Abstract
Quantitative mapping of the normal tissue dynamics of an entire developing mammalian organ has not been achieved so far but is essential to understand developmental processes and to provide quantitative data for computational modeling. We developed a four-dimensional (4D) imaging technique that can be used to quantitatively image tissue movements and dynamic GFP expression domains in a growing transgenic mouse limb by time-lapse optical projection tomography (OPT).
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Acknowledgements
We thank M. Logan (MRC National Institute for Medical Research) for providing the Scleraxis-GFP line, the Zeller lab for their time and helpful suggestions on the in vitro mouse limb bud culturing technique, L. Hay for technical support in the design and building of the 4D time-lapse OPT scanner, D.A. Kleinjan and V. van Heyningen for Pax6-GFP embryos, C. DeAngelis for performing the Fgf-8 in-situ hybridization, B. Pryce for sectioning the Scx-GFP limbs, J. Swoger for help with 3D reconstructions, and the Edinburgh Mouse Atlas Project (EMAP) for software to support the computational analysis. This project was supported as part of the EU Integrated Project grant 'Molecular Imaging' (to M.J.B.), by a research grant from the Human Frontier Science Program (to C.H.W.), by a grant from the Spanish Ministry of Science and Education (BFU2006-10978/BMC; to J.S.-E. and M.T.), by MRC and Centre for Genomic Regulation (to J.C. and J.S.) and by ICREA (to J.S.).
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Supplementary Text and Figures
Supplementary Note, Supplementary Methods (PDF 7122 kb)
Supplementary Movie 1
Raw data of microsphere landmarks at 0 hours. Movie showing the raw data obtained from multiple angles during an OPT scan at the first time-point for one of the fluorescent landmark-tracking experiments. (MOV 576 kb)
Supplementary Movie 2
Timelapse of microsphere landmarks over 6 hours. Timelapse movie from one angle of the same limb shown in Supplementary Movie 1. (MOV 225 kb)
Supplementary Movie 3
Annotated timelapse of microsphere landmarks over 6 hours. Annotated timelapse movie from one angle of the same limb shown in Supplementary Movie 1. (MOV 180 kb)
Supplementary Movie 4
Raw data of Scx-GFP expression pattern at 0 hours. Movie showing the raw data obtained from multiple angles during an OPT scan at the first time-point for one of the Scx-GFP imaging experiments. (MOV 725 kb)
Supplementary Movie 5
Raw data of Scx-GFP expression pattern after 19 hours. Movie showing the raw data obtained after 19 hours in culture for the same Scx-GFP imaging experiment shown in Supplementary Movie 4. (MOV 786 kb)
Supplementary Movie 6
4D timelapse of dynamic gene expression. Timelapse movie showing how the 3D domain of Scx-GFP expression changes over. (Same limb as shown in Supplementary Movie 4). All expression above a certain threshold level has been highlighted by the green isosurface. (MOV 578 kb)
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Boot, M., Westerberg, C., Sanz-Ezquerro, J. et al. In vitro whole-organ imaging: 4D quantification of growing mouse limb buds. Nat Methods 5, 609–612 (2008). https://doi.org/10.1038/nmeth.1219
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DOI: https://doi.org/10.1038/nmeth.1219
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