BUdR as an S-phase marker for quantitative studies of cytokinetic behaviour in the murine cerebral ventricular zone
Tóm tắt
BUdR incorporation into replicating DNA, detected immunohistochemically, is used as an S-phase marker in the proliferative cell populations of the cerebral wall of the mouse embryo on the 14th gestational day (E14). The analysis initiates a series of studies concerned with the cytokinetic behaviour and cell output of proliferative populations involved in neocortical histogenesis. On E14 there are two periventricular proliferative zones in the cerebral wall. These are the ventricular and subventricular zones. The ventricular zone is a pseudostratified epithelium. DNA replication occurs with the cell nucleus in the outer zone of the epithelium and mitoses at the ventricular surface. Prior applications of BUdR for studies of cytogenesis in the CNS have been extended in two principal ways: (1) basic fuchsin was used as counterstain for BUdR-negative nuclei and (2) labelling indices were determined separately in strata or bins, 10 μm in height, through the full depth of the ventricular zone and overlying cerebral wall. It was established that a single injection of 50 μg g−1 into the pregnant dam was associated with labelling of 100% of nuclei in S-phase over an interval extending from 15 min to at least 2.0 h after injection. The zone where nuclei are undergoing S-phase (S-phase zone) extends through the outer four bins of the ventricular zone. The method has high quantitative reproducibility with anse for labelling indices in bins within the S-phase zone less than 10% of the average values. Evidence is provided that BUdR incorporation is initiated with the nucleus in the outer aspect of the S-phase zone. The efficiency of incorporation of the marker is reduced as nuclei near the end of DNA replication and move to the inner aspect of the S-phase zone.
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