Analysis of rat vestibular hair cell development and regeneration using calretinin as an early marker

Abstract

Despite increased interest in inner ear hair cell regeneration, it is still unclear what exact mechanisms underlie hair cell regeneration in mammals because of our limited understanding of hair cell development and the lack of specific hair cell markers. In this report, we studied hair cell development using immunohistochemistry on sections prepared from embryonic day (E) 13 to postnatal day 7 rat inner ear tissues. Of many epithelial, neuronal, and glial markers, we found that calcium-binding protein antibodies recognizing calretinin, calmodulin, or parvalbumin labeled immature hair cells in rat vestibular end organs. In particular, calretinin antiserum labeled the initial differentiating hair cells at E15, a stage immediately after the terminal mitosis of hair cell progenitors. The selective immunoreactivity of post-mitotic presumptive hair cells, but not supporting cells or peripheral epithelial cells, was confirmed in utricular epithelial sheet cultures. Double labelling with calretinin and bromode-oxyuridine antibodies in long-term cultures showed that only a few mitotic utricular supposing cells became calretinin positive. Thus, although proliferation- mediated regeneration of new hair cells might directly contribute to hair cell regeneration in rat utricles after injury, it is very limited. In addition, double labeling with calretinin and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) revealed that differentiated hair cells underwent apoptosis during normal development at late embryonic and early postnatal stages in vivo and in vitro. Therefore, these experiments lay the groundwork for the time course of differentiation, regeneration, and apoptosis of mammalian vestibular hair cells. This work also suggests that calcium-binding proteins are useful markers for studies on inner ear hair cell differentiation and regeneration.