Hair cell development in vivo and in vitro: Analysis by using a monoclonal antibody specific to hair cells in the chick inner ear

Abstract

The purpose of this study was to establish a hair cell-specific marker and a convenient explant culture system for developing chick otocysts to facilitate in vivo and in vitro studies focusing on hair cell genesis in the inner ear. To achieve this, a hair cell-specific monoclonal antibody, 2A7, was generated by immunizing chick inner ear tissues to a mouse. Through the use of immunofluorescence and immunoelectron microscopy, it was shown that 2A7 immunoreactivity (2A7-IR) was primarily restricted to the apical region of inner ear hair cells, including stereocilia, kinocilia, apical membrane amongst the extending cilia, and superficial layer of the cuticular plate. Although the 2A7 antibody immunolabeled basically all of the hair cells in the posthatch chick inner ear, two different patterns of 2A7-IR were observed; hair cells located in the striolar region of the utricular macula, which consist of two distinct cell types identifiable on the basis of the type of nerve ending, Type I and II hair cells, showed labeling restricted to the basal end of the hair bundles. On the other hand, hair cells in the extrastriolar region, which are exclusively of Type II, showed labeling extending over virtually the entire length of the bundles. These findings raised the possibility that chick vestibular Type II hair cells, characterized by their bouton-type afferent nerve endings, can be divided into two subpopulations. Analysis of developing inner ear by using the 2A7 antibody revealed that this antibody also recognizes newly differentiated immature hair cells. Thus, the 2A7 antibody is able to recognize both immature and mature hair cells in vivo. The developmental potential of embryonic otocysts in vitro was then assessed by using explant cultures as a model. In this study, conventional otocyst explant cultures were modified by placing the tissues on floating polycarbonate filters on culture media, thereby allowing the easy manipulation of explants. In these cultures, 2A7-positive hair cells were differentiated from dividing precursor cells in vitro on the same schedule as in vivo. Furthermore, it was found that hair cells with both types of 2A7-IR were generated in culture as in vivo, indicating that a maturational process of hair cells also occurred. All these results as presented here suggest that the 2A7 monoclonal antibody as a hair cell-specific marker together with the culture system could be a potential tool in analysis of mechanisms underlying hair cell development.