Induced neural‐type differentiation in the cleavage‐arrested blastomere isolated from early ascidian embryos.

Abstract

SUMMARY1. Isolated blastomeres and pairs of blastomeres from 8-cell embryos of Halocynthia roretzi and Halocynthia aurantium were cleavage-arrested with cytochalasin B and cultured. Their differentiation was examined in terms of membrane excitability, immunoreactivity to an epidermis-specific monoclonal antibody (2C5), and the presence of acetylcholinesterase.2. The blastomeres that showed epidermal-type differentiation had Ca2+-dependent action potentials and membrane currents, and immunoreactivity to 2C5. The blastomeres that showed neural-type differentiation had Na+-, Ca2+-and TEAsensitive delayed K+ channels, and lacked immunoreactivity to 2C5.3. Cleavage-arrested anterior-animal blastomeres, a42, when cultured in isolation from an 8-cell embryo, differentiated exclusively into epidermal-type cells. However, when cultured in contact with anterior-vegetal blastomeres, A41, they mostly showed neural-type differentiation (seventeen out of twenty-four cells in H. roretzi).4. Reduction of the cytochalasin B concentration enhanced neural-type development of a4-2 blastomeres in contact with A4 1 blastomeres in H. aurantium, possibly by tightening the physical contact between the blastomeres.5. When a cleavage-arrested and isolated a4-2 blastomere was treated with 2%pronase at 10°C for 15 min at the time when sister control embryos reached the 32-cell stage, the blastomere underwent neural-type differentiation in a manner identical to that of a4 2 blastomeres contacted by A4 1 cells. 6. The period during which neural-type differentiation of a4 2blastomeres could be induced by treatment with pronase was from the 8-cell to the 110-cell stage. At the late gastrula stage neural-type differentiation of a4 2 blastomeres was not induced by pronase. The effective period for neural-type differentiation of a4 2 blastomeres in contact with A4 1 cells was between the 64-cell stage and late gastrula stage.Competence of the a4 2 blastomere to undergo neural-type differentiation decreased during gastrula stages, while the inducing ability of the A4-1 blastomere lasted longer.7. In a few cases the posterior-animal blastomere, b4-2, could also be induced to undergo neural-type differentiation after contact with A4-1 cells or after pronase treatment.8. The appearance of Na+ spikes in a42 blastomeres in contact with A4-1 cells was MS 7761 H. OKADO AND K. TAKAHASHI considered a manifestation of neural induction, similar in principle to the induction of ectoderm by the chorda-mesoderm in higher vertebrates.

Neural induction suppresses early expression of the inward‐rectifier K+ channel in the ascidian blastomere.

“…Previous work in our laboratory (Okado & Takahashi, 1990b) has shown that neural differentiation was induced by cell contact with the blastomere, A4-1, which contains the presumptive notochordal area, only if contact occurred before the late gastrula stage; the same critical period existed when neural differentiation was induced by protease (Okado & Takahashi, 1990b, 1993.…”

“…From previous studies (Okamura & Shidara, 1990a, b), it is known that most of the inward current is generated through the biexponentially inactivating-type Na+ channel, which is distinct from the fast-inactivating Na+ channel expressed at earlier stages. Long-lasting Ca2+ currents were also expressed in neural differentiation (Hirano & Takahashi, 1984;Okado & Takahashi, 1990b), although, in the solution we used, the contribution of the long-lasting-type Ca2+ current to the inward current was negligible, compared with that of the Na+ current. Outward currents were the currents through a TEA-sensitive type of the delayedrectifier K+ channel which can be distinguished from the channel of the musculartype cell on the basis of both kinetics and TEA sensitivity (Shidara & Okamura, 1991).…”

“…5A) and as large as that in protease-induced neurally differentiating cells. If a4-2 blastomeres are contacted with A4-1 blastomeres or treated with proteases after the late gastrula stage, neural induction does not occur and a4-2 blastomeres differentiate into epidermal-type cells (Okado & Takahashi, 1990b;1993). When the blastomere was treated with subtilisin at 15 h. later than the sensitive period for neural induction, the cell differentiated into an epidermal-type cell and exhibited the same level of inward-rectifier K+ channel current as that of cells not treated with protease (U in Fig.…”

“…Thus the suppression of transcription of the inward-rectifier K+ channel observed in the present experiment is not a factor for later differentiation but is a result of the determination of the neural cell type. (Okado & Takahashi, 1990b) and were cultured until 20 h after fertilization. The peak value of the inward current during voltage pulses stepped to -175 mV was measured.…”

“…In the later preparation, blastomeres cleavage-arrested by cytochalasin B can express tissue-specific markers corresponding to their cell fates, as first demonstrated in muscle lineage cells (Whittaker, 1973). The anterior ectodermal blastomere a4-2, isolated from the eight-cell embryo, will differentiate either into an epidermal-type cell or a neural-type cell under cleavage arrest, depending on the culture conditions (Okado & Takahashi, 1990b). If cultured in isolation under cleavage arrest, it becomes epidermis as evidenced by reactivity with epidermal-specific monoclonal antibodies and by the appearance of a long-lasting type Ca2+ current.…”