Occurrence of stapes-facial canal connectivity in adult <i>Pou3f4</i> and <i>Efnb2</i> single and compound mutant mice.

a<p> sample size refers to the number of ears analyzed.</p><p>* 0.02</p><p>** p<0.0001; all pairwise Fisher's exact tests were performed relative to the <i>Pou3f4</i>^Cre/Y; <i>Efnb2</i>^+/+ genotype. Statistical significance set at P<0.05.</p><p>Occurrence of stapes-facial canal connectivity in adult <i>Pou3f4</i> and <i>Efnb2</i> single and compound mutant mice.</p

Comparative gene and Cre-mediated reporter expression in the developing middle ear at E12.5.

<p>(<b>A, B</b>) <i>Efnb2</i> and <i>Pou3f4</i> 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 <i>Efnb2</i> in the arterial endothelium (A). Asterisks highlight the facial nerve dorsal to the stapes condensation. Arrowheads highlight a region of overlapping <i>Efnb2</i> and <i>Pou3f4</i> signal dorsal to the stapedial artery and surrounding the facial nerve. (<b>C, D</b>) 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. <i>cc</i>, otic capsule condensation; <i>cv</i>., cardinal vein; <i>oe</i>, otic epithelium; <i>ph./eu</i>., nascent pharynx/eustachian tube; <i>pom</i>, periotic mesenchyme; <i>sta</i>, stapes mesenchymal condensation; st art., stapedial artery; <i>sty</i>, styloid process mesenchymal condensation; <i>vii n</i>., VIIth nerve. (<b>E, F</b>) 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.</p

Comparative expression of <i>Efnb2</i>, <i>Pou3f4</i>, and <i>Sox9</i> at E13.5 and E16.5.

<p>Adjacent transverse sections through embryos at two developmental stages, hybridized to detect <i>Efnb2</i> (A,D), <i>Pou3f4</i> (B, E), or Sox9 (C, F). <i>c</i>, cartilaginous cochlear capsule; <i>cd</i>, cochlear duct; <i>eam</i>, external auditory canal; <i>eu</i>, nascent eustacian tube; <i>i</i>, incus; <i>m</i>, malleus; <i>ph</i>, pharynx, <i>spg</i>, spiral ganglion, <i>s</i>, stapes. Asterisk highlights the facial nerve. Double arrowheads in (A–C) highlight overlapping <i>Efnb2</i> and <i>Pou3f4</i> signals and relatively weak <i>Sox9</i> 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 <i>Efnb2</i> and <i>Pou3f4</i> signals at the forming S–V joint. Scale bar represents 200 microns in (A–C) and 250 microns in (D–F).</p

Enrichment of a putative Pou3f4 DNA binding site in anti-Pou3f4 middle ear ChIP eluate compared to IgY middle ear ChIP eluate.

<p>(<b>A</b>) Organization of the murine <i>Efnb2</i> 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>B</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.</p

Occurrence of stapes-facial canal or -styloid connectivity in <i>Pou3f4</i>^Cre/Y and <i>Efnb2</i> CKO mice.

a<p> sample size refers to the number of ears analyzed.</p><p>* P = 0.0365, compared to <i>Pou3f4</i>^Cre/Y neonate by Fisher's exact test. Statistical significance set at P<0.05.</p><p>Occurrence of stapes-facial canal or -styloid connectivity in <i>Pou3f4</i>^Cre/Y and <i>Efnb2</i> CKO mice.</p