A Spontaneous Mouse Strain with Cryptophthalmos
Authors: Michelle M. Curtain and Leah Rae Donahue
Source of Support: NIH-NEI grant EY015073 to Leah Rae Donahue
Mutation Symbol: ne
Mutation Name: No Eyelid
Strain of Origin: STOCK Tg(CAG-Bgeo/GFP)21Lbe/J X Cast/Ei
Current Strain Name: STOCK ne/J
Stock #: 006857 (view JAX® Mice Data Sheet for additional information including Price and Supply Information)
Phenotype Category: eye, skeleton, coat color, kidney
Origin and Description
No Eyelid was discovered in 2005 by Sandra Gray. It arose in the second generation of a research colony’s mapping cross consisting of a STOCK Tg(CAG-Bgeo/GFP)21Lbe/J (Stock #3920) X Cast/Ei background. Our original mutant had a missing eyelid in that the eye was covered over by fur with no indication of eyelid formation. Similar phenotypes expressed were closed eyelids and small eyes (See Photo 1, Photo 2, and Photo 3). Other phenotypes that arose in the colony were fused and malformed digits, malformed ear pinnae, slight discoloration of the fur on the head and missing kidneys; however these phenotypes are not fully penetrant. Malformed digits can appear in mice with and without eyelid abnormalities; discoloration of the fur and malformed ear pinnae sometimes appear in the eyelid mutants but not with regularity; the missing kidney phenotype has been observed only once. However, due to the similar phenotypes of the ne strain with the candidate gene Frem2, the inclusion of these not fully penetrant phenotypes should not be dismissed even though the missing or closed eyelid is the most predominant feature and what was used to map the ne mutation.
The colony is maintained by mating a +/ne by a ne/ne or the reciprocal. Male and female mutants are fertile but have small litters. A homozygote mated to a heterozygote, based on an average of ten litters, had 4.5 pups per litter. Ten litters of heterozygote by heterozygote matings averaged 5.7 pups per litter.
Genetic Analysis
No Eyelid is a recessive mutation determined by mating a ne/ne to a Cast/Ei inbred strain (Stock # 000928) then intercrossing F1 carriers. There were no mutants in the F1 mice (0/20) and about 11% (8/71) were affected in F2 mice
To determine a genetic map position, we mated a ne/ne to an A/J inbred (Stock #000646) and another ne/ne to a Balb/cBy inbred (Stock #000650). From each cross, sibling carriers were mated and mutants appeared in the F2 generation. Spleen and tail tips were collected from each set of mutants and stored at -80 degrees C. DNA was extracted using standard phenol extraction methods. Polymerase chain reaction was done with MIT or Research Genetics primer pairs. Twenty-three homozygotes were born out of 157 total F2 progeny from the two crosses. The mutation is located between markers D3Mit65 and D3Mit241. D3Mit65 is at 23.3 cM (according to MGI) or 50.4 Mb (according to Ensembl), and there was one recombination out of twenty meiosis or 5% recombination. There were two out of 18 for 11% recombination at D3Mit241 at 33 cM (MGI) or 66.3 Mb (Ensembl). There was no recombination (0/20) at D3Mit120 at 28 cM (MGI).
A candidate gene is Frem2 located at 53 Mb (Ensembl) or 29.9 cM (MGI). The Frem2 reported alleles have cryptophthalmos, microphthalmia, malformed ear pinnae, syndactyly, abnormal coat color, renal defects and heart defects. Our No Eyelid strain exhibits the eye, ear pinnae, coat color, renal and diget phenotypes with variable penetrace. The heart is normal in ne/ne mice up to two months of age; we have not looked at these organs in older mice.
Pathology
Using our standard pathology screen, a seven-week-old homozygote had a disorganized eye. The eye of a 26-day-old homozygote revealed the inner nuclear layer had dark cells that were either dying cells or had failed to develop. A three-week-old ne/ne female had no right kidney and both eyes had thin retinas. Another eight-week-old female homozygote had degeneration of the Organ of Corti at the tip of the cochlear. She also had subpial Rosenthal fibers and hypertrophic astrocytes in the ventral medulla and cervical spinal cord.
A clinical eye exam revealed a greater range of abnormalities in the eyes of mutants. In a four-month-old male mouse, both eyes had dermoid. A five-and-a-half month old mouse had a bloody white cornea. A two-and-a-half month old male had one eye with a retinal spot and the other with a broken cornea. Heterozygotes had normal eyes. Homozygotes from the mapping cross also revealed severe infection in the eyes with blood and cataracts; unaffected siblings were normal.
Hearing was assessed by auditory brainstem response (ABR) on four homozygotes and four unaffected mice all around two months old. Three of the homozygotes did not survive the test, maybe a response to the anesthesia, and the fourth mutant had normal hearing as did the unaffected mice.
Discussion
We have not proven or disproven our strain to be an allele of Frem2 , and we do not know if the digit, coat color, kidney and ear pinna phenotypes are part of the ne mutation. But if ne is another allele of the Frem2 gene, these phenotypes may be part of the mutation. Also, the variability of the ne eye pathology and phenotype may be explained by the myelencephalic blebs reported in some Frem2 strains during embryogenesis. We have not looked at embryos young enough to determine if ne also has these blebs or blisters; however, we looked at an E 17.5 litter where one embryo had an asymmetrical face in that one side was narrower than the other. Another from the same litter had one eye open at an age when eyelids should be covering the eye. These two examples may be due to myelencephalic blebs reported to occur earlier in development from E 11.5 to E 13.
Acknowledgements
We would like to thank Chantal Longo-Guess for ABR analysis; Norm Hawes for clinical eye evaluation, Coleen Marden for preparation of tissues for histological assessment; Rod Bronson, Ph.D, for pathological evaluation.
