Figure 1.
Schematic views of selected middle ear structures and otic capsule.
(A) Anatomy conforms to that of an excised mouse temporal bone at post-natal day 5, viewed laterally. Orientation within the head is indicated by the red box in A′, bottom right. R in axes = rostral. (B) Caudal view of the isolated adult stapes.
Figure 2.
Abnormalities of the stapedio-vestibular (S–V) joint and cochlear bony capsule in Pou3f4Cre/Y males.
(A–D) Views of the middle ear medial wall/cochlear bony capsule from control (A,C) and Pou3f4Cre/Y temporal bones (B,D) at 16 weeks of age. Soft tissue and bony structures, including the stapes head and crura, are dissected away for unobstructed views of the stapes footplate (fp) in oval window (ow) and cochlear bony capsule. rw, round window. (C,D) are magnified views of the fooplate/oval window in (A,B), respectively. Brackets in (D) highlight disarticulation at a severely affected mutant S–V joint with hypoplastic footplate. Asterisk highlights artifactual fracture of the footplate during dissection. Dotted line in (A) shows the course of the VIIth nerve within the facial canal. Arrows in (B) highlight translucence of the thin mutant cochlear bony capsule compared to wild-type; arrowheads in (B) highlight visibility of strial blood vessels and melanocytes through the mutant bony capsule. Axes in (B) apply to (A–D). (E, F, F′) Wild-type (E) and mutant (F, F′) S–V joint in histological section stained for elastic fibers (black), collagen (light pink), and nuclei. (F) corresponds to a bracketed regions of disarticulation in (D); (F′) corresponds to a region of mutant S–V joint where footplate and oval window are more closely apposed. Corresponding photos of wild-type and mutant are shown to scale.
Figure 3.
Stapes coalition in Pou3f4Cre/Y and Sox9-IRES-Cre-mediated Efnb2 CKO mice.
(A–D) Unstained preparations of the stapes in situ (A, B) and dissected (C, D) from Pou3f4+/Y wild-type (A, C) and Pou3f4Cre/Y mutant (B, D) males at 16 weeks of age. Arrowheads highlight the stapedial tendon. Arrows in (B, D) highlight a periosteal soft tissue bridge connecting the dorsal crus of the stapes and facial canal. (E, F) Alizarin red/alcian blue-stained preparation of wild-type (E) and mutant (F) stapes in situ at 16 weeks of age, showing a bony fusion of the dorsal crus to the facial canal in a mutant (arrow, F). Dotted line in (E) highlights the facial nerve. (G–I) Alcian blue/fast red-stained sagittal sections of P1 wild-type (G) and Pou3f4 mutants (H, I) at the level of the stapes head (sta), stapedius muscle (st m.), styloid process (sty), and facial nerve (vii). Arrow in (H) highlights a continuous perichondrium enveloping both the styloid process and the stapes. Arrowheads highlight insertion of the stapedial tendon into the stapes perichondrium. Asterisks highlight the cochlear capsule. (J) Photo of a dissected P0 wild-type stapes showing the level of section for (G,H,I,M,N). Schematic shows the plane of section. (K, L) Alizarin red/alcian blue-stained preparations from E19 wild-type (K) or Efnb2 CKO fetuses. (K) shows the wild-type otic capsule with middle ear structures dissected away for unobstructed view of the stapes (sta) and styloid process (arrowheads). White arrow highlights the lack of connection between normal stapes and styloid process. Dotted line hightlights a segment of the facial canal. (L) shows a magnified (relative to K) view of a dissected mutant stapes and styloid process; black arrow highlights the abnormal connection between these structures. (M, N) Alcian blue/fast red-stained sagittal sections from two different E19 Efnb2 CKO heads, showing full cartilage coalition of stapes and styloid process (N) or reduced distance/shared perichondrium between the two structures. Level of sections is comparable to (H, I). Axes near (M) and scale bar in (I) apply to all histological sections. Axes in (F) apply to (A, B, E, F, K, L). Scale bar in (D) applies to (A–F).
Table 1.
Occurrence of stapes-facial canal or -styloid connectivity in Pou3f4Cre/Y and Efnb2 CKO mice.
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 (A–C) 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 S–V joint. Scale bar represents 200 microns in (A–C) and 250 microns in (D–F).
Figure 5.
Comparative gene and Cre-mediated reporter expression in the developing middle ear at E12.5.
(A, B) Efnb2 and Pou3f4 gene expression domains, as shown in adjacent transverse sections through a wild-type E12.5 embryo. The stapedial artery, positioned roughly at the center of the stapes condensation, is marked by brown dots and by strong expression of Efnb2 in the arterial endothelium (A). Asterisks highlight the facial nerve dorsal to the stapes condensation. Arrowheads highlight a region of overlapping Efnb2 and Pou3f4 signal dorsal to the stapedial artery and surrounding the facial nerve. (C, D) Transverse section through a wild-type E12.5 embryo stained with alcian blue/nuclear fast red (C) and a scaled schematic drawing (D) of the stapes condensation and neighboring structures. White dot in (C) highlights the stapedial artery. Asterisks in (C,D) highlight the VIIth nerve. cc, otic capsule condensation; cv., cardinal vein; oe, otic epithelium; ph./eu., nascent pharynx/eustachian tube; pom, periotic mesenchyme; sta, stapes mesenchymal condensation; st art., stapedial artery; sty, styloid process mesenchymal condensation; vii n., VIIth nerve. (E, F) Sox9-IRES-Cre- (E) and Pou3f4-Cre- (F) mediated ROSA-YFP reporter expression in the region of the E12.5 nascent middle ear, as schematized in (D). (C–F) are shown to scale. Cre-positive cells or descendents of Cre-positive cells are labeled green. DAPI-positive nuclei are purple. White dots highlight the stapedial artery; asterisks highlight the VIIth nerve.
Table 2.
Occurrence of stapes-facial canal connectivity in adult Pou3f4 and Efnb2 single and compound mutant mice.
Figure 6.
Comparative expression of Sox9, Pou3f4, Epha4, and Ephb2 at E11.5.
(A–D) Adjacent transverse sections through a wild-type E11.5 embryo, hybridized to detect Sox9, Pou3f4, Epha4, or Ephb2. Mesenchyme that will form the stapes condensation is identified by Sox9 signal surrounding the stapedial artery (brown dots) and is located ventral to the facial nerve (asterisks). (E, F) Schematized spatial relationships between expression of Pou3f4 and either Epha4 (E) or Ephb2 (F). Asterisks denote VIIth nerve; brown dots denote the stapedial artery. cv, cardinal vein; oe, otic epithelium; s, stapes pre-condensation; i/m, incus/malleus pre-condensation. All panels are shown to scale.
Figure 7.
Comparative developmental expression of Epha4, Ephb2, Efnb2, Pou3f4, and Sox9 at the stapes condensation and neighboring structures.
(A–C) Transverse histological sections through wild-type embryos at E13, E13.5, and E14.5, stained with alcian blue/nuclear fast red. Asterisks highlight the VIIth nerve; brown dots or open arrow in (A) highlight the stapedial artery. cv, cardinal vein; s, stapes condensation; sty, styloid process condensation; oc, otic capsule cartilage; i, incus condensation; m, malleus cartilage. (D–Q) Developmental gene expression in wild-type embryos. Annotations are as in (A–C). Red and cyan dotted lines in (Q) highlight salient borders of Epha4 and Ephb2/Efnb2/Pou3f4 expression, respectively. (R) Pou3f4-Cre-mediated ROSA-YFP reporter expression at E14.5. Cre-positive cells or descendents of Cre-positive cells are labeled green. Section is counterstained with phalloidin (red) and DAPI (blue). All photos are shown to scale.
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, A′, B, B′) Transverse sections of control (A, A′) and Pou3f4Cre/Y mutant (B, B′) E13.0 littermates hybridized to detect Efnb2. Red arrowheads in (A′, B′) 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, C′, D, D′) Transverse sections of control (C, C′) and Sox9-IRES-Cre-mediated Efnb2 CKO (D, D′) E13.0 littermates hybridized to detect Pou3f4. (E, E′, F, F′) Transverse sections of control (E, E′) and Pou3f4Cre/Y mutant (F, F′) E14.5 littermates hybridized to detect Sox9. Arrowheads in (E′, F′) highlight an alteration in Sox9 patterning across genotypes. sty, styloid process condensation. Boxed regions of interest highlighting the stapes and surrounding structures in (A–F) are shown at 2x magnification in (A′–F′), respectively. Scale bar in (D) = 50 micron for (A–F), and 25 microns for (A′–F′). In all panels, white arrows highlight the stapedial artery, and asterisks highlight the VIIIth nerve. Axes in (C) apply to all photos.
Figure 9.
Enrichment of a putative Pou3f4 DNA binding site in anti-Pou3f4 middle ear ChIP eluate compared to IgY middle ear ChIP eluate.
(A) Organization of the murine Efnb2 genomic locus. Green triangles and box identify putative Pou3f4 DNA binding sites (ATTATTA motifs) at non-coding regions. Red triangle identifies the site of a selected negative control target lacking the ATTATTA motif. (B) Change in Pou3f4 DNA binding site abundance resulting from application of either anti-Pou3f4 and IgY IP, as assessed by qPCR and expressed in terms of fold changes for paired data (anti-Pou3f4 eluate/IgY eluate). Data for chromatin preparations from either E12.25 middle ear or E10.5 limb mesenchyme are shown. Each bar represents the range of fold change values obtained for sets of paired data (n = 8 Pou3f4 vs. IgY pulldown pairs, resulting from two qPCR trials with each of four independent chromatin preparations per tissue type; see Materials and Methods). Each horizontal line represents an average fold change.