Fig. 2. Defects in the inner and middle ear in Six1-deficient mice. (A,B) Transverse sections at the level of the pinna of the neonates. The semicircular canals and the endolymphatic sac are irregularly formed in the Six1^–/– neonates. The semicircular canals and the common crus are fused, forming a large cavity (B, arrowhead). (C-F) Wnt2b expression analysis by in situ hybridization. Expansion of the Wnt2b expression domain in the Six1^–/– embryos (E,F) compared with wild type (C,D) indicates that the enlarged region is the endolymphatic sac at E17.5 (E) and endolymphatic duct at E11.5 (F). In F, Wnt2b was expressed in the medial half of the otic vesicle, which corresponds to the enlarged endolymphatic duct as depicted in L (otic vesicle shown in F was flattened during the hybridization process). (G,H) Transverse sections at the cochlea level show complete loss of the cochlea in Six1^–/– neonates (asterisk). (I,J) Alcian blue/Alizarin red staining of neonatal skeletons revealed malformations of ossicles. (K,L) Transverse sections of wild-type (K) and Six1^–/– (L) embryos at E11.5. The cochlear region does not extend ventrally and the endolymphatic duct is dilated in Six1^–/– embryos. (M-P) Transverse sections of wild-type (M,O) and Six1^–/– (N,P) embryos at E12.5. The endolymphatic duct and canal plate are formed, but the morphology is abnormal in the Six1^–/– embryo (N). The cochlea is completely absent in Six1^–/– embryos (P, asterisks). (Q-T) Lateral views of the paint-filled inner ear of wild-type (Q) and Six1^–/– (R) E18.5 embryos and otic vesicles of wild-type (S) and Six1^–/– (T) E10.5 embryos. (R') Posterior view of the same inner ear as (R). Relative positions are aligned between wild type and Six1^–/–. The two ventrally protruding structures observed in (R and R') are the ventral ends of residual cavities of the canal plate-like structure and the endolymphatic duct. More than five Six1^–/– neonates or embryos at each stage were analyzed, and virtually the same results were obtained. co, cochlea; cp, canal plate; cpl, canal plate-like structure; d, dorsal; ed, endolymphatic duct; es, endolymphatic sac; in, incus; la, lateral; m, medial; ma, malleus; mc, Meckel's cartilage; ov, otic vesicle; sc, semicircular canals; st, stapes; tr, tympanic ring. Scale bars: 100 µm.