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Human Molecular Genetics Advance Access originally published online on December 20, 2007
Human Molecular Genetics 2008 17(6):844-853; doi:10.1093/hmg/ddm356
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Published by Oxford University Press 2007

Noggin heterozygous mice: an animal model for congenital conductive hearing loss in humans

Chan-Ho Hwang and Doris K. Wu*

Lab of Molecular Biology, National Institute on Deafness and Other Communication Disorders, 5 Research Court, Rm 2B34, Rockville, MD 20850, USA

* To whom correspondence should be addressed. Tel: +301 4024214; Fax: +301 4025475; Email: wud{at}nidcd.nih.gov

Received September 13, 2007; Accepted November 30, 2007


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 FUNDING
 REFERENCES
 
Conductive hearing loss occurs when sound waves are not relayed efficiently to the inner ear. Mutations of the NOGGIN (NOG) gene in humans are associated with several autosomal dominant disorders such as proximal symphalangism and multiple synostoses. These syndromes are characterized by skeletal defects and synostoses, which include conductive hearing loss. Noggin is an antagonist of bone morphogenetic proteins (BMPs), and balanced levels of BMPs and Noggin are required for proper skeletal formation. Depending on the genetic background, some of the Nog+/– mice display mild hearing loss, that is, conductive in nature. Since Noggin is a single exon gene, this data strongly suggest that the autosomal dominant disorders associated with NOG mutations are due to haploinsufficiency of NOGGIN. The conductive hearing loss in Nog+/– mice is caused by an ectopic bone bridge located between the stapes and the posterior wall of the tympanum, which affects the normal mobility of the ossicle. Our analyses suggest that the ectopic bone formation is caused by a failure of the stapes and styloid process to separate completely during development. This failure of bone separation in the Nog+/– mice reveals another consequence of chondrocyte hyperplasia due to unopposed Bmp activities in these mutants such as Bmp4 and Bmp14 (Gdf5). More importantly, these results establish Nog+/– mice as the first animal model for the study of conductive rather than neurosensory hearing loss that has direct relevance to human genetic disorders.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 FUNDING
 REFERENCES
 
The mammalian ear is comprised of three anatomical and functional compartments, the external, middle and inner ear. Environmental sounds collected by the auricle, travel through the external auditory canal and cause vibrations in the tympanic membrane. These vibrations are delivered to the inner ear via the three ossicles in the middle ear—malleus, incus and stapes. A major function of the middle ear is to amplify sound energies transferred from the external ear to the inner ear with minimal distortion (1). Hearing deficits due to failure of efficient sound conduction to the inner ear are known as conductive hearing loss.

Conductive hearing loss is associated with various human syndromes as well as isolated diseases. NOG is one of the genes associated with conductive hearing loss in humans. Mutations in NOG [(MIM 602991 [OMIM] )] have been linked to several autosomal dominant disorders such as proximal symphalangism [SYM1 (MIM 185800 [OMIM] )] (2), multiple synostoses [SYNS1 (MIM 186500 [OMIM] )] (2,3), tarsal/carpal coalition syndrome [TCC (MIM 186570 [OMIM] )] (4) and Teunissen–Cremers syndrome [(MIM 184460 [OMIM] )] (5). Common features among these syndromes are skeletal defects and synostoses, which include stapes ankylosis that leads to conductive hearing loss.

Nog is a single exon gene encoding a secreted molecule that forms a disulfide-linked homodimer. The dimerized polypeptides bind and antagonize the activities of bone morphogenetic proteins (BMPs). In the skeletal system, Nog is expressed in the condensing cartilage and immature chondrocytes. In Nog knockout embryos, cartilage condensations initiate normally but later develop hyperplasia resulting in oversized growth plates with no joint formation (6).

To establish an animal model for studying conductive hearing loss in patients with NOG mutations, we investigated the hearing ability of Nog+/– mice. We show that depending on the genetic background, some of the Nog heterozygous mice suffer hearing loss. We determined that hearing loss in these mice is conductive in nature due to extra bone formation in the stapes, impeding its mobility during sound transmission. In addition, our results suggest that failure of cartilage elements to separate during joint formation in Nog mutants may be another common consequence of unopposed BMP activities.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 FUNDING
 REFERENCES
 
Hearing loss in Nog+/– mice
We determined the hearing ability of the Nog+/– mice in a congenic C57BL/6J and a mixed C57BL/6J;FVB background by measuring their auditory brainstem response (ABR) at the ages of 7 weeks, 3 and 6 months old. None of the Nog+/– mice in a mixed C57BL/6J;FVB background showed any hearing loss (n= 18), whereas Nog+/– mice in a congenic C57BL/6J background showed a large range of hearing thresholds measured at 7–10 weeks of age (Fig. 1, black circles and gray triangles; n = 33). The differences in ABR thresholds between the Nog+/– mice and wild-type littermate controls were statistically significant at all frequencies tested (P < 0.001, t-test). Since the Nog+/– mice in a congenic C57BL/6J background displayed a wide range of hearing thresholds, we arbitrarily grouped specimens with hearing threshold shifts of >10 decibels (dB) at two or more different frequencies as a relatively more affected Group A. Thirteen heterozygous specimens fell into this category with one (n = 10/13) or both ears (n = 3/13) that fit this criterion (Fig. 1, black circles). The remaining Nog+/– mice that had hearing thresholds below the criterion were classified as Group B (Fig. 1, gray triangles; n = 20). No significant difference in hearing thresholds between mice in Group B and wild type was apparent at each frequency tested.


Figure 1
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  		Figure 1. Hearing thresholds of Nog+/– mice. Hearing thresholds of wild type (open squares), and Nog+/– mice in a congenic C57BL/6J background (black circles and gray triangles) at 7–10 weeks of age. Nog+/– mice whose hearing thresholds are more than 10 dB shifts at any two tested frequencies in one or both ears are represented by lines with black circles (Group A). The remaining Nog+/– mice are represented by lines with gray triangles (Group B).

 
In addition, Nog+/– mice that showed no significant threshold shifts at 10 weeks of age did not progressively develop hearing loss up to 1 year of age (n = 5). Beyond 1 year, age-related hearing loss developed in both wild-type and Nog+/– mice (Supplemental Materials, Fig. S1).

Middle ear anomaly in Nog+/– mice
To better understand the cause of hearing loss in Nog+/– mice, we examined the presence of hair cells in the cochlear duct of mice within Group A. Phalloidin staining showed the normal one row of inner hair cells and three rows of outer hair cells in these mice (n = 8, data not shown).

Upon examination of some of the middle ears within Group A, we observed an extra bone fragment between the posterior crus of the stapes and the posterior wall of tympanum (Fig. 2D; bracket). This extra bone was observed in all the Nog+/– ears analyzed within Group A that have hearing threshold shifts of >10 dB at two or more frequencies (n = 12/12). No extra bone fragment associated with the malleus or incus was evident. The extra bone was located at where the stapedius muscle is normally connected to the temporal bone in the wild type (Fig. 2A–D; arrowheads). Alizarin S red and Alcian blue stained the extra bone fragment well (Fig. 2F; bracket), but poorly for the stapedius muscle (Fig. 2E; arrowheads). We postulate that the extra bone fragment in the stapes interferes with the normal motions of the stapes and affects sound conduction to the inner ear.


Figure 2
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  		Figure 2. Middle ear anomaly in Nog+/– mice with hearing loss. (A,B,E and F) Specimens stained with Alcian blue and Alizarin S red. Bone is stained in red and cartilage in blue color. (C and D) Dissected and unstained middle ear specimens before fixation. (A) A lateral ventral view of a left middle ear in Nog+/– mouse with normal hearing. The malleus is connected to the incus via the malleoincudal joint (black arrows). The incus is connected to the head of the stapes via the stapedioincudal joint (white arrow). Footplate of the stapes lies on the oval window, and the stapedial artery passes through the obturator foramen of the stapes. Round window is located ventral to the oval window. (B) Normal structure of the stapes in Nog+/– mice in Group B. (C and E) A close-up view of the stapes after removal of the malleus and incus in wild type showing the stapedius muscle (arrowheads) connecting between the tubercle of the posterior crus to the temporal bone. (D and F) An extra bone fragment connecting between the stapes and the temporal bone (bracket) is present at the location of stapedius muscle in Nog+/– mice with hearing loss. Alizarin S red stains the extra bone in (F) but the stapedius muscle in (E) is poorly stained. All specimens shown are obtained from 3 to 6 months old mice. HL, hearing loss; I, incus; M, malleus; Of, obturator foramen; Ow, oval window; Rw, round window; Sa, stapedial artery; S, stapes; Ty, tympanic bone. Orientations: A, anterior; D, dorsal. Scale bar: 100 µm in (B) applies to (C–F).

 
Severity of threshold shifts correlates with the size of the extra bone fragment
A closer examination of the extra bone bridge within Group A indicated variability in size (n = 10). A majority of the specimens showed a rigid, extra bone bridge (n = 9) and a representative specimen is shown in Fig. 3A, which showed 18.0 dB mean threshold shift among three sequential frequencies, click, 8 and 16 kHz. In only one out of ten specimens examined, the mean threshold shift among the same three frequencies was lower at 11.4 dB and the extra bone was smaller and appears to consist of two pieces of bone (Fig. 3B). Specimens examined in the Nog+/– mice in Group B or unaffected ears of mice in Group A either showed extra bone fragments at the same locations that were not fused (Fig. 3C, brackets; n= 6) or a completely normal stapes (Fig. 3D; n = 3). These results indicate that there is an anatomical basis for the observed threshold shifts in Nog+/– mice. The extra bone bridge in the middle ear cavity in the Nog+/– mice was observed only in ears within Group A that met the criterion of >10 dB threshold shifts at two or more frequencies, indicating that these mice have compromised hearing. In addition, the variability in the size of the extra bone was correlated with the severity of hearing loss. The absence of ectopic bone bridge in Group B specimens is consistent with the lack of threshold shifts compared with wild type.


Figure 3
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  		Figure 3. Variability in the size of the extra bone in Nog+/– mice. (A) A representative of a majority of the samples in Nog+/– mice consisting of a large ectopic bone fragment (bracket) that connects the stapes and the adjacent temporal bone tightly. The mean threshold shift for this specimen among three sequential frequencies, click, 8 and 16 kHz, is 18.0 dB. (B) One specimen from Nog+/– mouse showing an ectopic bone fragment that appears to be made up of two pieces of bone (two brackets). The mean threshold shift for the same three frequencies tested is 11.4 dB, lower than the specimen in (A). (C and D) Middle ear specimens from Nog+/– mice showing ectopic bones that are not connected (C, brackets) or no ectopic bone formation (D). No significant threshold shifts are observed in these specimens. HL, hearing loss. Orientations: A, anterior; D, dorsal. Scale bar: 100 µm in (A) applies to (B– D).

 
Failure of proper separation of the stapes and styloid process leads to ectopic bone formation
We investigated the cause of ectopic bone formation by examining skeletal preparations of Nog+/– mice at embryonic and perinatal ages from embryonic day 15.5 (E15.5) to postnatal day 10 (P10) (Fig. 4A–D). The frequency of the ectopic bone detected among Nog+/– mice at these ages was similar to the adults (n = 5 litters, 6/12 heterozygotes). However, at E15.5, the ectopic bone fragment was located between the stapes and styloid process rather than the temporal bone (Fig. 4B; double arrow). This relationship was still evident at postnatal day 4 (Fig. 4D; white arrow). In the adult, the proximal region of the styloid process is no longer distinguishable from the temporal bone normally, which explains why the relationship of the ectopic bone between stapes and styloid process is not apparent in older specimens (Fig. 5B). Since, the stapes and styloid process are both second branchial arch derivatives, our results suggest that this ectopic bone formation in Nog+/– mice is caused by a failure of the developing stapes and styloid process to separate normally.


Figure 4
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  		Figure 4. Middle ear anomaly of Nog+/– mice at embryonic day 15.5 and postnatal day 4. Skeletal preparations of wild type (A and C) and Nog+/– (B and D) ears stained with Alcian blue and Alizarin S red at E15.5 (A and B) and P4 (C and D). (A and B) At E15.5, the stapes and styloid process are normally separate entities (A, arrow), but an extra bone bridge connecting the stapes to the styloid process is evident in some Nog+/– mice (B, double arrows). Inserts show higher magnifications of the ectopic bone region. (C and D) Similar ectopic bone is evident at P4 (D, white arrow). I, incus; M, malleus; S, stapes; Sp, styloid process. Orientations: A, anterior; D, dorsal. Scale bar: 1 mM in (B) applies to (A), 100 µm in (D) applies to (C).

 


Figure 5
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  		Figure 5. Nog null mice show fused ossicles. (A) A lateral view of the head of a wild-type mouse stained with Alizarin red S and Alcian blue. (B) Schematic diagram of normal middle ear structures. (C) A higher power view of (A) focusing on the middle ear. (D) Malformed middle ear structures of Nog null mouse. All ossicles are fused to each other. Stapes is underneath the malformed malleus and incus, and it cannot be seen from this view. (E–H) Acan and Gdf5 expression patterns in wild-type mouse at E17.5. Acan is expressed in the chondrocytes. Gdf5 is expressed in the incudostapedial joint (E and F; long arrow), annular ligament (E and F; short arrows), and malleoincudal joint (G and H; arrowhead). (I–L) Acan and Gdf5 expression patterns in sections of Nog null embryos at E17.5. There is an increase in chondrocyte-associated Acan expression but no evidence of joint-associated Gdf5 expression in Nog null mutants. There is no distinguishable annular ligament, but the stapedial artery is present, passing through a presumed obturator foramen. E, G, I and K are adjacent sections of F, H, J and L, respectively. The contours of the cartilages are outlined in (J) and (L). Hm, handle of malleus; I, incus; M, malleus; S, stapes; Sa, stapedial artery; Sp, styloid process. Orientations: A, anterior; L, lateral. Scale bar: 100 µm in (L) applies to (E–K).

 
Fused ossicles in Nog–/– embryos
Whole mount skeletal preparations of Nog–/– embryos at E17.5 showed overgrown and fused ossicles that were not easily identifiable as discrete entities (Fig. 5A—D). Further gene expression analyses on cryosections at the same age using Acan (aggrecan) as a marker for cartilage (Fig. 5E, G, I and K) and Gdf5 (growth differentiation factor 5) as a marker for prospective joints (Fig. 5F, H, J and L) confirmed the ossicle malformations observed in whole mounts. The size of each ossicle was enlarged compared with those in wild type (Fig. 5E, G, I and K). Gdf5, which is normally expressed in the malleoincudal joint (Fig. 5G and H; arrowhead), stapedioincudal joint (Fig. 5E and F; long arrow) and annular ligament (Fig. 5E and F; small arrows), were all absent in the Nog null embryos (Fig. 5J and L). This absence of Gdf5 expression is most likely secondary to the failure of joint formation to initiate, similar to other skeletal elements in Nog–/– embryos (6).

Relationships of Nog and Bmps during ossicle formation
Given the skeletal defects observed in Nog–/– embryos are attributed to unopposed Bmps activities (6,7), it is most likely that the ossicle defects in the middle ear are caused by similar mechanisms. To investigate which Bmps’ activities might be upregulated in the middle ear ossicles as a result of lack of Nog activities, we investigated the normal relationships between expression domains of Nog and several Bmps during middle ear development between E11.5 and E16.5. At E16.5 when the anatomy of the middle ear ossicles is clear (Fig. 6G), Bmp2, Bmp4 and Bmp7 are all dynamically expressed in the developing middle ear (Fig. 6D–F), and Gdf5 is expressed in various developing joints (Fig. 6B). Nog is expressed in chondrocytes of all prospective bones including, the malleus, incus, stapes and otic capsular bone (Fig. 6C; arrows). In addition, Nog is expressed in newly formed chondrocytes in prospective joints such as malleoincudal joint, annular ligament and stapedioincudal joint (Fig. 6C; arrowheads). These newly formed chondrocytes are not expressing Acan at this stage (Fig. 6A). Interestingly, Bmp2 expression is only detected in the inner but not the outer rim of the stapes (Fig. 6E; arrows). It is also expressed in mesenchyme surrounding the malleus and incus (Fig. 6E; double arrows). On the other hand, Bmp4 is expressed in the mesenchyme surrounding all three ossicles (Fig. 6D). Bmp7 shows similar expression pattern as Bmp4 but has more extensive mesenchymal expression domains than Bmp4 (Fig. 6F). Similar gene expression patterns are observed in the middle ears of Nog+/– embryos as wild type (Supplemental Materials, Fig. S2).


Figure 6
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  		Figure 6. Normal gene expression patterns during middle ear development at E16.5. Expression patterns of (A) Acan, (B) Gdf5, (C) Nog, (D) Bmp4, (E) Bmp2, (F) Bmp7 and (G) H&E staining in the developing wild-type middle ear. (A) Acan is highly expressed in the chondrocytes. (B) Gdf5 is expressed in the AL and SIJ. It is also expressed in some mesenchymal cells surrounding the developing ossicles. (C) Nog expression is present in chondrocytes of the malleus, incus, stapes and otic capsule (arrows), and in AL, malleoincudal joint and SIJ as well (arrowheads). (D) Bmp4 is expressed in the mesenchyme surrounding all three ossicles. (E) Bmp2 is expressed in the inner rim of the stapes (small arrows) and mesenchyme surrounding the malleus and the incus (double arrows). (F) Bmp7 expression pattern is very similar to that of Bmp4 but has more extensive mesenchymal expression than Bmp4. (G) The stapedius muscle that is negative for Sox9 is well stained with H&E. AL, annular ligament; I, incus; M, malleus; S, stapes; Sa, stapedial artery; SIJ, stapedioincudal joint; Sp, styloid process; Sm, stapedius muscle. Scale bar (G) = 100 µm and applies to all panels.

 
At E17.5, the stapes and styloid process are separate entities (Fig. 5C). Presumably, the two prospective cartilage elements separate at an earlier stage when the histology of the cartilage is not well defined (Fig. 7G). Therefore, we used Sox9, which is expressed in the condensing mesenchyme during initiation of cartilage formation, to identify the prospective styloid process and ossicles. From analyses of serial sections between E11.5 and E13.5 (n = 28), the stapes and styloid process appear connected at E11.5 (Fig. 7A, red arrowhead; n = 3) but are separate entities by E13.0 (n = 3). Figure 7B illustrates a specimen at E13 showing some weak Sox9 expression remaining between the two elements (Fig. 7B; small arrowheads). This distance between prospective styloid process and stapes continues to increase over time. These results suggest that the stapes and styloid process separates around E13 and we investigated which Bmps are expressed in this region at E12.5. At E12.5, all the prospective ossicles and styloid process are positive for Sox9 (Fig. 7C), and the prospective stapes and styloid process show signs of separation (n = 7). At this age, Gdf5 is highly expressed around the styloid process and future joint spaces of the ossicles (Fig. 7D). Nog is expressed in the stapes and styloid process (Fig. 7E), whereas Bmp4 only shows diffuse expression surrounding the malleus, incus and the styloid process, but no Bmp4 expression is associated with the developing stapes (Fig. 7F, double arrows; n = 5) until E13.5 (data not shown). Moreover, neither Bmp2 nor Bmp7 expression is detectable in the middle ear region at E12.5 (data not shown). Taken together, the expression patterns between E11.5 and E16.5 suggest that both Bmps and Nog are involved in ossicle formation, and Gdf5 and/or Bmp4 may play a more important role in modulating the separation between the stapes and styloid process than Bmp2 and Bmp7.


Figure 7
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  		Figure 7. Early development of the stapes and styloid process. Expression patterns of (A–C) Sox9, (D) Gdf5, (E) Nog, (F) Bmp4 in the developing wild-type middle ear at (A) E11.5, (B) E13.0 and (C–F) E12.5. The location of the styloid process is marked with a red arrowhead. (A) Prospective middle ear ossicles are positive for Sox9 at E11.5. The stapes and styloid process appear to be connected to each other at this age but are separate entities by E13. In one of the specimens at E13 (B), weak Sox9 expression connecting between two prospective cartilage elements is observed (arrowheads). (C–F) are 12 mM adjacent sections. (D) Gdf5 is highly expressed in the area adjacent to the styloid process and the joint spaces among the three ossicles. (E) Nog is expressed in the three ossicles and the styloid process, whereas Bmp4 only shows diffuse expression surrounding the malleus, incus and styloid process (F, double arrows). (G) H&E staining of the middle ear region at E12.5. Only mesenchymal condensation for the stapes and otic capsule is apparent. Co, cochlea; I, incus; M, malleus; S, stapes; Sp, styloid process; Scale bar in (G) =100 µm and applies to all panels.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 FUNDING
 REFERENCES
 
Hearing loss in Nog+/– mice is conductive in nature
Our results indicate that Nog+/– mice display conductive hearing loss. Several lines of evidence support this conclusion. First, no obvious sensory hair cell defect is evident based on phalloidin staining. Second, hearing loss due to conductive defects is usually milder than most cases of neurosensory deafness for both mice and humans. The average threshold shift of 18 dB observed in Nog+/– mice is considered mild, suggestive of conductive hearing loss. More importantly, the hearing loss detected in individual ears of the Nog+/– mice is fully correlated with the presence of ectopic bone formation in the stapes. In addition, the severity of hearing loss appears to be correlated with the size of the ectopic bone bridge as well.

Hearing loss in Nog+/– mice is dependent on their genetic background; only mice in a congenic C57BL/6J but not in a mixed C57BL/6J;FVB background shows this functional deficit. Among mice in C57BL/6J background, hearing loss is variable. Nevertheless, the high percentage of unilateral hearing loss among Nog+/– mice suggests that factors in addition to variability in genetic background are at play. Functional redundancy with other pathways in regulating bone formation and/or the stochastic nature of the Nog gene activity may contribute to the variable hearing loss phenotype as well (8,9).

A balance between Noggin and Bmps levels in middle ear ossicle formation
Bmps are multi-potent signaling peptides that are involved in tissue patterning and specification during embryogenesis although its name originated from its ability to induce bone and cartilage formation (10–12). Bmp signals are mediated by three type I (BMPR-IA, BMPR IB and Act-IA) and three type II (BMPR-II, ActR-II and ActR-IIB) receptors, and Smads 1, 5 and 8 are the immediate downstream molecules that mediate Bmp signaling (13). Bmps activities are known to be modulated in a dose-dependant manner by antagonists, such as Noggin, that bind Bmps with high affinity and prevent Bmp binding to their own receptors (14). Based on analyses of Nog–/– mice, Nog's main functions in mammals appear to be in the formation of posterior neural tube, somite differentiation and skeletal formation (15). In Nog–/– embryos, the skeletal elements of limbs and vertebrae are shorter and broader with absence of joints. These phenotypes are attributed to excess Bmp activities which enhance the recruitment of mesenchymal cells into cartilage, causing hyperplasia of cartilage frameworks and non-responsive to joint-inducing signals (6). The hypothesis of excess Bmp activities in Nog mutants is supported by the elevated levels of phosphorylated Smads 1, 5 and 8 in the joints of Nog+/– mice (16). Transgenic mouse models in which Bmp4 or Gdf5 is over-expressed in chondrocytes show skeletal defects similar to that of Nog–/– embryos (17,18). In addition, somite and axial skeletal defects in Nog null mutants are rescued with removal of one allele of Bmp4 (7). The limb defects in these compound mutants, however, did not improve, indicating that Noggin is antagonizing multiple Bmps including Gdf5, during chondrogenesis.

Bmps and Noggin may also regulate each other at the transcriptional level. For example, Bmp2 has been reported to induce the expression of Nog in osteoblasts in vitro (19). Conversely, mis-expression of Nog upregulates Bmp4 expression in the developing perichondrium (20). Furthermore, in the Nog–/– mutants, Bmp4 is ectopically expressed in the paraxial and axial mesoderm (7,15). While some of the above results might be attributable to Nog interfering with the auto-regulation of Bmp4, it is clear that balanced levels of Bmps and Noggin are important in patterning and specification of multiple organs during development.

Formation of middle ear ossicles is mediated by endochondral ossification, a process similar to those required for axial and limb skeleton formation. Similar to other developing skeletal elements, Bmp2, Bmp4 and Bmp7 are highly expressed in the mesenchyme surrounding each prospective ossicle, whereas Gdf5 is in the joints and Nog is in the chondrocytes (Fig. 6). Not surprisingly, the middle ear ossicles in Nog null embryos were also larger than normal and fused with no apparent joint formation, similar to defects in other skeletal elements, suggesting that balanced levels of Bmps and Noggin are required for ossicle formation (Fig. 5). Ectopic expression of Nog in neural crest cells of the second branchial arch results in the absence of the stapes, suggesting that too much Noggin affects the stapes formation (21). During the time that the stapes is presumably separating from the styloid process (or the second branchial arch proper), high Gdf5 as well as some Bmp4 hybridization signals were observed in the region. Since Noggin has been shown to bind Gdf5 and block Gdf5 effects in skeletogenesis similar to other Bmps (22), it is likely that the ectopic bone formation in the middle ear of Nog+/– mice is due to subtle but significant elevations of Bmp4 and/or Gdf5 activities.

Cause for extra bone formation in Nog+/– mice
The stapes is a derivative of neural crest cells in the second branchial arch, also known as the Reichert’s cartilage (23). Other derivatives of the Reichert’s cartilage include the styloid process of the temporal bone, the stylohyoid ligament and part of the hyoid bone (24). At maturation, the styloid process is incorporated as part of the temporal bone, and the stapes is connected to the temporal bone by the small stapedius muscle. Cell lineage tracing studies in mice indicate that the connective tissue surrounding the stapedius muscle is also derived from the second arch, along with the stapes and styloid process (25). The location of the extra bone observed in Nog+/– mice, between the posterior crura of the stapes and posterior wall of the tympanum, was at the same position where the stapedius muscle normally inserts. This extra bone fragment was connecting the stapes and styloid process at early developmental stages. These results together with the cell lineage tracing studies suggest that the location of the stapedius muscle is where the stapes anlage normally separates from the Reichert’s cartilage, and the extra bone observed in Nog+/– mice forms as a result of incomplete separation of stapes and styloid process during development, rather than de novo bone formation.

It is not clear whether this failure of prospective cartilage elements to separate properly applies to other skeletal defects in the Nog mutants. Presumably, the cavitation process during synovial joint formation, which is severely affected in Nog–/– mutants, could involve a similar separation process. It is difficult to evaluate this possibility in the Nog null embryos, since joint formation is failing at the initiation stage, before the cavitation begins. In the Nog+/– mice, carpal and tarsal fusions have been reported, but it is not clear if failure of cell separation contributed to these joint defects (26). Since the stapes and styloid process do not form a joint normally, it provides a clear example of an impediment of the prospective cartilages to separate due to chondrocyte hyperplasia. It is also a location where any subtle skeletal defect is easily exemplified by a functional deficit. Furthermore, the presence of strong Gdf5 expression at the time when the prospective styloid process and stapes elements separate raises the possibility that splitting of one prospective cartilage element into two may involve mechanisms, at least initially, similar to those required for forming a synovial joint.

Stapes formation in humans is similar to that in mice. A stapedial anlage becomes recognizable as a definite primordial structure at fifth week of fetal life (27,28). When the stapedial anlage separates from the Reichert’s cartilage, the two structures remain connected by the interhyale, which later develops into the stapedial tendon (28,29). Ectopic bones similar to those observed in Nog+/– mice have been described in human patients. In humans, this type of anomaly is referred as stapedial suprastructure fixation, but the genetic etiology of the disorder(s) has not been reported (29–31). Stapes ankylosis is the most common feature among human patients with NOG mutations, even though the precise anatomical defects in most cases have not been described in detail (5,32,33). Therefore, it is possible that failure of the stapedial anlage to separate from other parts of the Reichert’s cartilage may be one of the prevalent causes for stapes ankylosis in humans as well (29). This would also imply those cases of stapes ankylosis are congenital, which appears to hold true for most patients with NOG mutations. The exact age of onset of mild hearing loss, typical of most cases caused by conductive problems, is not usually clear since most patients do not complain about hearing loss until early school age (34).

Human syndromes associated with mutations of the NOG
The NOG is a small gene with a single exon, located on human chromosome 17q22. Mutations of NOG are associated with several autosomal dominant syndromes, each of which is characterized by a spectrum of specific skeletal defects and synostoses, including conductive hearing loss. For example, both SYM1 and SYNS1 patients, carrying missense mutations of NOG, have proximal interphalangeal joint fusions but only SYNS1 is associated with hip and vertebral fusions (2). In contrast, the main features of Teunissen–Cremers syndromes are hyperopia, stapes ankylosis and broad skeletal elements, but joint fusion is a rare occurrence (5). Mutations associated with this syndrome are mostly nonsense mutations resulting in a truncated protein. It is not clear whether the variable phenotypes among these different syndromes are related to the specific nature of the mutations. However, increasing evidence suggests that most, if not all, of these mutations result in haploinsufficiency of NOGGIN. First, expression of several forms of the mutant protein in vitro indicates that while some of the mutant proteins are deficient in forming homodimers, they do not interfere with the synthesis, secretion and dimerization of the wild-type protein (35). Second, even though Nog+/– mice were initially thought to be phenotypically normal (6), more recent studies showed that these mice have carpal and tarsal fusions that resemble some of the phenotypes reported in human patients (26). The conductive hearing loss phenotype described here for Nog+/– mice further supports the idea that the autosomal dominant phenotypes in human patients are due to dosage dependency of the NOGGIN protein.

An important clinical issue regarding patients with NOG mutations who underwent surgery to correct stapes ankylosis, showed disappointing hearing results within 2 years after successful surgery due to regrowth of bone over the stapes footplate (32,33). The cause of this phenomenon is not known. The affected patients may have a defect in healing and remodeling process of the middle ear skeleton, presumably due to excessive BMP signaling in their ossicles. In addition to being required for bone formation during embryogenesis, BMPs are also thought to be important during bone healing (36). Perhaps, a combination of surgery followed by Bmp antagonist therapy may be a viable clinical option for patients with NOG mutations, and Nog mutant mice will continue to serve as an excellent model for exploring these options.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
SUPPLEMENTARY MATERIAL
 FUNDING
 REFERENCES
 
Mice and genotyping
The Nogtm1Amc/+ (Nog+/–) mice (15) in a congenic C57BL/6J (B6.129S1—Nogtm1Amc) background were obtained from Dr Richard Harland (University of California at Berkeley). Nogtm1Amc/tm1Amc (Nog–/–) embryos were generated by timed mating of Nog+/– mice in a congenic C57BL/6J and a mixed C57BL/6J;FVB background. Genotyping was determined by PCR as previously described (15).

Hearing measurement
ABRs were recorded using Smart EP® (Intelligent Hearing System, FL, USA) and sub-dermal electrode needles. Auditory stimuli used were 47 µs clicks and 8-, 16-, 20- and 32-kHz tone pips. Responses were bandpass filtered between 30 and 5000 Hz and averaged from 512 to 1024 times. The hearing threshold was determined as the lowest stimulus level at which response peaks were repeatedly present. Distortion Product Otoacoustic Emission (DPOAE) was used as a quick screening for hearing function using DP2000® (Starkey Laboratory, MN, USA) and an acoustic probe (ER-10C; Etymotic Research, IL, USA). Two primary tones with frequency ratio f2/f1 = 1.2 were given at intensity levels of L1 (amplitude of f1) at 65 dB SPL and L2 at 55 dB SPL. The f2 was varied from 4 to 16 kHz to obtain the magnitude of the 2f1–f2 distortion product. The pass criterion of DPOAE was that the response levels were greater than noise levels among all frequencies tested. During ABR or DPOAE measurements, mice were anesthetized with an intra-peritoneal injection of 2.5% Tribromoethanol (Avertin®, Sigma-Aldrich, MO, USA) solution in phosphate-buffered saline given in 0.15 ml per 10 g body weight and kept on heating pad in a soundproof chamber. In mice showing a discrepancy of hearing thresholds between right and left ears, ABR values from the ear with poorer hearing ability were chosen as a representative hearing threshold for a given mouse.

Histological and skeletal preparation
Mice that showed hearing loss by ABR testing were sacrificed and the anatomy of their middle ears was examined between 3 and 6 months of age. Some dissected specimens were further processed for skeletal staining with Alizarin red and Alcian blue (37). Briefly, the specimens were treated with 100% ethanol for 5 days at room temperature, followed by acetone treatments for 3 days. Then the specimens were stained with Alizarin red S (0.005% solution) and Alcian blue (0.015% solution), followed by clearing with 1% potassium hydroxide solution.

In situ hybridization and H&E staining
Nog+/– and Nog–/– embryos between E12.5 and E17.5 were used for in situ hybridization analyses and H&E staining. For in situ hybridization, RNA probes for Bmp4 (38), Bmp2 (39), Bmp7 (39), Nog (6), Gdf5 (40), Acan (41) and Sox9 (42) were prepared as previously described. For H&E staining, 12 µm thickness cryosections were stained sequentially with hematoxylin (Gill’s formula, Vector Laboratory, CA, USA) and eosin Y (1%, Harleco, EM Diagnostic, Fisher Scientific, NJ, USA), followed by dehydration and mounting with Permount (Fisher Scientific, NJ, USA).


   SUPPLEMENTARY MATERIAL
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
FUNDING
 REFERENCES
 
Supplementary Material is available at HMG Online.


   FUNDING
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 FUNDING
REFERENCES
 
This work was supported by the NIDCD Intramural Program.


   ACKNOWLEDGEMENTS
 
We thank Dr Richard Harland for providing the Nog mutant mice and Drs Susan Sullivan and Robert Morell for critical reading of the manuscript. We also thank John Li for his assistance in conducting hearing tests in mice.

Conflict of Interest statement. None declared.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 FUNDING
 REFERENCES
 

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