Differentiation without cleavage (DWC) is traditionally initiated by adding excess KCl to seawater (Loeb, 1901; Lillie, 1902) . Exposure of the oocytes to 50-100 mM excess K+ in ASW (from a 2.5 M KCl stock) for 20 min followed by washing and culture in ASW results in nearly 100% of the oocytes undergoing DWC but has no deleterious effects on fertilized eggs, so these conditions are recommended.
Alternatively, fertilized eggs can be induced to undergo DWC by cleavage arrest using 0.1 µg/ml cytochalasin B (Eckberg, 1981a) from a 1 mg/ml stock in dimethyl sulfoxide. This procedure has the advantages that the embryos are diploid and have been activated naturally, by sperm.
Finally, polyspermic eggs also undergo DWC. We do not recommend this procedure for eliciting DWC as it is impossible either to obtain a culture in which >95% of the eggs are polyspermic or to have a consistent number of male pronuclei per egg.
Eggs undergoing DWC should be allowed to develop in stationary cultures, e.g., in Syracuse or Petri dishes. Cultures should be kept as dilute as is practical, as the eggs become ameboid. When the ameboid eggs make contact with one another, they tend to clump or fuse or both. Fused specimens are less likely to develop cilia.