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First published online 2 March 2005
doi: 10.1242/dev.01730

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Clonal analysis of the relationships between mechanosensory cells and the neurons that innervate them in the chicken ear

Department of Biological Sciences, Purdue University, West Lafayette, IN 47907, USA

View larger version (62K): [in a new window]   Fig. 1. Distribution of AP+ cells in all 278 ears (A), all sequenced picks from 29 ears (B) and representative infected ears (C-F). (A,B) Each row indicates an individual ear, sorted (A) by stage of virus delivery, shown along the left edge, and (B) by clonal relationships of interest. Vertical columns depict the parts of the ear. Within each row, gray regions are AP negative and black regions have one or more AP+ cells that did not yield clonal information (either because of lack of PCR product or because the samples were not picked). Red regions have one or more sequenced picks that are related to sequenced cells located in another region. In other words, the red bars show the distribution of clones with dispersed members. Blue regions have at least one sequenced pick that is not related to picks from other regions of the same ear. (C-F) Schematics showing the distribution of clones for four individual ears. Similar schematics are shown in subsequent figures. Within each ear, different symbols depict separate clones. Grey circles indicate sequenced picks unrelated to any other picks; i.e. each grey circle is a separate clone. Ears are oriented with medial to the left and dorsal to the top. Left ears are flipped for uniform orientation. Unsequenced AP+ cells are not indicated. Abbreviations: AG, acoustic ganglion and nerve; VG, vestibular ganglion and nerve; UM, utricular macula; SM, saccular macula; LM, lagenar macula; SC, superior crista; LC, lateral crista; PC, posterior crista; BP, basilar papilla; ULJ, utricle-lateral ampulla junction; ED, endolymphatic duct; ES, endolymphatic sac; NS-UT, non-sensory part of the utricle; NS-Sac, non-sensory part of the saccule; NS-CD, non-sensory part of the cochlear duct; SSC, semicircular canal and ampulla; mes, periotic mesenchyme; GG, geniculate ganglion; PTO, paratympanic organ; SE, sensory epithelium; NS, non-sensory epithelium. Cells in the proximal facial ganglion are included in VG due to difficulty in distinguishing the two structures.

View larger version (58K): [in a new window]   Fig. 2. Neurons and sensory cells are related in the middle ear. (A-C) Three examples containing a clone (red triangles) with members in both the geniculate ganglion and the paratympanic organ. See legend to Fig. 1 for details. Grey circles indicate sequenced picks unrelated to any other picks; i.e. each grey circle is a separate clone. (D-F) Histological images of two clones. Transverse sections with medial leftwards and dorsal upwards. GG, geniculate ganglion; PTO, paratympanic organ.

View larger version (97K): [in a new window]   Fig. 3. Neurons and sensory organs are related in the inner ear. (A-C) Three cases with clones composed of neural and sensory progeny (red triangles). E1,F1, etc. indicate the subsequent histological panels showing the red clone. (D) Reconstruction from serial sections, with AP+ sequenced picks (red) and AP+ cells not sequenced (purple) overlaid on the sensory parts (blue) of the utricle and lateral ampulla. Medial is leftwards and anterior is upwards. The approximate location of the striola is indicated by a curved black line. (E1-F2') AP+ cells in the vestibular (E1-E2') and auditory (F1-F2') ganglia, respectively. Adjacent panels show Hoechst nuclear staining. (G) Transverse section containing AP+ cells in both medial and lateral parts of utricular macula, oriented as in A, with higher magnifications in H (AP staining) and H' (NF160 immunostaining). UM, utricular macula; LC, lateral crista; SM, saccular macula.

View larger version (98K): [in a new window]   Fig. 4. Neurons and non-sensory cells are related in the inner ear. (A-D and E-H) Two separate examples. Each ear contains a clone (red triangles) containing AP+ cells in the acoustic ganglion and the non-sensory epithelium between the utricle and the lateral ampulla, the ULJ. Higher-power images depict the epithelial (B,F) and neuronal (C,D and G,H) members of the clones. Brackets indicate the positions of sensory epithelia. UM, utricular macula; LC, lateral crista.

View larger version (53K): [in a new window]   Fig. 5. Neurons in the auditory and vestibular ganglia are related, but satellite cells are not. (A-D) Satellite cells in the statoacoustic ganglion do not originate from the otic cup. (A) Electron micrograph of the acoustic ganglion at E10. False-coloring shows nuclei of a neuron (Nn) and a satellite cell (Ns) in green, the cytosol of the satellite cell in red, and the cytosol of the neuron in blue for ease of comparison to the fluorescent images in C and D. (B) Schematic diagram of the plasmid used for electroporation. (C) Confocal micrograph of E10 vestibular ganglion triple-labeled and false-colored for neurofilament 160 immunoreactivity (blue), phalloidin (red) and TOTO3+ DNA (green). (D) Confocal micrograph of E9 vestibular ganglion neurons labeled by electroporation. EYFP-positive nuclei (green), phalloidin (red), TOTO3 (blue) and neurofilaments (blue) are labeled. (E-G) Three ears with clones (red triangles) that span the two ganglia. (H-I) Histological detail of the picks indicated in E.

View larger version (21K): [in a new window]   Fig. 6. Summaries of: (A) observed lineages from otic cup injections; (B) gene expression patterns used to define neurogenic and sensory-competent regions on the ventral floor of the otic cup/otocyst of chicken and mouse; and (C) predicted fate map looking down onto the ventral floor of the otic cup/vesicle of the chicken, showing prosensory, neurogenic and non-sensory (white) domains. The primordia of the anterior and posterior crista are not shown in C because they would be expected to be located slightly more dorsally than the other primordia (Cole et al., 2000). Abbreviations: AG, acoustic ganglion and nerve; VG, vestibular ganglion and nerve; UM, utricular macula; SM, saccular macula; LM, lagenar macula; SC, superior crista; LC, lateral crista; PC, posterior crista; BP, basilar papilla; ULJ, utricle-lateral ampulla junction; ED, endolymphatic duct; ES, endolymphatic sac; NS-UT, non-sensory part of the utricle; NS-Sac, non-sensory part of the saccule; NS-CD, non-sensory part of the cochlear duct; SSC, semicircular canal and ampulla; mes, periotic mesenchyme; GG, geniculate ganglion; PTO, paratympanic organ.

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