Helicopter Ears: A Structural Mutation

 

Michelle M. Curtain and Leah Rae Donahue

Source of Support: This research was supported by NIH-NEI grant EY015073 to Leah Rae Donahue PI.

Mutation Symbol: He
Mutation Name: Helicopter Ears
Strain of Origin: B6129SF2/J
Current Strain Name: B6;129P2-He/J
Stock #: 007947 (view JAX® Mice Data Sheet for additional information including Price and Supply Information)
Phenotype Category: craniofacial, skeletal

Abstract

We report here an ear pinna mutation along with a skeletal phenotype.

Origin and Description

Helicopter Ears was discovered in a production colony of B6129SF2/J (Stock # 101045) mice in April 2006. This new spontaneous, dominant mutation is characterized by ear pinna angled outward from the head rather than upward. Mutants also have smaller bodies than littermate controls. (See Photo) Male and female heterozygotes are fertile and fertility appears unaffected even though mutants exhibit smaller genitals.

Genetic Analysis

Helicopter ears mutation was proven to be a dominant mutation when affected mice arose in the first generation of a +/He by C57BL/6J mating.

Using our standard mapping protocol, the Helicopter ears mutation was mapped by mating a female +/He with a Cast/Ei inbred male. Affected F1s were then backcrossed to another Cast/Ei. Affected N2 mice were collected and tissue was stored at -70 degrees C. DNA was extracted from either spleen or tail tips using standard phenol extraction methods. Polymerase chain reaction was done with MIT or Research Genetics primer pairs (MapPairs, from Research Genetics, Huntsville, Ala., or from Integrated DNA Technologies, Coralville, Ia.). Linkage cross analysis of 20 +/He mice showed no recombination near the centromere of Chromosome four. The three markers in this non-recombinant region are D4Mit316 at 5.2Mb, D4Mit235 at 8.3Mb and D4Mit181 at 9.5 Mb. The first recombination is at 24.5 Mb at marker D4Mit105 with 5% (1/20). Recombination increased at 45.1 Mb (D4Mit5) to 17.6% (3/17).

A preliminary look at homozygote viability was done. We mated +/He to a Cast/Ei inbred mouse and collected DNA from neonate and embryonic F2s. DNA from a one-week-old litter consisted four +/+ and eight +/He. DNA from E 14.5 typed for three wild types, five heterozygotes and two homozygotes. Results indicate that He/He are not born but are still in utero at E 14.5 though more numbers would be needed to confirm homozygote lethality.

Biological Characterization

-DEXA analysis of whole body aBMD and body composition of twelve-week-old mice:

Three-month-old male and female mutant and controls were collected and whole body, areal bone mineral density (aBMD), bone mineral content (BMC) and body composition (lean, fat and % fat mass) were assessed by PIXImus densitometry (GE LUNAR, Madison, WI) (Table 1-See protocol on this website). Overall +/He females differed significantly from +/He males and +/+ females. Female mutants had significantly less whole body BMD and BMC, fat, total mass, skull BMD and BMC. (graph 1 & graph 2) The composition and skeletal phenotype appears to mainly affect females.

-Craniofacial morphology of twelve-week-old mice:

Three-month-old male and female mutant and control skulls were collected and 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-See protocol this website) were made using digital calipers (Stoelting, Wood Dale, Ill) with previously established landmarks (Richtsmeier, 2000, see protocol this website). Female +/He had significantly less skull height and lower jaw length than female controls. Male mutants had significantly lower jaw length compared to male controls. Ear pinna was significantly reduced in both male and female mutants compared to same sex controls. (Graph 3)

Pathology

Using our standard pathology screen,  no lesions were seen in a three-month-old male mutant. Ear pinna was examined in a nine-month-old female mutant and it was histologically normal. Hearing was assessed by auditory brainstem response (ABR) on males and females.  Three mutants and four controls at 10-weeks-old had normal hearing; a different set of four mutants and two controls were all normal at two to three-months-old. A clinical eye exam  with three mutants and four controls of both sexes revealed no abnormalities, and an electroretinography (ERG) on a 54-week-old male mutant was normal.

Discussion

The Helicopter ear mutation affects both sexes equally in regard to the pinna phenotype, however with regard to the skeleton and body composition phenotype, females are more affected. Hearing is not impaired.

Acknowledgements

We would like to thank Jo-Lyn Harper for discovering this mutation; Heping Yu and 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 and Patricia Ward-Bailey for manuscript assistance and web publication.