While in toto imaging and image analysis methods have advanced the study of multicellular phenomena in development at single-cell resolution, little progress has been made in the design of tools to perturb complex tissues with comparable spatial and temporal resolution. Mutagenesis and RNAi are powerful tools for dissecting the genetic basis of developmental phenomena, yet neither offers the specificity or the temporal control required for dissecting the roles played by individual cells in coordinating developmental processes. Classical single cell perturbation techniques such as laser cell ablation and newer technologies built around photoactivatable reagents offer significant promise in filling this need. Their use to-date, however, remains limited by the challenge of reliably identifying target cells.
We have developed a platform for the real-time segmentation and tracking of single cells in 3D tissues to enable reproducible single cell perturbations at high throughput and without a need for cell-specific markers. ShootingStar can currently maintain real-time throughput on desktop hardware for C. elegans datasets until the onset of twitching and for D. rerio and D. melanogaster embryos containing on the order of thousands of cells. We have benchmarked this platform by the successive and parallel ablations of neural progenitors and neuronal support cells in the embryo of C. elegans. Specifically, we have performed parallel ablations of all 6 GLR cells to probe their role in nerve ring positioning. Ablated embryos exhibit no gross defects in nerve ring morphology or positioning but hatch late with the nerve ring exhibiting a strong anterior shift and evidence of defasciculation (N=3). While anatomical evidence has suggested that the GLR's play a role in nerve ring assembly, our observations instead suggest a role in maintaining nerve ring positioning during elongation and pharyngeal morphogenesis. While our applications to-date have focused on laser cell ablation, the architecture of ShootingStar allows for a diverse set of perturbations such as single-cell heatshock or the use of photoconvertible fluorophores to selectively label single cells on-demand. Current efforts are directed towards demonstrating these applications as well as the parallelization of cell tracking to enable real-time lineage tracing later in development for larger model organisms. The long-term goal of our work is to establish ShootingStar as a generalized platform for the study of single cell behaviors in complex multicellular environments in vitro and in vivo.