AceTree: a tool for visual analysis of Caenorhabditis elegans embryogenesis

Abstract

Background: The invariant lineage of the nematode Caenorhabditis elegans has potential as a powerful tool for the description of mutant phenotypes and gene expression patterns. We previously described procedures for the imaging and automatic extraction of the cell lineage from C. elegans embryos. That method uses time-lapse confocal imaging of a strain expressing histone-GFP fusions and a software package, StarryNite, processes the thousands of images and produces output files that describe the location and lineage relationship of each nucleus at each time point.

Results: We have developed a companion software package, AceTree, which links the images and the annotations using tree representations of the lineage. This facilitates curation and editing of the lineage. AceTree also contains powerful visualization and interpretive tools, such as space filling models and tree-based expression patterning, that can be used to extract biological significance from the data.

Conclusion: By pairing a fast lineaging program written in C with a user interface program written in Java we have produced a powerful software suite for exploring embryonic development.

Figure 1

AceTree main control and ImageWindow. The image window shows the annotated data for the four-cell stage embryo at the first time point of the series. The main control shows the JTree representation with cell ABa highlighted. The four founder cells, ABa, ABp, EMS, P2 can be seen in both the image window and the tree view. The tree also shows that a polar body has been found at this time point, called polar1. However it is not visible in plane 17, the plane shown in the image window.

Figure 2

View of nuclei data. Nuclei data from time points 4 and 5 are shown. The two divisions that occurred between times 4 and 5 can be traced from the successor flags. Nuclei location, size, and canonical names are also shown. These are the annotations produced by the automated lineaging software.

Figure 3

Tracking to the division of ABa. The image from plane 13 is annotated to show ABal as the current cell and a ghost representation of cell ABar which is centered at plane 19 and has no natural intersection in this plane. Correct tracking of divisions is a key challenge in automated lineaging.

Figure 4

Lineage curation using EditTraverse. The EditTraverse list box shows all divisions in the EMS lineage. As the user moves the highlight down the list, the ImageWindow tracks to the corresponding division. Here, cell Epr is about to divide.

Figure 5

Comparison of EMS lineages for two developmental series. The tree on the left is from a wild type embryo; that on the right is from an RNAi treated embryo with the lit-1 gene inhibited. This has caused the E lineage to adopt the fate of the MS lineage. The tree view shows this clearly.

Figure 6

Developmental comparison using space filling models. In both images, the time point corresponds to the point where the AB sublineage contains 64 cells. As in Figure 5, the wild type embryo is on the left and the embryo with lit-1 inhibited is on the right. The E lineage cells have not migrated to the interior in the RNAi embryo. Cell migration generally has not proceeded in the normal way. These are ventral views with anterior on the left. The nuclei are color-coded so the main lineages can be distinguished: ABa daughters in gray; ABp in white; E in red; MS in blue; C in yellow; D in pink; germ line in green.

Figure 7

An overview of the lineage with gene expression shown. This is a compact representation of the entire lineage of an embryo with the pha-4 gene tagged with a red fluorescent protein. The branches are color-coded by the strength of the red expression: gray if there is no expression, generally the case early in the development; green where there is a detectable low level of expression; red where expression is strong. Branch labels are offered for every tenth nucleus which is sufficient to identify the cell groupings where the gene is expressed: strongly in the E lineage, less so in some MS lineages and in some daughters of ABa. The tagged gene is only expressed in the latter stages of the embryonic development. This tree is interactive in AceTree so the user can bring up the image corresponding to any point by clicking on the branch in question.

Figure 8

Detailed view of a sublineage of interest. To visualize the expression of the tagged gene in the MSa sublineage a vertical tree representation can be used. Progression of expression can be followed by the color scheme, which goes from green to black to red as the intensity of expression increases. Expression is detected in several daughters of MSaa but not in daughters of MSap.

Figure 9

Showing gene expression using the space filling model. Two different views show the same time point, comparing the observed expression with that reported in the literature. These are ventral views with anterior on the left. The coloring scheme on the left characterizes the expression level of the tagged gene: cells expressing the gene are shown clearly with different shades of green and red using the scheme of Figures 7 and 8; non-expressing cells are grayed out. On the right, the sublineages reported in the literature to express the gene are shown with sublineage specific colors and cells not reported to be expressing are grayed out. The sublineage color scheme is: ABal in pink; ABar in blue; E in yellow; MSaa in magenta; MSap in cyan.

Figure 10

Using the space filling model to track embryonic rotation. The ABpl sublineage is shown in red; ABpr in blue; other cells are not shown. Each time point is just prior to the next round of cell divisions. A rotation of about 45 degrees of the axis between the two groups can be seen as development proceeds. Bear in mind that in a 3D view, cells closer to the viewer will appear larger than more distant cells.

Figure 11

Quantification of embryonic rotation. The angle of the line connecting the centroids of the ABal and ABar sublineages is tracked verses time on the left. The cell count verses time is on the right. After a rough beginning, the angle increases relatively smoothly after time point 30.