Small body and short ear pinna, a spontaneous mutation on chromosome 4 in the mouse

 

Michelle M. Curtain, Leah Rae Donahue

 Source of Support: NIH-NEI grant EY015073 to Leah Rae Donahue
Mutation Symbol: sbse
Mutation Name: small body and small ear
Strain of Origin: C57BL/6J
Current Strain Name: C57BL/6J-sbse/J

Stock #: 004246 (view JAX® Mice Data Sheet for additional information including Price and Supply Information)

Phenotype Category: craniofacial, skeletal, hydrocephaly, hearing

Origin and description

sbse is a spontaneous autosomal recessive mutation that arose on the C57BL/6J background in a production colony at the Jackson Laboratory in July 2001. The phenotype is a smaller body size along with smaller ear pinna and domed skull. (See Photo) Homozygotes live normal life spans and they are fertile.

Genetic analysis

sbse is inherited as a recessive mutation as shown by traditional linkage cross analysis. No visible mutants were seen in the F1 generation in which a mutant was mated to an unrelated C57BL/6J (0/29). The F2 generation showed the phenotype about 25% of the time, the expected Mendelian ratio (6/30).

For linkage analysis, a female sbse/sbse was mated to a male CAST/Ei. F1s were then intercrossed to produce F2 mutants. Spleens and tail tips were collected from 80 F2 sbse/sbse mice and were stored at -70C. DNA was extracted with the standard phenol method and the strain was mapped using our standard mapping protocol. The mutation is located near the centromere of Chromosome 4. The distal flank is at D4Mit316 (5.2 Mb) with 2/152 recombinants, or 1.3% recombination. There were no recombinants (0/152) on D4Mit149 at 3.6 Mb or D4Mit50 at 3.2 Mb.

A candidate gene Plag1 is located in our non-recombinant region at 3.8 Mb. Plag1 homozygous null mice display reduced male fertility, small seminal vesicles and ventral prostate, reduced litter size with female breeder mutants, reduced embryonic and postnatal growth, and delayed eyelid opening.  Our sbse exhibits some of these characteristics and are discussed in the Pathology and Biological Characterization sections. Our sbse strain has at least one characteristic that differs from the one known allele of Plag1 (Plag1tm1Wjmv ); our mutants have a hearing phenotype. Another reported Plag1 phenotype is a delayed eyelid opening, and this has not been confirmed in the sbse colony. We designed primers for the coding region of Plag1 ( http://frodo.wi.mit.edu/cgi-bin/primer3/primer3.cgi ). We compared sbse/sbse and C57BL/6J control sequence to the mouse sequence publicly available at ensembl.org/ PCR products were sequenced at The Jackson Laboratory core sequencing facility, and we did not find a mutation in the coding region.

Pathology

Tissues for histopathological examination were prepared from both sbse/sbse and controls. (Our standard pathology screen) There was severe hydrocephaly in an 11-week-old male mutant and mild hydrocephaly in a ten and 13-week-old mutant that may explain the domed skull. Due to the small bodies of the mutants, the pituitary gland of a 10-week-old mutant brother and sister were looked at but no abnormalities were found. A two-month-old sbse male had mild testicular degeneration and Plag1 is reported to have reduced male fertility.

Hearing was assessed by auditory brainstem response (ABR) threshold analysis (Zheng et al. 1999) on male and female mutants and controls, and mutants had age-related hearing loss.  At seven weeks, mutants had 20 db higher thresholds than controls. These same mutants at three months were 30 db higher and were deaf while the controls still had good hearing. Eyes of mutants and heterozygotes from both sexes at 4 weeks old were looked at with an ophthalmoscope. (eye protocols) There were no abnormalities.

Biological characterization

Reduced litter size

The reduced litter size reported in Plag1 females is also in our sabe strain. Upon examining ten litters from sbse/sbse females by +/sbse males, there was an average 4.3 pups per litter. To compare, male mutants mated with female carriers produced on average the same number of pups in a litter as heterozygote males mated with heterozygote females with both breeding schemes having 5.4 pups per litter when averaging ten litters from each.

Longitudinal weight study
Mice from eight different litters were collected and weighed at four, eight, 12 and 16 weeks of age (see Chart 1). Seven mice from each genotype (+/sbse and sbse/sbse) and sex (male and female) were represented. At each age, sbse/sbse males and females were about 70% the weight of same sex +/sbse mice. In conclusion, homozygotes did not catch up in size to the heterozygotes rather they grew within their own parameters. This may be similar to the reduced embryo size and postnatal growth in Plag1 mice.

DEXA analysis of whole body aBMD and body composition of twelve-week-old mice:
Whole body, areal bone mineral density (aBMD), bone mineral content (BMC) and body composition (lean and fat) were assessed by PIXImus densitometry (GE LUNAR, Madison, WI) (Table 1-See protocol on this website). sbse/sbse mice had significantly less fat, whole body BMD and BMC and skull BMC. For skull BMD, male sbse/sbse had significantly less than both male +/sbse and female sbse/sbse. Skull to body BMD was significantly greater with females of both genotypes compared to males of both genotypes. This skull to body BMD ratio appears more affected by sex and is due to females having greater skull BMD than males of the same genotype. (graph1 & graph 2)

Craniofacial morphology of twelve-week-old mice:

Skulls were prepared by incomplete maceration in potassium hydroxide, stained with alizarin red, and stored in undiluted glycerin (Green, 1952). Morphological measurements of the skull (Table 2) were made using digital calipers (Stoelting, Wood Dale, Ill) with previously established landmarks (Richtsmeier, 2000, see protocol this website). Skull and nose lengths, lower and upper jaw lengths, ear pinna, and skull length to width were all significantly less in sbse/sbse males and females than same sex +/sbse illustrating an overall smaller head in mutants. Additionally, the inner canthal of male homozygotes was significantly less than male heterozygotes indicating a slightly more proportionate smaller skull than females. However, skull to nose length ratio was greater in sbse/sbse mice compared to controls due to disproportionately shorter noses when compared to total skull length. Skull height to length ratios were significantly greater in sbse/sbse mice compared to same sex heterozytoges due to skull height in mutants being nearly the same as controls. Mutants heads are overall smaller but are disproportionate. (graph 3, graph 4, and graph 5.

Discussion

While we cannot confirm our sbse strain is a new allele of Plag1 without further sequencing and/or an allele test, sbse mice exhibit characteristics indicative of  a mutation in this gene

Acknowledgements

We would like to thank Sharon Colson for discovering the mutation; Coleen Marden for preparation of tissues for histological assessment; Rod Bronson, Ph.D, for pathological evaluation; Heping Yu for ABR analysis; Norm Hawes for clinical eye evaluation.