Measurement and 3D-visualization of cell-cycle length using double labelling with two thymidine analogues applied in early heart development

PLoS One. 2012;7(10):e47719. doi: 10.1371/journal.pone.0047719. Epub 2012 Oct 16.


Organ development is a complex spatial process in which local differences in cell proliferation rate play a key role. Understanding this role requires the measurement of the length of the cell cycle at every position of the three-dimensional (3D) structure. This measurement can be accomplished by exposing the developing embryo to two different thymidine analogues for two different durations immediately followed by tissue fixation. This paper presents a method and a dedicated computer program to measure the resulting labelling indices and subsequently calculate and visualize local cell cycle lengths within the 3D morphological context of a developing organ. By applying this method to the developing heart, we show a large difference in cell cycle lengths between the early heart tube and the adjacent mesenchyme of the pericardial wall. Later in development, a local increase in cell size was found to be associated with a decrease in cell cycle length in the region where the chamber myocardium starts to develop. The combined application of halogenated-thymidine double exposure and image processing enables the automated study of local cell cycle parameters in single specimens in a full 3D context. It can be applied in a wide range of research fields ranging from embryonic development to tissue regeneration and cancer research.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cell Cycle*
  • Chick Embryo
  • Computer Simulation
  • Heart / embryology*
  • Imaging, Three-Dimensional
  • Molecular Imaging
  • Myocardium / metabolism*
  • Organogenesis / physiology
  • Staining and Labeling
  • Thymidine / analogs & derivatives


  • Thymidine

Grants and funding

BAdB was supported by the EU seventh framework program project CHeartED (Health-F2-2008-223040). GvdB was supported by the Netherlands Heart foundation (NHS1996M002). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.