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

Abstract

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 ce...

Cell contact induces multiple types of electrical excitability from ascidian two-cell embryos that are cleavage arrested and contain all cell fate determinants

“…Since the 2C cell is naturally fated to become a half body, and thus its totipotency is similar to that of a fertilized egg, we expected the 2C cell to respond to various inductive stimuli and become any type of differentiated cells in a controllable way. The inductive stimulus tested was the cell contact with one of four cell types from a cleavage-arrested Halocynthia eight-cell embryo, in which we have demonstrated that major differentiation commitment to either epidermal or neural or notochordal or muscular type is initiated (32,33,51). This situation is similar to that of mammalian embryonic stem cells, differentiation of which is most successfully controlled when they were transplanted into early embryonic tissues (56).…”

“…These currents are to charge the neighboring cell membrane capacity from the voltage-clamped cell through gap junctional communication (GJC). The ramp potential change had a gradient of 2.0 mV/ms and was applied from a holding potential of Ϫ75 mV for 10 ms, keeping the test potential changes within Ϯ 20 mV until the end of the slope, as described previously (51). Care was taken not to evoke potential-dependent conductances.…”

“…We confirmed the final differentiation types of the cells by using electrophysiological techniques or by observing their morphological features. Differentiation types of 2C cells or contacted cell pairs were identified by their characteristic ionic currents recorded using conventional voltageclamp procedures (12,51). Under voltage-clamp conditions Na ϩ and A-type K ϩ , and/or delayed rectifier K ϩ currents represent neuronally differentiated cells, and inward rectifier K ϩ and Ca 2ϩ currents accompanied with neither Na ϩ nor delayed rectifier K ϩ currents represent epidermally differentiated cells.…”

“…All four types of cell pairs and a4.2-A4.1 cell pairs were separated from eight-cell embryos with a fine glass needle. After preparation, 2C cells from two-cell embryos and cell pairs from eight-cell embryos were kept in filtered seawater containing 2.0 mg/l cytochalasin B for 1 h. They were then transferred into seawater containing 0.2 mg/l cytochalasin B and placed on 1.0% agarosecoated microwells as described previously (51). Various sets of three cells (triplets) were then prepared by manipulating a pair of a4.2 cells (2C-aa triplet) or a pair of A4.1 cells (2C-AA triplet) or a pair of b4.2 cells (2C-bb triplet) or a pair of B4.1 cells (2C-BB triplet) in contact with a 2C cell in a microwell by using a fine glass needle at 6 -8 DHs of the 2C cell, and then cultured at 9.0 Ϯ 0.5°C.…”

“…The a4.2 cell has both neural and epidermal fates, and, if it is contacted and induced by the presumptive notochordal A4.1 cell in the cleavage-arrested pair, expresses neuronal ion channels and shows neuronal excitability that can be verified electrophysiologically or becomes the default epidermal cell (32,33,35). We also reported previously that the cleavagearrested anterior quadrant cell, A3, which is derived from a four-cell-stage embryo and is the parent cell of a4.2 and A4.1 cells in an eight-cell embryo, is multifated and can differentiate into any of three cell types (anterior neural, posterior neural, and epidermal cells) when it is contacted by various types of blastomeres from a cleavage-arrested eight-cell embryo or when either bFGF or subtilisin was applied (51).…”