Development of acetylcholinesterase during embryogenesis of the ascidian Ciona intestinalis

Abstract

We have characterized the embryonic muscle cell cholinesterase of the solitary ascidian, Ciona intestinalis (L.). The effects of selective enzyme inhibitors and the inhibition of enzyme activity at high concentrations of substrate suggest that the muscle cell enzyme is an acetylcholinesterase (E.C. 3.1.1.7). After gastrulation and before hatching, acetylcholinesterase activity increased 35- to 40-fold; after hatching (18 hours postfertilization) this activity continued to increase, leveling off at about 36 hours of development. Histochemical observations showed that before hatching acetylcholinesterase was located principally in the muscle cells of the tail and, after hatching, it began to develop in cells of the adult musculature and brain. Inhibition of protein syntnesis by puromycin and of RNA synthesis by actinomycin D, suggest that both protein and RNA synthesis were required for the increase in acetylcholinesterase activity observed in unhatched embryos. Although the continued increase in enzyme activity duirng embryonic development was sensitive to puromycin at all times tested, the actinomycin D sensitivity of this increase was restricted to a discrete time that was completed by about 11 hours of development.

Purification of Acetylcholinesterase from Sea Urchin (Hemicentrotus pulcherrimus) Embryos by Affinity Chromatography

“…The experiments using puromycin and Actinomycin D suggest that new RNA synthesis which begins sometime between the early and late gastrula stages is needed for the appearance of AchE activity (3,19,20,21). SATOH and IKEGAMI has suggested that the time of initiation of the enzyme development is determined by the clock mechanism closely associated with the DNA replication cycle (4, 5).…”

Development of translationally active mRNA for larval muscle acetylcholinesterase during ascidian embryogenesis.

“…Actinomycin D treatment begun before early gastrulation prevents enzyme formation but has less effect on the amount of enzvme activity produced the later it is added, finally causing no response when added after the middle tail formation stage (4,8 (12). Translationally active acetylcholinesterase mRNA was not found in RNA prepared from embryos before the 64-cell stage, but it was present in increasing amounts in RNA isolated from embryos between late gastrula and middle tail formation stages.…”

“…Actinomycin D treatment begun before early gastrulation prevents enzyme formation but has less effect on the amount of enzvme activity produced the later it is added, finally causing no response when added after the middle tail formation stage (4,8 (11) have used such an assay in Xenopus oocytes to study acetylcholinesterase mRNA in rat brain and Torpedo electric organ RNA preparations. We modified their technique to include an immunoprecipitation step with antibody prepared against purified ascidian acetylcholinesterase (12).…”

“…The enzyme is first seen histochemically in muscle rudiments of the ascidian neurula stage (1-3) and does not occur in other tissues at prehatching stages of embryonic development (4). Quantitative measurements reveal a very large increase in enzyme activity during the time from neural plate formation to hatching, an increase that is sensitive to inhibitors of protein and RNA synthesis (4). The results of various studies, both classical and recent, suggest a role for egg cytoplasmic determinants in regulating the occurrence of this acetylcholinesterase as well as other features of larval muscle differentiation (5).…”

Biosynthesis and secretion of catalytically active acetylcholinesterase in Xenopus oocytes microinjected with mRNA from rat brain and from Torpedo electric organ.

“…Meedel and Whittaker (17) have shown that increases in AcChoEase activity during the early stages of embryogenesis of the ascidian, Ciona intestinalis, are inhibited by actinomycin D and have suggested a possible role for mRNA production in the ontogeny of AcChoEase.…”