Using a differential interference contrast (DIC) microscope, which gives depth and contrast to transparent specimens, Sulston and Horvitz directly observed embryonic and post-embryonic development of the nematode Caenorhabditis elegans (C. elegans). Sulston and later Bob Horvitz identified the precise developmental map of C. elegans, a journey from fertilized zygote to an adult worm, and mapped the first developmental lineage of a multicellular organism . While analyzing the invariant C. elegans lineage, they made an intriguing observation, the temporally and developmentally regulated “programmed” cell death. It was evident that at specific developmental timing and based on certain developmental requirements, a number of cells died and disappeared surprisingly quickly. They interpreted the PCD as a series of morphological changes, especially in the cell nucleus, including bleb formation, refractivity increment, and finally cellular shrinkage and disappearance . The initiation and execution of apoptosis occur in three main steps: cells have to decide to undergo programmed cell death, the execution of apoptosis has to be regulated, and, finally, cells undergoing apoptosis have to be marked and eliminated by phagocytosis. (Video 1)
C. elegans N2 Bristol strain was used to capture live developmental apoptosis. Briefly, worms were propagated on a Nematode Growth Medium (NGM) plate seeded with OP50 (Escherichia coli). Ten microliters (10 μl) of M9 buffer were added to a coverslip. Early adult worms were picked and placed in the drop M9 buffer. Worms were cut at their midbodies by a needle to isolate embryos. Early-stage embryos were selected and placed on a Poly-l-lysine coated slide. The slide was then mounted and sealed to avoid evaporation. The Leica Thunder Imager microscope, equipped with DIC condenser prisms, was used to capture 50 z stacks (each 1.83 μm) every 90 seconds for 4 hours, during which several apoptotic cells were detected. Representative images of a corresponding time, focused on an apoptotic cell, were selected for the time-lapse movie.
This video is adapted from 10.3390/ijms222212466