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First published online December 11, 2006
doi: 10.1242/10.1242/jcs.03299

Journal of Cell Science 119, 5169-5177 (2006)
Published by The Company of Biologists 2006
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Cadherin-2 participates in the morphogenesis of the zebrafish inner ear

Sherry Babb-Clendenon1,*, Yu-chi Shen2,*, Qin Liu3, Katharyn E. Turner1, M. Susan Mills1, Greg W. Cook1, Caroline A. Miller4, Vincent H. Gattone, II4, Kate F. Barald2 and James A. Marrs1,{dagger}

1 Department of Medicine, Indiana University Medical Center, 950 West Walnut Street, Indianapolis, IN 46202, USA
2 Department of Cell and Developmental Biology, University of Michigan, Ann Arbor, MI 48109-0616, USA
3 Department of Biology, University of Akron, Akron, OH 44325
4 Department of Anatomy, Indiana University Medical Center, Indianapolis, IN 46202


Figure 1
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  		Fig. 1. Loss of Cdh2 affects kinocilia and cellular microtubule networks. Panels A and B are transverse sections tilted to include both the anterior and posterior maculae in the same view of 48 hpf embryos labeled with anti-Cdh2 antibody. In control embryos the anterior (am) and posterior maculae (pm), are strongly labeled by Cdh2 (A). Cdh2 immunolabeling of sensory patches in cdh2 MO-injected embryos is absent (B). Panels C and D are image volumes acquired by two-photon microscopy of 48 hour post-fertilization whole-mount zebrafish stained using anti-ß-catenin. Projections are 6 µm optical sections of a lateral view of the regions containing the anterior and posterior maculae (am and pm) in a control (C) and in a cdh2 MO-injected embryo. Rostral is left, dorsal is up and ventral is down. Panels E, F are projections of two optical sections through the anterior maculae of 120 hpf embryos labeled with anti-acetylated-tubulin (shown in green) to visualize kinocilia and Texas-Red-conjugated phalloidin (shown in white) to visualize hair cell sterociliary bundles. Well-formed kinocilia are present in hair cell bundles in control embryos (E), but reduced in cdh2 MO-injected embryos. This is especially evident in the insets in panels E and F, showing images of acetylated-tubulin-labeled posterior maculae from 48 hpf embryos using DAB/peroxidase detection (bottom insets), and a larger rendered volume from the image stack used to make panels E and F (upper inset; this volume can be viewed as rotating, three-dimensional rendered volumes in Movies 1 and 2, see supplementary material). Ciliary bundles in control embryos each correspond to a kinocilium. In cdh2 MO-injected embryos, hair cell bundles are associated with short kinocilia, or kinocilia are absent. Abbreviations: h, hindbrain. Bars: A-D, 50 µm; E-F, 10 µm.

 

Figure 2
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  		Fig. 2. Cellular junctions are maintained in cdh2 morphant. Transmission electron micrographs of anterior macula from 5 dpf ears from control (A-C) and cdh2 morphants (D-F) embryos show little or no change in cellular morphology (see text for details). Abbreviations: hb, hair cell bundles; hc, hair cell; sc, supporting cell. Bars: A, B, D, E, 5 µm; C,F, 100 nm.

 

Figure 3
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  		Fig. 3. Loss-of-function of Cdh2 does not inhibit otic induction. Whole-mount in situ hybridizations with a cldna (A,B), dlx3b (C,D), pax2a (E,F), and fgf8 (G-J) probes, early markers of otic induction, in wild-type control embryos (A,C,E), glo mutant embryos (B,D,F), control MO-injected embryos (G,I) and cdh2 MO-injected embryos (H,J). Panels are lateral views with anterior to the left and dorsal side up. A-H are 24 hpf embryos and I-J are 48 hpf embryos. Bar, 50 µm.

 

Figure 4
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  		Fig. 4. Statoacoustic ganglia are reduced in size in cdh2 mutants and knockdowns. Statoacoustic ganglia (SAg) were labeled with zn-5 in 36 hpf control (B), glo (C) and cdh2 MO-injected embryos. Circumferences of zn-5 labeled SAgs were measured (A). The average circumference of control SAgs (226.8 µm) was greater than that of cdh2 MO-injected embryos (192.3 µm, P<0.001) and of glo mutant embryos (176.2 µm, P<0.001). Average SAg circumference was smaller in the glo null mutant than in cdh2 MO-injected knockdowns (P<0.05).

 

Figure 5
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  		Fig. 5. Surface renderings of segmented inner ear volumes facilitate assessment of differences in morphology and allow unbiased measurement of inner ear volume. Panels A-F are optical sections acquired by two-photon microscopy of 48 hour post-fertilization zebrafish stained using Texas-Red-conjugated phalloidin. Lateral is up and rostral is to the right. Sections of the most ventral region of the ear (A,B) show that the posterior macula of the cdh2 MO-injected embryo (arrow in B) contains shorter stereociliary bundles compared with those of the control (arrow in A). Sections taken from more dorsal regions of the ears (E,F) show semicircular canals in the control embryo (E) which are absent in the cdh2 MO-injected embryo (F). To show the relationship of the optical sections to the surface rendered volumes, the optical sections from panels E and F are shown cutting through surface renderings of the volume of the inner ear in control (G) and in cdh2 MO-injected embryos (H). In the bottom panels, the entire volume of the inner ears are shown in control (I) and in cdh2 MO-injected embryos (J) relative to the most ventral optical sections. Panels K and L are shown to provide orientation of the rendered volume relative to the optical sections. Shown directly below are the surface renderings of inner ear volumes from a 48 hpf control (M,O,Q,S) and cdh2 MO-injected (N,P,R,T) embryos, first in the same orientations as in K and L, then rotated. Image volumes were segmented and volume measurements were generated using Amira software (Table 2). Semicircular canals pass through the control volume (M,O,Q,S), but are entirely absent from the cdh2 MO-injected embryo volume (N,P,R,T). In M-P, lateral is up, rostral is right and the view is of the dorsal side of the volume. In Q and R, dorsal is up, caudal is right and the view is of the lateral side. In S and T, dorsal is up caudal is right and the view is of the medial side. Bar, 50 µm.

 

Figure 6
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  		Fig. 6. Projection images of the inner ear reveal morphological distinctions between control and glo mutant embryos. Whole-mount embryos were stained with Texas-Red-conjugated phalloidin and image volumes were acquired by two-photon microscopy. Image projections were made from each volume that encompassed the region of the lateral, rostral and caudal epithelial projections into the otic vesicle. Each ear was classified by extent of epithelial projection formation and fusion (B, see Materials and methods for description of classification scheme). Control embryos (n=33) all were either category 1 or 2, with fully formed projections and fusion of at least the rostral and lateral epithelial pillars. glo mutant embryos (n=38) were spread across categories 2-5, with most lacking any fusion of epithelial pillars (A). Wild-type and glo mutant morphology were statistically different (P=8.6x10-9, df=4, Chi Square=43.39). Bar, 50 µm.

 

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© The Company of Biologists Ltd 2006