Pou3f4-Mediated Regulation of Ephrin-B2 Controls Temporal Bone Development in the Mouse
Wu DK (2014) Pou3f4-Mediated Regulation of Ephrin-B2 Controls Temporal Bone Development in the
Mouse. PLoS ONE 9(10): e109043. doi:10.1371/journal.pone.0109043
Pou3f4-Mediated Regulation of Ephrin-B2 Controls Temporal Bone Development in the Mouse
Steven Raft 0
Thomas M. Coate 0
Matthew W. Kelley 0
E. Bryan Crenshaw 0
Doris K. Wu 0
Andre van Wijnen, University of Massachusetts Medical, United States of America
0 1 Section on Sensory Cell Regeneration and Development, National Institute on Deafness and Other Communication Disorders, National Institutes of Health , Bethesda , Maryland, United States of America, 2 Laboratory of Cochlear Development, National Institute on Deafness and Other Communication Disorders, National Institutes of Health , Bethesda , Maryland, United States of America, 3 Children's Hospital of Philadelphia, University of Pennsylvania , Philadelphia, Pennsylvania , United States of America
The temporal bone encases conductive and sensorineural elements of the ear. Mutations of POU3F4 are associated with unique temporal bone abnormalities and X-linked mixed deafness (DFNX2/DFN3). However, the target genes and developmental processes controlled by POU3F4 transcription factor activity have remained largely uncharacterized. EphrinB2 (Efnb2) is a signaling molecule with well-documented effects on cell adhesion, proliferation, and migration. Our analyses of targeted mouse mutants revealed that Efnb2 loss-of-function phenocopies temporal bone abnormalities of Pou3f4 hemizygous null neonates: qualitatively identical malformations of the stapes, styloid process, internal auditory canal, and cochlear capsule were present in both mutants. Using failed/insufficient separation of the stapes and styloid process as a quantitative trait, we found that single gene Efnb2 loss-of-function and compound Pou3f4/Efnb2 loss-of-function caused a more severe phenotype than single gene Pou3f4 loss-of-function. Pou3f4 and Efnb2 gene expression domains overlapped at the site of impending stapes-styloid process separation and at subcapsular mesenchyme surrounding the cochlea; at both these sites, Efnb2 expression was attenuated in Pou3f4 hemizygous null mutants relative to control. Results of immunoprecipitation experiments using chromatin isolated from nascent middle ear mesenchyme supported the hypothesis of a physical association between Pou3f4 and specific non-coding sequence of Efnb2. We propose that Efnb2 is a target of Pou3f4 transcription factor activity and an effector of mesenchymal patterning during temporal bone development.
Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its
Supporting Information files.
Funding: Work was funded by the Intramural Program of the National Institute on Deafness and Other Communication Disorders. The funders had no role in
study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
Paired temporal bones at the sides and base of the skull house
conductive and sensori-neural components of the peripheral
auditory system . The tympanic part (middle ear) is air-filled
and contains structures that conduct sound energy to the cochlea:
the tympanic membrane, auditory ossicles (malleus, incus, stapes),
muscles, and ligaments (Fig. 1A). The petrous portion of the
temporal bone (bony capsule) encases inner ear sensory end organs
and neuronal ganglia within a labyrinthine space, and forms
canals for passage of cranial nerves and major vessels. It protects
the auditory-vestibular system and contributes to the architecture
of partitioned, fluid-filled spaces that comprise the inner ear.
Various parts of the temporal bone form by either
endochondral or intramembranous ossification . The auditory ossicles
and petrous portion form by endochondral ossification, and in
mice, these structures are cartilaginous at the time of birth. The
petrous portion and its developmental precursor (cartilaginous otic
capsule) derive from head mesoderm . The distal ossicles
(malleus and incus) derive from cranial neural crest . The stapes
is of mixed origin ; head and crura derive exclusively from
neural crest, and the footplate is a mixed derivative of neural crest
and head mesoderm (Fig. 1B).
Genetic classification of otologic conditions and identification of
pathogenic gene mutations provide insights into temporal bone
development. X-linked mixed (conductive and sensorineural)
progressive deafness with stapes fixation and perilymphatic gusher
(a profuse discharge of inner ear and cerebrospinal fluid) correlates
with unique temporal bone abnormalities (cochlear hypoplasia;
dilated internal auditory canal) upon radiological examination [6
9]. The genetic locus for this non-syndromic form of deafness
(DFNX2, formerly DFN3; OMIM 304400) maps to Xq21.1 
and is associated with mutations of coding or regulatory sequence
for the POU3F4 gene [11,12]. POU family transcription factors
bind to DNA in a sequence-specific manner through the bipartite
motif of POU homeodomain and POU-specific domains . In
the mouse, Pou3f4 is expressed in mesenchyme surrounding the
embryonic inner ear epithelium, and targeted mutagenesis of
Pou3f4 causes multiple developmental malformations of the ear,
including cochlear hypoplasia, thin cochlear bony capsule (petrous
temporal bone), dilated internal auditory canal, and malformed
stapes . Mice lacking Pou3f4 also show abnormalities of
specialized fibrocytes apposed to the bony cochlear wall, a
dramatically reduced endolymph potential, and profound
deafness, suggesting that loss of Pou3f4 disrupts cochlear fluid
homeostasis [17,18]. However, very little is known about target
genes that mediate the effects of Pou3f4 on ear development.
Ephrin-B2 (Efnb2) encodes a Type I transmembrane protein
that functions as a cell contact-dependent ligand for multiple Eph
receptor tyrosine kinases. Upon binding to a cognate Eph protein,
Efnb2 can also function cell-autonomously by signaling from its
cytoplasmic domain through SH2 and PDZ domain-containing
adaptor proteins [19,20]. Eph/ephrin-B signaling influences cell
migration, progenitor cell sorting, and tissue boundary formation
in the developing embryo through effects on cytoskeletal
organization and adhesion between adjacent cells [21,22]. Eph/
ephrin-B signaling also controls cell proliferation in the embryo
and adult stem cell niches of mice . Recent evidence
indicates that Pou3f4 transcription factor regulates Eph-ephrin
signaling: Pou3f4 in mesenchyme surrounding the cochlea binds to
specific non-coding sequence of Epha4 and is necessary for proper
expression of Epha4 , the product of which is a canonical
receptor for Efnb2 . This regulation is associated with proper
fasciculation of axons that innervate the cochlea . Here, we
report on temporal bone morphologies of single and compound
Pou3f4 and Efnb2 loss-of-function mutants, developmental gene
expression in wild type and mutant embryos, and
anti-Pou3f4mediated pull-down of Efnb2 sequence from chromatin of the
embryonic ear. The results provide evidence that Efnb2 is a direct
effector of Pou3f4 activity during temporal bone development.
Adult Pou3f4Cre/Y mice exhibit defects of the stapes, facial
canal, internal auditory canal, and bony capsule of the
Mutant mice wherein cre replaces the coding region of Pou3f4
(resulting in a null allele of Pou3f4) were previously characterized
with respect to spiral ganglion development . We analyzed
excised temporal bones from 1620 week old Pou3f4Cre/Y
hemizygous null males (n = 40) and wild-type male littermate
controls (n = 62). The stapedio-vestibular (SV) joint, which forms
a functional interface between middle and inner ear chambers,
comprises articular surfaces of the stapes footplate and oval
window of the inner ear bony capsule (Fig. 1A, B). At the SV
joint of wild-type controls, articular surfaces were closely apposed
and joined by elastic fibers; collectively, these fibers form the
annular ligament (Fig. 2A, C, E) . By contrast, all mutant
footplates were disarticulated from the oval window to varying
degrees (Fig. 2B, D), with both footplate hypoplasia and oval
window dysplasia contributing to the defect. Severely affected
floating footplates (Fig. 2D) were tethered to the oval window by
a thin elastin- and collagen-containing membrane (Fig. 2F). Less
affected regions of the mutant S-V joint showed disorganized
elastic fibers, irregular articular surfaces, and reduced thickness of
footplate and oval window bone compared to controls (Fig. 2E,
F9). The entire mutant cochlear bony labyrinth was abnormally
thin (Fig. 2A, B; arrows) and fragile upon dissection.
The facial canal is a bony sulcus running along the dorsal aspect
of the middle ear cavity; it contains the stapedius muscle and a
segment of the VIIth cranial nerve (Fig. 1A), tissues that mediate
the acoustic reflex and modify stapes mobility. Compared to
controls, Pou3f4Cre/Y mutant stapes were shifted dorsally and set
more closely to the facial canal (data not shown). In 45% of mutant
ears analyzed (18/40), a thin sheath of periostium connected the
dorsal crus of the stapes to an abnormal prominence on the lateral
wall of the facial canal (Fig. 3AD); this soft tissue bridge always
co-occurred with an intact stapedial tendon and did not grossly
limit stapes mobility as assessed by palpation. In 33% of mutant
ears analyzed (13/40), this same region of the dorsal crus was fused
to the lateral wall of the facial canal by a bone bridge (Fig. 3E, F),
causing an immobile stapes. All mutant ears with a bony coalition
lacked the stapedial tendon.
To assess the validity of scoring individual temporal bones in
generating data sets (denoted as occurrence of a defect, where
n = the number of temporal bone specimens), we analyzed
phenotypic penetrance by tabulating the prevalence of every
possible combination of stapes-facial canal connections (unilateral
vs. bilateral; soft tissue vs. bone) in the sample of Pou3f4Cre/Y
mutants (n = the number of mutant animals sampled; Table S1).
The distribution did not deviate significantly from the case where
each category is equally prevalent (P = 0.6526, Chi-square 3.308;
5 df). This supports the assumption that stapes-facial canal
connection type (soft or bone) and unilaterality/bilaterality of
the defect within an individual are independent events. No left- or
right-sided bias was noted among animals with a unilateral defect.
At the medial side of the temporal bone, the internal auditory
canal comprises three foramina for passage of VIIth and VIIIth
cranial nerve branches between the bony labyrinth and brain.
Pou3f4 null hemizygosity caused incomplete septation and
dilation of these foramina (Fig. S1A, B), as previously shown
. Together, these results extend previous characterizations
[14,15,30] of bony defects in the Pou3f4 mutant ear.
Most temporal bone defects of Pou3f4Cre/Y mice are
evident at the endochondral cartilage stage of
We next investigated the developmental origins of Pou3f4Cre/Y
temporal bone defects. Analyses of normally developing middle
ears between post-natal days 5 and 15 indicated that the lateral
wall of the facial canal is formed by fusion and growth of the
endochondral styloid process and intramembranous tympanic ring
(Fig. 1, S2). At perinatal stages, the styloid process is a rod of
cartilage extending from the dorso-lateral aspect of the inner ear
capsule (Fig. 3K, arrowheads; Fig S2A, D, G). To determine
whether bony fixation of the stapes and facial canal in Pou3f4Cre/
Y adults is caused by ossification of an abnormal cartilage
rudiment, we analyzed serial sections of Pou3f4Cre/Y (n = 22)
and wild-type (n = 17) temporal bones from E18.5-P0 littermates.
All wild-type ossicles were discrete, cartilaginous structures
enveloped by perichondrium and surrounded by loose
mesenchyme or a developing joint space (Fig. 3G, J). However, in 36%
of perinatal Pou3f4Cre/Y ears analyzed (8/22), the cartilaginous
dorsal crus and styloid process were connected (Fig. 3I), and the
stapedius muscle and tendon were present but dysmorphic
(Fig. 3G, I, arrowheads). In 32% of the perinatal mutant ears
analyzed (7/22), distance between the unconnected stapes and
styloid process was markedly reduced compared to control and a
common perichondrium enveloped both structures (Fig. 3H,
arrow). Thus, the Pou3f4Cre/Y perinatal-stage phenotype can be
categorized as a partially or fully coalesced stapes and styloid
process. The relative occurrence of these defects in our perinatal
stage sample did not differ from the relative occurrence of soft and
bone bridges identified in adult mutants (Table 1; Chi-square
1.152; df, 2; P = .5622; also see Table S1). Thus, the soft tissue
bridge and bone bridge abnormalities identified in adult
Pou3f4Cre/Y mice are likely consequences of perinatal close
proximity/common perichondrium and continuous cartilage
abnormalities, respectively. Other malformations evident in
neonatal Pou3f4Cre/Y mutants included a thin cochlear cartilage
capsule (Fig. 3GI, asterisks) and dilation/incomplete septation of
the internal auditory canal (Fig. S1D, E). However, we found no
perinatal cartilage abnormality that might account for SV joint
disarticulation observed in adult mutants (data not shown). Thus, a
subset of Pou3f4Cre/Y temporal bone malformations is manifested
at the cartilaginous stage of endochondral bone formation. These
results are consistent with immunohistochemical evidence of
Pou3f4 activity during early phases of periotic mesenchymal
patterning and/or condensation .
Efnb2 and Pou3f4 mRNA signals overlap in mesenchyme
surrounding the VIIth nerve, condensing stapes, spiral
ganglion, and cochlear duct
Periotic mesenchymal patterning and condensation in the
mouse are evident between stages E11 and E13.5 , and we
identified Efnb2 mRNA surrounding periotic mesenchymal
condensations during these and later stages of development
(Fig. 4A, D). At E13.5, Efnb2 and Pou3f4 mRNA expression
patterns in the developing middle ear were complementary and
partially overlapping. Overlap was noted in mesenchyme apposed
to the stapes footplate, within the developing SV joint, dorsal to
the stapes, and surrounding the VIIth nerve (Fig. 4A, B). Efnb2
and Pou3f4 signals also overlapped in sub-capsular mesenchyme
surrounding the cochlear duct and spiral ganglion, as previously
shown . Efnb2 and Pou3f4 expression was complementary
across the lateral wall of the cochlear capsule (Fig. 4A, B; denoted
by c). As previously shown [14,28,31], Pou3f4 was not expressed
in the developing inner ear epithelium, spiral ganglion, or
mesenchyme surrounding the incus and malleus, all of which
By RNA hybridization, we first detected Efnb2 signal in the
nascent middle ear between E11.5 and E12.5; at E12.5, Efnb2
and Pou3f4 signals overlapped in branchial arch mesenchyme
dorsal to the stapedial artery and surrounding the VIIth cranial
nerve (Fig. 5A, B). At E16.5, Pou3f4 signal intensity in
mesenchyme surrounding the stapes had decreased relative to
earlier stages, whereas Efnb2 signal within the developing SV
joint had intensified relative to earlier stages (Fig. 4A, B, D, E);
conversely, Efnb2 signal in E16.5 cochlear subcapsular
mesenchyme was weak compared to earlier stages, whereas Pou3f4
signal remained robust in mesenchyme surrounding the E16.5
cochlea and spiral ganglion.
In summary, Efnb2 and Pou3f4 mRNA signals show the
greatest degree of overlap between stages E12.5 and E13.5, as
periotic mesenchyme is patterned and condensed into
prechondrogenic rudiments (Fig. 5C, D). Overlap of signals between
stages E12.5 and E13.5 occurs at sites relevant to the Pou3f4Cre/Y
stapes phenotype described above.
Sox9-IRES-Cre-mediated inactivation of Efnb2
phenocopies cartilage abnormalities of the perinatal
To assess the function of Efnb2 in ossicular development, we
inactivated Efnb2 broadly in mesenchymal and epithelial
components of the ear using the Sox9-IRES-Cre driver (Fig. 5E; S3) .
Sox9-IRES-Cre+;Efnb2flox/null (Efnb2 CKO) pups were delivered
live, but died within 12 hours of birth. Alcian blue-stained whole
skeletal preparations of late-stage Efnb2 CKO fetuses had no
obvious axial, appendicular, or craniofacial deformities. However,
of 20 Efnb2 CKO ears analyzed at E19 by either histological or
whole mount cartilage/bone staining, 14 showed complete
coalition of the stapes and styloid process and 2 showed reduced
distance between stapes and styloid process cartilages and a shared
perichondrium (Fig. 3KN). These phenotypes were
morphologically indistinguishable from those of the perinatal Pou3f4Cre/Y
male (compare Fig. 3M, N and H, I). The total occurrence of all
identified stapes abnormalities in the fetal Efnb2 CKO sample
(Table 1, Total Affected; see also Table S1) did not differ from
that of the perinatal Pou3f4Cre/Y sample (P = 0.4913; Fishers
exact test). However, the occurrence of a fully connected
stapesstyloid cartilage bridge phenotype in the fetal Efnb2 CKO sample
was increased over that of the perinatal Pou3f4Cre/Y male by
roughly 2-fold (P = 0.0365; Fishers exact test). Other
endochondral cartilage-stage temporal bone defects observed in both Efnb2
CKO and Pou3f4Cre/Y mutants included a thin cochlear
cartilaginous capsule compared to control (Fig. 3G, H, I, M, N,
asterisks) and dilation/incomplete septation of the internal
auditory canal (Fig. S1DF). As with neonatal Pou3f4Cre/Y mice,
we found no abnormality at the developing SV joint in stage E19
Efnb2 CKO fetuses (data not shown). Distal ossicles (incus and
malleus) in the Efnb2 CKO were judged to be of normal size and
morphology. Together, these results indicate that
Sox9-IRES-Cremediated loss of Efnb2 phenocopies cartilage abnormalities of the
perinatal Pou3f4Cre/Y ear.
We next analyzed a sample of E19 fetuses homozygous for an
Efnb2 C-terminal truncation (ephrin-B2LacZ mice in ).
This allele produces an Efnb2:-galactosidase fusion protein that is
targeted to the plasma membrane and binds cognate EphB
receptors on adjacent cells, but is unable to transduce signals to the
cytoplasmic side of the membrane; thus, it can provide evidence of
Figure 4. Comparative expression of Efnb2, Pou3f4, and Sox9 at E13.5 and E16.5. Adjacent transverse sections through embryos at two
developmental stages, hybridized to detect Efnb2 (A,D), Pou3f4 (B, E), or Sox9 (C, F). c, cartilaginous cochlear capsule; cd, cochlear duct; eam, external
auditory canal; eu, nascent eustacian tube; i, incus; m, malleus; ph, pharynx, spg, spiral ganglion, s, stapes. Asterisk highlights the facial nerve. Double
arrowheads in (AC) highlight overlapping Efnb2 and Pou3f4 signals and relatively weak Sox9 signal dorsal to the stapes. Single arrowhead in (F)
highlights a lack of Sox9 signal in the corresponding region at E16.5. White arrowheads in (D, E) highlight overlapping Efnb2 and Pou3f4 signals at the
forming SV joint. Scale bar represents 200 microns in (AC) and 250 microns in (DF).
whether a phenotype is due to a specific loss of (forward) signaling
through an Eph receptor or (reverse) signaling through the Efnb2
C-terminus. Histological analyses of 7 ears from 5 Efnb2
Cterminal deletion homozygotes revealed neither a coalesced
stapesstyloid process nor reduced distance between the two structures,
and other components of the developing middle ear appeared
normal (Fig. S4). These results suggest that the Efnb2 C-terminus
is dispensable in forming separate stapes and styloid process
cartilages during development.
Compound loss of Efnb2 and Pou3f4 potentiates the
Pou3f4Cre/Y adult stapes phenotype
Recapitulation and increased severity of the perinatal
Pou3f4Cre/Y stapes-styloid defect in Sox9-IRES-Cre+;Efnb2flox/
null E19 fetuses prompted us to ask whether
Pou3f4Cre/Y;Efnb2flox/null compound mutant mice are viable and exhibit a more
severe middle ear phenotype than that of Pou3f4Cre/Y adult mice.
We therefore generated and analyzed the series of compound
mutant phenotypes listed in Table 2. Analyses of
Pou3f4Cre;ROSA-YFP mice revealed that this cross should inactivate
the floxed Efnb2 allele in subcapsular cochlear mesenchyme,
mesenchyme within the forming SV joint, and branchial arch
mesenchyme dorsal to the stapes and surrounding the VIIth nerve
(Fig. 5F; data not shown). Pou3f4-Cre-mediated Efnb2 CKO mice
were viable through at least 6 months of age. We therefore
analyzed the ears of 1620 week old adult male mice. This allowed
for rapid categorization of the stapes phenotype as either a soft
tissue bridge or bone bridge, as well as an assessment of the S-V
Bone or Cartilage Bridge
joint and internal auditory canal. All compound mutants were of
the same mixed genetic background as the Pou3f4Cre/Y males
described above (see Materials and Methods, Animals).
Table 2 shows genotypes and categorical data for the
stapesfacial canal abnormality. No such abnormalities were observed in
a sample of Efnb2 null heterozygote temporal bones (Pou3f4+/Y;
Efnb2null/+ or Pou3f4+/Y; Efnb2null/flox, n = 30), so these
genotypes were excluded from further analyses. A Chi-square test of
independence on the remaining data shown in Table 2 indicated
that the variables of genotype and phenotype are related
(Chisquare = 23.82; df = 6; P = 0.0006). Pair-wise comparisons
between Pou3f4Cre/Y; Efnb2+/+ and Pou3F4Cre/Y; Efnb2+/flox
or Pou3F4Cre/Y; Efnb2null/+ genotypes revealed no differences in
occurrence for any category (soft tissue bridge; bone bridge; and
total affected, ie., soft + bony connections), indicating that Efnb2
heterozygosity does not potentiate the Pou3f4 hemizygous null
stapes-facial canal phenotype. Comparisons between Pou3f4Cre/
Y;Efnb2+/+ and Pou3f4Cre/Y;Efnb2null/flox genotypes revealed
that the total occurrence of affected stapes (Total Affected) was
similar across genotypes (P = 0.1471; Fishers exact test). However,
the sample of mice lacking both genes had a roughly 2-fold
increase in occurrence of bony connections over that of
Pou3f4Cre/Y hemizygous null males (P = 0.0243; Fishers exact
test) and a decrease in soft tissue connections compared to
Pou3f4Cre/Y hemizygous null males (P ,0.0001; Fishers exact
test). In analyzing penetrance of phenotypic categories for the
Pou3f4Cre/Y;Efnb2null/flox sample, we found that the distribution
for all six possible categories does deviate from the case where each
category is equally prevalent (P = 0.0419, Chi-square: 11.53, 5 df),
but bilateral and unilateral bony defects (which dominate the
distribution) are equally prevalent in the sample (P .0.7232, by
Chi-square or Fishers exact test; Table S1). In summary,
compound Pou3f4 null hemizygosity;Efnb2 conditional null
homozygosity biases expressivity of the Pou3f4Cre/Y stapes-facial
canal defect toward the more severe form (bone bridge)
independently of its bilateral or unilateral configuration. Severity
of defects at the SV joint and internal auditory canal were judged
to be similar across Pou3f4Cre/Y;Efnb2+/+ and Pou3f4Cre/
Y;Efnb2null/flox genotypes (Fig. S1B, C; data not shown).
Eph receptor gene expression foreshadows borders of
distinct middle ear structures
As described in a previous section, we found no stapes-styloid
defects in a small sample of Efnb2 C-terminal deletion mutants, so
Efnb2 may function as a ligand to activate Eph receptor (forward)
signaling  in the present context. We surveyed the middle
ear rudiment for expression of Ephb1, Ephb2, Ephb3, Ephb4, and
Epha4, each of which encodes a cognate receptor for Efnb2. Sox9
hybridization signal, a marker of osteo-chondroprogenitor cells
, was used in conjunction with histological appearance and
anatomical landmarks to identify territories of nascent middle ear
rudiments. Of the set of Eph receptor genes tested, Ephb4 was
expressed widely throughout developing middle ear mesenchyme
between E11.5 and E13.5 (data not shown), whereas Epha4 and
Ephb2 were expressed in patterns that foreshadow borders of
distinct middle ear structures.
At E11.5, Epha4 mRNA signal marked a lateral region of the
nascent middle ear and appeared to overlap slightly with Pou3f4
signal in mesenchyme adjacent to the anterior cardinal vein
(Fig. 6AC, E). By E13, Epha4 signal had intensified lateral to the
stapes and VIIth nerve and formed a domain complementary to
that of Pou3f4 (Fig. 7A, D, M, P). By E14.5, Epha4 signal was
detected in loose mesenchyme surrounding the ossicular
condensations (Fig. 7C, F).
At E11.5, Ephb2 mRNA signal marked a medial region of the
nascent middle ear, with strong signal in mesenchyme dorsal to the
stapedial artery and surrounding the VIIth cranial nerve; this
overlapped with a portion of Pou3f4 signal (Fig. 6B, D, F). By
E13.5, strong Ephb2 signal surrounding the VIIth nerve had
resolved to a medial-lateral coursing stripe just dorsal to the stapes.
The Ephb2 stripe overlapped with Pou3f4 and Efnb2 signals at
E13.5 (Fig. 7H, K, N), prior to detectable cartilage matrix
deposition in the stapes condensation (Fig. 7B, C). At E14.5,
Ephb2 and Pou3f4 signals dorsal to the stapes were attenuated
relative to earlier stages (Fig. 7I, O). Pou3f4-Cre;ROSA-YFP
genetic labeling at E14.5 recapitulated the medial-lateral coursing
stripe dorsal to the stapes (Fig. 7R).
In summary, Epha4 mRNA signal distinguishes lateral middle
ear rudiments (incus, malleus) from the more medially situated
stapes, VIIth nerve, and cardinal vein; at E13.5, the Epha4
domain border defines an apparent plane at which the stapes head
and incus will articulate (Fig. 7Q, red dots, compare with Fig. 7E).
By contrast, Ephb2 mRNA signal distinguishes the stapes from
more dorsally situated rudiments, such as otic capsule surrounding
the vestibular canals, VIIth nerve, and styloid process (Fig. 7Q,
cyan dots, compare with Fig. 7H).
Pou3f4Cre/Y null hemizygosity dysregulates Efnb2 and
Sox9 expression dorsal to the stapes
The mutant phenotypes described above, together with
colocalization of Ephb2, Efnb2, and Pou3f4 in a stripe
distinguishing the nascent stapes from more dorsally located rudiments, led us
to ask whether Pou3f4 is required for normal expression of Efnb2,
Ephb2, or Epha4 in the developing middle ear. We hybridized
serial sections of E1313.5 Pou3f4Cre/Y mutant and Pou3f4+/Y
wild-type littermates with RNA probes and rated the extent and
intensity of signals in the region of interest. We found no consistent
differences in Epha4 (4 littermate pairs) or Ephb2 (6 littermate
pairs) signals across genotypes. However, all Pou3f4Cre/Y mutants
(from 6 littermate pairs) showed a reduced intensity of Efnb2
signal immediately dorsal to the stapes and surrounding the VIIth
nerve (Fig. 8A, A9, B, B9; S5), as well as in subcapsular
mesenchyme surrounding the cochlear epithelium and spiral
ganglion (Fig. S6). By contrast, Efnb2 signals at sites outside the
domain of Pou3f4 expression the stapedial artery or malleus/
incus rudiment, for example - were unchanged across genotypes
(Fig. 8A, A9, B, B9; S5). In a converse experiment, we assayed
Pou3f4 expression by RNA hybridization to E1313.5 Efnb2
CKO and control embryos (5 littermate pairs) and found no
differences in Pou3f4 signal across genotypes (Fig. 8C, C9,D, D9).
To determine whether attenuated Efnb2 signal dorsal to the
Pou3f4Cre/Y stapes correlates with failed separation of
Figure 8. Attenuated Efnb2 signal and altered patterning of Sox9 signal near the Pou3f4Cre/Y stapes condensation, but no apparent
change of Pou3f4 signal in the Efnb2 CKO. (A, A9, B, B9) Transverse sections of control (A, A9) and Pou3f4Cre/Y mutant (B, B9) E13.0 littermates
hybridized to detect Efnb2. Red arrowheads in (A9, B9) highlight the medial-lateral coursing stripe of Efnb2 signal dorsal to the stapes condensation. i/
m, Efnb2 signal at the incus and malleus rudiment. (C, C9, D, D9) Transverse sections of control (C, C9) and Sox9-IRES-Cre-mediated Efnb2 CKO (D, D9)
E13.0 littermates hybridized to detect Pou3f4. (E, E9, F, F9) Transverse sections of control (E, E9) and Pou3f4Cre/Y mutant (F, F9) E14.5 littermates
hybridized to detect Sox9. Arrowheads in (E9, F9) highlight an alteration in Sox9 patterning across genotypes. sty, styloid process condensation. Boxed
regions of interest highlighting the stapes and surrounding structures in (AF) are shown at 2x magnification in (A9F9), respectively. Scale bar in
(D) = 50 micron for (AF), and 25 microns for (A9F9). In all panels, white arrows highlight the stapedial artery, and asterisks highlight the VIIIth nerve.
Axes in (C) apply to all photos.
pre-cartilaginous mesenchymal condensations, we assessed Sox9
hybridization signal in Pou3f4Cre/Y mutant and Pou3f4+/Y
wildtype littermate pairs. In E12.5 wild-type embryos, the
chondrogenic marker Sox9 formed a continuous domain comprising
Meckels (1st branchial arch) cartilage, the malleus/incus territory,
stapes condensation, otic capsule condensation, styloid process
territory, and Reicherts (2nd branchial arch) condensation (Fig.
S7). Continuity of Sox9 expression across the stapes and styloid
process condensations was maintained until E13.5 (Fig. 4C, F;
black arrowheads), but all E14.5 wild-type embryos (4/4) showed a
sharply bordered region of Sox9 negativity dorsal to the stapes and
adjacent to the VIIth nerve (Fig. 8E, E9). E14.5 Pou3f4Cre/Y
mutants did not have a sharply bordered region of Sox9 negativity
dorsal to the stapes (0/4); instead, a continuous Sox9 signal of
graded intensity similar to the Sox9 pattern in normal E13.5 ears
- persisted in the E14.5 mutants (Fig. 8F, F9). In summary, these
results are consistent with a model in which Pou3f4 lies upstream
of Efnb2 in a genetic hierarchy, and attenuated Efnb2 expression
in the Pou3f4Cre/Y mutant is causal for insufficient separation of
stapes and styloid process rudiments prior to the onset of local
cartilage matrix deposition.
Pou3f4 protein associates with the Efnb2 locus in nascent
middle ear mesenchyme
To determine whether Pou3f4 physically interacts with the
Efnb2 locus in nascent middle ear mesenchyme, we first scanned
the Efnb2 genomic locus on mouse chromosome 8 for putative
Pou3f4 DNA binding/regulatory motifs . Six consensus motifs
(ATTATTA) were distributed across intronic regions of Efnb2
(Fig. 9A). Motifs at sites 3, 5, and 6 were fully conserved across two
or more species (mouse/rat or mouse/rat/human), and the site 1
motif, though non-conserved, was localized to an intronic region
of high phylogenetic conservation. We performed chromatin
immunoprecipitation (ChIP) on sites 1, 3, 5, 6, as well as a
nonconsensus site (NC), using purified chicken anti-Pou3f4 IgY
antibody and purified non-specific IgY as a negative control
condition . This was followed by SYBR-green-based qPCR
and absolute quantification (see Materials and Methods) to
measure concentrations of Efnb2 target sequences in the
antiPou3f4 and control IgY IPs. Chromatin was obtained from either
E12.25 middle ear mesenchyme or E10.5 limb mesenchyme, the
latter of which lacks detectable expression of Pou3f4 [31,38].
Differences in target sequence concentration across paired
experimental (anti-Pou3f4) and control (IgY) sample sets were
assessed for each site/tissue combination.
Site 1, 3, 5, 6, and NC target concentrations in anti-Pou3f4
(experimental) and IgY (control) eluates were statistically
indistinguishable using immunoprecipitated chromatin from limb mesenchyme
(Fig. 9B; data not shown). Likewise, using immunoprecipitated
chromatin from middle ear mesenchyme, site 1, 5, and NC target
concentrations in anti-Pou3f4 and IgY eluates were statistically
indistinguishable (Fig. 9B). Site 3 could not be evaluated statistically
in middle ear chromatin eluates due to lack of fluorescence signal
across 40 PCR cycles in roughly one-half of the assays. However,
experimental and control eluates of middle ear chromatin differed
significantly in their concentrations of target site 6 (p = 0.008;
Wilcoxon signed rank test), with anti-Pou3f4 eluates showing a 5.5
fold average enrichment over IgY eluates (Fig. 9B). These results
suggest that Pou3f4 transcription factor physically associates with
specific non-coding sequence of the Efnb2 locus during early stages of
middle ear development.
Pou3f4 and Efnb2 function within a common pathway to
promote temporal bone development
Our results indicate that Efnb2 lies downstream of Pou3f4 in a
genetic pathway governing embryonic-stage development of the
mouse temporal bone. Previous work demonstrates that Pou3f4
transcript and gene product are expressed in proximal branchial
mesenchyme (where the stapes and styloid process will form) and
in mesenchyme surrounding the nascent cochlea as early as E10.5
. Although Efnb2 is expressed in early migrating cranial
neural crest cells , we could not - by mRNA hybridization
identify Efnb2 transcript in mesenchyme surrounding the otic
epithelium until after E11.5. We found qualitatively identical
malformations of the stapes, styloid process, internal auditory
canal, and cochlear capsule in both Pou3f4Cre/Y and
(Sox9-IRESCre-mediated) Efnb2 CKO perinates, and between E12.5 and
E14.5 - Efnb2 and Pou3f4 expression overlapped in mesenchyme
that contributes to the formation of these structures. In
mesenchyme surrounding the E13 stapes and cochlear rudiments, Efnb2
expression was attenuated in the Pou3f4Cre/Y mutant compared
to control, but Pou3f4 expression appeared unaltered by
conditional loss of Efnb2. Conditional loss of Efnb2 (by
Sox9IRES-Cre) correlated with a 2-fold greater occurrence of full
stapes-styloid process coalition than that of Pou3f4Cre/Y mutants;
this is consistent with the finding that Efnb2 signal is attenuated
(rather than eliminated) in mesenchyme surrounding the
Pou3f4Cre/Y stapes rudiment. Adult double mutants lacking both
Pou3f4 and Efnb2 (Pou3f4Cre/Y;Efnb2flox/null) also displayed a
2fold increased occurrence of stapes-facial canal bony coalition over
adult Pou3f4Cre/Y single gene mutants. Thus, with respect to this
phenotypic feature, the effect of losing both genes appears to be
neither additive nor synergistic compared to loss of Efnb2 alone.
We also provide evidence that, at the developing mouse middle
ear, Efnb2 transcription is potentiated by a physical interaction
between Pou3f4 and the Efnb2 locus. Pou3f4 protein is localized
to nuclei of prospective middle ear mesenchyme cells by E11.5
. Using chromatin isolated from E12.25 nascent middle ear
mesenchyme, we found a 5.5 fold enrichment of specific intronic
sequence near the 39 end of Efnb2 in anti-Pou3f4 eluate compared
to IgY eluate. The relatively modest (though statistically
significant) magnitude of fold increase may be due to the following
factors. Our manual dissection undoubtedly captured a
heterogeneous isolate, with Pou3f4+:Efnb2+ cells forming a small minority
of cells from which chromatin was obtained. Efnb2 signal in the
region of interest (surrounding the stapes and within the domain of
Pou3f4) was attenuated but not entirely eliminated in the
Pou3f4Cre/Y mutant, indicating that Pou3f4 activity is not an
absolute requirement for Efnb2 expression at this site.
Transcriptional regulation of Efnb2 is complex, as evidenced by ChIP and
transactivation analyses of the 59 promoter region in cultured
mouse arterial endothelial cells . This study  revealed
strong transactivation of Efnb2 from the 59 promoter region by a
TALE homeodomain superfamily member, Meis1. In chicken
embryos of equivalent maturity to E12.513.5 mouse, Meis1 is
expressed broadly in proximal 2nd brachial arch mesenchyme
encompassing the stapes rudiment and surrounding structures
. In E14.5 mouse, Meis1 is strongly and widely expressed in
mesenchyme surrounding the ossicles . These considerations
suggest a model wherein region-specific potentiation of Efnb2
expression in periotic/branchial mesenchyme occurs through
specific binding of Pou3f4 to 39 intronic sequence.
We focused our ChIP and quantitative genetic analyses on the
middle ear (stapes-styloid/facial canal region in particular) because
the phenotype was efficiently scored by careful inspection of whole
temporal bone preparations, and this allowed adequate sample
sizes for statistical comparisons of multiple mutant genotypes.
However, additional qualitative similarities between Pou3f4 and
Efnb2 mutant phenotypes described here (e.g., thin cochlear
capsule; dilated internal auditory canal) and the finding of
attenuated Efnb2 mRNA signal in cochlear subcapsular mesenchyme of
Pou3f4Cre/Y mutants compared to that of control (Fig. S6) raise
the question of whether Pou3f4-mediated Efnb2 regulation has a
more comprehensive role in temporal bone development. A
previous study indicates that Epha4, encoding a cognate receptor
for Efnb2, is a direct target of Pou3f4 transcription factor within
cochlear sub-capsular mesenchyme . This regulatory
relationship is associated with mesenchyme-dependent fasciculation of
spiral (VIIIth) ganglion axons. It is therefore reasonable to ask
whether Pou3f4 directly coordinates temporal-spatial patterns of
transcription for cognate Eph and ephrin genes, the products of
which then mediate developmental events through ligand-receptor
interaction. By RNA in situ hybridization, we were unable to
detect altered expression of Ephb2 or Epha4 at the developing
Pou3f4Cre/Y middle ear. More sensitive techniques for assessing
change in Eph receptor transcript levels may be required in future
studies, as we found overlapping expression of Pou3f4 and Ephb2
in mesenchyme dorsal to the stapes and multiple Pou3f4 DNA
binding site motifs distributed across non-coding regions of the
Ephb2 gene (Coate and Raft, unpublished observations).
How the genetic regulatory mechanism proposed here affects
cellular behavior during ear development remains to be
determined. Genetic fate mapping in the mouse reveals that both
the stapes crura/head and styloid process derive from 2nd
pharyngeal arch neural crest [4,5]. Embryological studies of
humans  and rodents [46,47] indicate that discrete bony
elements of the middle ear originate by splitting of one or more
continuous mesenchymal condensations. We have shown that a
continuous domain of mesenchymal Sox9 gene expression
encompasses primordia of the ossicles, styloid process, and otic
capsule at stage E12.5. In Pou3f4Cre/Y mutant embryos,
attenuated Efnb2 mRNA signal correlated with failure to properly
pattern or downregulate Sox9 mRNA signal between the nascent
stapes and styloid process rudiments. It is therefore possible that
Eph-Efnb2 signaling promotes splitting of a continuous
mesenchymal condensation into separate elements through effects on
gene expression, cell adhesion and re-arrangement, or some
combination of these processes. Our evidence to date suggests that
the stapes-styloid phenotype of Sox9-IRES-Cre-mediated Efnb2
CKO embryos is caused by a loss of forward signaling through one
or more cognate Eph receptors, as we failed to find this phenotype
in perinatal mouse mutants lacking only the Efnb2 C-terminus.
Reverse genetics offers insight into the etiology of
DFNX2/DFN3 conductive hearing loss
Conductive hearing loss is typically due to inefficient transfer of
sound energy through the middle ear. However, the precise
anatomical bases for conductive hearing loss in humans with
POU3F4 mutations and DFNX2/DFN3 remain in question.
Early reports of DFNX2/DFN3-type patients cite congenital
fixation of the stapes footplate or absence of the annular ligament
as causing conductive hearing loss [48,49]. Other investigators
attribute reduced (or absent) stapes mobility to increased
perilymph fluid pressure within the bony capsule, which could
result from a widened internal auditory canal and abnormal
communication with the sub-dural space [6,50,51]. More recently,
it has been proposed that the dilated internal auditory canal and
other bony abnormalities characteristic of DFNX2/DFN3
dissipate acoustic energy at the level of the inner ear and amplify
boneconducted sounds [52,53]. This so-called third-window effect
confounds interpretation of routine audiometric testing and causes
inner ear conductive hearing loss, which can occur in the
presence of a normally functioning middle ear .
We found the internal auditory canal of perinatal Pou3f4Cre/Y
and Efnb2 CKO mutants to be dilated and dysmorphic at the
cartilaginous stage of temporal bone development, thus extending
previous findings on the adult Pou3f4 mutant temporal bone .
By contrast, we found no apparent malformation of the developing
stapes footplate/oval window in either mutant at perinatal stages,
suggesting that the footplate/oval window dysplasia of adult
Pou3f4 hemizygous null males arises during post-natal ossification
or bone remodeling. The stapes footplate/oval window dysplasia
observed in our sampling of adult Pou3f4Cre/Y and Pou3f4Cre/
Y;Efnb2flox/null temporal bones is consistent with previous
observations of Pou3f4 hemizygous null mice by Samadi et al.
, but is inconsistent with reports of stapes footplate fixation in
DFNX2/DFN3 patients [48,49]. Rather than a footplate fixation,
the Pou3f4Cre/Y, Pou3f4Cre/Y:Efnb2flox/null, and
Sox9-IRES-Cremediated Efnb2 CKO stapes was immobilized by a bony and/or
cartilaginous connection to the facial canal/styloid process. In
humans, this so-called supra-structure fixation of the stapes dorsal
crus and facial canal is rare and of unknown genetic etiology .
What might account for the apparent differences in stapes/oval
window/facial canal morphology across mice and humans lacking
Pou3f4/POU3F4 activity? Variation in the developmental
regulation of Pou3f4/POU3F4 expression is one likely
explanation. Breakpoint analyses of DFNX2/DFN3 microdeletions,
crossspecies non-coding sequence comparisons, and transgenic
activities of putative cis regulatory sequences suggest that the
temporal-spatial pattern of Pou3f4/POU3F4 expression during
ear development is a composite of multiple 59 cis-enhancer
activities; phylogenetically conserved enhancers are distributed
across a 1Mb region and respond differentially to major signaling
pathways in model organism transgenic assays [12,5759]. It is
also relevant that a supra-structure fixation of the stapes and
styloid process/facial canal has not previously been identified in
mice carrying other null alleles of Pou3f4 [14,15,17], and the
question of whether genetic background influences stapes
morphology in mice lacking Pou3f4 has been raised . Here
again, developmental gene expression requiring integration of
many signals across a vast 59 regulatory region may be susceptible
to background-specific modifiers. Finally, DFNX2/DFN3
anatomy may be incompletely characterized, given the small number of
cases that have been documented radiologically and lack of
postmortem temporal bone pathology studies.
Fixation of the stapes and styloid process/facial canal is found in
other targeted mouse mutants. Mice heterozygous for the BMP
antagonist noggin show genetic background-specific and
incompletely penetrant coalition of the stapes and styloid process/facial
canal without other apparent temporal bone defects . By
contrast, Wnt1-Cre-mediated inactivation of the endothelin-A
receptor can result in coalition of the stapes and styloid process in
the context of a severely dysmorphic middle ear and jaw . The
current model of early-stage jaw development places Endothelin-1
and BMP signaling upstream of several transcription factor
families (Dlx, Msx, Hand) that pattern dorso-ventral axes of the
1st and 2nd pharyngeal arches . Dlx transcription factors are
required for proper expression of another POU family
transcription factor gene, Pou3f3, in pharyngeal arch mesenchyme, and
Pou3f3 loss-of-function causes coalition of the stapes and styloid
process . Functional redundancy of Pou3f3 and Pou3f4 is
another reasonable explanation for the lack of a reported stapes
fixation in previous characterizations of Pou3f4 mutants, and may
also explain the variable expressivity/incomplete penetrance of
stapes-styloid process coalition in the sample of Pou3f4Cre/Y
mutants characterized here.
To date, no association exists between EFNB2 mutations and a
human disease or congenital defect. Complete loss of the human
gene activity may cause early embryonic lethality due to failed
remodeling of the primary embryonic vasculature into a system of
arteries and veins, as is the case in the mouse [39,64]. However, a
growing list of roles for Efnb2 in forming the murine
auditoryvestibular system [24,28,35,65,66] raises the possibility that
regulatory or hypomorphic mutations of EFNB2 underlie
functional variation in human hearing and balance.
Materials and Methods
Pou3f4 mutant females of mixed (B6/129:Swiss Webster:CD1)
genetic background, in which Cre is fused in frame with the
Pou3f4 start codon , were crossed to congenic C57BL/6
Efnb2LacZ/+ null heterozygote males  (Jax stock 006039) to
obtain F1 hybrids, which were maintained through 11 generations
of intercrosses. Sperm was cryopreserved from F1:N10 males
carrying both null alleles. A second Efnb2 allele, with exon1
flanked by loxP sites  (Jax stock 006042), was maintained in
the homozygous state on the C57BL/6 background as a separate
colony. All Pou3f4 mutant specimens (and controls) analyzed were
male progeny of F1 Pou3f4-Cre+;Efnb2+/+ or
Pou3f4-Cre+;Efnb2LacZ/+ hybrids crossed to congenic C57BL/6 Efnb2+/+ or
Efnb2flox/flox mice. Therefore all Pou3f4 mutants (and controls)
analyzed were F2 generation mixed:C57BL/6 hybrids. In some
experiments, Pou3f4-Cre+ mice were bred to C57BL/6
ROSAYFP reporter mice (Jax stock 006148). PCR assay of the Smcx and
Smcy genes  was used to determine the sex of embryos. A
separate congenic C57BL/6 breeding colony was used to generate
Sox9-IRES-Cre+;Efnb2LacZ/+ double heterozygous mice, which
were mated with Efnb2flox/flox mice to obtain congenic C57BL/6
Efnb2 CKO and control embryos, as previously described .
Fixed specimens of mixed 129/CD1 strain Efnb2 C-terminal
deletion homozygotes (ephrin-B2LacZ mice in ) and
wildtype littermate controls were kindly provided by Dr. Mark
Henkemeyer (UT Southwestern Medical Center). All PCR
genotyping of targeted alleles was conducted as previously
described. CD1 embryos were used for studies of normal gene
expression, histology, and chromatin isolation. All animal
experiments were carried out in strict accordance with
recommendations set forth in the Guide for the Care and Use of Laboratory
Animals of the National Institutes of Health. All protocols were
approved by the Investigational Animal Care and Use Committee
of the National Institute on Deafness and Other Communication
Tissue fixation and preparation
Pregnant dams were euthanized with an anesthetic
concentration of CO2 and cervical dislocation. For Alcian Blue/Nuclear
Fast Red histology at mid-gestational stages (E12E14.5) whole
embryos were immersed in methacarn fixative (6:3:1
methanol:chloroform:glacial Acetic Acid) for 1 to 5 hours, trimmed, rinsed in
methanol, cleared sequentially in methyl benzoate and xylene
(163060 minutes each), infiltrated with paraffin, and cut serially
in the transverse plane of the embryo at 7 micron thickness on a
Leica RM2145 microtome. For Alcian Blue/Nuclear Fast Red
histology at perinatal stages, heads were split mid-sagittally,
immersion-fixed overnight at 4 degrees Celsius in 4%
paraformaldehyde in PBS (pH 7.4), cryoprotected in 30% sucrose/PBS,
embedded in OCT compound (Tissue-Tek), and cut serially in the
sagittal plane at 7 micron thickness on a Leica CM 3050 S
cryostat. For Weigerts Resorcin Fuchin staining of the
stapediovestibular joint, adult temporal bones were dissected and
immersion-fixed overnight at 4 degrees Celsius in 4%
paraformaldehyde in PBS, decalcified in 0.1M EDTA in PBS, washed,
cleared sequentially in graded alcohols and xylene, infiltrated with
paraffin, and cut serially in the longitudinal plane of the ear at 7
micron thickness on a Leica RM2145 microtome. For anti-GFP
immunofluorescence and RNA in situ hybridization, mid-gestation
embryos were immersion-fixed overnight at 4 degrees Celsius in
4% paraformaldehyde in PBS, cryoprotected in 30% sucrose/
PBS, embedded in OCT medium, and cut serially in the
transverse plane at 7 micron thickness on a Leica CM 3050 S
cryostat. For whole mount Alcian Blue/Alizarin Red S staining of
mid-sagitally split perinatal or adult heads, skin and brain were
removed and specimens placed in 95% Ethanol for 35 days at
For Alcian Blue/Nuclear Fast Red histology, hydrated sections
were dipped in 3% acetic acid for 3 minutes, stained in 1% Alcian
Blue 8GX (Sigma A3157) at pH 2.5, washed in running water for
10 minutes, counterstained with Nuclear Fast Red (Vector
Laboratories H-3403) for 5 minutes, washed in tap water for 10
minutes, dehydrated, cleared, and mounted in Permount.
Weigerts Resorcin Fuchsin (EMS 26370) staining was carried
out according to manufacturers instructions. Anti-GFP
immunofluorescence on tissue sections was performed by standard
methods using a FITC-conjugated goat anti GFP antibody
(Gene-Tex 1:200), followed by counterstaining with
rhodaminephalloidin (Molecular Probes) and DAPI. RNA hybridzation to
tissue sections was performed in slide mailers using
digoxygeninlabeled probes in weakly acidic hybridization buffer (pH 4.5),
antidigoxigenin-AP Fab fragments (Roche 11093274910) in TBST
buffer, and the NBT/BCIP colorimetric substrate reaction in AP
buffer at pH 9.5. Whole mount Alcian Blue/Alizarin Red S
staining of cartilage and bone was performed according to the
method of McLeod .
RNA probes. Cre-mediated recombination at the floxed Efnb2
allele was validated with a 140bp exon 1-specific fragment
(NM_010111.5, nt 139-278) PCR cloned from C57BL/6 tail
DNA and ligated into pCR4-TOPO vector for in vitro
transcription. Wild-type Efnb2 was detected with a ,1 kb probe
transcribed from NM_010111.5, nt136-1188. Other cDNAs used
were as follows: Pou3f4 (NM_008901, a 511 base-pair NcoI
fragment), Sox9 (NM-011448, 500 base-pair fragment), Epha4
(NM_007936, nt2782-4242), Ephb2 (NM_010142), Ephb3
(NM_010143, nt535-1207), and Ephb4 (BC090839.1,
Histological and Anatomical Analyses
For qualitative comparisons of hybridization signals across
genotypes, mutant-control littermate pairs were serially sectioned
on the same day; each slide for a littermate pair contained mutant
and control sections and these were distributed in equal
proportions across the minimum number of slides needed to
uniformly sample entire domains of interest from every second
7micron serial section. Mutant and control sections were
randomized in their placement from slide to slide. Four separate
hybridization experiments were conducted, each containing one
or more littermate pair section sets probed for each of the
following genes: Efnb2, Ephb2, Epha4, Pou3f4, and Sox9.
Digital images of all sections in the area of the stapes were
uniformly captured for each littermate pair/probe set during a
single imaging session, and extent and intensity of signal in the
area of the mutant and control stapes was rated from raw TIFF
images. Scoring of defects at the stapes-styloid process or
stapesfacial canal in perinatal or adult ears was accomplished by partial
dissection and observation under a stereo-dissecting microscope
and digital capture of images on a Leica M205FA
stereomicroscope with motorized focusing. Statistical analyses of categorical
data (using Prism 5 software) were carried out using a two-sided
Fishers exact test for 262 contingencies or a Chi Square test for
data sets with 2 or more degrees of freedom. In the text,
occurrence denotes outcomes where n = the number of temporal
bones analyzed; penetrance denotes outcomes where n = the
number of animals analyzed.
Chromatin Immunoprecipitation (ChIP)-PCR
A sharpened stainless-steel wire was used to isolate blocks of
nascent middle ear mesenchyme from stage E12.25 CD1 mouse
embryos. We generated four independent biological samples, each
of which contained pooled tissue from eight ears. Each
independent sample was collected in ice-cold 1X PBS, fixed in 4%
paraformaldehyde for 20 minutes at room temperature, washed,
pelleted, and frozen. Chromatin was isolated from each pellet by
methods outlined in the Pierce Agarose ChIP Kit (Thermo
Scientific, Catalog #26156). Chromatin was treated with
micrococcal nuclease for 15 minutes at 37uC to produce DNA fragments
ranging in size from 200600 base pairs. Chromatin from E10.5
limb mesenchyme (where Pou3f4 is not expressed) [31,38] was
identically prepared. Each independent chromatin preparation
was divided equally by volume and incubated overnight at 4
degrees Celcius with either 10 mg whole IgY (Jackson
Immunoresearch, Catalog #003-000-003) or 10 mg anti Pou3f4-specific IgY
. Following incubation, Preciphen beads (Aves Labs, Catalog
#P-1010) were added to the antibody/chromatin mixture and
allowed to bind for 2 hours. After extensive rinsing in low- and
high-salt buffers, antibody/chromatin complexes were eluted from
the beads, treated with proteinase K, and column purified.
Concentrations of Efnb2 target sequences in IP eluates were
quantified by qPCR on an Applied Biosystems StepOne Realtime
PCR system using SYBR-green and the following primer sets
derived from mouse chromosome 8 sequence GRCm38.p2
C57BL/6J (NC_000074.6): a negative control site lacking the
Pou3f4 binding motif (non-consensus or NC site)
CTTGTTCCCAGTGTGGATGA (forward) and
ACCCCAAACAACTGAACCAG (reverse); site 1,
TTACGAATTGGACACTAACAAGC (forward) and TGGCCTGAAAAACAGGTTC
(reverse); site 3, ACACTAACAAGCCTCTTCTCCA (forward)
and TGCAGGAATATAAGTGGCCTGA (reverse); site 5,
TTTGGCTTTTCCTGGACATT (forward) and
GCCCAAGTTAATGCGTTTTC (reverse); site 6,
GACCTTGAGGCTCCTTTGC (forward) and GCAGAAACCCCGAAATGTAA
(reverse). The annealing temperature used for all reactions was 55uC,
with the exception of the site 3 primer pair reactions, which used
an annealing temperature of 60uC. All qPCR reactions comprised
40 cycles and resulted in a single product, as determined by melt
curve and gel electrophoretic analyses. Amplicon specificity was
validated by direct sequencing. For absolute quantifications of
template input to qPCR reactions, a standard curve was generated
for each primer pair using total genomic DNA (gDNA) from
pooled forelimb and hindlimb tissues of E10.5 CD1 mouse
embryos; gDNA was purified, fixed, nuclease-digested as described
above, and serially diluted for use in triplicate qPCR reactions.
Semi-log transformation and linear regression gave Pearson
correlation coefficients ranging from 2.963 to 2.999. Primer
efficiencies, defined by the equation 10[21/slope], ranged from
1.9 to 2.7. Concentrations of target site DNA from IgY and
antiPou3f4 pull-downs were computed from Ct values and standard
curve equations. Each of four independent chromatin preparations
(middle ear or limb) was assayed twice by qPCR in technical
triplicate, yielding 8 matched pairs of data (anti-Pou3f4 IP vs.
control IgY IP) for each target site tested. Paired data sets were
subjected to the Wilcoxon signed rank test (Prism 5 software), with
a cut-off for statistical significance at P,0.05.
Figure S1 Dilation and incomplete septation of the
internal auditory canal in Pou3f4Cre/Y, Pou3f4Cre/
Y;Efnb2flox/null, and Sox9-IRES-Cre+;Efnb2flox/null
mutants. (AC) Medial views of wild-type and mutant temporal
bones excised from 1620 week old adult mice and stained with
alizarin red/alcian blue, shown to scale. Bar = 100 microns.
Foramina of the internal auditory canal are highlighted by dotted
lines. Three foramina are evident in wild-type (A); there is no
septation of foramina ii (for superior vestibular VIIIth nerve) and
iii (for VIIth nerve) in the mutants (B,C). Foramen i conducts the
auditory VIIIth nerve branch). (DF) Medial views of wild-type
and mutant cartilaginous capsules from E19-P0 heads stained with
alizarin red/alcian blue, shown to scale. Bar = 100 microns.
Three foramina are evident in wild-type; arrow in (A) highlights
septation of foramina ii and iii. In mutants (E, F), the internal
auditory canal is enlarged compared to wild-type and there is no
septation of foramina ii and iii. Asterisk in (E) highlights cartilage
outside the focal plane of the capsule medial wall. Axes in A apply
to all photos. R = rostral.
Figure S2 Facial canal lateral wall is formed by fusion
and growth of the endochondral styloid process and
intramembranous tympanic ring. (AC) Lateral views of
excised, Alcian Blue/Alizarin Red-stained temporal bones from
wild-type mice at post-natal days 5, 8, and 15. Grey dots highlight
the tympanic ring dorsal edge at its apposition with the styloid
process (sty). The endochondral styloid process is cartilaginous and
stains blue at P5 and P8; the intramembranous tympanic ring (tr),
is ossified at birth and stains red at all stages shown. s, stapes; i,
incus; m, malleus. (DF) Magnified views of the boxed regions in
(AC), respectively. Arrows indicate apparent vectors of tympanic
ring growth; the tympanic ring is superficial to the styloid process
and its expansion fully obscures the styloid process by P15. (GI)
show ventral views of the specimens shown in (AC), respectively,
with tympanic ring (tr) and malleus (m) dissected away for
unobscured views of the stapes (s), facial canal (dotted line), and
styloid process (sty). Note the near-complete ossification of the
styloid process between stages P8 and P15.
Figure S3 Validation of Sox9-IRES-Cre-mediated
recombination at the Efnb2 locus. Sections of control (A) and
Efnb2 CKO littermates (B), showing the middle ear at stage
E14.5, hybridized with an Efnb2 exon1-specific probe. Signal
from the Efnb2 exon1 probe in developing ear and second
branchial arch tissues is markedly decreased in the mutant
compared to control. asterisk, VIIth nerve; s, stapes; m, malleus;
c, otic capsule; cd, cochlear duct; spg, spiral ganglion.
Figure S4 Homozygous deletion of the Efnb2
C-terminus has no apparent effect on stapes and styloid process
morphology. Sagittal sections of mixed 129/CD1 strain
wildtype control (A) and Efnb2C-del/C-del (B) littermates at stage E18.5,
stained with Toluidine Blue to reveal cartilage. Distance between
the stapes (sta) and styloid process (sty) is similar across genotypes.
Scale bar = 100 micrometers.
Figure S5 Efnb2 mRNA signal intensity is attenuated
relative to control in Pou3f4Cre/Y mutant mesenchyme
dorsal to the stapes at stages E1313.5. Representative
image data for 5 of 6 wild-type (AE) and mutant (A9E9)
littermate pairs, hybridized under controlled conditions to assay
for potential change in Efnb2 expression across genotypes.
Brackets in (A, A9) highlight mesenchyme dorsal to the stapes (s)
and surrounding the VIIth nerve (asterisk), where Efnb2
hybridization signal is specifically altered across genotypes.
Structures are identically framed in all panels. Efnb2 signals at
the malleus (m) and otic epithelium (oe) appear similar across
genotypes. Scale bar = 100 micrometers. The fifth of six
wildtype/mutant pairs analyzed is shown in the main body of the text
(Fig. 8A, A9, B, B9).
Figure S6 Efnb2 mRNA signal intensity is attenuated
relative to control in Pou3f4Cre/Y mutant sub-capsular
mesenchyme at stage E13. Transverse sections of control (A)
and Pou3f4Cre/Y (B) E13.0 littermates hybridized to detect Efnb2.
Red arrowheads highlight attenuation of Efnb2 hybridization
signal in mutant sub-capsular mesenchyme surrounding the
cochlea and spiral ganglion. Spiral ganglia are bounded by black
dotted lines; cochleae are encircled by solid white lines. Scale bar
= 100 micrometers.
Figure S7 Sox9 marks a continuous domain comprising
branchial arch cartilages, ossicles, styloid process, and
otic capsule at stage E12.5. (AF) Selected images from serial
transverse sections through the 1st and 2nd branchial arches of an
E12.5 embryo that were hybridized to detect Sox9 mRNA. Images
are arranged in an anterior to posterior sequence (A through F).
Note that Sox9 expression bridges all otic and branchial arch
rudiments specified. Meckels rudiment (Me), a dark-staining bar
in (A) is cartilaginous at this stage; all other rudiments are
mesenchymal condensations. Blue arrowheads in (C,D) highlight
Sox9 signal bridging the stapes (s) and styloid process (sty).
Asterisks in (C,D) highlight the VIIth cranial nerve. m/i, malleus/
incus condensation; oc, otic capsule condensation; m, caudal end
of the malleus/incus condensation in the 2nd arch; R, Reicherts
cartilage rudiment, ie, inner ear; hb, hindbrain; ph, pharynx.
The authors thank Drs. Robert Morell and Michael Hoa for critical
readings of the manuscript, Dr. Haruhiko Akiyama for providing the
Sox9IRES-Cre mouse, and Dr. Mark Henkemeyer for providing fixed tissue for
histological analyses of Efnb2 C-terminal deletion mutants and control
Conceived and designed the experiments: SR. Performed the experiments:
SR TMC. Analyzed the data: SR TMC. Contributed reagents/materials/
analysis tools: MWK EBC. Wrote the paper: SR. Designed ChIP
experiments: TMC SR. Assisted in study design: DKW.
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