Long-term consequences of Sox9 depletion on inner ear development

Abstract

The transcription factor Sox9 has been implicated in inner ear formation in several species. To investigate the long-term consequences of Sox9 depletion on inner ear development we analyzed the inner ear architecture of Sox9-depleted Xenopus tadpoles generated by injection of increasing amounts of Sox9 morpholino antisense oligonucleotides. We found that Sox9-depletion resulted in major defects in the development of vestibular structures, semicircular canals and utricle, while the ventrally located saccule was less severely affected in these embryos. Consistent with this phenotype, we observed a specific loss of the dorsal expression of Wnt3a expression in the otic vesicle of Sox9 morphants, associated with an increase in cell death and a reduction in cell proliferation in the region of the presumptive otic epithelium. We propose that, in addition to its early role in placode specification, Sox9 is also required for the maintenance of progenitors in the otic epithelium.

Figure 1. Morphological analysis of the inner ear of Sox9-depleted tadpoles at stage 52

Representative cases of the membranous labyrinth of Sox9-depleted tadpoles inner ears (Sox9MO; 2.5 ng) at stage 52 are shown after injection with latex paint. The overall architecture of the inner ear of these tadpoles has been divided into four classes; Class I: normal inner ear structure; Class II: abnormal semicircular canals and utricle; Class III: semicircular canals and utricle absent, saccule reduced in size; Class IV: no inner ear. aa, anterior ampula; asc, anterior semicircular canal; acr, anterior cristae recess; apr, amphibian papilla recess; bp, basilar papilla; ha, horizontal ampula; hsc, horizontal semicircular canal; psc, posterior semicircular canal; pcr, posterior cristae recess; s, saccule; u, utricle; um, utriclar macula. The scale bar represents 500 μm.

Figure 2. Morphological analysis of the inner ear of Sox9-depleted tadpoles at stage 48

Representative cases of the membranous labyrinth of Sox9-depleted tadpoles inner ears (Sox9MO; 2.5 ng) at stage 48 are shown after injection with latex paint. For phenotypic classes definition and abbreviations see Fig 1. The scale bar represents 500 μm.

Figure 3. Development of the inner ear sensory organs, otoconia and statoaccoustic ganglion in Sox9-depleted tadpoles at stage 45

(A) Representative cases of Bmp4 expression in the sensory organs of the inner ear of control and Sox9-depleted tadpoles (Sox9MO; 1 ng and 2.5 ng) at stage 45. The arrows indicate the position of the various sensory epithelium as they arise from anterior to posterior (left to right). AC, anterior crista; UM, utricular macula; HC, horizontal crista; SM, saccular macula; PC, posterior crista. The scale bar represents 100 μm. (B) Sensory epithelium of the saccular macula and associated hair cells and otoconia in control and Sox9-depleted embryos at stage 45. While hair cells and otoconia were missing in the inner ear of embryos injected with the higher dose of Sox9MO (2.5 ng), the statoaccoustic ganglion formed in its proper location in these embryos. The lower panels are higher magnification views of the boxed area shown in the upper panels. The scale bar represents 50 μm. MHC, hair cell; OTC, otoconia; SAG, statoaccoustic ganglion.

Figure 4. Sox9 depletion results in the loss of gene expression in the dorsal aspect of the otic vesicle

(A) The otic vesicle of embryos injected in one blastomere at the 8-cell stage with 2.5 ng of Sox9MO exhibited reduced Wnt3a and Pax2 expression associated with a dorsal expansion of Bmp4 and Sox2 at stage 35. The expression of Otx2 in the ventral aspect of the otic vesicle was also reduced but primarily along the antero-posterior axis. For whole-mount in situ hybridization, lateral views, dorsal to top. Anterior is to the right (control) or to the left (injected). Histology panels (dorsal to top) show transverse sections of Wnt3a- and Pax2-stained embryos. In the case of Bmp4-, Otx2- and Sox2-stained embryos longitudinal sections were performed. The arrows point to the otic vesicle on the injected side The scale bars represent 200 μm. (B) A three dimensional representation of the otic vesicle at stage 35 illustrates the changes in expression pattern of Wnt3a, Pax2, Bmp4, Sox2 and Otx2 in injected as compared to controls otocysts. The position of the dorso-ventral, antero-posterior and medio-lateral axis is indicated. D, dorsal; L, lateral; P, posterior.

Figure 5. Sox9 is expressed throughout the otic vesicle and in the developing semicircular canals

Developmental expression of Sox9 in the otocyst of wild type embryos by whole-mount in situ hybridization (A, B, D) and by in situ hybridization on section (C, E–I). The embryonic stage (Nieuwkoop and Faber, 1967) is indicated in the upper right corner of each panel. Sox9 is initially expressed throughout the entire otic epithelium (A–C) and then become progressively restricted to the dorsal and ventral aspects of the otic vesicle (D–H). At stage 45 (I) Sox9 is also detected in the protrusions of the developing semicircular canals (arrow). In all panels dorsal to top. For whole embryos staining anterior is to the right. In panels (A, B, D) the scale bar represents 300 μm. In panels (C, E–I) the scale bar represents 50 μm.

Figure 6. Sox9 is required for the survival of otic precursors

(A, B) Histological sections of embryos showing TUNEL staining at stage 27 (A) and stage 30 (B). The outlined areas demarcate the position of the otic vesicle, which is reduced in size on the injected side. Fluorescein-labeled TUNEL-positive cells are primarily found in tissues surrounding the otic vesicle. (C) Whole-mount TUNEL staining at stage 15. Lateral views, dorsal to top, anterior to the right (control side) or to the left (injected side). The circled areas (red) outline the approximate position of the prospective otic epithelium in which TUNEL-positive cells were counted. (D) Higher magnification views of the circled areas of the embryos injected with 2.5 ng Sox9MO shown in panel (C). In panels (A, B) the scale bars represent 50 μm. In panel (C) the scale bar represents 500 μm. (E) Graph illustrating the quantification of TUNEL-positive cells on control and injected sides in embryos that received unilateral injection of Sox9MO (1 ng, n=14; and 2.5 ng, n=13). (*) P-value is <0.003; (**) P-value is <0.001.

Figure 7. In the absence of Sox9 function otic precursors showed reduced proliferation

(A) Whole-mount phosphohistone H3 (PHH3) staining at stage 15. Lateral views, dorsal to top, anterior is to the right (control side) or to the left (injected side). The circled areas (red) outline the approximate position of the prospective otic epithelium in which PHH3-positive cells were counted. The scale bar represents 500 μm. (B) Higher magnification views of the circled areas of the embryos injected with 2.5 ng Sox9MO shown in panel (A). (C) Graph illustrating the quantification of PHH3-positive cells on control and injected sides in embryos that received unilateral injection of Sox9MO (1 ng, n=16; and 2.5 ng, n=15). (*) P-value is <0.0008; (**) P-value is <0.0003.