Analyses of paint-filled Foxg1−/− inner ears show a consistent phenotype of a single, anterior-positioned ampulla connecting to the anterior and lateral canals as previously reported (Fig. 1, arrow, n=36/36 ears; Pauley et al., 2006). The cochlear duct is shortened with only a single coil and an incomplete separation from the saccule. In addition to the reported phenotypes, some mutant embryos show a failure of posterior semicircular canal resorption (Fig. 1C, asterisk, n=6/36 ears, 16.7%, Table 1). None of these phenotypes are detected in the Foxg1 heterozygotes. Within the anterior ampulla of Foxg1−/− inner ear, a single crista is present in most cases (Fig. 2B, B', CA; n=22/24 ears, 91.7%), but occasionally two separate cristae can be found (Fig. 2C, C', CA1, CA2; n=2/24 ears, 8.3%). Normal anterior and posterior cristae have a non-sensory region, septum cruciatum, which divides the organ into two equal parts (Fig. 2D, G, Cr). However, the cruciatum within the mutant anterior crista is eccentrically located and does not bisect the sensory crista completely (Fig. 2E, F). The posterior crista shows similar malformed cruciatum (Fig. 2H, I). Both anterior and posterior cristae are misshapen and smaller in size, compared to wildtype (Fig. 2J). The average length of mutant anterior cristae is 31% less than wildtype (wildtype: 435.5 ± 9.6 μm (mean ± SEM), n=7; mutant: 302.0 ± 15.0 μm, n=7; P < 0.001; see Supp. Table S1, which is available online). In addition, the average length of mutant posterior cristae is reduced by 46% (wildtype: 413.0 ± 9.6 μm, n=6; mutant: 223.1 ± 17.3 μm, n=7; P < 0.001; Table S1).
Figure 1. Inner ear phenotypes of Foxg1−/− mice at E15.5. Paint-filled inner ears of wildtype (A) and Foxg1−/− (B, C) embryos. A: In wildtype inner ears, the anterior and lateral canals are connected to the anterior and lateral ampulla, respectively. The arrow points to the gap between the anterior and lateral ampullae. B, C:Foxg1−/− inner ears show a single, anterior-positioned, ampulla (A) connected to both anterior and lateral canals (arrow). The cochlear duct is shortened with only a single coil and an incomplete separation from the saccule. Some mutants show non-resorption of the posterior semicircular canal (asterisk in C). Orientations and scale bar in A apply to B and C. A, single ampulla; AA, anterior ampulla; asc, anterior semicircular canal; CC, common crus; Cd, cochlear duct; Ed, endolymphatic duct; LA, lateral ampulla; lsc, lateral semicircular canal; PA, posterior ampulla; psc, posterior semicircular canal; Sa, saccule; Ut, utricle.
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Figure 2. Crista phenotypes of Foxg1−/− inner ears at E18.5. Ventral views of dissected, unstained, membranous labyrinths of wildtype (A) and Foxg1−/− (B, C) embryos. A: Anterior and lateral cristae housed within each ampulla are connected to anterior and lateral semicircular canals, respectively. (B) A single crista or (C) two small cristae in Foxg1−/− ears are located within a single ampulla connecting to both anterior and lateral canals. A'–C': Schematic drawings of A–C, respectively. D–I: Phalloidin-labeled 3D-stacked confocal images of cristae. D: A wildtype anterior crista showing the septum cruciatum that divides the crista into two equal parts. E, F: Dysmorphic mutant cristae showing various size and shape. The septum cruciatum is eccentrically located and does not bisect the crista. G: Wildtype posterior crista with a septum cruciatum. H,I:Foxg1−/− posterior cristae showing variable size and shape, and abnormal cruciatum. Sometimes two cruciata are present (H). Insets in D–I show full-size cristae under lower magnification. J: Comparisons of crista lengths between wildtype and mutants (asterisk, P < 0.001). Scale bars in D apply to E–I. Orientations in A apply to B, C. AA, anterior ampulla; AC, anterior crista; asc, anterior semicircular canal; CA, mutant crista ampullaris within a single ampulla; Cr, cruciatum; LA, lateral ampulla; lsc, lateral semicircular canal; PC, posterior crista; UM, utricular macula; Ut, utricle.
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