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First published online 24 December 2003
doi: 10.1242/dev.00943

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Six1 controls patterning of the mouse otic vesicle

¹ Division of Biology, Center for Molecular Medicine, Jichi Medical School, Tochigi 329-0498, Japan
² Department of Molecular Neurobiology, Institute of Development, Aging and Cancer, Tohoku University, Sendai 980-8575, Japan
³ Division of Transgenic Technology, Center for Animal Resources and Development, Kumamoto University, Kumamoto 860-0811, Japan
⁴ Department of Otolaryngology, Tokyo Medical and Dental University, Tokyo 113-8519, Japan
⁵ Division of Nephrology, Department of Internal Medicine, Jichi Medical School, Tochigi 329-0498, Japan
⁶ Division of Cell Biology, Center for Experimental Medicine, Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan

View larger version (49K): [in a new window]   Fig. 1. Generation of Six1-deficient mice. (A) Targeting strategy of Six1. The Six1 gene consists of two exons (indicated by boxes), and the coding regions are marked in black. The entire coding regions were replaced with the EGFP gene (gfp) and the hygromycin-B-phosphotransferase gene (hph). Open arrowheads indicate the positions of PCR primers for genotyping. (B) Southern blot analyses of wild-type (+/+), heterozygous (+/–), and homozygous (–/–) mutant neonates. Tail DNA was digested with NcoI and hybridized to 5' probe (upper panel) and 3' probe (lower panel). The size of each band is indicated on the left side. (C) In situ hybridization to Six1 in E10.5 wild-type and homozygous embryos. Absence of Six1 mRNA was confirmed in the Six1-deficient embryo. dt, diphtheria toxin A gene; N, NcoI site. Scale bar: 1 mm.

View larger version (112K): [in a new window]   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.

View larger version (60K): [in a new window]   Fig. 4. Six1 expression pattern during inner ear development detected by in situ hybridization in the wild type (A-F) and by GFP luminescence in the heterozygotes (G-I) viewed laterally (A) and in transverse sections (B-I). (A) At E8.5, Six1 is weakly expressed in the otic placode (arrowhead) and the surrounding surface ectoderm. (B) At E9.5, Six1 is expressed in the invaginating otic pit and (C) in the whole region of the otic vesicle except the dorsalmost region. (D,G) At E10.5, Six1 is expressed in the ventral half of the otic vesicle. (E) At E11.5 and (F,H) E12.5, Six1 is expressed exclusively in the cochlea. (I) Expression of Six1 in the cochlea is maintained at E14.5 embryos. (J) A bright field image of the section in (I) stained with hematoxylin and eosin. More than three embryos at each stage were analyzed and virtually the same results obtained. d, dorsal; la, lateral. Scale bars: 100 µm.

View larger version (91K): [in a new window]   Fig. 3. Defects in the formation of the nose, thymus, kidney and skeletal muscles. Histological analyses of the wild-type (A,C,E,G) and the Six1–/– (B,D,F,H) neonates. (A,B) Transverse sections of the nasal region. Nasal cavities form complex, branched structures with nasal epithelia in the wild type (A), and a pair of simple round cavities with no nasal epithelia is seen in the Six1–/– neonates (B). (C,D) The thymus is prominent anterior to the heart in the wild type (C) but completely absent in the Six1–/– neonate (D, asterisk). (E,F) Kidney defects in Six1–/– mice. Note the bilateral renal aplasia (F, asterisks). (G,H) Abdominal transverse sections. Note severe reduction of skeletal muscle mass in Six1–/– neonates (H). More than five pairs of wild-type and Six1–/– neonates were analyzed and virtually the same results obtained except for the kidney (see text). ad, adrenal gland; kd, kidney; nc, nasal cavity; sc, spinal cord; th, thymus; ur, ureter. Scale bars: 1 mm.

View larger version (78K): [in a new window]   Fig. 5. Six1 specifies the expression domains of differentially expressed genes in the otic vesicle. Transverse section or whole-mount view of the otic vesicle of in situ hybridized wild type (A,C,E,G,I,K,M,O,Q,S,U,W) and Six1–/– embryos (B,D,F,H,J,L,N,P,R,T,V,X) of E10.5 (A-J,M-X) and E9.5 (K,L). (A,B) No Otx1 expression in the Six1–/– otic vesicle except for ectopic faint expression at the dorsal end. (C,D) Absence of Otx2 transcripts in the Six1–/– otic vesicle. (E,F) Lfng is expressed in the rostroventral region of the otic vesicle in the wild-type embryo but not in the Six1–/– embryo. (G,H) Fgf3 expression in the rostroventral region of the otic vesicle in the wild type is lost in the Six1–/– embryo. (I,J) Bmp4 expression in the wild-type otic vesicle (arrowheads) is lost in the Six1–/– otic vesicle. Staining in the ectoderm over the dorsal region of the otic vesicle (asterisk) has also disappeared. (K,L) Dlx5 is expressed dorsally in the wild type but in the whole region of the otic vesicle in the Six1–/– embryo. (M,N) Hmx3 expression domain is located only in the dorsolateral region in the wild type but is expanded ventrally in the Six1–/– embryo. (O,P) Dach1 expression is restricted to the dorsalmost region in the wild type, but the expression domain extends ventrally in the Six1–/– otic vesicle. Signals in the neighboring mesenchyme are also observed in the lower right side of the otic vesicle. (Q,R) Dach2 is expressed at the dorsal end of the otic vesicle in the wild type, but the expression domain of Dach2 is expanded ventrally along the lateral side in Six1–/–. (S,T) Pax2 is expressed in medial and ventral sides of the otic vesicle of both wild-type and Six1–/– embryos. (U,V) Eya1 is expressed in the ventral side of the wild-type and the Six1–/– otic vesicle. (W,X) Six4 expression in the ventral side of the otic vesicle is maintained in the Six1–/– embryo. More than three pairs of wild-type and Six1–/– embryos were analyzed and virtually the same results obtained. A-D and M-X: top, dorsal side (d); right, lateral side (la). E-L: top, dorsal side (d); right, anterior side (a).

View larger version (108K): [in a new window]   Fig. 6. Apoptosis and cell proliferation in the otic vesicle. (A-C) TUNEL method was used to examine apoptotic cell death in the otic vesicle of wild-type and Six1–/– embryos at E10.5 and E11.5. The results of TUNEL analysis for E11.5 otic vesicles of the wild type (A) and Six1–/– (B) are shown. For quantitative analysis, four to five pairs of wild-type and Six1–/– embryos were examined. For each embryo, apoptotic cell number was measured on three transverse sections passing through the central region of the otic vesicle and converted into the apoptotic cell number per 0.01 mm2. Their mean value for the three sections was adopted as the datum point for the otic vesicle of each embryo. The mean value of four or five embryos is shown with the standard deviation (C). Enhanced apoptosis was seen in the ventral and medial regions of the Six1–/– otic vesicle in comparison with the wild type in these stages. Statistical analysis was performed by Student's t-test. (D-F) Cell proliferation in the wild-type and the Six1–/– otic vesicle was assessed by BrdU incorporation. The results of immunohistochemistry for BrdU at E11.5 otic vesicles of wild-type (D) and Six1–/– (E) are shown. Quantitative analysis for BrdU-incorporated cell number was performed as described in TUNEL analysis (F). BrdU incorporation was considerably reduced in the Six1–/– ventral otic vesicle at E11.5. d, dorsal; la, lateral. Scale bars: 100 µm. *P<0.05; **P<0.005; ***P<0.01.

View larger version (57K): [in a new window]   Fig. 7. Expression patterns of Shh, Gli1, Ptch and Six1. (A-F) Expressions of Shh (A,B), Gli1 (C,D) and Ptch (E,F) in wild-type (A,C,E) and Six1–/– (B,D,F) embryos at E10.5. (G,H) Expression of Six1 in wild-type (G) and Shh–/– (H) otic vesicles at E10.5. Three pairs of wild-type and Six1–/– embryos and three pairs of wild-type and Shh–/– embryos were analyzed and virtually the same results obtained. d, dorsal; la, lateral. fp, floor plate; nc, notochord. Scale bars: 100 µm.

View larger version (38K): [in a new window]   Fig. 8. (A) Schematic representation of expression of genes in otic vesicles of the wild-type (left) and Six1–/– mice (right) at E10.5. In the otic vesicle of the wild-type, otic genes are expressed in the specified regions represented in different colors. In the wild-type otic vesicle, Six1 is expressed in the ventral half of the otic vesicle. In the otic vesicle of Six1–/–, the expression domains of Dlx5, Hmx3, Dach1 and Dach2 are expanded ventrally, and the expressions of the ventral marker genes (Otx1, Otx2, Fgf3 and Lfng) are lacking due to the absence of Six1. (B) Regulation of gene expression by Six1 in the ventral otic vesicle. Six1 activates the expression of ventral marker genes, Otx1, Otx2, Lfng and Fgf3, but represses dorsal marker genes, Dlx5, Hmx3, Dach1 and Dach2, and contributes to the patterning of the otic vesicle. D, dorsal; L, lateral; M, medial; V, ventral.