Objectives: Important roles of apoptosis have been suggested in odontogenesis, however, its mechanisms remain poorly understood. Cell death signals are communicated either by the intrinsic apoptotic machinery, switched mainly in mitochondria, or by the extrinsic ones, receptor mediated. Different caspases, intracellular cysteine proteases, are key components of apoptosis in both cases. Caspase-8 (extrinsic), caspase-9 (intrinsic), and caspase-3 (central step in both pathways) were investigated in correlation with dental apoptosis.
Methods: Knock-out mice in caspase-3 and caspase-9 (caspase-8 deficiency is lethal before tooth formation) and tooth explant cultures were applied to follow consequences of individual caspase deficiency/inhibition. E15.5 mice were used in knock-out study focused on apoptotic elimination of the primary enamel knot (PEK) at this stage. Explant cultures were treated with pharmaceutical inhibitors starting E13.5 up to 72 h. Appearance of apoptotic bodies and TUNEL test were criteria for apoptosis evaluation, and PCNA staining for proliferation within the tooth germs.
Results: No apoptotic bodies were found in the mutant tooth germs and TUNEL labelling showed negative or very weak staining. In caspase-9 deficient mice, no apoptosis was found in PEK, navel, stalk or surrounding tissues. All mutant mice showed the same PCNA staining pattern as the wild type mice. General inhibition of caspases in tooth germ cultures leads to lack of apoptotic features in the PEK, and confirms dental apoptosis as caspase dependent. Caspase-3 inhibition causes significant decrease of apoptotic cells within the PEK area after 24 h inhibition. Caspase-9 inhibition blocks caspase-3 activation as showed by immunohistochemistry and a decrease in apoptotic cell numbers was identified.
Conclusions: Despite knock-out and inhibitor mediated block of apoptosis in the PEK, the cells seem to disappear. Other forms of cell death or compensatory caspases might be involved. To check these hypotheses, TEM method and double/triple inhibitions will be applied.