Abstract
The 275 kD hair-cell antigen (HCA) is a protein that was originally identified using immunological techniques in the inner ears of early hatchling and adult chickens. The HCA is specifically associated with the apical surface of sensory hair cells; in the vestibular system the antigen is distributed over the entire stereocilia bundle, but in the auditory system it only extends a short distance up the shafts of the stereocilia. The objectives of this study were to ascertain when the HCA is first expressed during inner ear development, to compare the temporal and spatial patterns of HCA expression with those of neurite ingrowth, and to determine how the distribution of the antigen observed in the auditory system arises during development. Serial sections of otocysts from embryonic day (ED) 4 to ED7.5 (stages 24 to 32) were stained with a monoclonal antibody to the HCA and polyclonal antibodies to the neuron-glial cell adhesion molecule in order to analyse patterns of HCA expression and neurite ingrowth. Nerve fibres are first observed in the anterior pole of the otocyst at ED4.5 (stage 241, and in the evaginating basilar papilla by ED5 (stage 26). The HCA first appears within the vestibular system in the anterior pole of the otocyst at ED5 (stage 261, and within the auditory system in the distal end of the basilar papilla at ED6.5 (stage 29). Serial section analysis indicates that expression of the HCA is always limited to areas of the epithelium where nerve fibres are found, although the delay between the onset of innervation and the onset of HCA expression varies from one region of the otocyst to another. The growth of stereocilia bundles in the auditory system was studied from ED10 to 2 days after hatching in sections from the medial to distal regions of the basilar papilla double labelled with rhodamine phalloidin and monoclonal anti-HCA. At ED12 the stereocilia bundles are 1.7 ym high and the staining observed with both phalloidin and the antibody extend to the same maximum height above the apical surface of the hair cell. The maximum height that anti-HCA staining extends up the stereocilia bundle remains almost constant between ED12 and postnatal day 2, but between ED15 and ED18 the stereocilia bundle grows rapidly in height, with a membrane domain lacking the HCA forming at the distal ends of the stereocilia. The restricted distribution of the HCA observed on the apical surface of mature auditory hair cells in the basilar papilla is therefore generated during the final growth phase of the stereocilia bundle by the accumulation of HCA-free membrane at the distal ends of the stereocilia. The inner ear is a highly complex sensory organ that develops from a thickening of the head ectoderm, the otic placode, via inductive interactions with the underlying mesenchymal tissue and the adjacent rhombencephalon (Yntema, '33, '50; Waddington, '37; Harrison, '45). The otic placode, after forming the otocyst, gives rise to at least seven distinct cell types within the inner ear, including the neurons of the VIIIth ganglion and the mechanosensory hair cells that they innervate. There are seven discrete patches of sensory hair cells in the avian inner ear, six vestibular organs (three maculae and three ampullae), and one auditory organ known as the basilar papilla. The formation of the VIIIth ganglion, the differentiation of sensory hair cells, synaptogenesis, and the development of the stereocilia bundles in the chicken inner ear have been the subject of a considerable number of previous studies.