stage, the lens appeared fairly normal in structure with apparently normal fibers. However, vacuolation of fibers could be seen in the periphery. At 26 days, the fibers had lost their regular shape and were swollen and misshapen. Somewhat later (at 4-6 weeks), the whole lens (including the nucleus) was liquefying, and by 14 weeks only a vestige, consisting of the lens capsule with some epithelial cells, remained (Figure 2).
Phenotype of Coop/+ heterozygotes
The two most obvious features in Coop/+ heterozygotes were corneal opacity and small size of the eye (Figure 3). The severity of the corneal opacity was variable. When most severe, it was accompanied by obvious vascularization of the cornea. In the less severe cases, the iris appeared abnormal with attachments to the lens. The reduction in size of the eyes was also variable, and the phenotype could not be scored on eye size alone. However, mean eye weights of Coop/+ animals were clearly less than those of normal sibs. The mean eye weights of affected animals versus normal littermates aged 6-8 weeks being Coop/+ 18.4±0.43 mg (n=10) compared to 22.3±0.46 mg (n=9, t=6.256, df=36, p<0.0001) for unaffected animals. It is possible that in some animals the defects were so slight that they were scored as normal, since the proportion of mice scored as Coop/+ in backcrosses was less than 50%, namely 143/352 or 40.6%.
Mapping of Coop locus
A linkage backcross of Coop with the conventional marker nonagouti, a, revealed positive linkage. The offspring of crosses of Coop+/+a x +a/+a were: Coop+/+a, 44; Coop a/+a, 13; ++/+a, 7; and +a/+a, 32. This gives a linkage c2=32.7, p<0.0001 and a recombination percentage of 20.8±4.14%. Thus, the Coop locus is on Chr 2. However, the data did not indicate whether Coop lay proximal or distal to a. If proximal, the locus would lie close to that of Pax6, the gene mutated in small eye, Sey .
Phenotypes of homozygotes
Intercrosses of Coop/+ x Coop/+, made to detect Coop/Coop homozygotes, yielded no new phenotype, raising the possibility that homozygotes died prenatally. In addition, none of 14 tested affected animals from these crosses bred as homozygotes; the probability of this occurring by chance if Coop/Coop is viable is (2/3)14 or 0.00345.
To search for Coop/Coop homozygotes prenatally, Coop/+ females were mated to Coop/+ males and dissected when 12.5 to 18.5 days pregnant. Further Coop/+ females were mated to +/+ C3H/HeH males as controls. Among the fetuses from Coop/+ x Coop/+ crosses a new phenotype was detected. These fetuses lacked eyes and also nasal openings (Figure 4) and the phenotype strongly resembled that of Pax6sey/Pax6sey . Thus, these animals were regarded as putative Coop/Coop homozygotes. The frequency of this class showed good agreement with expectation based on a 3:1 ratio (Table 1; c2=0.30, p>0.5). No such animals were found among control crosses of Coop/+ x +/+ where homozygotes were not expected. Among the fetuses having eyes at 14.5 to 17.5 days from both types of crosses, some had visibly smaller eyes, with triangular lens apertures (Figure 4). These were considered to be Coop/+ heterozygotes, but they occurred with less than the expected frequency. This is in accord with the finding that, among postnatal animals, Coop/+ heterozygotes could not be scored on eye size only.
In view of the map position of the Coop locus on Chr 2 and the similarity of putative Coop/Coop to Pax6sey homozygotes, the possibility arose that Coop was allelic with Pax6. In order to test this, Coop/+ females were mated to males heterozygous for the deletion Sey4H, which includes deletion of the Pax6 locus (see Standard Ideogram/Anomaly Breakpoints of the Mouse 2000). Dissections of pregnant females revealed abnormal fetuses lacking eyes and nose and closely resembling those found among Coop/+ x Coop /+ crosses (Table 1 and Figure 4). This was taken as evidence that Coop involved a mutation in the Pax6 gene. In both the putative Coop/Coop and the Coop/Sey4H fetuses, small vestiges of eyes without any pigment could be seen. It is not clear whether this indicates a difference from the original Pax6sey/Pax6sey homozygotes.
In order to find the nature of the mutation, cDNA was prepared from fetuses showing the homozygous Coop/Coop phenotype and the sequence of the Pax6 coding region was compared with that of normal mice. A base-pair substitution C->T was found at position 1033 of the Mus musculus Pax6 mRNA for paired box protein (GenBank accession X63963). This led to a change of codon from CAG (glutamine) to TAG (stop). Thus, the mutation leads to a truncation of the protein between the homeobox and the proline/serine/threonine-rich region.
Further studies of cataract and curly whiskers, Ccw, have shown that the phenotype of the heterozygotes involves drastic degeneration of the lens. The lens appears to be formed normally, but its integrity can then not be maintained. Many different mutant genes which lead to cataract are known in the mouse [1,8-11]. In some of these mutants, early lens development is abnormal while others involve progressive abnormalities arising after lens formation. We have not found any description of lens degeneration resembling that seen in Ccw/+ mice. Thus, Ccw appears to be an example of a new type of cataract in the mouse.
New mapping data presented here provide a more precise position for the Ccw locus, relative to microsatellite markers, and this information should be valuable in cloning the gene. At present, this new information has not led to identification of any candidate genes. The phenotype of Ccw includes not only cataract, but also curly whiskers and early post-implantation lethality in homozygotes . The curly whiskers, like the cataract, appear to be of a new type. Most types of curly whiskers are scorable when the vibrissae first emerge at two to three days of age, but in Ccw/+ animals the phenotype is not scorable until 9-10 days. Because Ccw arose spontaneously, it is not possible to predict what type of DNA change (i.e., base-pair change, deletion, insertion, etc) is likely to be involved. It could be that there is a multi-locus deletion and that the different aspects of the phenotype are due to the loss of different genes. Another cataract mutant, dysgenetic lens, dyl, has been mapped nearby [12,13] and affected animals have defects in the forkhead gene Foxe3 . The possibility should be considered that the cataract phenotype of Ccw is due to a change in the same gene as dyl. However, the phenotype is different, as dyl is recessive, and the cataract results from failure of the lens to develop normally. In Ccw the lens develops normally and then degenerates. Moreover, although dyl maps near Ccw, it appears to lie at a more distal point, since it is 12 cM distal to Tyrp1. Ccw lies only 7 cM distal to Tyrp1. Thus, it seems unlikely that Ccw and dyl are allelic. At present, Ccw joins the list of mouse cataract mutants for which the underlying gene is not yet identified. Furthermore, no obvious candidate gene for the curly whiskers phenotype is located in this region of the mouse genetic map.
For the Coop mutant, on the other hand, the underlying mutation has been identified as a base-pair change in the Pax6 gene at position 1033, leading to a stop codon which causes premature termination of translation between the homeobox and proline/serine/threonine rich regions. Several Pax6 mutations are already known in the mouse. The first mutant allele, symbolized Pax6sey, involved a base-pair change  as did an ENU-induced allele, Pax6sey-Neu . Other alleles have involved multi-locus deletions, such as Pax6sey-H  and Sey4H (see Standard Ideogram/Anomaly Breakpoints of the Mouse 2000). The phenotypes of heterozygotes for the original Pax6sey and for the multi-locus deletions were similar, with small size of the eye as the striking feature. However, homozygotes for the deletion Pax6sey-H differ from those of Pax6sey/Pax6sey. Pax6sey-H/Pax6sey-H animals were found to die around the time of implantation while Pax6sey/Pax6sey survived to around birth, with absence of eyes and nose. This implies that the early lethality of Pax6sey-H/Pax6sey-H is due to a deleted gene other than Pax6. In Coop/+ heterozygotes, the phenotype is less severe than in Pax6sey/+ or Pax6sey-H/+. The reduction in size of the eye is not sufficient for reliable scoring, implying that Coop is not a null allele, but is a hypomorph with the truncated protein formed having some function. The phenotype of Coop/Coop homozygotes is very similar to that of Pax6sey/Pax6sey, however, with the possible exception of small vestiges of unpigmented eyes. In this, the allele differs from another hypomorphic allele Pax64Neu (Favor et al, unpublished data) in which the homozygotes had a less extreme phenotype than Pax6sey, and showed vestigial pigmented eyes. In the human, many different mutant alleles of PAX6 have been described, and the phenotypes observed in heterozygotes vary widely according to the nature of the mutation involved (see Human PAX6 Allelic Variant Database 2000). In the mouse, other alleles are being studied, and further investigation of all these alleles is likely to yield valuable information on the function of the Pax6 gene and protein.
We are grateful to M. Harrison for animal care, T. Hacker and colleagues for histology, A. Ford for photography, and Z. Tymowska-Lalanne for sequencing. This work was partly funded by European Union contract number CHRX-CT93-0181.
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