ACCURATE DETERMINATION OF THE TIME OF CELL BIRTH USING A SEQUENTIAL LABELING TECHNIQUE WITH [H-3] THYMIDINE AND BROMODEOXYURIDINE (WINDOW LABELING)

During tissue embryogenesis, precursor cells divide actively and eventually withdraw from the mitotic cycle before differentiation. Accurate information about the time of terminal mitosis ("birthdate") of precursors is of vital importance for studying relationships between cell proliferation and differentiation. Methods presently available for birthdate determination, based on "pulse" or "cumulative" labeling with either tritiated thymidine (3HT) or bromodeoxyuridine (BrDU) incorporated into DNA during the mitotic cycle, allow only the approximate timing of terminal mitosis. To overcome this limitation, we have developed a "window labeling" technique based on the sequential administration of 3HT and BrDU. Chick retinal precursor cell cultures were first exposed to 3HT and, after a specified time interval, also to BrDU. After 6 days the cultures were fixed and processed for BrDU immunocytochemistry and 3HT autoradiography. Three populations of cells could be easily identified: (a) unlabeled cells, representing post-mitotic cells before label exposure; (b) BrDU-labeled cells [either 3HT (+)/BrDU (+) or 3HT (-)/BrDU (+)], representing those that continue dividing after the addition of BrDU; and (c) "window-labeled" cells, 3HT (+)/BrDU (-), which are those undergoing their last round of DNA synthesis during the interval between 3HT and BrDU administration. Control experiments demonstrated that this method allows birthdate determinations with a resolution of hours or minutes and is essentially free of deleterious effects on precursor cell survival and differentiation.