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First published online 13 April 2005
doi: 10.1242/dev.01804

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Gbx2 is required for the morphogenesis of the mouse inner ear: a downstream candidate of hindbrain signaling

Laboratory of Molecular Biology, National Institutes on Deafness and Other Communication Disorders, National Institutes of Health, Bethesda, MD 20850, USA

View larger version (80K): [in a new window]   Fig. 1. Expression of Gbx2 in the developing mouse inner ear. (A) Dorsal view of a mouse embryo at 9.5 dpc probed for Gbx2 transcripts. Arrows indicate Gbx2 expression in the otocyst. Arrowheads indicate Gbx2 expression in the mid-hindbrain junction. (B) An enlarged lateral view of the right otocyst in A, showing Gbx2 hybridization signals in the dorsomedial half of the otocyst. (C) Gbx2 expression in the endolymphatic duct (ed) at 10.5 dpc. (D-G) Pairs of 12 µm adjacent sections probed for Gbx2 (D,F) and Bmp4 (E) or Lfng (G) transcripts. Arrows in E indicate Bmp4 expression in the presumptive cristae. Asterisks indicate Gbx2 expression in the neural tube. Approximate levels of sections for C-G are shown in the schematic diagram. Orientation in D applies to D-G; A, anterior; D, dorsal; L, lateral. Scale bar in G applies to D-G.

View larger version (52K): [in a new window]   Fig. 2. Paint-filled inner ears of Gbx2 mutants. Lateral views of right inner ears from heterozygous and homozygous Gbx2 embryos at 15.5 (A) and 11.5 dpc (B-E). (A) The control inner ear is shown on the left followed by four representative phenotypes, shown with increasing severity from Type I to IV. Type I: an enlarged membranous labyrinth lacking the endolymphatic duct (ed). Type II: absence of both the endolymphatic duct and common crus (cc, asterisk). The utricle (u) and saccule (s) are not easily discernible, and the cochlear duct is shortened. Type III: the inner ear is missing the anterior and posterior canals, in addition to phenotypes described for Type II. Type IV: a cystic inner ear with only the lateral canal and ampulla. (B) A normal inner ear at 11.5 dpc. (C-E) Gbx2 mutant ears with a normal (C), smaller (D) or non-existent (E) vertical canal pouch. asc, anterior semicircular canal; cc, common crus; cd, cochlear duct; ed, endolymphatic duct; es, endolymphatic sac; hp, horizontal canal pouch; lsc, lateral semicircular canal; psc, posterior semicircular canal; s, saccule; u, utricle; vp, vertical canal pouch. Scale bar in E applies to B-E.

View larger version (124K): [in a new window]   Fig. 3. Gene expression analyses of Gbx2 mutants. Whole mounts of heterozygous (A) and homozygous (B) Gbx2 embryos probed for Wnt2b transcripts at 10 dpc. Arrows indicate the presence or absence of Wnt2b expression in the otocyst. An arrowhead indicates Wnt2b expression in the eye. Inserts in A and B show +/– (A) and –/– (B) otocysts at 9.5 dpc that have undergone prolonged color development. (C-F) Cryosections probed for Dlx5 (C,D) and Hmx3 (E,F) transcripts at 9.5 dpc. Pairs of 12 µm adjacent sections from +/– (C,E) and –/– (D,F) Gbx2 inner ear. (C) Dlx5 is expressed in the entire dorsal region of a +/– otocyst, whereas Hmx3 is expressed only in the lateral region (E, arrows). (D,F) In a –/– otocyst, the medial expression domain of Dlx5 is absent, whereas both Dlx5 and Hmx3 are expressed in the lateral regions. Arrows in D-F indicate the borders of expression domains. Scale bars: in B, 100 µm for A,B; in D, 50 µm for C-F.

View larger version (92K): [in a new window]   Fig. 4. Ganglion formation in Gbx2-/- inner ears. Heterozygous (A,C,E,G) and homozygous (B,D,F,H) Gbx2 inner ears probed for Nf68 (A,B) Gata3 (C,D) and Neurod1 (E,F) transcripts, and apoptotic cells (G,H) at (A-D) 15.5, (E,F) 10.5 and (G,H) 9.5 dpc. (G,H) TUNEL analyses show an increased in apoptotic cells in the cochleo-vestibular ganglion (G,H, red arrowheads) of a –/– otocyst (H) compared with a +/– otocyst (G, black arrowheads). Arrows indicate a region of cell death within the otic epithelium of +/– and –/– inner ears. cvg, cochleo-vestibular ganglion; gg, geniculate ganglion; sg, spiral ganglion; vg, vestibular ganglion. Schematics on the right indicate the levels of sections. Scale bars: in D, 200 µm for A-D; in F, 100 µm for E,F; in H, 100 µm for G,H.

View larger version (85K): [in a new window]   Fig. 5. Sensory organ formation in Gbx2-/- inner ears. Sections of heterozygous (A,C,D,G,H) and homozygous (B,E,F,I,J) Gbx2 inner ears probed for Lfng or Myo15a transcripts at 10.5 (A,B) and 15.5 (C-J) dpc. (C-J) Pairs of 12 µm adjacent sections. Red lines indicate comparable Lfng expression domains between +/– and –/– ears. Lfng (E) and Myo15a (F) are expressed in the lateral crista (lc) and macula utriculi (mu) of a Type IV Gbx2-/- inner ear. Arrowheads in H indicate the sensory hair cells. Only one or two Lfng- (I) and Myo15a (J)-positive sensory patches are present in a Type II Gbx2-/- cochlear duct. Insert in J shows a higher power view of the Myo15a positive sensory region. Arrowheads indicate the region that is positive for both Lfng and Myo15a. Scale bars: in A, 100 µm for A,B; in C, 100 µm for C-F; in G, 100 µm for G-J.

View larger version (115K): [in a new window]   Fig. 6. Aberrant Otx2 expression in Gbx2 mutants. Otx2 (A,C,E,G) and Lfng (B,D,F,H) expression patterns in heterozygous (A-D) and homozygous (E-H) Gbx2 inner ears. (A,B and E,F) Comparable pairs of 12 µm adjacent sections dorsal to the pairs shown in C,D and G,H respectively. Otx2 and Lfng expression domains are complementary in heterozygous, but overlap with each other in homozygous Gbx2 otocysts (brackets). (I) The Otx2 expression domain expands medially in the growing cochlear duct of Gbx2-/- ears at 12 dpc (double arrows). (J-L) Comparable cochlear sections at 15.5 dpc. (J) Otx2 expression in the Reissner's membrane of a +/– cochlear duct (bracket). (K,L) Adjacent cochlear sections of Gbx2-/- mutants probed for Otx2 (K) and Lfng (L) transcripts. A bracket in K marks the normal Otx2 expression domain and arrows indicate ectopic Otx2 expression in the medial region. Arrows in L indicate a Lfng-positive sensory region in the cochlea, and arrowheads indicate the sensory region in the posterior crista. (M,N) Adjacent +/– inner ear sections at 10.5 dpc probed for Gbx2 (M) and Otx2 (N) transcripts. Arrows indicate the borders of expression domain for each gene, and double arrows indicate the lateral restriction of Otx2 expression domain. Schematics on the right indicate the levels of sections. Scale bars: in B, 100 µm for A-H; in L, 100 µm for L,K; in N, 50 µm for M,N.

View larger version (105K): [in a new window]   Fig. 7. Analysis of hindbrain markers in Gbx2-/- inner ears. Dorsal views of +/– (A,C,E) and –/– (B,D,F) Gbx2 embryos probed for Krox20 (A,B), Epha4 (C,D) and Hoxb1 (E,F) RNA transcripts at 9 dpc. In Gbx2-/- embryos, Krox20 (B) and Epha4 (D) expression domains are reduced in r5, and the anterior border of Hoxb1 expression domain (F) is aberrant. Arrows indicate the borders of otocysts. Scale bar in A: 100 µm for A-F.

View larger version (84K): [in a new window]   Fig. 8. Expression patterns of Fgf3 and kreisler/Mafb in Gbx2-/- embryos. Lateral (A,B) and dorsal (C,D) views of the kreisler/Mafb expression pattern in the hindbrain of Gbx2+/– (A,C) and –/– (B,D) embryos. (A,B) No obvious difference in kreisler/Mafb expression pattern is observed between +/– and –/– embryos before 9.0 dpc. (C,D) By 9.5 dpc, kreisler/Mafb is expressed weakly in r6 of +/– (C, arrowhead) and much more strongly in –/– (D) embryos. (E,F) Dorsal and (G,H) lateral views of the Fgf3 expression patterns in the hindbrains of heterozygous (E,G) and homozygous (F,H) Gbx2 mutants. (E,F) At 9.0 dpc, the Fgf3 expression domain extends beyond r5 and r6 into r4 and possibly includes the undefined region rostral to r4 in Gbx2-/- embryos. (G,H) By 9.5 dpc, Fgf3 expression is no longer detectable in the hindbrain (G) but is strong in r4 and rX (asterisk) and weaker in r6 (arrowhead) of Gbx2-/- embryos (H). The Fgf3 expression patterns in both otocysts (G,H) are comparable in the anteroventral lateral region. Arrows in C-H mark the anterior and posterior margin of the otocyst. sm, somite pairs. Scale bars: in A, 100 µm for A,B; in C, 100 µm for C,D; in E, 100 µm for E-H.

View larger version (38K): [in a new window]   Fig. 9. A summary of (A) gene expression changes in Gbx2 mutants and (B) proposed interactions of Gbx2 with other genes known to affect inner ear development. (A) Anterior views of a schematic Gbx2+/– and Gbx2-/- right otocyst. Gbx2 is expressed in the dorsomedial domain (blue); Lfng is expressed in the anteroventral lateral domain that expands medially (orange); and Otx2 is expressed in the ventral posterolateral domain (yellow) that is complementary to the Lfng domain and non-abutting to the Gbx2 domain. In a homozygous mutant, there is a loss of Wnt2b expression (purple) and Dlx5 expression dorsomedially (not shown), as well as an expansion of Otx2 expression domain ventromedially (yellow color). (B) Possible relationships of Gbx2 with other genes in the hindbrain and inner ear. Arrows do not imply direct interactions.