Quantitative semi-automated analysis of morphogenesis with single-cell resolution in complex embryos

Abstract

A quantitative understanding of tissue morphogenesis requires description of the movements of individual cells in space and over time. In transparent embryos, such as C. elegans, fluorescently labeled nuclei can be imaged in three-dimensional time-lapse (4D) movies and automatically tracked through early cleavage divisions up to ~350 nuclei. A similar analysis of later stages of C. elegans development has been challenging owing to the increased error rates of automated tracking of large numbers of densely packed nuclei. We present Nucleitracker4D, a freely available software solution for tracking nuclei in complex embryos that integrates automated tracking of nuclei in local searches with manual curation. Using these methods, we have been able to track >99% of all nuclei generated in the C. elegans embryo. Our analysis reveals that ventral enclosure of the epidermis is accompanied by complex coordinated migration of the neuronal substrate. We can efficiently track large numbers of migrating nuclei in 4D movies of zebrafish cardiac morphogenesis, suggesting that this approach is generally useful in situations in which the number, packing or dynamics of nuclei present challenges for automated tracking.

Setting Up a Simple Light Sheet Microscope for <em>In Toto</em> Imaging of <em>C. elegans</em> Development

“…We checked that under these conditions the embryos develop at normal speed and survive the imaging, consistent with earlier reports indicating that SPIM imaging causes only low phototoxicity levels in C. elegans embryos 6,7 .…”

“…We showed examples of time-lapse movies with sufficient temporal resolution to observe cell divisions and cell shape changes. The power of light sheet microscopy to study C. elegans development has also been recently illustrated by others 6,7,8 . Therefore, this technique will be very useful to study the morphogenesis and patterning of the C. elegans embryo.…”

SEGGA: a toolset for rapid automated analysis of epithelial cell polarity and dynamics

“…Although a tremendous amount of information about singlecell and collective cell behaviors can be obtained from live imaging studies, the availability of accurate and high-throughput methods for cell tracking and analysis is currently a rate-limiting step in making use of this information. Image analysis tools that detect cell nuclei can reliably track nuclear movements in C. elegans (Bao et al, 2006;Santella et al, 2010;Giurumescu et al, 2012), zebrafish (Keller et al, 2008), Drosophila (McMahon et al, 2008;Schindelin et al, 2012;Stegmaier et al, 2016) and mice (Lou et al, 2014). However, it is not possible to accurately determine cell shape and interactions from the positions of cell nuclei, as mathematical approaches that predict the outer contours of cells based on the locations of the cell centers often fail for cells that are elongated or irregular in shape, which are typical of developing epithelia Blankenship et al, 2006;Williams et al, 2014).…”

“…However, segmentation errors that lead to 1% untracked cells in each frame of a movie are predicted to interrupt more than half of all cell trajectories after 70 time points, making fully automated methods of limited use for long-term tracking. As an alternative strategy, several methods enable the user to inspect and manually correct the segmentation output (McMahon et al, 2008;Fernandez-Gonzalez and Zallen, 2011;Gelbart et al, 2012;Giurumescu et al, 2012;Mashburn et al, 2012;Barbier de Reuille et al, 2015;Cilla et al, 2015;MoralesNavarrete et al, 2015;Rozbicki et al, 2015). These methods have the potential to achieve high accuracy but require substantial effort to manually correct the segmentation at each time point, decreasing the throughput of these approaches.…”

Syndecan defines precise spindle orientation by modulating Wnt signaling in C. elegans

“…These observations used visual estimation of the division axis or of the positions of daughter cells. To quantitate ABar division orientation more precisely, we used the NucleiTracker4D software (Giurumescu et al, 2012) to track histone-GFP labeled nuclei in 4D movies (Fig. 3D,E).…”