Analysis of 4D DIC microscopic data to determine cell contacts in Caenorhabditis elegans embryos.
The Caenorhabditis elegans embryo is particularly amenable to microscopy and embryological studies because of its short developmental time, transparent shell, and nonpigmented cells. Identification of cell contacts is important for understanding how cells within the embryo can be polarized by their neighbors. This protocol describes a technique for identifying cell membranes and potential contacts between different blastomeres in the embryo and for following those contacts through development. This protocol involves manual segmentation of the membrane of each blastomere from four-dimensional (4D) differential interference contrast (DIC) data sets. Although this technique is described for use with C. elegans embryos, it will work with any 4D DIC data set. The recent development of a pleckstrin homology (PH)-domain tagged::GFP expressed in C. elegans embryos simplifies this analysis; however, many organisms lack appropriate green fluorescent protein (GFP) transgenes. The use of the GFP transgene improves on laser-mediated techniques for labeling embryonic cell membranes with fluorescent dye. Such methods require that a laser be used to carefully permeabilize the eggshell for entry of the dye into the embryo. The technique described in this protocol requires only easily obtainable 4D DIC data sets.
Timothy
T
Walston
Jeff
J
Hardin
D000818:Animals; D017173:Caenorhabditis elegans; D002448:Cell Adhesion; D002465:Cell Movement; D015509:Developmental Biology / Q000379:methods; D004625:Embryo, Nonmammalian / Q000033:anatomy & histology* / Q000166:cytology*; D021621:Imaging, Three-Dimensional / Q000379:methods*; D008857:Microscopy, Interference / Q000379:methods*; D018715:Microscopy, Video / Q000379:methods*