Establishment of hair bundle polarity and orientation in the developing vestibular system of the mouse

The morphological development of the vestibular maculae in the mouse was st udied in order to identify elements that may determine how hair-bundle pola rity is established. Utricles and saccules develop in parallel. Hair-bundle s first appear at embryonic day (E) 13.5. They are initially not polarised and have a kinocilium located at the centre of the cell surface surrounded by stereocilia. Polarisation is rapidly established as the kinocilium becom es eccentrically positioned. The orientation of these polarised bundles is initially not random. It varies systematically across the maculae and the g eneral orientation in utricles is the opposite of that in saccules. At E15. 5, in both maculae, hair-bundle orientation angles fall into two population s that differ by approximately 180 degrees defining a line of orientation r eversal, the position of which varies little during subsequent maturation. Many more immature hair bundles appear at E15.5 suggesting a second wave of hair cell differentiation is initiated. Otoconial membrane is produced sim ultaneously across the entire width of both maculae, indicating directional growth of the overlying extracellular matrix is unlikely to influence hair -bundle orientation. Growth of both maculae occurs asymmetrically, essentia lly outwards from the striola, but it is most pronounced after orientation is defined. Microtubules are prominent in hair cells at the earliest stages of their differentiation, but are oriented parallel to the long axis of th e cell and, thus, may not have a role in directing hair-bundle polarity. Mi crofilament assemblies that are aligned parallel to the apical surface and connect to the adherens junctions in supporting cells could provide a "fram ework" for hair-bundle orientation. The striated rootlets of ciliary centri oles that are aligned parallel to the cell surface with their tips associat ed with microfilament assemblies at adherens junctions were the only struct ural asymmetry identified that might influence the development of hair-bund le polarity.