A squash technique demonstrating embryonic nuclear cleavage of the nematode Caenorhabditis elegans

Abstract

A simple squash technique was developed which permits the observation of individual nuclei during embryogenesis of Caenorhabditis elegans. The technique consists of placing several two-cell stage embryos on a subbed slide into a droplet of M-9 salt buffer and incubating then in a sealed humidity chamber at 16.4°C for increasing time intervals. The embryos are then squashed, fixed, and stained with Hoechst 33258. Rate of cleavage at 25.0° is 1.8 times faster than that at 16.4°C. This yields superimposable growth curves upon correction for temperature. An initial lag in the rate of nuclear cleavage is followed by a burst of cell proliferation, which continues and then slows before 550-580 cells are produced at 4 to 5 hr at 25°C. The squash size increases with cell number and reaches a maximum at about the 400-cell stage when early morphogenesis begins. The second half of embryogenesis is characterized by histogenesis in which the cells are held more tightly together, individual nuclei become less distinct, and the squash size decreases to a minimum as a small worm is formed.