High Incidence of Non-Random Template Strand Segregation and Asymmetric Fate Determination In Dividing Stem Cells and their Progeny
Figure 2
Evidence of Co-Segregation of DNA Template Strands during Muscle Progenitor Cell Division
(A) Following muscle injury, CldU was administered to coincide with an early round of satellite cell division (~day 2 after injury), and IdU was administered to coincide with the subsequent round of DNA replication in the population. Cells were then isolated from muscle and plated singly on coated chamber slides, allowed to complete the current cell division, and then fixed. Cell pairs were analyzed for the expression of Syndecan-4 and Pax7, myogenic lineage markers [9,27], and for the marker of cell proliferation, Ki67. Representative pairs are shown on the left. The percentages of pairs that were myogenic (Syndecan-4+) and proliferating (Ki67+) were quantified and are presented graphically on the right.
(B) Cell pairs were immunostained for CldU and IdU. Shown is a representative photograph of an immunostained pair of cells, in which both daughter cells were labeled with the second label, IdU (green), but only one daughter inherited the first label, CldU (red).
(C) Quantification of asymmetric pairs. Almost half of the pairs derived from cells isolated from regenerating muscle showed asymmetric CldU staining as in (A) (all had symmetric IdU staining), and almost one third of the pairs of satellite cell progeny activated ex vivo in bulk myofiber explant cultures showed asymmetric inheritance of DNA label. Only a small percentage of pairs derived from established myoblast cultures (Mb in vitro) showed asymmetric inheritance of DNA label. Data represent mean ± the standard deviation (SD) (n = 4–7). SC*, progeny of activated satellite cells.
(D) Muscle was injured and labeled with BrdU on day 2 of regeneration, as with CldU above (C). On day 3, the satellite cell progeny were prepared, plated sparsely, and cytochalasin D was added to arrest cytokinesis. The top row shows a cell pair symmetrically inheriting BrdU-labeled DNA, and the bottom row shows an asymmetric pair. Similar percentages of cells showed asymmetric inheritance of template strands using this protocol as in (C).