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The lineaging of fluorescently-labeled Caenorhabditis elegans embryos with StarryNite and AceTree

Nature Protocols volume 1, pages 14681476 (2006) | Download Citation



Lineage analysis of Caenorhabditis elegans is a powerful tool for characterizing developmental phenotypes and embryonic gene-expression patterns. We present a detailed protocol for the lineaging of embryos by computational analysis of 4D images of embryos that ubiquitously express histone–GFP (green fluorescent protein) fusion proteins through the 350 cell stage followed by manual editing. We describe how to optimize imaging settings for this purpose, the use of the lineage-extraction software, StarryNite, and the lineage-editing software, AceTree. In addition, we describe a useful polymer bead mounting technique for C. elegans embryos that has several advantages compared with the standard agar pad mounting technique. The protocol requires about 1 h of user time spread over 2 days to generate the raw lineage, and an additional 2 or 4 h to edit the lineage to the 194- or 350-cell stage, respectively.

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This research is partly funded by the US National Institutes of Health. J.I.M. is a fellow of the Jane Coffin Childs Memorial Fund for Medical Research. Z.B. is a Damon Runyon Fellow supported by the Damon Runyon Cancer Research Foundation.

Author information


  1. Department of Genome Sciences, University of Washington Box 357730, Seattle, Washington 98195, USA.

    • John Isaac Murray
    • , Zhirong Bao
    • , Thomas J Boyle
    •  & Robert H Waterston


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Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Robert H Waterston.

Supplementary information


  1. 1.

    Supplementary Video 1

    12 cell-stage image stack. Each frame is a separate image plane starting at the top of the stack. In addition to the 12 cells at this stage, the polar body is visible as a smaller, more intense region of staining.

  2. 2.

    Supplementary Video 2

    100 cell-stage image stack.

  3. 3.

    Supplementary Video 3

    200 cell-stage image stack.

  4. 4.

    Supplementary Video 4

    350 cell-stage image stack.

  5. 5.

    Supplementary Video 5

    Full image series at plane 15. Each frame is a separate time point, 1 min apart. The images are from plane 15, near the center of the embryo.

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