RESEARCH ARTICLE Molecular Reproduction & Development (2015) Multispectral Labeling of Embryonic Cells with Lipophilic Carbocyanine Dyes MARIA VOLNOUKHIN y AND BRUCE P. BRANDHORST* Department of Molecular Biology and Biochemistry, University Drive Simon Fraser University, Burnaby, British Columbia, Canada SUMMARY Incubation of hatched Strongylocentrotus purpuratus sea urchin embryos or larvae with suspensions of the carbocyanine dyes DiI, DiO, and DiD resulted in the random labeling of membranes of some ectodermal epithelial cells and blastocoelar cells, producing a range of differentially colored cells that can be tracked during develop- ment. Simultaneous application of soluble Vybrant 1 preparations of the three dyes resulted in similar labeling of each cell. Dye labeling of the ectoderm was nearly eliminated by deciliation and some ciliated squamous epithelial cells adjacent to labelled cells were refractory to Vybrant 1 dye uptake irrespective of concentration or duration of treatment, together suggesting local variation in the properties of cell membranes or cilia. Furthermore, single cells possessing distinctive morphological features were detected. Mol. Reprod. Dev. 2015. ß 2015 Wiley Periodicals, Inc. Received 16 September 2014; Accepted 4 March 2015  Corresponding author: Department of Molecular Biology and Biochemistry 8888 University Drive Simon Fraser University Burnaby, BC Canada V3H3K3. E-mail: brandhor@sfu.ca y Deceased. Grant sponsor: Natural Sciences and Engineering Research Council of Canada Discovery Grant; Grant number: 45977 Published online in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/mrd.22477 INTRODUCTION Marking blastomeres by the application of particles or organic dyes onto living embryos is critical for the genera- tion of cell-lineage diagrams and fate maps in animals (reviewed by Kretzschmar and Watt, 2012). More recently, injection of dyes into embryonic blastomeres or the expres- sion of marker proteins have been used to track cell lineages and fates; such analysis has been further refined by the expression of fluorescent reporter proteins driven by specific transgenic promoters. Combined with modern fluorescence microscopic techniques, these cell-labeling approaches allow for the tracking of patches or clones of cells in live embryos over time and space, although individ- ual cells can be difficult to distinguish. The development of Brainbow for mouse embryos, for example, provides dis- tinctive multicolor labeling of adjacent neural cells, whose individual fates and spatial interactions can then be tracked using fluorescence microscopy (Livet et al., 2007). Brain- bow depends on Cre-mediated combinatorial expression of multiple recombinant fluorescent proteins in transgenic embryos; as such, this method has been modified and extended to provide multispectral marking of a range of cell types in Drosophila (Boulina et al., 2013; Worely et al., 2013), mice (Snippert et al., 2010), and zebrafish (Gupta and Poss, 2012; Pan et al., 2013), allowing clonal analyses at various developmental stages and facilitating axonal tracking in transparent embryos and larvae. While multispectral labeling of embryonic cells based on the induced recombination of fluorescent-protein trans- genes is a powerful tool, it depends on the creation and ß 2015 WILEY PERIODICALS, INC.