Cauliflower ear, a remutation to Bmp5
Jill Giggey, Joiel Bauschatz, Michelle Curtain, Julie Hurd and Leah Rae Donahue
Source of Support: This research was supported by a grant awarded to The Jackson Laboratory by The National Eye Institute titled "Gene Discovery For Craniofacial Disorders" (RO1 EY 015073 - Dr. Leah Rae Donahue, PI).
Mutation (allele) name: cauliflower ear
Mutation (allele) symbol: Bmp5<cfe-se7J>
Gene symbol: Bmp5
Strain of Origin: C.129S7-Gt(ROSA)26Sor/J
Current Strain Name: C.129S7-Gt(ROSA)26Sor-Bmp5cfe-se7J/J (cfe)
Stock Number: 005420
Phenotype Category: craniofacial
Discoverer: Phillip Russ
Origin and Description
We report here a new recessive mutation that arose spontaneously in the C.129S7-Gt(ROSA)26Sor/J colony, which we have named cauliflower ear (cfe). The recessive mutation was discovered at the Jackson Laboratory in 2000 in an Induced Mutant Resource colony. The homozygous mutation always affects both ears and is characterized by small, round ear pinnae with ridges along the perimeter. The stock is currently maintained by mating homozygotes to heterozygotes of the opposite sex. Both males and females are fertile; females have normal litter sizes and lactate frequently. The strain was tested for penetrance by mating two homozygous mice and was found to be 100% penetrant. The cfe mutation was mapped to chromosome 9 between the markers D9Mit11 and D9Mit259. A negative complementation test with homozygous Bmp5se/Bmp5se mice from the SeaGn/J strain determined that cfe is a remutation to Bmp5. Because of the ruffled ear pinnae of cfe/cfe mice compared to the smooth ear pinnae in Bmp5se/Bmp5se mice, we named this remutation cauliflower ear.
Genetic Analysis
Cauliflower ear is inherited as a recessive mutation as shown by traditional breeding experiments. For linkage analysis, a CAST/Ei male was mated to a homozygous cfe/cfe female. F1 hybrids were then intercrossed to produce F2 progeny. There were no visible mutants seen in the F1 generation (0/16) and 24% of F2 progeny were mutants (40/166). F2 progeny were observed for the cauliflower ear phenotype, and spleens and tail tips of affected mice were collected and stored at -70C for subsequent DNA typing to map the mutation. DNA was extracted from the tail tips by a standard hot sodium and Tris (HotSHOT) procedure (Truett, et al., 2000) and polymerase chain reaction was carried out with MIT primer pairs (MapPairs, Research Genetics, Huntsville Ala.). A genome scan began with markers near the Fgfr3 gene on Chromosome 5 because it displays defects within the inner ear, and the Bmp5 gene on Chromosome 9. Linkage of cfe on chromosome 9 was first detected with marker D9Mit164 located at 37.0 cM. Twenty-one DNA samples were then typed for additional markers until results showed linkage at 0% recombination with D9Mit11 at 48 cM (Mouse Genome Database, TJL). The positive allele test confirmed cfe to be a remutation in Bmp5.
Biological Characterization
A. DEXA Analysis of Whole Body BMD and Body Composition
Whole body weight assessed by PIXImus densitometry (GE LUNAR, Madison, WI) was not statistically significant different between mutants and controls in either females or males. Tail, femur, back and spine length were measured on six mutants and six controls in each sex, in order to determine a skeletal index (Table 1). Back length was measured from the first cervical vertebrae to the inferior surface of the ischial tuberosity. Spine length was measured from the first cervical vertebrae to the inferior surface of the fifth lumbar vertebrae. There were no significant differences found between mutants and controls. Ratios of tail length : back length, femur length : back length and spine length : back length were calculated (Table 2) and no significant differences were found. Whole body and skull x-rays of two male and two female mutants and controls were developed. After examination, there were no abnormalities found.
Whole body bone mineral density (BMD) and whole body bone mineral content (BMC) assessed by PIXImus (Table 3) were less in both female and male mutants than in controls. BMD was statistically significant in both males and females (Figure 1). However, BMC was not significant in either gender. There were no significant differences found with whole body lean and whole body fat between mutants and controls. However, homozygous cfe/cfe females have significantly less fat and lean than cfe/cfe males.
B. Craniofacial Morphology
Skulls of six male and six female mutants and controls were collected at twelve weeks of age, prepared by incomplete maceration in potassium hydroxide, stained with alizarin red, and stored in undiluted glycerin (Green, 1952). During the collection process, right ear pinnae were measured with digital hand calipers (Stoelting, Wood Dale, I11). Morphological measurements of the skull (Table 4) were also made using digital calipers (Stoelting, Wood Dale, Ill) with previously established landmarks (Richtsmeier, 2000). Both male and female mutants have shorter skull lengths as compared to controls (Figure 2) and were found to be statistically significant. Female mutants have statistically significant shorter nose lengths than controls (Figure 3). Male mutants have shorter nose lengths than controls, however, they are not significant. Male cfe/cfe mice have a greater skull height than male controls (Figure 4) and were statistically significant. However, female cfe/cfe mice have a lower skull height as compared to controls and are not significant. There is no difference in skull width among male and female mutants and controls. The upper and lower jaw lengths of both male and female mutants are shorter than controls (Figures 5 and 6). In females, the upper jaw length is not significant, but the lower jaw length is. In males, the upper jaw length is statistically significant and the lower jaw is not. Finally, measurements of the right ear pinna were significantly lower in mutants than controls for both males and females (Figure 7).
In female mutants and controls, the ratio skull length to nose length is statistically significant (Figure 8). However, it is not significant between male mutants and controls. The ratio skull height to skull length is significant in males but not in female mutants and controls (Figure 9). The ratio upper jaw to lower jaw in male and female mutants and controls is not statistically significant. Both male and female mutants and controls show statistically significant ratios between skull length to skull width (Figure 10). Finally, the skull height to skull width ratio is significant in male mutants and controls but not in females (Figure 11).
Table 1 : Skeletal Index Measurements of Twelve Week
C.129S7-Gt(ROSA)26Sov-Bmp5cfe-se7J/J Mice (n=6, mean +/- SEM)
Table 2 : Ratios of Skeletal Index Measurements of Twelve Week
C.129S7-Gt(ROSA)26Sov-Bmp5cfe-se7J/J (n=6, mean)
Table 3: PIXImus Densitometric Measurements of Twelve Week Old
C.129S7-Gt(ROSA)26Sov-Bmp5cfe-se7J/J (n=6, mean +/- SEM)
Table 4: Digital Caliber Measurements of Twelve Week
C.129S7-Gt(ROSA)26Sov-Bmp5cfe-se7J/J skulls
stained with Alzarin Red (n=6, mean +/- SEM)
Figure 1: BMD in Bmp5cfe-se7J/Bmp5cfe-se7J
Male and Female Mice Compared to Controls
Figure 2: Skull Length in Bmp5cfe-se7J/Bmp5cfe-se7J
Male and Female Mice Compared To Controls
Figure 3: Nose Length in Bmp5cfe-se7J/Bmp5cfe-se7J
Male and Female Mice Compared to Controls
Figure 4: Skull Height in Bmp5cfe-se7J/Bmp5cfe-se7J
Male and Female Mice Compared to Controls
Figure 5: Upper Jaw Length in Bmp5cfe-se7J/Bmp5cfe-se7J
Male and Female Mice Compared to Controls
Figure 6: Lower Jaw Length in Bmp5cfe-se7J/Bmp5cfe-se7J
Male and Female Mice Compared To Controls
Figure 7: Right Ear Pinna in Bmp5cfe-se7J/Bmp5cfe-se7J
Male and Female Mice Compared to Controls
Figure 8: Skull Length to Nose Length Ratio in Bmp5cfe-se7J/Bmp5cfe-se7J
Male and Female Mice Compared to Controls
Figure 9: Skull Height to Skull Length Ratio in Bmp5cfe-se7J/Bmp5cfe-se7J
Male and Female Mice Compared to Controls
Figure 10: Skull Length to Skull Width Ratio in Bmp5cfe-se7J/Bmp5cfe-se7J
Male and Female MiceCompared to Controls
Figure 11: Skull Height to Skull Width Ratio in Bmp5cfe-se7J/Bmp5cfe-se7J
Male and Female Mice Compared to Controls
C. Hearing tests
Hearing was assessed by ABR threshold analysis (Zheng et al. 1999) with two mutants and one control mouse at three months of age. The ABR results showed that both mutants and control mice have normal hearing.
D. Eye Examination
Two three month mutant and control male mice were examined by a slit lamp and indirect ophthalmoscope. Some mice showed an early onset of severe cataract, which may be progressive. Otherwise, eyes are normal.
Pathology
One male control and one female mutant were perfused at six weeks of age via cardiac infusion of Bouin?s fixative following admission of anesthesia. No lesions were found in any major organ.
Discussion
Bone morphogenetic protein (BMP) is a family of highly conserved, secreted proteins that affect differentiation, axis formation, growth control, and sexual development (Massague, 1990). It was found that mice carrying the recessive short ear mutation displayed defects within one of the eight BMP genes (Kingsley, 1992). Studies have shown that the classical short ear mutation presents with a nonsense mutation within the coding region of the BMP5 gene (King, 1994). Therefore, BMP5 plays a key role in skeletal patterning and soft tissue development (King, 1994).
Phenotypically, mice with the short ear mutation develop characteristic skeletal defects, including reduction of the external ear, loss of several small bones, alterations in size or shape of the xiphoid process, reduction of ventral processes at the sixth cervical vertebrae and deletion of one pair of ribs (Green and Green, 1946; Green, 1951,1968). Measurements of the skull show that short ear mice have a wider skull and a shorter nose than wild types (Kingsley, 1994). There are also a variety of soft tissue anomalies, including an increased frequency of misplaced ovaries, hydrotic kidneys, lung cysts, liver granulomas, and neuromuscular tail kinks that occur on inbred strains (Green, 1968). Various phenotypes have been reported for the short ear mutation, which suggests modifier genes are present in different mouse strains (Green, 1957).
The cauliflower ear mutation presents with a shortened external ear but with ruffles along the edges of the ear pinnae. This short ear allele is viable within this congenic strain and shows typical Mendelian ratios. There are no differences in body size or shape between the cauliflower mutant and wild type. There were no differences in the skeletal index measurements, including length of the femur, tail, back and spine. Finally, we have not found in our Bmp5cfe-se7J/Bmp5cfe-se7J mice any skeletal or soft-tissue abnormalities with routine pathological inspection.
The whole body bone mineral density and whole body bone mineral content is less in mutants than controls due to a disruption in skeletal patterning within the bones of BMP5 deficient mice. However, whole body BMD is significant and BMC is not, which is a result of the small sample size and the high standard error of measurement found within the BMC data. The skull length and nose length and the upper and lower jaw lengths of the cfe/cfe mice are shorter than controls in both genders. Skull length is significant in both gender, however, nose length is significant in females only. There is a significant difference in the lower jaw of females and the upper jaw is significant in males only. Male mutants have a greater skull height than female mutants and controls. There were no differences found with skull width between mutants and wild types. The skull height to skull length and skull height to skull width ratios are significant in males but not females. The skull length to nose length ratio is significant in females and not males. Both males and females have a significant skull length to skull width ratio. However, upper jaw and lower jaw ratios are not significant in either gender. Therefore, cfe/cfe mice of both genders display a shorter skull length, smaller ear pinnae, and shorter upper and lower jaws than controls. Females have a shorter nose length and a shorter skull height, while males have a greater skull height than females. Although the bodies are well proportioned in this strain, the skull measurements are different between male and female mutants and controls.
The morphological differences seen between the original short ear mutation and the cauliflower ear remutation may be due to differing genetic backgrounds. Further exploration of potential modifying genes contributed by various inbred backgrounds could help to elucidate the functional pathways of BMP5 and its role in skeletal and soft-tissue abnormalities. This could be of great use to determine the mechanisms responsible for human EPS (ear, patella, short stature) syndrome, which could be the human equivalent of BMP5 deficiency in the mouse (Lacombe, 1994).
Acknowledgements
We thank the following for their excellent expertise:
Phillip Russ, Discoverer
Colleen Marden, Pathology
Rod Bronson, Ph.D, Pathology Evaluation
Pat Ward-Bailey, Genetic Analysis
Jane Maynard, Tissue Preparation
Norm Hawes, Evaluation of Eyes
Heping Yu and Qing Yin Zheng, Evaluation of Ears
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