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First published online 23 March 2005
doi: 10.1242/dev.01796

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Role of the hindbrain in dorsoventral but not anteroposterior axial specification of the inner ear

¹ National Institute on Deafness and other Communication Disorders, 5 Research Court, Rockville, MD 20850, USA
² Division of Biology, 139-74, California Institute of Technology, Pasadena, CA 91125, USA

View larger version (64K): [in a new window]   Fig. 1. Respecification of the AP axis of the inner ear at E1.5. (A) The surgery for AP reversal. DiI crystals were spotted in the anterior region of a right otic cup before replacing a left donor otic cup. (B) Bright-field and (C) fluorescent micrographs showing DiI labeling in a transplanted otocyst 24 hours after surgery. Arrows indicate DiI labeling in the transplanted otocyst. (D,E) Similar Lfng expression patterns (arrows) in controls (D) and AP rotated otocysts (E). A, anterior; D, dorsal. Scale bar: 100 µm.

View larger version (75K): [in a new window]   Fig. 2. Maintenance of rhombomere identities after AP axial rotation of R4 to R7. The AP axis of the hindbrain, R4-R7, is reversed at E1.5. (A) An E1.5 embryo showing EphA4 expression in R3 and R5. The red rectangle indicates the region of hindbrain that is rotated. (B) EphA4-positive R5 is located next to the anterior half of the otocyst in controls. EphA4 is also expressed in R3 and otocysts. (C) EphA4 expression in R5 is relocated (R-R5) to the posterior half of the otocyst after AP rotation. (D,E) Hoxb1-positive R4 is located rostral to the otocyst in controls (D), but is relocated (R-R4) caudal to the otocyst after AP rotation (E). White and black brackets indicate otic locations. Scale bars: 200 µm in A; in B, 200 µm for B-E.

View larger version (66K): [in a new window]   Fig. 3. The AP axis of the inner ear is not affected by AP axial rotation of the hindbrain. (A-D) Lfng expression patterns in control (A,B) and R4-R7 rotated (C,D) embryos at E2.5. Arrows indicate the normal Lfng expression patterns. (E-H) NeuroD expression patterns in control (E,F) and R4-R7 rotated (G,H) embryos. Arrows indicate the normal NeuroD expression in the otic epithelium as well as delaminated neuroblasts. (I,J) Anatomy of the inner ear after rhombomere rotation (J) is similar to controls (I) at E7. AA, anterior ampulla; ASC, anterior semicircular canal; CC, common crus; CD, cochlear duct; ED/S, endolymphatic duct and sac; LA, lateral ampulla; LSC, lateral semicircular canal; PA, posterior ampulla; PSC, posterior semicircular canal. Scale bars: in A, 100 µm for A,C,E,G; in B, 100 µm for B,D,F,H; in I, 500 µm for I,J.

View larger version (84K): [in a new window]   Fig. 4. Effects of ablating ventral midline structures on inner ear development. Chicken embryos are operated in ovo at E1.5, and further incubated and harvested at E2.5 for the assessment of Shh expression using whole-mount in situ hybridization (A,D,G,J,M) or at E7 for anatomical analysis of the inner ear using the paint-fill technique (C,F,I,L,O). Representative histological sections from ablated regions are also shown (B,E,H,K,N). Whole-mount (A) and section (B) showing Shh expression in the floor plate (FP) and notochord (noto) of control embryos. (C) Normal inner ear of controls at E7. (D-F) Both the notochord and ventral neural tube, including the floor plate are ablated in the region between R4-R7. (D,E) Floor plate (arrow) and notochord (arrowheads) associated Shh expression are missing in the ablated region. (F) The lack of ventral inner ear structures in an ablated embryo (asterisk). (G-I) Only the notochord underneath R4-R7 is ablated. (G,H) Neural tube-associated (arrow) but not notochord-associated (arrowheads) Shh expression is present in the ablated region. (I) The inner ear morphology of a notochord-ablated embryo is normal. (J-L) A region of the neural tube between R4-R7 is ablated. (J,K) Neural tube-associated Shh expression is missing in the ablated region (arrow), whereas the notochord-associated Shh expression is not disrupted (arrowheads). (L) Dorsal inner ear structures (asterisk) are missing after neural tube removal. (M-O) Both the notochord and neural tube are ablated. (M,N) Shh expression is absent from the ablated region. (O) A rudimentary inner ear results when both the notochord and neural tube are removed. Black asterisks indicate the positions of otocysts. Scale bars: in A, 100 µm for A,D,G,J,M; in B, 100 µm for B,E,H,K,N; in C, 500 µm for C,F,I,L,O.

View larger version (69K): [in a new window]   Fig. 5. Effects of blocking SHH function on inner ear development. (A-D) Hybridoma cells expressing anti-SHH antibody (5E1) are pre-labeled with DiI, and implanted underneath the region of the hindbrain adjacent to the otocysts at E1.5. (A) A schematic diagram summarizing the procedure. (B) Bright-field and (C) fluorescent micrographs showing the location of implanted hybridoma cells (arrows) 24 hours after implantation. Asterisks in B,C,E,F indicate otocyst positions. (D) Ventral inner ear structures are missing in 5E1-implanted embryos (asterisk). (E-G) 5E1 cells are injected into the mesenchyme underneath the otocyst at E2.5 as indicated in E. (E) Bright-field and (F) fluorescent micrographs taken immediately after injection of 5E1 cells (arrows). (G) An inner ear injected with 5E1 at E2.5 lacks ventral structures (asterisk). Scale bars: in B, 500 µm for B,C; in E, 500 µm for E, F; in D, 500 µm for D,G.

View larger version (87K): [in a new window]   Fig. 6. Effects of DV axial rotation of the hindbrain/notochord on expression patterns of ventral otic genes. (A) DV rotation of the hindbrain and notochord between R4 and R7 at E1.5. (B,C) Shh expression pattern in whole-mount of control (B) and DV rotated hindbrain (C) embryos. (D,E) Shh-positive floor plate and notochord, which are normally located ventrally in control (D), are located dorsally in hindbrain rotated embryos (E, arrow). Red lines in B and C indicate the level of sections shown in D and E, respectively. (F-M) Expression patterns of NeuroD, Lfng, Six1 and Otx2 in control (F,H,J,L) and hindbrain rotated (G,I,K,M) embryos. NeuroD (G), Lfng (I) and Six1 (K) are downregulated in their normal expression domain ventrally and upregulated in the dorsoanterior aspect of the otocyst (arrows). (L,M) Otx2 expression is expanded dorsally (M) without the loss of its normal ventral domain (L) (brackets). An asterisk indicates the NeuroD-positive neuroblasts. Scale bars: in B, 100 µm for B,C; in D, 100 µm for D,E; in F, 100 µm for F-M.

View larger version (88K): [in a new window]   Fig. 7. Effect of DV axial rotation of the hindbrain on the expression of a dorsal otic gene, Gbx2. (A-C) Whole mounts and (D-F) sections of embryos showing expression patterns of Gbx2. Red lines in A-C indicate the level of sections shown in D-F. (A,D) Gbx2 expression in dorsomedial region of control otocyst. (B,C,E,F) Absence of Gbx2 expression in otocysts after DV rotation of the hindbrain (arrowheads). Arrows in C and F indicate Gbx2 expression in the ectopic otocyst that was translocated ventrally during neural tube rotation. D and V in D-F indicate the original dorsal and ventral axes of the neural tube, respectively. Rules in D and E indicate the relative changes in the distance between the dorsal neural tube and otocyst after neural tube rotation. Arrowheads in the insert in F indicate the basement membrane of the ectopic otic epithelial tissue. Scale bars: in A, 100 µm for A-C; in D, 100 µm for D-F.