Wnt-dependent regulation of inner ear morphogenesis is balanced by the opposing and supporting roles of Shh

Abstract

The inner ear is partitioned along its dorsal/ventral axis into vestibular and auditory organs, respectively. Gene expression studies suggest that this subdivision occurs within the otic vesicle, the tissue from which all inner ear structures are derived. While the specification of ventral otic fates is dependent on Shh secreted from the notochord, the nature of the signal responsible for dorsal otic development has not been described. In this study, we demonstrate that Wnt signaling is active in dorsal regions of the otic vesicle, where it functions to regulate the expression of genes (Dlx5/6 and Gbx2) necessary for vestibular morphogenesis. We further show that the source of Wnt impacting on dorsal otic development emanates from the dorsal hindbrain, and identify Wnt1 and Wnt3a as the specific ligands required for this function. The restriction of Wnt target genes to the dorsal otocyst is also influenced by Shh. Thus, a balance between Wnt and Shh signaling activities is key in distinguishing between vestibular and auditory cell types.

Figure 1.

Wnt/β-catenin activity within the developing inner ear. (a–f) Transverse sections through the otic epithelium of Topgal embryos stained with X-gal. Topgal expression is restricted to the dorsomedial region of the placode at 10 somites (10 s) (a), 12 somites (12 s) (b), and 17 somites (17 s) (c). Expression of Topgal extends throughout the dorsal otic vesicle at 21 somites (21 s) (d), 25 somites (25 s) (e), and 10.5 dpc (f). Doted lines in d mark the tips of the lateral and medial walls of the epithelia as they fuse. The black arrowhead points to the expression of Topgal soon after it expands to the lateral side of the otocyst. (g–i) Transverse sections through the otic vesicle of a 25-somite/9.5-dpc wild-type embryo stained with β-catenin antibody (red) and Dapi (green). Boxes mark the dorsal and ventral otic regions shown at higher magnification in h and i, respectively. White arrows point to representative cells showing nuclear β-catenin staining. White asterisk marks Topgal staining in the branchial arch. (ed) Ectoderm; (hb) hindbrain; (ncc) neural crest cells; (oc) otic cup; (op) otic placode; (ov) otic vesicle.

Figure 2.

Topgal and Dlx5 colocalize in the dorsal otocyst and are antagonized by Shh. Whole-mount staining for Dlx5 mRNA (a,b) and Topgal expression (c,d) at 8.5 dpc (a,c) and 9.5 dpc (b,d). Black arrowheads point to the otic epithelium. (e–r) Transverse sections through Topgal (e–g,k–n), Shh^–/–; Topgal (h–j), and ShhP1; Topgal (o–r) embryos at 10.5 dpc (e–j) and 11.5 dpc (k–r). Antibody staining for Dlx is in green (e,h,l,p), β-galactosidase in red (f,i,m,q), and merged channels in yellow (g,j,n,r). Note that Shh expression is normally absent in the wild-type otocyst (k) and ectopically expressed in the dorsal region of the ear in ShhP1 embryos (o). The graph shows a 33% increase in Dlx⁺; Topgal⁺ cells in ShhP1 embryos compared with wild type (p < 0.01, cell counts were normalized to wild type). (D) Dorsal; (L) lateral.

Figure 3.

Forced activation of the Wnt/β-catenin pathway by LiCl causes a ventral expansion of Wnt-responsive genes in the otic vesicle. (A) Transverse sections through otic explants stained for Topgal after being cultured for 24 h in the presence (panels b–f) or absence (panel a) of increasing concentrations of LiCl. Red arrowheads point to the expanded domain of Topgal in response to LiCl. (B) Gene expression analysis of otic explants cultured alone (panels a–f) or in the presence of 30 mM LiCl (panels g–l). The red brackets in panels g and h highlight the ectopic expression of Dlx5 and Gbx2, respectively. (D) Dorsal; (L) lateral.

Figure 4.

The dorsal neural tube is required for the expression of Wnt-responsive genes and the repression of Shh target genes in the dorsal otocyst. (a–c) Schematic representation of the manipulations performed on otic explants: untreated control explants (a), dorsal neural tube ablated explants (ΔdNT) (b), and dorsal neural tube ablated explants (c) cultured in the presence of 30 mM LiCl. (d–aa) Gene expression studies on otic explants cultured according to the conditions outlined in a–c. The numbers in each panel reflect the reproducibility of the expression pattern indicated. Arrowheads in y–aa point to the absence (black) or presence (red) of ectopic Gli1 expression along the lateral wall of the otocyst. (D) Dorsal; (L) lateral.

Figure 5.

Wnt1 and Wnt3a are required for the expression of Dlx5/6 and Gbx2 in the otic vesicle at 10.5 dpc. Transverse sections (a–l,q–bb) or lateral whole-mount views (m–p) of otic vesicles from wild-type (WT) (a,e,i,m,q,t,w,z), Wnt1^–/– (b,f,j,n,r,u, x,aa), Wnt3a^–/– (c,g,k,o), and Wnt1^–/–; Wnt3a^–/– (d,h,l,p,s,v,y,bb) embryos analyzed by RNA in situ hybridization (a–p,t–v) or antibody staining (q–s,w–bb) for the indicated markers. Red arrow in d highlights the loss of Dlx5 expression. The expression domain of Gbx2, marked by brackets (t,u), is lost in Wnt1^–/–; Wnt3a^–/– embryos (v). Images in s, v, y, and bb are magnified for better display of the expression patterns. The size ratio relative to wild type was unaltered for all other images. Bar, 50 μm. (D) Dorsal; (P) posterior; (L) lateral.

Figure 6.

Wnt1^–/–; Wnt3a^–/– embryos show aberrant inner ear morphology. Analysis of inner ears injected with latex paint from wild type (a), Wnt1^–/– (b), Wnt3a^–/– (c), and Wnt1^–/–; Wnt3a^–/– (d) embryos at 14.5 dpc. (aa) Anterior ampulla; (asc) anterior semicircular canal; (cc) common crus; (cd) cochlear duct; (cls) cochlear-like structure; (ed) endolymphatic duct; (la) lateral ampulla; (lsc) lateral semicircular canal; (pa) posterior ampulla; (psc) posterior semicircular canal; (s) saccule; (u) utricle.

Figure 7.

Fate mapping of Wnt-responsive cells in the inner ear. (a,b) TopCreERT2 expression in the inner ear was revealed by RNA in situ hybridization. Red arrow points to the ventral hinge point of the otic cup. (c–g) X-gal staining of inner ears from TopCreERT2; R26R/+ embryos administered with tamoxifen at 8.75 dpc. Cells that have responded to Wnt signaling are located along most of the medial and dorsal regions of the vesicle at 9.5 dpc (c) and 11.0 dpc (d). (e) Whole-mount view of the inner ear at 14.5 dpc. (f,g) Transverse sections through the ear shown in e revealing the contribution of Wnt-responsive cells to the vestibule (f) and medial wall of the cochlea (g). Red bracket marks the unstained lateral wall of the cochlea. (asc) Anterior semicircular canal; (cc) common crus; (cd) cochlear duct; (ed) endolymphatic duct; (lsc) lateral semicircular canal; (psc) posterior semicircular canal.

Figure 8.

Model depicting how inner ear polarity is generated in response to extracellular signals. Wnt signals secreted by the dorsal neural tube (dark green) are necessary and sufficient for the regulation of Dlx5/6 in the dorsal otocyst (light green). This contrasts with the function of Shh emanating from the notochord (blue), which specifies ventral otic cell fates by regulating the transcription of genes, including Pax2 and Ngn1 (light blue). The combination of Wnt and Shh signaling activities are required for the maintenance of Gbx2 along the dorsomedial side of the otocyst (green-blue shading). Shh also restricts Wnt pathway activation, and consequently Dlx5/6 expression, to dorsal regions of the otic vesicle. An additional signal secreted from the dorsal neural tube, possibly a Bmp family member (red), restricts Shh target genes to the ventromedial domain of the otocyst. A balance between these dorsal and ventral signals is key to promoting regional identity within the otic epithelium with the ultimate goal of coordinating the morphogenesis of vestibular and auditory organs.