The concentration of ACh exhibits distinct peaks during early cleavage divisions, but the major, sustained increases occur after the beginning of gastrulation, in tandem with transcription of zygotic genes and attendant rises in choline acetyltransferase, the enzyme that synthesizes ACh (Buznikov et al.
DMAE added to the embryos at the two-cell stage in concentrations of 100-400 [micro]M did not alter cleavage divisions or blastulation, but at very high concentrations (600-800 [micro]M), cleavages were inhibited (Figures 2 and 3).
Although cleavage divisions were affected only at very high DMAE concentrations, embryos treated with lower concentrations at the two-cell stage subsequently developed malformations (Figure 3): overabundance of mesenchyme-like cells that accumulated first in the vegetal half of the blastocoel, and later occupied all of the blastocoel, blocking or inhibiting gastrulation; absence of a second pair of arms in mid-pluteus, or sometimes shortening or absence of one arm of the first pair (Figure 3H).
In contrast to the lack of protection offered by ACh or choline for the effects of DMAE on cleavage divisions, we found that either agent, introduced 10-15 min before, simultaneously with, or 30-60 min after DMAE, provided protection against the delayed-onset disruption of the later developmental events (Figure 4).
On the other hand, it is clear from previous work that ACh does play a role in triggering cleavage divisions in sea urchins (Buznikov et al.
Here, we found a dose-dependent effect of DMAE that operated with a distinctly lower threshold than for inhibition of cleavage divisions, with the onset of anomalies shifting to later and later stages as the dose was lowered.