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Elife. 2019 Jan 21;8. pii: e44491. doi: 10.7554/eLife.44491.

Primed Track, high-fidelity lineage tracing in mouse pre-implantation embryos using primed conversion of photoconvertible proteins.

Author information

1
Department for Biosystems Science and Engineering (D-BSSE), ETH Zurich, Basel, Switzerland.
2
Department of Bioengineering, Imperial College London, London, United Kingdom.
3
Howard Hughes Medical Institute, Janelia Research Campus, Ashburn, United States.
4
Friedrich Miescher Institute for Biomedical Research (FMI), Basel, Switzerland.
5
Faculty of Sciences, University of Basel, Basel, Switzerland.
6
Center for Transgenic Models (CTM), University of Basel, Basel, Switzerland.
#
Contributed equally

Abstract

Accurate lineage reconstruction of mammalian pre-implantation development is essential for inferring the earliest cell fate decisions. Lineage tracing using global fluorescence labeling techniques is complicated by increasing cell density and rapid embryo rotation, which hampers automatic alignment and accurate cell tracking of obtained four-dimensional imaging data sets. Here, we exploit the advantageous properties of primed convertible fluorescent proteins (pr-pcFPs) to simultaneously visualize the global green and the photoconverted red population in order to minimize tracking uncertainties over prolonged time windows. Confined primed conversion of H2B-pr-mEosFP-labeled nuclei combined with light-sheet imaging greatly facilitates segmentation, classification, and tracking of individual nuclei from the 4-cell stage up to the blastocyst. Using green and red labels as fiducial markers, we computationally correct for rotational and translational drift, reduce overall data size, and accomplish high-fidelity lineage tracing even for increased imaging time intervals - addressing major concerns in the field of volumetric embryo imaging.

KEYWORDS:

developmental biology; embryo; lineage tracing; mouse; pre-implantation; primed conversion; volumetric imaging

Conflict of interest statement

MW, AP, LR, YK, PL, AP, PP No competing interests declared, MM Is an inventor on a provisional patent application filed by HHMI and ETH Zurich that describes pr-mEosFP. AB Is affiliated with Viventis Microscopy Sàrl. The author has no other competing interests to declare. PP Is an inventor on a patent application filed by ETH Zurich and Caltech that describes primed conversion. Is an inventor on a provisional patent application filed by HHMI and ETH Zurich that describes pr-mEosFP.

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