Transglutaminase and clusterin induction during normal and abnormal limb development in the mouse.

Apoptotic cell death is important in pattern formation in the limb. The purpose of this study was to investigate the relationship between the occurrence of apoptosis in the mouse limb during normal and abnormal development as well as the expression of tissue transglutaminase and clusterin, two proteins associated with apoptotic cell death. Mouse limb buds were cultured in vitro in the absence or presence of a potent teratogen, an activated analog of cyclophosphamide: 4-hydroperoxycyclophosphamide (1 or 10 micrograms/ml). Using whole-mount in situ DNA labeling and confocal microscopy, apoptotic cells were localized in the interdigital areas of control limbs after culture for 24 h. The number of apoptotic cells in the interdigital areas of the limbs was increased in the presence of 4-hydroperoxycyclophosphamide (1 microgram/ml). Exposure to a higher concentration (10 micrograms/ml) of 4-hydroperoxycyclophosphamide further increased the numbers of cells staining positively for apoptosis. The relative abundance of tissue transglutaminase increased 3-4-fold after 6 or 24 h of culture with either concentration of 4-hydroperoxycyclophosphamide; immunoreactive protein in drug-treated limbs decreased to control levels by 48 h. Transglutaminase immunoreactivity was localized in the interdigital areas of limbs 24 h after drug exposure. Clusterin immunoreactivity in the control limbs was weak. The abundance of clusterin was increased 3-4-fold in drug-treated limbs; this induction occurred only after 48 h of culture with 4-hydroperoxycyclophosphamide. Clusterin immunoreactivity in limbs after drug treatment for 48 h was localized to the interdigital areas; immunogold electron microscopy of clusterin expression showed a specific labeling in phagocytosed apoptotic bodies. Thus, the number of cells staining positively for apoptosis in the limb was greatly increased in the interdigital areas during abnormal limb development. The expression of both transglutaminase and clusterin was altered in areas of the limb undergoing apoptosis during abnormal limb development.