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First published online 27 July 2004
doi: 10.1242/dev.01292

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The development of semicircular canals in the inner ear: role of FGFs in sensory cristae

Weise Chang¹, John V. Brigande^2,*, Donna M. Fekete² and Doris K. Wu^1,

¹ National Institute on Deafness and Other Communication Disorders, Rockville, MD 20850, USA
² Department of Biological Sciences, Purdue University, West Lafayette, IN 47907, USA

View larger version (15K): [in a new window]   Fig. 1. A schematic diagram of the chicken inner ear from E3 to E7. Dotted lines represent the plane of a cross section through the vertical canals, shown above each stage. asc, anterior semicircular canal; cc, common crus; cd, cochlear duct; ed, endolymphatic duct; fp, fusion plate; psc, posterior semicircular canal; rd, resorption domain; vp, vertical canal pouch; A, anterior; D, dorsal.

View larger version (70K): [in a new window]   Fig. 2. Fate mapping the canal pouch. (A) Otic epithelia were labeled with DiI at the positions shown. Examples are shown at the time of injection on E4 (B,E,H) and again at E7 (C,D,F,G,I,J,L,M). Partially dissected E7 ears were imaged under fluorescence (C,F,I,L), combined fluorescence and bright field microscopy (D,G,J) or as a paint-fill for orientation (M). (K) Summary of fate mapping data. The number of specimens with DiI-labeled cells in the canals and common crus at E7 are shown in red and green, respectively. The common crus (long arrows), anterior canal (double arrows) and anterior ampulla (short arrows) are indicated. A region between the anterior ampulla and canal (flanked by arrowheads) is consistently unlabeled. See text for further details. Orientations in B applies to A, E and H. Orientations in D applies to C,F,G,I,J and L. M, medial; L, lateral; AA, anterior ampulla. Scale bars: in A, 200 µm; in B, 200 µm for E,H; in D, 200 µm for C,F,G,I,J,L.

View larger version (96K): [in a new window]   Fig. 3. Expression patterns of Bmp2 and Bmp4 in E3.5 canal pouches. (A) Arrows indicate the Bmp2-negative domain in the dorsal region of the canal pouch that will develop into the common crus. The ventral margins of the two Bmp2 expression domains (arrowheads in A) abut the Bmp4 sensory domains shown for another specimen in B. Arrowheads in B indicate Bmp4 expression in the mesenchyme. (C) Schematic showing the Bmp2 and Bmp4 expression domains superimposed. (D) Horizontal section through the lateral canal at E9. The Bmp2 hybridization signal is strong in the thick, outer rim, but weak in the thin, inner rim of the semicircular canals (arrowheads) and absent in the common crus (arrow). Orientations in C apply to A and B. Abbreviations are the same as for Fig. 1. AC, anterior crista; LC, lateral crista; lsc, lateral semicircular canal; PC, posterior crista. Scale bar in D: 100 µm.

View larger version (85K): [in a new window]   Fig. 5. Ectopic FGF treatments induce Bmp2 and Bmp7 expression in the canal pouch. Expression of each gene is indicated on the left of each panel; treatment group is indicated on the right. Expression is shown 24 hours after FGF2 bead implantations at E3 (B,D) and E4 (F,H,N), 3 days after RCAS-Fgf3 infection at E4 (J), or 24 hours after FGF10-heparin bead implantations at E3 (L). (A-D,K,L) Double arrows indicate differences in Bmp gene expression in the dorsal canal pouch (presumptive common crus domain) between treated and control specimens viewed in wholemounts. (E-H) The approximate level of each section is indicated in either A or B. (E-J) Arrows indicate Bmp2 expression normally evident in the rim of the prospective canal pouch; arrowheads indicate differences between experiments and controls in Bmp2 expression and epithelial thickness in the central part of the canal pouch. (M,N) Soho1 expression persists in the central region of the canal pouch (arrowhead) in both control and treated ears. Orientations in A applies to B-D,K,L; E applies to E-J,M,N. White arrows in B and L indicate the implanted beads. Scale bars: in E, 100 µm for E,F; in G, 100 µm for G,H; in J, 50 µm for I,J; in N, 50 µm for M,N.

View larger version (119K): [in a new window]   Fig. 4. Ectopic FGF treatments. Paint-filled inner ears were harvested on E7 (A,B) or E9 (C-J). Embryos were injected with viruses (A-F) or implanted with beads (G-J), as indicated in each panel. Viruses were delivered to the otocyst lumen on E2.5-3, except for the ear shown in E, which received injections into the periotic mesenchyme on E4. Beads were implanted on E4 (G,J) or E5 (H,I). White arrows denote defective regions and arrowheads are described in detail in the text. Black arrowheads in D and F indicate normal locations of the canal pouches. For illustration of the common crus phenotype, the endolymphatic duct was removed from the inner ears shown in E, H and I. Abbreviations are as in Fig. 1. AA, anterior ampulla; LA, lateral ampulla; PA, posterior ampulla. Orientations in C apply to all panels. Scale bars: in B, 100 µm for A,B; in G, 200 µm for C-J.

View larger version (125K): [in a new window]   Fig. 6. Noggin rescues the loss of common crus induced by FGFs. Paint-filled inner ears at E9 from (A) controls or (B-D) embryos implanted at E5 with beads as indicated. Arrows point to the region of the common crus. Arrowheads indicate truncation of canals. Asterisk denotes an ectopic epithelial stump. The endolymphatic ducts in C and D were removed prior to photography. Orientations in D apply to A-C. Scale bar in C: 200 µm for A,B,D.

View larger version (88K): [in a new window]   Fig. 7. Fate mapping of the FGF2-treated canal pouch. Otocysts were treated with either BSA (A-C) or FGF2 (D-F) at E4, and DiI was delivered to the 12 o'clock position of the canal pouch at E5; inner ears were harvested at E9. (G-I) FGF2 bead implantation and DiI injection were carried out at the same time, at E5. A, D and G show the location of DiI injection. The same three specimens shown in A, D and G are shown on E9 as fluorescent (B,E,H) or combined fluorescent and bright field images (C,F,I). DiI-labeled cells are associated with the common crus only in BSA-treated specimens (B,C; arrows), whereas some DiI-labeled cells in FGF2-treated specimens are associated with the canals (E,F,H,I; double arrows). Arrowheads indicate cells outside the membranous labyrinth and green arrows indicate the junction of the anterior and the posterior canals. A, anterior; D, dorsal; L, lateral; P, posterior. Scale bar in A: 200 µm for A-I.

View larger version (73K): [in a new window]   Fig. 8. Effects of FGF and SU5402 treatments prior to canal pouch formation. (A-F) Bmp2 expression in FGF-(B,D) or SU5402-treated (F) otocysts. A, C and E show left untreated ears of the same embryos that are shown in B, D and F, respectively. Otocysts implanted with an FGF2-soaked bead at E2.5 (B), infected with RCAS-Fgf3 at E2 (D), or implanted with SU5402-soaked beads at E3 (F) were analyzed 24 hours later. At E3-E3.5, Bmp2 expression in control inner ears is barely detectable (arrows, A,C), but it is upregulated with FGF2 and RCAS-Fgf3 treatments (white arrowhead in B and D). By E4, the two wedge-shaped Bmp2 expression domains are evident in the control ear (E), and this expression is downregulated in the SU5402-treated ear (F). The reduction of Bmp2 expression is more pronounced in the posterior domain (arrowhead) than the anterior domain (arrow). (G-J) Paint-filled inner ears treated with medium (G,I) and high (H,J) doses of SU5402 at E2.5 and harvested at E6 (G,H) or E9 (I,J). (G,I) A medium dose of SU5402 causes the loss of the posterior canal pouch at E6 (asterisk, G) and prevents formation of the posterior canal and ampulla at E9 (asterisk, I). (H,J) A high dose of SU5402 causes a severely deformed canal pouch at E6 (arrows, H) and absence of all three canals and ampullae, but the common crus is intact (arrows, J). Arrowhead in J indicates the presence of the anterior ampulla. Orientations in A apply to C and E; orientations in B apply to D and F; orientations in G apply to H-J.

View larger version (29K): [in a new window]   Fig. 9. Model demonstrating how FGFs originating from sensory primordia regulate semicircular canal and common crus formation in adjacent epithelium. (A) Progression of canal pouch development from E3.5 to E5.5. FGF3 and FGF10 emanating from sensory regions (black ovals) promote canal outgrowth by inducing a canal genesis zone (blue stars), possibly by activating Bmp2 expression (orange) and, potentially, other factors (X, Y and Z). Either through physical distance from the sources of FGFs or through other uncharacterized mechanisms, a FGF-negative, prospective common crus domain is established (light blue). This prospective common crus domain is Bmp2 and Bmp7 negative in the dorsal region. Two resorption domains (light gold ovals surrounded by gold dashes) are established on both sides of the common crus region in which the epithelia eventually disappear leaving behind two canals and the common crus (B). (C) A transient, exogenous dose of FGFs applied before E5 expands the BMP territory and prevents the normal resorption process. As the level of exogenous FGF diminishes over time, the resorption process resumes, and includes the rest of the epithelia in the common crus domain that failed to be properly specified. (D) Sustained FGF overexpression by RCAS blocks the resorption process so that the pouches remain open and a canal-rim fate is adopted.