Mosaic Cell Targeting for Visualization and Functional Characterization of Single-Cell Dynamics During Zebrafish Organogenesis

Animal morphogenesis relies on complex sets of cell behavior that elicit changes in the form and function of developing tissues, including cell growth and division, movement, shape changes, and death. To characterize these dynamics in vivo and define their respective contributions to specific morphogenetic events, approaches are needed to robustly mark and track cells across their lifetime in living embryos and functionally dissect regulatory mechanisms. Here, we detail a protocol for mosaic cell targeting in developing zebrafish embryos by combining Tol2 transgenesis with single-blastomere injections, thereby enabling titration of cell-labeling density for analysis of tissues, cell collectives, and single cells. Using the developing cardiovascular system as an example, we demonstrate that this method facilitates visual isolation of single cells within intact developing organs regardless of tissue depth and accessibility. Moreover, we show how this protocol can be expanded to incorporate gene editing strategies like CRISPR-Cas9 for highly specific perturbations of cell structure and function, while preserving wild-type developmental dynamics in surrounding cells and tissues.