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Physiol. Genomics 32: 311-321, 2008. First published November 6, 2007; doi:10.1152/physiolgenomics.00116.2007
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Received 25 May 2007; accepted in final form 2 November 2007.
Physiological Genomics 32:311-321 (2008)
1094-8341/08 $8.00 © 2008 American Physiological Society

An ENU-induced mutation in the Ankrd11 gene results in an osteopenia-like phenotype in the mouse mutant Yoda

Ivana Barbaric 1, Mark J. Perry 2, T. Neil Dear 3, Alexandra Rodrigues Da Costa 2, Daniela Salopek 4, Ana Marusic 4, Tertius Hough 3, Sara Wells 3, A. Jackie Hunter 5, Michael Cheeseman 1,3 and Steve D. M. Brown 1

1 MRC Mammalian Genetics Unit, Harwell
2 Department of Anatomy, University of Bristol, Bristol
3 MRC Mary Lyon Centre, Harwell, United Kingdom
4 Department of Anatomy, Zagreb University School of Medicine, Zagreb, Croatia
5 GlaxoSmithKline Pharmaceuticals, New Frontiers Science Park, Harlow, United Kingdom


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 DISCLOSURES
 REFERENCES
 
The mechanisms that regulate bone mass are important in a variety of complex diseases such as osteopenia and osteoporosis. Regulation of bone mass is a polygenic trait and is also influenced by various environmental and lifestyle factors, making analysis of the genetic basis difficult. As an effort toward identifying novel genes involved in regulation of bone mass, N-ethyl-N-nitrosourea (ENU) mutagenesis in mice has been utilized. Here we describe a mouse mutant termed Yoda that was identified in an ENU mutagenesis screen for dominantly acting mutations. Mice heterozygous for the Yoda mutation exhibit craniofacial abnormalities: shortened snouts, wider skulls, and deformed nasal bones, underlined by altered morphology of frontonasal sutures and failure of interfrontal suture to close. A major feature of the mutant is reduced bone mineral density. Homozygosity for the mutation results in embryonic lethality. Positional cloning of the locus identified a missense mutation in a highly conserved region of the ankyrin repeat domain 11 gene (Ankrd11). This gene has not been previously associated with bone metabolism and, thus, identifies a novel genetic regulator of bone homeostasis.

N-ethyl-N-nitrosourea; osteopenia; Ankrd11


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 DISCLOSURES
 REFERENCES
 
BONE DISORDERS SUCH AS OSTEOPENIA and osteoporosis have a major impact on health. Osteoporosis is a disease in which bone strength is compromised due to a decrease in bone mass and microarchitectural deterioration of bone tissue, resulting in increased fracture risk (3). Decreased bone mass that precedes osteoporosis is defined as osteopenia. The etiology of such disorders is complex. Multiple environmental, behavioral and nutritional factors are associated with a risk of bone loss, but there is also a clear evidence of a major genetic contribution (12, 20). However, the exact genetic components that contribute to the development of these disorders are largely unknown. To identify participating genes, various linkage and candidate gene association studies have been performed in human populations (21). These studies are complicated by the multifactorial, polygenic nature of the disorders. Due to such complexity, a great deal of research into the genetics of bone disorders is undertaken in animal models. Apart from analyzing inbred mouse strains, as well as overexpressing and inactivating candidate genes in mice, N-ethyl-N-nitrosourea (ENU) mutagenesis screens have been undertaken to identify novel genes and mutations that give rise to changes in bone metabolism (10, 16, 26).

We report the identification of the Yoda mutant in a dominant ENU mutagenesis screen (16). Heterozygous Yoda mice exhibit craniofacial abnormalities and reduced body size, as well as reduced bone mineral density (BMD). Homozygous Yoda mice die during embryogenesis. We have identified that Yoda mice carry a missense mutation in the ankyrin repeat domain 11 gene, Ankrd11. The Yoda mutant presents an important model for studying the pathogenic mechanisms and identifies Ankrd11 as a novel gene for studying the genetics of bone metabolism.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 DISCLOSURES
 REFERENCES
 
ENU mutagenesis.
Male BALB/cCrlAnn mice were mutagenized as described (16) and mated with C3H/HeH mice. G1 offspring were screened for abnormalities using the SHIRPA protocol. The Yoda mouse was identified in this screen.

Body length measurements.
Body length was measured from the tip of the nose to the beginning of the tail, using digital hand calipers (Mahr).

Cranial measurements.
Cranial measurements were taken using digital hand calipers (Mahr). The following distances on the skull were measured: skull length, nasal bones length, frontal bone length, parietal bone length and skull width, as indicated in Fig. 2C. These measurements were adapted from Richtsmeier et al. (22).

Alizarin red S and Alcian blue staining of mouse skulls.
Dissected skulls were placed in absolute ethanol for 2 days. Ethanol was then replaced with a solution containing 150 mg/l Alcian blue 8GX, 80% (vol/vol) ethanol and 20% (vol/vol) acetic acid. After 12–20 h, skulls were rinsed in absolute ethanol for 12 h. For initial clearing, samples were incubated in 2% (wt/vol) KOH for 6 h. This was followed by staining for 3 h in 2% (wt/vol) KOH containing 50 mg/l Alizarin red S monohydrate. Skulls were finally incubated in 2% (wt/vol) KOH for 12–20 h and stored in 25% (vol/vol) glycerol. All steps were performed at room temperature.

X-ray analysis.
Radiography was performed using a Faxitron digital X-ray system at 26 kV with average exposure time of 3 s. Digital images were obtained by using a fitted camera with a field of view 2 x 4 inches and 10 pixel/mm resolution. The Specimen program on the computer accompanying the Faxitron machine enabled instant viewing of X-ray images as well as storage of images as digital files.

DXA measurements.
Whole animals fixed in formalin were scanned by dual-energy X-ray absorptiometry (DXA) using a PIXImus scanner with PIXImus v1.44 software. For analysis of individual bone samples, excised right tibia and femur as well as lumbar vertebrae were cleaned and fixed in 70% (vol/vol) ethanol for 2 wk. BMD, bone mineral content and area of each bone were measured by DXA. The accuracy of this technique in measuring calcium content has been confirmed in earlier studies in which a highly significant correlation between femoral total bone mineral content and ash weight (r = 0.86, P < 0.0001) was found. The coefficient of variation for femoral BMD, obtained after scanning five times each with repositioning between scans, was 2.7%.

Peripheral quantitative computed tomography measurements.
Quantitative computed tomography was performed on excised femurs using a Stratec pQCT machine. Calibration of the machine was performed daily using a phantom sample provided by the manufacturer. Computed tomography (CT) speed used to obtain the scout view was 15 mm/s, and the slide distance was 0.5 mm. Measurements were obtained at two sites of each bone, representing 10 and 50% of femoral length as measured by planner image of scout view. A 0.5-mm-thick cross section was taken at these two sites, with a CT speed of 3 mm/s and a voxel size of 0.1 mm3. Analysis was performed using contour mode 3, peel mode 2, and separation mode 4. Threshold values were used to distinguish between the cortical shell from the soft tissue (300 mg/cm3), trabecular from (sub)cortical bone (700 mg/cm3), as well as to distinguish cortical bone along the endocortical surface (550 mg/cm3).

Bone histomorphometry.
Cancellous and cortical bone histomorphometry was performed at the distal femoral metaphysis and midfemoral diaphysis, respectively. Femora were freed from soft tissue and cut into proximal and distal halves. Both halves were fixed in 70% ethanol for 48 h and dehydrated through a graded series of alcohols: 80% ethanol, 90% ethanol followed by three changes of 100% ethanol for 24 h each. Femora were cleared in chloroform for 24 h, then placed for 24 h in 100% ethanol, and embedded without decalcification in LR White Hard Grade (London Resin, Reading, UK). Longitudinal sections of the distal femoral metaphyses were cut on a Reichert-Jung 2050 microtome (Heidelberg, Germany) with a "D" profile tungsten carbide knife, 7 µm sections were stained with 1% toluidine blue in 0.01 M citrate phosphate buffer, and 10 µm sections were mounted unstained in fluoromount (BDH; Laboratory Supplies, Poole, UK) for assessment of calcein and tetracycline hydrochloride labeling for new bone formation by fluorescent microscopy. Transverse sections of the immediate proximal end of the proximal portion of the femur were prepared for histomorphometric analysis of the middiaphysis, using a Reichert-Jung 2050 microtome as described above; 15 µm sections were stained with 1% toluidine blue in 0.01 M citrate phosphate buffer for bone area measurement.

Histomorphometric analysis of the distal femoral metaphysis was performed using transmitted and epifluorescent microscopy linked to a computer-assisted image analyzer (Osteomeasure; Osteometrics, Atlanta, GA). All sections were examined blind. For each animal two sampling sites, each with a standard area of 0.36 mm2, were analyzed. The proximal border of the proximal sampling site was situated 0.25 mm below the lowest part of the growth plate to exclude primary spongiosa as previously described (24). The second sampling site was situated immediately distal to the first. Both sites were centered within the shaft of the cortical bone for each section. All bone parameters were measured at 200-times magnification and assessed on two nonconsecutive longitudinal sections. Cancellous bone volume was examined on sections stained with toluidine blue for each animal and expressed as a percentage of total tissue volume (BV/TV).

Fluorochrome measurements were made on two nonconsecutive 10 µm sections per animal. The length of trabecular bone surface covered by double label (dlS) was expressed with reference to the total tissue volume (TV) as dlS/TV and as a percentage of the total length of cancellous bone surface (BS) as dlS/BS. The mineral apposition rate (MAR) was determined by dividing the mean distance between the fluorochrome labels by the 3-day interval between administration of the labels. The MAR values were not corrected for the obliquity of the plane of the section. The bone formation rate (BFR) was obtained from the product of MAR and either dlS/TV or dlS/BS giving BFR/TV and BFR/BS, respectively. Cortical bone parameters were assessed on cross-sections of the femoral diaphysis. Lengths of periosteal surface, endosteal surface, cortical bone area, and medullary area were analyzed on toluidine blue-stained sections.

Bone histomorphometry for osteoclast numbers.
Mouse tibiae were excised and the surrounding soft tissue was cleaned off. After fixation in 4% (wt/vol) paraformaldehyde, they were demineralized in 14% (wt/vol) EDTA (pH 7.4), dehydrated in a 70–100% (vol/vol) ethanol series, and embedded in paraffin. For each bone sample, osteoclasts were identified on four tartrate-resistant acid phosphatase-stained serial sections, counted using Osteomeasure software, and the mean of the four measurements was taken.

Clinical chemistry.
Blood samples were taken from superficial tail veins of mice, and plasma samples were analyzed on AU400 automated clinical chemistry analyzer (Olympus Diagnostic Systems) as described in Ref. 10. Leptin levels were measured using an ELISA-based kit (R&D Systems) according to the manufacturer's instructions.

Mapping the mutation.
Genome-wide low-resolution mapping was performed using DNA samples from 31 N2 C3H backcross animals that were identified as probable carriers of the Yoda mutation on the basis of their craniofacial phenotype. Genomic DNA samples isolated from tail biopsies of these animals were screened by PCR amplification and gel electrophoresis with 71 microsatellite markers spaced approximately equidistantly across the genome. Samples were genotyped as either homozygous C3H or heterozygous BALB/c-C3H for each marker. For finer mapping, crosses of Yoda mice with C57BL/6J were also set up, and further microsatellite markers polymorphic between either BALB/c and C3H or BALB/c and C57BL/6J were used to genotype Yoda mice that were recombinant in the critical region. Single nucleotide polymorphisms (SNPs) were genotyped by sequencing of PCR products amplified with primers flanking the SNP site. Sequencing of candidate genes involved designing primer pairs to amplify individual exons as well as flanking splice donor/acceptor sequences. All exons and splice sites in the critical region were sequenced using homozygous as well as heterozygous Yoda DNA, ensuring that no base changes were missed.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 DISCLOSURES
 REFERENCES
 
Gross morphological abnormalities in heterozygous Yoda mice.
The Yoda mouse was originally identified on the basis of a craniofacial abnormality in a visual screen of G1 offspring from ENU mutagenized BALB/c males crossed with wild-type C3H/HeH females (16). At weaning, heterozygous Yoda mice weighed less than the wild types, and this difference persisted into adult life (Supplementary Material, Fig. S1).1 Similarly, their body length was smaller from weaning, and this difference was also present in adult mice; the average body length of 6-mo-old Yoda mice was smaller by ~9% compared with their wild-type littermates (Fig. 1, A and B). However, histological sections of tibial growth plates and measurement of IGF-I level in plasma revealed no differences (data not shown).


Figure 1
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  		Fig. 1. Yoda mice have a reduced body size. A: typical example of the size difference between an adult Yoda mouse and a littermate control at 6 mo of age. B: body length of adult heterozygous Yoda males was 9% smaller compared with littermate controls. Mean body length of adult Yoda males (n = 9) was 106.4 ± 2.9 mm compared with 116.5 ± 1.4 mm for wild- type counterparts (n = 6). Values shown are means ± SD. *Student's t-test, P < 5 x 10–6.

 
Yoda mice had shorter (Fig. 2A) and wider (Fig. 2B) faces. These observations were reflected in significant differences between Yoda mice and littermate controls in the length of specific bones of the skull (Fig. 2, C and D). Given that Yoda mice were generally smaller than wild-type littermates it was to be expected that their skull length would also be smaller. Hence, to determine whether any of the cranial bones show disproportionate decrease or increase in length, cranial bone measurements were normalized against skull length for each animal. These ratios were then compared between Yoda and wild-type mice. The comparison revealed a significant decrease in normalized nasal bone length and frontal bone length as well as increase in normalized skull width and parietal bone length (Fig. 2E). These disproportions are evident as a stubby nose, wide-face appearance in Yoda mutants.


Figure 2
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  		Fig. 2. Craniofacial phenotype of Yoda mice. Yoda mice have a shorter (lateral view, A) and wider (dorsal view, B) face appearance compared with wild-type littermates. C: dorsal view of a mouse skull stained with Alizarin red S and Alcian blue, showing distances measured on dissected skulls. D: specific bone measurements on Yoda and wild-type littermate skulls. Significant differences are indicated. E: ratios of measurements in heterozygous Yoda mice for length of nasal bone:skull, parietal bone:skull, frontal bone:skull, and skull width:skull length. Values shown are means ± SD. Student's t-tests indicated significant differences in all of the measurements between the 2 groups. *P < 0.05, **P < 0.005, ***P < 0.001 with Bonferroni posttest correction factor.

 
Staining of skulls to visualize bone and cartilage revealed incomplete closure of the interfrontal suture (Fig. 3A), as well as a decreased interdigitation pattern of the frontonasal suture in Yoda heterozygotes (Fig. 3B). Furthermore, the morphology of the nasal bones of Yoda heterozygotes was altered, giving the skull a dome-shaped appearance (Fig. 3C). This is also clearly shown in the X-ray images of the skull (Fig. 3D).


Figure 3
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  		Fig. 3. Craniofacial anomalies in Yoda mice. A: example of an interfrontal suture that has failed to fuse (circled) in a 4-wk-old heterozygous Yoda mouse skull stained with Alizarin red S (staining bone) and Alcian blue (staining cartilage). Note the incomplete closure of the interfrontal suture in the close-up of the Yoda skull. Dorsal view of the skulls is shown. B: example of a nasofrontal suture (circled) that shows less interdigitation in a heterozygous Yoda mouse skull stained with Alizarin red S and Alcian blue. Dorsal view of the skulls is shown. C: lateral view of the skull showing the aberrant shape of the nasal bone in Yoda mice (indicated by arrow). D: X-ray image from lateral side of the skulls also showing the aberrant morphology of nasal bones.

 
Heterozygous Yoda mice have reduced BMD.
X-ray analysis suggested reduced bone mass in the bones of heterozygous Yoda mice, as they appeared more radiolucent than the wild-type bones (Fig. 4A). To investigate this further, whole body BMD was assessed using DXA. Heterozygous Yoda mice had lower whole body BMD (Fig. 4B). In an analysis of individual bones, a decrease in BMD was apparent in excised femora, tibiae, and lumbar vertebrae (L1–L6) from 12-wk-old as well as 15-mo-old heterozygous Yoda mice compared with their sex-matched wild-type littermates (Fig. 4, C, D, and E for femora, tibiae, and lumbar vertebrae, respectively). Bone mineral content (BMC) and bone area values are displayed in Table 1. All but three BMC values were significantly decreased in young and old Yoda mice compared with sex-matched littermates.


Figure 4
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  		Fig. 4. Heterozygous Yoda mice have reduced bone mineral density (BMD). A: typical example of an X-ray image of a femur from a 13-mo-old heterozygous Yoda mouse (N2 backcross of BALB/c to C3H) and sex-matched littermate control. The bone from the Yoda mouse appears more radiolucent than the wild-type control. B: whole body BMD measurement obtained by dual-energy X-ray absorptiometry (DXA) in 12-wk-old and 7-mo-old heterozygous Yoda mice (n = 5–12) and wild-type littermates (n = 5–15). C–E: DXA measurements of BMD in excised femora, tibiae, and lumbar vertebrae (respectively) from 12-wk and 15-mo-old heterozygous Yoda mice (n = 9–12; N4 BALB/c to C3H backcross) and wild-type littermates (n = 10–15). Values shown are means ± SD. Two-way ANOVA revealed the contribution of genotype as a source of variation, and these are displayed as % for each bone site. *P < 0.05, **P < 0.01, ***P < 0.001 by Bonferroni posttest following 2-way ANOVA.

 

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  		Table 1. BMC and bone area values as measured by DXA in young and old heterozygous Yoda mice and their wild-type littermates

 
The BMD differences observed by DXA analysis were further analyzed by peripheral quantitative computed tomography (pQCT). This also revealed a trend toward decreasing BMD in Yoda mice. Total BMD in femora of 3-mo-old Yoda females was decreased 5.5% compared with their wild-type littermates. This decrease became more pronounced as the mice aged and measured 27% at 7 mo but did not change significantly between 7 and 15 mo of age. Measurements for total, trabecular, and cortical components are shown in Table 2. In addition to these differences in Yoda females, a significant decrease in total BMD or cortical BMD was also seen in heterozygous Yoda males, at either 7 or 15 mo (Table 2). Overall, the loss of bone mass measured by DXA and pQCT appeared more pronounced in Yoda females than males, indicating sex-related aspects of the bone phenotype in Yoda mice.


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  		Table 2. pQCT analysis of femora from Yoda males and females and wild-type littermates

 
Interestingly, ageing of Yoda mice and littermate controls revealed that 80% of aged female Yoda mice exhibited spinal kyphosis that was particularly prominent by 20–22 mo of age (Fig. 5). X-ray analysis was used to determine whether vertebral compression, characteristic of advanced stages of osteoporosis, might account for this phenotype. However, the vertebrae did not exhibit obvious signs of compression in the affected region of the spine. Therefore, there may be an alternative etiology for this kyphosis. For example, alteration in the musculature surrounding the spine might result in such a phenotype.


Figure 5
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  		Fig. 5. Spinal kyphosis in older Yoda females. A 22-mo-old Yoda female exhibiting a hunched back (arrow) is shown. X-ray images did not reveal any obvious vertebral compressions in the spine of Yoda females.

 
Bone histomorphometry.
To explore further the bone phenotype revealed by DXA and pQCT, histomorphometry was performed on sections of the femur and tibia. Static histomorphometry in the distal femoral metaphysis showed no differences in cancellous bone mass (BV/TV%) between Yoda mice and their littermate controls (Table 3). There were also no differences between genotypes in any of the dynamic histomorphometric bone parameters at the proximal region of analysis. However, at the distal region a consistent decline was observed in all the dynamic parameters in Yoda mice. The amount of bone forming surface was decreased significantly (P < 0.05) in Yoda mice for both tissue volume (dLS/TV) and bone surface referents (dLS/BS). An apparent trend toward a reduction in MAR in Yoda mice resulted in a borderline significant (P = 0.06) decline in bone formation (BFR/TV and BFR/BS).


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  		Table 3. Cancellous bone histomorphometric data

 
To evaluate whether there are signs of high-turnover bone loss as the mice aged, we measured the numbers of osteoclasts in histological sections of tibiae from 15-mo-old Yoda mice and wild-type littermate controls. Yoda females had significantly fewer osteoclasts than their wild-type counterparts (P < 0.005), whereas the number of osteoclasts in Yoda males and wild-type controls did not differ significantly (Table 4).


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  		Table 4. Histomorphometric measurements of osteoclast numbers in tibiae of 15-mo-old Yoda mice and wild-type littermates

 
As radiographic images of femora (Fig. 4A) indicated reduction in cortical bone thickness, measurements of trabecular and cortical bone compartments were also performed. No significant differences were observed in the trabecular bone compartment (data not shown). Measurements of cortical bone included measuring the cortical bone area, medullary area, as well as the length of the periosteal and endosteal perimeters. Cortical bone area was reduced in female Yoda mice (P < 0.05) but not male Yoda mice compared with their wild-type littermates (Table 5). This coincided with periosteal perimeter being the same in male Yoda and wild types but decreased in female Yoda mice (P < 0.01). Both male and female Yoda mice had reduced enodosteal perimeters (P < 0.05 and P < 0.01, respectively), leading to reduced medullary areas (P < 0.01 and P < 0.001, respectively) compared with wild types.


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  		Table 5. Histomorphometric measurements of cortical bone in femora of 12-wk-old Yoda mice and wild-type littermates

 
Plasma biochemistry.
As an initial step in investigating whether there is a generalized metabolic disease underlying the bone phenotype or whether activity intrinsic to the bone cells is altered, markers related to bone metabolism, as well as some bone cell-specific markers, were assessed in the plasma of mutant and wild-type littermate mice. No consistent differences were apparent in a variety of selected mineral and protein parameters measured in plasma of heterozygous Yoda mice at various ages between 2 and 15 mo (phosphate, calcium, alkaline phosphatase, parathyroid hormone, osteocalcin, tartarate-resistant acid phosphatase 5b, 17-β-estradiol). The one exception was leptin, which was significantly lower in Yoda mice (15-mo-old mice; wild-type males = 8.3 ± 2.2 ng/ml, Yoda males = 5.2 ± 1.2 ng/ml, one-way ANOVA P = 0.001; wild-type females = 10.4 ± 4.3 ng/ml, Yoda females = 5.1 ± 2.7 ng/ml, one-way ANOVA P = 0.002). Despite the lower leptin levels in Yoda mice, no consistent differences in lean or fat mass were observed between Yoda and wild-type littermates at various time points examined by DXA (Supplementary Table S1).

Yoda homozygotes die during embryogenesis.
Homozygosity for the Yoda allele resulted in embryonic lethality, indicating that the mutation is semidominant. Yoda homozygous embryos died at approximately embryonic day 9.5, were small, and had failed to turn. There was a consistent presence of an allantoic remnant (Fig. 6), suggesting that chorioallantoic fusion had failed to occur. As the chorioallantoic fusion is required for proper development of the placenta (23), the lack of placental formation was possibly responsible for the death of the homozygous Yoda mutants.


Figure 6
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  		Fig. 6. The Yoda homozygous phenotype. A Yoda homozygous embryo (right) compared with a wild-type littermate (left) at embryonic day dE9.5. Yoda homozygous embryos are generally smaller and have not turned. Note the presence of a large remnant or cyst from the allantois (arrow).

 
Mapping of the Yoda mutation.
A positional cloning approach was used to identify the mutation underlying the Yoda phenotype. The Yoda mutation was generated on a BALB/c genetic background. Mice exhibiting the Yoda craniofacial phenotype were backcrossed to C3H/HeH wild-type mice and genotyped with a panel of microsatellite markers spanning the entire genome at regular intervals. A strong linkage was observed on chromosome 8 at marker D8Mit213 (logarithm of the odds score of 6.0 for a recombination fraction {Theta} = 0.1; Supplementary Material, Fig. S2). Subsequent fine mapping by backcrossing to either C3H/HeH or C57BL/6J mice and genotyping for further polymorphic markers on chromosome 8 narrowed the critical region containing the mutation to 625 Kb (Fig. 7A). Sequencing of all exons and splice sites in this region revealed a G/C to A/T transition in the predicted exon 11 of the Ankrd11 gene (Fig. 7B). This mutation leads to a nonconservative amino acid substitution of lysine for the original glutamate. The predicted Ankrd11 cDNA encodes a potential protein of 2,604 amino acids with several identifiable motifs in the protein, including an ankyrin repeat region at the NH2-terminal end of the protein (Fig. 8A) and a highly conserved COOH-terminal domain of Ankrd11 (PD416109 in ProDom database). Clustal W analysis of this 162 amino acid COOH-terminal domain of mouse Ankrd11 compared with probable vertebrate orthologs and invertebrate paralogs, and the closely related domain of Ankrd12, revealed very high conservation in all species examined (Fig. 8B). Significantly, all of the domains have conserved the glutamate residue that is mutated to lysine in Yoda. The mutation is substituting an acidic residue with a basic one and would be expected to have an effect on the role of that amino acid in the protein.


Figure 7
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  		Fig. 7. Genetic mapping and identification of the Yoda mutation. A: haplotype analysis of the 2 critical Yoda mice that narrowed the minimal region containing the mutation to 625 Kb. Due to the lack of polymorphic markers between BALB/c and C3H strains in the region between D8Mit322 marker and SNP Cp4, it was not possible to ascertain where exactly the recombination had occurred in the Mlc16 sample. B: sequence traces of part of the predicted exon 11 of Ankrd11. The mutation can be clearly seen as a change from G to A in the homozygote and as a double peak in the heterozygote. The mutation causes a nonconservative amino acid change from glutamate to lysine.

 

Figure 8
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  		Fig. 8. The Ankrd11 protein and the location of the amino acid mutation. A: schematic representation of domains identified in the Ankrd11 protein. B: Clustal W alignment of the conserved COOH-terminal domain from various species. An arrow indicates the conserved glutamate that is replaced by lysine in the Yoda mouse genome. Amino acids are colored by property and conservation. h, Human; m, mouse. C: model for the effect of the Yoda mutation on transcription from nuclear hormone receptors. NR, nuclear receptor; Ncoa, nuclear receptor coactivator; Pcaf, Cbp, histone acetyltransferases; Hdac, histone deacetylases. (Figure based in part on Ref. 13.)

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 DISCLOSURES
 REFERENCES
 
Phenotype of heterozygous Yoda mice.
The heterozygous Yoda mouse exhibits a complex phenotype with an abnormal cranial morphology, reduced body size and, notably, reduced BMD. The craniofacial deformity affects the skull vault of Yoda mice and consists of a shorter and wider skull, altered shape of nasal bones, an incompletely closed interfrontal suture, and altered morphology of the nasofrontal suture. Similar shortening of the snout as well as morphology of the nasal bones and nasofrontal suture were observed in the mice in which Tcfap2a transcription factor was depleted in a tissue-specific manner from the nasofrontal processes and limbs, and this phenotype was due to premature mineralization of the nasal bone fronts (15). These mice did not, however, possess an incompletely closed interfrontal suture evident in the Yoda mice.

Reduced BMD in Yoda mice at 12 wk of age indicated that they do not achieve the peak bone mass (defined as the greatest amount of bone obtained during skeletal growth) of their wild-type littermates. This is most likely due to decreases in osteoblast activity in Yoda mice at both the cancellous bone surface and the periosteal envelope of cortical bone, leading to a decline in BFR. Peak bone mass is a major factor involved in the pathogenesis of osteopenia and osteoporosis. Although it is recognized that the peak bone mass is influenced by genetic determinants, most of the genes involved remain to be identified (7, 14, 18). Peak bone mass is achieved by the end of adolescence (1, 27). With ageing, the rate of bone formation through the activity of osteoblasts cannot fully compensate for bone lost by resorption through the activity of osteoclasts, leading to bone loss. In the case of insufficient gain of peak bone mass, a normal age-related bone loss can lead to bone mass dropping below the value needed to maintain adequate mechanical bone strength, resulting in osteoporosis. Measurements of osteoclast numbers in aged mice revealed that heterozygous Yoda females also have a decreased number of osteoclasts compared with their littermate controls, indicating that the loss of bone mass in Yoda is likely due to an overall low bone turnover, i.e., both osteoblast and osteoclast activities are decreased. There is a clear sex difference, as osteoclast numbers are only reduced in female, not male, Yoda mice. Sex-related differences were also evident in the cortical bone phenotype of male and female Yoda mice compared with wild type. Further functional assays of osteoblast and osteoclast activity will be required to reveal the generation of this phenotype.

Sex-related differences in bone loss are also observed in humans, with more women being affected by osteoporosis than men. This is thought to be a consequence of a number of factors, including higher peak bone mass and larger bone size achieved by males, with both a larger diameter and greater cortical thickness in the long bones (25). Sex steroids are known to be major factors underlying these differences, and particularly important is estrogen. Notably, however, Yoda females did not have significantly reduced levels of estrogen.

Leptin levels were reduced in heterozygous Yoda mice, although no differences in fat mass were detected. Interestingly, previous studies showed that leptin is implicated in control of bone remodeling. However, the conclusions of its effects on bone remodeling are contradictory. Some studies reported that serum leptin acts as a potent inhibitor of bone formation in vivo and that increasing serum leptin levels leads to a reduction in bone mass (5, 6). Contrary to this, leptin promoted bone formation in vitro (9, 28) and also had a protective effect on ovariectomy-induced bone loss in rats (2). Whether leptin has a role in the bone phenotype of Yoda mice and what mechanism links the mutation in Ankrd11 to the observed decrease in leptin levels will be subject of further studies.

Mapping identified a mutation in the Ankrd11 gene.
The Yoda mutant is the first reported osteopenic mouse model to arise in any of the ENU mutagenesis programs for which the underlying mutation has been identified. Mapping using the recombinants uncovered the critical region of 625 Kb on chromosome 8. There is overwhelming evidence to indicate that the Ankrd11 mutation identified is causing the observed phenotype. Firstly, the coding regions and splice sites of all exons of all genes in the minimal region were sequenced, and only a single mutation (a missense mutation in the Ankrd11 gene) was identified. Secondly, even if there was an exon in a gene that had not been identified, the probability of there being more than one mutation in the coding region of a gene in this region is very small (P = 3 x 10–5) (11). Thirdly, it has become clear that virtually all mutations affect the protein coding region of genes, either through amino acid codon alteration or splice site mutations. Intergenic and intron mutations that have a phenotypic effect must be very rare as there are very few mutants of the many hundreds that have been mapped worldwide for which it was not possible to identify the mutations by sequencing coding regions of genes in the minimum interval. For example, in a recent study of ENU immunological mutants the mutations for all characterized mutants were identified and found to lie in the exons or splice sites of known or predicted genes (17). Fourthly, the probability that there is even another mutation in a noncoding region in the interval to which we have localized the Yoda phenotype is not high (binomial probability of 0.13 using a mutation rate of 1 nucleotide per 1.101 Mb) (19). Finally, the mutation identified in Ankrd11 gives rise to a nonconservative amino acid change in a residue that is highly conserved in many species from Drosophila to human, indicating the importance of that amino acid for the protein function. Thus, there is compelling evidence that we have identified the causative mutation of the Yoda phenotype.

Possible mechanism of action of the Yoda mutation.
The mouse Ankrd11 gene has not been previously studied, but the human ortholog was identified in a screen to detect proteins that interact with the NCOA3 nuclear receptor coactivator (31), which is involved in modulating the activity of nuclear hormone receptors (29). The COOH-terminal domain of ANKRD11 interacts not only with the nuclear receptor coactivators NCOA1, NCOA2, and NCOA3 (members of the p160/SRC family), but also with the histone deacetylases HDAC3, HDAC4, and HDAC5 (31). The Yoda mutation is located in a region that is able to interact with HDACs but lies outside of the region that interacts with nuclear receptor coactivators (31). Therefore, the Yoda mutation could conceivably reduce the ability of Ankrd11 to interact with HDACs but not with p160/SRC coactivators. This might result in an inability of the mutant Ankrd11 to inhibit hormone receptor-activated gene transcription through failure to recruit HDACs to the transcription complex (Fig. 8C).

Many nuclear hormone receptors are implicated in the pathogenesis of bone loss leading to osteoporosis (8). This is not surprising as hormone members of the nuclear receptor superfamily, acting as transcription factors, largely influence endocrine control of bone formation and resorption. These include receptors for steroid hormones (androgen, estrogen, glucocorticoid, mineralcorticoid, and progesterone), thyroid hormone, vitamin D, and retinoids. However, the influence of interacting proteins on the regulation of nuclear hormone receptor activity is yet to be fully understood, and mutants such as Yoda provide a means for studying such interactions. Identifying downstream targets of Ankrd11 should aid in uncovering the mechanisms that link the mutation in Ankrd11 with the phenotype of Yoda mice and reveal how this results in the observed sex-related differences. As steroids are responsible for determining male and female sexual development and maintenance of their adult phenotypes, it is possible that a mutation that perturbs regulation of steroid receptors could affect the way the phenotype develops in males vs. females. A sexually dimorphic phenotype has been observed in some mouse models in which a member of the nuclear receptor superfamily is overexpressed or inactivated (4, 30).

In conclusion, the Yoda mutation reveals the critical role of the Ankrd11 gene in embryogenesis, skeletogenesis, and bone turnover mechanisms. Elucidating in detail the molecular mechanisms by which the Yoda mutation leads to an osteopenic phenotype is expected to contribute to our understanding of the pathogenic processes involved in this disease.


   GRANTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
DISCLOSURES
 REFERENCES
 
This work was supported by the Medical Research Council, United Kingdom.


   DISCLOSURES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 DISCLOSURES
REFERENCES
 
Part of this work was funded by GlaxoSmithKline.


   ACKNOWLEDGMENTS
 
We thank Martin Fray and members of the FESA team for rederivation of Yoda mice; Zuzanna Tymowska-Lalanne, Debra Brooker, and Anne Southwell for assistance with genotyping; and Terry Hacker and Jim Humphreys for histology assistance.


   FOOTNOTES
 
Address for reprint requests and other correspondence: I. Barbaric, Department of Biomedical Science, University of Sheffield, Sheffield S10 2TN, UK (e-mail: I.Barbaric{at}sheffield.ac.uk).

Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).

1 The online version of this article contains supplemental material. Back


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 DISCLOSURES
 REFERENCES
 

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