Cleavage-arrested cell triplets from ascidian embryo differentiate into three cell types depending on cell combination and contact timing.

During early ascidian development, which is a prototype of early vertebrate development, anterior neuroectoderm cells (a4.2) from the eight-cell embryo are destined to become anterior neural structures including the brain vesicle, while presumptive notochordal neural cells (A4.1) become larval posterior neural structures including motoneurons. Whereas, an anterior quadrant cell (A3) of the four-cell embryo, from which both anterior neuroectoderm (a4.2) and notochordal neural cells (A4.1) are derived, has both fates. Cleavage-arrested cell triplets were prepared from the anterior quadrant cell and a pair of anterior neuroectoderm cells (A3-aa triplet) or a pair of presumptive notochordal neural cells (A3-AA triplet), and cultured in contact. Differentiation of cells in the triplet was determined electrophysiologically by observing cell type-specific currents. In the A3-aa triplet, when two neuroectoderm cells and an anterior quadrant cell were prepared from the same batch of embryos, all three cells in the triplet developed into neuronal cells in 60 % of cases, but in 40 % of cases all of them differentiated into epidermal cells. However, when the batch of embryos from which neuroectoderm cells were prepared was fertilized 3 h later than that from which the anterior quadrant cell was prepared all three cells in the triplet consistently became neuronal cells. In contrast, when the batch of embryos from which neuroectoderm cells were prepared was fertilized 3 h earlier, all three cells became epidermal. In the A3-AA triplet no switching of differentiation occurred and all three cells in the triplet differentiated into neuronal cells, although the amplitude of inward current was often small. In neuralized A3-aa triplets the spikes in the anterior quadrant cell were characteristically small in amplitude and brief in duration, suggesting the presence of A-currents, which is a characteristic feature of posterior neuronal differentiation. In contrast, the spikes in the anterior neuroectoderm cells were large in amplitude and long in duration, chracteristic to the anterior neuronal type. The majority of single isolated anterior quadrant cells became non-excitable. However, the minority was apparently autonomously neuralized to become the posterior neuronal type. In neuralized A3-AA triplets, the majority of anterior quadrant cells was induced to become the anterior neuronal type. When isolated anterior quadrant cells were neuralized with subtilisin, a protease, they also predominantly became the anterior neuronal type. While, in medium containing a fibroblast growth factor posterior neuralization of isolated anterior quadrant cells was facilitated, but the anterior neuronal type, although minor, appeared anew. These observations indicate that the multiple fates of the anterior quadrant cell expressed in vivo were effectively reproduced in this experimental condition at the single cell level. Interactive differentiation in this triplet system recapitulates not only fundamental neural induction of ascidian neuroectoderm cells, but also functional and positional specificity within the neuronal group.