Automated cell lineage tracing in Caenorhabditis elegans

Abstract

The invariant cell lineage and cell fate of Caenorhabditis elegans provide a unique opportunity to decode the molecular mechanisms of animal development. To exploit this opportunity, we have developed a system for automated cell lineage tracing during C. elegans embryogenesis, based on 3D, time-lapse imaging and automated image analysis. Using ubiquitously expressed histone-GFP fusion protein to label cells/nuclei and a confocal microscope, the imaging protocol captures embryogenesis at high spatial (31 planes at 1 microm apart) and temporal (every minute) resolution without apparent effects on development. A set of image analysis algorithms then automatically recognizes cells at each time point, tracks cell movements, divisions and deaths over time and assigns cell identities based on the canonical naming scheme. Starting from the four-cell stage (or earlier), our software, named starrynite, can trace the lineage up to the 350-cell stage in 25 min on a desktop computer. The few errors of automated lineaging can then be corrected in a few hours with a graphic interface that allows easy navigation of the images and the reported lineage tree. The system can be used to characterize lineage phenotypes of genes and/or extended to determine gene expression patterns in a living embryo at the single-cell level. We envision that this automation will make it practical to systematically decipher the developmental genes and pathways encoded in the genome of C. elegans.

Fig. 1.

A typical 2D image of an embryo with GFP–histone-labeled nuclei (green). The nuclei are annotated as determined by starrynite and acetree. Red circles represent the spherical models of identified nuclei (i.e., the intersections at the given plane; see Movie 2, which is published as supporting information on the PNAs web site). Note that MSaa is at metaphase and MSpa is at anaphase.

Fig. 2.

Cell movement is minimal under frequent imaging. Red circles mark nuclei of the current time point, and yellow circles mark nuclei from the previous time point. For each nucleus, the current position heavily overlaps with its previous position.

Fig. 3.

A wild-type lineage up to the 350-cell stage produced by automated analysis followed by minor editing. The tree agrees with the canonical lineage (1). The germline precursors Z2 and Z3 become too dim to be traced, resulting in two truncated branches in the tree (daughters of P4).

Fig. 4.

Benchmarks of starrynite. (A) Developmental time course of the C. elegans embryo. Red is based on the images of a real embryogenesis. The green part of the trace is an approximation based on ref. . We benchmark our algorithms at five stages of embryogenesis. (B) Error rates for false negatives, false positives during nuclear identification, and mismatches during nuclear tracking. Each point represents an image series tested. The first four stages were tested on 20 series, and the last stage was tested on 5 series. (C) Error rates for tracking cell divisions in the same test series as B, with stages c and d combined into one. A correctly tracked division is one for which the mother and two daughter cells are all correctly identified and matched. Each point represents an image series tested. Variation of total number of nuclei and divisions at each stage is due to variable developmental speed and imprecise order of certain cell divisions in different embryos.