Ameloblasts require active RhoA to generate normal dental enamel

RhoA plays a fundamental role in regulation of the actin cytoskeleton, intercellular attachment and cell proliferation. During amelogenesis, ameloblasts which produce the enamel proteins undergo dramatic cytoskeletal changes and RhoA protein level is upregulated. Transgenic mice were generated that express a dominant-negative RhoA transgene in ameloblasts using amelogenin gene regulatory sequences. Transgenic and WT molar tooth germs were incubated with NaF or NaCl in organ culture. F-actin stained with phalloidin was elevated significantly in WT ameloblasts treated with NaF compared to WT ameloblasts treated with NaCl or compared to transgenic ameloblasts treated with NaF, thereby confirming a block in the RhoA/ROCK pathway in the transgenic mice. Little difference in quantitative fluorescence (estimation of fluorosis) was observed between WT and transgenic incisors from mice provided NaF in their drinking water. We subsequently found reduced transgene expression in incisors compared to molars. Transgenic molar teeth had reduced amelogenin, E-cadherin and Ki67 compared to WT. Hypoplastic enamel in transgenic mice correlates with reduced expression of the enamel protein amelogenin, and E-cadherin and cell proliferation are regulated by RhoA in other tissues. Together these findings reveal deficits in molar ameloblast function when RhoA activity is inhibited