Abstract

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.

Highlights

  • To understand growth and morphogenesis during embryonic development it is essential to know local differences in morphogenetic parameters like cell size, cell cycle length and growth fraction

  • When the dividing cells can be assumed to be in a random phase of the cell cycle, the cell cycle length is constant and the population does not increase in size during the exposure time [8], the relation between labelling index, exposure time (Texp), cell cycle length (TC) and S-phase length (TS) can be described by a linear equation: LI = (TS+Texp)/TC [7]

  • The main assumptions are similar to those of the exposure of different specimens: the dividing cells are assumed to be in a random phase of the cell cycle, cell cycle length is constant and the population does not increase in size during the exposure time [8]

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Summary

Introduction

To understand growth and morphogenesis during embryonic development it is essential to know local differences in morphogenetic parameters like cell size, cell cycle length and growth fraction. The use of different exposure times enables the calculation of cell cycle length and S-phase length [4,7,8] In these studies, several embryos were exposed to a single radioactive or halogen-conjugated thymidine analogue for different lengths of time before sacrifice. When using a single thymidine analogue for estimation of local cell cycle lengths in a developing organ or embryo it is required to apply a 3D registration of the different specimens [7]. By exposing a single embryo to two distinct labels and different exposure times [9,10], the differential exposure time theorem (Fig. 1) for the estimation of cell cycle length can be applied to a single organ or specimen This approach has been applied in a study of the mouse limb bud in which local manual measurements of the cell cycle length were extrapolated to obtain a 3D pattern of the proliferation gradient [11]

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