Random distribution of labeled DNA-strands among daughter nuclei of LRCs.

<p>Analysis of the distribution of labeled DNA-strands among daughter nuclei of label-retaining cells (LRCs) on single cell level, using the actin-binding protein cytochalasin D. Cytochalasin D is an actin-binding protein that inhibits cytokinesis, while karyokinesis remains unaffected. Thereby, binucleate cells are created, which enables analysis of the distribution of DNA-strands among daughter nuclei on single cell level. (A): Scheme of the experimental set-up. Animals were pulsed continuously with EdU during embryonic development (day1–day5) and during post-embryonic development (day6–day11), followed by a chase time of 2 months in EdU-free medium. Subsequently animals were soaked in cytochalasin D for 7 days, EdU was visualized and DNA was stained with DAPI. (B): Visualization of binucleate LRCs in macerated cell suspensions. Fluorescence images of EdU (left), DAPI (middle), and interference contrast images (right) of binucleate LRCs, pulsed during embryonic (B1) and post-embryonic (B2, B3) development. Binucleate EdU⁺ cells display equivalent EdU-signal in both daughter nuclei. Abbreviations: EdU, 5-ethynyl-2′-deoxyuridine. Scale bars: 5 µm.