Morphological analysis of monolith. (A) A 1-day (day of development) +/+ embryo showing two otoliths (o) in the inner ear. (B) A 1-day mnl/mnl embryo with only one otolith in the inner ear. (C) Otoliths from similarly sized +/+ and mnl/mnl adults. Ventral surfaces that contact the sensory epithelia are shown. (D) Utriculus of a 4-day +/+ embryo with a normal otolith (o). (E) Utriculus of a 4-day mnl/mnl embryo with an abnormally small otolith (o). (F) Utriculus of a 4-day mnl/mnl embryo with no otolith. Hair cell cilia (hcc) are evident in the sensory epithelia of each utriculus. In A, B, and D–F, anterior is toward the left and dorsal is toward the top. The scale bar corresponds to 120 μm for A and B, 570 μm for C, and 15 μm for D–F.

Molecular analysis of monolith. (A–D) Whole mount in situ hybridizations of 24 hr +/+ (A), 24 hr mnl/mnl (B), 48 hr +/+ (C), and 48 hr mnl/mnl embryos (D) with riboprobe specific for dlx3. (E, F) Whole mount in situ hybridizations of 24 hr +/+ (E) and 24 hr mnl/mnl embryos (F) with riboprobe specific for msxC. (G, H) Whole mount in situ hybridizations of 24 hr +/+ (G) and 24 h mnl/mnl embryos (H) with riboprobe specific for msxD. All panels show dorsal views except C and D, which show lateral views. Anterior is toward the left in all panels. Abbreviations: a, pharyngeal arches; f, fin buds; n, nasal pits; o, otic vesicles. The scale bar corresponds to 100 μm for A, B, and E–H and 150 μm for C and D.

Chimeric analysis of monolith. One hundred ninety-eight +/+ → mnl/mnl chimeras were screened at 24 hr for otolith morphology (Table 1) and further analyzed by fluorescence imaging at 48 hr. (A) A normal ear with numerous labeled cells throughout the inner ear. The locations of cells that potentially facilitated phenotypic rescue could not be inferred from such widespread colonization patterns. (B) A normal ear in which a single labeled cell (or small group of cells) is visible in the anteroventral quadrant of the ear (arrow). (C) An abnormal ear with several labeled cells in the posterior half of the ear. The label visible near the anterodorsal border of the ear marks a neuron in the hindbrain. (D) Frequency and distribution of labeled cells in chimeric ears. The percentage of ears with labeled cells in the indicated quadrants is shown. After excluding ears with ubiquitous labeling, such as that shown in A, positions of labeled cells (dark spots) were projected onto maps representing 89 normal and 79 abnormal ears, respectively. The scale bar corresponds to 100 μm for A– C and 40 μm for D.

Sectional analysis of monolith. Sections of utricular sensory epithelia in histochemically stained 4-day chimeras. A number of chimeras initially analyzed by fluorescence imaging (Fig. 3) were fixed at 4 days, histochemically stained to visualize labeled +/+ cells, and sectioned. A total of 48 sectioned ears were analyzed. (A) Location of the utricular sensory epithelium within the ear. (B) The utricular sensory epithelium of a normal ear in which one hair cell and one support are labeled. The posterior edge of the utricular otolith is indicated. Of 21 normal ears that were sectioned, 15 contained labeled cells in the anteroventral epithelium; all 15 contained both labeled hair cells and labeled support cells in this region. For these ears, the average number (mean ± SD) of labeled cells visible in this region was 1.6 ± 0.7 hair cells, 1.3 ± 0.4 support cells, and 3.0 ± 1.1 total cells. (C) The utricular sensory epithelium of an abnormal ear in which one hair cell is labeled. A total of 8 abnormal ears with anteroventral fluorescence were sectioned; all contained histochemically stained hair cells in this region, but none contained labeled support cells. For these ears, the average number of labeled cells visible in this region was 2.0 ± 0.9 hair cells, 0 ± 0 support cells, and 2.2 ± 1.3 total cells. Nineteen abnormal ears in which no fluorescently labeled +/+ cells were detected in the anteroventral quadrant at 48 hr were also sectioned. Consistent with the early analysis, no histochemically stained +/+ cells were evident in any of these sections (not shown). Abbreviations: hc, hair cell; o, otolith; sc, support cell. In all panels, anterior is toward the left and dorsal is toward the top. The scale bar corresponds to 10 μm for B and C.

Acknowledgments
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Reprinted from Developmental Biology, 179(2), Riley, B.B. and Grunwald, D.J., A mutation in zebrafish affecting a localized cellular function required for normal ear development, 427-435, Copyright (1996) with permission from Elsevier. Full text @ Dev. Biol.