Park BY and Saint-Jeannet JP (2010), Long-term consequences of Sox9 depletion on inn...

XB-ART-41226

Dev Dyn 2010 Apr 01;2394:1102-12. doi: 10.1002/dvdy.22259.

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Long-term consequences of Sox9 depletion on inner ear development.

Park BY , Saint-Jeannet JP .

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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.

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Species referenced: Xenopus
Genes referenced: bmp4 lrp1 myh6 otc otx2 pax2 pc.1 sag sox2 sox9 sts wnt3a
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	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. 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. Scale bars = 100 mu m in A, 50 mu m in B. 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 eight-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 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 dorsoventral, anteroposterior, and mediolateral axis is indicated. D, dorsal; L, lateral; P, posterior. Scale bar = 200 mu m in A.
	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. A-H: 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). I: At stage 45, 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. Scale bars = 300 mu m in A,B,D; 50 mu m in C,E.I.
	FigureÂ 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. Scale bar = 500 Î¼m.
	FigureÂ 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 Figure 1. Scale bar = 500 Î¼m.

References [+] :

Bagheri-Fam, Long-range upstream and downstream enhancers control distinct subsets of the complex spatiotemporal Sox9 expression pattern. 2006, Pubmed