Proliferative and functional stages of rat ameloblast differentiation as revealed by combined immunocytochemistry against enamel matrix proteins and bromodeoxyuridine.

A double-staining immunocytochemical technique was used for the simultaneous detection, at the light- and electron-microscopical level, of proliferating bromodeoxyuridine (BrdU)-labelled cells and enamel protein (EP)-producing cells in the inner enamel epithelium (IEE) of rat tooth germ. BrdU-positive cells were found in the region of the IEE close to the cervical loop and never displayed EP-like immunoreactivity. BrdU-immunoreactivity was confined to the nucleus of replicating cells. In contrast, epithelial cells displaying EP-like immunoreactivity were found in the region of the forming dental cusp and were consistently BrdU-negative. EP-like immunoreactivity was detectable in the cytoplasmic compartments involved in the exocrine secretion pathway and in the extra-cellular matrix close to EP-immunoreactive cells. These data support the view that withdrawal from the cell cycle in the IEE is a temporal prerequisite for acquiring the functional competence of secreting EP. Moreover, cycling cells and secretory cells in the IEE constitute two separate compartments that are spatially defined, and that exhibit clear-cut staining patterns with respect to BrdU- and EP-immunoreactivity, respectively. We thus propose that BrdU-incorporation and EP-production may be used as specific markers of the differentiation of the IIE cells in studies of the possible role of growth factors, their receptors and oncoproteins in this tissue.