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

Maaike Welling,Periklis Pantazis,Aaron Ponti,Yumiko K Kawamura,Andrea Boni,Lluc Rullan Sabater,Prisca Liberali,Antoine HFM Peters,Manuel Alexander Mohr,Pawel Pelczar

doi:10.7554/elife.44491

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.

Highlights

Accurate lineage tracing and precise tracking of single cells in pre-implantation embryos are essential for a mechanistic understanding of the first cell fate decisions during mammalian development (Welling et al, 2016; Pantazis and Bollenbach, 2012)
Selective plane illumination microscopy (SPIM) has the potential to play a major role in achieving comprehensive, non-invasive imaging of mammalian pre-implantation development
We identified primed convertible mEosFP as the optimal fluorescent protein variant for in vivo primed conversion in the mouse embryo followed by long-term imaging

Summary

Introduction

Accurate lineage tracing and precise tracking of single cells in pre-implantation embryos are essential for a mechanistic understanding of the first cell fate decisions during mammalian development (Welling et al, 2016; Pantazis and Bollenbach, 2012). Selective plane illumination microscopy (SPIM) has the potential to play a major role in achieving comprehensive, non-invasive imaging of mammalian pre-implantation development. During these early steps of development, a major fraction of embryos (n = 9/19, 45% in this study) exhibit confounding rotational and spatial drift (Videos 1, 2 and 3), which often leads researchers to exclude these embryos from analysis, drastically decreasing efficiency, losing valuable data, and potentially biasing downstream results (Strnad et al, 2016; Motosugi et al, 2005). While high-imaging rates have helped to overcome these challenges for samples like zebrafish embryos, they demand increased data storage capacities.

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