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Human Molecular Genetics Advance Access originally published online on October 11, 2006
Human Molecular Genetics 2006 15(22):3273-3279; doi:10.1093/hmg/ddl403
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© The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

A mutation in the F-box gene, Fbxo11, causes otitis media in the Jeff mouse

Rachel E. Hardisty-Hughes1, Hilda Tateossian1, Susan A. Morse1, M. Rosario Romero1, Alice Middleton1, Zuzanna Tymowska-Lalanne1, A. Jackie Hunter2, Michael Cheeseman1,3 and Steve D.M. Brown1,*

1 MRC Mammalian Genetics Unit, Harwell, OX11 0RD, UK, 2 GlaxoSmithKline Pharmaceuticals, New Frontiers Science Park, Harlow CM19 5AW, UK and 3 MRC Mary Lyon Centre, Harwell, OX11 0RD, UK

* To whom correspondence should be addressed: s.brown{at}har.mrc.ac.uk

Received July 10, 2006; Accepted September 21, 2006


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Otitis media (OM), inflammation of the middle ear, is the most common cause of hearing impairment and surgery in children. Recurrent and chronic forms of OM are known to have a strong genetic component, but nothing is known of the underlying genes involved in the human population. We have previously identified a novel semi-dominant mouse mutant, Jeff, in which the heterozygotes develop chronic suppurative OM (Hardisty, R.E., Erven, A., Logan, K., Morse, S., Guionaud, S., Sancho-Oliver, S., Hunter, A.J., Brown, S.D. and Steel, K.P. (2003) The deaf mouse mutant Jeff (Jf) is a single gene model of otitis media. J. Assoc. Res. Otolaryngol., 4, 130–138.) and represent a model for chronic forms of OM in humans. We demonstrate here that Jeff carries a mutation in an F-box gene, Fbxo11. Fbxo11 is expressed in epithelial cells of the middle ears from late embryonic stages through to day 13 of postnatal life. In contrast to Jeff heterozygotes, Jeff homozygotes show cleft palate, facial clefting and perinatal lethality. We have also isolated and characterized an additional hypomorphic mutant allele, Mutt. Mutt heterozygotes do not develop OM but Mutt homozygotes also show facial clefting and cleft palate abnormalities. FBXO11 is one of the first molecules to be identified, contributing to the genetic aetiology of OM. In addition, the recessive effects of mutant alleles of Fbxo11 identify the gene as an important candidate for cleft palate studies in the human population.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Otitis media (OM), inflammation of the middle ear, is the most common cause of hearing impairment in children (1,2). In addition, it remains the commonest cause of surgery in children in the developed world. Acute episodes of OM are usually associated with middle ear infections by the bacterial pathogens Streptococcus pneumoniae and Haemophilus influenzae (3). However, prolonged stimulation of the inflammatory response accompanied by poor mucociliary response can lead to a persistent middle ear effusion (OME), and in many children, recurrent or chronic suppurative forms of the disease may develop. The prevalence of OM along with its recurrent and chronic nature underlies the frequency of tympanostomies undertaken in the affected children.

There is a variety of evidence suggesting a number of risk factors that predipose to the development of recurrent and chronic forms of OM, including poor mucociliary clearance, craniofacial abnormalities and the presence of an inflammatory stimulus, such as bacteria. However, evidence from studies in the human population and in mouse models demonstrates that there is a very significant genetic component predisposing to recurrent and chronic forms of OM (4–7). OM is a multifactorial disease and the underlying genetic determinants are likely to be complex (8). Several studies have focussed on studying human candidate genes and the association of polymorphisms with OM susceptibility (9). Although several inbred strains are predisposed to the development of OM, the genetic analysis of these strains is compounded by the complex genetic bases and the low penetrance of the phenotype. Moreover, there are a number of mouse mutants that demonstrate OM as part of a more complex syndrome with a wide spectrum of phenotypes. Our approach to identifying genes that are involved in the OM susceptibility has been to study highly penetrant mouse mutants that demonstrate OM in the absence of other diverse pathology and represent appropriate models for OM in the human population. These represent start points to uncover the genetic pathways involved in OM and as candidate genes for human association studies.

The Jeff mouse mutant was identified from a deafness screen as part of a large-scale ENU mouse mutagenesis programme (10). Jeff is a dominant mutant that in heterozygotes displays a conductive deafness due to the development of a chronic suppurative OM that develops at weaning and is associated with raised thresholds for a cochlear nerve response (4). Jeff heterozygotes are smaller than their wild-type littermates and have a mild craniofacial abnormality. In older Jeff heterozygote mice, hearing thresholds are raised beyond what might be expected of a simple conductive hearing impairment. Indeed, endocochlear potentials in these mice were abnormally low, suggesting that the mutation in older mice is associated with sensorineural hearing loss due to impaired strial function. There are many reports of middle ear disease in humans associated with a sensorineural component to the hearing loss. Overall, the disease pathology observed in Jeff indicates that the mutant is an appropriate model for OM in humans.

We have proceeded to identify the gene underlying the Jeff mutant. Jeff carries a mutation in the F-box gene, Fbxo11. In addition, we have isolated and characterized an additional ENU mutant allele at this locus, the Mutt mutation. Both Jeff and Mutt homozygotes demonstrate cleft palate defects, facial clefting and perinatal lethality. Fbxo11 represents an important candidate gene for the study of the genetic pathways involved in OM in the human population. In addition, the role of Fbxo11 in the development of the palatal shelves implicates that this as an important candidate for studies of cleft palate in the human population.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Mapping and identification of the Jeff mutation
The Jeff mutant is a semi-dominant mutation with the heterozygote previously described as having chronic proliferative OM (4). The Jeff mutation was mapped using backcrosses to an ~300 kb region of distal chromosome 17 flanked by the markers SNPMGUC17 and D17Mit1 (see Materials and Methods) (data not shown). On the basis of Ensembl predictions (http://www.ensembl.org), this region contains three genes and two pseudogenes. The three genes in the region are Fbxo11, Msh6 and a novel transcript, the 40S ribosomal s24 gene.

In order to identify the Jeff mutation, we carried out both denaturing high-performance liquid chromatography (DHPLC) screens and sequence analysis of all three genes, Fbxo11, Msh6 and s24. Ultimately, we sequenced all coding regions. This analysis revealed only one coding change in exon 13 of Fbxo11 (Fig. 1A), an A–T transversion at base 1472 causing a non-conservative glutamine to leucine change at amino acid 491. The change occurs in a highly conserved region of this protein that has been maintained through evolution (Fig. 1C). The Fbxo11 locus has one predicted transcript and encodes a protein of 850 amino acids, coded for by 22 exons. The 94 kDa protein consists of two carbohydrate-binding domains, an F-box motif and a zinc-finger domain (Fig. 1A).


Figure 4031
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  		Figure 1. Mutations in the Fbox gene, Fbxo11. (A) Protein structure of Fbxo11 from sequence predictions. The molecule consists of an F-box motif, two carbohydrate-binding (CASH) domains and a zinc finger domain (ZnF). The amino acid numbers constituting these domains are shown below the appropriate domain. The positions of the Jeff and Mutt mutations are also indicated (arrows). The position of peptides, flanking the Jeff mutation, used to raise a polyclonal antibody CIYVHEKGRGQFIEN (P1) and CPIVRHNKIHDGQHG (P2) is indicated (see also Materials and Methods). (B) Sequence lineups of the amino acids 238–251 (in the mouse) in different species. Note the change S–L in Mutt DNA (shown in red). (C) Sequence lineups of the amino acids 485–498 (in the mouse) in different species. Note the change Q–L in Jeff DNA (shown in red).

 
Characterization of the Jeff homozygous mutant phenotype
The mapping of the Jeff mutation allowed us to genotype and examine mice homozygous for the Jeff mutation. One hundred percent of Jeff homozygotes demonstrated perinatal lethality, dying at birth or within a few hours (n=36) due to respiratory problems (gasping for air and with air in their stomach). Homozygotes are born with upper eyelids open and show clefting of the hard or soft palate as well as facial clefting (Fig. 2B).


Figure 4032
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  		Figure 2. Coronal sections of the head (H&E staining) demonstrating the palate and facial phenotypes of late embryonic Jeff homozygotes and perinatal Mutt homozygotes and Jeff/Mutt compound heterozygotes that fail to thrive. Scale bar: 1 mM. Facial clefts are marked with a star and cleft palate is marked with an arrow. (A) E15.5 wild-type; (B) E15.5 Jeff homozygote with facial cleft and cleft palate; (C) 5 DAB wild-type, rostral section; (D) 5 DAB Mutt homozygote with facial cleft, rostral section; (E) 1 DAB compound Jeff/Mutt heterozygote with facial cleft, rostral section; (F) 5 DAB wild-type, caudal section; (G) 5 DAB Mutt homozygote with mild cleft palate, caudal section.

 
The original description of the OM phenotype in adult Jeff heterozygotes (4) was based on sagittal sections of bisected heads and this is not optimal to evaluate the possibility of cleft palate. In view of the finding of cleft palate in Jeff homozygous mice, the palate of Jeff heterozygotes was examined in coronal sections of the snout in a series of 17 Jf/+ mice 5, 13, 28, 56 and 120 DAB (days after birth). None had cleft palate and OM was clearly evident by 28 DAB onwards.

An additional Fbxo11 mutant allele: Mutt
We used DNA and sperm archives derived from ENU mutagenesis programmes (11) to identify an additional allele at the Fbxo11 locus. We screened the first seven exons of Fbxo11 employing heteroduplex analysis of 4200 mutant mice and identified a further point mutation in exon 7, leading to a non-conservative serine to leucine change, S244L (Fig. 1A) in a conserved region of the protein (Fig. 1B). We rederived this second allele of Fbxo11, Mutt, and examined the heterozygous and homozygous phenotype.

At 48 DAB a proportion of Mutt heterozygotes (13%, n=128) showed a reduced startle response to a toneburst of ~24 kHz, 90 dB SPL and a mild craniofacial abnormality, a shortened face (57%), similar to the craniofacial phenotype of the Jeff heterozygote (4) but did not have OM at 39–45 DAB (0/63 examined), suggesting that Mutt is a weaker hypomorphic allele of Fbxo11 in comparison to the Jeff mutation. However, similar to the Jeff mutation, we found that a small proportion of Mutt homozygotes (17%, n=52) showed perinatal lethality with these mice, demonstrating mild clefting of the palate along with facial clefting in some instances (Fig. 2D, 2 g). The small proportion of Mutt homozygotes demonstrating perinatal lethality, in comparison to the 100% lethality found in Jeff homozygotes, also indicates the hypomorphic nature of the Mutt allele. The surviving Mutt homozygotes demonstrated a marked craniofacial abnormality—short face (84%) and reduced hearing (42%)—using the 24 kHz, 90 dB SPL toneburst but did not have OM (0/17 examined).

Compound heterozygotes carrying both Jeff and Mutt alleles showed a similar phenotype to Jeff. About 88% of Jeff/Mutt mice (n=19) survived and demonstrated a shortened face, reduced hearing (in response to a click box) and OM. Thus, most of the compound heterozygotes recapitulated the Jeff heterozygous phenotype again indicating the hypomorphic nature of the Mutt mutation. However, importantly, a small proportion (12%, n=19) of the compound heterozygotes demonstrated facial clefting (Fig.2E) and failed to survive, similar to Jeff homozygotes and a proportion of the Mutt homozygotes (discussed earlier).

Expression of Fbxo11 during mouse development
Peptides flanking the Jeff mutation were used to raise an antibody to FBXO11 (Fig. 1A). Immunohistochemistry on paraffin sections was performed to study the expression of FBXO11 in wild-type embryonic tissue (from E8.5 to E18.5), in newborn, 4 DAB, 13 DAB and 21 DAB head tissue and in various adult tissues. At E8.5, we detected no labelling. At E9.5 and E10.5 (Fig. 3A), FBXO11 signal was restricted to the developing heart tissue. By E11.5 and E12.5, labelling was observed in the liver (Fig. 3B) that subsequently extended to the muscle by E13.5. By E14.5, labelling is still detected in the heart, liver and muscle, but signal is now seen in the developing secondary palate including the nasal, medial and oral epithelia of the palatal shelves, as they elevate above the tongue (Fig. 3C). At E15.5 and E16.5, the lung, kidney, heart, liver, muscle and adrenal gland are all labelled. At this time, fusion of the palate shelves has occurred, with signal confined to the nasal and oral epithelia. By E17.5, signal in the lung is confined to the bronchial epithelial cells (Fig. 3D) and expression is evident in the bone marrow, skin, tissue macrophages, osteoblasts, kidney, liver and spleen. At E18.5, bone marrow, liver, kidney and muscle are positive, but signal in heart and lung is beginning to fade out. At this time, labelling is first detected in the middle ear epithelium (Fig. 3E). At the new born stage, labelling is strong in the middle ear and confined to the mucin secreting cells, as well as persisting in bone marrow (Fig. 3F), kidney and liver. Middle ear expression persists in postnatal head tissue at 4 DAB (Fig. 3 g) and 13 DAB (Fig. 3 h) and has declined by 21 DAB (data not shown). Expression of FBXO11 therefore occurs in the middle ear just preceding the period OM is evident in mice. In adult, tissue expression is seen in the alveolar macrophages of the lung, the glomeruli and the collecting tubules of the kidney, the midbrain, the heart and the muscle (data not shown).


Figure 4033
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  		Figure 3. Expression of FBXO11 in wild-type tissues at various developmental and adult stages of the mouse. Scale bar: 50 µm. (A) E10.5 heart tissue expression, is in developing cardiac cells (arrow); (B) E11.5 liver tissue, expression is in haematopoietic cells (arrow); (C) E14.5 palate, expression is in the margins of the palatal shelves (PS) (arrows); (D) E17.5, bronchial epithelial cells (arrow): B, bronchiole; (E) E18.5, middle ear epithelial cells (arrow); (F) new-born stage, bone marrow; (G) 4 DAB, middle ear epithelium (arrow); (H) 13 DAB, middle ear epithelium (arrow). MEC, middle ear cavity.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
The Jeff mouse mutant is a model of chronic OM in the human population. We have therefore proceeded to map and identify the mutation underlying the Jeff mutant. The Jeff mutant carries a mutation in the F-box protein, Fbxo11. FBXO11 is expressed in the middle ear epithelium just preceding the period OM is evident in the Jeff mouse. We have also isolated and characterized an additional mutant allele at the Fbxo11 locus, the Mutt mutation. The Mutt allele does not develop OM, suggesting it is a hypomorphic allele at the Fbxo11 locus. However, like the Jeff mutation, a proportion of Mutt homozygotes show facial clefting and cleft palate. At E14.5, FBXO11 is expressed in the margins of the palatal shelves. The mutational and expression analysis of the Fbxo11 gene identifies a new locus involved with cleft palate and facial clefts in the mouse (12–15). It will be important to investigate the contribution of this F-box protein and its substrate to cleft palate defects in the human population.

Fbxo11 is one member of a large family of proteins involved with ubiquitination. Much of the targeted protein ubiquitination that occurs in eukaryotic organisms is performed by cullin-based E3 ubiquitin ligases, which form a superfamily of modular E3s (16). The best understood cullin-based E3 is the SCF ubiquitin ligase (17–19) composed of a modular E3 core, containing CUL1 and RBX1, SKP1 and a member of the F-box family of proteins (20–25). The interaction of the F-box protein with SKP1 occurs via the F-box motif, an approximately 40 amino acid motif first identified in yeast and human cyclin-F. F-box proteins also contain further interaction domains that bind ubiquitination targets. A recent study (16) identified 74 mouse genes encoding recognizable F-box motifs subdivided into three subsets: FBXL (containing leucine rich repeats), FBXW (containing WD40 motifs) and FBXO (proteins that contain an F-box and an ‘other’ identifiable motif) of which there are 47 members to date (16,26). A fragment of FBXO11 was originally identified in a differential expression analysis of cultured melanocytes from generalized vitiligo patients versus control cells (27). This cDNA which they called VIT1 was found to be absent in melanocytes from vitiligo patients. Recently, FBXO11/PRMT9 was identified as an arginine methyltransferase with a structure different from all other known protein arginine methyltransferases (28), but a potentially diverse set of targets (29).

Very little is known of the function of most F-box proteins in disease and development but there are examples of proteins from all three subclasses playing pivotal roles. It is interesting to surmise, however, on the role that ubiqutination and protein turnover might play in regulating signalling networks involved in epithelial inflammatory events in the middle ear and the involvement of FBXO11. Nontypeable H. influenzae (NTHi) is often associated with middle ear infections and acute episodes of OM. NTHi activates an intricate array of host epithelial signalling networks (30) that contribute to the pathogenesis of the disease. One signalling pathway, by example, leads to the activation of NF-{kappa}B and up-regulation of cytokines IL-1ß, IL-8, TNF{alpha} as well as the mucin, MUC2. NF-{kappa}B pathways are the sites of several control points involving ubquitination (31). For example, NF-{kappa}B is maintained in an inactive state by binding to the protein I{kappa}B. Following phosphorylation of I{kappa}B, I{kappa}B is targeted for ubiquitination and degradation by the proteosome, thus freeing NF-{kappa}B for entry into the nucleus (32). It will be important to identify the protein substrates of FBXO11 that are targeted for ubiquitination and to identify whether they impact on the known signalling pathways that are involved in epithelial inflammatory responses in the middle ear.

The characterization of Fbxo11 as a major gene involved in susceptibility to OM identifies a further function for this class of proteins. In addition, it provides an important locus for candidate gene studies in the human population. Indeed, it is noteworthy that our initial studies of FBXO11 SNPs in human OM families have uncovered nominal evidence of association, indicating the genetic involvement of human FBXO11 in chronic OM with effusion and recurrent OM (33).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Mice and husbandry
Mice were housed in conventional cages and were provided with food and water ad libitum and maintained according to home office and ethical regulations. Sentinel health screening from this old MRC Harwell mouse house showed the presence of the following FELASA listed agents (http://www.felasa.org) (34): MHV (judged by histology to be enteropathic strains), Adenovirus II and TMEV, none of which is primary respiratory pathogens. Intestinal flagellates, pinworms and the opportunist respiratory pathogen Pasteurella pneumotropica were also common isolates. Non-FELASA listed bacteria isolated from the nasopharynx of sentinel mice included Staphylococcus spp., Staphylococcus aureus, Alpha-haemolytic streptococci and other Streptococcus spp. (but not S. pneumoniae). The Jeff colony has subsequently been rederived by embryo transfer into a new MRC Harwell SPF facility, the Mary Lyon Centre, and is maintained on the C57BL/6J background. The stocks are free of FELASA listed agents, but the nasopharynx of sentinel mice has similar non-FELASA list streptococcal and staphylococcal flora.

Genetic mapping
ENU mutagenesis was carried out on a BALB/c background and males outcrossed to C3H/HeN (10). For inheritance testing, affected F1 individuals were backcrossed to C3H/HeN. We originally mapped the Jeff mutation to chromosome 17, based on 30 affected individuals (4). To further increase the resolution of this genetic map, 920 meioses were generated. Mapping was further enhanced by a second backcross [(Jf/+xC57BL/6J)x(C57BL/6J)]. Markers and primer sequences are available on request from the authors.

Denaturing high-performance liquid chromatography
Mutation detection was performed using a transgenomic wave machine, utilizing DHPLC. The system was run according to manufacturer's instructions (Transgenomic). Exons to be screened were amplified using primers placed in flanking introns. DNAs from a Jf/+ mouse and a BALB/c (+/+) mouse were amplified for each exon using Taq polymerase (ABgene), at an annealing temperature of 55°C. Following amplification, heteroduplexes were formed by thermocycling.

Sequencing
PCR products from Jf/+ and BALB/c DNA were purified using QIAquick PCR purification kit (Qiagen). Direct sequencing was performed using Applied Biosystem's Bigdye Terminator v3.1 cycle sequencing mix and sequenced on an ABI prism 377 DNA sequencer according to manufacturer's instructions.

Genotyping
Genotyping for Jeff mice was performed by PCR amplification of the exon containing the mutation followed by digest with BclI. Amplification of exon 13 using primers 5'-TGC CTG ATG TAA AAA TTA CTC CAC-3' and 5'-TCT CTA GGG ATC AGG CAC ATC-3' yields a product of 199 bp. In the presence of the mutation, a BclI restriction site is introduced giving two bands of 132 and 67 bp.

To genotype Mutt mice, genomic DNA was used to PCR amplify the region containing the mutation with primers 5-' biotin TTC AGA GCC TTC CAT GAA CAC G-3' and 5'-NNN CCT GGC AAG GTT GCA GAC-AA-3'. The PCR product (77 bp) and primer 5'-TCA TCA TTG AGA ACA CTA GA-3' were used for subsequent pyrosequencing SNP analysis to identify differences in sequences.

Fbxo11 polyclonal antibody
A polyclonal antibody against mouse FBXO11 was produced by Covalab UK (www.covalab.com) using two peptides as antigens. The peptide sequences were 419CIYVHEKGRGQFIEN433 and 497CPIVRHNKIHDGQHG511 and they lay in the central unique part of the mouse Fbxo11 protein. Both peptides were injected together into rabbits. The serum from the immunized animals was collected and the antibody was purified by affinity chromatography using the peptides. Affinity-purified antibody was tested on western blot using whole mouse head lysates where it recognizes bands of ~46, 32 and 26 kDa. Various cell lines showed bands of similar sizes by western blot. Since the antibody does not detect full-length protein, we tested its specificity in various ways. First, pre-incubation of the affinity-purified antibody with increasing amounts of the peptides used as antigens for its production gradually abolished the signal detected by western blot and immunohistochemistry. Secondly, following immunoprecipitation with the purified antibody, the 46 kDa band was digested with trypsin and identified as FBXO11 by peptide mass fingerprint (data not shown). In addition, we transfected Cos-7 cells with a plasmid containing the mouse FBXO11 sequence tagged to the Xpress epitope (Invitrogen). In lysates from transfected Cos-7 cells, anti-Fbxo11 polyclonal antibody recognizes an identical band to that detected by anti-Xpress antibody and of the expected size (~94 kDa).

Western blotting
Mouse head tissue was homogenized at 4°C in lysis buffer (50 mM HEPES, pH 7.4, 10% Triton X-100, 50 mM sodium phosphate, 10 mM EDTA, 10 mM sodium fluoride, 10 mM sodium orthovanadate, 2 mM benzamidine and a protease inhibitor cocktail). Homogenate was solubilized on ice for 1 h and centrifuged at 4°C, first at 3000 rpm for 15 min and then at 14 000 for 1 h at 4°C. The supernatant was run in a 4–12% acrylamide gel (Invitrogen NuPAGE) and then the gel was blotted onto a PVDF membrane. The membrane was incubated in a blocking solution containing PBS, 0.1% Tween-20 and -5% skimmed milk for 1 h at room temperature. After blocking, the membrane was incubated with anti-Fbxo11 antibody at 7.5 µg/ml in blocking solution for 1 h at room temperature. After four washes in PBS/0.1% Tween-20, the membrane was incubated with anti-rabbit IgG conjugated to HRP for 1 h at room temperature and washed as before. The bands labelled by the antibody were detected by ECL-Plus (GE Healthcare).

Histology
Whole embryos (E8.5–E18.5), new born mice, adult heads (4, 13 and 21 DAB) and various adult tissues were fixed in 10% buffered formaldehyde, decalcified in EDTA (embryos E14.5–18.5 for 3–5 days and adult heads for 4 weeks) and embedded in paraffin. Four-micrometer-thick transverse and coronal sections were obtained, de-paraffinized in xylene substitute and rehydrated via a graded ethanol solutions. For morphological observations, sections were stained with H&E. The ears of 39–45 DAB Mutt mice on the C3H/HeN background (+/+ n=17, Mutt/+ n=63, Mutt/Mutt n=17) and double heterozygotes (Jf/Mutt n=17) were surveyed for OM. The ears and palates were surveyed in SPF Jeff heterozygotes on C57BL/6J background (two 5 DAB, two 13 DAB, three 28 DAB, five 56 DAB, five 120 DAB). These heads were decalcified 24–48 h with Immunocal (Decal Corp. Tallman, NY, USA) and stained with H&E.

Immunostaining
For immunohistochemical analysis, the avidin–biotin complex (ABC) method was used. Endogenous peroxidase activity was quenched with 3% hydrogen peroxide in isopropanol for 20 min. The slides were pre-treated by boiling in a microwave in 1 mM EDTA, pH 8, for E8.5–13.5 embryos and in water, for E14.5–18.5 embryos and all adult tissues, for 14 min, cooled at RT for 20 min and rinsed with phosphate-buffered saline. The immunostaining was performed using a DAKO autostainer at room temperature. To inhibit non-specific endogenous biotin staining, the DAKO Biotin Blocking System was used (DAKO, X0590). A blocking solution of 10% swine serum (DAKO, X0901) was used for 1 h. Fbxo11 antibody incubations were conducted for 1 h using a 1:200 dilution. Biotinylated swine anti-rabbit antibody (DAKO, E0353) and ChemMate Detection Kit (DAKO, K5001) were used to develop the specific signals. Negative control sections were stained with the FBXO11 antibody previously incubated with the blocking peptides and processed identically. The slides were counterstained with haematoxylin.


   ACKNOWLEDGEMENTS
 
This work was funded by the MRC and by the European Commission under contract number QLG2-CT-2002-00930. The authors would like to thank Caroline Barker, Adele Seymour, Jennifer Corrigan and Terry Hacker for histology services.

Conflict of Interest statement. None declared.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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