What a couple of dimensions can do for you: Comparative developmental studies using 4D microscopy--examples from...

The development of an organism consists of processes occurring in space and time. To analyze this 4-dimensional development in embryogenesis, an appropriate method should be chosen. We present here a sophisticated method, 4D microscopy (3D time-lapse microscopy), initially developed to analyze the cell lineage of wild-type and mutant embryos of the nematode Caenorhabditis elegans. Our method records the entire development of an embryo and allows detailed analyses of events such as cleavage, cell migration, cell death (apoptosis), and cell differentiation during development. The 4D microscopy system has 3 main parts: a motorized microscope, trigger software, and a database that facilitates the analysis of recordings. Adopting the 4D microscopy technique for uses beyond the analysis of C. elegans makes it possible to discern the cell lineage of other small embryos. Our method fills a gap in the study of the development of diverse organisms that are impossible to observe with fluorescent labeling techniques using single blastomeres. The use of this technique to investigate the development of organisms such as tardigrades, acoelomorphs, rotifers, and gastrotrichs provides fresh insight into the evolution of developmental processes and the phylogenetic relationships between such taxa. Using tardigrade development as an example, we demonstrate that the use of 4D microscopy can reveal new characters and corroborate or disapprove old characters. We discuss the results in the light of recent phylogenetic hypotheses regarding the Arthropoda and their probable sister group, the Cycloneuralia, which together form the Ecdysozoa.